The glycine receptor in the mutant mouse spastic (spa): strychnine binding characteristics and pharmacology

There is a marked deficit in the binding of the glycine receptor antagonist strychnine to the CNS of the mutant mouse spastic. The characteristics and pharmacology of [3H]strychnine binding to washed homogenates of spastic and littermate control spinal cord and brainstem were investigated to determine the nature of this defect. The maximal binding of [3H]strychnine to spastic homogenates is approximately 20% the value obtained from littermate control homogenates; the affinity of [3H]strychnine binding is approximately 25% lower than littermate control values. The pharmacology of [3H]strychnine binding has the same rank order of potency in spastic and littermate control mice; however, there are small differences in the potency of several compounds in spastic vs littermate control animals. These results indicate that the glycine receptor alteration seen in the spastic mutant mouse is primarily due to a decrease in receptor number. They also suggest that the pharmacological characteristics of the glycine receptor in spastic are different from littermate control. The data do not permit a distinction between whether the strychnine binding sites found in spastic represent the same population seen in littermate control animals or are a subpopulation that is spared by the mutation.     

Binding characteristics of [3H]opioid ligands to active opioid binding sites solubilized from rat brain membranes by glycodeoxycholate and NaCl: the recovery of binding activity by dilution

This paper describes the binding properties of [3H]peptidergic opioid ligands to binding sites solubilized from rat brain membranes by the treatment with 0.125% sodium glycodeoxycholate and 1 M NaCl. The highest amount of the specific binding of [3H]-[D-Ala2-, Met5]enkephalinamide was obtainable when 10-fold diluted solubilized preparations were incubated in the presence of 0.1 mM MnCl2 and 100 mM NaCl at 0 degree C (on ice) for 3 h. With this assay condition, the significant binding of following [3H]opioid ligands, which have been thought to be selective for receptor types, was also observed: [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin (mu-type), [3H]-[D-Ala2, D-Leu5]enkephalin (delta-type) and [3H]dynorphin1-9 (kappa-type). The number of binding sites in solubilized preparations for each [3H]ligand corresponded to 40-50% recovery of original membrane-bound binding sites. The Scatchard plot of the concentration-saturation binding curve showed only one class of binding sites, with a high affinity for each [3H]ligand. Apparent dissociation constants between solubilized receptors and [3H]ligands were the same as membrane-bound ones, but the ligand specificity for each receptor-type, which was examined by binding inhibition tests with unlabeled ligands, decreased. Present results indicate that heterogeneous opioid receptors in rat brain membranes seem to be transformed into less heterogeneous forms through the treatment with glycodeoxycholate and NaCl and the dilution process.     

Some properties of solubilized GABA receptor

gamma-Aminobutyric acid (GABA) receptor was solubilized from synaptic membrane of the rat brain by various detergents. Nonidet P-40, a non-ionic detergent, was found to be an effective solubilizing agent, since it caused no interference on the receptor binding assay, yielded a [3H]muscimol binding protein with a high specific activity and no aggregation, and preserved good stability of the solubilized fraction. Ammonium sulfate precipitation of the solubilized supernatant significantly increased the binding of [3H]muscimol to GABA receptor, possibly by removing heat-stable and small molecular inhibitory substances. The specific [3H]muscimol binding to the soluble fraction obtained by Nonidet P-40 treatment and subsequent ammonium sulfate precipitation, was saturable with KD 13 and 64 nM, and Bmax 3.4 and 1.8 pmol/mg protein, respectively. The enhancement of the [3H]muscimol binding by diazepam as found in synaptic membrane was also detected in the soluble fraction. Molecular weight of the [3H]muscimol binding site was determined by gel filtration on Sephadex G-200 and was calculated to be 270,000 daltons. This value was identical with that of the [3H]flunitrazepam binding site which appeared in the same solubilized fraction. These results indicate that the properties of solubilized GABA receptor are identical to those of membrane-bound GABA receptor. Furthermore, the present results suggest that both GABA and benzodiazepine receptors may reside on the same macromolecule in synaptic membrane.     

Autoradiographic localization of high affinity GABA, benzodiazepine, dopaminergic, adrenergic and muscarinic cholinergic receptors in the rat, monkey and human retina

High affinity gamma-aminobutyric acid, benzodiazepine, strychnine (glycine), dopamine, spirodecanone, alpha 1-adrenergic, alpha 2-adrenergic, beta-adrenergic and muscarinic cholinergic binding sites were localized by semiquantitative autoradiography in rat and, in some instances, in monkey and human retinae using [3H]muscimol, [3H]flunitrazepam, [3H]strychnine, [3H]spiperone, [3H]prazosin, [3H]para-aminoclonidine, [3H]dihydroalprenolol and [3H]quinuclidinyl benzylate, respectively. In nearly every case, the inner plexiform layer (IP) contained a high receptor density. The distribution of alpha 1 sites was unusual in that binding was concentrated in the outer plexiform layer (OP). Dopaminergic and, to a lesser extent, beta-adrenergic binding was diffusely distributed in the outer nuclear layer, the OP, the inner nuclear layer and the IP. The ganglion cell layer displayed significant benzodiazepine binding. The intraretinal distribution of pre- and postsynaptic markers of these neurotransmitters is discussed.     

Picrotoxinin and diazepam bind to two distinct proteins: further evidence that pentobarbital may act at the picrotoxinin site

alpha-[3H]Dihydropicrotoxinin (DHP) and [3H]diazepam binding proteins were solubilized from rat brain membranes with 1% Lubrol-Px. Gel filtration of the Lubrol-solubilized fraction revealed that [3H]DHP and [3H]diazepam bind to two distinct peaks with apparent molecular weights of 185,000 and 61,000, respectively. The signal-to-noise ratio of [3H]DHP binding to 185,000-dalton fractions was improved significantly. [3H]DHP bound to the 185,000-dalton fraction with two binding constants. Muscimol and pentobarbital, while enhancing [3H]diazepam binding to membrane and crude Lubrol-solubilized fractions, failed to enhance [3H]diazepam binding to the 61,000-dalton fraction. Pentobarbital inhibited the binding of [3H]DHP to the 185,000-dalton fraction with an IC50 value of 60 +/- 12 micro M. The binding of [3H]DHP alos was inhibited by several depressant and convulsant drugs which affect gamma-aminobutyric acid (GABA)-mediated transmission. These results provide strong evidence that picrotoxinin and diazepam bind to two distinct proteins and that pentobarbital may act at the picrotoxinin-sensitive site of the benzodiazepine . GABA receptor . ionophore complex.     

Strychnine binding associated with synaptic glycine receptors in rat spinal cord membranes: Ionic influences

Ammonium salts of some anions decrease the potency of glycine in inhibiting [³H]strychnine binding associated with synaptic glycine receptors. A correspondence exists between the ability of the ammonium salts of anions to increase the IC₅₀ of glycine in inhibiting the [³H]strychnine binding, their capacity to reduce the [³H] strychnine binding itself, and their capacity to reverse inhibitory postsynaptic potentials. The decrease of [³H]strychnine binding in the presence of chloride is abolished by sodium, while the decrease of the potency of glycine in inhibiting [³H]strychnine is not. Binding of [³H]strychnine is influenced by monovalent cations in a biphasic fashion. Concentrations of Li⁺, K⁺, and Na⁺ up to 150 mM decrease [³H]strychnine binding, while higher concentrations of the cations increase [³H]strychnine binding. Inhibition by glycine of [³H]strychnine binding is enhanced by low concentrations of these cations.     

Age-dependent changes in brain glycine concentration and strychnine-induced seizures in the rat

Glycine levels and receptor binding were measured in the medulla and spinal cord of 2-month, 10-month, and 24-month-old Fischer 344 rats. The behavioral response to the administration of the glycine antagonist, strychnine, was also evaluated in 2- and 24-month-old animals to investigate the relevance of these parameters to the susceptibility to seizures. Significant reductions in glycine in both the spinal cord and medulla occurred from 2 to 24 months of age. The glycine precursors, serine and threonine, were decreased only in the spinal cord. [3H]Strychnine binding was also decreased by 38% and 34% in the medulla and spinal cord, respectively, of 24-month-old rats compared to 2-month-olds. [3H]GABA binding was similarly reduced while no age-related changes in [3H]diazepam binding in the spinal cord were detected. Comparison of 2- and 24-month-old animals after systemic injection of 1.75 mg/kg strychnine showed that senescent animals have a higher incidence of seizures and mortality compared to young animals. Decreases in glycinergic neurotransmission may lower strychnine seizure threshold in the aged animal.     

Reduction of [3H]muscimol binding sites in rat dorsal spinal cord after neonatal capsaicin treatment

Two-day-old rats were pretreated with 50 mg/kg of capsaicin. After 3--4 months, specific binding of [3H]muscimol and [3H]strychnine was measured in membrane preparations from dorsal spinal cord. A 20-30% decrease of the number of [3H]muscimol binding sites was observed after capsaicin treatment. In contrast, [3H]strychnine binding was unchanged. The results provide indirect evidence for a presynaptic location of GABA receptors on capsaicin-sensitive primary afferent neurons.     

Solubilization of peripheral benzodiazepine-binding sites from rat kidney

The ability of a variety of detergents to solubilize peripheral benzodiazepine-binding sites from rat kidney was tested. Of all the detergents tested, only digitonin was found to be suitable for solubilization. This detergent solubilized 21% of the binding activity; 47% was inactivated, and 32% remained in the pellet. Specific binding of [3H]Ro 5-4864 to membrane-bound and solubilized peripheral benzodiazepine-binding sites was saturable, yielding a linear Scatchard plot (r = 0.96). KD values obtained for the membrane-bound and solubilized peripheral benzodiazepine binding sites were 3.9 +/- 0.4 nM and 5.4 +/- 0.4 nM, respectively. Respective Bmax values were 4.6 +/- 0.5 and 1.9 +/- 0.2 pmol/mg of protein. The KD value for the solubilized material obtained from kinetic experiments was 5.3 +/- 0.6 nM. The potency of PK 11195, Ro 5-4864, diazepam, flurazepam, chlordiazepoxide, Ro 15-1788, methyl-beta-carboline-3-carboxylate, and clonazepam to displace bound [3H]Ro 5-4864 from peripheral binding sites was similar in the membrane-bound and the soluble states. Most of the binding activity of the solubilized binding sites was destroyed by heating at 60 degrees C for 30 min or by treatment with 2 M guanidinium chloride or 4 M urea. More than 95% of the binding activity of the solubilized binding sites was retained after 18 hr at 4 degrees C, and more than 60% was retained after 4 days at the same temperature. These results indicate that the binding characteristics of peripheral benzodiazepine-binding sites extant in the membrane-bound state are retained after solubilization.     

Chronic GABA exposure down-regulates GABA-benzodiazepine receptor-ionophore complex in cultured cerebral cortical neurons.

Cerebral cortical cultured neurons were characterized for GABA-benzodiazepine (BZ) receptor complex, and the effect of chronic exposure of cortical neurons to GABA on GABA-BZ receptor system was investigated. In the intact cells, the [3H]flunitrazepam binding was rapid and saturable, with an apparent Kd of 4.2 +/- 1.5 nM and Bmax of 776 +/- 54 fmol/mg protein. Specifically bound [3H]flunitrazepam was displaced in a concentration-dependent manner by various BZ receptor ligands such as Ro15-1788, DMCM, Ro15-4513, clonazepam, alprazolam, diazepam and zolpidem, and enhanced by GABA, muscimol and pentobarbital. GABA induced enhancement of 36Cl-influx in a concentration-dependent manner (EC50 = 9 +/- 2 microM). Chronic exposure of the cultured neurons to GABA resulted in a reduced [3H]flunitrazepam, [3H]GABA, [3H]Ro15-1788, [3H]Ro15-4513 and [35S]TBPS binding, a reduced enhancement of [3H]flunitrazepam binding by GABA, and a reduced GABA-induced 36Cl-influx susceptible to reversal by concomitant exposure of the cultures to R 5135, a GABAA-receptor antagonist. These findings indicate that cerebral cortical cultured neurons provide an ideal model to study GABA-BZ receptor complex using binding and 36Cl-influx assays, and chronic exposure of cortical cultures to GABA leads to a down-regulation of GABA-BZ receptor system. It is a GABAA receptor-mediated slow process.     

Solubilization and properties of a benzodiazepine receptor from calf cortex

A putative benzodiazepine receptor was solubilized from calf cortex by the use of sodium deoxycholate as an 10.9S entity containing subunits of which at least part are of 51 K molecular weight. The solubilized receptor retains its high affinity for [3H]flunitrazepam. The affinity for various other drugs, including benzodiazepines and xanthine derivatives, was also not significantly altered. GABAergic modulation of the receptor affinity for [3H]flunitrazepam was diminished but still detectable.     

Benzodiazepine receptors in human brain: characterization, subcellular localization and solubilization

1. Benzodiazepine receptors have been characterized in human brain mainly using [3H]-Ro 15-1788 and [3H]-flunitrazepam. Both ligands present a very high affinity for the receptor sites (Kd values of 0.56 and 1.53 nM respectively). 2. GABA enhanced the affinity of [3H]-flunitrazepam and [3H]-diazepam, but not that of [3H]-Ro 15-1788 and [3H]-methyl-beta-carboline 3-carboxylate for their specific binding sites as well in cerebral as in cerebellar human cortex. 3. Subcellular distribution of the benzodiazepine receptors revealed a main synaptosomal localization in human cerebral cortex, cerebellum and striatum. 4. Solubilized benzodiazepine receptors were obtained using 0.5% sodium deoxycholate and were characterized with [3H]-Ro 15-1788. The solubilized receptors are still coupled to GABA receptors since the [3H]-flunitrazepam specific binding was enhanced in the presence of micromolar concentrations of GABA.     

Aging: Effect on the interaction of ethanol and pentobarbital with the benzodiazepine-GABA receptor-ionophore complex

Benzodiazepine receptor binding and modulation by pentobarbital and ethanol was studied using the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate to solubilize the gamma-aminobutyric acid (GABA)-benzodiazepine receptor-ionophore complex from the brains of Fischer 344 rats of 3-4, 12-15 and more than 28 months of age. The affinity of the benzodiazepine binding site was significantly lower in the young rats compared to either the mature or senescent animals. However, no age-related changes in the maximum number of benzodiazepine binding sites or GABA concentrations occurred in the detergent extract. Pentobarbital produced practically identical dose-dependent enhancement of [3H]flunitrazepam specific binding in all 3 age groups. In contrast, ethanol between 0.1 and 200 microM failed to produce a dose-dependent effect on [3H]flunitrazepam specific binding in any age group. The effect of pentobarbital and ethanol on [35S]t-butyl-bicyclophosphorothionate [( 35S]TBPS) specific binding to the picrotoxinin binding site was examined in the above solubilized receptor/ionophore complex under the same binding conditions. Both sedative-hypnotics produced a dose-dependent decrease in [35S]TBPS specific binding. However, pentobarbital was over 10,000 times more potent. It appears that ethanol may not enhance [3H]flunitrazepam specific binding in this solubilized preparation because of its weak action at the picrotoxinin binding site.     

Two distinct solubilized benzodiazepine receptors: differential modulation by ions

The modulation of solubilized type 1 and type 2 benzodiazepine receptors from cow brain by gamma-aminobutyric acid (GABA), divalent cations, and anions has been evaluated. GABA stimulates [3H] flunitrazepam binding of both receptor subtypes, whereas divalent cations and anions selectively stimulate solubilized type 2 receptors. Of numerous anions examined, only chloride, bromide, and iodide enhance [3H] flunitrazepam binding. Chloride and bromide increase mainly receptor affinity for [3H]flunitrazepam, whereas iodide largely influences Bmax values. Divalent cations also stimulate soluble type 2 receptors. Calcium, zinc, manganese, barium, and magnesium have similar potencies in enhancing [3H]flunitrazepam binding, whereas copper and nickel are about 4 to 5 times more potent. The 2- to 3-fold increase in type 2 receptor binding by divalent cations involves change in numbers of binding sites. Effects of combinations of GABA, calcium, and chloride suggest that they may exert their modulating effects on type 2 receptors through different mechanisms. GABA, calcium, and chloride also protect [3H]flunitrazepam binding from heat inactivation, indicating a close link in the native state between the GABA, ions, and the benzodiazepine recognition sites. Since physiologic concentrations of calcium and chloride influence type 2 receptors, these ions may be involved in those pharmacologic effects of benzodiazepines mediated by type 2 sites.     

Regulation of glycine receptor binding in the mouse hypoglossal nucleus in response to axotomy

Recent studies have shown that muscarinic receptors in brain hypoglossal nuclei exhibit a loss of specific ligand binding in response to axotomy of the hypoglossal nerve. The mouse hypoglossal nucleus contains a high level of receptors for the inhibitory neurotransmitter, glycine; the ligand [3H]strychnine binds to the glycine receptor with high affinity. In the present study [3H]strychnine binding in mouse hypoglossal nuclei was examined at 1 to 150 days after unilateral lesions of the hypoglossal nerve. Brains were sectioned on a cryostat, thaw-mounted onto microscope slides, incubated with [3H]strychnine and processed for light microscopic autoradiography. Receptor density was assessed by counting silver grains in photomicrographs of operated and control nuclei. During the first 25 days after axotomy grain density fell to 50 percent of that of the control nucleus. After this time grain density slowly increased, returning to control levels by 150 days post lesion. These data indicate that glycine receptors on the axotomized cells of the hypoglossal nucleus are lost when connection with the target muscles of the tongue is interrupted, and that the receptors reappear when the hypoglossal nerve regenerates. It is suggested that excitatory and inhibitory neurotransmitter receptor systems may be regulated in a coordinated fashion by the functional state of the motoneuron.     

Binding characteristics of [H]opioid ligands to active opioid binding sites solubilized from rat brain membranes by glycodeoxycholate and NaCl: the recovery of binding activity by dilution

This paper describes the binding properties of [³H]peptidergic opioi ligands to binding sites solubilized from rat brain membranes by the treatment with 0.125% sodium glycodeoxycholate and 1 M NaCl. The highest amount of the specific binding of [³H]-[d-Ala²-, Met⁵]enkephalinamide was obtainable when 10-fold diluted solubilized preparations were incubated in the presence of 0.1 mM MnCl₂ and 100 mM NaCl at 0 °C (on ice) for 3 h. With this assay condition, the significant binding of followint [³H]opioid ligands, which have been thought to be selective for receptor types, was also observed: [³H]-[d-A;a², MePhe⁴, Gly-ol⁵]enkephalin (μ-type), [³H]-[d-Ala²,d-Leu⁵]enkephalin (δ-type) and [³H]dynorphin_1–9 (K-type). The number of binding sites in solubilized preparations for each [³H]ligand corresponded to 40–50% recovery of original membrane-bound binding sites. The Scatchard plot of the concentration-saturation binding curve showed only one class of binding sites, with a high affinity for each [³H]ligand. Apparent dissociation constants between solubilized receptors and [³H]ligands were the same as membrane-bound ones, but the ligand specificity for each receptor-type, which was examined by binding inhibition tests with unlabeled ligands, decreased. Present results indicate that heterogeneous opioid receptors in rat brain membranes seem to be transformed into less heterogeneous forms through the treatment with glycogdeoxycholate and NaCl and the dilution process.     

Protein kinases regulate glycine receptor binding in brain stem auditory nuclei after unilateral cochlear ablation

Glycinergic synaptic inhibition is part of acoustic information processing in brain stem auditory pathways and contributes to the regulation of neuronal excitation. We found previously that unilateral cochlear ablation (UCA) in young adult guinea pigs decreased [3H]strychnine binding activity in several brain stem auditory nuclei. This study determined if the UCA-induced deficit could be regulated by protein kinase C (PKC), protein kinase A (PKA) or Ca2+/calmodulin-dependent protein kinase II (CaMKII). The specific binding of [3H]strychnine was measured in slices of the dorsal (DCN), posteroventral (PVCN) and anteroventral (AVCN) cochlear nucleus (CN), the lateral (LSO) and medial (MSO) superior olive, and the inferior colliculus (IC) 145 days after UCA. Tissues from age-matched unlesioned animals served as controls. UCA induced deficits in specific binding in the AVCN, PVCN, and LSO on the ablated side and in the MSO bilaterally. These deficits were reversed by 3 microM phorbol 1,2-dibutyrate, a PKC activator, or 0.2 mM dibutyryl-cAMP, a PKA activator. However, 50 nM Ro31-8220, a PKC inhibitor, and 2 microM H-89, a PKA inhibitor, had no effect in unlesioned controls and after UCA. In contrast, 4 microM KN-93, a CaMKII inhibitor, relieved or reversed the UCA-induced binding deficits and elevated binding in the IC. These findings suggest that a UCA-induced down-regulation of glycine receptor synthesis may have occurred via reduced phosphorylation of proteins that control receptor synthesis; this effect was reversed by diminishing CaMKII activity or increasing PKC and PKA activity.     

Glycine receptor internalization by protein kinases activation

Although glycine-induced currents in the central nervous system have been proven to be modulated by protein kinases A (PKA) and C (PKC), the mechanism is not well understood. In order to better comprehend the mechanism involved in this phenomenon, we tested the PKA and PKC activation effect on the specific [(3) H]glycine and [(3) H]strychnine binding to postsynaptic glycine receptor (GlyR) in intact rat retina. The specific binding constituted about 20% of the total radioligand binding. Kinetic analysis of the specific binding exhibited a sigmoidal behavior with three glycine and two strychnine binding sites and affinities of 212 nM for [(3) H]glycine and 50 nM for [(3) H]strychnine. Specific radioligand binding was decreased (60-85%) by PKA and PKC activation, an effect that was blocked by specific kinases inhibitors, as well as by cytochalasin D. GlyR expressed in the plasma membrane decreased about 50% in response to kinases activation, which was consistent with an increase of the receptor in the microsomal fraction when PKA was activated. Moreover, immunoprecipitation studies indicated that these kinases lead to a time-dependent receptor phosphorylation. Our results suggest that in retina, GlyR is cross-regulated by G protein-coupled receptors, activating PKA and PKC.     

Glutamate release induced by activation of glycine and GABA transporters in spinal cord is enhanced in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a progressive and fatal neurodegenerative disease, involving both upper and lower motor neurons, the cause of which is obscure, although glutamate (GLU)-induced excitotoxicity has been suggested to play a major role. We studied the release of [3H]d-aspartate ([3H]d-ASP) and endogenous glutamate evoked by glycine (GLY) or GABA from spinal cord synaptosomes in mice expressing a mutant form of human SOD1 with a Gly93Ala substitution ([SOD1-G93A(+)]), a transgenic model of amyotrophic lateral sclerosis, in mice expressing the non-mutated form of human SOD1 [SOD1+], and in non-transgenic littermates [SOD1(-)/G93A(-)]. In parallel experiments, we also studied the release of [3H]GABA evoked by GLY and that of [3H]GLY evoked by GABA. Mutant mice were killed at advanced phase of pathology or during the pre-symptomatic period. In SOD1(-)/G93A(-) or SOD1(+) mice GLY evoked [3H]d-ASP and [3H]GABA release, while GABA caused [3H]d-ASP, but not [3H]GLY, release. The GLY-evoked release of [3H]d-ASP, but not that of [3H]GABA, and the GABA-evoked [3H]d-ASP release, but not that of [3H]GLY, were more pronounced in SOD1-G93A(+) than in SOD1(+) or SOD1(-)/G93A(-) mice. Furthermore, the excessive potentiation of [3H]d-ASP by GLY or GABA was already present in asymptomatic 30-40 day-old SOD1-G93A(+) mice. The releases of endogenous glutamate and GABA also were enhanced by GLY and the GLY-evoked release of endogenous glutamate, but not of endogenous GABA, was higher in SOD1-G93A(+) than in control animals. Potentiation of the spontaneous amino acid release is likely to be mediated by activation of a GLY or a GABA transporter, since the effect of GLY was counteracted by the GLY transporter blocker glycyldodecylamide but not by the GLY receptor antagonists strychnine and 5,7-dichlorokynurenate while the effect of GABA was diminished by the GABA transporter blocker SKF89976-A but not by the GABA receptor antagonists SR9531 and CGP52432. It is concluded that the glutamate release machinery seems excessively functional in SOD1-G93A(+) animals.     

Glycine high affinity uptake and strychnine binding associated with glycine receptors in the frog central nervous system

Accumulation of [3H]glycine into synaptosomal fractions occurs by high affinity systems in cerebral cortex, optic tectum, brain stem and spinal cord of the frog. Specific [3H]strychnine binding which appears associated with postsynaptic glycine receptors is also demonstrable in these regions. By contrast, only very low levels of strychnine binding and high affinity glycine uptake occur in higher centers of the rat central nervous system. The relative potencies of small neutral amino acids in competing for [3H]strychnine binding are similar in frog brain and spinal cord. No evidence for a high affinity accumulation of [3H]taurine by synaptosomal fractions of frog spinal cord can be demonstrated. These observations favor glycine rather than taurine as an inhibitory transmitter in frog spinal cord. Moreover, these findings suggest that glycine may have a synaptic role in higher brain centers in the frog.