Bidirectional valence coding in amygdala intercalated clusters: A neural substrate for the opponent-process theory of motivation.

Processing emotionally meaningful stimuli and eliciting appropriate valence-specific behavior in response is a critical brain function for survival. Thus, how positive and negative valence are represented in neural circuits and how corresponding neural substrates interact to cooperatively select appropriate behavioral output are fundamental questions. In previous work, we identified that two amygdala intercalated clusters show opposite response selectivity to fear- and anxiety-inducing stimuli - negative valence (Hagihara et al., 2021). Here, we further show that the two clusters also exhibit distinctly different representations of stimuli with positive valence, demonstrating a broader role of the amygdala intercalated system beyond fear and anxiety. Together with the mutually inhibitory connectivity between the two clusters, our findings suggest that they serve as an ideal neural substrate for the integrated processing of valence for the selection of behavioral output.

Adjuvant Analgesics in Acute Pain - Evaluation of Efficacy.

PURPOSE OF THE REVIEW: Acute postoperative pain impacts a significant number of patients and is associated with various complications, such as a higher occurrence of chronic postsurgical pain as well as increased morbidity and mortality. RECENT FINDINGS: Opioids are often used to manage severe pain, but they come with serious adverse effects, such as sedation, respiratory depression, postoperative nausea and vomiting, and impaired bowel function. Therefore, most enhanced recovery after surgery protocols promote multimodal analgesia, which includes adjuvant analgesics, to provide optimal pain control. In this article, we aim to offer a comprehensive review of the contemporary literature on adjuvant analgesics in the management of acute pain, especially in the perioperative setting. Adjuvant analgesics have proven efficacy in treating postoperative pain and reducing need for opioids. While ketamine is an established option for opioid-dependent patients, magnesium and α2-agonists have, in addition to their analgetic effect, the potential to attenuate hemodynamic responses, which make them especially useful in painful laparoscopic procedures. Furthermore, α2-agonists and dexamethasone can extend the analgesic effect of regional anesthesia techniques. However, findings for lidocaine remain inconclusive.

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.

Disynaptic specificity of serial information flow for conditioned fear.

Memory encoding and retrieval rely on specific interactions across multiple brain areas. Although connections between individual brain areas have been extensively studied, the anatomical and functional specificity of neuronal circuit organization underlying information transfer across multiple brain areas remains unclear. Here, we combine transsynaptic viral tracing, optogenetic manipulations, and calcium dynamics recordings to dissect the multisynaptic functional connectivity of the amygdala. We identify a distinct basolateral amygdala (BLA) subpopulation that connects disynaptically to the periaqueductal gray (PAG) via the central amygdala (CeA). This disynaptic pathway serves as a core circuit element necessary for the learning and expression of conditioned fear and exhibits learning-related plasticity. Together, our findings demonstrate the utility of multisynaptic approaches for functional circuit analysis and indicate that disynaptic specificity may be a general feature of neuronal circuit organization.

Compartmentalized dendritic plasticity during associative learning.

Experience-dependent changes in behavior are mediated by long-term functional modifications in brain circuits. Activity-dependent plasticity of synaptic input is a major underlying cellular process. Although we have a detailed understanding of synaptic and dendritic plasticity in vitro, little is known about the functional and plastic properties of active dendrites in behaving animals. Using deep brain two-photon Ca2+ imaging, we investigated how sensory responses in amygdala principal neurons develop upon classical fear conditioning, a form of associative learning. Fear conditioning induced differential plasticity in dendrites and somas regulated by compartment-specific inhibition. Our results indicate that learning-induced plasticity can be uncoupled between soma and dendrites, reflecting distinct synaptic and microcircuit-level mechanisms that increase the computational capacity of amygdala circuits.

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.

Intercalated amygdala clusters orchestrate a switch in fear state.

Adaptive behaviour necessitates the formation of memories for fearful events, but also that these memories can be extinguished. Effective extinction prevents excessive and persistent reactions to perceived threat, as can occur in anxiety and 'trauma- and stressor-related' disorders1. However, although there is evidence that fear learning and extinction are mediated by distinct neural circuits, the nature of the interaction between these circuits remains poorly understood2-6. Here, through a combination of in vivo calcium imaging, functional manipulations, and slice physiology, we show that distinct inhibitory clusters of intercalated neurons (ITCs) in the mouse amygdala exert diametrically opposed roles during the acquisition and retrieval of fear extinction memory. Furthermore, we find that the ITC clusters antagonize one another through mutual synaptic inhibition and differentially access functionally distinct cortical- and midbrain-projecting amygdala output pathways. Our findings show that the balance of activity between ITC clusters represents a unique regulatory motif that orchestrates a distributed neural circuitry, which in turn regulates the switch between high- and low-fear states. These findings suggest that the ITCs have a broader role in a range of amygdala functions and associated brain states that underpins the capacity to adapt to salient environmental demands.

Efficient optogenetic silencing of neurotransmitter release with a mosquito rhodopsin.

Information is carried between brain regions through neurotransmitter release from axonal presynaptic terminals. Understanding the functional roles of defined neuronal projection pathways requires temporally precise manipulation of their activity. However, existing inhibitory optogenetic tools have low efficacy and off-target effects when applied to presynaptic terminals, while chemogenetic tools are difficult to control in space and time. Here, we show that a targeting-enhanced mosquito homolog of the vertebrate encephalopsin (eOPN3) can effectively suppress synaptic transmission through the Gi/o signaling pathway. Brief illumination of presynaptic terminals expressing eOPN3 triggers a lasting suppression of synaptic output that recovers spontaneously within minutes in vitro and in vivo. In freely moving mice, eOPN3-mediated suppression of dopaminergic nigrostriatal afferents induces a reversible ipsiversive rotational bias. We conclude that eOPN3 can be used to selectively suppress neurotransmitter release at presynaptic terminals with high spatiotemporal precision, opening new avenues for functional interrogation of long-range neuronal circuits in vivo.

State-dependent encoding of exploratory behaviour in the amygdala.

The behaviour of an animal is determined by metabolic, emotional and social factors1,2. Depending on its state, an animal will focus on avoiding threats, foraging for food or on social interactions, and will display the appropriate behavioural repertoire3. Moreover, survival and reproduction depend on the ability of an animal to adapt to changes in the environment by prioritizing the appropriate state4. Although these states are thought to be associated with particular functional configurations of large-brain systems5,6, the underlying principles are poorly understood. Here we use deep-brain calcium imaging of mice engaged in spatial or social exploration to investigate how these processes are represented at the neuronal population level in the basolateral amygdala, which is a region of the brain that integrates emotional, social and metabolic information. We demonstrate that the basolateral amygdala encodes engagement in exploratory behaviour by means of two large, functionally anticorrelated ensembles that exhibit slow dynamics. We found that spatial and social exploration were encoded by orthogonal pairs of ensembles with stable and hierarchical allocation of neurons according to the saliency of the stimulus. These findings reveal that the basolateral amygdala acts as a low-dimensional, but context-dependent, hierarchical classifier that encodes state-dependent behavioural repertoires. This computational function may have a fundamental role in the regulation of internal states in health and disease.

Adaptive disinhibitory gating by VIP interneurons permits associative learning.

Learning drives behavioral adaptations necessary for survival. While plasticity of excitatory projection neurons during associative learning has been extensively studied, little is known about the contributions of local interneurons. Using fear conditioning as a model for associative learning, we found that behaviorally relevant, salient stimuli cause learning by tapping into a local microcircuit consisting of precisely connected subtypes of inhibitory interneurons. By employing deep-brain calcium imaging and optogenetics, we demonstrate that vasoactive intestinal peptide (VIP)-expressing interneurons in the basolateral amygdala are activated by aversive events and provide a mandatory disinhibitory signal for associative learning. Notably, VIP interneuron responses during learning are strongly modulated by expectations. Our findings indicate that VIP interneurons are a central component of a dynamic circuit motif that mediates adaptive disinhibitory gating to specifically learn about unexpected, salient events, thereby ensuring appropriate behavioral adaptations.

Structural and Functional Remodeling of Amygdala GABAergic Synapses in Associative Fear Learning.

Associative learning is thought to involve different forms of activity-dependent synaptic plasticity. Although previous studies have mostly focused on learning-related changes occurring at excitatory glutamatergic synapses, we found that associative learning, such as fear conditioning, also entails long-lasting functional and structural plasticity of GABAergic synapses onto pyramidal neurons of the murine basal amygdala. Fear conditioning-mediated structural remodeling of GABAergic synapses was associated with a change in mIPSC kinetics and an increase in the fraction of synaptic benzodiazepine-sensitive (BZD) GABAA receptors containing the α2 subunit without altering the intrasynaptic distribution and overall amount of BZD-GABAA receptors. These structural and functional synaptic changes were partly reversed by extinction training. These findings provide evidence that associative learning, such as Pavlovian fear conditioning and extinction, sculpts inhibitory synapses to regulate inhibition of active neuronal networks, a process that may tune amygdala circuit responses to threats.

Amygdala ensembles encode behavioral states.

Internal states, including affective or homeostatic states, are important behavioral motivators. The amygdala regulates motivated behaviors, yet how distinct states are represented in amygdala circuits is unknown. By longitudinally imaging neural calcium dynamics in freely moving mice across different environments, we identified opponent changes in activity levels of two major, nonoverlapping populations of basal amygdala principal neurons. This population signature does not report global anxiety but predicts switches between exploratory and nonexploratory, defensive states. Moreover, the amygdala separately processes external stimuli and internal states and broadcasts state information via several output pathways to larger brain networks. Our findings extend the concept of thalamocortical "brain-state" coding to include affective and exploratory states and provide an entry point into the state dependency of brain function and behavior in defined circuits.

Encoding of Conditioned Taste Aversion in Cortico-Amygdala Circuits.

Avoidance of potentially toxic food by means of conditioned taste aversion is critical for survival of many animals. However, the underlying neuronal mechanisms are poorly understood. Here, using two-photon calcium imaging of defined gustatory cortex neurons in vivo, we show that conditioned taste aversion dynamically shifts neuronal population coding by stimulus-specific recruitment of neurons that project to the basolateral amygdala.

Author Correction: Transneuronal propagation of mutant huntingtin contributes to non-cell autonomous pathology in neurons.

In the version of this article initially published, the catalog numbers for BoNT A and B were given in the Methods section as T0195 and T5644; the correct numbers are B8776 and B6403. The error has been corrected in the HTML and PDF versions of the article.

New perspectives on central amygdala function.

The central nucleus of the amygdala (CEA) is a striatum-like structure orchestrating a diverse set of adaptive behaviors, including defensive and appetitive responses [1-3]. Studies using anatomical, electrophysiological, imaging and optogenetic approaches revealed that the CEA network consists of recurrent inhibitory circuits comprised of precisely connected functionally and genetically defined cell types that can select and control specific behavioral outputs [3,4,5•,6•,7-9,11,12]. While bivalent functionality of the CEA in adaptive behavior has been clearly demonstrated, we are just beginning to understand to which degree individual CEA circuit elements are functionally segregated or overlapping. Importantly, recent studies seem to suggest that optogenetic manipulations of the same, or overlapping cell populations can give rise to distinct, or sometimes even opposite, behavioral phenotypes [5•,6•,9-12]. In this review, we discuss recent progress in our understanding of how defined CEA circuits can control defensive and appetitive behaviors, and how seemingly contradictory results could point to an integrated concept of CEA function.

Hyperactivity of Anterior Cingulate Cortex Areas 24a/24b Drives Chronic Pain-Induced Anxiodepressive-like Consequences.

Pain associates both sensory and emotional aversive components, and often leads to anxiety and depression when it becomes chronic. Here, we characterized, in a mouse model, the long-term development of these sensory and aversive components as well as anxiodepressive-like consequences of neuropathic pain and determined their electrophysiological impact on the anterior cingulate cortex (ACC, cortical areas 24a/24b). We show that these symptoms of neuropathic pain evolve and recover in different time courses following nerve injury in male mice. In vivo electrophysiological recordings evidence an increased firing rate and bursting activity within the ACC when anxiodepressive-like consequences developed, and this hyperactivity persists beyond the period of mechanical hypersensitivity. Whole-cell patch-clamp recordings also support ACC hyperactivity, as shown by increased excitatory postsynaptic transmission and contribution of NMDA receptors. Optogenetic inhibition of the ACC hyperactivity was sufficient to alleviate the aversive and anxiodepressive-like consequences of neuropathic pain, indicating that these consequences are underpinned by ACC hyperactivity.SIGNIFICANCE STATEMENT Chronic pain is frequently comorbid with mood disorders, such as anxiety and depression. It has been shown that it is possible to model this comorbidity in animal models by taking into consideration the time factor. In this study, we aimed at determining the dynamic of different components and consequences of chronic pain, and correlated them with electrophysiological alterations. By combining electrophysiological, optogenetic, and behavioral analyses in a mouse model of neuropathic pain, we show that the mechanical hypersensitivity, ongoing pain, anxiodepressive consequences, and their recoveries do not necessarily exhibit temporal synchrony during chronic pain processing, and that the hyperactivity of the anterior cingulate cortex is essential for driving the emotional impact of neuropathic pain.

Amygdala Inhibitory Circuits Regulate Associative Fear Conditioning.

Associative memory formation is essential for an animal's survival by ensuring adaptive behavioral responses in an ever-changing environment. This is particularly important under conditions of immediate threats such as in fear learning. One of the key brain regions involved in associative fear learning is the amygdala. The basolateral amygdala is the main entry site for sensory information to the amygdala complex, and local plasticity in excitatory basolateral amygdala principal neurons is considered to be crucial for learning of conditioned fear responses. However, activity and plasticity of excitatory circuits are tightly controlled by local inhibitory interneurons in a spatially and temporally defined manner. In this review, we provide an updated view on how distinct interneuron subtypes in the basolateral amygdala contribute to the acquisition and extinction of conditioned fear memories.

Central amygdala circuits modulate food consumption through a positive-valence mechanism.

The complex behaviors underlying reward seeking and consumption are integral to organism survival. The hypothalamus and mesolimbic dopamine system are key mediators of these behaviors, yet regulation of appetitive and consummatory behaviors outside of these regions is poorly understood. The central nucleus of the amygdala (CeA) has been implicated in feeding and reward, but the neurons and circuit mechanisms that positively regulate these behaviors remain unclear. Here, we defined the neuronal mechanisms by which CeA neurons promote food consumption. Using in vivo activity manipulations and Ca2+ imaging in mice, we found that GABAergic serotonin receptor 2a (Htr2a)-expressing CeA neurons modulate food consumption, promote positive reinforcement and are active in vivo during eating. We demonstrated electrophysiologically, anatomically and behaviorally that intra-CeA and long-range circuit mechanisms underlie these behaviors. Finally, we showed that CeAHtr2a neurons receive inputs from feeding-relevant brain regions. Our results illustrate how defined CeA neural circuits positively regulate food consumption.

Neural ensemble dynamics underlying a long-term associative memory.

The brain's ability to associate different stimuli is vital for long-term memory, but how neural ensembles encode associative memories is unknown. Here we studied how cell ensembles in the basal and lateral amygdala encode associations between conditioned and unconditioned stimuli (CS and US, respectively). Using a miniature fluorescence microscope, we tracked the Ca2+ dynamics of ensembles of amygdalar neurons during fear learning and extinction over 6 days in behaving mice. Fear conditioning induced both up- and down-regulation of individual cells' CS-evoked responses. This bi-directional plasticity mainly occurred after conditioning, and reshaped the neural ensemble representation of the CS to become more similar to the US representation. During extinction training with repetitive CS presentations, the CS representation became more distinctive without reverting to its original form. Throughout the experiments, the strength of the ensemble-encoded CS-US association predicted the level of behavioural conditioning in each mouse. These findings support a supervised learning model in which activation of the US representation guides the transformation of the CS representation.

NMDA-receptor-dependent plasticity in the bed nucleus of the stria terminalis triggers long-term anxiolysis.

Anxiety is controlled by multiple neuronal circuits that share robust and reciprocal connections with the bed nucleus of the stria terminalis (BNST), a key structure controlling negative emotional states. However, it remains unknown how the BNST integrates diverse inputs to modulate anxiety. In this study, we evaluated the contribution of infralimbic cortex (ILCx) and ventral subiculum/CA1 (vSUB/CA1) inputs in regulating BNST activity at the single-cell level. Using trans-synaptic tracing from single-electroporated neurons and in vivo recordings, we show that vSUB/CA1 stimulation promotes opposite forms of in vivo plasticity at the single-cell level in the anteromedial part of the BNST (amBNST). We find that an NMDA-receptor-dependent homosynaptic long-term potentiation is instrumental for anxiolysis. These findings suggest that the vSUB/CA1-driven LTP in the amBNST is involved in eliciting an appropriate response to anxiogenic context and dysfunction of this compensatory mechanism may underlie pathologic anxiety states.