Episodic Social Stress-Escalated Cocaine Self-Administration: Role of Phasic and Tonic Corticotropin Releasing Factor in the Anterior and Posterior Ventral Tegmental Area.

Intermittent social defeat stress escalates later cocaine self-administration. Reward and stress both activate ventral tegmental area (VTA) dopamine neurons, increasing downstream extracellular dopamine concentration in the medial prefrontal cortex and nucleus accumbens. The stress neuropeptide corticotropin releasing factor (CRF) and its receptors (CRF-R1, CRF-R2) are located in the VTA and influence dopaminergic activity. These experiments explore how CRF release and the activation of its receptors within the VTA both during and after stress influence later cocaine self-administration in rats.In vivo microdialysis of CRF in the VTA demonstrated that CRF is phasically released in the posterior VTA (pVTA) during acute defeat, but, with repeated defeat, CRF is recruited into the anterior VTA (aVTA) and CRF tone is increased in both subregions. Intra-VTA antagonism of CRF-R1 in the pVTA and CRF-R2 in the aVTA during each social defeat prevented escalated cocaine self-administration in a 24 h "binge." VTA CRF continues to influence cocaine seeking in stressed animals long after social defeat exposure. Unlike nonstressed controls, previously stressed rats show significant cocaine seeking after 15 d of forced abstinence. Previously stressed rats continue to express elevated CRF tone within the VTA and antagonism of pVTA CRF-R1 or aVTA CRF-R2 reverses cocaine seeking. In conclusion, these experiments demonstrate neuroadaptive changes in tonic and phasic CRF with repeated stress, that CRF release during stress may contribute to later escalated cocaine taking, and that persistently elevated CRF tone in the VTA may drive later cocaine seeking through increased activation of pVTA CRF-R1 and aVTA CRF-R2. SIGNIFICANCE STATEMENT: Corticotropin releasing factor (CRF) within the ventral tegmental area (VTA) has emerged as a likely candidate molecule underlying the fundamental link between stress history and escalated drug self-administration. However, the nature of CRF release in the VTA during acute and repeated stress, as well as its role in enduring neuroadaptations driving later drug taking and seeking, are poorly understood. These experiments explore how CRF is released and interacts with its receptors in specific regions of the VTA both during and after stress to fuel later escalated cocaine taking and seeking behavior. Understanding these acute and persistent changes to the VTA CRF system may lead to better therapeutic interventions for addiction.

Effects of Gabra2 Point Mutations on Alcohol Intake: Increased Binge-Like and Blunted Chronic Drinking by Mice.

BACKGROUND: Alcohol use disorders are associated with single-nucleotide polymorphisms in GABRA2, the gene encoding the GABAA receptor α2-subunit in humans. Deficient GABAergic functioning is linked to impulse control disorders, intermittent explosive disorder, and to drug abuse and dependence, yet it remains unclear whether α2-containing GABAA receptor sensitivity to endogenous ligands is involved in excessive alcohol drinking. METHODS: Male wild-type (Wt) C57BL/6J and point-mutated mice rendered insensitive to GABAergic modulation by benzodiazepines (BZD; H101R), allopregnanolone (ALLO) or tetrahydrodeoxycorticosterone (THDOC; Q241M), or high concentrations of ethanol (EtOH) (S270H/L277A) at α2-containing GABAA receptors were assessed for their binge-like, moderate, or escalated chronic drinking using drinking in the dark, continuous access (CA) and intermittent access (IA) to alcohol protocols, respectively. Social approach by mutant and Wt mice in forced alcohol abstinence was compared to approach by EtOH-naïve controls. Social deficits in forced abstinence were treated with allopregnanolone (0, 3.0, 10.0 mg/kg, intraperitoneal [i.p.]) or midazolam (0, 0.56, 1.0 mg/kg, i.p.). RESULTS: Mice with BZD-insensitive α2-containing GABAA receptors (H101R) escalated their binge-like drinking. Mutants harboring the Q241M point substitution in Gabra2 showed blunted chronic intake in the CA and IA protocols. S270H/L277A mutants consumed excessive amounts of alcohol but, unlike wild-types, they did not show forced abstinence-induced social deficits. CONCLUSIONS: These findings suggest a role for: (i) H101 in species-typical binge-like drinking, (ii) Q241 in escalated chronic drinking, and (iii) S270 and/or L277 in the development of forced abstinence-associated social deficits. Clinical findings report reduced BZD-binding sites in the cortex of dependent patients; the present findings suggest a specific role for BZD-sensitive α2-containing receptors. In addition, amino acid residue 241 in Gabra2 is necessary for positive modulation and activation of GABAA receptors by ALLO and THDOC; we postulate that neurosteroid action on α2-containing receptor may be necessary for escalated chronic EtOH intake.

Dissociation of µ-opioid receptor and CRF-R1 antagonist effects on escalated ethanol consumption and mPFC serotonin in C57BL/6J mice.

Both the opioid antagonist naltrexone and corticotropin-releasing factor type-1 receptor (CRF-R1) antagonists have been investigated for the treatment of alcoholism. The current study examines the combination of naltrexone and CP154526 to reduce intermittent access ethanol drinking [intermittent access to alcohol (IAA)] in C57BL/6J male mice, and if these compounds reduce drinking via serotonergic mechanisms in the dorsal raphe nucleus (DRN). Systemic injections and chronic intracerebroventricular infusions of naltrexone, CP154526 or CP376395 transiently decreased IAA drinking. Immunohistochemistry revealed CRF-R1 or µ-opioid receptor immunoreactivity was co-localized in tryptophan hydroxylase (TPH)-immunoreactive neurons as well as non-TPH neurons in the DRN. Mice with a history of IAA or continuous access to alcohol were microinjected with artificial cerebral spinal fluid, naltrexone, CP154526 or the combination into the DRN or the median raphe nucleus (MRN). Either intra-DRN naltrexone or CP154526 reduced IAA in the initial 2 hours of fluid access, but the combination did not additively suppress IAA, suggesting a common mechanism via which these two compounds affect intermittent drinking. These alcohol-reducing effects were localized to the DRN of IAA drinkers, as intra-MRN injections only significantly suppressed water drinking, and continuous access drinkers were not affected by CRF-R1 antagonism. Extracellular serotonin was measured in the medial prefrontal cortex (mPFC) using in vivo microdialysis after intra-DRN microinjections in another group of mice. Intra-DRN CP154526 increased serotonin impulse flow to the mPFC while naltrexone did not. This suggests the mPFC may not be an essential location to intermittent drinking, as evidenced by different effects on serotonin signaling to the forebrain yet similar behavioral findings.

Social stress and CRF-dopamine interactions in the VTA: role in long-term escalation of cocaine self-administration.

The nature of neuroadaptations in the genesis of escalated cocaine taking remains a topic of considerable interest. Intermittent social defeat stress induces both locomotor and dopaminergic cross-sensitization to cocaine, as well as escalated cocaine self-administration. The current study examines the role of corticotropin releasing factor receptor subtypes 1 and 2 (CRFR1, CRFR2) within the ventral tegmental area (VTA) during social defeat stress. This study investigated whether injecting either a CRFR1 or CRFR2 antagonist directly into the VTA before each social defeat would prevent the development of later (1) locomotor sensitization, (2) dopaminergic sensitization, and (3) escalated cocaine self-administration in rats. CRFR1 antagonist CP376395 (50 or 500 ng/side), CRFR2 antagonist Astressin2-B (100 or 1000 ng/side), or vehicle (aCSF) was microinjected into the VTA 20 min before social defeat stress (or handling) on days 1, 4, 7, and 10. Ten days later, rats were injected with cocaine (10 mg/kg, i.p.) and assessed for either locomotor sensitization, measured by walking activity, or dopaminergic sensitization, measured by extracellular dopamine (DA) in the nucleus accumbens shell (NAcSh) through in vivo microdialysis. Locomotor sensitization testing was followed by intravenous cocaine self-administration. Intra-VTA antagonism of CRFR1, but not CRFR2, inhibited the induction of locomotor cross-sensitization to cocaine, whereas both prevented dopaminergic cross-sensitization and escalated cocaine self-administration during a 24 h "binge." This may suggest dissociation between locomotor sensitization and cocaine taking. These data also suggest that interactions between CRF and VTA DA neurons projecting to the NAcSh are essential for the development of dopaminergic cross-sensitization to cocaine.

Corticotropin Releasing Factor Binding Protein and CRF2 Receptors in the Ventral Tegmental Area: Modulation of Ethanol Binge Drinking in C57BL/6J Mice.

BACKGROUND: Most studies with corticotropin releasing factor (CRF) and ethanol (EtOH) consumption have focused on CRF type 1 (CRF1 ) receptors; less is known about other components of the CRF system, such as the CRF type 2 (CRF2 ) receptors and the CRF binding protein (CRFBP). In humans, several nucleotide polymorphisms in the CRFBP gene have been associated with EtOH abuse. METHODS: The role of the CRFBP within the ventral tegmental area (VTA) and the central nucleus of the amygdala (CeA) was investigated in C57BL/6J mice exposed to an EtOH binge drinking paradigm (drinking in the dark [DID]), or to a dependence-producing drinking protocol (2-bottle choice, intermittent access to alcohol [IAA]) for 4 weeks. Potential interactions between VTA CRFBP and CRF2 receptors on EtOH binge drinking were also assessed. Mice were microinjected with the CRFBP antagonist CRF fragment 6-33 (CRF6-33 ) into the VTA or CeA, or with the CRF2 antagonist astressin-2B (A2B) alone or in combination with CRF6-33 into the VTA, and had access to 20% (w/v) EtOH for 4 hours (DID). Separate cohorts of mice received vehicle and doses of CRF6-33 into the VTA or CeA and had access to EtOH/water for 24 hours (IAA). Blood EtOH concentrations (BECs) were measured, and signs of withdrawal by handling-induced convulsions were determined. RESULTS: Intra-VTA CRF6-33 and A2B reduced EtOH intake dose dependently in mice during DID. Furthermore, a combination of a subeffective dose of CRF6-33 and a lower dose of A2B promoted additive effects in attenuating EtOH binge drinking. Intra-VTA CRF6-33 did not affect EtOH consumption in mice given IAA, and intra-CeA CRF6-33 did not change alcohol consumption in both models of drinking. DID and IAA promoted pharmacologically relevant BECs; however, only mice given IAA exhibited convulsive events during withdrawal. CONCLUSIONS: These findings suggest that VTA CRFBP is involved in the initial stages of escalated EtOH drinking by mechanisms that may involve CRF2 receptors.

GABA(B) receptor modulation of serotonin neurons in the dorsal raphé nucleus and escalation of aggression in mice.

The serotonin (5-HT) system in the brain has been studied more than any other neurotransmitter for its role in the neurobiological basis of aggression. However, which mechanisms modulate the 5-HT system to promote escalated aggression is not clear. We here explore the role of GABAergic modulation in the raphé nuclei, from which most 5-HT in the forebrain originates, on escalated aggression in male mice. Pharmacological activation of GABA(B), but not GABA(A), receptors in the dorsal raphé nucleus (DRN) escalated aggressive behaviors. In contrast, GABA agonists did not escalate aggressive behaviors after microinjection into the median raphé nucleus. The aggression-heightening effect of the GABA(B) agonist baclofen depended on the activation of 5-HT neurons in the DRN because it was blocked by coadministration of the 5-HT(1A) agonist 8-OH-DPAT [((+/-)-8-hydroxy-2-(di-n-propylamino)tetralin) hydrobromide] (DPAT), which acts on autoreceptors and inhibits 5-HT neural activity. In vivo microdialysis showed that GABA(B) activation in the DRN increased extracellular 5-HT level in the medial prefrontal cortex. This may be attributable to an indirect action via presynaptic GABA(B) receptors. The presynaptic GABA(B) receptors suppress Ca(2+) channel activity and inhibit neurotransmission, and the coadministration of N-type Ca(2+) channel blocker facilitated the effect of baclofen. These findings suggest that the indirect disinhibition of 5-HT neuron activity by presynaptic GABA(B) receptors on non-5-HT neurons in the DRN is one of the neurobiological mechanisms of escalated aggression.

Persistent escalation of alcohol drinking in C57BL/6J mice with intermittent access to 20% ethanol.

BACKGROUND: Intermittent access (IA) to drugs of abuse, as opposed to continuous access, is hypothesized to induce a kindling-type transition from moderate to escalated use, leading to dependence. Intermittent 24-hour cycles of ethanol access and deprivation can generate high levels of voluntary ethanol drinking in rats. METHODS: The current study uses C57BL/6J mice (B6) in an IA to 20% ethanol protocol to escalate ethanol drinking levels. Adult male and female B6 mice were given IA to 20% ethanol on alternating days of the week with water available ad libitum. Ethanol consumption during the initial 2 hours of access was compared with a short-term, limited access "binge" drinking procedure, similar to drinking-in-the-dark (DID). B6 mice were also assessed for ethanol dependence with handling-induced convulsion, a reliable measure of withdrawal severity. RESULTS: After 3 weeks, male mice given IA to ethanol achieved high stable levels of ethanol drinking in excess of 20 g/kg/24 h, reaching above 100 mg/dl blood ethanol concentrations, and showed a significantly higher ethanol preference than mice given continuous access to ethanol. Also, mice given IA drank about twice as much as DID mice in the initial 2-hour access period. B6 mice that underwent the IA protocol for longer periods of time displayed more severe signs of alcohol withdrawal. Additionally, female B6 mice were given IA to ethanol and drank significantly more than males (ca. 30 g/kg/24 h). DISCUSSION: The IA method in B6 mice is advantageous because it induces escalated, voluntary, and preferential per os ethanol intake, behavior that may mimic a cardinal feature of human alcohol dependence, though the exact nature and site of ethanol acting in the brain and blood as a result of IA has yet to be determined.

Persistent increase of I.V. cocaine self-administration in a subgroup of C57BL/6J male mice after social defeat stress.

RATIONALE: Stressful life experiences can persistently increase the motivation for, and consumption of, intensely rewarding stimuli, like cocaine, over time. In rodents, intermittent versus continuous exposure to social stress engenders opposing changes to reward-related behavior, as measured by consumption of sucrose and cocaine. OBJECTIVE: The present study examines if the effects of intermittent versus continuous social stress on cocaine self-administration in mice parallel those seen in rats. METHODS: Both forms of social stress involve a brief daily physical confrontation with an aggressive resident for 10 consecutive days. Continuous social stress involves constant visual and olfactory exposure to an aggressive resident via habitation in a protected portion of the resident's home cage, while exposure to an aggressive resident during intermittent social stress is limited to a single, physical encounter per day. Implementing a femoral vein catheterization method for the first time in mice, we determined divergent changes to intravenous cocaine self-administration. RESULTS: Modestly increased cocaine self-administration after intermittent social stress was confirmed. In a subset of animals, continuous social stress in mice substantially increased cocaine self-administration and sucrose intake. By stark contrast, another subpopulation had substantial attenuation of cocaine self-administration and sucrose intake after continuous social stress. CONCLUSIONS: Bimodal divergence in responding for rewarding stimuli including cocaine after social stress experience likely reflects two opposing forms of coping to continuous social stress that promote either a sensitization or attenuation of reward-seeking.

Fighting Females: Neural and Behavioral Consequences of Social Defeat Stress in Female Mice.

BACKGROUND: Despite the twofold higher prevalence of major depressive and posttraumatic stress disorders in women compared with men, most clinical and preclinical studies have focused on male subjects. We used an ethological murine model to study several cardinal symptoms of affective disorders in the female targets of female aggression. METHODS: Intact Swiss Webster (CFW) female resident mice were housed with castrated male mice and tested for aggression toward female intruders. For 10 days, aggressive CFW female residents defeated C57BL/6J (B6) female intruders during 5-minute encounters. Measures of corticosterone, c-Fos activation in hypothalamic and limbic structures, and species-typical behaviors were collected from defeated and control females. Ketamine (20 mg/kg) was tested for its potential to reverse stress-induced social deficits. RESULTS: Housed with a castrated male mouse, most intact resident CFW females readily attacked unfamiliar B6 female intruders, inflicting >40 bites in a 5-minute encounter. Compared with controls, defeated B6 females exhibited elevated plasma corticosterone and increased c-Fos activation in the medial amygdala, ventral lateral septum, ventromedial hypothalamus, and hypothalamic paraventricular nucleus. Chronically defeated females also showed vigilance-like behavior and deficits in social interactions, novel object investigation, and nesting. The duration of social interactions increased 24 hours after chronically defeated female mice received a systemic dose of ketamine. CONCLUSIONS: These findings demonstrate that CFW female mice living with male conspecifics can be used as aggressive residents in an ethological model of female social defeat stress. These novel behavioral methods will encourage further studies of sex-specific neural, physiological, and behavioral adaptations to chronic stress and the biological bases for interfemale aggression.

The Urge to Fight: Persistent Escalation by Alcohol and Role of NMDA Receptors in Mice.

Alcohol drinking, in some individuals, culminates in pathologically aggressive and violent behaviors. Alcohol can escalate the urge to fight, despite causing disruptions in fighting performance. When orally administered under several dosing conditions the current study examined in a mouse model if repeated alcohol escalates the motivation to fight, the execution of fighting performance, or both. Specifically, seven daily administrations of alcohol (0, 1.8, or 2.2 g/kg) determined if changes in the motivation to initiate aggressive acts occur with, or without, shifts in the severity of fighting behavior. Responding under the control of a fixed interval (FI) schedule for aggression reinforcements across the initial daily sessions indicated the development of tolerance to alcohol's sedative effect. By day 7, alcohol augmented FI response rates for aggression rewards. While alcohol escalated the motivation to fight, fighting performance remained suppressed across the entire 7 days. Augmented FI responding for aggression rewards in response to a low dose of alcohol (1.0 g/kg) proved to be persistent, as we observed sensitized rates of responding for more than a month after alcohol pretreatment. In addition, this sensitization of motivated aggression did not occur with a general enhancement of motor activity. Antagonism of NMDA or AMPA receptors with ketamine, dizocilpine, or NBQX during later challenges with alcohol were largely serenic without having any notable impact on the expression of alcohol-escalated rates of FI responding. The current dissociation of appetitive and performance measures indicates that discrete neural mechanisms controlling aggressive arousal can be distinctly sensitized by alcohol.

A Role for Prefrontal Cortical NMDA Receptors in Murine Alcohol-Heightened Aggression.

Alcohol is associated with nearly half of all violent crimes committed in the United States; yet, a potential neural basis for this type of pathological aggression remains elusive. Alcohol may act on N-methyl-d-aspartate receptors (NMDARs) within cortical circuits to impede processing and to promote aggression. Here, male mice were characterized as alcohol-heightened (AHAs) or alcohol non-heightened aggressors (ANAs) during resident-intruder confrontations after self-administering 1.0 g/kg alcohol (6% w/v) or water. Alcohol produced a pathological-like pattern of aggression in AHAs; these mice shifted their bites to more vulnerable locations on the body of a submissive animal, including the anterior back and ventrum after consuming alcohol. In addition, through immunoblotting, we found that AHAs overexpressed the NMDAR GluN2D subunit in the prefrontal cortex (PFC) as compared to ANAs while the two phenotypes expressed similar levels of GluN1, GluN2A and GluN2B. After identifying several behavioral and molecular characteristics that distinguish AHAs from ANAs, we tested additional mice for their aggression following preferential antagonism of GluN2D-containing NMDARs. In these experiments, groups of AHAs and ANAs self-administered 1.0 g/kg alcohol (6% w/v) or water before receiving intraperitoneal (i.p.) doses of ketamine or memantine, or infusions of memantine directly into the prelimbic (PLmPFC) or infralimbic medial PFC (ILmPFC). Moderate doses of IP ketamine, IP memantine, or intra-PLmPFC memantine increased aggression in AHAs, but only in the absence of alcohol. Prior alcohol intake blocked the pro-aggressive effects of ketamine or memantine. In contrast, only memantine, administered systemically or intra-PLmPFC, interacted with prior alcohol intake to escalate aggression in ANAs. Intra-ILmPFC memantine had no effect on aggression in either AHAs or ANAs. In sum, this work illustrates a potential role of GluN2D-containing NMDARs in the PLmPFC in alcohol-heightened aggression. GluN2D-containing NMDARs are highly expressed on cortical parvalbumin-containing interneurons, suggesting that, in a subset of individuals, alcohol may functionally alter signal integration within cortical microcircuits to dysregulate threat reactivity and promote aggression. This work suggests that targeting GluN2D-NMDARs may be of use in reducing the impact of alcohol-related violence in the human population.

Persistent escalation of alcohol consumption by mice exposed to brief episodes of social defeat stress: suppression by CRF-R1 antagonism.

RATIONALE: Episodic bouts of social stress can precede the initiation, escalation, or relapse to disordered alcohol intake. Social stress may engender neuroadaptations in the hypothalamic-pituitary-adrenal (HPA) axis and in extrahypothalamic stress circuitry to promote the escalation of alcohol intake. OBJECTIVES: We aimed to (1) confirm a pattern of escalated drinking in socially defeated mice and to (2) test drugs that target distinct aspects of the HPA axis and extrahypothalamic neural substrates for their effectiveness in reducing murine, stress-escalated drinking. METHODS: Male C57BL/6J (B6) mice were socially defeated by resident Swiss-derived males for ten consecutive days receiving 30 bites/day. Ten days after the final defeat, cohorts of B6 mice received continuous or intermittent access to 20% EtOH (w/v) and water. After 4 weeks of drinking, mice were injected with weekly, systemic doses of the CRF-R1 antagonist, CP376395; the glucocorticoid receptor antagonist, mifepristone; the 11-beta-hydroxylase inhibitor, metyrapone; or the 5-alpha-reductase inhibitor, finasteride. RESULTS: Prior to drug treatments, defeated mice reliably consumed more EtOH than non-defeated controls, and mice given alcohol intermittently consumed more EtOH than those with continuous access. CP376395 (17-30 mg/kg) reduced continuous, but not intermittent EtOH intake (g/kg) in socially defeated mice. Mifepristone (100 mg/kg), however, increased drinking by defeated mice with intermittent access to alcohol while reducing drinking during continuous access. When administered finasteride (100 mg/kg) or metyrapone (50 mg/kg), all mice reduced their EtOH intake while increasing their water consumption. CONCLUSIONS: Mice with a history of episodic social defeat stress were selectively sensitive to the effects of CRF-R1 antagonism, suggesting that CRF-R1 may be a potential target for treating alcohol use disorders in individuals who escalate their drinking after exposure to repeated bouts of psychosocial stress. Future studies will clarify how social defeat stress may alter the expression of extrahypothalamic CRF-R1 and glucocorticoid receptors.

MK-801 infusions to the ventral tegmental area and ventromedial hypothalamus produce opposite effects on lordosis of hormone-primed rats.

Progesterone initiates female sexual behavior of rodents (lordosis) through actions at intracellular progestin receptors in the ventromedial hypothalamus. Progesterone's metabolite, 5alpha-pregnan-3alpha-ol-20-one, mediates the intensity and duration of lordosis through its actions at GABA(A) receptors in the ventral tegmental area. Whether progestins can influence sexual behavior through actions that involve N-methyl-D-aspartate receptors (NMDARs) in the ventromedial hypothalamus and ventral tegmental area was investigated. The current study examines the effect of bilateral ventral tegmental area or ventromedial hypothalamus infusions of the non-competitive NMDAR antagonist (+)-MK-801 hydrogen maleate (MK-801; 0, 20, or 200 ng) on lordosis, motor activity, and NMDA R1 subtype (NMDAR1) immunoreactivity in estradiol benzoate (10 microg)+progesterone (50 microg)- and estradiol benzoate+vehicle primed rats. Compared to vehicle infusions, infusions of MK-801 to the ventral tegmental area facilitated lordosis of estradiol benzoate (10 microg)+progesterone (50 microg)- and estradiol benzoate+vehicle primed rats. Infusions of MK-801 to the ventromedial hypothalamus inhibited lordosis of estradiol benzoate (10 microg)+progesterone (50 microg)- and estradiol benzoate+vehicle primed rats, compared to vehicle. There was no effect of MK-801 infusions to the ventral tegmental area or the ventromedial hypothalamus on motor behavior. Immunocytochemistry for NMDAR1 revealed MK-801 (200 ng) infusions to the ventral tegmental area or ventromedial hypothalamus of estradiol benzoate (10 microg)+progesterone (50 microg)- or estradiol benzoate+vehicle primed rats significantly reduced the number of darkly stained NMDAR1-immunoreactive cells, compared to vehicle infusions. These data suggest NMDARs may be important in the mediation of hormonal actions in both the ventral tegmental area and the ventromedial hypothalamus for sexual receptivity of rodents, but in different ways.

Prevention and reversal of social stress-escalated cocaine self-administration in mice by intra-VTA CRFR1 antagonism.

BACKGROUND: A history of brief intermittent social defeat stress can escalate cocaine self-administration and induce long-term adaptations in the mesolimbic dopamine system. Extra-hypothalamic corticotrophin releasing factor (CRF) has been shown to be closely associated with stress-induced escalation of drug use. How repeated stress modulates CRF release in the ventral tegmental area (VTA) and the roles of CRF receptors during different phases of stress-induced cocaine self-administration remain to be defined. OBJECTIVE: The current study examines the roles of CRF and CRF receptor 1 (CRFR1) in escalated intravenous cocaine self-administration after exposure to social defeat stress in mice. METHODS AND RESULTS: First, CRFR1 antagonist (CP 376,395, 15 mg/kg, i.p.) given 30 min prior to each social defeat episode prevented later escalated cocaine self-administration. When CP 376,395 (5 and 15 mg/kg, i.p.) was administered 10 days after the last episode of social stress, the escalation of cocaine intake was dose-dependently reversed. Moreover, socially defeated mice showed increased CRF release in the VTA compared to controls. To further explore the role of CRFR1, CP 376,395 (0.5 and 1 µg/0.2 µl) was infused directly into the VTA before the cocaine self-administration session. Intra-VTA antagonism of CRFR1 was sufficient to reverse social defeat stress-escalated cocaine self-administration. CONCLUSION: These findings suggest that CRF and CRFR1 exert multiple roles in the response to social stress that are relevant to escalated cocaine self-administration.

Escalated cocaine "binges" in rats: enduring effects of social defeat stress or intra-VTA CRF.

RATIONALE: Exposure to intermittent social defeat stress elicits corticotropin releasing factor (CRF) release into the VTA and induces long-term modulation of mesocorticolimbic dopamine activity in rats. These adaptations are associated with an intense cocaine-taking phenotype, which is prevented by CRF receptor antagonists. OBJECTIVE: The present studies examine whether infusion of CRF into the VTA is sufficient to escalate cocaine-taking behavior, in the absence of social defeat experience. Additionally, we aimed to characterize changes in cocaine valuation that may promote binge-like cocaine intake. METHODS: Male Long-Evans rats were microinjected into the VTA with CRF (50 or 500 ng/side), vehicle, or subjected to social defeat stress, intermittently over 10 days. Animals were then trained to self-administer IV cocaine (FR5). Economic demand for cocaine was evaluated using a within-session behavioral-economics threshold procedure, which was followed by a 24-h extended access "binge." RESULTS: Rats that experienced social defeat or received intra-VTA CRF microinfusions (50 ng) both took significantly more cocaine than controls over the 24-h binge but showed distinct patterns of intake. Behavioral economic analysis revealed that individual demand for cocaine strongly predicts binge-like consumption, and demand elasticity (i.e. α) is augmented by intra-VTA CRF, but not by social defeat. The effects of CRF on cocaine-taking were also prevented by intra-VTA pretreatment with CP376395, but not Astressin-2B. CONCLUSIONS: Repeated infusion of CRF into the VTA persistently alters cocaine valuation and intensifies binge-like drug intake in a CRF-R1-dependent manner. Conversely, the persistent pattern of cocaine bingeing induced by social defeat stress may suggest impaired inhibitory control, independent of reward valuation.

CRF type 1 receptor antagonism in ventral tegmental area of adolescent rats during social defeat: prevention of escalated cocaine self-administration in adulthood and behavioral adaptations during adolescence.

BACKGROUND: Activation of corticotropin-releasing factor type 1 receptors (CRF-R1) in the ventral tegmental area (VTA) represents a critical mechanism for social defeat to escalate cocaine self-administration in adult rats. OBJECTIVE: We determined the acute effect of a CRF-R1 antagonist (CP376395) microinfusion into the VTA prior to each episode of social defeat in adolescent rats and determined whether this drug treatment could prevent later escalation of cocaine taking in early adulthood. METHODS: Rats were implanted with bilateral cannulae aimed at the VTA 5 days before the first social defeat. Bilateral microinfusion of CP376395 (500 ng/side) or vehicle occurred 20 min before each episode of social defeat on postnatal days (P) 35, 38, 41, and 44. Behavior was quantified on P35 and P44. On P57, rats were implanted with intra-jugular catheters, and subsequent cocaine self-administration was analyzed. RESULTS: CP376395-treated adolescent rats walked less and were attacked more slowly but were socially investigated more than vehicle-treated adolescents. Vehicle-treated rats showed increased social and decreased non-social exploration from P35 to P44, while CP376395-treated rats did not. Socially defeated, vehicle-treated adolescents took more cocaine during a 24-h unlimited access binge during adulthood. The latency to supine posture on P44 was inversely correlated with later cocaine self-administration during fixed and progressive ratio schedules of reinforcement and during the binge. CONCLUSIONS: CP376395 treatment in adolescence blocked escalation of cocaine taking in adulthood. Episodes of social defeat stress engender neuroadaptation in CRF-R1s in the VTA that alter coping with social stress and that persist into adulthood.

Social stress-escalated intermittent alcohol drinking: modulation by CRF-R1 in the ventral tegmental area and accumbal dopamine in mice.

RATIONALE: Excessive alcohol (EtOH) drinking is difficult to model in animals despite the extensive human literature demonstrating that stress increases EtOH consumption. OBJECTIVE: The current experiments show escalations in voluntary EtOH drinking caused by a history of social defeat stress and intermittent access to EtOH in C57BL/6J mice compared to non-stressed mice given intermittent EtOH or continuous EtOH. To explore a mechanistic link between stress and drinking, we studied the role of corticotropin-releasing factor type-1 receptors (CRF-R1) in the dopamine-rich ventral tegmental area (VTA). RESULTS: Intra-VTA infusions of a CRF-R1 antagonist, CP376395, infused into the VTA dose-dependently and selectively reduced intermittent EtOH intake in stressed and non-stressed mice, but not in mice given continuous EtOH. In contrast, intra-VTA infusions of the CRF-R2 antagonist astressin2B non-specifically suppressed both EtOH and H2O drinking in the stressed group without effects in the non-stressed mice. Using in vivo microdialysis in the nucleus accumbens (NAc) shell, we observed that stressed mice drinking EtOH intermittently had elevated levels of tonic dopamine concentrations compared to non-stressed drinking mice. Also, VTA CP376395 potentiated dopamine output to the NAc only in the stressed group causing further elevations of dopamine post-infusion. CONCLUSIONS: These findings illustrate a role for extrahypothalamic CRF-R1 as especially important for stress-escalated EtOH drinking beyond schedule-escalated EtOH drinking. CRF-R1 may be a mechanism for balancing the dysregulation of stress and reward in alcohol use disorders.

Maladaptive choices by defeated rats: link between rapid approach to social threat and escalated cocaine self-administration.

RATIONALE: Intermittent social defeat stress engenders persistent neuroadaptations and can result in later increased cocaine taking and seeking. However, there are individual differences in stress-escalated cocaine self-administration behavior, which may be a direct result of individual differences in the manner in which rats experience social defeat stress. OBJECTIVE: The present study dissected the discrete behavioral phases of social defeat and analyzed which behavioral characteristics may be predictive of subsequent cocaine self-administration. METHODS: Male Long-Evans rats underwent nine intermittent social defeat episodes over 21 days in a three-compartment apparatus permitting approach to and escape from a confrontation with an aggressive resident rat. Rats then self-administered intravenous cocaine, which culminated in a 24-h unlimited access "binge." Behaviors during social defeat and cocaine self-administration were evaluated by principal component analysis (PCA). RESULTS: PCA revealed that the latency to enter the threatening environment was highly predictive of later cocaine self-administration during the 24-h binge. This behavior was not associated with other cocaine-predictive traits, such as reactivity to novelty in an open field, saccharin preference, and motor impulsivity. Additionally, there was no effect of latency to enter a threatening environment on physiological measures of stress, including plasma corticosterone and corticotropin releasing factor (CRF) in the extended amygdala. However, latency to enter the threatening environment was negatively correlated with brain-derived neurotropic factor (BDNF) and its receptor, tyrosine kinase B (TrkB) in the hippocampus. CONCLUSION: These data suggest that latency to enter a threatening environment is a novel behavioral characteristic predictive of later cocaine self-administration.

Escalated aggression as a reward: corticosterone and GABA(A) receptor positive modulators in mice.

RATIONALE: Individuals seek out the opportunity to fight, but the mechanisms behind this positively reinforcing effect of aggression have yet to be understood. OBJECTIVES: The aims of this study were to (1) describe behavioral and corticosterone elevations that occur in aggressive mice conditioned to respond for the opportunity to fight another mouse, (2) determine if corticosterone elevations are necessary for operant responding and escalated aggression, and (3) determine if corticosterone elevations alter the aggression-heightening effects of gamma-aminobutyric acid (GABA)(A) receptor positive modulators. METHODS AND RESULTS: Aggressive male CFW mice were conditioned to respond under the control of a fixed-interval 10-min (FI10) schedule that reinforced their operant behavior by the presentation of an intruder mouse into their home cage. After the FI10, aggressive behavior was ca. 75% higher than the species-typical levels of fighting and plasma corticosterone was more than twice as high after briefly fighting and/or responding on the FI10 schedule. Inhibition of corticosterone synthesis by metyrapone (30-100 mg/kg) reduced both conditioned responding as well as the aggressive behavior after the FI. Although the benzodiazepine midazolam (0.3-3 mg/kg) heightened species-typical aggressive behavior, it did not increase the high level of aggression engendered by the FI schedule. However, midazolam (0.3 mg/kg) and the neurosteroid allopregnanolone (17 mg/kg) both heightened aggression when given after corticosterone synthesis inhibition by metyrapone (56 mg/kg). CONCLUSIONS: These data suggest that corticosterone elevations are required for responding that is motivated by aggressive behavior and for escalated aggression that follows this responding. Corticosterone elevations also appear to inhibit the aggression heightening effect of GABA(A) receptor positive modulators.

Benzodiazepines and heightened aggressive behavior in rats: reduction by GABA(A)/alpha(1) receptor antagonists.

RATIONALE: Positive modulators of the benzodiazepine/GABA(A) receptor complex can heighten aggressive behavior; the GABA(A)/alpha(1) subunit may play a critical role in benzodiazepine-modulated aggressive behavior. OBJECTIVE: The carboline derivatives, beta-CCt and 3-PBC, antagonists with preferential action at the GABA(A) receptors with alpha(1) subunits, may antagonize benzodiazepine-heightened aggression, thus implicating the alpha(1) subunit in heightened aggression. METHODS: The GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4c]-pyridin-3-ol (THIP) (0.01-3.0 mg/kg), and the benzodiazepine receptor agonists midazolam (0.3-3.0 mg/kg) and triazolam (0.003-3.0 mg/kg) were administered to adult male resident rats to assess the drugs' effects on their aggressive behavior toward an intruder. Then beta-CCt (0.3-10.0 mg/kg) and 3-PBC (0.3-17.0 mg/kg) were each administered in conjunction with midazolam. The salient elements of aggressive and non-aggressive behavior were measured by analyzing video recordings and encoding each behavioral act and posture in terms of its frequency and duration of occurrence. RESULTS: Midazolam significantly increased the duration of aggressive behaviors at 1.0 and 1.7 mg/kg, and triazolam increased attack bite frequency at 0.03 mg/kg, both implicating GABA(A) receptors with benzodiazepine binding sites in aggressive behavior. In the present dose range, THIP did not affect any behaviors. The broad-spectrum benzodiazepine antagonist, flumazenil (1.0 mg/kg), antagonized the aggression-heightening effects of midazolam. beta-CCt (0.3-10.0 mg/kg) and 3-PBC (0.3-17.0 mg/kg) also antagonized the aggression-heightening effects of midazolam (1.0 mg/kg). CONCLUSIONS: These results implicate both the GABA(A) gamma and alpha(1) subunits in benzodiazepine-heightened aggression.