Voltage imaging in the olfactory bulb using transgenic mouse lines expressing the genetically encoded voltage indicator ArcLight.

Genetically encoded voltage indicators (GEVIs) allow optical recordings of membrane potential changes in defined cell populations. Transgenic reporter animals that facilitate precise and repeatable targeting with high expression levels would further the use of GEVIs in the in vivo mammalian brain. However, the literature on developing and applying transgenic mouse lines as vehicles for GEVI expression is limited. Here we report the first in vivo experiments using a transgenic reporter mouse for the GEVI ArcLight, which utilizes a Cre/tTA dependent expression system (TIGRE 1.0). We developed two mouse lines with ArcLight expression restricted to either olfactory receptor neurons, or a subpopulation of interneurons located in the granule and glomerular layers in the olfactory bulb. The ArcLight expression in these lines was sufficient for in vivo imaging of odorant responses in single trials using epifluorescence and 2-photon imaging. The voltage responses were odor-specific and concentration-dependent, which supported earlier studies about perceptual transformations carried out by the bulb that used calcium sensors of neural activity. This study demonstrates that the ArcLight transgenic line is a flexible genetic tool that can be used to record the neuronal electrical activity of different cell types with a signal-to-noise ratio that is comparable to previous reports using viral transduction.

A collection of genetic mouse lines and related tools for inducible and reversible intersectional mis-expression.

Thanks to many advances in genetic manipulation, mouse models have become very powerful in their ability to interrogate biological processes. In order to precisely target expression of a gene of interest to particular cell types, intersectional genetic approaches using two promoter/enhancers unique to a cell type are ideal. Within these methodologies, variants that add temporal control of gene expression are the most powerful. We describe the development, validation and application of an intersectional approach that involves three transgenes, requiring the intersection of two promoter/enhancers to target gene expression to precise cell types. Furthermore, the approach uses available lines expressing tTA/rTA to control the timing of gene expression based on whether doxycycline is absent or present, respectively. We also show that the approach can be extended to other animal models, using chicken embryos. We generated three mouse lines targeted at the Tigre (Igs7) locus with TRE-loxP-tdTomato-loxP upstream of three genes (p21, DTA and Ctgf), and combined them with Cre and tTA/rtTA lines that target expression to the cerebellum and limbs. Our tools will facilitate unraveling biological questions in multiple fields and organisms.

Genetic Identification of Vagal Sensory Neurons That Control Feeding.

Energy homeostasis requires precise measurement of the quantity and quality of ingested food. The vagus nerve innervates the gut and can detect diverse interoceptive cues, but the identity of the key sensory neurons and corresponding signals that regulate food intake remains unknown. Here, we use an approach for target-specific, single-cell RNA sequencing to generate a map of the vagal cell types that innervate the gastrointestinal tract. We show that unique molecular markers identify vagal neurons with distinct innervation patterns, sensory endings, and function. Surprisingly, we find that food intake is most sensitive to stimulation of mechanoreceptors in the intestine, whereas nutrient-activated mucosal afferents have no effect. Peripheral manipulations combined with central recordings reveal that intestinal mechanoreceptors, but not other cell types, potently and durably inhibit hunger-promoting AgRP neurons in the hypothalamus. These findings identify a key role for intestinal mechanoreceptors in the regulation of feeding.

Voltage imaging and optogenetics reveal behaviour-dependent changes in hippocampal dynamics.

A technology that simultaneously records membrane potential from multiple neurons in behaving animals will have a transformative effect on neuroscience research1,2. Genetically encoded voltage indicators are a promising tool for these purposes; however, these have so far been limited to single-cell recordings with a marginal signal-to-noise ratio in vivo3-5. Here we developed improved near-infrared voltage indicators, high-speed microscopes and targeted gene expression schemes that enabled simultaneous in vivo recordings of supra- and subthreshold voltage dynamics in multiple neurons in the hippocampus of behaving mice. The reporters revealed subcellular details of back-propagating action potentials and correlations in subthreshold voltage between multiple cells. In combination with stimulation using optogenetics, the reporters revealed changes in neuronal excitability that were dependent on the behavioural state, reflecting the interplay of excitatory and inhibitory synaptic inputs. These tools open the possibility for detailed explorations of network dynamics in the context of behaviour. Fig. 1 PHOTOACTIVATED QUASAR3 (PAQUASAR3) REPORTS NEURONAL ACTIVITY IN VIVO.: a, Schematic of the paQuasAr3 construct. b, Photoactivation by blue light enhanced voltage signals excited by red light in cultured neurons that expressed paQuasAr3 (representative example of n = 4 cells). c, Model of the photocycle of paQuasAr3. d, Confocal images of sparsely expressed paQuasAr3 in brain slices. Scale bars, 50 µm. Representative images, experiments were repeated in n = 3 mice. e, Simultaneous fluorescence and patch-clamp recordings from a neuron expressing paQuasAr3 in acute brain slice. Top, magnification of boxed regions. Schematic shows brain slice, patch pipette and microscope objective. f, Simultaneous fluorescence and patch-clamp recordings of inhibitory post synaptic potentials in an L2-3 neuron induced by electrical stimulation of L5-6 in acute slice. g, Normalized change in fluorescence (ΔF/F) and SNR of optically recorded post-synaptic potentials (PSPs) as a function of the amplitude of the post-synaptic potentials. The voltage sensitivity was ΔF/F = 40 ± 1.7% per 100 mV. The SNR was 0.93 ± 0.07 per 1 mV in a 1-kHz bandwidth (n = 42 post-synaptic potentials from 5 cells, data are mean ± s.d.). Schematic shows brain slice, patch pipette, field stimulation electrodes and microscope objective. h, Optical measurements of paQuasAr3 fluorescence in the CA1 region of the hippocampus (top) and glomerular layer of the olfactory bulb (bottom) of anaesthetized mice (representative traces from n = 7 CA1 cells and n = 13 olfactory bulb cells, n = 3 mice). Schematics show microscope objective and the imaged brain region. i, STA fluorescence from 88 spikes in a CA1 oriens neuron. j, Frames from the STA video showing the delay in the back-propagating action potential in the dendrites relative to the soma. k, Sub-Nyquist fitting of the action potential delay and width shows electrical compartmentalization in the dendrites. Experiments in k-m were repeated in n = 2 cells from n = 2 mice.

A gut-to-brain signal of fluid osmolarity controls thirst satiation.

Satiation is the process by which eating and drinking reduce appetite. For thirst, oropharyngeal cues have a critical role in driving satiation by reporting to the brain the volume of fluid that has been ingested1-12. By contrast, the mechanisms that relay the osmolarity of ingested fluids remain poorly understood. Here we show that the water and salt content of the gastrointestinal tract are precisely measured and then rapidly communicated to the brain to control drinking behaviour in mice. We demonstrate that this osmosensory signal is necessary and sufficient for satiation during normal drinking, involves the vagus nerve and is transmitted to key forebrain neurons that control thirst and vasopressin secretion. Using microendoscopic imaging, we show that individual neurons compute homeostatic need by integrating this gastrointestinal osmosensory information with oropharyngeal and blood-borne signals. These findings reveal how the fluid homeostasis system monitors the osmolarity of ingested fluids to dynamically control drinking behaviour.

Shared and distinct transcriptomic cell types across neocortical areas.

The neocortex contains a multitude of cell types that are segregated into layers and functionally distinct areas. To investigate the diversity of cell types across the mouse neocortex, here we analysed 23,822 cells from two areas at distant poles of the mouse neocortex: the primary visual cortex and the anterior lateral motor cortex. We define 133 transcriptomic cell types by deep, single-cell RNA sequencing. Nearly all types of GABA (γ-aminobutyric acid)-containing neurons are shared across both areas, whereas most types of glutamatergic neurons were found in one of the two areas. By combining single-cell RNA sequencing and retrograde labelling, we match transcriptomic types of glutamatergic neurons to their long-range projection specificity. Our study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct areas of the adult mouse cortex.

A Suite of Transgenic Driver and Reporter Mouse Lines with Enhanced Brain-Cell-Type Targeting and Functionality.

Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

Cell-nonautonomous local and systemic responses to cell arrest enable long-bone catch-up growth in developing mice.

Catch-up growth after insults to growing organs is paramount to achieving robust body proportions. In fly larvae, injury to individual tissues is followed by local and systemic compensatory mechanisms that allow the damaged tissue to regain normal proportions with other tissues. In vertebrates, local catch-up growth has been described after transient reduction of bone growth, but the underlying cellular responses are controversial. We developed an approach to study catch-up growth in foetal mice in which mosaic expression of the cell cycle suppressor p21 is induced in the cartilage cells (chondrocytes) that drive long-bone elongation. By specifically targeting p21 expression to left hindlimb chondrocytes, the right limb serves as an internal control. Unexpectedly, left-right limb symmetry remained normal, revealing deployment of compensatory mechanisms. Above a certain threshold of insult, an orchestrated response was triggered involving local enhancement of bone growth and systemic growth reduction that ensured that body proportions were maintained. The local response entailed hyperproliferation of spared left limb chondrocytes that was associated with reduced chondrocyte density. The systemic effect involved impaired placental function and IGF signalling, revealing bone-placenta communication. Therefore, vertebrates, like invertebrates, can mount coordinated local and systemic responses to developmental insults that ensure that normal body proportions are maintained.

Generation of a whole-brain atlas for the cholinergic system and mesoscopic projectome analysis of basal forebrain cholinergic neurons.

The cholinergic system in the brain plays crucial roles in regulating sensory and motor functions as well as cognitive behaviors by modulating neuronal activity. Understanding the organization of the cholinergic system requires a complete map of cholinergic neurons and their axon arborizations throughout the entire brain at the level of single neurons. Here, we report a comprehensive whole-brain atlas of the cholinergic system originating from various cortical and subcortical regions of the mouse brain. Using genetically labeled cholinergic neurons together with whole-brain reconstruction of optical images at 2-µm resolution, we obtained quantification of the number and soma volume of cholinergic neurons in 22 brain areas. Furthermore, by reconstructing the complete axonal arbors of fluorescently labeled single neurons from a subregion of the basal forebrain at 1-µm resolution, we found that their projections to the forebrain and midbrain showed neuronal subgroups with distinct projection specificity and diverse arbor distribution within the same projection area. These results suggest the existence of distinct subtypes of cholinergic neurons that serve different regulatory functions in the brain and illustrate the usefulness of complete reconstruction of neuronal distribution and axon projections at the mesoscopic level.

Local processing in neurites of VGluT3-expressing amacrine cells differentially organizes visual information.

Neurons receive synaptic inputs on extensive neurite arbors. How information is organized across arbors and how local processing in neurites contributes to circuit function is mostly unknown. Here, we used two-photon Ca2+ imaging to study visual processing in VGluT3-expressing amacrine cells (VG3-ACs) in the mouse retina. Contrast preferences (ON vs. OFF) varied across VG3-AC arbors depending on the laminar position of neurites, with ON responses preferring larger stimuli than OFF responses. Although arbors of neighboring cells overlap extensively, imaging population activity revealed continuous topographic maps of visual space in the VG3-AC plexus. All VG3-AC neurites responded strongly to object motion, but remained silent during global image motion. Thus, VG3-AC arbors limit vertical and lateral integration of contrast and location information, respectively. We propose that this local processing enables the dense VG3-AC plexus to contribute precise object motion signals to diverse targets without distorting target-specific contrast preferences and spatial receptive fields.

An R-CaMP1.07 reporter mouse for cell-type-specific expression of a sensitive red fluorescent calcium indicator.

Genetically encoded calcium indicators (GECIs) enable imaging of in vivo brain cell activity with high sensitivity and specificity. In contrast to viral infection or in utero electroporation, indicator expression in transgenic reporter lines is induced noninvasively, reliably, and homogenously. Recently, Cre/tTA-dependent reporter mice were introduced, which provide high-level expression of green fluorescent GECIs in a cell-type-specific and inducible manner when crossed with Cre and tTA driver mice. Here, we generated and characterized the first red-shifted GECI reporter line of this type using R-CaMP1.07, a red fluorescent indicator that is efficiently two-photon excited above 1000 nm. By crossing the new R-CaMP1.07 reporter line to Cre lines driving layer-specific expression in neocortex we demonstrate its high fidelity for reporting action potential firing in vivo, long-term stability over months, and versatile use for functional imaging of excitatory neurons across all cortical layers, especially in the previously difficult to access layers 4 and 6.

Dynamics of embryonic stem cell differentiation inferred from single-cell transcriptomics show a series of transitions through discrete cell states.

The complexity of gene regulatory networks that lead multipotent cells to acquire different cell fates makes a quantitative understanding of differentiation challenging. Using a statistical framework to analyze single-cell transcriptomics data, we infer the gene expression dynamics of early mouse embryonic stem (mES) cell differentiation, uncovering discrete transitions across nine cell states. We validate the predicted transitions across discrete states using flow cytometry. Moreover, using live-cell microscopy, we show that individual cells undergo abrupt transitions from a naïve to primed pluripotent state. Using the inferred discrete cell states to build a probabilistic model for the underlying gene regulatory network, we further predict and experimentally verify that these states have unique response to perturbations, thus defining them functionally. Our study provides a framework to infer the dynamics of differentiation from single cell transcriptomics data and to build predictive models of the gene regulatory networks that drive the sequence of cell fate decisions during development.

Genetically Targeted All-Optical Electrophysiology with a Transgenic Cre-Dependent Optopatch Mouse.

Recent advances in optogenetics have enabled simultaneous optical perturbation and optical readout of membrane potential in diverse cell types. Here, we develop and characterize a Cre-dependent transgenic Optopatch2 mouse line that we call Floxopatch. The animals expressed a blue-shifted channelrhodopsin, CheRiff, and a near infrared Archaerhodopsin-derived voltage indicator, QuasAr2, via targeted knock-in at the rosa26 locus. In Optopatch-expressing animals, we tested for overall health, genetically targeted expression, and function of the optogenetic components. In offspring of Floxopatch mice crossed with a variety of Cre driver lines, we observed spontaneous and optically evoked activity in vitro in acute brain slices and in vivo in somatosensory ganglia. Cell-type-specific expression allowed classification and characterization of neuronal subtypes based on their firing patterns. The Floxopatch mouse line is a useful tool for fast and sensitive characterization of neural activity in genetically specified cell types in intact tissue. SIGNIFICANCE STATEMENT: Optical recordings of neural activity offer the promise of rapid and spatially resolved mapping of neural function. Calcium imaging has been widely applied in this mode, but is insensitive to the details of action potential waveforms and subthreshold events. Simultaneous optical perturbation and optical readout of single-cell electrical activity ("Optopatch") has been demonstrated in cultured neurons and in organotypic brain slices, but not in acute brain slices or in vivo Here, we describe a transgenic mouse in which expression of Optopatch constructs is controlled by the Cre-recombinase enzyme. This animal enables fast and robust optical measurements of single-cell electrical excitability in acute brain slices and in somatosensory ganglia in vivo, opening the door to rapid optical mapping of neuronal excitability.

Adult mouse cortical cell taxonomy revealed by single cell transcriptomics.

Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. We constructed a cellular taxonomy of one cortical region, primary visual cortex, in adult mice on the basis of single-cell RNA sequencing. We identified 49 transcriptomic cell types, including 23 GABAergic, 19 glutamatergic and 7 non-neuronal types. We also analyzed cell type-specific mRNA processing and characterized genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we found that some of our transcriptomic cell types displayed specific and differential electrophysiological and axon projection properties, thereby confirming that the single-cell transcriptomic signatures can be associated with specific cellular properties.

Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance.

An increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types. However, current approaches for this are still limited in functionality and specificity. Here we utilize several intersectional strategies to generate multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity. We developed driver and double reporter mouse lines and viral vectors using the Cre/Flp and Cre/Dre double recombinase systems and established a new, retargetable genomic locus, TIGRE, which allowed the generation of a large set of Cre/tTA-dependent reporter lines expressing fluorescent proteins, genetically encoded calcium, voltage, or glutamate indicators, and optogenetic effectors, all at substantially higher levels than before. High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws. These novel transgenic lines greatly expand the ability to monitor and manipulate neuronal activities with increased specificity.

Experience-dependent remodeling of basket cell networks in the dentate gyrus.

The structural organization of neural circuits is strongly influenced by experience, but the underlying mechanisms are incompletely understood. We found that, in the developing dentate gyrus (DG), excitatory drive promotes the somatic innervation of principal granule cells (GCs) by parvalbumin (PV)-positive basket cells. In contrast, presynaptic differentiation of GCs and interneuron subtypes that inhibit GC dendrites is largely resistant to loss of glutamatergic neurotransmission. The networks of PV basket cells in the DG are regulated by vesicular release from projection entorhinal cortical neurons and, at least in part, by NMDA receptors in interneurons. Finally, we present evidence that glutamatergic inputs and NMDA receptors regulate these networks through a presynaptic mechanism that appears to control the branching of interneuron axons. Our results provide insights into how cortical activity tunes the inhibition in a subcortical circuit and reveal new principles of interneuron plasticity.

Anatomical characterization of Cre driver mice for neural circuit mapping and manipulation.

Significant advances in circuit-level analyses of the brain require tools that allow for labeling, modulation of gene expression, and monitoring and manipulation of cellular activity in specific cell types and/or anatomical regions. Large-scale projects and individual laboratories have produced hundreds of gene-specific promoter-driven Cre mouse lines invaluable for enabling genetic access to subpopulations of cells in the brain. However, the potential utility of each line may not be fully realized without systematic whole brain characterization of transgene expression patterns. We established a high-throughput in situ hybridization (ISH), imaging and data processing pipeline to describe whole brain gene expression patterns in Cre driver mice. Currently, anatomical data from over 100 Cre driver lines are publicly available via the Allen Institute's Transgenic Characterization database, which can be used to assist researchers in choosing the appropriate Cre drivers for functional, molecular, or connectional studies of different regions and/or cell types in the brain.

Scalable control of mounting and attack by Esr1+ neurons in the ventromedial hypothalamus.

Social behaviours, such as aggression or mating, proceed through a series of appetitive and consummatory phases that are associated with increasing levels of arousal. How such escalation is encoded in the brain, and linked to behavioural action selection, remains an unsolved problem in neuroscience. The ventrolateral subdivision of the murine ventromedial hypothalamus (VMHvl) contains neurons whose activity increases during male-male and male-female social encounters. Non-cell-type-specific optogenetic activation of this region elicited attack behaviour, but not mounting. We have identified a subset of VMHvl neurons marked by the oestrogen receptor 1 (Esr1), and investigated their role in male social behaviour. Optogenetic manipulations indicated that Esr1(+) (but not Esr1(-)) neurons are sufficient to initiate attack, and that their activity is continuously required during ongoing agonistic behaviour. Surprisingly, weaker optogenetic activation of these neurons promoted mounting behaviour, rather than attack, towards both males and females, as well as sniffing and close investigation. Increasing photostimulation intensity could promote a transition from close investigation and mounting to attack, within a single social encounter. Importantly, time-resolved optogenetic inhibition experiments revealed requirements for Esr1(+) neurons in both the appetitive (investigative) and the consummatory phases of social interactions. Combined optogenetic activation and calcium imaging experiments in vitro, as well as c-Fos analysis in vivo, indicated that increasing photostimulation intensity increases both the number of active neurons and the average level of activity per neuron. These data suggest that Esr1(+) neurons in VMHvl control the progression of a social encounter from its appetitive through its consummatory phases, in a scalable manner that reflects the number or type of active neurons in the population.

Olfactory cortical neurons read out a relative time code in the olfactory bulb.

Odor stimulation evokes complex spatiotemporal activity in the olfactory bulb, suggesting that both the identity of activated neurons and the timing of their activity convey information about odors. However, whether and how downstream neurons decipher these temporal patterns remains unknown. We addressed this question by measuring the spiking activity of downstream neurons while optogenetically stimulating two foci in the olfactory bulb with varying relative timing in mice. We found that the overall spike rates of piriform cortex neurons (PCNs) were sensitive to the relative timing of activation. Posterior PCNs showed higher sensitivity to relative input times than neurons in the anterior piriform cortex. In contrast, olfactory bulb neurons rarely showed such sensitivity. Thus, the brain can transform a relative time code in the periphery into a firing rate-based representation in central brain areas, providing evidence for the relevance of a relative time-based code in the olfactory bulb.

A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo.

Fluorescent calcium indicator proteins, such as GCaMP3, allow imaging of activity in genetically defined neuronal populations. GCaMP3 can be expressed using various gene delivery methods, such as viral infection or electroporation. However, these methods are invasive and provide inhomogeneous and nonstationary expression. Here, we developed a genetic reporter mouse, Ai38, which expresses GCaMP3 in a Cre-dependent manner from the ROSA26 locus, driven by a strong CAG promoter. Crossing Ai38 with appropriate Cre mice produced robust GCaMP3 expression in defined cell populations in the retina, cortex, and cerebellum. In the primary visual cortex, visually evoked GCaMP3 signals showed normal orientation and direction selectivity. GCaMP3 signals were rapid, compared with virally expressed GCaMP3 and synthetic calcium indicators. In the retina, Ai38 allowed imaging spontaneous calcium waves in starburst amacrine cells during development, and light-evoked responses in ganglion cells in adult tissue. Our results show that the Ai38 reporter mouse provides a flexible method for targeted expression of GCaMP3.