Spinal Ventral Horn Neurons Research Articles

Transsynaptic degeneration of ventral horn motor neurons exists but plays a minor role in lower motor system dysfunction in acute ischemic rats.

As a leading cause of mortality and long-term disability, acute ischemic stroke can produce far-reaching pathophysiological consequences. Accumulating evidence has demonstrated abnormalities in the lower motor system following stroke, while the existence of Transsynaptic degeneration of contralateral spinal cord ventral horn (VH) neurons is still debated. Using a rat model of acute ischemic stroke, we analyzed spinal cord VH neuron counts contralaterally and ipsilaterally after stroke with immunofluorescence staining. Furthermore, we estimated the overall lower motor unit abnormalities after stroke by simultaneously measuring the modified neurological severity score (mNSS), compound muscle action potential (CMAP) amplitude, repetitive nerve stimulation (RNS), spinal cord VH neuron counts, and the corresponding muscle fiber morphology. The activation status of microglia and extracellular signal-regulated kinase 1/2 (ERK 1/2) in the spinal cord VH was also assessed. At 7 days after stroke, the contralateral CMAP amplitudes declined to a nadir indicating lower motor function damage, and significant muscle disuse atrophy was observed on the same side; meanwhile, the VH neurons remained intact. At 14 days after focal stroke, lower motor function recovered with alleviated muscle disuse atrophy, while transsynaptic degeneration occurred on the contralateral side with elevated activation of ERK 1/2, along with the occurrence of neurogenic muscle atrophy. No apparent decrement of CMAP amplitude was observed with RNS during the whole experimental process. This study offered an overview of changes in the lower motor system in experimental ischemic rats. We demonstrated that transsynaptic degeneration of contralateral VH neurons occurred when lower motor function significantly recovered, which indicated the minor role of transsynaptic degeneration in lower motor dysfunction during the acute and subacute phases of focal ischemic stroke.

Orexin-A inhibits γ-aminobutyric acid current of neonatal rat spinal cord ventral horn neurons by activating OX1R, OX2R and Ca2+-independent PKC

To investigate the effect of orexin-A on the functionality of ionotropic γ-aminobutyric acid (GABA) receptors in spinal cord ventral horn neurons and its mechanisms. The spinal cord containing the lumbosacral enlargement was isolated from neonatal SD rats (7-12 days old) and sliced. The slices were digested with papain (in 0.18 g/30 mL artificial cerebrospinal fluid) for 40-60 min, and the ventral horn neurons were separated acutely using fire-polished Pasteur pipettes. After the cells adhered to the bottom of Petri dishes, patch-clamp experiments combined with pharmacological methods were performed to test the effects of orexin-A on GABA currents of the neurons treated with SB334867 (a selective OX1R antagonist), TCSOX229 (a selective OX2R antagonist), Bis-Ⅳ (a PKC inhibitor), PMA (a PKC agonist), Rp-cAMP (a PKA inhibitor), or BAPTA (Ca2+ chelator). The isolated neurons maintained good morphologies with diverse shapes of cell body and long protrusions. Treatment with orexin-A significantly inhibited the amplitude of GABA-induced current (P < 0.001, n=49) with an inhibition rate of (67.48±12.50)%. SB334867 and TCSOX229, when applied simultaneously, completely abolished the suppressive effect of orexin-A on the GABA currents (P=0.93, n=6), and their separate use partially relieved the suppressive effect of orexin-A (P=0.001, n=8; P=0.02, n=8). The application of Bis-Ⅳ also abolished the suppressive effect of orexin-A on GABA currents (P=0.31, n=5). PMA mimicked the effect of orexin-A in these neurons and significantly inhibited GABA currents with an inhibition rate of (60.79±10.94)%, and the application of orexin-A did not cause further suppression of GABA currents in PMA-treated neurons (P=0.15, n=6). Orexin-A was still capable of suppressing GABA currents in Rp-cAMP-treated neurons (P=0.001, n=5). The extracellular Ca2+-free solution (P=0.004, n=8) or the presence of BAPTA (P=0.04, n=7) did not significantly affect the inhibitory effect of orexin-A on GABA currents. Orexin-A inhibits GABA currents in the ventral horn neurons of rat spinal cord probably by activating OX1R, OX2R and Ca2+-independent PKC.

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.

Etomidate reduces excitability of the neurons and suppresses the function of nAChR ventral horn in the spinal cord of neonatal rats

To investigate the effects of etomidate on electrophysiological properties and nicotinic acetylcholine receptors (nAChRs) of ventral horn neurons in the spinal cord. The spinal cord containing lumbosacral enlargement was isolated from 19 neonatal SD rats aged 7-12 days. The spinal cord were sliced and digested with papain (0.18 g/30 mL artificial cerebrospinal fluid) and incubated for 40 min. At the ventral horn, acute mechanical separation of neurons was performed with fire-polished Pasteur pipettes, and perforated patch-clamp recordings combined with pharmacological methods were employed on the adherent healthy neurons. In current-clamp mode, the spontaneous action potential (AP) of the ventral horn neurons in the spinal cord was recorded. The effects of pretreatment with different concentrations of etomidate on AP recorded in the ventral horn neurons were examined. In the voltage-clamp mode, nicotine was applied to induce inward currents in the ventral horn neurons, and the effect of pretreatment with etomidate on the inward currents induced by nicotine were examined with different etomidate concentrations, different holding potentials and different use time. The isolated ventral horn neurons were in good condition with large diverse somata and intact processes. The isolated spinal ventral horn neurons (n=21) had spontaneous action potentials, and were continuously perfused for 2 min with 0.3, 3.0 and 30.0 μmol/L etomidate. Compared with those before administration, the AP amplitude, spike potential amplitude and overshoot were concentration-dependently suppressed (P &lt; 0.01), and spontaneous discharge frequency was obviously reduced (P &lt; 0.01, n=12). The APs of the other 9 neurons were completely abolished by etomidate at 3.0 or 30 μmol/L. At the same holding potential (VH=-70 mV), pretreatment with 0.3, 3.0 or 30.0 μmol/L etomidate for 2 min concentration-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine (P &lt; 0.01, n=7). At the holding potentials of - 30, - 50, and - 70 mV, pretreatment with 30.0 μmol/L etomidate for 2 min voltage-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine (P &lt; 0.01, n=6 for each holding potential). During the 6 min of 30.0 μmol/L etomidate pretreatment, the clamped cells were exposed to 0.4 mmol/L nicotine for 4 times at 0, 2, 4, and 6 min (each exposure time was 2 s), and the nicotinic current amplitude decreased gradually as the number of exposures increased. But at the same concentration, two nicotine exposures (one at the beginning and the other at the end of the 6 min pretreatment) resulted in a significantly lower inhibition rate compared with 4 nicotine exposures (P &lt; 0.01, n=6). etomidate reduces the excitability of the spinal ventral neurons in a concentration-dependent manner and suppresses the function of nAChR in a concentration-, voltage-, and use-dependent manner.

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 β-adrenoceptors of presynaptic neurons. Moreover, NA induced an inward current through the activation of postsynapticα1- and β-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.

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. 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. 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'-(β-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. 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.

Effects of Melatonin on the Behavioral Indices and Structural Characteristics of Cerebral and Spinal Neurons of Rats with Experimental Hemiparkinsonism

In adult Wistar rats, a model of experimental hemiparkinsonism (EHP) was induced by stereotaxic microinjection of 8.0 μg 6-hydroxydopamine (6-HODA) into the left forebrain lateral ascending bundle. In such animals, we studied behavioral indices in the open field and elevated plus maze tests and also structural changes in the cerebral substantia nigra and spinal L5 ventral horn neurons. Six groups of animals were formed (in each group, n = 8): intact rats (I), animals that obtained microinjections of saline into the above lateral bundle (sham-injected, ShI), two groups with weak and intense motor EHP manifestations (frequency of circulatory movements in the apomorphine test less than one and six and more per 1 min, groups HP<1 and HP≥6, respectively), and two groups of rats with weak and intense EHP manifestations, which obtained daily i.p. injections of 10 mg/kg melatonin during 18 days (groups HP<1+M and HP≥6+M). In the open-field test, we observed in groups HP<1 and HP≥6 significant suppression of horizontal motor activity (very intense in the latter of the above-mentioned groups) and also significant weakening of vertical activity (number of rearings). Changes in the number of defecation acts were less expressed. In the elevated plus maze, manifestations of a significantly increased anxiety level were observed in rats of the above two groups. In the substantia nigra of animals with EHP, a considerable part of the neurons demonstrated clear signs of negative structural modifications; the number of neurons with the normal structure was considerably smaller than in the control. Analogous negative morphological changes were found in the lumbar segment of the spinal cord. Course injections of melatonin into rats with EHP noticeable weakened negative shifts in both open-field behavioral indices and level of anxiety (according to estimates in the elevated plus maze). Under the action of melatonin, morphological characteristics of neurons and gliocytes in the substantia nigra and spinal cord of rats with EHP demonstrated appreciable normalization. The mechanisms underlying functionaland structural disorders in the CNS of animals after central microinjections of the 6-HODA neurotoxin and also neuroprotective effects of melatonin, which provide weakening of such disorders, are discussed.

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

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.

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 α4β2 nicotinic receptor antagonist, dihydro-β-erythroidine hydrobromide (DhβE), 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 α4β2 AChRs are localized at postsynaptic sites in the spinal ventral horn, non-α4β2 and non-α7 nAChRs are located presynaptically, and nAChRs enhance excitatory synaptic transmission in the spinal ventral horn.

Effects of succinylcholine in an organotypic spinal cord-skeletal muscle coculture of embryonic mice

Intoxication with organophosphorus compounds is an important clinical problem worldwide. Although the core treatments – atropine, oximes and diazepam – are defined, high case fatalities were reported for intoxication with organophosphorus insecticides. In particular the role of oximes is not completely understood since they might benefit only patients poisoned by specific pesticides or patients with moderate poisoning and few randomised trials of such poisoning have been performed. This justifies the need for new in vitro test-systems like cocultures of spinal cord and muscle tissue, which have been recently introduced. However this test-system is not yet fully characterized. In order to estimate the applicability of cocultures of spinal cord and muscle tissue their sensitivity to succinylcholine (di-acetylcholine), a depolarizing muscle relaxant in clinical use, was tested.The test-system evaluated in the current study showed sensitivity to succinylcholine with an EC50 as low as 1μM thereby being close to the EC50 value in adult human patients (2.6μM). Furthermore, action potential activity of spinal ventral horn neurons was not altered by succinylcholine. The latter observations strongly suggest that our preparation well predicts the qualitative and quantitative actions of novel drugs targeting the neuromuscular system in vivo. In summary, cocultures of spinal cord and muscle tissue seem to be a valid test-system for the development and investigation of new oximes. Moreover, practical aspects like transport over long distances to further laboratories, the opportunity to conduct long-term studies and the reduction of animal usage display further advantages of its use.

Effect of Xenon on Excitatory and Inhibitory Transmission in Rat Spinal Ventral Horn Neurons

The minimum alveolar concentration is determined in the spinal cord rather than in the brain. Xenon inhibits glutamatergic excitatory synaptic transmission in the dorsal horn neurons. However, its actions in the ventral horn neurons have not been investigated. The effects of 50 or 75% xenon on excitatory and inhibitory synaptic transmission were examined in the spinal lamina IX neurons of neonatal rats by using a whole cell patch clamp technique. Fifty percent xenon inhibited the α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid-induced currents (amplitudes = 72 ± 9% and integrated area = 73 ± 13% of the control values), and α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid receptor-mediated electrically evoked excitatory postsynaptic currents (amplitudes = 69 ± 13% of the control values). Seventy-five percent xenon similarly inhibited α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid-induced currents. However, xenon had no effect on the N-methyl-D-aspartate-induced currents or N-methyl-D-aspartate receptor-mediated electrically evoked excitatory postsynaptic currents. Xenon decreased the amplitude, but not the frequency, of miniature excitatory postsynaptic currents. There were no discernible effects on the currents induced by γ-aminobutyric acid or glycine or on miniature inhibitory postsynaptic currents. Xenon inhibits α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid receptor-mediated glutamatergic excitatory transmission in the spinal lamina IX neurons via a postsynaptic mechanism. In contrast, there are no substantial effects on N-methyl-D-aspartate receptor-mediated or inhibitory synaptic transmission. The suppressive effects on excitatory synaptic transmission in the ventral horn neurons partly account for the mechanism behind xenon's ability to produce immobility in response to noxious stimuli and to determine the minimum alveolar concentration.

Excitation of rat spinal ventral horn neurons by purinergic P2X and P2Y receptor activation

ATPγS, a nonhydrolyzable ATP analog, was found to dose-dependently generate an inward current at a holding potential of −70 mV (EC 50 = 43 μM) in lamina IX neurons of rat spinal cord slices using the whole-cell patch-clamp technique. This inward current had an extrapolated reversal potential of −9 mV and was resistant to the Na +-channel blocker tetrodotoxin, glutamate-receptor antagonists or nominally Ca 2+-free medium. ATPγS also increased the frequency and amplitude of glutamatergic spontaneous excitatory postsynaptic current (sEPSC); this action was dose-dependent and sensitive to tetrodotoxin. Unlike ATPγS, the P2X-receptor agonist, BzATP or α,β-methylene ATP, did not change holding currents, but the current response produced by ATPγS disappeared in the presence of the P2-receptor antagonist PPADS. The sEPSC frequency and amplitude increase was observed with α,β-methylene ATP, but not with the P2Y-receptor agonist, 2-methylthio ADP, UTP or UDP. The current response by ATPγS was suppressed by the addition of GDPβS into the patch-pipette solution. As for ATPγS, 2-methylthio ADP produced an inward current, while UTP and UDP had no effect on holding currents. The P2Y 1-receptor antagonist MRS2179 inhibited the ATPγS-induced inward current, but did not affect the sEPSC frequency and amplitude increase produced by ATPγS. These data indicate that extracellular ATP increases the excitability of lamina IX neurons by membrane depolarization (probably through non-selective cation-channel activation) and spontaneous excitatory transmission enhancement, which may be mediated by P2Y 1 and P2X receptors, respectively. This finding supports the idea that purinergic receptor antagonists provide a therapy for spinal cord injury.

Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research

RNA interference (RNAi) is gaining acceptance as a potential therapeutic strategy against peripheral disease, and several clinical trials are already underway with 21-mer small-interfering RNA (siRNA) as the active pharmaceutical agent. However, for central affliction like pain, such innovating therapies are limited but nevertheless crucial to improve pain research and management. We demonstrate here the proof-of-concept of the use of 27-mer Dicer-substrate siRNA (DsiRNA) for silencing targets related to CNS disorders such as pain states. Indeed, low dose DsiRNA (0.005 mg/kg) was highly efficient in reducing the expression of the neurotensin receptor-2 (NTS2, a G-protein-coupled receptor (GPCR) involved in ascending nociception) in rat spinal cord through intrathecal (IT) administration formulated with the cationic lipid i-Fect. Along with specific decrease in NTS2 mRNA and protein, our results show a significant alteration in the analgesic effect of a selective-NTS2 agonist, reaching 93% inhibition up to 3-4 days after administration of DsiRNA. In order to ensure that these findings were not biased by unsuspected off-target effects (OTEs), we also demonstrated that treatment with a second NTS2-specific DsiRNA also reversed NTS2-induced antinociception, and that NTS2-specific 27-mer duplexes did not alter signaling through NTS1, a closely related receptor. Altogether, DsiRNAi represents a potent tool for dissecting nociceptive pathways and could further lead to a new class of central active drugs.

Hypothermia Suppresses Excitatory Synaptic Transmission and Neuronal Death Induced by Experimental Ischemia in Spinal Ventral Horn Neurons

Whole-cell patch-clamp recordings were performed from the ventral horn neurons obtained from the rat spinal cord slices. This study investigated the effects of hypothermia on excitatory synaptic transmission and ischemia-induced neuronal death. Hypothermia has long been recognized as a promising physical strategy against both ischemic and traumatic spinal cord injuries. However, the mechanism of hypothermia-mediated neuroprotective action in the spinal cord is still not fully understood at the single cell level. Whole-cell patch-clamp recordings were performed from ventral horn neurons obtained from the spinal cord slices. Ischemia was simulated by superfusing an oxygen- and glucose-deprived medium [ischemia simulating medium (ISM)]. When the temperature of the superfusing artificial cerebrospinal fluid solution was changed from normothermia (36 degrees C) to hypothermia (32 degrees C, 28 degrees C, and 24 degrees C), the frequency of spontaneous excitatory postsynaptic currents was significantly decreased in a temperature-dependent manner. Surperfusing the ISM generated an agonal inward current which consisted of a slow and subsequent rapid inward current in all of the neurons tested. The latencies of the slow and rapid inward currents after the ISM exposures were significantly longer at hypothermia than at normothermia. Hypothermia decreased the slope of the ISM-induced slow inward current, although it did not affect the slope of the rapid inward current. Moreover, the glutamate receptor antagonists slightly prolonged the latencies of the slow and rapid inward currents that were induced by ISM and significantly decreased their slopes. These results suggest that hypothermia reduces the excitatory synaptic activities and ischemic neuronal death in the spinal ventral horn. This finding may help in achieving a better understanding of the mechanisms of hypothermia-mediated neuroprotection in the spinal cord.

Selective Vulnerability to Ischemia in the Rat Spinal Cord

Whole-cell patch-clamp recordings were performed from ventral horn (VH) and dorsal horn (DH) neurons obtained from the rat spinal cord slices. This study investigated which is more vulnerable to ischemia, spinal VH neurons or DH neurons. Spinal cord ischemia or injury sometimes causes a greater loss of motor function than of sensory function in patients. However, it is difficult to evaluate whether spinal motor neurons are more vulnerable than sensory neurons because of the anatomic complexity and a variety of physiologic factors in the spinal cord. Whole-cell patch-clamp recordings were performed from VH and DH neurons obtained from the spinal cord slices. Ischemia was simulated by superfusing an oxygen- and glucose-deprived medium (ischemia simulating medium [ISM]). Perfusion with ISM generated an agonal depolarization in all VH and DH neurons recorded in current-clamp mode. Following ISM superfusion, an agonal inward current was produced at a holding potential of -70 mV in all VH and DH neurons tested in voltage-clamp mode. The agonal inward current consisted of a slow and subsequent rapid inward current. The average latency of the rapid inward currents after ISM exposures in VH neurons was significantly shorter than that in DH neurons. The average amplitude of the agonal inward currents in VH neurons was significantly bigger than that of DH neurons. Moreover, the recovery ratio by the reintroduction of oxygen and glucose in VH neurons was smaller than that in DH neurons. These results suggest that VH neurons are more vulnerable to ischemia than DH neurons. This finding may help in achieving a better understanding of the difference between motor and sensory disturbance in spinal cord ischemia or injury patients.

Effects of isoflurane and enflurane on GABAA and glycine receptors contribute equally to depressant actions on spinal ventral horn neurones in rats

Volatile anaesthetics are widely used agents in clinical anaesthesia, although their mechanism of action is poorly understood. In particular, the dominant molecular mechanisms by which volatile anaesthetics depress spinal neurones and thereby mediate spinal effects such as immobility have recently become a matter of dispute. As GABAA and glycine receptors are potential candidates we investigated the impact of both receptor systems in mediating the depressant effects of isoflurane and enflurane on spinal neurones in rats. The effects of isoflurane and enflurane on spontaneous action potential firing were investigated by extracellular voltage recordings from ventral horn interneurones in cultured spinal cord tissue slices obtained from embryonic rats (E 14-15). Isoflurane and enflurane reduced spontaneous action potential firing. Concentrations causing half-maximal effects (isoflurane: 0.17 mM; enflurane: 0.50 mM) were less than EC50-immobility (isoflurane: 0.32 mM; enflurane: 0.62 mM). Effects of isoflurane were mediated by 39% by glycine receptors and 36% by GABAA receptors. The effects of enflurane were mediated 26% by GABAA receptors and 29% by glycine receptors. These results demonstrate that the effects of isoflurane and enflurane on GABAA and glycine receptors contribute almost equally to their depressant actions on spinal ventral horn neurones in rats. The fraction of inhibition mediated by both receptor systems differs between specific volatile anaesthetics. Our data argue against the theory that a dominant molecular mechanism accounts for spinal effects of volatile anaesthetics.

Presynaptic Angiotensin II AT1 Receptors Enhance Inhibitory and Excitatory Synaptic Neurotransmission to Motoneurons and Other Ventral Horn Neurons in Neonatal Rat Spinal Cord

In neonatal spinal cord, we previously reported that exogenous angiotensin II (ANG II) acts at postsynaptic AT1 receptors to depolarize neonatal rat spinal ventral horn neurons in vitro. This study evaluated an associated increase in synaptic activity. Patch clamp recordings revealed that 38/81 thoracolumbar (T7-L5) motoneurons responded to bath applied ANG II (0.3-1 microM; 30 s) with a prolonged (5-10 min) and reversible increase in spontaneous postsynaptic activity, selectively blockable with Losartan (n = 5) but not PD123319 (n = 5). ANG-II-induced events included both spontaneous inhibitory (IPSCs; n = 6) and excitatory postsynaptic currents (EPSCs; n = 5). While most ANG induced events were tetrodotoxin-sensitive, ANG induced a significant tetrodotoxin-resistant increase in frequency but not amplitude of miniature IPSCs (n = 7/13 cells) and EPSCs (n = 2/7 cells). In 35/77 unidentified neurons, ANG II also induced a tetrodotoxin-sensitive and prolonged increase in their spontaneous synaptic activity that featured both IPSCs (n = 5) and EPSCs (n = 4) when tested in the presence of selective amino acid receptor antagonists. When tested in the presence of tetrodotoxin, ANG II was noted to induce a significant increase in the frequency but not the amplitude of mIPSCs (n = 9) and mEPSCs (n = 8). ANG also increased spontaneous motor activity from isolated mouse lumbar ventral rootlets. Collectively, these observations support the existence of a wide pre- and postsynaptic distribution of ANG II AT1 receptors in neonatal ventral spinal cord that are capable of influencing both inhibitory and excitatory neurotransmission.

ErbB transmembrane tyrosine kinase receptors are expressed by sensory and motor neurons projecting into sciatic nerve.

Adult spinal cord motor and dorsal root ganglion (DRG) sensory neurons express multiple neuregulin-1 (NRG-1) isoforms that act as axon-associated factors promoting neuromuscular junction formation and Schwann cell proliferation and differentiation. NRG-1 isoforms are also expressed by muscle and Schwann cells, suggesting that motor and sensory neurons are themselves acted on by NRG-1 isoforms produced by their peripheral targets. To test this hypothesis, we examined the expression of the NRG-1 receptor subunits erbB2, erbB3, and erbB4 in rat lumbar DRG and spinal cord. All three erbB receptors are expressed in these tissues. Sciatic nerve transection, an injury that induces Schwann cell expression of NRG-1, alters erbB expression in DRG and cord. Virtually all DRG neurons are erbB2- and erbB3-immunoreactive, with erbB4 also detectable in many neurons. In spinal cord white matter, erbB2 and erbB4 antibodies produce dense punctate staining, whereas the erbB3 antibody primarily labels glial cell bodies. Spinal cord dorsal and ventral horn neurons, including alpha-motor neurons, exhibit erbB2, erbB3, and erbB4 immunoreactivity. Spinal cord ventral horn also contains a population of small erbB3+/S100beta+/GFAP- cells (GFAP-negative astrocytes or oligodendrocytes). We conclude that sensory and motor neurons projecting into sciatic nerve express multiple erbB receptors and are potentially NRG-1 responsive.

Monoamines increase the excitability of spinal neurones in the neonatal rat by hyperpolarizing the threshold for action potential production.

During fictive locomotion in the adult decerebrate cat, motoneurone excitability is increased by a hyperpolarization of the threshold potential at which an action potential is elicited (V(th)). This lowering of V(th) occurs at the onset of fictive locomotion, is evident for the first action potential elicited and is presumably caused by a neuromodulatory process. The present study tests the hypothesis that the monoamines serotonin (5-HT) and noradrenaline (NA) can hyperpolarize neuronal V(th). The neonatal rat isolated spinal cord preparation and whole-cell recording techniques were used to examine the effects of bath-applied 5-HT and NA on the V(th) of spinal ventral horn neurones. In the majority of lumbar ventral horn neurones, 5-HT (13/26) and NA (10/16) induced a hyperpolarization of V(th) ranging from -2 to -8 mV. 5-HT and NA had similar effects on V(th) for individual neurones. This hyperpolarization of V(th) was not due to a reduction of an accommodative process, and could be seen without changes in membrane potential or membrane resistance. These data reveal a previously unknown action of 5-HT and NA, hyperpolarization of V(th) of spinal neurones, a process that would facilitate both neuronal recruitment and firing.

Quantitative investigation of calcium signals for locomotor pattern generation in the lamprey spinal cord.

Locomotor pattern generation requires the network coordination of spinal ventral horn neurons acting in concert with the oscillatory properties of individual neurons. In the spinal cord, N-methyl-d-aspartate (NMDA) activates neuronal oscillators that are believed to rely on Ca(2+) entry to the cytosol through voltage-operated Ca(2+) channels and synaptically activated NMDA receptors. Ca(2+) signaling in lamprey ventral horn neurons thus plays a determinant role in the regulation of the intrinsic membrane properties and network synaptic interaction generating spinal locomotor neural pattern activity. We have characterized aspects of this signaling quantitatively for the first time. Resting Ca(2+) concentrations were between 87 and 120 nM. Ca(2+) concentration measured during fictive locomotion increased from soma to distal dendrites [from 208 +/- 27 (SE) nM in the soma to 335 +/- 41 nM in the proximal dendrites to 457 +/- 68 nM in the distal dendrites]. We sought to determine the temporal and spatial properties of Ca(2+) oscillations, imaged with Ca(2+)-sensitive dyes and correlated with fluctuations in membrane potential, during lamprey fictive locomotion. The Ca(2+) signals recorded in the dendrites showed a great deal of spatial heterogeneity. Rapid changes in Ca(2+)-induced fluorescence coincided with action potentials, which initiated significant Ca(2+) transients distributed throughout the neurons. Ca(2+) entry to the cytosol coincided with the depolarizing phase of the locomotor rhythm. During fictive locomotion, larger Ca(2+) oscillations were recorded in dendrites compared with somata in motoneurons and premotor interneurons. Ca(2+) fluctuations were barely detected with dyes of lower affinity providing alternative empirical evidence that Ca(2+) responses are limited to hundreds of nanomolars during fictive locomotion.