Rat Lumbar Motoneurons Research Articles

Ia EPSPs in rat spinal motoneurons are potentiated after a 5-week whole body vibration.

Whole body vibration (WBV) is often applied as an alternative method for strength training or to prevent muscle force decrease. In this study, we evaluated the influence of WBV on Ia monosynaptic input from muscle spindles because the tonic vibration reflex is responsible for the enhancement of muscle activity observed after WBV. The aim was to investigate whether repeated activation of muscle spindles during WBV may result in altered synaptic excitation of motoneurons. WBV was performed on adult male Wistar rats, 5 days/wk, for 5 wk, and each daily session consisted of four 30-s runs of vibration at 50 Hz. Fast-type medial gastrocnemius motoneurons were investigated intracellularly in deeply anesthetized animals in the experimental (n = 7, 34 motoneurons) and control (n = 7, 32 motoneurons) groups. Monosynaptic Ia excitatory postsynaptic potentials (EPSPs) were evoked by electrical stimulation of afferent fibers from the synergistic lateral gastrocnemius and soleus muscles. Data were analyzed using a mixed linear model. The central latencies of EPSPs were 0.45-0.9 ms with no differences in the mean values between the analyzed groups (P = 0.291). WBV induced an increase of the mean EPSP amplitude by 28% (P = 0.025), correlated with the resting membrane potential and input resistance, and a shortening of the mean EPSP rise time by 11% (P = 0.012). The potentiation of synaptic excitation of motoneurons was not accompanied by changes of passive membrane properties, pointing to synaptic plasticity. This indicates that WBV may support rehabilitation or training processes aimed at increasing muscle strength on the basis of increased motoneuronal drive.NEW & NOTEWORTHY The study provides new information on neuronal plasticity following repeatedly exerted mechanical loading. We demonstrate in electrophysiological experiments on rat lumbar motoneurons that low-volume whole body vibration applied systematically for 5 wk potentiates synaptic excitation from primary muscle afferents. The adaptive changes are expressed by higher amplitudes and shorter rise times of monosynaptic EPSPs evoked in motoneurons of the vibrated group compared with the control.

Calpain fosters the hyperexcitability of motoneurons after spinal cord injury and leads to spasticity.

Up-regulation of the persistent sodium current (INaP) and down-regulation of the potassium/chloride extruder KCC2 lead to spasticity after spinal cord injury (SCI). We here identified calpain as the driver of the up- and down-regulation of INaP and KCC2, respectively, in neonatal rat lumbar motoneurons. Few days after SCI, neonatal rats developed behavioral signs of spasticity with the emergence of both hyperreflexia and abnormal involuntary muscle contractions on hindlimbs. At the same time, in vitro isolated lumbar spinal cords became hyperreflexive and displayed numerous spontaneous motor outputs. Calpain-I expression paralleled with a proteolysis of voltage-gated sodium (Nav) channels and KCC2. Acute inhibition of calpains reduced this proteolysis, restored the motoneuronal expression of Nav and KCC2, normalized INaP and KCC2 function, and curtailed spasticity. In sum, by up- and down-regulating INaP and KCC2, the calpain-mediated proteolysis of Nav and KCC2 drives the hyperexcitability of motoneurons which leads to spasticity after SCI.

Neuroprotective action of bacterial melanin in rats after corticospinal tract lesions

Experiments were performed on 48 albino rats. Part of the experimental animals were initially trained to a balancing instrumental conditioned reflex (ICR). Unilateral bulbar pyramidotomy performed in all rats caused contralateral hemiparesis. On the next day following the operation 24 rats were injected intramuscularly with bacterial melanin solution. 12 of these rats were initially trained to ICR. Recovery periods of ICR and paralyzed hindlimb movements were registered for melanin injected rats (n=24) and for operated rats, not treated with melanin (n=24). In rats injected with bacterial melanin the posttraumatic recovery is shorter than in animals not treated with melanin. The fastest and complete recovery was registered in rats initially trained to ICR and injected after the operation with bacterial melanin. Electrophysiological experiments were performed in transected animals treated with melanin, transected animals without melanin treatment and intact animals. Spiking activity of motoneurons was registered in lumbar motoneurons of rats in response to high frequency stimulation above the corticospinal tract transection. Spiking activity was very similar in motoneurons of melanin injected and intact or non operated animals. In animals, not dosed with bacterial melanin after the operation, areactivity or no change in firing rate was registered in response to stimulus. Stimulation of the corticospinal tract of melanin injected rats produced potentiation of the motoneuronal firing rate and is an evidence of regeneration in corticospinal tract. Similarity in spiking activity of intact and melanin injected rats shows the recovery of conductance in pyramidal tract. Morphohistochemical examination was carried out to confirm the results of behavioral and electrophysiological experiments. Medulla slices were prepared to trace the regeneration of nerve fibers. Examination of transection area revealed that bacterial melanin increases vascularization, dilates the capillaries in nervous tissue and stimulates the process of sprouting.

Differential Plasticity of the GABAergic and Glycinergic Synaptic Transmission to Rat Lumbar Motoneurons after Spinal Cord Injury

Maturation of inhibitory postsynaptic transmission onto motoneurons in the rat occurs during the perinatal period, a time window during which pathways arising from the brainstem reach the lumbar enlargement of the spinal cord. There is a developmental switch in miniature IPSCs (mIPSCs) from predominantly long-duration GABAergic to short-duration glycinergic events. We investigated the effects of a complete neonatal [postnatal day 0 (P0)] spinal cord transection (SCT) on the expression of Glycine and GABA(A) receptor subunits (GlyR and GABA(A)R subunits) in lumbar motoneurons. In control rats, the density of GlyR increased from P1 to P7 to reach a plateau, whereas that of GABA(A)R subunits dropped during the same period. In P7 animals with neonatal SCT (SCT-P7), the GlyR densities were unchanged compared with controls of the same age, while the developmental downregulation of GABA(A)R was prevented. Whole-cell patch-clamp recordings of mIPSCs performed in lumbar motoneurons at P7 revealed that the decay time constant of miniature IPSCs and the proportion of GABAergic events significantly increased after SCT. After daily injections of the 5-HT(2)R agonist DOI, GABA(A)R immunolabeling on SCT-P7 motoneurons dropped down to values reported in control-P7, while GlyR labeling remained stable. A SCT made at P5 significantly upregulated the expression of GABA(A)R 1 week later with little, if any, influence on GlyR. We conclude that the plasticity of GlyR is independent of supraspinal influences whereas that of GABA(A)R is markedly influenced by descending pathways, in particular serotoninergic projections.

The Time Course of Transmitter at Glycinergic Synapses onto Motoneurons

The concentration of transmitter in the synaptic cleft and its clearance time are one of the main determinants of synaptic strength. We estimated the time course of glycine at rat lumbar motoneurons synapses in spinal cord slices by recording synaptic currents in the presence of a low-affinity competitive antagonist at glycine receptors [2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium (SR-95531)]. Data were analyzed by using the established activation mechanism for glycine receptors and our measurements of SR-95531 binding rates. We show that this technique alone is not sufficient to determine simultaneously the peak concentration of transmitter and its clearance time. However, we found that block of the glial glycine transporter prolongs the glycine transient. This observation puts additional constraints on the range of possible values of the time course of glycine, indicating that glycine reaches a peak concentration of 2.2-3.5 mM and is cleared from the cleft with a time constant of 0.6-0.9 ms.

Expression of nuclear receptor coactivators in androgen-responsive and -unresponsive motoneurons

Adult rat lumbar motoneurons in the spinal nucleus of the bulbocavernosus (SNB) respond to androgens with an increase in soma size. This response is mediated by the androgen receptor (AR) in these motoneurons. Interestingly, other lumbar motoneurons in the rat possess the AR, yet do not respond to androgens in this fashion. This paradox suggests the existence and participation of nuclear receptor coregulators in conferring direct androgen-responsiveness to select motoneurons in the adult rat spinal cord. Nuclear receptor coregulators have received much attention recently for their proposed role in enhancing or repressing the transcriptional activity of steroid hormone receptors. The present study used immunocytochemistry to identify a number of nuclear receptor coactivators that are expressed by adult lumbar motoneurons: SRC-1, SRC-2, CBP, p300, and cJUN. Results of this study indicate that all five of these coactivators are abundantly expressed in the androgen-responsive SNB, and in two adjacent motor pools, the androgen-responsive dorsolateral nucleus (DLN), and the androgen- unresponsive retrodorsolateral nucleus (RDLN). While we detected significant regional differences for only SRC-1 and cJUN, the SNB consistently contained the highest percentage of immunoreactive motoneurons for all five cofactors examined. Our results indicate five different putative cofactors have the potential to participate in motoneuronal responses to androgens, since their distribution overlaps well with the distribution of ARs in these motoneurons.

Passive exercise and fetal spinal cord transplant both help to restore motoneuronal properties after spinal cord transection in rats

Spinal cord transection influences the properties of motoneurons and muscles below the lesion, but the effects of interventions that conserve muscle mass of the paralyzed limbs on these motoneuronal changes are unknown. We examined the electrophysiological properties of rat lumbar motoneurons following spinal cord transection, and the effects of two interventions shown previously to significantly attenuate the associated hindlimb muscle atrophy. Adult rats receiving a complete thoracic spinal cord transection (T-10) were divided into three groups receiving: (1) no further treatment; (2) passive cycling exercise for 5 days/week; or (3) acute transplantation of fetal spinal cord tissue. Intracellular recording of motoneurons was carried out 4-5 weeks following transection. Transection led to a significant change in the rhythmic firing patterns of motoneurons in response to injected currents, as well as a decrease in the resting membrane potential and spike trigger level. Transplants of fetal tissue and cycling exercise each attenuated these changes, the latter having a stronger effect on maintenance of motoneuron properties, coinciding with the reported maintenance of structural and biochemical features of hindlimb muscles. The mechanisms by which these distinct treatments affect motoneuron properties remain to be uncovered, but these changes in motoneuron excitability are consistent with influences on ion conductances at or near the initial segment. The results may support a therapeutic role for passive limb manipulation and transplant of stem cells in slowing the deleterious responses of motoneurons to spinal cord injury, such that they remain more viable for subsequent alternative strategies.

Variations in oxidative enzyme type profiles among prenatal rat lumbar motoneurons

We have used cytochrome oxidase histochemical staining to evaluate whether immature rat lumbar motoneurons show intrinsic separation into high or low oxidative enzyme types. Relative oxidative enzyme levels are frequently used to help differentiate between muscle fibres of various types and to differentiate between mature neurons. Here we show a wide variation in motoneuron cytochrome oxidase levels from prenatal times, although the range of staining levels as measured densitometrically is greater for mature than for prenatal animals. We find variation in cytochrome oxidase levels among motoneurons prior to the formation of mature patterns of connectivity or electrical activity, and conclude therefore that this differentiation is unlikely to have arisen by differential usage and probably arose as a function of cell lineage.

Ontogeny of androgen receptor immunoreactivity in lumbar motoneurons and in the sexually dimorphic levator ani muscle of male rats

We documented the ontogeny of androgen receptor (AR) immunoreactivity for rat lumbar motoneurons of the sexually dimorphic motor pools, the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN), and for the sexually monomorphic retrodorsolateral nucleus (RDLN). We also assessed the ontogeny of AR immunoreactivity in the rat sexually dimorphic levator ani (LA), which is a target muscle for SNB motoneurons. Lumbar spinal cords and LA muscles from gonadally intact males at ages postnatal days (P)7, P10, and P14 and as adults were incubated with the rabbit antiserum PG-21. Half of the prepubertal males (P7–P14) received 200 μg of testosterone propionate (TP) 2 hours prior to death to enhance immunodetection of ARs. We found that SNB motoneurons developed AR immunoreactivity first and achieved adult levels by P10. In contrast, the number of RDLN motoneurons with AR-immunopositive nuclei during development remained well below the adult number. Development of AR immunoreactivity in the DLN shared characteristics with both the SNB and the RDLN. AR immunoreactivity developed in some DLN motoneurons by P10, although the percentage of labelled motoneurons remained below that in adulthood. Acute TP treatment significantly increased the number of SNB motoneurons with AR-positive nuclei at P7. The LA showed a robust pattern of AR immunostaining from P7 to adulthood. Immunostaining was present only in nuclei and constituted only a subpopulation of the nuclei present in muscle. The present results confirm and extend previous results based on steroid autoradiography and steroid binding assays regarding regional and developmental differences in the expression of ARs. J. Comp. Neurol. 379:88-98, 1997. © 1997 Wiley-Liss, Inc.

Ontogeny of androgen receptor immunoreactivity in lumbar motoneurons and in the sexually dimorphic levator ani muscle of male rats

We documented the ontogeny of androgen receptor (AR) immunoreactivity for rat lumbar motoneurons of the sexually dimorphic motor pools, the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN), and for the sexually monomorphic retrodorsolateral nucleus (RDLN). We also assessed the ontogeny of AR immunoreactivity in the rat sexually dimorphic levator ani (LA), which is a target muscle for SNB motoneurons. Lumbar spinal cords and LA muscles from gonadally intact males at ages postnatal days (P)7, P10, and P14 and as adults were incubated with the rabbit antiserum PG-21. Half of the prepubertal males (P7-P14) received 200 micrograms of testosterone propionate (TP) 2 hours prior to death to enhance immunodetection of ARs. We found that SNB motoneurons developed AR immunoreactivity at first and achieved adult levels by P10. In contrast, the number of RDLN motoneurons with AR-immunopositive nuclei during development remained well below the adult number. Development of AR immunoreactivity in the DLN shared characteristics with both the SNB and the RDLN. AR immunoreactivity developed in some DLN motoneurons by P10, although the percentage of labelled motoneurons remained below that in adulthood. Acute TP treatment significantly increased the number of SNB motoneurons with AR-positive nuclei at P7. The LA showed a robust pattern of AR immunostaining from P7 to adulthood. Immunostaining was present only in nuclei and constituted only a subpopulation of the nuclei present in muscle. The present results confirm and extend previous results based on steroid autoradiography and steroid binding assays regarding regional and developmental differences in the expression of ARs.

Glutamatergic and non-glutamatergic responses evoked in neonatal rat lumbar motoneurons on stimulation of the lateroventral spinal cord surface

The effects on lumbar motoneurons of thoracic cord stimulation were investigated in the neonatal rat hemisected spinal cord vitro using intracellular recording. Four responses were evoked—a fast, excitatory postsynaptic potential, a second component to the fast excitatory postsynaptic potential, a fast inhibitory postsynaptic potential and a slow excitatory postsynaptic potential. The fast (CNQX-sensitive) excitatory postsynaptic potential was probably monosynaptic, was blocked by CNQX, (10μM) and showed a frequency-dependent run-down at stimulation frequencies between 0.1 and 1 Hz. A slower component to the fast excitatory postsynaptic potential ((±)-2-amino-5-phosphono-valeric acid-sensitive excitatory postsynaptic potential) was blocked by (±)-2-amino-5-phosphonovaleric acid (50 μM). Following fast excitatory postsynaptic potential blockade with both CNQX and (±)-2-amino-5-phospho-novaleric acid, a fast inhibitory postsynaptic potential was revealed. This reversed at a membrane potential close to resting and was incompletely blocked by either bicuculline (30 μM) or strychnine (10 μM). The slow excitatory postsynaptic potential was a delayed depolarization associated with a small increase in input resistance (20%) and was insensitive to block by CNQX and/or (±)-2-amino-5-phosphonovaleric acid. It increased in amplitude on membrane depolarization and decreased on hyperpolarization and was potentiated by cocaine (3μM) and citalopram (0.1μM), but not by desipramine (5μM). The slow excitatory postsynaptic potential was blocked by ketanserin (1μM) and by LY 53857 (1μM). It is concluded that a non-glutamatergic transmitter is involved in generating the slow excitatory postsynaptic potential possibly 5-hydroxytryptamine acting at 5-hydroxytryptamine 2 receptors.

Ontogeny of calcitonin gene-related peptide immunoreactivity in rat lumbar motoneurons: delayed appearance and sexual dimorphism in the spinal nucleus of the bulbocavernosus.

Immunoreactivity for calcitonin gene-related peptide (CGRP) has been observed in both adult and embryonic rat motoneurons. However, the developmental pattern of CGRP expression in motoneurons has not been systematically examined and the role of CGRP in neuromuscular development is poorly understood. We have mapped the ontogeny of CGRP-like immunoreactivity in three motoneuron pools of the rat lumbar spinal cord from birth through adulthood. Immunoreactivity was uniformly high in lateral horn motoneurons (the retrodorsolateral nucleus) of males and females at all ages examined. The majority of motoneurons of the dorsolateral nucleus also were positive throughout postnatal development although the percentage of positive motoneurons was slightly higher in males than in females. In contrast, virtually no motoneurons of the spinal nucleus of the bulbocavernosus, located in the medial ventral horn, were positive for CGRP in neonatal rats. CGRP-like immunoreactivity was delayed in this nucleus until approximately postnatal day 6 in males and day 27 in females. Because these three motoneuronal nuclei are differentially sensitive to early androgen and differ with respect to the timing of several developmental milestones, these observations have implications for the regulation and possible roles of CGRP in developing motor systems.

Calcitonin gene-related peptide staining intensity is reduced in rat lumbar motoneurons after spinal cord transection: a quantitative immunocytochemical study

The expression of Calcitonin gene-related peptide (CGRP) has been demonstrated in motoneurons of several species. We have investigated in adult rats the influence of transection of the spinal cord on CGRP immunoreactivity of motoneurons located below the section. Quantitative analysis has been performed with computer-assisted image analysis. As early as 48 h after the section, CGRP immunoreactivity is modified, and the reduction is maximal after one month. Then, both the number of immunoreactive cells and the intensity of staining increase until the 5th month. It is concluded that the expression of CGRP is under the influence of supraspinal afferents to the motoneuron.

Brainstem projections to lumbar motoneurons in rat—II. An ultrastructural study by means of the anterograde transport of wheat germ agglutinin coupled to horseradish peroxidase and using the tetramethyl benzidine reaction

The descending projections from the ventrolateral medial reticular formation to the lumbar motoneuronal cell groups were studied in rat at the ultrastructural level using the anterograde transport of wheat germ agglutinin coupled to horseradish peroxidase in combination with the chromogen tetramethyl benzidine. The tissue containing the horseradish peroxidase reaction products was processed for electron microscopy using the method of chemical dehydration. In the ultrathin sections of the lumbar motoneuronal cell groups reaction products were easily recognized by their electron-dense crystal-like structure. These crystals were mostly found in terminals (66%) and to a lesser extent in axons (10%) and dendrites (10%). In the lumbar motoneuronal cell groups six different types of terminals were distinguished. It was found that F-type terminal profiles most frequently contained crystals, whereas fewer S- and G-types did so. The various findings are basically the same as those of a similar ultrastructural autoradiographic study with [ 3H]leucine as a tracer, except for the labelling of dendrites, which is discussed. It is concluded that the horseradish peroxidase technique employed in the present study can be reliably used for anterograde tracing at the ultrastructural level. Its advantages and disadvantages in comparison with the electron microscopical autoradiographic technique are discussed.

Brainstem projections to lumbar motoneurons in rat—I. An ultrastructural study using autoradiography and the combination of autoradiography and horseradish peroxidase histochemistry

In 11 rats the descending projections from the ventrolateral medullary medial reticular formation, the medullary raphe nuclei and the area of the nucleus coeruleus and subcoeruleus to lumbar motoneuronal cell groups were studied by means of electron microscopical autoradiography after [ 3H]leucine injections in the respective brainstem areas. The distribution of the transported radioactivity in the autoradiographs was determined using the circle method [Williams (1977), in Practical Methods in Electron Microscopy, Vol. 6, pp. 85–173] which showed that the vast majority of the silver grains was located over terminal profiles. In the motoneuronal cell groups six different types of terminals were distinguished. After injections in the ventrolateral medial reticular formation the majority of the silver grains was located over F-type terminal profiles while many fewer silver grains were found over S- and G-types. After injections in the raphe nuclei and the adjoining medial reticular formation approximately equal numbers of silver grains were found over F- and G-type terminals while fewer were found over S-type. A small proportion of silver grains was present over C-type terminals and only after injections in the ventrolateral medial reticular formation. After [ 3H]leucine injections in the area of the nucleus coeruleus and subcoeruleus the majority of silver grains were located over E- and S-type terminals whereas relatively few were located over F-type terminals. The E-type terminal, which has not been described before in the motoneuronal cell groups, is characterized by the fact that it contains relatively small vesicles and occasionally elongated or canaliculi-like structures. In the three groups of experiments approximately 40–50% of the labelled S- and F-type terminal profiles established synaptic contacts, but only approximately 10% of the labelled E- and G-type terminal profiles did so. In all cases these synaptic contacts were established mainly with proximal dendrites. In the autoradiographs some of the silver grains were concentrated into clusters. The vast majority of these clusters, consisting of six or more silver grains, were centred over terminal profiles. The differential distribution of these clusters over the different types of terminal profiles in the various experiments was roughly the same as found by means of the circle method. In two rats [ 3H]leucine injections in the ventrolateral medial reticular formation were combined with horseradish peroxidase injections in the ipsilateral hindleg muscles, resulting in retrograde labelling of the corresponding motoneurons as visualized by means of the tetramethyl benzidine incubation method. In the electron microscopical autoradiographs only terminals carrying clusters of six or more silver grains were studied. More than 50% of the postsynaptic structures which were contacted by these cluster-labelled terminal profiles contained horseradish peroxidase reaction products. The findings in these various experiments indicated that fibres descending from the ventrolateral medial reticular formation, the caudal raphe nuclei and the area of the nucleus coeruleus and subcoeruleus to the motoneuronal cell groups carry different combinations of types of terminals which contact mainly proximal dendrites. The findings after combining [ 3H]leucine injections in the ventrolateral medial reticular formation with horseradish peroxidase injections in the hindleg muscles demonstrated that the vast majority of the terminals originating from neurons in the medial reticular formation actually contact motoneurons.

Synaptic processes evoked by intracortical microstimulation in lumbar motoneurons of rats

Experiments with intracortical microstimulation and intracellular recording from motoneurons of the rat hind limb showed that synaptic effects due to activation of pyramidal neurons of the motor cortex and transmitted along the pyramidal tract are exclusively polysynaptic in character. Mainly excitatory effects were found in motoneurons of flexors and extensors, and of distal and proximal muscles. The minimal intensity of intracortical stimulation required for synaptic excitation of α-motoneurons is 5–10 µA. Low-threshold synaptic effects in lumbar motoneurons and movements of the hind limbs are evoked from the same zones.