A line attractor encoding a persistent internal state requires neuropeptide signaling.

Internal states drive survival behaviors, but their neural implementation is poorly understood. Recently, we identified a line attractor in the ventromedial hypothalamus (VMH) that represents a state of aggressiveness. Line attractors can be implemented by recurrent connectivity or neuromodulatory signaling, but evidence for the latter is scant. Here, we demonstrate that neuropeptidergic signaling is necessary for line attractor dynamics in this system by using cell-type-specific CRISPR-Cas9-based gene editing combined with single-cell calcium imaging. Co-disruption of receptors for oxytocin and vasopressin in adult VMH Esr1+ neurons that control aggression diminished attack, reduced persistent neural activity, and eliminated line attractor dynamics while only slightly reducing overall neural activity and sex- or behavior-specific tuning. These data identify a requisite role for neuropeptidergic signaling in implementing a behaviorally relevant line attractor in mammals. Our approach should facilitate mechanistic studies in neuroscience that bridge different levels of biological function and abstraction.

An approximate line attractor in the hypothalamus encodes an aggressive state.

The hypothalamus regulates innate social behaviors, including mating and aggression. These behaviors can be evoked by optogenetic stimulation of specific neuronal subpopulations within MPOA and VMHvl, respectively. Here, we perform dynamical systems modeling of population neuronal activity in these nuclei during social behaviors. In VMHvl, unsupervised analysis identified a dominant dimension of neural activity with a large time constant (>50 s), generating an approximate line attractor in neural state space. Progression of the neural trajectory along this attractor was correlated with an escalation of agonistic behavior, suggesting that it may encode a scalable state of aggressiveness. Consistent with this, individual differences in the magnitude of the integration dimension time constant were strongly correlated with differences in aggressiveness. In contrast, approximate line attractors were not observed in MPOA during mating; instead, neurons with fast dynamics were tuned to specific actions. Thus, different hypothalamic nuclei employ distinct neural population codes to represent similar social behaviors.

A genetically tractable jellyfish model for systems and evolutionary neuroscience.

Jellyfish are radially symmetric organisms without a brain that arose more than 500 million years ago. They achieve organismal behaviors through coordinated interactions between autonomously functioning body parts. Jellyfish neurons have been studied electrophysiologically, but not at the systems level. We introduce Clytia hemisphaerica as a transparent, genetically tractable jellyfish model for systems and evolutionary neuroscience. We generate stable F1 transgenic lines for cell-type-specific conditional ablation and whole-organism GCaMP imaging. Using these tools and computational analyses, we find that an apparently diffuse network of RFamide-expressing umbrellar neurons is functionally subdivided into a series of spatially localized subassemblies whose synchronous activation controls directional food transfer from the tentacles to the mouth. These data reveal an unanticipated degree of structured neural organization in this species. Clytia affords a platform for systems-level studies of neural function, behavior, and evolution within a clade of marine organisms with growing ecological and economic importance.

A circuit logic for sexually shared and dimorphic aggressive behaviors in Drosophila.

Aggression involves both sexually monomorphic and dimorphic actions. How the brain implements these two types of actions is poorly understood. We have identified three cell types that regulate aggression in Drosophila: one type is sexually shared, and the other two are sex specific. Shared common aggression-promoting (CAP) neurons mediate aggressive approach in both sexes, whereas functionally downstream dimorphic but homologous cell types, called male-specific aggression-promoting (MAP) neurons in males and fpC1 in females, control dimorphic attack. These symmetric circuits underlie the divergence of male and female aggressive behaviors, from their monomorphic appetitive/motivational to their dimorphic consummatory phases. The strength of the monomorphic → dimorphic functional connection is increased by social isolation in both sexes, suggesting that it may be a locus for isolation-dependent enhancement of aggression. Together, these findings reveal a circuit logic for the neural control of behaviors that include both sexually monomorphic and dimorphic actions, which may generalize to other organisms.

Multimodal Analysis of Cell Types in a Hypothalamic Node Controlling Social Behavior.

The ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) contains ∼4,000 neurons that project to multiple targets and control innate social behaviors including aggression and mounting. However, the number of cell types in VMHvl and their relationship to connectivity and behavioral function are unknown. We performed single-cell RNA sequencing using two independent platforms-SMART-seq (∼4,500 neurons) and 10x (∼78,000 neurons)-and investigated correspondence between transcriptomic identity and axonal projections or behavioral activation, respectively. Canonical correlation analysis (CCA) identified 17 transcriptomic types (T-types), including several sexually dimorphic clusters, the majority of which were validated by seqFISH. Immediate early gene analysis identified T-types exhibiting preferential responses to intruder males versus females but only rare examples of behavior-specific activation. Unexpectedly, many VMHvl T-types comprise a mixed population of neurons with different projection target preferences. Overall our analysis revealed that, surprisingly, few VMHvl T-types exhibit a clear correspondence with behavior-specific activation and connectivity.

The Neuropeptide Tac2 Controls a Distributed Brain State Induced by Chronic Social Isolation Stress.

Chronic social isolation causes severe psychological effects in humans, but their neural bases remain poorly understood. 2 weeks (but not 24 hr) of social isolation stress (SIS) caused multiple behavioral changes in mice and induced brain-wide upregulation of the neuropeptide tachykinin 2 (Tac2)/neurokinin B (NkB). Systemic administration of an Nk3R antagonist prevented virtually all of the behavioral effects of chronic SIS. Conversely, enhancing NkB expression and release phenocopied SIS in group-housed mice, promoting aggression and converting stimulus-locked defensive behaviors to persistent responses. Multiplexed analysis of Tac2/NkB function in multiple brain areas revealed dissociable, region-specific requirements for both the peptide and its receptor in different SIS-induced behavioral changes. Thus, Tac2 coordinates a pleiotropic brain state caused by SIS via a distributed mode of action. These data reveal the profound effects of prolonged social isolation on brain chemistry and function and suggest potential new therapeutic applications for Nk3R antagonists.

Encoding of female mating dynamics by a hypothalamic line attractor.

Females exhibit complex, dynamic behaviours during mating with variable sexual receptivity depending on hormonal status1-4. However, how their brains encode the dynamics of mating and receptivity remains largely unknown. The ventromedial hypothalamus, ventrolateral subdivision contains oestrogen receptor type 1-positive neurons that control mating receptivity in female mice5,6. Here, unsupervised dynamical system analysis of calcium imaging data from these neurons during mating uncovered a dimension with slow ramping activity, generating a line attractor in neural state space. Neural perturbations in behaving females demonstrated relaxation of population activity back into the attractor. During mating, population activity integrated male cues to ramp up along this attractor, peaking just before ejaculation. Activity in the attractor dimension was positively correlated with the degree of receptivity. Longitudinal imaging revealed that attractor dynamics appear and disappear across the oestrus cycle and are hormone dependent. These observations suggest that a hypothalamic line attractor encodes a persistent, escalating state of female sexual arousal or drive during mating. They also demonstrate that attractors can be reversibly modulated by hormonal status, on a timescale of days.

Causal evidence of a line attractor encoding an affective state.

Continuous attractors are an emergent property of neural population dynamics that have been hypothesized to encode continuous variables such as head direction and eye position1-4. In mammals, direct evidence of neural implementation of a continuous attractor has been hindered by the challenge of targeting perturbations to specific neurons within contributing ensembles2,3. Dynamical systems modelling has revealed that neurons in the hypothalamus exhibit approximate line-attractor dynamics in male mice during aggressive encounters5. We have previously hypothesized that these dynamics may encode the variable intensity and persistence of an aggressive internal state. Here we report that these neurons also showed line-attractor dynamics in head-fixed mice observing aggression6. This allowed us to identify and manipulate line-attractor-contributing neurons using two-photon calcium imaging and holographic optogenetic perturbations. On-manifold perturbations yielded integration of optogenetic stimulation pulses and persistent activity that drove the system along the line attractor, while transient off-manifold perturbations were followed by rapid relaxation back into the attractor. Furthermore, single-cell stimulation and imaging revealed selective functional connectivity among attractor-contributing neurons. Notably, individual differences among mice in line-attractor stability were correlated with the degree of functional connectivity among attractor-contributing neurons. Mechanistic recurrent neural network modelling indicated that dense subnetwork connectivity and slow neurotransmission7 best recapitulate our empirical findings. Our work bridges circuit and manifold levels3, providing causal evidence of continuous attractor dynamics encoding an affective internal state in the mammalian hypothalamus.

A framework for studying emotions across species.

Since the 19th century, there has been disagreement over the fundamental question of whether "emotions" are cause or consequence of their associated behaviors. This question of causation is most directly addressable in genetically tractable model organisms, including invertebrates such as Drosophila. Yet there is ongoing debate about whether such species even have "emotions," as emotions are typically defined with reference to human behavior and neuroanatomy. Here, we argue that emotional behaviors are a class of behaviors that express internal emotion states. These emotion states exhibit certain general functional and adaptive properties that apply across any specific human emotions like fear or anger, as well as across phylogeny. These general properties, which can be thought of as "emotion primitives," can be modeled and studied in evolutionarily distant model organisms, allowing functional dissection of their mechanistic bases and tests of their causal relationships to behavior. More generally, our approach not only aims at better integration of such studies in model organisms with studies of emotion in humans, but also suggests a revision of how emotion should be operationalized within psychology and psychiatry.

Antagonistic control of social versus repetitive self-grooming behaviors by separable amygdala neuronal subsets.

Animals display a range of innate social behaviors that play essential roles in survival and reproduction. While the medial amygdala (MeA) has been implicated in prototypic social behaviors such as aggression, the circuit-level mechanisms controlling such behaviors are not well understood. Using cell-type-specific functional manipulations, we find that distinct neuronal populations in the MeA control different social and asocial behaviors. A GABAergic subpopulation promotes aggression and two other social behaviors, while neighboring glutamatergic neurons promote repetitive self-grooming, an asocial behavior. Moreover, this glutamatergic subpopulation inhibits social interactions independently of its effect to promote self-grooming, while the GABAergic subpopulation inhibits self-grooming, even in a nonsocial context. These data suggest that social versus repetitive asocial behaviors are controlled in an antagonistic manner by inhibitory versus excitatory amygdala subpopulations, respectively. These findings provide a framework for understanding circuit-level mechanisms underlying opponency between innate behaviors, with implications for their perturbation in psychiatric disorders.

Control of stress-induced persistent anxiety by an extra-amygdala septohypothalamic circuit.

The extended amygdala has dominated research on the neural circuitry of fear and anxiety, but the septohippocampal axis also plays an important role. The lateral septum (LS) is thought to suppress fear and anxiety through its outputs to the hypothalamus. However, this structure has not yet been dissected using modern tools. The type 2 CRF receptor (Crfr2) marks a subset of LS neurons whose functional connectivity we have investigated using optogenetics. Crfr2(+) cells include GABAergic projection neurons that connect with the anterior hypothalamus. Surprisingly, we find that these LS outputs enhance stress-induced behavioral measures of anxiety. Furthermore, transient activation of Crfr2(+) neurons promotes, while inhibition suppresses, persistent anxious behaviors. LS Crfr2(+) outputs also positively regulate circulating corticosteroid levels. These data identify a subset of LS projection neurons that promote, rather than suppress, stress-induced behavioral and endocrinological dimensions of persistent anxiety states and provide a cellular point of entry to LS circuitry.

Tachykinin-expressing neurons control male-specific aggressive arousal in Drosophila.

Males of most species are more aggressive than females, but the neural mechanisms underlying this dimorphism are not clear. Here, we identify a neuron and a gene that control the higher level of aggression characteristic of Drosophila melanogaster males. Males, but not females, contain a small cluster of FruM(+) neurons that express the neuropeptide tachykinin (Tk). Activation and silencing of these neurons increased and decreased, respectively, intermale aggression without affecting male-female courtship behavior. Mutations in both Tk and a candidate receptor, Takr86C, suppressed the effect of neuronal activation, whereas overexpression of Tk potentiated it. Tk neuron activation overcame reduced aggressiveness caused by eliminating a variety of sensory or contextual cues, suggesting that it promotes aggressive arousal or motivation. Tachykinin/Substance P has been implicated in aggression in mammals, including humans. Thus, the higher aggressiveness of Drosophila males reflects the sexually dimorphic expression of a neuropeptide that controls agonistic behaviors across phylogeny.

Transformations of neural representations in a social behaviour network.

Mating and aggression are innate social behaviours that are controlled by subcortical circuits in the extended amygdala and hypothalamus1-4. The bed nucleus of the stria terminalis (BNSTpr) is a node that receives input encoding sex-specific olfactory cues from the medial amygdala5,6, and which in turn projects to hypothalamic nuclei that control mating7-9 (medial preoptic area (MPOA)) and aggression9-14 (ventromedial hypothalamus, ventrolateral subdivision (VMHvl)), respectively15. Previous studies have demonstrated that male aromatase-positive BNSTpr neurons are required for mounting and attack, and may identify conspecific sex according to their overall level of activity16. However, neural representations in BNSTpr, their function and their transformations in the hypothalamus have not been characterized. Here we performed calcium imaging17,18 of male BNSTprEsr1 neurons during social behaviours. We identify distinct populations of female- versus male-tuned neurons in BNSTpr, with the former outnumbering the latter by around two to one, similar to the medial amygdala and MPOA but opposite to VMHvl, in which male-tuned neurons predominate6,9,19. Chemogenetic silencing of BNSTprEsr1 neurons while imaging MPOAEsr1 or VMHvlEsr1 neurons in behaving animals showed, unexpectedly, that the male-dominant sex-tuning bias in VMHvl was inverted to female-dominant whereas a switch from sniff- to mount-selective neurons during mating was attenuated in MPOA. Our data also indicate that BNSTprEsr1 neurons are not essential for conspecific sex identification. Rather, they control the transition from appetitive to consummatory phases of male social behaviours by shaping sex- and behaviour-specific neural representations in the hypothalamus.

Sensory representation and detection mechanisms of gut osmolality change.

Ingested food and water stimulate sensory systems in the oropharyngeal and gastrointestinal areas before absorption1,2. These sensory signals modulate brain appetite circuits in a feed-forward manner3-5. Emerging evidence suggests that osmolality sensing in the gut rapidly inhibits thirst neurons upon water intake. Nevertheless, it remains unclear how peripheral sensory neurons detect visceral osmolality changes, and how they modulate thirst. Here we use optical and electrical recording combined with genetic approaches to visualize osmolality responses from sensory ganglion neurons. Gut hypotonic stimuli activate a dedicated vagal population distinct from mechanical-, hypertonic- or nutrient-sensitive neurons. We demonstrate that hypotonic responses are mediated by vagal afferents innervating the hepatic portal area (HPA), through which most water and nutrients are absorbed. Eliminating sensory inputs from this area selectively abolished hypotonic but not mechanical responses in vagal neurons. Recording from forebrain thirst neurons and behavioural analyses show that HPA-derived osmolality signals are required for feed-forward thirst satiation and drinking termination. Notably, HPA-innervating vagal afferents do not sense osmolality itself. Instead, these responses are mediated partly by vasoactive intestinal peptide secreted after water ingestion. Together, our results reveal visceral hypoosmolality as an important vagal sensory modality, and that intestinal osmolality change is translated into hormonal signals to regulate thirst circuit activity through the HPA pathway.

Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing.

Behavior cannot be predicted from a "connectome" because the brain contains a chemical "map" of neuromodulation superimposed upon its synaptic connectivity map. Neuromodulation changes how neural circuits process information in different states, such as hunger or arousal. Here we describe a genetically based method to map, in an unbiased and brain-wide manner, sites of neuromodulation under different conditions in the Drosophila brain. This method, and genetic perturbations, reveal that the well-known effect of hunger to enhance behavioral sensitivity to sugar is mediated, at least in part, by the release of dopamine onto primary gustatory sensory neurons, which enhances sugar-evoked calcium influx. These data reinforce the concept that sensory neurons constitute an important locus for state-dependent gain control of behavior and introduce a methodology that can be extended to other neuromodulators and model organisms.

Distinct hypothalamic control of same- and opposite-sex mounting behaviour in mice.

Animal behaviours that are superficially similar can express different intents in different contexts, but how this flexibility is achieved at the level of neural circuits is not understood. For example, males of many species can exhibit mounting behaviour towards same- or opposite-sex conspecifics1, but it is unclear whether the intent and neural encoding of these behaviours are similar or different. Here we show that female- and male-directed mounting in male laboratory mice are distinguishable by the presence or absence of ultrasonic vocalizations (USVs)2-4, respectively. These and additional behavioural data suggest that most male-directed mounting is aggressive, although in rare cases it can be sexual. We investigated whether USV+ and USV- mounting use the same or distinct hypothalamic neural substrates. Micro-endoscopic imaging of neurons positive for oestrogen receptor 1 (ESR1) in either the medial preoptic area (MPOA) or the ventromedial hypothalamus, ventrolateral subdivision (VMHvl) revealed distinct patterns of neuronal activity during USV+ and USV- mounting, and the type of mounting could be decoded from population activity in either region. Intersectional optogenetic stimulation of MPOA neurons that express ESR1 and vesicular GABA transporter (VGAT) (MPOAESR1∩VGAT neurons) robustly promoted USV+ mounting, and converted male-directed attack to mounting with USVs. By contrast, stimulation of VMHvl neurons that express ESR1 (VMHvlESR1 neurons) promoted USV- mounting, and inhibited the USVs evoked by female urine. Terminal stimulation experiments suggest that these complementary inhibitory effects are mediated by reciprocal projections between the MPOA and VMHvl. Together, these data identify a hypothalamic subpopulation that is genetically enriched for neurons that causally induce a male reproductive behavioural state, and indicate that reproductive and aggressive states are represented by distinct population codes distributed between MPOAESR1 and VMHvlESR1 neurons, respectively. Thus, similar behaviours that express different internal states are encoded by distinct hypothalamic neuronal populations.

Stimulus-specific hypothalamic encoding of a persistent defensive state.

Persistent neural activity in cortical, hippocampal, and motor networks has been described as mediating working memory for transiently encountered stimuli1,2. Internal emotional states, such as fear, also persist following exposure to an inciting stimulus3, but it is unclear whether slow neural dynamics are involved in this process. Neurons in the dorsomedial and central subdivisions of the ventromedial hypothalamus (VMHdm/c) that express the nuclear receptor protein NR5A1 (also known as SF1) are necessary for defensive responses to predators in mice4-7. Optogenetic activation of these neurons, referred to here as VMHdmSF1 neurons, elicits defensive behaviours that outlast stimulation5,8, which suggests the induction of a persistent internal state of fear or anxiety. Here we show that in response to naturalistic threatening stimuli, VMHdmSF1 neurons in mice exhibit activity that lasts for many tens of seconds. This persistent activity was correlated with, and required for, persistent defensive behaviour in an open-field assay, and depended on neurotransmitter release from VMHdmSF1 neurons. Stimulation and calcium imaging in acute slices showed that there is local excitatory connectivity between VMHdmSF1 neurons. Microendoscopic calcium imaging of VMHdmSF1 neurons revealed that persistent activity at the population level reflects heterogeneous dynamics among individual cells. Unexpectedly, distinct but overlapping VMHdmSF1 subpopulations were persistently activated by different modalities of threatening stimulus. Computational modelling suggests that neither recurrent excitation nor slow-acting neuromodulators alone can account for persistent activity that maintains stimulus identity. Our results show that stimulus-specific slow neural dynamics in the hypothalamus, on a time scale orders of magnitude longer than that of working memory in the cortex9,10, contribute to a persistent emotional state.

Sensory neuron-specific GPCR Mrgprs are itch receptors mediating chloroquine-induced pruritus.

The cellular and molecular mechanisms mediating histamine-independent itch in primary sensory neurons are largely unknown. Itch induced by chloroquine (CQ) is a common side effect of this widely used antimalarial drug. Here, we show that Mrgprs, a family of G protein-coupled receptors expressed exclusively in peripheral sensory neurons, function as itch receptors. Mice lacking a cluster of Mrgpr genes display significant deficits in itch induced by CQ but not histamine. CQ directly excites sensory neurons in an Mrgpr-dependent manner. CQ specifically activates mouse MrgprA3 and human MrgprX1. Loss- and gain-of-function studies demonstrate that MrgprA3 is required for CQ responsiveness in mice. Furthermore, MrgprA3-expressing neurons respond to histamine and coexpress gastrin-releasing peptide, a peptide involved in itch sensation, and MrgprC11. Activation of these neurons with the MrgprC11-specific agonist BAM8-22 induces itch in wild-type but not mutant mice. Therefore, Mrgprs may provide molecular access to itch-selective neurons and constitute novel targets for itch therapeutics.

Identification of positionally distinct astrocyte subtypes whose identities are specified by a homeodomain code.

Astrocytes constitute the most abundant cell type in the central nervous system (CNS) and play diverse functional roles, but the ontogenetic origins of this phenotypic diversity are poorly understood. We have investigated whether positional identity, a fundamental organizing principle governing the generation of neuronal subtype diversity, is also relevant to astrocyte diversification. We identified three positionally distinct subtypes of white-matter astrocytes (WMA) in the spinal cord, which can be distinguished by the combinatorial expression of Reelin and Slit1. These astrocyte subtypes derive from progenitor domains expressing the homeodomain transcription factors Pax6 and Nkx6.1, respectively. Loss- and gain-of-function experiments indicate that the positional identity of these astrocyte subtypes is controlled by Pax6 and Nkx6.1 in a combinatorial manner. Thus, positional identity is an organizing principle underlying astrocyte, as well as neuronal, subtype diversification and is controlled by a homeodomain transcriptional code whose elements are reutilized following the specification of neuronal identity earlier in development.