Optogenetic dissection of a behavioural module in the vertebrate spinal cord.

Locomotion relies on neural networks called central pattern generators (CPGs) that generate periodic motor commands for rhythmic movements. In vertebrates, the excitatory synaptic drive for inducing the spinal CPG can originate from either supraspinal glutamatergic inputs or from within the spinal cord. Here we identify a spinal input to the CPG that drives spontaneous locomotion using a combination of intersectional gene expression and optogenetics in zebrafish larvae. The photo-stimulation of one specific cell type was sufficient to induce a symmetrical tail beating sequence that mimics spontaneous slow forward swimming. This neuron is the Kolmer-Agduhr cell, which extends cilia into the central cerebrospinal-fluid-containing canal of the spinal cord and has an ipsilateral ascending axon that terminates in a series of consecutive segments. Genetically silencing Kolmer-Agduhr cells reduced the frequency of spontaneous free swimming, indicating that activity of Kolmer-Agduhr cells provides necessary tone for spontaneous forward swimming. Kolmer-Agduhr cells have been known for over 75 years, but their function has been mysterious. Our results reveal that during early development in zebrafish these cells provide a positive drive to the spinal CPG for spontaneous locomotion.

Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells.

One of the limitations on imaging fluorescent proteins within living cells is that they are usually present in small numbers and need to be detected over a large background. We have developed the means to isolate specific fluorescence signals from background by using lock-in detection of the modulated fluorescence of a class of optical probe termed "optical switches." This optical lock-in detection (OLID) approach involves modulating the fluorescence emission of the probe through deterministic, optical control of its fluorescent and nonfluorescent states, and subsequently applying a lock-in detection method to isolate the modulated signal of interest from nonmodulated background signals. Cross-correlation analysis provides a measure of correlation between the total fluorescence emission within single pixels of an image detected over several cycles of optical switching and a reference waveform detected within the same image over the same switching cycles. This approach to imaging provides a means to selectively detect the emission from optical switch probes among a larger population of conventional fluorescent probes and is compatible with conventional microscopes. OLID using nitrospirobenzopyran-based probes and the genetically encoded Dronpa fluorescent protein are shown to generate high-contrast images of specific structures and proteins in labeled cells in cultured and explanted neurons and in live Xenopus embryos and zebrafish larvae.

Emergence of patterned activity in the developing zebrafish spinal cord.

BACKGROUND: Developing neural networks display spontaneous and correlated rhythmic bursts of action potentials that are essential for circuit refinement. In the spinal cord, it is poorly understood how correlated activity is acquired and how its emergence relates to the formation of the spinal central pattern generator (CPG), the circuit that mediates rhythmic behaviors like walking and swimming. It is also unknown whether early, uncorrelated activity is necessary for the formation of the coordinated CPG. RESULTS: Time-lapse imaging in the intact zebrafish embryo with the genetically encoded calcium indicator GCaMP3 revealed a rapid transition from slow, sporadic activity to fast, ipsilaterally correlated, and contralaterally anticorrelated activity, characteristic of the spinal CPG. Ipsilateral correlations were acquired through the coalescence of local microcircuits. Brief optical manipulation of activity with the light-driven pump halorhodopsin revealed that the transition to correlated activity was associated with a strengthening of ipsilateral connections, likely mediated by gap junctions. Contralateral antagonism increased in strength at the same time. The transition to coordinated activity was disrupted by long-term optical inhibition of sporadic activity in motoneurons and ventral longitudinal descending interneurons and resulted in more neurons exhibiting uncoordinated activity patterns at later time points. CONCLUSIONS: These findings show that the CPG in the zebrafish spinal cord emerges directly from a sporadically active network as functional connectivity strengthens between local and then more distal neurons. These results also reveal that early, sporadic activity in a subset of ventral spinal neurons is required for the integration of maturing neurons into the coordinated CPG network.