Control of respiratory motor pattern by sensory neurons in spinal cord of lamprey

Summary Extracellular stimulation over the dorsal funiculus in the spinal cord of lampreys was found to selectively activate prolonged episodes of fictive arousal respiration (Figs. 1, 3). The induced episodes showed comparable increases in cycle frequency and motoneuron burst duration to the spontaneous arousal pattern observed in isolated brain preparations (Fig. 2). Intracellular stimulation of primary sensory neurons with axons in the dorsal funiculus, called dorsal cells, also elicited the arousal pattern (Fig. 4). Mechanoreceptive dorsal cells respond to cutaneous stimulation. When mechanical stimuli were applied to the skin of intact lampreys (Fig. 6) or to lampreys with ipsilateral vagotomy, arousal respiration was induced (Figs. 7, 8). Bilateral, but not unilateral, trigeminal lesion blocked dorsal cell induction of the arousal response (Fig. 5). Spontaneous arousal respiration was recorded from intact, unrestrained lampreys (Fig. 9). These results suggest that fictive arousal respiration is the in vitro correlate of natural arousal respiration in lampreys, and that one mechanism leading to arousal respiration may be the activity of sensory dorsal cells. A model for respiratory motor pattern switching in lamprey is proposed. The model suggests that the normal and arousal patterns are produced by separately engaging rostral or caudal pattern generators in the medulla, rather than by modifying one pattern generator (Fig. 10).     

Coordinated network functioning in the spinal cord: An evolutionary perspective

The successful achievement of harmonious locomotor movement results from the integrated operation of all body segments. Here, we will review current knowledge on the functional organization of spinal networks involved in mammalian locomotion. Attention will not simply be restricted to hindlimb muscle control, but by also considering the necessarily coordinated activation of trunk and forelimb muscles, we will try to demonstrate that while there has been a progressive increase in locomotor system complexity during evolution, many basic organizational features have been preserved across the spectrum from lower vertebrates through to humans. Concerning the organization of axial neuronal networks that control trunk muscles, it has been found across the vertebrate range that during locomotor movement a motor wave travels longitudinally in the spinal cord via the coupling of rhythmic segmental networks. For hindlimb activation it has been found in all species studied that the rostral lumbar segments contain the key elements for pattern generation. We also showed that rhythmic arm movements are under the control of cervical forelimb generators in quadrupeds as well as in human. Finally, it is highlighted that the coordination of quadrupedal movements during locomotion derives principally from an asymmetrical coordinating influence occurring in the caudo-rostral direction from the lumbar hindlimb networks.     

Survival motor neuron protein reduction deregulates autophagy in spinal cord motoneurons in vitro

Spinal muscular atrophy (SMA) is a genetic disorder characterized by degeneration of spinal cord motoneurons (MNs), resulting in muscular atrophy and weakness. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene and decreased SMN protein. SMN is ubiquitously expressed and has a general role in the assembly of small nuclear ribonucleoproteins and pre-mRNA splicing requirements. SMN reduction causes neurite degeneration and cell death without classical apoptotic features, but the direct events leading to SMN degeneration in SMA are still unknown. Autophagy is a conserved lysosomal protein degradation pathway whose precise roles in neurodegenerative diseases remain largely unknown. In particular, it is unclear whether autophagosome accumulation is protective or destructive, but the accumulation of autophagosomes in the neuritic beadings observed in several neurite degeneration models suggests a close relationship between the autophagic process and neurite collapse. In the present work, we describe an increase in the levels of the autophagy markers including autophagosomes, Beclin1 and light chain (LC)3-II proteins in cultured mouse spinal cord MNs from two SMA cellular models, suggesting an upregulation of the autophagy process in Smn (murine survival motor neuron protein)-reduced MNs. Overexpression of Bcl-x_L counteracts LC3-II increase, contributing to the hypothesis that the protective role of Bcl-x_L observed in some SMA models may be mediated by its role in autophagy inhibition. Our in vitro experimental data indicate an upregulation in the autophagy process and autophagosome accumulation in the pathogenesis of SMA, thus providing a valuable clue in understanding the mechanisms of axonal degeneration and a possible therapeutic target in the treatment of SMA.     

Mechanical properties of the lamprey spinal cord: Uniaxial loading and physiological strain

During spinal cord injury, nerves suffer a strain beyond their physiological limits which damages and disrupts their structure. Research has been done to measure the modulus of the spinal cord and surrounding tissue; however the relationship between strain and spinal cord fibers is still unclear. In this work, our objective is to measure the stress–strain response of the spinal cord in vivo and in vitro and model this response as a function of the number of fibers. We used the larvae lamprey (Petromyzon Marinus), a model for spinal cord regeneration and animal locomotion. We found that physiologically the spinal cord is pre-stressed to a longitudinal strain of 10% and this strain increases to 15% during swimming. Tensile measurements show that uniaxial, longitudinal loading is independent of the meninges. Stress values for uniaxial strains below 18%, are homogeneous through the length of the body. However, for higher uniaxial strains the Head section shows more resistance to longitudinal loading than the Tail. These data, together with the number of fibers obtained from histological sections were used in a composite-material model to obtain the properties of the spinal cord fibers (2.4 MPa) and matrix (0.017 MPa) to uniaxial longitudinal loading. This model allowed us to approximate the percentage of fibers in the spinal cord, establishing a relationship between uniaxial longitudinal strains and spinal cord composition. We showed that there is a proportional relationship between the number of fibers and the properties of the spinal cord at large uniaxial strains.     

Neuroprotective effects of toll-like receptor 4 antagonism in spinal cord cultures and in a mouse model of motor neuron degeneration

Sustained inflammatory reactions are common pathological events associated with neuron loss in neurodegenerative diseases. Reported evidence suggests that Toll-like receptor 4 (TLR4) is a key player of neuroinflammation in several neurodegenerative diseases. However, the mechanisms by which TLR4 mediates neurotoxic signals remain poorly understood. We investigated the role of TLR4 in in vitro and in vivo settings of motor neuron degeneration. Using primary cultures from mouse spinal cords, we characterized both the proinflammatory and neurotoxic effects of TLR4 activation with lipopolysaccharide (activation of microglial cells, release of proinflammatory cytokines and motor neuron death) and the protective effects of a cyanobacteria-derived TLR4 antagonist (VB3323). With the use of TLR4-deficient cells, a critical role of the microglial component with functionally active TLR4 emerged in this setting. The in vivo experiments were carried out in a mouse model of spontaneous motor neuron degeneration, the wobbler mouse, where we preliminarily confirmed a protective effect of TLR4 antagonism. Compared with vehicle- and riluzole-treated mice, those chronically treated with VB3323 showed a decrease in microglial activation and morphological alterations of spinal cord neurons and a better performance in the paw abnormality and grip-strength tests. Taken together, our data add new understanding of the role of TLR4 in mediating neurotoxicity in the spinal cord and suggest that TLR4 antagonists could be considered in future studies as candidate protective agents for motor neurons in degenerative diseases.     

A change in the ultrastructure of frog spinal cord and motor neuron synapses during prolonged activation]

Motoneuron axosomatic synapses (AS) were shown to be larger than axodendritic (AD) synapses under anaesthesia. A 10 minute-long electrical stimulation both by low and high frequency caused enlargement of AS and AD synapses, it being the more pronounced the higher was the activation frequency. In all cases changes the AS synapses were more marked than those of AD synapses. On the bases of morphological and physiological data it was concluded that the changes of the synapse dimentions did not affect the synaptic transmission.     

Evaluation of lumbar enlargement motor neuron excitability: Comparison between H-reflex and transcutaneous spinal cord stimulation

Multisegmental muscle responses (MMR) are reflexes in the leg muscles evoked by transcutaneous electrical spinal cord stimulation over the Th11–Th12 vertebrae. We have used MMR to evaluate the excitability of lumbosacral motor neurons in individuals having paraplegia of low limbs. Ten individuals were tested using H-reflexes and MMR bilaterally before (n ₀ = 20) and during 4-weeks course of rehabilitation (n=76). The H-reflex and MMR of m. gastrocnemius lateralis were obtained in: 15 and 13 cases out of 20, respectively. Both reflexes were recorded in 11 and were absent in 3 cases, matched up to 70% of recordings. In dynamic, the both methods were 100% reproducible and the responses’ amplitude varied in similar directions in 67% of records. The data confirm the validity and reproducibility of the MMR for evaluation of the motor neurons excitability in lumbosacral cord. The H-reflex magnitude shows moderate correlation with MMR in m. gastrocnemius lateralis (r = 0.59, p < 0.001), and weak correlation with MMR in mm. rectus femoris, biceps femoris, and tibialis anterior (r < 0.40, p < 0.001). These findings do not allow extrapolate the results from the H-reflex measurement on the state of lumbosacral cord on the whole. At the same time, measurements of the MMR allow estimate simultaneously the excitability of motor pools innervating several muscle groups. This gives the possibility to assess the functional state of the motor neurons in the lumbosacral cord for clinical and experimental studies, including the spinal cord damage.     

The ultrastructural characteristics of the motor neuron synaptic organization in the spinal cord of the frog Rana ridibunda

Electron microscopic studies on the spinal motor nuclei in amphibians indicate significant diversity in chemical synapses formed on motoneurones by axonal endings of supra- and intraspinal systems. High ultrastructural specialization was observed among axosomatic, axodendritic and axoaxonal synapses. Several types of axo-spine synapses and axodentritic synaptic complexes of the "glomerular" type were revealed. New data on ultrastructural peculiarities of chemical synapses presented in this paper, together with earlier detailed data on morphologically mixed and electrotonic synapses, increase our knowledge of evolutionary trends in synaptic organization of motoneurones in the spinal cord and suggest the existence of a complex mechanism of integration of synaptic influences in the spinal cord of lower vertebrates.     

Time-related changes of motor unit properties in the rat medial gastrocnemius muscle after the spinal cord injury. II. Effects of a spinal cord hemisection

The contractile properties of motor units (MUs) were investigated in the medial gastrocnemius (MG) muscle in rats after the spinal cord hemisection at a low thoracic level. Hemisected animals were divided into 4 groups: 14, 30, 90 and 180 days after injury. Intact rats formed a control group. The mass of the MG muscle did not change significantly after spinal cord hemisection, hind limb locomotor pattern was almost unchanged starting from two weeks after injury, but contractile properties of MUs were however altered. Contraction time (CT) and half-relaxation time (HRT) of MUs were prolonged in all investigated groups of hemisected rats. The twitch-to-tetanus ratio (Tw/Tet) of fast MUs after the spinal cord hemisection increased. For slow MUs Tw/Tet values did not change in the early stage after the injury, but significantly decreased in rats 90 and 180 days after hemisection. As a result of hemisection the fatigue resistance especially of slow and fast resistant MU types was reduced, as well as fatigue index (Fat I) calculated for the whole examined population of MUs decreased progressively with the time. After spinal cord hemisection a reduced number of fast MUs presented the sag at frequencies 30 and 40 Hz, however more of them revealed sag in 20 Hz tetanus in comparison to control group. Due to considerable changes in twitch contraction time and disappearance of sag effect in unfused tetani of some MUs in hemisected animals, the classification of MUs in all groups of rats was based on the 20 Hz tetanus index (20 Hz Tet I) but not on the standard criteria usually applied for MUs classification. MU type differentiations demonstrated some clear changes in MG muscle composition in hemisected animals consisting of an increase in the proportion of slow MUs (likely due to an increased participation of the studied muscle in tonic antigravity activity) together with an increase in the percentage of fast fatigable MUs.     

Extensive fusion of mitochondria in spinal cord motor neurons

The relative roles played by trafficking, fission and fusion in the dynamics of mitochondria in neurons have not been fully elucidated. In the present study, a slow widespread redistribution of mitochondria within cultured spinal cord motor neurons was observed as a result of extensive organelle fusion. Mitochondria were labeled with a photoconvertible fluorescent protein (mitoKaede) that is red-shifted following brief irradiation with blue light. The behavior of these selectively labeled mitochondria was followed by live fluorescence imaging. Marking mitochondria within the cell soma revealed a complete mixing, within 18 hours, of these organelles with mitochondria coming from the surrounding neurites. Fusion of juxtaposed mitochondria was directly observed in neuritic processes at least 200 microns from the cell body. Within 24 hours, photoconverted mitoKaede was dispersed to all of the mitochondria in the portion of neurite under observation. When time lapse imaging over minutes was combined with long-term observation of marked mitochondria, moving organelles that traversed the field of view did not initially contain photoconverted protein, but after several hours organelles in motion contained both fluorescent proteins, coincident with widespread fusion of all of the mitochondria within the length of neurite under observation. These observations suggest that there is a widespread exchange of mitochondrial components throughout a neuron as a result of organelle fusion.     

Time-related changes of motor unit properties in the rat medial gastrocnemius muscle after spinal cord injury. I. Effects of total spinal cord transection

The contractile properties of motor units (MUs) were electrophysiologically investigated in the medial gastrocnemius (MG) muscle in 17 Wistar three-month-old female rats: 14, 30, 90 and 180 days after the total transection of the thoracic spinal cord and compared to those in intact (control) rats. A sag phenomenon, regularly observed in unfused tetani of fast units in intact animals at 40 Hz stimulation, almost completely disappeared in spinal rats. Therefore, the MUs of intact and spinal rats were classified as fast or slow types basing on 20 Hz tetanus index, the value of which was lower or equal 2.0 for fast and higher than 2.0 for slow MUs. The MUs composition of MG muscle changed with time after the spinal cord transection: an increasing proportion of fast fatigable (FF) units starting one month after injury and a disappearance of slow (S) units within the three months were observed. In all MUs investigated the twitch contraction and half-relaxation time were significantly prolonged after injury (p < 0.01, Mann–Whitney U-test). Moreover, a decrease of the fatigue index for fast resistant (FR) and slow MUs was observed in subsequent groups of spinal rats. No significant changes were found between twitch forces in all MU types of spinal animals (p > 0.05). However, due to a decrease of the maximal tetanic force, a significant rise of the twitch-to-tetanus ratio of all MUs in spinal rats was detected (p < 0.01). The considerable reduction of ability to potentiate the force was noticed for fast, especially FF type MUs. In conclusion, the spinal cord transection leads to changes in the proportion of the three MU types in rat MG muscle. The majority of changes in MUs’ contractile properties were developed progressively with time after the spinal cord injury. However, the most intensive alterations of twitch-time parameters were observed in rats one month after the transection.     

The influence of glycine and related compounds on spinal cord injury-induced spasticity

Spasticity is a frequent and complex sequel to spinal cord injury. The neurochemical basis for the origin of spasticity is largely unknown. Glycine is among the most abundant neurotransmitters in the spinal cord. However, the role of glycine and related compounds in spasticity have received little attention. An ischemic spinal cord injury was created in rabbits, by an intraaortic balloon occlusion technique, which produced lower limb spasticity. A catheter was inserted into the cisterna magna and the spinal cord was bathed with 100 M solutions of glycine, strychnine,d-serine, -alanine, MK-801, or artificial CSF for 4 hours at a rate of 10 l/min. H-reflexes were monitored before and during infusion by stimulating the posterior tibial nerve and recording from the plantar surface of the foot. Glycine,d-serine, and MK-801 depressed the H wave, strychnine produced a heightened H wave, and -alanine caused no significant changes. These results indicate that glycine and related compounds may influence spasticity.