EPHA4 and V2 interneurons in the mammalian locomotor network

Central pattern generators (CPGs) are neural networks that can execute halfautomated movements without supraspinal or sensory input. Hindlimb locomotion in mammals is dependent upon such a CPG which is located ventrally in the spinal cord lumbar enlargement. The key features of mammalian locomotion are ipsilateral excitatory interneurons which execute rhythm generation, as well as commissural inhibitory interneurons which execute left-right coordination and flexor-extensor coordination. Little is known about the ipsilateral excitatory interneurons in the mammalian CPG, and our goal was to identify and describe the role of such excitatory, ipsilateral interneurons.

First we identified the EphA4-positive neurons as a key component of the mammalian CPG, a component which normally is restricted to one side by the signaling induced by the interaction of the axon guidance molecules ephrinB3 and EphA4. We found that in EphA4 and ephrinB3 null mice, CPG neurons aberrantly cross the midline. This results in an abnormal synchronized bilateral coordination ( hopping ) due to an increased crossover of excitatory, normally EphA4-expressing neurons, which make connections to the CPG and thus override the normal alternating cross-coordination in the locomotion system.

Next, we directly demonstrated that EphA4-positive neurons are rhythmically active and that a subset of these are ipsilaterally projecting, excitatory interneurons with projections onto motor neurons. This positions them as an identified excitatory interneuron group in the mammalian CPG and suggests that the group may be defined by the combined expression of EphA4 and glutamate release.

We then went on to look for a possible overlap between EphA4-positive interneurons and the developmentally defined group of ventral V2 interneurons in the spinal cord. We found that the vast majority of V2 interneurons express EphA4 and further characterized the V2 interneurons as segmental ipsilaterally projecting, excitatory (V2a) or inhibitory (V2b) interneurons located in a position which suggests that they are members of the mammalian CPG. Furthermore, we show that there are more EphA4-expressing interneurons than what can be accounted for by V2 interneurons and motor neurons, and the aberrant crossing of processes from EphA4-positive neurons in ephA4LacZ-/- mice does not originate from the V2a population.

Finally, we used a mouse model where excitatory ipsilaterally projecting, Chx10- positive (V2a), interneurons are specifically ablated. In the absence of Chx10 neurons, the locomotor burst activity displayed increased variation, but flexor-extensor coordination was unaffected while left-right alternation was disrupted. Evidence for a direct excitatory input of V2a interneurons onto commissural interneurons was provided by anatomical tracing studies. Among the commissural interneurons contacted were the V0 (Evx1 positive) interneurons which are involved in left-right alternation. These observations point to an essential role for V2a interneurons in the control of left-right alternation.

Together, work presented in this thesis has identified essential components of the mammalian CPG for walking, leading us towards a better understanding of the fundamental principles for the organization of the mammalian locomotor network. Our hope is that our contribution will help to improve clinical neuro-rehabilitation of spinal cord injured patients.

List of scientific papers

I. Kullander K, Butt SJ, Lebret JM, Lundfald L, Restrepo CE, Rydström A, Klein R, Kiehn O (2003). Role of EphA4 and EphrinB3 in local neuronal circuits that control walking. Science. 299(5614): 1889-92.
https://pubmed.ncbi.nlm.nih.gov/12649481

II. Butt SJ, Lundfald L, Kiehn O (2005). EphA4 defines a class of excitatory locomotor-related interneurons. Proc Natl Acad Sci U S A. 102(39): 14098-103.
https://pubmed.ncbi.nlm.nih.gov/16172411

III. Lundfald L, Restrepo CE, Butt SJ, Peng CY, Droho S, Endo T, Zeilhofer HU, Sharma K, Kiehn O (2007). Phenotype of V2-derived interneurons and their relationship to the axon guidance molecule EphA4 in the developing mouse spinal cord. Eur J Neurosci. 26(11): 2989-3002.
https://pubmed.ncbi.nlm.nih.gov/18028107

IV. Crone SA, Quinlan KA, Zagoraiou L, Droho S, Restrepo CE, Lundfald L, Endo T, Setlak J, Jessell TM, Kiehn O, Sharma K (2008). Genetic ablation of V2a ipsilateral interneurons disrupts left-right locomotor coordination in mammalian spinal cord. Neuron. [Accepted]
https://pubmed.ncbi.nlm.nih.gov/18940589