EZH2-dependent epigenetic reprogramming in the central nucleus of amygdala regulates adult anxiety in both sexes after adolescent alcohol exposure.

Alcohol use and anxiety disorders occur in both males and females, but despite sharing similar presentation and classical symptoms, the prevalence of alcohol use disorder (AUD) is lower in females. While anxiety is a symptom and comorbidity shared by both sexes, the common underlying mechanism that leads to AUD and the subsequent development of anxiety is still understudied. Using a rodent model of adolescent intermittent ethanol (AIE) exposure in both sexes, we investigated the epigenetic mechanism mediated by enhancer of zeste 2 (EZH2), a histone methyltransferase, in regulating both the expression of activity-regulated cytoskeleton-associated protein (Arc) and an anxiety-like phenotype in adulthood. Here, we report that EZH2 protein levels were significantly higher in PKC-δ positive GABAergic neurons in the central nucleus of amygdala (CeA) of adult male and female rats after AIE. Reducing protein and mRNA levels of EZH2 using siRNA infusion in the CeA prevented AIE-induced anxiety-like behavior, increased H3K27me3, decreased H3K27ac at the Arc synaptic activity response element (SARE) site, and restored deficits in Arc mRNA and protein expression in both male and female adult rats. Our data indicate that an EZH2-mediated epigenetic mechanism in the CeA plays an important role in regulating anxiety-like behavior and Arc expression after AIE in both male and female rats in adulthood. This study suggests that EZH2 may serve as a tractable drug target for the treatment of adult psychopathology after adolescent alcohol exposure.

Chemogenetic Manipulation of Amygdala Kappa Opioid Receptor Neurons Modulates Amygdala Neuronal Activity and Neuropathic Pain Behaviors.

Neuroplasticity in the central nucleus of the amygdala (CeA) plays a key role in the modulation of pain and its aversive component. The dynorphin/kappa opioid receptor (KOR) system in the amygdala is critical for averse-affective behaviors in pain conditions, but its mechanisms are not well understood. Here, we used chemogenetic manipulations of amygdala KOR-expressing neurons to analyze the behavioral consequences in a chronic neuropathic pain model. For the chemogenetic inhibition or activation of KOR neurons in the CeA, a Cre-inducible viral vector encoding Gi-DREADD (hM4Di) or Gq-DREADD (hM3Dq) was injected stereotaxically into the right CeA of transgenic KOR-Cre mice. The chemogenetic inhibition of KOR neurons expressing hM4Di with a selective DREADD actuator (deschloroclozapine, DCZ) in sham control mice significantly decreased inhibitory transmission, resulting in a shift of inhibition/excitation balance to promote excitation and induced pain behaviors. The chemogenetic activation of KOR neurons expressing hM3Dq with DCZ in neuropathic mice significantly increased inhibitory transmission, decreased excitability, and decreased neuropathic pain behaviors. These data suggest that amygdala KOR neurons modulate pain behaviors by exerting an inhibitory tone on downstream CeA neurons. Therefore, activation of these interneurons or blockade of inhibitory KOR signaling in these neurons could restore control of amygdala output and mitigate pain.

Activation of the CXCR4 Receptor by Chemokine CXCL12 Increases the Excitability of Neurons in the Rat Central Amygdala.

Primarily regarded as immune proteins, chemokines are emerging as a family of molecules serving neuromodulatory functions in the developing and adult brain. Among them, CXCL12 is constitutively and widely expressed in the CNS, where it was shown to act on cellular, synaptic, network, and behavioral levels. Its receptor, CXCR4, is abundant in the amygdala, a brain structure involved in pathophysiology of anxiety disorders. Dysregulation of CXCL12/CXCR4 signaling has been implicated in anxiety-related behaviors. Here we demonstrate that exogenous CXCL12 at 2 nM but not at 5 nM increased neuronal excitability in the lateral division of the rat central amygdala (CeL) which was evident in the Late-Firing but not Regular-Spiking neurons. These effects were blocked by AMD3100, a CXCR4 antagonist. Moreover, CXCL12 increased the excitability of the neurons of the basolateral amygdala (BLA) that is known to project to the CeL. However, CXCL12 increased neither the spontaneous excitatory nor spontaneous inhibitory synaptic transmission in the CeL. In summary, the data reveal specific activation of Late-Firing CeL cells along with BLA neurons by CXCL12 and suggest that this chemokine may alter information processing by the amygdala that likely contributes to anxiety and fear conditioning.

Presynaptic inhibition of excitatory synaptic transmission from the calcitonin gene-related peptide-containing parabrachial neurons to the central amygdala in mice - unexpected influence of systemic inflammation thereon.

The monosynaptic connection from the lateral parabrachial nucleus (LPB) to the central amygdala (CeA) serves as a fundamental pathway for transmitting nociceptive signals to the brain. The LPB receives nociceptive information from the dorsal horn and spinal trigeminal nucleus and sends it to the "nociceptive" CeA, which modulates pain-associated emotions and nociceptive sensitivity. To elucidate the role of densely expressed mu-opioid receptors (MORs) within this pathway, we investigated the effects of exogenously applied opioids on LPB-CeA synaptic transmission, employing optogenetics in mice expressing channelrhodopsin-2 in LPB neurons with calcitonin gene-related peptide (CGRP). A MOR agonist ([D-Ala2,N-Me-Phe4,Glycinol5]-enkephalin, DAMGO) significantly reduced the amplitude of light-evoked excitatory postsynaptic currents (leEPSCs), in a manner negatively correlated with an increase in the paired-pulse ratio. An antagonist of MORs significantly attenuated these effects. Notably, this antagonist significantly increased leEPSC amplitude when applied alone, an effect further amplified in mice subjected to lipopolysaccharide injection 2 h before brain isolation, yet not observed at the 24-h mark. We conclude that opioids could shut off the ascending nociceptive signal at the LPB-CeA synapse through presynaptic mechanisms. Moreover, this gating process might be modulated by endogenous opioids, and the innate immune system influences this modulation.

Development of activity-based anorexia requires PKC-δ neurons in two central extended amygdala nuclei.

Anorexia nervosa (AN) is a serious psychiatric disease, but the neural mechanisms underlying its development are unclear. A subpopulation of amygdala neurons, marked by expression of protein kinase C-delta (PKC-δ), has previously been shown to regulate diverse anorexigenic signals. Here, we demonstrate that these neurons regulate development of activity-based anorexia (ABA), a common animal model for AN. PKC-δ neurons are located in two nuclei of the central extended amygdala (EAc): the central nucleus (CeA) and oval region of the bed nucleus of the stria terminalis (ovBNST). Simultaneous ablation of CeAPKC-δ and ovBNSTPKC-δ neurons prevents ABA, but ablating PKC-δ neurons in the CeA or ovBNST alone is not sufficient. Correspondingly, PKC-δ neurons in both nuclei show increased activity with ABA development. Our study shows how neurons in the amygdala regulate ABA by impacting both feeding and wheel activity behaviors and support a complex heterogeneous etiology of AN.

Nausea-induced suppression of feeding is mediated by central amygdala Dlk1-expressing neurons.

The motivation to eat is suppressed by satiety and aversive stimuli such as nausea. The neural circuit mechanisms of appetite suppression by nausea are not well understood. Pkcδ neurons in the lateral subdivision of the central amygdala (CeA) suppress feeding in response to satiety signals and nausea. Here, we characterized neurons enriched in the medial subdivision (CeM) of the CeA marked by expression of Dlk1. CeADlk1 neurons are activated by nausea, but not satiety, and specifically suppress feeding induced by nausea. Artificial activation of CeADlk1 neurons suppresses drinking and social interactions, suggesting a broader function in attenuating motivational behavior. CeADlk1 neurons form projections to many brain regions and exert their anorexigenic activity by inhibition of neurons of the parabrachial nucleus. CeADlk1 neurons are inhibited by appetitive CeA neurons, but also receive long-range monosynaptic inputs from multiple brain regions. Our results illustrate a CeA circuit that regulates nausea-induced feeding suppression.

GABA release from central amygdala neurotensin neurons differentially modulates ethanol consumption in male and female mice.

The central nucleus of the amygdala is known to play key roles in alcohol use and affect. Neurotensin neurons in the central nucleus of the amygdala have been shown to regulate alcohol drinking in male mice. However, little is known about which neurotransmitters released by these cells drive alcohol consumption or whether these cells drive alcohol consumption in female mice. Here we show that knockdown of GABA release from central amygdala neurotensin neurons using a Nts-cre-dependent vGAT-shRNA-based AAV strategy reduces alcohol drinking in male, but not female, mice. This manipulation did not impact avoidance behavior, except in a fasted novelty-suppressed feeding test, in which vGAT shRNA mice demonstrated increased latency to feed on a familiar high-value food reward, an effect driven by male mice. In contrast, vGAT shRNA female mice showed heightened sensitivity to thermal stimulation. These data show a role for GABA release from central amygdala neurotensin neurons in modulating consumption of rewarding substances in different motivational states.

Effects of sub-chronic CRH administration into the hypothalamic paraventricular and central amygdala nuclei in male rats with a focus on food intake biomarkers.

CRH neurons are found in the paraventricular nucleus(PVN) and central amygdala(CeA) nuclei. This study investigated the effects of sub-chronic CRH administration into the PVN and CeA nuclei on food intake biomarkers in rats divided into five groups: control, two shams, and two CRH-PVN and CRH-CeA groups(receiving CRH in nuclei for seven days). The CRH-PVN group had significantly higher cumulative food intake and food intake trends than the CRH-CeA group. The CRH-CeA and CRH-PVN groups exhibited significant increases in food intake during hours 1 and 2, respectively. Moreover, to be time-dependent, food intake is modulated by different brain nuclei. The CRH signaling pathway appeared to be activated later in the PVN than CeA. Both groups exhibited significantly higher leptin levels, the CRH-PVN group exhibited higher ghrelin levels and lower glucose levels. Repetitive administration of CRH into the PVN and CeA significantly reduced body weight differences. CRH administration into the PVN affected both leptin and ghrelin levels, but ghrelin had a greater impact on glucose variations and cumulative food intake than leptin. Finally, CRH administration into the PVN and CeA likely activated the HPA axis, and the CeA had a greater impact on the stress circuit than on food intake behavior.

Traumatic Stress-Induced Increases in Anxiety-like Behavior and Alcohol Self-Administration Are Mediated by Central Amygdala CRF1 Neurons That Project to the Lateral Hypothalamus.

Avoidance stress coping, defined as persistent internal and/or external avoidance of stress-related stimuli, is a key feature of anxiety- and stress-related disorders, and contributes to increases in alcohol misuse after stress exposure. Previous work using a rat model of predator odor stress avoidance identified corticotropin-releasing factor (CRF) signaling via CRF Type 1 receptors (CRF1) in the CeA, as well as CeA projections to the lateral hypothalamus (LH) as key mediators of conditioned avoidance of stress-paired contexts and/or increased alcohol drinking after stress. Here, we report that CRF1-expressing CeA cells that project to the LH are preferentially activated in male and female rats that show persistent avoidance of predator odor stress-paired contexts (termed Avoider rats), and that chemogenetic inhibition of these cells rescues stress-induced increases in anxiety-like behavior and alcohol self-administration in male and female Avoider rats. Using slice electrophysiology, we found that prior predator odor stress exposure blunts inhibitory synaptic transmission and increases synaptic drive in CRF1 CeA-LH cells. In addition, we found that CRF bath application reduces synaptic drive in CRF1 CeA-LH cells in Non-Avoiders only. Collectively, these data show that CRF1 CeA-LH cells contribute to stress-induced increases in anxiety-like behavior and alcohol self-administration in male and female Avoider rats.SIGNIFICANCE STATEMENT Stress may lead to a variety of behavioral and physiological negative consequences, and better understanding of the neurobiological mechanisms that contribute to negative stress effects may lead to improved prevention and treatment strategies. This study, performed in laboratory rats, shows that animals that exhibit avoidance stress coping go on to develop heightened anxiety-like behavior and alcohol self-administration, and that these behaviors can be rescued by inhibiting the activity of a specific population of neurons in the central amygdala. This study also describes stress-induced physiological changes in these neurons that may contribute to their role in promoting increased anxiety and alcohol self-administration.

Divergent Population-Specific Effects of Chronic Ethanol Exposure on Excitability and Inhibitory Transmission in Male and Female Rat Central Amygdala.

The central nucleus of the amygdala (CeA) is implicated in alcohol use disorder (AUD) and AUD-associated plasticity. The CeA is a primarily GABAergic nucleus that is subdivided into lateral and medial compartments with genetically diverse subpopulations. GABAA receptors are heteromeric pentamers with subunits conferring distinct physiological characteristics. GABAA receptor signaling in the CeA has been implicated in ethanol-associated plasticity; however, population-specific changes in inhibitory signaling and subunit expression remain unclear. Here, we combined electrophysiology with single-cell gene expression analysis of population markers and GABAA receptor subunits to examine population-specific changes in inhibitory control in male and female rats following chronic ethanol exposure. We found that chronic ethanol exposure and withdrawal produced global changes in GABAA receptor subunit expression at the transcript and protein levels, increased excitability in female CeA neurons, and increased inhibitory synaptic transmission in male CeA neurons. When we examined CeA neurons at the single-cell level we found heterogenous populations, as previously reported. We observed ethanol-induced increases in excitability only in somatostatin neurons in the CeA of females, decreases in excitability only in the protein kinase C delta (PKCd) population in males, and ethanol-induced increases in inhibitory transmission in male PKCd and calbindin 2-expressing CeA neurons. There were no population-specific differences in GABAA receptor (Gabr) subunits in males but reduced GabrA5 expression in female somatostatin neurons. Collectively, these findings suggest that defined CeA populations display differential ethanol sensitivity in males and females, which may play a role in sex differences in vulnerability to AUD or expression of AUD pathology.SIGNIFICANCE STATEMENT The CeA is involved in the effects of ethanol in the brain; however, the population-specific changes in CeA activity remain unclear. We used recordings of CeA neuronal activity and single-cell gene expression to examine population-specific changes in inhibitory control in male and female rats following chronic ethanol exposure and found sex- and population-specific effects of chronic ethanol exposure and withdrawal. Specifically, female CeA neurons displayed increased excitability in the somatostatin CeA population, whereas male CeA neurons displayed increased inhibitory control in both PKCd and calbindin populations and decreased excitability in the PKCd population. These findings identify CeA populations that display differential sensitivity to ethanol exposure, which may contribute to sex differences in vulnerability to alcohol use disorder.

IL-18 Signaling in the Rat Central Amygdala Is Disrupted in a Comorbid Model of Post-Traumatic Stress and Alcohol Use Disorder.

Alcohol use disorder (AUD) and anxiety disorders are frequently comorbid and share dysregulated neuroimmune-related pathways. Here, we used our established rat model of comorbid post-traumatic stress disorder (PTSD)/AUD to characterize the interleukin 18 (IL-18) system in the central amygdala (CeA). Male and female rats underwent novel (NOV) and familiar (FAM) shock stress, or no stress (unstressed controls; CTL) followed by voluntary alcohol drinking and PTSD-related behaviors, then all received renewed alcohol access prior to the experiments. In situ hybridization revealed that the number of CeA positive cells for Il18 mRNA increased, while for Il18bp decreased in both male and female FAM stressed rats versus CTL. No changes were observed in Il18r1 expression across groups. Ex vivo electrophysiology showed that IL-18 reduced GABAA-mediated miniature inhibitory postsynaptic currents (mIPSCs) frequencies in CTL, suggesting reduced CeA GABA release, regardless of sex. Notably, this presynaptic effect of IL-18 was lost in both NOV and FAM males, while it persisted in NOV and FAM females. IL-18 decreased mIPSC amplitude in CTL female rats, suggesting postsynaptic effects. Overall, our results suggest that stress in rats with alcohol access impacts CeA IL-18-system expression and, in sex-related fashion, IL-18's modulatory function at GABA synapses.

Stimulation of lateral parabrachial (LPB) to central amygdala (CeA) pituitary adenylate cyclase-activating polypeptide (PACAP) neurons induces anxiety-like behavior and mechanical allodynia.

BACKGROUND: Anxiety disorders are the most prevalent psychiatric disorders, and they are highly comorbid with chronic pain conditions. The central nucleus of the amygdala (CeA) is known not only for its role in the regulation of anxiety but also as an important site for the negative affective dimension of pain. Pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide whose terminals are abundant in the CeA, is strongly implicated in the stress response as well as in pain processing. Here, using Cre-dependent viral vectors, we explored in greater detail the role of the PACAP projection to the CeA that originates in the lateral parabrachial nucleus (LPB). METHODS: We first performed a circuit mapping experiment by injecting an anterograde Cre-dependent virus expressing a fluorescent reporter in the LPB of PACAP-Cre mice and observing their projections. Then, we used a chemogenetic approach (a Cre-dependent Designer Receptors Activated by Designer Drugs, DREADDs) to assess the effects of the direct stimulation of the PACAP LPB to CeA projection on general locomotor activity, anxiety-like behavior (using a defensive withdrawal test), and mechanical pain sensitivity (using the von Frey test). RESULTS: We found that the CeA, together with other areas, is one of the major downstream projection targets of PACAP neurons originating in the lateral parabrachial nucleus (LPB). In the DREADD experiment, we then found that the selective activation of this neuronal pathway is sufficient to increase both anxiety-like behavior and mechanical pain sensitivity in mice, without affecting general locomotor activity. CONCLUSION: In conclusion, our data suggest that the dysregulation of this circuit may contribute to a variety of anxiety disorders and chronic pain states, and that PACAP may represent an important therapeutic target for the treatment of these conditions.

Somatostatin signaling modulates binge drinking behavior via the central nucleus of the amygdala.

Somatostatin (SST) is a neuropeptide widely expressed in the central nervous system with dense expression in limbic regions such as the extended amygdala. It has recently gained attention for playing a role in modulating alcohol use disorders and co-morbid neuropsychiatric disorders. However, the role of SST in the central nucleus of the amygdala (CeA), a key region for neuropeptide regulation of alcohol and anxiety related behaviors, in alcohol consumption has not been assessed. In this work we perform an initial examination of the interaction between the CeA SST system and binge ethanol intake. Binge intake is a dangerous pattern of excessive ethanol consumption associated with health complications and the transition into alcohol dependence. We use the Drinking in the Dark (DID) model of binge intake in C57BL/6J male and female mice to examine: 1) the impact of 3 DID cycles on CeA SST expression; 2) the effect of intra-CeA SST injection on binge-like ethanol consumption; and 3) if the SST receptor 2 or 4 (SST2R or SST4R) mediate any effect on consumption. Our results show binge-like ethanol intake decreases SST expression in the CeA, but not neighboring basolateral amygdala. We further found intra-SST CeA administration reduces binge ethanol intake. This decrease was replicated by the administration of an SST4R agonist. These effects were not sex-dependent. Overall, this work lends further support for SST playing a role in alcohol related behaviors and as a potential therapeutic target.

Corticotropin-releasing hormone neurons in the central nucleus of amygdala are required for chronic stress-induced hypertension.

AIMS: Chronic stress is a well-known risk factor for the development of hypertension. However, the underlying mechanisms remain unclear. Corticotropin-releasing hormone (CRH) neurons in the central nucleus of the amygdala (CeA) are involved in the autonomic responses to chronic stress. Here, we determined the role of CeA-CRH neurons in chronic stress-induced hypertension. METHODS AND RESULTS: Borderline hypertensive rats (BHRs) and Wistar-Kyoto (WKY) rats were subjected to chronic unpredictable stress (CUS). Firing activity and M-currents of CeA-CRH neurons were assessed, and a CRH-Cre-directed chemogenetic approach was used to suppress CeA-CRH neurons. CUS induced a sustained elevation of arterial blood pressure (ABP) and heart rate (HR) in BHRs, while in WKY rats, CUS-induced increases in ABP and HR quickly returned to baseline levels after CUS ended. CeA-CRH neurons displayed significantly higher firing activities in CUS-treated BHRs than unstressed BHRs. Selectively suppressing CeA-CRH neurons by chemogenetic approach attenuated CUS-induced hypertension and decreased elevated sympathetic outflow in CUS-treated BHRs. Also, CUS significantly decreased protein and mRNA levels of Kv7.2 and Kv7.3 channels in the CeA of BHRs. M-currents in CeA-CRH neurons were significantly decreased in CUS-treated BHRs compared with unstressed BHRs. Blocking Kv7 channel with its blocker XE-991 increased the excitability of CeA-CRH neurons in unstressed BHRs but not in CUS-treated BHRs. Microinjection of XE-991 into the CeA increased sympathetic outflow and ABP in unstressed BHRs but not in CUS-treated BHRs. CONCLUSIONS: CeA-CRH neurons are required for chronic stress-induced sustained hypertension. The hyperactivity of CeA-CRH neurons may be due to impaired Kv7 channel activity, which represents a new mechanism involved in chronic stress-induced hypertension.

DNMT1 Mediates Chronic Pain-Related Depression by Inhibiting GABAergic Neuronal Activation in the Central Amygdala.

BACKGROUND: Chronic pain can induce depressive emotion. DNA methyltransferases (DNMTs) have been shown to be involved in the development of chronic pain and depression. However, the role and mechanism of DNMTs in chronic pain-induced depression are not well understood. METHODS: In well-established spared nerve injury (SNI)-induced chronic pain-related depression models, the expression of DNMTs and the functional roles and underlying mechanisms of DNMT1 in central amygdala (CeA) GABAergic (gamma-aminobutyric acidergic) neurons were investigated using molecular, pharmacological, electrophysiological, optogenetic, and chemogenetic techniques and behavioral tests. RESULTS: DNMT1, but not DNMT3a or DNMT3b, was upregulated in the CeA of rats with SNI-induced chronic pain-depression. Inhibition of DNMT1 by 5-Aza or viral knockdown of DNMT1 in GABAergic neurons in the CeA effectively ameliorated the depression-like behaviors induced by chronic pain. The DNMT1 action was associated with methylation at the CpG-rich Gad1 promoter and GAD67 downregulation, leading to a decrease of GABAergic neuronal activity. Optogenetic activation of GABAergic neurons in the CeA improved SNI-induced depression-like behaviors. Moreover, optogenetic or chemogenetic inhibition of GABAergic neurons in the CeA reversed DNMT1 knockdown-induced improvement of depression-like behaviors in SNI mice. CONCLUSIONS: Our findings suggest that DNMT1 is involved in the development of chronic pain-related depression by epigenetic repression of GAD67, leading to the inhibition of GABAergic neuronal activation. This study indicates that DNMT1 could be a potential target for the treatment of chronic pain-related depression.

BNST PKCδ neurons are activated by specific aversive conditions to promote anxiety-like behavior.

The bed nucleus of the stria terminalis (BNST) is a critical mediator of stress responses and anxiety-like behaviors. Neurons expressing protein kinase C delta (BNSTPKCδ) are an abundant but understudied subpopulation implicated in inhibiting feeding, but which have conflicting reports about their role in anxiety-like behaviors. We have previously shown that expression of PKCδ is dynamically regulated by stress and that BNSTPKCδ cells are recruited during bouts of active stress coping. Here, we first show that in vivo activation of this population is mildly aversive. This aversion was insensitive to prior restraint stress exposure. Further investigation revealed that unlike other BNST subpopulations, BNSTPKCδ cells do not exhibit increased cfos expression following restraint stress. Ex vivo current clamp recordings also indicate they are resistant to firing. To elucidate their afferent control, we next used rabies tracing with whole-brain imaging and channelrhodopsin-assisted circuit mapping, finding that BNSTPKCδ cells receive abundant input from affective, arousal, and sensory regions including the basolateral amygdala (BLA) paraventricular thalamus (PVT) and central amygdala PKCδ-expressing cells (CeAPKCδ). Given these findings, we used in vivo optogenetics and fiber photometry to further examine BNSTPKCδ cells in the context of stress and anxiety-like behavior. We found that BNSTPKCδ cell activity is associated with increased anxiety-like behavior in the elevated plus maze, increases following footshock, and unlike other BNST subpopulations, does not desensitize to repeated stress exposure. Taken together, we propose a model in which BNSTPKCδ cells may serve as threat detectors, integrating exteroceptive and interoceptive information to inform stress coping behaviors.

Distinct populations of corticotropin-releasing factor (CRF) neurons mediate divergent yet complementary defensive behaviors in response to a threat.

Defensive behaviors in response to a threat are shared across the animal kingdom. Active (fleeing, sheltering) or passive (freezing, avoiding) defensive responses are adaptive and facilitate survival. Selecting appropriate defensive strategy depends on intensity, proximity, temporal threat threshold, and past experiences. Hypothalamic corticotropin-releasing factor (CRF) is a major driver of an acute stress response, whereas extrahypothalamic CRF mediates stress-related affective behaviors. In this review, we shift the focus from a monolithic role of CRF as an anxiogenic peptide to comprehensively dissecting contributions of distinct populations of CRF neurons in mediating defensive behaviors. Direct interrogation of CRF neurons of the central amygdala (CeA) or the bed nucleus of the stria terminalis (BNST) show they drive unconditioned defensive responses, such as vigilance and avoidance of open spaces. Although both populations also contribute to learned fear responses in familiar, threatening contexts, CeA-CRF neurons are particularly attuned to the ever-changing environment. Depending on threat intensities, they facilitate discrimination of salient stimuli predicting manageable threats, and prevent their generalization. Finally, hypothalamic CRF neurons mediate initial threat assessment and active defense such as escape to shelter. Overall, these three major populations of CRF neurons demonstrate divergent, yet complementary contributions to the versatile defense system: heightened vigilance, discriminating salient threats, and active escape, representing three legs of the defense tripod. Despite the 'CRF exhaustion' in the field of affective neuroscience, understanding contributions of specific CRF neurons during adaptive defensive behaviors is needed in order to understand the implications of their dysregulation in fear- and anxiety-related psychiatric disorders. This article is part of the Special Issue on "Fear, Anxiety and PTSD".

Water deprivation induces hypoactivity in rats independently of oxytocin receptor signaling at the central amygdala.

Introduction: Vasopressin (AVP) and oxytocin (OXT) are neuropeptides produced by magnocellular neurons (MCNs) of the hypothalamus and secreted through neurohypophysis to defend mammals against dehydration. It was recently demonstrated that MCNs also project to limbic structures, modulating several behavioral responses. Methods and Results: We found that 24 h of water deprivation (WD) or salt loading (SL) did not change exploration or anxiety-like behaviors in the elevated plus maze (EPM) test. However, rats deprived of water for 48 h showed reduced exploration of open field and the closed arms of EPM, indicating hypoactivity during night time. We evaluated mRNA expression of glutamate decarboxylase 1 (Gad1), vesicular glutamate transporter 2 (Slc17a6), AVP (Avpr1a) and OXT (Oxtr) receptors in the lateral habenula (LHb), basolateral (BLA) and central (CeA) amygdala after 48 h of WD or SL. WD, but not SL, increased Oxtr mRNA expression in the CeA. Bilateral pharmacological inhibition of OXTR function in the CeA with the OXTR antagonist L-371,257 was performed to evaluate its possible role in regulating the EPM exploration or water intake induced by WD. The blockade of OXTR in the CeA did not reverse the hypoactivity response in the EPM, nor did it change water intake induced in 48-h water-deprived rats. Discussion: We found that WD modulates exploratory activity in rats, but this response is not mediated by oxytocin receptor signaling to the CeA, despite the upregulated Oxtr mRNA expression in that structure after WD for 48 h.

Central amygdala angiotensin type 1 receptor (Agtr1) expressing neurons contribute to fear extinction.

The renin-angiotensin system (RAS) has been linked to the pathophysiology of posttraumatic stress disorder (PTSD) however, the underlying neurobiological mechanism(s) remain elusive. Here we utilized angiotensin II receptor type 1 (AT1R) transgenic mice combined with neuroanatomical, behavioral, and electrophysiological approaches, to examine the role of the central amygdala (CeA) expressing AT1R neurons in fear and anxiety-related behavior. Within the major amygdala subdivisions, AT1R+ neurons were localized to gamma-aminobutyric acid (GABA) expressing neurons in the lateral division of the central amygdala (CeL), and the majority of them were identified as protein kinase C-δ positive (PKCδ+) neurons. Following CeA-AT1R deletion using cre-expressing lentiviral delivery in AT1R-Flox mice, generalized anxiety and locomotor activity as well as the acquisition of conditioned fear were unaltered while the acquisition of extinction learning, as measured by percent freezing behavior, was significantly enhanced. During electrophysiological recordings of CeL-AT1R+ neurons, the application of angiotensin II (1 µm) increased the amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) and decreased the excitability of CeL-AT1R+ neurons. Overall, these findings demonstrate that CeL-AT1R-expressing neurons play a role in fear extinction, potentially through facilitated CeL-AT1R+ GABAergic inhibition. These results provide new evidence for mechanisms of angiotensinergic neuromodulation of the CeL and its role in fear extinction and may aid in further advancing targeted novel therapies for improving maladaptive fear learning processes associated with PTSD.