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.

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.

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.

Inhibitory transmission in the bed nucleus of the stria terminalis in male and female mice following morphine withdrawal.

The United States is experiencing an opioid crisis imposing enormous fiscal and societal costs and driving the staggering overdose death rate. While prescription opioid analgesics are essential for treating acute pain, cessation of use in individuals with a physical dependence induces an aversive withdrawal syndrome that promotes continued drug use to alleviate/avoid these symptoms. Additionally, repeated bouts of withdrawal often lead to an increased propensity for relapse. Understanding the neurobiology underlying withdrawal is essential for providing novel treatment options to alleviate physiological and affective components accompanying the cessation of opiate use. Here, we administered morphine and precipitated withdrawal with naloxone to investigate behavioral and cellular responses in C57BL/6J male and female mice. Following 3 days of administration, both male and female mice demonstrated sensitized withdrawal symptoms. Since the bed nucleus of the stria terminalis (BNST) plays a role in mediating withdrawal-associated behaviors, we examined plastic changes in inhibitory synaptic transmission within this structure 24 hours following the final precipitated withdrawal. In male mice, morphine withdrawal increased spontaneous GABAergic signaling compared with controls. In contrast, morphine withdrawal decreased spontaneous GABAergic signaling in female mice. Intriguingly, these opposing GABAergic effects were contingent upon activity-dependent dynamics within the ex vivo slice. Our findings suggest that male and female mice exhibit some divergent cellular responses in the BNST following morphine withdrawal, and alterations in BNST inhibitory signaling may contribute to the expression of behaviors following opioid withdrawal.

Alcohol consumption increases basal extracellular glutamate in the nucleus accumbens core of Sprague-Dawley rats without increasing spontaneous glutamate release.

Glutamate neurotransmission in the nucleus accumbens core (NAc) mediates ethanol consumption. Previous studies using non-contingent and voluntary alcohol administration in inbred rodents have reported increased basal extracellular glutamate levels in the NAc. Here, we assessed basal glutamate levels in the NAc following intermittent alcohol consumption in male Sprague-Dawley rats that had access to ethanol for 7 weeks on alternating days. We found increased basal NAc glutamate at 24 h withdrawal from ethanol and thus sought to identify the source of this glutamate. To do so, we employed a combination of microdialysis, slice electrophysiology and western blotting. Reverse dialysis of the voltage-gated sodium channel blocker tetrodotoxin did not affect glutamate levels in either group. Electrophysiological recordings in slices made after 24 h withdrawal revealed a decrease in spontaneous excitatory postsynaptic current (sEPSC) frequency relative to controls, with no change in sEPSC amplitude. No change in metabotropic glutamate receptor 2/3 (mGlu2/3) function was detected as bath application of the mGlu2/3 agonist LY379268 decreased spontaneous and miniature EPSC frequency in slices from both control and ethanol-consuming rats. The increase in basal glutamate was not associated with changes in the surface expression of GLT-1, however, a decrease in slope of the no-net-flux dialysis function was observed following ethanol consumption, indicating a potential decrease in glutamate reuptake. Taken together, these findings indicate that the increase in basal extracellular glutamate occurring after chronic ethanol consumption is not mediated by an increase in action potential-dependent glutamate release or a failure of mGlu2/3 autoreceptors to regulate such release.

Angiotensin type 1a receptors in the paraventricular nucleus of the hypothalamus protect against diet-induced obesity.

Obesity is associated with increased levels of angiotensin-II (Ang-II), which activates angiotensin type 1a receptors (AT1a) to influence cardiovascular function and energy homeostasis. To test the hypothesis that specific AT1a within the brain control these processes, we used the Cre/lox system to delete AT1a from the paraventricular nucleus of the hypothalamus (PVN) of mice. PVN AT1a deletion did not affect body mass or adiposity when mice were maintained on standard chow. However, maintenance on a high-fat diet revealed a gene by environment interaction whereby mice lacking AT1a in the PVN had increased food intake and decreased energy expenditure that augmented body mass and adiposity relative to controls. Despite this increased adiposity, PVN AT1a deletion reduced systolic blood pressure, suggesting that this receptor population mediates the positive correlation between adiposity and blood pressure. Gene expression studies revealed that PVN AT1a deletion decreased hypothalamic expression of corticotrophin-releasing hormone and oxytocin, neuropeptides known to control food intake and sympathetic nervous system activity. Whole-cell patch-clamp recordings confirmed that PVN AT1a deletion eliminates responsiveness of PVN parvocellular neurons to Ang-II, and suggest that Ang-II responsiveness is increased in obese wild-type mice. Central inflammation is associated with metabolic and cardiovascular disorders and PVN AT1a deletion reduced indices of hypothalamic inflammation. Collectively, these studies demonstrate that PVN AT1a regulate energy balance during environmental challenges that promote metabolic and cardiovascular pathologies. The implication is that the elevated Ang-II that accompanies obesity serves as a negative feedback signal that activates PVN neurons to alleviate weight gain.

Dopamine D2 receptors in the bed nucleus of the stria terminalis modulate alcohol-related behaviors.

Dysregulation of the dopamine (DA) system is a hallmark of substance abuse disorders, including alcohol use disorder (AUD). Of the DA receptor subtypes, the DA D2 receptors (D2Rs) play a key role in the reinforcing effects of alcohol. D2Rs are expressed in numerous brain regions associated with the regulation of appetitive behaviors. One such region is the bed nucleus of the stria terminalis (BNST), which has been linked to the development and maintenance of AUD. Recently, we identified alcohol withdrawal-related neuroadaptations in the periaqueductal gray/dorsal raphe to BNST DA circuit in male mice. However, the role of D2R-expressing BNST neurons in voluntary alcohol consumption is not well characterized. In this study, we used a CRISPR-Cas9-based viral approach, to selectively reduce the expression of D2Rs in BNST VGAT neurons and interrogated the impact of BNST D2Rs in alcohol-related behaviors. In male mice, reduced D2R expression potentiated the stimulatory effects of alcohol and increased voluntary consumption of 20% w/v alcohol in a two-bottle choice intermittent access paradigm. This effect was not specific to alcohol, as D2R deletion also increased sucrose intake in male mice. Interestingly, cell-specific deletion of BNST D2Rs in female mice did not alter alcohol-related behaviors but lowered the threshold for mechanical pain sensitivity. Collectively, our findings suggest a role for postsynaptic BNST D2Rs in the modulation of sex-specific behavioral responses to alcohol and sucrose.

Activation of the dorsal septum increases alcohol consumption in male C57BL/6J mice.

Binge drinking is a common pattern of excessive alcohol consumption associated with Alcohol Use Disorder (AUD) and unraveling the neurocircuitry that promotes this type of drinking is critical to the development of novel therapeutic interventions. The septal region was once a focal point of alcohol research yet has seen limited study over the last decade in relation to binge drinking. Numerous studies point to involvement of the dorsal septum (dSep) in excessive drinking and withdrawal, but few studies have manipulated this region in the context of binge drinking behavior. The present experiments were primarily designed to determine the effect of chemogenetic manipulation of the dSep on binge-like alcohol drinking in male and female C57BL/6J mice. Mice received bilateral infusion of AAVs harboring hM4Di, hM3Dq, or mCherry into the dSep and subjects were challenged with systemic administration of clozapine-N-oxide (CNO) and vehicle in the context of binge-like alcohol consumption, locomotor activity, and sucrose drinking. CNO-mediated activation (hM3Dq) of the dSep resulted in increased binge-like alcohol consumption, locomotor activity, and sucrose intake in males. DSep activation promoted sucrose drinking in female mice, but alcohol intake and locomotor activity were unaffected. Conversely, silencing (hM4Di) of the dSep modestly decreased locomotor activity in males and did not influence alcohol or sucrose intake in either sex. These data support a role for the dSep in promoting binge-like drinking behavior in a sex-dependent fashion and suggests a broad role for the region in the modulation of general appetitive behaviors and locomotor activity.

Periaqueductal gray/dorsal raphe dopamine neurons contribute to sex differences in pain-related behaviors.

Sex differences in pain severity, response, and pathological susceptibility are widely reported, but the neural mechanisms that contribute to these outcomes remain poorly understood. Here we show that dopamine (DA) neurons in the ventrolateral periaqueductal gray/dorsal raphe (vlPAG/DR) differentially regulate pain-related behaviors in male and female mice through projections to the bed nucleus of the stria terminalis (BNST). We find that activation of vlPAG/DRDA+ neurons or vlPAG/DRDA+ terminals in the BNST reduces nociceptive sensitivity during naive and inflammatory pain states in male mice, whereas activation of this pathway in female mice leads to increased locomotion in the presence of salient stimuli. We additionally use slice physiology and genetic editing approaches to demonstrate that vlPAG/DRDA+ projections to the BNST drive sex-specific responses to pain through DA signaling, providing evidence of a novel ascending circuit for pain relief in males and contextual locomotor response in females.

Endogenous oxytocin inhibits hypothalamic corticotrophin-releasing hormone neurones following acute hypernatraemia.

Significant prior evidence indicates that centrally acting oxytocin robustly modulates stress responsiveness and anxiety-like behaviour, although the neural mechanisms behind these effects are not entirely understood. A plausible neural basis for oxytocin-mediated stress reduction is via inhibition of corticotrophin-releasing hormone (CRH) neurones in the paraventricular nucleus of the hypothalamus (PVN) that regulate activation of the hypothalamic-pituitary-adrenal axis. Previously, we have shown that, following s.c. injection of 2.0 mol L-1 NaCl, oxytocin synthesising neurones are activated in the rat PVN, an oxytocin receptor (Oxtr)-dependent inhibitory tone develops on a subset of parvocellular neurones and stress-mediated increases in plasma corticosterone levels are blunted. In the present study, we utilised transgenic male CRH-reporter mice to selectively target PVN CRH neurones for whole-cell recordings. These experiments reveal that acute salt loading produces tonic inhibition of PVN CRH neurones through a mechanism that is largely independent of synaptic activity. Further studies reveal that a subset of CRH neurones within the PVN synthesise mRNA for Oxtr(s). Salt induced Oxtr-dependent inhibitory tone was eliminated in individual PVN CRH neurones filled with GDP-ß-S. Additional electrophysiological studies suggest that reduced excitability of PVN CRH neurones in salt-loaded animals is associated with increased activation of inwardly rectifying potassium channels. Nevertheless, substantial effort to recapitulate the core effects of salt loading by activating Oxtr(s) with an exogenous agonist produced mixed results. Collectively, these results enhance our understanding of how oxytocin receptor-mediated signalling modulates the function of CRH neurones in the PVN.

Chronic intermittent ethanol exposure dysregulates a GABAergic microcircuit in the bed nucleus of the stria terminalis.

Neuroadaptations in brain regions that regulate emotional and reward-seeking behaviors have been suggested to contribute to pathological behaviors associated with alcohol-use disorder. One such region is the bed nucleus of the stria terminalis (BNST), which has been linked to both alcohol consumption and alcohol withdrawal-induced anxiety and depression. Recently, we identified a GABAergic microcircuit in the BNST that regulates anxiety-like behavior. In the present study, we examined how chronic alcohol exposure alters this BNST GABAergic microcircuit in mice. We selectively targeted neurons expressing corticotropin releasing factor (CRF) using a CRF-reporter mouse line and combined retrograde labeling to identify BNST projections to the ventral tegmental area (VTA) and lateral hypothalamus (LH). Following 72 h of withdrawal from four weekly cycles of chronic intermittent ethanol (CIE) vapor exposure, the excitability of a sub-population of putative local CRF neurons that did not project to either VTA or LH (CRFnon-VTA/LH neurons) was increased. Withdrawal from CIE also increased excitability of non-CRF BNST neurons that project to both LH and VTA (BNSTnon-CRF-proj neurons). Furthermore, both populations of neurons had a reduction in spontaneous EPSC amplitude while frequency was unaltered. Withdrawal from chronic alcohol was accompanied by a significant increase in spontaneous IPSC frequency selectively in the BNSTnon-CRF-proj neurons. Together, these data suggest that withdrawal from chronic ethanol dysregulates local CRF-GABAergic microcircuit to inhibit anxiolytic outputs of the BNST which may contribute to enhanced anxiety during alcohol withdrawal and drive alcohol-seeking behavior. This article is part of the special issue on 'Neuropeptides'.

The kappa opioid receptor modulates GABA neuron excitability and synaptic transmission in midbrainprojections from the insular cortex.

As an integrative hub, the insular cortex (IC) translates external cues into interoceptive states that generate complex physiological, affective, and behavioral responses. However, the precise circuit and signaling mechanisms in the IC that modulate these processes are unknown. Here, we describe a midbrain-projecting microcircuit in the medial aspect of the agranular IC that signals through the Gαi/o-coupled kappa opioid receptor (KOR) and its endogenous ligand dynorphin (Dyn). Within this microcircuit, Dyn is robustly expressed in layer 2/3, while KOR is localized to deep layer 5, which sends a long-range projection to the substantia nigra (SN). Using ex vivo electrophysiology, we evaluated the functional impact of KOR signaling in layer 5 of the IC. We found that bath application of dynorphin decreased GABA release and increased glutamate release on IC-SN neurons, but did not alter their excitability. Conversely, dynorphin decreased the excitability of GABA neurons without altering synaptic transmission. Pretreatment with the KOR antagonist nor-BNI blocked the effects of dynorphin in IC-SN neurons and GABA neurons, indicating that the changes in synaptic transmission and excitability were selectively mediated through KOR. Selective inhibition of IC GABA neurons using a KOR-derived DREADD recapitulated these effects. This work provides insight into IC microcircuitry and indicates that Dyn/KOR signaling may act to directly reduce activity of layer 5 GABA neurons. In turn, KOR-driven inhibition of GABA promotes disinhibition of IC-SN neurons, which can modulate downstream circuits. Our findings present a potential mechanism whereby chronic upregulation of IC Dyn/KOR signaling can lead to altered subcortical function and downstream activity.

Alcohol drinking alters stress response to predator odor via BNST kappa opioid receptor signaling in male mice.

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.

The connection between stress and alcohol use is highly complex. On one hand, there is the idea of having a drink to 'steady the nerves'. On the other hand, in alcoholics, abnormal responses to stress often accompany heavy drinking. In this case, it remains unknown whether stress cause excessive drinking, or vice versa. Areas of the brain that normally help respond to stress work differently in long-term, heavy drinkers. One example is a structure called the bed nucleus of the stria terminalis (BNST), which is over-active in anxiety disorders and is also associated with some of the symptoms of alcohol withdrawal. The mechanism behind both problems is thought to be a specific 'signaling system' that is activated by a small molecule called dynorphin. Previous research into the effects of dynorphin was performed either in the context of alcoholism or of anxiety disorders, but it was not known if there was a connection between the two. Therefore, Hwa et al. wanted to determine how prolonged alcohol use might affect responses to stress, and whether dynorphin signaling plays a role. To model long-term alcohol use in the laboratory, a group of mice was given free access to alcohol every other day, ensuring that they developed the mouse equivalent of a drinking habit. After six weeks, these 'heavy drinkers' went through a period of abstinence, mimicking alcohol withdrawal. Then, the mice were stressed by exposing them to a chemical that smelled like a fox, one of the mice's predators in the wild. When mice smell predators, they normally respond by fleeing from the area and digging up debris to defend itself. As expected, the control mice in this study, which did not drink alcohol, did just that. In contrast, the heavy drinkers largely ignored the predator scent by not digging and even spent time hanging around the area that smelled like the predator. Blocking dynorphin-induced signaling in the alcoholic mice, either using a drug or by deleting the gene that codes for dynorphin, reset the stress response to normal, allowing these mice to avoid the predator and dig as normal. Furthermore, measuring the electrical activity in the brain revealed that the BNST was abnormally active in alcohol-drinking mice, driven by signals from another part of the brain, the prefrontal cortex. This reveals part of the circuitry in the brain responsible for the connection between alcohol withdrawal and the stress response. These results shed new light on the biological mechanisms underpinning the relationship between alcohol use and stress. In the future, these could be used to determine why heavy drinking can overlap with anxiety disorders, or to develop new treatments that would help recovering alcoholics cope better with stress.

Ethanol-induced conditioned place preference and aversion differentially alter plasticity in the bed nucleus of stria terminalis.

Contextual cues associated with drugs of abuse, such as ethanol, can trigger craving and drug-seeking behavior. Pavlovian procedures, such as place conditioning, have been widely used to study the rewarding/aversive properties of drugs and the association between environmental cues and drug seeking. Previous research has shown that ethanol as an unconditioned stimulus can induce a strong conditioned place preference (CPP) or aversion (CPA) in rodents. However, the neural mechanisms underlying ethanol-induced reward and aversion have not been thoroughly investigated. The bed nucleus of the stria terminalis (BNST), an integral part of the extended amygdala, is engaged by both rewarding and aversive stimuli and plays a role in ethanol-seeking behavior. Here, we used ex-vivo slice physiology to probe learning-induced synaptic plasticity in the BNST following ethanol-induced CPP and CPA. Male DBA/2 J mice (2-3 months old) were conditioned using previously reported ethanol-induced CPP/CPA procedures. Ethanol-induced CPP was associated with increased neuronal excitability in the ventral BNST (vBNST). Conversely, ethanol-induced CPA resulted in a significant decrease in spontaneous glutamatergic transmission without alterations in GABAergic signaling. Ethanol-CPA also led to a significant increase in the paired-pulse ratio at excitatory synapses, suggestive of a decrease in presynaptic glutamate release. Collectively, these data demonstrate that the vBNST is involved in the modulation of contextual learning associated with both the rewarding and the aversive properties of ethanol in mice.

Predator odor increases avoidance and glutamatergic synaptic transmission in the prelimbic cortex via corticotropin-releasing factor receptor 1 signaling.

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 H2O 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 H2O. Slice physiology recordings were performed in layers 2/3 and 5 of the PL and IL, following TMT, vanilla, or H2O exposure. In TMT mice, but not vanilla or H2O 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 H2O mice following bath application of 300 nM CRF, but only H2O 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 H2O 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.

Dynorphin-kappa opioid receptor activity in the central amygdala modulates binge-like alcohol drinking in mice.

Although previous research has demonstrated a role for kappa opioid receptor-mediated signaling in escalated alcohol consumption associated with dependence and stress exposure, involvement of the dynorphin/kappa opioid receptor (DYN/KOR) system in binge-like drinking has not been fully explored. Here we used pharmacological and chemogenetic approaches to examine the influence of DYN/KOR signaling on alcohol consumption in the drinking-in-the-dark (DID) model of binge-like drinking. Systemic administration of the KOR agonist U50,488 increased binge-like drinking (Experiment 1) while, conversely, systemic administration of the KOR antagonist nor-BNI reduced drinking in the DID model (Experiment 2). These effects of systemic KOR manipulation were selective for alcohol as neither drug influenced consumption of sucrose in the DID paradigm (Experiment 3). In Experiment 4, administration of the long-acting KOR antagonist nor-BNI into the central nucleus of the amygdala (CeA) decreased alcohol intake. Next, targeted "silencing" of DYN+ neurons in the CeA was accomplished using a chemogenetic strategy. Cre-dependent viral expression in DYN+ neurons was confirmed in CeA of Pdyn-IRES-Cre mice and functionality of an inhibitory (hM4Di) DREADD was validated (Experiment 5). Activating the inhibitory DREADD by CNO injection reduced binge-like alcohol drinking, but CNO injection did not alter alcohol intake in mice that were treated with control virus (Experiment 6). Collectively, these results demonstrate that DYN/KOR signaling in the CeA contributes to excessive alcohol consumption in a binge-drinking model.

Stress-Induced Alterations of Norepinephrine Release in the Bed Nucleus of the Stria Terminalis of Mice.

Stress can drive adaptive changes to maintain survival during threatening stimuli. Chronic stress exposure, however, may result in pathological adaptations. A key neurotransmitter involved in stress signaling is norepinephrine. Previous studies show that acute stress elevates norepinephrine levels in the bed nucleus of the stria terminalis (BNST), a critical node regulating anxiety and upstream of stress responses. Here, we use mice expressing channelrhodopsin in norepinephrine neurons to selectively activate terminals in the BNST, and measure norepinephrine release with optogenetics-assisted fast-scan cyclic voltammetry (FSCV). We demonstrate that while corticosterone habituates to chronic restraint stress, cFos activation of medullary norepinephrine neurons shows equivalent activation under both acute and chronic stress conditions. Mice exposed to a single restraint session show an identical optically stimulated norepinephrine release profile compared to that of unexposed mice. Mice experiencing 5 days of restraint stress, however, show elevated norepinephrine release across multiple stimulation parameters, and reduced sensitivity to the α2-adrenergic receptor (AR) antagonist idazoxan. These data are the first to examine norepinephrine release in the BNST to tonic and phasic stimulation frequencies, and confirm that repeated stress alters autoreceptor sensitivity.