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.     

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.     

The cord stretch receptors in the abdominal nerve cord of the crayfish Cherax destructor: physiology and relationships

The physiology and relationships of tonic cord stretch receptor neurons in the crayfish Cherax destructor were examined with intracellular and extracellular recording. Cord stretch evoked slow depolarisations leading to action potentials in tonic cord stretch receptor neurons. Intermittent post-synaptic potentials were also seen in cord stretch receptor neurons but were not the primary cause of the action potentials. Cord stretch still evoked action potentials in cord stretch receptor neurons when all synaptic activity, monitored at another known chemical synapse, was blocked using high [Mg(2+)] and low [Ca(2+)] in the bath. One source of facilitating excitatory post-synaptic potentials in the cord stretch receptor neurons was from mechanosensory hairs on the dorsal abdominal surface. Tonic cord stretch receptor neuron activity was associated with an increase in the activity of the abdominal slow extensor inhibitor motor neuron and at least one abdominal flexor excitor motor neuron in its segment, and reduced activity in the abdominal slow flexor inhibitor motor neuron. Activation of individual cord stretch receptor neurons produced a local resistance reflex. Cord stretch, activating many receptors, produced several other outcomes. One was the "extensor state" described in earlier literature. The tonic cord stretch receptor neurons of Cherax destructor appear to be stretch-sensitive interneurons that receive inputs from other elements of the abdominal control system and mediate polysynaptic reflex activity in postural motor neurons.     

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。     

Lack of evidence for cell death among avian spinal cord interneurons during normal development and following removal of targets and afferents.

Chick embryos and posthatched chicks were examined at several ages for the presence of pyknotic interneurons in the lumbar spinal cord. Because no pyknotic interneurons were found, direct cell counts of healthy interneurons were carried out and a comparison made between early- and late-stage embryos and hatchlings. There was no decrease in the number of interneurons in the ventral intermediate gray matter of the spinal cord between embryonic day (E) 8 and 2 weeks posthatching (PH) or in the dorsal horn between E10 and 2 weeks PH. To study whether interneuron survival is regulated by targets or afferents, a situation known to exist in other developing neural populations, early embryos were subjected to (1) removal of one limb, resulting in the loss of lateral motor column motoneurons and dorsal root ganglion sensory afferents; (2) transection of the thoracic spinal cord, thereby removing both descending afferents and rostral targets of spinal interneurons, or (3) a combination of the two operations. No reductions in interneuron numbers were found as a result of these operations. Furthermore, morphometric analysis also revealed no change in neuronal size following these experimental manipulations. By contrast, there was a slight decrease in the total area of spinal gray matter that was most prominent in the dorsal region following limb bud removal. Our results indicate (1) that spinal interneurons fail to exhibit the massive naturally occurring death of postmitotic neurons that has been observed for several other populations of spinal neurons, and (2) spinal interneurons appear to be relatively resistant to induced cell death following the removal of substantial numbers of afferent inputs and targets.     

Locomotion induced by epidural stimulation in a decerebrated cat after spinal cord injury

The effect of partial and complete spinal cord transection (Th7–Th8) on locomotor activity evoked in decerebrated cats by electrical epidural stimulation (segment L5, 80–100 μA, 0.5 ms at 5 Hz) has been investigated. Transection of dorsal columns did not substantially influence the locomotion. Disruption of the ventral spinal quadrant resulted in deterioration and instability of the locomotor rhythm. Injury to lateral or medial descending motor systems led to redistribution of the tone in antagonist muscles. Locomotion could be evoked by epidural stimulation within 20 h after complete transection of the spinal cord. The restoration of polysynaptic components in EMG responses correlated with recovery of the stepping function. The data obtained confirm that initiation of locomotion under epidural stimulation is caused by direct action on intraspinal systems responsible for locomotor regulation. With intact or partially injured spinal cord, this effect is under the influence of supraspinal motor systems correcting and stabilizing the evoked locomotor pattern.     

Lack of evidence for cell death among avian spinal cord interneurons during normal development and following removal of targets and afferents

Chick embryos and posthatched chicks were examined at several ages for the presence of pyknotic interneurons in the lumbar spinal cord. Because no pyknotic interneurons were found, direct cell counts of healthy interneurons were carried out and a comparison made between early-and late-stage embryos and hatchlings. There was no decrease in the number of interneurons in the ventral intermediate gray matter of the spinal cord between embryonic day (E) 8 and 2 weeks posthatching (PH) or in the dorsal horn between E10 and 2 weeks PH. To study whether interneuron survival is regulated by targets or afferents, a situation known to exist in other developing neural populations, early embryos were subjected to (1) removal of one limb, resulting in the loss of lateral motor column motoneurons and dorsal root ganglion sensory afferents; (2) transection of the thoracic spinal cord, thereby removing both descending afferents and rostral targets of spinal interneurons, or (3) a combination of the two operations. No reductions in interneuron numbers were found as a result of these operations. Furthermore, morphometric analysis also revaled no change in neuronal size following these experimental manipulations. By contrast, there was a slight decrease in the total area of spinal gray matter that was most prominent in the dorsal region following limb bud removal. Our results indicate (1) that spinal interneurons fail to exhibit the massive naturally occurring death of postmitotic neurons that has been observed for several other populations of spinal neurons, and (2) spinal interneurons appear to be relatively resistant to induced cell death following the removal of substantial numbers of afferent inputs and targets.     

Probing the locomotor conundrum: descending the 'V' interneuron ladder

The assembly of neuronal circuits involved in locomotor control in the mammalian spinal cord is influenced by genetic programs specifying four ventral (V) interneuron populations (V0-V3). In this issue of Neuron, Crone et al. and Zhang et al. make use of genetic tools to map connectivity patterns and to abolish the function of V2a and V3 interneurons. The absence of V2a interneurons reveals defects in left-right alternation during locomotion, whereas ablation of either V2a or V3 interneurons leads to disturbances in the precision and reliability of the motor output.     

Development of spinal motor networks in the chick embryo.

We have examined the cellular and synaptic mechanisms underlying the genesis of alternating motor activity in the developing spinal cord of the chick embryo. Experiments were performed on the isolated lumbosacral cord maintained in vitro. Intracellular and whole cell patch clamp recordings obtained from sartorius (primarily a hip flexor) and femorotibialis (a knee extensor) motoneurons showed that both classes of cell are depolarized simultaneously during each cycle of motor activity. Sartorius motoneurons generally fire two bursts/cycle, whereas femorotibialis motoneurons discharge throughout their depolarization, with peak activity between the sartorius bursts. Voltage clamp recordings revealed that inhibitory and excitatory synaptic currents are responsible for the depolarization of sartorius motoneurons, whereas femorotibialis motoneurons are activated principally by excitatory currents. Early in development, the dominant synaptic currents in rhythmically active sartorius motoneurons appear to be inhibitory so that firing is restricted to a single, brief burst at the beginning of each cycle. In E7-E13 embryos, lumbosacral motor activity could be evoked following stimulation in the brainstem, even when the brachial and cervical cord was bathed in a reduced calcium solution to block chemical synaptic transmission. These findings suggest that functional descending connections from the brainstem to the lumbar cord are present by E7, although activation of ascending axons or electrical synapses cannot be eliminated. Ablation, optical, and immunocytochemical experiments were performed to characterize the interneuronal network responsible for the synaptic activation of motoneurons. Ablation experiments were used to show that the essential interneuronal elements required for the rhythmic alternation are in the ventral part of the cord. This observation was supported by real-time Fura-2 imaging of the neuronal calcium transients accompanying motor activity, which revealed that a high proportion of rhythmically active cells are located in the ventrolateral part of the cord and that activity could begin in this region. The fluorescence transients in the majority of neurons, including motoneurons, occurred in phase with ventral root or muscle nerve activity, implying synchronized neuronal action in the rhythm generating network. Immunocytochemical experiments were performed in E14-E16 embryos to localize putative inhibitory interneurons that might be involved in the genesis or patterning of motor activity. The results revealed a pattern similar to that seen in other vertebrates with the dorsal horn containing neurons with gamma-aminobutyric acid (GABA)-like immunoreactivity and the ventral and intermediate regions containing neurons with glycine-like immunoreactivity.     

Distributions of active spinal cord neurons during swimming and scratching motor patterns

The spinal cord can generate motor patterns underlying several kinds of limb movements. Many spinal interneurons are multifunctional, contributing to multiple limb movements, but others are specialized. It is unclear whether anatomical distributions of activated neurons differ for different limb movements. We examined distributions of activated neurons for locomotion and scratching using an activity-dependent dye. Adult turtles were stimulated to generate repeatedly forward swimming, rostral scratching, pocket scratching, or caudal scratching motor patterns, while sulforhodamine 101 was applied to the spinal cord. Sulforhodamine-labeled neurons were widely distributed rostrocaudally, dorsoventrally, and mediolaterally after each motor pattern, concentrated bilaterally in the deep dorsal horn, the lateral intermediate zone, and the dorsal to middle ventral horn. Labeled neurons were common in all hindlimb enlargement segments and the pre-enlargement segment following swimming and scratching, but a significantly higher percentage were in the rostral segments following swimming than rostral scratching. These findings suggest that largely the same spinal regions are activated during swimming and scratching, but there are some differences that may indicate locations of behaviorally specialized neurons. Finally, the substantial inter-animal variability following a single kind of motor pattern may indicate that essentially the same motor output is generated by anatomically variable networks.     

AMINO ACID AND SUBSTANCE P CONTENTS IN SPINAL CORD OF CATS WITH EXPERIMENTAL HIND-LIMB RIGIDITY PRODUCED BY OCCLUSION OF SPINAL CORD BLOOD SUPPLY

Abstract— Experimental hind-limb rigidity of spinal origin was produced in cats by temporary occlusion of thoracic aorta and internal mammary arteries. In the lumbar segments (L6- S1) of these rigid cats, the monosynaptic reflex recorded from ventral roots was enhanced whereas the polysynaptic reflexes as well as the dorsal root reflexes were almost abolished. On morphological examination of the lumbar spinal cord, the number of interneurons was greatly reduced, whereas the small sized cells, presumably glial cells, were increased by about two times. Ventral horn motoneurons were also reduced. The lumbar spinal cords of the rigid cats were analysed for amino acid and substance P contents. Four major amino acids, aspartate, glutamate, glycine and GABA, were definitely reduced in both grey and white matter except that the glutamate level in the dorsal white was within the normal range. Content and distribution pattern of substance P were not altered in the lumbar cord of the rigid cats. These results are consistent with the notions that GABA occurs in the dorsal horn interneurons subserving primary afferent depolarisation, and that substance P is concentrated in primary afferent fibre terminals. The implications of the decrease of aspartate, glutamate and glycine in the spinal cord of rigid cats are discussed.     

Flexibility in the patterning and control of axial locomotor networks in lamprey

In lower vertebrates, locomotor burst generators for axial muscles generally produce unitary bursts that alternate between the two sides of the body. In lamprey, a lower vertebrate, locomotor activity in the axial ventral roots of the isolated spinal cord can exhibit flexibility in the timings of bursts to dorsally-located myotomal muscle fibers versus ventrally-located myotomal muscle fibers. These episodes of decreased synchrony can occur spontaneously, especially in the rostral spinal cord where the propagating body waves of swimming originate. Application of serotonin, an endogenous spinal neurotransmitter known to presynaptically inhibit excitatory synapses in lamprey, can promote decreased synchrony of dorsal-ventral bursting. These observations suggest the possible existence of dorsal and ventral locomotor networks with modifiable coupling strength between them. Intracellular recordings of motoneurons during locomotor activity provide some support for this model. Pairs of motoneurons innervating myotomal muscle fibers of similar ipsilateral dorsoventral location tend to have higher correlations of fast synaptic activity during fictive locomotion than do pairs of motoneurons innervating myotomes of different ipsilateral dorsoventral locations, suggesting their control by different populations of premotor interneurons. Further, these different motoneuron pools receive different patterns of excitatory and inhibitory inputs from individual reticulospinal neurons, conveyed in part by different sets of premotor interneurons. Perhaps, then, the locomotor network of the lamprey is not simply a unitary burst generator on each side of the spinal cord that activates all ipsilateral body muscles simultaneously. Instead, the burst generator on each side may comprise at least two coupled burst generators, one controlling motoneurons innervating dorsal body muscles and one controlling motoneurons innervating ventral body muscles. The coupling strength between these two ipsilateral burst generators may be modifiable and weakening when greater swimming maneuverability is required. Variable coupling of intrasegmental burst generators in the lamprey may be a precursor to the variable coupling of burst generators observed in the control of locomotion in the joints of limbed vertebrates.     

QUANTITATIVE HISTOCHEMISTRY OF γ-AMINOBUTYRIC ACID IN CAT SPINAL CORD WITH SPECIAL REFERENCE TO PRESYNAPTIC INHIBITION

Abstract— A method is described for quantifying the GABA distribution in cat spinal cord at 200–500 μn resolution. Isolated spinal cord (L5–S1) was frozen and sectioned at about 150 μm thickness. The frozen tissue section was cut into 200 or 500 μm square blocks. The GABA content of each square tissue block was determined by enzymic micromethods and GABA distribution was mapped quantitatively. Average GABA concentrations were: 0·4 mmol/l. in white matter, 1·2 mmol/l. in ventral horn and 1·7 mmol/l. in dorsal horn. The highest concentrations of GABA (2–3 mmol/l.) were found in the dorsolateral part of dorsal horn. In order to destroy the interneurons of dorsal horn, the blood vessels supplying the dorsal horn of the lumbar enlargement were unilaterally cauterized. Seven to 30 days after operation, both the size of dorsal root potential and the GABA level in the dorsal horn were markedly reduced on the cauterized side. These results suggest that GABA is highly concentrated in the interneurons of dorsal horn and functions as a transmitter of presynaptic inhibition.     

Changes in segmental reflex responses during stimulation of locus coeruleus and potentiation of the action of catecholamines

Repetitive stimulation of the locus coeruleus (up to 150 µA in strength) was accompanied by marked weakening of the inhibitory action of flexor reflex afferents and of the reciprocal inhibitory action on extensor motoneurons. Meanwhile stimulation of this sort had no significant effect on direct inhibition of flexor and extensor motoneurons, on the facilitatory action of flexor reflex afferents and the reciprocal inhibitory action on flexor motoneurons and also on dorsal root potentials. Intravenously injected pyrogallol had a similar action, but its effect was much weaker after spinalization of the animals or blocking of spinal cord conduction by cold. Enhancement of the monosynaptic reflex, which also was observed after injection of pyrogallol, was characterized by different temporal parameters; the intensity of this effect was unaffected both by spinalization and by cold block. These data, and also the results of experiments with partial divisions of the spinal cord, suggest that the effects of stimulation of the locus coeruleus are the result of activity of a descending coerulo-spinal tract, running in the ventral quadrant of the spinal cord.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 1, pp. 39–47, January–February, 1981.     

Regional Distribution of Immunoreactive-Thyrotrophin-Releasing Hormone and Substance P, and Indoleamines in Human Spinal Cord

The regional distributions of thyrotrophin-releasing hormone (TRH) and substance P in postmortem human spinal cord were determined by radioimmunoassay in fresh tissue taken from 22 patients who died without known neurological disease. Dorsal, ventral, and intermediolateral spinal cord regions were obtained from different segmental levels (lumbar L1, 2, 3, and 4; thoracic groups T1-3, T4-6, T7-9, and T10-12) together with selective regions of grey matter of lumbar spinal cord. The effects on peptide levels of the age of the patient, the postmortem time interval, and freezing the tissue samples prior to assay were assessed. Levels of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were determined in regional lumbar and thoracic tissue using HPLC with electrochemical detection. Substance P was found in the highest concentration in the dorsal spinal cord, with no significant segmental differences. In contrast, TRH was present in higher levels in the ventral rather than the dorsal spinal cord, with segmental differences. There was a significant difference in the 5-HT/5-HIAA ratio between dorsal and ventral spinal cord, with the highest ratio in the ventral spinal cord. There were no significant differences in substance P, TRH, or 5-HT levels in spinal cords between 5 and 20 h postmortem or from patients aged between 65 and 90 years. Freezing the tissue (-80 degrees C for 24 h) prior to assay significantly reduced TRH and substance P levels compared to samples assayed immediately without prior freezing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pathfinding during spinal tract formation in the chick-quail chimera analysed by species-specific monoclonal antibodies.

In order to analyse the spinal tract formation at early stages of development in avian embryos, chick-quail spinal cord chimeras were prepared and species-specific monoclonal antibodies (MAb) were developed. MAbs CN, QN and CQN uniquely stained chick, quail, and both chick and quail nervous tissues, respectively. All three antibodies appeared to bind to the same membrane molecule, but to different epitopes. Cord reversal revealed the features of axonal growth of both cord interneurons and dorsal root ganglion cells. Quail cord interneurons grew along an originally ventral marginal layer in the quail cord transplanted in a reversed position, then turned toward the ventral side at the boundary between the graft and the host, and grew along the host chick ventral marginal layer. Central axons of dorsal root ganglia were restricted to the ventrolateral region of the cord which originally formed the dorsal funiculus. These results suggest that cord interneurons and dorsal root ganglion cells actively select to grow along specific regions of the cord and that spinal tract formation appears to be determined by cord cells, and not by sclerotome cells.     

Olig3 Is Not Involved in the Ventral Patterning of Spinal Cord

At embryonic stages, Olig3 is initially expressed in the dorsal-most region of the spinal cord, but later in the ventral marginal zone as well. Previous studies indicated that Olig3 controlled the patterning of dorsal spinal cord and loss of Olig3 function led to the re-specification of dI2 and dI3 neurons into dI4 interneurons. However, the role of Olig3 in regulating the development of ventral spinal cord has remained unknown. BrdU labeling demonstrated that ventral Olig3 was expressed in the post-mitotic neurons and Olig3+ cells seen at late embryonic stages were born at the earlier stage but remained in the marginal zone throughout embryogenesis. Loss-of-function and gain-of-function experiment indicated that Nkx2.2 regulated the expression of Olig3 in V3 interneurons. However, Olig3 mutation didn’t apparently affect the generation and migration of ventral neurons. These findings suggest that Olig3 plays different roles in regulating the development of dorsal and ventral spinal cord.     

Fictive hindlimb motor patterns evoked by AMPA and NMDA in turtle spinal cord-hindlimb nerve preparations.

Application of the glutamate agonists alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA, 5-10 microM), or N-methyl-D-aspartate (NMDA, 50-100 microM) to the turtle spinal cord produced fictive hindlimb motor patterns in low-spinal immobilized animals (in vivo) and in isolated spinal cord-hindlimb nerve preparations (in vitro). For in vivo experiments, drugs were applied onto the dorsal surface of 2-4 adjacent spinal cord segments in and near the anterior hindlimb enlargement. Motor output was recorded unilaterally or bilaterally from hindlimb muscle nerves. AMPA elicited vigorous motor patterns in vivo that included strict hip flexor-extensor and right-left alternation. In most turtles, the monoarticular knee extensor nerve FT-KE was active during the HE phase of AMPA evoked burst cycles, similar to the timing of pocket scratch motor patterns. NMDA was less effective in vivo, typically producing only weak and irregular bursting from hip nerves and little or no knee extensor (KE) discharge. Sensory stimulation of a rostral scratch reflex in vivo could reset an ongoing AMPA-evoked motor rhythm, indicating that cutaneous reflex pathways interact centrally with the chemically activated rhythm generator. Most in vitro preparations consisted of six segments of spinal cord, including the entire 5-segment hindlimb enlargement (D8-S2) and the segment immediately anterior to the enlargement (D7), with attached hindlimb nerves. In contrast to in vivo experiments, in vitro preparations exhibited highly regular, long-lasting motor rhythms when NMDA was superfused over the spinal cord. AMPA also produced rhythmic motor patterns in vitro, but these lasted only a few minutes before they were replaced with tonic discharge. FT-KE timing during in vitro chemically elicited activity was similar to that of sensory-evoked pocket scratch motor patterns. Some NMDA-evoked rhythmicity persisted even in 3-segment (D6-D8) and 1-segment (D8) in vitro preparations, demonstrating that neural mechanisms for chemically activated rhythmogenesis reside even in a single segment of the hindlimb enlargement.