Locomotor-Like Rhythms in a Genetically Distinct Cluster of Interneurons in the Mammalian Spinal Cord

Abstract

Electrophysiological and morphological properties of genetically identified spinal interneurons were examined to elucidate their possible contribution to locomotor-like rhythmic activity in 1- to 4-day-old mice. In the transgenic mice used in our study, the HB9 promotor controlled the expression of the reporter gene enhanced green fluorescent protein (eGFP), giving rise to GFP+ motoneurons and ventral interneurons. However, only motoneurons and a small group of bipolar, GFP+ interneurons expressed the HB9 protein. The HB9(+)/GFP+ interneurons were clustered close to the medial surface in lamina VIII along segments L1-L3. The correlation between activity pattern in these visually identified interneurons and motoneuron output was examined using simultaneous whole cell and ventral root recordings. Neurochemically induced rhythmic membrane depolarizations in HB9/GFP interneurons were synchronous with ventral root rhythms, indicating that the interneurons received synaptic inputs from rhythm-generating networks. The frequency of excitatory postsynaptic currents significantly increased during ventral root bursts, but there was no change in the frequency of inhibitory postsynaptic currents during the cycle period. These data implied that HB9/GFP interneurons received primarily excitatory inputs from rhythmogenic interneurons. Neurobiotin-filled axon terminals were in close apposition to other neurons in the cluster and to motoneuron dendrites, raising the possibility that HB9/GFP interneurons formed synaptic connections with each other and with motoneurons. The expression of the vesicular glutamate transporter 2 in axon terminals of HB9/GFP interneurons indicated that these were glutamatergic interneurons. Our findings suggest that the visually identified HB9/GFP interneurons are premotor excitatory interneurons and putative constituents of networks generating locomotor rhythms in the mammalian spinal cord.

Strategies for delineating spinal locomotor rhythm-generating networks and the possible role of Hb9 interneurones in rhythmogenesis

“…Interestingly, we have found that, in addition to GFP expression in motoneurones, there is a population of small, clustered glutamatergic GFP positive interneurones located in medial lamina VIII (and ventral lamina X) bordering on the ventral commissure Fig. 1A, B; see also Hinckley et al, 2005). By crossing Hb9::GFP mice with Hb9 nlslacZ/+ knock-in mice, we demonstrated that the GFP positive neurones in medial lamina VIII were indeed Hb9 positive.…”

“…1I). In addition, GFP positive neurones in this area in the hemisected spinal cord are rhythmically active during drug-induced rhythmic activity in the newborn mouse (Hinckley et al, 2005, Fig. 1I).…”

“…However, some evidence has been presented that GFP positive neurones in this region send processes to the motor pools (Hinckley et al, 2005). This remains an unresolved question.…”

“…The use of molecular biological approaches to alter the expression or activity of these specific classes of interneurones has allowed for the identification and study of populations of neurones derived from these domains (Gosgnach et al, 2006;Lanuza et al, 2004). Furthermore, by using fluorescent proteins which are driven by the promoters for these transcription factors, it is possible to study and define these populations anatomically (Alvarez et al, 2005) or physiologically (Hinckley et al, 2005;Wilson et al, 2005) in the post-natal animal. Importantly, this has led to the ability to study the intrinsic properties of identified populations of neurones .…”

“…Given that Hb9 interneurones fulfill many of the criteria necessary for locomotor rhythmogenic neurones (see above), including that they are conditional endogenous bursters (see above, and Wilson et al, 2005), that during rhythmic activity they appear to burst in phase with ventral root activity (Hinckley et al, 2005), that locomotor rhythmogenesis seems to arise in large part from the part of the spinal cord which produces flexor-related bursting activity, L2 (Cazalets et al, 1995;Cowley and Schmidt, 1997;Kjaerulff and Kiehn, 1996;Marcoux and Rossignol, 2000), and that the rhythm generator may not be organised in a symmetric flexor-extensor half-centre network, we would propose the possibility that the locomotor network may be arranged as illustrated in Fig. 3.…”