Mediaccedilatildeo CRFeacutergica do comportamento defensivo de camundongos

Ricardo L Nunes-de-Souza (souzarnfcfarunespbr) Lab Neuropsicofarmacologia Faculdade de Ciecircncias Farmacecircuticas UNESP

1 Gramado 04 de setembro de 2015

Aversive Stimuli

(innate learned nociceptive)

Defense

System +

Recuperative Behavior eg grooming licking

Defensive Behavior eg Avoidance Escape Defensive upright Freezing

+

Modified from Siegfried et al Neurosci amp Biobehav Rev 1990

The role of pain and defense systems

-

2

Pain System (Nociceptive Intensity

Detector)

+

+

Blanchard amp Blanchard 1990

Aversive Stimuli

(innate learned nociceptive)

Defense

System

+

Recuperative Behavior eg grooming licking

Defensive Behavior eg Avoidance Escape Defensive upright Freezing

+

Modified from Siegfried et al Neurosci amp Biobehav Rev 1990

The role of pain and defense systems

-

3

Pain System (Nociceptive Intensity

Detector)

+

+

Walter Rudolf Hess 1881-1973

4

Hormonal Changes eg ACTH

Brain Defense System

Nociceptive Stimuli

Innate Danger Stimuli

Learned Danger Stimuli

Periaqueductal Gray

Hypothalamus

Pituitary

Fight amp Flight

Freezing

Neurovegetative

Activation

Antinociception

Amygdala

(Modified from Graeff amp Guimaratildees 2012)

Chemical or electrical

stimulation

5

6

Social Conflict Test Intruder-Resident Model

Resident (aggressive)

Intruder (defensive upright)

Tail-flick test

0

2

4

6

8

Intruder

Resident

Tail-

flic

lk la

ten

cy (

sec)

0 20 40 60 80 100 Bites

Modified from Miczek et al 1982

Stress-induced analgesia (or antinociception)

7

Mecanismos e Sistemas Envolvidos na Modulaccedilatildeo da Analgesia

induzida pelo Estresse

8

Hormonal Changes eg ACTH

Brain Defense System

Nociceptive Stimuli

Innate Danger Stimuli

Learned Danger Stimuli

Periaqueductal Gray

Hypothalamus

Pituitary

Fight amp Flight Freezing

Neurovegetative Activation

Antinociception

Amygdala

(Modified from Graeff amp Guimaratildees 2012) 9

Eur J Pharmacol 1988 Sep 23154(3)271-6 The NMDA receptor central role in pain inhibition in rat periaqueductal gray Jacquet YF Author information Abstract An injection of the excitatory amino acid analogue N-methyl-D-aspartate (NMDA) in the rat periaqueductal gray resulted in potent analgesia A prior injection of the NMDA antagonist (-)-2-amino-7-phosphonoheptanoate (D-AP7) antagonized this action indicating a receptor-mediated action NMDA given with morphine potentiated the morphine analgesia while D-AP7 blocked morphine analgesia These results delineate for the first time a functional role for the NMDA receptor in the control of pain in the mammalian central nervous system

Os receptores NMDA da PAG poderiam estar envolvidos na analgesia induzida pelo estresse

NMDA

10

Aversive Stimuli

(innate learned nociceptive)

Defense

System +

Recuperative Behavior eg grooming licking

Defensive Behavior eg Avoidance Escape Defensive upright Freezing

+

Modified from Siegfried et al Neurosci amp Biobehav Rev 1990

The role of pain and defense systems

-

2

Pain System (Nociceptive Intensity

Detector)

+

+

Blanchard amp Blanchard 1990

Aversive Stimuli

(innate learned nociceptive)

Defense

System

+

Recuperative Behavior eg grooming licking

Defensive Behavior eg Avoidance Escape Defensive upright Freezing

+

Modified from Siegfried et al Neurosci amp Biobehav Rev 1990

The role of pain and defense systems

-

3

Pain System (Nociceptive Intensity

Detector)

+

+

Walter Rudolf Hess 1881-1973

4

Hormonal Changes eg ACTH

Brain Defense System

Nociceptive Stimuli

Innate Danger Stimuli

Learned Danger Stimuli

Periaqueductal Gray

Hypothalamus

Pituitary

Fight amp Flight

Freezing

Neurovegetative

Activation

Antinociception

Amygdala

(Modified from Graeff amp Guimaratildees 2012)

Chemical or electrical

stimulation

5

6

Social Conflict Test Intruder-Resident Model

Resident (aggressive)

Intruder (defensive upright)

Tail-flick test

0

2

4

6

8

Intruder

Resident

Tail-

flic

lk la

ten

cy (

sec)

0 20 40 60 80 100 Bites

Modified from Miczek et al 1982

Stress-induced analgesia (or antinociception)

7

Mecanismos e Sistemas Envolvidos na Modulaccedilatildeo da Analgesia

induzida pelo Estresse

8

Hormonal Changes eg ACTH

Brain Defense System

Nociceptive Stimuli

Innate Danger Stimuli

Learned Danger Stimuli

Periaqueductal Gray

Hypothalamus

Pituitary

Fight amp Flight Freezing

Neurovegetative Activation

Antinociception

Amygdala

(Modified from Graeff amp Guimaratildees 2012) 9

Eur J Pharmacol 1988 Sep 23154(3)271-6 The NMDA receptor central role in pain inhibition in rat periaqueductal gray Jacquet YF Author information Abstract An injection of the excitatory amino acid analogue N-methyl-D-aspartate (NMDA) in the rat periaqueductal gray resulted in potent analgesia A prior injection of the NMDA antagonist (-)-2-amino-7-phosphonoheptanoate (D-AP7) antagonized this action indicating a receptor-mediated action NMDA given with morphine potentiated the morphine analgesia while D-AP7 blocked morphine analgesia These results delineate for the first time a functional role for the NMDA receptor in the control of pain in the mammalian central nervous system

Os receptores NMDA da PAG poderiam estar envolvidos na analgesia induzida pelo estresse

NMDA

10

Aversive Stimuli

(innate learned nociceptive)

Defense

System

+

Recuperative Behavior eg grooming licking

Defensive Behavior eg Avoidance Escape Defensive upright Freezing

+

Modified from Siegfried et al Neurosci amp Biobehav Rev 1990

The role of pain and defense systems

-

3

Pain System (Nociceptive Intensity

Detector)

+

+

Walter Rudolf Hess 1881-1973

4

Hormonal Changes eg ACTH

Brain Defense System

Nociceptive Stimuli

Innate Danger Stimuli

Learned Danger Stimuli

Periaqueductal Gray

Hypothalamus

Pituitary

Fight amp Flight

Freezing

Neurovegetative

Activation

Antinociception

Amygdala

(Modified from Graeff amp Guimaratildees 2012)

Chemical or electrical

stimulation

5

6

Social Conflict Test Intruder-Resident Model

Resident (aggressive)

Intruder (defensive upright)

Tail-flick test

0

2

4

6

8

Intruder

Resident

Tail-

flic

lk la

ten

cy (

sec)

0 20 40 60 80 100 Bites

Modified from Miczek et al 1982

Stress-induced analgesia (or antinociception)

7

Mecanismos e Sistemas Envolvidos na Modulaccedilatildeo da Analgesia

induzida pelo Estresse

8

Hormonal Changes eg ACTH

Brain Defense System

Nociceptive Stimuli

Innate Danger Stimuli

Learned Danger Stimuli

Periaqueductal Gray

Hypothalamus

Pituitary

Fight amp Flight Freezing

Neurovegetative Activation

Antinociception

Amygdala

(Modified from Graeff amp Guimaratildees 2012) 9

Eur J Pharmacol 1988 Sep 23154(3)271-6 The NMDA receptor central role in pain inhibition in rat periaqueductal gray Jacquet YF Author information Abstract An injection of the excitatory amino acid analogue N-methyl-D-aspartate (NMDA) in the rat periaqueductal gray resulted in potent analgesia A prior injection of the NMDA antagonist (-)-2-amino-7-phosphonoheptanoate (D-AP7) antagonized this action indicating a receptor-mediated action NMDA given with morphine potentiated the morphine analgesia while D-AP7 blocked morphine analgesia These results delineate for the first time a functional role for the NMDA receptor in the control of pain in the mammalian central nervous system

Os receptores NMDA da PAG poderiam estar envolvidos na analgesia induzida pelo estresse

NMDA

10

Walter Rudolf Hess 1881-1973

4

Hormonal Changes eg ACTH

Brain Defense System

Nociceptive Stimuli

Innate Danger Stimuli

Learned Danger Stimuli

Periaqueductal Gray

Hypothalamus

Pituitary

Fight amp Flight

Freezing

Neurovegetative

Activation

Antinociception

Amygdala

(Modified from Graeff amp Guimaratildees 2012)

Chemical or electrical

stimulation

5

6

Social Conflict Test Intruder-Resident Model

Resident (aggressive)

Intruder (defensive upright)

Tail-flick test

0

2

4

6

8

Intruder

Resident

Tail-

flic

lk la

ten

cy (

sec)

0 20 40 60 80 100 Bites

Modified from Miczek et al 1982

Stress-induced analgesia (or antinociception)

7

Mecanismos e Sistemas Envolvidos na Modulaccedilatildeo da Analgesia

induzida pelo Estresse

8

Hormonal Changes eg ACTH

Brain Defense System

Nociceptive Stimuli

Innate Danger Stimuli

Learned Danger Stimuli

Periaqueductal Gray

Hypothalamus

Pituitary

Fight amp Flight Freezing

Neurovegetative Activation

Antinociception

Amygdala

(Modified from Graeff amp Guimaratildees 2012) 9

Eur J Pharmacol 1988 Sep 23154(3)271-6 The NMDA receptor central role in pain inhibition in rat periaqueductal gray Jacquet YF Author information Abstract An injection of the excitatory amino acid analogue N-methyl-D-aspartate (NMDA) in the rat periaqueductal gray resulted in potent analgesia A prior injection of the NMDA antagonist (-)-2-amino-7-phosphonoheptanoate (D-AP7) antagonized this action indicating a receptor-mediated action NMDA given with morphine potentiated the morphine analgesia while D-AP7 blocked morphine analgesia These results delineate for the first time a functional role for the NMDA receptor in the control of pain in the mammalian central nervous system

Os receptores NMDA da PAG poderiam estar envolvidos na analgesia induzida pelo estresse

NMDA

10

Hormonal Changes eg ACTH

Brain Defense System

Nociceptive Stimuli

Innate Danger Stimuli

Learned Danger Stimuli

Periaqueductal Gray

Hypothalamus

Pituitary

Fight amp Flight

Freezing

Neurovegetative

Activation

Antinociception

Amygdala

(Modified from Graeff amp Guimaratildees 2012)

Chemical or electrical

stimulation

5

6

Social Conflict Test Intruder-Resident Model

Resident (aggressive)

Intruder (defensive upright)

Tail-flick test

0

2

4

6

8

Intruder

Resident

Tail-

flic

lk la

ten

cy (

sec)

0 20 40 60 80 100 Bites

Modified from Miczek et al 1982

Stress-induced analgesia (or antinociception)

7

Mecanismos e Sistemas Envolvidos na Modulaccedilatildeo da Analgesia

induzida pelo Estresse

8

Hormonal Changes eg ACTH

Brain Defense System

Nociceptive Stimuli

Innate Danger Stimuli

Learned Danger Stimuli

Periaqueductal Gray

Hypothalamus

Pituitary

Fight amp Flight Freezing

Neurovegetative Activation

Antinociception

Amygdala

(Modified from Graeff amp Guimaratildees 2012) 9

Eur J Pharmacol 1988 Sep 23154(3)271-6 The NMDA receptor central role in pain inhibition in rat periaqueductal gray Jacquet YF Author information Abstract An injection of the excitatory amino acid analogue N-methyl-D-aspartate (NMDA) in the rat periaqueductal gray resulted in potent analgesia A prior injection of the NMDA antagonist (-)-2-amino-7-phosphonoheptanoate (D-AP7) antagonized this action indicating a receptor-mediated action NMDA given with morphine potentiated the morphine analgesia while D-AP7 blocked morphine analgesia These results delineate for the first time a functional role for the NMDA receptor in the control of pain in the mammalian central nervous system

Os receptores NMDA da PAG poderiam estar envolvidos na analgesia induzida pelo estresse

NMDA

10

6

Social Conflict Test Intruder-Resident Model

Resident (aggressive)

Intruder (defensive upright)

Tail-flick test

0

2

4

6

8

Intruder

Resident

Tail-

flic

lk la

ten

cy (

sec)

0 20 40 60 80 100 Bites

Modified from Miczek et al 1982

Stress-induced analgesia (or antinociception)

7

Mecanismos e Sistemas Envolvidos na Modulaccedilatildeo da Analgesia

induzida pelo Estresse

8

Hormonal Changes eg ACTH

Brain Defense System

Nociceptive Stimuli

Innate Danger Stimuli

Learned Danger Stimuli

Periaqueductal Gray

Hypothalamus

Pituitary

Fight amp Flight Freezing

Neurovegetative Activation

Antinociception

Amygdala

(Modified from Graeff amp Guimaratildees 2012) 9

Eur J Pharmacol 1988 Sep 23154(3)271-6 The NMDA receptor central role in pain inhibition in rat periaqueductal gray Jacquet YF Author information Abstract An injection of the excitatory amino acid analogue N-methyl-D-aspartate (NMDA) in the rat periaqueductal gray resulted in potent analgesia A prior injection of the NMDA antagonist (-)-2-amino-7-phosphonoheptanoate (D-AP7) antagonized this action indicating a receptor-mediated action NMDA given with morphine potentiated the morphine analgesia while D-AP7 blocked morphine analgesia These results delineate for the first time a functional role for the NMDA receptor in the control of pain in the mammalian central nervous system

Os receptores NMDA da PAG poderiam estar envolvidos na analgesia induzida pelo estresse

NMDA

10

Social Conflict Test Intruder-Resident Model

Resident (aggressive)

Intruder (defensive upright)

Tail-flick test

0

2

4

6

8

Intruder

Resident

Tail-

flic

lk la

ten

cy (

sec)

0 20 40 60 80 100 Bites

Modified from Miczek et al 1982

Stress-induced analgesia (or antinociception)

7

Mecanismos e Sistemas Envolvidos na Modulaccedilatildeo da Analgesia

induzida pelo Estresse

8

Hormonal Changes eg ACTH

Brain Defense System

Nociceptive Stimuli

Innate Danger Stimuli

Learned Danger Stimuli

Periaqueductal Gray

Hypothalamus

Pituitary

Fight amp Flight Freezing

Neurovegetative Activation

Antinociception

Amygdala

(Modified from Graeff amp Guimaratildees 2012) 9

Eur J Pharmacol 1988 Sep 23154(3)271-6 The NMDA receptor central role in pain inhibition in rat periaqueductal gray Jacquet YF Author information Abstract An injection of the excitatory amino acid analogue N-methyl-D-aspartate (NMDA) in the rat periaqueductal gray resulted in potent analgesia A prior injection of the NMDA antagonist (-)-2-amino-7-phosphonoheptanoate (D-AP7) antagonized this action indicating a receptor-mediated action NMDA given with morphine potentiated the morphine analgesia while D-AP7 blocked morphine analgesia These results delineate for the first time a functional role for the NMDA receptor in the control of pain in the mammalian central nervous system

Os receptores NMDA da PAG poderiam estar envolvidos na analgesia induzida pelo estresse

NMDA

10

Mecanismos e Sistemas Envolvidos na Modulaccedilatildeo da Analgesia

induzida pelo Estresse

8

Hormonal Changes eg ACTH

Brain Defense System

Nociceptive Stimuli

Innate Danger Stimuli

Learned Danger Stimuli

Periaqueductal Gray

Hypothalamus

Pituitary

Fight amp Flight Freezing

Neurovegetative Activation

Antinociception

Amygdala

(Modified from Graeff amp Guimaratildees 2012) 9

Eur J Pharmacol 1988 Sep 23154(3)271-6 The NMDA receptor central role in pain inhibition in rat periaqueductal gray Jacquet YF Author information Abstract An injection of the excitatory amino acid analogue N-methyl-D-aspartate (NMDA) in the rat periaqueductal gray resulted in potent analgesia A prior injection of the NMDA antagonist (-)-2-amino-7-phosphonoheptanoate (D-AP7) antagonized this action indicating a receptor-mediated action NMDA given with morphine potentiated the morphine analgesia while D-AP7 blocked morphine analgesia These results delineate for the first time a functional role for the NMDA receptor in the control of pain in the mammalian central nervous system

Os receptores NMDA da PAG poderiam estar envolvidos na analgesia induzida pelo estresse

NMDA

10

Hormonal Changes eg ACTH

Brain Defense System

Nociceptive Stimuli

Innate Danger Stimuli

Learned Danger Stimuli

Periaqueductal Gray

Hypothalamus

Pituitary

Fight amp Flight Freezing

Neurovegetative Activation

Antinociception

Amygdala

(Modified from Graeff amp Guimaratildees 2012) 9

Eur J Pharmacol 1988 Sep 23154(3)271-6 The NMDA receptor central role in pain inhibition in rat periaqueductal gray Jacquet YF Author information Abstract An injection of the excitatory amino acid analogue N-methyl-D-aspartate (NMDA) in the rat periaqueductal gray resulted in potent analgesia A prior injection of the NMDA antagonist (-)-2-amino-7-phosphonoheptanoate (D-AP7) antagonized this action indicating a receptor-mediated action NMDA given with morphine potentiated the morphine analgesia while D-AP7 blocked morphine analgesia These results delineate for the first time a functional role for the NMDA receptor in the control of pain in the mammalian central nervous system

Os receptores NMDA da PAG poderiam estar envolvidos na analgesia induzida pelo estresse

NMDA

10

Eur J Pharmacol 1988 Sep 23154(3)271-6 The NMDA receptor central role in pain inhibition in rat periaqueductal gray Jacquet YF Author information Abstract An injection of the excitatory amino acid analogue N-methyl-D-aspartate (NMDA) in the rat periaqueductal gray resulted in potent analgesia A prior injection of the NMDA antagonist (-)-2-amino-7-phosphonoheptanoate (D-AP7) antagonized this action indicating a receptor-mediated action NMDA given with morphine potentiated the morphine analgesia while D-AP7 blocked morphine analgesia These results delineate for the first time a functional role for the NMDA receptor in the control of pain in the mammalian central nervous system

Os receptores NMDA da PAG poderiam estar envolvidos na analgesia induzida pelo estresse

NMDA

10

11

Bert Siegfried

(modified from Siegfried amp Nunes de Souza Eur J Pharmacol 168239-242 1989)

00

10

20

30

40

50

Tail-

Flic

k La

ten

cy (

s)

PAG NMDA Receptor amp Stress-Induced Analgesia

C Nx (20) AP-7 (20) C Nx (20) AP-7 (20)

Aggressive Interaction

12

NPLA nNOS inhibitor X

NMDA receptor activation amp NO synthesis

13

NPLA nNOS inhibitor 14

Formalin Test

First Phase Second Phase 15

NPLA nNOS inhibitor

16

bull NPLA intra-PAG atenuou o comportamento de defesa e a antinocicepccedilatildeo induzidos pelo NMDA sugerindo que NO exerce um papel importante no controle tanto da ansiedade como da antinocicepccedilatildeo

bull Tais respostas dependem da ativaccedilatildeo de outros neurotransmissores

NMDA e NO

17

Os efeitos do NO dependem de outros neurotransmissores

18

CRF times Defensive Reactions (or anxiety-like responses)

ICV injections of Ucn (1-3) produce inconsistent effects on anxiety No effect

ICV injections of CRF produce anxiogenic-like effects Anxiety (eg Bale amp Vale Annu Rev Pharmacol Toxicol 2004 Hauger et al CNS Neurol Disord Drug Targets 2006)

CRF receptor affinities CRF1 CRF=Ucn1gtgtgtUcn2Ucn3

CRF2 Ucn 1 Ucn 2 Ucn 3 gtgt CRF (eg Tacheacute amp Brunnhuber Ann NYAcad Sci 2008)

NO times Fator Liberador de Corticotrofina (CRF)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Distribution of (a) CRH Ucn Ucn II and Ucn III mRNA and (b) CRH1 and CRH2 mRNA in a sagittal section of the rodent brain (modified from Reul amp Holsboer Dialogues Clin Neurosci 2002)

Endogenous ligands

CRF1 amp CRF2 Receptors

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Anxiety-like behavior

Spatio-Temporal Measures

eg time spent in surface tunnel and home

cage

Ethological Measures eg risk assessment

(SAP stretched attend postures) freezing

burying

Animal Tests of Anxiety

Elevated Plus-Maze

Anxiety indices open arm entries open

arm time

General activity closed arm entries

Surface

RAT EXPOSURE TEST

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

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O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

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50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

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30

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60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

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10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

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20

25

30

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40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

(modified from Siegfried amp Nunes de Souza Eur J Pharmacol 168239-242 1989)

00

10

20

30

40

50

Tail-

Flic

k La

ten

cy (

s)

PAG NMDA Receptor amp Stress-Induced Analgesia

C Nx (20) AP-7 (20) C Nx (20) AP-7 (20)

Aggressive Interaction

12

NPLA nNOS inhibitor X

NMDA receptor activation amp NO synthesis

13

NPLA nNOS inhibitor 14

Formalin Test

First Phase Second Phase 15

NPLA nNOS inhibitor

16

bull NPLA intra-PAG atenuou o comportamento de defesa e a antinocicepccedilatildeo induzidos pelo NMDA sugerindo que NO exerce um papel importante no controle tanto da ansiedade como da antinocicepccedilatildeo

bull Tais respostas dependem da ativaccedilatildeo de outros neurotransmissores

NMDA e NO

17

Os efeitos do NO dependem de outros neurotransmissores

18

CRF times Defensive Reactions (or anxiety-like responses)

ICV injections of Ucn (1-3) produce inconsistent effects on anxiety No effect

ICV injections of CRF produce anxiogenic-like effects Anxiety (eg Bale amp Vale Annu Rev Pharmacol Toxicol 2004 Hauger et al CNS Neurol Disord Drug Targets 2006)

CRF receptor affinities CRF1 CRF=Ucn1gtgtgtUcn2Ucn3

CRF2 Ucn 1 Ucn 2 Ucn 3 gtgt CRF (eg Tacheacute amp Brunnhuber Ann NYAcad Sci 2008)

NO times Fator Liberador de Corticotrofina (CRF)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Distribution of (a) CRH Ucn Ucn II and Ucn III mRNA and (b) CRH1 and CRH2 mRNA in a sagittal section of the rodent brain (modified from Reul amp Holsboer Dialogues Clin Neurosci 2002)

Endogenous ligands

CRF1 amp CRF2 Receptors

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Anxiety-like behavior

Spatio-Temporal Measures

eg time spent in surface tunnel and home

cage

Ethological Measures eg risk assessment

(SAP stretched attend postures) freezing

burying

Animal Tests of Anxiety

Elevated Plus-Maze

Anxiety indices open arm entries open

arm time

General activity closed arm entries

Surface

RAT EXPOSURE TEST

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

NPLA nNOS inhibitor X

NMDA receptor activation amp NO synthesis

13

NPLA nNOS inhibitor 14

Formalin Test

First Phase Second Phase 15

NPLA nNOS inhibitor

16

bull NPLA intra-PAG atenuou o comportamento de defesa e a antinocicepccedilatildeo induzidos pelo NMDA sugerindo que NO exerce um papel importante no controle tanto da ansiedade como da antinocicepccedilatildeo

bull Tais respostas dependem da ativaccedilatildeo de outros neurotransmissores

NMDA e NO

17

Os efeitos do NO dependem de outros neurotransmissores

18

CRF times Defensive Reactions (or anxiety-like responses)

ICV injections of Ucn (1-3) produce inconsistent effects on anxiety No effect

ICV injections of CRF produce anxiogenic-like effects Anxiety (eg Bale amp Vale Annu Rev Pharmacol Toxicol 2004 Hauger et al CNS Neurol Disord Drug Targets 2006)

CRF receptor affinities CRF1 CRF=Ucn1gtgtgtUcn2Ucn3

CRF2 Ucn 1 Ucn 2 Ucn 3 gtgt CRF (eg Tacheacute amp Brunnhuber Ann NYAcad Sci 2008)

NO times Fator Liberador de Corticotrofina (CRF)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Distribution of (a) CRH Ucn Ucn II and Ucn III mRNA and (b) CRH1 and CRH2 mRNA in a sagittal section of the rodent brain (modified from Reul amp Holsboer Dialogues Clin Neurosci 2002)

Endogenous ligands

CRF1 amp CRF2 Receptors

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Anxiety-like behavior

Spatio-Temporal Measures

eg time spent in surface tunnel and home

cage

Ethological Measures eg risk assessment

(SAP stretched attend postures) freezing

burying

Animal Tests of Anxiety

Elevated Plus-Maze

Anxiety indices open arm entries open

arm time

General activity closed arm entries

Surface

RAT EXPOSURE TEST

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

NPLA nNOS inhibitor 14

Formalin Test

First Phase Second Phase 15

NPLA nNOS inhibitor

16

bull NPLA intra-PAG atenuou o comportamento de defesa e a antinocicepccedilatildeo induzidos pelo NMDA sugerindo que NO exerce um papel importante no controle tanto da ansiedade como da antinocicepccedilatildeo

bull Tais respostas dependem da ativaccedilatildeo de outros neurotransmissores

NMDA e NO

17

Os efeitos do NO dependem de outros neurotransmissores

18

CRF times Defensive Reactions (or anxiety-like responses)

ICV injections of Ucn (1-3) produce inconsistent effects on anxiety No effect

ICV injections of CRF produce anxiogenic-like effects Anxiety (eg Bale amp Vale Annu Rev Pharmacol Toxicol 2004 Hauger et al CNS Neurol Disord Drug Targets 2006)

CRF receptor affinities CRF1 CRF=Ucn1gtgtgtUcn2Ucn3

CRF2 Ucn 1 Ucn 2 Ucn 3 gtgt CRF (eg Tacheacute amp Brunnhuber Ann NYAcad Sci 2008)

NO times Fator Liberador de Corticotrofina (CRF)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Distribution of (a) CRH Ucn Ucn II and Ucn III mRNA and (b) CRH1 and CRH2 mRNA in a sagittal section of the rodent brain (modified from Reul amp Holsboer Dialogues Clin Neurosci 2002)

Endogenous ligands

CRF1 amp CRF2 Receptors

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Anxiety-like behavior

Spatio-Temporal Measures

eg time spent in surface tunnel and home

cage

Ethological Measures eg risk assessment

(SAP stretched attend postures) freezing

burying

Animal Tests of Anxiety

Elevated Plus-Maze

Anxiety indices open arm entries open

arm time

General activity closed arm entries

Surface

RAT EXPOSURE TEST

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Formalin Test

First Phase Second Phase 15

NPLA nNOS inhibitor

16

bull NPLA intra-PAG atenuou o comportamento de defesa e a antinocicepccedilatildeo induzidos pelo NMDA sugerindo que NO exerce um papel importante no controle tanto da ansiedade como da antinocicepccedilatildeo

bull Tais respostas dependem da ativaccedilatildeo de outros neurotransmissores

NMDA e NO

17

Os efeitos do NO dependem de outros neurotransmissores

18

CRF times Defensive Reactions (or anxiety-like responses)

ICV injections of Ucn (1-3) produce inconsistent effects on anxiety No effect

ICV injections of CRF produce anxiogenic-like effects Anxiety (eg Bale amp Vale Annu Rev Pharmacol Toxicol 2004 Hauger et al CNS Neurol Disord Drug Targets 2006)

CRF receptor affinities CRF1 CRF=Ucn1gtgtgtUcn2Ucn3

CRF2 Ucn 1 Ucn 2 Ucn 3 gtgt CRF (eg Tacheacute amp Brunnhuber Ann NYAcad Sci 2008)

NO times Fator Liberador de Corticotrofina (CRF)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Distribution of (a) CRH Ucn Ucn II and Ucn III mRNA and (b) CRH1 and CRH2 mRNA in a sagittal section of the rodent brain (modified from Reul amp Holsboer Dialogues Clin Neurosci 2002)

Endogenous ligands

CRF1 amp CRF2 Receptors

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Anxiety-like behavior

Spatio-Temporal Measures

eg time spent in surface tunnel and home

cage

Ethological Measures eg risk assessment

(SAP stretched attend postures) freezing

burying

Animal Tests of Anxiety

Elevated Plus-Maze

Anxiety indices open arm entries open

arm time

General activity closed arm entries

Surface

RAT EXPOSURE TEST

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

NPLA nNOS inhibitor

16

bull NPLA intra-PAG atenuou o comportamento de defesa e a antinocicepccedilatildeo induzidos pelo NMDA sugerindo que NO exerce um papel importante no controle tanto da ansiedade como da antinocicepccedilatildeo

bull Tais respostas dependem da ativaccedilatildeo de outros neurotransmissores

NMDA e NO

17

Os efeitos do NO dependem de outros neurotransmissores

18

CRF times Defensive Reactions (or anxiety-like responses)

ICV injections of Ucn (1-3) produce inconsistent effects on anxiety No effect

ICV injections of CRF produce anxiogenic-like effects Anxiety (eg Bale amp Vale Annu Rev Pharmacol Toxicol 2004 Hauger et al CNS Neurol Disord Drug Targets 2006)

CRF receptor affinities CRF1 CRF=Ucn1gtgtgtUcn2Ucn3

CRF2 Ucn 1 Ucn 2 Ucn 3 gtgt CRF (eg Tacheacute amp Brunnhuber Ann NYAcad Sci 2008)

NO times Fator Liberador de Corticotrofina (CRF)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Distribution of (a) CRH Ucn Ucn II and Ucn III mRNA and (b) CRH1 and CRH2 mRNA in a sagittal section of the rodent brain (modified from Reul amp Holsboer Dialogues Clin Neurosci 2002)

Endogenous ligands

CRF1 amp CRF2 Receptors

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Anxiety-like behavior

Spatio-Temporal Measures

eg time spent in surface tunnel and home

cage

Ethological Measures eg risk assessment

(SAP stretched attend postures) freezing

burying

Animal Tests of Anxiety

Elevated Plus-Maze

Anxiety indices open arm entries open

arm time

General activity closed arm entries

Surface

RAT EXPOSURE TEST

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

bull NPLA intra-PAG atenuou o comportamento de defesa e a antinocicepccedilatildeo induzidos pelo NMDA sugerindo que NO exerce um papel importante no controle tanto da ansiedade como da antinocicepccedilatildeo

bull Tais respostas dependem da ativaccedilatildeo de outros neurotransmissores

NMDA e NO

17

Os efeitos do NO dependem de outros neurotransmissores

18

CRF times Defensive Reactions (or anxiety-like responses)

ICV injections of Ucn (1-3) produce inconsistent effects on anxiety No effect

ICV injections of CRF produce anxiogenic-like effects Anxiety (eg Bale amp Vale Annu Rev Pharmacol Toxicol 2004 Hauger et al CNS Neurol Disord Drug Targets 2006)

CRF receptor affinities CRF1 CRF=Ucn1gtgtgtUcn2Ucn3

CRF2 Ucn 1 Ucn 2 Ucn 3 gtgt CRF (eg Tacheacute amp Brunnhuber Ann NYAcad Sci 2008)

NO times Fator Liberador de Corticotrofina (CRF)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Distribution of (a) CRH Ucn Ucn II and Ucn III mRNA and (b) CRH1 and CRH2 mRNA in a sagittal section of the rodent brain (modified from Reul amp Holsboer Dialogues Clin Neurosci 2002)

Endogenous ligands

CRF1 amp CRF2 Receptors

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Anxiety-like behavior

Spatio-Temporal Measures

eg time spent in surface tunnel and home

cage

Ethological Measures eg risk assessment

(SAP stretched attend postures) freezing

burying

Animal Tests of Anxiety

Elevated Plus-Maze

Anxiety indices open arm entries open

arm time

General activity closed arm entries

Surface

RAT EXPOSURE TEST

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Os efeitos do NO dependem de outros neurotransmissores

18

CRF times Defensive Reactions (or anxiety-like responses)

ICV injections of Ucn (1-3) produce inconsistent effects on anxiety No effect

ICV injections of CRF produce anxiogenic-like effects Anxiety (eg Bale amp Vale Annu Rev Pharmacol Toxicol 2004 Hauger et al CNS Neurol Disord Drug Targets 2006)

CRF receptor affinities CRF1 CRF=Ucn1gtgtgtUcn2Ucn3

CRF2 Ucn 1 Ucn 2 Ucn 3 gtgt CRF (eg Tacheacute amp Brunnhuber Ann NYAcad Sci 2008)

NO times Fator Liberador de Corticotrofina (CRF)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Distribution of (a) CRH Ucn Ucn II and Ucn III mRNA and (b) CRH1 and CRH2 mRNA in a sagittal section of the rodent brain (modified from Reul amp Holsboer Dialogues Clin Neurosci 2002)

Endogenous ligands

CRF1 amp CRF2 Receptors

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Anxiety-like behavior

Spatio-Temporal Measures

eg time spent in surface tunnel and home

cage

Ethological Measures eg risk assessment

(SAP stretched attend postures) freezing

burying

Animal Tests of Anxiety

Elevated Plus-Maze

Anxiety indices open arm entries open

arm time

General activity closed arm entries

Surface

RAT EXPOSURE TEST

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

CRF times Defensive Reactions (or anxiety-like responses)

ICV injections of Ucn (1-3) produce inconsistent effects on anxiety No effect

ICV injections of CRF produce anxiogenic-like effects Anxiety (eg Bale amp Vale Annu Rev Pharmacol Toxicol 2004 Hauger et al CNS Neurol Disord Drug Targets 2006)

CRF receptor affinities CRF1 CRF=Ucn1gtgtgtUcn2Ucn3

CRF2 Ucn 1 Ucn 2 Ucn 3 gtgt CRF (eg Tacheacute amp Brunnhuber Ann NYAcad Sci 2008)

NO times Fator Liberador de Corticotrofina (CRF)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Distribution of (a) CRH Ucn Ucn II and Ucn III mRNA and (b) CRH1 and CRH2 mRNA in a sagittal section of the rodent brain (modified from Reul amp Holsboer Dialogues Clin Neurosci 2002)

Endogenous ligands

CRF1 amp CRF2 Receptors

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Anxiety-like behavior

Spatio-Temporal Measures

eg time spent in surface tunnel and home

cage

Ethological Measures eg risk assessment

(SAP stretched attend postures) freezing

burying

Animal Tests of Anxiety

Elevated Plus-Maze

Anxiety indices open arm entries open

arm time

General activity closed arm entries

Surface

RAT EXPOSURE TEST

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Distribution of (a) CRH Ucn Ucn II and Ucn III mRNA and (b) CRH1 and CRH2 mRNA in a sagittal section of the rodent brain (modified from Reul amp Holsboer Dialogues Clin Neurosci 2002)

Endogenous ligands

CRF1 amp CRF2 Receptors

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Anxiety-like behavior

Spatio-Temporal Measures

eg time spent in surface tunnel and home

cage

Ethological Measures eg risk assessment

(SAP stretched attend postures) freezing

burying

Animal Tests of Anxiety

Elevated Plus-Maze

Anxiety indices open arm entries open

arm time

General activity closed arm entries

Surface

RAT EXPOSURE TEST

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Distribution of (a) CRH Ucn Ucn II and Ucn III mRNA and (b) CRH1 and CRH2 mRNA in a sagittal section of the rodent brain (modified from Reul amp Holsboer Dialogues Clin Neurosci 2002)

Endogenous ligands

CRF1 amp CRF2 Receptors

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Anxiety-like behavior

Spatio-Temporal Measures

eg time spent in surface tunnel and home

cage

Ethological Measures eg risk assessment

(SAP stretched attend postures) freezing

burying

Animal Tests of Anxiety

Elevated Plus-Maze

Anxiety indices open arm entries open

arm time

General activity closed arm entries

Surface

RAT EXPOSURE TEST

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Anxiety-like behavior

Spatio-Temporal Measures

eg time spent in surface tunnel and home

cage

Ethological Measures eg risk assessment

(SAP stretched attend postures) freezing

burying

Animal Tests of Anxiety

Elevated Plus-Maze

Anxiety indices open arm entries open

arm time

General activity closed arm entries

Surface

RAT EXPOSURE TEST

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull Hipotaacutelamo bull PAG

Anxiety-like behavior

Spatio-Temporal Measures

eg time spent in surface tunnel and home

cage

Ethological Measures eg risk assessment

(SAP stretched attend postures) freezing

burying

Animal Tests of Anxiety

Elevated Plus-Maze

Anxiety indices open arm entries open

arm time

General activity closed arm entries

Surface

RAT EXPOSURE TEST

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Anxiety-like behavior

Spatio-Temporal Measures

eg time spent in surface tunnel and home

cage

Ethological Measures eg risk assessment

(SAP stretched attend postures) freezing

burying

Animal Tests of Anxiety

Elevated Plus-Maze

Anxiety indices open arm entries open

arm time

General activity closed arm entries

Surface

RAT EXPOSURE TEST

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

RAT EXPOSURE TEST

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bullAmiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

E F Carvalho-Netto

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Elevated Plus-Maze

NBI 27914 CFR1 receptor antagonist ASV-30 CFR2 receptor antagonist

TT Miguel

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

NOC-9 NO donor NBI 27914 CFR1 receptor antagonist

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

bull A ativaccedilatildeo de receptores CRF1 da PAG aumenta a ansiedade em camundongos expostos ao LCE e RET

bull O bloqueio de receptores CRF2 da PAG natildeo altera os efeitos ansiogecircnicos do CRF

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Os efeitos aversivos induzidos pelo NO satildeo

bloqueados com antagonista de receptores CRF1 na PAG

O NO parece facilitar a liberaccedilatildeo de CRF na PAG

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

0

5

10

15

20

25

30

35

40

45

O

pe

n a

rm

Entries Time

Control 375 75 150

CRF (pmol)

Cipriano et al in preparation

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

34

0

10

20

30

40

50

60

70

salina 4 8 16 24

b

raccedilo

s a

bert

os

LCE

entradas tempo

ucn3 (pmol)

Urocortin 3 CRFr2 agonist

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

CRFr2 antagonist Cipriano et al in preparation

0

10

20

30

40

50

60

70

saline 0375 075 15 1 3

O

pen

Arm

EPM

Entries Time

CP376395 (nmol) ASV30 (nmol)

0

2

4

6

8

10

12

14

saline 0375 075 15 1 3

Clo

sed

arm

en

trie

s

CP376395 (nmol) ASV30 (nmol)

CRFr1 antagonist

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Os receptores CRF1 localizados na Amiacutedala (mas natildeo na PAG) exercem um papel

tocircnico na modulaccedilatildeo da ansiedade em camundongos expostos ao LCE

bull Bloqueio ou ativaccedilatildeo de CRF2 da amiacutedala natildeo altera a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 da Amiacutedala provoca ansiogecircnese em camundongos expostos ao

LCE

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Aonde o CRF induz efeitos ansiogecircnicos no SNC Estruturas de interesse bull mPFC bull BNST

bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

ldquoPredator specific c-fos mRNA induction was observed in several brain regions comprising the brain defense circuit (BNST medial region of the ventromedial nucleus and dorsal premammillary nucleusrdquo) Helmer et al Endocrinol 2003 ldquoElectrical stimulation of the BNST elicits behavioral and endocrine changes which are similar to those induced by environmental stressors (Casada and Dafny 1991 Dunn 1987)rdquo ldquoPharmacological inactivation of the BNST decreases the expression of conditioned or unconditioned responses induced by aversive situations (Sahuque et al 2006)rdquo

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

0

5

10

15

20

25

30

35

40

Vehicle 1875 375 75

O

pe

n a

rm

Intra-BNST treatment

Entries Time

NOC-9 (nmol01 microl)

0

5

10

15

20

Vehicle 075 150 30

Clo

sed

Arm

En

trie

s

EPM

0

10

20

30

40

Vehicle 075 150 30

O

pe

n A

rm

CP 376395 (nmol) Intra-BNST treatment

Entries Time

Faria et al submitted

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Faria et al submitted

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

41

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

42

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

43

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

44

bull Bloqueio per se CRF1 no BNST natildeo altera ansiedade em camundongos no LCE

bull Ativaccedilatildeo nitreacutergica do BNST provoca ansiogecircnese em camundongos no LCE

bull Efeitos ansiogecircnicos do NO satildeo atenuados pelo bloqueio de CRF1

bull Congelamento induzido pelo NO natildeo eacute alterado pelo bloqueio CRF1

bull Ansiedade no LCE x Congelamento mecanismos diferentes

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Aonde o CRF induz efeitos ansiogecircnicos no SNC

Estruturas de interesse bull mPFC bull BNST bull Amiacutedala bull PAG

Schematic illustration of the distribution of CRH1 and CRH2α in the brain (Steckler amp Holsboer Biol Psychiatry 1999)

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

46

TT Miguel

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

CP 376395 antagonista seletivo dos receptores CRF1

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

CRF receptors Intracellular Pathways

Major intracellular pathways for signal transduction by CRF1 and CRF2 receptors (from Hauger et al CNS Neurol Disord Drug Targets 2006)

X H-89 PKA inhibitor

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

0

10

20

30

40

50

60

O

pen

arm

Intra-mPFC Treatment (nmol02 microl)

Entries Time

Vehicle 125 25 50 H-89 (nmol02 microl)

0

2

4

6

8

10

12

14

16

Clo

se

d A

rm E

ntr

ies

Elevated Plus Maze

Miguel et al 2014

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

0

10

20

30

40

50

60

1 2 3 4

O

pe

n A

rm

Intra-mPFC Treatment

Entries Time

Vehicle CRF Vehicle CRF

Vehicle H-89

Miguel et al 2014

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a

resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a ansiedade no LCE

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

bull Ativaccedilatildeo de CRF1 do CPFm aumenta a resposta de defesa no LCE

bull Bloqueio de CRF1 do CPFm atenua a

ansiedade no LCE

Os receptores CRF1 localizados na Amiacutedala e CPFm (mas natildeo na PAG e BNST)

modulam tonicamente o comportamento defensivo de camundongos expostos ao

LCE

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol

Clo

sed

Arm

En

trie

s

RIGHT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Resultados Recentes

Ativaccedilatildeo Nitreacutergica do CPFm Direito

NS Costa

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

0

2

4

6

8

10

12

14

Vehicle NOC-9 375 nmol NOC-9 750 nmol

Clo

sed

Arm

En

trie

s

Bilateral mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol NOC-9 750 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Bilateral)

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

0

2

4

6

8

10

12

14

16

18

Vehicle NOC-9 375 nmol C

lose

d A

rm E

ntr

ies

LEFT mPFC - Anxiety Measures

0

10

20

30

40

50

60

Vehicle NOC-9 375 nmol

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Ativaccedilatildeo Nitreacutergica do

CPFm (Esquerdo)

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

0

2

4

6

8

10

12

14

16

18

Vehicle CoCl2 C

lose

d A

rm E

ntr

ies

LEFT mPFC - CoCl2 - Anxiety Measures

0

10

20

30

40

50

60

Vehicle CoCl2

O

pe

n A

rm

Entries

Time

Costa et al in preparation

Inibiccedilatildeo do CPFm

Esquerdo

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

58

Estresse amp Ansiedade

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

LCE

0

5

10

15

20

25

30

controle estressado

b

raccedilo

s ab

ert

os entradas tempo

0

2

4

6

8

10

12

14

controle estressado

en

trad

as b

raccedilo

s fe

chad

os

0

5

10

15

20

25

30

35

40

CONTROLE RESTRAINT

B

raccedilo

s A

be

rto

s

Entradas

Tempo

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Cirurgia estereotaacutexica

5 a 7 dias

Injeccedilatildeo Veiacuteculo

Exposiccedilatildeo ao LCE

10min

CoCl2 inibidor sinaacuteptico

Estresse

Contenccedilatildeo 30rsquo

Derrota postura de submissatildeo

24h ou

Inibiccedilatildeo do CPFm Esquerdo times Estresse

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

0

10

20

30

40

50

60

Saline CoCl2 Saline CoCl2 Saline CoCl2

Entries

Time

O

pe

n A

rm

______________ no stress

______________ restraint

______________ defeat

Costa et al in preparation

Left mPFC Inhibition times Stress Anxiety 24h

MA Vicente

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard

Graduate Students Ana Claudia Cipriano Diego C Mascarenhas Matheus P Faria Nathaacutelia Santos Costa Tatiani Sorregotti Undergraduate Students

Carolina Trovarelli Daniela Freitas Guilherme Tinoco Rafael Luis Ferreira

Postdoctor Karina S Gomes Maria Adrielle Vicente Tarciso T Miguel (UFO) Eduardo F Carvalho-Netto (Novartis)

Financial Support FAPESP CNPq CAPES School of Pharmaceutical Sciences - UNESP

Research Team

Collaborators

Technicians Elisabete Z P Lepera Rosana F P Silva

University Zurich

Bert Siegfried

61

University Hawaii

Caroline amp Bob Blanchard