Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning.

Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.

A distinct cortical code for socially learned threat.

Animals can learn about sources of danger while minimizing their own risk by observing how others respond to threats. However, the distinct neural mechanisms by which threats are learned through social observation (known as observational fear learning1-4 (OFL)) to generate behavioural responses specific to such threats remain poorly understood. The dorsomedial prefrontal cortex (dmPFC) performs several key functions that may underlie OFL, including processing of social information and disambiguation of threat cues5-11. Here we show that dmPFC is recruited and required for OFL in mice. Using cellular-resolution microendoscopic calcium imaging, we demonstrate that dmPFC neurons code for observational fear and do so in a manner that is distinct from direct experience. We find that dmPFC neuronal activity predicts upcoming switches between freezing and moving state elicited by threat. By combining neuronal circuit mapping, calcium imaging, electrophysiological recordings and optogenetics, we show that dmPFC projections to the midbrain periaqueductal grey (PAG) constrain observer freezing, and that amygdalar and hippocampal inputs to dmPFC opposingly modulate observer freezing. Together our findings reveal that dmPFC neurons compute a distinct code for observational fear and coordinate long-range neural circuits to select behavioural responses.

Chronic Ethanol Exposure Modulates Periaqueductal Gray to Extended Amygdala Dopamine Circuit.

The bed nucleus of the stria terminalis (BNST) is a component of the extended amygdala that regulates motivated behavior and affective states and plays an integral role in the development of alcohol-use disorder (AUD). The dorsal subdivision of the BNST (dBNST) receives dense dopaminergic input from the ventrolateral periaqueductal gray (vlPAG)/dorsal raphe (DR). To date, no studies have examined the effects of chronic alcohol on this circuit. Here, we used chronic intermittent ethanol exposure (CIE), a well-established rodent model of AUD, to functionally interrogate the vlPAG/DR-BNST dopamine (DA) circuit during acute withdrawal. We selectively targeted vlPAG/DRDA neurons in tyrosine hydroxylase-expressing transgenic adult male mice. Using ex vivo electrophysiology, we found hyperexcitability of vlPAG/DRDA neurons in CIE-treated mice. Further, using optogenetic approaches to target vlPAG/DRDA terminals in the dBNST, we revealed a CIE-mediated shift in the vlPAG/DR-driven excitatory-inhibitory (E/I) ratio to a hyperexcitable state in dBNST. Additionally, to quantify the effect of CIE on endogenous DA signaling, we coupled optogenetics with fast-scan cyclic voltammetry to measure pathway-specific DA release in dBNST. CIE-treated mice had significantly reduced signal half-life, suggestive of faster clearance of DA signaling. CIE treatment also altered the ratio of vlPAG/DRDA-driven cellular inhibition and excitation of a subset of dBNST neurons. Overall, our findings suggest a dysregulation of vlPAG/DR to BNST dopamine circuit, which may contribute to pathophysiological phenotypes associated with AUD.SIGNIFICANCE STATEMENT The dorsal bed nucleus of the stria terminalis (dBNST) is highly implicated in the pathophysiology of alcohol-use disorder and receives dopaminergic inputs from ventrolateral periaqueductal gray/dorsal raphe regions (vlPAG/DR). The present study highlights the plasticity within the vlPAG/DR to dBNST dopamine (DA) circuit during acute withdrawal from chronic ethanol exposure. More specifically, our data reveal that chronic ethanol strengthens vlPAG/DR-dBNST glutamatergic transmission while altering both DA transmission and dopamine-mediated cellular inhibition of dBNST neurons. The net result is a shift toward a hyperexcitable state in dBNST activity. Together, our findings suggest chronic ethanol may promote withdrawal-related plasticity by dysregulating the vlPAG/DR-dBNST DA circuit.

Insula Dynorphin and Kappa Opioid Receptor Systems Regulate Alcohol Drinking in a Sex-Specific Manner in Mice.

Alcohol use disorder is complex and multifaceted, involving the coordination of multiple signaling systems across numerous brain regions. Previous work has indicated that both the insular cortex and dynorphin (DYN)/kappa opioid receptor (KOR) systems contribute to excessive alcohol use. More recently, we identified a microcircuit in the medial aspect of the insular cortex that signals through DYN/KOR. Here, we explored the role of insula DYN/KOR circuit components on alcohol intake in a long-term intermittent access (IA) procedure. Using a combination of conditional knock-out strategies and site-directed pharmacology, we discovered distinct and sex-specific roles for insula DYN and KOR in alcohol drinking and related behavior. Our findings show that insula DYN deletion blocked escalated consumption and decreased the overall intake of and preference for alcohol in male and female mice. This effect was specific to alcohol in male mice, as DYN deletion did not impact sucrose intake. Further, insula KOR antagonism reduced alcohol intake and preference during the early phase of IA in male mice only. Alcohol consumption was not affected by insula KOR knockout in either sex. In addition, we found that long-term IA decreased the intrinsic excitability of DYN and deep layer pyramidal neurons (DLPNs) in the insula of male mice. Excitatory synaptic transmission was also impacted by IA, as it drove an increase in excitatory synaptic drive in both DYN neurons and DLPNs. Combined, our findings suggest there is a dynamic interplay between excessive alcohol consumption and insula DYN/KOR microcircuitry.SIGNIFICANCE STATEMENT The insular cortex is a complex region that serves as an integratory hub for sensory inputs. In our previous work, we identified a microcircuit in the insula that signals through the kappa opioid receptor (KOR) and its endogenous ligand dynorphin (DYN). Both the insula and DYN/KOR systems have been implicated in excessive alcohol use and alcohol use disorder (AUD). Here, we use converging approaches to determine how insula DYN/KOR microcircuit components contribute to escalated alcohol consumption. Our findings show that insula DYN/KOR systems regulate distinct phases of alcohol consumption in a sex-specific manner, which may contribute to the progression to AUD.

Impact and Role of Hypothalamic Corticotropin Releasing Hormone Neurons in Withdrawal from Chronic Alcohol Consumption in Female and Male Mice.

Worldwide, alcohol use and abuse are a leading risk of mortality, causing 5.3% of all deaths (World Health Organization, 2022). The endocrine stress system, initiated by the peripheral release of corticotropin releasing hormone (CRH) from primarily glutamatergic neurons in the paraventricular nucleus of the hypothalamus (PVN), is profoundly linked with alcohol use, abuse, and relapse (Blaine and Sinha, 2017). These PVN CRH-releasing (PVNCRH) neurons are essential for peripheral and central stress responses (Rasiah et al., 2023), but little is known about how alcohol affects these neurons. Here, we show that two-bottle choice alcohol consumption blunts the endocrine-mediated corticosterone response to stress during acute withdrawal in female mice. Conversely, using slice electrophysiology, we demonstrate that acute withdrawal engenders a hyperexcitable phenotype of PVNCRH neurons in females that is accompanied by increased glutamatergic transmission in both male and female mice. GABAergic synaptic transmission was unaffected by alcohol history. We then tested whether chemogenetic inhibition of PVNCRH neurons would restore stress response in female mice with a history of alcohol drinking in the looming disk test, which mimics an approaching predator threat. Accordingly, inhibition of PVNCRH neurons reduced active escape in hM4Di alcohol history mice only. This study indicates that stress-responsive PVNCRH neurons in females are particularly affected by a history of alcohol consumption. Interestingly, women have indicated an increase in heavy alcohol use to cope with stress (Rodriguez et al., 2020), perhaps pointing to a potential underlying mechanism in alcohol-mediated changes to PVNCRH neurons that alter stress response.SIGNIFICANCE STATEMENT Paraventricular nucleus of the hypothalamus neurons that release corticotropin releasing hormone (PVNCRH) are vital for stress response. These neurons have been understudied in relation to alcohol and withdrawal despite profound relations between stress, alcohol use disorders (AUD), and relapse. In this study, we use a variety of techniques to show that acute withdrawal from a history of alcohol impacts peripheral stress response, PVNCRH neurons, and behavior. Specifically, PVNCRH are in a hyperactive state during withdrawal, which drives an increase in active stress coping behaviors in female mice only. Understanding how alcohol use and withdrawal affects stress responding PVNCRH neurons may contribute to finding new potential targets for the treatment of alcohol use disorder.

Coordination of social behaviors by the bed nucleus of the stria terminalis.

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.

Kappa opioid receptor and dynorphin signaling in the central amygdala regulates alcohol intake.

Excessive alcohol drinking has been shown to modify brain circuitry to predispose individuals for future alcohol abuse. Previous studies have implicated the central nucleus of the amygdala (CeA) as an important site for mediating the somatic symptoms of withdrawal and for regulating alcohol intake. In addition, recent work has established a role for both the Kappa Opioid Receptor (KOR) and its endogenous ligand dynorphin in mediating these processes. However, it is unclear whether these effects are due to dynorphin or KOR arising from within the CeA itself or other input brain regions. To directly examine the role of preprodynorphin (PDYN) and KOR expression in CeA neurons, we performed region-specific conditional knockout of these genes and assessed the effects on the Drinking in the Dark (DID) and Intermittent Access (IA) paradigms. Conditional gene knockout resulted in sex-specific responses wherein PDYN knockout decreased alcohol drinking in both male and female mice, whereas KOR knockout decreased drinking in males only. We also found that neither PDYN nor KOR knockout protected against anxiety caused by alcohol drinking. Lastly, a history of alcohol drinking did not alter synaptic transmission in PDYN neurons in the CeA of either sex, but excitability of PDYN neurons was increased in male mice only. Taken together, our findings indicate that PDYN and KOR signaling in the CeA plays an important role in regulating excessive alcohol consumption and highlight the need for future studies to examine how this is mediated through downstream effector regions.

Chronic alcohol consumption alters home-cage behaviors and responses to ethologically relevant predator tasks in mice.

BACKGROUND: Alcohol withdrawal is a key component of severe alcohol use disorder. Animal models of alcohol withdrawal tend to focus on traditional anxiety/stress tests. While these have been essential to advancing our understanding of the biology of alcohol withdrawal, abrupt cessation of drinking following heavy alcohol consumption can also trigger withdrawal-related affective states that impact responses to a variety of life events and stressors. To this end, we show that behaviors in a variety of tasks that differ in task demand and intensity are altered during withdrawal in male and female mice after voluntary alcohol access. METHODS: Male and female miceunderwent six weeks of intermittent two-bottle choice alcohol exposure followed by behavioral tests. The tests included-Home cage: low-stress baseline environment to measure spontaneous natural behaviors; Open field: anxiety-inducing bright novel environment; Looming disc: arena with a protective hut where mice are exposed to a series of discs that mimic an overhead advancing predator, and Robogator-simulated predator task: forced foraging behavioral choice in the presence of an advancing robot predator that "attacks" when mice are near a food pellet in a large open arena. RESULTS: A history of alcohol exposure impacted behaviors in these tasks in a sex-dependent manner. In the home cage, alcohol induced reductions in digging and heightened stress coping through an increase in grooming time. In males, increased rearing yielded greater vigilance/exploration in a familiar environment. The open-field test revealed an anxiety phenotype in both male and female mice exposed to alcohol. Male mice showed no behavioral alterations to the looming disc task, while females exposed to alcohol showed greater escape responses than water controls, indicative of active stress-response behaviors. In males, the Robogator task revealed a hesitant/avoidant phenotype in alcohol-exposed mice under greater task demands. CONCLUSIONS: Few drugs show robust evidence of efficacy in clinical trials for alcohol withdrawal. Understanding how withdrawal alters a variety of behaviors in both males and females that are linked to stress coping can increase our understanding of alcohol misuse and aid in developing better medications for treating individuals with AUD.

Serotonin engages an anxiety and fear-promoting circuit in the extended amygdala.

Serotonin (also known as 5-hydroxytryptamine (5-HT)) is a neurotransmitter that has an essential role in the regulation of emotion. However, the precise circuits have not yet been defined through which aversive states are orchestrated by 5-HT. Here we show that 5-HT from the dorsal raphe nucleus (5-HTDRN) enhances fear and anxiety and activates a subpopulation of corticotropin-releasing factor (CRF) neurons in the bed nucleus of the stria terminalis (CRFBNST) in mice. Specifically, 5-HTDRN projections to the BNST, via actions at 5-HT2C receptors (5-HT2CRs), engage a CRFBNST inhibitory microcircuit that silences anxiolytic BNST outputs to the ventral tegmental area and lateral hypothalamus. Furthermore, we demonstrate that this CRFBNST inhibitory circuit underlies aversive behaviour following acute exposure to selective serotonin reuptake inhibitors (SSRIs). This early aversive effect is mediated via the corticotrophin-releasing factor type 1 receptor (CRF1R, also known as CRHR1), given that CRF1R antagonism is sufficient to prevent acute SSRI-induced enhancements in aversive learning. These results reveal an essential 5-HTDRN→CRFBNST circuit governing fear and anxiety, and provide a potential mechanistic explanation for the clinical observation of early adverse events to SSRI treatment in some patients with anxiety disorders.

An isotropic EPI database and analytical pipelines for rat brain resting-state fMRI.

Resting-state functional magnetic resonance imaging (fMRI) has drastically expanded the scope of brain research by advancing our knowledge about the topologies, dynamics, and interspecies translatability of functional brain networks. Several databases have been developed and shared in accordance with recent key initiatives in the rodent fMRI community to enhance the transparency, reproducibility, and interpretability of data acquired at various sites. Despite these pioneering efforts, one notable challenge preventing efficient standardization in the field is the customary choice of anisotropic echo planar imaging (EPI) schemes with limited spatial coverage. Imaging with anisotropic resolution and/or reduced brain coverage has significant shortcomings including reduced registration accuracy and increased deviation in brain feature detection. Here we proposed a high-spatial-resolution (0.4 mm), isotropic, whole-brain EPI protocol for the rat brain using a horizontal slicing scheme that can maintain a functionally relevant repetition time (TR), avoid high gradient duty cycles, and offer unequivocal whole-brain coverage. Using this protocol, we acquired resting-state EPI fMRI data from 87 healthy rats under the widely used dexmedetomidine sedation supplemented with low-dose isoflurane on a 9.4 T MRI system. We developed an EPI template that closely approximates the Paxinos and Watson's rat brain coordinate system and demonstrated its ability to improve the accuracy of group-level approaches and streamline fMRI data pre-processing. Using this database, we employed a multi-scale dictionary-learning approach to identify reliable spatiotemporal features representing rat brain intrinsic activity. Subsequently, we performed k-means clustering on those features to obtain spatially discrete, functional regions of interest (ROIs). Using Euclidean-based hierarchical clustering and modularity-based partitioning, we identified the topological organizations of the rat brain. Additionally, the identified group-level FC network appeared robust across strains and sexes. The "triple-network" commonly adapted in human fMRI were resembled in the rat brain. Through this work, we disseminate raw and pre-processed isotropic EPI data, a rat brain EPI template, as well as identified functional ROIs and networks in standardized rat brain coordinates. We also make our analytical pipelines and scripts publicly available, with the hope of facilitating rat brain resting-state fMRI study standardization.

Tumor necrosis factor-α modulates GABAergic and dopaminergic neurons in the ventrolateral periaqueductal gray of female mice.

Neuroimmune signaling is increasingly identified as a critical component of various illnesses, including chronic pain, substance use disorder, and depression. However, the underlying neural mechanisms remain unclear. Proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), may play a role by modulating synaptic function and long-term plasticity. The midbrain structure periaqueductal gray (PAG) plays a well-established role in pain processing, and although TNF-α inhibitors have emerged as a therapeutic strategy for pain-related disorders, the impact of TNF-α on PAG neuronal activity has not been thoroughly characterized. Recent studies have identified subpopulations of ventrolateral PAG (vlPAG) with opposing effects on nociception, with dopamine (DA) neurons driving pain relief in contrast to GABA neurons. Therefore, we used slice physiology to examine the impact of TNF-α on neuronal activity of both these subpopulations. We focused on female mice since the PAG is a sexually dimorphic region and most studies use male subjects, limiting our understanding of mechanistic variations in females. We selectively targeted GABA and DA neurons using transgenic reporter lines. Following exposure to TNF-α, there was an increase in excitability of GABA neurons along with a reduction in glutamatergic synaptic transmission. In DA neurons, TNF-α exposure resulted in a robust decrease in excitability along with a modest reduction in glutamatergic synaptic transmission. Interestingly, TNF-α had no effect on inhibitory transmission onto DA neurons. Collectively, these data suggest that TNF-α differentially affects the function of GABA and DA neurons in female mice and enhances our understanding of how TNF-α-mediated signaling modulates vlPAG function.NEW & NOTEWORTHY This study describes the effects of TNF-α on two distinct subpopulations of neurons in the vlPAG. We show that TNF-α alters both neuronal excitability and glutamatergic synaptic transmission on GABA and dopamine neurons within the vlPAG of female mice. This provides critical new information on the role of TNF-α in the potential modulation of pain, since activation of vlPAG GABA neurons drives nociception, whereas activation of dopamine neurons drives analgesia.

Correction: Cocaine- and stress-primed reinstatement of drug-associated memories elicit differential behavioral and frontostriatal circuit activity patterns via recruitment of L-type Ca2+ channels.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Cocaine- and stress-primed reinstatement of drug-associated memories elicit differential behavioral and frontostriatal circuit activity patterns via recruitment of L-type Ca2+ channels.

Cocaine-associated memories are critical drivers of relapse in cocaine-dependent individuals that can be evoked by exposure to cocaine or stress. Whether these environmental stimuli recruit similar molecular and circuit-level mechanisms to promote relapse remains largely unknown. Here, using cocaine- and stress-primed reinstatement of cocaine conditioned place preference to model drug-associated memories, we find that cocaine drives reinstatement by increasing the duration that mice spend in the previously cocaine-paired context whereas stress increases the number of entries into this context. Importantly, both forms of reinstatement require Cav1.2 L-type Ca2+ channels (LTCCs) in cells of the prelimbic cortex that project to the nucleus accumbens core (PrL→NAcC). Utilizing fiber photometry to measure circuit activity in vivo in conjunction with the LTCC blocker, isradipine, we find that LTCCs drive differential recruitment of the PrL→ NAcC pathway during cocaine- and stress-primed reinstatement. While cocaine selectively activates PrL→NAcC cells prior to entry into the cocaine-paired chamber, a measure that is predictive of duration in that chamber, stress increases persistent activity of this projection, which correlates with entries into the cocaine-paired chamber. Using projection-specific chemogenetic manipulations, we show that PrL→NAcC activity is required for both cocaine- and stress-primed reinstatement, and that activation of this projection in Cav1.2-deficient mice restores reinstatement. These data indicate that LTCCs are a common mediator of cocaine- and stress-primed reinstatement. However, they engage different patterns of behavior and PrL→NAcC projection activity depending on the environmental stimuli. These findings establish a framework to further study how different environmental experiences can drive relapse, and supports further exploration of isradipine, an FDA-approved LTCC blocker, as a potential therapeutic for the prevention of relapse in cocaine-dependent individuals.

Low-dose alcohol: Interoceptive and molecular effects and the role of dentate gyrus in rats.

Alcohol abuse and dependence are world-wide health problems. Most research on alcohol use focuses on the consequences of moderate to high levels of alcohol. However, even at low concentrations, alcohol is capable of producing effects in the brain that can ultimately affect behavior. The current studies seek to understand the effects of low-dose alcohol (blood alcohol levels of ≤10mM). To do so, these experiments utilize a combination of behavioral and molecular techniques to (1) assess the ability of the interoceptive effects of a low dose of alcohol to gain control over goal-tracking behavior in a Pavlovian discrimination task, (2) determine brain regional differences in cellular activity via expression of immediate early genes (IEGs), and (3) assess the role of the dentate gyrus in modulating sensitivity to the interoceptive effects of a low dose of alcohol. Here, we show that intragastric administration of a dose of 0.8 g/kg alcohol produces blood alcohol levels ≤10mM in both male and female Long-Evans rats and can readily be trained as a Pavlovian interoceptive drug cue. In rats trained on this procedure, this dose of alcohol also modulates expression of the IEGs c-Fos and Arc in brain regions known to modulate expression of alcohol interoceptive effects. Finally, pharmacological inactivation of the dentate gyrus with GABA agonists baclofen and muscimol disrupted the ability of a low dose of alcohol to serve as an interoceptive cue. Together, these findings demonstrate behavioral and molecular consequences of low-dose alcohol.

Distinct extended amygdala circuits for divergent motivational states.

The co-morbidity of anxiety and dysfunctional reward processing in illnesses such as addiction and depression suggests that common neural circuitry contributes to these disparate neuropsychiatric symptoms. The extended amygdala, including the bed nucleus of the stria terminalis (BNST), modulates fear and anxiety, but also projects to the ventral tegmental area (VTA), a region implicated in reward and aversion, thus providing a candidate neural substrate for integrating diverse emotional states. However, the precise functional connectivity between distinct BNST projection neurons and their postsynaptic targets in the VTA, as well as the role of this circuit in controlling motivational states, have not been described. Here we record and manipulate the activity of genetically and neurochemically identified VTA-projecting BNST neurons in freely behaving mice. Collectively, aversive stimuli exposure produced heterogeneous firing patterns in VTA-projecting BNST neurons. By contrast, in vivo optically identified glutamatergic projection neurons displayed a net enhancement of activity to aversive stimuli, whereas the firing rate of identified GABAergic (γ-aminobutyric acid-containing) projection neurons was suppressed. Channelrhodopsin-2-assisted circuit mapping revealed that both BNST glutamatergic and GABAergic projections preferentially innervate postsynaptic non-dopaminergic VTA neurons, thus providing a mechanistic framework for in vivo circuit perturbations. In vivo photostimulation of BNST glutamatergic projections resulted in aversive and anxiogenic behavioural phenotypes. Conversely, activation of BNST GABAergic projections produced rewarding and anxiolytic phenotypes, which were also recapitulated by direct inhibition of VTA GABAergic neurons. These data demonstrate that functionally opposing BNST to VTA circuits regulate rewarding and aversive motivational states, and may serve as a crucial circuit node for bidirectionally normalizing maladaptive behaviours.

Potent and Selective Peptide-based Inhibition of the G Protein Gαq.

In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gαq binds its two major classes of effectors, the phospholipase C (PLC)-ß isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gαq within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gαq in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gαq A representative peptide was specific for active Gαq because it did not bind inactive Gαq or other classes of active Gα subunits and did not inhibit the activation of PLC-ß3 by Gß1γ2 In contrast, the peptide robustly prevented activation of PLC-ß3 or p63RhoGEF by Gαq; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gαq in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gαq-dependent activation of PLC-ß3 at least as effectively as a dominant-negative form of full-length PLC-ß3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gαq in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gαq in cells.

Chronic EtOH effects on putative measures of compulsive behavior in mice.

Addictions, including alcohol use disorders, are characterized by the loss of control over drug seeking and consumption, but the neural circuits and signaling mechanisms responsible for the transition from controlled use to uncontrolled abuse remain incompletely understood. Prior studies have shown that 'compulsive-like' behaviors in rodents, for example, persistent responding for ethanol (EtOH) despite punishment, are increased after chronic exposure to EtOH. The main goal of the current study was to assess the effects of chronic intermittent EtOH (CIE) exposure on multiple, putative measures of compulsive-like EtOH seeking in C57BL/6 J mice. Mice were exposed to two or four weekly cycles of CIE and then, post-withdrawal, tested for progressive ratio responding for EtOH, sustained responding during signaled EtOH unavailability and (footshock) punished suppression of responding for EtOH. Results showed that mice exposed to CIE exhibited attenuated suppression of EtOH seeking during punishment, as compared with air-exposed controls. By contrast, CIE exposure affected neither punished food reward-seeking behavior, nor other putative measures of compulsive-like EtOH seeking. Ex vivo reverse transcription polymerase chain reaction analysis of brain tissue found reduced sensitivity to punished EtOH seeking after CIE exposure was accompanied by a significant increase in gene expression of the GluN1 and GluN2A subunits of the N-methyl-d-aspartate receptor, specifically in the medial orbitofrontal cortex. Moreover, slice electrophysiological analysis revealed increased N-methyl-d-aspartate receptor-mediated currents in the orbitofrontal cortex after CIE exposure in test-naïve mice. Collectively, the current findings add to the growing body of evidence demonstrating that chronic exposure to EtOH fosters resistance to punished EtOH seeking in association with adaptations in cortical glutamatergic transmission.

Serotonin's Complex Role in Alcoholism: Implications for Treatment and Future Research.

Current pharmacological treatments for alcohol dependence have focused on reducing alcohol consumption, but to date there are few treatments that also address the negative affective symptoms during acute and protracted alcohol withdrawal which are often exacerbated in people with comorbid anxiety and depression. Selective serotonin reuptake inhibitors (SSRIs) are sometimes prescribed to ameliorate these symptoms but can exacerbate anxiety and cravings in a select group of patients. In this critical review, we discuss recent literature describing an association between alcohol dependence, the SERT linked polymorphic region (5-HTTLPR), and pharmacological response to SSRIs. Given the heterogeneity in responsiveness to serotonergic drugs across the spectrum of alcoholic subtypes, we assess the contribution of specific 5-HT circuits to discrete endophenotypes of alcohol dependence. 5-HT circuits play a distinctive role in reward, stress, and executive function which may account for the variation in response to serotonergic drugs. New optogenetic and chemogenetic methods for dissecting 5-HT circuits in alcohol dependence may provide clues leading to more effective pharmacotherapies. Although our current understanding of the role of 5-HT systems in alcohol dependence is incomplete, there is some evidence to suggest that 5-HT3 receptor antagonists are effective in people with the L/L genotype of the 5-HTTLPR polymorphism while SSRIs may be more beneficial to people with the S/L or S/S genotype. Studies that assess the impact of serotonin transporter polymorphisms on 5-HT circuit function and the subsequent development of alcohol use disorders will be an important step forward in treating alcohol dependence.

Effects of chronic alcohol consumption on neuronal function in the non-human primate BNST.

Alterations in hypothalamic-pituitary-adrenal axis function contribute to many of the adverse behavioral effects of chronic voluntary alcohol drinking, including alcohol dependence and mood disorders; limbic brain structures such as the bed nucleus of the stria terminalis (BNST) may be key sites for these effects. Here, we measured circulating levels of several steroid hormones and performed whole-cell electrophysiological recordings from acutely prepared BNST slices of male rhesus monkeys allowed to self-administer alcohol for 12 months or a control solution. Initial comparisons revealed that BNST neurons in alcohol-drinking monkeys had decreased membrane resistance, increased frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) with no change in spontaneous excitatory postsynaptic currents (sEPSCs). We then used a combined variable cluster analysis and linear mixed model statistical approach to determine whether specific factors including stress and sex hormones, age and measures of alcohol consumption and intoxication are related to these BNST measures. Modeling results showed that specific measures of alcohol consumption and stress-related hormone levels predicted differences in membrane conductance in BNST neurons. Distinct groups of adrenal stress hormones were negatively associated with the frequency of sIPSCs and sEPSCs, and alcohol drinking measures and basal neuronal membrane properties were additional positive predictors of inhibitory, but not excitatory, PSCs. The amplitude of sEPSCs was highly positively correlated with age, independent of other variables. Together, these results suggest that chronic voluntary alcohol consumption strongly influences limbic function in non-human primates, potentially via interactions with or modulation by other physiological variables, including stress steroid hormones and age.

Retrieval of an ethanol-conditioned taste aversion promotes GABAergic plasticity in the insular cortex.

Blunted sensitivity to ethanol's aversive effects can increase motivation to consume ethanol; yet, the neurobiological circuits responsible for encoding these aversive properties are not fully understood. Plasticity in cells projecting from the insular cortex (IC) to the basolateral amygdala (BLA) is critical for taste aversion learning and retrieval, suggesting this circuit's potential involvement in modulating the aversive properties of ethanol. Here, we tested the hypothesis that GABAergic activity onto IC-BLA projections would be facilitated following the retrieval of an ethanol-conditioned taste aversion (CTA). Consistent with this hypothesis, frequency of mIPSCs was increased following retrieval of an ethanol-CTA across cell layers in IC-BLA projection neurons. This increase in GABAergic plasticity occurred in both a circuit-specific and learning-dependent manner. Additionally, local inhibitory inputs onto layer 2/3 IC-BLA projection neurons were greater in number and strength following ethanol-CTA. Finally, DREADD-mediated inhibition of IC parvalbumin-expressing cells blunted the retrieval of ethanol-CTA in male, but not female, mice. Collectively, this work implicates a circuit-specific and learning-dependent increase in GABAergic tone following retrieval of an ethanol-CTA, thereby advancing our understanding of how the aversive effects of ethanol are encoded in the brain.