Brief Social Defeat Stress: Long Lasting Effects on Cocaine Taking During a Binge and Zif268 mRNA Expression in the Amygdala and Prefrontal Cortex Herbert E Covington, Takefumi Kikusui, Justin Goodhue, Ella M Nikulina, Ronald P Hammer Jr., and Klaus A Miczek

Materials and Methods Male Long-Evans rats Male Long-Evans rats g g Individually housed in clear acrylic chambers Individually housed in clear acrylic chambers Cellulose pellet bedding Cellulose pellet bedding Free access to food and water Free access to food and water Reversed light cycle Reversed light cycle Removable stainless steel mesh panels Removable stainless steel mesh panels

Materials and Methods Controlled temperature (21 +/- 1C) Controlled temperature (21 +/- 1C) Controlled humidity (35-40%) Controlled humidity (35-40%) Each chamber served at various phases of the experimental protocol as the home cage Each chamber served at various phases of the experimental protocol as the home cage Habituated to saline injections for 3 days prior to measurements Habituated to saline injections for 3 days prior to measurements

Social Stress Episode First phase: Experimental rat’s home cage initially placed within an aggressive resident’s cage where the intruder was exposed to the resident’s species-typical threats for 10 minutes through a protective screen First phase: Experimental rat’s home cage initially placed within an aggressive resident’s cage where the intruder was exposed to the resident’s species-typical threats for 10 minutes through a protective screen Second phase: Experimental intruder was removed from their protective home cage, and a confrontation persisted until the attacks by the resident resulted in defeat Second phase: Experimental intruder was removed from their protective home cage, and a confrontation persisted until the attacks by the resident resulted in defeat

Social Stress Episode Defeat= display of 4 seconds of supine posture by the intruder, or a maximum duration of 5 minutes Defeat= display of 4 seconds of supine posture by the intruder, or a maximum duration of 5 minutes Third Phase: Experimental rat was exposed for 10 min to the resident’s threats behind the protective screen of its cage Third Phase: Experimental rat was exposed for 10 min to the resident’s threats behind the protective screen of its cage

Induction of behavioral sensitization Establish pre-stress baseline motor activity 1 day prior to episode Establish pre-stress baseline motor activity 1 day prior to episode Injected with saline Injected with saline Video samples collected at 5-10 minutes, and minutes after injections Video samples collected at 5-10 minutes, and minutes after injections

Expression of behavioral sensitization Each defeat stressed and unstressed rats’ locomotor response to a cocaine (10mg/kg) challenge on day 20 (10 days after the last defeat) Each defeat stressed and unstressed rats’ locomotor response to a cocaine (10mg/kg) challenge on day 20 (10 days after the last defeat) Stressed and unstressed control rats received an injection of amphetamine (1.0mg/kg) on experimental day 70 Stressed and unstressed control rats received an injection of amphetamine (1.0mg/kg) on experimental day 70 Saline inj administered 15 min prior to the stimulant drug challenge Saline inj administered 15 min prior to the stimulant drug challenge Determines baseline measurements of locomotor activity Determines baseline measurements of locomotor activity

Expression of behavioral sensitization Immediately after baseline, administered stimulant challenge Immediately after baseline, administered stimulant challenge Administered between 11:00 and 14:00 Administered between 11:00 and 14:00 5 min video samples were collected 5-10 min after saline 5 min video samples were collected 5-10 min after saline 5 min video samples were collected 5-10 min and min after stimulant challenges 5 min video samples were collected 5-10 min and min after stimulant challenges

Expression of behavioral sensitization Observed the frequency and analyzed each 5 min sample Observed the frequency and analyzed each 5 min sample Walking Walking Rearing Rearing Sniffing Sniffing Grooming Grooming Digging Digging Inactivity Inactivity

Experiment 1: Cocaine Self- Administration Implanted catheters into the right jugular vein Implanted catheters into the right jugular vein Recovered for 5 days after surgery Recovered for 5 days after surgery One wall of chamber had two retractable levers One wall of chamber had two retractable levers Self administer cocaine (0.75mg/kg/infusion) Self administer cocaine (0.75mg/kg/infusion)

Experiment 1 Locomotor activity was monitored from 1h before binge until 1h after Locomotor activity was monitored from 1h before binge until 1h after Prolonged protocol lower dose cocaine (0.3mg/kg/infusion) Prolonged protocol lower dose cocaine (0.3mg/kg/infusion) Start at 19:00 for 24h Start at 19:00 for 24h Red light at the start of light phase Red light at the start of light phase

Experiment 1 No priming No priming Fixed ratio of 1 with a 30 second time out Fixed ratio of 1 with a 30 second time out Initiated at 10:00 Initiated at 10:00 Terminated after 15 infusions or 5 hours of access Terminated after 15 infusions or 5 hours of access 10 additional days on a FR 5 reinforcement for 15 infusions 10 additional days on a FR 5 reinforcement for 15 infusions Continuous access to cocaine during 24h binge Continuous access to cocaine during 24h binge

Experiment 2: Zif 268 mRNA Expression in the mPFC, NAc, Dorsal Striatum, and Amygdala Challenge inj of d-amphetamine (1.0mg/kg) on experimental day 70 Challenge inj of d-amphetamine (1.0mg/kg) on experimental day 70 Tissue collected for Zif 268 mRNA (Egr 1 ) expression analysis Tissue collected for Zif 268 mRNA (Egr 1 ) expression analysis Stress-sensitizing protocol on zif268 mRNA expression within 7 days of stress exposure Stress-sensitizing protocol on zif268 mRNA expression within 7 days of stress exposure

Neuronal zif268 expression measurements prelimbic and infralimbic cortex prelimbic and infralimbic cortex NAc core and shell NAc core and shell Dorsal striatum Dorsal striatum Medial, central, and basolateral amygdaloid nuclei Medial, central, and basolateral amygdaloid nuclei

Results: Experiment 1 T-tests T-tests Kaplan-Meier Survival Analysis Kaplan-Meier Survival Analysis Two-way ANOVA Two-way ANOVA Bonferroni correction Bonferroni correction

Results: Experiment 1 No significant differences found in behavior other than walking No significant differences found in behavior other than walking In cocaine challenge stressed rats walked more than unstressed In cocaine challenge stressed rats walked more than unstressed No significant difference in acquisition of self administration between stressed and unstressed rats No significant difference in acquisition of self administration between stressed and unstressed rats

Results: Experiment 1 Stressed rats self-administered considerably more total cocaine than unstressed rats Stressed rats self-administered considerably more total cocaine than unstressed rats Unstressed rats took in less cocaine during light cycle Unstressed rats took in less cocaine during light cycle Stressed rats self-administered cocaine continuously Stressed rats self-administered cocaine continuously

Results: Experiment 1 Total horizontal activity and stereotyped sniffing increased when cocaine available Total horizontal activity and stereotyped sniffing increased when cocaine available Stressed rats showed more horizontal and stereotyped behavior during light phase Stressed rats showed more horizontal and stereotyped behavior during light phase No difference in behavior during dark phase between the groups No difference in behavior during dark phase between the groups

Results: Experiment 2 Amphetamine challenge on day 70 Amphetamine challenge on day days after last defeat stress 60 days after last defeat stress Increased walking behavior for stressed rats Increased walking behavior for stressed rats Interaction seen between stress and amphetamine challenge in zif268 mRNA expression in CeA Interaction seen between stress and amphetamine challenge in zif268 mRNA expression in CeA Greater in stressed rats Greater in stressed rats

Results: Experiment 2 Amphetamine significantly reduced zif268 mRNA expression in CeA in stressed rats Amphetamine significantly reduced zif268 mRNA expression in CeA in stressed rats Reduced zif268 mRNA expression in MeA and infralimbic cortex in stressed rats Reduced zif268 mRNA expression in MeA and infralimbic cortex in stressed rats Increased zif268 mRNA expression in MeA in unstressed rats Increased zif268 mRNA expression in MeA in unstressed rats

Results: Experiment 2 No interaction between stress and amphetamine on zif268 mRNA expression in the BLA, NAc shell or core, or dorsal striatum No interaction between stress and amphetamine on zif268 mRNA expression in the BLA, NAc shell or core, or dorsal striatum No difference in zif268 mRNA between rats in the amygdala, cortex, accumbens, or dorsal striatum No difference in zif268 mRNA between rats in the amygdala, cortex, accumbens, or dorsal striatum Zif268 reduction in infralimbic 7 days after social defeat Zif268 reduction in infralimbic 7 days after social defeat

Social stress and sensitization An episode of social defeat once every 72h over 10 days sensitized the motor response to cocaine or amphetamine for at least 2 months An episode of social defeat once every 72h over 10 days sensitized the motor response to cocaine or amphetamine for at least 2 months Sensitize specific behavioral, physiological, and neural responses to future challenges Sensitize specific behavioral, physiological, and neural responses to future challenges Intruders continue to show robust hyperthermia and tachycardia Intruders continue to show robust hyperthermia and tachycardia

Sensitization and self- administration Important feature is episodic nature of the manipulations Important feature is episodic nature of the manipulations Stress-sensitization increases continuous cocaine self-administration behavior Stress-sensitization increases continuous cocaine self-administration behavior Prolonged access supports neural, endocrine, autonomic, and behavioral adaptations Prolonged access supports neural, endocrine, autonomic, and behavioral adaptations Access to cocaine exceeding 12-16h reveals a transition to behavioral dysregulation Access to cocaine exceeding 12-16h reveals a transition to behavioral dysregulation

Patterns for self-administration Disruption in physiological and behavioral homeostasis during binge Disruption in physiological and behavioral homeostasis during binge Stressed rats had increased cocaine intake Stressed rats had increased cocaine intake Low doses of cocaine follows circadian patterns-intake during dark cycle Low doses of cocaine follows circadian patterns-intake during dark cycle High doses eliminate circadian cycles High doses eliminate circadian cycles Stressed rats had no decrease in cocaine Stressed rats had no decrease in cocaine

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

Discussion: Zif268 Long-lasting changes in the amount of zif268 mRNA expression in the mPFC and amygdala Long-lasting changes in the amount of zif268 mRNA expression in the mPFC and amygdala Episodic stress induced increases of DA and glutamate in mPFC Episodic stress induced increases of DA and glutamate in mPFC Amygdala and cortex adapt to stress- induced DA release Amygdala and cortex adapt to stress- induced DA release

Discussion: Zif268 Reduction in activity of amygdala neurons is suggested to correlate with cocaine craving Reduction in activity of amygdala neurons is suggested to correlate with cocaine craving Stress + Amphetamine decreases zif268 expressed in amygdala neurons Stress + Amphetamine decreases zif268 expressed in amygdala neurons Amphetamine increases Fos expressed in amygdala neurons Amphetamine increases Fos expressed in amygdala neurons

Possible reasons for difference in expression of Zif268 and Fos Functional role of Zif268 and Fos may differ within the same cell Functional role of Zif268 and Fos may differ within the same cell Discrete populations of neurons may either express elevated Fos or reduced zif268 Discrete populations of neurons may either express elevated Fos or reduced zif268

Conclusions Behavioral sensitization to social stress Behavioral sensitization to social stress Compulsive drug taking Compulsive drug taking Stress manipulations alter mPFC, CeA, and MeA Stress manipulations alter mPFC, CeA, and MeA

Intermittent pulses of stress Long-term intracellular effects Long-term intracellular effects Locomotor sensitization Locomotor sensitization Goal directed behavioral responses Goal directed behavioral responses Behavioral responses to stimulant administration Behavioral responses to stimulant administration Intense cocaine self-administration Intense cocaine self-administration

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