Coherence analysis of the calcium activity of putative astrocytic and neuronal cells on the L5 ventral horn and neural output in activated lumbar CPG networks.

Astrocytes are thought to play a crucial role in providing structure to the spinal cord and maintaining efficient synaptic function and metabolism because their fine processes envelop the synapses of neurons and form many neuronal networks within the central nervous system (CNS). To investigate whether putative astrocytes and putative neurons distributed on the ventral horn play a role in the modulation of lumbar locomotor central pattern generator (CPG) networks, we used extracellular recording and optical imaging techniques and recorded the neural output from the left L5 ventral root and the calcium activity of putative astrocytes and neurons in the L5 ventral horn at the same time when activating an isolated L1-L5 spinal cord preparation from rats aged 0-2 days. Optical measurements detected cells that showed a fluorescence intensity change under all experimental conditions, namely, (1) 5-HT + NMDA, (2) TTX, and (3) TTX + Low K+. These cells were semiautomatically identified using an in-house MATLAB-based program, as putative astrocytes and neurons according to the cell classification, i.e., increased or decreased fluorescence intensity change (ΔF/F0), and subjective judgment based on their soma size. Coherence and its phase were calculated according to the calcium activity of the putative astrocytes and putative neurons, and neural output was calculated during fictive locomotion with in-house MATLAB-based programs. We found that the number of putative astrocytes activated by applying low K+ tends not to differ from that activated by applying the protease-activated receptor 1 (PAR1) selective agonist TFLLR-NH2 (TFLLR). Moreover, the calcium activity of several putative astrocytes and neurons synchronized with locomotor-like activity at a frequency range below 0.5 Hz and the time lag between peaks of cellular calcium activity and locomotor-like activity ranged from -1000 to + 1000 ms. These findings presumably indicates that these putative astrocytes and neurons in the left L5 ventral horn require -1000 to + 1000 ms to communicate with lumbar CPG networks and maintain efficient synaptic function and metabolism in activated lumbar CPG networks. This finding suggests the possibility that putative astrocytic and neuronal cells in the L5 ventral horn contribute to generating the rhythms and patterns of locomotor-like activity by activated CPG networks in the first to fifth lumbar spinal cord.

Bisperoxovanadium promotes motor neuron survival and neuromuscular innervation in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN) disease, with no present cure. The progressive loss of MNs is the hallmark of ALS. We have previously shown the therapeutic effects of the phosphatase and tensin homolog (PTEN) inhibitor, potassium bisperoxo (picolinato) vanadium (bpV[pic]), in models of neurological injury and demonstrated significant neuroprotective effects on MN survival. However, accumulating evidence suggests PTEN is detrimental for MN survival in ALS. Therefore, we hypothesized that treating the mutant superoxide dismutase 1 G93A (mSOD1G93A) mouse model of ALS during motor neuron degeneration and an in vitro model of mSOD1G93A motor neuron injury with bpV(pic) would prevent motor neuron loss. To test our hypothesis, we treated mSOD1G93A mice intraperitoneally daily with 400 µg/kg bpV(pic) from 70 to 90 days of age. Immunolabeled MNs and microglial reactivity were analyzed in lumbar spinal cord tissue, and bpV(pic) treatment significantly ameliorated ventral horn motor neuron loss in mSOD1G93A mice (p = 0.003) while not significantly altering microglial reactivity (p = 0.701). Treatment with bpV(pic) also significantly increased neuromuscular innervation (p = 0.018) but did not affect muscle atrophy. We also cultured motor neuron-like NSC-34 cells transfected with a plasmid to overexpress mutant SOD1G93A and starved them in serum-free medium for 24 h with and without bpV(pic) and downstream inhibitor of Akt signaling, LY294002. In vitro, bpV(pic) improved neuronal viability, and Akt inhibition reversed this protective effect (p < 0.05). In conclusion, our study indicates systemic bpV(pic) treatment could be a valuable neuroprotective therapy for ALS.

Long-Term Suppression of c-Jun and nNOS Preserves Ultrastructural Features of Lower Motor Neurons and Forelimb Function after Brachial Plexus Roots Avulsion.

Brachial plexus root avulsions cause debilitating upper limb paralysis. Short-term neuroprotective treatments have reported preservation of motor neurons and function in model animals while reports of long-term benefits of such treatments are scarce, especially the morphological sequelae. This morphological study investigated the long-term suppression of c-Jun- and neuronal nitric oxide synthase (nNOS) (neuroprotective treatments for one month) on the motor neuron survival, ultrastructural features of lower motor neurons, and forelimb function at six months after brachial plexus roots avulsion. Neuroprotective treatments reduced oxidative stress and preserved ventral horn motor neurons at the end of the 28-day treatment period relative to vehicle treated ones. Motor neuron sparing was associated with suppression of c-Jun, nNOS, and pro-apoptotic proteins Bim and caspases at this time point. Following 6 months of survival, neutral red staining revealed a significant loss of most of the motor neurons and ventral horn atrophy in the avulsed C6, 7, and 8 cervical segments among the vehicle-treated rats (n = 4). However, rats that received neuroprotective treatments c-Jun JNK inhibitor, SP600125 (n = 4) and a selective inhibitor of nNOS, 7-nitroindazole (n = 4), retained over half of their motor neurons in the ipsilateral avulsed side compared. Myelinated axons in the avulsed ventral horns of vehicle-treated rats were smaller but numerous compared to the intact contralateral ventral horns or neuroprotective-treated groups. In the neuroprotective treatment groups, there was the preservation of myelin thickness around large-caliber axons. Ultrastructural evaluation also confirmed the preservation of organelles including mitochondria and synapses in the two groups that received neuroprotective treatments compared with vehicle controls. Also, forelimb functional evaluation demonstrated that neuroprotective treatments improved functional abilities in the rats. In conclusion, neuroprotective treatments aimed at suppressing degenerative c-Jun and nNOS attenuated apoptosis, provided long-term preservation of motor neurons, their organelles, ventral horn size, and forelimb function.

Orexin-A potentiates glycine currents by activating OX1R and IP3/Ca2+/PKC signaling pathways in spinal cord ventral horn neurons.

Orexin-A/B modulates multiple physical functions by activating their receptors (OX1R and OX2R), but its effects in the spinal cord motor control remain unknown. Using acute separation (by digestive enzyme) of cells and patch-clamp recordings, we aimed to investigate the effect and mechanisms of orexin-A on the glycine receptors in the spinal cord ventral horn neurons. Orexin-A potentiated the glycine currents by activating OX1R. In Ca2+-free extracellular solution, orexin-A still increased the glycine currents. While, the orexin-A-induced potentiation was blocked when Ca2+ was chelated by internal infusion of BAPTA, and the orexin-A effect was abolished by the IP3 receptor antagonists heparin and Xe-C. The PKC inhibitor Bis-IV nullified the orexin-A effect. In addition, orexin-A did not cause a further enhancement of the glycine currents after bath application of the PKC activator PMA. In conclusion, after OX1R is activated, a distinct IP3/Ca2+-dependent PKC signaling pathway, is likely responsible for the orexin-A potentiation on glycine currents in the spinal cord ventral horn neurons.

Blocking CTGF/CCN2 reverses neural fibrosis and sensorimotor declines in a rat model of overuse-induced median mononeuropathy.

Encapsulation of median nerves is a hallmark of overuse-induced median mononeuropathy and contributes to functional declines. We tested if an antibody against CTGF/CCN2 (termed FG-3019 or Pamrevlumab) reduces established neural fibrosis and sensorimotor declines in a clinically relevant rodent model of overuse in which median mononeuropathy develops. Young adult female rats performed a high repetition high force (HRHF) lever-pulling task for 18 weeks. Rats were then euthanised at 18 weeks (HRHF untreated), or rested and systemically treated for 6 weeks with either an anti-CCN2 monoclonal antibody (HRHF-Rest/FG-3019) or IgG (HRHF-Rest/IgG), with results compared with nontask control rats. Neuropathology was evident in HRHF-untreated and HRHF-Rest/IgG rats as increased perineural collagen deposition and degraded myelin basic protein (dMBP) in median nerves, and increased substance P in lower cervical dorsal root ganglia (DRG), compared with controls. Both groups showed functional declines, specifically, decreased sensory conduction velocity in median nerves, noxious cold temperature hypersensitivity, and grip strength declines, compared with controls. There were also increases of ATF3-immunopositive nuclei in ventral horn neurons in HRHF-untreated rats, compared with controls (which showed none). FG-3019-treated rats showed no increase above control levels of perineural collagen or dMBP in median nerves, Substance P in lower cervical DRGs, or ATF3-immunopositive nuclei in ventral horns, and similar median nerve conduction velocities and thermal sensitivity, compared with controls. We hypothesize that neural fibrotic processes underpin the sensorimotor declines by compressing or impeding median nerves during movement, and that inhibiting fibrosis using an anti-CCN2 treatment reverses these effects.

Mechanisms of noradrenergic modulation of synaptic transmission and neuronal excitability in ventral horn neurons of the rat spinal cord.

Noradrenaline (NA) modulates the spinal motor networks for locomotion and facilitates neuroplasticity, possibly assisting neuronal network activation and neuroplasticity in the recovery phase of spinal cord injuries. However, neither the effects nor the mechanisms of NA on synaptic transmission and neuronal excitability in spinal ventral horn (VH) neurons are well characterized, especially in rats aged 7 postnatal days or older. To gain insight into NA regulation of VH neuronal activity, we used a whole-cell patch-clamp approach in late neonatal rats (postnatal day 7-15). In voltage-clamp recordings at -70 mV, NA increased the frequency and amplitude of excitatory postsynaptic currents via the activation of somatic α1- and ß-adrenoceptors of presynaptic neurons. Moreover, NA induced an inward current through the activation of postsynapticα1- and ß-adrenoceptors. At a holding potential of 0 mV, NA also increased frequency and amplitude of both GABAergic and glycinergic inhibitory postsynaptic currents via the activation of somatic adrenoceptors in presynaptic neurons. In current-clamp recordings, NA depolarized resting membrane potentials and increased the firing frequency of action potentials in VH neurons, indicating that it enhances the excitability of these neurons. Our findings provide new insights that establish NA-based pharmacological therapy as an effective method to activate neuronal networks of the spinal VH in the recovery phase of spinal cord injuries.

Molecular mechanisms of the antispasticity effects of baclofen on spinal ventral horn neurons.

BACKGROUND: Baclofen is a lipophilic γ-aminobutyric acid (GABA) derivative that exhibits strong intrinsic activity and a high affinity for GABAB receptors. Intrathecal baclofen therapy has been used as an antispasticity and muscle relaxant drug in the clinical treatment of patients with severe spasticity. However, the cellular mechanisms of the antispasticity effects of baclofen on the ventral horn neurons of the spinal cord are unknown. OBJECTIVE: We examined the action of baclofen on excitatory synaptic transmission in ventral horn neurons in the rat spinal cord by whole-cell patch-clamp recordings. RESULTS: Baclofen significantly reduced the frequency and amplitude of miniature excitatory postsynaptic currents. The reduction in miniature excitatory postsynaptic current frequency was particularly strong, indicating presynaptic inhibition by baclofen. Moreover, baclofen-induced outward currents in all neurons tested. The baclofen-induced outward currents persisted in the presence of tetrodotoxin and glutamate receptor antagonists and were diminished in the presence of the postsynaptic intracellular K channel blocker cesium sulfate and the G-protein inhibitor guanosine 5'-(ß-thio)diphosphate trilithium salt. These results indicate direct postsynaptic depression mediated by G-protein-activated K channels by GABAB receptors on ventral horn neurons. The baclofen-induced outward currents and the inhibitory effects on spontaneous excitatory postsynaptic currents were blocked by the selective GABAB receptor antagonist CGP35348. CONCLUSION: Baclofen may have both presynaptic and postsynaptic capacity to inhibit synaptic transmission in ventral horn neurons by GABAB receptors. These cellular mechanisms may induce the antispasticity effects of intrathecal baclofen therapy in the spinal cord.

l-Theanine and NEP1-40 promote nerve regeneration and functional recovery after brachial plexus root avulsion.

Brachial plexus root avulsion causes severe sequelae Treatments and prognosis face many problems, including inflammatory reaction, oxidative damage, and myelin related inhibitory effect. l-Theanine has anti-inflammatory, anti-oxidative, and neuroprotective effects. NEP1-40 competitively inhibits Nogo-66 receptor (NgR1) promotes axonal regeneration. Forty-eight Sprague-Dawley rats were randomly assigned into four groups to establish an animal model of brachial plexus root avulsion. Inflammation and oxidative damage were evaluated by spectrophotometry and motor function of the upper limbs was assessed via Terzis grooming test after modeling. Immunofluorescence and hematoxylin and eosin staining were utilized to determine the content of reactive oxygen species, activation of microglial cells, neuroprotection, and nerve regeneration. Compared with the control group, the L-Theanine + NEP1-40 group had significantly decreased myeloperoxidase, malondialdehyde, interleukin-6, reactive oxygen species, and microglial cells, significantly increased score on the Terzis grooming test, increased motor neuron content, and thickened muscle fibers, increased area, and appearance of large and clear motor endplate structures. The results of this study suggest that l-Theanine combined with NEP1-40significantly promoted nerve regeneration after brachial plexus root avulsion, and may be a potential treatment for promoting nerve regeneration. Possible mechanisms underlying these results are alleviation of oxidative damage and inflammatory responses in the injured area and antagonism of myelin inhibition.

Triggering of Autophagy by Baicalein in Response to Apoptosis after Spinal Cord Injury: Possible Involvement of the PI3K Activation.

High level apoptosis induced by spinal cord injury (SCI) evokes serious damage because of the loss and dysfunction of motor neurons. Our previous studies showed that inhibition of autophagy evokes the activation of apoptosis. Interestingly, Baicalein, a medicine with anti-apoptosis activity that is derived from the roots of herb Scutellaria baicalensis, largely induces autophagy by activating phosphatidylinositol 3-kinase (PI3K). In this study, we investigated the effects of intraperitoneal injection of Baicalein on autophagy and apoptosis in SCI mice and evaluated the relationship between autophagy and apoptosis. We demonstrated that Baicalein promoted the functional recovery of motor neurons at 7 d after SCI. In addition, Baicalein enhanced neuronal autophagy and the autophagy-related factor PI3K, while inhibiting the p62 protein. Baicalein treatment decreased neuronal apoptosis at 7 d after SCI. Moreover, when inhibiting autophagy, apoptosis was upgraded by Baicalein treatment after injury. Thus, Baicalein attenuated SCI by inducing autophagy to reduce apoptosis in neurons potentially via activating PI3K.

Monoamine oxidase-B inhibitor protects degenerating spinal neurons, enhances nerve regeneration and functional recovery in sciatic nerve crush injury model.

Monoamine oxidase-B (MAOB), a flavin adenine dinucleotide (FAD), is an enzyme which catalyzes the oxidation of amines. MAOB is proposed to play a major role in the pathogenesis of neurodegeneration through the production of reactive oxygen species (ROS) and neurotoxins. The present study was designed to outline the effects of the MAOB inhibitor (MAOB-I) on neuroprotection of spinal neurons, regeneration of sciatic nerve fibers, and recovery of sensory-motor functions in the sciatic nerve crush injury model. Male Wistar rats (4-months-old) were assigned to i) Naïve (N), ii) Sham (S), iii) Sciatic nerve crush and treated with saline (CRUSH + SALINE) and iv) Sciatic nerve crush and treated with MAOB inhibitor (CRUSH + MAOB-I) groups (n = 10/group). In groups iii and iv, the crush injury was produced by crushing the sciatic nerve followed by treatment with saline or MAOB-I (Selegiline® 2.5 mg/kg) intraperitoneally for 10 days. Behavioral tests were conducted from week 1 to week 6. At the end of the study, sciatic nerve and lumbar spinal cord were examined by immunohistochemistry, light and electron microscopy. MAOB-I treatment showed significant improvement in sensory and motor functions compared to saline treatment (p < 0.05-0.001) in injured nerves. The morphological study showed a significantly increased number of nerve fibers in sciatic nerve distal to the site of injury (p < 0.05), with better myelination pattern in CRUSH + MAOB-I treated group compared to CRUSH + SALINE group. Spinal cord ventral horns showed a significant increase in the number of NeuN-immunoreactive neurons in the MAOB-I treated group compared to Saline treated group (p < 0.01). MAOB-I has a significant potential for protecting the degenerating spinal cord neurons and enhancing the regeneration of injured sciatic nerve fibers following crush injury.

Endomorphin-2 Decreases Excitatory Synaptic Transmission in the Spinal Ventral Horn of the Rat.

Motor impairment is one of the serious side-effects of morphine, which is an exogenous agonist of the µ-opioid receptor (MOR) as well as a widely used analgesic drug in clinical practice for chronic pain treatment. Endomorphins (EMs, including EM-1 and EM-2), the most effective and specific endogenous agonists of the MOR, exert more potent analgesia in acute and neuropathic pain than other opiates, such as morphine. Although EMs had fewer side-effects comparing to other opiates, motor impairment was still one unwanted reaction which limited its clinical application. In order to prevent and treat the motor impairment, it is critical to reveal the neural mechanisms underlying such locomotion disorder. The purpose of the present study was to reveal the neural mechanisms underlying the effects of EM-2 on the activity of motoneurons in the spinal ventral horn. First, we examine the distribution of EM-2-immunoreactive (IR) primary afferent fibers and their synaptic connections with the motoneurons innervating the skeletal muscles of the lower limb revealed by sciatic nerve retrograde tracing. The results showed that EM-2-IR fibers and terminals were sparsely observed in lamina IX and they formed symmetric synaptic connections with the motoneurons within lamina IX of the spinal ventral horn. Then, whole-cell patch-clamp technique was used to observe the effects of EM-2 on the spontaneous excitatory postsynaptic current (sEPSC) of motoneurons in lamina IX. The results showed that EM-2 could decrease both the frequency and amplitude of the sEPSC of the motoneurons in lamina IX, which was reversed by the MOR antagonist CTOP. These results indicate that EM-2-IR fibers originated from primary afferent fibers form symmetric synaptic connections with motoneurons innervating skeletal muscles of the lower limbs in lamina IX of the spinal ventral horn and EM-2 might exert inhibitory effects on the activities of these motoneurons through both presynaptic and postsynaptic mechanisms.

Effect of physical exercise and anabolic steroid treatment on spinal motoneurons and surrounding glia of wild-type and ALS mice.

Motoneuron degeneration is the hallmark of amyotrophic lateral sclerosis (ALS). The cause and predisposing factors for sporadic ALS are still unknown. Exposure to a specific environmental risk factors in subjects with a susceptibility genotype may increase the risk of the disease. The role of physical activity and the use of anabolic steroids are still debated in epidemiological studies on patients and murine models of ALS. To assess at the cellular level the role (beneficial or detrimental) of physical exercise and the use of anabolic steroid, we here investigated, in SOD1(G93A) (mSOD1) mice and wild-type littermates, changes in the ventral horn after regular exercise, treatment with the anabolic androgenic steroid 19-nortestosterone (nandrolone), and their combination, compared with matched control sedentary mice. The experiments were pursued for several weeks until symptom onset in mSOD1 mice. Lumbar motoneurons, astrocytes and microglia were analyzed. In wild-type mice, cytological alterations of motoneurons were observed especially after nandrolone treatment. The following main findings were observed in treated mSOD1 mice versus untreated ones: i) nandrolone treatment markedly enhanced motoneuron loss; this detrimental effect was reverted by the combination with exercise, resulting in increased motoneuron survival; ii) astrocytic activation was most marked after nandrolone treatment when motoneuron damage was most severe; iii) microglia activation was most marked after physical exercise when motoneuron damage was less severe. The results indicate a vulnerability of mSOD1 motoneurons to nandrolone treatment, a potential neuroprotective effect of physical exercise, and a modulation by glial cells in the ALS murine model in the examined paradigms.

Generation of Spinal Motor Neurons from Human Pluripotent Stem Cells.

Human embryonic stem cells (ESCs) are characterized by their unique ability to self-renew indefinitely, as well as to differentiate into any cell type of the human body. Induced pluripotent stem cells (iPSCs) share these salient characteristics with ESCs and can easily be generated from any given individual by reprogramming somatic cell types such as fibroblasts or blood cells. The spinal motor neuron (MN) is a specialized neuronal subtype that synapses with muscle to control movement. Here, we present a method to generate functional, postmitotic, spinal motor neurons through the directed differentiation of ESCs and iPSCs by the use of small molecules. These cells can be utilized to study the development and function of human motor neurons in healthy and disease states.

Creating Interactions between Tissue-Engineered Skeletal Muscle and the Peripheral Nervous System.

Effective models of mammalian tissues must allow and encourage physiologically (mimetic) correct interactions between co-cultured cell types in order to produce culture microenvironments as similar as possible to those that would normally occur in vivo. In the case of skeletal muscle, the development of such a culture model, integrating multiple relevant cell types within a biomimetic scaffold, would be of significant benefit for investigations into the development, functional performance, and pathophysiology of skeletal muscle tissue. Although some work has been published regarding the behaviour of in vitro muscle models co-cultured with organotypic slices of CNS tissue or with stem cell-derived neurospheres, little investigation has so far been made regarding the potential to maintain isolated motor neurons within a 3D biomimetic skeletal muscle culture platform. Here, we review the current state of the art for engineering neuromuscular contacts in vitro and provide original data detailing the development of a 3D collagen-based model for the co-culture of primary muscle cells and motor neurons. The devised culture system promotes increased myoblast differentiation, forming arrays of parallel, aligned myotubes on which areas of nerve-muscle contact can be detected by immunostaining for pre- and post-synaptic proteins. Quantitative RT-PCR results indicate that motor neuron presence has a positive effect on myotube maturation, suggesting neural incorporation influences muscle development and maturation in vitro. The importance of this work is discussed in relation to other published neuromuscular co-culture platforms along with possible future directions for the field.

Hydrogen peroxide modulates neuronal excitability and membrane properties in ventral horn neurons of the rat spinal cord.

Hydrogen peroxide (H2O2), a reactive oxygen species, is an important signaling molecule for synaptic and neuronal activity in the central nervous system; it is produced excessively in brain ischemia and spinal cord injury. Although H2O2-mediated modulations of synaptic transmission have been reported in ventral horn (VH) neurons of the rat spinal cord, the effects of H2O2 on neuronal excitability and membrane properties remain poorly understood. Accordingly, the present study investigated such effects using a whole-cell patch-clamp technique. The bath-application of H2O2 decreased neuronal excitability accompanied by decreased input resistance, firing frequency, and action potential amplitude and by increased rheobase. These H2O2-mediated changes were induced by activation of extrasynaptic, but not synaptic, GABAA receptors. Indeed, GABAergic tonic currents were enhanced by H2O2. On the other hand, the amplitude of medium and slow afterhyperpolarization (mAHP and sAHP), which plays important roles in controlling neuronal excitability and is mediated by small-conductance calcium-activated potassium (SK) channels, was significantly decreased by H2O2. When extrasynaptic GABAA receptors were completely blocked, these decreases of mAHP and sAHP persisted, and H2O2 increased excitability, suggesting that H2O2 per se might have the potential to increase neuronal excitability via decreased SK channel conductance. These findings indicate that activating extrasynaptic GABAA receptors or SK channels may attenuate acute neuronal damage caused by H2O2-induced hyperexcitability and therefore represent a novel therapeutic target for the prevention and treatment of H2O2-induced motor neuron disorders.

High-mobility group box 1 induces neuron autophagy in a rat spinal root avulsion model.

Autophagy, a tightly regulated lysosome-dependent catabolic pathway, is implicated in various pathological states in the nervous system. High-mobility group box 1 (HMGB1) is an inflammatory mediator known to be released into the local microenvironment from damaged cells. However, whether autophagy is induced and exogenous HMGB1 is involved in the process of spinal root avulsion remain unclear. Here, we investigated the induction effect of autophagy and the possible role of HMGB1 during spinal root avulsion. It was found that autophagy was activated in the anterior horn of the spinal cord as represented by the increased expression of the autophagic marker microtubule-associated protein light chain 3-II (LC3-II), degradation of sequestosome 1 (p62), and formation of autophagosomes, and that autophagy was inhibited after intraperitoneal injection of anti-HMGB1-neutralizing antibodies in the rat spinal root avulsion model. In addition, HMGB1-induced autophagy and activated mitogen-activated protein kinases (MAPKs) in primary spinal neurons, including c-Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase (ERK), and p38MAPK. Inhibition of JNK or ERK activity significantly blocked the effect of HMGB1-induced autophagy in primary spinal neurons. Finally, HMGB1-induced autophagy increased cell viability in primary spinal neurons under oxygen-glucose deprivation conditions. The above results suggest that HMGB1 is a critical regulator of autophagy and HMGB1-induced autophagy plays an important role in protecting spinal neurons against injury, which may provide new insights into the pathophysiological process of spinal root avulsion.

Hydrogen peroxide modulates synaptic transmission in ventral horn neurons of the rat spinal cord.

KEY POINTS: Excessive production of reactive oxygen species (ROS) is implicated in many central nervous system disorders; however, the physiological role of ROS in spinal ventral horn (VH) neurons remains poorly understood. We investigated how pathological levels of H2O2, an abundant ROS, regulate synaptic transmission in VH neurons of rats using a whole-cell patch clamp approach. H2O2 increased the release of glutamate and GABA from presynaptic terminals. The increase in glutamate release involved N-type voltage-gated calcium channels (VGCCs), ryanodine receptors (RyRs), and inositol trisphosphate receptors (IP3 Rs); the increase in GABA release, which inhibited glutamatergic transmission, involved IP3 R. Inhibiting N-type VGCCs and RyRs attenuates excitotoxicity resulting from increased glutamatergic activity while preserving the neuroprotective effects of GABA, and may represent a novel strategy for treating H2O2-induced motor neuron disorders resulting from trauma or ischaemia-reperfusion injury. Excessive production of reactive oxygen species (ROS) is a critical component of the cellular and molecular pathophysiology of many central nervous system (CNS) disorders, including trauma, ischaemia-reperfusion injury, and neurodegenerative diseases. Hydrogen peroxide (H2O2), an abundant ROS, modulates synaptic transmission and contributes to neuronal damage in the CNS; however, the pathophysiological role of H2O2 in spinal cord ventral horn (VH) neurons remains poorly understood, despite reports that these neurons are highly vulnerable to oxidative stress and ischaemia. This was investigated in the present study using a whole-cell patch clamp approach in rats. We found that exogenous application of H2O2 increased the release of glutamate from excitatory presynaptic terminals and γ-aminobutyric acid (GABA) from inhibitory presynaptic terminals. The increase of glutamate release was induced in part by an increase in Ca(2+) influx through N-type voltage-gated calcium channels (VGCCs) as well as by ryanodine receptor (RyR)- and inositol trisphosphate receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER). In inhibitory presynaptic neurons, increased IP3 R-mediated Ca(2+) release from the ER increased GABAergic transmission, which served to rescue VH neurons from excessive release of glutamate from presynaptic terminals. These findings indicate that inhibiting N-type VGCCs or RyRs may attenuate excitotoxicity resulting from increased glutamatergic activity while preserving the neuroprotective effects of GABA, and may therefore represent a novel and targeted strategy for preventing and treating H2O2-induced motor neuron disorders.

Paclitaxel Inhibits Expression of Neuronal Nitric Oxide Synthase and Prevents Mitochondrial Dysfunction in Spinal Ventral Horn in Rats After C7 Spinal Root Avulsion.

AIM: This study evaluated the neuroprotective effect of intrathecally infused paclitaxel in the prevention of motoneuron death and mitochondrial dysfunction following brachial plexus avulsion injury. MATERIAL AND METHODS: Brachial root avulsion injury was induced in Sprague-Dawley rats. The Paclitaxel treatment group (n = 32) received a 5-d intrathecal infusion of paclitaxel (256 ng/d) via a micro infusion pump, whereas the Control group (n = 32) received normal saline. The cervical cord was harvested at survival times of 1, 2, 4, and 6 wk (n = 8 each). The number of surviving and nNOS-positive motoneurons at the injury level in the ventral horn was determined with NADPH-d histochemistry. Mitochondrial function at this location was measured with CcO histochemistry and densitometry. An independent t-test was applied to detect differences between the study groups at specific survival times. RESULTS: The Paclitaxel treatment group showed a significant relative reduction in nNOS expression at 2, 4, and 6 wk, and significantly improved mitochondrial function at 4 and 6 wk. Motoneuron survival was significantly increased at 2, 4, and 6 wk. CONCLUSION: Paclitaxel has a significant neuroprotective effect against spinal motoneuron degeneration following brachial plexus avulsion injury, which involves inhibition of nNOS expression and prevention of mitochondrial dysfunction.

Synaptic modulation of excitatory synaptic transmission by nicotinic acetylcholine receptors in spinal ventral horn neurons.

Nicotinic acetylcholine receptors (nAChRs) are distributed widely in the central nervous system and play important roles in higher brain functions, including learning, memory, and recognition. However, functions of the cholinergic system in spinal motoneurons remain poorly understood. In this study, we investigated the actions of presynaptic and postsynaptic nAChRs in spinal ventral horn neurons by performing whole-cell patch-clamp recordings on lumbar slices from male rats. The application of nicotine or acetylcholine generated slow inward currents and increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs). Slow inward currents by acetylcholine or nicotine were not inhibited by tetrodotoxin (TTX) or glutamate receptor antagonists. In the presence of TTX, the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) were also increased by acetylcholine or nicotine. A selective α4ß2 nicotinic receptor antagonist, dihydro-ß-erythroidine hydrobromide (DhßE), significantly decreased nicotine-induced inward currents without affecting the enhancement of sEPSCs and mEPSCs. In addition, a selective α7 nicotinic receptor antagonist, methyllycaconitine, did not affect either nicotine-induced inward currents or the enhancement of sEPSCs and mEPSCs. These results suggest that α4ß2 AChRs are localized at postsynaptic sites in the spinal ventral horn, non-α4ß2 and non-α7 nAChRs are located presynaptically, and nAChRs enhance excitatory synaptic transmission in the spinal ventral horn.

Propofol modulates phasic and tonic GABAergic currents in spinal ventral horn interneurones.

BACKGROUND: Surgical interventions like skin incisions trigger withdrawal reflexes which require motor neurones and local circuit interneurones in the spinal ventral horn. This region plays a key role in mediating immobilizing properties of the GABAergic anaesthetic propofol. However, it is unclear how propofol modulates GABA(A) receptors in the spinal ventral horn and whether tonic or phasic inhibition is involved. METHODS: Organotypic spinal cord tissue slices were prepared from mice. Whole-cell recordings were performed for quantifying effects of propofol on GABA(A) receptor-mediated phasic transmission and tonic conductance. RESULTS: Propofol increased GABAergic phasic transmission by a prolongation of the decay time constant in a concentration-dependent manner. The amount of the charge transferred per inhibitory post-synaptic current, described by the area under the curve, was significantly augmented by 1 µM propofol (P<0.01). A GABA(A) receptor-mediated tonic current was not induced by 1 µM propofol but at a concentration of 5 µM (P<0.05). CONCLUSIONS: Propofol depresses ventral horn interneurones predominantly by phasic rather than by tonic GABA(A) receptor-mediated inhibition. However, the present results suggest that the involvement of a tonic inhibition might contribute to the efficacy of propofol to depress nociceptive reflexes at high concentrations of the anaesthetic.