Bed Nucleus of the Stria Terminalis (BNST) neurons containing the serotonin 5HT2c receptor modulate operant alcohol self-administration behavior in mice.

The serotonin 5HT2c 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 5HT2c exerts its effects on specific alcohol-related behaviors, especially in females. Here, we investigated the effects of site-specific manipulation of the 5HT2c 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 5HT2c 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 5HT2c-containing neurons (BNST5HT2c) 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 BNST5HT2c did not alter operant sucrose responding or motivation for sucrose in either sex. We then performed cell-type specific anterograde tracing, which revealed that BNST5HT2c 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 BNST5HT2c inhibition on vlPAG activity. We show that chemogenetic inhibition of BNST5HT2c reduces vlPAG cFos in both sexes, but that this reduction is more robust in males. Together these findings suggest that BNST5HT2c neurons, and to a small extent the BNST 5HT2c receptor, serve to promote aversive responses to alcohol consumption, potentially through sex-dependent disinhibition of vlPAG neurons.

Bed Nucleus of the Stria Terminalis (BNST) neurons containing the serotonin 5HT2c receptor modulate operant alcohol self-administration behavior in mice.

The serotonin 5HT2c 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 5HT2c exerts its effects on specific alcohol-related behaviors, especially in females. Here, we investigated the effects of site-specific manipulation of the 5HT2c 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 5HT2c 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 5HT2c-containing neurons (BNST5HT2c) 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 BNST5HT2c did not alter operant sucrose responding or motivation for sucrose in either sex. We then performed cell-type specific anterograde tracing, which revealed that BNST5HT2c 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 BNST5HT2c inhibition on vlPAG activity. We show that chemogenetic inhibition of BNST5HT2c reduces vlPAG cFos in both sexes, but that this reduction is more robust in males. Together these findings suggest that BNST5HT2c neurons, and to a small extent the BNST 5HT2c receptor, serve to promote aversive responses to alcohol consumption, potentially through sex-dependent disinhibition of vlPAG neurons.

Alcohol Dependence Modifies Brain Networks Activated During Withdrawal and Reaccess: A c-Fos-Based Analysis in Mice.

BACKGROUND: High-level alcohol consumption causes neuroplastic changes in the brain that promote pathological drinking behavior. Some of these changes have been characterized in defined brain circuits and cell types, but unbiased approaches are needed to explore broader patterns of adaptations. METHODS: We used whole-brain c-Fos mapping and network analysis to assess patterns of neuronal activity during alcohol withdrawal and following reaccess in a well-characterized model of alcohol dependence. Mice underwent 4 cycles of chronic intermittent ethanol to increase voluntary alcohol consumption, and a subset underwent forced swim stress to further escalate consumption. Brains were collected either 24 hours (withdrawal) or immediately following a 1-hour period of alcohol reaccess. c-fos counts were obtained for 110 brain regions using iDISCO and ClearMap. Then, we classified mice as high or low drinkers and used graph theory to identify changes in network properties associated with high-drinking behavior. RESULTS: During withdrawal, chronic intermittent ethanol mice displayed widespread increased c-Fos expression relative to air-exposed mice, independent of forced swim stress. Reaccess drinking reversed this increase. Network modularity, a measure of segregation into communities, was increased in high-drinking mice after alcohol reaccess relative to withdrawal. The cortical amygdala showed increased cross-community coactivation during withdrawal in high-drinking mice, and cortical amygdala silencing in chronic intermittent ethanol mice reduced voluntary drinking. CONCLUSIONS: Alcohol withdrawal in dependent mice causes changes in brain network organization that are attenuated by reaccess drinking. Olfactory brain regions, including the cortical amygdala, drive some of these changes and may play an important but underappreciated role in alcohol dependence.

The effects of fatty acid amide hydrolase inhibition and monoacylglycerol lipase inhibition on habit formation in mice.

Emerging data indicate that endocannabinoid signaling is critical to the formation of habitual behavior. Previous work demonstrated that antagonism of cannabinoid receptor type 1 (CB1R) with AM251 during operant training impairs habit formation, but it is not known if this behavioral effect is specific to disrupted signaling of the endocannabinoid ligands anandamide or 2-arachidonoyl glycerol (2-AG). Here, we used selective pharmacological compounds during operant training to determine the impact of fatty acid amide hydrolase (FAAH) inhibition to increase anandamide (and other n-acylethanolamines) or monoacylglycerol lipase (MAGL) inhibition to increase 2-AG levels on the formation of habitual behaviors in mice using a food-reinforced contingency degradation procedure. We found, contrary to our hypothesis, that inhibition of FAAH and of MAGL disrupted the formation of habits. Next, AM251 was administered during training to verify that impaired habit formation could be assessed using contingency degradation. AM251-exposed mice responded at lower rates during training and at higher rates in the test. To understand the inconsistency with published data, we performed a proof-of-principle dose-response experiment to compare AM251 in our vehicle-solution to the published vehicle-suspension on response rates. We found consistent reductions in response rate with increasing doses of AM251 in solution and an inconsistent dose-response relationship with AM251 in suspension. Together, our data suggest that further characterization of the role of CB1R signaling in the formation of habitual responding is warranted and that augmenting endocannabinoids may have clinical utility for prophylactically preventing aberrant habit formation such as that hypothesized to occur in substance use disorders.

Saracatinib Fails to Reduce Alcohol-Seeking and Consumption in Mice and Human Participants.

More effective treatments to reduce pathological alcohol drinking are needed. The glutamatergic system and the NMDA receptor (NMDAR), in particular, are implicated in behavioral and molecular consequences of chronic alcohol use, making the NMDAR a promising target for novel pharmacotherapeutics. Ethanol exposure upregulates Fyn, a protein tyrosine kinase that indirectly modulates NMDAR signaling by phosphorylating the NR2B subunit. The Src/Fyn kinase inhibitor saracatinib (AZD0530) reduces ethanol self-administration and enhances extinction of goal-directed ethanol-seeking in mice. However, less is known regarding how saracatinib affects habitual ethanol-seeking. Moreover, no prior studies have assessed the effects of Src/Fyn kinase inhibitors on alcohol-seeking or consumption in human participants. Here, we tested the effects of saracatinib on alcohol consumption and craving/seeking in two species, including the first trial of an Src/Fyn kinase inhibitor to reduce drinking in humans. Eighteen male C57BL/6NCrl mice underwent operant conditioning on a variable interval schedule to induce habitual responding for 10% ethanol/0.1% saccharin. Next, mice received 5 mg/kg saracatinib or vehicle 2 h or 30 min prior to contingency degradation to measure habitual responding. In the human study, 50 non-treatment seeking human participants who drank heavily and met DSM-IV criteria for alcohol abuse or dependence were randomized to receive 125 mg/day saracatinib (n = 33) or placebo (n = 17). Alcohol Drinking Paradigms (ADP) were completed in a controlled research setting: before and after 7-8 days of treatment. Each ADP involved consumption of a priming drink of alcohol (0.03 mg%) followed by ad libitum access (3 h) to 12 additional drinks (0.015 g%); the number of drinks consumed and craving (Alcohol Urge Questionnaire) were recorded. In mice, saracatinib did not affect habitual ethanol seeking or consumption at either time point. In human participants, no significant effects of saracatinib on alcohol craving or consumption were identified. These results in mice and humans suggest that Fyn kinase inhibition using saracatinib, at the doses tested here, may not reduce alcohol consumption or craving/seeking among those habitually consuming alcohol, in contrast to reports of positive effects of saracatinib in individuals that seek ethanol in a goal-directed manner. Nevertheless, future studies should confirm these negative findings using additional doses and schedules of saracatinib administration.

A Gut Feeling about Dopamine.

In this issue of Neuron, Fernandes et al. (2020) compare intra-gastric sugar and non-caloric sweetener to investigate how post-ingestive effects can be reinforcing, revealing a role for the hepatic vagus nerve in transforming sugar sensing by the gut into behavioral reinforcement via midbrain dopamine neuron responses.

Endocannabinoid contributions to alcohol habits and motivation: Relevance to treatment.

Individuals with alcohol use disorder exhibit compulsive habitual behaviors that are thought to be, in part, a consequence of chronic and persistent use of alcohol. The endocannabinoid system plays a critical role in habit learning and in ethanol self-administration, but the role of this neuromodulatory system in the expression of habitual alcohol seeking is unknown. Here, we investigated the role of the endocannabinoid system in established alcohol habits using contingency degradation in male C57BL/6 mice. We found that administration of the novel diacyl glycerol lipase inhibitor DO34, which decreases the biosynthesis of the endocannabinoid 2-arachidonoyl glycerol (2-AG), reduced habitual responding for ethanol and ethanol approach behaviors. Moreover, administration of the endocannabinoid transport inhibitor AM404 or the cannabinoid receptor type 1 antagonist AM251 produced similar reductions in habitual responding for ethanol and ethanol approach behaviors. Notably, AM404 was also able to reduce ethanol seeking and consumption in mice that were insensitive to lithium chloride-induced devaluation of ethanol. Conversely, administration of JZL184, a monoacyl glycerol lipase inhibitor that increases levels of 2-AG, increased motivation to respond for ethanol on a progressive ratio schedule of reinforcement. These results demonstrate an important role for endocannabinoid signaling in the motivation to seek ethanol, in ethanol-motivated habits, and suggest that pharmacological manipulations of endocannabinoid signaling could be effective therapeutics for treating alcohol use disorder.

Sex-Dependent Modulation of Anxiety and Fear by 5-HT1A Receptors in the Bed Nucleus of the Stria Terminalis.

Serotonin (5-hydroxytryptamine; 5-HT) coordinates behavioral responses to stress through a variety of presynaptic and postsynaptic receptors distributed across functionally diverse neuronal networks in the central nervous system. Efferent 5-HT projections from the dorsal raphe nucleus (DRN) to the bed nucleus of the stria terminalis (BNST) are generally thought to enhance anxiety and aversive learning by activating 5-HT2C receptor (5-HT2CR) signaling in the BNST, although an opposing role for postsynaptic 5-HT1A receptors has recently been suggested. In the present study, we sought to delineate a role for postsynaptic 5-HT1A receptors in the BNST in aversive behaviors using a conditional knockdown of the 5-HT1A receptor. Both males and females were tested to dissect out sex-specific effects. We found that male mice have significantly reduced fear memory recall relative to female mice and inactivation of 5-HT1A receptor in the BNST increases contextual fear conditioning in male mice so that they resemble the females. This coincided with an increase in neuronal excitability in males, suggesting that 5-HT1A receptor deletion may enhance contextual fear recall by disinhibiting fear memory circuits in the BNST. Interestingly, 5-HT1A receptor knockdown did not significantly alter anxiety-like behavior in male or female mice, which is in agreement with previous findings that anxiety and fear are modulated by dissociable circuits in the BNST. Overall, these results suggest that BNST 5-HT1A receptors do not significantly alter behavior under basal conditions, but can act as a molecular brake that buffer against excessive activation of aversive circuits in more threatening contexts.

Bi-directional modulation of food habit expression by the endocannabinoid system.

The compulsive, habitual behaviors that have been observed in individuals diagnosed with substance use disorders may be due to disruptions in the neural circuits that mediate goal-directed actions. The endocannabinoid system has been shown to play a critical role in habit learning, but the role of this neuromodulatory system in habit expression is unclear. Here, we investigated the role of the endocannabinoid system in established habitual actions using contingency degradation in male C57BL/6 mice. We found that administration of the endocannabinoid transport inhibitor AM404 reduced habitual responding for food and that antagonism of cannabinoid receptor type 1 (CB1), but not transient receptor potential cation subfamily V (TRPV1), receptors produced a similar reduction in habitual responding. Moreover, pharmacological stimulation of CB1 receptors increased habitual responding for food. Co-administration of an enzyme inhibitor that selectively increases the endocannabinoid 2-arachidonoyl glycerol (2-AG) with AM404 partially restored habitual responding for food. Together, these findings demonstrate an important role for the endocannabinoid system in the expression of habits and provide novel insights into potential pharmacological strategies for reducing habitual behaviors in mental disorders.

Medial prefrontal D1 dopamine neurons control food intake.

Although the prefrontal cortex influences motivated behavior, its role in food intake remains unclear. Here, we demonstrate a role for D1-type dopamine receptor-expressing neurons in the medial prefrontal cortex (mPFC) in the regulation of feeding. Food intake increases activity in D1 neurons of the mPFC in mice, and optogenetic photostimulation of D1 neurons increases feeding. Conversely, inhibition of D1 neurons decreases intake. Stimulation-based mapping of prefrontal D1 neuron projections implicates the medial basolateral amygdala (mBLA) as a downstream target of these afferents. mBLA neurons activated by prefrontal D1 stimulation are CaMKII positive and closely juxtaposed to prefrontal D1 axon terminals. Finally, photostimulating these axons in the mBLA is sufficient to increase feeding, recapitulating the effects of mPFC D1 stimulation. These data describe a new circuit for top-down control of food intake.

The role of sleep in forming a memory representation of a two-dimensional space.

There is ample evidence from human and animal models that sleep contributes to the consolidation of newly learned information. The precise role of sleep for integrating information into interconnected memory representations is less well understood. Building on prior findings that following sleep (as compared to wakefulness) people are better able to draw inferences across learned associations in a simple hierarchy, we ask how sleep helps consolidate relationships in a more complex representational space. We taught 60 subjects spatial relationships between pairs of buildings, which (unknown to participants) formed a two-dimensional grid. Critically, participants were only taught a subset of the many possible spatial relations, which allowed them to potentially infer the remainder. After a 12 h period that either did or did not include a normal period of sleep, participants returned to the lab. We examined the quality of each participant's map of the two-dimensional space, and their knowledge of relative distances between buildings. After 12 h with sleep, subjects could more accurately map the full space than subjects who experienced only wakefulness. The incorporation of untaught, but inferable, associations was particularly improved. We further found that participants' distance judgment performance related to self-reported navigational style, but only after sleep. These findings demonstrate that consolidation over a night of sleep begins to integrate relations into an interconnected complex representation, in a way that supports spatial relational inference.

From habits to self-regulation: how do we change?

The Yale Cognitive Science department hosted the conference "From Habits to Self-Regulation: How Do We Change?" on November 4 and 5, 2011, to showcase current research on self-control in cognitive science, psychology, and neuroscience. The conference included a panel discussion by four philosophers who gave context for the scope and limitations of research on self-control. The common theme concerning the best method to attain lasting change included becoming aware of what one wants to change, increasing commitment to the goal of change, and imagining all of the potential problems and solutions to those problems.

Hippocampal leptin signaling reduces food intake and modulates food-related memory processing.

The increase in obesity prevalence highlights the need for a more comprehensive understanding of the neural systems controlling food intake; one that extends beyond food intake driven by metabolic need and considers that driven by higher-order cognitive factors. The hippocampus, a brain structure involved in learning and memory function, has recently been linked with food intake control. Here we examine whether administration of the adiposity hormone leptin to the dorsal and ventral sub-regions of the hippocampus influences food intake and memory for food. Leptin (0.1 µg) delivered bilaterally to the ventral hippocampus suppressed food intake and body weight measured 24 h after administration; a higher dose (0.4 µg) was needed to suppress intake following dorsal hippocampal delivery. Leptin administration to the ventral but not dorsal hippocampus blocked the expression of a conditioned place preference for food and increased the latency to run for food in an operant runway paradigm. Additionally, ventral but not dorsal hippocampal leptin delivery suppressed memory consolidation for the spatial location of food, whereas hippocampal leptin delivery had no effect on memory consolidation in a non-spatial appetitive response paradigm. Collectively these findings indicate that ventral hippocampal leptin signaling contributes to the inhibition of food-related memories elicited by contextual stimuli. To conclude, the results support a role for hippocampal leptin signaling in the control of food intake and food-related memory processing.