brief social defeat stress: long lasting effects on cocaine taking during a binge and zif268 mrna...

Brief Social Defeat Stress: Brief Social Defeat Stress: Long Lasting Effects on Long Lasting Effects on Cocaine Cocaine TakingTaking During a During a Binge and Zif268 mRNA Binge and Zif268 mRNA

Expression in the Amygdala Expression in the Amygdala and Prefrontal Cortexand Prefrontal CortexHerbert E Covington, Takefumi Herbert E Covington, Takefumi Kikusui, Justin Goodhue, Ella M Kikusui, Justin Goodhue, Ella M

Nikulina, Ronald P Hammer Jr., and Nikulina, Ronald P Hammer Jr., and Klaus A MiczekKlaus A Miczek

Materials and MethodsMaterials and Methods

Male Long-Evans ratsMale Long-Evans rats 225-250g225-250g Individually housed in clear acrylic Individually housed in clear acrylic

chamberschambers Cellulose pellet beddingCellulose pellet bedding Free access to food and waterFree access to food and water Reversed light cycleReversed light cycle Removable stainless steel mesh panelsRemovable stainless steel mesh panels

Materials and MethodsMaterials and Methods

Controlled temperature (21 +/- 1C)Controlled temperature (21 +/- 1C) Controlled humidity (35-40%)Controlled humidity (35-40%) Each chamber served at various Each chamber served at various

phases of the experimental protocol phases of the experimental protocol as the home cageas the home cage

Habituated to saline injections for 3 Habituated to saline injections for 3 days prior to measurementsdays prior to measurements

Social Stress EpisodeSocial Stress Episode

First phase: Experimental rat’s home cage First phase: Experimental rat’s home cage initially placed within an aggressive initially placed within an aggressive resident’s cage where the intruder was resident’s cage where the intruder was exposed to the resident’s species-typical exposed to the resident’s species-typical threats for 10 minutes through a protective threats for 10 minutes through a protective screenscreen

Second phase: Experimental intruder was Second phase: Experimental intruder was removed from their protective home cage, removed from their protective home cage, and a confrontation persisted until the and a confrontation persisted until the attacks by the resident resulted in defeatattacks by the resident resulted in defeat

Social Stress EpisodeSocial Stress Episode

Defeat= display of 4 seconds of Defeat= display of 4 seconds of supine posture by the intruder, or a supine posture by the intruder, or a maximum duration of 5 minutesmaximum duration of 5 minutes

Third Phase: Experimental rat was Third Phase: Experimental rat was exposed for 10 min to the resident’s exposed for 10 min to the resident’s threats behind the protective screen threats behind the protective screen of its cageof its cage

Induction of behavioral Induction of behavioral sensitizationsensitization

Establish pre-stress baseline motor Establish pre-stress baseline motor activity 1 day prior to episodeactivity 1 day prior to episode

Injected with salineInjected with saline Video samples collected at 5-10 Video samples collected at 5-10

minutes, and 25-30 minutes after minutes, and 25-30 minutes after injectionsinjections

Expression of behavioral Expression of behavioral sensitizationsensitization

Each defeat stressed and unstressed rats’ Each defeat stressed and unstressed rats’ locomotor response to a cocaine locomotor response to a cocaine (10mg/kg) challenge on day 20 (10 days (10mg/kg) challenge on day 20 (10 days after the last defeat)after the last defeat)

Stressed and unstressed control rats Stressed and unstressed control rats received an injection of amphetamine received an injection of amphetamine (1.0mg/kg) on experimental day 70(1.0mg/kg) on experimental day 70

Saline inj administered 15 min prior to the Saline inj administered 15 min prior to the stimulant drug challengestimulant drug challenge

Determines baseline measurements of Determines baseline measurements of locomotor activitylocomotor activity


Immediately after baseline, Immediately after baseline, administered stimulant challengeadministered stimulant challenge

Administered between 11:00 and Administered between 11:00 and 14:0014:00

5 min video samples were collected 5-5 min video samples were collected 5-10 min after saline10 min after saline

5 min video samples were collected 5-5 min video samples were collected 5-10 min and 25-30 min after stimulant 10 min and 25-30 min after stimulant challengeschallenges


Observed the frequency and analyzed Observed the frequency and analyzed each 5 min sampleeach 5 min sample

WalkingWalking Rearing Rearing SniffingSniffing GroomingGrooming DiggingDigging InactivityInactivity

Experiment 1: Cocaine Self-Experiment 1: Cocaine Self-AdministrationAdministration

Implanted catheters into the right Implanted catheters into the right jugular veinjugular vein

Recovered for 5 days after surgeryRecovered for 5 days after surgery One wall of chamber had two One wall of chamber had two

retractable leversretractable levers Self administer cocaine Self administer cocaine

(0.75mg/kg/infusion)(0.75mg/kg/infusion)

Experiment 1Experiment 1

Locomotor activity was monitored Locomotor activity was monitored from 1h before binge until 1h afterfrom 1h before binge until 1h after

Prolonged protocol lower dose Prolonged protocol lower dose cocaine (0.3mg/kg/infusion)cocaine (0.3mg/kg/infusion)

Start at 19:00 for 24hStart at 19:00 for 24h Red light at the start of light phaseRed light at the start of light phase

Experiment 1Experiment 1

No primingNo priming Fixed ratio of 1 with a 30 second time outFixed ratio of 1 with a 30 second time out Initiated at 10:00Initiated at 10:00 Terminated after 15 infusions or 5 hours of Terminated after 15 infusions or 5 hours of

accessaccess 10 additional days on a FR 5 reinforcement 10 additional days on a FR 5 reinforcement

for 15 infusionsfor 15 infusions Continuous access to cocaine during 24h Continuous access to cocaine during 24h

bingebinge

Experiment 2: Experiment 2: ZifZif268268 mRNA mRNA Expression in the mPFC, NAc, Expression in the mPFC, NAc,

Dorsal Striatum, and AmygdalaDorsal Striatum, and Amygdala

Challenge inj of d-amphetamine Challenge inj of d-amphetamine (1.0mg/kg) on experimental day 70(1.0mg/kg) on experimental day 70

Tissue collected for Tissue collected for ZifZif268268 mRNA mRNA ((EgrEgr11) expression analysis) expression analysis

Stress-sensitizing protocol on zif268 Stress-sensitizing protocol on zif268 mRNA expression within 7 days of mRNA expression within 7 days of stress exposurestress exposure

Neuronal zif268 expression Neuronal zif268 expression measurementsmeasurements

prelimbic and infralimbic cortexprelimbic and infralimbic cortex NAc core and shellNAc core and shell Dorsal striatumDorsal striatum Medial, central, and basolateral Medial, central, and basolateral

amygdaloid nucleiamygdaloid nuclei

Results: Experiment 1Results: Experiment 1

T-tests T-tests Kaplan-Meier Survival Analysis Kaplan-Meier Survival Analysis Two-way ANOVATwo-way ANOVA Bonferroni correctionBonferroni correction


No significant differences found in No significant differences found in behavior other than walkingbehavior other than walking

In cocaine challenge stressed rats In cocaine challenge stressed rats walked more than unstressedwalked more than unstressed

No significant difference in No significant difference in acquisition of self administration acquisition of self administration between stressed and unstressed between stressed and unstressed ratsrats


Stressed rats self-administered Stressed rats self-administered considerably more total cocaine than considerably more total cocaine than unstressed ratsunstressed rats

Unstressed rats took in less cocaine Unstressed rats took in less cocaine during light cycleduring light cycle

Stressed rats self-administered Stressed rats self-administered cocaine continuously cocaine continuously


Total horizontal activity and Total horizontal activity and stereotyped sniffing increased when stereotyped sniffing increased when cocaine availablecocaine available

Stressed rats showed more Stressed rats showed more horizontal and stereotyped behavior horizontal and stereotyped behavior during light phaseduring light phase

No difference in behavior during dark No difference in behavior during dark phase between the groupsphase between the groups


Amphetamine challenge on day 70Amphetamine challenge on day 70 60 days after last defeat stress60 days after last defeat stress Increased walking behavior for Increased walking behavior for

stressed ratsstressed rats Interaction seen between stress and Interaction seen between stress and

amphetamine challenge in zif268 amphetamine challenge in zif268 mRNA expression in CeAmRNA expression in CeA

Greater in stressed ratsGreater in stressed rats


Amphetamine significantly reduced Amphetamine significantly reduced zif268 mRNA expression in CeA in zif268 mRNA expression in CeA in stressed ratsstressed rats

Reduced zif268 mRNA expression in Reduced zif268 mRNA expression in MeA and infralimbic cortex in MeA and infralimbic cortex in stressed ratsstressed rats

Increased zif268 mRNA expression in Increased zif268 mRNA expression in MeA in unstressed ratsMeA in unstressed rats


No interaction between stress and No interaction between stress and amphetamine on zif268 mRNA expression amphetamine on zif268 mRNA expression in the BLA, NAc shell or core, or dorsal in the BLA, NAc shell or core, or dorsal striatumstriatum

No difference in zif268 mRNA between rats No difference in zif268 mRNA between rats in the amygdala, cortex, accumbens, or in the amygdala, cortex, accumbens, or dorsal striatumdorsal striatum

Zif268 reduction in infralimbic 7 days after Zif268 reduction in infralimbic 7 days after social defeatsocial defeat

Social stress and Social stress and sensitizationsensitization

An episode of social defeat once An episode of social defeat once every 72h over 10 days sensitized every 72h over 10 days sensitized the motor response to cocaine or the motor response to cocaine or amphetamine for at least 2 monthsamphetamine for at least 2 months

Sensitize specific behavioral, Sensitize specific behavioral, physiological, and neural responses physiological, and neural responses to future challengesto future challenges

Intruders continue to show robust Intruders continue to show robust hyperthermia and tachycardia hyperthermia and tachycardia

Sensitization and self-Sensitization and self-administrationadministration

Important feature is episodic nature of the Important feature is episodic nature of the manipulationsmanipulations

Stress-sensitization increases continuous Stress-sensitization increases continuous cocaine self-administration behaviorcocaine self-administration behavior

Prolonged access supports neural, endocrine, Prolonged access supports neural, endocrine, autonomic, and behavioral adaptations autonomic, and behavioral adaptations

Access to cocaine exceeding 12-16h reveals Access to cocaine exceeding 12-16h reveals a transition to behavioral dysregulationa transition to behavioral dysregulation

Patterns for self-Patterns for self-administrationadministration

Disruption in physiological and Disruption in physiological and behavioral homeostasis during bingebehavioral homeostasis during binge

Stressed rats had increased cocaine Stressed rats had increased cocaine intakeintake

Low doses of cocaine follows circadian Low doses of cocaine follows circadian patterns-intake during dark cyclepatterns-intake during dark cycle

High doses eliminate circadian cyclesHigh doses eliminate circadian cycles Stressed rats had no decrease in cocaine Stressed rats had no decrease in cocaine

DiscussionDiscussion

Stress has an immediate activational Stress has an immediate activational effect on drug takingeffect on drug taking

Discussion: Zif268Discussion: Zif268

Long-lasting changes in the amount Long-lasting changes in the amount of zif268 mRNA expression in the of zif268 mRNA expression in the mPFC and amygdalamPFC and amygdala

Episodic stress induced increases of Episodic stress induced increases of DA and glutamate in mPFCDA and glutamate in mPFC

Amygdala and cortex adapt to stress-Amygdala and cortex adapt to stress-induced DA releaseinduced DA release

Discussion: Zif268Discussion: Zif268

Reduction in activity of amygdala Reduction in activity of amygdala neurons is suggested to correlate neurons is suggested to correlate with cocaine cravingwith cocaine craving

Stress + Amphetamine decreases Stress + Amphetamine decreases zif268 expressed in amygdala zif268 expressed in amygdala neuronsneurons

Amphetamine increases Fos Amphetamine increases Fos expressed in amygdala neuronsexpressed in amygdala neurons

Possible reasons for difference Possible reasons for difference in expression of Zif268 and Fosin expression of Zif268 and Fos

Functional role of Zif268 and Fos Functional role of Zif268 and Fos may differ within the same cellmay differ within the same cell

Discrete populations of neurons may Discrete populations of neurons may either express elevated Fos or either express elevated Fos or reduced zif268reduced zif268

ConclusionsConclusions

Behavioral sensitization to social Behavioral sensitization to social stressstress

Compulsive drug takingCompulsive drug taking Stress manipulations alter mPFC, Stress manipulations alter mPFC,

CeA, and MeACeA, and MeA

Intermittent pulses of stressIntermittent pulses of stress

Long-term intracellular effectsLong-term intracellular effects Locomotor sensitizationLocomotor sensitization Goal directed behavioral responsesGoal directed behavioral responses Behavioral responses to stimulant Behavioral responses to stimulant

administrationadministration Intense cocaine self-administrationIntense cocaine self-administration

THE ENDTHE END

brief social defeat stress: long lasting effects on cocaine taking during a binge and zif268 mrna...

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