Physiological, anatomical and genetic identification of CPG neurons in the developing mammalian spinal cord
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.
References (164)
- et al.
Projection from excitatory C3–C4 propriospinal neurones to spinocerebellar and spinoreticular neurones in the C6–Th1 segments of the cat
Neurosci. Res.
(1990) - et al.
Postnatal development of locomotion in the laboratory rat
Anim. Behav.
(1975) The roles of spinal interneurons and motoneurons in the lamprey locomotor network
Prog. Brain Res.
(1999)- et al.
The neuronal network for locomotion in the lamprey spinal cord: evidence for the involvement of commissural interneurons
J. Physiol. (Paris)
(1995) - et al.
Functional identification of interneurons responsible for left–right coordination of hindlimbs in mammals
Neuron
(2003) - et al.
Organization of left–right coordination in the mammalian locomotor network
Brain Res. Rev.
(2002) - et al.
Cytochemical characteristics of cat spinal neurons activated during fictive locomotion
Brain Res. Bull.
(1995) - et al.
Distribution of 5-hydroxytryptamine-immunoreactive boutons on immunohistochemically-identified Renshaw cells in cat and rat lumbar spinal cord
Brain Res.
(1999) - et al.
Ubiquity of motor networks in the spinal cord of vertebrates
Brain Res. Bull.
(2000) - et al.
Two types of motor rhythm induced by NMDA and amines in an in vitro spinal cord preparation of neonatal rat
Neurosci. Lett.
(1990)
Variability as a characteristic of immature motor systems: an electromyographic study of swimming in the newborn rat
Behav. Brain Res.
A comparison of motor patterns induced by N-methyl-d-aspartate, acetylcholine and serotonin in the in vitro neonatal rat spinal cord
Neurosci. Lett.
Some limitations of ventral root recordings for monitoring locomotion in the in vitro neonatal rat spinal cord preparation
Neurosci. Lett.
Activity of muscle spindle afferents during scratching in the cat
Brain Res.
Neuronal patterning: making stripes in the spinal cord
Curr. Biol.
The formation of sensorimotor circuits
Curr. Opin. Neurobiol.
Neural networks that co-ordinate locomotion and body orientation in lamprey
Trends Neurosci.
Contralaterally projecting lamina VIII interneurones in middle lumbar segments in the cat
Brain Res.
Labelling of midlumbar neurones projecting to cat hindlimb motoneurones by transneuronal transport of a horseradish peroxidase conjugate
Neurosci. Lett.
Gap junctions and motor behavior
Trends Neurosci.
Plateau properties in mammalian spinal interneurons during transmitter-induced locomotor activity
Neuroscience
Contributions of intrinsic motor neuron properties to the production of rhythmic motor output in the mammalian spinal cord
Brain Res. Bull.
In vivo neuronal tracing with GFP-TTC gene delivery
Mol. Cell. Neurosci.
Current concepts in neuroanatomical tracing
Prog. Neurobiol.
Cell-type specific organization of glycine receptor clusters in the mammalian spinal cord
J. Comp. Neurol.
Distribution of cholinergic contacts on Renshaw cells in the rat spinal cord: a light microscopic study
J. Physiol.
Neuronal control of swimming locomotion: analysis of the pteropod mollusc Clione and embryos of the amphibian Xenopus
Trends Neurosci.
Postsynaptic expression of tetanus toxin light chain blocks synaptogenesis in Drosophila
Curr. Biol.
Altered electrical properties in Drosophila neurons developing without synaptic transmission
J. Neurosci.
Pharmacological block of the electrogenic sodium pump disrupts rhythmic bursting induced by strychnine and bicuculline in the neonatal rat spinal cord
J. Neurophysiol.
Modulation of the spinal network for locomotion by substance P in the neonatal rat
Exp. Brain Res.
Interaction between disinhibited bursting and fictive locomotor patterns in the rat isolated spinal cord
J. Neurophysiol.
Characterization of reliability of spike timing in spinal interneurons during oscillating inputs
J. Neurophysiol.
The respective contribution of lumbar segments to the generation of locomotion in the isolated spinal cord of newborn rat
Eur. J. Neurosci.
Synaptic targets of commissural interneurons in the lumbar spinal cord of neonatal rats
J. Comp. Neurol.
Spatiotemporal pattern of motoneuron activation in the rostral lumbar and the sacral segments during locomotor-like activity in the neonatal mouse spinal cord
J. Neurosci.
Localization of rhythmogenic networks responsible for spontaneous bursts induced by strychnine and bicuculline in the rat isolated spinal cord
J. Neurosci.
Spontaneous rhythmic bursts induced by pharmacological block of inhibition in lumbar motoneurons of the neonatal rat spinal cord
J. Neurophysiol.
Transneuronal tracing of diverse CNS circuits by Cre-mediated induction of wheat germ agglutinin in transgenic mice
Proc. Natl. Acad. Sci. U.S.A.
The intrinsic factors in the act of progression in the mammal
Proc. R. Soc. Lond. B: Biol.
Identification of interneurons with contralateral, caudal axons in the lamprey spinal cord: synaptic interactions and morphology
J. Neurophysiol.
Electrophysiological properties of identified classes of lamprey spinal neurons
J. Neurophysiol.
Lamprey spinal interneurons and their roles in swimming activity
Brain Behav. E
Activities of spinal neurons during brain stem-dependent fictive swimming in lamprey
J. Neurophysiol.
Functionality of commissural interneurons in the hindlimb central pattern generator of the neonatal rat
Abstr. Soc. Neurosci.
Firing properties of identified interneuron populations in the mammalian hindlimb central pattern generator
J. Neurosci.
Fast and slow locomotor burst generation in the hemi-spinal cord of the lamprey
J. Neurophysiol.
Calbindin D28k expression in immunohistochemically identified Renshaw cells
NeuroReport
Activation of the central pattern generators for locomotion by serotonin and excitatory amino acids in neonatal rat
J. Physiol.
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