The initiation and coordination of activity in limb muscles are the main functions of neural circuits that control locomotion. Commissural neurons connect locomotor circuits on the two sides of the spinal cord, and represent the known neural substrate for left-right coordination. Here we demonstrate that a group of ipsilateral interneurons, V2a interneurons, plays an essential role in the control of left-right alternation. In the absence of V2a interneurons, the spinal cord fails to exhibit consistent left-right alternation. Locomotor burst activity shows increased variability, but flexor-extensor coordination is unaffected. Anatomical tracing studies reveal a direct excitatory input of V2a interneurons onto commissural interneurons, including a set of molecularly defined V0 neurons that drive left-right alternation. Our findings imply that the neural substrate for left-right coordination consists of at least two components; commissural neurons and a class of ipsilateral interneurons that activate commissural pathways.

Local circuits in the spinal cord that generate locomotion are termed central pattern generators (CPGs). These provide coordinated bilateral control over the normal limb alternation that underlies walking. The molecules that organize the mammalian CPG are unknown. Isolated spinal cords from mice lacking either the EphA4 receptor or its ligand ephrinB3 have lost left-right limb alternation and instead exhibit synchrony. We identified EphA4-positive neurons as an excitatory component of the locomotor CPG. Our study shows that dramatic locomotor changes can occur as a consequence of local genetic rewiring and identifies genes required for the development of normal locomotor behavior.

The ventral spinal cord consists of interneuron groups arising from distinct, genetically defined, progenitor domains along the dorsoventral axis. Many of these interneuron groups settle in the ventral spinal cord which, in mammals, contains the central pattern generator for locomotion. In order to better understand the locomotor networks, we have used different transgenic mice for anatomical characterization of one of these interneuron groups, called V2 interneurons. Neurons in this group are either V2a interneurons marked by the postmitotic expression of the transcription factor Chx10, or V2b interneurons which express the transcription factors Gata2 and Gata3. We found that all V2a and most V2b interneurons were ipsilaterally projecting in embryos as well as in newborns. V2a interneurons were for the most part glutamatergic while V2b interneurons were mainly GABAergic or glycinergic. Furthermore, we demonstrated that a large proportion of V2 interneurons expressed the axon guidance molecule EphA4, a molecule previously shown to be important for correct organization of locomotor networks. We also showed that V2 interneurons and motor neurons alone did not account for all EphA4-expressing neurons in the spinal cord. Together, these findings enable a better interpretation of neural networks underlying locomotion, and open up the search for as yet unknown components of the mammalian central pattern generator.

Commissural interneurons (CINs) are a necessary component of central pattern generators (CPGs) for locomotion because they mediate the coordination of left and right muscle activity. The projection patterns and relative locations of different classes of CINs in the ventromedial part of the rodent lumbar cord have been described (Eide et al. [1999] J Comp Neurol 403:332-345; Stokke et al. [2002] J Comp Neurol 446:349-359; Nissen et al. [2005] J Comp Neurol 483:30-47). However, the distribution and relative prevalence of different CIN neurotransmitter phenotypes in the ventral region of the mammalian spinal cord where the locomotor CPG is localized is unknown. In this study we describe the relative proportions and anatomical locations of putative inhibitory and excitatory CINs in the lumbar spinal cord of newborn mice. To directly visualize potential neurotransmitter phenotypes we combined retrograde labeling of CINs with in situ hybridization against the glycine transporter, GlyT2, or the vesicular glutamate transporter, vGluT2, in wildtype mice and in transgenic mice expressing eGFP driven by the promoters of glutamic acid decarboxylase (GAD) 65, GAD67, or GlyT2. Our study shows that putative glycinergic, GABAergic, and glutamatergic CINs are expressed in almost equal numbers, with a small proportion of CINs coexpressing GlyT2 and GAD67::eGFP, indicating a putative combined glycinergic/GABAergic phenotype. These different CIN phenotypes were intermingled in laminas VII and VIII. Our results suggest that glycinergic, GABAergic, and glutamatergic CINs are the principal CIN phenotypes in the CPG region of the lumbar spinal cord in the newborn mouse. We compare these results to descriptions of CIN neurotransmitter phenotypes in other vertebrate species.

Relatively little is known about the interneurons that constitute the mammalian locomotor central pattern generator and how they interact to produce behavior. A potential avenue of research is to identify genetic markers specific to interneuron populations that will assist further exploration of the role of these cells in the network. One such marker is the EphA4 axon guidance receptor. EphA4-null mice display an abnormal rabbit-like hopping gait that is thought to be the result of synchronization of the normally alternating, bilateral locomotor network via aberrant crossed connections. In this study, we have performed whole-cell patch clamp on EphA4-positive interneurons in the flexor region (L2) of the locomotor network. We provide evidence that although EphA4 positive interneurons are not entirely a homogeneous population, most of them fire in a rhythmic manner. Moreover, a subset of these interneurons provide direct excitation to ipsilateral motor neurons as determined by spike-triggered averaging of the local ventral root DC trace. Our findings substantiate the role of EphA4-positive interneurons as significant components of the ipsilateral locomotor network and describe a group of putative excitatory central pattern generator neurons.ephrin ͉ synaptic transmission ͉ axon guidance ͉ locomotion A dvances in transgenic technologies have greatly facilitated our understanding of the development and function of neural networks (1, 2). These techniques allow incorporation of molecular markers such as ␤-galactosidase (␤-gal) or green fluorescent protein (GFP) under the control of selective promoters to provide important means of identifying and targeting specific neuronal populations (3-7). Moreover, knockouts of fate-determining transcription factors (8) or transmitter systems active during development (9) provide a powerful tool to investigate the overall structure of a network and how it is assembled during development. Such studies are particularly relevant in mammalian systems where it has been an immense task to characterize the principle constituents of neural networks from both developmental genetics (1) and physiological (10-12) perspectives. A recent example of a genetic loss-of-function that is related to a distinct abnormal behavioral phenotype is the rabbit-like hopping gait exhibited in mice that have a targeted deletion of the axon guidance molecules EphA4 and ephrinB3 (13)(14)(15). This pronounced phenotype could be reproduced in isolated spinal cords from mutant mice, suggesting that the neuronal network controlling locomotion, also called the central pattern generator (CPG), is genetically reconfigured in the mutants (16). The experiments demonstrated that the hopping gait in mutants was related to an increase in midline crossing of axons originating from EphA4-expressing neurons in the spinal cord. Moreover, the experiments showed that a large proportion of glutamatergic excitatory cells in the ventral spinal cord expressed EphA4. This finding led us to hypothesize that a group of EphA4 neurons ...