Are novel treatments for brain disorders hiding in plain sight?

Psychiatric diseases are strongly influenced by genetics, but genetically guided treatments have been slow to develop, and precise molecular mechanisms remain mysterious. Although individual locations in the genome tend to not contribute powerfully to psychiatric disease incidence, genome-wide association studies (GWAS) have now successfully linked hundreds of specific genetic loci to psychiatric disorders [1-3]. Here, building upon results from well-powered GWAS of four phenotypes relevant to psychiatry, we motivate an exploratory workflow leading from GWAS screening, through causal testing in animal models using methods such as optogenetics, to new therapies in human beings. We focus on schizophrenia and the dopamine D2 receptor (DRD2), hot flashes and the neurokinin B receptor (TACR3), cigarette smoking and receptors bound by nicotine (CHRNA5, CHRNA3, CHRNB4), and alcohol use and enzymes that help to break down alcohol (ADH1B, ADH1C, ADH7). A single genomic locus may not powerfully determine disease at the level of the population, but the same locus may nevertheless represent a potent treatment target suitable for population-wide therapeutic approaches.

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.

Maturation and circuit integration of transplanted human cortical organoids.

Self-organizing neural organoids represent a promising in vitro platform with which to model human development and disease1-5. However, organoids lack the connectivity that exists in vivo, which limits maturation and makes integration with other circuits that control behaviour impossible. Here we show that human stem cell-derived cortical organoids transplanted into the somatosensory cortex of newborn athymic rats develop mature cell types that integrate into sensory and motivation-related circuits. MRI reveals post-transplantation organoid growth across multiple stem cell lines and animals, whereas single-nucleus profiling shows progression of corticogenesis and the emergence of activity-dependent transcriptional programs. Indeed, transplanted cortical neurons display more complex morphological, synaptic and intrinsic membrane properties than their in vitro counterparts, which enables the discovery of defects in neurons derived from individuals with Timothy syndrome. Anatomical and functional tracings show that transplanted organoids receive thalamocortical and corticocortical inputs, and in vivo recordings of neural activity demonstrate that these inputs can produce sensory responses in human cells. Finally, cortical organoids extend axons throughout the rat brain and their optogenetic activation can drive reward-seeking behaviour. Thus, transplanted human cortical neurons mature and engage host circuits that control behaviour. We anticipate that this approach will be useful for detecting circuit-level phenotypes in patient-derived cells that cannot otherwise be uncovered.

A functional cellular framework for sex and estrous cycle-dependent gene expression and behavior.

Sex hormones exert a profound influence on gendered behaviors. How individual sex hormone-responsive neuronal populations regulate diverse sex-typical behaviors is unclear. We performed orthogonal, genetically targeted sequencing of four estrogen receptor 1-expressing (Esr1+) populations and identified 1,415 genes expressed differentially between sexes or estrous states. Unique subsets of these genes were distributed across all 137 transcriptomically defined Esr1+ cell types, including estrous stage-specific ones, that comprise the four populations. We used differentially expressed genes labeling single Esr1+ cell types as entry points to functionally characterize two such cell types, BNSTprTac1/Esr1 and VMHvlCckar/Esr1. We observed that these two cell types, but not the other Esr1+ cell types in these populations, are essential for sex recognition in males and mating in females, respectively. Furthermore, VMHvlCckar/Esr1 cell type projections are distinct from those of other VMHvlEsr1 cell types. Together, projection and functional specialization of dimorphic cell types enables sex hormone-responsive populations to regulate diverse social behaviors.

Deep brain optogenetics without intracranial surgery.

Achieving temporally precise, noninvasive control over specific neural cell types in the deep brain would advance the study of nervous system function. Here we use the potent channelrhodopsin ChRmine to achieve transcranial photoactivation of defined neural circuits, including midbrain and brainstem structures, at unprecedented depths of up to 7 mm with millisecond precision. Using systemic viral delivery of ChRmine, we demonstrate behavioral modulation without surgery, enabling implant-free deep brain optogenetics.

Comprehensive Dual- and Triple-Feature Intersectional Single-Vector Delivery of Diverse Functional Payloads to Cells of Behaving Mammals.

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 in 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 Ca2+ indicators) has thus far fallen short of versatile targeting to cells jointly defined by many individually selected features. Here, 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.

Cortical Observation by Synchronous Multifocal Optical Sampling Reveals Widespread Population Encoding of Actions.

To advance the measurement of distributed neuronal population representations of targeted motor actions on single trials, we developed an optical method (COSMOS) for tracking neural activity in a largely uncharacterized spatiotemporal regime. COSMOS allowed simultaneous recording of neural dynamics at ∼30 Hz from over a thousand near-cellular resolution neuronal sources spread across the entire dorsal neocortex of awake, behaving mice during a three-option lick-to-target task. We identified spatially distributed neuronal population representations spanning the dorsal cortex that precisely encoded ongoing motor actions on single trials. Neuronal correlations measured at video rate using unaveraged, whole-session data had localized spatial structure, whereas trial-averaged data exhibited widespread correlations. Separable modes of neural activity encoded history-guided motor plans, with similar population dynamics in individual areas throughout cortex. These initial experiments illustrate how COSMOS enables investigation of large-scale cortical dynamics and that information about motor actions is widely shared between areas, potentially underlying distributed computations.

Activity in grafted human iPS cell-derived cortical neurons integrated in stroke-injured rat brain regulates motor behavior.

Stem cell transplantation can improve behavioral recovery after stroke in animal models but whether stem cell-derived neurons become functionally integrated into stroke-injured brain circuitry is poorly understood. Here we show that intracortically grafted human induced pluripotent stem (iPS) cell-derived cortical neurons send widespread axonal projections to both hemispheres of rats with ischemic lesions in the cerebral cortex. Using rabies virus-based transsynaptic tracing, we find that at 6 mo after transplantation, host neurons in the contralateral somatosensory cortex receive monosynaptic inputs from grafted neurons. Immunoelectron microscopy demonstrates myelination of the graft-derived axons in the corpus callosum and that their terminals form excitatory, glutamatergic synapses on host cortical neurons. We show that the stroke-induced asymmetry in a sensorimotor (cylinder) test is reversed by transplantation. Light-induced inhibition of halorhodopsin-expressing, grafted neurons does not recreate the impairment, indicating that its reversal is not due to neuronal activity in the graft. However, we find bilateral decrease of motor performance in the cylinder test after light-induced inhibition of either grafted or endogenous halorhodopsin-expressing cortical neurons, located in the same area, and after inhibition of endogenous halorhodopsin-expressing cortical neurons by exposure of their axons to light on the contralateral side. Our data indicate that activity in the grafted neurons, probably mediated through transcallosal connections to the contralateral hemisphere, is involved in maintaining normal motor function. This is an example of functional integration of efferent projections from grafted neurons into the stroke-affected brain's neural circuitry, which raises the possibility that such repair might be achievable also in humans affected by stroke.

Development of an optogenetic toolkit for neural circuit dissection in squirrel monkeys.

Optogenetic tools have opened a rich experimental landscape for understanding neural function and disease. Here, we present the first validation of eight optogenetic constructs driven by recombinant adeno-associated virus (AAV) vectors and a WGA-Cre based dual injection strategy for projection targeting in a widely-used New World primate model, the common squirrel monkey Saimiri sciureus. We observed opsin expression around the local injection site and in axonal projections to downstream regions, as well as transduction to thalamic neurons, resembling expression patterns observed in macaques. Optical stimulation drove strong, reliable excitatory responses in local neural populations for two depolarizing opsins in anesthetized monkeys. Finally, we observed continued, healthy opsin expression for at least one year. These data suggest that optogenetic tools can be readily applied in squirrel monkeys, an important first step in enabling precise, targeted manipulation of neural circuits in these highly trainable, cognitively sophisticated animals. In conjunction with similar approaches in macaques and marmosets, optogenetic manipulation of neural circuits in squirrel monkeys will provide functional, comparative insights into neural circuits which subserve dextrous motor control as well as other adaptive behaviors across the primate lineage. Additionally, development of these tools in squirrel monkeys, a well-established model system for several human neurological diseases, can aid in identifying novel treatment strategies.

Publisher Correction: Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity.

In this Article originally published, owing to a technical error, author affiliations were incorrectly assigned in the HTML version; the PDF was correct. These errors have now been corrected.

Developmental Dysfunction of VIP Interneurons Impairs Cortical Circuits.

GABAergic interneurons play important roles in cortical circuit development. However, there are multiple populations of interneurons and their respective developmental contributions remain poorly explored. Neuregulin 1 (NRG1) and its interneuron-specific receptor ERBB4 are critical genes for interneuron maturation. Using a conditional ErbB4 deletion, we tested the role of vasoactive intestinal peptide (VIP)-expressing interneurons in the postnatal maturation of cortical circuits in vivo. ErbB4 removal from VIP interneurons during development leads to changes in their activity, along with severe dysregulation of cortical temporal organization and state dependence. These alterations emerge during adolescence, and mature animals in which VIP interneurons lack ErbB4 exhibit reduced cortical responses to sensory stimuli and impaired sensory learning. Our data support a key role for VIP interneurons in cortical circuit development and suggest a possible contribution to pathophysiology in neurodevelopmental disorders. These findings provide a new perspective on the role of GABAergic interneuron diversity in cortical development. VIDEO ABSTRACT.

Brain-Derived Neurotrophic Factor in the Mesolimbic Reward Circuitry Mediates Nociception in Chronic Neuropathic Pain.

BACKGROUND: The mesolimbic reward system plays a critical role in modulating nociception; however, its underlying molecular, cellular, and neural circuitry mechanisms remain unknown. METHODS: Chronic constrictive injury (CCI) of the sciatic nerve was used to model neuropathic pain. Projection-specific in vitro recordings in mouse brain slices and in vivo recordings from anesthetized animals were used to measure firing of dopaminergic neurons in the ventral tegmental area (VTA). The role of VTA-nucleus accumbens (NAc) circuitry in nociceptive regulation was assessed using optogenetic and pharmacological manipulations, and the underlying molecular mechanisms were investigated by Western blotting, enzyme-linked immunosorbent assays, and conditional knockdown techniques. RESULTS: c-Fos expression in and firing of contralateral VTA-NAc dopaminergic neurons were elevated in CCI mice, and optogenetic inhibition of these neurons reversed CCI-induced thermal hyperalgesia. CCI increased the expression of brain-derived neurotrophic factor (BDNF) protein but not messenger RNA in the contralateral NAc. This increase was reversed by pharmacological inhibition of VTA dopaminergic neuron activity, which induced an antinociceptive effect that was neutralized by injecting exogenous BDNF into the NAc. Moreover, inhibition of BDNF synthesis in the VTA with anisomycin or selective knockdown of BDNF in the VTA-NAc pathway was antinociceptive in CCI mice. CONCLUSIONS: These results reveal a novel mechanism of nociceptive modulation in the mesolimbic reward circuitry and provide new insight into the neural circuits involved in the processing of nociceptive information.

CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.

Optimal distribution of heterogeneous organelles and cell types within an organ is essential for physiological processes. Unique for the ovary, hormonally regulated folliculogenesis, ovulation, luteal formation/regression and associated vasculature changes lead to tissue remodeling during each reproductive cycle. Using the CLARITY approach and marker immunostaining, we identified individual follicles and corpora lutea in intact ovaries. Monitoring lifetime changes in follicle populations showed age-dependent decreases in total follicles and percentages of advanced follicles. Follicle development from primordial to preovulatory stage was characterized by 3 × 105-fold increases in volume, decreases in roundness, and decreased clustering of same stage follicles. Construction of follicle-vasculature relationship maps indicated age- and gonadotropin-dependent increases in vasculature and branching surrounding follicles. Heterozygous mutant mice with deletion of hypoxia-response element in the vascular endothelial growth factor A (VEGFA) promoter showed defective ovarian vasculature and decreased ovulatory responses. Unilateral intrabursal injection of axitinib, an inhibitor of VEGF receptors, retarded neo-angiogenesis that was associated with defective ovulation in treated ovaries. Our approach uncovers unique features of ovarian architecture and essential roles of vasculature in organizing follicles to allow future studies on normal and diseased human ovaries. Similar approaches could also reveal roles of neo-angiogenesis during embryonic development and tumorigenesis.

Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity.

Intratumoral heterogeneity is a critical factor when diagnosing and treating patients with cancer. Marked differences in the genetic and epigenetic backgrounds of cancer cells have been revealed by advances in genome sequencing, yet little is known about the phenotypic landscape and the spatial distribution of intratumoral heterogeneity within solid tumours. Here, we show that three-dimensional light-sheet microscopy of cleared solid tumours can identify unique patterns of phenotypic heterogeneity, in the epithelial-to-mesenchymal transition and in angiogenesis, at single-cell resolution in whole formalin-fixed paraffin-embedded (FFPE) biopsy samples. We also show that cleared FFPE samples can be re-embedded in paraffin after examination for future use, and that our tumour-phenotyping pipeline can determine tumour stage and stratify patient prognosis from clinical samples with higher accuracy than current diagnostic methods, thus facilitating the design of more efficient cancer therapies.

In Vivo Interrogation of Spinal Mechanosensory Circuits.

Spinal dorsal horn circuits receive, process, and transmit somatosensory information. To understand how specific components of these circuits contribute to behavior, it is critical to be able to directly modulate their activity in unanesthetized in vivo conditions. Here, we develop experimental tools that enable optogenetic control of spinal circuitry in freely moving mice using commonly available materials. We use these tools to examine mechanosensory processing in the spinal cord and observe that optogenetic activation of somatostatin-positive interneurons facilitates both mechanosensory and itch-related behavior, while reversible chemogenetic inhibition of these neurons suppresses mechanosensation. These results extend recent findings regarding the processing of mechanosensory information in the spinal cord and indicate the potential for activity-induced release of the somatostatin neuropeptide to affect processing of itch. The spinal implant approach we describe here is likely to enable a wide range of studies to elucidate spinal circuits underlying pain, touch, itch, and movement.

Optogenetic and chemogenetic strategies for sustained inhibition of pain.

Spatially targeted, genetically-specific strategies for sustained inhibition of nociceptors may help transform pain science and clinical management. Previous optogenetic strategies to inhibit pain have required constant illumination, and chemogenetic approaches in the periphery have not been shown to inhibit pain. Here, we show that the step-function inhibitory channelrhodopsin, SwiChR, can be used to persistently inhibit pain for long periods of time through infrequent transdermally delivered light pulses, reducing required light exposure by >98% and resolving a long-standing limitation in optogenetic inhibition. We demonstrate that the viral expression of the hM4D receptor in small-diameter primary afferent nociceptor enables chemogenetic inhibition of mechanical and thermal nociception thresholds. Finally, we develop optoPAIN, an optogenetic platform to non-invasively assess changes in pain sensitivity, and use this technique to examine pharmacological and chemogenetic inhibition of pain.

LSPS/Optogenetics to Improve Synaptic Connectivity Mapping: Unmasking the Role of Basket Cell-Mediated Feedforward Inhibition.

Neocortical pyramidal cells (PYRs) receive synaptic inputs from many types of GABAergic interneurons. Connections between parvalbumin (PV)-positive, fast-spiking interneurons ("PV cells") and PYRs are characterized by perisomatic synapses and high-amplitude, short-latency IPSCs. Here, we present novel methods to study the functional influence of PV cells on layer 5 PYRs using optogenetics combined with laser-scanning photostimulation (LSPS). First, we examined the strength and spatial distribution of PV-to-PYR inputs. To that end, the fast channelrhodopsin variant AAV5-EF1α-DIO-hChR2(E123T)-eYFP (ChETA) was expressed in PV cells in somatosensory cortex of mice using an adeno-associated virus-based viral construct. Focal blue illumination (100-150 µm half-width) was directed through the microscope objective to excite PV cells along a spatial grid covering layers 2-6, while IPSCs were recorded in layer 5 PYRs. The resulting optogenetic input maps showed evoked PV cell inputs originating from an ∼500-µm-diameter area surrounding the recorded PYR. Evoked IPSCs had the short-latency/high-amplitude characteristic of PV cell inputs. Second, we investigated how PV cell activity modulates PYR output in response to synaptic excitation. We expressed halorhodopsin (eNpHR3.0) in PV cells using the same strategy as for ChETA. Yellow illumination hyperpolarized eNpHR3.0-expressing PV cells, effectively preventing action potential generation and thus decreasing the inhibition of downstream targets. Synaptic input maps onto layer 5 PYRs were acquired using standard glutamate-photolysis LSPS either with or without full-field yellow illumination to silence PV cells. The resulting IPSC input maps selectively lacked short-latency perisomatic inputs, while EPSC input maps showed increased connectivity, particularly from upper layers. This indicates that glutamate uncaging LSPS-based excitatory synaptic maps will consistently underestimate connectivity.

Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity.

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near -65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼ 15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor-based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure-function relationships of the light-gated pore.

Optogenetic Stimulation of Neural Grafts Enhances Neurotransmission and Downregulates the Inflammatory Response in Experimental Stroke Model.

Compelling evidence suggests that transplantation of neural stem cells (NSCs) from multiple sources ameliorates motor deficits after stroke. However, it is currently unknown to what extent the electrophysiological activity of grafted NSC progeny participates in the improvement of motor deficits and whether excitatory phenotypes of the grafted cells are beneficial or deleterious to sensorimotor performances. To address this question, we used optogenetic tools to drive the excitatory outputs of the grafted NSCs and assess the impact on local circuitry and sensorimotor performance. We genetically engineered NSCs to express the Channelrhodopsin-2 (ChR2), a light-gated cation channel that evokes neuronal depolarization and initiation of action potentials with precise temporal control to light stimulation. To test the function of these cells in a stroke model, rats were subjected to an ischemic stroke and grafted with ChR2-NSCs. The grafted NSCs identified with a human-specific nuclear marker survived in the peri-infarct tissue and coexpressed the ChR2 transgene with the neuronal markers TuJ1 and NeuN. Gene expression analysis in stimulated versus vehicle-treated animals showed a differential upregulation of transcripts involved in neurotransmission, neuronal differentiation, regeneration, axonal guidance, and synaptic plasticity. Interestingly, genes involved in the inflammatory response were significantly downregulated. Behavioral analysis demonstrated that chronic optogenetic stimulation of the ChR2-NSCs enhanced forelimb use on the stroke-affected side and motor activity in an open field test. Together these data suggest that excitatory stimulation of grafted NSCs elicits beneficial effects in experimental stroke model through cell replacement and non-cell replacement, anti-inflammatory/neurotrophic effects.

Striatal Cholinergic Interneurons Control Motor Behavior and Basal Ganglia Function in Experimental Parkinsonism.

Despite evidence showing that anticholinergic drugs are of clinical relevance in Parkinson's disease (PD), the causal role of striatal cholinergic interneurons (CINs) in PD pathophysiology remains elusive. Here, we show that optogenetic inhibition of CINs alleviates motor deficits in PD mouse models, providing direct demonstration for their implication in parkinsonian motor dysfunctions. As neural correlates, CIN inhibition in parkinsonian mice differentially impacts the excitability of striatal D1 and D2 medium spiny neurons, normalizes pathological bursting activity in the main basal ganglia output structure, and increases the functional weight of the direct striatonigral pathway in cortical information processing. By contrast, CIN inhibition in non-lesioned mice does not affect locomotor activity, equally modulates medium spiny neuron excitability, and does not modify spontaneous or cortically driven activity in the basal ganglia output, suggesting that the role of these interneurons in motor function is highly dependent on dopamine tone.