Persistent sodium currents and repetitive firing in motoneurons of the sacrocaudal spinal cord of adult rats

Months after sacral spinal transection in rats (chronic spinal rats), motoneurons below the injury exhibit large, low-threshold persistent inward currents (PICs), composed of persistent sodium currents (Na PICs) and persistent calcium currents (Ca PICs). Here, we studied whether motoneurons of normal adult rats also exhibited Na and Ca PICs when the spinal cord was acutely transected at the sacral level (acute spinal rats) and examined the role of the Na PIC in firing behavior. Intracellular recordings were obtained from motoneurons of acute and chronic spinal rats while the whole sacrocaudal spinal cord was maintained in vitro. Compared with chronic spinal rats, motoneurons of acute spinal rats were more difficult to activate because the input resistance was 22% lower and resting membrane potential was hyperpolarized 4.1 mV further below firing threshold (-50.9 +/- 6.2 mV). In acute spinal rats, during a slow voltage ramp, a PIC was activated subthreshold to the spike (at -57.2 +/- 5.0 mV) and reached a peak current of 1.11 +/- 1.21 nA. This PIC was less than one-half the size of that in chronic spinal rats (2.79 +/- 0.94 nA) and usually was not large enough to produce bistable behavior (plateau potentials and self-sustained firing not present), unlike in chronic spinal rats. The PIC was composed of two components: a TTX-sensitive Na PIC (0.44 +/- 0.36 nA) and a nimodipine-sensitive Ca PIC (0.78 +/- 0.82 nA). Both were smaller than in chronic spinal rats (but with similar Na/Ca ratio). The presence of the Na PIC was critical for normal repetitive firing, because no detectable Na PIC was found in the few motoneurons that could not fire repetitively during a slow ramp current injection and motoneurons that had large Na PICs more readily produced repetitive firing and had lower minimum firing rates compared with neurons with small Na PICs. Furthermore, when the Na PIC was selectively blocked with riluzole, steady repetitive firing was eliminated, even though transient firing could be evoked on a rapid current step and the spike itself was unaffected. In summary, only small Ca and Na PICs occur in acute spinal motoneurons, but the Na PIC is essential for steady repetitive firing. We discuss how availability of monoamines may explain the variability in Na PICs and firing in the normal and spinal animals.     

Differential regulation of alpha-calcitonin gene-related peptide and preprocholecystokinin messenger RNA expression in alpha-motoneurons: effects of testosterone and inactivity induced factors.

alpha-Calcitonin gene-related peptide expression in alpha-motoneurons is regulated by spinal cord transection, axotomy and testosterone, but to date there are no studies which examine the regulation of cholecystokinin expression in motoneurons. In the present study, we compared the regulation of preprocholecystokinin and alpha-calcitonin gene-related peptide messenger RNA levels in motoneurons of the spinal nucleus of the bulbocavernosus. Previously, we demonstrated that manipulations which decrease activity in target muscles of the spinal nucleus of the bulbocavernosus motoneurons increase alpha-calcitonin gene-related peptide message and peptide levels in spinal nucleus of the bulbocavernosus motoneurons. This muscle-nerve interaction is mediated by a soluble factor which is increased by castration. We now report that decreasing plasma testosterone levels decreased preprocholecystokinin messenger RNA levels. Testosterone replacement at the time of castration restored preprocholecystokinin messenger RNA levels to intact values. Injections of crude extracts prepared from denervated bulbocavernosus/levator ani into the homologous muscles of gonadally intact rats increased the levels of alpha-calcitonin gene-related peptide messenger RNA in spinal nucleus of the bulbocavernosus motoneurons. The levels of preprocholecystokinin messenger RNA did not differ in rats injected with denervated bulbocavernosus/levator ani extract or buffer, both of which were significantly higher than in intact, untreated rats. The results of the present experiments imply that levels of preprocholecystokinin and alpha-calcitonin gene-related peptide messenger ribonucleic acid are differentially regulated in spinal nucleus of the bulbocavernosus motoneurons.     

Glatiramer acetate positively influences spinal motoneuron survival and synaptic plasticity after ventral root avulsion

Avulsion of ventral roots induces degeneration of most axotomized motoneurons. At present there are no effective strategies to prevent such neuronal loss and to preserve the affected spinal circuits. Interestingly, changes in the spinal cord network also occur during the course of the experimental model of multiple sclerosis (experimental autoimmune encephalomyelitis—EAE). Glatiramer acetate (GA) significantly reduces the seriousness of the symptoms during the exacerbation of EAE. However, little is known about its effects on motoneurons. In the present study, we investigated whether GA has an influence on synapse plasticity and glial reaction after ventral root avulsion (VRA). Lewis rats were subjected to the avulsion of lumbar ventral roots and treated with GA. The animals were sacrificed after 14 days of treatment and the spinal cords processed for immunohistochemistry. A correlation between the synaptic changes and glial activation was obtained by performing immunolabeling against synaptophysin, GFAP and Iba-1. GA treatment preserved synaptophysin labeling, and significantly reduced the glial reaction in the area surrounding the axotomized motoneurons. After ventral root avulsion, GA treatment was also neuroprotective. The present results indicate that the immunomodulator GA has an influence on the stability of nerve terminals in the spinal cord, which may in turn contribute to future treatment strategies after proximal lesions to spinal motoneurons.     

肌源神经营养因子(MDNF)对体外培养的大鼠脊髓运动神经元缺氧时p53表达的影响

本研究目的是观察缺氧时肌源神经营养因子(MDNF)对体外培养的大鼠脊髓运动神经元p53蛋白表达的影响。从成鼠和胎鼠骨骼肌中提取MDNF。在胎鼠脊髓运动神经元培养第7 d时将其分成对照组和实验组(包括成鼠MDNF组和胎鼠MDNF组),取100μl MDNF(0.1μg/ml)加入培养大鼠脊髓运动神经元的培养液中,对照组加入等量的PBS。然后用液体石蜡封闭液面造成神经元缺氧损伤,缺氧3 d后,应用Nissl染色、原位末端标记、免疫组化方法,对损伤的大鼠脊髓运动神经元进行检测。结果表明:培养的大鼠脊髓运动神经元缺氧3 d后,经MDNF孵育可使脊髓运动神经元TUNEL、p53表达均较对照组明显减弱,神经元损伤程度减轻,神经元存活数明显高于对照组,且胎鼠MDNF的效果更好。提示:大鼠脊髓运动神经元缺氧后可出现神经细胞凋亡,但胎鼠MDNF较成鼠MDNF更能减弱缺氧时脊髓运动神经元p53的表达,抑制缺氧后神经元的死亡。     

Hyperthermia induces gene expression of heat shock protein 70 and phosphorylation of mitogen activated protein kinases in the rat cerebellum

The highly polysialylated neural cell adhesion molecule (PSA-NCAM) is recognized as a marker of neurogenesis or neural plasticity in adult nervous system. PSA-NCAM expression was examined in the spinal cord of transgenic mice harboring a mutant Cu/Zn superoxide dismutase (SOD1) gene. Immunohistochemistry showed a progressive expression of PSA-NCAM in surviving motoneurons of spinal ventral horns from an early and presymptomatic stage (25 weeks) before significant loss of ventral horn neurons, while no detectable PSA-NCAM in the ventral horn of non-transgenic littermates during the ageing process. The present data suggest that a specific expression of PSA-NCAM may be involved in the survival of spinal motoneurons under pathological conditions such as amyotrophic lateral sclerosis.     

Distribution of median, ulnar and radial motoneurons in the monkey spinal cord: a retrograde triple-labeling study.

Topographic distribution of motoneurons innervating hand muscles through the median (Mn), ulnar (Ul), or radial (Rd) nerves was examined using a retrograde multiple-labeling technique in the macaque monkey. The Mn and Ul motoneurons, i.e. flexor motoneurons, were distributed from C6 to T2 and from C7 to T2 segments of the spinal cord, respectively, while the Rd motoneurons, i.e. extensor motoneurons, were distributed from C4 to T2. The present study further revealed partial intermingling of the cell bodies and partial overlap of the dendritic fields among the motoneurons projecting through different nerves, indicating that subregions of motoneuronal pool participate in coordination between the flexor and extensor, or among the flexor muscles. It was suggested that there exists a control mechanism for precise hand movements in the spinal cord.     

Rescue of rat spinal motoneurons from avulsion-induced cell death by intrathecal administration of IGF-I and Cerebrolysin.

Ventral root avulsion results in the loss of motoneurons in the corresponding spinal cord segment. In the present experiments we have tested effects of insulin-like growth factor-I (IGF-I) and Cerebrolysin on survival of avulsed motoneurons after their chronic intrathecal administration in the adult rats. We have found that avulsion of the C5 ventral roots results in significant loss of motoneurons in the same spinal cord segment due mainly to apoptosis. In comparison to the untreated control rats, the amount of motoneuron survival in avulsed ventral horn was significantly higher after 4 weeks intrathecal administration of IGF-I or Cerebrolysin. No significant differences were observed between effects of IGF-I and Cerebrolysin in our experimental model. The results suggest that both IGF-I and Cerebrolysin can reduce avulsion-induced loss of adult rat motoneurons.     

Sexual dimorphism of the motoneurons in the human spinal cord

There have been few morphometric studies on the size of the motoneurons of the human spinal cord. The purpose of the present study is to report the differences in the size of the motoneurons between males and females in the human spinal cord. We examined numbers and transverse cell body areas of the motoneurons of the anterior horn using 16 male and 21 female human spinal cords at levels C5 and L3. The sizes of the motoneurons were larger in males than in females, but their numbers were practically the same. These results can be of great importance in setting standard values for the understanding of morphological and functional correlations.     

Monoaminergic and GABAergic terminations in phrenic nucleus of rat identified by immunohistochemical labeling

The termination patterns of axons in the phrenic nucleus immunoreactive to synthetic enzymes for catecholamines and for serotonin and GABA were studied in rats. Spinal cord tissue in which phrenic motoneurons were retrogradely labeled with horseradish peroxidase was incubated with antisera against dopamine beta-hydroxylase, phenylethanolamine-N-methyltransferase, 5-hydroxytryptamine, and GABA to identify presumptive terminations of monoaminergic and GABAergic neurons onto identified phrenic motoneurons. In the C3 to C5 spinal cord, 5-hydroxytryptamine-, dopamine beta-hydroxylase- and GABA-like positive terminals with varicosities formed a dense network, with presumptive synaptic contacts on dendrites and somas of phrenic motoneurons. A similar pattern of terminations was also observed in adjacent (non-respiratory muscle) motoneuron pools. There were fewer phenylethanolamine-N-methyltransferase-positive terminal arborizations in the cervical spinal cord compared to thoracic spinal cord; phenylethanolamine-N-methyltransferase terminals were not seen in the vicinity of phrenic motoneurons. These results suggest that phrenic motoneuronal activity is influenced by multiple supraspinal inputs utilizing different neurotransmitters. These transmitters also mediate inputs to other (nearby) spinal motoneurons and thus are not unique for signal transmission to phrenic motoneurons.     

Exogenous brain-derived neurotrophic factor regulates the synaptic composition of axonally lesioned and normal adult rat motoneurons

Brain-derived neurotrophic factor has previously been shown to promote survival and axonal regeneration in injured spinal motoneurons and, also, to modulate synaptic transmission and regulate the density of synaptic innervation in a variety of neurons. The present light and electron microscopic study demonstrates synaptotrophic effects of exogenously applied brain-derived neurotrophic factor on the synaptic composition of both normal and axonally lesioned adult rat spinal motoneurons. After L5-L6 ventral root avulsion, a massive loss of all types of boutons occurred on the somata of the lesioned motoneurons which persisted for at least 12 weeks postoperatively. We found that (i) intrathecal infusion of brain-derived neurotrophic factor during the first postoperative week did not prevent the synaptic detachment and activation of glial cells; (ii) prolonged treatment for four weeks restored synaptic covering and significantly reduced microglial reaction; (iii) the synaptotrophic effect remained significant for at least eight weeks after cessation of the treatment; (iv) brain-derived neurotrophic factor mainly supported F-type boutons with presumably inhibitory function, while it had little effect on S-type boutons associated with excitatory action; and (v) in normal unlesioned motoneurons, four weeks of treatment with brain-derived neurotrophic factor induced sprouting of F-type boutons, a loss of S-type boutons and motoneuron atrophy.The present data show that exogenous neurotrophins not only help to restore synaptic circuitry in axonally injured motoneurons, but also strongly influence the synaptic composition in normal motoneurons.     

Serotonin facilitates a persistent calcium current in motoneurons of rats with and without chronic spinal cord injury

In the months after spinal cord transection, motoneurons in the rat spinal cord develop large persistent inward currents (PICs) that are responsible for muscle spasticity. These PICs are mediated by low-threshold TTX-sensitive sodium currents (Na PIC) and L-type calcium currents (Ca PIC). Recently, the Na PIC was shown to become supersensitive to serotonin (5-HT) after chronic injury. In the present paper, a similar change in the sensitivity of the Ca PIC to 5-HT was investigated after injury. The whole sacrocaudal spinal cord from acute spinal rats and spastic chronic spinal rats (S2 level transection 2 mo previously) was studied in vitro. Intracellular recordings were made from motoneurons and slow voltages ramps were applied to measure PICs. TTX was used to block the Na PIC. For motoneurons of chronic spinal rats, a low dose of 5-HT (1 microM) significantly lowered the threshold of the Ca PIC from -56.7 +/- 6.0 to -63.1 +/- 7.1 mV and increased the amplitude of the Ca PIC from 2.4 +/- 1.0 to 3.0 +/- 0.73 nA. Higher doses of 5-HT acted similarly. For motoneurons of acute spinal rats, low doses of 5-HT had no significant effects, whereas a high dose (about 30 microM) significantly lowered the threshold of the L-Ca PIC from -58.5 +/- 14.8 to -62.5 +/- 3.6 mV and increased the amplitude of the Ca PIC from 0.69 +/- 1.05 to 1.27 +/- 1.1 nA. Thus Ca PICs in motoneurons are about 30-fold supersensitive to 5-HT in chronic spinal rats. The 5-HT-induced facilitation of the Ca PIC was blocked by nimodipine, not by the I(h) current blocker Cs(+) (3 mM) or the SK current blocker apamin (0.15 microM), and it lasted for hours after the removal of 5-HT from the nCSF, even increasing initially after removing 5-HT. The effects of 5-HT make motoneurons more excitable and ultimately lead to larger, more easily activated plateaus and self-sustained firing. The supersensitivity to 5-HT suggests the small amounts of endogenous 5-HT below the injury in a chronic spinal rat may act on supersensitive receptors to produce large Ca PICs and ultimately enable muscle spasms.     

Repeated stimuli for axonal growth causes motoneuron death in adult rats: the effect of botulinum toxin followed by partial denervation

Axons of motoneurons to tibialis anterior and extensor digitorum longus muscles of adult rats were induced to sprout by injecting botulinum toxin into them, by partial denervation or by a combination of the two procedures. Ten weeks later, the number of motoneurons innervating the control and operated tibialis anterior and extensor digitorum longus muscles was established by retrograde labelling with horseradish peroxidase. In the same preparations, the motoneurons were also stained with a Nissl stain (gallocyanin) to reveal motoneurons in the sciatic pool. Examination of the spinal cords from animals treated with botulinum toxin showed that the number of retrogradely labelled cells and those stained with gallocyanin in the ventral horn on the treated compared to the control side was unchanged. In rats that had their L4 spinal nerve sectioned on one side, the number of retrogradely labelled cells on the operated side was 48+/-3% (n = 5) of that present in the control unoperated ventral horn. Thus, just over half the innervation was removed by cutting the L4 spinal nerve. Counts made from gallocyanin-stained sections showed that 94+/-4% (n = 5) of motoneurons were present in the ventral horn on the operated side. Thus, section of the L4 spinal nerve did not lead to any death of motoneurons. In rats that had their muscles injected with botulinum toxin three weeks prior to partial denervation, the number of retrogradely labelled cells was reduced from 48+/-3% (n = 5) to 35+/-4% (n = 5). Moreover, only 67+/-5% (n = 5) of motoneurons stained with gallocyanin, suggesting that a proportion of motoneurons died after this combined procedure. This result was supported by experiments in which motor unit numbers in extensor digitorum longus muscles were determined by measurements of stepwise increments of force in response to stimulation of the motor nerve with increasing stimulus intensity. In partially denervated extensor digitorum longus muscles, 16.6+/-0.7 (n = 5) motor units could be identified, and in animals treated with botulinum toxin prior to partial denervation only 13.3+/-0.9 (n = 3) motor units were present. Taken together, these results show that treatment with botulinum toxin followed by partial denervation causes motoneuron death in adult rats.     

Synaptic inputs from low threshold afferents of trunk muscles to motoneurons innervating the longissimus lumborum muscle in the spinal cat

(1) We studied the reflex actions of group I and II afferents to longissimus lumborum (Long) motoneurons in the L1–L5 spinal segments from the epaxial muscle, m. Long, and the hypaxial muscle, m. obliquus externus abdominus (OEA). (2) Postsynaptic potentials (PSPs) recorded from 140 Long motoneurons in 30 spinal cats were analyzed. Under the present experimental conditions, the stimulation of Long and OEA nerves at an intensity below 1.5 times threshold (T) activated only group I muscle afferents, while stimulations at 2–5T activated group II muscle afferents as well. (3) The incidence of PSPs was related to the proximity of the spinal segments of the nerves stimulated to the spinal segment of the motoneurons; the shorter the distance the larger the PSPs and higher incidence of PSPs. The Long motoneurons received group I afferent input mainly from the same and adjacent segments, and received group II afferent inputs from a wider range of segments. (4) A short (i.e., less than 1.0 ms) latency of excitatory PSPs (EPSPs) evoked by ipsilateral group I afferents of Long at the same or adjacent segment indicated a monosynaptic connection. In general, the central latencies became longer as the distance between spinal segments of stimulated nerves and motoneurons increased. Major PSP components were produced by polysynaptic neuronal pathways. The spatial facilitation between PSPs evoked by afferents of different nerves (i.e., ipsilateral Long (iLong) and contralateral Long (cLong) of the same segment; iLongs of different segments; and iLong and iOEA of the same segment) indicated that they shared common interneurons. (5) Although iLong and iOEA muscle afferents produced predominantly EPSPs, and contralateral muscle afferents elicited predominantly IPSPs in Long motoneurons at each spinal segment, the patterns of convergence from Long and OEA muscle afferents of different spinal segments and of different sides differed considerably among motoneurons. (6) These findings demonstrated various input patterns of individual motoneurons within the same motoneuron pool, which might reflect the complexity of neuronal control of the back muscles for various trunk movements, including lateral and dorsal bending, rotating, and fixation of the trunk. Electronic Publication     

Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons

Motoneurons represent a specialized class of neurons essential for the control of body movement. Motoneuron loss is the cause of a wide range of neurological disorders including amyotrophic lateral sclerosis and spinal muscular atrophy. Embryonic stem cells are a promising cell source for the study and potential treatment of motoneuron diseases. Here, we present a novel in vitro protocol of the directed differentiation of human embryonic stem cells (hESCs) into engraftable motoneurons. Neural induction of hESCs was induced on MS5 stromal feeders, resulting in the formation of neural rosettes. In response to sonic hedgehog and retinoic acid, neural rosettes were efficiently directed into spinal motoneurons with appropriate in vitro morphological, physiological, and biochemical properties. Global gene expression analysis was used as an unbiased measure to confirm motoneuron identity and type. Transplantation of motoneuron progeny into the developing chick embryo resulted in robust engraftment, maintenance of motoneuron phenotype, and long-distance axonal projections into peripheral host tissues. Transplantation into the adult rat spinal cord yielded neural grafts comprising a large number of human motoneurons with outgrowth of choline acetyltransferase positive fibers. These data provide evidence for in vivo survival of hESC-derived motoneurons, a key requirement in the development of hESC-based cell therapy in motoneuron disease. Disclosure of potential conflicts of interest is found at the end of this article.     

Targeting functional subtypes of spinal motoneurons and skeletal muscle fibers in vivo by intramuscular injection of adenoviral and adeno-associated viral vectors

We report that functional subtypes of spinal motoneurons and skeletal muscle fibers can be selectively transduced using replication-defective adenoviral (ADV) or adeno-associated (AAV) viral vectors. After intramuscular injection in adult rodents, ADV vectors transduced both fast-twitch and slow-twitch skeletal muscle fibers. Intramuscular injection of ADV vectors also caused transduction of spinal motoneurons and dorsal root ganglion cells. However, only neurons innervating the injected muscle were transduced, as shown by co-injection of a retrograde axonal tracer. In adult male rats it is therefore possible to transduce fast or slow spinal motoneurons and muscle fibers selectively since in these animals, the extensor digitorum longus and soleus muscles contain almost exclusively fast or slow motor units, respectively. In rats, AAV vectors transduced muscle fibers in the predominantly fast extensor digitorum longus but not in the predominantly slow soleus muscle. We did not observe any transduction of spinal motoneurons following intramuscular injection of AAV vectors. These results show that physiologically and clinically important subpopulations of cells in the neuromuscular system can be selectively transduced by viral vectors.     

Differential synaptic effects on physiological flexor hindlimb motoneurons from cutaneous nerve inputs in spinal cat.

1. We previously demonstrated in the spinal cat that superficial peroneal cutaneous nerve stimulation produced strong reflex contraction in tibialis anterior (TA) and semitendinosus (St) muscles but unexpectedly produced mixed effects in another physiological flexor muscle, extensor digitorum longus (EDL). The goal of the present study was to further characterize the organization of ipsilateral cutaneous reflexes by examining the postsynaptic potentials (PSPs) produced in St, TA, and EDL motoneurons by superficial peroneal and saphenous nerve stimulation in decerebrate, spinal cats. 2. In TA and St motoneurons, low-intensity cutaneous nerve stimulation that activated only large (A alpha) fibers [i.e., approximately 2-3 times threshold (T)], typically produced biphasic PSPs consisting of an initial excitatory phase and subsequent inhibitory phase (EPSP, IPSP). Increasing the stimulus intensity to activate both large (A alpha) and small (A delta) myelinated cutaneous fibers supramaximally (15-45 T) tended to enhance later excitatory components in TA and St motoneurons. 3. In EDL motoneurons, 2-3 T stimulation of the superficial peroneal nerve evoked initial inhibition (of variable magnitude) in 7/10 EDL motoneurons tested, with either excitation (n = 2) or mixed effects (n = 1) observed in the remaining EDL motoneurons. Saphenous nerve stimuli produced excitation either alone, or preceded by an inhibitory phase in EDL. Increasing the stimulus intensity enhanced later inhibitory influences from superficial peroneal and excitatory influences both from superficial peroneal and saphenous nerve inputs in EDL motoneurons. 4. Short-latency (less than 1.8 ms) EPSPs were observed in a few motoneurons in all reflex pathways examined, except for EPSPs in EDL motoneurons evoked by saphenous stimulation. IPSPs with central latencies less than 1.8 ms were also produced by both saphenous (TA, n = 1; EDL, n = 2) and superficial peroneal (EDL, n = 4) nerve stimulation. 5. The results, in comparison with other reports employing spinal and nonspinal preparations, suggest that removal of influences from higher centers reveals inhibitory circuits from the superficial peroneal and saphenous nerves to EDL motoneurons in the spinal preparation. The inhibitory inputs observed are thought to reflect the activation of "specialized" reflex pathways. Additionally, the demonstration of short-latency EPSPs and IPSPs suggest that the minimal linkage in both the excitatory and inhibitory cutaneous reflex pathways examined is disynaptic. The results are discussed in relation to previous studies on classically conditioned flexion reflex facilitation in spinal cat.     

Dense transient receptor potential cation channel, vanilloid family, type 2 (TRPV2) immunoreactivity defines a subset of motoneurons in the dorsal lateral nucleus of the spinal cord, the nucleus ambiguus and the trigeminal motor nucleus in rat

The transient receptor potential cation channel, vanilloid family, type 2 (TRPV2) is a member of the TRPV family of proteins and is a homologue of the capsaicin/vanilloid receptor (transient receptor potential cation channel, vanilloid family, type 1, TRPV1). Like TRPV1, TRPV2 is expressed in a subset of dorsal root ganglia (DRG) neurons that project to superficial laminae of the spinal cord dorsal horn. Because noxious heat (>52 degrees C) activates TRPV2 in transfected cells this channel has been implicated in the processing of high intensity thermal pain messages in vivo. In contrast to TRPV1, however, which is restricted to small diameter DRG neurons, there is significant TRPV2 immunoreactivity in a variety of CNS regions. The present report focuses on a subset of neurons in the brainstem and spinal cord of the rat including the dorsal lateral nucleus (DLN) of the spinal cord, the nucleus ambiguus, and the motor trigeminal nucleus. Double label immunocytochemistry with markers of motoneurons, combined with retrograde labeling, established that these cells are, in fact, motoneurons. With the exception of their smaller diameter, these cells did not differ from other motoneurons, which are only lightly TRPV2-immunoreactive. As for the majority of DLN neurons, the densely-labeled populations co-express androgen receptor and follow normal DLN ontogeny. The functional significance of the very intense TRPV2 expression in these three distinct spinal cord and brainstem motoneurons groups remains to be determined.     

Expression of GABA-immunoreactivity by spinal motoneurons of some vertebrates

Electrophysiological and biochemical investigations have shown that gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the vertebrate central nervous system. However, the present study shows that some motoneurons located in the spinal cord of young chickens and adult monkeys display a GABA-like immunoreactivity. The expression of GABA immunoreactivity in vertebrate motoneurons suggests that this inhibitory amino acid is colocalized with acetylcholine and could play a role in the neuromuscular transmission.     

Reversal of the late phase of spike frequency adaptation in cat spinal motoneurons during fictive locomotion

In spinal motoneurons, late spike frequency adaptation (SFA) is defined as the slowing of the firing rate over tens of seconds and can be seen during sustained or intermittent current injection. Although the function of late SFA is not known, it may result in a decrease in force production over time, or muscle fatigue. Because locomotion can persist for long periods of time without fatigue, late SFA was studied using intracellular recordings from adult cat motoneurons during fictive locomotion. Of eight lumbar motoneurons studied, all showed late adaptation during control conditions, but none demonstrated late adaptation during locomotor activity. The most consistent properties that correlated with the presence or absence of late SFA were those related to availability of fast, inactivating sodium channels, particularly action potential rate of rise. Evidence of the reversal of late SFA during locomotion was present for several minutes following locomotor trials, consistent with the suggestion that SFA is modulated through slow metabotropic pathways. The abolition of late adaptation in spinal motoneurons during fictive locomotion is an example of a state-dependent change in the "intrinsic" properties of mammalian motoneurons. This change contributes to increased excitability of motoneurons during locomotion and results in robust firing during sustained locomotion.     

Viral delivery of NR2D subunits reduces Mg2+ block of NMDA receptor and restores NT-3-induced potentiation of AMPA-kainate responses in maturing rat motoneurons

N-methyl-D-aspartate (NMDA) responsiveness of motoneurons declines during the initial 2 postnatal weeks due to increasing Mg2+ block of NMDA receptors. Using gene chip analyses, RT-PCR, and immunochemistry, we have shown that the NR2D subunit of the NMDA receptor (NMDAR), known to confer resistance to Mg2+ block, also declines in motoneurons during this period. We injected a viral construct (HSVnr2d) into the lumbar spinal cord on postnatal day 2 in an attempt to restore NMDAR function in motoneurons during the second postnatal week. Following HSVnr2d injection, we detected elevated levels of NR2D mRNA in spinal cord samples and NR2D protein specifically in motoneurons. These molecular changes were associated with marked functional alterations whereby NMDAR-mediated responses in motoneurons associated with both dorsal root (DR) and ventrolateral funiculus (VLF) inputs returned to values observed at E18 due to decreased Mg2+ blockade. Viruses carrying the beta-galactosidase gene did not induce these effects. NT-3 is known to potentiate AMPA-kainate responses in motoneurons if the response has an NMDAR-mediated component and thus is normally ineffective during the second postnatal week. Restoration of NMDAR-mediated responsiveness in the second postnatal week was accompanied by a return of the ability of neurotrophin-3 (NT-3) to potentiate the AMPA-kainate responses produced by both DR and VLF synaptic inputs. We conclude that delivery of the gene for a specific NMDA subunit can restore properties characteristic of younger animals to spinal cord motoneurons. This approach might be useful for enhancing the function of fibers surviving in the damaged spinal cord.