Coding of locomotor phase in populations of neurons in rostral and caudal segments of the neonatal rat lumbar spinal cord

Several experiments have demonstrated that rostral segments of the vertebrate lumbar spinal cord produce a rhythmic motor output more readily and of better quality than caudal segments. Here we examine how this rostrocaudal gradient of rhythmogenic capability is reflected in the spike activity of neurons in the rostral (L(2)) and caudal (L(5)) lumbar spinal cord of the neonatal rat. The spike activity of interneurons in the ventromedial cord, a region necessary for the production of locomotion, was recorded intracellularly with patch electrodes and extracellularly with tetrodes during pharmacologically induced locomotion. Both L(2) and L(5) neurons tended to be active in phase with their homologous ventral root. L(5) neurons, however, had a wider distribution of their preferred phases of activity throughout the locomotor cycle than L(2) neurons. The strength of modulation of the activity of individual L(2) neurons was also larger than that of L(5) neurons. These differences resulted in a stronger rhythmic signal from the L(2) neuronal population than from the L(5) population. These results demonstrate that the rhythmogenic capability of each spinal segment was reflected in the activity of interneurons located in the same segment. In addition to paralleling the rostrocaudal gradient of rhythmogenic capability, these results further suggest a colocalization of motoneurons and their associated interneurons involved in the production of locomotion.     

Growing corticospinal axons by-pass lesions of neonatal rat spinal cord

The anterograde transport of horseradish peroxidase was used to label newly growing corticospinal axons after they had entered lesioned regions of the neonatal rat spinal cord. Two types of lesions were made at thoracic and lumbar levels before the arrival of the first corticospinal axons. (1) Thermal lesions were produced by the brief application of a heated rod to the vertebral column and could destroy the normal growth path over several spinal segments. Corticospinal axons, when successful in growing distal to thermal lesions, did so at the same rate as in normal controls and retaining their normal relative positions and morphology, especially fasciculation. (2) Surgical lesions were produced by cutting the spinal cord and were limited to one segment but could result in a barrier in the normal growth path composed of a cyst or glial scar. Corticospinal axons that succeeded in growing distal to a surgical lesion did so by being deflected to unusual positions, became defasciculated, and sometimes their normal growth rate was slowed. That corticospinal axons could in many instances grow past the two types lf lesion suggests that a morphologically stereotyped glial scaffolding is not necessary for axon growth. The role of fasciculation in normal axon growth is highlighted by the disparate effects of the two types of lesion.     

Norepinephrine depolarizes lateral horn cells of neonatal rat spinal cord in vitro

Superfusion of norepinephrine (NE) (1-50 microM) onto lateral horn cells, including antidromically identified sympathetic preganglionic neurons (SPNs), situated in thin transverse neonatal rat thoracolumbar spinal cord slices caused a membrane depolarization and repetitive cell discharges. The NE depolarization was associated with an increase in membrane resistance, and the response became smaller upon conditioning hyperpolarization; a clear reversal of polarity, however, was not observed. Pretreating the slices with phentolamine and prazosin but not yohimbine or propranolol prevented the depolarizing effect of NE. This finding, in conjunction with the evidence of the presence of noradrenergic fibers in the spinal cord, suggests that NE may serve as an excitatory neurotransmitter to neurons of the lateral horn.     

大鼠脊髓星形胶质细胞培养方法的改良

目的:改进大鼠脊髓星形胶质细胞的体外培养方法,为研究脊髓星形胶质细胞的生物学作用及脊髓相关疾病提供实验模型.方法:新生1～2d的SD乳鼠,无菌操作下游离整段脊柱,将脊髓从椎管中冲出,机械吹打制成单细胞悬液,差速黏附处理以去除成纤维细胞成分.利用GFAP免疫荧光显色对培养细胞进行鉴定.结果:经原代和传代培养,获得的星形胶质细胞纯化率高达99%以上.结论:采用本实验室改进的方法,操作简便快捷,培养的大鼠脊髓星形胶质细胞生长良好,纯度高,为后续实验对脊髓星形胶质细胞的生物学作用研究奠定实验基础.     

Effects of noradrenaline on locomotor rhythm-generating networks in the isolated neonatal rat spinal cord.

We have studied the effects of the biogenic amine noradrenaline (NA) on motor activity in the isolated neonatal rat spinal cord. The motor output was recorded with suction electrodes from the lumbar ventral roots. When applied on its own, NA (0.5-50 microM) elicited either no measurable root activity, or activity of a highly variable nature. When present, the NA-induced activity consisted of either low levels of unpatterned tonic discharges, or an often irregular, slow rhythm that displayed a high degree of synchrony between antagonistic motor pools. Finally, in a few cases, NA induced a slow locomotor-like rhythm, in which activity alternated between the left and right sides, and between rostral and caudal roots on the same side. As shown previously, stable locomotor activity could be induced by bath application of N-methyl-D-aspartate (NMDA; 4-8.5 microM) and/or serotonin (5-HT; 4-20 microM). NA modulated this activity by decreasing the cycle frequency and increasing the ventral root burst duration. These effects were dose dependent in the concentration range 1-5 microM. In contrast, at no concentration tested did NA have consistent effects on burst amplitudes or on the background activity of the ongoing rhythm. Moreover, NA did not obviously affect the left/right and rostrocaudal alternation of the NMDA/5-HT rhythm. The NMDA/5-HT locomotor rhythm sometimes displayed a time-dependent breakdown in coordination, ultimately resulting in tonic ventral root activity. However, the addition of NA to the NMDA/5-HT saline could reinstate a well-coordinated locomotor rhythm. We conclude that exogenously applied NA can elicit tonic activity or can trigger a slow, irregular and often synchronous motor pattern. When NA is applied during ongoing locomotor activity, the amine has a distinct slowing effect on the rhythm while preserving the normal coordination between flexors and extensors. The ability of NA to "rescue" rhythmic locomotor activity after its time-dependent deterioration suggests that the amine may be important in the maintenance of rhythmic motor activity.     

Activity-related extracellular potassium transients in the neonatal rat spinal cord: an in vitro study

Transient increases and decreases in extracellular potassium (delta[K+]o) were recorded from the gray matter of hemisected, neonatal rat spinal cords isolated from 3, 4, 9- and 10-day-old pups. delta[K+]o were evoked in both the ventral and dorsal regions of the gray matter by electrical stimulation. In the ventral horn, repetitive stimulation of the ventral root was required to elicit detectable delta[K+]o. By contrast, single dorsal root stimuli evoked clear delta[K+]o. In the dorsal horn, single orthodromic stimuli elicited delta[K+]o as large as 4-5 mM from a baseline of 4.5 mM. With repetitive stimulation the [K+]o reached, but never exceeded, a ceiling of 10-11 mM. Undershoots were seen only after repetitive stimulation. Spontaneous delta[K+]o were observed in the ventral horn and were well correlated with ventral root activity. Spontaneous delta[K+]o were rare in the dorsal cord, but were recorded after bath application of apamin or tetraethylammonium. The magnitude and distribution of evoked K+ transients and postsynaptic components of the evoked field potential were well correlated in both the dorsal and the ventral gray matter. delta[K+]o were reversibly blocked by 1 mM CdCl2 in the bath and diminished by 1 mM BaCl2. Bath application of mephenesin, apamin or tetraethylammonium diminished evoked delta[K+]o in a stimulus-dependent manner. In apamin and tetraethylammonium, decreases from control responses were largest with high intensity stimulation, the opposite was the case with mephenesin. These results are interpreted in terms of the spinal circuits activated by high- and low-intensity electrical stimulation. We conclude that activity-related delta[K+]o in neonatal spinal cord are large enough to modulate neuronal electrical activity and the [K+]o is well regulated compared to other immature CNS regions studied. Thus, local increases in [K+]o may, by modulating neuronal activity, play a role in neonatal spinal cord developmental processes.     

A single re-implanted ventral root exerts neurotropic effects over multiple spinal cord segments in the adult rat

Spinal cord injuries, particularly traumatic injuries to the conus medullaris and cauda equina, are typically complex and involve multiple segmental levels. Implantation of avulsed ventral roots into the spinal cord as a repair strategy has been shown to be neuroprotective and promote axonal regeneration by spinal cord neurons into an implanted root. However, it is not well known over what distance in the spinal cord an implanted ventral root can exert its neurotropic effect. Here, we investigated whether an avulsed L6 ventral root acutely implanted into the rat spinal cord after a four level (L5–S2) unilateral ventral root avulsion injury may exert neurotropic effects on autonomic and motor neurons over multiple spinal cord segments at 6 weeks postoperatively. Using retrograde labeling techniques and stereological quantification methods, we demonstrate that autonomic and motor neurons from all four lesioned spinal cord segments, spanning more than an 8 mm rostro-caudal distance, reinnervated the one implanted root. The rostro-caudal distribution suggested a gradient of neurotropism, where the axotomized neurons closest to the implanted site had the highest probability of root reinnervation. These results suggest that implantation of a single ventral root may provide neurotropic effects to injured neurons at the site of lesion as well as in the adjacent spinal cord segments. Our findings may be of translational research interest for the development of surgical repair strategies after multi-level conus medullaris and cauda equina injuries, in which fewer ventral roots than spinal cord segments may be available for implantation.     

Fictive locomotor patterns generated by tetraethylammonium application to the neonatal rat spinal cord in vitro

Intrinsic spinal networks generate a locomotor rhythm characterized by alternating electrical discharges from flexor and extensor motor pools. Because this process is preserved in the rat isolated spinal cord, this preparation in vitro may be a useful model to explore methods to reactivate locomotor networks damaged by spinal injury. The present electrophysiological investigation examined whether the broad spectrum potassium channel blocker tetraethylammonium could generate locomotor-like patterns. Low (50-500 microM) concentrations of tetraethylammonium induced irregular, synchronous discharges incompatible with locomotion. Higher concentrations (1-10 mM) evoked alternating discharges between flexor and extensor motor pools, plus large depolarization of motoneurons with spike broadening. The alternating discharges were superimposed on slow, shallow waves of synchronous depolarization. Rhythmic alternating patterns were suppressed by blockers of glutamate, GABA(A) and glycine receptors, disclosing a background of depolarizing bursts inhibited by antagonism of group I metabotropic glutamate receptors. Furthermore, tetraethylammonium also evoked irregular discharges on dorsal roots. Rhythmic alternating patterns elicited by tetraethylammonium on ventral roots were relatively stereotypic, had limited synergy with fictive locomotion induced by dorsal root stimuli, and were not accelerated by 4-aminopyridine. Horizontal section of the spinal cord preserved irregular ventral root discharges and dorsal root discharges, demonstrating that the action of tetraethylammonium on spinal networks was fundamentally different from that of 4-aminopyridine. These results show that a potassium channel blocker such as tetraethylammonium could activate fictive locomotion in the rat isolated spinal cord, although the pattern quality lacked certain features like frequency modulation and strong synergy with other inputs to locomotor networks.     

Lumbar spinal cord explants from neonatal rat display age-related decrease of outgrowth in culture

Monosodium glutamate was administered subcutaneously to male neonate rats, and the effects on cell number and cytoarchitecture of third-layer pyramidal neurons from the prefrontal cerebral cortex were studied in the adult. Monosodium glutamate treatment (4 mg/g of body weight, on post-natal days 1, 3, 5 and 7) resulted in fewer neurons, and shorter and less ramified dendritic processes, than those observed in control animals. Both density and proportional shapes of dendritic spines were not modified. We propose a dual effect of neonatal exposure to glutamate: an excitotoxic effect leading to cell death, and; a secondary neuroprotective effect, arising from the proliferation of glial cells and their subsequent uptake of glutamate, that favors the survival of the remaining neurons, and leads to a further hypotrophic effect on their dendritic processes.     

Radial glial cells derived from the neonatal rat spinal cord: morphological and immunocytochemical characterization

Radial glial cells are transiently bipolar cells in the developing central nervous system, best known for their role in guiding migrating neurons. The aim of the present study was to investigate phenotypic characteristics of these bipolar precursor cells in a mixed glial cell culture system derived from the rat neonatal spinal cord. Morphological characterization was assessed by cell-specific immunocytochemical markers (nestin, vimentin, 3CB2) and transmission electron microscopy. Our study yielded substantial evidence showing that the bipolar cells exhibit immunocytochemical and ultrastructural features of radial glial cells. Immunohistochemistry of the neonatal rat spinal cord using the same cell-specific markers suggested these cells are likely derived from the subependymal zone, ventral commissure, and dorsomedial septum. We believe our data recommend this mixed glial culture system to be a valuable tool in studying radial glial cells in vitro.     

Brainstem modulation of locomotion in the neonatal mouse spinal cord

During development, descending projections to the spinal cord are immature. Available data suggest that even though these projections are not fully formed, they contribute to activation of spinal circuitry and promote development of network function. Here we examine the modulation of sacrocaudal afferent-evoked locomotor activity by descending pathways. We first examined the effects of brainstem transection on the afferent evoked locomotor-like rhythm using an isolated brainstem–spinal cord preparation of the mouse. Transection increased the frequency and stability of the locomotor-like rhythm while the phase remained unchanged. We then made histologically verified lesions of the ventrolateral funiculus and observed similar effects on the stability and frequency of the locomotor rhythm. We next tested whether these effects were due to downstream effects of the transection. A split-bath was constructed between the brainstem and spinal cord. Neural activity was suppressed in the brainstem compartment using cooled high sucrose solutions. This manipulation led to a reversible change in frequency and stability that mirrored our findings using lesion approaches. Our findings suggest that spontaneous brainstem activity contributes to the ongoing modulation of afferent-evoked locomotor patterns during early postnatal development. Our work suggests that some of the essential circuits necessary to modulate and control locomotion are at least partly functional before the onset of weight-bearing locomotion.     

Voltage-sensitivity of motoneuron NMDA receptor channels is modulated by serotonin in the neonatal rat spinal cord

Both N-methyl-D-aspartate (NMDA) and serotonin (5-HT) receptors contribute to the generation of rhythmic motor patterns in the rat spinal cord. Co-application of these chemicals is more effective at producing locomotor-like activity than either neurochemical alone. In addition, NMDA application to rat spinal motoneurons, synaptically isolated in tetrodotoxin, induces nonlinear membrane behavior that results in voltage oscillations which can be blocked by 5-HT antagonists. However, the mechanisms underlying NMDA and 5-HT receptor interactions pertinent to motor rhythm production remain to be determined. In the present study, an in vitro neonatal rat spinal cord preparation was used to examine whether NMDA receptor-mediated nonlinear membrane voltage is modulated by 5-HT. Whole-cell recordings of spinal motoneurons demonstrated that 5-HT shifts the region of NMDA receptor-dependent negative slope conductance (RNSC) of the current-voltage relationship to more hyperpolarized potentials and enhances whole-cell inward current. The influence of 5-HT on the RNSC was similar to the effect on the RNSC of decreasing the extracellular Mg(2+)concentration. The results suggest that 5-HT may modulate this form of membrane voltage nonlinearity by regulating Mg(2+) blockade of the NMDA ionophore.     

Interplay between neuromodulator-induced switching of short-term plasticity at sensorimotor synapses in the neonatal rat spinal cord

In the present study, we investigated the modulation of short-term depression (STD) at synapses between sensory afferents and rat motoneurons by serotonin, dopamine and noradrenaline. STD was elicited with trains of 15 stimuli at 1, 5 and 10 Hz and investigated using whole-cell voltage-clamp recordings from identified motoneurons in the neonatal rat spinal cord in vitro . STD was differentially modulated by the amines. Dopamine was effective at all stimulation frequencies, whereas serotonin affected STD only during 5 and 10 Hz stimulus trains and noradrenaline during 1 and 5 Hz trains. Dopamine and serotonin homogenized the degree of depression observed with the different stimulation modalities, in contrast to noradrenaline, which amplified the rate differences. The different modulatory profiles observed with the amines were partly due to GABAergic interneuron activity. In the presence of GABA_A and GABA_B receptor antagonists, the rate and/or kinetics of STD did not vary with the stimulation frequency in contrast to the control condition, and noradrenaline failed to alter either synaptic amplitude or STD, suggesting indirect actions. Dopamine and serotonin strongly decreased STD and converted depression to facilitation at 5 and 10 Hz during the blockade of the GABAergic receptors in 50% of the neurons tested. Altogether, these results show that STD expressed at sensorimotor synapses in the neonatal rat not only is a function of the frequency of afferent firing but also closely depends on the neuromodulatory state of these connections, with a major contribution from GABAergic transmission.     

GABAergic inactivation of the central pattern generators for locomotion in isolated neonatal rat spinal cord.

1. Experiments were performed using an isolated brainstem-spinal cord preparation from newborn rats, in order to study the GABAergic control of the spinal neuronal networks that generate locomotor rhythms in mammals. Locomotor-like activities were recorded in the ventral roots, and the various neurochemical compounds were added to the superfusion saline. 2. Bath application of GABA suppressed in a dose-dependent manner the motor activity induced by an excitatory amino acid N-methyl-D,L-aspartate (NMA). Both the GABAA agonist muscimol and the GABAB agonist baclofen mimicked the effects of GABA, since they either slowed down or stopped the rhythmic activity. 3. Experiments were performed in which the lumbar compartment was superfused separately from the brainstem. Chemical activation of the brainstem by NMA alone failed to induce locomotor-like activity. When GABAA (bicuculline) and GABAB (phaclofen) antagonists were simultaneously bath applied to the lumbar spinal cord, however, locomotor-like activity was induced. 4. The GABA uptake inhibitors nipecotic acid and guvacine suppressed the rhythmic motor pattern induced by NMA in a dose-dependent manner. The effects of nipecotic acid were reversed by bicuculline and phaclofen. 5. Bicuculline added during NMA-induced locomotor-like activity greatly increased both the frequency and amplitude of motor bursts, while phaclofen modified only the frequency. 6. The motor pattern depended on the balance between activatory and inactivatory influences, since the rhythmic patterns recorded with low doses of NMA associated with high doses of bicuculline were similar to those elicited by higher doses of NMA associated with low doses of bicuculline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonergic fibers induce a long-lasting inhibition of monosynaptic reflex in the neonatal rat spinal cord.

The transmitter mechanism of a long-lasting descending inhibition of the monosynaptic reflex was investigated in the isolated spinal cord of the neonatal rat. The monosynaptic reflex elicited by dorsal root stimulation was recorded extracellularly from a lumbar ventral root (L3-L5). Electrical stimulation of the upper thoracic part of the hemisected cord caused an inhibition lasting about 40 s of the monosynaptic reflex. This descending inhibition was markedly attenuated by perfusing the spinal cord with reserpine (1 microM) or 5,7-dihydroxytryptamine (10 microM) for 2-6 h. The perfusion with reserpine (1 microM) for 4 h significantly decreased the contents of 5-hydroxytryptamine, dopamine, and norepinephrine of the neonatal rat spinal cord, whereas the perfusion with 5,7-dihydroxytryptamine (10 microM) for 4 h decreased the contents of 5-hydroxytryptamine and dopamine. The descending inhibition was markedly potentiated by a 5-hydroxytryptamine uptake blocker, citalopram (10 nM), and was blocked by a 5-hydroxytryptamine antagonist, ketanserin (10-100 nM). Application of 5-hydroxytryptamine to the spinal cord induced an inhibition of the monosynaptic reflex, a later part of which was blocked by ketanserin. Ketanserin also moderately blocked inhibitions of the monosynaptic reflex caused by norepinephrine and dopamine. Phentolamine (10 microM) abolished the depressant actions of norepinephrine and dopamine, but did not affect that of 5-hydroxytryptamine or the descending inhibition. These results strongly suggest the involvement of 5-hydroxytryptamine, but not dopamine nor norepinephrine, in the descending inhibition. Besides ketanserin, the descending inhibition was blocked by ritanserin, haloperidol, and pipamperone, which have affinities to 5-hydroxytryptamine2 receptors, and also by spiperone and methiothepin, which are antagonists at both 5-hydroxytryptamine1 and 5-hydroxytryptamine2 receptors (all 1 microM). On the other hand, a 5-hydroxytryptamine1C and 5-hydroxytryptamine2 antagonist, mesulergine (1 microM), and 5-hydroxytryptamine3 antagonists, ICS 205-930 and quipazine (both 1 microM), did not depress either the descending inhibition or the 5-hydroxytryptamine-evoked inhibition of the monosynaptic reflex. The results with these antagonists favor the involvement of 5-hydroxytryptamine2 receptors although the results with mesulergine disagree with this notion. 5-Hydroxytryptamine1 agonists, such as 8-hydroxy-2-(di-n-propylamino)tetralin, buspirone, and 5-carboxyamidotryptamine, and a 5-hydroxytryptamine3 agonist, 2-methyl-5-hydroxytryptamine, induced a long-lasting inhibition of the monosynaptic reflex, which was blocked by ketanserin whereas a 5-hydroxytryptamine2 agonist, S-(+)-alpha-methyl-5-hydroxytryptamine, evoked a biphasic inhibition, in which only the later component was blocked by ketanserin.(ABSTRACT TRUNCATED AT 400 WORDS)