Motor neurons control locomotor circuit function retrogradely via gap junctions

Abstract

Motor neurons are the final stage of neural processing for the execution of motor behaviours. Traditionally, motor neurons have been viewed as the ‘final common pathway’, serving as passive recipients merely conveying to the muscles the final motor program generated by upstream interneuron circuits1,2. Here we reveal an unforeseen role of motor neurons in controlling the locomotor circuit function via gap junctions in zebrafish. These gap junctions mediate a retrograde analogue propagation of voltage fluctuations from motor neurons to control the synaptic release and recruitment of the upstream V2a interneurons that drive locomotion. Selective inhibition of motor neurons during ongoing locomotion de-recruits V2a interneurons and strongly influences locomotor circuit function. Rather than acting as separate units, gap junctions unite motor neurons and V2a interneurons into functional ensembles endowed with a retrograde analogue computation essential for locomotor rhythm generation. These results show that motor neurons are not a passive recipient of motor commands but an integral component of the neural circuits responsible for motor behaviour.

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Figure 1: Existence of gap junctions between V2a interneurons and motor neurons.
Figure 2: Motor neurons control the strength of synaptic transmission and the firing threshold of V2a interneurons.
Figure 3: Motor neurons control the recruitment of V2a interneurons during locomotion.
Figure 4: Optogenetic inhibition of motor neurons decreases the frequency of locomotion.

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Acknowledgements

We thank C. Wyart, S. Grillner, O. Kiehn and G. Silberberg as well as the members of our lab for comments on an early version of the manuscript. We are grateful to A. Baradel, C. Wyart, S. Higashijima and H. Baier for providing the zebrafish lines used in this study. This study was supported by grants from the Swedish Research Council, Karolinska Institute and the Swedish Brain Foundation.

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Authors

Contributions

J.S., K.A. and A.E.M. initiated the project and designed the experiments. J.S. performed all the electrophysiological and optogenetic experiments. K.A. performed the anatomical and contributed to the electrophysiological experiments. E.R.B. contributed to the anatomical and electrophysiological experiments. All the authors contributed to data analysis, discussed the results and participated in writing the manuscript.

Corresponding author

Correspondence to Abdeljabbar El Manira.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Co-existence of electrical and chemical synapses.

a, Representative recording of a V2a interneuron and a motor neuron connected with mixed synapses (n = 11 from 11 zebrafish). EPSP amplitude induced in the motor neuron by stimulation of the V2a interneuron was reduced by blockers of ionotropic glutamate receptors (AP-5 and NBQX) leaving only an electrical event. b, Stimulation of the motor neuron also induced an electrical event in the V2a interneuron that was insensitive to the glutamate receptor blockers. c, Changes in the amplitude of the synaptic events in motor neurons and V2a interneurons induced by glutamate receptor blockers (*P < 0.05, two-tailed Student’s t-test, error bars denote s.e.m.). d, Representative recording of a V2a interneuron and a motor neuron (n = 12 from 12 zebrafish) in which blockade of chemical synaptic transmission with cadmium decreased the EPSP amplitude leaving only an electrical event. e, Cadmium did not affect the amplitude of the electrical events induced in the V2a interneuron by stimulation of the motor neuron. f, Effect of cadmium on the synaptic events in motor neurons and V2a interneurons (*P < 0.05, two-tailed Student’s t-test, error bars denote s.e.m). g, Representative recording of a V2a interneuron and a motor neuron connected with chemical synapses (n = 11 from 11 zebrafish). Blockade of ionotropic glutamate receptors completely abolished the V2a interneuron-induced EPSP in the motor neuron. h, In this pair, stimulation of the motor neuron did not induce any electrical event in the V2a interneuron. i, Effects of glutamate blockers and cadmium on the synaptic events induced in motor neurons and V2a interneurons (***P < 0.001, two-tailed Student’s t-test, error bars denote s.e.m.).

Extended Data Figure 2 Co-localization of connexin 35/36 with putative synaptic sites between V2a interneurons and motor neurons.

a, Representative recording of a V2a interneuron and a motor neuron connected with mixed synapses that were filled with neurobiotin (n = 7 from 7 zebrafish). b, Reconstruction of the recorded neurons revealed a zone of close apposition between V2a interneuron axon collaterals and motor neuron dendrites. c, Connexin 35/36 (Cx) was co-localized with close appositions between V2a interneuron axons and motor neuron dendrites. d, Representative recording of a V2a interneuron and a motor neuron connected only with chemical synapses that were filled with neurobiotin (n = 3 from 3 zebrafish). e, Reconstruction of the recorded pair revealed close appositions between V2a interneuron axon collaterals and motor neuron dendrites. f, In pairs only connected with chemical synapses no co-localization of connexin 35/36 was observed with close appositions between V2a interneuron axons and motor neuron dendrites. g, The position of motor neuron cell bodies relative to those of the V2a interneurons (0 position on both axes) in pairs connected with mixed or only with chemical synapses. h, Scatter plot showing the coupling strength (electrical component/EPSP amplitude) in pairs connected with mixed or only with chemical synapses. The amplitude of the electrical component was determined after blockade of chemical synaptic transmission with ionotropic glutamate receptor antagonists or cadmium.

Extended Data Figure 3 Electrical coupling between motor neurons.

a, Injection of neurobiotin into a single motor neuron (red) resulted in dye coupling of other motor neurons. b, c, Representative recording of a pair of motor neurons (MN1 and MN2) displaying bidirectional electrical coupling (n = 11 from 11 zebrafish). d, Reconstruction of the recorded motor neuron pair in b and c revealed a zone of dendro-dendritic close apposition. e, Connexin 35/36 (Cx) co-localized very closely to the motor neuron dendrites. f, Injection of neurobiotin into a single V2a interneuron (red) resulted in dye coupling of other V2a interneurons (n = 6 from 6 zebrafish). fi, fii, Higher magnifications of the dashed boxes in f.

Extended Data Figure 4 Yellow light had no effect in control animals without NpHR expression.

a, Representative recordings showing that yellow light stimulation did not induce any change in the membrane potential of the motor neurons and the V2a interneurons. b, Representative recording of a V2a interneuron from a control zebrafish without NpHR–mCherry expression. Yellow light stimulation over two spinal segments had no effect on the rhythmic swimming-related activity in the V2a interneuron (n = 9 from 9 zebrafish). c, Plot showing the lack of effect of the yellow light stimulation on the swimming-related activity frequency (n = 9, P > 0.05, two-tailed Student’s t-test). d, Graph showing the lack of effect of the yellow light on the swimming-related activity duration (n = 9, P > 0.05, two-tailed Student’s t-test).

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Song, J., Ampatzis, K., Björnfors, E. et al. Motor neurons control locomotor circuit function retrogradely via gap junctions. Nature 529, 399–402 (2016). https://doi.org/10.1038/nature16497

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