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.     

Involvement of GABA and glycine in recurrent inhibition of spinal motoneurons.

1. Recurrent inhibitory postsynaptic potentials (IPSPs) were recorded intracellularly from chloride-loaded motoneurons in the isolated lumbar spinal cord of neonatal rats (day 5-day 12). This in vitro preparation exhibited an intact and functional recurrent inhibitory pathway that displayed characteristics previously described for this pathway in other species. 2. Although strychnine (1-5 microM) depressed the chloride-dependent recurrent synaptic potentials evoked by ventral root stimulation by 48.2 +/- 2.7% (mean +/- SE, n = 13), confirming that part of the recurrent IPSP is mediated by a glycinergic mechanism, in every case a residual strychnine-resistant synaptic potential was observed. 3. The gamma-aminobutyric acid (GABA) antagonist bicuculline, in low concentrations (2-10 microM), depressed the recurrent synaptic potentials in a dose-dependent manner by 27.0 +/- 4.3% (range 0-49%, n = 19). Application of bicuculline almost eliminated the strychnine-resistant component of the IPSP. However, in some motoneurons, a small synaptic potential remained after combined application of strychnine and bicuculline. 4. The selective antagonists of GABA uptake, (+/-)-nipecotic acid (1 mM) and guvacine (1 mM), increased the amplitude of recurrent synaptic potentials in 12 of 16 motoneurons by 37.2 +/- 7.2% (range 12.6-84.2%). 5. The excitatory amino acid antagonists kynurenic acid (1 mM), 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX (10 microM)] and 6,7-dinitroquinoxaline-2,3-dione (10 microM) potentiated recurrent synaptic potentials in 5 of 7 motoneurons. However, CNQX (10-15 microM) in the presence of strychnine and bicuculline virtually abolished the synaptic potential remaining after application of the inhibitory amino acid antagonists. It is concluded that ventral root stimulation evokes a small excitatory amino acid-mediated synaptic potential in neonatal rat motoneurons. 6. An antidromic synaptic potential due to electrotonic coupling between motoneurons was unaffected by changes in membrane potential, chloride loading, or antagonists of glycine, GABA, excitatory amino acid, and acetylcholine receptors. 7. The results suggest that a major portion of the strychnine-resistant component of the IPSP is mediated by a GABAergic mechanism. It is concluded that both glycinergic and GABAergic mechanisms play a role in recurrent inhibition of motoneurons in the mammalian spinal cord. It is unknown whether these inhibitory amino acids are released by a single pool of Renshaw cells or by neurochemically distinct populations.     

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.     

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.     

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.     

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.     

Relative contribution by GABA or glycine to Cl(-)-mediated synaptic transmission on rat hypoglossal motoneurons in vitro

The relative contribution by GABA and glycine to synaptic transmission of motoneurons was investigated using an hypoglossus nucleus slice preparation from neonatal rats. Spontaneous, miniature, or electrically evoked postsynaptic currents (sPSCs, mPSCs, ePSCs, respectively) mediated by glycine or GABA were recorded under whole cell voltage clamp after blocking excitatory glutamatergic transmission with kynurenic acid. The overall majority of Cl(-)-mediated sPSCs was glycinergic, while only one-third was GABAergic; 70 +/- 10% of mPSCs were glycinergic while 22 +/- 8% were GABAergic. Tetrodotoxin (TTX) application dramatically reduced the frequency (and slightly the amplitude) of GABAergic events without changing frequency or amplitude of glycinergic sPSCs. These results indicate that, unlike spontaneous GABAergic transmission, glycine-mediated neurotransmission was essentially independent of network activity. There was a consistent difference in the kinetics of GABAergic and glycinergic responses as GABAergic events had significantly slower rise and decay times than glycinergic ones. Such a difference was always present whenever sPSCs, mPSCs, or ePSCs were measured. Finally, GABAergic and glycinergic mPSCs were differentially modulated by activation of glutamate metabotropic receptors (mGluRs), which are abundant in the hypoglossus nucleus. In fact, the broad-spectrum mGluR agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (50 microM), which in control solution increased the frequency of both GABAergic and glycinergic sPSCs, enhanced the frequency of glycinergic mPSCs only. These results indicate that on brain stem motoneurons, Cl(-)-mediated synaptic transmission is mainly due to glycine rather than GABA and that GABAergic and glycinergic events differ in terms of kinetics and pharmacological sensitivity to mGluR activation or TTX.     

The distribution of GABA and glycine response in the mouse brain using Xenopus oocytes

Injection of mRNA from different regions of the central nervous system (CNS) of the mouse into the Xenopus oocyte caused a difference in the relative glycine to gamma-aminobutyric acid (GABA) response of the oocyte. Glycine caused the response in oocytes injected with brainstem mRNA but hardly in oocytes injected with cerebrum mRNA and in oocytes injected with cerebellum mRNA. In contrast, GABA caused the response in oocytes injected with mRNA from each of the 3 parts of the CNS. These obvious regional differences may reflect the distribution of the receptors to each plausible neurotransmitter in the CNS.     

Depolarizing actions of GABA and glycine on amphibian retinal horizontal cells.

1. The effects of inhibitory amino acid transmitters on horizontal cells in the superfused amphibian retina were studied by the use of conventional intracellular recording techniques. 2. Gamma-aminobutyric acid (GABA) caused a calcium-independent depolarization of horizontal cells in mud puppy and tiger salamander. This action was mimicked by muscimol but not baclofen (BAC) and blocked by bicuculline and picrotoxin (PTX), matching the GABAa receptor profile. 3. The purported GABA uptake inhibitors nipecotate (NPA) and guvacine (GUV) acted as GABAa agonists, having pharmacological properties very similar to GABA itself. These agents also activated receptors of amacrine and ganglion cells, causing membrane polarizations similar to GABA. Concentrations of these analogues that did not activate the GABAa receptor (submillimolar) did not lower the effective dose of GABA, even after prolonged application. 4. Glycine (GLY) also depolarized horizontal cells, but only in approximately 25% of the horizontal cells was the amplitude of the depolarization as great as GABA. The glycine response was blocked by both strychnine (STR, 10 microM) and PTX (100 microM). In contrast, the action of GABA was unaffected by STR. 5. Ion substitution and channel-blocking agents indicated that the effects of applied GABA and GLY were independent of both external sodium and calcium. 6. The results suggest that GABA receptors on horizontal cells may act 1) as a positive feedback system to modulate the light response and 2) as a mechanism for chemical coupling between horizontal cells.     

Role of synaptic inputs in determining input resistance of developing brain stem motoneurons

The contribution of synaptic input to input resistance was examined in 208 developing genioglossal motoneurons in 3 postnatal age groups (5-7 day, 13-16 day, and 18-24 day) using sharp electrode recording in a slice preparation of the rat brain stem. High magnesium (Mg(2+); 6 mM) media generated significant increases (21-38%) in both the input resistance (R(n)) and the first time constant (tau(0)) that were reversible. A large percent of the conductance blocked by high Mg(2+) was also sensitive to tetrodotoxin (TTX). Little increase in resistance was attained by adding blockers of specific amino acid (glutamate, glycine, and GABA) transmission over that obtained with the high Mg(2+). Comparing across age groups, there was a significantly larger percent change in R(n) with the addition of high Mg(2+) at postnatal days 13 to 15 (P13-15; 36%) than that found at P5-6 (21%). Spontaneous postsynaptic potentials were sensitive to the combined application of glycine receptor antagonist, strychnine, and the GABA(A) receptor antagonist, bicuculline. Application of either 10 microM strychnine or bicuculline separately produced a reversible increase in both R(n) and tau(0). Addition of 10 microM bicuculline to a strychnine perfusate failed to further increase either R(n) or tau(0). The strychnine/bicuculline-sensitive component of the total synaptic conductance increased with age so that this form of neurotransmission constituted the majority (>60%) of the observed percent decrease in R(n) and tau(0) in the oldest age group. The proportion of change in tau(0) relative to R(n) following strychnine or high magnesium perfusate varied widely from cell to cell and from age to age without pattern. Based on a model from the literature, this pattern indicates a nonselective distribution of the blocked synaptic conductances over the cell body and dendrites. Taken together, the fast inhibitory synapses (glycine, GABA(A)) play a greater role in determining cell excitability in developing brain stem motoneurons as postnatal development progresses. These findings suggest that synaptically mediated conductances effect the membrane behavior of developing motoneurons.     

Role of potassium conductances in determining input resistance of developing brain stem motoneurons

The role of potassium conductances in determining input resistance was studied in 166 genioglossal (GG) motoneurons using sharp electrode recording in brain stem slices of the rats aged 5-7 days, 13-15 days, and 19-24 days postnatal (P). A high magnesium (Mg(2+); 6 mM) perfusate was used to block calcium-mediated synaptic release while intracellular or extracellular cesium (Cs(+)) and/or extracellular tetraethylammonium (TEA) or barium (Ba(2+)) were used to block potassium conductances. In all cases, the addition of TEA to the high Mg(2+) perfusate generated a larger increase in both input resistance (R(n)) and the first membrane time constant (tau(0)) than did high Mg(2+) alone indicating a substantial nonsynaptic contribution to input resistance. With intracellular injection of Cs(+), GG motoneurons with lower resistance (<40 MOmega), on the average, showed a larger percent increase in R(n) than cells with higher resistance (>40 MOmega). There was also a significant increase in the effect of internal Cs(+) on R(n) and tau(0) with age. The largest percent increase (67%) in the tau(0) due to intracellular Cs(+) occurred at P13-15, a developmental stage characterized by a large reduction in specific membrane resistance. Addition of external Cs(+) blocked conductances (further increasing R(n) and tau(0)) beyond those blocked by the TEA perfusate. Substitution of external calcium with 2 mM barium chloride produced a significant increase in both R(n) and tau(0) at all ages studied. The addition of either intracellular Cs(+) or extracellular Ba(2+) created a depolarization shift of the membrane potential. The amount of injected current required to maintain the membrane potential was negatively correlated with the control R(n) of the cell at most ages. Thus low resistance cells had, on the average, more Cs(+)- and Ba(2+)-sensitive channels than their high resistance counterparts. There was also a disproportionately larger percent increase in tau(0) as compared with R(n) for both internal Cs(+) and external Ba(2+). Based on a model by Redman and colleagues, it might be suggested that the majority of these potassium conductances underlying membrane resistance are initially located in the distal dendrites but become more uniformly distributed over the motoneuron surface in the oldest animals.     

Development of specific connectivity between premotor neurons and motoneurons in the brain stem and spinal cord

Astounding progress has been made during the past decade in understanding the general principles governing the development of the nervous system. An area of prime physiological interest that is being elucidated is how the neural circuitry that governs movement is established. The concerted application of molecular biological, anatomical, and electrophysiological techniques to this problem is yielding gratifying insight into how motoneuron, interneuron, and sensory neuron identities are determined, how these different neuron types establish specific axonal projections, and how they recognize and synapse upon each other in patterns that enable the nervous system to exercise precise control over skeletal musculature. This review is an attempt to convey to the physiologist some of the exciting discoveries that have been made, within a context that is intended to link molecular mechanism to behavioral realization. The focus is restricted to the development of monosynaptic connections onto skeletal motoneurons. Principal topics include the inductive mechanisms that pattern the placement and differentiation of motoneurons, Ia sensory afferents, and premotor interneurons; the molecular guidance mechanisms that pattern the projection of premotor axons in the brain stem and spinal cord; and the precision with which initial synaptic connections onto motoneurons are established, with emphasis on the relative roles played by cellular recognition versus electrical activity. It is hoped that this review will provide a guide to understanding both the existing literature and the advances that await this rapidly developing topic.     

Release of GABA and taurine from brain slices.

In brain slices the mechanisms of release of GABA have been extensively studied, but those of taurine markedly less. The knowledge acquired from studies on GABA is, nevertheless, still fragmentary, not to speak of that obtained from the few studies on taurine, and firm conclusions are difficult, even impossible, to draw. This is mainly due to methodological matters, such as the diversity and pitfalls of the techniques applied. Brain slices are relatively easy to prepare and they represent a preparation that may most closely reflect relations prevailing in vivo, since the tissue structure and cellular integrity are largely preserved. In our opinion the most recommendable method at present is to superfuse freely floating agitated slices in continuously oxygenated medium. Taurine is metabolically rather inert in the brain, whereas the metabolism of GABA must be taken into account in all release studies. The use of inhibitors of GABA catabolism is discouraged, however, since a block in GABA metabolism may distort relations between different releasable pools of GABA in tissue. It is not known for sure how well, and homogeneously, incubation of slices with radioactive taurine labels the releasable pools but at least in the case of GABA there may prevail differences in the behavior of labeled and endogenous GABA. It is suggested therefore that the results obtained with radioactive GABA or taurine should be frequently checked and confirmed by analyzing the release of respective endogenous compounds. The spontaneous efflux of both GABA and taurine from brain slices is very slow. The magnitude of stimulation of GABA release by homoexchange is greater than that of taurine under the same experimental conditions. However, the release of both amino acids is generally enhanced by a great number of structural analogs, the most potent being those which are simultaneously the most potent inhibitors of uptake. This may result in part from inhibition of reuptake of amino acid molecules released from slices but the findings may also signify that the efflux of GABA and taurine is at least partially mediated by the membrane carriers operating in an outward direction. It is thus advisable not to interpret that stimulation of release in the presence of uptake inhibitors solely results from the block of reuptake of exocytotically released molecules, since changes in the carrier-mediated transport are also likely to occur upon stimulation. The electrical and K+ stimulation evoke the release of both GABA and taurine. The evoked release of GABA is several-fold greater than that of taurine in slices from the adult brain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanisms of GABA and glycine depolarization-induced calcium transients in rat dorsal horn neurons.

1. The mechanisms and effects of GABA- and glycine-evoked depolarization were studied in cultured rat dorsal horn neurons using indo-1 recordings of [Ca2+]i and patch clamp recordings in conventional whole-cell or perforated-patch mode. 2. Application of GABA to unclamped neurons caused [Ca2+]i increases that were dose dependent and exhibited GABAA receptor pharmacology. Calcium entered the neurons via high-threshold voltage-gated calcium channels (conotoxin and nimodipine sensitive). 3. In perforated-patch recordings employing cation-selective ionophores, GABAA receptor activation depolarized 123 of 132 cells to membrane potentials as depolarized as -33 mV (mean -50 mV in all 132 cells, +12 mV above resting potential). The ionic basis of the depolarization was determined by extracellular ion substitution; increased anionic conductance could account fully for the results. 4. Glycine, acting at a strychnine-sensitive receptor, also caused Ca2+ entry into these neurons through voltage-gated Ca2+ channels. Glycine and GABA both evoked [Ca2+]i responses in the same cells and the responses were highly correlated in amplitude. Glycine also depolarized all five cells tested with perforated recording. Each of the five cells was also depolarized by muscimol to a value similar to that obtained for glycine. 5. Both the depolarization and the increases in [Ca2+]i caused by GABA and glycine could potentially play a role in processes of development and differentiation and sensory transmission in the spinal cord dorsal horn.     

Developmental switch from GABA to glycine release in single central synaptic terminals

Early in postnatal development, inhibitory inputs to rat lateral superior olive (LSO) neurons change from releasing predominantly GABA to releasing predominantly glycine into the synapse. Here we show that spontaneous miniature inhibitory postsynaptic currents (mIPSCs) also change from GABAergic to glycinergic over the first two postnatal weeks. Many 'mixed' mIPSCs, resulting from co-release of glycine and GABA from the same vesicles, are seen during this transition. Immunohistochemistry showed that a large number of terminals contained both GABA and glycine at postnatal day 8 (P8). By P14, both the content of GABA in these mixed terminals and the contribution of GABA to the mixed mIPSCs had decreased. The content of glycine in terminals increased over the same period. Our results indicate that switching from GABAergic to glycinergic inputs to the LSO may occur at the level of a single presynaptic terminal. This demonstrates a new form of developmental plasticity at the level of a single central synapse.     

Effects of GABA receptor antagonists on retinal glycine receptors and on homomeric glycine receptor alpha subunits

Glycinergic and GABAergic inhibition are juxtaposed at one retinal synaptic layer yet likely perform different functions. These functions have usually been evaluated using receptor antagonists. In examining retinal glycine receptors, we were surprised to find that commonly used concentrations of GABA antagonists blocked significant fractions of the glycine current. In retinal amacrine and ganglion cells, the competitive GABAA receptor antagonists (bicuculline and SR95531) were also competitive GlyR antagonists. Picrotoxinin produced a noncompetitive inhibition of retinal GlyRs. [1,2,5,6-tetrahydropyridine-4-yl] methylphosphinic acid, the GABACR antagonist, did not inhibit glycine receptors. All three GABAA receptor antagonists were competitive inhibitors of homomeric alpha1 or alpha2 GlyRs expressed in human embryonic kidney cells (HEK293) cells. Interestingly, bicuculline was much more effective at alpha2 GlyRs and might be used to separate glycine receptor subtypes. Thus commonly used concentrations of GABA antagonists do not unambiguously differentiate GABA and glycine pathways. Picrotoxinin inhibition of GABAC receptors requires two amino acids in the second transmembrane region (TM2): 2' serine and 6' threonine. Although TM2 regions in GABA and glycine receptors are highly homologous, neither 2' serine nor 6' threonine is essential for picrotoxinin sensitivity in glycine receptors.     

Effect of glycine and glycine receptor antagonists on NMDA-induced brain injury

In postnatal day 7 rats, a unilateral intrastriatal injection of 12.5 nmol of N-methyl-D-aspartate (NMDA) reproducibly injures the ipsilateral striatum, adjacent hippocampus and overlying cortex. The severity of injury can be quantified by comparing cerebral hemisphere weights in animals sacrificed 5 days after the injection. Co-injection of NMDA and the glycine receptor antagonists kynurenic acid (KYN) or 7-chlorokynurenic acid (7-CKA) reduced the severity of NMDA-induced damage in a dose-dependent fashion. One hundred nmol of KYN with 12.5 nmol of NMDA reduced average % damage from 19.3 +/- 0.9% (n = 9) to 2.3 +/- 0.5% (n = 6), P less than 0.001, ANOVA. Co-injection of 40 nmol of 7-CKA with 12.5 nmol of NMDA (n = 6) reduced average % damage from 17.1 +/- 1.6% (n = 15) to 3.0 +/- 0.6%, P less than 0.001, ANOVA. Concurrent injection of 1000 nmol glycine with 5 nmol NMDA did not increase the extent of NMDA-induced damage. Our results demonstrate that glycine receptor antagonists attenuate NMDA-induced brain injury in vivo.