Virus stamping for targeted single-cell infection in vitro and in vivo.

Genetic engineering by viral infection of single cells is useful to study complex systems such as the brain. However, available methods for infecting single cells have drawbacks that limit their applications. Here we describe 'virus stamping', in which viruses are reversibly bound to a delivery vehicle-a functionalized glass pipette tip or magnetic nanoparticles in a pipette-that is brought into physical contact with the target cell on a surface or in tissue, using mechanical or magnetic forces. Different single cells in the same tissue can be infected with different viruses and an individual cell can be simultaneously infected with different viruses. We use rabies, lenti, herpes simplex, and adeno-associated viruses to drive expression of fluorescent markers or a calcium indicator in target cells in cell culture, mouse retina, human brain organoid, and the brains of live mice. Virus stamping provides a versatile solution for targeted single-cell infection of diverse cell types, both in vitro and in vivo.

The first stage of cardinal direction selectivity is localized to the dendrites of retinal ganglion cells.

Inferring the direction of image motion is a fundamental component of visual computation and essential for visually guided behavior. In the retina, the direction of image motion is computed in four cardinal directions, but it is not known at which circuit location along the flow of visual information the cardinal direction selectivity first appears. We recorded the concerted activity of the neuronal circuit elements of single direction-selective (DS) retinal ganglion cells at subcellular resolution by combining GCaMP3-functionalized transsynaptic viral tracing and two-photon imaging. While the visually evoked activity of the dendritic segments of the DS cells were direction selective, direction-selective activity was absent in the axon terminals of bipolar cells. Furthermore, the glutamate input to DS cells, recorded using a genetically encoded glutamate sensor, also lacked direction selectivity. Therefore, the first stage in which extraction of a cardinal motion direction occurs is the dendrites of DS cells.

Ambient illumination toggles a neuronal circuit switch in the retina and visual perception at cone threshold.

VIDEO ABSTRACT: Gradual changes in the sensory environment can lead to abrupt changes in brain computations and perception. However, mechanistic understanding of the mediating microcircuits is missing. By sliding through light levels from starlight to daylight, we identify retinal ganglion cell types in the mouse that abruptly and reversibly switch the weighting of center and surround interactions in their receptive field around cone threshold. Two-photon-targeted recordings and genetic and viral tracing experiments revealed that the circuit element responsible for the switch is a large inhibitory neuron that provides direct inhibition to ganglion cells. Our experiments suggest that weak excitatory input via electrical synapses together with the spiking threshold in inhibitory cells act as a switch. We also reveal a switch-like component in the spatial integration properties of human vision at cone threshold. This work demonstrates that circuits in the retina can quickly and reversibly switch between two distinct states, implementing distinct perceptual regimes at different light levels.

Spatially asymmetric reorganization of inhibition establishes a motion-sensitive circuit.

Spatial asymmetries in neural connectivity have an important role in creating basic building blocks of neuronal processing. A key circuit module of directionally selective (DS) retinal ganglion cells is a spatially asymmetric inhibitory input from starburst amacrine cells. It is not known how and when this circuit asymmetry is established during development. Here we photostimulate mouse starburst cells targeted with channelrhodopsin-2 (refs 6-8) while recording from a single genetically labelled type of DS cell. We follow the spatial distribution of synaptic strengths between starburst and DS cells during early postnatal development before these neurons can respond to a physiological light stimulus, and confirm connectivity by monosynaptically restricted trans-synaptic rabies viral tracing. We show that asymmetry develops rapidly over a 2-day period through an intermediate state in which random or symmetric synaptic connections have been established. The development of asymmetry involves the spatially selective reorganization of inhibitory synaptic inputs. Intriguingly, the spatial distribution of excitatory synaptic inputs from starburst cells is significantly more symmetric than that of the inhibitory inputs at the end of this developmental period. Our work demonstrates a rapid developmental switch from a symmetric to asymmetric input distribution for inhibition in the neural circuit of a principal cell.

Genetically timed, activity-sensor and rainbow transsynaptic viral tools.

We developed retrograde, transsynaptic pseudorabies viruses (PRVs) with genetically encoded activity sensors that optically report the activity of connected neurons among spatially intermingled neurons in the brain. Next we engineered PRVs to express two differentially colored fluorescent proteins in a time-shifted manner to define a time period early after infection to investigate neural activity. Finally we used multiple-colored PRVs to differentiate and dissect the complex architecture of brain regions.

Local retinal circuits of melanopsin-containing ganglion cells identified by transsynaptic viral tracing.

Intrinsically photosensitive melanopsin-containing retinal ganglion cells (ipRGCs) control important physiological processes, including the circadian rhythm, the pupillary reflex, and the suppression of locomotor behavior (reviewed in [1]). ipRGCs are also activated by classical photoreceptors, the rods and cones, through local retinal circuits [2, 3]. ipRGCs can be transsynaptically labeled through the pupillary-reflex circuit with the derivatives of the Bartha strain of the alphaherpesvirus pseudorabies virus(PRV) [4, 5] that express GFP [6-12]. Bartha-strain derivatives spread only in the retrograde direction [13]. There is evidence that infected cells function normally for a while during GFP expression [7]. Here we combine transsynaptic PRV labeling, two-photon laser microscopy, and electrophysiological techniques to trace the local circuit of different ipRGC subtypes in the mouse retina and record light-evoked activity from the transsynaptically labeled ganglion cells. First, we show that ipRGCs are connected by monostratified amacrine cells that provide strong inhibition from classical-photoreceptor-driven circuits. Second, we show evidence that dopaminergic interplexiform cells are synaptically connected to ipRGCs. The latter finding provides a circuitry link between light-dark adaptation and ipRGC function.

Predominance of supraspinal innervation of the left ovary.

In our previous studies using the viral transneuronal tracing technique we demonstrated the spinal and supraspinal components of the ovarian innervation. Since increasing number of data indicate the presence of morphological and functional laterality in the control of gonadal functions, we aimed to investigate whether cerebral structures trans-synaptically involved in the innervation of the ovary exhibit asymmetry or not. In one of the studies the left or the right ovary was injected with the red fluorescent protein expressing pseudorabies virus and the number of infected "red" autofluorescent neurons from the right and the left ovary was compared. In another study in order to have distinct labeling of cell groups connected with the right- and left-sided ovary in the same animal, a dual viral labeling was applied. The left- and right-sided ovary were inoculated with genetically engineered pseudorabies virus expressing a red fluorescent protein or a green fluorescent protein gene. Viral infection of brain nuclei including the dorsal vagal nucleus, caudal raphe nuclei, A5 noradrenergic cell group, hypothalamic paraventricular nucleus, from the left ovary in each case was enhanced when compared with labeling from the right gonad. Data suggest a predominance in the supraspinal innervation of the left ovary.