Meghan E. Flanigan

Binge alcohol drinking is a public health concern that can dramatically increase the risk for development of alcohol use disorder (AUD). Continued alcohol drinking in the face of negative consequences is another key feature of AUD. A better understanding of the neural circuitry that regulates these behaviors could provide insight as to novel treatments for AUD. Serotonin is a neurotransmitter that has been implicated in alcohol consumption in both human studies and animal models. The orbitofrontal cortex (OFC) is a brain region that both receives serotonergic input from the dorsal raphe and has been implicated in AUD. However, how volitional alcohol consumption impacts serotonin signaling within the OFC and how this contributes to alcohol related behaviors is unknown. Here, we show that a history of alcohol consumption alters the ability of 5-HT to hyperpolarize OFC pyramidal neurons in mice and monkeys. Consistent with this, a history of binge alcohol consumption decreases the expression of the 5-HT _1A but not 5-HT _2A receptor in the OFC from mice. Next, we show that deletion of the 5-HT _1A receptor from the OFC increased alcohol intake and preference in male, but not female mice. Finally, we found that 5-HT _1A receptor deletion led to increased quinine-adulterated alcohol intake, a measure of aversion-resistant drinking, in both male and female mice. Altogether, we identified serotonin signaling in the OFC as key target for modulation of binge and compulsive alcohol consumption. SIGNIFICANCE STATEMENT: Binge alcohol drinking is a public health concern that increases the risk for development of alcohol use disorder (AUD). To develop novel treatments for AUD, there is a need to better understand the neural circuitry that regulates binge alcohol consumption. Serotonin (5-HT) is a neuromodulator implicated in alcohol consumption in human studies and animal models. The orbitofrontal cortex (OFC) is a brain region that both receives 5-HT input and has been implicated in AUD. Here, we show that a history of alcohol consumption alters 5-HT signaling in the OFC of mice and monkeys and that alterations in 5-HT signaling increases alcohol consumption. Altogether, we identified 5-HT signaling in the OFC as a key target for modulating alcohol consumption.

Negative emotional contagion—witnessing others in distress—affects an individual’s emotional responsivity. However, whether it shapes coping strategies when facing future threats remains unknown. We found that mice that briefly observe a conspecific being harmed become resilient, withstanding behavioral despair after an adverse experience. Photometric recordings during negative emotional contagion revealed increased serotonin (5-HT) release in the lateral habenula. Whereas 5-HT and emotional contagion reduced habenular burst firing, limiting 5-HT synthesis prevented burst plasticity. Enhancing raphe-to-habenula 5-HT was sufficient to recapitulate resilience. In contrast, reducing 5-HT release in the habenula made witnessing a conspecific in distress ineffective to promote the resilient phenotype after adversity. These findings reveal that 5-HT supports vicarious emotions and leads to resilience by tuning definite patterns of habenular neuronal activity.

The serotonin 5HT_2c receptor has been widely implicated in the pathophysiology of alcohol use disorder (AUD), particularly alcohol seeking and the affective consequences of chronic alcohol consumption. However, little is known about the brain sites in which 5HT_2c exerts its effects on specific alcohol-related behaviors, especially in females. Here, we investigated the effects of site-specific manipulation of the 5HT_2c receptor system in the BNST on operant alcohol self-administration behaviors in adult mice of both sexes, including the acquisition and maintenance of fixed-ratio responding, motivation for alcohol (progressive ratio), and quinine-adulterated responding for alcohol on a fixed-ratio schedule (punished alcohol seeking). Knockdown of 5HT_2c in the BNST did not affect the acquisition or maintenance of operant alcohol self-administration, nor did it affect progressive ratio responding for alcohol. This manipulation had only a subtle effect on responding for quinine alcohol selectively in females. On the other hand, chemogenetic inhibition of BNST 5HT_2c-containing neurons (BNST^5HT2c) increased operant alcohol self-administration behavior in both sexes on day 2, but not day 9, of testing. It also increased operant responding for 1000 μM quinine-adulterated alcohol selectively in males. Importantly, chemogenetic inhibition of BNST^5HT2c did not alter operant sucrose responding or motivation for sucrose in either sex. We then performed cell-type specific anterograde tracing, which revealed that BNST^5HT2c project to similar regions in males and females, many of which have been previously implicated in AUD. We next used chemogenetics and quantification of the immediate early gene cFos to characterize the functional influence of BNST^5HT2c inhibition on vlPAG activity. We show that chemogenetic inhibition of BNST^5HT2c reduces vlPAG cFos in both sexes, but that this reduction is more robust in males. Together these findings suggest that BNST^5HT2c neurons, and to a small extent the BNST 5HT_2c receptor, serve to promote aversive responses to alcohol consumption, potentially through sex-dependent disinhibition of vlPAG neurons.

The serotonin 5HT _2c receptor has been widely implicated in the pathophysiology of alcohol use disorder (AUD), particularly alcohol seeking and the affective consequences of chronic alcohol consumption. However, little is known about the brain sites in which 5HT _2c exerts its effects on specific alcohol-related behaviors, especially in females. Here, we investigated the effects of site-specific manipulation of the 5HT _2c receptor system in the BNST on operant alcohol self-administration behaviors in adult mice of both sexes, including the acquisition and maintenance of fixed-ratio responding, motivation for alcohol (progressive ratio), and quinine-adulterated responding for alcohol on a fixed-ratio schedule (punished alcohol seeking). Knockdown of 5HT _2c in the BNST did not affect the acquisition or maintenance of operant alcohol self-administration, nor did it affect progressive ratio responding for alcohol. This manipulation had only a subtle effect on responding for quinine alcohol selectively in females. On the other hand, chemogenetic inhibition of BNST 5HT _2c -containing neurons (BNST ^5HT2c ) increased operant alcohol self-administration behavior in both sexes on day 2, but not day 9, of testing. It also increased operant responding for 1000 μM quinine-adulterated alcohol selectively in males. Importantly, chemogenetic inhibition of BNST ^5HT2c did not alter operant sucrose responding or motivation for sucrose in either sex. We then performed cell-type specific anterograde tracing, which revealed that BNST ^5HT2c project to similar regions in males and females, many of which have been previously implicated in AUD. We next used chemogenetics and quantification of the immediate early gene cFos to characterize the functional influence of BNST ^5HT2c inhibition on vlPAG activity. We show that chemogenetic inhibition of BNST ^5HT2c reduces vlPAG cFos in both sexes, but that this reduction is more robust in males. Together these findings suggest that BNST ^5HT2c neurons, and to a small extent the BNST 5HT _2c receptor, serve to promote aversive responses to alcohol consumption, potentially through sex-dependent disinhibition of vlPAG neurons.

Summary: Serotonin 5HT _2c receptors have been implicated in the pathophysiology of both mood disorders and alcohol use disorder, but the circuits mediating the effects of systemic pharmacological manipulations of this receptor on behavior have not been identified. Binge alcohol consumption induces discrete social and arousal disturbances in human populations, which are thought to promote increased drinking. However, whether models of binge drinking in rodents can induce these same long-term negative behavioral symptoms is unknown. In this study, we employed multiple anatomical, physiological, and behavioral approaches to identify two populations of neurons expressing serotonin 5HT _2c receptors, one in the lateral habenula (LHb _5HT2c ) and one in the bed nucleus of the stria terminalis (BNST _5HT2c ), that display coordinated in-vivo responses to social, arousal, and alcohol-related stimuli and are physiologically modulated by binge alcohol consumption in a sex-specific manner. Critically, these physiological changes were associated with sex-specific behavioral disturbances that closely mirror social and arousal symptoms observed in humans during abstinence from binge drinking. Specifically, we observed that one week of abstinence from three weeks of binge alcohol drinking induced social recognition deficits in females and potentiated acoustic startle responses in males. While both populations of neurons (BNST and LHb) and the 5HT _2c receptor itself contribute to the sex-specific effects of alcohol on social and arousal behaviors to some degree, the primary causal mechanism underlying these phenomena appears to be excessive activation of LHb _5HT2c neurons. These findings may have implications for the development of sex-specific treatments for mood and alcohol use disorders targeting the brain’s serotonin system.

Hwa LS, Neira S, Flanigan ME, Stanhope CM, Pina MM, Pati D, Hon OJ, Yu W, Kokush E, Calloway R, Boyt K, Kash TL. 2020. Alcohol drinking alters stressresponse to predator odor via BNST kappa opioid receptor signaling in male mice. eLife 9:59709. doi: 10.7554/eLife.59709

Heightened aggressive behavior is considered as one of the central symptoms of many neuropsychiatric disorders including autism, schizophrenia, and dementia. The consequences of aggression pose a heavy burden on patients and their families and clinicians. Unfortunately, we have limited treatment options for aggression and lack mechanistic insight into the causes of aggression needed to inform new efforts in drug discovery and development. Levels of proinflammatory cytokines in the periphery or cerebrospinal fluid were previously reported to correlate with aggressive traits in humans. However, it is still unknown whether cytokines affect brain circuits to modulate aggression. Here, we examined the functional role of interleukin 1β (IL-1β) in mediating individual differences in aggression using a resident-intruder mouse model. We found that nonaggressive mice exhibit higher levels of IL-1β in the dorsal raphe nucleus (DRN), the major source of forebrain serotonin (5-HT), compared to aggressive mice. We then examined the effect of pharmacological antagonism and viral-mediated gene knockdown of the receptors for IL-1 within the DRN and found that both treatments consistently increased aggressive behavior of male mice. Aggressive mice also exhibited higher c-Fos expression in 5-HT neurons in the DRN compared to nonaggressive mice. In line with these findings, deletion of IL-1 receptor in the DRN enhanced c-Fos expression in 5-HT neurons during aggressive encounters, suggesting that modulation of 5-HT neuronal activity by IL-1β signaling in the DRN controls expression of aggressive behavior.

The bed nucleus of the stria terminalis (BNST) is a sexually dimorphic, neuropeptide‐rich node of the extended amygdala that has been implicated in responses to stress, drugs of abuse, and natural rewards. Its function is dysregulated in neuropsychiatric disorders that are characterized by stress‐ or drug‐induced alterations in mood, arousal, motivation, and social behavior. However, compared to the BNST’s role in mood, arousal, and motivation, its role in social behavior has remained relatively understudied. Moreover, the precise cell types and circuits underlying the BNST’s role in social behavior have only recently begun to be explored using modern neuroscience techniques. Here, we systematically review the existing literature investigating the neurobiological substrates within the BNST that contribute to the coordination of various sex‐dependent and sex‐independent social behavioral repertoires, focusing largely on pharmacological and circuit‐based behavioral studies in rodents. We suggest that the BNST coordinates social behavior by promoting appropriate assessment of social contexts to select relevant behavioral outputs and that disruption of socially relevant BNST systems by stress and drugs of abuse may be an important factor in the development of social dysfunction in neuropsychiatric disorders.

Maladaptive responses to stress are a hallmark of alcohol use disorder, but the mechanisms that underlie this are not well characterized. Here, we show that kappa opioid receptor signaling in the bed nucleus of the stria terminalis (BNST) is a critical molecular substrate underlying abnormal stress responses to predator odor following heavy alcohol drinking. Exposure to predator odor during protracted withdrawal from intermittent alcohol drinking resulted in enhanced prefrontal cortex (PFC)-driven excitation of prodynorphin-containing neurons in the BNST. Furthermore, deletion of prodynorphin in the BNST and chemogenetic inhibition of the PFC-BNST pathway restored abnormal responses to predator odor in alcohol-exposed mice. These findings suggest that increased corticolimbic drive may promote abnormal stress behavioral responses to predator odor during protracted withdrawal. Various nodes of this PFC-BNST dynorphin-related circuit may serve as potential targets for potential therapeutic mediation as well as biomarkers of negative responses to stress following heavy alcohol drinking.