AMPA-evoked acetylcholine release from cultured spinal cord motoneurons and its inhibition by GABA and glycine

The release of [(3)H]acetylcholine evoked by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and its inhibition mediated by GABA(A) and glycine receptors were studied in superfused cultured rat embryo spinal cord motoneurons prelabeled with [(3)H]choline. AMPA elicited tritium release, possibly representing [(3)H]acetylcholine release in a concentration-dependent manner. The release was external Ca(2+)-dependent and was sensitive to Cd(2+) ions, omega-conotoxin GVIA and omega-conotoxin MVIIC, but not to nifedipine, suggesting the involvement of N-, P/Q-, but not L-type Ca(2+) channels. The AMPA effect was insensitive to tetrodotoxin. The glutamate receptors involved are AMPA type since the AMPA-evoked [(3)H]acetylcholine release was blocked by LY303070 and was potentiated by the antidesensitizing agent cyclothiazide. Muscimol inhibited completely the AMPA effect on [(3)H]acetylcholine release; muscimol was potentiated by diazepam and antagonized by SR95531, indicating the involvement of benzodiazepine-sensitive GABA(A) receptors. Glycine, acting at strychnine-sensitive receptors, also inhibited the effect of AMPA, but only in part. The inhibitory effects of muscimol and glycine are additive.We conclude that glutamate can act at AMPA receptors sited on spinal motoneurons to evoke release of acetylcholine. GABA and glycine, possibly released as cotransmitters from spinal interneurons, inhibit glutamate-evoked acetylcholine release by activating GABA(A) and glycine receptors on motoneurons.     

GABA and glycine co-release optimizes functional inhibition in rat brainstem motoneurons in vitro

Whole-cell patch clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs) were obtained in identified abducens motoneurons (aMns) from young rats (P5-P13). Three types of mIPSC were distinguished according to their kinetics and their sensitivity to receptor antagonists: faster decaying events mediated by glycine receptors (glyRs), slower decaying events mediated by GABA_A receptors (GABA_ARs), and mIPSCs displaying two components corresponding to GABA and glycine co-release. Dual component events accounted for ≈30 % of mIPSCs, independently of the rat's age and were also identified during evoked transmitter release. In contrast, the kinetics of glyR- and GABA_AR-mediated mIPSCs became faster during development. Monosynaptic inhibitory postsynaptic potentials (IPSPs) were able to fully inhibit motoneuron discharge elicited by current pulses. When the GABA_AR-mediated component or the glyR-mediated component of the IPSP was blocked, the inhibition of motoneuron firing was reduced. The 20-80 % rise time and duration of GABA_AR-mediated IPSPs were significantly longer than those mediated by glyRs. The time window of inhibition for each component was determined using single postsynaptic action potentials elicited with various delays from the onset of the IPSP. GlyR-mediated IPSPs induced fast transient inhibition whereas GABA_AR-mediated IPSPs induced slow sustained suppression of firing. Using a modelling approach, we found that the two components summated non-linearly. We conclude that in developing aMns, co-release of GABA and glycine determines the strength and timing of inhibition through non-linear interactions between the two components, thus optimizing inhibition of motoneuron function.     

Cotransmission of GABA and glycine to brain stem motoneurons.

Using whole cell patch-clamp recording in a rat brain stem slice preparation, we found that gamma-aminobutyric acid (GABA) and glycine act as cotransmitters to hypoglossal motoneurons (HMs). Focal application of GABA and glycine onto a single HM revealed that GABAA and glycine receptors are present on the same neuron. To demonstrate that HMs receive both GABAergic and glycinergic synaptic inputs, we simultaneously recorded GABAA- and glycine-receptor-mediated spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in single HMs. GABAergic and glycinergic mIPSCs were differentiated based on their kinetics and modulation by pentobarbital. Specifically, GABAA-receptor-mediated events decayed more slowly than glycine-receptor-mediated events. GABAergic response decay kinetics were prolonged by pentobarbital, whereas glycinergic response decay kinetics remained unchanged. The distinct kinetics of the glycine- and GABAA-receptor-mediated synaptic events allowed us to record dual component mIPSCs, mIPSCs that are mediated by both receptor types. These data suggest that GABA and glycine are colocalized in the same presynaptic vesicle and are coreleased from presynaptic terminals opposed to motoneurons.     

Three types of inhibitory miniature potentials in frog spinal cord motoneurons: Possible GABA and glycine cotransmission

Miniature inhibitory postsynaptic potentials (mIPSP) of motoneurons in isolated frog spinal cord were recorded in conditions of blockade of the conduction of nerve spikes and ionotropic glutamate receptors (TTX, 1 μM, CNQX, 25 μM, D-AP5, 50 μM). Three types of mIPSP were identified: those with fast and slow time characteristics and mIPSP with two-component decays. Two-component mIPSP accounted for 8.7% of all selected responses, fast mIPSP for 64.5%, and slow mIPSP for 26.8%. Blockade of GABA_A receptors with bicuculline (20 μM) led to decreases in the numbers of slow and two-component mIPSP and an increase in the number of mIPSP with fast kinetics. Strychnine (1 μM), a blocker of glycine receptors, led to a reduction in the number of fast receptors and an increase in the number of slow potentials. These data suggest that frog spinal cord motoneurons have three types of inhibitory mIPSP, mediated by GABA, glycine, and simultaneous release of these two transmitters from the same presynaptic terminals. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 92, No. 1, pp. 18–26, January, 2006.     

Location and magnitude of conductance changes produced by Renshaw recurrent inhibition in spinal motoneurons

The mean location of Renshaw synapses on spinal motoneurons and their synaptic conductance were estimated from changes in impedance magnitude produced by sustained recurrent inhibition. Motoneuron impedance was determined by injecting quasi-white noise current into lumbosacral motoneurons of pentobarbital-anesthetized cats. Synaptic location and conductance were estimated by comparing observed impedance changes to simulation results obtained using standard motoneuron models and compartmental models fit to each impedance function. Estimated synaptic locations ranged from 0.10 to 0.41lambda, with a mean of 0.19 or 0.24lambda, depending on the estimation method. Average dendritic path length was 262 microm. Average synaptic conductance was 23 to 27 nS (range: 6.7 to 57.9 nS), corresponding to conductance changes of 78 to 88% of resting membrane conductance. Estimated accuracy was supported by consistency using different estimation methods, agreement with Fyffe's 1991 morphological data, and comparisons of observed and simulated recurrent IPSP amplitudes. Synaptic location, but not synaptic conductance, was correlated with rheobase, a measure of motoneuron excitability. Synaptic conductance did not depend on synaptic location. A regression analysis demonstrated that synaptic conductance and cell impedance were the principal factors determining recurrent IPSP amplitude. Simulations using the observed values and locations of Renshaw conductance demonstrate that recurrent inhibition can require as much as an additional 14 to 18% sustained excitatory synaptic conductance to depolarize motoneurons sufficiently to activate somatic or dendritic inward currents and recruit motoneurons or amplify excitatory synaptic currents.     

GABA(B) modulation of GABA(A) and glycine receptor-mediated synaptic currents in hypoglossal motoneurons

We studied the effects of GABA(B) receptor activation on either glycine or GABA(A) receptor-mediated synaptic transmission to hypoglossal motoneurons (HMs, P8-13) using a rat brainstem slice preparation. Activation of GABA(B) receptors with baclofen, a GABA(B) receptor agonist, inhibited the amplitude of evoked glycine and GABA(A) receptor-mediated inhibitory postsynaptic currents. Additionally, with blockade of postsynaptic GABA(B) receptors baclofen decreased the frequency of both glycine and GABA(A) receptor-mediated spontaneous miniature inhibitory postsynaptic currents (mIPSCs), indicating a presynaptic site of action. Conversely, the GABA(B) receptor antagonist CGP 35348 increased the frequency of glycine receptor-mediated mIPSCs. Application of the GABA transport blocker SKF 89976A decreased the frequency of glycinergic mIPSCs. Lastly, we compared the effects of baclofen on the frequency of glycine and GABA(A) receptor-mediated mIPSC during HM development. At increased postnatal ages (P8-13 versus P1-3) mIPSC frequency was more strongly reduced by baclofen. These results show that presynaptic GABA(B) receptors inhibits glycinergic and GABAergic synaptic transmission to HMs, and the presynaptic sensitivity to baclofen is increased in P8-13 versus P1-3 HMs. Further, endogenous GABA is capable of modulating inhibitory synaptic transmission to HMs.     

GABA A mediated inhibition and post-inspiratory pattern of laryngeal constrictor motoneurons in rat

Laryngeal constrictor motoneurons (LCMN) are activated during post-inspiration and act to slow expiratory airflow. However, little is known about how this phasic activity is generated. Here, we investigated the electrophysiological responses of identified LCMN to local application of GABA and bicuculline methiodide (BIC) in 14 anaesthetised Sprague-Dawley rats. During extracellular recordings, GABA iontophoresis (0.5M) strongly inhibited LCMN (n=6). Interestingly, BIC iontophoresis (5 mM) reduced, rather than increased, LCMN post-inspiratory activity (5 out of 6). Furthermore, intracellular recording revealed that BIC reduced not only the hyperpolarisation of the LCMN during inspiration (2.5+/-1.4 mV before and 1.5+/-0.4 mV after the BIC, P=0.05, n=5), but also the depolarisation during post-inspiration (3.0+/-1.3 mV before and 1.6+/-0.4 mV after the BIC, P=0.02, n=5). Our results demonstrate for the first time that the inspiratory inhibition of LCMN is primarily mediated by GABA(A) receptors. A possible involvement of a post-inhibitory rebound mechanism is discussed to explain how blockade of an inspiratory inhibition would affect LCMN excitability during post-inspiration.     

Involvement of retinoic acid and its receptor beta in differentiation of motoneurons in chick spinal cord.

Retinoic acid is known to play an important role during development of central nervous system. In order to clarify function of retinoic acid during the development, we investigated expression pattern of the chick retinoic acid receptor subtype beta gene by an in situ hybridization method. We found that expression of the beta gene is localized in neural tube at stages 16-20, then is turned to be restricted to developing motoneurons at stages 23-29. These results suggested that retinoic acid and its receptor beta are involved in differentiation of the motoneurons in spinal cord.     

Activation of group I metabotropic glutamate receptors depresses recurrent inhibition of motoneurons in the neonatal rat spinal cord in vitro

A variety of experimental studies have demonstrated the neuroprotective effects of melatonin, based on its antioxidant activity. In a prospective randomized study, the effects of melatonin were investigated in experimental head trauma-induced oxidative stress in rabbits. The experimental study was performed on 30 rabbits. The animals were divided into three groups. Group I (sham procedure): a right parietal craniotomy was performed on each animal, and the dura mater was left intact. Group II: experimental brain trauma (EBT) was performed on each animal using a 1 cm inner diameter × 10 cm long glass tube, through which a 20 g weight (0.5 cm diameter) was dropped onto the brain at the craniotomy site, causing a contusional head trauma. Group III: the same EBT model was performed, but 2.5 mg/kg melatonin was injected intraperitoneally four times (total dose 10 mg/kg); these injections were performed 20 min before the operation, during the trauma, 1 h later and 2 h later. The rabbits were sacrificed after the EBT at 24 h after the brain trauma. The activities of the three principal antioxidant enzymes—catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px)—were determined, and the levels of malondialdehyde (MDA), a product of lipid peroxidation, and glutathione (GSH) were measured in brain homogenates. MDA levels were found to be higher in the EBT group than in the EBT+melatonin group or the sham procedure group. The SOD activity was found to be higher in the EBT group than in the sham procedure group. Enzymatic parameters (except for SOD) were significantly higher in melatonin-treated animals than in EBT animals. GSH levels in melatonin-treated animals were decreased compared with EBT animals. In conclusion, the data indicate that melatonin protects against free radical-mediated oxidative changes in brain tissue by boosting antioxidant enzymes, and in particular lowering lipid peroxidation in rabbits with EBT.     

Spatial distribution of recurrent inhibitory synapses on spinal motoneurons in the cat

1. Intracellular staining of Renshaw cells and alpha motoneurons was used to determine the spatial distribution of recurrent inhibitory synapses on spinal motoneurons in the cat. In each experiment, a Renshaw cell and one or more possible target motoneurons were labeled with horseradish peroxidase after physiological identification. 2. Paris of labeled neurons were reconstructed and measured at the light microscopic level. As defined by light microscopy, presumed synaptic contacts between nine Renshaw cells and 10 postsynaptic motoneurons were observed. On average, each Renshaw cell made three synaptic contacts (range 1-9) on each motoneuron. 3. Electron microscopic confirmation of several presumed contacts provided evidence that the appositions identified by light microscopic criteria are genuine contacts between Renshaw cell boutons and the labeled motoneuron. 4. All of the identified synapses observed in these experiments were located on motoneuron dendrites, between 65 and 706 microns from the soma. Use of a simplified cable model indicated that the synapses are electrotonically close to the soma, the average location being approximately 0.25 length constants from the soma (range 0.04-0.82 lambda). 5. These observations provide direct evidence to support the hypothesis that Renshaw cell synapses on motoneurons are located on the dendrites and not on the cell body (whereas reciprocal inhibitory synapses, from Ia inhibitory interneurons, are predominantly located on the soma). The functional significance of the observed distribution of Renshaw inhibitory synapses is discussed. One possibility is that the recurrent inhibitory pathway selectively inhibits particular dendritic inputs.     

Differential effects of ethanol on GABA(A) and glycine receptor-mediated synaptic currents in brain stem motoneurons

Ethanol potentiates glycinergic synaptic transmission to hypoglossal motoneurons (HMs). This effect on glycinergic transmission changes with postnatal development in that juvenile HMs (P9-13) are more sensitive to ethanol than neonate HMs (P1-3). We have now extended our previous study to investigate ethanol modulation of synaptic GABA(A) receptors (GABA(A)Rs), because both GABA and glycine mediate inhibitory synaptic transmission to brain stem motoneurons. We tested the effects of ethanol on GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) recorded from neonate and juvenile rat HMs in an in vitro slice preparation. Bath application of 30 mM ethanol had no significant effect on the GABAergic mIPSC amplitude or frequency recorded at either age. At 100 mM, ethanol significantly decreased the GABAergic mIPSC amplitude recorded from neonate (6 +/- 3%, P < 0.05) and juvenile (16 +/- 3%, P < 0.01) HMs. The same concentration of ethanol increased the GABAergic mIPSC frequency recorded from neonate (64 +/- 17%, P < 0.05) and juvenile (40 +/- 15%, n.s.) HMs. In contrast, 100 mM ethanol robustly potentiated glycinergic mIPSC amplitude in neonate (31 +/- 3%, P < 0.0001) and juvenile (41 +/- 7%, P < 0.001) HMs. These results suggest that glycine receptors are more sensitive to modulation by ethanol than GABA(A) receptors and that 100 mM ethanol has the opposite effect on GABA(A)R-mediated currents in juvenile HMs, that is, inhibition rather than enhancement. Further, comparing ethanol's effects on GABAergic mIPSC amplitude and frequency, ethanol modulates GABAergic synaptic transmission to HMs differentially. Presynaptically, ethanol enhances mIPSC frequency while postsynaptically it decreases mIPSC amplitude.     

Co-localization of choline acetyltransferase and postsynaptic glycine receptors in motoneurons of rat spinal cord demonstrated by immunocytochemistry

Male rats were perfused with paraformaldehyde and picric acid. The cervical spinal cord was cryosectioned and immunostained with a monoclonal antibody against the postsynaptic receptor for the neurotransmitter glycine. The anterior horn contained glycinoceptive neurons of varying morphology. Cholinergic cells were identified in the same tissue sections when subsequently immunostained with a monoclonal antibody against choline acetyltransferase, the biosynthetic enzyme of acetylcholine. Immunoreactivity for the glycine receptor was detected in the plasma membrane and for choline acetyltransferase in the perikaryal cytoplasm of identical anterior horn cells, classified as small, medium and large motoneurons. This suggests that motoneurons have receptors for glycine on their cell surface.     

Distribution of effective synaptic currents underlying recurrent inhibition in cat triceps surae motoneurons

1. Steady-state recurrent (Renshaw) inhibitory postsynaptic potentials (RIPSPs) were evoked in cat triceps surae motoneurons by stimulating the heteronymous muscle nerve at 100 Hz after dorsal root section. The effective synaptic currents (i.e., the net synaptic current measured at the soma, IN) underlying these inhibitory potentials were measured with a modified voltage-clamp technique. 2. The average value of the effective synaptic currents measured in medial gastrocnemius (MG) motoneurons was 0.4 nA. There was no significant correlation between the IN measured in individual cells and motoneuron input resistance (RN), rheobase (IR), duration of the spike afterhyperpolarization (AHPt1/2), or putative motor-unit type, although the steady-state inhibitory post-synaptic potential (IPSP) amplitudes were correlated with all of these parameters. 3. Steady-state recurrent inhibition was accompanied by a small (3.5%, on average) decrease in the resting input resistance of the motoneurons. The small magnitude of this measured change supports the hypothesis of Burke et al. that the site of synaptic contact between Renshaw cells and motoneurons is somewhat distal to the cell soma. 4. The absence of a differential distribution of the effective synaptic currents generated by Renshaw cells within the MG pool does not support the idea that recurrent inhibition mediates a selective reduction of the firing of small, low-threshold motoneurons by large, high-threshold motoneurons. The small amplitude of the effective synaptic currents we measured suggests that the contribution of recurrent inhibition to the direct modulation of motoneuron firing rate is subtle and that it is perhaps principally involved in the fine control and smooth production of muscle force.     

The action of ammonium on postsynaptic inhibition of cat spinal motoneurons

Summary Potential changes were investigated which follow the intra- and extracellular electrophoretic injection of NH₄ ⁺ and also the intravenous application of neutral ammonium salts. The common effect found in all kinds of application is a depolarizing shift of the IPSP-equilibrium potential (E_IPSP) towards the resting potential. Inhibitory transmission is preserved but produces considerably reduced hyperpolarizing potential changes at normal resting potentials. In addition, ammonium ions produce short term shifts of resting and overshoot potentials with intracellular application. The latter changes can be explained under the assumption that NH₄ ⁺ partly substitutes for Na⁺ as for the action potential generation.The observed reversible reduction of E_IPSP has high sensitivity towards extracellular and systemic NH₄ ⁺ application as compared to intracellular injection. The time course of the depolarizing shift of E_IPSP, especially its restitution, was shortest in extracellular application. It is suggested that ammonium release from the internal site produces the long term effects observed on the IPSP after intracellular injection. The externally effective dosages correspond to intracerebral NH₄ ⁺-concentrations which are reported for preconvulsive states of various kinds of metabolically induced epilepsies.We thank Miss C. Lueg for careful technical assistance.     

Plasticity of recurrent inhibitory reflexes in cat spinal motoneurons following peripheral nerve injury

Summary Chronic axotomy of a peripheral motor nerve in cat causes a gradual reduction in the number of intramedullary axon collaterals originating from the axotomized motoneurons (Havton and Kellerth 1984, 1989). This axon collateral elimination would be expected to reduce the amount of recurrent inhibitory reflex actions mediated by these cells. The aim of the present study was to investigate the recurrent inhibition originating from axotomized motoneurons and, also, to see whether the elimination of axon collaterals from the axotomized neurons might induce secondary compensatory changes in the recurrent inhibitory pathways originating from synergistic non-lesioned motoneurons. The results, which were obtained by means of intracellular recordings and monosynaptic reflex testing, indicate that postoperative enhancement of reflex actions may take place in the recurrent inhibitory pathways persisting in the axotomized motoneurons as well as in those originating from synergistic nonlesioned motoneurons. It is suggested that the site of compensatory enhancement is at the synaptic reflex contacts between the motoraxon collaterals and the inhibitory Renshaw interneurons.     

Synaptic organization in teleost spinal motoneurons.

Glass microelectrodes were inserted into motoneurons innervating pectoral fin muscles to record action and synaptic potentials, evoked by electrical stimulation of ventral and dorsal roots, and the medulla oblongata. Ventral root stimulation evoked a small depolarizing response which had properties compatible with those of the EPSP; its amplitude changes were graded, being increased by membrane hyperpolarization and decreased by high frequency repetitive stimulation. The latency of the response was sufficiently longer than that of the antidromic spike to allow for a monosynaptic delay. Stimulation of the dorsal root produced EPSPs with relatively long latencies, suggesting mediation by a polysynaptic pathway. EPSPs with short latencies were evoked by stimulation of the medulla oblongata, indicating the presence of a monosynaptic excitatory connection. Action potentials, recorded from peripheral nerve after stimulation of the medulla oblongata, were facilitated by conditioning volleys via ventral roots. This facilitation was blocked by dihydro-beta-erythroidine hydrobromide and atropine sulphate, indicating the cholinergic nature of the EPSP of ventral root origin. The conduction velocities of motor axons and of the ventral roots fibers responsible for production of EPSPs were about the same. The EPSP of ventral root origin had a slower rising time course and lesser sensitivity to shifts of membrane potential than the EPSP of medulla oblongata origin, suggesting that the sites of generation of the former EPSP were on the peripheral dendrites. From the above results, it was concluded that the EPSP of ventral root origin was mediated by recurrent axon collaterals of motoneurons.