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.     

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.     

Coordination of Fictive Motor Activity in the Larval Zebrafish Is Generated by Non-Segmental Mechanisms

The cellular and network basis for most vertebrate locomotor central pattern generators (CPGs) is incompletely characterized, but organizational models based on known CPG architectures have been proposed. Segmental models propose that each spinal segment contains a circuit that controls local coordination and sends longer projections to coordinate activity between segments. Unsegmented/continuous models propose that patterned motor output is driven by gradients of neurons and synapses that do not have segmental boundaries. We tested these ideas in the larval zebrafish, an animal that swims in discrete episodes, each of which is composed of coordinated motor bursts that progress rostrocaudally and alternate from side to side. We perturbed the spinal cord using spinal transections or strychnine application and measured the effect on fictive motor output. Spinal transections eliminated episode structure, and reduced both rostrocaudal and side-to-side coordination. Preparations with fewer intact segments were more severely affected, and preparations consisting of midbody and caudal segments were more severely affected than those consisting of rostral segments. In reduced preparations with the same number of intact spinal segments, side-to-side coordination was more severely disrupted than rostrocaudal coordination. Reducing glycine receptor signaling with strychnine reversibly disrupted both rostrocaudal and side-to-side coordination in spinalized larvae without disrupting episodic structure. Both spinal transection and strychnine decreased the stability of the motor rhythm, but this effect was not causal in reducing coordination. These results are inconsistent with a segmented model of the spinal cord and are better explained by a continuous model in which motor neuron coordination is controlled by segment-spanning microcircuits.     

Partial cloning and expression of mRNA coding choline acetyltransferase in the spinal cord of the goldfish, Carassius auratus

Choline acetyltransferase (ChAT, EC 2.3.1.6) synthesizes a neurotransmitter, acetylcholine in cholinergic neurons. ChAT is considered to be the most specific marker for cholinergic neurons. To obtain a better marker of the neurons, as the first step, we isolated a partial ChAT cDNA from the goldfish (Carassius auratus) brain by RT-PCR methods. The partial cDNA of the goldfish ChAT was composed of 718 nucleotides. The amino acid sequence of the goldfish ChAT is approximately 70% identical to those of mammalian and chicken ChAT. Northern blot analysis demonstrated that ChAT mRNA was expressed in the brain and the spinal cord of the goldfish, and much abundant in the spinal cord. In the spinal cord of the goldfish, ChAT-positive neurons were detected mainly in the ventral horn by in situ hybridization. In addition, fluorescence in situ hybridization combined with a retrograde labeling by using True Blue demonstrated ChAT mRNA positive neurons were exactly motoneurons. In the cord, putative presynaptic sympathetic neurons were also labeled.     

Cholinergic Receptor Alterations in the Brain Stem of Spinal Cord Injured Rats

Cholinergic receptors in upper motor neurons of brain stem control locomotion and coordination. Present study unravels cholinergic alterations in brain stem during spinal cord injury to understand signalling pathway changes which may be associated with spinal cord injury mediated motor deficits. We evaluated cholinergic function in brain stem by studying the expression of choline acetyl transferase and acetylcholine esterase. We quantified metabotropic muscarinic cholinergic receptors by receptor assays for total muscarinic, muscarinic M1 and M3 receptor subunits, gene expression studies using Real Time PCR and confocal imaging using FITC tagged secondary antibodies. The gene expression of ionotropic nicotinic cholinergic receptors and confocal imaging were also studied. The results from our study showed metabolic disturbance in cholinergic pathway as choline acetyl transferase is down regulated and acetylcholine esterase is up regulated in spinal cord injury group. The significant decrease in muscarinic receptors showed by decreased receptor number along with down regulated gene expression and confocal imaging accounts for dysfunction of metabotropic acetylcholine receptors in spinal cord injury group. Ionotropic acetylcholine receptor alterations were evident from the decreased gene expression of alpha 7 nicotinic acetylcholine receptors and confocal imaging. The motor coordination was analysed by Grid walk test which showed an increased foot slips in spinal cord injured rats. The significant reduction in brain stem cholinergic function might have intensified the motor dysfunction and locomotor disabilities.     

Functional regeneration and recovery of locomotor activity in spinally transected lamprey.

Spinally transected lamprey recovery locomotor function within 3-6 weeks, and recovery is due, in part, to functional regeneration of neural pathways in the central nervous system (CNS). Our data demonstrate for the first time in the lamprey that descending axons arising from brainstem command neurons can functionally regenerate and restore locomotor initiation below a healed spinal transection site. Immediately after behavioral recovery (3-6 weeks) the locomotor pattern was incomplete but returned to normal during the remainder of the recovery period (6-40 weeks). Initially, the extent of regeneration of descending axons was limited but increased to at least 30-50 mm at recovery times of 24-40 weeks. Regenerated giant Muller axons do not contribute significantly to recovery of locomotor function; rather, regenerated axons of smaller reticulospinal neurons appear to restore locomotor initiation. The restoration of locomotor coordination across a spinal lesion is dependent on two mechanisms: regeneration of spinal coordinating neurons and mechanosensory inputs. Comparisons are made to spinal cord regeneration in other lower vertebrates and to the relative lack of CNS regeneration and behavioral recovery in higher vertebrates.     

Differential susceptibilities of spinal cord neurons to retinoic acid-induced survival and differentiation

In a previous study, we demonstrated trophic effects of vitamin A and its active metabolite, retinoic acid (RA), on perinatal rat spinal cord neurons and astrocytes in vitro. We now report that RA increases the survival of cholinergic neurons without affecting that of GABAergic neurons. These results were supported by measured levels of acetylcholinesterase (AChE), choline acetyltransferase (ChAT), and glutamic acid decarboxylase (GAD) activities, key enzymes of acetylcholine and gamma-aminobutyric acid metabolism, respectively, which showed RA-induced increases in AChE and ChAT levels but no elevations of GAD activity. In contrast to these phenotype-specific effects, most neurons showed RA-induced increases in neuritic outgrowth, density, and silver impregnation. Taken together, these results demonstrate neurotransmitter-specific and generalized effects of RA on developing CNS neurons.     

Multiple Neurotrophic Factors from Skeletal Muscle: Demonstration of Effects of Basic Fibroblast Growth Factor and Comparisons with the 22-Kilodalton Choline Acetyltransferase Development Factor

Extracts of skeletal muscle contain chromatographically distinct molecules that enhance the cholinergic development of cultured embryonic rat spinal cord neurons. We have recently purified a 20-22 kilodalton anionic polypeptide choline acetyltransferase (ChAT) development factor (CDF) from rat skeletal muscle extracts that stimulates the development of ChAT activity in rat spinal cord cultures. The maximum increase in the level of ChAT activity achieved by this factor, however, is less than that achieved by the addition of the crude extract. We now show that muscle extract also contains mitogenic activity that is immunologically related to basic fibroblast growth factor (bFGF) and also that recombinant bFGF stimulates ChAT development in rat spinal cord cultures. bFGF, however, differs from CDF in its physiochemical, chromatographic, and immunological properties and by its action on nonneuronal cells. Individually, CDF and bFGF each enhance the level of ChAT activity in rat spinal cord cultures two- to threefold after 2 days of treatment. However, their combined actions result in a five- to sixfold enhancement of ChAT activity, suggesting that they are affecting cholinergic development through different means. The demonstration that extracts of rat skeletal muscle contain two biochemically and immunologically distinct polypeptides, with additive effects on cultured embryonic spinal cord neurons, provides additional evidence for the involvement of multiple target-derived neurotrophic factors in the regulation of cholinergic development.     

Rescue of motoneurons from cell death by a purified skeletal muscle polypeptide: effects of the ChAT development factor, CDF

Rat skeletal muscle contains a 22 kd polypeptide that increases the level of choline acetyltransferase (ChAT) activity in cultures of embryonic rat spinal cord neurons and has been purified to homogeneity. The application of this factor, ChAT development factor or CDF, to developing chick embryos during the period of naturally occurring motoneuron cell death significantly increased the survival of motoneurons but did not affect the survival of dorsal root ganglion neurons or sympathetic preganglionic neurons (column of Terni). These results provide the first demonstration that an isolated, skeletal muscle-derived molecule can selectively enhance the survival of motoneurons in vivo and suggest that CDF may function in vivo to regulate the survival and development of motoneurons.     

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.     

Generation of locomotion rhythms without inhibition in vertebrates: the search for pacemaker neurons

Locomotion rhythms are thought to be generated by neurons in the central-pattern-generator (CPG) circuit in the spinal cord. Synaptic connections in the CPG and pacemaker properties in certain CPG neurons, both may contribute to generation of the rhythms. In the half-center model proposed by Graham Brown a century ago, reciprocal inhibition plays a critical role. However, in all vertebrate preparations examined, rhythmic motor bursts can be induced when inhibition is blocked in the spinal cord. Without inhibition, neuronal pacemaker properties may become more important in generation of the rhythms. Pacemaker properties have been found in motoneurons and some premotor interneurons in different vertebrates and they can be dependent on N-Methyl-d-aspartate (NMDA) receptors (NMDAR) or rely on other ionic currents like persistent inward currents. In the swimming circuit of the hatchling Xenopus tadpole, there is substantial evidence that emergent network properties can give rise to swimming rhythms. During fictive swimming, excitatory interneurons (dINs) in the caudal hindbrain fire earliest on each swimming cycle and their spikes drive the firing of other CPG neurons. Regenerative dIN firing itself relies on reciprocal inhibition and background excitation. We now find that the activation of NMDARs can change dINs from firing singly at rest to current injection to firing repetitively at swimming frequencies. When action potentials are blocked, some intrinsic membrane potential oscillations at about 10 Hz are revealed, which may underlie repetitive dIN firing during NMDAR activation. In confirmation of this, dIN repetitive firing persists in NMDA when synaptic transmission is blocked by Cd(2+). When inhibition is blocked, only dINs and motoneurons are functional in the spinal circuit. We propose that the conditional intrinsic NMDAR-dependent pacemaker firing of dINs can drive the production of swimming-like rhythms without the participation of inhibitory neurotransmission.     

Cholinergic receptor alterations in the cerebral cortex of spinal cord injured rat

Many areas of the cerebral cortex process sensory information or coordinate motor output necessary for control of movement. Disturbances in cortical cholinergic system can affect locomotor coordination. Spinal cord injury causes severe motor impairment and disturbances in cholinergic signalling can aggravate the situation. Considering the impact of cortical cholinergic firing in locomotion, we focussed the study in understanding the cholinergic alterations in cerebral cortex during spinal cord injury. The gene expression of key enzymes in cholinergic pathway - acetylcholine esterase and choline acetyl transferase showed significant upregulation in the cerebral cortex of spinal cord injured group compared to control with the fold increase in expression of acetylcholine esterase prominently higher than cholineacetyl transferase. The decreased muscarinic receptor density and reduced immunostaining of muscarinic receptor subtypes along with down regulated gene expression of muscarinic M1 and M3 receptor subtypes accounts for dysfunction of metabotropic acetylcholine receptors in spinal cord injury group. Ionotropic acetylcholine receptor alterations were evident from the decreased gene expression of alpha 7 nicotinic receptors and reduced immunostaining of alpha 7 nicotinic receptors in confocal imaging. Our data pin points the disturbances in cortical cholinergic function due to spinal cord injury; which can augment the locomotor deficits. This can be taken into account while devising a proper therapeutic approach to manage spinal cord injury.     

Effects of dorsal root section and occlusion of dorsal spinal artery on the neurotransmitter candidates in rat spinal cord

In order to obtain further evidence of putative neurotransmitters in primary sensory neurons and interneurons in the dorsal spinal cord, we have studied the effects of unilateral section of dorsal roots and unilateral occlusion of the dorsal spinal artery on cholinergic enzyme activity and on selected amino acid levels in the spinal cord. One week after sectioning dorsal roots from caudal cervical (C₇) to cranial thoracic (T₂) levels, the specific activity of choline acetyltransferase (ChAT) was significantly decreased and acetylcholinesterase (AChE) showed a tendency to decrease in the dorsal quadrant on the operated side of the spinal cord. Dorsal root sectioning had little effect on the levels of free glutamic acid or other amino acids in the dorsal spinal cord. These results suggest that primary sensory neurons may include some cholinergic axons, and that levels of putative amino acid transmitters are not regulated by materials supplied by axonal transport from the dorsal root ganglia. By contrast, one week following unilateral occlusion of the dorsal spinal artery, the activities of ChAT and AChE were unchanged in the operated quadrant of the spinal cord, while decreases of Asp, Glu, and GABA, and an increase in Tau were detected. These findings are consistent with the proposals that such amino acids, but not ACh, may function as neurotransmitter candidates in interneurons of the dorsal spinal cord.Abbreviation used ACh acetylcholine - AChE acetylcholinesterase - Asp aspartic acid - ChAT choline acetyltransferase - GABA -aminobutyric acid - Glu glutamic acid - Gly glycine - SP substance P - Tau taurine     

Development and neuromodulation of spinal locomotor networks in the metamorphosing frog

Metamorphosis in the anuran frog, Xenopus laevis, involves profound structural and functional transformations in most of the organism's physiological systems as it encounters a complete alteration in body plan, habitat, mode of respiration and diet. The metamorphic process also involves a transition in locomotory strategy from axial-based undulatory swimming using alternating contractions of left and right trunk muscles, to bilaterally-synchronous kicking of the newly developed hindlimbs in the young adult. At critical stages during this behavioural switch, functional larval and adult locomotor systems co-exist in the same animal, implying a progressive and dynamic reconfiguration of underlying spinal circuitry and neuronal properties as limbs are added and the tail regresses. To elucidate the neurobiological basis of this developmental process, we use electrophysiological, pharmacological and neuroanatomical approaches to study isolated in vitro brain stem/spinal cord preparations at different metamorphic stages. Our data show that the emergence of secondary limb motor circuitry, as it supersedes the primary larval network, spans a developmental period when limb circuitry is present but not functional, functional but co-opted into the axial network, functionally separable from the axial network, and ultimately alone after axial circuitry disappears with tail resorption. Furthermore, recent experiments on spontaneously active in vitro preparations from intermediate metamorphic stage animals have revealed that the biogenic amines serotonin (5-HT) and noradrenaline (NA) exert short-term adaptive control over circuit activity and inter-network coordination: whereas bath-applied 5-HT couples axial and appendicular rhythms into a single unified pattern, NA has an opposite decoupling effect. Moreover, the progressive and region-specific appearance of spinal cord neurons that contain another neuromodulator, nitric oxide (NO), suggests it plays a role in the maturation of limb locomotor circuitry. In summary, during Xenopus metamorphosis the network responsible for limb movements is progressively segregated from an axial precursor, and supra- and intra-spinal modulatory inputs are likely to play crucial roles in both its functional flexibility and maturation.     

Synaptic patterning of left-right alternation in a computational model of the rodent hindlimb central pattern generator

Establishing, maintaining, and modifying the phase relationships between extensor and flexor muscle groups is essential for central pattern generators in the spinal cord to coordinate the hindlimbs well enough to produce the basic walking rhythm. This paper investigates a simplified computational model for the spinal hindlimb central pattern generator (CPG) that is abstracted from experimental data from the rodent spinal cord. This model produces locomotor-like activity with appropriate phase relationships in which right and left muscle groups alternate while extensor and flexor muscle groups alternate. Convergence to this locomotor pattern is slow, however, and the range of parameter values for which the model produces appropriate output is relatively narrow. We examine these aspects of the model’s coordination of left-right activity through investigation of successively more complicated subnetworks, focusing on the role of the synaptic architecture in shaping motoneuron phasing. We find unexpected sensitivity in the phase response properties of individual neurons in response to stimulation and a need for high levels of both inhibition and excitation to achieve the walking rhythm. In the absence of cross-cord excitation, equal levels of ipsilateral and contralateral inhibition result in a strong preference for hopping over walking. Inhibition alone can produce the walking rhythm, but contralateral inhibition must be much stronger than ipsilateral inhibition. Cross-cord excitatory connections significantly enhance convergence to the walking rhythm, which is achieved most rapidly with strong crossed excitation and greater contralateral than ipsilateral inhibition. We discuss the implications of these results for CPG architectures based on unit burst generators.     

Intersegmental coordinating system of the lamprey central pattern generator for locomotion

Summary In the lamprey,Ichthyomyzon unicuspis, the wave of activity required for normal swimming movements can be generated by a central pattern generator (CPG) residing in the spinal cord. A constant phase coupling between spinal segments can be organized by intersegmental coordinating neurons intrinsic to the cord. The rostral and caudal segmental oscillators of the CPG have different preferred frequencies when separated from each other. Therefore the system must maintain the segmental oscillators of the locomotor CPG at a single common frequency and with the proper relative timing. Using selective lesions and a split-bath, it is demonstrated that the coordinating system is comprised of at least 3 subsystems, short-axon systems in the lateral and medial tracts and a long axon system in the lateral tracts. Each alone can sustain relatively stable coordinated activity.Abbreviations CPG central pattern generator - NMDA N-methyl-D-aspartate - VR ventral root