Central Nucleus Of The Amygdala Neurons Research Articles

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

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

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.

Paraventricular nucleus-central amygdala oxytocinergic projection modulates pain-related anxiety-like behaviors in mice.

Anxiety disorders associated with pain are a common health problem. However, the underlying mechanisms remain poorly understood. We aimed to investigate the role of paraventricular nucleus (PVN)-central nucleus of the amygdala (CeA) oxytocinergic projections in anxiety-like behaviors induced by inflammatory pain. After inflammatory pain induction by complete Freund's adjuvant (CFA), mice underwent elevated plus maze, light-dark transition test, and marble burying test to examine the anxiety-like behaviors. Chemogenetic, optogenetic, and fiber photometry recordings were used to modulate and record the activity of the oxytocinergic projections of the PVN-CeA. The key results are as follows: inflammatory pain-induced anxiety-like behaviors in mice accompanied by decreased activity of PVN oxytocin neurons. Chemogenetic activation of PVN oxytocin neurons prevented pain-related anxiety-like behaviors, whereas inhibition of PVN oxytocin neurons induced anxiety-like behaviors in naïve mice. PVN oxytocin neurons projected directly to the CeA, and microinjection of oxytocin into the CeA blocked anxiety-like behaviors. Inflammatory pain also decreased the activity of CeA neurons, and optogenetic activation of PVNoxytocin -CeA circuit prevented anxiety-like behavior in response to inflammatory pain. The results of our study suggest that oxytocin has anti-anxiety effects and provide novel insights into the role of PVNoxytocin -CeA projections in the regulation of anxiety-like behaviors induced by inflammatory pain.

Ethanol Metabolite, Acetate, Increases Excitability of the Central Nucleus of Amygdala Neurons through Activation of NMDA Receptors.

The central nucleus of the amygdala (CeA) is a key brain region involved in emotional and stressor responses due to its many projections to autonomic regulatory centers. It is also a primary site of action from ethanol consumption. However, the influence of active metabolites of ethanol such as acetate on the CeA neural circuitry has yet to be elucidated. Here, we investigated the effect of acetate on CeA neurons with the axon projecting to the rostral ventrolateral medulla (CeA-RVLM), as well as quantified cytosolic calcium responses in primary neuronal cultures. Whole-cell patch-clamp recordings in brain slices containing autonomic CeA-RVLM neurons revealed a dose-dependent increase in neuronal excitability in response to acetate. N-Methyl-d-aspartate receptor (NMDAR) antagonists suppressed the acetate-induced increase in CeA-RVLM neuronal excitability and memantine suppressed the direct activation of NMDAR-dependent inward currents by acetate in brain slices. We observed that acetate increased cytosolic Ca2+ in a time-dependent manner in primary neuronal cell cultures. The acetate enhancement of calcium signaling was abolished by memantine. Computational modeling of acetic acid at NMDAR/NR1 glutamatergic and glycinergic sites suggests potential active site interactions. These findings suggest that within the CeA, acetate is excitatory at least partially through activation of NMDAR, which may underlie the impact of ethanol consumption on autonomic circuitry.

Multimodal mapping of cell types and projections in the central nucleus of the amygdala.

The central nucleus of the amygdala (CEA) is a brain region that integrates external and internal sensory information and executes innate and adaptive behaviors through distinct output pathways. Despite its complex functions, the diversity of molecularly defined neuronal types in the CEA and their contributions to major axonal projection targets have not been examined systematically. Here, we performed single-cell RNA-sequencing (scRNA-seq) to classify molecularly defined cell types in the CEA and identified marker genes to map the location of these neuronal types using expansion-assisted iterative fluorescence in situ hybridization (EASI-FISH). We developed new methods to integrate EASI-FISH with 5-plex retrograde axonal labeling to determine the spatial, morphological, and connectivity properties of ~30,000 molecularly defined CEA neurons. Our study revealed spatiomolecular organization of the CEA, with medial and lateral CEA associated with distinct molecularly defined cell families. We also found a long-range axon projection network from the CEA, where target regions receive inputs from multiple molecularly defined cell types. Axon collateralization was found primarily among projections to hindbrain targets, which are distinct from forebrain projections. This resource reports marker gene combinations for molecularly defined cell types and axon-projection types, which will be useful for selective interrogation of these neuronal populations to study their contributions to the diverse functions of the CEA.

Cellular mechanisms underlying central sensitization in a mouse model of chronic muscle pain.

Chronic pain disorders are often associated with negative emotions, including anxiety and depression. The central nucleus of the amygdala (CeA) has emerged as an integrative hub for nociceptive and affective components during central pain development. Prior adverse injuries are precipitating factors thought to transform nociceptors into a primed state for chronic pain. However, the cellular basis underlying the primed state and the subsequent development of chronic pain remains unknown. Here, we investigated the cellular and synaptic alterations of the CeA in a mouse model of chronic muscle pain. In these mice, local infusion of pregabalin, a clinically approved drug for fibromyalgia and other chronic pain disorders, into the CeA or chemogenetic inactivation of the somatostatin-expressing CeA (CeA-SST) neurons during the priming phase prevented the chronification of pain. Further, electrophysiological recording revealed that the CeA-SST neurons had increased excitatory synaptic drive and enhanced neuronal excitability in the chronic pain states. Finally, either chemogenetic inactivation of the CeA-SST neurons or pharmacological suppression of the nociceptive afferents from the brainstem to the CeA-SST neurons alleviated chronic pain and anxio-depressive symptoms. These data raise the possibility of targeting treatments to CeA-SST neurons to prevent central pain sensitization.

Implication of the Central Nucleus of the Amygdala in Cardiovascular Regulation and Limiting Maximum Exercise Performance During High-intensity Exercise in Rats

To date, the mechanism of central fatigue during high-intensity exercise has remained unclear. Here we elucidate the central mechanisms of cardiovascular regulation during high-intensity exercise with a focus on the hypothesis that amygdala activation acts to limit maximum exercise performance. In the first of three experiments, we probed the involvement of the central nucleus of the amygdala (CeA) in such regulation. Wistar rats were subjected to a maximum exercise test and their total running time and cardiovascular responses were compared before and after bilateral CeA lesions. Next, probing the role of central pathways, we tested whether high-intensity exercise activated neurons in CeA and/or the hypothalamic paraventricular nucleus (PVN) that project to the nucleus tractus solitarius (NTS). Finally, to understand the potential autonomic mechanisms affecting maximum exercise performance, we measured the cardiovascular responses in anesthetized rats to electrical microstimulation of the CeA, PVN, or both. We have found that (1) CeA lesions resulted in an increase in the total exercise time and the time at which an abrupt increase in arterial pressure appeared, indicating an apparent suppression of fatigue. (2) We confirmed that high-intensity exercise activated both the PVN-NTS and CeA-NTS pathways. Moreover, we discovered that (3) while stimulation of the CeA or PVN alone both induced pressor responses, their simultaneous stimulation also increased muscle vascular resistance. These results are evidence that cardiovascular responses during high-intensity exercise are affected by CeA activation, which acts to limit maximum exercise performance, and may implicate autonomic control modulating the PVN-NTS pathway via the CeA.

GABAergic CaMKIIα+ Amygdala Output Attenuates Pain and Modulates Emotional-Motivational Behavior via Parabrachial Inhibition.

Pain and emotion are strongly regulated by neurons in the central nucleus of the amygdala (CeA), a major output of the limbic system; yet, the neuronal signaling pathways underlying this modulation are incompletely understood. Here, we characterized a subpopulation of CeA neurons that express the CaMKIIα gene (CeACAM neurons) and project to the lateral parabrachial nucleus (LPBN), a brainstem region known for its critical role in distributing nociceptive and other aversive signals throughout the brain. In male Sprague Dawley rats, we show that CeACAM-LPBN neurons are GABAergic and mostly express somatostatin. In anaesthetized rats, optogenetic stimulation of CeACAM-LPBN projections inhibited responses of LPBN neurons evoked by electrical activation of Aδ- and C-fiber primary afferents; this inhibition could be blocked by intra-LPBN application of the GABAA receptor antagonist bicuculline. CeACAM-LPBN stimulation also dampened LPBN responses to noxious mechanical, thermal, and chemical stimuli. In behaving rats, optogenetic stimulation of CeACAM-LPBN projections attenuated nocifensive responses to mechanical pressure and radiant heat, disrupted the ability of a noxious shock to drive aversive learning, reduced the defensive behaviors of thigmotaxis and freezing, induced place preference, and promoted food consumption in sated rats. Thus, we suggest that CeACAM-LPBN projections mediate a form of analgesia that is accompanied by a shift toward the positive-appetitive pole of the emotional-motivational continuum. Since the affective state of pain patients strongly influences their prognosis, we envision that recruitment of this pathway in a clinical setting could potentially promote pain resilience and recovery.SIGNIFICANCE STATEMENT Pain and emotion interact on multiple levels of the nervous system. Both positive and negative emotion may have analgesic effects. However, while the neuronal mechanisms underlying "stress-induced analgesia" have been the focus of many studies, the neuronal substrates underlying analgesia accompanied by appetitive emotional-motivational states have received far less attention. The current study focuses on a subpopulation of amygdala neurons that form inhibitory synapses within the brainstem lateral parabrachial nucleus (LPBN). We show that activation of these amygdalo-parabrachial projections inhibits pain processing, while also reducing behaviors related to negative affect and enhancing behaviors related to positive affect. We propose that recruitment of this pathway would benefit pain patients, many of whom suffer from psychological comorbidities such as anxiety and depression.

Sex Differences in CGRP Regulation and Function in the Amygdala in a Rat Model of Neuropathic Pain

The amygdala has emerged as a key player in the emotional response to pain and pain modulation. The lateral and capsular regions of the central nucleus of the amygdala (CeA) represent the “nociceptive amygdala” due to their high content of neurons that process pain-related information. These CeA divisions are the targets of the spino-parabrachio-amygdaloid pain pathway, which is the predominant source of calcitonin gene-related peptide (CGRP) within the amygdala. Changes in lateral and capsular CeA neurons have previously been observed in pain models, and synaptic plasticity in these areas has been linked to pain-related behavior. CGRP has been demonstrated to play an important role in peripheral and spinal mechanisms, and in pain-related amygdala plasticity in male rats in an acute arthritis pain model. However, the role of CGRP in chronic neuropathic pain-related amygdala function and behaviors remains to be determined for both male and female rats. Here we tested the hypothesis that the CGRP1 receptor is involved in neuropathic pain-related amygdala activity, and that blockade of this receptor can inhibit neuropathic pain behaviors in both sexes. CGRP mRNA expression levels in the CeA of male rats were upregulated at the acute stage of the spinal nerve ligation (SNL) model of neuropathic pain, whereas female rats had significantly higher CGRP and CGRP receptor component expression at the chronic stage. A CGRP1 receptor antagonist (CGRP 8-37) administered into the CeA in chronic neuropathic rats reduced mechanical hypersensitivity (von Frey and paw compression tests) in both sexes but showed female-predominant effects on emotional-affective responses (ultrasonic vocalizations) and anxiety-like behaviors (open field test). CGRP 8-37 inhibited the activity of CeA output neurons assessed with calcium imaging in brain slices from chronic neuropathic pain rats. Together, these findings may suggest that CGRP1 receptors in the CeA are involved in neuropathic pain-related amygdala activity and contribute to sensory aspects in both sexes but to emotional-affective pain responses predominantly in females. The sexually dimorphic function of CGRP in the amygdala would make CGRP1 receptors a potential therapeutic target for neuropathic pain relief, particularly in females in chronic pain conditions.

Topographic organization underlies intrinsic and morphological heterogeneity of central amygdala neurons expressing corticotropin‐releasing hormone

The central nucleus of the amygdala (CeA) network consists of a heterogeneous population of inhibitory GABAergic neurons distributed across distinct subregions. While the specific roles for molecularly defined CeA neurons have been extensively studied, our understanding of functional heterogeneity within classes of molecularly distinct CeA neurons remains incomplete. In addition, manipulation of genetically defined CeA neurons has produced inconsistent behavioral results potentially due to broad targeting across CeA subregions. Therefore, elucidating heterogeneity within molecularly defined neurons in subdivisions of the CeA is pivotal for gaining a complete understanding of how CeA circuits function. Here, we used a multifaceted approach involving transgenic reporter mice, brain slice electrophysiology, and neuronal morphology to dissect the heterogeneity of corticotropin‐releasing hormone (CRH) neurons in topographically distinct subregions of the CeA. Our results revealed that intrinsic and morphological properties of CRH‐expressing (CRH+) neurons in the lateral (CeL) and medial (CeM) subdivisions of the CeA were significantly different. We found that CeL‐CRH+ neurons are relatively homogeneous in morphology and firing profile. Conversely, CeM‐CRH+ neurons displayed heterogeneous electrophysiological and morphological phenotypes. Overall, these results show phenotypic differences between CRH+ neurons in CeL and CeM.

Lateral habenula-projecting central amygdala circuits expressing GABA and NPY Y1 receptor modulate binge-like ethanol intake in mice

The central nucleus of the amygdala (CeA) is a critical brain region in the integration of emotional behaviors and is one of the major output areas of the amygdaloid complex. The CeA is composed of GABAergic interneurons and projection neurons which co-express a range of peptides including neuropeptide Y (NPY). Importantly, GABA and NPY signaling, via the NPY Y1 receptor (Y1R), in the CeA modulate binge-like ethanol intake in rodents and these systems undergo neuroplastic alterations following a history of ethanol consumption. Here we assessed the roles of GABAergic and Y1R+ circuits arising from the CeA and innervating the lateral habenula (LHb), a brain region that modulates the aversive properties of ethanol, in modulating binge-like ethanol intake in mice using “drinking in the dark” (DID) procedures. Using an anterograde cre-inducible reporter virus we established the CeA → LHb circuit in male and female vgat-ires-cre and NPY1r-cre mice. Next, we found that chemogenetic silencing of both the GABAergic or Y1R+ CeA → LHb circuit significantly blunted binge-like intake of a 20% ethanol solution but this same procedure failed to alter the consumption of a 3% sucrose solution. Finally, one, 4-day cycle of DID failed to alter basal or effects of ethanol or NPY on inhibitory transmission in Y1R+ CeA → LHb neurons. The present results suggest that blunting GABAergic tone in LHb-projecting CeA neurons may represent a new approach to preventing the development of AUDs. Drugs that target NPY Y1Rs are potential attractive targets.

Regulation of dark period sleep by the Amygdala: A microinjection and optogenetics study

The central nucleus of the amygdala (CNA) projects to brainstem regions that generate and regulate rapid eye movement sleep (REM). We used optogenetics to assess the influence of CNA inputs into reticularis pontis oralis (RPO), pedunculopontine tegmentum (PPT) and nucleus subcoeruleus (SubC) on dark period sleep. We compared these results to effects of microinjections into CNA of the GABAA agonist, muscimol (MUS, inhibition of cell bodies) and tetrodotoxin (TTX, inhibition of cell bodies and fibers of passage). For optogenetics, male Wistar rats received excitatory (AAV5-EF1a-DIO -hChR2(H134R)-EYFP) or inhibitory (AAV-EF1a-DIO-eNpHR3.0-EYFP; DIO-eNpHR3.0) opsins into CNA and AAV5-EF1a-mCherry-IRES-WGA-Cre into RPO, PPT, or SubC. This enabled only CNA neurons synaptically connected to each region to express opsin. Optic cannulae for light delivery into CNA and electrodes for determining sleep were implanted. Sleep was recorded with and without blue or amber light stimulation of CNA. Separate rats received MUS or TTX into CNA prior to recording sleep. Optogenetic activation of CNA neurons projecting to RPO enhanced REM and did not alter non-REM (NREM) whereas activation of CNA neurons projecting to PPT or SubC did not significantly affect sleep. Inhibition of CNA neurons projecting to any region did not significantly alter sleep. TTX inactivation of CNA decreased REM and increased NREM whereas muscimol inactivation did not significantly alter sleep. Thus, the amygdala can regulate decreases and increases in REM, and RPO is important for CNA promotion of REM. Fibers passing through CNA, likely from the basolateral nucleus of the amygdala, also play a role in regulating sleep.

A Distinct Metabolically Defined Central Nucleus Circuit Bidirectionally Controls Anxiety-Related Behaviors.

Anxiety disorders are debilitating psychiatric diseases that affect ∼16% of the world's population. Although it has been proposed that the central nucleus of the amygdala (CeA) plays a role in anxiety, the molecular and circuit mechanisms through which CeA neurons modulate anxiety-related behaviors are largely uncharacterized. Soluble epoxide hydrolase (sEH) is a key enzyme in the metabolism of polyunsaturated fatty acids (PUFAs), and has been shown to play a role in psychiatric disorders. Here, we reported that sEH was enriched in neurons in the CeA and regulated anxiety-related behaviors in adult male mice. Deletion of sEH in CeA neurons but not astrocytes induced anxiety-like behaviors. Mechanistic studies indicated that sEH was required for maintaining the the excitability of sEH positive neurons (sEHCeA neurons) in the CeA. Using chemogenetic manipulations, we found that sEHCeA neurons bidirectionally regulated anxiety-related behaviors. Notably, we identified that sEHCeA neurons directly projected to the bed nucleus of the stria terminalis (BNST; sEHCeA-BNST). Optogenetic activation and inhibition of the sEHCeA-BNST pathway produced anxiolytic and anxiogenic effects, respectively. In summary, our studies reveal a set of molecular and circuit mechanisms of sEHCeA neurons underlying anxiety.SIGNIFICANCE STATEMENT Soluble epoxide hydrolase (sEH), a key enzyme that catalyzes the degradation of EETs, is shown to play a key role in mood disorders. It is well known that sEH is mostly localized in astrocytes in the prefrontal cortex and regulates depressive-like behaviors. Notably, sEH is also expressed in central nucleus of the amygdala (CeA) neurons. While the CeA has been studied for its role in the regulation of anxiety, the molecular and circuit mechanism is quite complex. In the present study, we explored a previously unknown cellular and circuitry mechanism that guides sEHCeA neurons response to anxiety. Our findings reveal a critical role of sEH in the CeA, sEHCeA neurons and CeA-bed nucleus of the stria terminalis (BNST) pathway in regulation of anxiety-related behaviors.

Dissecting a disynaptic central amygdala-parasubthalamic nucleus neural circuit that mediates cholecystokinin-induced eating suppression

ObjectiveCholecystokinin (CCK) plays a critical role in regulating eating and metabolism. Previous studies have mapped a multi-synapse neural pathway from the vagus nerve to the central nucleus of the amygdala (CEA) that mediates the anorexigenic effect of CCK. However, the neural circuit downstream of the CEA is still unknown due to the complexity of the neurons in the CEA. Here we sought to determine this circuit using a novel approach. MethodsIt has been established that a specific population of CEA neurons, marked by protein kinase C-delta (PKC-δ), mediates the anorexigenic effect of CCK by inhibiting other CEA inhibitory neurons. Taking advantage of this circuit, we dissected the neural circuit using a unique approach based on the idea that neurons downstream of the CEA should be disinhibited by CEAPKC-δ+ neurons while being activated by CCK. We also used optogenetic assisted electrophysiology circuit mapping and in vivo chemogenetic manipulation methods to determine the circuit structure and function. ResultsWe found that neurons in the parasubthalamic nucleus (PSTh) are activated by the activation of CEAPKC-δ+ neurons and by the peripheral administration of CCK. We demonstrated that CEAPKC-δ+ neurons inhibit the PSTh-projecting CEA neurons; accordingly, the PSTh neurons can be disynaptically disinhibited or “activated” by CEAPKC-δ+ neurons. Finally, we showed that chemogenetic silencing of the PSTh neurons effectively attenuates the eating suppression induced by CCK. ConclusionsOur results identified a disynaptic CEA-PSTh neural circuit that mediates the anorexigenic effect of CCK and thus provide an important neural mechanism of how CCK suppresses eating.

Anatomical and Functional Characterization of Central Amygdala Glucagon-Like Peptide 1 Receptor Expressing Neurons.

Glucagon-like peptide 1 receptors (GLP-1Rs) are highly expressed in the brain and are responsible for mediating the acute anorexigenic actions of widely prescribed GLP-1R agonists. Neurobiological efforts to localize the hypophagic effects of GLP-1R agonists in the brain have mainly focused on the hypothalamus and hindbrain. In this study, we performed a deep anatomical and neurophysiological characterization of GLP-1Rs in the central nucleus of the amygdala (CeA). At an mRNA level, we found that Glp1r is diffusely coexpressed in known CeA subpopulations like protein kinase c δ (Prkcd), somatostatin (Sst), or tachykinin2 (Tac2). At a cellular level, we used Glp1r-Cre mice and viral Cre-dependent tracing to map the anatomical positions of GLP-1R cells across the rostral-caudal axis of the CeA and in CeA subdivisions. We found that Glp1rCeA cells are highly enriched in the medial subdivision of the CeA (CeM). Using whole cell patch clamp electrophysiology, we found that Glp1rCeA neurons are characterized by the presence of Ih-like currents and resemble a low threshold bursting neuronal subtype in response to hyperpolarizing and depolarizing current injections. We observed sex differences in the magnitude of Ih-like currents and membrane capacitance. At rest, we observed that nearly half of Glp1rCeA neurons are spontaneously active. We observed that active and inactive neurons display significant differences in excitability even when normalized to an identical holding potential. Our data are the first to deeply characterize the pattern of Glp1r in the CeA and study the neurophysiological characteristics of CeA neurons expressing Glp1r. Future studies leveraging these data will be important to understanding the impact of GLP-1R agonists on feeding and motivation.

Chronic restraint stress induces anxiety-like behavior and remodeling of dendritic spines in the central nucleus of the amygdala

Previous studies have shown that the anxiogenic effects of chronic stress do not correlate with dendritic remodeling in the central nucleus of the amygdala (CeA). We analyzed the effect of chronic restraint stress (CRS; 20 min/day for 14 days), relative to control (CTRL) conditions on anxiety-like behavior in the elevated plus maze (EPM) and the open field tests, and dendritic morphology, dendritic spine density and spine type numbers in pyramidal neurons of the CeA. Reversal of CRS-induced effects was explored in animals allowed a 14-day stress-free recovery after treatments. CRS decreased the frequency and time in the open arms and increased the anxiety index in the EPM, and reduced visits and time in the center of the open field. Morphological assays in these animals revealed no effect of CRS on dendritic complexity in CeA neurons; however, a decrease in dendritic spine density together with decreased and increased amounts of mushroom and thin spines, respectively, was detected. Subsequent to a stress-free recovery, a significant reduction in open arm entries together with an increased anxiety index was detected in CRS-exposed animals; open field parameters did not change significantly. A decreased density of total dendritic spines, in parallel with higher and lower numbers of thin and stubby spines, respectively, was observed in CeA neurons. Results suggest that CRS-induced anxiety-like behavior might be accounted for by a reduction in synaptic connectivity of the CeA. This effect, which is long lasting, could mediate the persisting anxiogenic effects of chronic stress after exposure to it has ended.

Input-dependent synaptic suppression by pregabalin in the central amygdala in male mice with inflammatory pain

Pregabalin (PGB) is a synthetic amino acid compound most widely prescribed for chronic peripheral and central neuropathic pain. PGB is a ligand for the α2δ1 subunit of voltage-dependent calcium channels, and its binding reduces neurotransmitter release and thus inhibits synaptic transmission. The central nucleus of the amygdala (CeA) is a kernel site for the enhanced nociception-emotion link in chronic pain. The nociceptive information is conveyed to the CeA via the following two pathways: 1) the pathway arising from the basolateral amygdala (BLA), which carries nociceptive information mediated by the thalamocortical system, and 2) that arising from the external part of the pontine lateral parabrachial nucleus (LPB), that forms the final route of the spino-parabrachio-amygdaloid pathway that conveys nociceptive information directly from the superficial layer of the spinal dorsal horn. We compared the effects of PGB on the excitatory postsynaptic currents of neurons in the right CeA in response to electrical stimulation of BLA and LPB pathways using the whole-cell patch-clamp technique. Inflammatory pain was induced by intraplantar injection of formalin solution at the left hind paw.At eight hours post-formalin, PGB reduced EPSCs amplitude of the BLA-to-CeA synaptic transmission, accompanied by a significant increase in the PPR, suggesting a decreased release probability from the presynaptic terminals. In addition, these effects of PGB were only seen in inflammatory conditions. PGB did not affect the synaptic transmission at the LPB-to-CeA pathway, even in formalin-treated mice. These results suggest PGB improves not simply the aberrantly enhanced nociception but also various pain-associated cognitive and affective consequences in patients with chronic nociplastic pain.

Oxytocin antagonist induced visceral pain and corticotropin-releasing hormone neuronal activation in the central nucleus of the amygdala during colorectal distention in mice

Activation of neurons containing oxytocin and corticotropin-releasing hormone (CRH) in the paraventricular nucleus (PVN) of the hypothalamus, the anterior cingulate cortex (ACC), and the central nucleus of the amygdala (CeA) during colorectal distention (CRD) is likely to play a crucial role in animal models of irritable bowel syndrome (IBS). Earlier studies in rodents showed that the microbiome is involved in social behavior via oxytocin expression in the brain. However, the detailed mechanism of visceral sensation and oxytocin is largely unknown. We tested the following hypotheses: (1) that oxytocin neurons in the PVN are activated by CRD, and (2) that the activation of oxytocin neurons by CRD is related to anxiety-like behavior, visceral perception, and an activation of CRH CeA neurons or ACC neurons. Oxytocin antagonist caused visceral hypersensitivity and anxiety-like behavior. In the PVN, oxytocin neurons were activated by CRD. Noxious CRD activated the CeA, basolateral nucleus of the amygdala (BLA), and ACC. High-dose oxytocin antagonist suppressed ACC activity and activated CRH CeA neurons. These results support our hypotheses. Oxytocin likely regulates CRH CeA neurons in an inhibitory manner and the ACC in an excitatory manner. Further research into the interaction of oxytocin and CRH in visceral pain and anxiety is warranted.

Differential efferent projections of GABAergic neurons in the basolateral and central nucleus of amygdala in mice

The Basolateral amygdala (BLA) and central nucleus of the amygdala (CEA) have been proved to play a key role in the control of anxiety, stress and fear-related behaviors. BLA is a cortex-like complex consisting of both γ-aminobutyric acidergic (GABAergic) interneurons and glutamatergic neurons. The CEA is a striatum-like output of the amygdala, consisting almost exclusively of GABAergic medium spiny neurons. In this study, we explored the morphology and axonal projections of the GABAergic neurons in BLA and CEA, using conditional anterograde axonal tracing, immunohistochemistry, and VGAT-Cre transgenic mice to further understand their functional roles. We found that the axonal projections of GABAergic neurons from the BLA mainly distributed to the forebrain, whilst GABAergic neurons from the CEA distributed to the forebrain, midbrain and brainstem. In the forebrain, the axonal projections of GABAergic neurons from the BLA projected to the anterior olfactory nucleus, the cerebral cortex, the septum, the striatum, the thalamus, the amygdala and the hippocampus. The axonal projections of GABAergic neurons from the CEA distributed to the nuclei of the prefrontal cortex, the bed nucleus of the stria terminalis, the hypothalamus and the thalamus. In the midbrain and brainstem, the axonal projections of GABAergic neurons from the CEA were found in the periaqueductal gray, the substantia nigra, and the locus coeruleus. These data reveal the neuroanatomical basis for exploring the function of GABAergic neurons in the BLA and CEA, particularly during the processing of fear-related behavior.

Phenotyping CCL2 Containing Central Amygdala Neurons Controlling Alcohol Withdrawal-Induced Anxiety.

Chemokines such as chemokine (C-C motif) ligand 2 (CCL2) play a role in several behaviors, including anxiety-like behavior, but whether neurons are an important source of CCL2 for behavior and how neuronal CCL2 may work to affect behavior are still debated. When a herpes simplex virus (HSV) vector was used to knockdown CCL2 mRNA in neurons of the central nucleus of the amygdala (CeA) in rats experiencing multiple withdrawals from low dose ethanol, anxiety-like behavior appeared in the social interaction task. To examine this finding further Fractalkine (CX3CL1), a chemokine that is often found to have an opposing function to CCL2 was measured in these rats. Both alcohol withdrawal and CCL2 knockdown increased the levels of the anti-inflammatory protein CX3CL1. The combination of alcohol withdrawal and CCL2 knockdown decreased CX3CL1 and may alter pro-inflammatory/anti-inflammatory balance, and thus highlights the potential importance of CCL2 and CCL2/CX3CL1 balance in anxiety. To find a mechanism by which neuronal chemokines like CCL2 could affect behavior, retrograde tracing with fluorescent nanobeads was done in two brain regions associated with anxiety the bed nucleus of the stria terminalis (BNST) and the ventral periaqueductal gray (VPAG). These studies identified CeA projection neurons to these brain regions that contain CCL2. To demonstrate that CCL2 can be transported via axons to downstream brain regions, the axonal transport blocker, colchicine, was given and 24 h later, the accumulation of CCL2 in CeA neuronal cell bodies was found. Finally, CCL2 in CeA neurons was localized to the synapse using confocal microscopy with enhanced resolution following deconvolution and electron microscopy, which along with the other evidence suggests that CCL2 may be transported down axons in CeA neurons and released from nerve terminals perhaps into brain regions like the BNST and VPAG to affect behaviors such as anxiety. These results suggest that neurons are an important target for chemokine research related to behavior.