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Development of central pattern generating circuits
Development of central pattern generating circuits Eve Marder and Kristina J Rehm The networks that generate rhythmic motor patterns in invertebrates and vertebrates are ideal for studying the mechanisms by which functional circuits are formed during development. Rhythmic motor patterns and movements are seen embryonically, before they are needed for behavior; recent work suggests that activity in immature spinal cord networks is important for circuit formation and transmitter specification. Despite significant advances in describing the patterns of transcription factor expression in both invertebrate nervous systems and vertebrate spinal cord, a real understanding of how central pattern generators develop is hindered by our lack of knowledge of the organization of these circuits in adults. Addresses Volen Center and Biology Department, MS 013, Brandeis University, Waltham, Massachusetts 02454-9110, USA Corresponding author: Marder, Eve ([email protected])
Current Opinion in Neurobiology 2005, 15:86–93 This review comes from a themed issue on Development Edited by Jane Dodd and Alex L Kolodkin Available online 26th January 2005 0959-4388/$ – see front matter # 2005 Elsevier Ltd. All rights reserved.
patterns they generate are easy to record, and second, the behaviors that they control are important for the animal [2]. For example, the central pattern generating circuits that control breathing, feeding, and locomotion are necessary for animals to survive after they hatch or are born. Unfortunately, the few systems in which central pattern generating circuits are relatively well understood [3,4] are not ideal for developmental studies, and little is known about the identity of the neurons and their connections in the central pattern generating circuits of those animals commonly used for developmental and molecular studies. Nonetheless, considerable progress on numerous fronts suggests that studying circuit construction using motor circuits that generate rhythmic motor patterns will be extremely fruitful in years to come, and some of these first steps are described below.
Spontaneous motor activity in the embryo Embryos move before animals hatch or are born. For example, chick embryos show spontaneous leg movements [5]; the spinal cords and brainstems of chick, rat and mouse embryos show rhythmic activity [6,7,8,9, 10,11,12]; Drosophila melanogaster embryos generate peristaltic movements similar to those observed in the larvae [13]; and the stomatogastric nervous system of embryonic lobsters generates rhythmic motor patterns [14–16,17] before the animal hatches and starts feeding.
How are functional circuits constructed during development? Answering this question requires a circuit or set of circuits with known and measurable functions, and access to those circuits at different times in development. Despite many recent and remarkable advances in developmental biology, understanding how circuits are formed has been hampered by our relatively rudimentary knowledge of the organization of most circuits in the adult nervous system. In many brain regions it is difficult to define, except in the most general of terms, what the circuit does, what the cell types in the circuit are, and how the neurons are connected. For this reason, much developmental work on circuits has focused on primary sensory areas endowed with orderly topographic maps, such as visual cortex [1], where both functional and anatomical correlates of circuit function can be studied.
What functions are served by these embryonic movements? Are these spontaneous rhythmic discharges produced by precociously wired up central pattern generators, or are they bursts of activity produced by networks that are immature and differ significantly from those seen in the adult? In some preparations there is evidence indicating that the earliest rhythmic motor discharges might be produced by an immature network that is supplanted by the one that will operate later in the animal’s life. In other preparations the basic backbone of the adult circuit appears very early in embryonic development, and developmental differences in motor patterns could be attributable to alterations in sensory and neuromodulatory inputs. Obviously, in some ways this is a false dichotomy, because all circuits must be constructed de novo during development, but it does focus our attention on the question of whether or not there is an extended period during embryonic development in which motor activity is produced by different network architectures and/or different cellular and synaptic mechanisms from those operating later in life.
Motor circuits that produce rhythmic movements are also amenable to developmental study because first, the motor
The earliest movements generated by the chick spinal cord are generated by a circuit different from that which
DOI 10.1016/j.conb.2005.01.011
Introduction
Current Opinion in Neurobiology 2005, 15:86–93
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Development of central pattern generating circuits Marder and Rehm 87
functions later in life. This is evident because in the adult spinal cord motor neurons are driven by central pattern generating circuits that consist of interneurons. By contrast, the circuitry generating the early spinal cord activity depends heavily on cholinergic excitatory connections, presumably from collaterals of the motor neurons themselves [18]. Later in development glutamate is the predominant excitatory transmitter used in the central pattern generating interneuronal circuitry that drives movement. If cholinergic transmission is pharmacologically blocked early in development, the embryonic circuit is briefly disrupted but rhythmic activity returns, and is thereafter dependent on GABAergic transmission [6]. Recordings made systematically from the embryonic mouse and rat spinal cords at different days of development show that the patterns of activity change during an extended period of time [9,12], both in their expression and in the neurotransmitters that mediate excitatory transmission. An elegant recent study by Hanson and Landmesser [7] studied the mouse spinal cord at E11 to E14, before all of the motor neurons have reached their targets. Two types of spontaneous episodes were observed, major episodes that propagated throughout the cord, and more local activity. Nicotinic cholinergic antagonists slowed the frequency of bursting activity, demonstrating again that much of the early rhythmic activity appears to be a function of a circuit depending heavily on cholinergic transmission from the motor neurons themselves. What functions do these precocious motor patterns and movements serve? The most obvious answer is that the activity produced in these developing circuits is needed to generate activity-dependent tuning or wiring signals. Recent exciting work in the chick showed that chronic in ovo application of drugs that block glycine and g-aminobutyric acid (GABA)-mediated synapses altered the patterns of spontaneous activity, resulting in pathfinding errors and produced misplaced motor neuron somata [8]. Additionally, these picrotoxin treatments altered the expression of polysialic acid (PSA) and downregulated EphA4 from dorsally projecting axons. Before this demonstration, the consensus was that early pathfinding and outgrowth decisions would be primarily genetically encoded, and that activity would be more important for later processes of synapse stabilization and fine-tuning of circuitry [19]. The importance of activity for specification of neurotransmitter phenotype in the developing spinal cord is the subject of another valuable new study [20]. Although there is a large amount of literature describing factors that influence neurotransmitter phenotype in the sympathetic nervous system [21], the authors of this study characterize the specific patterns of Ca2+ spikes in several classes of neurons in the Xenopus laevis spinal cord, and demonstrate www.sciencedirect.com
that changes in these activity patterns result in neurotransmitter phenotype switches without affecting the expression of markers of cell identity. The authors view this as an homeostatic response because decreased activity led to an increased number of neurons expressing excitatory transmitters and a decreased number of neurons expressing inhibitory transmitters and vice versa [20].
Genetic markers and identification of pattern generating interneurons Much of our insight into the fundamental mechanisms underlying pattern generation has come from small invertebrate circuits in which it was possible to identify neurons easily on the basis of electrophysiological and anatomical criteria [3,22]. The ability to identify individual or small classes of neurons made it possible to study their connectivity and their role in generating motor patterns. The same strategy has been partially successful in several vertebrate preparations such as the tadpole [23] and lamprey [24]. However, even in the simplest spinal cords identifying neurons and establishing their properties and connectivity is far more difficult than it was any of the classic invertebrate preparations such as the leech heartbeat system, the stomatogastric ganglion, or the Tritonia swim system [3]. Until recently, identification of interneuron classes in the spinal cord depended on electrophysiological criteria, augmented by immunocytochemistry and conventional anatomical tracing methods. Using these methods, progress has been made in identifying interneuronal classes in the lamprey, amphibian, and cat spinal cords [23,24,25,26,27]. With the advent of new molecular tools, there is the exciting potential that these tools will be helpful not only for understanding early developmental events [28,29,30] but also for aiding neuronal identification and revealing patterns of connectivity in the spinal cord. This has led to increased interest in the organization of the rodent and zebrafish spinal cords [31,32] and numerous studies using both classical anatomical and physiological methods [33,34,35–38] and molecular tools are appearing [31,39,40,41,42–44,45,46,47,48]. These studies include attempts to use transcription factor expression to identify neuronal populations in the spinal cord [31,39,40,41,42–44,45,46,49,50]. That said, transcription factor expression studies have not yet yielded cell identifications with the requisite precision for the construction of the detailed functional wiring diagrams needed to understand the operation of spinal cord circuitry and motor pattern generation. A notable exception is a recent study looking at two transcription factors important in Renshaw cell development [47]. In studies on small invertebrate pattern generators, the function of individual neurons or circuit elements is studied by perturbing the activity of a neuron [51] and/ Current Opinion in Neurobiology 2005, 15:86–93
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or by deleting it from the circuit [52] to see the effects of these manipulations on the motor patterns produced. Electrophysiological perturbations of the activity of single neurons in the spinal cord are unlikely to be particularly useful in altering the output of a central pattern generator comprised of many neurons. Nonetheless, in principle, genetic approaches enable the manipulation or deletion of whole classes of spinal cord interneurons in order to determine their role in pattern generation. For example, Lanuza et al. [46] studied the locomotory motor patterns produced by mice mutant for the Dbx1 homeobox gene and found disturbed patterns of hindlimb left–right alternation, suggesting that the ventral interneurons that express this gene are important for controlling left–right alternation. Mice mutant for the EphA4 receptor or its ligand ephrinB3 also show defects in left–right alternation [53] that can be restored pharmacologically. These studies are only the first of many using molecular and genetic tools that will undoubtedly aid in identifying the classes of spinal cord interneurons and their function both in development and in the adult. Another use of genetic tools is to add neurons to a circuit during development. Two new studies in the zebrafish [54,55] demonstrate that additional Mauthner neurons incorporate into and function in the developing escape circuit. The authors suggest that incorporation of duplicated neurons could be a mechanism for circuit evolution [54,55].
The roles of sensory and descending inputs on the development of central pattern generating circuits In the course of normal behavior, central pattern generating networks receive inputs from sensory and descending neuromodulatory inputs, which adapt the motor patterns produced by central pattern generating circuits to the animal’s needs [4]. Therefore, it is interesting to ask what roles these inputs might take in the development of central pattern generating circuits. A recent study in Drosophila [13] showed that altered but functional embryonic and larval motor patterns were produced by animals that developed in the absence of most of their sensory inputs. This study was remarkable both because it had been assumed previously that loss of the sensory neurons would perturb early pathfinding and target selections and because it showed clearly the relative independence of the central pattern generating mechanisms from the phasic sensory drive. Similar alterations in movements were seen in mutants lacking only the chordotonal organs [56], suggesting that the chordotonal organs provide a major input to the central pattern generating circuitry that produces larval movements. In Drosophila, the dendritic arbors of identified motor neurons are found in specific regions of the neuropil, presumably correctly placed to receive appropriate synaptic inputs from central pattern generating neurons [57]. Genetic methods are Current Opinion in Neurobiology 2005, 15:86–93
now being employed to reveal the identity of neurons that influence locomotory programs [58], to enable intracellular recordings from identified neurons in Drosophila [59], or to study the effects of sensory neurons on locomotion [60]. The roles that neuromodulatory inputs could take in the development and maturation of central pattern generating circuits have been studied in numerous preparations. In the crustacean stomatogastric nervous system, the central pattern generating circuit consists largely of connections among the motor neurons themselves. Midway through embryonic development in this system the full complement of neurons is present and, although the embryonic motor patterns differ from those in the adult, under appropriate neuromodulatory conditions adult-like motor patterns can be elicited from the embryonic network [14,15,61]. One of the salient take-home messages from these studies is that the neuromodulatory complement of identified sensory and descending neuromodulatory inputs changes during embryonic and larval stages [62–65] and that receptors and transporters for a neuromodulator might be present before the neuromodulatory substance itself is present in sensory or projection neurons [17]. Interestingly, although many of the actions of neuromodulators appear to be unchanged through embryonic, larval and adult life [65,66], other neuromodulatory actions change during development [17]. Recent studies of the maturation of rodent locomotory systems suggest that appropriate neuromodulatory drive might be crucial for the function [67–69] and maturation of central pattern generating circuits. In addition to being a modulator of embryonic and larval spinal cord central pattern generators [69–71], serotonin might also be important in shaping the formation of the central pattern generating circuits required for locomotion [72,73]. Specifically, when descending serotonergic pathways in the medulla are removed, spinal interneurons express a serotonergic phenotype, and serotonin appears to influence the time course of the development of inhibitory pathways in the spinal cord [72]. Recent work on the factors that influence the development of serotonergic neurons [74] should increase our ability to manipulate this neuromodulatory system more precisely in development. In mammals it is crucial that the respiratory network is functional when the animal is born, or death ensues within a few minutes [75]. The preBo¨ tzinger complex (preBo¨ tC) contains neurons that are thought to provide a pacemaker for respiration [76] and lesions of these neurons disturb or disrupt breathing [77]. PreBo¨ tC neurons express the transcription factor MafB and animals with mutations in the gene that encodes this factor die at birth [78]. A new study has investigated the influence of descending noradrenergic neurons on the development of the mouse respiratory system [79]. In mice mutant for www.sciencedirect.com
Development of central pattern generating circuits Marder and Rehm 89
the Phox2A gene, the noradrenergic A6 neurons fail to develop (these neurons ordinarily provide modulatory drive to the preBo¨ tC), and these animals die at birth. In vitro preparations from these animals show altered responses to hypoxia and to norepinephrine. Moreover, gestational treatments of wild type animals with a noradrenergic antagonist produced significantly altered respiratory rhythms [79], suggesting that the neuromodulator is required for correct maturation of the network.
Reconfiguring circuits as a consequence of changes in body plan During development and growth all animals change size and shape. Although in mammals these changes are continuous and gradual, many other animals undergo stage-specific abrupt changes in body plan as a consequence of molts and metamorphoses. For example, as tadpoles metamorphose into frogs, they lose their tail and acquire legs, and holometabolous insects such as Drosophila and Manduca sexta undergo behavioral and body plan changes as they are transformed from the crawling larval stages into flying adults with wings and legs. There is a great deal known about the steroid-controlled remodeling events in insect metamorphosis [80,81], both at the neuromuscular junction [82,83] and in the nerve cord [84]. A recent study demonstrates that steroidinduced dendritic regression accompanied by synapse loss in Manduca can be attenuated by electrical activity [85]. Thus, even in an animal in which stereotyped alterations in the nervous system accompany alterations in body plan, activity might influence the formation of the new circuits. Amphibian metamorphosis is associated with a complete reorganization of the animal’s locomotory systems. In a fascinating new study, Combes et al. [86] record from isolated spinal cord and brainstem preparations before, during and after metamorphosis. Not surprisingly, recordings from ventral roots of pre-metamorphic tadpoles revealed motor outputs consistent with swimming, whereas postmetamorphic animals expressed motor patterns consistent with kicking. Recordings from animals in the midst of metamorphosis revealed two motor patterns with distinct periods that sometimes occurred separately and sometimes occurred together. These data suggest that during metamorphosis the circuits for both behaviors are functional in the spinal cord [86], creating the opportunity to understand the mechanisms by which these circuits are reconfigured during a major maturational event. Not only do the locomotory circuits change during amphibian metamorphosis but frogs also make a transition from gill-breathing to lung-breathing [87]. The circuit required for the adult behavior is present early in development, and is actively inhibited before needed [87], as is thought www.sciencedirect.com
to be the case in several other preparations [15,88]. That said, numerous changes occur in the amphibian respiratory pattern generators during metamorphosis [89].
Conclusions Although molecular and genetic tools have the potential to facilitate manipulations of developing motor systems in both vertebrate and invertebrate preparations, they cannot entirely replace the slow slogging effort needed to understand how rhythmic motor patterns in adult animals are produced. Sadly, to date, those organisms best suited for the study of the mechanisms underlying the organization and function of central pattern generators are not those best suited for studying their development. Conversely, very little is known about the organization of motor circuits in those organisms ideally suited for molecular and genetic manipulations during early development. The defined motor patterns produced by both vertebrate and invertebrate central pattern generators enable extremely accurate assessment of altered network function. Ongoing work on the classic preparations used to study mechanisms of central pattern generation will continue to inform new studies on genetic and molecular development of central pattern generators, and make possible understanding these circuits in organisms with large numbers of neurons.
Acknowledgements This work was supported by NS 17813 (E Marder) and an Individual NRSA award NS 050479 (K Rehm).
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Current Opinion in Neurobiology 2005, 15:86–93
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33. Nissen UV, Mochida H, Glover JC: Development of projection specific interneurons and projection neurons in the embryonic mouse and rat spinal cord. C Comp Neurol 2004, in press. This is a careful description of the development of spinal cord interneurons in rats and mice using classical anatomical and immunocytochemical methods. 34. Butt SJ, Kiehn O: Functional identification of interneurons responsible for left-right coordination of hindlimbs in mammals. Neuron 2003, 38:953-963. In this study, the authors subdivide a population of descending commissural interneurons into four functionally different classes on the basis of their synaptic actions on motor neurons. The authors go on to study the action of each of these classes during fictive motor patterns and suggest some hypotheses about their roles in the organization of locomotion. 35. Butt SJ, Harris-Warrick RM, Kiehn O: Firing properties of identified interneuron populations in the mammalian hindlimb central pattern generator. J Neurosci 2002, 22:9961-9971. 36. Birinyi A, Viszokay K, Weber I, Kiehn O, Antal M: Synaptic targets of commissural interneurons in the lumbar spinal cord of neonatal rats. J Comp Neurol 2003, 461:429-440. 37. Kadison SR, Kaprielian Z: Diversity of contralateral commissural projections in the embryonic rodent spinal cord. J Comp Neurol 2004, 472:411-422. 38. Szucs P, Odeh F, Szokol K, Antal M: Neurons with distinctive firing patterns, morphology and distribution in laminae V-VII of the neonatal rat lumbar spinal cord. Eur J Neurosci 2003, 17:537-544. 39. Dasen JS, Liu JP, Jessell TM: Motor neuron columnar fate imposed by sequential phases of Hox-c activity. Nature 2003, 425:926-933. 40. Sockanathan S, Perlmann T, Jessell TM: Retinoid receptor signaling in postmitotic motor neurons regulates rostrocaudal positional identity and axonal projection pattern. Neuron 2003, 40:97-111. In this study, the authors show that retinoid receptor activation is important in specifying motor neuron subtypes. This suggests a regionally restricted role for retinoid signaling. 41. Novitch BG, Wichterle H, Jessell TM, Sockanathan S: A requirement for retinoic acid-mediated transcriptional activation in ventral neural patterning and motor neuron specification. Neuron 2003, 40:81-95. In this study, the authors investigate the roles of retinoid signaling in neuronal specification in the ventral spinal cord. 42. William CM, Tanabe Y: Jessell TM: Regulation of motor neuron subtype identity by repressor activity of Mnx class homeodomain proteins. Development 2003, 130:1523-1536. 43. Helmbacher F, Dessaud E, Arber S, deLapeyriere O, Henderson CE, Klein R, Maina F: Met signaling is required for recruitment of motor neurons to PEA3-positive motor pools. Neuron 2003, 39:767-777. 44. Lin AW, Carpenter EM: Hoxa10 and Hoxd10 coordinately regulate lumbar motor neuron patterning. J Neurobiol 2003, 56:328-337. 45. Kiehn O, Kullander K: Central pattern generators deciphered by molecular genetics. Neuron 2004, 41:317-321. An excellent review of new advances in the use of genetic and molecular tools to understand the organization of mammalian central pattern generating networks. 46. Lanuza GM, Gosgnach S, Pierani A, Jessell TM, Goulding M: Genetic identification of spinal interneurons that coordinate left-right locomotor activity necessary for walking movements. Neuron 2004, 42:375-386. A subset of commissural spinal interneurons express the homeobox gene Dbx1. Mutant mice without these ventral interneurons do not show normal patterns of left–right alternation, suggesting that these neurons are part of the central pattern generating circuit in normal locomotion. 47. Sapir T, Geiman EJ, Wang Z, Velasquez T, Mitsui S, Yoshihara Y, Frank E, Alvarez FJ, Goulding M: Pax6 and Engrailed 1 regulate two distinct aspects of Renshaw cell development. J Neurosci 2004, 24:1255-1264. In this study, the authors provide evidence that a subset of V1 interneurons become Renshaw cells, one of the best-defined populations of www.sciencedirect.com
spinal cord interneurons. Pax6 (paired box gene 6) is required for early Renshaw cell development, and Engrailed 1 (En1) is important for synapse formation between Renshaw cells and motor neurons. 48. Millen KJ, Millonig JH, Hatten ME: Roof plate and dorsal spinal cord dl1 interneuron development in the dreher mutant mouse. Dev Biol 2004, 270:382-392. Inductive signals from the roof plate control the production of dorsal interneurons in the spinal cord. In this study, the authors show that a small number of neurons express Lmx1a at E8.5 in the lateral neural plate. Lmx1a expression is restricted to the roof plate when the neural tube closes. In dreher mice drJdrJ, although non-functional Lmx1a is correctly expressed at E8.5-E9.5, it disappears by E10.5. As it does, Bmp expression fails, and the generation and differentiation of the dorsal-most spinal cord interneurons is abnormal. 49. Higashijima S, Masino MA, Mandel G, Fetcho JR: Engrailed-1 expression marks a primitive class of inhibitory spinal interneuron. J Neurosci 2004, 24:5827-5839. This study demonstrates the utility (in some cases) of transcription factors for cell identification in the zebrafish spinal cord. Engrailed-1 expression marks a class of ascending interneurons, called circumferential ascending (CiA) interneurons that express the glycine transporter 2 gene. The CiA neurons are rhythmically active during swimming and make monosynaptic connections with other neurons. Individual Engrailed-1-positive cells function in both sensory gating and motor pattern generation. 50. Thaler JP, Koo SJ, Kania A, Lettieri K, Andrews S, Cox C, Jessell TM, Pfaff SL: A postmitotic role for Isl-class LIM homeodomain proteins in the assignment of visceral spinal motor neuron identity. Neuron 2004, 41:337-350. 51. Weimann JM, Marder E: Switching neurons are integral members of multiple oscillatory networks. Curr Biol 1994, 4:896-902. 52. Miller JP, Selverston AI: Mechanisms underlying pattern generation in lobster stomatogastric ganglion as determined by selective inactivation of identified neurons. IV. Network properties of pyloric system. J Neurophysiol 1982, 48:1416-1432. 53. Kullander K, Butt SJ, Lebret JM, Lundfald L, Restrepo CE, Rydstrom A, Klein R, Kiehn O: Role of EphA4 and EphrinB3 in local neuronal circuits that control walking. Science 2003, 299:1889-1892. In this study, the authors characterize the motor patterns generated by mice lacking either ephrinB3 or the EphA4 receptor. Isolated spinal cords from newborn mice were studied. Bilateral ventral roots displayed an abnormal synchronous rhythm instead of the normal left–right alternation seen in wild type mice of the same age. Left–right alternation is supposed to be mediated by commissural interneurons that cross the midline. In the mutant mice additional fibers were seen to cross the midline, suggesting that these aberrant connections were responsible, at least in part, for the abnormal motor patterns. 54. Hale ME, Kheirbek MA, Schriefer JE, Prince VE: Hox gene misexpression and cell-specific lesions reveal functionality of homeotically transformed neurons. J Neurosci 2004, 24:3070-3076. The authors asked whether or not ectopic Mauthner cells made by manipulations of the Hox genes could integrate into spinal circuitry and participate in the escape response of larval zebrafish. Using calcium imaging the authors found that the homeotically transformed neurons responded to startle stimuli and that lesions of the normal Mauthner cells did not decrease escape performance when the ectopic cells were present. From these data the authors suggest that the incorporation of functionally redundant neurons into circuits could provide a substrate for circuit evolution. 55. Liu KS, Gray M, Otto SJ, Fetcho JR, Beattie CE: Mutations in deadly seven/notch1a reveal developmental plasticity in the escape response circuit. J Neurosci 2003, 23:8159-8166. Supernumerary Mauthner cells result from mutations in the zebrafish deadly seven/notch1a (des) gene. In this study the authors investigate whether or not and how these additional Mauthner neurons become wired into the escape circuit. Using Ca2+ imaging, the authors find that the Mauthner cells in desb420 mutants were active during an escape response and that the escape response in mutant larvae was similar to that in wild type fish. The authors argue that excess Mauthner cells are incorporated into the escape–response circuit in such a way to produce a normal response. Current Opinion in Neurobiology 2005, 15:86–93
92 Development
56. Caldwell JC, Miller MM, Wing S, Soll DR, Eberl DF: Dynamic analysis of larval locomotion in Drosophila chordotonal organ mutants. Proc Natl Acad Sci USA 2003, 100:16053-16058. Mutants lacking chordotonal organs show altered larval movements, similar to those generated in animals lacking almost all sensory neurons, suggesting that the chordotonal organs play a major part in the production of normal larval movements. 57. Landgraf M, Jeffrey V, Fujioka M, Jaynes JB, Bate M: Embryonic origins of a motor system: motor dendrites form a myotopic map in Drosophila. PLoS Biol 2003, 1:E41. The authors present a description of the development of the structure of motor dendrites in the larval Drosophila. 58. Suster ML, Martin JR, Sung C, Robinow S: Targeted expression of tetanus toxin reveals sets of neurons involved in larval locomotion in Drosophila. J Neurobiol 2003, 55:233-246. In this study, the authors blocked transmitter release from defined populations of larval Drosophila neurons using targeted tetanus toxin expression. Expression of toxin in dopaminergic and serotonergic neurons altered turning. 59. Choi JC, Park D, Griffith LC: Electrophysiological and morphological characterization of identified motor neurons in the Drosophila third instar larva central nervous system. J Neurophysiol 2004, 91:2353-2365. The authors used genetic methods to express green fluorescent protein (GFP) in subsets of neurons, and then electrophysiological recordings and dye-fills of individual neurons were made. This is an important paper because it is one of the first to obtain voltage clamp recordings from identified neurons in the fly. 60. Ainsley JA, Pettus JM, Bosenko D, Gerstein CE, Zinkevich N, Anderson MG, Adams CM, Welsh MJ, Johnson WA: Enhanced locomotion caused by loss of the Drosophila DEG/ENaC protein Pickpocket1. Curr Biol 2003, 13:1557-1563. Pickpocket1 (PPK1) is a Drosophila subunit of the epithelial sodium channel (ENaC) that is found in dendritic (md) sensory neurons in the larval body wall and in some bipolar neurons in the brain. ppk1 null mutant larvae showed normal touch sensation and md neuron morphology but altered crawling. 61. Fe´ nelon VS, Le Feuvre Y, Meyrand P: Phylogenetic, ontogenetic and adult adaptive plasticity of rhythmic neural networks: a common neuromodulatory mechanism? J Comp Physiol A Neuroethol Sens Neural Behav Physiol 2004, 190:691-705. The authors present a comprehensive review of the development of the stomatogastric nervous system and compare it with other preparations. 62. Fe´ nelon VS, Kilman V, Meyrand P, Marder E: Sequential developmental acquisition of neuromodulatory inputs to a central pattern-generating network. J Comp Neurol 1999, 408:335-351. 63. Kilman VL, Fe´ nelon V, Richards KS, Thirumalai V, Meyrand P, Marder E: Sequential developmental acquisition of cotransmitters in identified sensory neurons of the stomatogastric nervous system of the lobsters, Homarus americanus and Homarus gammarus. J Comp Neurol 1999, 408:318-334. 64. Le Feuvre Y, Fe´ nelon VS, Meyrand P: Ontogeny of modulatory inputs to motor networks: early established projection and progressive neurotransmitter acquisition. J Neurosci 2001, 21:1313-1326. 65. Pulver SR, Thirumalai V, Richards KS, Marder E: Dopamine and histamine in the developing stomatogastric system of the lobster Homarus americanus. J Comp Neurol 2003, 462:400-414. 66. Richards KS, Marder E: The actions of crustacean cardioactive peptide on adult and developing stomatogastric ganglion motor patterns. J Neurobiol 2000, 44:31-44. 67. Merrywest SD, McDearmid JR, Kjaerulff O, Kiehn O, Sillar KT: Mechanisms underlying the noradrenergic modulation of longitudinal coordination during swimming in Xenopus laevis tadpoles. Eur J Neurosci 2003, 17:1013-1022. 68. Pearson SA, Mouihate A, Pittman QJ, Whelan PJ: Peptidergic activation of locomotor pattern generators in the neonatal spinal cord. J Neurosci 2003, 23:10154-10163. Current Opinion in Neurobiology 2005, 15:86–93
69. Madriaga MA, McPhee LC, Chersa T, Christie KJ, Whelan PJ: Modulation of locomotor activity by multiple 5-HT and dopaminergic receptor subtypes in the neonatal mouse spinal cord. J Neurophysiol 2004, 92:1566-1576. 70. Brustein E, Chong M, Holmqvist B, Drapeau P: Serotonin patterns locomotor network activity in the developing zebrafish by modulating quiescent periods. J Neurobiol 2003, 57:303-322. 71. McLean DL, Sillar KT: Divergent actions of serotonin receptor activation during fictive swimming in frog embryos. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 2004, 190:391-402. 72. Branchereau P, Chapron J, Meyrand P: Descending 5-hydroxytryptamine raphe inputs repress the expression of serotonergic neurons and slow the maturation of inhibitory systems in mouse embryonic spinal cord. J Neurosci 2002, 22:2598-2606. 73. Cazalets JR, Gardette M, Hilaire G: Locomotor network maturation is transiently delayed in the MAOA- deficient mouse. J Neurophysiol 2000, 83:2468-2470. 74. Ding YQ, Marklund U, Yuan W, Yin J, Wegman L, Ericson J, Deneris E, Johnson RL, Chen ZF: Lmx1b is essential for the development of serotonergic neurons. Nat Neurosci 2003, 6:933-938. This is a study of the mechanisms that influence the development of serotonin neurons in the raphe nuclei in mice. A LIM homeodomaincontaining gene Lmx1b, is required for the development of the neurons that contain 5-HT. 75. Viemari JC, Burnet H, Bevengut M, Hilaire G: Perinatal maturation of the mouse respiratory rhythm-generator: in vivo and in vitro studies. Eur J Neurosci 2003, 17:1233-1244. This study shows that two days before birth mice are unable to survive when surgically delivered despite the fact that slice preparations from these animals indicate that respiratory rhythms can be produced. These slice preparations respond differently from those from older animals. 76. Smith JC, Ellenberger HH, Ballanyi K, Richter DW, Feldman JL: Pre-Bo¨ tzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 1991, 254:726-729. 77. Gray PA, Janczewski WA, Mellen N, McCrimmon DR, Feldman JL: Normal breathing requires preBotzinger complex neurokinin-1 receptor-expressing neurons. Nat Neurosci 2001, 4:927-930. 78. Blanchi B, Kelly LM, Viemari JC, Lafon I, Burnet H, Bevengut M, Tillmanns S, Daniel L, Graf T, Hilaire G et al.: MafB deficiency causes defective respiratory rhythmogenesis and fatal central apnea at birth. Nat Neurosci 2003, 6:1091-1100. Mice deficient for the transcription factor MafB die at birth and slices made from these animals show altered respiratory rhythms. MafB is expressed in neurons found in the preBo¨ tzC, suggesting that proper formation of the preBo¨ tzC requires MafB action. 79. Viemari JC, Bevengut M, Burnet H, Coulon P, Pequignot JM, Tiveron MC, Hilaire G: Phox2a gene, A6 neurons, and noradrenaline are essential for development of normal respiratory rhythm in mice. J Neurosci 2004, 24:928-937. The Phox2a gene encodes a homeodomain protein required for the development of noradrenergic A6 neurons. Mice fetuses mutant for this gene had impaired in vivo ventilation, and slices from these animals showed alterations in respiratory rhythms and responses to hypoxia and norepinephrine. Treatments with the adrenergic antagonist prazosin produced effects similar to those shown in the mutants. These data are interpreted to mean that normal development requires noradrenergic inputs at the appropriate time. 80. Consoulas C, Duch C, Bayline RJ, Levine RB: Behavioral transformations during metamorphosis: remodeling of neural and motor systems. Brain Res Bull 2000, 53:571-583. 81. Weeks JC: Thinking globally, acting locally: steroid hormone regulation of the dendritic architecture, synaptic connectivity and death of an individual neuron. Prog Neurobiol 2003, 70:421-442. www.sciencedirect.com
Development of central pattern generating circuits Marder and Rehm 93
An excellent review of steroid reconfiguration of the nervous system in Manduca sexta. 82. Hebbar S, Fernandes JJ: Pruning of motor neuron branches establishes the DLM innervation pattern in Drosophila. J Neurobiol 2004, 60:499-516. In Drosophila, larval muscles are replaced by adult muscles at metamorphosis. In this study, the authors describe the dorsal longitudinal muscle (DLM) innervation pattern through metamorphosis in wild type and in flies carrying several mutations. 83. Duch C, Mentel T: Stage-specific activity patterns affect motoneuron axonal retraction and outgrowth during the metamorphosis of Manduca sexta. Eur J Neurosci 2003, 17:945-962. During metamorphosis many larval muscles and sensory neurons are replaced, but many motor neurons persist and are remodeled in the adult. In Manduca sexta, the formation of adult dorsal longitudinal flight muscle is accompanied by retraction of the axonal terminals and dendrites of the larval motor neurons, muscle degeneration, and myoblast accumulation. These structural changes have been attributed to steroid action. This study investigates whether or not activity-dependent mechanisms are also important. Precociously simulating the motor neurons with ecdysislike patterns resulted in growth of their terminals, suggesting that steroids might act together with activity-dependent mechanisms during postembryonic alterations at the neuromuscular junction. 84. Gray JR, Weeks JC: Steroid-induced dendritic regression reduces anatomical contacts between neurons during synaptic weakening and the developmental loss of a behavior. J Neurosci 2003, 23:1406-1415. Steroid hormones influence neuronal structure in many preparations. In larvae of Manduca sexta, each abdominal proleg has an array of mechanosensory hairs that are innervated by a planta hair sensory neuron. Planta hair sensory neurons elicit monosynaptic excitatory post-synaptic potentials (EPSPs) onto accessory planta retractor motor neurons that mediate a withdrawal reflex behavior that is lost when the animal pupates. An ecdysteriod prepupal peak triggers the regression of motor neuron dendrites and a large reduction in the amplitude of EPSPs evoked by planta hair sensory neurons. The authors conclude that steroid-induced regression of motor neuron dendrites physically disconnects the sensory neurons and the motor neurons, resulting in the loss of the proleg withdrawal reflex.
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85. Duch C, Mentel T: Activity affects dendritic shape and synapse elimination during steroid controlled dendritic retraction in Manduca sexta. J Neurosci 2004, 24:9826-9837. In this study, the authors demonstrate that enhanced electrical activity of the MN5 neuron decreases dendritic regression and loss of synaptic connections normally triggered by steroids. The authors suggest that activity can modify otherwise rigidly defined hormonally triggered developmental sequences. 86. Combes D, Merrywest SD, Simmers J, Sillar KT: Developmental segregation of spinal networks driving axial- and hindlimb-based locomotion in metamorphosing Xenopus laevis. J Physiol 2004, 559:17-24. This is a remarkable study describing the motor patterns produced in Xenopus as swimming tadpoles make the transition from axial-based swimming to limbed propulsion. During this switch the larval and adult motor systems function in the same animal. The authors of this study developed isolated preparations of the spinal cord and brainstem at varying times during these transitions. Extracellular ventral root recordings from the spinal cord of pre-metamorphic tadpoles showed motor patterns corresponding to larval axial swimming. Older, postmetamorphic animals expressed bilaterally synchronous hindlimb flexion– extension kicks. In vitro recordings from stages in which the tail and the limbs are both functional showed two distinct motor patterns, corresponding to bipedal extension–flexion cycles and larval-like swimming. The authors take these data to indicate that during metamorphosis separate networks coexist in the spinal cord and remain functional after isolation in vitro, thus enabling the opportunity to study changes in the nervous system required to adapt to changes in body plan. 87. Straus C, Wilson RJ, Remmers JE: Developmental disinhibition: turning off inhibition turns on breathing in vertebrates. J Neurobiol 2000, 45:75-83. 88. Bentley DR, Hoy RR: Postembryonic development of adult motor patterns in crickets: a neural analysis. Science 1970, 170:1409-1411. 89. Winmill RE, Hedrick MS: Gap junction blockade with carbenoxolone differentially affects fictive breathing in larval and adult bullfrogs. Respir Physiol Neurobiol 2003, 138:239-251.
Current Opinion in Neurobiology 2005, 15:86–93
Current Opinion in Neurobiology 2005, 15:86–93 This review comes from a themed issue on Development Edited by Jane Dodd and Alex L Kolodkin Available online 26th January 2005 0959-4388/$ – see front matter # 2005 Elsevier Ltd. All rights reserved.
patterns they generate are easy to record, and second, the behaviors that they control are important for the animal [2]. For example, the central pattern generating circuits that control breathing, feeding, and locomotion are necessary for animals to survive after they hatch or are born. Unfortunately, the few systems in which central pattern generating circuits are relatively well understood [3,4] are not ideal for developmental studies, and little is known about the identity of the neurons and their connections in the central pattern generating circuits of those animals commonly used for developmental and molecular studies. Nonetheless, considerable progress on numerous fronts suggests that studying circuit construction using motor circuits that generate rhythmic motor patterns will be extremely fruitful in years to come, and some of these first steps are described below.
Spontaneous motor activity in the embryo Embryos move before animals hatch or are born. For example, chick embryos show spontaneous leg movements [5]; the spinal cords and brainstems of chick, rat and mouse embryos show rhythmic activity [6,7,8,9, 10,11,12]; Drosophila melanogaster embryos generate peristaltic movements similar to those observed in the larvae [13]; and the stomatogastric nervous system of embryonic lobsters generates rhythmic motor patterns [14–16,17] before the animal hatches and starts feeding.
How are functional circuits constructed during development? Answering this question requires a circuit or set of circuits with known and measurable functions, and access to those circuits at different times in development. Despite many recent and remarkable advances in developmental biology, understanding how circuits are formed has been hampered by our relatively rudimentary knowledge of the organization of most circuits in the adult nervous system. In many brain regions it is difficult to define, except in the most general of terms, what the circuit does, what the cell types in the circuit are, and how the neurons are connected. For this reason, much developmental work on circuits has focused on primary sensory areas endowed with orderly topographic maps, such as visual cortex [1], where both functional and anatomical correlates of circuit function can be studied.
What functions are served by these embryonic movements? Are these spontaneous rhythmic discharges produced by precociously wired up central pattern generators, or are they bursts of activity produced by networks that are immature and differ significantly from those seen in the adult? In some preparations there is evidence indicating that the earliest rhythmic motor discharges might be produced by an immature network that is supplanted by the one that will operate later in the animal’s life. In other preparations the basic backbone of the adult circuit appears very early in embryonic development, and developmental differences in motor patterns could be attributable to alterations in sensory and neuromodulatory inputs. Obviously, in some ways this is a false dichotomy, because all circuits must be constructed de novo during development, but it does focus our attention on the question of whether or not there is an extended period during embryonic development in which motor activity is produced by different network architectures and/or different cellular and synaptic mechanisms from those operating later in life.
Motor circuits that produce rhythmic movements are also amenable to developmental study because first, the motor
The earliest movements generated by the chick spinal cord are generated by a circuit different from that which
DOI 10.1016/j.conb.2005.01.011
Introduction
Current Opinion in Neurobiology 2005, 15:86–93
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Development of central pattern generating circuits Marder and Rehm 87
functions later in life. This is evident because in the adult spinal cord motor neurons are driven by central pattern generating circuits that consist of interneurons. By contrast, the circuitry generating the early spinal cord activity depends heavily on cholinergic excitatory connections, presumably from collaterals of the motor neurons themselves [18]. Later in development glutamate is the predominant excitatory transmitter used in the central pattern generating interneuronal circuitry that drives movement. If cholinergic transmission is pharmacologically blocked early in development, the embryonic circuit is briefly disrupted but rhythmic activity returns, and is thereafter dependent on GABAergic transmission [6]. Recordings made systematically from the embryonic mouse and rat spinal cords at different days of development show that the patterns of activity change during an extended period of time [9,12], both in their expression and in the neurotransmitters that mediate excitatory transmission. An elegant recent study by Hanson and Landmesser [7] studied the mouse spinal cord at E11 to E14, before all of the motor neurons have reached their targets. Two types of spontaneous episodes were observed, major episodes that propagated throughout the cord, and more local activity. Nicotinic cholinergic antagonists slowed the frequency of bursting activity, demonstrating again that much of the early rhythmic activity appears to be a function of a circuit depending heavily on cholinergic transmission from the motor neurons themselves. What functions do these precocious motor patterns and movements serve? The most obvious answer is that the activity produced in these developing circuits is needed to generate activity-dependent tuning or wiring signals. Recent exciting work in the chick showed that chronic in ovo application of drugs that block glycine and g-aminobutyric acid (GABA)-mediated synapses altered the patterns of spontaneous activity, resulting in pathfinding errors and produced misplaced motor neuron somata [8]. Additionally, these picrotoxin treatments altered the expression of polysialic acid (PSA) and downregulated EphA4 from dorsally projecting axons. Before this demonstration, the consensus was that early pathfinding and outgrowth decisions would be primarily genetically encoded, and that activity would be more important for later processes of synapse stabilization and fine-tuning of circuitry [19]. The importance of activity for specification of neurotransmitter phenotype in the developing spinal cord is the subject of another valuable new study [20]. Although there is a large amount of literature describing factors that influence neurotransmitter phenotype in the sympathetic nervous system [21], the authors of this study characterize the specific patterns of Ca2+ spikes in several classes of neurons in the Xenopus laevis spinal cord, and demonstrate www.sciencedirect.com
that changes in these activity patterns result in neurotransmitter phenotype switches without affecting the expression of markers of cell identity. The authors view this as an homeostatic response because decreased activity led to an increased number of neurons expressing excitatory transmitters and a decreased number of neurons expressing inhibitory transmitters and vice versa [20].
Genetic markers and identification of pattern generating interneurons Much of our insight into the fundamental mechanisms underlying pattern generation has come from small invertebrate circuits in which it was possible to identify neurons easily on the basis of electrophysiological and anatomical criteria [3,22]. The ability to identify individual or small classes of neurons made it possible to study their connectivity and their role in generating motor patterns. The same strategy has been partially successful in several vertebrate preparations such as the tadpole [23] and lamprey [24]. However, even in the simplest spinal cords identifying neurons and establishing their properties and connectivity is far more difficult than it was any of the classic invertebrate preparations such as the leech heartbeat system, the stomatogastric ganglion, or the Tritonia swim system [3]. Until recently, identification of interneuron classes in the spinal cord depended on electrophysiological criteria, augmented by immunocytochemistry and conventional anatomical tracing methods. Using these methods, progress has been made in identifying interneuronal classes in the lamprey, amphibian, and cat spinal cords [23,24,25,26,27]. With the advent of new molecular tools, there is the exciting potential that these tools will be helpful not only for understanding early developmental events [28,29,30] but also for aiding neuronal identification and revealing patterns of connectivity in the spinal cord. This has led to increased interest in the organization of the rodent and zebrafish spinal cords [31,32] and numerous studies using both classical anatomical and physiological methods [33,34,35–38] and molecular tools are appearing [31,39,40,41,42–44,45,46,47,48]. These studies include attempts to use transcription factor expression to identify neuronal populations in the spinal cord [31,39,40,41,42–44,45,46,49,50]. That said, transcription factor expression studies have not yet yielded cell identifications with the requisite precision for the construction of the detailed functional wiring diagrams needed to understand the operation of spinal cord circuitry and motor pattern generation. A notable exception is a recent study looking at two transcription factors important in Renshaw cell development [47]. In studies on small invertebrate pattern generators, the function of individual neurons or circuit elements is studied by perturbing the activity of a neuron [51] and/ Current Opinion in Neurobiology 2005, 15:86–93
88 Development
or by deleting it from the circuit [52] to see the effects of these manipulations on the motor patterns produced. Electrophysiological perturbations of the activity of single neurons in the spinal cord are unlikely to be particularly useful in altering the output of a central pattern generator comprised of many neurons. Nonetheless, in principle, genetic approaches enable the manipulation or deletion of whole classes of spinal cord interneurons in order to determine their role in pattern generation. For example, Lanuza et al. [46] studied the locomotory motor patterns produced by mice mutant for the Dbx1 homeobox gene and found disturbed patterns of hindlimb left–right alternation, suggesting that the ventral interneurons that express this gene are important for controlling left–right alternation. Mice mutant for the EphA4 receptor or its ligand ephrinB3 also show defects in left–right alternation [53] that can be restored pharmacologically. These studies are only the first of many using molecular and genetic tools that will undoubtedly aid in identifying the classes of spinal cord interneurons and their function both in development and in the adult. Another use of genetic tools is to add neurons to a circuit during development. Two new studies in the zebrafish [54,55] demonstrate that additional Mauthner neurons incorporate into and function in the developing escape circuit. The authors suggest that incorporation of duplicated neurons could be a mechanism for circuit evolution [54,55].
The roles of sensory and descending inputs on the development of central pattern generating circuits In the course of normal behavior, central pattern generating networks receive inputs from sensory and descending neuromodulatory inputs, which adapt the motor patterns produced by central pattern generating circuits to the animal’s needs [4]. Therefore, it is interesting to ask what roles these inputs might take in the development of central pattern generating circuits. A recent study in Drosophila [13] showed that altered but functional embryonic and larval motor patterns were produced by animals that developed in the absence of most of their sensory inputs. This study was remarkable both because it had been assumed previously that loss of the sensory neurons would perturb early pathfinding and target selections and because it showed clearly the relative independence of the central pattern generating mechanisms from the phasic sensory drive. Similar alterations in movements were seen in mutants lacking only the chordotonal organs [56], suggesting that the chordotonal organs provide a major input to the central pattern generating circuitry that produces larval movements. In Drosophila, the dendritic arbors of identified motor neurons are found in specific regions of the neuropil, presumably correctly placed to receive appropriate synaptic inputs from central pattern generating neurons [57]. Genetic methods are Current Opinion in Neurobiology 2005, 15:86–93
now being employed to reveal the identity of neurons that influence locomotory programs [58], to enable intracellular recordings from identified neurons in Drosophila [59], or to study the effects of sensory neurons on locomotion [60]. The roles that neuromodulatory inputs could take in the development and maturation of central pattern generating circuits have been studied in numerous preparations. In the crustacean stomatogastric nervous system, the central pattern generating circuit consists largely of connections among the motor neurons themselves. Midway through embryonic development in this system the full complement of neurons is present and, although the embryonic motor patterns differ from those in the adult, under appropriate neuromodulatory conditions adult-like motor patterns can be elicited from the embryonic network [14,15,61]. One of the salient take-home messages from these studies is that the neuromodulatory complement of identified sensory and descending neuromodulatory inputs changes during embryonic and larval stages [62–65] and that receptors and transporters for a neuromodulator might be present before the neuromodulatory substance itself is present in sensory or projection neurons [17]. Interestingly, although many of the actions of neuromodulators appear to be unchanged through embryonic, larval and adult life [65,66], other neuromodulatory actions change during development [17]. Recent studies of the maturation of rodent locomotory systems suggest that appropriate neuromodulatory drive might be crucial for the function [67–69] and maturation of central pattern generating circuits. In addition to being a modulator of embryonic and larval spinal cord central pattern generators [69–71], serotonin might also be important in shaping the formation of the central pattern generating circuits required for locomotion [72,73]. Specifically, when descending serotonergic pathways in the medulla are removed, spinal interneurons express a serotonergic phenotype, and serotonin appears to influence the time course of the development of inhibitory pathways in the spinal cord [72]. Recent work on the factors that influence the development of serotonergic neurons [74] should increase our ability to manipulate this neuromodulatory system more precisely in development. In mammals it is crucial that the respiratory network is functional when the animal is born, or death ensues within a few minutes [75]. The preBo¨ tzinger complex (preBo¨ tC) contains neurons that are thought to provide a pacemaker for respiration [76] and lesions of these neurons disturb or disrupt breathing [77]. PreBo¨ tC neurons express the transcription factor MafB and animals with mutations in the gene that encodes this factor die at birth [78]. A new study has investigated the influence of descending noradrenergic neurons on the development of the mouse respiratory system [79]. In mice mutant for www.sciencedirect.com
Development of central pattern generating circuits Marder and Rehm 89
the Phox2A gene, the noradrenergic A6 neurons fail to develop (these neurons ordinarily provide modulatory drive to the preBo¨ tC), and these animals die at birth. In vitro preparations from these animals show altered responses to hypoxia and to norepinephrine. Moreover, gestational treatments of wild type animals with a noradrenergic antagonist produced significantly altered respiratory rhythms [79], suggesting that the neuromodulator is required for correct maturation of the network.
Reconfiguring circuits as a consequence of changes in body plan During development and growth all animals change size and shape. Although in mammals these changes are continuous and gradual, many other animals undergo stage-specific abrupt changes in body plan as a consequence of molts and metamorphoses. For example, as tadpoles metamorphose into frogs, they lose their tail and acquire legs, and holometabolous insects such as Drosophila and Manduca sexta undergo behavioral and body plan changes as they are transformed from the crawling larval stages into flying adults with wings and legs. There is a great deal known about the steroid-controlled remodeling events in insect metamorphosis [80,81], both at the neuromuscular junction [82,83] and in the nerve cord [84]. A recent study demonstrates that steroidinduced dendritic regression accompanied by synapse loss in Manduca can be attenuated by electrical activity [85]. Thus, even in an animal in which stereotyped alterations in the nervous system accompany alterations in body plan, activity might influence the formation of the new circuits. Amphibian metamorphosis is associated with a complete reorganization of the animal’s locomotory systems. In a fascinating new study, Combes et al. [86] record from isolated spinal cord and brainstem preparations before, during and after metamorphosis. Not surprisingly, recordings from ventral roots of pre-metamorphic tadpoles revealed motor outputs consistent with swimming, whereas postmetamorphic animals expressed motor patterns consistent with kicking. Recordings from animals in the midst of metamorphosis revealed two motor patterns with distinct periods that sometimes occurred separately and sometimes occurred together. These data suggest that during metamorphosis the circuits for both behaviors are functional in the spinal cord [86], creating the opportunity to understand the mechanisms by which these circuits are reconfigured during a major maturational event. Not only do the locomotory circuits change during amphibian metamorphosis but frogs also make a transition from gill-breathing to lung-breathing [87]. The circuit required for the adult behavior is present early in development, and is actively inhibited before needed [87], as is thought www.sciencedirect.com
to be the case in several other preparations [15,88]. That said, numerous changes occur in the amphibian respiratory pattern generators during metamorphosis [89].
Conclusions Although molecular and genetic tools have the potential to facilitate manipulations of developing motor systems in both vertebrate and invertebrate preparations, they cannot entirely replace the slow slogging effort needed to understand how rhythmic motor patterns in adult animals are produced. Sadly, to date, those organisms best suited for the study of the mechanisms underlying the organization and function of central pattern generators are not those best suited for studying their development. Conversely, very little is known about the organization of motor circuits in those organisms ideally suited for molecular and genetic manipulations during early development. The defined motor patterns produced by both vertebrate and invertebrate central pattern generators enable extremely accurate assessment of altered network function. Ongoing work on the classic preparations used to study mechanisms of central pattern generation will continue to inform new studies on genetic and molecular development of central pattern generators, and make possible understanding these circuits in organisms with large numbers of neurons.
Acknowledgements This work was supported by NS 17813 (E Marder) and an Individual NRSA award NS 050479 (K Rehm).
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An excellent review of steroid reconfiguration of the nervous system in Manduca sexta. 82. Hebbar S, Fernandes JJ: Pruning of motor neuron branches establishes the DLM innervation pattern in Drosophila. J Neurobiol 2004, 60:499-516. In Drosophila, larval muscles are replaced by adult muscles at metamorphosis. In this study, the authors describe the dorsal longitudinal muscle (DLM) innervation pattern through metamorphosis in wild type and in flies carrying several mutations. 83. Duch C, Mentel T: Stage-specific activity patterns affect motoneuron axonal retraction and outgrowth during the metamorphosis of Manduca sexta. Eur J Neurosci 2003, 17:945-962. During metamorphosis many larval muscles and sensory neurons are replaced, but many motor neurons persist and are remodeled in the adult. In Manduca sexta, the formation of adult dorsal longitudinal flight muscle is accompanied by retraction of the axonal terminals and dendrites of the larval motor neurons, muscle degeneration, and myoblast accumulation. These structural changes have been attributed to steroid action. This study investigates whether or not activity-dependent mechanisms are also important. Precociously simulating the motor neurons with ecdysislike patterns resulted in growth of their terminals, suggesting that steroids might act together with activity-dependent mechanisms during postembryonic alterations at the neuromuscular junction. 84. Gray JR, Weeks JC: Steroid-induced dendritic regression reduces anatomical contacts between neurons during synaptic weakening and the developmental loss of a behavior. J Neurosci 2003, 23:1406-1415. Steroid hormones influence neuronal structure in many preparations. In larvae of Manduca sexta, each abdominal proleg has an array of mechanosensory hairs that are innervated by a planta hair sensory neuron. Planta hair sensory neurons elicit monosynaptic excitatory post-synaptic potentials (EPSPs) onto accessory planta retractor motor neurons that mediate a withdrawal reflex behavior that is lost when the animal pupates. An ecdysteriod prepupal peak triggers the regression of motor neuron dendrites and a large reduction in the amplitude of EPSPs evoked by planta hair sensory neurons. The authors conclude that steroid-induced regression of motor neuron dendrites physically disconnects the sensory neurons and the motor neurons, resulting in the loss of the proleg withdrawal reflex.
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85. Duch C, Mentel T: Activity affects dendritic shape and synapse elimination during steroid controlled dendritic retraction in Manduca sexta. J Neurosci 2004, 24:9826-9837. In this study, the authors demonstrate that enhanced electrical activity of the MN5 neuron decreases dendritic regression and loss of synaptic connections normally triggered by steroids. The authors suggest that activity can modify otherwise rigidly defined hormonally triggered developmental sequences. 86. Combes D, Merrywest SD, Simmers J, Sillar KT: Developmental segregation of spinal networks driving axial- and hindlimb-based locomotion in metamorphosing Xenopus laevis. J Physiol 2004, 559:17-24. This is a remarkable study describing the motor patterns produced in Xenopus as swimming tadpoles make the transition from axial-based swimming to limbed propulsion. During this switch the larval and adult motor systems function in the same animal. The authors of this study developed isolated preparations of the spinal cord and brainstem at varying times during these transitions. Extracellular ventral root recordings from the spinal cord of pre-metamorphic tadpoles showed motor patterns corresponding to larval axial swimming. Older, postmetamorphic animals expressed bilaterally synchronous hindlimb flexion– extension kicks. In vitro recordings from stages in which the tail and the limbs are both functional showed two distinct motor patterns, corresponding to bipedal extension–flexion cycles and larval-like swimming. The authors take these data to indicate that during metamorphosis separate networks coexist in the spinal cord and remain functional after isolation in vitro, thus enabling the opportunity to study changes in the nervous system required to adapt to changes in body plan. 87. Straus C, Wilson RJ, Remmers JE: Developmental disinhibition: turning off inhibition turns on breathing in vertebrates. J Neurobiol 2000, 45:75-83. 88. Bentley DR, Hoy RR: Postembryonic development of adult motor patterns in crickets: a neural analysis. Science 1970, 170:1409-1411. 89. Winmill RE, Hedrick MS: Gap junction blockade with carbenoxolone differentially affects fictive breathing in larval and adult bullfrogs. Respir Physiol Neurobiol 2003, 138:239-251.
Current Opinion in Neurobiology 2005, 15:86–93