Sex differences in the Activity of Basolateral Amygdalar Neurons that Project to the Bed Nucleus of the Stria Terminalis and their Role in Anticipatory Anxiety.

Abnormal fear and anxiety can manifest as psychiatric disorders. The bed nucleus of the stria terminalis (BNST) is implicated in sustained responding to, or anticipation of, an aversive event which can be expressed as anticipatory anxiety. The basolateral amygdala (BLA) is also active during anticipatory anxiety and sends projections to the BNST. However, little is known about the role for BLA neurons that project to BNST (BLA-BNST) in anticipatory anxiety in rodents. To address this, we tested if chemogenetic inactivation of the BLA-BNST pathway attenuates sustained conditioned responses produced by anticipation of an aversive stimulus. For comparison, we also assessed BLA-BNST inactivation during social interaction, which is sensitive to unlearned anxiety. We found that BLA-BNST inactivation reduced conditioned sustained freezing and increased social behaviors, but surprisingly, only in males. To determine if sex differences in BLA-BNST neuronal activity contribute to the differences in behavior, we used in vivo and ex vivo electrophysiological approaches. In males, BLA-BNST projection neurons were more active and excitable which coincided with a smaller after-hyperpolarization current (I AHP) compared to other BLA neurons; whereas in females, BLA-BNST neurons were less excitable and had larger I AHP compared to other BLA neurons. These findings demonstrate that activity of BLA-BNST neurons mediates conditioned anticipatory anxiety-like behavior in males. The lack of a role of BLA-BNST in females in this behavior, possibly due to low excitability of these neurons, also highlights the need for caution when generalizing the role of specific neurocircuits in fear and anxiety. Significance Statement :Anxiety disorders disproportionately affect women. This hints towards sex differences within anxiety neurocircuitry, yet most of our understanding is derived from male rodents. Furthermore, debilitating anticipation of adverse events is among the most severe anxiety symptoms, but little is known about anticipatory anxiety neurocircuitry. Here we demonstrated that BLA-BNST activity is required for anticipatory anxiety to a prolonged aversive cue, but only in males. Moreover, BLA-BNST neurons are hypoactive and less excitable in females. These results uncover BLA-BNST as a key component of anticipatory anxiety circuitry, and cellular differences may explain the sex-dependent role of this circuit. Uncovering this disparity provides evidence that the assumed basic circuitry of an anxiety behavior might not readily transpose from males to females.

Repeated stress induces a pro-inflammatory state, increases amygdala neuronal and microglial activation, and causes anxiety in adult male rats.

A link exists between immune function and psychiatric conditions, particularly depressive and anxiety disorders. Psychological stress is a powerful trigger for these disorders and stress influences immune state. However, the nature of peripheral immune changes after stress conflicts across studies, perhaps due to the focus on few measures of pro-inflammatory or anti-inflammatory processes. The basolateral amygdala (BLA) is critical for emotion, and plays an important role in the effects of stress on anxiety. As such, it may be a primary central nervous system (CNS) mediator for the effects of peripheral immune changes on anxiety after stress. Therefore, this study aimed to delineate the influence of stress on peripheral pro-inflammatory and anti-inflammatory aspects, BLA immune activation, and its impact on BLA neuronal activity. To produce a more encompassing view of peripheral immune changes, this study used a less restrictive approach to categorize and group peripheral immune changes. We found that repeated social defeat stress in adult male Sprague-Dawley rats increased the frequencies of mature T-cells positive for intracellular type 2-like cytokine and serum pro-inflammatory cytokines. Principal component analysis and hierarchical clustering was used to guide grouping of T-cells and cytokines, producing unique profiles. Stress shifted the balance towards a specific set that included mostly type 2-like T-cells and pro-inflammatory cytokines. Within the CNS component, repeated stress caused an increase of activated microglia in the BLA, increased anxiety-like behaviors across several assays, and increased BLA neuronal firing in vivo that was prevented by blockade of microglia activation. Because repeated stress can trigger anxiety states by actions in the BLA, and altered immune function can trigger anxiety, these results suggest that repeated stress may trigger anxiety-like behaviors by inducing a pro-inflammatory state in the periphery and the BLA. These results begin to uncover how stress may recruit the immune system to alter the function of brain regions critical to emotion.

NPY2 Receptors Reduce Tonic Action Potential-Independent GABAB Currents in the Basolateral Amygdala.

Although NPY has potent anxiolytic actions within the BLA, selective activation of BLA NPY Y2 receptors (Y2Rs) acutely increases anxiety by an unknown mechanism. Using ex vivo male rat brain slice electrophysiology, we show that the selective Y2R agonist, [ahx5-24]NPY, reduced the frequency of GABAA-mediated mIPSCs in BLA principal neurons (PNs). [ahx5-24]NPY also reduced tonic activation of GABAB receptors (GABABR), which increased PN excitability through inhibition of a tonic, inwardly rectifying potassium current (KIR ). Surprisingly, Y2R-sensitive GABABR currents were action potential-independent, persisting after treatment with TTX. Additionally, the Ca2+-dependent, slow afterhyperpolarizing K+ current (IsAHP ) was enhanced in approximately half of the Y2R-sensitive PNs, possibly from enhanced Ca2+ influx, permitted by reduced GABABR tone. In male and female mice expressing tdTomato in Y2R-mRNA cells (tdT-Y2R mice), immunohistochemistry revealed that BLA somatostatin interneurons express Y2Rs, as do a significant subset of BLA PNs. In tdT-Y2R mice, [ahx5-24]NPY increased excitability and suppressed the KIR in nearly all BLA PNs independent of tdT-Y2R fluorescence, consistent with presynaptic Y2Rs on somatostatin interneurons mediating the above effects. However, only tdT-Y2R-expressing PNs responded to [ahx5-24]NPY with an enhancement of the IsAHP Ultimately, increased PN excitability via acute Y2R activation likely correlates with enhanced BLA output, consistent with reported Y2R-mediated anxiogenesis. Furthermore, we demonstrate the following: (1) a novel mechanism whereby activity-independent GABA release can powerfully dampen BLA neuronal excitability via postsynaptic GABABRs; and (2) that this tonic inhibition can be interrupted by neuromodulation, here by NPY via Y2Rs.SIGNIFICANCE STATEMENT Within the BLA, NPY is potently anxiolytic. However, selective activation of NPY2 receptors (Y2Rs) increases anxiety by an unknown mechanism. We show that activation of BLA Y2Rs decreases tonic GABA release onto BLA principal neurons, probably from Y2R-expressing somatostatin interneurons, some of which coexpress NPY. This increases principal neuron excitability by reducing GABAB receptor (GABABR)-mediated activation of G-protein-coupled, inwardly rectifying K+ currents. Tonic, Y2R-sensitive GABABR currents unexpectedly persisted in the absence of action potential firing, revealing, to our knowledge, the first report of substantial, activity-independent GABABR activation. Ultimately, we provide a plausible explanation for Y2R-mediated anxiogenesis in vivo and describe a novel and modulatable means of damping neuronal excitability.

Sex- and Estrus-Dependent Differences in Rat Basolateral Amygdala.

Depression and anxiety are diagnosed almost twice as often in women, and the symptomology differs in men and women and is sensitive to sex hormones. The basolateral amygdala (BLA) contributes to emotion-related behaviors that differ between males and females and across the reproductive cycle. This hints at sex- or estrus-dependent features of BLA function, about which very little is known. The purpose of this study was to test whether there are sex differences or estrous cyclicity in rat BLA physiology and to determine their mechanistic correlates. We found substantial sex differences in the activity of neurons in lateral nuclei (LAT) and basal nuclei (BA) of the BLA that were associated with greater excitatory synaptic input in females. We also found strong differences in the activity of LAT and BA neurons across the estrous cycle. These differences were associated with a shift in the inhibition-excitation balance such that LAT had relatively greater inhibition during proestrus which paralleled more rapid cued fear extinction. In contrast, BA had relatively greater inhibition during diestrus that paralleled more rapid contextual fear extinction. These results are the first to demonstrate sex differences in BLA neuronal activity and the impact of estrous cyclicity on these measures. The shift between LAT and BA predominance across the estrous cycle provides a simple construct for understanding the effects of the estrous cycle on BLA-dependent behaviors. These results provide a novel framework to understand the cyclicity of emotional memory and highlight the importance of considering ovarian cycle when studying the BLA of females.SIGNIFICANCE STATEMENT There are differences in emotional responses and many psychiatric symptoms between males and females. This may point to sex differences in limbic brain regions. Here we demonstrate sex differences in neuronal activity in one key limbic region, the basolateral amygdala (BLA), whose activity fluctuates across the estrous cycle due to a shift in the balance of inhibition and excitation across two BLA regions, the lateral and basal nuclei. By uncovering this push-pull shift between lateral and basal nuclei, these results help to explain disparate findings about the effects of biological sex and estrous cyclicity on emotion and provide a framework for understanding fluctuations in emotional memory and psychiatric symptoms.

Involvement of neuropeptide Y Y1 receptors in the regulation of neuroendocrine corticotropin-releasing hormone neuronal activity.

The neuroendocrine parvocellular CRH neurons in the paraventricular nucleus (PVN) of the hypothalamus are the main integrators of neural inputs that initiate hypothalamic-pituitary-adrenal (HPA) axis activation. Neuropeptide Y (NPY) expression is prominent within the PVN, and previous reports indicated that NPY stimulates CRH mRNA levels. The purpose of these studies was to examine the participation of NPY receptors in HPA axis activation and determine whether neuroendocrine CRH neurons express NPY receptor immunoreactivity. Infusion of 0.5 nmol NPY into the third ventricle increased plasma corticosterone levels in conscious rats, with the peak of hormone levels occurring 30 min after injection. This increase was prevented by pretreatment with the Y1 receptor antagonist BIBP3226. Immunohistochemistry showed that CRH-immunoreactive neurons coexpressed Y1 receptor immunoreactivity (Y1r-ir) in the PVN, and a majority of these neurons (88.8%) were neuroendocrine as determined by ip injections of FluoroGold. Bilateral infusion of the Y1/Y5 agonist, [leu(31)pro(34)]NPY (110 pmol), into the PVN increased c-Fos and phosphorylated cAMP response element-binding protein expression and elevated plasma corticosterone levels. Increased expression of c-Fos and phosphorylated cAMP response element-binding protein was observed in populations of CRH/Y1r-ir cells. The current findings present a comprehensive study of NPY Y1 receptor distribution and activation with respect to CRH neurons in the PVN. The expression of NPY Y1r-ir by neuroendocrine CRH cells suggests that alterations in NPY release and subsequent activation of NPY Y1 receptors plays an important role in the regulation of the HPA.

Influence of dehydration on the expression of neuropeptide Y Y1 receptors in hypothalamic magnocellular neurons.

Regulation of vasopressin (VP) and oxytocin (OT) secretion involves integration of neural signals from hypothalamic osmoreceptors, ascending catecholaminergic and peptidergic cell groups in the brain stem, and local and autoregulatory afferents. Neuropeptide Y (NPY) is one factor that stimulates the release of VP and OT from the supraoptic (SON) and paraventricular nuclei of the hypothalamus via activation of Y1 receptors (Y1R). The current studies were designed to assess the regulation and distribution of NPY Y1R expression in the SON of male rats that were either given 2% NaCl drinking water (24-72 h) or water deprived (48 h). Subjecting male rats to these conditions resulted in significant increases in both the number of cells expressing Y1R immunoreactivity (ir) and the amount of Y1R protein per cell within the SON. Y1R immunoreactivity was increased in the magnocellular but not medial parvocellular paraventricular nuclei, and Y1R mRNA levels were increased in the SON of salt-loaded rats. Subpopulations of both VP and OT cells in the hypothalamus express Y1R immunoreactivity and a greater percentage of VP-ir cells express Y1R after salt loading. To control for potential effects of dehydration-induced anorexia, a group of euhydrate animals was pair fed with animals consuming 2% NaCl. No detectable change in Y1R expression was observed in the SON of pair-fed animals, even though body weights were significantly lower than controls. These data demonstrate that NPY Y1R gene and protein expression are increased in the SON of salt-loaded and water-deprived animals and provide a mechanism whereby NPY can support VP/OT release during prolonged challenges to fluid homeostasis.

Comparative distribution of neuropeptide Y Y1 and Y5 receptors in the rat brain by using immunohistochemistry.

Neuropeptide Y (NPY) Y1 and Y5 receptor subtypes mediate many of NPY's diverse actions in the central nervous system. The present studies use polyclonal antibodies directed against the Y1 and Y5 receptors to map and compare the relative distribution of these NPY receptor subtypes within the rat brain. Antibody specificity was assessed by using Western analysis, preadsorption of the antibody with peptide, and preimmune serum controls. Immunostaining for the Y1 and Y5 receptor subtypes was present throughout the rostral-caudal aspect of the brain with many regions expressing both subtypes: cerebral cortex, hippocampus, hypothalamus, thalamus, amygdala, and brainstem. Further studies using double-label immunocytochemistry indicate that Y1R immunoreactivity (-ir) and Y5R-ir are colocalized in the cerebral cortex and caudate putamen. Y1 receptor ir was evident in the central amygdala, whereas both Y1- and Y5-immunoreactive cells and fibers were present in the basolateral amygdala. Corresponding with the physiology of NPY in the hypothalamus, both Y1R- and Y5R-ir was present within the paraventricular (PVN), supraoptic, arcuate nuclei, and lateral hypothalamus. In the PVN, Y5R-ir and Y1R-ir were detected in cells and fibers of the parvo- and magnocellular divisions. Intense immunostaining for these receptors was observed within the locus coeruleus, A1-5 and C1-3 nuclei, subnuclei of the trigeminal nerve and nucleus tractus solitarius. These data provide a detailed and comparative mapping of Y1 and Y5 receptor subtypes within cell bodies and nerve fibers in the brain which, together with physiological and electrophysiological studies, provide a better understanding of NPY neural circuitries.