A 'tool box' for deciphering neuronal circuits in the developing chick spinal cord.
Bottom Line: Here we present a circuit-deciphering 'tool box' for fast, reliable and cheap genetic targeting of neuronal circuits in the developing spinal cord of the chick.We demonstrate targeting of motoneurons and spinal interneurons, mapping of axonal trajectories and synaptic targeting in both single and populations of spinal interneurons, and viral vector-mediated labeling of pre-motoneurons.We also demonstrate fluorescent imaging of the activity pattern of defined spinal neurons during rhythmic motor behavior, and assess the role of channel rhodopsin-targeted population of interneurons in rhythmic behavior using specific photoactivation.
Affiliation: Department of Medical Neurobiology, IMRIC, Hebrew University Medical School, Jerusalem, Israel.Show MeSH
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Mentions: The calcium indicator GCaMP3 was cloned into a Cre-dependent PiggyBac vector. Targeting to motoneurons was attained using electroporation of the conditional GCaMP3 plasmid (Figure 6A) into E3 chick neural tube along with ubiquitously-expressed PBase and Cre under the control of the chick motoneuron-specific enhancer of the TAG1/Axonin1 gene (19) (Table 1, Figure 6B). Electroporated embryos were sacrificed at E12; the spinal cords were isolated and mounted in an in vitro chamber equipped for combined optical imaging of the targeted neurons and electrophysiological recording of the motor output. Fluorophores were excited at the appropriate wavelength, and images were acquired from the ventral or lateral surface of the cord using epifluorescence microscopy and a cooled 14bit CCD camera, before and during motor rhythm produced by electrical stimulation (Figure 6C). Figure 6C shows that the stimulation elicited an alternating flexor-extensor rhythm recorded from the satrorius and femorotibialis nerves, respectively, and that the simultaneous calcium imaging and electrophysiological recordings could be used to resolve the activity patterns of flexor and extensor motoneurons during the motor rhythm (Figure 6C and Supplementary Movie 1).
Affiliation: Department of Medical Neurobiology, IMRIC, Hebrew University Medical School, Jerusalem, Israel.