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.

PRESYNAPTIC NETWORKS. Single-cell-initiated monosynaptic tracing reveals layer-specific cortical network modules.

Individual cortical neurons can selectively respond to specific environmental features, such as visual motion or faces. How this relates to the selectivity of the presynaptic network across cortical layers remains unclear. We used single-cell-initiated, monosynaptically restricted retrograde transsynaptic tracing with rabies viruses expressing GCaMP6s to image, in vivo, the visual motion-evoked activity of individual layer 2/3 pyramidal neurons and their presynaptic networks across layers in mouse primary visual cortex. Neurons within each layer exhibited similar motion direction preferences, forming layer-specific functional modules. In one-third of the networks, the layer modules were locked to the direction preference of the postsynaptic neuron, whereas for other networks the direction preference varied by layer. Thus, there exist feature-locked and feature-variant cortical networks.

Integration of binocular optic flow in cervical neck motor neurons of the fly.

Global visual motion elicits an optomotor response of the eye that stabilizes the visual input on the retina. Here, we analyzed the neck motor system of the blowfly to understand binocular integration of visual motion information underlying a head optomotor response. We identified and characterized two cervical nerve motor neurons (called CNMN6 and CNMN7) tuned precisely to an optic flow corresponding to pitch movements of the head. By means of double recordings and dye coupling, we determined that these neurons are connected ipsilaterally to two vertical system cells (VS2 and VS3), and contralaterally to one horizontal system cell (HSS). In addition, CNMN7 turned out to be connected to the ipsilateral CNMN6 and to its contralateral counterpart. To analyze a potential function of this circuit, we performed behavioral experiments and found that the optomotor pitch response of the fly head was only observable when both eyes were intact. Thus, this neural circuit performs two visuomotor transformations: first, by integrating binocular visual information it enhances the tuning to the optic flow resulting from pitch movements of the head, and second it could assure an even head declination by coordinating the activity of the CNMN7 neurons on both sides.

Central gating of fly optomotor response.

We study the integration of multisensory and central input at the level of an identified fly motoneuron, the ventral cervical nerve motoneuron (VCNM) cell, which controls head movements of the animal. We show that this neuron receives input from a central neuron signaling flight activity, from two identified wide-field motion-sensitive neurons, from the wind-sensitive Johnston organ on the antennae, and from the campaniform sensillae of the halteres. We find that visual motion alone leads to only subthreshold responses. Only when it is combined with flight activity or wind stimuli does the VCNM respond to visual motion by modulating its spike activity in a directionally selective way. This nonlinear enhancement of visual responsiveness in the VCNM by central activity is reflected at the behavioral level, when compensatory head movements are measured in response to visual motion. While head movements of flies have only a small amplitude when flies are at rest, the response amplitude is increased by a factor of 30-40 during flight.

Robust coding of ego-motion in descending neurons of the fly.

In many species, motion-sensitive neurons responding to optic flow at higher processing stages are well characterized; however, less is known how this representation of ego-motion is further transformed into an appropriate motor response. Here, we analyzed in the blowfly Calliphora vicina the visuomotor transformation from motion-sensitive neurons in the lobula plate [V2 and vertical system (VS) cells] onto premotor descending neurons [descending neurons of the ocellar and vertical system (DNOVS) cells] feeding into the motor circuit of the fly thoracic ganglion. We found that each of these cells is tuned to rotation of the fly around a particular body axis. Comparing the responses of presynaptic and postsynaptic cells revealed that DNOVS cells have approximately the same tuning widths as V2 and VS cells. However, DNOVS signals cells are less corrupted by fluctuations arising from the spatial structure of the visual input than their presynaptic elements. This leads to a more robust representation of ego-motion at the level of descending neurons. Thus, when moving from lobula plate cells to descending neurons, the selectivity for a particular optic flow remains unaltered, but the robustness of the representation increases.

Local and global motion preferences in descending neurons of the fly.

For a moving animal, optic flow is an important source of information about its ego-motion. In flies, the processing of optic flow is performed by motion sensitive tangential cells in the lobula plate. Amongst them, cells of the vertical system (VS cells) have receptive fields with similarities to optic flows generated during rotations around different body axes. Their output signals are further processed by pre-motor descending neurons. Here, we investigate the local motion preferences of two descending neurons called descending neurons of the ocellar and vertical system (DNOVS1 and DNOVS2). Using an LED arena subtending 240 degrees x 95 degrees of visual space, we mapped the receptive fields of DNOVS1 and DNOVS2 as well as those of their presynaptic elements, i.e. VS cells 1-10 and V2. The receptive field of DNOVS1 can be predicted in detail from the receptive fields of those VS cells that are most strongly coupled to the cell. The receptive field of DNOVS2 is a combination of V2 and VS cells receptive fields. Predicting the global motion preferences from the receptive field revealed a linear spatial integration in DNOVS1 and a superlinear spatial integration in DNOVS2. In addition, the superlinear integration of V2 output is necessary for DNOVS2 to differentiate between a roll rotation and a lift translation of the fly.

Nonlinear integration of binocular optic flow by DNOVS2, a descending neuron of the fly.

For visual orientation and course stabilization, flies rely heavily on the optic flow perceived by the animal during flight. The processing of optic flow is performed in motion-sensitive tangential cells of the lobula plate, which are well described with respect to their visual response properties and the connectivity among them. However, little is known about the postsynaptic descending neurons, which convey motion information to the motor circuits in the thoracic ganglion. Here we investigate the physiology and connectivity of an identified premotor descending neuron, called DNOVS2 (for descending neuron of the ocellar and vertical system). We find that DNOVS2 is tuned in a supralinear way to rotation around the longitudinal body axis. Experiments involving stimulation of the ipsilateral and the contralateral eye indicate that ipsilateral computation of motion information is modified nonlinearly by motion information from the contralateral eye. Performing double recordings of DNOVS2 and lobula plate tangential cells, we find that DNOVS2 is connected ipsilaterally to a subset of vertical-sensitive cells. From the contralateral eye, DNOVS2 receives input most likely from V2, a heterolateral spiking neuron. This specific neural circuit is sufficient for the tuning of DNOVS2, making it probably an important element in optomotor roll movements of the head and body around the fly's longitudinal axis.

Integration of lobula plate output signals by DNOVS1, an identified premotor descending neuron.

Many motion-sensitive tangential cells of the lobula plate in blowflies are well described with respect to their visual response properties and the connectivity among them. They have large and complex receptive fields with different preferred directions in different parts of their receptive fields matching the optic flow that occurs during various flight maneuvers. However, much less is known about how tangential cells connect to postsynaptic neurons descending to the motor circuits in the thoracic ganglion and how optic flow is represented in these downstream neurons. Here we describe the physiology and the connectivity of a prominent descending neuron called DNOVS1 (for descending neurons of the ocellar and vertical system). We find that DNOVS1 is electrically coupled to a subset of vertical system cells. The specific wiring leads to a preference of DNOVS1 for rotational flow fields around a particular body axis. In addition, DNOVS1 receives input from interneurons connected to the ocelli.

Functional neuroanatomy of the rhinophore of Aplysia punctata.

BACKGROUND: For marine snails, olfaction represents a crucial sensory modality for long-distance reception, as auditory and visual information is limited. The posterior tentacle of Aplysia, the rhinophore, is a chemosensory organ and several behavioural studies showed that the rhinophores can detect pheromones, initiate orientation and locomotion toward food. However the functional neuroanatomy of the rhinophore is not yet clear. Here we apply serotonin-immunohistochemistry and fluorescent markers in combination with confocal microscopy as well as optical recording techniques to elucidate the structure and function of the rhinophore of the sea slug Aplysia punctata. RESULTS: With anatomical techniques an overview of the neuroanatomical organization of the rhinophore is presented. Labelling with propidium iodide revealed one layer of cell nuclei in the sensory epithelium and densely packed cell nuclei beneath the groove of the rhinophore, which extends to about two third of the total length of the rhinophore. Serotonin immunoreactivity was found within the olfactory glomeruli underneath the epithelium as well as in the rhinophore ganglion. Retrograde tracing from the rhinophore ganglion with 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA) demonstrated the connection of glomeruli with the ganglion. Around 36 glomeruli (mean diameter 49 microm) were counted in a single rhinophore. Fluorimetric measurements of intracellular Ca2+ levels using Fura-2 AM loading revealed Ca2+-responses within the rhinophore ganglion to stimulation with amino acids. Bath application of different amino acids revealed differential responses at different positions within the rhinophore ganglion. CONCLUSION: Our neuroanatomical study revealed the number and position of glomeruli in the rhinophore and the rhinophore ganglion as processing stage of sensory information. Serotonin-immunoreactive processes were found extensively within the rhinophore, but was not detected within any peripheral cell body. Amino acids were used as olfactory stimuli in optical recordings and induced sensory responses in the rhinophore ganglion. The complexity of changes in intracellular Ca2+-levels indicates, that processing of odour information takes place within the rhinophore ganglion. Our neuroanatomical and functional studies of the rhinophore open up a new avenue to analyze the olfactory system in Aplysia.