Change in the balance of excitatory and inhibitory midline fiber crossing as an explanation for the hopping phenotype in EphA4 knockout mice.

Neuronal networks in the spinal cord termed central pattern generators (CPGs) are responsible for the generation of rhythmic movements, such as walking. The axon guidance molecule EphA4 has been suggested to play a role in the configuration of spinal CPG networks in mammals. In EphA4 knockout (EphA4-KO) mice, the normal alternating walking pattern is replaced by a rabbit-like hopping gait, which can be reproduced when locomotor-like activity is induced in the isolated spinal cord. This hopping phenotype has been explained by an abnormal midline crossing of ipsilateral axons. Here, we investigated the nature of this overcrossing in heterozygous EphA4 (EphA4(lacZ/+) ) mice that showed normal alternating gait and homozygous EphA4 (EphA4(lacZ/lacZ) ) mice with hopping gait. Localized lesions showed that the hopping phenotype is maintained by fibers crossing in the ventral commissure. Using transgenic mouse lines in which glutamatergic, GABAergic and glycinergic neurons are intrinsically labeled, we showed a significant increase in the number of crossing excitatory ß-galactosidase-positive neurons and a decrease in the number of inhibitory neurons crossing the midline in EphA4(lacZ/lacZ) mice compared with EphA4(lacZ/+) mice. These results show that the hopping phenotype is the result of a change in the balance between excitatory and inhibitory signals across the midline and that EphA4-positive neurons play an essential role in the mammalian CPG.

Transmitter-phenotypes of commissural interneurons in the lumbar spinal cord of newborn mice.

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.

Genetic ablation of V2a ipsilateral interneurons disrupts left-right locomotor coordination in mammalian spinal cord.

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.

Excitatory components of the mammalian locomotor CPG.

Locomotion in mammals is to a large degree controlled directly by intrinsic spinal networks, called central pattern generators (CPGs). The overall function of these networks is governed by interaction between inhibitory and excitatory neurons. In the present review, we will discuss recent findings addressing the role of excitatory synaptic transmission for network function including the role of specific excitatory neuronal populations in coordinating muscle activity and in generating rhythmic activity.

Phenotype of V2-derived interneurons and their relationship to the axon guidance molecule EphA4 in the developing mouse spinal cord.

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.

EphA4 defines a class of excitatory locomotor-related interneurons.

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.

ShcA regulates neurite outgrowth stimulated by neural cell adhesion molecule but not by fibroblast growth factor 2: evidence for a distinct fibroblast growth factor receptor response to neural cell adhesion molecule activation.

Homophilic binding in trans of the neural cell adhesion molecule (NCAM) mediates adhesion between cells and leads, via activation of intracellular signaling cascades, to neurite outgrowth in primary neurons as well as in the neuronal cell line PC12. NCAM mediates neurite extension in PC12 cells by two principal routes of signaling: NCAM/Fyn and NCAM/fibroblast growth factor receptor (FGFR), respectively. Previous studies have shown that activation of mitogen-activated protein kinases is a pivotal point of convergence in NCAM signaling, but the mechanisms behind this activation are not clear. Here, we investigated the involvement of adaptor proteins in NCAM and fibroblast growth factor 2 (FGF2)-mediated neurite outgrowth in the PC12-E2 cell line. We found that both FGFR substrate-2 and Grb2 play important roles in NCAM as well as in FGF2-stimulated events. In contrast, the docking protein ShcA was pivotal to neurite outgrowth induced by NCAM, but not by FGF2, in PC12 cells. Moreover, in rat cerebellar granule neurons, phosphorylation of ShcA was stimulated by an NCAM mimicking peptide, but not by FGF2. This activation was blocked by inhibitors of both FGFR and Fyn, indicating that NCAM activates FGFR signaling in a manner distinct from FGF2 stimulation, and regulates ShcA phosphorylation by the concerted efforts of the NCAM/FGFR as well as the NCAM/Fyn signaling pathway.

Role of EphA4 and EphrinB3 in local neuronal circuits that control walking.

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.