Alteration of serotonin release response in the central nucleus of the amygdala to noxious and non-noxious mechanical stimulation in a neuropathic pain model rat.

Previously, we found that serotonin (5-HT) release in the central nucleus of the amygdala (CeA) of anesthetized rats decreases in response to innocuous stroking of the skin, irrespective of stimulus laterality, but increases in response to noxious pinching applied to a hindlimb contralateral to the 5-HT measurement site. The aim of the present study was to determine whether intra-CeA 5-HT release responses to cutaneous stimulation were altered in an animal model of neuropathic pain induced by ligation of the left L5 spinal nerve. In anesthetized neuropathic pain model rats, stroking of the left hindlimb increased 5-HT release in the CeA, whereas stroking of the right hindlimb decreased it. Meanwhile, pinching of the left hindlimb increased intra-CeA 5-HT release irrespective of stimulus laterality. In conclusion, the present study demonstrated that intra-CeA 5-HT release responses to cutaneous stimulation are altered in an animal model of neuropathic pain.

Top-down control of flight by a non-canonical cortico-amygdala pathway.

Survival requires the selection of appropriate behaviour in response to threats, and dysregulated defensive reactions are associated with psychiatric illnesses such as post-traumatic stress and panic disorder1. Threat-induced behaviours, including freezing and flight, are controlled by neuronal circuits in the central amygdala (CeA)2; however, the source of neuronal excitation of the CeA that contributes to high-intensity defensive responses is unknown. Here we used a combination of neuroanatomical mapping, in vivo calcium imaging, functional manipulations and electrophysiology to characterize a previously unknown projection from the dorsal peduncular (DP) prefrontal cortex to the CeA. DP-to-CeA neurons are glutamatergic and specifically target the medial CeA, the main amygdalar output nucleus mediating conditioned responses to threat. Using a behavioural paradigm that elicits both conditioned freezing and flight, we found that CeA-projecting DP neurons are activated by high-intensity threats in a context-dependent manner. Functional manipulations revealed that the DP-to-CeA pathway is necessary and sufficient for both avoidance behaviour and flight. Furthermore, we found that DP neurons synapse onto neurons within the medial CeA that project to midbrain flight centres. These results elucidate a non-canonical top-down pathway regulating defensive responses.

Immune sensing of food allergens promotes avoidance behaviour.

In addition to its canonical function of protection from pathogens, the immune system can also alter behaviour1,2. The scope and mechanisms of behavioural modifications by the immune system are not yet well understood. Here, using mouse models of food allergy, we show that allergic sensitization drives antigen-specific avoidance behaviour. Allergen ingestion activates brain areas involved in the response to aversive stimuli, including the nucleus of tractus solitarius, parabrachial nucleus and central amygdala. Allergen avoidance requires immunoglobulin E (IgE) antibodies and mast cells but precedes the development of gut allergic inflammation. The ability of allergen-specific IgE and mast cells to promote avoidance requires cysteinyl leukotrienes and growth and differentiation factor 15. Finally, a comparison of C57BL/6 and BALB/c mouse strains revealed a strong effect of the genetic background on the avoidance behaviour. These findings thus point to antigen-specific behavioural modifications that probably evolved to promote niche selection to avoid unfavourable environments.

Plastic and stimulus-specific coding of salient events in the central amygdala.

The central amygdala (CeA) is implicated in a range of mental processes including attention, motivation, memory formation and extinction and in behaviours driven by either aversive or appetitive stimuli1-7. How it participates in these divergent functions remains elusive. Here we show that somatostatin-expressing (Sst+) CeA neurons, which mediate much of CeA functions3,6,8-10, generate experience-dependent and stimulus-specific evaluative signals essential for learning. The population responses of these neurons in mice encode the identities of a wide range of salient stimuli, with the responses of separate subpopulations selectively representing the stimuli that have contrasting valences, sensory modalities or physical properties (for example, shock and water reward). These signals scale with stimulus intensity, undergo pronounced amplification and transformation during learning, and are required for both reward and aversive learning. Notably, these signals contribute to the responses of dopamine neurons to reward and reward prediction error, but not to their responses to aversive stimuli. In line with this, Sst+ CeA neuron outputs to dopamine areas are required for reward learning, but are dispensable for aversive learning. Our results suggest that Sst+ CeA neurons selectively process information about differing salient events for evaluation during learning, supporting the diverse roles of the CeA. In particular, the information for dopamine neurons facilitates reward evaluation.

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.

[Neurons in central nucleus of the amygdala modulates social distance and behavior].

Central nucleus of the amygdala (CeA) has been known as an output region of emotional processing, such as fear emotion. However, recent development of technology allows us to dissect CeA neurons to find the additional role of CeA in feeding behavior, anxiety behavior and pain regulation. On the other hand, neuropeptide B/W receptor 1 expressing neurons (NPBWR1 neurons) have been known to localize in the CeA, but their physiological role is still unclear. In this review, I will introduce the recent findings about the variety of neurons in the CeA and explain the role of NPBWR1 neurons in the regulation of social behavior.

The central extended amygdala guides survival-relevant tradeoffs: Implications for understanding common psychiatric disorders.

To thrive in challenging environments, individuals must pursue rewards while avoiding threats. Extensive studies in animals and humans have identified the central extended amygdala (EAc)-which includes the central nucleus of the amygdala (Ce) and bed nucleus of the stria terminalis (BST)-as a conserved substrate for defensive behavior. These studies suggest the EAc influences defensive responding and assembles fearful and anxious states. This has led to the proliferation of a view that the EAc is fundamentally a defensive substrate. Yet mechanistic work in animals has implicated the EAc in numerous appetitive and consummatory processes, yielding fresh insights into the microcircuitry of survival- and emotion-relevant response selection. Coupled with the EAc's centrality in a conserved network of brain regions that encode multisensory environmental and interoceptive information, these findings suggest a broader role for the EAc as an arbiter of survival- and emotion-relevant tradeoffs for action selection. Determining how the EAc optimizes these tradeoffs promises to improve our understanding of common psychiatric illnesses such as anxiety, depression, alcohol- and substance-use disorders, and anhedonia.

The central nucleus of the amygdala and the construction of defensive modes across the threat-imminence continuum.

In nature, animals display defensive behaviors that reflect the spatiotemporal distance of threats. Laboratory-based paradigms that elicit specific defensive responses in rodents have provided valuable insight into the brain mechanisms that mediate the construction of defensive modes with varying degrees of threat imminence. In this Review, we discuss accumulating evidence that the central nucleus of the amygdala (CeA) plays a key role in this process. Specifically, we propose that the mutually inhibitory circuits of the CeA use a winner-takes-all strategy that supports transitioning across defensive modes and the execution of specific defensive behaviors to previously formed threat associations. Our proposal provides a conceptual framework in which seemingly divergent observations regarding CeA function can be interpreted and identifies various areas of priority for future research.

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.

Encoding of Environmental Cues in Central Amygdala Neurons during Foraging.

To successfully forage in an environment filled with rewards and threats, animals need to rely on familiar structures of their environment that signal food availability. The central amygdala (CeA) is known to mediate a panoply of consummatory and defensive behaviors, yet how specific activity patterns within CeA subpopulations guide optimal choices is not completely understood. In a paradigm of appetitive conditioning in which mice freely forage for food across a continuum of cues, we found that two major subpopulations of CeA neurons, Somatostatin-positive (CeASst) and protein kinase Cδ-positive (CeAPKCδ) neurons, can assign motivational properties to environmental cues. Although the proportion of food responsive cells was higher within CeASst than CeAPKCδ neurons, only the activities of CeAPKCδ, but not CeASst, neurons were required for learning of contextual food cues. Our findings point to a model in which CeAPKCδ neurons may incorporate stimulus salience together with sensory features of the environment to encode memory of the goal location.SIGNIFICANCE STATEMENT The CeA has a very important role in the formation of memories that associate sensory information with aversive or rewarding representation. Here, we used a conditioned place preference paradigm, where freely moving mice learn to associate external cues with food availability, to investigate the roles of CeA neuron subpopulations. We found that CeASst and CeAPKCδ neurons encoded environmental cues during foraging but only the activities of CeAPKCδ neurons were required for learning of contextual food cues.

Overview of the central amygdala role in feeding behaviour.

The neural regulation of feeding behaviour, as an essential factor for survival, is an important research area today. Feeding behaviour and other lifestyle habits play a major role in optimising health and obesity control. Feeding behaviour is physiologically controlled through processes associated with energy and nutrient needs. Different brain nuclei are involved in the neural regulation of feeding behaviours. Therefore, understanding the function of these brain nuclei helps develop feeding control methods. Among important brain nuclei, there is scant literature on the central amygdala (CeA) nucleus and feeding behaviour. The CeA is one of the critical brain regions that play a significant role in various physiological and behavioural responses, such as emotional states, reward processing, energy balance and feeding behaviour. It contains γ-aminobutyric acid neurons. Also, it is the major output region of the amygdaloidal complex. Moreover, the CeA is also involved in multiple molecular and biochemical factors and has extensive connections with other brain nuclei and their neurotransmitters, highlighting its role in feeding behaviour. This review aims to highlight the significance of the CeA nucleus on food consumption by its interaction with the performance of reward, digestive and emotional systems.

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.

Central amygdala micro-circuits mediate fear extinction.

Fear extinction is an adaptive process whereby defensive responses are attenuated following repeated experience of prior fear-related stimuli without harm. The formation of extinction memories involves interactions between various corticolimbic structures, resulting in reduced central amygdala (CEA) output. Recent studies show, however, the CEA is not merely an output relay of fear responses but contains multiple neuronal subpopulations that interact to calibrate levels of fear responding. Here, by integrating behavioural, in vivo electrophysiological, anatomical and optogenetic approaches in mice we demonstrate that fear extinction produces reversible, stimulus- and context-specific changes in neuronal responses to conditioned stimuli in functionally and genetically defined cell types in the lateral (CEl) and medial (CEm) CEA. Moreover, we show these alterations are absent when extinction is deficient and that selective silencing of protein kinase C delta-expressing (PKCδ) CEl neurons impairs fear extinction. Our findings identify CEA inhibitory microcircuits that act as critical elements within the brain networks mediating fear extinction.

Ionic and signaling mechanisms involved in neurotensin-mediated excitation of central amygdala neurons.

Neurotensin (NT) serves as a neuromodulator in the brain where it regulates a variety of physiological functions. Whereas the central amygdala (CeA) expresses NT peptide and NTS1 receptors and application of NT has been shown to excite CeA neurons, the underlying cellular and molecular mechanisms have not been determined. We found that activation of NTS1 receptors increased the neuronal excitability of the lateral nucleus (CeL) of CeA. Both phospholipase Cß (PLCß) and phosphatidylinositol 4,5-bisphosphate (PIP2) depletion were required, whereas intracellular Ca2+ release and PKC were unnecessary for NT-elicited excitation of CeL neurons. NT increased the input resistance and time constants of CeL neurons, suggesting that NT excites CeL neurons by decreasing a membrane conductance. Depressions of the inwardly rectifying K+ (Kir) channels including both the Kir2 subfamily and the GIRK channels were required for NT-elicited excitation of CeL neurons. Activation of NTS1 receptors in the CeL led to GABAergic inhibition of medial nucleus of CeA neurons, suggesting that NT modulates the network activity in the amygdala. Our results may provide a cellular and molecular mechanism to explain the physiological functions of NT in vivo.

Agent-based modeling of the central amygdala and pain using cell-type specific physiological parameters.

The amygdala is a brain area involved in emotional regulation and pain. Over the course of the last 20 years, multiple researchers have studied sensory and motor connections within the amygdala in trying to understand the ultimate role of this structure in pain perception and descending control of pain. A number of investigators have been using cell-type specific manipulations to probe the underlying circuitry of the amygdala. As data have accumulated in this research space, we recognized a critical need for a single framework to integrate these data and evaluate emergent system-level responses. In this manuscript, we present an agent-based computational model of two distinct inhibitory neuron populations in the amygdala, those that express protein kinase C delta (PKCδ) and those that express somatostatin (SOM). We utilized a network of neural links to simulate connectivity and the transmission of inhibitory signals between neurons. Type-specific parameters describing the response of these neurons to noxious stimuli were estimated from published physiological and immunological data as well as our own wet-lab experiments. The model outputs an abstract measure of pain, which is calculated in terms of the cumulative pro-nociceptive and anti-nociceptive activity across neurons in both hemispheres of the amygdala. Results demonstrate the ability of the model to produce changes in pain that are consistent with published studies and highlight the importance of several model parameters. In particular, we found that the relative proportion of PKCδ and SOM neurons within each hemisphere is a key parameter in predicting pain and we explored model predictions for three possible values of this parameter. We compared model predictions of pain to data from our earlier behavioral studies and found areas of similarity as well as distinctions between the data sets. These differences, in particular, suggest a number of wet-lab experiments that could be done in the future.

Dynamic integration and segregation of amygdala subregional functional circuits linking to physiological arousal.

The dynamical organization of brain networks is essential to support human cognition and emotion for rapid adaption to ever-changing environment. As the core nodes of emotion-related brain circuitry, the basolateral amygdala (BLA) and centromedial amygdala (CMA) as two major amygdalar nuclei, are recognized to play distinct roles in affective functions and internal states, via their unique connections with cortical and subcortical structures in rodents. However, little is known how the dynamical organization of emotion-related brain circuitry reflects internal autonomic responses in humans. Using resting-state functional magnetic resonance imaging (fMRI) with K-means clustering approach in a total of 79 young healthy individuals (cohort 1: 42; cohort 2: 37), we identified two distinct states of BLA- and CMA-based intrinsic connectivity patterns, with one state (integration) showing generally stronger BLA- and CMA-based intrinsic connectivity with multiple brain networks, while the other (segregation) exhibiting weaker yet dissociable connectivity patterns. In an independent cohort 2 of fMRI data with concurrent recording of skin conductance, we replicated two similar dynamic states and further found higher skin conductance level in the integration than segregation state. Moreover, machine learning-based Elastic-net regression analyses revealed that time-varying BLA and CMA intrinsic connectivity with distinct network configurations yield higher predictive values for spontaneous fluctuations of skin conductance level in the integration than segregation state. Our findings highlight dynamic functional organization of emotion-related amygdala nuclei circuits and networks and its links to spontaneous autonomic arousal in humans.

Sex differences in fear memory consolidation via Tac2 signaling in mice.

Memory formation is key for brain functioning. Uncovering the memory mechanisms is helping us to better understand neural processes in health and disease. Moreover, more specific treatments for fear-related disorders such as posttraumatic stress disorder and phobias may help to decrease their negative impact on mental health. In this line, the Tachykinin 2 (Tac2) pathway in the central amygdala (CeA) has been shown to be sufficient and necessary for the modulation of fear memory consolidation. CeA-Tac2 antagonism and its pharmacogenetic temporal inhibition impair fear memory in male mice. Surprisingly, we demonstrate here the opposite effect of Tac2 blockade on enhancing fear memory consolidation in females. Furthermore, we show that CeA-testosterone in males, CeA-estradiol in females and Akt/GSK3ß/ß-Catenin signaling both mediate the opposite-sex differential Tac2 pathway regulation of fear memory.

Oxytocin excites BNST interneurons and inhibits BNST output neurons to the central amygdala.

The dorsolateral bed nucleus of the stria terminalis (BNSTDL) has high expression of oxytocin (OT) receptors (OTR), which were shown to facilitate cued fear. However, the role of OTR in the modulation of BNSTDL activity remains elusive. BNSTDL contains GABA-ergic neurons classified based on intrinsic membrane properties into three types. Using in vitro patch-clamp recordings in male rats, we demonstrate that OT selectively excites and increases spontaneous firing rate of Type I BNSTDL neurons. As a consequence, OT increases the frequency, but not amplitude, of spontaneous inhibitory post-synaptic currents (sIPSCs) selectively in Type II neurons, an effect abolished by OTR antagonist or tetrodotoxin, and reduces spontaneous firing rate in these neurons. These results suggest an indirect effect of OT in Type II neurons, which is mediated via OT-induced increase in firing of Type I interneurons. As Type II BNSTDL neurons were shown projecting to the central amygdala (CeA), we also recorded from retrogradely labeled BNST→CeA neurons and we show that OT increases the frequency of sIPSC in these Type II BNST→CeA output neurons. In contrast, in Type III neurons, OT reduces the amplitude, but not frequency, of both sIPSCs and evoked IPSCs via a postsynaptic mechanism without changing their intrinsic excitability. We present a model of fine-tuned modulation of BNSTDL activity by OT, which selectively excites BNSTDL interneurons and inhibits Type II BNST→CeA output neurons. These results suggest that OTR in the BNST might facilitate cued fear by inhibiting the BNST→CeA neurons.

Central amygdala circuits in valence and salience processing.

Behavioral responses to environmental stimuli are dictated by the affective valence of the stimulus, good (positive valence) or bad (negative valence). These stimuli can innately elicit an affective response that promotes approach or avoidance behavior. In addition to innately valenced stimuli, valence can also be assigned to initially neutral stimuli through associative learning. A stimulus of a given valence can vary in salience depending on the strength of the stimulus, the underlying state of the animal, and the context of the stimulus presentation. Salience endows the stimulus with the ability to direct attention and elicit preparatory responses to mount an incentive-based motivated behavior. The central nucleus of the amygdala (CeA) has emerged as an early integration point for valence and salience detection to engage preparatory autonomic responses and behavioral posturing in response to both aversive and appetitive stimuli. There are numerous cell types in the CeA that are involved in valence and salience processing through a variety of connections, and we will review the recent progress that has been made in identifying these circuit elements and their roles in these processes.

Incentive motivation: 'wanting' roles of central amygdala circuitry.

The central nucleus of amygdala (CeA) mediates positively-valenced reward motivation as well as negatively-valenced fear. Optogenetic or neurochemical stimulation of CeA circuitry can generate intense incentive motivation to pursue and consume a paired natural food, sex, or addictive drug reward, and even create maladaptive 'wanting what hurts' such as attraction to a shock rod. Evidence indicates CeA stimulations selectively amplify incentive motivation ('wanting') but not hedonic impact ('liking') of the same reward. Further, valence flips can occur for CeA contributions to motivational salience. That is, CeA stimulation can promote either incentive motivation or fearful motivation, even in the same individual, depending on situation. These findings may carry implications for understanding CeA roles in neuropsychiatric disorders involving aberrant motivational salience, ranging from addiction to paranoia and anxiety disorders.