Adult Spinal Motoneurons Research Articles

Adult spinal motoneurons change their neurotransmitter phenotype to control locomotion

A particularly essential determinant of a neuron's functionality is its neurotransmitter phenotype. While the prevailing view is that neurotransmitter phenotypes are fixed and determined early during development, a growing body of evidence suggests that neurons retain the ability to switch between different neurotransmitters. However, such changes are considered unlikely in motoneurons due to their crucial functional role in animals' behavior. Here we describe the expression and dynamics of glutamatergic neurotransmission in the adult zebrafish spinal motoneuron circuit assembly. We demonstrate that part of the fast motoneurons retain the ability to switch their neurotransmitter phenotype under physiological (exercise/training) and pathophysiological (spinal cord injury) conditions to corelease glutamate in the neuromuscular junctions to enhance animals' motor output. Our findings suggest that motoneuron neurotransmitter switching is an important plasticity-bestowing mechanism in the reconfiguration of spinal circuits that control movements.

Erythropoietin Attenuates the Apoptosis of Adult Neurons After Brachial Plexus Root Avulsion by Downregulating JNK Phosphorylation and c-Jun Expression and Inhibiting c-PARP Cleavage.

In the present study, the effects of erythropoietin (EPO) on preventing adult neurons from apoptosis (introduced by brachial plexus avulsion) were examined, and the mechanism was analyzed. Fifty injury rat models were established in this study by using micro-hemostat forceps to pull out brachial plexus root from the intervertebral foramen in supine position. These models were divided into EPO group (avulsion + 1000U/kg subcutaneously on alternate days) and control group (avulsion + normal saline). C5-T1 spinal cord was harvested at days 1, 2, 4, 7, and 14. Compared with the control group, the apoptosis of spinal motoneurons was significantly decreased on days 4 and 7 in the EPO group, which was also approved by TUNEL examination results. The detection of p-JNK and expression of c-Jun and cleavage of cleaved PARP (c-PARP) were also examined by immunohistochemistry and were increased immediately at day 1, and peaked at day 2, day 2, and day 4 in control group, respectively. However, the amounts were decreased and delayed by EPO treatment significantly at the same time points. In conclusion, the apoptosis of adult spinal motorneurons was associated with JNK phosphorylation, c-Jun expression, and caspase activity, and EPO-mediated neuronal protective effect is proved by downregulating the JNK phosphorylation and c-Jun expression and inhibiting of c-PARP cleavage.

Enhancement of cultured adult motor neuron survival with cold pre-incubation

The aim of the study was to extend the survival of adult spinal motor neurons in serum free culture. Anterior half of the spinal cord was removed from young adult mice and dissociated. Cultured cells attempted to extend neurites within hours of incubation at 37°C and died within 24h. To prevent this early regenerative activity, thus to decrease the metabolic requirements of the neurons, cultures were transferred to 4°C immediately after they were set and kept there for 3 days. Preparations were then taken to 37°C where they lived up to 8 days. Some neurons continued to extend neurites until the day they died. To understand whether the enhancement of survival involves new protein synthesis, transcription and translation were blocked during cold pre-incubation, which shortened the half life of neurons but not changed the maximum survival period. In conclusion this study has shown that, in the serum-free cultures, the survival of adult spinal motor neurons can be significantly enhanced by cold pre-incubation whose effect seems to depend largely on a reduction in the metabolic activity and less on new protein synthesis.

Microarray analysis of gene expression patterns in adult spinal motoneurons after different types of axonal injuries

Three experimental models of axonal injuries in adult rat spinal motoneurons were established to investigate changes of gene expression in response to such injuries. We took advantage of cDNA microarray analysis to determine the differential expression of genes in injured motoneurons following distal axotomy or root avulsion in the absence or presence of BDNF. The major finding was that, in response to proximal axonal injury (avulsion), expression of genes that are known to facilitate neuronal survival and axonal regeneration (e.g., IGFRII, PI3K, IGFBP-6, GSTs, GalR2) were down-regulated; but following treatment with BDNF they were up-regulated. In addition, the expression of genes known to be involved in apoptosis and DNA damage (e.g., ANX5, TS, ALR) were down-regulated in BDNF-treated animals with avulsion. Furthermore, many functional families of genes previously shown to play roles in the pathophysiology of axonal injury, including SNAP-25A, SV2B, Ras-related ras3a/4b, ERK1/2, 14-3-3 proteins, proteasome proteins, oncogenes, GAP-43, and NMDAR1, were altered after either distal axotomy or avulsion injury. Some of the changes in gene expression, including Lim-2, FRAG1, GlaR2, GSTs, ALR, TS, ANX3/5, and nhe1/2, are first reported here in injured motoneurons. The differential expression of genes identified by the expression arrays was confirmed by gene-specific RT-PCR for eight genes (GAP-43, IGFR II, Lim-2, MIF, NDAP1, TS, PCC3, and FRAG1) and by in situ hybridization for Lim-2. These results suggest that abnormal regulation of particular biochemical pathways may induce motoneuron death after ventral root avulsion in adult animals. This study presents an approach for selecting specific genes and their products that may be involved in motoneuron degeneration following axonal injuries.

Antisense oligos to neuronal nitric oxide synthase aggravate motoneuron death induced by spinal root avulsion in adult rat

The present study used nitric oxide synthase (nNOS) antisense oligos (nNOS AS-ODN) to assess the role of nNOS in motoneuron death induced by spinal root avulsion. A right seventh cervical (C7) spinal root avulsion was performed on adult male Sprague–Dawley rats. Two weeks later, FITC-labeled random oligos (FITC-R-ODN), nNOS AS-ODN, R-ODN or TE buffer was applied to the lesioned side of the C7 spinal segment and refreshed every 3 days. FITC-R-ODN was first detected inside the injured motoneurons at 10 h, accumulated to a maximum by 24 h and faded out from 72 h. Following avulsion, nNOS AS-ODN decreased the number of nNOS-positive motoneurons in the lesioned segment compared either with buffer ( P < 0.001 at 15 days, 3 and 4 weeks post-injury) or with R-ODN control ( P = 0.002 at 15 days, P < 0.001 at 3 and 4 weeks post-injury). Interestingly, nNOS AS-ODN also decreased the number of surviving motoneurons compared either with buffer ( P = 0.005 at 15 days, P < 0.001 at 3 or 4 weeks) or with R-ODN control ( P < 0.001 at 3 or 4 weeks). Meanwhile, there were no significant differences between R-ODN and buffer control either in the number of nNOS-positive motoneurons ( P = 0.245 at 15 days, P = 0.089 at 3 weeks and P = 0.162 at 4 weeks) or in the number of surviving motoneurons ( P = 0.426 at 15 days, P = 0.321 at 3 weeks or P = 0.344 at 4 weeks). These findings indicate that nNOS AS-ODN, applied from 2 weeks after avulsion, aggravates the motoneuron death due to root avulsion by specifically down-regulating nNOS gene expression and that the expression of nNOS in adult spinal motoneurons in response to root avulsion may play a beneficial role in the survival of injured neurons.

Delayed implantation of a peripheral nerve graft reduces motoneuron survival but does not affect regeneration following spinal root avulsion in adult rats.

Adult spinal motoneurons can regenerate their axons into a peripheral nerve (PN) graft following root avulsion injury if the graft is implanted immediately after the lesion is induced. The present study was designed to determine how avulsed motoneurons respond to a PN graft if implantation takes place a few days to a few weeks later. Survival, regeneration, and gene expression changes of injured motoneurons after delayed PN graft implantation were studied. The survival rates of spinal motoneurons were 78%, 65%, 57%, or 53% if a PN graft was implanted immediately, 1, 2, or 3 weeks after root avulsion, respectively. Interestingly, most of the surviving motoneurons were able to regenerate their axons into the graft regardless of the delay. All regenerating motoneurons expressed p75, but not nNOS, while all motoneurons that failed to regenerate expressed nNOS, but not p75. p75 and nNOS may, therefore, be used as markers for success or failure to regenerate axons. In the group with immediate graft implantation, 85% of the surviving motoneurons extended axons into the PN graft, while in the groups in which implantation was delayed 1, 2, or 3 weeks, 84%, 82%, and 83% of the surviving motoneurons, respectively, were found to have regenerated into the grafts. These findings indicate that avulsed spinal motoneurons retain the ability to regenerate for at least 3 weeks, and perhaps for as long as they survive. Therefore, the delayed implantation of a PN graft after root avulsion may provide a continued conducive environment to support regeneration.

5-HT1A receptors increase excitability of spinal motoneurons by inhibiting a TASK-1-like K+ current in the adult turtle

The modulatory effects of serotonin mediated by 5-HT1A receptors in adult spinal motoneurons were investigated by intracellular recordings in a slice preparation from the turtle. In current-clamp mode, activation of 5-HT1A receptors by 8-OH-DPAT led to depolarization and an increase in input resistance in most motoneurons but caused hyperpolarization and a decrease in input resistance in the remaining smaller fraction of cells. When slices were preincubated in medium containing the 5-HT1A receptor antagonist WAY-100635, 8-OH-DPAT had no effect. In voltage-clamp mode, with 1 mm CsCl in the bathing medium, 8-OH-DPAT consistently inhibited a leak current that was sensitive to extracellular acidification and anandamide, a TASK-1 channel blocker. In medium with a low pH, as in the presence of anandamide, 8-OH-DPAT had no effect. Our results show that activation of 5-HT1A receptors contributes to the excitatory effect of serotonin on spinal motoneurons by inhibition of a TASK-1 potassium channel leading to depolarization and increased input resistance.

5-HT1A receptors increase excitability of spinal motoneurons by inhibiting a TASK-1-like K+ current in the adult turtle.

The modulatory effects of serotonin mediated by 5-HT1A receptors in adult spinal motoneurons were investigated by intracellular recordings in a slice preparation from the turtle. In current-clamp mode, activation of 5-HT1A receptors by 8-OH-DPAT led to depolarization and an increase in input resistance in most motoneurons but caused hyperpolarization and a decrease in input resistance in the remaining smaller fraction of cells. When slices were preincubated in medium containing the 5-HT1A receptor antagonist WAY-100635, 8-OH-DPAT had no effect. In voltage-clamp mode, with 1 mM CsCl in the bathing medium, 8-OH-DPAT consistently inhibited a leak current that was sensitive to extracellular acidification and anandamide, a TASK-1 channel blocker. In medium with a low pH, as in the presence of anandamide, 8-OH-DPAT had no effect. Our results show that activation of 5-HT1A receptors contributes to the excitatory effect of serotonin on spinal motoneurons by inhibition of a TASK-1 potassium channel leading to depolarization and increased input resistance.

Caspase inhibitors promote the survival of avulsed spinal motoneurons in neonatal rats

Following ventral root avulsion in neonatal animals, the degeneration of spinal motoneurons occurs by an apoptotic-like morphological pathway. In adult animals, however, the mechanism of degeneration of injured motoneurons is still controversial. Because caspases are important mediators of apoptosis, we have investigated the effects of the caspase inhibitors, benzyloxycarbonyl-Asp(OMe)fluoromethylketone (Boc-D-FMK), and N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO) on the survival of neonatal and adult spinal motoneurons after root avulsion of the C7 spinal cord. In the control neonatal animals, virtually all motoneurons had degenerated by 7 days following root avulsion. Treatment with either 0.5 microg Boc-D-FMK or 1 microg Ac-DEVD-CHO enhanced the survival of motoneurons to 80% and 85% for up to 2 weeks post-injury. By 21 days post-injury, 70% of avulsed motoneurons were still present after Boc-D-FMK treatment, whereas all avulsed motoneurons died after treatment with Ac-DEVD-CHO. In adult animals, neither inhibitor was neuroprotective for motoneurons following root avulsion. In summary, the inhibition of caspases effectively rescued avulsed neonatal motoneurons which are died by apoptotic pathway. By contrast, because caspase inhibitors failed to rescue injured motoneurons in adult animals, their death may occur by a non-apoptotic pathway.

Developmental changes in expression of myotonic dystrophy protein kinase in the rat central nervous system.

Myotonic dystrophy protein kinase (DMPK) is the protein product of the genetic locus associated with myotonic dystrophy, in which alterations of muscle excitability, cardiac conduction defects, mental retardation, and cognitive deficiencies are inherited as an autosomal dominant trait. DMPK belongs to a novel protein serine/threonine kinase family, but its regulation and physiological functions have not been specified. In a first step toward understanding the functions of DMPK in the central nervous system, we have characterized its localization and developmental pattern of expression in rat brain and spinal cord by using a monospecific rabbit antiserum produced against bacterially expressed DMPK. Expression of DMPK begins after birth and increases gradually to peak at postnatal day 21 with antibody labeling of neuronal cell types in many regions. After postnatal day 21 and proceeding to the adult, the pattern of expression becomes more restricted, with localization to certain regions or cell groups in the central nervous system. Electron microscopy reveals localization within adult spinal motor neurons to the endoplasmic reticulum and dendritic microtubules. The adult localizations suggest that DMPK may function in membrane trafficking and secretion within neurons associated with cognition, memory, and motor control.

Effects of neurotransplants and BDNF on the survival and regeneration of injured adult spinal motoneurons.

We compared the effects of peripheral nerve grafts, embryonic spinal cord transplants and brain-derived neurotrophic factor (BDNF) on the survival and axon regeneration of adult rat spinal motor neurons undergoing retrograde degeneration after ventral root avulsion. Following implantation into the dorsolateral funiculus of the injured spinal cord segment, neither a peripheral nerve graft nor a combination of peripheral nerve graft with embryonic spinal cord transplant could prevent the retrograde motor neuron degeneration induced by ventral root avulsion. However, intrathecal infusion of BDNF promoted long-term survival of the lesioned motor neurons and induced abundant motor axon regeneration from the avulsion zone along the spinal cord surface towards the BDNF source. A combination of ventral root reconstitution and BDNF treatment might therefore be a promising means for the support of both motor neuron survival and guided motor axon regeneration after ventral root lesions.

Influence of voltage-sensitive dendritic conductances on bistable firing and effective synaptic current in cat spinal motoneurons in vivo.

1. After application of the noradrenergic alpha 1 agonist methoxamine, muscle spindle Ia input evoked bistable firing patterns (i.e., persistent discharges after Ia input ceased) in adult spinal motoneurons in the decerebrate cat preparation. These bistable discharge patterns were compared with the Ia currents generated in voltage-clamp conditions. 2. During voltage clamp at depolarized holding potentials, the Ia effective synaptic current underwent strong amplification. In those cells with strong bistable firing, a prolonged tail current followed the Ia input. Because the voltage clamp held the behavior of somatic conductances constant, these data indicate that voltage-sensitive conductances in the motoneuron dendrites made an important contribution to bistable firing.

Brain-derived neurotrophic factor promotes survival and blocks nitric oxide synthase expression in adult rat spinal motoneurons after ventral root avulsion

In adult spinal motoneurons, retrograde cell death is induced by ventral root avulsion. A lethal effect of nitric oxide has been implicated, since nitric oxide synthase (NOS) is expressed in the motoneurons destined to die. Our study investigates the effects of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) on the retrograde cell death and NOS expression of adult rat spinal motoneurons. Following ventral root avulsion and 4 weeks of continuous treatment, BDNF, but not CNTF, was found to prevent cell death and NOS expression in the lesioned motoneurons. This suggests a therapeutic potential for BDNF in the adult nervous system, possibly through blockage of nitric oxide synthesis.

BDNF and NT-4/5 exert neurotrophic influences on injured adult spinal motor neurons

Adult motor neurons, like their immature antecedents, express the mRNA for the signaling receptor for brain-derived neurotrophic factor (BDNF) and for neurotrophin-4/5 (NT-4/5). However, while both BDNF and NT-4/5 support the survival of axotomized developing spinal motor neurons in vitro or in vivo, it is not known whether these factors continue to influence spinal motor neurons in adulthood. The present study tests if BDNF or NT-4/5 modulate the reactive responses of adult spinal motor neurons to nerve injury. We utilize sciatic nerve transection to axotomize the spinal motor neurons that form the retrodorsal lateral nucleus (RDLN) and show that, after axotomy, RDLN motor neurons lose ChAT immunoreactivity and also reexpress p75Ingfr, the low affinity receptor for all neurotrophin family members. Treatment with BDNF or NT-4/5 alters these effects of sciatic nerve transection. Both BDNF and NT-4/5 attenuate the loss of ChAT expression in axotomized RDLN motor neurons; thus, as compared to vehicle treatments, BDNF and NT-4/5 produce statistically significant increases in the optical density of ChAT immunostaining. Furthermore, BDNF and NT-4/5 also significantly increase the RDLN reexpression of p75Ingfr after sciatic nerve transection. Interestingly, essentially identical increases in RDLN ChAT and p75Ingfr immunostaining are produced by sciatic nerve crush injuries in the absence of exogenous neurotrophin treatment. These data show that treatment with exogenous BDNF and NT-4/5 changes the response of adult spinal motor neurons to sciatic nerve transection. Furthermore, these neurotrophins elicit reactive responses in axotomized motor neurons that mimic those produced by endogenous agents in regenerating crushed peripheral nerve.

Adult Spinal Motoneurons Remain Viable Despite Prolonged Absence of Functional Synaptic Contact with Muscle

Several rat medial gastrocnemius (MG) motor axons were allowed to regenerate into normally innervated muscle. Under these conditions, synapse formation is known to be prevented by the existence of the original innervation of the host muscle. A study was made of the ability of the implanted spinal motoneurons to acquire and retrogradely transport horseradish peroxidase (HRP) injected into the host muscle at various postoperative intervals. HRP-labeled MG motoneurons on the implanted side were observed at postoperative intervals as long as 290 days. A comparison of the number of labeled MG motoneurons on the implanted side versus the number on the unoperated, control side indicated no significant differences. At all investigated postoperative intervals except the earliest (7 DPO), a significant decrease in the mean MG motoneuron soma cross-sectional area was observed relative to the unoperated, control side. Analysis of labeled motoneuron size distributions showed that postoperative atrophy of larger, presumably alpha, motoneurons occurred at a significantly faster rate than in smaller, presumably gamma, motoneurons. These results demonstrate that axotomized adult spinal motoneurons survive and remain viable for prolonged periods when denied the opportunity to reinnervate muscle but do so in an atrophied state. The results indicate further that alpha and gamma motoneurons differ quantitatively in their responses to peripheral axotomy.