Elongation factor 2 and fragile X mental retardation protein control the dynamic translation of Arc/Arg3.1 essential for mGluR-LTD

The initiation and coordination of activity in limb muscles are the main functions of neural circuits that control locomotion. Commissural neurons connect locomotor circuits on the two sides of the spinal cord, and represent the known neural substrate for left-right coordination. Here we demonstrate that a group of ipsilateral interneurons, V2a interneurons, plays an essential role in the control of left-right alternation. In the absence of V2a interneurons, the spinal cord fails to exhibit consistent left-right alternation. Locomotor burst activity shows increased variability, but flexor-extensor coordination is unaffected. Anatomical tracing studies reveal a direct excitatory input of V2a interneurons onto commissural interneurons, including a set of molecularly defined V0 neurons that drive left-right alternation. Our findings imply that the neural substrate for left-right coordination consists of at least two components; commissural neurons and a class of ipsilateral interneurons that activate commissural pathways.     

Functional identification of interneurons responsible for left-right coordination of hindlimbs in mammals

Local neuronal networks that are responsible for walking are poorly characterized in mammals. Using an innovative approach to identify interneuron inputs onto motorneuron populations in a neonatal rodent spinal cord preparation, we have investigated the network responsible for left-right coordination of the hindlimbs. We demonstrate how commissural interneurons (CINs), whose axons traverse the midline to innervate contralateral neurons, are organized such that distinct flexor and extensor centers in the rostral lumbar spinal cord define activity in both flexor and extensor caudal motor pools. In addition, the nature of some connections are reconfigured on switching from rest to locomotion via a mechanism that might be associated with synaptic plasticity in the spinal cord. These results from identified pattern-generating interneurons demonstrate how interneuron populations create an effective network to underlie behavior in mammals.     

CTCF regulates ataxin-7 expression through promotion of a convergently transcribed, antisense noncoding RNA

Neural networks in the spinal cord control two basic features of locomotor movements: rhythm generation and pattern generation. Rhythm generation is generally considered to be dependent on glutamatergic excitatory neurons. Pattern generation involves neural circuits controlling left-right alternation, which has been described in great detail, and flexor-extensor alternation, which remains poorly understood. Here, we use a mouse model in which glutamatergic neurotransmission has been ablated in the locomotor region of the spinal cord. The isolated in?vitro spinal cord from these mice produces locomotor-like activity-when stimulated with neuroactive substances-with prominent flexor-extensor alternation. Under these conditions, unlike in control mice, networks of inhibitory interneurons generate the rhythmic activity. In the absence of glutamatergic synaptic transmission, the flexor-extensor alternation appears to be generated by Ia inhibitory interneurons, which mediate reciprocal inhibition from muscle proprioceptors to antagonist motor neurons. Our study defines a minimal inhibitory network that is needed to produce flexor-extensor alternation during locomotion.     

Genetic identification of spinal interneurons that coordinate left-right locomotor activity necessary for walking movements

The sequential stepping of left and right limbs is a fundamental motor behavior that underlies walking movements. This relatively simple locomotor behavior is generated by the rhythmic activity of motor neurons under the control of spinal neural networks known as central pattern generators (CPGs) that comprise multiple interneuron cell types. Little, however, is known about the identity and contribution of defined interneuronal populations to mammalian locomotor behaviors. We show a discrete subset of commissural spinal interneurons, whose fate is controlled by the activity of the homeobox gene Dbx1, has a critical role in controlling the left-right alternation of motor neurons innervating hindlimb muscles. Dbx1 mutant mice lacking these ventral interneurons exhibit an increased incidence of cobursting between left and right flexor/extensor motor neurons during drug-induced locomotion. Together, these findings identify Dbx1-dependent interneurons as key components of the spinal locomotor circuits that control stepping movements in mammals.     

Effects of corticospinal tract stimulation on renal sympathetic nerve activity in rats with intact and chronically lesioned spinal cords

Sympathetic preganglionic neurons and interneurons are closely apposed (presumably synapsed upon) by corticospinal tract (CST) axons. Sprouting of the thoracic CST rostral to lumbar spinal cord injuries (SCI) substantially increases the incidence of these appositions. To test our hypothesis that these additional synapses would increase CST control of sympathetic activity after SCI, we measured the effects of electrical stimulation of the CST on renal sympathetic nerve activity (RSNA) and arterial pressure (AP) in alpha-chloralose-anesthetized rats with either chronically intact or chronically lesioned spinal cords. Stimuli were delivered to the CST at intensities between 25-150 muA and frequencies between 25 and 75 Hz. Stimulation of the CST at the midcervical level decreased RSNA and AP. These decreases were not mediated by direct projections of the CST to the thoracic spinal cord because we could still elicit them by midcervical stimulation after acute lesions of the CST at caudal cervical levels. In contrast, caudal thoracic CST stimulation increased RSNA and AP. Neither the responses to cervical nor thoracic stimulation were affected by chronic lumbar SCI. These data show that the CST mediates decreases in RSNA via a cervical spinal system but excites spinal sympathetic neurons at caudal thoracic levels. Because chronic lumber spinal cord injury affected responses evoked from neither the cervical nor thoracic CST, we conclude that lesion-induced or regeneration-induced formation of new synapses between the CST and sympathetic neurons may not affect cardiovascular regulation.     

Mechanisms of Left-Right Coordination in Mammalian Locomotor Pattern Generation Circuits: A Mathematical Modeling View

The locomotor gait in limbed animals is defined by the left-right leg coordination and locomotor speed. Coordination between left and right neural activities in the spinal cord controlling left and right legs is provided by commissural interneurons (CINs). Several CIN types have been genetically identified, including the excitatory V3 and excitatory and inhibitory V0 types. Recent studies demonstrated that genetic elimination of all V0 CINs caused switching from a normal left-right alternating activity to a left-right synchronized “hopping” pattern. Furthermore, ablation of only the inhibitory V0 CINs (V0_D subtype) resulted in a lack of left-right alternation at low locomotor frequencies and retaining this alternation at high frequencies, whereas selective ablation of the excitatory V0 neurons (V0_V subtype) maintained the left–right alternation at low frequencies and switched to a hopping pattern at high frequencies. To analyze these findings, we developed a simplified mathematical model of neural circuits consisting of four pacemaker neurons representing left and right, flexor and extensor rhythm-generating centers interacting via commissural pathways representing V3, V0_D, and V0_V CINs. The locomotor frequency was controlled by a parameter defining the excitation of neurons and commissural pathways mimicking the effects of N-methyl-D-aspartate on locomotor frequency in isolated rodent spinal cord preparations. The model demonstrated a typical left-right alternating pattern under control conditions, switching to a hopping activity at any frequency after removing both V0 connections, a synchronized pattern at low frequencies with alternation at high frequencies after removing only V0_D connections, and an alternating pattern at low frequencies with hopping at high frequencies after removing only V0_V connections. We used bifurcation theory and fast-slow decomposition methods to analyze network behavior in the above regimes and transitions between them. The model reproduced, and suggested explanation for, a series of experimental phenomena and generated predictions available for experimental testing.     

Partly shared spinal cord networks for locomotion and scratching

Animals produce a variety of behaviors using a limited number of muscles and motor neurons. Rhythmic behaviors are often generated in basic form by networks of neurons within the central nervous system, or central pattern generators (CPGs). It is known from several invertebrates that different rhythmic behaviors involving the same muscles and motor neurons can be generated by a single CPG, multiple separate CPGs, or partly overlapping CPGs. Much less is known about how vertebrates generate multiple, rhythmic behaviors involving the same muscles. The spinal cord of limbed vertebrates contains CPGs for locomotion and multiple forms of scratching. We investigated the extent of sharing of CPGs for hind limb locomotion and for scratching. We used the spinal cord of adult red-eared turtles. Animals were immobilized to remove movement-related sensory feedback and were spinally transected to remove input from the brain. We took two approaches. First, we monitored individual spinal cord interneurons (i.e., neurons that are in between sensory neurons and motor neurons) during generation of each kind of rhythmic output of motor neurons (i.e., each motor pattern). Many spinal cord interneurons were rhythmically activated during the motor patterns for forward swimming and all three forms of scratching. Some of these scratch/swim interneurons had physiological and morphological properties consistent with their playing a role in the generation of motor patterns for all of these rhythmic behaviors. Other spinal cord interneurons, however, were rhythmically activated during scratching motor patterns but inhibited during swimming motor patterns. Thus, locomotion and scratching may be generated by partly shared spinal cord CPGs. Second, we delivered swim-evoking and scratch-evoking stimuli simultaneously and monitored the resulting motor patterns. Simultaneous stimulation could cause interactions of scratch inputs with subthreshold swim inputs to produce normal swimming, acceleration of the swimming rhythm, scratch-swim hybrid cycles, or complete cessation of the rhythm. The type of effect obtained depended on the level of swim-evoking stimulation. These effects suggest that swim-evoking and scratch-evoking inputs can interact strongly in the spinal cord to modify the rhythm and pattern of motor output. Collectively, the single-neuron recordings and the results of simultaneous stimulation suggest that important elements of the generation of rhythms and patterns are shared between locomotion and scratching in limbed vertebrates.     

DCC mediated axon guidance of spinal interneurons is essential for normal locomotor central pattern generator function

Coordinated limb rhythmic movements take place through organized signaling in local spinal cord neuronal networks. The establishment of these circuitries during development is dependent on the correct guidance of axons to their targets. It has previously been shown that the well-known axon guidance molecule netrin-1 is required for configuring the circuitry that provides left-right alternating coordination in fictive locomotion. The attraction of commissural axons to the midline in response to netrin-1 has been shown to involve the netrin-1 receptor DCC (deleted in Colorectal Cancer). However, the role of DCC for the establishment of CPG coordination has not yet been resolved. We show that mice carrying a null mutation of DCC displayed an uncoordinated left-right activity during fictive locomotion accompanied by a loss of interneuronal subpopulations originating from commissural progenitors. Thus, DCC plays a crucial role in the formation of spinal neuronal circuitry coordinating left-right activities. Together with the previously published results from netrin-1 deficient mice, the data presented in this study suggest a role for the most ventral originating V3 interneurons in synchronous activities over the midline. Further, it provides evidence that axon crossing in the spinal cord is more intricately controlled than in previously suggested models of DCC-netrin-1 interaction.     

Neuroanatomical and functional analysis of neural tube formation in notochordless Xenopus embryos; laterality of the ventral spinal cord is lost.

Notochordless Xenopus embryos were produced by u.v. irradiation of the uncleaved fertilized egg. The spinal cords were examined using intermediate filament staining for glial cells, retrograde HRP staining for neuronal morphology and an anti-glycinergic antibody to reveal commissural cells and axons. The floorplate cells of the normal cord appear to be absent and their position along the ventral midline of the cord is occupied by motor neurones, Kolmer-Agduhr cells, radial glial cells and a ventrally placed marginal zone containing the longitudinal axons. Motor neurone number is reduced to 15% of control values, and the sensory extramedullary cell number is increased twentyfold. Commissural axons are still able to cross the ventral cord but do so at abnormal angles and some commissural axons continue to grow circumferentially up the contralateral side of the cord rather than turning to grow longitudinally. Extracellular electrophysiological recordings from motor axons reveal that the normal alternation of locomotor activity on the left and right side of the embryo is lost in notochordless animals. These results suggest that the notochord and/or the normal floor plate structure are important for the development of the laterality of spinal cord connections and may influence motor neurone proliferation or differentiation.     

Adjacent pioneer commissural interneuron growth cones switch from contact avoidance to axon fasciculation after midline crossing

Commissural interneurons (CI) of the vertebrate spinal cord are guided ventrally toward the floor plate, but subsequently cross the midline and select a longitudinal fascicle at specific dorsal-ventral (D-V) positions. We examined at high resolution the detailed behaviors of individual pathfinding CI growth cones on the ipsilateral and contralateral sides of the spinal cord of living Xenopus embryos. We find that pre-crossing CI growth cones exhibit distinct pathfinding behaviors compared to post-crossing axons and that the behavioral switch occurs immediately upon crossing to the contralateral side. Groups of pioneer commissural axons typically extend simultaneously toward the ventral midline following discrete paths with separation between adjacent commissurals apparently maintained through contact inhibition. In contrast, shortly after crossing the midline, commissural axons turn longitudinally and begin to fasciculate with other crossed CIs. However, growth cones of crossed commissurals often select their final D-V longitudinal track through a series of rapid step-like dorsal adjustments that may be due to differential fasciculation with longitudinal axons. Together, our results suggest that guidance of commissural axons is controlled in part through interactions among CIs that switch rapidly from avoidance to fasciculation after midline crossing.     

Calcium imaging of network function in the developing spinal cord

We have used calcium imaging to visualize the spatiotemporal organization of activity generated by in vitro spinal cord preparations of the developing chick embryo and the neonatal mouse. During each episode of spontaneous activity, we found that chick spinal neurons were activated rhythmically and synchronously throughout the transverse extent of the spinal cord. At the onset of a spontaneous episode, optical activity originated in the ventrolateral part of the cord. Back-labeling of spinal interneurons with calcium dyes suggested that this ventrolateral initiation was mediated by activation of a class of interneurons, located dorsomedial to the motor nucleus, that receive direct monosynaptic input from motoneurons. Studies of locomotor-like activity in the anterior lumbar segments of the neonatal mouse cord revealed the existence of a rostrocaudal wave in the oscillatory component of each cycle of rhythmic motoneuron activity. This finding raises the possibility that the activation of mammalian motoneurons during locomotion may share some of the same rostrocaudally organized mechanisms that evolved to control swimming in fishes.     

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.Download video file.^{(145M, mp4)}     

Ultrastructural study of neurotensin immunoreactivity in the superficial laminae of the dorsal horn of the rat

Neurotensin immunoreactivity was identified in cell bodies, dendrites, spines, axons, terminals and varicosities in superficial laminae of rat spinal cord with the electron microscope. Unlabeled terminals synapsed with neurotensin-immunoreactive cell bodies, dendrites and spines. Presynaptic terminals contained round or pleomorphic vesicles and generally made symmetrical contacts with medium-sized neurotensin-containing dendrites in outer lamina II, and asymmetrical or symmetrical contacts with large and small dendrites and spines in inner lamina II. Neurotensin immunoreactive axons were unmyelinated, and their terminals were presynaptic to unlabeled dendrites and spines in laminae I and II. Terminals contained small, round, clear vesciles (31 nm) and occasional large granular vesicles (78 nm). Contacts in outer lamina II were evenly distributed among dendrites of various sizes and spines, whereas the majority of labeled terminals in inner lamina II made contacts onto small dendrites and spines. These findings indicate that neurotensin effects in rat spinal cord are mediated by axodendritic synapses, and that neurotensin cells at the inner and outer borders of lamina II contact dendrites of efferent neurons or other interneurons in the dorsal horn.     

Perturbation of neuronal differentiation and axon guidance in the spinal cord of mouse embryos lacking a floor plate: analysis of Danforth's short-tail mutation.

The floor plate of the vertebrate nervous system has been implicated in the guidance of commissural axons at the ventral midline. Experiments in chick have also suggested that at earlier stages of development the floor plate induces the differentiation of motor neurons and other neurons of the ventral spinal cord. Here we have examined the development of the spinal cord in a mouse mutant, Danforth's short-tail, in which the floor plate is absent from caudal regions of the neuraxis. In affected regions of the spinal cord, commissural axons exhibited aberrant projection patterns as they reached and crossed the ventral midline. In addition, motor neurons were absent or markedly reduced in number in regions of the spinal cord lacking a floor plate. Our results suggest that the floor plate is indeed an intermediate target in the projection of commissural axons and support the idea that several different mechanisms operate in concert in the guidance of axons to their cellular targets in the developing nervous system. In addition, these experiments suggest that the mechanisms that govern the differentiation of the floor plate and other ventral cell types in the neural tube are common to mammals and lower vertebrates.     

Development, neurochemical properties, and axonal projections of a population of last-order premotor interneurons in the white matter of the chick lumbosacral spinal cord

There is general agreement that last-order premotor interneurons-a set of neurons that integrate activities generated by the spinal motor apparatus, sensory information and volleys arising from higher motor centres, and transmit the integrated signals to motoneurons through monosynaptic contacts-play crucial roles in the initiation and maintenance of spinal motor activities. Here, we demonstrate the development, neurochemical properties, and axonal projections of a unique group of last-order premotor interneurons within the ventrolateral aspect of the lateral funiculus of the chick lumbosacral spinal cord. Neurons expressing immunoreactivity for neuron-specific enolase were first detected in the ventrolateral white matter at embryonic day 9 (E9). The numbers of immunoreactive neurons were significantly increased at E10-E12, while most of them were gradually concentrated in small segmentally arranged nuclei (referred to as major nuclei of Hofmann) protruding from the white matter in a necklace like fashion dorsal to the ventral roots. The major nuclei of Hofmann became more prominent at E12-E16, but substantial numbers of cells were still located within the ventrolateral white matter (referred to as minor nucleus of Hofmann). The distribution of immunoreactive neurons achieved by E16 was maintained during later developmental stages and was also characteristic of adult animals. After injection of Phaseolus vulgaris-leucoagglutinin unilaterally into the minor nucleus of Hofmann, labeled fibres were detected in the ventrolateral white matter ipsilateral to the injection site. Ascending and descending fibres were revealed throughout the entire rostro-caudal length of the lumbosacral spinal cord. Axon terminals were predominantly found within the lateral motor column and the ventral regions of lamina VII ipsilateral to the injection site. Several axon varicosities made close appositions with somata and dendrites of motoneurons, which were identified as synaptic contacts in a consecutive electron microscopic study. With the postembedding immunogold method, 21 of 97 labeled terminals investigated were immunoreactive for glycine and 2 of them showed immunoreactivity for gamma-aminobutyric acid (GABA). The axon trajectories of neurons within the minor nucleus of Hofmann suggest that some of these cells might represent a population of last-order premotor interneurons. J. Exp. Zool. 286:157-172, 2000.     

谷氨酸转运抑制剂对器官型培养脊髓片的影响

观察谷氨酸转运体抑制剂苏一羟天冬氨酸(Threo-hydroxyaspartate,THA)对器官型培养的脊髓片的影响,探讨谷氨酸在运动神经元损伤中的作用。取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,在培养液中加入不同浓度THA(50μmol／L、100μmol／L、5001μmol／L),用神经元的特异性免疫组化染色剂SMI-32,非磷酸化神经丝标记物,对脊髓腹角α运动神经元进行鉴定,用单克隆抗钙网膜蛋白(calretinin)抗体对背角中间神经元进行记数,测定培养液中乳酸脱氢酶(LDH)的含量,并与对照组比较。结果显示对照组α运动神经元数目恒定,THA可以引起剂量依赖性的培养液中LDH含量增高和α运动神经元数目减少,而脊髓背角的中间神经元损伤相对较轻,其中THA100μmol／L组在体外培养4周后出现类似于肌萎缩侧索硬化(ALS)的病理改变：α运动神经元数目较对照组明显减少,而脊髓背角的中间神经元数目无显著变化。细胞外谷氨酸增高主要对运动神经元造成损伤,脊髓运动神经元较感觉神经元对谷氨酸的兴奋毒作用更加敏感。