Organization of the gravity-sensing system in zebrafish.

Motor circuits develop in sequence from those governing fast movements to those governing slow. Here we examine whether upstream sensory circuits are organized by similar principles. Using serial-section electron microscopy in larval zebrafish, we generated a complete map of the gravity-sensing (utricular) system spanning from the inner ear to the brainstem. We find that both sensory tuning and developmental sequence are organizing principles of vestibular topography. Patterned rostrocaudal innervation from hair cells to afferents creates an anatomically inferred directional tuning map in the utricular ganglion, forming segregated pathways for rostral and caudal tilt. Furthermore, the mediolateral axis of the ganglion is linked to both developmental sequence and neuronal temporal dynamics. Early-born pathways carrying phasic information preferentially excite fast escape circuits, whereas later-born pathways carrying tonic signals excite slower postural and oculomotor circuits. These results demonstrate that vestibular circuits are organized by tuning direction and dynamics, aligning them with downstream motor circuits and behaviors.

Central Vestibular Tuning Arises from Patterned Convergence of Otolith Afferents.

As sensory information moves through the brain, higher-order areas exhibit more complex tuning than lower areas. Though models predict that complexity arises via convergent inputs from neurons with diverse response properties, in most vertebrate systems, convergence has only been inferred rather than tested directly. Here, we measure sensory computations in zebrafish vestibular neurons across multiple axes in vivo. We establish that whole-cell physiological recordings reveal tuning of individual vestibular afferent inputs and their postsynaptic targets. Strong, sparse synaptic inputs can be distinguished by their amplitudes, permitting analysis of afferent convergence in vivo. An independent approach, serial-section electron microscopy, supports the inferred connectivity. We find that afferents with similar or differing preferred directions converge on central vestibular neurons, conferring more simple or complex tuning, respectively. Together, these results provide a direct, quantifiable demonstration of feedforward input convergence in vivo.

In vitro imaging of retinal whole mounts.

Neuronal circuits of the vertebrate retina are organized into stereotyped laminae. This orderly arrangement makes the retina an ideal model system for imaging studies aimed at understanding how circuits assemble during development. In particular, live-cell imaging techniques are readily applied to the developing retina to monitor dynamic changes over time in cell structure and connectivity. Such imaging studies have collectively revealed novel strategies by which retinal neurons contact their presynaptic and postsynaptic partners to establish synaptic connections. We describe here the procedures developed in our laboratory for confocal and multiphoton live-cell imaging of the developing retina using in vitro retinal explants. Retinas can be removed from the eye and kept in culture conditions for several days with limited disruption to the retinal circuit. The explanted retina is amenable to a variety of labeling techniques and provides a large, flat, unobstructed surface that is ideal for optical imaging experiments. This protocol describes procedures for mounting and imaging the isolated mouse retina. The same general procedure, with only minor modification (composition of culture medium), has been used to image retinas from a variety of vertebrates (e.g., chick, ferret, and rabbit).

In vivo imaging of zebrafish retina.

Neuronal circuits of the vertebrate retina are organized into stereotyped laminae. This orderly arrangement makes the retina an ideal model system for imaging studies aimed at understanding how circuits assemble during development. In particular, live-cell imaging techniques are readily applied to the developing retina to monitor dynamic changes over time in cell structure and connectivity. Such imaging studies have collectively revealed novel strategies by which retinal neurons contact their presynaptic and postsynaptic partners to establish synaptic connections. We describe here the procedures developed in our laboratory for confocal and multiphoton live-cell imaging of the developing retina using in vivo preparations. Zebrafish larvae are an ideal specimen for in vivo imaging experiments as they can be made to remain transparent throughout development. Isolated retinal cells can be readily labeled by DNA injection into the one-cell staged embryo, or via transplantation of fluorescently labeled cells from stable transgenics.

Development of cell type-specific connectivity patterns of converging excitatory axons in the retina.

To integrate information from different presynaptic cell types, dendrites receive distinct patterns of synapses from converging axons. How different afferents in vivo establish specific connectivity patterns with the same dendrite is poorly understood. Here, we examine the synaptic development of three glutamatergic bipolar cell types converging onto a common postsynaptic retinal ganglion cell. We find that after axons and dendrites target appropriate synaptic layers, patterns of connections among these neurons diverge through selective changes in the conversion of axo-dendritic appositions to synapses. This process is differentially regulated by neurotransmission, which is required for the shift from single to multisynaptic appositions of one bipolar cell type but not for maintenance and elimination, respectively, of connections from the other two types. Thus, synaptic specificity among converging excitatory inputs in the retina emerges via differential synaptic maturation of axo-dendritic appositions and is shaped by neurotransmission in a cell type-dependent manner.

Ballistic labeling with fluorescent dyes and indicators.

Neuronal cell labeling is fundamental to investigations of the nervous system. Labeling of cells in live or fixed tissue with dyes or ion indicators using ballistic approaches has recently been developed for the study of neuronal architecture and function. In this approach, dye-coated particles are propelled into cells by a pulse of pressurized helium. This unit provides step-by-step protocols for coating tungsten particles with fluorescent or indicator dyes and for delivering these particles into cells and tissue. The major advantage of the ballistic method of dye delivery is that large populations of neurons can be rapidly labeled within a piece of live or fixed tissue. Advantages and limitations of the approach are discussed and technical advice is provided.