The mesencephalic locomotor region recruits V2a reticulospinal neurons to drive forward locomotion in larval zebrafish

The mesencephalic locomotor region (MLR) is a brain stem area whose stimulation triggers graded forward locomotion. How MLR neurons recruit downstream vsx2+ (V2a) reticulospinal neurons (RSNs) is poorly understood. Here, to overcome this challenge, we uncovered the locus of MLR in transparent larval zebrafish and show that the MLR locus is distinct from the nucleus of the medial longitudinal fasciculus. MLR stimulations reliably elicit forward locomotion of controlled duration and frequency. MLR neurons recruit V2a RSNs via projections onto somata in pontine and retropontine areas, and onto dendrites in the medulla. High-speed volumetric imaging of neuronal activity reveals that strongly MLR-coupled RSNs are active for steering or forward swimming, whereas weakly MLR-coupled medullary RSNs encode the duration and frequency of the forward component. Our study demonstrates how MLR neurons recruit specific V2a RSNs to control the kinematics of forward locomotion and suggests conservation of the motor functions of V2a RSNs across vertebrates.


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Sample-size was estimated base on 3R principles Data exclusions In figure 5 there was one fish in which we applied intensities of stimulation in steps of 0.5µA while the other fish were stimulated with steps of 1uA.We subsequently ignored the data points of stimulation intensities at 0.5, 1.5 and 2.5µA for this particular fish.Some stimulation trials were excluded from multiple linear analysis performed in figure 6 based on the number of isolated forward swim bouts that could be extracted from the tail angle trace.Only the trials for which at least 2 clear forward bouts could be extracted were used.For figure 7, Out of 11 enucleated larvae recorded, we kept for the analysis 3 larvae that produced at least 50 swim bouts and at least one bout of each category (forward, left turn, right turn).The exclusion criteria were not pre-established.

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At least two measures were taken to verify the reproducibility of the experimental findings.In figure 5 there was one fish in which we applied intensities of stimulation in steps of 0.5µA while the other fish were stimulated with steps of 1uA.We subsequently ignored the data points of stimulation intensities at 0.5, 1.5 and 2.5µA for this particular fish.All replication attempts were successful.
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The primary antibodies used in combination here have been tested separately resulting in similar labeling of the same neuronal structures.Removing the primary antibodies from the procedure while keeping everything else the same did not produce any labeling of neuronal structures in our material.The rabbit anti-DBH from this study was successfully used in zebrafish by other authors to label locus coeruleus neurons specifically1,2.In our material, only locus coeruleus neurons were labeled with this antibody.Alternatively, the rabbit anti-DBH from this study was replaced with a rabbit anti-tyrosine hydroxylase (RRID: AB_390204, cat.number AB152, lot: 3031639, diluted 1:400, Millipore).The resulting labeling of the locus coeruleus neurons was similar in every aspect to the one obtained with the DBH antibody.The goat anti-ChAT used in this study has been used with success on a wide range of vertebrate species, including zebrafish, to label cholinergic neurons1,3,4,5.

nature portfolio | reporting summary
March 2021

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