Physiological, anatomical and genetic identification of CPG neurons in the developing mammalian spinal cord

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Abstract

The basic motor patterns underlying rhythmic limb movements during locomotion are generated by neuronal networks located within the spinal cord. These networks are called Central Pattern Generators (CPGs). Isolated spinal cord preparations from newborn rats and mice have become increasingly important for understanding the organization of the CPG in the mammalian spinal cord. Early studies using these preparations have focused on the overall network structure and the localization of the CPG. In this review we concentrate on recent experiments aimed at identifying and characterizing CPG-interneurons in the rodent. These experiments include the organization and function of descending commissural interneurons (dCINs) in the hindlimb CPG of the neonatal rat, as well as the role of Ephrin receptor A4 (EphA4) and its Ephrin ligand B3 (EphrinB3), in the construction of the mammalian locomotor network. These latter experiments have defined EphA4 as a molecular marker for mammalian excitatory hindlimb CPG neurons. We also review genetic approaches that can be applied to the mouse spinal cord. These include methods for identifying sub-populations of neurons by genetically encoded reporters, techniques to trace network connectivity with cell-specific genetically encoded tracers, and ways to selectively ablate or eliminate neuron populations from the CPG. We propose that by applying a multidisciplinary approach it will be possible to understand the network structure of the mammalian locomotor CPG. Such an understanding will be instrumental in devising new therapeutic strategies for patients with spinal cord injury.

Introduction

Classical experiments in the beginning of the last century determined that the spinal cord has an intrinsic rhythmogenic capability (Brown, 1911). These experiments originally performed in the dog showed that the spinal cord in vertebrates contains sufficient neuronal elements to produce rhythmic movements, such as locomotion and swimming, in the absence of sensory inputs. The networks responsible for the rhythmic movements are often called Central Pattern Generators (CPGs). These networks generate both the rhythm as well as the correct patterns of activities. In two non-mammalian vertebrate species, the lamprey and the Xenopus tadpole, the constituent neurons of the CPGs for swimming have been identified and their connections described (Arshavsky Yu et al., 1993, Grillner et al., 1995, Roberts et al., 2000). The advances in understanding the organization of the tadpole and lamprey locomotor CPGs has been greatly facilitated by the fact that these experiments can be performed under in vitro conditions, which are ideal for studying the electrophysiological and pharmacological properties of CPG elements and their connections. This has incited interest in developing viable mammalian spinal cord preparations where a similar network analysis can be applied. The isolated rodent spinal cord is such a preparation, and its introduction to the locomotor field (Kudo and Yamada, 1987, Smith and Feldman, 1987) has generated a wave of new studies of the locomotor networks of the mammalian spinal cord. These studies are released from the restriction imposed by the in vivo conditions of the cat spinal cord, which for decades was the dominant preparation used to study mammalian locomotion. Moreover, the viability of the mouse to genetic manipulation (Lewandoski, 2001, Yu and Bradley, 2001), as well as its use in elucidating the embryonic development and organization of the mammalian spinal cord, has further increased the interest in the rodent spinal cord as a model system for understanding the cellular organization of the mammalian locomotor CPG. Several recent reviews have covered different aspects of the CPG organization in rodents (Butt et al., 2002b, Cazalets and Bertrand, 2000, Cazalets et al., 1998, Kiehn et al., 1997, Kiehn and Kjaerulff, 1998, Kiehn et al., 2000, Kiehn and Tresch, 2002, Kudo and Nishimaru, 1998, Schmidt and Jordan, 2000, Sillar et al., 1997, Vinay et al., 2002). In the present review we focus on experiments that aim at identifying CPG neurons.

Section snippets

The isolated neonatal rodent spinal cord: a model system for studying mammalian locomotion

The spinal cords from newborn rats (0 to about 7 days old) are easily isolated and can survive in vitro for extended periods of time. This preparation was originally developed to study spinal reflex pathways and their pharmacological regulation (Otsuka and Konishi, 1974); however, in the eighties Kudo and Yamada (1987) and Smith and Feldman (1987) first used the preparation to demonstrate that exposure of the cord to a N-methyl-d-aspartic acid (NMDA)-receptor agonist resulted in rhythmic

Localization of the hindlimb CPG

One of the first steps necessary for analyzing the locomotor CPG is to determine its precise anatomical location in the spinal cord. This has been done in the neonatal rat by lesion/isolation studies and by applying locomotor-inducing drugs to restricted areas of the cord. These studies are ideal to perform in vitro. The results of these experiments as well as their comparison to findings in other vertebrates including the cat, turtle, chick and mudpuppy have been reviewed extensively

The black box approach can reveal the overall network structure

The network structure of CPGs was initially inferred from recordings of the activity in motor neurons or interneurons (Grillner, 1975, Grillner, 1985) and by performing pharmacological intervention experiments. This black box approach has also been applied to the rodent CPG and has given some information about the overall structure of the CPG.

Intracellular recordings from lumbar motor neurons in the neonatal rat have shown that the predominant synaptic pattern impinging onto those cells from

Commissural interneurons: the first identified group of CPG-neurons in the rodent spinal cord

Opening the box requires intracellular recordings from interneurons. The small size (10–15 μm) of the interneurons in the neonatal rodent spinal cord makes them, however, largely inaccessible to sharp electrode recordings (MacLean et al., 1995). The introduction of tight-seal whole cell recordings to the cord has alleviated this problem and made recordings from interneurons readily available. Initially, recordings were performed from rhythmically active unidentified neurons in the ventromedial

Identification of excitatory CPG neurons in the rodent spinal cord

Ipsilateral projecting glutamatergic excitatory interneurons (EIs) are the most likely sources for rhythm generation in the tadpole and lamprey swimming CPGs. They provide the drive to other ipsilaterally located CPG neurons and motor neurons.

Until now, limited knowledge has accumulated about the identity of EIs in the mammalian locomotor CPGs. Several groups have identified rhythmically active interneurons in locomotor related group I afferent pathways (Gossard et al., 1994, Hultborn, 2001,

New methods to identify mammalian CPG neurons and their connectivity

In the previous section we described experiments that show that genetic knockout experiments can be a useful tool in studying CPG organization and co-ordination in the mammalian spinal cord. In this section we will briefly consider some other genetic methods for identifying CPG neurons. All of these methods will have to be applied in combination with classical electrophysiological methods to obtain a functional understanding of the neurons in the network.

Conclusions

The isolated rodent spinal cord preparation has proven to be a useful model in which to study the spinal networks generating locomotion in mammals. Early experiments in this preparation have primarily addressed questions regarding the neurotransmitter control and localization of the CPG, as well as the overall CPG structure. Recent studies are now aimed at identifying populations of CPG neurons and resolving their function in the network. The anatomical and electrophysiological characterization

Acknowledgements

The research in Ole Kiehn’s Laboratory is supported by the NIH, Human Frontier Science Program, the Swedish Research Council, and Karolinska Institutet. We thank our colleagues for many discussions of some of the work presented here. We would like to thank David Schmitt for his helpful comments.

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