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Motor Neurons Control Retrogradely via Gap Junctions


Nature  529, 399–402 (21 January 2016)

Motor neurons control locomotor circuit function retrogradely via gap junctions

Jianren Song,

Department of Neuroscience, Karolinska Institute, 171 77 Stockholm, Sweden


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 circuits. 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.

The existence of electrical coupling between motor neurons and the premotor excitatory V2a interneurons was examined using three complementary sets of experiments in the adult zebrafish. First, we show the occurrence of dye coupling via gap junctions from motor neurons to V2a interneurons following the injection of neurobiotin into muscles or intracellularly into single motor neurons. However, practically no dye coupling was seen between motor neurons and the excitatory commissural (V0v) interneurons or the inhibitory glycinergic interneurons. Second, we reveal, using immunohistochemistry, that connexin 35/36 was co-localized with close appositions between motor neuron dendrites and V2a interneuron axons. Moreover, connexin 35/36 punctae on motor neuron dendrites were co-localized with most of the synaptophysin-labelled presynaptic terminals. Finally, the occurrence of bidirectional electrical coupling was confirmed electrophysiologically using whole-cell paired recordings from V2a interneurons and motor neurons (n = 57 pairs, 57 zebrafish. We previously showed that V2a interneurons are subdivided into three sub-classes that are selectively connected to slow, intermediate and fast motor neurons and form three separate modules. Electrical coupling occurred between pairs of the same module as well as between pairs of adjacent modules.

Together the above results show that gap junctions occur between presynaptic terminals of V2a interneurons and dendrites of motor neurons exclusively in pairs connected with chemical synapses. This enables backward propagation of electrical signals from motor neurons to the upstream excitatory V2a interneurons.

In addition to their mixed electrical and chemical connections with V2a interneurons, motor neurons are also electrically coupled to each other

V2a interneurons play an important role in generating locomotor rhythm and driving motor neuron activity. While motor neurons have traditionally been excluded from contributing to rhythm generation, their strong influence on V2a interneurons prompted us to reassess their role in the locomotor central pattern generator.

These results uncover a prominent role of motor neurons, extending their influence retrogradely to control the recruitment threshold and firing activity of the V2a interneurons—a pivotal interneuron component of the rhythm generator circuit.

Motor neurons are traditionally considered passive recipients of motor programs generated by upstream interneuron circuits. Here we reveal an unforeseen role of motor neurons that extends their influence to premotor excitatory interneurons and embeds them within the core circuit generating the locomotor rhythm. Thus far, the only established route for motor neuron influence in the spinal cord was via Renshaw cells, which produce recurrent inhibition but do not contribute to the rhythm generation. We now demonstrate a novel and direct path via gap junctions that mediates a simultaneous integration of ongoing activity of motor neurons by upstream excitatory interneurons. The existence of electrical coupling between motor neurons and the excitatory V2a interneurons suggests a major revision of the construction of locomotor circuits. The phasic change of the membrane potential of motor neurons during each locomotor cycle is immediately transferred to upstream excitatory interneurons and affects their recruitment and synaptic release. Rather than acting only as feed-forward units, the bidirectional transfer of electrical signals via electrical coupling ties motor neurons and interneurons into functional ensembles endowed with an analogue processing essential for the elaboration of the locomotor rhythm. Indeed, motor neurons serve more complex functions and might use additional mechanisms in conjunction with gap junctions to influence the function of neural circuits for motor behaviour.

Mixed synapses are widespread in vertebrates, including mammals. Indeed, there is growing evidence of the presence of glutamatergic mixed electrical and chemical synapses in the mammalian nervous system. An important function ascribed to electrical coupling via gap junctions is to synchronize the activity of neuronal ensembles. However, retrograde intercellular communication via gap junctions has been shown in sensory systems of fish and crustaceans, and can lead to synchronization of the activity of sensory axons. In addition, gap junctions can synchronize the activity of interneurons and motor neurons to produce rhythmic activity. Anatomical substrates exist in the adult mammalian spinal cord to enable retrograde influence from motor neurons onto the premotor locomotor circuit as we have now revealed in the adult zebrafish. Therefore we propose that motor neurons have a strong and immediate influence on the locomotor circuit and are not merely passive recipients of motor commands but rather an integral component of the spinal circuit generating the locomotor rhythm.

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