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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, suggesting that VGluT2+VGaT+ neurons are integrators of signals related to aversive outcomes but not of aversion-induced behavioral kinematics. We interpret these results such that VTA VGluT2+VGaT+ neurons may integrate diverse inputs related to the detection and processing of motivationally-salient outcomes.

There is no question that genetically encoded tools have revolutionized neuroscience. These include optically modulated tools for writing-in (optogenetics) and reading-out (calcium, voltage, and neurotransmitter indicators) neural activity as well as precision expression of these reagents using virally mediated delivery. With few exceptions, these powerful approaches are derived from naturally occurring molecules that are sourced from diverse organisms that span all kingdoms of life. Successful expression of genetic tools in standard neuroscience model organisms requires optimizing gene structure, taking into account differences in both protein translation and trafficking. Myriad approaches have resolved these two challenges, resulting in order-of-magnitude increases in functional expression. In this chapter, we focus on synthesizing prior experience in successfully enabling the transition of genes across kingdoms with a goal of facilitating the production of the next generation of molecular tools for neuroscience. We then provide a detailed protocol that allows expression and testing of novel genetically encoded tools in mammalian cell lines and primary cultured neurons.

The COVID-19 pandemic compelled fast adaptation of telehealth to addiction treatment services. This study aims to examine the feasibility and effectiveness of transitioning an in-person hospital addiction consult service (ACS) to telehealth. The Stanford Hospital ACS adapted to the pandemic by transforming an in-person ACS to a telehealth ACS. We compared 30-day readmission rates in patients with and without an addiction medicine consult pre-pandemic (in-person ACS) and during the pandemic (telehealth ACS). The ACS completed 370 and 473 unique patient consults in the year preceding (in-person consults) and during the pandemic (telehealth consults) respectively. Patients seen by telehealth ACS had decreased 30-day readmission rates consistent with those seen before COVID-19. A telehealth ACS is feasible and effective in the in-patient setting. Telehealth ACS holds promise to extend the reach of substance use disorder evaluation and treatment in underserved areas.

From genetics to material to behavior Introducing new genes into an organism can endow new biochemical functions or change the patterns of existing functions, but extending these manipulations to structure at the tissue level is challenging. Combining genetic engineering and polymer chemistry, Liu et al. directly leveraged complex cellular architectures of living organisms to synthesize, fabricate, and assemble bioelectronic materials (see the Perspective by Otto and Schmidt). An engineered enzyme expressed in genetically targeted neurons synthesized conductive polymers in tissues of freely moving animals. These polymers enabled modulation of membrane properties in specific neuron populations and manipulation of behavior in living animals. Science , this issue p. 1372 ; see also p. 1303

Significance Since the invention of optogenetics, using light to control the activity of individual neurons and dissect the complex neural circuits has been a powerful tool for neuroscience owing to the high temporal precision and neuron-type specificity. However, one of the major challenges of optogenetics is the invasive delivery of light sources such as fiber optics inside the brain of live animals due to limited tissue penetration of photons. Here, we report a method termed “sono-optogenetics,” which provides minimally invasive optogenetic neuromodulation in the brain without any scalp incision, craniotomy, or brain implant. Sono-optogenetics delivers nanoscopic light sources via the endogenous blood circulation and provides millisecond-timescale switching of light emission for optogenetic neuromodulation via brain-penetrant focused ultrasound., Optogenetics, which uses visible light to control the cells genetically modified with light-gated ion channels, is a powerful tool for precise deconstruction of neural circuitry with neuron-subtype specificity. However, due to limited tissue penetration of visible light, invasive craniotomy and intracranial implantation of tethered optical fibers are usually required for in vivo optogenetic modulation. Here we report mechanoluminescent nanoparticles that can act as local light sources in the brain when triggered by brain-penetrant focused ultrasound (FUS) through intact scalp and skull. Mechanoluminescent nanoparticles can be delivered into the blood circulation via i.v. injection, recharged by 400-nm photoexcitation light in superficial blood vessels during circulation, and turned on by FUS to emit 470-nm light repetitively in the intact brain for optogenetic stimulation. Unlike the conventional “outside-in” approaches of optogenetics with fiber implantation, our method provides an “inside-out” approach to deliver nanoscopic light emitters via the intrinsic circulatory system and switch them on and off at any time and location of interest in the brain without extravasation through a minimally invasive ultrasound interface.

Encoding and predicting aversive events are critical functions of circuits that support survival and emotional well-being. Maladaptive circuit changes in emotional valence processing can underlie the pathophysiology of affective disorders. The lateral habenula (LHb) has been linked to aversion and mood regulation through modulation of the dopamine and serotonin systems. We have defined the identity and function of glutamatergic (Vglut2) control of the LHb, comparing the role of inputs originating in the globus pallidus internal segment (GPi), and lateral hypothalamic area (LHA), respectively. We found that LHb-projecting LHA neurons, and not the proposed GABA/glutamate co-releasing GPi neurons, are responsible for encoding negative value. Monosynaptic rabies tracing of the presynaptic organization revealed a predominantly limbic input onto LHA Vglut2 neurons, while sensorimotor inputs were more prominent onto GABA/glutamate co-releasing GPi neurons. We further recorded the activity of LHA Vglut2 neurons, by imaging calcium dynamics in response to appetitive versus aversive events in conditioning paradigms. LHA Vglut2 neurons formed activity clusters representing distinct reward or aversion signals, including a population that responded to mild foot shocks and predicted aversive events. We found that the LHb-projecting LHA Vglut2 neurons encode negative valence and rapidly develop a prediction signal for negative events. These findings establish the glutamatergic LHA-LHb circuit as a critical node in value processing.

The lateral hypothalamus (LH), together with multiple neuromodulatory systems of the brain, such as the dorsal raphe nucleus (DR), is implicated in arousal, yet interactions between these systems are just beginning to be explored. Using a combination of viral tracing, circuit mapping, electrophysiological recordings from identified neurons, and combinatorial optogenetics in mice, we show that GABAergic neurons in the LH selectively inhibit GABAergic neurons in the DR, resulting in increased firing of a substantial fraction of its neurons that ultimately promotes arousal. These DRGABAneurons are wake active and project to multiple brain areas involved in the control of arousal, including the LH, where their specific activation potently influences local network activity leading to arousal from sleep. Our results show how mutual inhibitory projections between the LH and the DR promote wakefulness and suggest a complex arousal control by intimate interactions between long-range connections and local circuit dynamics.SIGNIFICANCE STATEMENT:Multiple brain systems including the lateral hypothalamus and raphe serotonergic system are involved in the regulation of the sleep/wake cycle, yet the interaction between these systems have remained elusive. Here we show that mutual disinhibition mediated by long range inhibitory projections between these brain areas can promote wakefulness. The main importance of this work relies in revealing the interaction between a brain area involved in autonomic regulation and another in controlling higher brain functions including reward, patience, mood and sensory coding.

Summary The lateral hypothalamic area (LHA) regulates feeding- and reward-related behavior, but because of its molecular and anatomical heterogeneity, the functions of defined neuronal populations are largely unclear. Glutamatergic neurons within the LHA (LHAVglut2) negatively regulate feeding and appetitive behavior. However, this population comprises transcriptionally distinct and functionally diverse neurons that project to diverse brain regions, including the lateral habenula (LHb) and ventral tegmental area (VTA). To resolve the function of distinct LHAVglut2 populations, we systematically compared projections to the LHb and VTA using viral tracing, single-cell sequencing, electrophysiology, and in vivo calcium imaging. LHAVglut2 neurons projecting to the LHb or VTA are anatomically, transcriptionally, electrophysiologically, and functionally distinct. While both populations encode appetitive and aversive stimuli, LHb projecting neurons are especially sensitive to satiety state and feeding hormones. These data illuminate the functional heterogeneity of LHAVglut2 neurons, suggesting that reward and aversion are differentially processed in divergent efferent pathways.

The lateral hypothalamus (LH), together with multiple neuromodulatory systems of the brain, such as the dorsal raphé nucleus (DR), is implicated in arousal, yet interactions between these systems are just beginning to be explored. Using a combination of viral tracing, circuit mapping, electrophysiological recordings from identified neurons and combinatorial optogenetics in mice, we show that GABAergic neurons in the LH selectively inhibit GABAergic neurons in the DR resulting in increased firing of a substantial fraction of its neurons that ultimately promotes arousal. These DRGABA neurons are wake active and project to multiple brain areas involved in the control of arousal including the LH, where their specific activation potently influences local network activity leading to arousal from sleep. Our results show how mutual inhibitory projections between the LH and the DR promote wakefulness and suggest a complex arousal control by intimate interactions between long-range connections and local circuit dynamics.

SUMMARYThe ability to record transient cellular events in the DNA or RNA of cells would enable precise, large-scale analysis, selection, and reprogramming of heterogeneous cell populations. Here we report a molecular technology for stable genetic tagging of cells that exhibit activity-related increases in intracellular calcium concentration (FLiCRE). We used FLiCRE to transcriptionally label activated neural ensembles in the nucleus accumbens of the mouse brain during brief stimulation of aversive inputs. Using single-cell RNA sequencing, we detected FLiCRE transcripts among the endogenous transcriptome, providing simultaneous readout of both cell-type and calcium activation history. We identified a cell-type in the nucleus accumbens activated downstream of long-range excitatory projections. Taking advantage of FLiCRE’s modular design, we expressed an optogenetic channel selectively in this cell-type, and showed that direct recruitment of this otherwise genetically-inaccessible population elicits behavioral aversion. The specificity and minute-resolution of FLiCRE enables molecularly-informed characterization, manipulation, and reprogramming of activated cellular ensembles.

Both the resolution and dimensionality with which biologists can characterize cell types have expanded dramatically in recent years, and intersectional consideration of such features (e.g. multiple gene-expression and anatomical parameters) is increasingly understood to be essential. At the same time, genetically-targeted technology for writing-in and reading-out activity patterns for cells within living organisms has enabled causal investigation in physiology and behavior; however, cell-type-specific delivery of these tools (including microbial opsins for optogenetics and genetically-encoded Ca(2+) indicators) has thus far fallen short of versatile targeting to cells jointly defined by many individually-selected features. In this Resource, we develop a comprehensive intersectional targeting toolbox, including 39 novel vectors for joint-feature-targeted delivery of 13 molecular payloads (including opsins, indicators, and fluorophores), systematic approaches for development and optimization of new intersectional tools, hardware for in vivo monitoring of expression dynamics, and the first versatile single-virus tools (Triplesect) that enable targeting of triply-defined cell types.

The ability to record transient cellular events in the DNA or RNA of cells would enable precise, large-scale analysis, selection, and reprogramming of heterogeneous cell populations. Here, we report a molecular technology for stable genetic tagging of cells that exhibit activity-related increases in intracellular calcium concentration (FLiCRE). We used FLiCRE to transcriptionally label activated neural ensembles in the nucleus accumbens of the mouse brain during brief stimulation of aversive inputs. Using single-cell RNA sequencing, we detected FLiCRE transcripts among the endogenous transcriptome, providing simultaneous readout of both cell-type and calcium activation history. We identified a cell type in the nucleus accumbens activated downstream of long-range excitatory projections. Taking advantage of FLiCRE's modular design, we expressed an optogenetic channel selectively in this cell type and showed that direct recruitment of this otherwise genetically inaccessible population elicits behavioral aversion. The specificity and minute resolution of FLiCRE enables molecularly informed characterization, manipulation, and reprogramming of activated cellular ensembles.

The naturally occurring channelrhodopsin variant anion channelrhodopsin-1 (ACR1), discovered in the cryptophyte algae Guillardia theta, exhibits large light-gated anion conductance and high anion selectivity when expressed in heterologous settings, properties that support its use as an optogenetic tool to inhibit neuronal firing with light. However, molecular insight into ACR1 is lacking owing to the absence of structural information underlying light-gated anion conductance. Here we present the crystal structure of G. theta ACR1 at 2.9 Å resolution. The structure reveals unusual architectural features that span the extracellular domain, retinal-binding pocket, Schiff-base region, and anion-conduction pathway. Together with electrophysiological and spectroscopic analyses, these findings reveal the fundamental molecular basis of naturally occurring light-gated anion conductance, and provide a framework for designing the next generation of optogenetic tools.

Both designed and natural anion-conducting channelrhodopsins (dACRs and nACRs, respectively) have been widely applied in optogenetics (enabling selective inhibition of target-cell activity during animal behaviour studies), but each class exhibits performance limitations, underscoring trade-offs in channel structure-function relationships. Therefore, molecular and structural insights into dACRs and nACRs will be critical not only for understanding the fundamental mechanisms of these light-gated anion channels, but also to create next-generation optogenetic tools. Here we report crystal structures of the dACR iC++, along with spectroscopic, electrophysiological and computational analyses that provide unexpected insights into pH dependence, substrate recognition, channel gating and ion selectivity of both dACRs and nACRs. These results enabled us to create an anion-conducting channelrhodopsin integrating the key features of large photocurrent and fast kinetics alongside exclusive anion selectivity.

Water deprivation produces a drive to seek and consume water. How neural activity creates this motivation remains poorly understood. We used activity-dependent genetic labeling to characterize neurons activated by water deprivation in the hypothalamic median preoptic nucleus (MnPO). Single-cell transcriptional profiling revealed that dehydration-activated MnPO neurons consist of a single excitatory cell type. After optogenetic activation of these neurons, mice drank water and performed an operant lever-pressing task for water reward with rates that scaled with stimulation frequency. This stimulation was aversive, and instrumentally pausing stimulation could reinforce lever-pressing. Activity of these neurons gradually decreased over the course of an operant session. Thus, the activity of dehydration-activated MnPO neurons establishes a scalable, persistent, and aversive internal state that dynamically controls thirst-motivated behavior.

SUMMARY Dopamine (DA) neurons in the ventral tier of the substantia nigra pars compacta (SNc) degenerate prominently in Parkinson’s disease, while those in the dorsal tier are relatively spared. Defining the molecular, functional, and developmental characteristics of each SNc tier is crucial to understand their distinct susceptibility. We demonstrate that Sox6 expression distinguishes ventrally and dorsally biased DA neuron populations in the SNc. The Sox6+ population in the ventral SNc includes an Aldh1a1+ subset and is enriched in gene pathways that underpin vulnerability. Sox6+ neurons project to the dorsal striatum and show activity correlated with acceleration. Sox6− neurons project to the medial, ventral, and caudal striatum and respond to rewards. Moreover, we show that this adult division is encoded early in development. Overall, our work demonstrates a dual origin of the SNc that results in DA neuron cohorts with distinct molecular profiles, projections, and functions., Graphical Abstract, In brief In this study, Pereira Luppi et al. refine the landscape of midbrain dopamine neurons through Sox6 lineage analysis to shed light on dorsal and ventral substantia nigra pars compacta from molecular, anatomical, functional, and developmental perspectives.

Survival in threatening situations depends on the selection and rapid execution of an appropriate active or passive defensive response, yet the underlying brain circuitry is not understood. Here we use circuit-based optogenetic, in vivo and in vitro electrophysiological, and neuroanatomical tracing methods to define midbrain periaqueductal grey circuits for specific defensive behaviours. We identify an inhibitory pathway from the central nucleus of the amygdala to the ventrolateral periaqueductal grey that produces freezing by disinhibition of ventrolateral periaqueductal grey excitatory outputs to pre-motor targets in the magnocellular nucleus of the medulla. In addition, we provide evidence for anatomical and functional interaction of this freezing pathway with long-range and local circuits mediating flight. Our data define the neuronal circuitry underlying the execution of freezing, an evolutionarily conserved defensive behaviour, which is expressed by many species including fish, rodents and primates. In humans, dysregulation of this 'survival circuit' has been implicated in anxiety-related disorders.

SUMMARY Ventral tegmental area (VTA) neurons play roles in reward and aversion. We recently discovered that the VTA has neurons that co-transmit glutamate and GABA (glutamate-GABA co-transmitting neurons), transmit glutamate without GABA (glutamate-transmitting neurons), or transmit GABA without glutamate (GABA-transmitting neurons). However, the functions of these VTA cell types in motivated behavior are unclear. To identify the functions of these VTA cell types, we combine recombinase mouse lines with INTRSECT2.0 vectors to selectively target these neurons. We find that VTA cell types have unique signaling patterns for reward, aversion, and learned cues. Whereas VTA glutamate-transmitting neurons signal cues predicting reward, VTA GABA-transmitting neurons signal cues predicting the absence of reward, and glutamate-GABA co-transmitting neurons signal rewarding and aversive outcomes without signaling learned cues related to those outcomes. Thus, we demonstrate that genetically defined subclasses of VTA glutamate and GABA neurons signal different aspects of motivated behavior., Graphical Abstract, In Brief Root et al. examine ventral tegmental area (VTA) neurons that release glutamate without GABA, GABA without glutamate, or both glutamate and GABA. Cell types have differential projection densities and unique neuronal activity profiles related to cues predicting rewarding, nonreward, or aversive outcomes and in the receipt of predicted rewards.

Summary: Parvalbumin (PV)-expressing GABAergic neurons are the largest class of inhibitory neocortical cells. We visualize brain-wide, monosynaptic inputs to PV neurons in mouse barrel cortex. We develop intersectional rabies virus tracing to specifically target GABAergic PV cells and exclude a small fraction of excitatory PV cells from our starter population. Local inputs are mainly from layer (L) IV and excitatory cells. A small number of inhibitory inputs originate from LI neurons, which connect to LII/III PV neurons. Long-range inputs originate mainly from other sensory cortices and the thalamus. In visual cortex, most transsynaptically labeled neurons are located in LIV, which contains a molecularly mixed population of projection neurons with putative functional similarity to LIII neurons. This study expands our knowledge of the brain-wide circuits in which PV neurons are embedded and introduces intersectional rabies virus tracing as an applicable tool to dissect the circuitry of more clearly defined cell types. : Hafner et al. develop constructs for intersectional rabies virus tracing to map brain-wide, monosynaptic inputs to cortical inhibitory parvalbumin cells. They find extensive local innervation by excitatory cells, inputs from inhibitory cells in layer I, and a strong contribution of layer IV projection neurons to cortical long-range inputs. Keywords: parvalbumin, GABAergic neuron, barrel cortex, rabies tracing, intersectional, Cre, Flp

Studying intact systems with simultaneous local precision and global scope is a fundamental challenge in biology. This familiar trade-off leads to important conceptual and experimental difficulties in psychiatric disease research and throughout the study of complex biological systems. Part of a solution may arise from optogenetics: the combination of genetic and optical methods to achieve gain- or loss-of-function of temporally defined events in specific cells embedded within intact living tissue or organisms. Such precise causal control within the functioning intact system can be achieved via introduction of genes that confer to cells both light-detection capability and specific effector function. A broad array of optogenetic tools and neuroscience applications have driven the wide adoption of optogenetics as a standard approach in neuroscience.

Alterations in the balance between neuronal excitation and inhibition (E:I balance) have been implicated in the neural circuit activity–based processes that contribute to autism phenotypes. We investigated whether acutely reducing E:I balance in mouse brain could correct deficits in social behavior. We used mice lacking the CNTNAP2 gene, which has been implicated in autism, and achieved a temporally precise reduction in E:I balance in the medial prefrontal cortex (mPFC) either by optogenetically increasing the excitability of inhibitory parvalbumin (PV) neurons or decreasing the excitability of excitatory pyramidal neurons. Surprisingly, both of these distinct, real-time, and reversible optogenetic modulations acutely rescued deficits in social behavior and hyperactivity in adult mice lacking CNTNAP2. Using fiber photometry, we discovered that native mPFC PV neuronal activity differed between CNTNAP2 knockout and wild-type mice. During social interactions with other mice, PV neuron activity increased in wild-type mice compared to interactions with a novel object, whereas this difference was not observed in CNTNAP2 knockout mice. Together, these results suggest that real-time modulation of E:I balance in the mouse prefrontal cortex can rescue social behavior deficits reminiscent of autism phenotypes.

As the power of genetically encoded interventional and observational tools for neuroscience expands, the boundaries of experimental design are increasingly defined by limits in selectively expressing these tools in relevant cell types. Single-recombinase-dependent expression systems have been widely used as a means to restrict gene expression based on single features by combining recombinase-dependent viruses with recombinase-expressing transgenic animals. This protocol details how to create INTRSECT constructs and use multiple recombinases to achieve targeting of a desired gene to subsets of neurons that are defined by multiple genetic and/or topological features. This method includes the design and utilization of both viruses and transgenic animals: these tools are inherently flexible and modular and may be used in different combinations to achieve the desired gene expression pattern. © 2017 by John Wiley & Sons, Inc.

SUMMARYBoth the lateral and the central nuclei of the amygdala are required for adaptive behavioral responses to environmental cues predicting threats. While experience-driven synaptic plasticity in the lateral amygdala is thought to underlie the formation of association between a sensory stimulus and an ensuing threat, how the central amygdala participates in such learning process remains unclear. Here we show that a specific class of central amygdala neurons, the protein kinase C-δ-expressing neurons, is essential for the synaptic plasticity underlying learning in the lateral amygdala, as it is required for lateral amygdala neurons to respond to unconditioned stimulus, and furthermore carries information about the unconditioned stimulus to instruct learning. Our results uncover an amygdala functional organization that may play a key role in diverse learning processes.

Experience-driven synaptic plasticity in the lateral amygdala is thought to underlie the formation of associations between sensory stimuli and an ensuing threat. However, how the central amygdala participates in such a learning process remains unclear. Here we show that PKC-δ-expressing central amygdala neurons are essential for the synaptic plasticity underlying learning in the lateral amygdala, as they convey information about the unconditioned stimulus to lateral amygdala neurons during fear conditioning.

Clinical and research efforts have focused on promoting functional recovery after stroke. Brain stimulation strategies are particularly promising because they allow direct manipulation of the target area's excitability. However, elucidating the cell type and mechanisms mediating recovery has been difficult because existing stimulation techniques nonspecifically target all cell types near the stimulated site. To circumvent these barriers, we used optogenetics to selectively activate neurons that express channelrhodopsin 2 and demonstrated that selective neuronal stimulations in the ipsilesional primary motor cortex (iM1) can promote functional recovery. Stroke mice that received repeated neuronal stimulations exhibited significant improvement in cerebral blood flow and the neurovascular coupling response, as well as increased expression of activity-dependent neurotrophins in the contralesional cortex, including brain-derived neurotrophic factor, nerve growth factor, and neurotrophin 3. Western analysis also indicated that stimulated mice exhibited a significant increase in the expression of a plasticity marker growth-associated protein 43. Moreover, iM1 neuronal stimulations promoted functional recovery, as stimulated stroke mice showed faster weight gain and performed significantly better in sensory-motor behavior tests. Interestingly, stimulations in normal nonstroke mice did not alter motor behavior or neurotrophin expression, suggesting that the prorecovery effect of selective neuronal stimulations is dependent on the poststroke environment. These results demonstrate that stimulation of neurons in the stroke hemisphere is sufficient to promote recovery.

Precisely defining the roles of specific cell types is an intriguing and challenging frontier in the study of intact biological systems, and has stimulated the rapid development of genetically-encoded observation and control tools. However, targeting these tools with adequate specificity remains challenging: most cell types are best defined by the intersection of two or more features such as active promoter elements, location, and connectivity. Here we have combined recombinase tools with engineered introns to achieve expression of genetically-encoded payloads conditional upon multiple cell-type features, using Boolean logical operations all governed by a single versatile vector. We use this approach to target intersectionally-specified populations of inhibitory interneurons in mammalian hippocampus and neurons of the ventral tegmental area defined by both genetic and wiring properties. This flexible and modular approach may expand the application of genetically-encoded interventional and observational tools for intact-systems biology.

SummarySocial interaction is a complex behavior essential for many species and is impaired in major neuropsychiatric disorders. Pharmacological studies have implicated certain neurotransmitter systems in social behavior, but circuit-level understanding of endogenous neural activity during social interaction is lacking. We therefore developed and applied a new methodology, termed fiber photometry, to optically record natural neural activity in genetically and connectivity-defined projections to elucidate the real-time role of specified pathways in mammalian behavior. Fiber photometry revealed that activity dynamics of a ventral tegmental area (VTA)-to-nucleus accumbens (NAc) projection could encode and predict key features of social, but not novel object, interaction. Consistent with this observation, optogenetic control of cells specifically contributing to this projection was sufficient to modulate social behavior, which was mediated by type 1 dopamine receptor signaling downstream in the NAc. Direct observation of deep projection-specific activity in this way captures a fundamental and previously inaccessible dimension of mammalian circuit dynamics.

There is growing evidence that interneurons (INs) orchestrate neural activity and plasticity in corticoamygdala circuits to regulate fear behaviors. However, defining the precise role of cholecystokinin-expressing INs (CCK INs) remains elusive due to the technical challenge of parsing this population from CCK-expressing principal neurons (CCK PNs). Here, we used an intersectional genetic strategy in CCK-Cre;Dlx5/6-Flpe double-transgenic mice to study the anatomical, molecular and electrophysiological properties of CCK INs in the basal amygdala (BA) and optogenetically manipulate these cells during fear extinction. Electrophysiological recordings confirmed that this strategy targeted GABAergic cells and that a significant proportion expressed functional cannabinoid CB1 receptors; a defining characteristic of CCK-expressing basket cells. However, immunostaining showed that subsets of the genetically-targeted cells expressed either neuropeptide Y (NPY; 29%) or parvalbumin (PV; 17%), but not somatostatin (SOM) or Ca2+/calmodulin-dependent protein kinase II (CaMKII)-α. Further morphological and electrophysiological analyses showed that four IN types could be identified among the EYFP-expressing cells: CCK/cannabinoid receptor type 1 (CB1R)-expressing basket cells, neurogliaform cells, PV+ basket cells, and PV+ axo-axonic cells. At the behavioral level,in vivooptogenetic photostimulation of the targeted population during extinction acquisition led to reduced freezing on a light-free extinction retrieval test, indicating extinction memory facilitation; whereas photosilencing was without effect. Conversely, non-selective (i.e., inclusive of INs and PNs) photostimulation or photosilencing of CCK-targeted cells, using CCK-Cre single-transgenic mice, impaired extinction. These data reveal an unexpectedly high degree of phenotypic complexity in a unique population of extinction-modulating BA INs.

Diverse optogenetic tools have allowed versatile control over neural activity. Many depolarizing and hyperpolarizing tools have now been developed in multiple laboratories and tested across different preparations, presenting opportunities but also making it difficult to draw direct comparisons. This challenge has been compounded by the dependence of performance on parameters such as vector, promoter, expression time, illumination, cell type and many other variables. As a result, it has become increasingly complicated for end users to select the optimal reagents for their experimental needs. For a rapidly growing field, critical figures of merit should be formalized both to establish a framework for further development and so that end users can readily understand how these standardized parameters translate into performance. Here we systematically compared microbial opsins under matched experimental conditions to extract essential principles and identify key parameters for the conduct, design and interpretation of experiments involving optogenetic techniques.

The capture and utilization of light is an exquisitely evolved process. The single-component microbial opsins, although more limited than multicomponent cascades in processing, display unparalleled compactness and speed. Recent advances in understanding microbial opsins have been driven by molecular engineering for optogenetics and by comparative genomics. Here we provide a Primer on these light-activated ion channels and pumps, describe a group of opsins bridging prior categories, and explore the convergence of molecular engineering and genomic discovery for the utilization and understanding of these remarkable molecular machines.

Cortico-thalamo-cortical circuits mediate sensation and generate neural network oscillations associated with slow-wave sleep and various epilepsies. Cortical input to sensory thalamus is thought to mainly evoke feed-forward synaptic inhibition of thalamocortical (TC) cells via reticular thalamic nucleus (nRT) neurons, especially during oscillations. This relies on a stronger synaptic strength in the cortico-nRT pathway than in the cortico-TC pathway, allowing the feed-forward inhibition of TC cells to overcome direct cortico-TC excitation. We found a systemic and specific reduction in strength in GluA4-deficient (Gria4(-/-)) mice of one excitatory synapse of the rhythmogenic cortico-thalamo-cortical system, the cortico-nRT projection, and observed that the oscillations could still be initiated by cortical inputs via the cortico-TC-nRT-TC pathway. These results reveal a previously unknown mode of cortico-thalamo-cortical transmission, bypassing direct cortico-nRT excitation, and describe a mechanism for pathological oscillation generation. This mode could be active under other circumstances, representing a previously unknown channel of cortico-thalamo-cortical information processing.

Severe behavioural deficits in psychiatric diseases such as autism and schizophrenia have been hypothesized to arise from elevations in the cellular balance of excitation and inhibition (E/I balance) within neural microcircuitry. This hypothesis could unify diverse streams of pathophysiological and genetic evidence, but has not been susceptible to direct testing. Here we design and use several novel optogenetic tools to causally investigate the cellular E/I balance hypothesis in freely moving mammals, and explore the associated circuit physiology. Elevation, but not reduction, of cellular E/I balance within the mouse medial prefrontal cortex was found to elicit a profound impairment in cellular information processing, associated with specific behavioural impairments and increased high-frequency power in the 30–80 Hz range, which have both been observed in clinical conditions in humans. Consistent with the E/I balance hypothesis, compensatory elevation of inhibitory cell excitability partially rescued social deficits caused by E/I balance elevation. These results provide support for the elevated cellular E/I balance hypothesis of severe neuropsychiatric disease-related symptoms. One model for the cellular disturbances underlying social and emotional deficits in disorders such as autism and schizophrenia is an imbalance in excitatory and inhibitory activity in certain neural systems. This idea has not been directly testable so far, but testability comes a little closer with the development of two optogenetic tools that have different spectral and temporal characteristics, thereby allowing selective control of two intermingled populations of neurons. Use of these new opsins shows that increasing relative excitation in mouse prefrontal cortex impairs social and learning behaviours. This provides support for the elevated cellular excitatory/inhibitory balance hypothesis of certain neuropsychiatric symptoms.

Genetically encoded, single-component optogenetic tools have made a significant impact on neuroscience, enabling specific modulation of selected cells within complex neural tissues. As the optogenetic toolbox contents grow and diversify, the opportunities for neuroscience continue to grow. In this review, we outline the development of currently available single-component optogenetic tools and summarize the application of various optogenetic tools in diverse model organisms.

Anxiety, a sustained state of heightened apprehension in the absence of immediate threat, becomes severely debilitating in disease states1. Anxiety disorders represent the most common of psychiatric diseases (28% lifetime prevalence)2, and contribute to the etiology of major depression and substance abuse3,4. Although it has been proposed that the amygdala, a brain region important for emotional processing5–8, has a role in anxiety9–13, neural mechanisms that control anxiety remain unclear. Here we explore neural circuits underlying anxiety-related behaviors by using optogenetics with two-photon microscopy, anxiety assays in freely moving mice, and electrophysiology. With the capability of optogenetics14–16 to control not only cell types but also specific connections between cells, we observed that temporally precise optogenetic stimulation of basolateral amygdala (BLA) terminals in the central nucleus of the amygdala (CeA-achieved by viral transduction of the BLA with a codon-optimized channelrhodopsin followed by restricted illumination in downstream CeA - exerted an acute, reversible anxiolytic effect. Conversely, selective optogenetic inhibition of the same projection with a third-generation halorhodopsin15 (eNpHR3.0) increased anxiety-related behaviors. Importantly, these effects were not observed with direct optogenetic control of BLA somata, possibly owing to recruitment of antagonistic downstream structures. Together, these results implicate specific BLA-CeA projections as critical circuit elements for acute anxiety control in the mammalian brain, and demonstrate the importance of optogenetically targeting defined projections, beyond simply targeting cell types, in the study of circuit function relevant to neuropsychiatric disease.

The use of light-activated modulation techniques, such as optogenetics, is growing in popularity for enabling basic neuroscience research. It is also being explored for advancing more applied applications like therapeutic neuromodulation. However, current hardware systems are generally limited to acute measurements or require external tethering of the system to the light source. This paper presents an implantable prototype for use in techniques that modulate neurological state through optically-activated channels and compounds. The prototype system employs a three chip custom IC architecture to manage information flow into the neural substrate, while also handling power dissipation and providing a chronic barrier to the tissue interface. In addition to covering the details of the IC architecture, we discuss system level design constraints and solutions, and in-vitro test results using our prototype system with an optogenetic model. Potential technical limitations for the broader adoption of these techniques will also be considered.

SummaryIn vivo experimental evidence indicates that acute neuronal activation increases Aβ release from presynaptic terminals, whereas long-term effects of chronic synaptic activation on Aβ pathology remain unclear. To address this issue, we adopted optogenetics and transduced stabilized step-function opsin, a channelrhodopsin engineered to elicit a long-lasting neuronal hyperexcitability, into the hippocampal perforant pathway of APP transgenic mice. In vivo microdialysis revealed a ∼24% increase in the hippocampal interstitial fluid Aβ42 levels immediately after acute light activation. Five months of chronic optogenetic stimulation increased Aβ burden specifically in the projection area of the perforant pathway (i.e., outer molecular layer of the dentate gyrus) of the stimulated side by ∼2.5-fold compared with that in the contralateral side. Epileptic seizures were observed during the course of chronic stimulation, which might have partly contributed to the Aβ pathology. These findings implicate functional abnormalities of specific neuronal circuitry in Aβ pathology and Alzheimer disease.

Objective: Functional recovery after stroke has been observed and is currently attributed to both brain remodeling and plasticity. One form of cortical reorganization involves the balance of interhemispheric interactions between ipsilesional and contralesional cortex. Stimulation of ipsilesional primary motor cortex (iM1) has been shown to be beneficial, however, the role of the contralesional M1 (cM1) remains controversial. Recently we showed that optogenetic stimulations of iM1 post-stroke promote functional recovery. In this study, we investigate the role of contralesional cortex in recovery by optogenetically stimulating iM1 or cM1 and examine the involvement of activity-dependent neurotrophins. Methods: Thy-1-ChR2-YFP line-18 transgenic male mice were used. Mice underwent stereotaxic surgery to implant a fiber cannula in either iM1 or cM1, followed by an intraluminal middle cerebral artery suture occlusion. Optogenetic stimulation began at day5 post-stroke and continued until day14 post-stroke. Sensorimotor behavior tests were used to assess their recovery at day 0, 2, 7, 10 and 14 post-stroke. Mice were sacrificed at day15 post-stroke and neurotrophin expressions were examined using quantitative PCR. Results: Repeated iM1 stimulations promoted functional recovery at day14 post-stroke, with improved motor performance on the rotating beam test (p Conclusion: Our data suggest that activity-dependent neurotrophins in the contralesional cortex may be an important mechanism mediating stroke recovery. Current studies include specific stimulation and inhibition of the iM1 or cM1 post-stroke to elucidate the neurocircuitry mediating stroke recovery. In addition, the expression of neurotrophins will be examined in these studies to elucidate their role in the recovery process.

Studying intact biological systems with both local precision and global scope is a fundamental challenge, especially in psychiatric disease research. Part of a solution may arise from the technology of optogenetics: the combination of genetic and optical methods to control events in specific cells embedded within intact living tissue or organisms. For example, “opsin” genes can be introduced into mammalian neurons to mediate millisecond-precision and reliable elicitation or inhibition of neural activity in response to light pulses. This approach has now been used to control neuronal activity in a wide range of animals, resulting in insights into fundamental aspects of circuit function, as well as circuit dysfunction and treatment in pathological states. Here we review the current state of optogenetics for neuroscience and psychiatry, and address the evolving challenges and future opportunities. Despite both rapid growth and wide scope of applications, fundamental challenges remain to be addressed.

INTRODUCTIONMembers of the microbial opsin gene family have emerged recently as crucial tools for “optogenetics,” a new neuroscience technology. “Optogenetics” can be defined as the integration of optics and genetics to control well-defined events (such as action potentials) within specified cells (such as a targeted class of projection neurons) in living tissues (such as the brains of freely behaving mammals). In this article, we focus on the diversity of the microbial opsin genes and the structure–function properties of their corresponding proteins.

Parkinson's disease (PD) is an incurable age-related neurodegenerative disorder affecting both the central and peripheral nervous systems. Although common, the etiology of PD remains poorly understood. Genetic studies infer that the disease results from a complex interaction between genetics and environment and there is growing evidence that PD may represent a constellation of diseases with overlapping yet distinct underlying mechanisms. Novel clinical approaches will require a better understanding of the mechanisms at work within an individual as well as methods to identify the specific array of mechanisms that have contributed to the disease. Induced pluripotent stem cell (iPSC) strategies provide an opportunity to directly study the affected neuronal subtypes in a given patient. Here we report the generation of iPSC-derived midbrain dopaminergic neurons from a patient with a triplication in the α-synuclein gene (SNCA). We observed that the iPSCs readily differentiated into functional neurons. Importantly, the PD-affected line exhibited disease-related phenotypes in culture: accumulation of α-synuclein, inherent overexpression of markers of oxidative stress, and sensitivity to peroxide induced oxidative stress. These findings show that the dominantly-acting PD mutation is intrinsically capable of perturbing normal cell function in culture and confirm that these features reflect, at least in part, a cell autonomous disease process that is independent of exposure to the entire complexity of the diseased brain.

Despite a rapidly-growing scientific and clinical brain imaging literature based on functional magnetic resonance imaging (fMRI) using blood oxygenation level-dependent (BOLD) signals, it remains controversial whether BOLD signals in a particular region can be caused by activation of local excitatory neurons. This difficult question is central to the interpretation and utility of BOLD, with major significance for fMRI studies in basic research and clinical applications. Using a novel integrated technology unifying optogenetic control of inputs with high-field fMRI signal readouts, we show here that specific stimulation of local CaMKIIalpha-expressing excitatory neurons, either in the neocortex or thalamus, elicits positive BOLD signals at the stimulus location with classical kinetics. We also show that optogenetic fMRI (of MRI) allows visualization of the causal effects of specific cell types defined not only by genetic identity and cell body location, but also by axonal projection target. Finally, we show that of MRI within the living and intact mammalian brain reveals BOLD signals in downstream targets distant from the stimulus, indicating that this approach can be used to map the global effects of controlling a local cell population. In this respect, unlike both conventional fMRI studies based on correlations and fMRI with electrical stimulation that will also directly drive afferent and nearby axons, this of MRI approach provides causal information about the global circuits recruited by defined local neuronal activity patterns. Together these findings provide an empirical foundation for the widely-used fMRI BOLD signal, and the features of of MRI define a potent tool that may be suitable for functional circuit analysis as well as global phenotyping of dysfunctional circuitry.

In the study of complex mammalian behaviours, technological limitations have prevented spatiotemporally precise control over intracellular signalling processes. Here we report the development of a versatile family of genetically encoded optical tools ('optoXRs') that leverage common structure-function relationships among G-protein-coupled receptors (GPCRs) to recruit and control, with high spatiotemporal precision, receptor-initiated biochemical signalling pathways. In particular, we have developed and characterized two optoXRs that selectively recruit distinct, targeted signalling pathways in response to light. The two optoXRs exerted opposing effects on spike firing in nucleus accumbens in vivo, and precisely timed optoXR photostimulation in nucleus accumbens by itself sufficed to drive conditioned place preference in freely moving mice. The optoXR approach allows testing of hypotheses regarding the causal impact of biochemical signalling in behaving mammals, in a targetable and temporally precise manner.

Human embryonic stem cells possess the unique ability to differentiate into any adult cell type. Recent advances in the understanding of stem cell biology make new applications possible for stem cell based technology. Of note, it is now possible to reprogram terminally differentiated human somatic cells into pluripotent cells that are functionally equivalent to embryonic stem cells. These induced pluripotent cells may become the substrate for future disease models and cell-based therapies. In addition, novel techniques for genetic manipulation have increased the ease with which genes can be modified into stem cells. In this review, we describe these novel technologies as well as developments in the understanding of basic biology of stem cell pluripotency and differentiation.