Hindbrain modules differentially transform activity of single collicular neurons to coordinate movements.

Seemingly simple behaviors such as swatting a mosquito or glancing at a signpost involve the precise coordination of multiple body parts. Neural control of coordinated movements is widely thought to entail transforming a desired overall displacement into displacements for each body part. Here we reveal a different logic implemented in the mouse gaze system. Stimulating superior colliculus (SC) elicits head movements with stereotyped displacements but eye movements with stereotyped endpoints. This is achieved by individual SC neurons whose branched axons innervate modules in medulla and pons that drive head movements with stereotyped displacements and eye movements with stereotyped endpoints, respectively. Thus, single neurons specify a mixture of endpoints and displacements for different body parts, not overall displacement, with displacements for different body parts computed at distinct anatomical stages. Our study establishes an approach for unraveling motor hierarchies and identifies a logic for coordinating movements and the resulting pose.

Orientation columns in the mouse superior colliculus.

More than twenty types of retinal ganglion cells conduct visual information from the eye to the rest of the brain. Each retinal ganglion cell type tessellates the retina in a regular mosaic, so that every point in visual space is processed for visual primitives such as contrast and motion. This information flows to two principal brain centres: the visual cortex and the superior colliculus. The superior colliculus plays an evolutionarily conserved role in visual behaviours, but its functional architecture is poorly understood. Here we report on population recordings of visual responses from neurons in the mouse superior colliculus. Many neurons respond preferentially to lines of a certain orientation or movement axis. We show that cells with similar orientation preferences form large patches that span the vertical thickness of the retinorecipient layers. This organization is strikingly different from the randomly interspersed orientation preferences in the mouse's visual cortex; instead, it resembles the orientation columns observed in the visual cortices of large mammals. Notably, adjacent superior colliculus orientation columns have only limited receptive field overlap. This is in contrast to the organization of visual cortex, where each point in the visual field activates neurons with all preferred orientations. Instead, the superior colliculus favours specific contour orientations within ∼30° regions of the visual field, a finding with implications for behavioural responses mediated by this brain centre.

A hub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans.

Innate social behaviours emerge from neuronal circuits that interpret sensory information on the basis of an individual's own genotype, sex and experience. The regulated aggregation behaviour of the nematode Caenorhabditis elegans, a simple animal with only 302 neurons, is an attractive system to analyse these circuits. Wild social strains of C. elegans aggregate in the presence of specific sensory cues, but solitary strains do not. Here we identify the RMG inter/motor neuron as the hub of a regulated circuit that controls aggregation and related behaviours. RMG is the central site of action of the neuropeptide receptor gene npr-1, which distinguishes solitary strains (high npr-1 activity) from wild social strains (low npr-1 activity); high RMG activity is essential for all aspects of social behaviour. Anatomical gap junctions connect RMG to several classes of sensory neurons known to promote aggregation, and to ASK sensory neurons, which are implicated in male attraction to hermaphrodite pheromones. We find that ASK neurons respond directly to pheromones, and that high RMG activity enhances ASK responses in social strains, causing hermaphrodite attraction to pheromones at concentrations that repel solitary hermaphrodites. The coordination of social behaviours by RMG suggests an anatomical hub-and-spoke model for sensory integration in aggregation, and points to functions for related circuit motifs in the C. elegans wiring diagram.

GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems.

The identification of synaptic partners is challenging in dense nerve bundles, where many processes occupy regions beneath the resolution of conventional light microscopy. To address this difficulty, we have developed GRASP, a system to label membrane contacts and synapses between two cells in living animals. Two complementary fragments of GFP are expressed on different cells, tethered to extracellular domains of transmembrane carrier proteins. When the complementary GFP fragments are fused to ubiquitous transmembrane proteins, GFP fluorescence appears uniformly along membrane contacts between the two cells. When one or both GFP fragments are fused to synaptic transmembrane proteins, GFP fluorescence is tightly localized to synapses. GRASP marks known synaptic contacts in C. elegans, correctly identifies changes in mutants with altered synaptic specificity, and can uncover new information about synaptic locations as confirmed by electron microscopy. GRASP may prove particularly useful for defining connectivity in complex nervous systems.

Transcriptional profiling of mouse projection neurons with VECTORseq.

Existing techniques for transcriptional profiling of projection neurons could be applied to only one neuronal population per experiment. To increase throughput, we developed VECTORseq, which repurposes retrogradely infecting viruses to deliver multiplexable RNA barcodes, enabling projection anatomy to be read out in single-cell datasets. In this protocol, we describe the delivery of viral barcodes to mouse brain to label different projection neurons. We then detail single-cell or nuclei isolation for sequencing, followed by the analysis of single-cell sequencing data. For complete details on the use and execution of this protocol, please refer to Cheung et al. (2021).

Superior colliculus drives stimulus-evoked directionally biased saccades and attempted head movements in head-fixed mice.

Animals investigate their environments by directing their gaze towards salient stimuli. In the prevailing view, mouse gaze shifts entail head rotations followed by brainstem-mediated eye movements, including saccades to reset the eyes. These 'recentering' saccades are attributed to head movement-related vestibular cues. However, microstimulating mouse superior colliculus (SC) elicits directed head and eye movements resembling SC-dependent sensory-guided gaze shifts in other species, suggesting that mouse gaze shifts may be more flexible than has been recognized. We investigated this possibility by tracking eye and attempted head movements in a head-fixed preparation that eliminates head movement-related sensory cues. We found tactile stimuli evoke directionally biased saccades coincident with attempted head rotations. Differences in saccade endpoints across stimuli are associated with distinct stimulus-dependent relationships between initial eye position and saccade direction and amplitude. Optogenetic perturbations revealed SC drives these gaze shifts. Thus, head-fixed mice make sensory-guided, SC-dependent gaze shifts involving coincident, directionally biased saccades and attempted head movements. Our findings uncover flexibility in mouse gaze shifts and provide a foundation for studying head-eye coupling.

Virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq) defines projection neurons involved in sensorimotor integration.

Behavior arises from concerted activity throughout the brain. Consequently, a major focus of modern neuroscience is defining the physiology and behavioral roles of projection neurons linking different brain areas. Single-cell RNA sequencing has facilitated these efforts by revealing molecular determinants of cellular physiology and markers that enable genetically targeted perturbations such as optogenetics, but existing methods for sequencing defined projection populations are low throughput, painstaking, and costly. We developed a straightforward, multiplexed approach, virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq). VECTORseq repurposes commercial retrogradely infecting viruses typically used to express functional transgenes (e.g., recombinases and fluorescent proteins) by treating viral transgene mRNA as barcodes within single-cell datasets. VECTORseq is compatible with different viral families, resolves multiple populations with different projection targets in one sequencing run, and identifies cortical and subcortical excitatory and inhibitory projection populations. Our study provides a roadmap for high-throughput identification of neuronal subtypes based on connectivity.

Soma-Targeted Imaging of Neural Circuits by Ribosome Tethering.

Neuroscience relies on techniques for imaging the structure and dynamics of neural circuits, but the cell bodies of individual neurons are often obscured by overlapping fluorescence from axons and dendrites in surrounding neuropil. Here, we describe two strategies for using the ribosome to restrict the expression of fluorescent proteins to the neuronal soma. We show first that a ribosome-tethered nanobody can be used to trap GFP in the cell body, thereby enabling direct visualization of previously undetectable GFP fluorescence. We then design a ribosome-tethered GCaMP for imaging calcium dynamics. We show that this reporter faithfully tracks somatic calcium dynamics in the mouse brain while eliminating cross-talk between neurons caused by contaminating neuropil. In worms, this reporter enables whole-brain imaging with faster kinetics and brighter fluorescence than commonly used nuclear GCaMPs. These two approaches provide a general way to enhance the specificity of imaging in neurobiology.

New technologies for imaging synaptic partners.

Understanding the brain will require unraveling its synaptic circuitry, but methods that can reliably identify connected neurons are often excruciatingly slow. Although light microscopy can provide much higher throughput, synapses are smaller than the diffraction limit and cannot readily be assigned to particular presynaptic and postsynaptic cells without specialized labeling methods. Here we review the ongoing development of techniques that allow direct imaging of neural networks by specifically marking connected cells or their synapses.

Caenorhabditis elegans SID-2 is required for environmental RNA interference.

In plants and in the nematode Caenorhabditis elegans, an RNAi signal can trigger gene silencing in cells distant from the site where silencing is initiated. In plants, this signal is known to be a form of dsRNA, and the signal is most likely a form of dsRNA in C. elegans as well. Furthermore, in C. elegans, dsRNA present in the environment or expressed in ingested bacteria is sufficient to trigger RNAi (environmental RNAi). Ingestion and soaking delivery of dsRNA has also been described for other invertebrates. Here we report the identification and characterization of SID-2, an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans. SID-2, when expressed in the environmental RNAi defective species Caenorhabditis briggsae, confers environmental RNAi.

Analysis of mouse embryonic patterning and morphogenesis by forward genetics.

Many aspects of the genetic control of mammalian embryogenesis cannot be extrapolated from other animals. Taking a forward genetic approach, we have induced recessive mutations by treatment of mice with ethylnitrosourea and have identified 43 mutations that affect early morphogenesis and patterning, including 38 genes that have not been studied previously. The molecular lesions responsible for 14 mutations were identified, including mutations in nine genes that had not been characterized previously. Some mutations affect vertebrate-specific components of conserved signaling pathways; for example, at least five mutations affect previously uncharacterized regulators of the Sonic hedgehog (Shh) pathway. Approximately half of all of the mutations affect the initial establishment of the body plan, and several of these produce phenotypes that have not been described previously. A large fraction of the genes identified affect cell migration, cellular organization, and cell structure. The findings indicate that phenotype-based genetic screens provide a direct and unbiased method to identify essential regulators of mammalian development.

Transport of dsRNA into cells by the transmembrane protein SID-1.

RNA interference (RNAi) spreads systemically in plants and nematodes to silence gene expression distant from the site of initiation. We previously identified a gene, sid-1, essential for systemic but not cell-autonomous RNAi in Caenorhabditis elegans. Here, we demonstrate that SID-1 is a multispan transmembrane protein that sensitizes Drosophila cells to soaking RNAi with a potency that is dependent on double-stranded RNA (dsRNA) length. Further analyses revealed that SID-1 enables passive cellular uptake of dsRNA. These data indicate that systemic RNAi in C. elegans involves SID-1-mediated intercellular transport of dsRNA.