Neocortical long-range inhibition promotes cortical synchrony and sleep.

Behavioral states such as sleep and wake are highly correlated with specific patterns of rhythmic activity in the cortex. During low arousal states such as slow wave sleep, the cortex is synchronized and dominated by low frequency rhythms coordinated across multiple regions. Although recent evidence suggests that GABAergic inhibitory neurons are key players in cortical state modulation, the in vivo circuit mechanisms coordinating synchronized activity among local and distant neocortical networks are not well understood. Here, we show that somatostatin and chondrolectin co-expressing cells (Sst-Chodl cells), a sparse and unique class of neocortical inhibitory neurons, are selectively active during low arousal states and are largely silent during periods of high arousal. In contrast to other neocortical inhibitory neurons, we show these neurons have long-range axons that project across neocortical areas. Activation of Sst-Chodl cells is sufficient to promote synchronized cortical states characteristic of low arousal, with increased spike co-firing and low frequency brain rhythms, and to alter behavioral states by promoting sleep. Contrary to the prevailing belief that sleep is exclusively driven by subcortical mechanisms, our findings reveal that these long-range inhibitory neurons not only track changes in behavioral state but are sufficient to induce both sleep-like cortical states and sleep behavior, establishing a crucial circuit component in regulating behavioral states.

The parasubthalamic nucleus refeeding ensemble delays feeding initiation and hastens water drinking.

The parasubthalamic nucleus (PSTN) is activated by refeeding after food deprivation and several PSTN subpopulations have been shown to suppress feeding. However, no study to date directly addressed the role of PSTN neurons activated upon food access in the control of ensuing food consumption. Here we identify consumption latency as a sensitive behavioral indicator of PSTN activity, and show that, in hungry mice, the ensemble of refeeding-activated PSTN neurons drastically increases the latency to initiate refeeding with both familiar and a novel, familiar food, but does not control the amount of food consumed. In thirsty mice, this ensemble also delays sucrose consumption but accelerates water consumption, possibly reflecting anticipatory prandial thirst, with again no influence on the amount of fluid consumed. We next sought to identify which subpopulations of PSTN neurons might be driving these latency effects, using cell-type and pathway-specific chemogenetic manipulations. Our results suggest a prominent role of PSTN Tac1 neurons projecting to the central amygdala in the hindrance of feeding initiation. While PSTN Crh neurons also delay the latency of hungry mice to ingest familiar foods, they surprisingly promote the consumption of novel, palatable substances. Furthermore, PSTN Crh neurons projecting to the bed nucleus of the stria terminalis accelerate rehydration in thirsty mice. Our results demonstrate the key role of endogenous PSTN activity in the control of feeding and drinking initiation and delineate specific circuits mediating these effects, which may have relevance for eating disorders.

Salience signaling and stimulus scaling of ventral tegmental area glutamate neuron subtypes.

Ventral tegmental area (VTA) glutamatergic neurons participate in reward, aversion, drug-seeking, and stress. Subsets of VTA VGluT2+ neurons are capable of co-transmitting glutamate and GABA (VGluT2+VGaT+ neurons), transmitting glutamate without GABA (VGluT2+VGaT- neurons), or co-transmitting glutamate and dopamine (VGluT2+TH+ neurons), but whether these molecularly distinct subpopulations show behavior-related differences is not wholly understood. We identified that neuronal activity of each VGluT2+ subpopulation is sensitive to reward value but signaled this in different ways. The phasic maximum activity of VGluT2+VGaT+ neurons increased with sucrose concentration, whereas VGluT2+VGaT- neurons increased maximum and sustained activity with sucrose concentration, and VGluT2+TH+ neurons increased sustained but not maximum activity with sucrose concentration. Additionally, VGluT2+ subpopulations signaled consummatory preferences in different ways. VGluT2+VGaT- neurons and VGluT2+TH+ neurons showed a signaling preference for a behaviorally-preferred fat reward over sucrose, but in temporally-distinct ways. In contrast, VGluT2+VGaT+ neurons uniquely signaled a less behaviorally-preferred sucrose reward compared with fat. Further experiments suggested that VGluT2+VGaT+ consummatory reward-related activity was related to sweetness, partially modulated by hunger state, and not dependent on caloric content or behavioral preference. All VGluT2+ subtypes increased neuronal activity following aversive stimuli but VGluT2+VGaT+ neurons uniquely scaled their magnitude and sustained activity with footshock intensity. Optogenetic activation of VGluT2+VGaT+ neurons during low intensity footshock enhanced fear-related behavior without inducing place preference or aversion. We interpret these data such that VTA glutamatergic subpopulations signal different elements of rewarding and aversive experiences and highlight the unique role of VTA VGluT2+VGaT+ neurons in enhancing the salience of behavioral experiences.

Mimicking opioid analgesia in cortical pain circuits.

The anterior cingulate cortex plays a pivotal role in the cognitive and affective aspects of pain perception. Both endogenous and exogenous opioid signaling within the cingulate mitigate cortical nociception, reducing pain unpleasantness. However, the specific functional and molecular identities of cells mediating opioid analgesia in the cingulate remain elusive. Given the complexity of pain as a sensory and emotional experience, and the richness of ethological pain-related behaviors, we developed a standardized, deep-learning platform for deconstructing the behavior dynamics associated with the affective component of pain in mice-LUPE (Light aUtomated Pain Evaluator). LUPE removes human bias in behavior quantification and accelerated analysis from weeks to hours, which we leveraged to discover that morphine altered attentional and motivational pain behaviors akin to affective analgesia in humans. Through activity-dependent genetics and single-nuclei RNA sequencing, we identified specific ensembles of nociceptive cingulate neuron-types expressing mu-opioid receptors. Tuning receptor expression in these cells bidirectionally modulated morphine analgesia. Moreover, we employed a synthetic opioid receptor promoter-driven approach for cell-type specific optical and chemical genetic viral therapies to mimic morphine's pain-relieving effects in the cingulate, without reinforcement. This approach offers a novel strategy for precision pain management by targeting a key nociceptive cortical circuit with on-demand, non-addictive, and effective analgesia.

Prefrontal cortical dynorphin peptidergic transmission constrains threat-driven behavioral and network states.

Prefrontal cortical (PFC) circuits provide top-down control of threat reactivity. This includes ventromedial PFC (vmPFC) circuitry, which plays a role in suppressing fear-related behavioral states. Dynorphin (Dyn) has been implicated in mediating negative affect and maladaptive behaviors induced by severe threats and is expressed in limbic circuits, including the vmPFC. However, there is a critical knowledge gap in our understanding of how vmPFC Dyn-expressing neurons and Dyn transmission detect threats and regulate expression of defensive behaviors. Here, we demonstrate that Dyn cells are broadly activated by threats and release Dyn locally in the vmPFC to limit passive defensive behaviors. We further demonstrate that vmPFC Dyn-mediated signaling promotes a switch of vmPFC networks to a fear-related state. In conclusion, we reveal a previously unknown role of vmPFC Dyn neurons and Dyn neuropeptidergic transmission in suppressing defensive behaviors in response to threats via state-driven changes in vmPFC networks.

Retrograde endocannabinoid signaling at inhibitory synapses in vivo.

Endocannabinoid (eCB)-mediated suppression of inhibitory synapses has been hypothesized, but this has not yet been demonstrated to occur in vivo because of the difficulty in tracking eCB dynamics and synaptic plasticity during behavior. In mice navigating a linear track, we observed location-specific eCB signaling in hippocampal CA1 place cells, and this was detected both in the postsynaptic membrane and the presynaptic inhibitory axons. All-optical in vivo investigation of synaptic responses revealed that postsynaptic depolarization was followed by a suppression of inhibitory synaptic potentials. Furthermore, interneuron-specific cannabinoid receptor deletion altered place cell tuning. Therefore, rapid, postsynaptic, activity-dependent eCB signaling modulates inhibitory synapses on a timescale of seconds during behavior.

Prefrontal cortical dynorphin peptidergic transmission constrains threat-driven behavioral and network states.

Prefrontal cortical (PFC) circuits provide top-down control of threat reactivity. This includes ventromedial PFC (vmPFC) circuitry, which plays a role in suppressing fear-related behavioral states. Dynorphin (Dyn) has been implicated in mediating negative affect and mal-adaptive behaviors induced by severe threats and is expressed in limbic circuits, including the vmPFC. However, there is a critical knowledge gap in our understanding of how vmPFC Dyn-expressing neurons and Dyn transmission detect threats and regulate expression of defensive behaviors. Here, we demonstrate that Dyn cells are broadly activated by threats and release Dyn locally in the vmPFC to limit passive defensive behaviors. We further demonstrate that vmPFC Dyn-mediated signaling promotes a switch of vmPFC networks to a fear-related state. In conclusion, we reveal a previously unknown role of vmPFC Dyn neurons and Dyn neuropeptidergic transmission in suppressing defensive behaviors in response to threats via state-driven changes in vmPFC networks.

Sexually dimorphic mechanisms of VGLUT-mediated protection from dopaminergic neurodegeneration.

Parkinson's disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. We discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT+ DA neurons in mice and flies identified candidate genes that we functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. We discovered transcription factors modulating dVGLUT-dependent DA neuroprotection and identified dj-1ß as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health.

Human OPRM1 and murine Oprm1 promoter driven viral constructs for genetic access to µ-opioidergic cell types.

With concurrent global epidemics of chronic pain and opioid use disorders, there is a critical need to identify, target and manipulate specific cell populations expressing the mu-opioid receptor (MOR). However, available tools and transgenic models for gaining long-term genetic access to MOR+ neural cell types and circuits involved in modulating pain, analgesia and addiction across species are limited. To address this, we developed a catalog of MOR promoter (MORp) based constructs packaged into adeno-associated viral vectors that drive transgene expression in MOR+ cells. MORp constructs designed from promoter regions upstream of the mouse Oprm1 gene (mMORp) were validated for transduction efficiency and selectivity in endogenous MOR+ neurons in the brain, spinal cord, and periphery of mice, with additional studies revealing robust expression in rats, shrews, and human induced pluripotent stem cell (iPSC)-derived nociceptors. The use of mMORp for in vivo fiber photometry, behavioral chemogenetics, and intersectional genetic strategies is also demonstrated. Lastly, a human designed MORp (hMORp) efficiently transduced macaque cortical OPRM1+ cells. Together, our MORp toolkit provides researchers cell type specific genetic access to target and functionally manipulate mu-opioidergic neurons across a range of vertebrate species and translational models for pain, addiction, and neuropsychiatric disorders.

Optoα1AR activation in astrocytes modulates basal hippocampal synaptic excitation and inhibition in a stimulation-specific manner.

Astrocytes play active roles at synapses and can monitor, respond, and adapt to local synaptic activity. While there is abundant evidence that astrocytes modulate excitatory transmission in the hippocampus, evidence for astrocytic modulation of hippocampal synaptic inhibition remains more limited. Furthermore, to better investigate roles for astrocytes in modulating synaptic transmission, more tools that can selectively activate native G protein signaling pathways in astrocytes with both spatial and temporal precision are needed. Here, we utilized AAV8-GFAP-Optoα1AR-eYFP (Optoα1AR), a viral vector that enables activation of Gq signaling in astrocytes via light-sensitive α1-adrenergic receptors. To determine if stimulating astrocytic Optoα1AR modulates hippocampal synaptic transmission, recordings were made in CA1 pyramidal cells with surrounding astrocytes expressing Optoα1AR, channelrhodopsin (ChR2), or GFP. Both high-frequency (20 Hz, 45-ms light pulses, 5 mW, 5 min) and low-frequency (0.5 Hz, 1-s pulses at increasing 1, 5, and 10 mW intensities, 90 s per intensity) blue light stimulation were tested. 20 Hz Optoα1AR stimulation increased both inhibitory and excitatory postsynaptic current (IPSC and EPSC) frequency, and the effect on miniature IPSCs (mIPSCs) was largely reversible within 20 min. However, low-frequency stimulation of Optoα1AR did not modulate either IPSCs or EPSCs, suggesting that astrocytic Gq -dependent modulation of basal synaptic transmission in the hippocampus is stimulation-dependent. By contrast, low-frequency stimulation of astrocytic ChR2 was effective in increasing both synaptic excitation and inhibition. Together, these data demonstrate that Optoα1AR activation in astrocytes changes basal GABAergic and glutamatergic transmission, but only following high-frequency stimulation, highlighting the importance of temporal dynamics when using optical tools to manipulate astrocyte function.

Unique functional responses differentially map onto genetic subtypes of dopamine neurons.

Dopamine neurons are characterized by their response to unexpected rewards, but they also fire during movement and aversive stimuli. Dopamine neuron diversity has been observed based on molecular expression profiles; however, whether different functions map onto such genetic subtypes remains unclear. In this study, we established that three genetic dopamine neuron subtypes within the substantia nigra pars compacta, characterized by the expression of Slc17a6 (Vglut2), Calb1 and Anxa1, each have a unique set of responses to rewards, aversive stimuli and accelerations and decelerations, and these signaling patterns are highly correlated between somas and axons within subtypes. Remarkably, reward responses were almost entirely absent in the Anxa1+ subtype, which instead displayed acceleration-correlated signaling. Our findings establish a connection between functional and genetic dopamine neuron subtypes and demonstrate that molecular expression patterns can serve as a common framework to dissect dopaminergic functions.

Structural basis for ion selectivity in potassium-selective channelrhodopsins.

KCR channelrhodopsins (K+-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K+ selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K+ selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K+ selectivity; rather than forming the symmetrical filter of canonical K+ channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K+ selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K+ selectivity also provides a framework for next-generation optogenetics.

Orbitofrontal cortex control of striatum leads economic decision-making.

Animals must continually evaluate stimuli in their environment to decide which opportunities to pursue, and in many cases these decisions can be understood in fundamentally economic terms. Although several brain regions have been individually implicated in these processes, the brain-wide mechanisms relating these regions in decision-making are unclear. Using an economic decision-making task adapted for rats, we find that neural activity in both of two connected brain regions, the ventrolateral orbitofrontal cortex (OFC) and the dorsomedial striatum (DMS), was required for economic decision-making. Relevant neural activity in both brain regions was strikingly similar, dominated by the spatial features of the decision-making process. However, the neural encoding of choice direction in OFC preceded that of DMS, and this temporal relationship was strongly correlated with choice accuracy. Furthermore, activity specifically in the OFC projection to the DMS was required for appropriate economic decision-making. These results demonstrate that choice information in the OFC is relayed to the DMS to lead accurate economic decision-making.

Genetically targeted chemical assembly of polymers specifically localized extracellularly to surface membranes of living neurons.

Multicellular biological systems, particularly living neural networks, exhibit highly complex organization properties that pose difficulties for building cell-specific biocompatible interfaces. We previously developed an approach to genetically program cells to assemble structures that modify electrical properties of neurons in situ, opening up the possibility of building minimally invasive cell-specific structures and interfaces. However, the efficiency and biocompatibility of this approach were challenged by limited membrane targeting of the constructed materials. Here, we design a method for highly localized expression of enzymes targeted to the plasma membrane of primary neurons, with minimal intracellular retention. Next, we show that polymers synthesized in situ by this approach form dense extracellular clusters selectively on the targeted cell membrane and that neurons remain viable after polymerization. Last, we show generalizability of this method across a range of design strategies. This platform can be readily extended to incorporate a broad diversity of materials onto specific cell membranes within tissues and may further enable next-generation biological interfaces.

Monosynaptic inputs to ventral tegmental area glutamate and GABA co-transmitting neurons.

A unique population of ventral tegmental area (VTA) neurons co-transmits glutamate and GABA as well as functionally signals rewarding and aversive outcomes. However, the circuit inputs to VTA VGluT2+VGaT+ neurons are unknown, limiting our understanding of the functional capabilities of these neurons. To identify the inputs to VTA VGluT2+VGaT+ neurons, we coupled monosynaptic rabies tracing with intersectional genetic targeting of VTA VGluT2+VGaT+ neurons in mice. We found that VTA VGluT2+VGaT+ neurons received diverse brain-wide inputs. The largest numbers of monosynaptic inputs to VTA VGluT2+VGaT+ neurons were from superior colliculus, lateral hypothalamus, midbrain reticular nucleus, and periaqueductal gray, whereas the densest inputs relative to brain region volume were from dorsal raphe nucleus, lateral habenula, and ventral tegmental area. Based on these and prior data, we hypothesized that lateral hypothalamus and superior colliculus inputs were glutamatergic neurons. Optical activation of glutamatergic lateral hypothalamus neurons robustly activated VTA VGluT2+VGaT+ neurons regardless of stimulation frequency and resulted in flee-like ambulatory behavior. In contrast, optical activation of glutamatergic superior colliculus neurons activated VTA VGluT2+VGaT+ neurons for a brief period of time at high stimulation frequency and resulted in head rotation and arrested ambulatory behavior (freezing). For both pathways, behaviors induced by stimulation were uncorrelated with VTA VGluT2+VGaT+ neuron activity. However, stimulation of glutamatergic lateral hypothalamus neurons, but not glutamatergic superior colliculus neurons, was associated with VTA VGluT2+VGaT+ footshock-induced activity. We interpret these results such that inputs to VTA VGluT2+VGaT+ neurons may integrate diverse signals related to the detection and processing of motivationally-salient outcomes. Further, VTA VGluT2+VGaT+ neurons may signal threat-related outcomes, possibly via input from lateral hypothalamus glutamate neurons, but not threat-induced behavioral kinematics.

Cardiogenic control of affective behavioural state.

Emotional states influence bodily physiology, as exemplified in the top-down process by which anxiety causes faster beating of the heart1-3. However, whether an increased heart rate might itself induce anxiety or fear responses is unclear3-8. Physiological theories of emotion, proposed over a century ago, have considered that in general, there could be an important and even dominant flow of information from the body to the brain9. Here, to formally test this idea, we developed a noninvasive optogenetic pacemaker for precise, cell-type-specific control of cardiac rhythms of up to 900 beats per minute in freely moving mice, enabled by a wearable micro-LED harness and the systemic viral delivery of a potent pump-like channelrhodopsin. We found that optically evoked tachycardia potently enhanced anxiety-like behaviour, but crucially only in risky contexts, indicating that both central (brain) and peripheral (body) processes may be involved in the development of emotional states. To identify potential mechanisms, we used whole-brain activity screening and electrophysiology to find brain regions that were activated by imposed cardiac rhythms. We identified the posterior insular cortex as a potential mediator of bottom-up cardiac interoceptive processing, and found that optogenetic inhibition of this brain region attenuated the anxiety-like behaviour that was induced by optical cardiac pacing. Together, these findings reveal that cells of both the body and the brain must be considered together to understand the origins of emotional or affective states. More broadly, our results define a generalizable approach for noninvasive, temporally precise functional investigations of joint organism-wide interactions among targeted cells during behaviour.

All-optical physiology resolves a synaptic basis for behavioral timescale plasticity.

Learning has been associated with modifications of synaptic and circuit properties, but the precise changes storing information in mammals have remained largely unclear. We combined genetically targeted voltage imaging with targeted optogenetic activation and silencing of pre- and post-synaptic neurons to study the mechanisms underlying hippocampal behavioral timescale plasticity. In mice navigating a virtual-reality environment, targeted optogenetic activation of individual CA1 cells at specific places induced stable representations of these places in the targeted cells. Optical elicitation, recording, and modulation of synaptic transmission in behaving mice revealed that activity in presynaptic CA2/3 cells was required for the induction of plasticity in CA1 and, furthermore, that during induction of these place fields in single CA1 cells, synaptic input from CA2/3 onto these same cells was potentiated. These results reveal synaptic implementation of hippocampal behavioral timescale plasticity and define a methodology to resolve synaptic plasticity during learning and memory in behaving mammals.

Activity of a direct VTA to ventral pallidum GABA pathway encodes unconditioned reward value and sustains motivation for reward.

Dopamine signaling from the ventral tegmental area (VTA) plays critical roles in reward-related behaviors, but less is known about the functions of neighboring VTA GABAergic neurons. We show here that a primary target of VTA GABA projection neurons is the ventral pallidum (VP). Activity of VTA-to-VP-projecting GABA neurons correlates consistently with size and palatability of the reward and does not change following cue learning, providing a direct measure of reward value. Chemogenetic stimulation of this GABA projection increased activity of a subset of VP neurons that were active while mice were seeking reward. Optogenetic stimulation of this pathway improved performance in a cue-reward task and maintained motivation to work for reward over days. This VTA GABA projection provides information about reward value directly to the VP, likely distinct from the prediction error signal carried by VTA dopamine neurons.

Cell-type-specific population dynamics of diverse reward computations.

Computational analysis of cellular activity has developed largely independently of modern transcriptomic cell typology, but integrating these approaches may be essential for full insight into cellular-level mechanisms underlying brain function and dysfunction. Applying this approach to the habenula (a structure with diverse, intermingled molecular, anatomical, and computational features), we identified encoding of reward-predictive cues and reward outcomes in distinct genetically defined neural populations, including TH+ cells and Tac1+ cells. Data from genetically targeted recordings were used to train an optimized nonlinear dynamical systems model and revealed activity dynamics consistent with a line attractor. High-density, cell-type-specific electrophysiological recordings and optogenetic perturbation provided supporting evidence for this model. Reverse-engineering predicted how Tac1+ cells might integrate reward history, which was complemented by in vivo experimentation. This integrated approach describes a process by which data-driven computational models of population activity can generate and frame actionable hypotheses for cell-type-specific investigation in biological systems.