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.

Mechanisms of CD40-dependent cDC1 licensing beyond costimulation.

CD40 signaling in classical type 1 dendritic cells (cDC1s) is required for CD8 T cell-mediated tumor rejection, but the underlying mechanisms are incompletely understood. Here, we identified CD40-induced genes in cDC1s, including Cd70, Tnfsf9, Ptgs2 and Bcl2l1, and examined their contributions to anti-tumor immunity. cDC1-specific inactivation of CD70 and COX-2, and global CD27 inactivation, only partially impaired tumor rejection or tumor-specific CD8 T cell expansion. Loss of 4-1BB, alone or in Cd27-/- mice, did not further impair anti-tumor immunity. However, cDC1-specific CD40 inactivation reduced cDC1 mitochondrial transmembrane potential and increased caspase activation in tumor-draining lymph nodes, reducing migratory cDC1 numbers in vivo. Similar impairments occurred during in vitro antigen presentation by Cd40-/- cDC1s to CD8+ T cells, which were reversed by re-expression of Bcl2l1. Thus, CD40 signaling in cDC1s not only induces costimulatory ligands for CD8+ T cells but also induces Bcl2l1 that sustains cDC1 survival during priming of anti-tumor responses.

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.

Cis interactions in the Irf8 locus regulate stage-dependent enhancer activation.

Individual elements within a superenhancer can act in a cooperative or temporal manner, but the underlying mechanisms remain obscure. We recently identified an Irf8 superenhancer, within which different elements act at distinct stages of type 1 classical dendritic cell (cDC1) development. The +41-kb Irf8 enhancer is required for pre-cDC1 specification, while the +32-kb Irf8 enhancer acts to support subsequent cDC1 maturation. Here, we found that compound heterozygous Δ32/Δ41 mice, lacking the +32- and +41-kb enhancers on different chromosomes, show normal pre-cDC1 specification but, surprisingly, completely lack mature cDC1 development, suggesting cis dependence of the +32-kb enhancer on the +41-kb enhancer. Transcription of the +32-kb Irf8 enhancer-associated long noncoding RNA (lncRNA) Gm39266 is also dependent on the +41-kb enhancer. However, cDC1 development in mice remained intact when Gm39266 transcripts were eliminated by CRISPR/Cas9-mediated deletion of lncRNA promoters and when transcription across the +32-kb enhancer was blocked by premature polyadenylation. We showed that chromatin accessibility and BATF3 binding at the +32-kb enhancer were dependent on a functional +41-kb enhancer located in cis Thus, the +41-kb Irf8 enhancer controls the subsequent activation of the +32-kb Irf8 enhancer in a manner that is independent of associated lncRNA transcription.

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.

High Amount of Transcription Factor IRF8 Engages AP1-IRF Composite Elements in Enhancers to Direct Type 1 Conventional Dendritic Cell Identity.

Development and function of conventional dendritic cell (cDC) subsets, cDC1 and cDC2, depend on transcription factors (TFs) IRF8 and IRF4, respectively. Since IRF8 and IRF4 can each interact with TF BATF3 at AP1-IRF composite elements (AICEs) and with TF PU.1 at Ets-IRF composite elements (EICEs), it is unclear how these factors exert divergent actions. Here, we determined the basis for distinct effects of IRF8 and IRF4 in cDC development. Genes expressed commonly by cDC1 and cDC2 used EICE-dependent enhancers that were redundantly activated by low amounts of either IRF4 or IRF8. By contrast, cDC1-specific genes relied on AICE-dependent enhancers, which required high IRF concentrations, but were activated by either IRF4 or IRF8. IRF8 was specifically required only by a minority of cDC1-specific genes, such as Xcr1, which could distinguish between IRF8 and IRF4 DNA-binding domains. Thus, these results explain how BATF3-dependent Irf8 autoactivation underlies emergence of the cDC1-specific transcriptional program.

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.

Early Release Science of the exoplanet WASP-39b with JWST NIRCam.

Measuring the metallicity and carbon-to-oxygen (C/O) ratio in exoplanet atmospheres is a fundamental step towards constraining the dominant chemical processes at work and, if in equilibrium, revealing planet formation histories. Transmission spectroscopy (for example, refs. 1,2) provides the necessary means by constraining the abundances of oxygen- and carbon-bearing species; however, this requires broad wavelength coverage, moderate spectral resolution and high precision, which, together, are not achievable with previous observatories. Now that JWST has commenced science operations, we are able to observe exoplanets at previously uncharted wavelengths and spectral resolutions. Here we report time-series observations of the transiting exoplanet WASP-39b using JWST's Near InfraRed Camera (NIRCam). The long-wavelength spectroscopic and short-wavelength photometric light curves span 2.0-4.0 micrometres, exhibit minimal systematics and reveal well defined molecular absorption features in the planet's spectrum. Specifically, we detect gaseous water in the atmosphere and place an upper limit on the abundance of methane. The otherwise prominent carbon dioxide feature at 2.8 micrometres is largely masked by water. The best-fit chemical equilibrium models favour an atmospheric metallicity of 1-100-times solar (that is, an enrichment of elements heavier than helium relative to the Sun) and a substellar C/O ratio. The inferred high metallicity and low C/O ratio may indicate significant accretion of solid materials during planet formation (for example, refs. 3,4,) or disequilibrium processes in the upper atmosphere (for example, refs. 5,6).

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.