stress acutely promotes calcium-dependent glutaminergic synaptic plasticity in vta via differential...

0 10 20 300.5

1.0

1.5

2.0Phe

CRFPhe 0.5 µM+CRF

Phe 1µM+CRF

Time(min)

I K(Ca)

(nor

m.)

0.5 µM

0.5 µM

+CRF

1 µM

1 µM+C

RF0

10

20

30

40

50

60

I K(Ca)

faci

litat

ion

(%

0 10 20 30 40 500.5

1.0

1.5

2.0

Phe 0.5 µM Phe 0.5 µM + CRF

LTP

Time (min)

NM

DA

R E

PS

C (n

orm

.)

Phe 0.

5 µM

Phe 0.

5 µM +

CRF0

10

20

30

40

50

***

I K(Ca)

faci

litat

ion

(%)

-20 20 40 60

-20

20

40

60

r2 = 0.65

IK(Ca) facilitation (%)

NM

DA

R-L

TP (%

)

Phe 0.

5 µM

Phe 0.

5 µM +

CRF0

10

20

30

40

50

*

NM

DA

R-L

TP (%

)

0 10 20 30 40 500.5

1.0

1.5

2.0 IP3 alone IP3 + CRF

LTP

Time (min)

NM

DA

R E

PS

C (n

orm

.)

0 10 20 30 40 500

20

40

60

80

100

120

1

2IP3+

CRF

LTP

Time (min)

NM

DA

R E

PS

C (p

A)

0 nM 100 nM0

10

20

30

40

[CRF]

I K(Ca)

faci

litat

ion

(%)

0 nM 100 nM0

10

20

30

40

[CRF]

NM

DA

R-L

TP (%

)

-20 20 40

-20

20

40r2=0.47


NM

DA

R-L

TP (%

)

0 10 20 30 40 500.5

1.0

1.5

2.0

CRF 100nMControl

LTP

CRF

Time (min)

NM

DA

R E

PS

C (n

orm

.)

0 10 20 30 40 500

50

100

12

LTP

CRF100 nM

Time (min)

NM

DA

R E

PS

C (p

A)

0 10 20 300.5

1.0

1.5

2.0

100 nM300 nM

CRF 30 nM

Time(min)

I K(Ca)

(nor

m.)

CRF100 nM

0 10 20 300.5

1.0

1.5

2.0

1st EPSC2nd EPSC

Time(min)

NM

DA

R E

PS

C (n

orm

.)

0.5 µM 1 µM0

10

20

30

40

50

60

**

[Phe]

I K(Ca)

faci

litat

ion

(%)

-20 20 40 60 80

20

40

60

80

r2=0.45


NM

DA

R-L

TP (%

)

0.5 µM 1 µM0

10

20

30

40

50

60

**

[Phe]

NM

DA

R L

TP (%

)

0 10 20 30 40 500

50

100

12

LTP

Phe1 µM

Time (min)N

MD

AR

EP

SC

(pA

)

0 10 20 30 40 500.5

1.0

1.5

2.0

Phe 1 µMPhe 0.5 µMPhe

LTP

Time (min)

NM

DA

R E

PS

C (n

orm

.)

0 10 20 300.5

1.0

1.5

2.0Phe 0.5 µM

1 µM

Time (min)

I K(C

a) (n

orm

.)

0.5 µM 1 µM0

10

20

30

40

50

60

**

[Phe]

I K(C

a) fa

cilit

atio

n (%

)

0 10 20 300.5

1.0

1.5

2.0

1st EPSCPhe1 µM 2nd EPSC

Time(min)

NM

DA

R E

PS

C (n

orm

.)

1st EPSC 2nd EPSC0

10

20

30

40

50

60

[Phe]

NM

DA

R E

PS

C in

crea

se (%

)

References 1.  Sinha R. Chronic stress, drug use, and vulnerability to addiction. Ann.N.Y.Acad.Sci., 2008. 2.  Kauer JA and Malenka RC. Synaptic plasticity and addiction. Nat Rev Neurosci., 2007. 3.  Sombers L. et al. Synaptic overflow of dopamine in the nucleus accumbens arises from neuronal activity in the ventral tegmental area. J Neurosci.,

2009. 4.  Harnett MT. et al. Burst-timing-dependent plasticity of NMDA receptor-mediated transmission in midbrain dopamine neurons. Neuron., 2009. 5.  Refojo D. et al. Glutamatergic and dopaminergic neurons mediate anxiogenic and anxiolytic effects of CRHR1. Science, 2011. 6.  Grenhoff J. et al. Alpha 1-adrenergic effects on dopamine neurons recorded intracellularly in the rat midbrain slice. Eur J Neurosci., 1995. 7.  Cui G. et al. Diferential regulation of action potential- and metabotropic glutamate receptor-induced Ca2+ signals by inositol 1,4,5-trisphosphate in

dopaminergic neurons. J Neurosci., 2007.

Background -  Stressful life experiences are well-known risk factors for the development of drug addiction1. -  Addictive drugs are thought to hijack the synaptic plasticity mechanisms in brain circuits involved in reward learning, specially the dopaminergic system in the ventral tegmental area (VTA)2. -  Activation of NMDA receptors (NMDARs) is necessary for dopamine (DA) neuron burst firing in response to drugs and drug-conditioned cues3. Therefore, long term potentiation (LTP) of NMDARs in DA neurons may contribute to the increased motivational valence of drug-associated cues. -  Mechanistically, NMDAR-LTP induction requires burst-evoked Ca2+ induced-Ca2+ release (CICR) amplified by preceding production of IP3 due to metabotropic glutamate receptors activation4. -  Norepinephrine (NE) and corticotropin releasing factor (CRF) release in the VTA following acute stress5,6 can quickly modulate CICR through α1 adrenoreceptors (α1ARs) or CRF2 receptors (CRF2Rs), respectively.

Hypothesis -  In vitro, acute activation of α1ARs and CRF2Rs would enhance the induction of NMDAR-LTP in DA neurons through IP3Rs modulation (see scheme below). -  In vivo, an acute stressor applied right before conditioning would enhance the acquisition of conditioned placed preference (CPP), a widely used model of reward conditioning.

Jorge Tovar-Diaz, Matthew Pomrenze, Russell J Kan & Hitoshi Morikawa

Stress acutely promotes calcium-dependent glutamatergic synaptic plasticity in VTA via differential actions of CRF and norepinephrine

Methodology -  In vitro -  Whole-cell voltage clamp recordings were performed in VTA DA neurons (naïve male Sprague Dawley rats, 4-6 weeks old). Cells were held at -55 mV in all recordings. -  Ca2+ signals were indirectly measured through IK(Ca) currents. We have previously demonstrated that IK(Ca) is generated by unclamped action potentials and is TTX and apamine sensitive7. -  Excitatory postsynaptic currents (EPSCs) where induced with a bipolar electrode. NMDAR-mediated EPSCs were pharmacologically isolated with a cocktail of AMPA, GABAA, GABAB, and D2 blockers and recorded in low Mg++ aCSF. -  Different concentrations of phenylephrine (Phe) and CRF were directly perfused in the bath. -  LTP protocol. We used a “simultaneous pairing” protocol, where presynaptic stimulation and postsynaptic burst firing were delivered at the same time. Ten pairings every 20 sec (bipolar electrode stimulation, 30 stim at 30 Hz) paired with burst firing (5 unclamped action potentials at 20 Hz). -  Caged IP3 was loaded in the recording pipette and uncaged with UV light. - In vivo -  Social defeat stress was performed by direct exposure (5 min) to a highly territorial resident male, followed by protected exposure through a perforated plastic wall (25 min). -  Conditioned place preference (CPP). Ten min after social defeat, rats were injected with cocaine (5 or 10 mg/kg, i.p.) and placed in the conditioning box.

Results

Conclusions -  In vitro - Activation of α1ARs increases CICR through IP3 production, enabling induction of NMDAR-LTP by a simultaneous pairing protocol. -  Activation of CRF2Rs facilitates CICR and thus NMDAR-LTP, but only when IP3 is provided from another source. -  CRF2Rs and α1ARs work in a cooperative manner through IP3-dependent CICR to promote NMDAR-LTP. - In vivo - Acute social defeat stress enhances the acquisition of cocaine-CPP only at low doses of the drug. - Intra-VTA atagonisms of CRF/adrenergic transmission prevents the effect of stress on CPP acquisiton..

Funded by NIH grants DA015687 and AA015521

1. α1AR activation facilitates NMDAR-LTP by increasing Ca2+ release from internal stores

Model of acute stress enhancement of NMDAR-LTP in DA neurons!

1)  Sustained presynaptic stimulation induces

IP3 production through mGluRs activation."2)  Burst firing induces Ca2+ influx through

voltage gated Ca2+ channels."3)  Acting as a coincident detector, IP3R-

mediated CICR enables NMDAR-LTP."4)  Acute activation of α1ARs increases IP3

production, while CRF2Rs increases IP3Rs sensitivity, thus facilitating NMDAR-LTP induction."

In vivo, an acute stress applied previous to conditioning, could increase the valence of the cues to be associated with the reward, thus strengthening their conditioning."

2. CRF alone does not affect Ca2+ release nor NMDAR-LTP

3. CRF increases IP3-induced Ca2+ release, thus facilitating NMDAR-LTP

4. CRF increases α1AR-induced Ca2+ release, thus facilitating NMDAR-LTP

5. Acute stress enhances cocaine-CPP, effect prevented by intra-VTA blockage of CRF2Rs and α1ARs

Time course of CRF effect on a) IK(Ca), n= 5 (30 nM), 8 (100 nM) and 4 cells (300 nM) and b) NMDAR EPSC, n= 5 cells (100 nM). c) RepresentaJve experiment and summary Jme course of CRF (100 nM) on NMDAR-‐LTP (n= 10 cells). d) Summary bar graphs of CRF 100 nM effect on IK(Ca) and NMDAR-‐LTP.

a) Time course, average bar graph and representaJve traces of an acJon potenJal-‐induced IK(Ca) alone (black), IP3-‐facilitated (blue) and IP3+CRF-‐facilitated (red)(n= 8 cells). **p<0.009 paired t test. b) IP3-‐induced (n= 8 cells) and IP3+CRF-‐induced (n= 11 cells) NMDAR-‐LTP summary Jme course and representaJve experiment. c) Summary bar graphs of IP3-‐induced and IP3+CRF-‐induced IK(Ca) facilitaJon (***p<0.001 unpaired t test) and NMDAR-‐LTP (***p<0.001 unpaired t test) and their correlaJon. IP3 [10mM] was loaded in the recording pipet and acJvated with UV light (1mW*100ms) through the microscope objecJve.

Acute social defeat enables low dose-‐cocaine condiJoning. a) Change in percentage of Jme spent in the drug-‐paired side a_er condiJoning with low or high doses of cocaine. *** p < 0.005 two-‐way ANOVA. n= 8rats/group. b) A mix of antagonists K41498/prazosin (30 µM/30 µM in 0.3µl) prevents the effect of stress on CPP acquisiJon. Preliminary result (n= 4 rats each group).

a) b)

c)

d)

a)

c)

b)

d)

a) Phenylephrine (Phe)-‐induced increase in IK(Ca), Jme course and summary bar graph. n= 7 (0.5 µM) and 9 cells (1 µM). ** p<0.005, t test. b) Phe does not affect basal NMDAR EPSC amplitude. n= 5 cells. c) RepresentaJve experiment and d) Summary Jme graph of of Phe-‐induced NMDAR-‐LTP. n= 7 (0.5 µM) and 10 (1 µM) cells. e) Summary bar graphs of Phe 1 µM on IK(Ca) and NMDAR-‐LTP and their correlaJon. **p<0.002 t test.

a)

b)

c)

e) c)

a) Time course, summary bar graphs and representaJve traces of CRF effect on Phe-‐facilitated IK(Ca). n= 8 (Phe 0.5µM) and 9 (1µM) cells. b) LTP summary Jme course, bar graphs and correlaJon of CRF effect on Phe-‐induced IK(Ca) facilitaJon and NMDAR-‐LTP. *p<0.03 and ***p<0.007 unpaired t test.

100 pA

100 ms

Time (s)

10 pA

500 ms

10 pA

50 ms

1

2

10 pA

50 ms

1

2

20 pA

50 ms

50 pA

100 ms

a)

b)

mGluR

G

Ca2+ Store

IP3

IP3 R

IP3

PLC

NMDR - LTP

mGluR

Gq

PLC

VGCC

Glu

G q

q

Phasic firing

Afferent stimulation

NM DAR

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+Ca2+

IP3

qIP3

Ca2+

Ca2+Ca2+

G s

PKA

CRF2R1 AR

Acute stress

NE CRF

NE CRF

2+P

GluGlu

Glu

Glu

1

2

3

4

Stim

Burst

Stim

Burst

100 pA

200 ms

IK(Ca) alone

IK(Ca) + IP3 flash

IK(Ca) + IP3 flash + CRF

20 pA

20 ms

1

2

IP3

CRF

0 10 20 300.5

1.0

1.5

2.0

Time (min)

I K(Ca)

(nor

m.)

IP3 IP3 + CRF0

10

20

30

40

50

60**

I K(Ca)

faci

litat

ion

(%)

IP3 IP3 + CRF0

10

20

30

40

50

60

***

I K(Ca)

faci

litat

ion

(%)

0 20 40 60 80 1000

20

40

60

80

100

r2=0.64


NM

DA

R-L

TP (%

)

IP3 IP3 + CRF0

10

20

30

40

50

60 ***

NM

DA

R-L

TP (%

)

50 pA

100 ms

IK(Ca) alone

IK(Ca) + Phe

IK(Ca) + Phe + CRF

Pre Post Pre Post45

50

55

60

65

70 StressControl

***

10 mg/kg 5 mg/kg

Pre

fere

nce

for

coca

ine-

paire

d si

de (%

)

Pre Post45

50

55

60

65

70 CRF2/!1 antagonists

PBS

5 mg/kg

Pre

fere

nce

for

coca

ine-

paire

d si

de (%

)