Control of goal-directed and inflexible actions by dorsal striatal melanocortin systems, in coordination with the central nucleus of the amygdala.

The dorsomedial striatum (DMS) is associated with flexible goal seeking, as opposed to routinized habits. Whether local mechanisms brake this function, for instance when habits may be adaptive, is incompletely understood. We find that a sub-population of dopamine D1 receptor-containing striatal neurons express the melanocortin-4 receptor (MC4R) for α-melanocyte stimulating hormone. These neurons within the DMS are necessary and sufficient for controlling the capacity of mice to flexibly adjust actions based on the likelihood that they will be rewarded. In investigating MC4R function, we found that it suppresses immediate-early gene levels in the DMS and concurrently, flexible goal seeking. MC4R+ neurons receive input from the central nucleus of the amygdala, and behavioral experiments indicate that they are functionally integrated into an amygdalo-striatal circuit that suppresses action flexibility in favor of routine. Publicly available spatial transcriptomics datasets were analyzed for gene transcript correlates of Mc4r expression across the striatal subregions, revealing considerable co-variation in dorsal structures. This insight led to the discovery that the function of MC4R in the dorsolateral striatum complements that in the DMS, in this case suppressing habit-like behavior. Altogether, our findings suggest that striatal MC4R controls the capacity for goal-directed and inflexible actions alike.

Central amygdala contributes to stimulus facilitation and pre-stimulus vigilance during cerebellar learning.

Our previous studies found that the central amygdala (CeA) modulates cerebellum-dependent eyeblink conditioning (EBC) using muscimol inactivation. We also found that CeA inactivation decreases cerebellar neuronal activity during the conditional stimulus (CS) from the start of training. Based on these findings, we hypothesized that the CeA facilitates CS input to the cerebellum. The current study tested the CS facilitation hypothesis using optogenetic inhibition with archaerhodopsin (Arch) and excitation with channelrhodopsin (ChR2) of the CeA during EBC in male rats. Optogenetic manipulations were administered during the 400 ms tone CS or during a 400 ms pre-CS period. As predicted by the CS facilitation hypothesis CeA inhibition during the CS impaired EBC and CeA excitation during the CS facilitated EBC. Unexpectedly, CeA inhibition just prior to the CS also impaired EBC, while CeA excitation during the pre-CS pathway did not facilitate EBC. The results suggest that the CeA contributes to CS facilitation and vigilance during the pre-CS period. These putative functions of the CeA may be mediated through separate output pathways from the CeA to the cerebellum.

Recruitment of hippocampal and thalamic pathways to the central amygdala in the control of feeding behavior under novelty.

It is adaptive to restrict eating under uncertainty, such as during habituation to novel foods and unfamiliar environments. However, sustained restrictive eating can become maladaptive. Currently, the neural substrates of restrictive eating are poorly understood. Using a model of feeding avoidance under novelty, our recent study identified forebrain activation patterns and found evidence that the central nucleus of the amygdala (CEA) is a core integrating node. The current study analyzed the activity of CEA inputs in male and female rats to determine if specific pathways are recruited during feeding under novelty. Recruitment of direct inputs from the paraventricular nucleus of the thalamus (PVT), the infralimbic cortex (ILA), the agranular insular cortex (AI), the hippocampal ventral field CA1, and the bed nucleus of the stria terminals (BST) was assessed with combined retrograde tract tracing and Fos induction analysis. The study found that during consumption of a novel food in a novel environment, larger number of neurons within the PVTp and the CA1 that send monosynaptic inputs to the CEA were recruited compared to controls that consumed familiar food in a familiar environment. The ILA, AI, and BST inputs to the CEA were similarly recruited across conditions. There were no sex differences in activation of any of the pathways analyzed. These results suggest that the PVTp-CEA and CA1-CEA pathways underlie feeding inhibition during novelty and could be potential sites of malfunction in excessive food avoidance.

Deciphering the role of brainstem glycinergic neurons during startle and prepulse inhibition.

Prepulse inhibition (PPI) of the auditory startle response, a key measure of sensorimotor gating, diminishes with age and is impaired in various neurological conditions. While PPI deficits are often associated with cognitive impairments, their reversal is routinely used in experimental systems for antipsychotic drug screening. Yet, the cellular and circuit-level mechanisms of PPI remain unclear, even under non-pathological conditions. We recently showed that brainstem neurons located in the caudal pontine reticular nucleus (PnC) expressing the glycine transporter type 2 (GlyT2±) receive inputs from the central nucleus of the amygdala (CeA) and contribute to PPI but via an uncharted pathway. Here, using tract-tracing, immunohistochemistry and in vitro optogenetic manipulations coupled to field electrophysiological recordings, we reveal the neuroanatomical distribution of GlyT2± PnC neurons and PnC-projecting CeA glutamatergic neurons and we provide mechanistic insights on how these glutamatergic inputs suppress auditory neurotransmission in PnC sections. Additionally, in vivo experiments using GlyT2-Cre mice confirm that optogenetic activation of GlyT2± PnC neurons enhances PPI and is sufficient to induce PPI in young mice, emphasizing their role. However, in older mice, PPI decline is not further influenced by inhibiting GlyT2± neurons. This study highlights the importance of GlyT2± PnC neurons in PPI and underscores their diminished activity in age-related PPI decline.

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.

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.

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.

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.