Background Intermittent access 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 intermittent access to 20% ethanol protocol to escalate ethanol drinking levels. Adult male and female B6 mice were given intermittent access 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 to 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 (HIC), a reliable measure of withdrawal severity. Results After 3 weeks, male mice given intermittent access to ethanol achieved high stable levels of ethanol drinking in excess of 20 g/kg/24h, reaching above 100 mg/dl BEC, and showed a significantly higher ethanol preference than mice given continuous access to ethanol. Also, mice given intermittent access drank about twice as much as DID mice in the initial 2-hour access period. B6 mice that underwent the intermittent access protocol for longer periods of time displayed more severe signs of alcohol withdrawal. Additionally, female B6 mice were given intermittent access to ethanol and drank significantly more than males (ca. 30 g/kg/24h). Discussion The intermittent access 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 intermittent access has yet to be determined.

Summary Escalated aggression can have devastating societal consequences, yet underlying neurobiological mechanisms are poorly understood. Here we show significantly increased inter-male mouse aggression when neurotransmission is constitutively blocked from either of two subsets of serotonergic, Pet1+ neurons – one identified by dopamine receptor D1(Drd1a)::cre driven activity perinatally, the other by Drd2::cre from pre-adolescence onward. Blocking neurotransmission from other Pet1+ neuron subsets of similar size and/or overlapping anatomical domains had no effect on aggression compared to controls, suggesting subtype-specific serotonergic neuron influences on aggression. Using established and novel intersectional genetic tools, we further characterized these subtypes across multiple parameters, showing both overlapping and distinct features in axonal projection targets, gene expression, electrophysiological properties, and effects on non-aggressive behaviors. Notably, Drd2::cre-marked 5-HT neurons exhibited D2-dependent inhibitory responses to dopamine in slices, suggesting direct and specific interplay between inhibitory dopaminergic signaling and a serotonergic subpopulation. Thus, we identify specific serotonergic modules that shape aggression.

Rationale Stress experiences have been shown to be a risk factor for alcohol abuse in humans; however, a reliable mouse model using episodic social stress has yet to be developed. Objectives The current studies investigated the effects of mild and moderate social defeat protocols on plasma corticosterone, voluntary alcohol drinking, and motivation to drink alcohol. Methods Outbred CFW mice were socially defeated for 10 days during which the intruder mouse underwent mild (15 bites: mean = 1.5 min), or moderate (30 bites: mean = 3.8 min) stress. Plasma corticosterone was measured on days 1 and 10 of the defeat. Ethanol drinking during continuous access to alcohol was measured 10 days following the defeat or 10 days prior to, during and 20 days after the defeat. Motivation to drink was determined using a PR operant conditioning schedule during intermittent access to ethanol. Results Plasma corticosterone was elevated in both stress groups on days 1 and 10. Ethanol consumption and preference following moderate social stress was higher than both the mild stress group and controls. Mice with previously acquired ethanol drinking showed decreased ethanol consumption during the moderate stress followed by an increase 20 days post-defeat. Moderately stressed mice also showed escalated ethanol intake (11g/kg/day) and ethanol self-administration during a schedule of intermittent access to alcohol. Conclusion Social defeat experiences of moderate intensity and duration led to increased ethanol drinking and preference in CFW mice. Ongoing work investigates the interaction between glucocorticoids and dopaminergic systems as neural mechanisms for stress-escalated alcohol consumption.

Background Most studies with corticotropin releasing factor (CRF) and ethanol 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 ethanol 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 ethanol binge drinking paradigm (drinking-in-the-dark, DID), or to a dependence-producing drinking protocol (two-bottle choice, intermittent access to alcohol, IAA) for 4 weeks. Potential interactions between VTA CRFBP and CRF2 receptors on ethanol binge drinking were also assessed. Mice were microinjected with the CRFBP antagonist CRF6–33 into the VTA or CeA, or with the CRF2 antagonist Astressin2-B (A2B) alone or in combination with CRF6–33 into the VTA, and had access to 20% (w/v) ethanol for 4 h (DID). Separate cohorts of mice received vehicle and doses of CRF6–33 into the VTA or CeA and had access to ethanol/water for 24 h (IAA). Blood ethanol concentrations (BECs) were measured, and signs of withdrawal by handling-induced convulsion were determined. Results Intra-VTA CRF6–33 and A2B reduced ethanol intake dose-dependently in mice during DID. Furthermore, a combination of a sub-effective dose of CRF6–33 and a lower dose of A2B promoted additive effects in attenuating ethanol binge drinking. Intra-VTA CRF6–33 did not affect ethanol 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 ethanol drinking by mechanisms that may involve CRF2 receptors.

Rationale Manipulation of the stress neuropeptide corticotropin-releasing factor (CRF), specifically central antagonism of the type-1 receptors (CRF-R1), effectively reduces alcoholic-like ethanol drinking in rodents. Escalated consumption is largely controlled by neurocircuitry that is important for reward and affect such as the ventral tegmental area (VTA) and the dorsal raphé nucleus (DRN). Objective The current studies investigated the role of CRF-R1 within the VTA and DRN, and their relation to escalated ethanol drinking in two species. An additional goal was to explore whether high alcohol-drinking individuals would be more affected by CRF-R1 antagonism than low alcohol-drinking individuals. Methods With a two-bottle choice drinking procedure, adult male C57BL/6J mice and Long-Evans rats were given 24 hr access to 20% ethanol and water on an intermittent schedule. Rats and mice were implanted with cannulae targeting the VTA or DRN. Doses of the CRF-R1 antagonist CP-154,526 were microinfused to modulate drinking of ethanol and water over the course of 24 hr. Results In both mice and rats, intra-VTA CP-154,526 selectively decreased ethanol intake while identical doses (0.3 and 0.6 μg) infused intra-DRN reduced both ethanol and water drinking. Long-Evans rats displayed a range of individual differences for ethanol preference, and CP-154,526 suppressed ethanol drinking in the high-preferring animals regardless of brain site manipulation. Conclusions The current findings confirm previous studies that blockade of CRF-R1 efficaciously reduces escalated drinking while also suggesting that the effects of intermittent access on alcohol consumption may require CRF interaction with dopamine in the VTA.

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 shell (NAc), 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.

Acute exposure to a salient stressor, such as in post-traumatic stress disorder, can have lasting impacts upon an individual and society. To study stress in rodents, some naturalistic methods have included acute exposure to a predator odor, such as the synthetic fox odor 2,4,5, trimethyl-3-thiazoline (TMT). These experiments explore the stress-related behaviors and cortical activity induced by TMT exposure in adult male C57BL/6J mice and the influence of the stress neuropeptide corticotropin-releasing factor (CRF) on these responses. Compared to H 2 O and a novel odorant, vanilla, mice exposed to TMT in the home cage showed increased avoidance and defensive burying indicative of evident stress responses. Consistent with stress-induced activation of the medial prefrontal cortex (mPFC), we found that the prelimbic (PL) and infralimbic (IL) subregions of the mPFC had elevated c-Fos immunolabeling after TMT and vanilla compared to H 2 O. Slice physiology recordings were performed in layers 2/3 and 5 of the PL and IL, following TMT, vanilla, or H 2 O exposure. In TMT mice, but not vanilla or H 2 O mice, PL layers 2/3 showed heightened spontaneous excitatory post-synaptic currents and synaptic drive, suggesting TMT enhanced excitatory transmission. Synaptic drive in PL was increased in both TMT and H 2 O mice following bath application of 300 nM CRF, but only H 2 O mice increased excitatory currents with 100 nM CRF, suggesting dose-effect curve shifts in TMT mice. Further, systemic pretreatment with the CRF-R1 antagonist CP154526 and bath application with the CRF-R1 antagonist NBI27914 reduced excitatory transmission in TMT mice, but not H 2 O mice. CP154526 also reduced stress-reactive behaviors induced by TMT. Taken together, these findings suggest that exposure to TMT leads to CRF-R1 driven changes in behavior and changes in synaptic function in layer 2/3 neurons in the PL, which are consistent with previous findings that CRF-R1 in the mPFC plays an important role in predator odor-related behaviors.Neuropsychopharmacology (2019) 44:766-775; https://doi.

Sex differences in chronic pain and alcohol abuse are not well understood. The development of rodent models is imperative for investigating the underlying changes behind these pathological states. In the present study, we investigated whether hind paw treatment with the inflammatory agent Complete Freund’s Adjuvant (CFA) could generate hyperalgesia and alter alcohol consumption in male and female C57BL/6J mice. CFA treatment led to greater nociceptive sensitivity for both sexes in the Hargreaves test, and increased alcohol drinking for males in a continuous access two-bottle choice (CA2BC) paradigm. Regardless of treatment, female mice exhibited greater alcohol drinking than males. Following a 2-hour terminal drinking session, CFA treatment failed to produce changes in alcohol drinking, blood ethanol concentration (BEC), and plasma corticosterone (CORT) for both sexes. 2-hr alcohol consumption and CORT was higher in females than males, irrespective of CFA treatment. Taken together, these findings have established that male mice are more susceptible to escalations in alcohol drinking when undergoing pain, despite higher levels of total alcohol drinking and CORT in females. Furthermore, the exposure of CFA-treated C57BL/6J mice to the CA2BC drinking paradigm has proven to be a useful model for studying the relationship between chronic pain and alcohol abuse. Future applications of the CFA/CA2BC model should incorporate manipulations of stress signaling and other related biological systems to improve our mechanistic understanding of pain and alcohol interactions.