Action of γ-aminobutyric acid and glycine on red nucleus neurones

Summary GABA and glycine were applied iontophoretically in the red nucleus. Their effects were tested upon focal potentials in extracellular records, and upon the cell membrane (polarization, conductance, amplitude of spike and synaptic potentials) of rubrospinal neurons in intracellular records. Both GABA and glycine reduced the amplitude of focal potentials, hyperpolarized the membrane and increased its conductance. Quantitatively GABA actions were clearly more pronounced than glycine actions, based on the comparison of iontophoretic currents.Supported by: Deutsche Forschungsgemeinschaft (Grant Br 242/7).     

Modulation of inhibitory glycine receptors in cultured embryonic mouse hippocampal neurons by zinc, thiol containing redox agents and carnosine

Modulation of inhibitory glycine receptors by zinc (Zn(2+)) and endogenous redox agents such as glutathione may alter inhibition in the mammalian brain. Despite the abundance of Zn(2+) in the hippocampus and its ability to modulate glycine receptors, few studies have examined Zn(2+) modulation of hippocampal glycine receptors. Whether redox agents modulate hippocampal glycine receptors also remains unknown. This study examined Zn(2+) and redox modulation of glycine receptor-mediated currents in cultured embryonic mouse hippocampal neurons using whole-cell recordings. Zn(2+) concentrations below 10 microM potentiated currents elicited by low glycine, beta-alanine, and taurine concentrations by 300-400%. Zn(2+) concentrations above 300 microM produced nearly complete inhibition. Potentiating Zn(2+) concentrations shifted the dose-response curves for the three agonists to the left and decreased the Hill coefficient for glycine and beta-alanine but not taurine. Inhibiting Zn(2+) concentrations shifted the dose-response curves for glycine and beta-alanine to the right but reduced the maximum taurine response. Histidine residues may participate in potentiation because diethyl pyrocarbonate and pH 5.4 diminished Zn(2+) enhancement of glycine currents. pH 5.4 diminished Zn(2+) block of glycine currents, but diethyl pyrocarbonate did not. These findings indicate that separate sites mediate Zn(2+) potentiation and inhibition. The redox agents glutathione, dithiothreitol, tris(2-carboxyethyl)phosphine, and 5,5'-dithiobis(2-nitrobenzoic acid) did not alter glycine currents by a redox mechanism. However, glutathione and dithiothreitol interfered with the effects of Zn(2+) on glycine currents by chelating it. Carnosine had similar effects. Thus, Zn(2+) and thiol containing redox agents that chelate Zn(2+) modulate hippocampal glycine receptors with the mechanism of Zn(2+) modulation being agonist dependent.     

Enhanced release of inhibitory and excitatory transmitter quanta in the crayfish neuromuscular junction by glycine and GABA

High concentrations of glycine (greater than or equal to 0.1 mol/l) applied to the bathing medium of voltage-clamped muscle fibres elicited high rates of spontaneous inhibitory miniature currents (sIPSCs) which were identified by means of the noise analysis technique. The rate of spontaneous excitatory miniature currents (sEPSCs) was not raised appreciably in presence of these high concentrations of glycine. Contrary to the effect of glycine, high concentrations of GABA (greater than or equal to 0.1 mol/l) desensitized inhibitory postsynaptic receptors and induced high rates of sEPSCs. When, in addition to GABA, glycine was also applied at high concentrations, the discharge of sEPSCs was suppressed. Moreover, glycine reduced significantly the current noise intensity elicited by activation of excitatory postsynaptic membrane channels with 1.10(-4) mol/l glutamate. The experiments suggest an inhibitory action of glycine on the excitatory receptor--channel complex in the postsynaptic membrane.     

Nongenomic regulation of glutamatergic neurotransmission in hippocampus by thyroid hormones

Thyroid hormones (THs) are well known for their genomic effects but recently attention has focused also on their nongenomic actions as rapid modulators of membrane receptors. Here we show that thyroxine (T4) and 3,3',5'-l-triiodothyronine (T3) rapidly decrease N-methyl-d-aspartate (NMDA)-evoked currents in rat hippocampal cultures with potency in the micromolar range. The effect is not mediated by glutamate or glycine binding sites as an increase in agonist or glycine concentration does not alter TH potencies. Furthermore THs' effect on NMDA receptors is independent of voltage and of subunit composition. The mechanism of THs' antagonistic effect does not involve PKC phosphorylation of NMDA receptors since neither blocking nor stimulating PKC changed THs' modulation. T3, but not T4, inhibits also kainate-evoked currents in hippocampal neurons in culture. In hippocampal pyramidal neurons in slice, T3, but not T4, significantly reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting their amplitude and decay. In cultured rat cortical neurons THs prevented glutamate-induced neuronal death at concentrations similar to those effective on glutamatergic receptors. Taken together our data show for the first time that THs can rapidly affect ionotropic glutamatergic receptors in hippocampal neurons, an effect that could have an important role in their modulation of brain function in physiological and pathological states.     

Glycine transporter type 1 blockade changes NMDA receptor-mediated responses and LTP in hippocampal CA1 pyramidal cells by altering extracellular glycine levels

Long-term potentiation (LTP) in the hippocampal CA1 region requires the activation of NMDA receptors (NMDARs). NMDAR activation in turn requires membrane depolarization as well as the binding of glutamate and its coagonist glycine. Previous pharmacological studies suggest that the glycine transporter type 1 (GlyT1) maintains subsaturating concentrations of glycine at synaptic NMDARs. Antagonists of GlyT1 increase levels of glycine in the synaptic cleft and, like direct glycine site agonists, can augment NMDAR currents and NMDAR-mediated functions such as LTP. In addition, stimulation of the glycine site initiates signalling through the NMDAR complex, priming the receptors for clathrin-dependent endocytosis. We have used a new potent GlyT1 antagonist, CP-802,079, with whole-cell patch-clamp recordings in acute rat hippocampal slices to determine the effect of GlyT1 blockade on LTP. Reverse microdialysis experiments in the hippocampus of awake, freely moving rats, showed that this drug elevated only the extracellular concentration of glycine. We found that CP-802,079, sarcosine and glycine significantly increased the amplitude of the NMDAR currents and LTP. In contrast, application of higher concentrations of CP-802,079 and glycine slightly reduced NMDAR currents and did not increase LTP. Overall, these data suggest that the level of glycine present in the synaptic cleft tightly regulates the NMDAR activity. This level is kept below the 'set point' of the NMDAR internalization priming mechanism by the presence of GlyT1-dependent uptake.     

The kinetics of inhibition of rat recombinant heteromeric α1β glycine receptors by the low-affinity antagonist SR-95531

The GABA_A antagonist SR-95531 (gabazine) is known to block glycine receptors, albeit with low affinity. We have studied the effect of SR-95531 on rat recombinant α1β glycine receptors expressed in human embryonic kidney (HEK293) cells by recording macroscopic currents elicited by rapid glycine application to outside-out patches. SR-95531 has a fast unbinding rate ( k _offSR, about 3000 s⁻¹) and this means that the time course of its unbinding is comparable to the expected time course of the transmitter in the cleft. We also found that equilibrium applications of SR-95531 reduced the response to brief glycine applications by an amount inversely proportional to the duration of glycine application. The fast unbinding rate of SR-95531 from the glycine receptor will make it useful for establishing the time course of glycine concentration at glycinergic synapses.     

Modulation of glycinergic synaptic current kinetics by octanol in mouse hypoglossal motoneurons

Octanol-induced changes in the kinetics of glycinergic inhibitory postsynaptic currents (IPSCs) were investigated by whole-cell recording from hypoglossal motoneurons in mouse brainstem slices. Octanol (1 mM) prolonged the decay time constants (tau(decay)) of stimulus-evoked IPSCs (e-IPSCs) by 202+/-67% (SE). The depression of e-IPSC amplitudes was dose-dependent with an EC50 of 475 microM. Octanol also reduced the amplitude and prolonged the decay time constant of glycinergic currents evoked by local pressure ejection of glycine (I(gly)). Replacement of extracellular Na+ by choline and application of the specific glycine transporter GLYT1 inhibitor, sarcosine, lengthened tau(decay) of I(gly), but did not change the decay time constants of e-IPSCs. Intracellular acidification by the weak organic acid salt sodium propionate (30 mM) reduced the e-IPSC amplitude by 22+/-9% and prolonged tau by 18+/-6%. Sodium propionate also prolonged the decay time constants of I(gly) by 28+/-11%. The observed effects on decay kinetics were much smaller than those caused by octanol. The data show that octanol prolongs the decay time course of glycinergic synaptic currents by mechanisms independent of glycine uptake or intracellular acidification. We conclude that the effects were most probably due to direct action on postsynaptic glycine receptors.     

Interactions between GABA and glycine at inhibitory amino acid receptors on rat olfactory bulb neurons

Whole cell voltage-clamp electrophysiology was used to examine interactions between GABA and glycine at inhibitory amino acid receptors on rat olfactory bulb neurons in primary culture. Membrane currents evoked by GABA and glycine were selectively inhibited by low concentrations of bicuculline and strychnine, respectively, suggesting that they activate pharmacologically distinct receptors. However, GABA- and glycine-mediated currents showed cross-inhibition when the two amino acids were applied sequentially. Application of one amino acid inhibited the response to immediate subsequent application of the other. In the majority of neurons, GABA inhibited subsequent glycine-evoked currents and glycine inhibited subsequent GABA-evoked currents. In a small proportion of neurons, however, GABA inhibited glycine-evoked currents but glycine had little effect on GABA-evoked currents. The reverse was true in other neurons, suggesting that alterations in chloride gradients alone did not account for the cross-inhibition. Furthermore, no cross-inhibition was observed between GABA- and glycine-evoked currents in some neurons. The amplitude of the current evoked by the coapplication of saturating concentrations of GABA and glycine in these neurons was nearly the sum of the currents evoked by GABA and glycine alone. In contrast, the currents were not additive in neurons demonstrating cross-inhibition. These results suggest that olfactory bulb neurons heterogeneously express a population of inhibitory amino acid receptors that can bind either GABA or glycine. Interactions between GABA and glycine at inhibitory amino acid receptors may provide a mechanism to modulate inhibitory synaptic transmission.     

A novel physiological mechanism of glycine-induced immunomodulation: Na+-coupled amino acid transporter currents in cultured brain macrophages

Glycine is known to modulate immune cell responses. However, the physiological mechanisms underlying inhibitory effects of glycine on macrophages are not well understood. Here we show that glycine is capable of inducing inward currents in brain macrophages (microglia). In contrast to glycine, the glycine receptor agonist taurine failed to elicit currents. Glycine-evoked currents of brain macrophages were unaffected by strychnine, Cl⁻-free extracellular solution, N -[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine (NFPS) and amoxapine, but were abolished upon omission of extracellular Na⁺. Furthermore, glycine caused increases in the intracellular Na⁺ concentration and pronounced membrane depolarization. Glycine-evoked depolarization was Na⁺ dependent and occurred independently of the intracellular Cl⁻ concentration. Similarly to glycine, glutamine and α-(methylamino)isobutyric acid (MeAIB) elicited inward currents in brain macrophages. In the presence of either glutamine or MeAIB, glycine-induced currents were inhibited. It is concluded that neither functional glycine receptors nor glycine transporters are expressed in brain macrophages. We suggest that glycine mediates its effects by activation of system A Na⁺-coupled neutral amino acid transporters.     

Pre- and postsynaptic modulation of glycinergic and gabaergic transmission by muscarinic receptors on rat hypoglossal motoneurons in vitro

The motor output of hypoglossal motoneurons to tongue muscles takes place in concert with the respiratory rhythm and is determined by the balance between excitatory glutamatergic transmission and inhibitory transmission mediated by glycine or GABA. The relative contribution by these transmitters is a phasic phenomenon modulated by other transmitters. We examined how metabotropic muscarinic receptors, widely expressed in the brainstem where they excite cranial motor nuclei, might influence synaptic activity mediated by GABA or glycine. For this purpose, using thin slices of the neonatal rat brainstem, we recorded (under whole-cell patch clamp) glycinergic or GABAergic responses from visually identified hypoglossal motoneurons after pharmacological block of glutamatergic transmission. Muscarine inhibited spontaneous and electrically induced events mediated by GABA or glycine. The amplitude of glycinergic miniature inhibitory postsynaptic currents was slightly reduced by muscarine, while GABAergic miniature inhibitory postsynaptic currents were unaffected. Motoneuron currents induced by focally applied GABA and glycine were depressed by muscarine with stronger reduction in glycine-mediated responses. Histochemical observations indicated the presence of M1, M2 and M5 subtypes of muscarinic receptors in the neonatal hypoglossal nucleus. These results suggest that muscarine potently depressed inhibitory neurotransmission on brainstem motoneurons, and that this action was exerted via preterminal and extrasynaptic receptors. Since the large reduction in inhibitory neurotransmission may contribute to overall excitation of brainstem motoneurons by muscarinic receptors, these data might help to understand the central components of action of antimuscarinic agents in preanesthetic medication or against motion sickness.     

Ethanol dual modulatory actions on spontaneous postsynaptic currents in spinal motoneurons

Recently we have shown that acute ethanol (EtOH) exposure suppresses dorsal root-evoked synaptic potentials in spinal motoneurons. To examine the synaptic mechanisms underlying the reduced excitatory activity, EtOH actions on properties of action potential-independent miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) were studied in spinal motoneurons of newborn rats. Properties of mEPSCs generated by activation of N-methyl-D-aspartate receptors (NMDARs) and non-NMDA receptors and of mIPSCs mediated by glycine and gamma-aminobutyric acid-A receptors (GlyR and GABA(A)R) were examined during acute exposure to 70 and 200 mM EtOH. In the presence of 70 mM EtOH, the frequency of NMDAR- and non-NMDAR-mediated mEPSCs decreased to 53 +/- 5 and 45 +/- 7% (means +/- SE) of control values, respectively. In contrast, the frequency of GlyR- and GABA(A)R-mediated mIPSCs increased to 138 +/- 15 and 167 +/- 23% of control, respectively. Based on the quantal theory of transmitter release, changes in the frequency of miniature currents are correlated with changes in transmitter release, suggesting that EtOH decreased presynaptic glutamate release and increased the release of both glycine and GABA. EtOH did not change the amplitude or rise and decay times of either mEPSCs or mIPSCs, indicating that the presynaptic changes were not associated with changes in the properties of postsynaptic receptors/channels. Acute exposure to 200 mM EtOH increased mIPSC frequency two- to threefold, significantly higher than the increase induced by 70 mM EtOH. However, the decrease in mEPSC frequency was similar to that observed in 70 mM EtOH. Those findings implied that the regulatory effect of EtOH on glycine and GABA release was dose-dependent. Exposure to the higher EtOH concentration had opposite actions on mEPSC and mIPSC amplitudes: it attenuated the amplitude of NMDAR- and non-NMDAR-mediated mEPSCs to ~80% of control and increased GlyR- and GABA(A)R-mediated mIPSC amplitude by ~20%. EtOH-induced changes in the amplitude of postsynaptic currents were not associated with changes in their basic kinetic properties. Our data suggested that in spinal networks of newborn rats, EtOH was more effective in modulating the release of excitatory and inhibitory neurotransmitters than changing the properties of their receptors/channels.     

γ-Aminobutyric acid and inhibition in the septal nuclei of the rat

1. The electrophoretic application of gamma-aminobutyrate (GABA) and glycine to septal neurones inhibited their discharge, but the currents required to cause equivalent degrees of inhibition were always smaller for GABA than for glycine.2. The actions of GABA but not those of glycine were antagonized by bicuculline and not by strychnine and vice versa.3. The recurrent collateral inhibition of lateral septal neurones and the direct inhibition of medial cells evoked by stimulation of the fimbria were blocked by bicuculline but not by strychnine.4. The bursting patterns of discharge of laterally situated medial septal neurones was disrupted by bicuculline, but required a longer application of the drug than was needed to block the direct fimbrial inhibition. It is suggested that this disruption is due to diffusion of the drug to affect the inhibitory interneurones of the lateral septum.5. It is concluded that the inhibitory processes in the septum involve GABA as the synaptic transmitter.     

Glycine Receptors and Glycinergic Synaptic Input at the Axon Terminals of Mammalian Retinal Rod Bipolar Cells

We investigated the properties of glycine receptors and glycinergic synaptic inputs at the axon terminals of rod bipolar cells (RBCs) in rats by patch-clamp recording. Glycine currents recorded from isolated axon terminals were larger than those from isolated somata/dendrites; this was confirmed by puffing glycine onto these two regions in retinal slices. The current density at terminal endings was more than one order of magnitude higher than the density at somatic/dendritic regions. Glycine currents from isolated terminals and isolated somata/dendrites showed similar sensitivity to picrotoxinin blockade. Single-channel opening recorded from isolated terminals and somata/dendrites displayed a similar main-state conductance of ≈46 pS. Application of glycine effectively suppressed depolarization-evoked increases in intracellular Ca²⁺ at the terminals. In the presence of GABA_A and GABA_C antagonists, strychnine-sensitive chloride currents were evoked in RBCs in retinal slices by puffing kainate onto the inner plexiform layer. No such currents were observed if the recorded RBCs did not retain axon terminals or if Ca²⁺ was replaced by Co²⁺ in the extracellular solution. The currents displayed discrete miniature-like events, which were partially blocked by tetrodotoxin. Consistent with early studies in the rabbit and mouse, this study demonstrates that glycine receptors are highly concentrated at the axon terminals of rat RBCs. The pharmacological and physiological properties of glycine receptors located in the axon terminal and somatic/dendritic regions, however, appear to be the same. This study provides evidence for the existence of functional glycinergic synaptic input at the axon terminals of RBCs, suggesting that glycine receptors may play a role in modulating bipolar cell synaptic transmission.     

Creatine as a compatible osmolyte in muscle cells exposed to hypertonic stress

Glycinergic interplexiform cells provide a feedback signal from the inner retina to the outer retina. To determine if cones receive such a signal, glycine was applied on cultured porcine cone photoreceptors recorded with the patch clamp technique. A minor population of cone photoreceptors was found to generate large currents in response to puff application of glycine. These currents reversed close to the calculated equilibrium potential for chloride ions. These glycine-elicited currents were sensitive to strychnine but not to picrotoxin consistent with the expression of α–β-heteromeric glycine receptors. Glycine receptors were also activated by taurine and β-alanine. The glycine receptor antibody mAb4a labelled a minority of the cone photoreceptors identified by an antibody specific for cone arrestin. Finally, expression of the β subunit of the glycine receptor was demonstrated by single cell RT-PCR in a similar proportion (∼13%) of cone photoreceptors freshly isolated by lectin-panning. The identity of cone photoreceptors was assessed by their specific expression of the cone arrestin mRNA. The population of cone photoreceptors expressing the glycine receptor was not correlated to a specific colour-sensitive subtype as demonstrated by single cell RT-PCR experiments using primers for S opsin, cone arrestin and glycine receptor β subunit. This glycine receptor expression in a minority of cones defines a new cone population suggesting an unexpected role for glycine in the visual information processing in the outer retina.     

Modulation of human GABArho1 receptors by taurine

A study was made of the effects of taurine on GABArho1 receptors expressed in Xenopus oocytes. The EC(50) and reversal potentials for GABA, taurine and glycine currents were 2.3+/-0.4 microM (-25+/-0.9 mV), 5+/-0.8mM (-27+/-0.4 mV) and 7+/-0.5mM (-22+/-0.6 mV), respectively. Co-application of GABA and taurine, revealed a taurine concentration-dependent biphasic-modulation of the receptor: at 0.3-30 microM taurine potentiated the GABA-currents, whereas at 0.3-30 mM the GABA-currents were reduced. In contrast glycine potentiated the GABA-currents at all concentrations tested. TPMPA, a GABA(C) specific receptor antagonist, also blocked effectively and reversibly the taurine and glycine currents. Finally, lanthanum and zinc modulated the currents generated by the three amino acids. Taurine is abundant in the retina and our observations suggest that taurine may play an important role modulating the retinal GABAergic transmission.     

The effects of glycine and GABA on isolated horizontal cells from the salamander retina.

1. Horizontal cells, identified by their morphology, were isolated from the salamander retina and examined in whole cell patch clamp. 2. All cells showed large outward currents activating positive to about -50 mV, and a minority of cells showed fast, tetrodotoxin-suppressible Na+ currents. Slow inward currents that might shape the light responses were never observed. 3. All cells showed conductance increases to both gamma-aminobutyric acid (GABA) and glycine that were completely blocked by bicuculline and strychnine, respectively. No cross-blocking by these antagonists was observed. Partial replacements of Cl- with large, impermeant anions indicated that both GABA- and glycine-evoked currents were carried by Cl- ions. 4. Responses to both GABA and glycine desensitized strongly with time constants of approximately 2 s. 5. Responses to glutamate were not enhanced by glycine. Similarly, responses to GABA were not enhanced by glutamate. 6. GABA-mediated synaptic interactions between horizontal cells may account for the changes in the kinetics of horizontal cell light responses seen when glycine is applied to the intact retina.     

Frequency-dependent modulation of glycine receptor activation recorded from the zebrafish larvae hindbrain

In vertebrates, most glycinergic inhibitory neurons discharge phasically at a relatively low frequency. Such a pattern of glycine liberation from presynaptic terminals may affect the kinetics of post-synaptic glycine receptors. To examine this influence, we have analyzed the behavior of glycine receptors in response to repetitive stimulation at frequencies at which consecutive outside-out currents did not superimpose (0.5-4 Hz). Neurotransmitter release was mimicked on outside-out patches from zebrafish hindbrain Mauthner cells using fast flow application techniques. The amplitude of outside-out currents evoked by short (1 ms) repetitive applications of a saturating concentration (3 mM) of glycine remained unchanged for application frequencies1 Hz. Glycine-evoked current simulations using a simple Markov model describing zebrafish glycine receptor kinetic behavior, indicates that this down-regulation of glycine receptor efficacy is due to a progressive accumulation of the receptors in a long lasting desensitization state. Our simulations suggest that this down-regulation can occur even when spontaneous inhibitory currents were generated randomly at a frequency>1 Hz.     

Group II metabotropic glutamate receptor modulation of excitatory transmission in rat subthalamic nucleus

Patch pipettes were used to record currents in whole-cell configuration to study the effects of group II metabotropic glutamate receptor (mGluR) stimulation on synaptic transmission in slices of rat subthalamic nucleus. Evoked glutamatergic excitatory postsynaptic currents (EPSCs) were reversibly reduced by the selective group II mGluR agonist (2' S ,2' R ,3' R )-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) in a concentration-dependent manner, with an IC₅₀ of 0.19 ± 0.05 µ m . DCG IV (1 µ m ) had no effect on inhibitory postsynaptic currents mediated by GABA. DCG IV-induced inhibition of EPSCs was reversed by the selective group II mGluR antagonist LY 341495 (100 n m ) and mimicked by another selective group II agonist (2 S ,1' S ,2' S )-2-(carboxycyclopropyl)glycine ( l -CCG-I). Inhibition of EPSC amplitude by DCG IV and l -CCG-I was associated with an increase in the paired-pulse ratio of EPSCs. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (2 µ m ) reduced the inhibitory effect of DCG IV on EPSCs. However, the response to DCG IV was not affected by the protein kinase A (PKA) activator forskolin (20 µ m ), by the adenylyl cyclase inhibitor MDL 12230A (20 µ m ), or by the phosphodiesterase inhibitor Ro 20–1724 (50 µ m ). DCG IV-induced inhibition of EPSCs was reduced by the non-selective protein kinase inhibitors H-7 (100 µ m ), H-8 (50 µ m ) and HA-1004 (100 µ m ). These results suggest that group II mGluR stimulation acts presynaptically to inhibit glutamate release by a PKC-dependent mechanism in the subthalamic nucleus.     

Effects of glycine on the crayfish neuromuscular junction

Glycine applied in the bathing medium at concentrations exceeding 0.1 mol/l elicited high rates of spontaneous inhibitory postsynaptic currents (sIPSCs) in crayfish neuromuscular junctions. This effect of glycine was reversible within seconds. In several experiments on application of 0.5 mol/l glycine the rate of sIPSCs immediately increased to about 10 kHz and thereafter declined exponentially with time constants of between 10 and 20 s. This resulted in a release of about 140,000-200,000 inhibitory quanta per trial. When the readily releasable pool of transmitter had been so depleted by glycine, it was necessary to superfuse the preparation with normal solution for 5-10 min in order to be able to again evoke a high rate of sIPSCs. A similar effect of glycine on spontaneous release was also observed in some preparations which had been previously bathed in zero Ca2+ solution for up to 45 min. Addition of 25 mmol/l Mg2+ to the bathing fluid did not block the glycine evoked release of transmitter. However, in sodium-free superfusions the increase in the rate of sIPSCs induced by glycine was reduced. In the presence of 0.5 mol/l glycine no excitatory miniature currents (sEPSCs) were observed, in fact, glycine depressed excitatory synaptic transmission. In addition to the increasing the rate of sIPSCs, high concentrations of glycine evoked 'giant' sIPSCs (gsIPSCs). They were about 10-15 times larger than the normal sIPSCs and occurred at rates lower than 3 Hz, irrespective of whether the bathing medium contained sodium or not. However, in sodium-free superfusions the time constants of the decay of gsIPSCs were prolonged by a factor 2-3. These results suggest that glycine elicited sIPSCs and gsIPSCs by different mechanisms. Possible mechanisms which might explain the effects of glycine on release of inhibitory transmitter are discussed.     

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.