No abstract

The properties of mammalian spinal interneurons that underlie rhythmic locomotor networks remain poorly described. Using postnatal transgenic mice in which expression of green fluorescent protein is driven by the promoter for the homeodomain transcription factor Hb9, as well as Hb9 -lacZ knock-in mice, we describe a novel population of glutamatergic interneurons located adjacent to the ventral commissure from cervical to midlumbar spinal cord levels. Hb9 ϩ interneurons exhibit strong postinhibitory rebound and demonstrate pronounced membrane potential oscillations in response to chemical stimuli that induce locomotor activity. These data provide a molecular and physiological delineation of a small population of ventral spinal interneurons that exhibit homogeneous electrophysiological features, the properties of which suggest that they are candidate locomotor rhythm-generating interneurons.

Despite significant advances in our understanding of pattern generation in invertebrates and lower vertebrates, there have been barriers to the application of the principles learned to the definition of networks underlying mammalian locomotion. Major difficulties have arisen in identifying spinal interneurones in preparations which allow study of neuronal intrinsic properties and the role of identified interneurones in locomotor networks. Recent genetic technologies in which selective expression of fluorescent proteins in specific populations of mouse spinal neurones have provided new avenues of investigation. In this review, we focus on the generation of locomotor rhythm and outline criteria that rhythm-generating neurones might be expected to fulfill. We then examine the extent to which a recently identified population of spinal interneurones, Hb9 interneurones, fulfill these criteria. Finally, we suggest that Hb9 interneurones could be involved in an asymmetric model of locomotor rhythmogenesis through projections of electrotonically coupled rhythmgenerating modules to flexor pattern formation half-centres. The principles learned from studying this population of interneurones have led to strategies to systematically evaluate neurones that may be involved in locomotor rhythmogenesis.

Spinal neurons and networks that generate rhythmic locomotor activity remain incompletely defined, prompting the use of molecular biological strategies to label populations of neurons in the postnatal mouse. During spinal cord development, expression of Lhx3 in the absence of Isl1 specifies a V2 interneuronal fate. In this study, postnatal V2-derived interneurons were identified by yellow fluorescent protein (YFP) expression in the double-transgenic offspring of Lhx3Cre/+ x thy1-loxP-stop-loxP-YFP mice. While some motoneurons were labelled, several populations of interneurons predominantly located in lamina VII could also be distinguished. Small interneurons were located throughout the spinal cord whereas larger interneurons were concentrated in the lumbar enlargement. Some V2-derived interneurons were propriospinal, with axons that bifurcated in the lateral funiculus. V2-derived interneurons gave rise to populations of both excitatory and inhibitory interneurons in approximately equal proportions, as demonstrated by in situ hybridization with VGLUT2 mRNA. Immunohistochemical studies revealed YFP+ boutons throughout the spinal cord. Both glutamatergic and glycinergic YFP+ boutons were observed in lamina IX where many apposed motoneuron somata. GABAergic YFP+ boutons were also observed in lamina IX, and they did not form P-boutons. At P0, more than half of the YFP+ interneurons expressed Chx10 and thus were derived from the V2a subclass. In adult mice, there was an increase in Fos expression in V2-derived interneurons following locomotion, indicating that these neurons are active during this behaviour. The heterogeneity of V2-derived interneurons in adult mice indicates that physiologically distinct subpopulations, including last-order interneurons, arise from these embryonically defined neurons.

Following birth, when mammals are relatively immobile, significant development of the motor system facilitates weight bearing and locomotion. Prominent cholinergic C-terminals develop on somata and proximal dendrites of spinal motoneurons during this time period. It is hypothesized that these terminals are essential in regulating motoneuron excitability and thus their development contributes to motor system maturation. Therefore, the development of pre- and postsynaptic components of the C-terminal synapse on motoneurons in mice during the early postnatal period was investigated. Fluorescence immunohistochemical studies revealed that developmental increases in punctate labeling of presynaptic cholinergic terminals, as visualized by vesicular acetylcholine transporter immunoreactivity (VAChT-IR) corresponded to the progressive expression and spatial restriction of immunoreactivity for the calcium channel subunit alpha(1)2.2 (N-type) located presynaptically and the muscarinic type 2 acetylcholine receptor situated postsynaptically. In addition, clustering of immunoreactivity for the potassium channel subunit K(V)2.1 occurred within the early postnatal period in concert and colocalized with the maturation of the C-terminals. The time course of development of these components of the C-terminal synapse corresponds to the maturation of the motor system that enables the animal to locomote in an adult-like fashion.