We used confocal microscopy to examine the morphology of spinal interneurons in living larval zebrafish with the aim of providing a morphological foundation for generating functional hypotheses. Interneurons were retrogradely labeled by injections of fluorescent dextrans into the spinal cord, and the three-dimensional morphology of living cells was reconstructed from confocal optical sections through the transparent fish. At least eight types of interneurons are present in the spinal cord of larval zebrafish; four of these are described here for the first time. The newly discovered cell types include classes of commissural neurons with axons that ascend, descend, and bifurcate in the contralateral spinal cord. Our reexamination of previously described cell types revealed functionally relevant features of their morphology, such as undescribed commissural axons, as well as the relationships between the trajectories of the axons of interneurons and the descending Mauthner axons. In addition to describing neurons, we surveyed their morphology at multiple positions along the spinal cord and found longitudinal changes in their distribution and sizes. For example, some cell types increase in size from rostral to caudal, whereas others decrease. Our observations lead to predictions of the roles of some of these interneurons in motor circuits. These predictions can be tested with the combination of functional imaging, single-cell ablation, and genetic approaches that make zebrafish a powerful model system for studying neuronal circuits.

Most studies of spinal interneurons in vertebrate motor circuits have focused on the activity of interneurons in a single motor behavior. As a result, relatively little is known about the extent to which particular classes of spinal interneurons participate in different behaviors. Similarities between the morphology and connections of interneurons activated in swimming and escape movements in different fish and amphibians led to the hypothesis that spinal interneurons might be shared by these behaviors. To test this hypothesis, we took advantage of the optical transparency of zebrafish larvae and developed a new preparation in which we could use confocal calcium imaging to monitor the activity of individual identified interneurons noninvasively, while we simultaneously filmed the movements of the fish with a high-speed digital camera. With this approach, we could directly examine the involvement of individual interneurons in different motor behaviors. Our work revealed unexpected differences in the interneurons activated in swimming and escape behaviors. The observations lead to predictions of different behavioral roles for particular classes of spinal interneurons that can eventually be tested directly in zebrafish by using laser ablations or mutant lines with interneuronal deficits.