Presynaptic sensor and silencer of peptidergic transmission reveal neuropeptides as primary transmitters in pontine fear circuit.

Neurons produce and release neuropeptides to communicate with one another. Despite their importance in brain function, circuit-based mechanisms of peptidergic transmission are poorly understood, primarily due to the lack of tools for monitoring and manipulating neuropeptide release in vivo. Here, we report the development of two genetically encoded tools for investigating peptidergic transmission in behaving mice: a genetically encoded large dense core vesicle (LDCV) sensor that detects presynaptic neuropeptide release and a genetically encoded silencer that specifically degrades neuropeptides inside LDCVs. Using these tools, we show that neuropeptides, not glutamate, encode the unconditioned stimulus in the parabrachial-to-amygdalar threat pathway during Pavlovian threat learning. We also show that neuropeptides play important roles in encoding positive valence and suppressing conditioned threat response in the amygdala-to-parabrachial endogenous opioidergic circuit. These results show that our sensor and silencer for presynaptic peptidergic transmission are reliable tools to investigate neuropeptidergic systems in awake, behaving animals.

Coordinating brain-distributed network activities in memory resistant to extinction.

Certain memories resist extinction to continue invigorating maladaptive actions. The robustness of these memories could depend on their widely distributed implementation across populations of neurons in multiple brain regions. However, how dispersed neuronal activities are collectively organized to underpin a persistent memory-guided behavior remains unknown. To investigate this, we simultaneously monitored the prefrontal cortex, nucleus accumbens, amygdala, hippocampus, and ventral tegmental area (VTA) of the mouse brain from initial recall to post-extinction renewal of a memory involving cocaine experience. We uncover a higher-order pattern of short-lived beta-frequency (15-25 Hz) activities that are transiently coordinated across these networks during memory retrieval. The output of a divergent pathway from upstream VTA glutamatergic neurons, paced by a slower (4-Hz) oscillation, actuates this multi-network beta-band coactivation; its closed-loop phase-informed suppression prevents renewal of cocaine-biased behavior. Binding brain-distributed neural activities in this temporally structured manner may constitute an organizational principle of robust memory expression.

Stress-sensitive neural circuits change the gut microbiome via duodenal glands.

Negative psychological states impact immunity by altering the gut microbiome. However, the relationship between brain states and microbiome composition remains unclear. We show that Brunner's glands in the duodenum couple stress-sensitive brain circuits to bacterial homeostasis. Brunner's glands mediated the enrichment of gut Lactobacillus species in response to vagus nerve stimulation. Cell-specific ablation of the glands markedly suppressed Lactobacilli counts and heightened vulnerability to infection. In the forebrain, we mapped a vagally mediated, polysynaptic circuit connecting the central nucleus of the amygdala to Brunner's glands. Chronic stress suppressed central amygdala activity and phenocopied the effects of gland lesions. Conversely, excitation of either the central amygdala or parasympathetic vagal neurons activated Brunner's glands and reversed the effects of stress on the gut microbiome and immunity. The findings revealed a tractable brain-body mechanism linking psychological states to host defense.

Top-Down Control of Sweet and Bitter Taste in the Mammalian Brain.

Hardwired circuits encoding innate responses have emerged as an essential feature of the mammalian brain. Sweet and bitter evoke opposing predetermined behaviors. Sweet drives appetitive responses and consumption of energy-rich food sources, whereas bitter prevents ingestion of toxic chemicals. Here we identified and characterized the neurons in the brainstem that transmit sweet and bitter signals from the tongue to the cortex. Next we examined how the brain modulates this hardwired circuit to control taste behaviors. We dissect the basis for bitter-evoked suppression of sweet taste and show that the taste cortex and amygdala exert strong positive and negative feedback onto incoming bitter and sweet signals in the brainstem. Finally we demonstrate that blocking the feedback markedly alters responses to ethologically relevant taste stimuli. These results illustrate how hardwired circuits can be finely regulated by top-down control and reveal the neural basis of an indispensable behavioral response for all animals.

Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning.

Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.

Primate Amygdala Neurons Simulate Decision Processes of Social Partners.

By observing their social partners, primates learn about reward values of objects. Here, we show that monkeys' amygdala neurons derive object values from observation and use these values to simulate a partner monkey's decision process. While monkeys alternated making reward-based choices, amygdala neurons encoded object-specific values learned from observation. Dynamic activities converted these values to representations of the recorded monkey's own choices. Surprisingly, the same activity patterns unfolded spontaneously before partner's choices in separate neurons, as if these neurons simulated the partner's decision-making. These "simulation neurons" encoded signatures of mutual-inhibitory decision computation, including value comparisons and value-to-choice conversions, resulting in accurate predictions of partner's choices. Population decoding identified differential contributions of amygdala subnuclei. Biophysical modeling of amygdala circuits showed that simulation neurons emerge naturally from convergence between object-value neurons and self-other neurons. By simulating decision computations during observation, these neurons could allow primates to reconstruct their social partners' mental states.

Sexually Dimorphic Control of Parenting Behavior by the Medial Amygdala.

Social behaviors, including behaviors directed toward young offspring, exhibit striking sex differences. Understanding how these sexually dimorphic behaviors are regulated at the level of circuits and transcriptomes will provide insights into neural mechanisms of sex-specific behaviors. Here, we uncover a sexually dimorphic role of the medial amygdala (MeA) in governing parental and infanticidal behaviors. Contrary to traditional views, activation of GABAergic neurons in the MeA promotes parental behavior in females, while activation of this population in males differentially promotes parental versus infanticidal behavior in an activity-level-dependent manner. Through single-cell transcriptomic analysis, we found that molecular sex differences in the MeA are specifically represented in GABAergic neurons. Collectively, these results establish crucial roles for the MeA as a key node in the neural circuitry underlying pup-directed behaviors and provide important insight into the connection between sex differences across transcriptomes, cells, and circuits in regulating sexually dimorphic behavior.

Limbic Neurons Shape Sex Recognition and Social Behavior in Sexually Naive Males.

Sexually naive animals have to distinguish between the sexes because they show species-typical interactions with males and females without meaningful prior experience. However, central neural pathways in naive mammals that recognize sex of other individuals remain poorly characterized. We examined the role of the principal component of the bed nucleus of stria terminalis (BNSTpr), a limbic center, in social interactions in mice. We find that activity of aromatase-expressing BNSTpr (AB) neurons appears to encode sex of other animals and subsequent displays of mating in sexually naive males. Silencing these neurons in males eliminates preference for female pheromones and abrogates mating success, whereas activating them even transiently promotes male-male mating. Surprisingly, female AB neurons do not appear to control sex recognition, mating, or maternal aggression. In summary, AB neurons represent sex of other animals and govern ensuing social behaviors in sexually naive males.

Stress-Induced Metabolic Disorder in Peripheral CD4+ T Cells Leads to Anxiety-like Behavior.

Physical or mental stress leads to neuroplasticity in the brain and increases the risk of depression and anxiety. Stress exposure causes the dysfunction of peripheral T lymphocytes. However, the pathological role and underlying regulatory mechanism of peripheral T lymphocytes in mood disorders have not been well established. Here, we show that the lack of CD4+ T cells protects mice from stress-induced anxiety-like behavior. Physical stress-induced leukotriene B4 triggers severe mitochondrial fission in CD4+ T cells, which further leads to a variety of behavioral abnormalities including anxiety, depression, and social disorders. Metabolomic profiles and single-cell transcriptome reveal that CD4+ T cell-derived xanthine acts on oligodendrocytes in the left amygdala via adenosine receptor A1. Mitochondrial fission promotes the de novo synthesis of purine via interferon regulatory factor 1 accumulation in CD4+ T cells. Our study implicates a critical link between a purine metabolic disorder in CD4+ T cells and stress-driven anxiety-like behavior.

A Tradeoff in the Neural Code across Regions and Species.

Many evolutionary years separate humans and macaques, and although the amygdala and cingulate cortex evolved to enable emotion and cognition in both, an evident functional gap exists. Although they were traditionally attributed to differential neuroanatomy, functional differences might also arise from coding mechanisms. Here we find that human neurons better utilize information capacity (efficient coding) than macaque neurons in both regions, and that cingulate neurons are more efficient than amygdala neurons in both species. In contrast, we find more overlap in the neural vocabulary and more synchronized activity (robustness coding) in monkeys in both regions and in the amygdala of both species. Our findings demonstrate a tradeoff between robustness and efficiency across species and regions. We suggest that this tradeoff can contribute to differential cognitive functions between species and underlie the complementary roles of the amygdala and the cingulate cortex. In turn, it can contribute to fragility underlying human psychopathologies.

An Amygdala-Hippocampus Subnetwork that Encodes Variation in Human Mood.

Human brain networks that encode variation in mood on naturalistic timescales remain largely unexplored. Here we combine multi-site, semi-chronic, intracranial electroencephalography recordings from the human limbic system with machine learning methods to discover a brain subnetwork that correlates with variation in individual subjects' self-reported mood over days. First we defined the subnetworks that influence intrinsic brain dynamics by identifying regions that showed coordinated changes in spectral coherence. The most common subnetwork, found in 13 of 21 subjects, was characterized by ß-frequency coherence (13-30 Hz) between the amygdala and hippocampus. Increased variability of this subnetwork correlated with worsening mood across these 13 subjects. Moreover, these subjects had significantly higher trait anxiety than the 8 of 21 for whom this amygdala-hippocampus subnetwork was absent. These results demonstrate an approach for extracting network-behavior relationships from complex datasets, and they reveal a conserved subnetwork associated with a psychological trait that significantly influences intrinsic brain dynamics and encodes fluctuations in mood.

Anatomically Defined and Functionally Distinct Dorsal Raphe Serotonin Sub-systems.

The dorsal raphe (DR) constitutes a major serotonergic input to the forebrain and modulates diverse functions and brain states, including mood, anxiety, and sensory and motor functions. Most functional studies to date have treated DR serotonin neurons as a single population. Using viral-genetic methods, we found that subcortical- and cortical-projecting serotonin neurons have distinct cell-body distributions within the DR and differentially co-express a vesicular glutamate transporter. Further, amygdala- and frontal-cortex-projecting DR serotonin neurons have largely complementary whole-brain collateralization patterns, receive biased inputs from presynaptic partners, and exhibit opposite responses to aversive stimuli. Gain- and loss-of-function experiments suggest that amygdala-projecting DR serotonin neurons promote anxiety-like behavior, whereas frontal-cortex-projecting neurons promote active coping in the face of challenge. These results provide compelling evidence that the DR serotonin system contains parallel sub-systems that differ in input and output connectivity, physiological response properties, and behavioral functions.

Corticoamygdala Transfer of Socially Derived Information Gates Observational Learning.

Observational learning is a powerful survival tool allowing individuals to learn about threat-predictive stimuli without directly experiencing the pairing of the predictive cue and punishment. This ability has been linked to the anterior cingulate cortex (ACC) and the basolateral amygdala (BLA). To investigate how information is encoded and transmitted through this circuit, we performed electrophysiological recordings in mice observing a demonstrator mouse undergo associative fear conditioning and found that BLA-projecting ACC (ACC→BLA) neurons preferentially encode socially derived aversive cue information. Inhibition of ACC→BLA alters real-time amygdala representation of the aversive cue during observational conditioning. Selective inhibition of the ACC→BLA projection impaired acquisition, but not expression, of observational fear conditioning. We show that information derived from observation about the aversive value of the cue is transmitted from the ACC to the BLA and that this routing of information is critically instructive for observational fear conditioning. VIDEO ABSTRACT.

Neuronal Representation of Social Information in the Medial Amygdala of Awake Behaving Mice.

The medial amygdala (MeA) plays a critical role in processing species- and sex-specific signals that trigger social and defensive behaviors. However, the principles by which this deep brain structure encodes social information is poorly understood. We used a miniature microscope to image the Ca2+ dynamics of large neural ensembles in awake behaving mice and tracked the responses of MeA neurons over several months. These recordings revealed spatially intermingled subsets of MeA neurons with distinct temporal dynamics. The encoding of social information in the MeA differed between males and females and relied on information from both individual cells and neuronal populations. By performing long-term Ca2+ imaging across different social contexts, we found that sexual experience triggers lasting and sex-specific changes in MeA activity, which, in males, involve signaling by oxytocin. These findings reveal basic principles underlying the brain's representation of social information and its modulation by intrinsic and extrinsic factors.

Combined Social and Spatial Coding in a Descending Projection from the Prefrontal Cortex.

Social behaviors are crucial to all mammals. Although the prelimbic cortex (PL, part of medial prefrontal cortex) has been implicated in social behavior, it is not clear which neurons are relevant or how they contribute. We found that PL contains anatomically and molecularly distinct subpopulations that target three downstream regions that have been implicated in social behavior: the nucleus accumbens (NAc), amygdala, and ventral tegmental area. Activation of NAc-projecting PL neurons (PL-NAc), but not the other subpopulations, decreased the preference for a social target. To determine what information PL-NAc neurons convey, we selectively recorded from them and found that individual neurons were active during social investigation, but only in specific spatial locations. Spatially specific manipulation of these neurons bidirectionally regulated the formation of a social-spatial association. Thus, the unexpected combination of social and spatial information within the PL-NAc may contribute to social behavior by supporting social-spatial learning.

Distinct Hippocampal Pathways Mediate Dissociable Roles of Context in Memory Retrieval.

Memories about sensory experiences are tightly linked to the context in which they were formed. Memory contextualization is fundamental for the selection of appropriate behavioral reactions needed for survival, yet the underlying neuronal circuits are poorly understood. By combining trans-synaptic viral tracing and optogenetic manipulation, we found that the ventral hippocampus (vHC) and the amygdala, two key brain structures encoding context and emotional experiences, interact via multiple parallel pathways. A projection from the vHC to the basal amygdala mediates fear behavior elicited by a conditioned context, whereas a parallel projection from a distinct subset of vHC neurons onto midbrain-projecting neurons in the central amygdala is necessary for context-dependent retrieval of cued fear memories. Our findings demonstrate that two fundamentally distinct roles of context in fear memory retrieval are processed by distinct vHC output pathways, thereby allowing for the formation of robust contextual fear memories while preserving context-dependent behavioral flexibility.

Htr2a-Expressing Cells in the Central Amygdala Control the Hierarchy between Innate and Learned Fear.

Fear is induced by innate and learned mechanisms involving separate pathways. Here, we used an olfactory-mediated innate-fear versus learned-fear paradigm to investigate how these pathways are integrated. Notably, prior presentation of innate-fear stimuli inhibited learned-freezing response, but not vice versa. Whole-brain mapping and pharmacological screening indicated that serotonin-2A receptor (Htr2a)-expressing cells in the central amygdala (CeA) control both innate and learned freezing, but in opposing directions. In vivo fiber photometry analyses in freely moving mice indicated that innate but not learned-fear stimuli suppressed the activity of Htr2a-expressing CeA cells. Artificial inactivation of these cells upregulated innate-freezing response and downregulated learned-freezing response. Thus, Htr2a-expressing CeA cells serve as a hierarchy generator, prioritizing innate fear over learned fear.

Elucidating an Affective Pain Circuit that Creates a Threat Memory.

Animals learn to avoid harmful situations by associating a neutral stimulus with a painful one, resulting in a stable threat memory. In mammals, this form of learning requires the amygdala. Although pain is the main driver of aversive learning, the mechanism that transmits pain signals to the amygdala is not well resolved. Here, we show that neurons expressing calcitonin gene-related peptide (CGRP) in the parabrachial nucleus are critical for relaying pain signals to the central nucleus of amygdala and that this pathway may transduce the affective motivational aspects of pain. Genetic silencing of CGRP neurons blocks pain responses and memory formation, whereas their optogenetic stimulation produces defensive responses and a threat memory. The pain-recipient neurons in the central amygdala expressing CGRP receptors are also critical for establishing a threat memory. The identification of the neural circuit conveying affective pain signals may be pertinent for treating pain conditions with psychiatric comorbidities.

The cortical amygdala consolidates a socially transmitted long-term memory.

Social communication guides decision-making, which is essential for survival. Social transmission of food preference (STFP) is an ecologically relevant memory paradigm in which an animal learns a desirable food odour from another animal in a social context, creating a long-term memory1,2. How food-preference memory is acquired, consolidated and stored is unclear. Here we show that the posteromedial nucleus of the cortical amygdala (COApm) serves as a computational centre in long-term STFP memory consolidation by integrating social and sensory olfactory inputs. Blocking synaptic signalling by the COApm-based circuit selectively abolished STFP memory consolidation without impairing memory acquisition, storage or recall. COApm-mediated STFP memory consolidation depends on synaptic inputs from the accessory olfactory bulb and on synaptic outputs to the anterior olfactory nucleus. STFP memory consolidation requires protein synthesis, suggesting a gene-expression mechanism. Deep single-cell and spatially resolved transcriptomics revealed robust but distinct gene-expression signatures induced by STFP memory formation in the COApm that are consistent with synapse restructuring. Our data thus define a neural circuit for the consolidation of a socially communicated long-term memory, thereby mechanistically distinguishing protein-synthesis-dependent memory consolidation from memory acquisition, storage or retrieval.

Abstract representations emerge in human hippocampal neurons during inference.

Humans have the remarkable cognitive capacity to rapidly adapt to changing environments. Central to this capacity is the ability to form high-level, abstract representations that take advantage of regularities in the world to support generalization1. However, little is known about how these representations are encoded in populations of neurons, how they emerge through learning and how they relate to behaviour2,3. Here we characterized the representational geometry of populations of neurons (single units) recorded in the hippocampus, amygdala, medial frontal cortex and ventral temporal cortex of neurosurgical patients performing an inferential reasoning task. We found that only the neural representations formed in the hippocampus simultaneously encode several task variables in an abstract, or disentangled, format. This representational geometry is uniquely observed after patients learn to perform inference, and consists of disentangled directly observable and discovered latent task variables. Learning to perform inference by trial and error or through verbal instructions led to the formation of hippocampal representations with similar geometric properties. The observed relation between representational format and inference behaviour suggests that abstract and disentangled representational geometries are important for complex cognition.