Optogenetic Activation of Excitatory Premotor Interneurons Is Sufficient to Generate Coordinated Locomotor Activity in Larval Zebrafish

Abstract

Neural networks in the spinal cord can generate locomotion in the absence of rhythmic input from higher brain structures or sensory feedback because they contain an intrinsic source of excitation. However, the molecular identity of the spinal interneurons underlying the excitatory drive within the locomotor circuit has remained unclear. Using optogenetics, we show that activation of a molecularly defined class of ipsilateral premotor interneurons elicits locomotion. These interneurons represent the excitatory module of the locomotor networks and are sufficient to produce a coordinated swimming pattern in zebrafish. They correspond to the V2a interneuron class and express the transcription factor Chx10. They produce sufficient excitatory drive within the spinal networks to generate coordinated locomotor activity. Therefore, our results define the V2a interneurons as the excitatory module within the spinal locomotor networks that is sufficient to initiate and maintain locomotor activity.

Systematic Shifts in the Balance of Excitation and Inhibition Coordinate the Activity of Axial Motor Pools at Different Speeds of Locomotion

“…1 A, B). Whole-cell patch and peripheral motor nerve recordings were performed as described previously (Drapeau et al, 1999;Masino and Fetcho, 2005), using a modified patch solution to perform voltage-clamp recordings (see below). Briefly, larvae were first anesthetized in MS-222 and then immobilized in ␣-bungarotoxin (Sigma-Aldrich), both of which were dissolved in extracellular solution (1 mg/ml).…”

“…To more reliably evoke a broader range of swim frequencies than those observed spontaneously, we delivered a brief electrical stimulus to the skin to mimic tactile input (Clarke and Roberts, 1984). The highest frequencies within a bout tend to occur immediately following the stimulus (McLean et al, 2008); however, increases in frequency within this period could be achieved by increasing the intensity of the electrical stimulus (2-10 V, 0.1 ms). Recruitment patterns during evoked decelerations in swimming frequency and spontaneous accelerations are virtually indistinguishable (McLean et al, 2008).…”

“…A growing body of work in mice is beginning to reveal the spinal mechanisms responsible for gait transitions, which involve differences in the contribution of genetically identifiable excitatory and inhibitory premotor populations (Gosgnach et al, 2006;Crone et al, 2009;Zhong et al, 2011;Talpalar et al, 2013). However, one of the first demonstrations of switches in spinal circuits during increases in locomotor speed was provided by studies of swimming in larval zebrafish (McLean et al, 2008). In particular, subsets of premotor excitatory neurons originating from the P0 progenitor domain, called V0-eD neurons (Satou et al, 2012), are active at slow speeds, but these cells are then inhibited at faster speeds as a new excitatory population arising from the P2 progenitor domain, called V2a neurons (Kimura et al, 2006), is engaged.…”

“…At the fastest speeds, cells within the V2a population are in turn silenced, as others are activated. While speed-dependent differences in appendicular premotor populations are relatively intuitive, given requisite differences in interlimb coordination (Berkowitz et al, 2010), it is unclear why this occurs during axially based locomotion, where smooth gradations in tail beat amplitudes and frequencies generate faster speeds of swimming (McLean et al, 2008;Green et al, 2011).…”

“…Our recent work studying the biophysical properties of axial motoneurons in larval zebrafish has revealed remarkable diversity related to recruitment order. Motoneurons are recruited from the bottom of spinal cord up, with progressively larger, lower Rin motoneurons added to the active pool as swimming frequency increases (McLean et al, 2007). The recruitment order of motoneurons is associated with systematic differences in nonlinear membrane properties that match Rin.…”