Lara S. Hwa

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.

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

RationaleExcessive alcohol (EtOH) drinking is difficult to model in animals despite the extensive human literature demonstrating that stress increases EtOH consumption.ObjectiveThe 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).ResultsIntra-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.ConclusionsThese 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.

Alcohol and violence: neuropeptidergic modulation of monoamine systems

Neurobiological processes underlying the epidemiologically established link between alcohol and several types of social, aggressive, and violent behavior remain poorly understood. Acute low doses of alcohol, as well as withdrawal from long‐term alcohol use, may lead to escalated aggressive behavior in a subset of individuals. An urgent task will be to disentangle the host of interacting genetic and environmental risk factors in individuals who are predisposed to engage in escalated aggressive behavior. The modulation of 5‐hydroxytryptamine impulse flow by gamma‐aminobutyric acid (GABA) and glutamate, acting via distinct ionotropic and metabotropic receptor subtypes in the dorsal raphe nucleus during alcohol consumption, is of critical significance in the suppression and escalation of aggressive behavior. In anticipation and reaction to aggressive behavior, neuropeptides such as corticotropin‐releasing factor, neuropeptide Y, opioid peptides, and vasopressin interact with monoamines, GABA, and glutamate to attenuate and amplify aggressive behavior in alcohol‐consuming individuals. These neuromodulators represent novel molecular targets for intervention that await clinical validation. Intermittent episodes of brief social defeat during aggressive confrontations are sufficient to cause long‐lasting neuroadaptations that can lead to the escalation of alcohol consumption.

Escalated aggression in animal models: shedding new light on mesocorticolimbic circuits

Recent developments promise to significantly advance the understudied behavioral and neurobiology of aggression: (1) Animal models that capture essential features of human violence and callousness have been developed. These models range from mice that have been selectively bred for short attack latencies, monogamous prairie voles, and glucocorticoid-compromised rats to rodents and non-human primates that escalate their aggression after consuming or when withdrawing from alcohol. (2) Optogenetic stimulation and viral vector-based approaches have begun to identify overlapping and distinctive neural microcircuits and intracellular molecules for adaptive vs. excessive, maladaptive aggressive behavior in several rodent models. Projections from hypothalamic and mesencephalic neurons to the medial prefrontal cortex contain microcircuits that appear pivotal for the escalation of aggression.

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.

Prevention of Alcohol-Heightened Aggression by CRF-R1 Antagonists in Mice: Critical Role for DRN-PFC Serotonin Pathway

Alcohol can escalate aggressive behavior in a significant subgroup of rodents, humans, and nonhuman primates. The present study investigated whether blockade of corticotropin-releasing factor receptor type 1 (CRF-R1) could prevent the emergence of alcohol-heightened aggression in mice. The serotonin (5-HT) pathway from the dorsal raphe nucleus (DRN) to the medial prefrontal cortex (mPFC) by CRF-R1 was investigated as a possible target for the prevention of alcohol-heightened aggressive behavior. Male CFW mice that reliably exhibited aggressive behaviors after consuming 1 g/kg of alcohol received systemic or intra-DRN administration of CRF-R1 antagonists, CP-154,526 or MTIP, before a confrontation with a male conspecific. Blockade of DRN CRF-R1 receptors with both antagonists significantly reduced only alcohol-heightened aggression, whereas systemic administration reduced both alcohol-heightened and species-typical aggression. Next, a 5-HT1A agonist, 8-OH-DPAT, was coadministered with CP-154,526 into the DRN to temporarily disrupt 5-HT activity. This manipulation abolished the antiaggressive effects of intra-DRN CP-154,526. In the mPFC, in vivo microdialysis revealed that extracellular 5-HT levels were increased in mice that consumed alcohol and were then injected with CP-154,526, both systemically or intra-DRN. Neither alcohol nor CP-154,526 alone affected 5-HT release in the mPFC. The present results suggest the DRN as a critical site for CRF-R1 to modulate alcohol-heightened aggression via action on the serotonergic DRN–PFC pathway.