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Assembly of Motor Circuits in the Spinal Cord: Driven to Function by Genetic and Experience-Dependent Mechanisms
Abstract
Motor circuits in the spinal cord integrate information from various sensory and descending pathways to control appropriate motor behavior. Recent work has revealed that target-derived retrograde signaling mechanisms act to influence sequential assembly of motor circuits through combinatorial action of genetic and experience-driven programs. These parallel activities imprint somatotopic information at the level of the spinal cord in precisely interconnected circuits and equip animals with motor circuits capable of reacting to changing demands throughout life.
... Peripherally, group Ia afferents project axons to muscle spindles, which provide feedback to the spinal cord about the state of muscle contraction and limb position (Maier, 1997;Windhorst, 2007). Recent studies have revealed the molecular mechanisms guiding proprioceptive sensory afferent projections to the ventral spinal cord and the formation of specific connections within their target regions (Arber, 2012;Chen et al., 2003;Ladle et al., 2007;Levine et al., 2012;Catela et al., 2015). Once these monosynaptic sensory-motor circuits are formed, however, we know little about how they are maintained over the lifespan of an animal. ...
... Monosynaptic sensory-motor circuits have been extensively studied using electrophysiology, mouse genetics, and molecular approaches since the 1950s (Brown, 1981;Arber, 2012;Ladle et al., 2007;Catela et al., 2015). However, how these and all other circuits in the nervous system are properly maintained over the lifespan of an animal is unclear. ...
In contrast to our knowledge of mechanisms governing circuit formation, our understanding of how neural circuits are maintained is limited. Here, we show that Dicer, an RNaseIII protein required for processing microRNAs (miRNAs), is essential for maintenance of the spinal monosynaptic stretch reflex circuit in which group Ia proprioceptive sensory neurons form direct connections with motor neurons. In postnatal mice lacking Dicer in proprioceptor sensory neurons, there are no obvious defects in specificity or formation of monosynaptic sensory-motor connections. However, these circuits degrade through synapse loss and retraction of proprioceptive axonal projections from the ventral spinal cord. Peripheral terminals are also impaired without retracting from muscle targets. Interestingly, despite these central and peripheral axonal defects, proprioceptive neurons survive in the absence of Dicer-processed miRNAs. These findings reveal that Dicer, through its production of mature miRNAs, plays a key role in the maintenance of monosynaptic sensory-motor circuits.
... In addition, the size and morphology of motor neurons were not changed either in db/db mice in comparison to controls as indicated in Figure 7C, suggesting that motor neurons are relatively more resistant during diabetes progression. Motor neurons within the ventral spinal cord integrate diverse synaptic inputs from the supraspinal descending pathways, spinal interneurons, and peripheral sensory feedback to generate the rhythm and pattern of locomotion and mediate the whole-body movement (35,36). These inputs are mainly divided into two categories: excitatory inputs from glutamatergic neurons and inhibitory inputs from GABAergic or glycinergic neurons. ...
Diabetic neuropathy is the most common complication of diabetes and lacks effective treatments. Although sensory dysfunction during the early stages of diabetes has been extensively studied in various animal models, the functional and morphological alterations in sensory and motor systems during late stages of diabetes remain largely unexplored. In the current work, we examined the influence of diabetes on sensory and motor function as well as morphological changes in late stages of diabetes. The obese diabetic Lepr db/db mice (db/db) were used for behavioral assessments and subsequent morphological examinations. The db/db mice exhibited severe sensory and motor behavioral defects at the age of 32 weeks, including significantly higher mechanical withdrawal threshold and thermal latency of hindpaws compared with age-matched nondiabetic control animals. The impaired response to noxious stimuli was mainly associated with the remarkable loss of epidermal sensory fibers, particularly CGRP-positive nociceptive fibers. Unexpectedly, the area of CGRP-positive terminals in the spinal dorsal horn was dramatically increased in diabetic mice, which was presumably associated with microglial activation. In addition, the db/db mice showed significantly more foot slips and took longer time during the beam-walking examination compared with controls. Meanwhile, the running duration in the rotarod test was markedly reduced in db/db mice. The observed sensorimotor deficits and motor dysfunction were largely attributed to abnormal sensory feedback and muscle atrophy as well as attenuated neuromuscular transmission in aged diabetic mice. Morphological analysis of neuromuscular junctions (NMJs) demonstrated partial denervation of NMJs and obvious fragmentation of acetylcholine receptors (AChRs). Intrafusal muscle atrophy and abnormal muscle spindle innervation were also detected in db/db mice. Additionally, the number of VGLUT1-positive excitatory boutons on motor neurons was profoundly increased in aged diabetic mice as compared to controls. Nevertheless, inhibitory synaptic inputs onto motor neurons were similar between the two groups. This excitation-inhibition imbalance in synaptic transmission might be implicated in the disturbed locomotion. Collectively, these results suggest that severe sensory and motor deficits are present in late stages of diabetes. This study contributes to our understanding of mechanisms underlying neurological dysfunction during diabetes progression and helps to identify novel therapeutic interventions for patients with diabetic neuropathy.
... Although accumulating evidence suggests that sensorymotor connections are established by positional and morphological factors of both axons and dendrites (40,41), a role for cell-to-cell recognition in the establishment and refinement of homonymous connections has not been entirely ruled out (46). The distinguishing feature of homonymous connections that may enable cell-to-cell recognition is the common peripheral muscle target for both the proprioceptive afferents and motor neurons (47). In mutants lacking the HOXD9-11 transcription factors, quadriceps motor neurons have normal cell body positions and dendritic arbors, yet these motor neurons receive aberrant synaptic inputs from adductor sensory afferents (31). ...
Presynaptic inputs determine the pattern of activation of postsynaptic neurons in a neural circuit. Molecular and genetic pathways that regulate the selective formation of subsets of presynaptic inputs are largely unknown, despite significant understanding of the general process of synaptogenesis. In this study, we have begun to identify such factors using the spinal monosynaptic stretch reflex circuit as a model system. In this neuronal circuit, Ia proprioceptive afferents establish monosynaptic connections with spinal motor neurons that project to the same muscle (termed homonymous connections) or muscles with related or synergistic function. However, monosynaptic connections are not formed with motor neurons innervating muscles with antagonistic functions. The ETS transcription factor ER81 (also known as ETV1) is expressed by all proprioceptive afferents, but only a small set of motor neuron pools in the lumbar spinal cord of the mouse. Here we use conditional mouse genetic techniques to eliminate Er81 expression selectively from motor neurons. We find that ablation of Er81 in motor neurons reduces synaptic inputs from proprioceptive afferents conveying information from homonymous and synergistic muscles, with no change observed in the connectivity pattern from antagonistic proprioceptive afferents. In summary, these findings suggest a role for ER81 in defined motor neuron pools to control the assembly of specific presynaptic inputs and thereby influence the profile of activation of these motor neurons.
... However, we still lack a comprehensive picture of spinal MN molecular heterogeneity in mammalian embryos. Therefore, we decided to perform single-cell RNA sequencing (scRNA-seq) on mouse spinal MNs at embryonic day 13.5 (E13.5) (Fig. 1b), a stage at which these neurons have acquired pool identities and established axon innervations into target muscles 16 . Given that MNs represent only a tiny fraction of all spinal cells, using the entire tissue as a source would not be an optimal approach for in-depth characterizations of MN diversity 15 . ...
Spinal motor neurons (MNs) integrate sensory stimuli and brain commands to generate movements. In vertebrates, the molecular identities of the cardinal MN types such as those innervating limb versus trunk muscles are well elucidated. Yet the identities of finer subtypes within these cell populations that innervate individual muscle groups remain enigmatic. Here we investigate heterogeneity in mouse MNs using single-cell transcriptomics. Among limb-innervating MNs, we reveal a diverse neuropeptide code for delineating putative motor pool identities. Additionally, we uncover that axial MNs are subdivided into three molecularly distinct subtypes, defined by mediolaterally-biased Satb2, Nr2f2 or Bcl11b expression patterns with different axon guidance signatures. These three subtypes are present in chicken and human embryos, suggesting a conserved axial MN expression pattern across higher vertebrates. Overall, our study provides a molecular resource of spinal MN types and paves the way towards deciphering how neuronal subtypes evolved to accommodate vertebrate motor behaviors.
... 23 While segregating, MNs start projecting their axons out of the spinal cord to innervate different targets. [24][25][26] At later embryonic stages, MNs receive inputs from afferent sensory neurons and interneurons, [27][28][29] while they themselves also start establishing synapses with the targeted muscles by forming neuromuscular junctions. 30 Initially discovered as axon guidance cues, 31 comprises secreted proteins that also regulate vascular processes. ...
How the vascular and neural compartment cooperate to achieve such a complex and highly specialized structure as the central nervous system is still unclear. Here, we reveal a crosstalk between motor neurons (MNs) and endothelial cells (ECs), necessary for the coordinated development of MNs. By analyzing cell-to-cell interaction profiles of the mouse developing spinal cord, we uncovered semaphorin 3C (Sema3C) and PlexinD1 as a communication axis between MNs and ECs. Using cell-specific knockout mice and in vitro assays, we demonstrate that removal of Sema3C in MNs, or its receptor PlexinD1 in ECs, results in premature and aberrant vascularization of MN columns. Those vascular defects impair MN axon exit from the spinal cord. Impaired PlexinD1 signaling in ECs also causes MN maturation defects at later stages. This study highlights the importance of a timely and spatially controlled communication between MNs and ECs for proper spinal cord development.
... There, the trophic factors initiate a host of morphological changes to the cell that can influence motor skills and ability (Kaspar et al., 2003;Song et al., 2016). These effects are also likely important during embryonic development (Connor and Smith, 1994;Ladle et al., 2007;Li et al., 2008); thus, at least, it seems plausible that AR action at the level of the muscle during the development similarly helps shape the circuitry in the spinal cord that is necessary to generate foot flags. ...
Sex steroids play an important role in regulation of the vertebrate reproductive phenotype. This is because sex steroids not only activate sexual behaviors that mediate copulation, courtship, and aggression, but they also help guide the development of neural and muscular systems that underlie these traits. Many biologists have therefore described the effects of sex steroid action on reproductive behavior as both “activational” and “organizational,” respectively. Here, we focus on these phenomena from an evolutionary standpoint, highlighting that we know relatively little about the way that organizational effects evolve in the natural world to support the adaptation and diversification of reproductive behavior. We first review the evidence that such effects do in fact evolve to mediate the evolution of sexual behavior. We then introduce an emerging animal model – the foot-flagging frog, Staurois parvus – that will be useful to study how sex hormones shape neuromotor development necessary for sexual displays. The foot flag is nothing more than a waving display that males use to compete for access to female mates, and thus the neural circuits that control its production are likely laid down when limb control systems arise during the developmental transition from tadpole to frog. We provide data that highlights how sex steroids might organize foot-flagging behavior through its putative underlying mechanisms. Overall, we anticipate that future studies of foot-flagging frogs will open a powerful window from which to see how sex steroids influence the neuromotor systems to help germinate circuits that drive signaling behavior. In this way, our aim is to bring attention to the important frontier of endocrinological regulation of evolutionary developmental biology (endo-evo-devo) and its relationship to behavior.
... does not exclude a role for genetically defined differentiation, guidance and connectivity (10). Spatial gradients of cellular differentiation in the rostrocaudal and mediolateral axes appear to account for gradients in the density of sensory feedback from and to specific muscles (2,42). Genetic differentiation and mutual recognition between cell-types presumably underlie general rules for connectivity such as the unique projection of recurrent collaterals from motoneurons onto inhibitory Renshaw interneurons (24,87). ...
Control of musculoskeletal systems depends on integration of voluntary commands and somatosensory feedback in the complex neural circuits of the spinal cord. Particular connectivity patterns have been identified experimentally, and it has been suggested that these may result from the wide variety of transcriptional types that have been observed in spinal interneurons. We ask instead whether the details of these connectivity patterns (and perhaps many others) can arise as a consequence of Hebbian adaptation during early development. We constructed an anatomically simplified model plant system with realistic muscles and sensors and connected it to a recurrent, random neuronal network consisting of both excitatory and inhibitory neurons endowed with Hebbian learning rules. We then generated a wide set of randomized muscle twitches typical of those described during fetal development and allowed the network to learn. Multiple simulations consistently resulted in diverse and stable patterns of activity and connectivity that included subsets of the interneurons that were similar to 'archetypical' interneurons described in the literature. We also found that such learning led to an increased degree of cooperativity between interneurons when performing larger limb movements on which it had not been trained. Hebbian learning gives rise to rich sets of diverse interneurons whose connectivity reflects the mechanical properties of the plant. At least some of the transcriptomic diversity may reflect the effects of this process rather than the cause of the connectivity. Such a learning process seems better suited to respond to the musculoskeletal mutations that underlie the evolution of new species.
... In the spinal cord, functional circuits produce movements with precise timing, duration, and amplitude to adjust to changes in the environment (Arber, 2017;Brownstone and Bui, 2010;Büschges et al., 2011;Fetcho and McLean, 2010;Goulding, 2009;Grillner and Jessell, 2009;Hayashi et al., 2018;Kiehn, 2016;Roberts et al., 2010). The assembly of these circuits is defined early during development through processes that specify the constituent neurons' identity and connectivity (Blankenship and Feller, 2010;Drapeau et al., 2002;Goulding and Pfaff, 2005;Ladle et al., 2007;Meng and Heckscher, 2021;Saint-Amant and Drapeau, 2000;Wan et al., 2019). Two recent studies in C. elegans and Drosophila have revealed that, during maturation, the decision-making circuitry is maintained unchanged, whereas sensory and motor pathways show age-dependent structural changes (Lee and Doe, 2021;Witvliet et al., 2021). ...
Locomotion is mediated by spinal circuits that generate movements with a precise coordination and vigor. The assembly of these circuits is defined early during development; however, whether their organization and function remain invariant throughout development is unclear. Here, we show that the first established fast circuit between two dorsally located V2a interneuron types and the four primary motoneurons undergoes major transformation in adult zebrafish compared with what was reported in larvae. There is a loss of existing connections and establishment of new connections combined with alterations in the mode, plasticity, and strength of synaptic transmission. In addition, we show that this circuit no longer serves as a swim rhythm generator, but instead its components become embedded within the spinal escape circuit and control propulsion following the initial escape turn. Our results thus reveal significant changes in the organization and function of a motor circuit as animals develop toward adulthood.
... Spinal circuits for movements are established early in development, as evidenced by movements in utero. Spontaneous activity within spinal circuits provides the excitatory drive for these early movements, supplemented by sensory afferents that enter the spinal cord at later stages of embryonic development (Naka, 1964;Ozaki and Snider, 1997;Ladle et al., 2007). Spinal circuits can generate rhythmic and patterned muscle activity underlying locomotion at embryonic stages (Altman and Sudarshan, 1975;Branchereau et al., 2000;Talpalar et al., 2011). ...
Primitive reflexes are evident shortly after birth. Many of these reflexes disappear during postnatal development as part of the maturation of motor control. This study investigates the changes of connectivity related to sensory integration by spinal dI3 interneurons during the time in which the palmar grasp reflex gradually disappears in postnatal mice pups. Our results reveal an increase in GAD65/67-labeled terminals to perisomatic Vglut1-labeled sensory inputs contacting cervical and lumbar dI3 interneurons between postnatal day 3 and day 25. In contrast, there were no changes in the number of perisomatic Vglut1-labeled sensory inputs to lumbar and cervical dI3 interneurons other than a decrease between postnatal day 15 and day 25. Changes in postsynaptic GAD65/67-labeled inputs to dI3 interneurons were inconsistent with a role in the sustained loss of the grasp reflex. These results suggest a possible link between the maturation of hand grasp during postnatal development and increased presynaptic inhibition of sensory inputs to dI3 interneurons.
... S1). At P0, spinal neurons have been generated and the basic functional features of adult spinal circuitry have been established, but expression persists for many developmental markers of neuronal subtype diversity (2,9,10,18). Using marker analyses, 6743 cells passed quality control and were identified as neurons (Fig. 1A). ...
Neuronal identities
Neurons of the mouse spinal cord can be identified by any of several metrics, including what neurotransmitters they use, what cells they connect to, where they are located, and what neuroprogenitor gave rise to them. Osseward et al. generated a different metric, genetic signatures, and identified classes of local and projection neurons that were otherwise heterogeneous by other classification systems. With this focus on a cell's genetic signature, its neurotransmitter phenotype, which is accessible by a variety of transcriptional routes, can be seen as a parallel to convergent evolution in development.
Science , this issue p. 385
... The generation of spontaneous activity is one of the important roles of acetylcholine during the early phase of neural development. Propagating wave-like activity in the brain-spinal cord (the activity studied in the present experiment) and that in the retina (termed the retinal wave) (Wong, 1999) is mediated by nAChRs during a specific period of development, which is later replaced by glutamatergic regulation (Nakayama et al., 1999;Ren and Greer, 2003;Ladle et al., 2007;Mochida et al., 2009;Momose-Sato et al., 2012b;Wong, 1999). Regarding nAChR subtypes, previous studies reported that correlated wave activity in the brain-spinal cord was more dominantly dependent on nAChRs containing non-7 ...
Correlated spontaneous activity propagating over a wide region of the central nervous system is expressed during a specific period of embryonic development. We previously demonstrated using an optical imaging technique with a voltage-sensitive dye that this wave-like activity, which we referred to as the depolarization wave, is fundamentally involved in the early process of synaptic network formation. We found that the in ovo application of bicuculline/strychnine or d-tubocurarine, which blocked the neurotransmitters mediating the wave, significantly reduced functional synaptic expression in the brainstem sensory nucleus. This result, particularly for d-tubocurarine, an antagonist of nicotinic acetylcholine receptors, suggested that prenatal nicotine exposure associated with maternal smoking affects the development of neural circuit formation by interfering with the correlated wave. In the present study, we tested this hypothesis by examining the effects of nicotine on the correlated activity and assessing the chronic action of nicotine in ovo on functional synaptic expression along the vagal sensory pathway. In ovo observations of chick embryo behavior and electrical recording using in vitro preparations showed that the application of nicotine transiently increased embryonic movements and electrical bursts associated with the wave, but subsequently inhibited these activities, suggesting that the dominant action of the drug was to inhibit the wave. Optical imaging with the voltage-sensitive dye showed that the chronic exposure to nicotine in ovo markedly reduced functional synaptic expression in the higher-order sensory nucleus of the vagus nerve, the parabrachial nucleus. The results suggest that prenatal nicotine exposure disrupts the initial formation of the neural circuitry by inhibiting correlated spontaneous wave activity.
... Les études ayant porté sur la transmission chimique impliquée dans la génération de l'activité spontanée des circuits spinaux embryonnaires chez plusieurs espèces (poulet, rat et souris) ont abouti à un modèle dans lequel l'activation du circuit spinal peut être subdivisée en deux phases , Ladle et al. 2007). ...
Comme beaucoup de réseaux neuronaux en développement, la moelle épinière génère spontanément une activité neuronale synchronisée récurrente qui intervient dans de nombreux aspects de maturation du circuit moteur. Au cours d'une période embryonnaire initiale (E12,5-E14,5 chez la souris), cette activité est majoritairement sous le contrôle de la libération d’acétylcholine, de GABA et de glycine. Dans les motoneurones, cette activité se caractérise par des dépolarisations géantes (GDPs) évoquées principalement par une libération de GABA, cette dernière étant régulée par l’activation de récepteurs cholinergiques, suggérant une boucle récurrente entre les motoneurones et les interneurones GABAergiques. Les mécanismes de libération de ces neurotransmetteurs et l’identification des premiers interneurones GABAergiques fonctionnels interagissant avec les motoneurones sont inconnus. Nous montrons qu’à E12,5, les cellules de Renshaw (CRs), expriment du GABA et génèrent aussi des GDPs, pouvant évoquer une décharge répétée de potentiels d’action ou de « potentiels en plateau ». Différents profils de décharge intrinsèques des CRs, en réponse à l’injection de courant, ont été observés et classés en « 1 PA », « train de PA », « potentiels en plateau ». De manière surprenante, entre E12,5 et E14,5, alors que l’excitabilité intrinsèque des MNs augmente classiquement, l’excitabilité intrinsèque des CRs régresse transitoirement. Nous montrons que les rapports des conductances sodiques persistantes et les conductances potassiques voltage-dépendantes jouent un rôle majeur dans la détermination des profils de décharge et peuvent expliquer la régression d’excitabilité intrinsèque des CRs.
... Spinal neuroplasticity refers to the ability of spinal neural circuits to make physiological, anatomical, and functional changes in response to a stimulus (Baker-Herman et al., 2004;Cadotte et al., 2012). The neuroplastic nature of spinal synapses is crucial for the development of neural relay circuits during the embryonic and adolescence stage (Ladle et al., 2007). However, irregular synaptic alterations following injury or disease can lead to pain and spasticity (Cadotte et al., 2012;Cadotte and Fehlings, 2013). ...
Traumatic spinal cord injury (SCI) impedes signal transmission by disrupting both the local neurons and their surrounding synaptic connections. Although the majority of SCI patients retain spared neural tissue at the injury site, they predominantly suffer from complete autonomic and sensorimotor dysfunction. While there have been significant advances in the characterization of the spared neural tissue following SCI, the functional role of injury-induced interneuronal plasticity remains elusive. In healthy individuals, spinal interneurons are responsible for relaying signals to coordinate both sympathetic and parasympathetic functions. However, the spontaneous synaptic loss following injury alters these intricate interneuronal networks in the spinal cord. Here, we propose the synaptopathy hypothesis of SCI based on recent findings regarding the maladaptive role of synaptic changes amongst the interneurons. These maladaptive consequences include circuit inactivation, neuropathic pain, spasticity, and autonomic dysreflexia. Recent preclinical advances have uncovered the therapeutic potential of spinal interneurons in activating the dormant relay circuits to restore sensorimotor function. This review will survey the diverse role of spinal interneurons in SCI pathogenesis as well as treatment strategies to target spinal interneurons.
... The research focus historically has been on spinal motor neuron development (Jessell, 2000;Ladle et al., 2007;Purves, 1988). Yet, among the most functionally diverse populations of motor neurons are those of the medullary vagal motor nuclei, which consist of neurons located in the nucleus ambiguus (nAmb) and dorsal motor nucleus of the vagus (DMV). ...
The vagal motor nucleus ambiguus (nAmb) innervates the intrinsic muscles of the larynx, providing direct motor control over vocal production in humans and rodents. Here, we demonstrate that early developmental signaling through the MET receptor tyrosine kinase (MET) is required for proper formation of the nAmb. Embryonic deletion of Met in the developing brainstem resulted in a loss of one-third of motor neurons in the nAmb. While the remaining neurons were able to establish connections with target muscles in the larynx, advanced signal processing analyses revealed severe deficits in ultrasonic vocalization in early postnatal life. Abnormal vocalization patterns persisted into adulthood in the majority of mice tested. Interestingly, 28% of adult mice recovered the ability to vocalize demonstrating heterogeneity in circuit restitution. Together, the data establish MET as a factor necessary for development of a specific subset of neurons in the nAmb required for normal ultrasonic vocalization.
... Spinal neuron distribution along the DV and mediolateral (ML) axes constitute a critical feature of microcircuit organization and functionality (Ladle et al., 2007;Tripodi et al., 2011). Indeed, proper cell body position of dorsal inhibitory INs along the ML axis is crucial for the establishment of their sensorimotor connectivity (Hilde et al., 2016) while the distribution of distinct Lbx1-positive premotor (Goetz et al., 2015) or V1 IN subsets (Bikoff et al., 2016) constrains patterns of input from sensory and motor neurons. ...
Spinal dorsal interneurons, which are generated during embryonic development, relay and process sensory inputs from the periphery to the central nervous system. Proper integration of these cells into neuronal circuitry depends on their correct positioning within the spinal parenchyma. Molecular cues that control neuronal migration have been extensively characterized but the genetic programs that regulate their production remain poorly investigated. Onecut (OC) transcription factors have been shown to control the migration of the dorsal interneurons (dINs) during spinal cord development. Here, we report that the OC factors moderate the expression of Pou2f2, a transcription factor essential for B-cell differentiation, in spinal dINs. Overexpression or inactivation of Pou2f2 leads to alterations in the differentiation of dI2, dI3 and Phox2a-positive dI5 populations and to defects in the distribution of dI2-dI6 interneurons. Thus, an OC-Pou2f2 genetic cascade regulates adequate diversification and distribution of dINs during embryonic development.
... Patterns of S-M connection are largely conserved across limbed vertebrates (Hasan and Stuart, 1988;Hongo et al., 1984;Lichtman et al., 1984;Mendelson and Frank, 1991), and they are established early in development, primarily in an activity-independent manner (Mears and Frank, 1997;Mendelsohn et al., 2015). This has prompted suggestions that cell-cell recognition underlies the selectivity of these connections (Ladle et al., 2007). But, with the exception of a single repellent mechanism, in which plexinD1 + sensory axons are precluded from forming monosynaptic connections with sema3E + motor neurons (Fukuhara et al., 2013;Pecho-Vrieseling et al., 2009), molecular mediators of S-M recognition have not been defined in this system. ...
Proprioceptive sensory axons in the spinal cord form selective connections with motor neuron partners, but the strategies that confer such selectivity remain uncertain. We show that muscle-specific sensory axons project to motor neurons along topographically organized angular trajectories and that motor pools exhibit diverse dendritic arbors. On the basis of spatial constraints on axo-dendritic interactions, we propose positional strategies that can account for sensory-motor connectivity and synaptic organization. These strategies rely on two patterning principles. First, the degree of axo-dendritic overlap reduces the number of potential post-synaptic partners. Second, a close correlation between the small angle of axo-dendritic approach and the formation of synaptic clusters imposes specificity of connections when sensory axons intersect multiple motor pools with overlapping dendritic arbors. Our study identifies positional strategies with prominent roles in the organization of spinal sensory-motor circuits.
... Indeed, the clustering and dorsoventral settling position of motor neuron pools serve as a determinant of the pattern of sensory input specificity (Sürmeli et al., 2011). Similarly, position of dorsal interneuron (dINs) along the mediolateral axis in lamina V determines their connectivity with sensory afferents (Ladle et al., 2007;Tripodi et al., 2011;Hilde et al., 2016). Furthermore, positional distinctions among V1 interneuron subsets (Bikoff et al., 2016) or Lbx1-derived premotor interneurons (Goetz et al., 2015) constrain patterns of input from sensory and motor neurons. ...
During embryonic development, the dorsal spinal cord generates numerous interneuron populations eventually involved in motor circuits or in sensory networks that integrate and transmit sensory inputs from the periphery. The molecular mechanisms that regulate the specification of these multiple dorsal neuronal populations have been extensively characterized. In contrast, the factors that contribute to their diversification into smaller specialized subsets and those that control the specific distribution of each population in the developing spinal cord remain unknown. Here, we demonstrate that the Onecut transcription factors, namely Hepatocyte Nuclear Factor-6 (HNF-6) (or OC-1), OC-2 and OC-3, regulate the diversification and the distribution of spinal dorsal interneuron (dINs). Onecut proteins are dynamically and differentially distributed in spinal dINs during differentiation and migration. Analyzes of mutant embryos devoid of Onecut factors in the developing spinal cord evidenced a requirement in Onecut proteins for proper production of a specific subset of dI5 interneurons. In addition, the distribution of dI3, dI5 and dI6 interneuron populations was altered. Hence, Onecut transcription factors control genetic programs that contribute to the regulation of spinal dIN diversification and distribution during embryonic development.
... First, why do blood vessels remain outside MN columns for a specific developmental window? During E9.5 till E12.5 differentiated MNs are migrating into MN columns, MN cell bodies are clustering into columns, are establishing synaptic contacts and are extending their axons out of the SC to innervate specific muscles 54 . Thus, the presence of vascular sprouts could interfere in these processes. ...
Formation of a precise vascular network within the central nervous system is of critical importance to assure delivery of oxygen and nutrients and for accurate functionality of neuronal networks. Vascularization of the spinal cord is a highly stereotypical process. However, the guidance cues controlling blood vessel patterning in this organ remain largely unknown. Here we describe a new neuro-vascular communication mechanism that controls vessel guidance in the developing spinal cord. We show that motor neuron columns remain avascular during a developmental time window, despite expressing high levels of the pro-angiogenic vascular endothelial growth factor (VEGF). We describe that motor neurons express the VEGF trapping receptor sFlt1 via a Neuropilin-1-dependent mechanism. Using a VEGF gain-of-function approach in mice and a motor neuron-specific sFlt1 loss-of-function approach in chicken, we show that motor neurons control blood vessel patterning by an autocrine mechanism that titrates motor neuron-derived VEGF via their own expression of sFlt1.
... In mouse and rat embryos, it has been reported that spontaneous activity undergoes two types of developmental changes in pharmacological substrates. One is a switching of the dominant contributor from nicotinic acetylcholine receptors to glutamate receptors, and the other is the change in GABA/glycinergic responses from depolarizing/excitatory to hyperpolarizing/inhibitory (Nakayama et al., 1999;Ren and Greer, 2003;Myers et al., 2005;Ladle et al., 2007;Momose-Sato et al., 2012b). In the chick embryo, the dominant neuronal response to GABA and glycine is depolarizing/excitatory at least until E8 in the hindbrain (Momose-Sato et al., 1998) and E10-E11 in the spinal cord Gonzalez-Islas and Wenner, 2006). ...
Spontaneous activity in the developing central nervous system occurs before the brain responds to external sensory inputs, and appears in the hindbrain and spinal cord as rhythmic electrical discharges of cranial and spinal nerves. This spontaneous activity recruits a large population of neurons and propagates like a wave over a wide region of the central nervous system. Here, we review spontaneous activity in the chick hindbrain by focusing on this large-scale synchronized activity. Asynchronous activity that is expressed earlier than the above mentioned synchronized activity and activity originating in midline serotonergic neurons are also briefly mentioned.
... The development of nIII motoneuron identity is controversial. One proposal for how individual mo-toneurons come to acquire their identity is post-hoc self-identification following random extraocular muscle innervation (Glover, 2003), consistent with target-derived signals regulating motoneuron connectivity (Ladle et al., 2007) but not with a stereotyped order of subpopulation birth (Shaw and Alley, 1981). Others propose that a caudal-to-rostral order of differentiation of both extraocular muscle and motoneuron groups allows each newly-born pair to match (Evinger, 1988), although this conflicts with the observed order of motor nucleus development (Altman and Bayer, 1981;Varela-Echavarría et al., 1996). ...
Both spatial and temporal cues determine the fate of immature neurons. A major challenge at the interface of developmental and systems neuroscience is to relate this spatiotemporal trajectory of maturation to circuit-level functional organization. This study examined the development of two extraocular motor nuclei (nIII and nIV), structures in which a motoneuron's identity, or choice of muscle partner, defines its behavioral role. We used retro-orbital dye fills, in combination with fluorescent markers for motoneuron location and birthdate, to probe spatial and temporal organization of the oculomotor (nIII) and trochlear (nIV) nuclei in the larval zebrafish. We described a dorsoventral organization of the four nIII motoneuron pools, in which inferior and medial rectus motoneurons occupy dorsal nIII, while inferior oblique and superior rectus motoneurons occupy distinct divisions of ventral nIII. Dorsal nIII motoneurons are, moreover, born before motoneurons of ventral nIII and nIV. Order of neurogenesis can therefore account for the dorsoventral organization of nIII and may play a primary role in determining motoneuron identity. We propose that the temporal development of extraocular motoneurons plays a key role in assembling a functional oculomotor circuit. This article is protected by copyright. All rights reserved.
... Along with neuron survival, target-released NGF also regulates axon growth and retraction (Campenot 1977), synapse and neural circuit formation (Ladle et al. 2007;Sharma et al. 2010), and expression of neurotransmitters (Luo et al. 2007;Patel et al. 2003). In vitro studies, which use compartmentalized chambers, have shown that NGF applied directly to distal axons acts locally to support their extension; however, when only cell bodies are exposed to NGF, neurons fail to extend axons into a compartment that lacks NGF (Campenot 1977). ...
The physiology of NGF is extremely complex, and although the study of this neurotrophin began more than 60 years ago, it is far from being concluded. NGF, its precursor molecule pro-NGF, and their different receptor systems (i.e., TrkA, p75NTR, and sortilin) have key roles in the development and adult physiology of both the nervous and immune systems. Although the NGF receptor system and the pathways activated are similar for all types of cells sensitive to NGF, the effects exerted during embryonic differentiation and in committed mature cells are strikingly different and sometimes opposite. Bearing in mind the pleiotropic effects of NGF, alterations in its expression and synthesis, as well as variations in the types of receptor available and in their respective levels of expression, may have profound effects and play multiple roles in the development and progression of several diseases. In recent years, the use of NGF or of inhibitors of its receptors has been prospected as a therapeutic tool in a variety of neurological diseases and injuries. In this review, we outline the different roles played by the NGF system in various moments of nervous and immune system differentiation and physiology, from embryonic development to aging. The data collected over the past decades indicate that NGF activities are highly integrated among systems and are necessary for the maintenance of homeostasis. Further, more integrated and multidisciplinary studies should take into consideration these multiple and interactive aspects of NGF physiology in order to design new therapeutic strategies based on the manipulation of NGF and its intracellular pathways.
... During embryogenesis, distinct sensory neuron subpopulations are specified, which can be distinguished also based on the myelination status of their axons. Myelinated larger-caliber axons (Ab-type) are associated with low-threshold mechanoreceptors, while thinly myelinated intermediate-sized axons (Adtype) and nonmyelinated small-caliber axons (C-type nociceptors) transmit painful stimuli, for example, noxious heat or cold (Dyck and Thomas, 1993;Goulding, 2009;Ladle et al., 2007). ...
... This stereotyped organization of motorneurons has long been thought to be a possible substrate for facilitating the connectivity of pre-motor inputs that control and coordinate movement . The formation of the musculotopic motor map is intrinsically programmed by an intricate genetic system that specifies the subtype identity of motorneurons and controls soma migration, axon targeting, dendritic pattern, and sensory connectivity (Dasen and Jessell 2009;Ladle et al., 2007;Bonanomi and Pfaff, 2010). How these complementary positional and genetic factors influence the wiring of inputs to control the fine pattern and coordination of motorneuron firing to achieve complex motor behaviors remains poorly understood. ...
The coordination of multi-muscle movements originates in the circuitry that regulates the firing patterns of spinal motorneurons. Sensory neurons rely on the musculotopic organization of motorneurons to establish orderly connections, prompting us to examine whether the intraspinal circuitry that coordinates motor activity likewise uses cell position as an internal wiring reference. We generated a motorneuron-specific GCaMP6f mouse line and employed two-photon imaging to monitor the activity of lumbar motorneurons. We show that the central pattern generator neural network coordinately drives rhythmic columnar-specific motorneuron bursts at distinct phases of the locomotor cycle. Using multiple genetic strategies to perturb the subtype identity and orderly position of motorneurons, we found that neurons retained their rhythmic activity-but cell position was decoupled from the normal phasing pattern underlying flexion and extension. These findings suggest a hierarchical basis of motor circuit formation that relies on increasingly stringent matching of neuronal identity and position.
Copyright © 2015 Elsevier Inc. All rights reserved.
... In addition, there is a temporal influence of development on these spatial coordinates such that distinct cell fates emerge at different times during development. This yields a four dimensional system for establishing spinal neuron cell fate that has been reviewed extensively (Jessell, 2000;Jankowska, 2001;Lee and Pfaff, 2001;Muroyama et al., 2002;Helms and Johnson, 2003;Goulding and Pfaff, 2005;Kiehn, 2006;Ladle et al., 2007;Stepien and Arber, 2008;Dasen and Jessell, 2009;Goulding, 2009;Grillner and Jessell, 2009;Hegarty et al., 2013). ...
The spinal cord of vertebrate animals is comprised of intrinsic circuits that are capable of sensing the environment and generating complex motor behaviors. There are two major perspectives for understanding the biology of this complicated structure. The first approaches the spinal cord from the point of view of function and is based on classic and ongoing research in electrophysiology, adult behavior, and spinal cord injury. The second view considers the spinal cord from a developmental perspective and is founded mostly on gene expression and gain-of-function and loss-of-function genetic experiments. Together these studies have uncovered functional classes of neurons and their lineage relationships. In this review, we summarize our knowledge of developmental classes, with an eye toward understanding the functional roles of each group.
... SNA is a network-driven phenomenon that depends on chemical neurotransmission between spinal neurons O'Donovan et al., 1998). The neurotransmitter receptors that SNA is dependent upon change during development (Ladle et al., 2007;Momose-Sato and Sato, 2013). At early stages in chick, rat, and mouse spinal development, nicotinic cholinergic transmission is thought to drive the expression of SNA, while excitatory GABA A receptor activation also contributes; later in embryonic development, glutamatergic transmission becomes increasingly important while nicotinic transmission contributes, but is less critical for the expression of spinal SNA (Sernagor et al., 1995;Chub and O'Donovan, 1998;Milner and Landmesser, 1999;Wenner and O'Donovan, 2001;Hanson and Landmesser, 2003;Ren and Greer, 2003;Mochida et al., 2009;Momose-Sato et al., 2012;Czarnecki et al., 2014). ...
Synaptically driven spontaneous network activity (SNA) is observed in virtually all developing networks. Recurrently connected spinal circuits express SNA, which drives fetal movements during a period of development when GABA is depolarizing and excitatory. Blockade of nicotinic acetylcholine receptor (nAChR) activation impairs the expression of SNA and the development of the motor system. It is mechanistically unclear how nicotinic transmission influences SNA, and in this study we tested several mechanisms that could underlie the regulation of SNA by nAChRs. We find evidence that is consistent with our previous work suggesting that cholinergically-driven Renshaw cells can initiate episodes of SNA. While Renshaw cells receive strong nicotinic synaptic input, we see very little evidence suggesting other spinal interneurons or motoneurons receive nicotinic input. Rather, we found that nAChR activation tonically enhanced evoked and spontaneous presynaptic release of GABA in the embryonic spinal cord. Enhanced spontaneous and/or evoked release could contribute to increased SNA frequency. Finally, our study suggests that blockade of nAChRs can reduce the frequency of SNA by reducing probability of GABAergic release. This result suggests that the baseline frequency of SNA is maintained through elevated GABA release driven by tonically active nAChRs. Nicotinic receptors regulate GABAergic transmission and SNA, which are critically important for the proper development of the embryonic network. Therefore, our results provide a better mechanistic framework for understanding the motor consequences of fetal nicotine exposure. This article is protected by copyright. All rights reserved.
© 2015 Wiley Periodicals, Inc.
... DRGs and spinal cords at lumbar levels. We used E14.5-16.5 embryos since sensorymotor connections are already detected in the lumbar region as early as E17.5 [9,15] suggesting that molecules involved in sensory-motor specificity would be expressed earlier by proprioceptive sensory and/or motor neurons. Sensory-motor circuits are formed between presynaptic proprioceptive sensory neurons (marked by the expression of Pv, a specific proprioceptive marker [16,17]) (Fig. 1A, 1C), and postsynaptic motor neurons in the ventral spinal cord (identified by Islet-1) (Fig. 1A-1B). ...
Cell adhesion molecules belonging to the immunoglobulin superfamily (IgSF) control synaptic specificity through hetero- or homophilic interactions in different regions of the nervous system. In the developing spinal cord, monosynaptic connections of exquisite specificity form between proprioceptive sensory neurons and motor neurons, however, it is not known whether IgSF molecules participate in regulating this process. To determine whether IgSF molecules influence the establishment of synaptic specificity in sensory-motor circuits, we examined the expression of 157 IgSF genes in the developing dorsal root ganglion (DRG) and spinal cord by in situ hybridization assays. We find that many IgSF genes are expressed by sensory and motor neurons in the mouse developing DRG and spinal cord. For instance, Alcam, Mcam, and Ocam are expressed by a subset of motor neurons in the ventral spinal cord. Further analyses show that Ocam is expressed by obturator but not quadriceps motor neurons, suggesting that Ocam may regulate sensory-motor specificity in these sensory-motor reflex arcs. Electrophysiological analysis shows no obvious defects in synaptic specificity of monosynaptic sensory-motor connections involving obturator and quadriceps motor neurons in Ocam mutant mice. Since a subset of Ocam+ motor neurons also express Alcam, Alcam or other functionally redundant IgSF molecules may compensate for Ocam in controlling sensory-motor specificity. Taken together, these results reveal that IgSF molecules are broadly expressed by sensory and motor neurons during development, and that Ocam and other IgSF molecules may have redundant functions in controlling the specificity of sensory-motor circuits.
... Complex movements require the control of individual muscles in a collaborating manner. This coordination relies on a highly organized circuitry between SNs, association neurons, and SpMNs as reviewed by Ladle et al. (2007). In a perspective of regeneration therapies, SpMNs with the correct identity should insert in a pre-existing neuronal circuitry. ...
Motor neurons (MNs) are neuronal cells located in the central nervous system (CNS) controlling a variety of downstream targets. This function infers the existence of MN subtypes matching the identity of the targets they innervate. To illustrate the mechanism involved in the generation of cellular diversity and the acquisition of specific identity, this review will focus on spinal MNs (SpMNs) that have been the core of significant work and discoveries during the last decades. SpMNs are responsible for the contraction of effector muscles in the periphery. Humans possess more than 500 different skeletal muscles capable to work in a precise time and space coordination to generate complex movements such as walking or grasping. To ensure such refined coordination, SpMNs must retain the identity of the muscle they innervate. Within the last two decades, scientists around the world have produced considerable efforts to elucidate several critical steps of SpMNs differentiation. During development, SpMNs emerge from dividing progenitor cells located in the medial portion of the ventral neural tube. MN identities are established by patterning cues working in cooperation with intrinsic sets of transcription factors. As the embryo develop, MNs further differentiate in a stepwise manner to form compact anatomical groups termed pools connecting to a unique muscle target. MN pools are not homogeneous and comprise subtypes according to the muscle fibers they innervate. This article aims to provide a global view of MN classification as well as an up-to-date review of the molecular mechanisms involved in the generation of SpMN diversity. Remaining conundrums will be discussed since a complete understanding of those mechanisms constitutes the foundation required for the elaboration of prospective MN regeneration therapies.
... The neuromuscular system provides rapid and coordinated force generation, whereby the number and firing rate of recruited motor units are systematically adjusted to meet environmental demands (Monster and Chan, 1977;Henneman and Mendell, 1981;Clamann, 1993;Cope and Sokoloff, 1999). Indeed, the elegant simplicity with which animals navigate their environment relies on neural circuitry that is inherently modifiable, and the ability to perform a variety of motor tasks while responding quickly to unexpected perturbations and threats is essential for individual survival (Ladle et al., 2007;Miri et al., 2013). Control of α-MN repetitive firing properties is a therefore highly conserved and critical adaption of mammalian and non-mammalian species alike, and identifying the responsible spinal circuits has been of essential importance in our understanding of neuromuscular function and dysfunction (Miles and Sillar, 2011). ...
C-boutons are important cholinergic modulatory loci for state-dependent alterations in motoneuron firing rate. m2 receptors are concentrated postsynaptic to C-boutons, and m2 receptor activation increases motoneuron excitability by reducing the action potential afterhyperpolarization. Here, using an intensive review of the current literature as well as data from our laboratory, we illustrate that C-bouton postsynaptic sites comprise a unique structural/functional domain containing appropriate cellular machinery (a "signaling ensemble") for cholinergic regulation of outward K(+) currents. Moreover, synaptic reorganization at these critical sites has been observed in a variety of pathologic states. Yet despite recent advances, there are still great challenges for understanding the role of C-bouton regulation and dysregulation in human health and disease. The development of new therapeutic interventions for devastating neurological conditions will rely on a complete understanding of the molecular mechanisms that underlie these complex synapses. Therefore, to close this review, we propose a comprehensive hypothetical mechanism for the cholinergic modification of α-MN excitability at C-bouton synapses, based on findings in several well-characterized neuronal systems.
... From this time point on, cell bodies within the spinal cord can be labeled retrogradely from specifi c muscles within the limb (Fig. 4c ). Furthermore, intraspinal connections between motor neurons and afferent branches of sensory neurons, or spinal interneurons are formed during later embryonal development [ 33 ]. In perinatal animals, the labeling procedure is basically the same as described above; however, the skin was sealed right after application of the tracer using histoacrylic glue and the incubation time was elongated to 20-24 h. ...
The ability to exert complex locomotor behaviors requires precise guidance of sensory and motor fibers to the extremities, resulting in the formation of precise peripheral networks. Interactions of growing axons with their environment and co-extending nerve fibers have been shown to critically contribute to the establishment of neuronal projections to the limb, governing accurate fasciculation of heterotypic fiber systems and mediating the stereotypic dorsal-ventral guidance decisions of growing motor axons at the base of the limb. Here we provide a detailed methodology to quantitate selective fasciculation of axon tracts at specific choice points as well as guidance fidelity of motor neurons projecting to the dorsal or ventral limb during embryonal development. Immunohistochemical staining of whole-mount embryo preparations was employed to analyze patterned growth of axons towards the plexus region, and selective branching beyond this choice point. Retrograde tracing of motor neurons from the dorsal or ventral limb mesenchyme served to analyze stereotypical dorsal-ventral guidance decisions of motor neurons localized in the medial and lateral aspects of the lateral motor column (LMC). These methods therefore provide a valuable tool to reliably quantify sensory-motor fasciculation and stereotypic guidance events during early embryonal development.
... These motor behaviors require the production of a reciprocating pattern of motor impulses to antagonist groups of flexor and extensor muscles (Sherrington, 1893;Grillner, 1975). Multiple studies have shown that flexor-extensor alternation is an intrinsic property of the locomotor central pattern generator (CPG) in limbed animals (Brown, 1911;Eccles et al., 1956;Goulding, 2009;Grillner, 1975;Grillner and Jessell, 2009;Kiehn, 2006;Ladle et al., 2007). However, efforts to identify the interneuron (IN) cell types that secure flexor-extensor alternation have met with limited success, and because of this we still know very little about the overall organization of the locomotor CPG in limbed vertebrates. ...
Reciprocal activation of flexor and extensor muscles constitutes the fundamental mechanism that tetrapod vertebrates use for locomotion and limb-driven reflex behaviors. This aspect of motor coordination is controlled by inhibitory neurons in the spinal cord; however, the identity of the spinal interneurons that serve this function is not known. Here, we show that the production of an alternating flexor-extensor motor rhythm depends on the composite activities of two classes of ventrally located inhibitory neurons, V1 and V2b interneurons (INs). Abrogating V1 and V2b IN-derived neurotransmission in the isolated spinal cord results in a synchronous pattern of L2 flexor-related and L5 extensor-related locomotor activity. Mice lacking V1 and V2b inhibition are unable to articulate their limb joints and display marked deficits in limb-driven reflex movements. Taken together, these findings identify V1- and V2b-derived neurons as the core interneuronal components of the limb central pattern generator (CPG) that coordinate flexor-extensor motor activity.
... One neural system in which the cellular origins of synaptic specificity have been examined in some detail is the spinal monosynaptic stretch reflex circuit. Here, the fine pattern of sensory-motor connectivity has been defined through a combination of anatomical and physiological studies (Brown, 1981;Ladle et al., 2007). In this circuit, group Ia proprioceptive afferent fibers make strong connections with motor neurons supplying the same muscle and weaker connections with motor neurons supplying synergistic muscles (Eccles et al., 1957;Frank and Mendelson, 1990). ...
In mammalian spinal cord, group Ia proprioceptive afferents form selective monosynaptic connections with a select group of motor pool targets. The extent to which sensory recognition of motor neurons contributes to the selectivity of sensory-motor connections remains unclear. We show here that proprioceptive sensory afferents that express PlexinD1 avoid forming monosynaptic connections with neurons in Sema3E(+) motor pools yet are able to form direct connections with neurons in Sema3E(off) motor pools. Anatomical and electrophysiological analysis of mice in which Sema3E-PlexinD1 signaling has been deregulated or inactivated genetically reveals that repellent signaling underlies aspects of the specificity of monosynaptic sensory-motor connectivity in these reflex arcs. A semaphorin-based system of motor neuron recognition and repulsion therefore contributes to the formation of specific sensory-motor connections in mammalian spinal cord.
... 10,000) in its nervous system and excellent genetic tools for selective manipulation of single neuron types [1,2]. Furthermore, the organization of somatosensory afferents and motor neuron dendrites in the larval ventral nerve cord not only resembles their organization in adult flies and other insects [3,4], but also the organization of the vertebrate spinal cord [5,6]. ...
All organisms react to noxious and mechanical stimuli but we still lack a complete understanding of cellular and molecular mechanisms by which somatosensory information is transformed into appropriate motor outputs. The small number of neurons and excellent genetic tools make Drosophila larva an especially tractable model system in which to address this problem. We developed high throughput assays with which we can simultaneously expose more than 1,000 larvae per man-hour to precisely timed noxious heat, vibration, air current, or optogenetic stimuli. Using this hardware in combination with custom software we characterized larval reactions to somatosensory stimuli in far greater detail than possible previously. Each stimulus evoked a distinctive escape strategy that consisted of multiple actions. The escape strategy was context-dependent. Using our system we confirmed that the nociceptive class IV multidendritic neurons were involved in the reactions to noxious heat. Chordotonal (ch) neurons were necessary for normal modulation of head casting, crawling and hunching, in response to mechanical stimuli. Consistent with this we observed increases in calcium transients in response to vibration in ch neurons. Optogenetic activation of ch neurons was sufficient to evoke head casting and crawling. These studies significantly increase our understanding of the functional roles of larval ch neurons. More generally, our system and the detailed description of wild type reactions to somatosensory stimuli provide a basis for systematic identification of neurons and genes underlying these behaviors.
... For example, in the nematode Caenorhabditis elegans, NMJs comprise a mixture of excitatory cholinergic and inhibitory GABAergic synapses [10][11][12][13]. The excitatory NMJs of dipteran insects (i.e., Drosophila) and chordates (i.e., vertebrate) utilize glutamate and ACh, respectively [14][15][16]. The expectation that neurotransmitter expression is tightly regulated at NMJs has been partly met by the identification of transcription factors of the LIM domain and homeodomain (HD domain) families, which are differentially expressed in motor neurons, where they orchestrate the developmental decisions of which neurotransmitter to express [17][18][19]. ...
The human brain comprises approximately 100 billion neurons that express a diverse, and often subtype-specific, set of neurotransmitters and voltage-gated ion channels. Given this enormous complexity, a fundamental question is how is this achieved? The acquisition of neurotransmitter phenotype was viewed as being set by developmental programs 'hard wired' into the genome. By contrast, the expression of neuron-specific ion channels was considered to be highly dynamic (i.e., 'soft wired') and shaped largely by activity-dependent mechanisms. Recent evidence blurs this distinction by showing that neurotransmitter phenotype can be altered by activity and that neuron type-specific ion channel expression can be set, and perhaps limited by, developmental programs. Better understanding of these early regulatory mechanisms may offer new avenues to avert the behavioral changes that are characteristic of many mental illnesses.
This chapter discusses Renshaw cells, which are part of the recurrent feedback inhibition loop located in the spinal cord’s ventral horn. This loop regulates the firing of motoneuron signals for muscle contraction. The chapter outlines the various state feedback output responses required to lessen tremors and enhance the impacted population’s force-muscle activation response. Simulation model investigation reveals such frequency responses. Renshaw cells orchestrate the recurrent feedback inhibition by synchronizing oscillations and strengthening muscular action at frequencies over 20 Hz. They also eliminate oscillations and tremors in the muscles at about 10 Hz. This emphasizes how beneficial Renshaw cells are at reducing physiological tremors.
Those studying neural systems within the brain have historically assumed that lower-level processes in the spinal cord act in a mechanical manner, to relay afferent signals and execute motor commands. From this view, abstracting temporal and environmental relations is the province of the brain. Here we review work conducted over the last 50 years that challenges this perspective, demonstrating that mechanisms within the spinal cord can organize coordinated behavior (stepping), induce a lasting change in how pain (nociceptive) signals are processed, abstract stimulus–stimulus (Pavlovian) and response-outcome (instrumental) relations, and infer whether stimuli occur in a random or regular manner. The mechanisms that underlie these processes depend upon signal pathways (e.g., NMDA receptor mediated plasticity) analogous to those implicated in brain-dependent learning and memory. New data show that spinal cord injury (SCI) can enable plasticity within the spinal cord by reducing the inhibitory effect of GABA. It is suggested that the signals relayed to the brain may contain information about environmental relations and that spinal cord systems can coordinate action in response to descending signals from the brain. We further suggest that the study of stimulus processing, learning, memory, and cognitive-like processing in the spinal cord can inform our views of brain function, providing an attractive model system. Most importantly, the work has revealed new avenues of treatment for those that have suffered a SCI.
The embryological development of the extracranial neuraxis represents a complex interplay between local processes occurring in a temporally organized manner, which reflects neural tube formation, proliferation of progenitor cells, and production of individual cell types. These processes reflect a highly coordinated chain of morphogenetic steps occurring in sequence with other parts of the developing nervous system, notably supraspinal structures, which are linked by spinofugal and petal projections, and the evolving peripheral target providing the target and source of afferent and efferent linkages with specific neuraxial substrates. This chapter reviews these linkages, the biology associated with the trophic targeting of these linkages, and the time course of the development process of these coordinated events.
Recent spinal cord literature abounds with descriptions of genetic preprogramming and the molecular control of circuit formation. In this paper we explore to what extent circuit formation based on learning rather than preprogramming could explain some prominent aspects of the spinal cord connectivity patterns observed in animals. To test this we developed an artificial organism with a basic musculoskeletal system and proprioceptive sensors, connected to a neural network. We adjusted the initially randomized gains in the neural network according to a Hebbian plasticity rule while exercising the model system with spontaneous muscle activity patterns similar to those observed during early fetal development. The resulting connection matrices support functional self-organization of the mature pattern of Ia to motoneuron connectivity in the spinal circuitry. More coordinated muscle activity patterns such as observed later during neonatal locomotion impaired projection selectivity. These findings imply a generic functionality of a musculoskeletal system to imprint important aspects of its mechanical dynamics onto a neural network, without specific preprogramming other than setting a critical period for the formation and maturation of this general pattern of connectivity. Such functionality would facilitate the successful evolution of new species with altered musculoskeletal anatomy and it may help to explain patterns of connectivity and associated reflexes that appear during abnormal development.
Spinal motor neurons (MNs) integrate sensory stimuli and brain commands to generate motor movements in vertebrates. Distinct MN populations and their diversity has long been hypothesized to co-evolve with motor circuit to provide the neural basis from undulatory to ambulatory locomotion during aquatic-to-terrestrial transition of vertebrates. However, how these subtypes are evolved remains largely enigmatic. Using single-cell transcriptomics, we investigate heterogeneity in mouse MNs and discover novel segment-specific subtypes. Among limb-innervating MNs, we reveal a diverse neuropeptide code for delineating putative motor pool identities. We further uncovered that axial MNs are subdivided by three conserved and molecularly distinct subpopulations, defined by Satb2, Nr2f2 or Bcl11b expression. Although axial MNs are conserved from cephalochordates to humans, subtype diversity becomes prominent in land animals and appears to continue evolving in humans. Overall, our study provides a unified classification system for spinal MNs and paves the way towards deciphering how neuronal subtypes are evolved.
The specificity of monosynaptic connections between proprioceptive sensory neurons and their recipient spinal motor neurons depends on multiple factors, including motor neuron positioning and dendrite morphology, axon projection patterns of proprioceptive sensory neurons in the spinal cord, and the ligand-receptor molecules involved in cell-to-cell recognition. However, with few exceptions, the transcription factors engaged in this process are poorly characterized. We show here, that members of the HoxD family of transcription factors play a critical role in the specificity of monosynaptic sensory-motor connections. Mice lacking Hoxd9, Hoxd10, and Hoxd11 exhibit defects in locomotion but have no obvious defects in motor neuron positioning or dendrite morphology through the medio-lateral and rostro-caudal axes. However, we found that quadriceps motor neurons in these mice show aberrant axon development and receive inappropriate inputs from proprioceptive sensory axons innervating the obturator muscle. These genetic studies demonstrate that the HoxD transcription factors play an integral role in the synaptic specificity of monosynaptic sensory-motor connections in the developing spinal cord.
Axons and their branches are generated to allow a neuron to connect with synaptic targets. This chapter provides a comprehensive overview of the molecular and cellular mechanisms underlying the development of these structures. Specific topics include cell biological mechanisms (cytoskeleton and membrane trafficking), extracellular regulation (neurotrophins, guidance cues, cell adhesion, and neural activity), and intracellular signaling mechanisms.
Throughout his scientific career, Tom Jessell pioneered the spinal cord as a model system to study the molecular programs of neural specification, axon guidance, and connection specificity. His contributions to these fields and more broadly to that of developmental neuroscience will continue to inspire and define many generations of researchers. It is challenging to capture all of Tom’s findings in one essay, and therefore, here we wish to briefly highlight his contributions to the problem of connection specificity, with a focus on the spinal sensory-motor reflex circuit. In particular, emphasis will be placed on discoveries from his laboratory that revealed a significant role of positional strategies in establishing selective sensory-motor connections. This work introduced novel principles of neuronal connectivity that may apply to how precise circuit wiring occurs throughout the nervous system.
Discovered little more than a decade ago, optogenetics - a revolutionary technique combining genetic and optical methods to observe and control the function of neurons - is now a widely used research tool. Optogenetics-driven research has led to insights into Parkinson's disease and other neurological and psychiatric disorders. With contributions from leaders and innovators from both academia and industry, this volume explores the discovery and application of optogenetics, from the basic science to its potential clinical use. Chapters cover a range of optogenetics applications, including for brain circuits, plasticity, memory, learning, sleep, vision and neurodegenerative and neuropsychiatric diseases. Providing authoritative coverage of the huge potential that optogenetics research carries, this is an ideal resource for researchers and graduate students, as well as for those working in the biotechnology and pharmaceutical industries and in a clinical setting.
The peripheral somatosensory system overproduces neurons early in development followed by a period of cell death during final target innervation. The decision to survive or die in somatosensory neurons of the dorsal root ganglion (DRG) is mediated by target derived neurotrophic factors and their cognate receptors. Subsets of peripheral somatosensory neurons can be crudely defined by the neurotrophic receptors that they express: peptidergic nociceptors (TrkA+), non‐peptidergic nociceptors (Ret+), mechanoreceptors (Ret+ or TrkB+), and proprioceptors (TrkC+). A direct comparison of early developmental timing between these subsets has not been performed. Here we characterized the accumulation and death of TrkA, B, C, and Ret+ neurons in the DRG as a function of developmental time. We find that TrkB, TrkC, and Ret‐expressing neurons in the DRG complete developmental cell death prior to TrkA‐expressing neurons. Given the broadly defined roles of the neurotrophin receptor p75NTR in augmenting neurotrophic signaling in sensory neurons, we investigated its role in supporting the survival of these distinct subpopulations. We find that TrkA+, TrkB+, and TrkC+ sensory neuron subpopulations require p75NTR for survival, but proliferating progenitors do not. These data demonstrate how diverging sensory neurons undergo successive waves of cell death and how p75NTR represses the magnitude, but not developmental window of this culling. This article is protected by copyright. All rights reserved.
Unlabelled:
Spinal reflex circuit development requires the precise regulation of axon trajectories, synaptic specificity, and synapse formation. Of these three crucial steps, the molecular mechanisms underlying synapse formation between group Ia proprioceptive sensory neurons and motor neurons is the least understood. Here, we show that the Rho GTPase Cdc42 controls synapse formation in monosynaptic sensory-motor connections in presynaptic, but not postsynaptic, neurons. In mice lacking Cdc42 in presynaptic sensory neurons, proprioceptive sensory axons appropriately reach the ventral spinal cord, but significantly fewer synapses are formed with motor neurons compared with wild-type mice. Concordantly, electrophysiological analyses show diminished EPSP amplitudes in monosynaptic sensory-motor circuits in these mutants. Temporally targeted deletion of Cdc42 in sensory neurons after sensory-motor circuit establishment reveals that Cdc42 does not affect synaptic transmission. Furthermore, addition of the synaptic organizers, neuroligins, induces presynaptic differentiation of wild-type, but not Cdc42-deficient, proprioceptive sensory neurons in vitro Together, our findings demonstrate that Cdc42 in presynaptic neurons is required for synapse formation in monosynaptic sensory-motor circuits.
Significance statement:
Group Ia proprioceptive sensory neurons form direct synapses with motor neurons, but the molecular mechanisms underlying synapse formation in these monosynaptic sensory-motor connections are unknown. We show that deleting Cdc42 in sensory neurons does not affect proprioceptive sensory axon targeting because axons reach the ventral spinal cord appropriately, but these neurons form significantly fewer presynaptic terminals on motor neurons. Electrophysiological analysis further shows that EPSPs are decreased in these mice. Finally, we demonstrate that Cdc42 is involved in neuroligin-dependent presynaptic differentiation of proprioceptive sensory neurons in vitro These data suggest that Cdc42 in presynaptic sensory neurons is essential for proper synapse formation in the development of monosynaptic sensory-motor circuits.
Controlling muscle function is essential for human behaviour and survival, thus, impairment of motor function and muscle paralysis can severely impact quality of life and may be immediately life-threatening, as occurs in many cases of traumatic spinal cord injury (SCI) and in patients with amyotrophic lateral sclerosis (ALS). Repairing damaged spinal motor circuits, in either SCI or ALS, currently remains an elusive goal. Therefore alternative strategies are needed to artificially control muscle function and thereby enable essential motor tasks. This review focuses on recent advances towards restoring motor function, with a particular focus on stem cell-derived neuronal engraftment strategies, optogenetic control of motor function and the potential future translational application of these approaches.
One striking feature of the nervous system is the long processes or the axons extending out from the soma. They transmit electric signals that are collected at the dendrites and integrated at the axonal hillock. Each neuron has only one axon, which can reach their synaptic targets as far as 1 m away in humans. To connect with multiple targets, branches sprout at different locations, either along the axonal shaft as interstitial collaterals or at the nerve endings as terminal arbors.
The functional organization of the vertebrate central nervous system (CNS) during the early phase of development has long been unclear, because conventional electrophysiological means have several technical limitations. First, early embryonic neurons are small and fragile, and the application of microelectrodes is often difficult. Second, the simultaneous recording of electrical activity from multiple sites is limited, and as a consequence, response patterns of neural networks cannot be assessed. Optical recording techniques with voltage-sensitive dyes have overcome these obstacles and provided a new approach to the analysis of the functional development/organization of the CNS. In this review, we provide detailed information concerning the recording of optical signals in the embryonic nervous system. After outlining methodological considerations, we present examples of recent progress in optical studies on the embryonic nervous system with special emphasis on two topics. The first is the study of how synapse networks form in specific neuronal circuits. The second is the study of non-specific correlated wave activity, which is considered to play a fundamental role in neural development. These studies clearly demonstrate the utility of fast voltage-sensitive dye imaging as a powerful tool for elucidating the functional organization of the vertebrate embryonic CNS.
Intrafusal fibers of muscle spindles are innervated in the central region by afferent sensory axons and at both polar regions by efferent γ-motoneurons. We previously demonstrated that both neuron–muscle contact sites contain cholinergic synapse-like specialisation, including aggregates of the nicotinic acetylcholine receptor (AChR). In this study we tested the hypothesis that agrin and its receptor complex (consisting of LRP4 and the tyrosine kinase MuSK) are involved in the aggregation of AChRs in muscle spindles, similar to their role at the neuromuscular junction. We show that agrin, MuSK and LRP4 are concentrated at the contact site between the intrafusal fibers and the sensory- and γ-motoneuron, respectively, and that they are expressed in the cell bodies of proprioceptive neurons in dorsal root ganglia. Moreover, agrin and LRP4, but not MuSK, are expressed in γ-motoneuron cell bodies in the ventral horn of the spinal cord. In agrin- and in MuSK-deficient mice, AChR aggregates are absent from the polar regions. In contrast, the subcellular concentration of AChRs in the central region where the sensory neuron contacts the intrafusal muscle fiber is apparently unaffected. Skeletal muscle-specific expression of miniagrin in agrin−/− mice in vivo is sufficient to restore the formation of γ-motoneuron endplates. These results show that agrin and MuSK are major determinants during the formation of γ-motoneuron endplates but appear dispensable for the aggregation of AChRs at the central region. Our results therefore suggest different molecular mechanisms for AChR clustering within two domains of intrafusal fibers.
Muscle spindles are complex stretch-sensitive mechanoreceptors. They consist of specialized skeletal muscle fibers, called intrafusal fibers, which are innervated in the central (equatorial) region by afferent sensory axons and in both polar regions by efferent γ-motoneurons. We show that AChRs are concentrated at the γ-motoneuron endplate as well as in the equatorial region where they colocalize with the sensory nerve ending. In addition to the AChRs, the contact site between sensory nerve ending and intrafusal muscle fiber contains a high concentration of choline acetyltransferase, vesicular acetylcholine transporter and the AChR-associated protein rapsyn. Moreover, bassoon, a component of the presynaptic cytomatrix involved in synaptic vesicle exocytosis, is present in γ-motoneuron endplates but also in the sensory nerve terminal. Finally, we demonstrate that during postnatal development of the γ-motoneuron endplate, the AChR subunit stoichiometry changes from the γ-subunit-containing fetal AChRs to the ε-subunit-containing adult AChRs, similar and approximately in parallel to the postnatal subunit maturation at the neuromuscular junction. In contrast, despite the onset of ε-subunit expression during postnatal development the γ-subunit remains detectable in the equatorial region by subunit-specific antibodies as well as by analysis of muscle spindles from mice with genetically-labeled AChR γ-subunits. These results demonstrate an unusual maturation of the AChR subunit composition at the annulospiral endings and suggest that in addition to the recently described glutamatergic secretory system, the sensory nerve terminals are also specialized for cholinergic synaptic transmission, synaptic vesicle storage and exocytosis.
A remarkable feature of nervous system development is the ability of axons emerging from newly formed neurons to traverse, by cellular scale, colossal distances to appropriate targets. The earliest axons achieve this in an essentially axon-free environment, but the vast majority of axons eventually grow along a scaffold of nerve tracts created by earlier extending axons. Signal exchange between sequentially or simultaneously extending axons may well represent the predominant mode of axonal navigation, but proportionally few efforts have so far been directed at deciphering the underlying mechanisms. This review intends to provide a conceptual update on the cellular and molecular principles driving axon-axon interactions, with emphasis on those contributing to the fidelity of axonal navigation, sorting and connectivity during nerve and circuit assembly.
In adult vertebrates, sensory neurons innervating stretch-sensitive muscle spindles make monosynaptic excitatory connections with specific subsets of motoneurons in the spinal cord. Spindle afferents (Ia fibers) make the strongest connections with motoneurons supplying the same (homonymous) muscle but make few or no connections with motoneurons supplying antagonistic or functionally unrelated muscles. In lower vertebrates these connections are specific from the time they first are formed, but there is comparatively little information about how these reflex connections form in mammals. We therefore studied the pattern of these synaptic connections during postnatal development in mice. Intracellular recordings were made from identified hindlimb motoneurons in an isolated spinal cord preparation, and monosynaptic inputs from Ia fibers in identified hindlimb muscle nerves were measured at different times during the first postnatal week. The pattern of connections was specific throughout this period. Ia fibers made strong connections with homonymous motoneurons but only weak connections with antagonistic motoneurons at every time point examined, from P0 through P7. Even when muscle nerves were stimulated at only 0.1 Hz, the pattern of connections was still highly specific, arguing against a special subpopulation of labile inappropriate connections. The absence of appreciable rearrangements in the pattern of these connections during the first postnatal week is, therefore, analogous to the situation in lower vertebrates, suggesting that mechanisms responsible for establishing this specificity have been conserved during evolution.
Motor function depends on the formation of selective connections between sensory and motor neurons and their muscle targets. The molecular basis of the specificity inherent in this sensory-motor circuit remains unclear. We show that motor neuron pools and subsets of muscle sensory afferents can be defined by the expression of ETS genes, notably PEA3 and ER81. There is a matching in PEA3 and ER81 expression by functionally interconnected sensory and motor neurons. ETS gene expression by motor and sensory neurons fails to occur after limb ablation, suggesting that their expression is coordinated by signals from the periphery. ETS genes may therefore participate in the development of selective sensory-motor circuits in the spinal cord.
Patterned spontaneous electrical activity has been demonstrated in a number of developing neural circuits and has been proposed to play a role in refining connectivity once axons reach their targets. Using an isolated spinal cord preparation, we have found that chick lumbosacral motor axons exhibit highly regular bursts of activity from embryonic day 4 (E4) (stage 24-25), shortly after they exit the spinal cord and while still en route toward their target muscles. Similar bursts could be evoked by stimulating descending pathways at cervical or thoracic levels. Unlike older embryonic cord circuits, the major excitatory transmitter driving activity was not glutamate but acetylcholine, acting primarily though nicotinic non-alpha7 receptors. The circuit driving bursting was surprisingly robust and plastic, because bursting was only transiently blocked by cholinergic antagonists, and following recovery, was now driven by GABAergic inputs. Permanent blockade of spontaneous activity was only achieved by a combination of cholinergic antagonists and bicuculline, a GABAA antagonist. The early occurrence of patterned motor activity suggests that it could be playing a role in either peripheral pathfinding or spinal cord circuit formation and maturation. Finally, the characteristic differences in burst parameters already evident between different motoneuron pools at E4 would require that the combination of transcription factors responsible for specifying pool identity to have acted even earlier.
Recent studies indicate a modular organization of the nociceptive withdrawal reflex system. Each module has a characteristic receptive field, closely matching the withdrawal movement caused by its effector muscle. In the rat, the strength of the sensory input to each module is tuned during the first postnatal weeks, i.e., erroneous spinal connections are depressed, and adequate connections are strengthened. To clarify if this tuning is dependent on supraspinal structures, the effect of a complete neonatal spinal cord transection on the postnatal tuning of withdrawal reflexes was studied. The nociceptive receptive fields of single hindlimb muscles and compound withdrawal reflexes were examined in decerebrate unanesthetized and awake rats, respectively. Noxious thermal CO(2) laser stimulation was used to evoke reflex responses. Neonatal spinal cord transection resulted in a disrupted reflex organization in the adult rat, resembling that previously found in neonatal rats. The receptive fields of single hindlimb muscles exhibited abnormal distribution of sensitivity not matching the withdrawal action of the effector muscles. Likewise, the composite nocifensive movements, as documented in the awake rat, often resulted in erroneous movements toward the stimulus. It is concluded that withdrawal reflexes do not become functionally adapted in rats spinalized at birth. These findings suggest a critical role for supraspinal systems in the postnatal tuning of spinal nociceptive systems.
Interneuronal gap junctional coupling is a hallmark of neural development whose functional significance is poorly understood. We have characterized the extent of electrical coupling and dye coupling and patterns of gap junction protein expression in lumbar spinal motor neurons of neonatal rats. Intracellular recordings showed that neonatal motor neurons are transiently electrically coupled and that electrical coupling is reversibly abolished by halothane, a gap junction blocker. Iontophoretic injection of Neurobiotin, a low molecular weight compound that passes across most gap junctions, into single motor neurons resulted in clusters of many labeled motor neurons at postnatal day 0 (P0)-P2, and single labeled motor neurons after P7. The compact distribution of dye-labeled motor neurons suggested that, after birth, gap junctional coupling is spatially restricted. RT-PCR, in situ hybridization, and immunostaining showed that motor neurons express five connexins, Cx36, Cx37, Cx40, Cx43, and Cx45, a repertoire distinct from that expressed by other neurons or glia. Although all five connexins are widely expressed among motor neurons in embryonic and neonatal life, Cx36, Cx37, and Cx43 continue to be expressed in many adult motor neurons, and expression of Cx45, and in particular Cx40, decreases after birth. The disappearance of electrical and dye coupling despite the persistent expression of several gap junction proteins suggests that gap junctional communication among motor neurons may be modulated by mechanisms that affect gap junction assembly, permeability, or open state.
A number of homeodomain transcription factors have been implicated in controlling the differentiation of various types of neurons including spinal motoneurons. Some of these proteins are also expressed in spinal interneurons, but their function is unknown. Progress in understanding the role of transcription factors in interneuronal development has been slow because the synaptic connections of interneurons, which in part define their identity, are difficult to establish. Using whole cell recording in the isolated spinal cord of chick embryos, we assessed the synaptic connections of lumbosacral interneurons expressing the Engrailed-1 (En1) transcription factor. Specifically we established whether En1-expressing interneurons made direct connections with motoneurons and whether they constitute a single interneuron class. Cells were labeled with biocytin and subsequently processed for En1 immunoreactivity. Our findings indicate that the connections of En1-expressing cells with motoneurons and with sensory afferents were diverse, suggesting that the population was heterogeneous. In addition, the synaptic connections we tested were similar in interneurons that expressed the En1 protein and in many that did not. The majority of sampled En1 cells did, however, exhibit a direct synaptic connection to motoneurons that is likely to be GABAergic. Because our physiological methods underestimate the number of direct connections with motoneurons, it is possible that the great majority, perhaps all, En1-expressing cells make direct synaptic connections with motoneurons. Our results raise the possibility that En1 could be involved in interneuron-motoneuron connectivity but that its expression is not restricted to a distinct functional subclass of ventral interneuron. These findings constrain hypotheses about the role of En-1 in interneuron development and function.
We examined the ability of the isolated lumbosacral spinal cord of the neonatal mouse (P0-7) to generate rhythmic motor activity under several different conditions. In the absence of electrical or pharmacological stimulation, we recorded several patterns of spontaneous ventral root depolarization and discharge. Spontaneous, alternating discharge between contralateral ventral roots could occur two to three times over a 10-min interval. We also observed other patterns, including left-right synchrony and rhythmic activity restricted to one side of the cord. Trains of stimuli delivered to the lumbar/coccygeal dorsal roots or the sural nerve reliably evoked episodes of rhythmic activity. During these evoked episodes, rhythmic ventral root discharges could occur on one side of the cord or could alternate from side to side. Bath application of a combination of N-methyl-D,L-aspartate (NMA), serotonin, and dopamine produced rhythmic activity that could last for several hours. Under these conditions, the discharge recorded from the left and right L(1)-L(3) ventral roots alternated. In the L(4)-L(5) segments, the discharge had two peaks in each cycle, coincident with discharge of the ipsilateral and contralateral L(1)-L(3) roots. The L(6) ventral root discharge alternated with that recorded from the ipsilateral L(1)-L(3) roots. We established that the drug-induced rhythm was locomotor-like by recording an alternating pattern of discharge between ipsilateral flexor and extensor hindlimb muscle nerves. In addition, by recording simultaneously from ventral roots and muscle nerves, we established that ankle flexor discharge was in phase with ipsilateral L(1)/L(2) ventral root discharge, while extensor discharge was in phase with ipsilateral L(6) ventral root discharge. Rhythmic patterns of ventral root discharge were preserved following mid-sagittal section of the spinal cord, demonstrating that reciprocal inhibitory connections between the left and right sides of the cord are not essential for rhythmogenesis in the neonatal mouse cord. Blocking N-methyl-D-aspartate receptors, in both the intact and the hemisected preparation, revealed that these receptors contribute to but are not essential for rhythmogenesis. In contrast, the rhythm was abolished following blockade of kainate/AMPA receptors with 6-cyano-7-nitroquinoxalene-2,3-dione. These findings demonstrate that the isolated mouse spinal cord can produce a variety of coordinated activities, including locomotor-like activity. The ability to study these behaviors under a variety of different conditions offers promise for future studies of rhythmogenic mechanisms in this preparation.
Different aspects of spinal locomotor organization have been studied in the mouse during embryonic and neonatal development using in vitro preparations of isolated lumbosacral cords. The first consideration was the embryonic development of an alternating bilateral pattern. From embryonic day (E) 12, perfusion of serotonin could induce relatively synchronous lumbar bursts across the cord. Bilateral activity became progressively alternate at E15 due to the appearance of glycinergic inhibitory interactions (revealed by strychnine application). Strictly alternating patterns were expressed at E18 and were maintained after birth. In a second step, we investigated cellular properties involved in lumbar rhythmogenesis in postnatal day 0-2 preparations which displayed spontaneous locomotor-like activity. Perfusion of receptor antagonists showed the co-operative involvement of N-methyl-D-aspartate (NMDA)- and non-NMDA-receptors for excitatory amino acids-mediated operation of locomotor networks. In a final step we investigated the localization of locomotor networks within the lumbar cord. Data obtained from preparations exhibiting spontaneous or Mg2+-free induced bursts revealed that the networks are present throughout the lumbar cord and that rhythmogenesis is distributed throughout all segmental levels.
Spinal motoneurons have long been the focus of studies of synaptic action and membrane properties, and the general firing properties and morphological features of both alpha and gamma motoneurons are well-known.
Recent advances in techniques, especially the intraneuronal injection of the enzyme horseradish peroxidase, have led to a new era in our understanding of spinal cord structure and function. Input to the cord is precisely organized: the primary afferent fibres from different types of receptors distribute their anatomically specific collaterals to particular parts of the dorsal horn, afferent fibres from the skin lay down a precise somatotopic map, input to the dorsal horn from descending systems is also distributed in a localized way. The neurones of the dorsal horn are varied in both structure and function, even so some quite specific cell types can be identified and the dendritic trees may respect laminar boundaries as determined cytoarchitectonically (although the majority of neurones have dendrites that cut across these boundaries). The output pathways from the dorsal horn are many and various, but again they arise from cells in definite parts of the dorsal horn. The dorsal horn must be considered as a well-organized, and complex, part of the central nervous system. It cannot be considered as a structural or functional unit but is made up of many interacting parts that process input from the primary afferent fibres, from other levels of the spinal cord and from many descending pathways from the brain.
Much of the current knowledge and thinking about the cellular and synaptic neuroanatomy of the spinal cord is summarized in this volume. This reviewer welcomed particularly the inclusion of several chapters which are oriented toward comparative anatomy. As Eccles notes in his brief preface, the neuroanatomists cast frequent side glances toward physiological data. In fact, a number of the contributors are themselves members of the physiological fraternity. R. Nieuwenhuys (Amsterdam) and J. H. R. Schoen (Leiden) have chapters on the comparative anatomy of the spinal cord and of the descending fiber systems, respectively. Cytoarchitechtonics and synaptology are detailed by B. Rexed (Uppsala), the motor neuron pools by G. J. Romanes (Edinburgh), the volume and surface of spinal neurons by J. P. Schade (Amsterdam), and neuronal interdependence by S. Gelfan (New York). J. M. Sprague and H. Ha (Philadelphia) present data on the sites of impingement of dorsal root fibers and
Development of neuronal circuits generating locomotor activity was studied using an isolated lumbar spinal cord preparation from fetal and neonatal rats. Bath application of N-methyl-d-aspartate (NMDA) or 5-HT evoked patterned motor activity resembling that seen during normal fictive locomotion on embryonic day (E) 20.5. Glycine-mediated inhibition was essential to the formation of this coordinated motor activity. In preparations from fetuses at the earlier stages (E14.5-E16.5), we observed spontaneous motoneuronal activity and chemically induced rhythmic bursts, which were synchronized on the two sides in the corresponding ventral roots. The spontaneous activity was not blocked by kynurenate, the glutamate receptor blocker, although it was completely abolished by strychnine, the glycine receptor antagonist. A brief application of glycine evoked excitatory responses resembling the spontaneous bursts in both time course and amplitude. It is concluded that glycine functions transiently as excitatory transmitters at these stages. These results suggest that functional change in glycine-induced responses during development plays an important role in differentiation of the neuronal circuits generating locomotion.
The development of central projections of sensory neurons in lumbosacral dorsal root ganglia (DRGs) was examined by using horseradish peroxidase labeling techniques in chick embryos from stage 23 (E4) to stage 39 (E13). Our results show that primary afferents reach the spinal cord by stage 23. Afferent axons extend in the primordium of the dorsal funiculus for several segments rostral and caudal to their segment of entry for over 24 hours before invading the gray matter at stage 28 (E6). Sensory fibers grow into the vicinity of motoneuron dendrites by stage 32 (E7.5), about the time that reflexes and apparent monosynaptic EPSPs can first be elicited. Dense projections into the dorsal laminae of the spinal cord, presumably representing cutaneous afferents, appear somewhat later, at about stage 39 (E13), when the segmental projection pattern begins to resemble the mature pattern.
The paper presents a method for ultrastructural analysis and description of neuronal architecture and synaptology of cat spinal alpha-motoneurons from complete series of consecutive ultrathin sections through the cell body and proximal parts of the dendrites. The method implies that sections are selected for analysis only at certain constant intervals in the series. The occurrence of boutons of different morphological types on the neuronal surface was expressed by their percentage covering of the neuronal membrane. The neuronal surface was divided into a number of compartments and the synaptic covering was calculated separately for each compartment. An interval of 6 micrometer between the sections was used for these calculations, and the obtained values for synaptic covering were found not to differ significantly from those obtained in controls at 3 micrometer intervals. The number and location of individual large boutons (C- and M-types) were studied at 3 micrometer section intervals, and the escape of boutons connected to this procedure was estimated from control observations at 1 micrometer intervals. It is concluded that detailed information on neuronal synaptology can be obtained with this method, which will be used in three subsequent studies on functionally identified and intracellularly stained cat alpha-motoneurons.
1. The enzyme horseradish peroxidase (HRP) was injected into single Ib muscle afferent fibres in anaesthetized cats. Subsequently, histochemistry allowed the morphology of the axons and their collaterals in the lumbosacral spinal cord to be determined. 2. Eleven Ib axons were stained, seven from lateral gastrocneminus-soleus, one from medial gastrocnemius and three from muscles innervated by the posterior tibial nerve. Ten of the axons were traced into the dorsal roots and all but one (from the posterior tibial nerve) bifurcated upon entering the cord. Between 5.1 and 9.9 mm of each axon was stained and the fibres gave off eighty-four collaterals at intervals of 100-2300 micron, at an average spacing of about 900 micron. The spacing between collaterals on the (finer) descending axon branches was generally less than the intervals between collaterals on ascending branches. 3. All Ib collaterals had a characteristic morphology. The collaterals coursed cranially on a direct path through the dorsal horn to lamina IV or V before branching. They arborized widely in the intermediate region, mainly in lamina VI and in the dorsal part of lamina VII. Occasionally, less extensive arborizations were seen more dorsally in lamina IV and V. The rostro-caudal extent of individual collateral arborizations was limited to 200-400 micron and there was no overlap between adjacent collaterals. Each terminal arborization gave rise to 56-384 boutons, mainly of them 'en passant' type. 4. The results are discussed in relation to previous anatomical and electrophysiological studies.
1. The enzyme horseradish peroxidase (HRP) was injected into single Ia muscle afferent fibres in anaesthetized cats. Subsequent histochemistry allowed the morphology of the axons and their collaterals in the lumbosacral spinal cord to be determined. 2. Fifteen Ia axons were stained, four from medial gastrocnemius, four from lateral gastrocnemius-soleus and seven from muscles innervated by the posterior tibial nerve. All thirteen axons that could be traced into the dorsal roots bifurcated upon entering the cord. Between 4 and 11 mm of axons were stained and they gave off eighty seven collaterals over distances between 3 and 9 mm. Collaterals were given off at intervals of 100-2600 micron at an average spacing of about 1000 micron. 3. All Ia collaterals had a characteristic morphology. After leaving the parent axon they ran ventrally to lamina VI and then ventrolaterally to the motor nuclei. The collaterals coursed cranially from their point of origin to the motor nuclei so that their lamina VI terminations were about 100-300 micro caudal to their terminations in motor nuclei. Terminal arborizations were limited to three sites; lamina VI (the intermediate region), lamina VII (the Ia inhibitory interneurone region) and lamina IX (the motor nuclei). The three sets of terminals had characteristic arborizations and bouton arrangements. 4. The results are discussed in relation to previous anatomical studies. In particular the present results suggest that a single Ia collateral makes contact with many more motoneurones than has previously been suggested in fact with fifty to sixty rather than with about ten.
An autoradiographic analysis of the time and sites of origin, and the migration and settling patterns of neurons was made in the spinal cord of the mouse. The neurons originated on days 10--14 of gestation with temporal gradients along the ventrodorsal and rostrocaudal axes. The motor neurons originated on days 10 and 11 of gestation; the neurons in the intermediate gray region originated on days 11--14 of gestation; the neurons of the head of the dorsal horn originated on days 12--14 of gestation. The neurons that originated on days 10 and 11 originated and migrated primarily from the basal plate, and they settled in the adjacent regions of the intermediate zone; those neurons formed on days 12--14 originated and migrated primarily from the alar plate, and it was concluded that these neuroblasts similarly settled in the adjacent regions of the intermediate zone. Extraventricular proliferation, which presumably signaled the initial stages of gliogenesis, was first observed on day 12 of gestation. This study supports the classical idea of the mosaic pattern of neurogenesis in the embryonic spinal cord.
1. Retrograde transport of horseradish peroxidase was used to map the initial projection patterns of lumbosacral motoneurones to the embryonic chick hind limb. 2. The stage 28 segmental projection pattern to each of the four primary muscle masses was characteristic and indistinguishable from the stage 36 projection pattern to the sum of the muscles derived from that mass. In addition, the adductor motoneurone pool was found to be similar in position (both rostro-caudal and mediolateral) at stages 29, 30, 32, 33 1/2 and 36. 3. Therefore axons from lumbosacral motoneurones project for the most part only to appropriate regions from early times shortly after they grow into the limb bud. Furthermore, the attainment of the segmental projection pattern occurs prior to the normal time of, and therefore without the aid of, cell death. This conclusion was supported by electrophysiological recordings made from muscle nerves. 4. A regionalization of the projection patterns within a single muscle mass could be shown both anatomically and physiologically prior to the cleavage of the mass into individual muscles and the projections were in a general way appropriate for the muscles derived from those regions. 5. Therefore the process of muscle cleavage does not in itself create the specific projection patterns observed, and motoneurone axons appear to grow to and to ramify and make synapses only within regions which correspond to their adult muscles. 6. Finally, the termination site of each motoneurone axon in the early limb was found to be tightly correlated in a somatotopic fashion with the position occupied by its soma in the cord. This suggests that some feature of the motoneurone related to its position may be of importance in achieving the specific projection patterns observed.
1. The motor nuclei supplying many of the hind limb muscles were localized in late chick embryos (stage 36-37; 10-11 days) by utilizing the technique of retrograde transport of horseradish peroxidase. 2. Each nucleus was found to be localized in a characteristic position in both the rostro-caudal and transverse plane of the spinal cord with only slight individual variation. 3. Each motor nucleus consisted of an elongate, coherent cluster of labelled cells, with few cells occurring outside the cluster. Thus, there did not appear to be extensive overlap of nuclei nor extensive intermingling of motoneurones projecting to different muscles. 4. The position of a motor nucleus in the transverse plane was not correlated with whether its muscle was used as an extensor or flexor; nor were adjacent nuclei necessarily co-activated during normal unrestrained walking movements as deduced from e.m.g. recordings. The position of a motor nucleus also was not correlated in a topographical manner with the adult position in the limb of the muscle to which it projected. 5. Further, while no correlation was found between the rostrocaudal position of a motor nucleus and the embryonic muscle mass from which its muscle was derived, such a relationship existed for the medio-lateral position; all muscles arising from the dorsal muscle mass, regardless of their function or adult position, were innervated by laterally situated motoneurones, all muscles arising from the ventral muscle mass by medially situated motoneurones. 6. It is concluded that motoneurone position is most closely correlated with ontogenetic events presumaeriphery. It can also be inferred that the central connexions onto motoneurones, responsible for their proper activation, cannot be achieved by a simple mechanism based largely on the position of the motoneurone soma.
An autoradiographic determination of the time of origin of the lateral motor columns (LMC) of the chick embryo has been made. The first motor neurons of the brachial LMC are born at stage 15; the earliest birthdates of lumbar LMC neurons are at stage 17. At least 95% of the motor neurons of both brachial and lumbar columns are produced by stage 23 (4 days). The remaining 5% of the motor neurons are produced during the next two days. A clear rostrocaudal gradient of motor neuron production is seen beoth between the brachial and lumbar LMCs and within the LMCs themselves. The LMCs are assembled in a mediolateral sequence: the early-born motor neurons settle medially, the later-born motor neurons settle more laterally. Observations were made of other large early-born neurons which remain permanently in the dorsal gray of the spinal cord.
The spatial organization of the cutaneous input to hindlimb withdrawal reflexes was studied in spinalized, decerebrated, unanesthetized rats. Reflex activity in plantar flexors of the digits, pronators of the foot, dorsiflexors of the digits, and/or the ankle and flexors of the knee was recorded with electromyographic techniques for up to 12 h after spinalization. Graded mechanical (pinch) and thermal stimulation (CO2 laser) of the skin were used. Reflexes were absent ("spinal shock") during approximately 10-20 min after spinalization. The reflex thresholds for pinch and CO2 laser stimulation then decreased considerably during the following 5-8 h. After this time, even mild pressure (less than 0.1 N/mm2) on the skin was sufficient to evoke a reflex in most muscles. During the period from about 0.5-3 h after spinalization, the nociceptive receptive field of each muscle usually corresponded to the area of the skin withdrawn by the muscle. Maximal responses were evoked from the area of the receptive field maximally withdrawn. During this period, responses to innocuous pinch were evoked mainly from the most sensitive area of the receptive fields. Concomitant with the decrease in reflex thresholds, the nociceptive receptive fields expanded for all muscles, often to include areas of the skin not withdrawn by the muscles. For most muscles, reflexes on tactile stimuli were eventually elicited from the entire receptive fields. The receptive fields for thermonociceptive and mechanonociceptive inputs were similar in most muscles. The interossei muscles were exceptional in that they responded very weakly to thermal stimulation. It is concluded that there are neuronal networks in the spinal cord that translate cutaneous nociceptive and tactile input into a withdrawal. However, the control exerted by descending pathways is necessary to maintain a functionally adequate excitability in these reflex pathways and an appropriate size for their receptive fields.
1. Intracellular staining of Renshaw cells and alpha motoneurons was used to determine the spatial distribution of recurrent inhibitory synapses on spinal motoneurons in the cat. In each experiment, a Renshaw cell and one or more possible target motoneurons were labeled with horseradish peroxidase after physiological identification. 2. Paris of labeled neurons were reconstructed and measured at the light microscopic level. As defined by light microscopy, presumed synaptic contacts between nine Renshaw cells and 10 postsynaptic motoneurons were observed. On average, each Renshaw cell made three synaptic contacts (range 1-9) on each motoneuron. 3. Electron microscopic confirmation of several presumed contacts provided evidence that the appositions identified by light microscopic criteria are genuine contacts between Renshaw cell boutons and the labeled motoneuron. 4. All of the identified synapses observed in these experiments were located on motoneuron dendrites, between 65 and 706 microns from the soma. Use of a simplified cable model indicated that the synapses are electrotonically close to the soma, the average location being approximately 0.25 length constants from the soma (range 0.04-0.82 lambda). 5. These observations provide direct evidence to support the hypothesis that Renshaw cell synapses on motoneurons are located on the dendrites and not on the cell body (whereas reciprocal inhibitory synapses, from Ia inhibitory interneurons, are predominantly located on the soma). The functional significance of the observed distribution of Renshaw inhibitory synapses is discussed. One possibility is that the recurrent inhibitory pathway selectively inhibits particular dendritic inputs.
1. The organization of the nociceptive hindlimb withdrawal reflexes was investigated in 93 halothane/nitrous oxide anesthetized rats. Electromyographical techniques were used to record reflex activity in single motor units. 2. Most of the hindlimb muscles were activated by noxious mechanical stimulation of the skin of the ipsilateral hindlimb. These were the plantar flexors of the digits, the pronators of the paw, the dorsiflexors and the plantar flexors of the ankle, the flexors of the knee, the flexors of the hip and the adductors. By grading the stimulus intensity it was shown that all these muscles received input from cutaneous nociceptors. 3. Noxious stimulation of the skin failed to activate the obturator, knee extensors and m. tibialis posterior and, in most rats tested, m. semimembranosus and m. adductor magnus. The plantar flexors of the ankle, while exhibiting a clear nocireceptive field in all rats tested, had a high threshold and responded much more weakly than the dorsiflexors of the ankle. Thus, responses in muscles which oppose gravity in the standing position were either very weak or absent. 4. The present study shows that each of the activated hindlimb muscles has a highly organized nocireceptive field on the skin, which is related to the withdrawal movement caused by the muscle itself. Each of the muscles normally causes the withdrawal of its receptive field when the foot is on the ground. The skin area most effectively withdrawn, in this situation, corresponds to the most sensitive area of the nocireceptive field. However, with the exception of the plantar flexors of the digits and/or the ankle, each of the hindlimb muscles also withdraws the major parts of their receptive fields when the foot is off the ground. The locations of the nocireceptive fields were independent of the position of the hindlimb. These characteristics of the nociceptive withdrawal reflexes are the basis for their "local sign" (Sherrington 1906). 5. The threshold and the time course of reflex activation were different in different muscles. However, muscles with a similar action; the plantar flexors of the digits, the pronators of the paw, the dorsiflexors of the digits, the flexors of the knee and the adductors, respectively, had similar thresholds and time courses. Furthermore, the threshold and latency of activation of each muscle increased towards the border of its nocireceptive field, reflecting a decreasing sensitivity. These findings explain the progressive recruitment of muscles during increasing strength of noxious stimulation, termed "irradiation" (Sherrington 1906).(ABSTRACT TRUNCATED AT 400 WORDS)
This review will summarize a number of quantitative characteristics especially of Renshaw cells and the motor axon-Renshaw cell connections. A side-effect will be the demonstration of gaps in our knowledge and data base which will hopefully incite further experimental work
The morphology of dendritic trees (dendroarchitecture) of motor neurons innervating specific hindlimb muscles (motoneuron pools, MNP) was studied in the chick spinal cord. Motoneurons were labelled by intramuscular injections of horseradish peroxidase conjugated with cholera toxin subunit B. MNPs of posterior iliotibial and femorotibial muscles were located at the dorsolateral part of lateral motor column of lumbosacral segments (LS) 1-4 and 1-3, respectively. Although the dendritic profiles of femorotibialis motoneurons were fewer than those of posterior iliotibialis, these two MNPs had a similar distribution pattern of dendrites. Dendritic profiles were about equally distributed in the gray and white matter. Dendrites from the MNP of posterior iliotibialis radiated in all directions. A large number of dendrites penetrated into the white matter, and some even reached to the subpial regions of the lateral funiculus. One array of dendrites that projected dorsomedialwards extended to the base of the posterior horn. MNPs of both the iliofibularis (LS 4-7) and caudilioflexorius (LS 6-8) had dendritic trees with similar distribution patterns. There were two main arrays of dendritic extensions; one along the dorsal, and another along the ventral border of the lateral motor column. Dendrites from the iliofibularis and caudilioflexorius motoneurons were located more frequently in the white matter than in the gray matter. A large number of dendrites extended in all directions from the MNP of the adductor muscle, which was located in the medial region of lateral motor column of LS 1-2. The distribution of dendrites from a few other MNPs was also examined.(ABSTRACT TRUNCATED AT 250 WORDS)
alpha-Motoneurons innervating the triceps surae and short plantar muscles were stained intracellularly with horseradish peroxidase (HRP) in 0-44-day-old kittens and adult cats. The terminal arborizations of the recurrent axon collaterals in the spinal cord were studied in the light microscope (LM). The short plantar motoneurons lacked axon collaterals in all age groups. With a few exceptions in the youngest kittens (0-1 days of age), the projection field of the axon collaterals of triceps surae motoneurons did not change during development. The exceptional motoneurons had axon collaterals projecting ventromedial to the adult termination areas in Rexed's laminae VII and IX. Within all parts of the projection field, there was a substantial postnatal reduction in the number of axon collateral swellings, interpreted as synaptic terminals, and a total elimination of short and thin axonal processes without swellings. The findings are discussed in relation to earlier demonstrated loss of synaptic terminals on the motoneurons and elimination of polyneuronal innervation of muscle fibers postnatally.
Recent advances in techniques, especially the intraneuronal injection of the enzyme horseradish peroxidase, have led to a new ear in our understanding of spinal cord structure and function. Input to the cord is precisely organized: the primary afferent fibres from different types of receptors distribute their anatomically specific collaterals to particular parts of the dorsal horn, afferent fibres from the skin lay down a precise somatotopic map, input to the dorsal horn from descending systems is also distributed in a localized way. The neurones of the dorsal horn are varied in both structure and function, even so some quite specific cell types can be identified and the dendritic trees may respect laminar boundaries as determined cytoarchitectonically (although the majority of neurones have dendrites that cut across these boundaries). The output pathways from the dorsal horn are many and various, but again they arise from cells in definite parts of the dorsal horn. The dorsal horn must be considered as a well-organized, and complex, part of the central nervous system. It cannot be considered as a structural or functional unit but is made up of many interacting parts that process input from the primary afferent fibres, from other levels of the spinal cord and from many descending pathways from the brain.
Motoneuron pools supplying principal muscles of the shoulder girdle and wing were localized in the chicks 2-7 days post hatching with the use of retrograde axonic transport of horse radish peroxidase (HRP). HRP was injected into selected muscles and the animals sacrificed after 24 h survival. Labelled motoneuron pools representing individual muscles were found to be clustered in longitudinal columns along the brachial spinal cord segments 13-16. Muscles derived from the dorsal muscle mass were innervated by motoneurons located in the ventro-lateral portion of the horn, while those originating from the ventral muscle mass received innervation from neurons occupying the dorsomedial portion within the ventral horn of the spinal cord. The rostrocaudal extent of the motoneuron pools could be correlated with the proximodistal position of wing muscles. The observed orderly topographical relationships between clusters of motoneurons of the brachial spinal cord and the muscles they innervate will be used as baseline data for experiments where the limb innervation is perturbed.
1. The enzyme horseradish peroxidase was injected into identified lumbosacral alpha-motoneurones and Group Ia afferent fibres in cats anaesthetized with chloralose and paralysed with gallamine triethiodide. Subsequent histological examination allowed the determination of (a) the extent of the motoneuronal dendritic trees, (b) the number and location of Ia synapses upon the motoneurones. 2. alpha-motoneurones had seven to eighteen primary dendrites and each produced daughter branches up to the fourth to the sixth order. At dendritic bifurcations Rall's 3/2 Power Law was obeyed. There was little or no dendritic tapering up to about 800 micrometers from the soma. Beyond this distance, however, there was considerable tapering. 3. Horseradish peroxidase injections revealed that motoneuronal dendrites are much longer than previously thought. Individual dendrites could be traced for up to 1600 micrometers from the soma and dendritic trees were usually 2-3 mm from tip to tip. Nearly all the motoneurones had dendrites that entered the white matter of the cord. Dendrites could also reach as far dorsally as laminae V and VI. 4. Ia synapses upon motoneuronal somata were examined in cords counterstained with cresyl violet or methylene green. About 10% of Ia boutons in lamina IX were on somata and each Ia collateral terminated on 3.66 motoneuronal somata or the most proximal (30 micrometer) dendrites, with on average about two contacts per motoneurone. 5. Ten Ia afferent fibre-motoneurone pairs were injected with horseradish peroxidase. The following conclusions were drawn: (i) only one collateral of any given Ia axon makes contact with a motoneurone even though other collaterals from the same axon might pass through the dendritic tree, (ii) usually all contacts made between a Ia fibre and a motoneurone are at about the same geometrical distance from the soma, even when on different dendrites, (iii) between two and five contacts are made upon the dendritic tree (average 3.4) at distances of between 20 and 820 micrometers from the soma. 6. The results are discussed in relation to previous anatomical and electrophysiological work.
Recent experiments have shown that certain motoneurons do selectively innervate particular groups of limb muscles after the normal relationships between nerves and muscles have been perturbed in the embryo. This chapter describes the anatomical organization of the motor pools supplying limb muscles and shows how this organization can be viewed as a continuous representation of the position of the muscle precursors within the developing limb bud. It discusses the development of limb innervation at two levels of precision. The chapter considers the problem of how specific patterns of connections are formed between the clusters of motoneurons that make up the lateral motor columns and the limb muscles. This includes consideration of both normal development and experimental perturbations of the normal relationships between motor pools and muscle. A major issue is the possible relationship between pattern formation and cell death. The chapter also attempts to synthesize the present knowledge about the development of limb innervation and propose a theory to account for the experimental observations. It deals with the development of limb innervation in several species, but concentrates on the chick embryo because more detailed information is available about the chick than about other species.
1. The distribution of terminals from vestibulospinal (VS) axons on the dendritic trees of neck motoneurons was determined by combining the anterograde transport of Phaseolus Vulgaris Leucoagglutinin (PHA-L) with intracellular staining techniques and three-dimensional reconstruction methods. 2. Contacts between VS axon terminals and dendrites were arranged in a nonuniform pattern that depended on the orientation (i.e., rostro-caudal vs. dorsolateral) of the dendrites. 3. This innervation pattern satisfies a critical structural condition necessary for selective nonlinear interactions between pairs of simultaneously active inputs to motoneurons.
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The withdrawal reflex pathways to hindlimb muscles have an elaborate spatial organization in the rat. In short, the distribution of sensitivity within the cutaneous receptive field of a single muscle has a spatial pattern that is a mirror image of the spatial pattern of the withdrawal of the skin surface ensuing on contraction in the respective muscle. In the present study, a search for neurones encoding the specific spatial input-output relationship of withdrawal reflexes to single muscles was made in the lumbosacral spinal cord in halothane/nitrous oxide-anaesthetized rats. The cutaneous receptive fields of 147 dorsal horn neurones in the L4-5 segments receiving a nociceptive input and a convergent input from A and C fibres from the hindpaw were studied. The spatial pattern of the response amplitude within the receptive fields of 118 neurones was quantitatively compared with those of withdrawal reflexes to single muscles. Response patterns exhibiting a high similarity to those of withdrawal reflexes to single muscles were found in 27 neurones located in the deep dorsal horn. Twenty-six of these belonged to class 2 (responding to tactile and nociceptive input) and one belonged to class 3 (responding only to nociceptive input). None of the neurones tested (n = 20) with reflex-like response patterns could be antidromically driven from the upper cervical cord, suggesting that they were spinal interneurones. With some overlap, putative interneurones of the withdrawal reflexes to the plantar flexors of the digits, the plantar flexors of the ankle, the pronators, the dorsiflexors of the ankle, and a flexor of the knee, were found in succession in a mediolateral direction.(ABSTRACT TRUNCATED AT 250 WORDS)
1. The postnatal development of nociceptive withdrawal reflexes was studied. In awake intact rats, forelimb, hindlimb and tail reflexes were recorded on videotape. In decerebrate spinal rats, electromyography (EMG) was used to record nociceptive withdrawal reflexes in musculi extensor digitorum longus (EDL), peronei, gastrocnemius-soleus (G-S) and biceps posterior-semitendinosus (BP-ST). Thermal (short-lasting CO2 laser pulses) and mechanical stimulation were used. 2. In adults, nociceptive withdrawal reflexes were typically well directed and reflex pathways to single hindlimb muscles had functionally adapted receptive fields. By contrast, at postnatal day (P) 1-7, the nociceptive withdrawal reflexes were often inappropriate, sometimes producing movements towards the stimulation, and EMG recordings revealed unadapted variable receptive fields. With increasing age, the nociceptive withdrawal reflexes progressively became well directed, thus producing localized withdrawal. Both withdrawal movements and spatial organization of the receptive fields were adult-like at P20-25. 3. Up to P25, reflex thresholds were more or less constant in both intact awake rats and spinal decerebrate rats, except in G-S in which no nociceptive withdrawal reflexes were evoked from P20 on. After P25, mechanical, but not thermal, thresholds increased dramatically. 4. EMG recordings revealed that during the first three postnatal weeks, the latency of the CO2 laser-evoked nociceptive withdrawal reflexes decreased significantly in peronei and BP-ST, but not in EDL, and thereafter increased significantly in peronei, BP-ST and EDL. The magnitude of the nociceptive withdrawal reflexes in these muscles increased markedly between P7 and P20 and showed little change thereafter. 5. Possible mechanisms underlying the postnatal tuning of the nociceptive withdrawal reflexes are discussed.
Eight functionally identified group Ia muscle afferents from triceps surae or plantaris muscles were labeled intraaxonally with horseradish peroxidase (HRP) in seven adult cats. Subsequently, HRP was injected into two to six homonymous or heteronymous α-motoneurons per animal (total = 22), each identified by motor unit type and located near the site of afferent injection. The complete trajectories of labeled afferents were reconstructed, and putative synaptic contacts on HRP-labeled motoneurons were identified at high magnification. Dendritic paths from each contact were also mapped and measured. A total of 24 contact systems (the combination of a group Ia afferent and a postsynaptic motoneuron) were reconstructed, of which 17 were homonymous, and seven were heteronymous.
The formation of laminar-specific projections is a key event in the development of appropriate neuronal connections in many regions of the central nervous system. In order to provide a framework for defining functions of molecules related to spinal laminar targeting of dorsal root ganglion neurons in mice, we have characterized the initial trajectories of sensory axons in relation to the maturation of their target laminae in the spinal cord. We show that morphological and biochemical differentiation of distinct clusters of neurons in the dorsal region of the spinal cord precedes initial collateral branching from sensory axons. Between embryonic day (E) 12.5 and E13.5, sensory axons develop swelling ("nodes") along their entire intraspinal extent and elaborate interstitial collateral branches from these nodes. Collaterals from the different classes of sensory axons then penetrate the gray matter of the spinal cord sequentially. Each class of sensory axons projects directly to its target lamina, never branching into inappropriate laminae en route. Some cutaneous afferents traverse the entire width of the spinal cord to reach superficial laminae on the contralateral side, strictly avoiding both the ventral spinal cord and inappropriate laminae of the deep dorsal horn. The pathways taken by developing sensory afferents are compatible with the idea that cells in inappropriate laminae exert inhibitory influences on sensory axons which regulate their laminar specificity.
Operant conditioning of the H reflex, the electrical analogue of the spinal stretch reflex, in freely moving rats is a relatively simple model for studying long-term supraspinal control over spinal cord function. Motivated by food reward, rats can gradually increase or decrease the soleus H reflex. This study is the first effort to determine which spinal cord pathways convey the descending influence from supraspinal structures that changes the H reflex. In anesthetized Sprague-Dawley rats, the entire dorsal column (DC), which includes the main corticospinal tract, or the right lateral column (LC) was transected by electrocautery. Animals recovered quickly and the minimal transient effects of transection on the right soleus H reflex disappeared within 16 days. Beginning at least 18 days after transection, 12 rats were exposed to the HRdown-conditioning mode, in which reward was given when the H reflex of the right soleus muscle was below a criterion value. In seven LC rats exposed to the HRdown mode, the H reflex fell to 71 +/- 8% (mean +/- SE) of its initial value. In six of the seven, conditioning was successful (i.e., decrease to < or = 80%). These results were comparable with those previously obtained from normal rats. In contrast, in five DC rats exposed to the HRdown mode, the H reflex at the end of exposure was 106 +/- 12% of its initial value. In none of these rats was HRdown-conditioning successful. DC rats differed significantly from normal and LC rats in both final H reflex values and number successful. In five DC and three LC rats that continued under control conditions over 30-78 days, the H reflex at the end of the period was 98 +/- 4% and 100 +/- 8%, respectively, of its initial value, indicating that DC or LC transection itself did not lead to gradual increase or decrease in the H reflex. The results indicate that the DC, containing the main corticospinal tract, is essential for HRdown-conditioning, whereas the ipsilateral LC, containing the main rubrospinal, vestibulospinal, and reticulospinal tracts, is not essential. Combined with the known muscular specificity of conditioning, these results suggest that the main corticospinal tract is essential for HRdown-conditioning. The DC ascending tract might also be necessary. The respective roles of the DC descending and ascending tracts, and transection effects on HRup-conditioning and on the maintenance of both HRup- and HRdown-conditioning after they have occurred, remain to be defined.
The neuromotor conservatism hypothesis predicts that neuromotor patterns in homologous tetrapod muscles are conserved evolutionarily despite the musculoskeletal modifications of vertebrate limbs. A complete description of the anatomical organization of the neurons innervating homologous limb muscles is a prerequisite to any test of the neuromotor conservatism hypothesis. This study uses the retrograde neuronal tracer WGA-HRP to selectively label the motor neuron pools of seven homologous forelimb muscles in mice (Mus musculus) and iguanas (Iguana iguana): Mm. pectoralis, spinodeltoideus, biceps brachii, lateral and long heads of triceps brachii, and the supraspinatus and infraspinatus (in mice) or their reptilian homolog, the supracoracoideus (in iguanas). In vertebrates, motoneurons are arranged in longitudinal columns of cells in the ventral horn of the spinal cord. Mouse motor pools average 1,952 microns in length, except the pectoralis pool which averaged 2,949 microns in length. Iguana pools average 3,196 microns in length. The number of neurons per pool ranged from 70-199 in mice and from 58-114 neurons in iguanas. In both iguanas and mice the motor pools for the spinodeltoids, biceps, and the supracoracoideus (or its mammalian homologs) lie anterior to the pectoralis and triceps motor pools. In the transverse plane, the pectoralis pool lies medial to those of the triceps. The pools of the biceps and spinodeltoids are located dorsal and lateral to those of the pectoralis and supracoracoideus (or its homologs in mammals). The resulting motor pool maps support the hypothesis that the anatomical organization of motoneurons in ancestral reptiles has been retained in these two tetrapod descendents.
Double immunofluorescence was utilized to determine whether Renshaw cells contain calbindin D28k immunoreactivity. Renshaw cells were identified by their characteristic expression patterns of gephyrin immunoreactivity in sections of rat and cat lumbar spinal cord. In the rat, all neurons classified as Renshaw cells (n = 487) also contained calbindin D28k-immunoreactivity, and all calbindin D28k-immunoreactive cells located in the ventral-most region of lamina VII expressed the characteristic gephyrin labeling and morphology of Renshaw cells. In the cat, fewer than half of the Renshaw cells (47%; n = 128) were double-labeled. In both species, occasional calbindin D28k-immunoreactive Renshaw cells were identified within motor nuclei in lamina IX. The distinctive immunolabeling of Renshaw cells allowed us to estimate that there are about 250 Renshaw cells in each ventral horn of the fourth lumbar segment of rat spinal cord, and about 750 cells per ventral horn in the L6 segment of the cat. We conclude that the functional properties of Renshaw cells, including their ability to fire action potentials at high rates, likely require specific homeostatic mechanisms including strong intracellular calcium buffering, the precise mechanisms of which may vary between species.
Muscle spindles are skeletal muscle sensory organs that provide axial and limb position information (proprioception) to the central nervous system. Spindles consist of encapsulated muscle fibers (intrafusal fibers) that are innervated by specialized motor and sensory axons. Although the molecular mechanisms involved in spindle ontogeny are poorly understood, the innervation of a subset of developing myotubes (type I) by peripheral sensory afferents (group Ia) is a critical event for inducing intrafusal fiber differentiation and subsequent spindle formation. The Egr family of zinc-finger transcription factors, whose members include Egr1 (NGFI-A), Egr2 (Krox-20), Egr3 and Egr4 (NGFI-C), are thought to regulate critical genetic programs involved in cellular growth and differentiation (refs 4-8, and W.G.T. et al., manuscript submitted). Mice deficient in Egr3 were generated by gene targeting and had gait ataxia, increased frequency of perinatal mortality, scoliosis, resting tremors and ptosis. Although extrafusal skeletal muscle fibers appeared normal, Egr3-deficient animals lacked muscle spindles, a finding that is consistent with their profound gait ataxia. Egr3 was highly expressed in developing muscle spindles, but not in Ia afferent neurons or their terminals during developmental periods that coincided with the induction of spindle morphogenesis by sensory afferent axons. These results indicate that type I myotubes are dependent upon Egr3-mediated transcription for proper spindle development.
Development of neuronal circuits generating locomotor activity was studied using an isolated lumbar spinal cord preparation from fetal and neonatal rats. Bath application of N-methyl-D-aspartate (NMDA) or 5-HT evoked patterned motor activity resembling that seen during normal fictive locomotion on embryonic day (E) 20.5. Glycine-mediated inhibition was essential to the formation of this coordinated motor activity. In preparations from fetuses at the earlier stages (E14.5-E16.5), we observed spontaneous motoneuronal activity and chemically induced rhythmic bursts, which were synchronized on the two sides in the corresponding ventral roots. The spontaneous activity was not blocked by kynurenate, the glutamate receptor blocker, although it was completely abolished by strychnine, the glycine receptor antagonist. A brief application of glycine evoked excitatory responses resembling the spontaneous bursts in both time course and amplitude. It is concluded that glycine functions transiently as excitatory transmitters at these stages. These results suggest that functional change in glycine-induced responses during development plays an important role in differentiation of the neuronal circuits generating locomotion.
Spontaneous neuronal activity has been detected in many parts of the developing vertebrate nervous system. Recent studies suggest that this activity depends on properties that are probably shared by all developing networks. Of particular importance is the high excitability of recurrently connected, developing networks and the presence of activity-induced transient depression of network excitability. In the spinal cord, it has been proposed that the interaction of these properties gives rise to spontaneous, periodic activity.
The nervous system is evolutionarily conservative compared to the peripheral appendages that it controls. However, species-specific behaviors may have arisen from very small changes in neuronal circuits. In particular, changes in neuromodulatory systems may allow multifunctional circuits to produce different sets of behaviors in closely related species. Recently, it was demonstrated that even species differences in complex social behavior may be attributed to a change in the promoter region of a single gene regulating a neuromodulatory action.
The fidelity of impulse propagation through the complex axonal tree en route to the various target cells of that fiber is an important question in neurobiology. Anatomists can trace pathways, but if impulses fail to propagate down to the terminals to release transmitter onto the target cell, there is a significant 'disconnect' between anatomy and physiology. These issues have been studied at length in the spinal cord of the cat where it has proven possible to examine the connections made by afferent fibers on motoneurons under different stimulus conditions. EPSP amplitude varies systematically during high frequency stimulation of the afferents according to the identity of the target motoneuron. This variation is a function of the state of the motoneuron's relation to its peripheral target. It changes after motoneuron axotomy and recovers with reinnervation of the periphery. Neurotrophins delivered to the axotomized motor axons fail to induce recovery. Chronic stimulation of the motor nerve alters muscle properties with coordinated changes in properties of the synapses on motoneurons innervating the stimulated muscle. We cannot yet definitively establish the mechanisms determining synaptic behavior during high frequency stimulation. However, the retrograde regulation of these properties suggests that it is an important variable and thus is worthy of intensive further study.
The connections formed between sensory and motor neurons (MNs) play a critical role in the control of motor behavior. During development, the axons of proprioceptive sensory neurons project into the spinal cord and form both direct and indirect connections with MNs. Two ETS transcription factors, ER81 and PEA3, are expressed by developing proprioceptive neurons and MNs, raising the possibility that these genes are involved in the formation of sensory-motor connections. Er81 mutant mice exhibit a severe motor discoordination, yet the specification of MNs and induction of muscle spindles occurs normally. The motor defect in Er81 mutants results from a failure of group Ia proprioceptive afferents to form a discrete termination zone in the ventral spinal cord. As a consequence there is a dramatic reduction in the formation of direct connections between proprioceptive afferents and MNs. ER81 therefore controls a late step in the establishment of functional sensory-motor circuitry in the developing spinal cord.
Spinal interneurons help to coordinate motor behavior. During spinal cord development, distinct classes of interneurons are generated from progenitor cells located at different positions within the ventral neural tube. V0 and V1 interneurons derive from adjacent progenitor domains that are distinguished by expression of the homeodomain proteins Dbx1 and Dbx2. The spatially restricted expression of Dbx1 has a critical role in establishing the distinction in V0 and V1 neuronal fate. In Dbx1 mutant mice, neural progenitors fail to generate V0 neurons and instead give rise to interneurons that express many characteristics of V1 neurons-their transcription factor profile, neurotransmitter phenotype, migratory pattern, and aspects of their axonal trajectory. Thus, a single progenitor homeodomain transcription factor coordinates many of the differentiated properties of one class of interneurons generated in the ventral spinal cord.
Neural circuits are assembled with remarkable precision during embryonic
development, and the selectivity inherent in their formation helps to define
the behavioural repertoire of the mature organism. In the vertebrate central
nervous system, this developmental program begins with the differentiation
of distinct classes of neurons from progenitor cells located at defined positions
within the neural tube. The mechanisms that specify the identity of neural
cells have been examined in many regions of the nervous system and reveal
a high degree of conservation in the specification of cell fate by key signalling
molecules.
The pathway mediating the monosynaptic stretch reflex has served as an important model system for studies of plasticity in the spinal cord. Its usefulness is extended by evidence that neurotrophins, particularly neurotrophin-3 (NT-3), which has been shown to promote spinal axon elongation, can modulate the efficacy of the muscle spindle-motoneurone connection both after peripheral nerve injury and during development. The findings summarized here emphasize the potential for neurotrophins to modify function of both damaged and undamaged neurones. It is important to recognize that these effects may be functionally detrimental as well as beneficial.