Neural basis for fasting activation of the hypothalamic-pituitary-adrenal axis.

Fasting initiates a multitude of adaptations to allow survival. Activation of the hypothalamic-pituitary-adrenal (HPA) axis and subsequent release of glucocorticoid hormones is a key response that mobilizes fuel stores to meet energy demands1-5. Despite the importance of the HPA axis response, the neural mechanisms that drive its activation during energy deficit are unknown. Here, we show that fasting-activated hypothalamic agouti-related peptide (AgRP)-expressing neurons trigger and are essential for fasting-induced HPA axis activation. AgRP neurons do so through projections to the paraventricular hypothalamus (PVH), where, in a mechanism not previously described for AgRP neurons, they presynaptically inhibit the terminals of tonically active GABAergic afferents from the bed nucleus of the stria terminalis (BNST) that otherwise restrain activity of corticotrophin-releasing hormone (CRH)-expressing neurons. This disinhibition of PVHCrh neurons requires γ-aminobutyric acid (GABA)/GABA-B receptor signalling and potently activates the HPA axis. Notably, stimulation of the HPA axis by AgRP neurons is independent of their induction of hunger, showing that these canonical 'hunger neurons' drive many distinctly different adaptations to the fasted state. Together, our findings identify the neural basis for fasting-induced HPA axis activation and uncover a unique means by which AgRP neurons activate downstream neurons: through presynaptic inhibition of GABAergic afferents. Given the potency of this disinhibition of tonically active BNST afferents, other activators of the HPA axis, such as psychological stress, may also work by reducing BNST inhibitory tone onto PVHCrh neurons.

5-HT modulation of a medial septal circuit tunes social memory stability.

Social memory-the ability to recognize and remember familiar conspecifics-is critical for the survival of an animal in its social group1,2. The dorsal CA2 (dCA2)3-5 and ventral CA1 (vCA1)6 subregions of the hippocampus, and their projection targets6,7, have important roles in social memory. However, the relevant extrahippocampal input regions remain poorly defined. Here we identify the medial septum (MS) as a dCA2 input region that is critical for social memory and reveal that modulation of the MS by serotonin (5-HT) bidirectionally controls social memory formation, thereby affecting memory stability. Novel social interactions increase activity in dCA2-projecting MS neurons and induce plasticity at glutamatergic synapses from MS neurons onto dCA2 pyramidal neurons. The activity of dCA2-projecting MS cells is enhanced by the neuromodulator 5-HT acting on 5-HT1B receptors. Moreover, optogenetic manipulation of median raphe 5-HT terminals in the MS bidirectionally regulates social memory stability. This work expands our understanding of the neural mechanisms by which social interactions lead to social memory and provides evidence that 5-HT has a critical role in promoting not only prosocial behaviours8,9, but also social memory, by influencing distinct target structures.

Diverse intracellular signaling pathways mediate the effects of neurotensin on the excitability of type II neurons in the rat dorsolateral bed nucleus of the stria terminalis.

We examined the effects of neurotensin (NTS) on the excitability of type II neurons in the rat dorsolateral bed nucleus of the stria terminalis (dlBNST) using whole-cell patch-clamp electrophysiology. Bath-application of NTS depolarized type II dlBNST neurons. Analyses of the steady-state I-V relationships implied that the depolarizing effect of NTS is due to potassium conductance blocking. The depolarizing effect of NTS was abolished in the presence of a PLC inhibitor, but not affected by a protein kinase C inhibitor. In the presence of a CaMKII inhibitor, NTS showed depolarizing effects via the increase in non-selective cation conductance in addition to the decrease in potassium conductance. Unexpectedly, in the presence of a PKA inhibitor, NTS hyperpolarized type II dlBNST neurons. These results reveal that diverse signaling pathways mediate the effects of NTS on the excitability of type II dlBNST neurons. The elevation of intracellular Ca2+ levels via the inositol phosphate-mediated signaling activates both Ca2+-dependent adenylate cyclase (AC) and CaMKII. Activation of the AC-cAMP-PKA pathway exerts depolarizing effects on type II dlBNST neurons by decreasing potassium conductance and increasing non-selective cation conductance, whereas activation of the CaMKII pathway exerts hyperpolarizing effects on dlBNST neurons by decreasing non-selective cation conductance.

Cell-type diversity in the bed nucleus of the stria terminalis to regulate motivated behaviors.

Over the past few decades, the bed nucleus of the stria terminalis (BNST) gained popularity as a unique brain region involved in regulating motivated behaviors related to neuropsychiatric disorders. The BNST, a component of the extended amygdala, consists of a variety of subnuclei and neuronal ensembles. Multiple studies have highlighted the BNST as playing a fundamental role in integrating information by interfacing with other brain regions to regulate distinct aspects of motivated behaviors associated with stress, anxiety, depression, and decision-making. However, due to the high molecular heterogeneity found within BNST neurons, the precise mechanisms by which this region regulates distinct motivational states remains largely unclear. Single-cell RNA sequencing data have revealed that the BNST consists of multiple genetically identifiable cell-type clusters. Contemporary tools can therefore be leveraged to target and study such cell-types and elucidate their precise functional role. In this review, we discuss the different subsets of neurons found in the BNST, their anatomical distribution, and what is currently known about BNST cell-types in regulating motivated behaviors.

Corticotropin-releasing factor neurons in the bed nucleus of the stria terminalis exhibit sex-specific pain encoding in mice.

The bed nucleus of the stria terminalis (BNST) plays an emerging role in pain regulation. Pharmacological studies have found that inhibiting corticotropin-releasing factor (CRF) signaling in the BNST can selectively mitigate the sensory and affective-motivational components of pain. However, mechanistic insight on the source of CRF that drives BNST responses to these harmful experiences remains unknown. In the present study, we used a series of genetic approaches to show that CRF in the BNST is engaged in the processing and modulation of pain. We conducted cell-type specific in vivo calcium imaging in CRF-Cre mice and found robust and synchronized recruitment of BNSTCRF neurons during acute exposures to noxious heat. Distinct patterns of recruitment were observed by sex, as the magnitude and timing of heat responsive activity in BNSTCRF neurons differed for male and female mice. We then used a viral approach in Floxed-CRF mice to selectively reduce CRF expression in the BNST and found it decreased nociceptive sensitivity for both sexes and increased paw attending for females. Together, these findings reveal that CRF in the BNST influences multiple facets of the pain experience to impact the sex-specific expression of pain-related behaviors.

Oxytocin and vasopressin within the ventral and dorsal lateral septum modulate aggression in female rats.

In contrast to male rats, aggression in virgin female rats has been rarely studied. Here, we established a rat model of enhanced aggression in females using a combination of social isolation and aggression-training to specifically investigate the involvement of the oxytocin (OXT) and arginine vasopressin (AVP) systems within the lateral septum (LS). Using neuropharmacological, optogenetic, chemogenetic as well as microdialysis approaches, we revealed that enhanced OXT release within the ventral LS (vLS), combined with reduced AVP release within the dorsal LS (dLS), is required for aggression in female rats. Accordingly, increased activity of putative OXT receptor-positive neurons in the vLS, and decreased activity of putative AVP receptor-positive neurons in the dLS, are likely to underly aggression in female rats. Finally, in vitro activation of OXT receptors in the vLS increased tonic GABAergic inhibition of dLS neurons. Overall, our data suggest a model showing that septal release of OXT and AVP differentially affects aggression in females by modulating the inhibitory tone within LS sub-networks.

Optimization of whole-brain rabies virus tracing technology for small cell populations.

The lateral hypothalamus (LH) is critically involved in the regulation of homeostatic energy balance. Some neurons in the LH express receptors for leptin (LepRb), a hormone known to increase energy expenditure and decrease energy intake. However, the neuroanatomical inputs to LepRb-expressing LH neurons remain unknown. We used rabies virus tracing technology to map these inputs, but encountered non-specific tracing. To optimize this technology for a minor cell population (LepRb is not ubiquitously expressed in LH), we used LepRb-Cre mice and assessed how different titers of the avian tumor virus receptor A (TVA) helper virus affected rabies tracing efficiency and specificity. We found that rabies expression is dependent on TVA receptor expression, and that leakiness of TVA receptors is dependent on the titer of TVA virus used. We concluded that a titer of 1.0-3.0 × 107 genomic copies per µl of the TVA virus is optimal for rabies tracing. Next, we successfully applied modified rabies virus tracing technology to map inputs to LepRb-expressing LH neurons. We discovered that other neurons in the LH itself, the periventricular hypothalamic nucleus (Pe), the posterior hypothalamic nucleus (PH), the bed nucleus of the stria terminalis (BNST), and the paraventricular hypothalamic nucleus (PVN) are the most prominent input areas to LepRb-expressing LH neurons.

Dorsal hippocampus- and ACC-projecting medial septum neurons differentially contribute to the recollection of episodic-like memory.

Episodic memory refers to the recollection of previous experiences containing specific temporal, spatial, and emotional information. The ability to recollect episodic memory requires coordination of multiple brain regions, including the hippocampus (HPC) and the cingulate cortex. While the afferents into HPC and cingulate cortex that orchestrate the episodic memory remain unclear. The medial septum (MS), one of the anatomical location of cholinergic centers, innervates not only the dorsal HPC (dHPC), but also the cingulate and entorhinal cortices. By using "What-Where-When" episodic-like memory (ELM) behavioral model and viral tracing, we found that MS neurons projected to dHPC and anterior cingulate cortex (ACC), which exerted distinct impacts on ELM recollection. Chemogenetic inhibition of the dHPC-projecting MS neurons disrupted "What-Where-When" ELM recollection as well as object location, object-in-place, and recency recognition memories recollection, while chemogenetic inhibition of the ACC-projecting MS neurons only disrupted "What-Where-When" ELM recollection. Moreover, neither dHPC- nor ACC-projecting MS neurons were involved in novel object recognition memory recollection or locomotor activity. Immunostaining showed that ACC- and dHPC-projecting MS neurons are partially overlapped populations. These findings reveal an unsuspected division of ELM processing and provide the potential mechanism that the recollection of episodic memory need the coordination of MS neurons projecting to dHPC and ACC.

Effects of neural estrogen receptor beta deletion on social and mood-related behaviors and underlying mechanisms in male mice.

Estradiol derived from neural aromatization of testosterone plays a key role in the organization and activation of neural structures underlying male behaviors. This study evaluated the contribution of the estrogen receptor (ER) ß in estradiol-induced modulation of social and mood-related behaviors by using mice lacking the ERß gene in the nervous system. Mutant males exhibited reduced social interaction with same-sex congeners and impaired aggressive behavior. They also displayed increased locomotor activity, and reduced or unaffected anxiety-state level in three paradigms. However, when mice were exposed to unescapable stress in the forced swim and tail suspension tests, they spent more time immobile and a reduced time in swimming and climbing. These behavioral alterations were associated with unaffected circadian and restraint stress-induced corticosterone levels, and unchanged number of tryptophan hydroxylase 2-immunoreactive neurons in the dorsal raphe. By contrast, reduced mRNA levels of oxytocin and arginine-vasopressin were observed in the bed nucleus of stria terminalis, whereas no changes were detected in the hypothalamic paraventricular nucleus. The neural ERß is thus involved to different extent levels in social and mood-related behaviors, with a particular action on oxytocin and arginine-vasopressin signaling pathways of the bed nucleus of stria terminalis, yet the involvement of other brain areas cannot be excluded.

Bed nuclei of the stria terminalis modulate memory consolidation via glucocorticoid-dependent and -independent circuits.

There is extensive evidence that glucocorticoid hormones enhance memory consolidation, helping to ensure that emotionally significant events are well remembered. Prior findings suggest that the anteroventral region of bed nuclei of the stria terminalis (avBST) regulates glucocorticoid release, suggesting the potential for avBST activity to influence memory consolidation following an emotionally arousing learning event. To investigate this issue, male Sprague-Dawley rats underwent inhibitory avoidance training and repeated measurement of stress hormones, immediately followed by optogenetic manipulations of either the avBST or its projections to downstream regions, and 48 h later were tested for retention. The results indicate that avBST inhibition augmented posttraining pituitary-adrenal output and enhanced the memory for inhibitory avoidance training. Pretreatment with a glucocorticoid synthesis inhibitor blocked the memory enhancement as well as the potentiated corticosterone response, indicating the dependence of the memory enhancement on glucocorticoid release during the immediate posttraining period. In contrast, posttraining avBST stimulation decreased retention yet had no effect on stress hormonal output. Subsequent experiments revealed that inhibition of avBST input to the paraventricular hypothalamus enhanced stress hormonal output and subsequent retention, whereas stimulation did not affect either. Conversely, stimulation-but not inhibition-of avBST input to the ventrolateral periaqueductal gray impaired consolidation, whereas neither manipulation affected glucocorticoid secretion. These findings indicate that divergent pathways from the avBST are responsible for the mnemonic effects of avBST inhibition versus stimulation and do so via glucocorticoid-dependent and -independent mechanisms, respectively.

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.

Optogenetic gamma stimulation rescues memory impairments in an Alzheimer's disease mouse model.

Slow gamma oscillations (30-60 Hz) correlate with retrieval of spatial memory. Altered slow gamma oscillations have been observed in Alzheimer's disease. Here, we use the J20-APP AD mouse model that displays spatial memory loss as well as reduced slow gamma amplitude and phase-amplitude coupling to theta oscillations phase. To restore gamma oscillations in the hippocampus, we used optogenetics to activate medial septal parvalbumin neurons at different frequencies. We show that optogenetic stimulation of parvalbumin neurons at 40 Hz (but not 80 Hz) restores hippocampal slow gamma oscillations amplitude, and phase-amplitude coupling of the J20 AD mouse model. Restoration of slow gamma oscillations during retrieval rescued spatial memory in mice despite significant plaque deposition. These results support the role of slow gamma oscillations in memory and suggest that optogenetic stimulation of medial septal parvalbumin neurons at 40 Hz could provide a novel strategy for treating memory deficits in AD.

Hypothalamic neuronal circuits regulating hunger-induced taste modification.

The gustatory system plays a critical role in sensing appetitive and aversive taste stimuli for evaluating food quality. Although taste preference is known to change depending on internal states such as hunger, a mechanistic insight remains unclear. Here, we examine the neuronal mechanisms regulating hunger-induced taste modification. Starved mice exhibit an increased preference for sweetness and tolerance for aversive taste. This hunger-induced taste modification is recapitulated by selective activation of orexigenic Agouti-related peptide (AgRP)-expressing neurons in the hypothalamus projecting to the lateral hypothalamus, but not to other regions. Glutamatergic, but not GABAergic, neurons in the lateral hypothalamus function as downstream neurons of AgRP neurons. Importantly, these neurons play a key role in modulating preferences for both appetitive and aversive tastes by using distinct pathways projecting to the lateral septum or the lateral habenula, respectively. Our results suggest that these hypothalamic circuits would be important for optimizing feeding behavior under fasting.

TMEM16B Calcium-Activated Chloride Channels Regulate Action Potential Firing in Lateral Septum and Aggression in Male Mice.

The lateral septum (LS) plays an important role in regulating aggression. It is well recognized that LS lesions lead to a dramatic increase in aggressive behaviors. A better understanding of LS neurophysiology and its functional output is therefore important to assess LS involvement in regulating aggression. The LS is a heterogeneous structure that maintains inputs and outputs with multiple brain regions, and is also divided into subregions that innervate one another. Thus, it is challenging to identify the exact cell type and projections for characterization. In this study, we determined the expression pattern of the calcium-activated chloride channel, TMEM16B, in the LS of both male and female mice. We then investigated the physiological contribution of the calcium-activated chloride channel to LS neuronal signaling. By performing whole-cell patch-clamp recording, we showed that TMEM16B alters neurotransmitter release at the hippocampal-LS synapse, and regulates spike frequency and spike frequency adaptation in subpopulations of LS neurons. We further demonstrated that loss of TMEM16B function promotes lengthened displays of aggressive behaviors by male mice during the resident intruder paradigm. In conclusion, our findings suggest that TMEM16B function contributes to neuronal excitability in subpopulations of LS neurons and the regulation of aggression in male mice.SIGNIFICANCE STATEMENT Aggression is a behavior that arose evolutionarily from the necessity to compete for limited resources and survival. One particular brain region involved in aggression is the lateral septum (LS). In this study, we characterized the expression of the TMEM16B calcium-activated chloride channel in the LS and showed that TMEM16B regulates the action potential firing frequency of LS neurons. We discovered that loss of TMEM16B function lengthens the displays of aggressive behaviors in male mice. These findings suggest that TMEM16B plays an important role in regulating LS neuronal excitability and behaviors associated with LS function, thereby contributing to our understanding of how the LS may regulate aggression.

The Lateral Hypothalamic and BNST GABAergic Projections to the Anterior Ventrolateral Periaqueductal Gray Regulate Feeding.

Overeating is a serious issue in modern society, causing many health problems, including obesity. Although the hypothalamus has been previously identified as the key brain structure that regulates body weight homeostasis, the downstream pathways and non-canonical neural circuitry involved in feeding behavior remain largely uncharacterized. Here, we discover that suppressing the activity of GABAergic cells in the anterior ventrolateral periaqueductal gray (vlPAG), whether directly or through long-projection GABAergic inputs from either the bed nucleus of the stria terminalis (BNST) or the lateral hypothalamus (LH), is sufficient to promptly induce feeding behavior in well-fed mice. In contrast, optogenetic activation of these cells interrupts food intake in starved mice. Long-term chemogenetic manipulation of vlPAG GABAergic cell activity elicits a corresponding change in mouse body weight. Our studies reveal distinct midbrain GABAergic pathways and highlight an important role of GABAergic cells in the anterior vlPAG in feeding behavior.

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice.

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

A bed nucleus of stria terminalis microcircuit regulating inflammation-associated modulation of feeding.

Loss of appetite or anorexia associated with inflammation impairs quality of life and increases morbidity in many diseases. However, the exact neural mechanism that mediates inflammation-associated anorexia is still poorly understood. Here we identified a population of neurons, marked by the expression of protein kinase C-delta, in the oval region of the bed nucleus of the stria terminalis (BNST), which are activated by various inflammatory signals. Silencing of these neurons attenuates the anorexia caused by these inflammatory signals. Our results demonstrate that these neurons mediate bidirectional control of general feeding behaviors. These neurons inhibit the lateral hypothalamus-projecting neurons in the ventrolateral part of BNST to regulate feeding, receive inputs from the canonical feeding regions of arcuate nucleus and parabrachial nucleus. Our data therefore define a BNST microcircuit that might coordinate canonical feeding centers to regulate food intake, which could offer therapeutic targets for feeding-related diseases such as anorexia and obesity.

Single-Cell Multi-omic Integration Compares and Contrasts Features of Brain Cell Identity.

Defining cell types requires integrating diverse single-cell measurements from multiple experiments and biological contexts. To flexibly model single-cell datasets, we developed LIGER, an algorithm that delineates shared and dataset-specific features of cell identity. We applied it to four diverse and challenging analyses of human and mouse brain cells. First, we defined region-specific and sexually dimorphic gene expression in the mouse bed nucleus of the stria terminalis. Second, we analyzed expression in the human substantia nigra, comparing cell states in specific donors and relating cell types to those in the mouse. Third, we integrated in situ and single-cell expression data to spatially locate fine subtypes of cells present in the mouse frontal cortex. Finally, we jointly defined mouse cortical cell types using single-cell RNA-seq and DNA methylation profiles, revealing putative mechanisms of cell-type-specific epigenomic regulation. Integrative analyses using LIGER promise to accelerate investigations of cell-type definition, gene regulation, and disease states.

Characterization of the relaxin family peptide receptor 3 system in the mouse bed nucleus of the stria terminalis.

The bed nucleus of the stria terminalis (BNST) is a critical node involved in stress and reward-related behaviors. Relaxin family peptide receptor 3 (RXFP3) signaling in the BNST has been implicated in stress-induced alcohol seeking behavior. However, the neurochemical phenotype and connectivity of BNST RXFP3-expressing (RXFP3+) cells have yet to be elucidated. We interrogated the molecular signature and electrophysiological properties of BNST RXFP3+ neurons using a RXFP3-Cre reporter mouse line. BNST RXFP3+ cells are circumscribed to the dorsal BNST (dBNST) and are neurochemically heterogeneous, comprising a mix of inhibitory and excitatory neurons. Immunohistochemistry revealed that ~48% of BNST RXFP3+ neurons are GABAergic, and a quarter of these co-express the calcium-binding protein, calbindin. A subset of BNST RXFP3+ cells (~41%) co-express CaMKIIα, suggesting this subpopulation of BNST RXFP3+ neurons are excitatory. Corroborating this, RNAscope® revealed that ~35% of BNST RXFP3+ cells express vVGluT2 mRNA, indicating a subpopulation of RXFP3+ neurons are glutamatergic. RXFP3+ neurons show direct hyperpolarization to bath application of a selective RXFP3 agonist, RXFP3-A2, while around 50% of cells were depolarised by exogenous corticotrophin releasing factor. In behaviorally naive mice the majority of RXFP3+ neurons were Type II cells exhibiting Ih and T type calcium mediated currents. However, chronic swim stress caused persistent plasticity, decreasing the proportion of neurons that express these channels. These studies are the first to characterize the BNST RXFP3 system in mouse and lay the foundation for future functional studies appraising the role of the murine BNST RXFP3 system in more complex behaviors.

Inactivation of a CRF-dependent amygdalofugal pathway reverses addiction-like behaviors in alcohol-dependent rats.

The activation of a neuronal ensemble in the central nucleus of the amygdala (CeA) during alcohol withdrawal has been hypothesized to induce high levels of alcohol drinking in dependent rats. In the present study we describe that the CeA neuronal ensemble that is activated by withdrawal from chronic alcohol exposure contains ~80% corticotropin-releasing factor (CRF) neurons and that the optogenetic inactivation of these CeA CRF+ neurons prevents recruitment of the neuronal ensemble, decreases the escalation of alcohol drinking, and decreases the intensity of somatic signs of withdrawal. Optogenetic dissection of the downstream neuronal pathways demonstrates that the reversal of addiction-like behaviors is observed after the inhibition of CeA CRF projections to the bed nucleus of the stria terminalis (BNST) and that inhibition of the CRFCeA-BNST pathway is mediated by inhibition of the CRF-CRF1 system and inhibition of BNST cell firing. These results suggest that the CRFCeA-BNST pathway could be targeted for the treatment of excessive drinking in alcohol use disorder.