EphA4 defines a class of excitatory locomotor-related interneurons

Abstract

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 ...

Myelin-derived ephrinB3 restricts axonal regeneration and recovery after adult CNS injury

“…The long fasciculated corticospinal tract develops normally in ephrinB3 −/− mice, but segmental corticospinal tract (CST) projections cross the midline inappropriately after leaving the bundled dorsalCST, before reaching caudal synaptic targets. However, the abnormal bilateral synchronization of hindlimb gait observed in ephrinB3 −/− mice does not depend on misrouted CST fibers but reflects excess midline crossing of EphA4-expressing segmental interneurons of the central locomotor pattern generator in the spinal cord (27)(28)(29).…”

Activity of Renshaw Cells during Locomotor-Like Rhythmic Activity in the Isolated Spinal Cord of Neonatal Mice

“…The source of these EPSCs is therefore more likely to be either ipsilaterally projecting excitatory interneurons in the locomotor network or MNs, which have been shown recently to release glutamate as well as acetylcholine onto RCs (Mentis et al, 2005;Nishimaru et al, 2005). As for the former possibility, two populations of excitatory, ipsilaterally projecting, putative CPG interneurons have been identified recently: interneurons that express the transcription factor HB9 (Hinckley et al, 2005;Wilson et al, 2005) and a subset of neurons expressing the axon guidance marker EphA4 (Butt et al, 2005). Both types of neurons fire during the peak of the ipsilateral ventral root burst, ideal for providing direct or indirect excitation of RCs.…”

“…Electrical recordings and stimulations of the ventral roots were performed with glass suction electrodes placed in close proximity to the exit point of the ventral roots. Locomotor-like rhythmic activity was evoked by bath application of NMDA (5-7.5 M) in combination with serotonin (5-HT) (7-10 M) as described previously (Kullander et al, 2003;Butt et al, 2005). Motor neuron activity was monitored in the second lumbar segment (L2) ventral root.…”

“…In all experiments, we used heterozygote GAD67-GFP knock-in mice (Tamamaki et al, 2003) aged postnatal day 0 -4. The animals were anesthetized with isoflurane, and the spinal cords were removed as described previously (Kullander et al, 2003;Butt et al, 2005). For recording, the dissected spinal cord was placed in a chamber perfused with oxygenated Ringer's solution [in mM: 128 NaCl, 4.69 KCl, 25 NaHCO 3 , 1.18 KH 2 PO 4 , 1.25 MgSO 4 , 2.5 CaCl 2 , and 22 glucose (aerated with 5% CO 2 in O 2 )] at room temperature (20 -23°C).…”

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

“…However, neither the precise connectivity of locomotor-related spinal interneurones nor their intrinsic properties have been defined. That is, despite the demonstration that many ventral interneurones are rhythmically active during locomotor activity in the cat (Angel et al, 2005;Baev et al, 1979;Gossard et al, 1994;Huang et al, 2000;Jankowska et al, 1967a;Matsuyama et al, 2004;Shefchyk et al, 1990) and rodent (Butt and Kiehn, 2003;Butt et al, 2005;Kiehn et al, 1996;Tresch and Kiehn, 1999;Zhong et al, 2006) little is known about the connectivity of these neurones…”