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.     

Release of [3H]gamma-aminobutyric acid from glial (Müller) cells of the rat retina: effects of K+, veratridine, and ethylenediamine

In several neural systems, glial cells appear to take up and release gamma-aminobutyric acid (GABA) upon depolarization. We have studied the release of [3H]GABA from Müller (glial) cells in the rat retina by a double isotope-labeling technique in which Müller cells are preloaded with 3H-GABA while a population of neurons is prelabeled with [14C]glycine. By autoradiography, we have confirmed that [3H]GABA is taken up by the radially oriented Müller cells, whereas [3H]glycine is accumulated by a subset of amacrine cells (neurons). Using the double-labeling procedure, we have examined the effects of two depolarizing agents, high K+ and veratridine, and the GABA mimetic, ethylenediamine, on transmitter release from glial cells and neurons simultaneously. We found the following. (1) Depolarization with 56 mM K+ released both [3H]GABA and [14C]glycine. About 70 to 80% of this release was blocked in Ca2+-free medium. (2) Veratridine (10 microM) also released both of the transmitters. This release was strongly inhibited by 100 nM tetrodotoxin or 1mM procaine. Under Ca2+-free conditions, less than 20% isotope release was observed. (3) Ethylenediamine released [3H]GABA readily, whereas little [14C]glycine release was observed. Removal of Ca2+ had no significant effect on transmitter release. Furthermore, in Na+-free medium ethylenediamine failed to induce [3H] GABA or [14C]glycine release. These results suggest that high K+ and veratridine release [3H]GABA from Müller cells by a Ca2+-dependent process. Ethylenediamine, on the other hand, appears to induce [3H]GABA release by a Ca2+-independent, carrier-mediated exchange mechanism.     

Localization of high affinity [3H]glycine transport sites in the cerebellar cortex

A study was made of [3H]glycine uptake sites in a preparation greatly enriched in large pieces of the cerebellar glomeruli (glomerulus particles) and in morphologically well preserved slices of rat cerebellum. Electron microscopic autoradiography revealed that of the neurones in the cerebellar cortex only Golgi cells transported [3H]glycine at the low concentration used. Glial cells also took up [3H]glycine but to a lesser extent than the Golgi neurons. It was also confirmed that under comparable conditions Golgi cells transport [3H]GABA. Kinetic studies utilizing the Golgi axon terminal-containing glomerulus particles showed that glycine is a weak non-competitive inhibitor of [3H]GABA uptake (Ki over 600 microM vs the Kt of about 20 microM) and that GABA is an even weaker inhibitor of [3H]glycine uptake. These observations indicated that glycine and GABA do not share the same carrier. Quantitative electron microscopic autoradiography showed that the uptake of the two amino acids, in terms of the unit area of labelled Golgi axon terminals, was not additive. In contrast, their uptake in terms of unit protein was strictly additive. These observations, the first relating to unit volume and the latter to the total volume of Golgi terminals, are consistent with the view that there are two biochemically separate populations of Golgi neurons, one transporting glycine the other GABA. Saturable [3H]strychnine binding was detected in the preparations of glomerulus particles, but in comparison with those from the spinal cord the affinity was lower and [3H]strychnine was not displaced by glycine. Available information on glycine receptors, however, suggest that this should not exclude the possibility of strychnine resistant glycine receptors in the rat cerebellum.     

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.     

Localization of high affinity [H]glycine transport sites in the cerebellar cortex

A study was made of [³H]glycine uptake sites in a preparation greatly enriched in large pieces of the cerebellar glomeruli (glomerulus particles) and in morphologically well preserved slices of rat cerebellum. Electron microscopic autoradiography revealed that of the neurones in the cerebellar cortex only Golgi cells transported [³H]glycine at the low concentration used. Glial cells also took up [³H]glycine but to a lesser extent than the Golgi neurons. It was also confirmed that under comparable conditions Golgi cells transport [³H]GABA. Kinetic studies utilizing the Golgi axon terminal-containing glomerulus particles showed that glycine is a weak non-competitive inhibitor of [³H]GABA uptake (K_i over 600 μM vs theK_t of about 20 μM) and that GABA is an even weaker inhibitor of [³H]glycine uptake. These observations indicated that glycine and GABA do not share the same carrier. Quantitative electron microscopic autoradiography showed that the uptake of the two amino acids, in terms of the unit area of labelled Golgi axon terminals, was not additive. In contrast, their uptake in terms of unit protein was strictly additive. These observations, the first relating to unit volume and the latter to the total volume of Golgi terminals, are consistent with the view that there are two biochemically separate populations of Golgi neurons, one transporting glycine the other GABA. Saturable [³H]strychnine binding was detected in the preparations of glomerulus particles, but in comparison with those from the spinal cord the affinity was lower and [³H]strychnine was not displaced by glycine. Available information on glycine receptors, however, suggest that this should not exclude the possibility of strychnine resistant glycine receptors in the rat cerebellum.     

Prolonged depolarizing potentials of the neurons in a strychninized isolated strip of the cat cerebral cortex

Reactions of isolated cortical slab neurons to the supracortical application of strychnine were investigated with intracellular registration in experiments on unanaesthetized and immobilized cats. It was shown that some neurons demonstrated prolonged depolarizing potentials (PDP) spontaneously and as reactions to single intracortical electrical stimuli. The development of these potentials could be a result of transformation of the reaction of the "paroxysmal depolarizing shift (PDS)--hyperpolarization" type, where hyperpolarizations were replaced by depolarizing potentials. A gradual increase of depolarizing afterpotentials resulted in DDP generation. These transformations, as a rule, were accompanied by amplification of the summary epileptiform activity in an isolated cortical slab. The suggestion was made that the DDP generation was determined by an increase in the Ca(++)-conductance of the neuronal membrane in an isolated cortical slab with the intensification of paroxysmal reactions.     

Physiological identification of GABA as the inhibitory transmitter for mammalian cortical neurons in cell culture

(1) Rat cortical neurons grown in dissociated cell culture exhibit IPSPs which appear to be generated by an increase in membrane conductance to chloride.

(2) The neurons are all sensitive to GABA in micromolar concentrations and GABA mimics the inhibitory transmitter.

(3) The neurons are much less sensitive to glycine and insensitive to taurine.

(4) Bicuculline and strychnine both block essentially all IPSPs and at the same concentrations block GABA effects.

(5) It is concluded that GABA is the main, or only, inhibitory transmitter utilized by the cortical neurons in vitro. The relevance of this conclusion to in situ transmitter identification is discussed.

Reduction of [H]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.     

Membrane and synaptic properties of nucleus tractus solitarius neurons projecting to the caudal ventrolateral medulla

Projections from the nucleus of tractus solitarius (NTS) to the caudal ventrolateral medulla (CVLM) are important in mediating autonomic reflexes. However, little is known about the cellular properties of the CVLM-projecting NTS neurons. In this study, the CVLM-projecting NTS neurons were retrogradely labeled by fluorescent microspheres injected into the CVLM. Whole cell voltage- and current-clamp recordings were performed on labeled NTS neurons in coronal brainstem slices. Compared with unlabeled neurons, the labeled NTS neurons had more depolarized resting membrane potentials, larger input resistance, and higher firing activity in response to depolarizing currents. Bath application of an ionotropic glutamate receptor antagonist kynurenic acid and a non-NMDA receptor antagonist CNQX significantly decreased the firing activity in the majority of labeled NTS neurons. In contrast, an NMDA receptor antagonist AP5 failed to alter the firing activity in labeled neurons tested. While the glycine receptor antagonist strychnine had no effect on the firing activity, blockade of GABA(A)receptors with bicuculline significantly increased the firing rate in the majority of labeled NTS neurons. Furthermore, CNQX blocked the majority of spontaneous excitatory postsynaptic currents (EPSCs) and evoked EPSCs elicited by stimulation of the tractus solitarius. The residual spontaneous and evoked EPSCs were abolished by the nicotinic receptor antagonist mecamylamine and the purinergic P2X receptor antagonist iso-PPADS. Finally, while bicuculline completely blocked the miniature inhibitory postsynaptic currents (IPSCs), the spontaneous and evoked IPSCs were abolished by a combination of bicuculline and strychnine in labeled NTS neurons. Collectively, these data suggest that the CVLM-projecting neurons are a population of neurons with distinctive membrane properties.     

Transmitters and reticulospinal neurons

Glycine and GABA strongly depress the firing of reticulospinal neurons, glycine being more effective than GABA. The effects of strychnine and bicuculline methochloride on synaptic inhibition and on the actions of these amino acids are consistent with the earlier evidence that both glycine and GABA are inhibitory transmitters in the medullary reticular formation. However, although strychnine is consistently a relatively specific antagonist of glycine, bicuculline methochloride is far less specific in distinguishing between glycine and GABA on reticulospinal neurons. Studies of synaptic inhibition in the brain stem using bicuculline methochloride alone are therefore to be interpreted with caution. Both convulsants should be tested on the actions of glycine and GABA before conclusions about transmitter identity are made. Large propertions of reticulospinal neurons appear to be unaffected by ACh, 5-HT, NA, and DA. Of the amine-sensitive reticulospinal neurons, ACh and DA usually depress firing whereas 5-HT and NA usually cause excitation. These effects, however, are generally weak (especially excitation), and it is difficult to draw firm conclusions about the possible functional significance of these substances on reticulospinal neurons.     

Putative acidic amino acid transmitters in the cerebellum I. Depolarization-induced release

In the present investigation we studied the autoradiographic localization and the characteristics of the depolarization-induced release of acidic amino acids in in vitro rat cerebellar preparations. Light microscopy autoradiography of cerebellar slices preincubated in the presence of the non-metabolized glutamate analogue D-[3H]aspartate showed a large accumulation of radioactivity over glial cells, and very little labelling of the granule cells, whose putative neurotransmitter may be glutamate. In spite of its predominant localization in glia, D-[3H]aspartate (and [14C]glutamate) was released from cerebellar slices depolarized with high [K+] in a Ca2+-dependent way, and the release elicited by veratrine was prevented by TTX. These findings, together with the observation that freshly isolated or cultured glial cells did not show any Ca2+-dependent, depolarization-induced release of D-[3H]aspartate, suggest that the radioactive amino acid released from slices has a neuronal origin. The high [K+]-induced release of exogenous radioactive acidic amino acids from superfused cerebellar synaptosomal preparations exhibited, as best, a modest Ca2+-dependence, a result probably due to the existence of a substantial non-Ca2+-dependent release of the amino acid from glial fragments contaminating the preparation. However, both the K+-evoked release of endogenous glutamate, and that of [14C]glutamate previously synthesized from [14C]glutamine were largely Ca2+-dependent, suggesting that nerve endings are the main sites involved in the stimulus-coupled secretion. In the experiments in which synaptosomes had been prelabelled with [14C]glutamine, a study of the specific radioactivity of the glutamate released and of that present in synaptosomes at the beginning and at the end of superfusion period provided evidence in favour of a preferential release of the newly synthesized [14C]glutamate. In contrast to glutamate, endogenous aspartate was not released in a Ca2+-dependent manner, and the efflux of newly formed [14C]aspartate was only slightly potentiated by Ca2+, which suggests that glutamate and aspartate are not released from the same sites. Studies on preparations (slices and synaptosomes) from immature, 8-day-old cerebella showed that neither the K+-evoked release of D-[3H]aspartate, nor that of endogenous glutamate was Ca2+-dependent. In conclusion, the data presented are consistent with the proposition that glutamate has a neurotransmitter role in the cerebellum.U     

Inhibition of spinal or hypoglossal motoneurons of the newborn rat by glycine or GABA

The function of GABA or glycine during early postnatal development remains controversial as their action is reported as either excitatory or inhibitory. The present study addressed the question of the functional role of GABA or glycine on rat motoneurons shortly after birth. For this purpose, using in vitro preparations from immature rats (postnatal age, P0-P4 days), we recorded from lumbar spinal motoneurons and hypoglossal motoneurons. All data were obtained under current clamp conditions (recording with potassium methylsulphate containing electrodes) from cells at about -70 mV resting potential. On spinal motoneurons we used the glycinergic and GABAergic recurrent postsysnaptic potential (PSP) mediated by Renshaw cells to assess its impact on excitatory synaptic inputs from dorsal afferent fibres. Despite its depolarizing nature, the recurrent PSP consistently inhibited synaptic excitation of lumbar motoneurons. On hypoglossal motoneurons, exogenously applied GABA or glycine produced depolarization with decreased input resistance. This response was always associated with inhibition of cell firing induced by intracellular current pulses. Even when the membrane potential was repolarized to resting level in the presence of GABA or glycine, hypoglossal motoneurons failed to generate spikes. Conversely, similar depolarization produced by glutamate consistently facilitated spike firing. GABAergic and glycinergic synaptic potentials evoked by focal stimulation of the reticular formation inhibited firing and/or increased firing latency in the majority of hypoglossal motoneurons. These results indicate that, immediately after birth, rat motoneurons were inhibited by synaptically released or exogenously applied GABA or glycine.     

Early development of glycine- and GABA-mediated synapses in rat spinal cord.

Motoneuron responses to the inhibitory amino acids glycine and GABA, and the contribution of inhibitory synapses to developing sensorimotor synapses were studied in rat spinal cords during the last week in utero. In differentiating motoneurons, glycine and GABA induced Cl(-)-dependent membrane depolarizations and large decreases in membrane resistance. These responses gradually decreased during embryonic development, and at birth they were significantly smaller than in embryos. In motoneurons of embryos and neonates, dorsal root stimulation produced only depolarizing potentials, some of which reversed at -50 mV membrane potential. Reduction of extracellular Cl- concentrations increased the amplitude of these potentials, suggesting that they are generated by Cl- current. Contribution of Cl(-)-dependent potentials to compound dorsal root-evoked potentials was studied by determining the effects of glycine and GABA antagonists on them. In motoneurons of embryos at days 16-17 of gestation (D16-D17), strychnine or bicuculline blocked dorsal root-evoked potentials. This suppression was neither the result of a decrease in neuronal excitability nor the inhibition of glutamate receptors. Strychnine-evoked depression was not blocked by atropine, indicating that it was not due to disinhibition of muscarinic synapses. By D19, strychnine and bicuculline significantly increased dorsal root-evoked potentials rather than blocking them. This reversed function did not result from an increase in neuronal excitability or changes in the specificity of strychnine and bicuculline antagonism. The number of glycine- and GABA-immunoreactive cells increased 20% between D17 and D19. The number of immunoreactive cells and fibers significantly increased in the motor nuclei and dorsal horn laminae. These morphological changes may contribute to establishment of new synaptic contacts on motoneurons, thus changing the actions of strychnine and bicuculline on dorsal root-evoked potentials.     

Actions of Glycine on Non-dopaminergic Neurons of the Rat Substantia Nigra

The effects of glycine on non-dopaminergic cells in rat substantia nigra pars compacta and pars reticulata maintained in vitro were investigated using intracellular recording techniques. Glycine, superfused at a concentration between 30 μM and 1 mM, reversibly blocked the spontaneous firing of these neurons. The inhibition of firing discharge was associated with a hyperpolarization of the membrane (potassium acetate-filled electrodes) and an increase in conductance. Under voltage-clamp experiments (holding potential between -57 and -65 mV), glycine produced an outward response which reversed polarity at about -74 mV. However, when the recording electrodes were filled with KCI, the glycinergic response was mainly depolarizing/inward and reversed at about -43 mV. Thus, it appeared to be due to an increase in chloride permeability. Furthermore, the effects of glycine were reversibly antagonized by strychnine (between 300 nM and 1 μM). Our findings demonstrate that glycine is a potent inhibitory agent on non-dopaminergic cells of the substantia pars compacta and pars reticulata that acts by activating strychnine-sensitive receptors.     

Active and Passive Membrane Properties of Spinal Cord Neurons that Are Rhythmically Active during Swimming in Xenopus Embryos

Cellular properties have been examined in ventrally located Xenopus spinal cord neurons that are rhythmically active during fictive swimming and presumed to be motoneurons. Resting potentials and input resistances of such neurons are - 75 +/- 2 mV (mean +/- standard error) and 118 +/- 17 M ohm respectively. Most cells fire a single impulse, 0.5 to 2.0 ms in duration and 48.5 +/- 1.8 mV in amplitude, in response to a depolarizing current step. A minority fire several spikes of diminishing amplitude to more strongly depolarizing current. Cells held above spike, threshold fire on rebound from brief hyperpolarizing pulses. Spikes are blocked by 0.1 to 1.0 microM tetrodotoxin (TTX) and are therefore Na+-dependent. Current/voltage (I/V) plots to injected current are approximately linear near the resting potential but become non-linear at more depolarized levels. Cells recorded in TTX with CsCI-filled microelectrodes show a linearized I/V plot at depolarized membrane potentials suggesting the normal presence of a voltage-dependent K+ conductance activated at relatively depolarized levels. Most cells recorded in this way but without TTX fire long trains of spikes of near constant amplitude, pointing to a role of the K+ conductance in limiting firing in normal cells. Spike blockage with TTX reveals, in some cells, a transient depolarizing Cd2+-sensitive and therefore presumably Ca2+-dependent potential that increases in amplitude with depolarization. Cells in TTX, Cd2+, and strychnine, and recorded with CsCI-filled microelectrodes to block active conductances respond to hyperpolarizing current steps with a two component exponential response. The cell time constant (tau0) obtained from the longer of these by exponential peeling is relatively long (mean 15.7 ms). These findings contribute to an increased understanding of the cellular properties involved in spinal rhythm generation in this simple vertebrate.     

Protein pattern alterations in hippocampal and cortical cells as a function of training in rats

This study reports changes in the protein pattern and incorporation of l-[U-¹⁴C]-leucine in brain cells of the hippocampus and sensorimotor cortex of rats. The following subcellular fractions were analyzed by SDS-acrylamide gel electrophoresis: plasma membranes, synaptosomal membranes and synaptic mitochondria.Recurring reversal training gave an increased synthesis of synaptosomal membrane proteins with mol. wt. 35,000–45,000 and 60,000 and 100,000 in trained animals compared to active controls. Lesser changes were observed in plasma membrane and synaptic mitochondria fractions. Of the brain areas studied, the hippocampal synaptosomal fraction showed an initial, temporary response, and the cortical cell fractions responded subsequently. Judged from the time sequence of the protein response, it seems that recurrent reversal training induces a change in synaptic protein towards higher molecular weights, suggesting that these changes reflect a modification of the distribution of synaptic protein.     

Impact of STZ‐induced hyperglycemia and insulin‐induced hypoglycemia in plasma amino acids and cortical synaptosomal neurotransmitters

In this work, we evaluated the effects of streptozotocin (STZ)‐induced hyperglycemia and an acute episode of insulin‐induced hypoglycemia in plasma amino acids and cortical neurotransmitters. For that purpose, we used citrate (vehicle)‐treated Wistar rats, STZ‐treated rats [i.p., 50 mg/kg body weight], and STZ‐treated rats injected with insulin [s.c., dose adjusted with blood glucose levels] 1 h prior to sacrifice to induce an acute episode of hypoglycemia. Plasma was collected for determination of amino acids levels. In addition, cortical synaptosomal preparations were obtained and the total levels of neurotransmitters, levels of aspartate, glutamate, taurine, and GABA released by the action of KCl, iodoacetic acid (IAA), ouabain, and veratridine, membrane potential and ATP levels were evaluated. Compared with control rats, plasma from hypoglycemic rats presented increased levels of aspartate, glutamate, glutamine, and taurine whereas GABA levels were decreased in STZ and hypoglycemic rats. Similarly, glutamate and taurine levels were increased in hypoglycemic synaptosomes while GABA decreased in hypoglycemic and STZ‐diabetic synaptosomes. The depolarizing agent KCl promoted an increase in aspartate, glutamate, and taurine release from hypoglycemic synaptosomes. The highest release of neurotransmitters occurred in the presence of veratridine and ouabain, two other depolarizing agents, in all groups of experimental animals. However, a higher release of glutamate was observed in the diabetic and hypoglycemic synaptosomes. No alterations were observed in synaptosomal membrane potential and ATP levels. These results show that in the presence of a metabolic insult a higher release of excitatory amino acids occurs, which may underlay the neuronal injury observed in type 1 diabetic patients under insulin therapy. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

Is picolinic acid a glycine agonist at strychnine-sensitive receptors?

By means of unit recording and electrophoretic application, the effect of picolinic acid on feline spinal interneurons in situ was studied in comparison with glycine. Picolinic acid inhibited neuronal firing in 60% of neurons and in some cases the inhibitory actions were antagonized by strychnine. Inhibition of firing by glycine, which was also strychnine-sensitive, was reduced in case of concomitant administration of picolinic acid. These results suggest that picolinic acid might act as a glycine agonist at strychnine-sensitive receptors.     

Biochemical correlates of GABA function in rat cortical neurons in culture

Serial biochemical studies of a rat cortical tissue culture system in which synapses regularly form showed that γ-aminobutyric acid (GABA) is present in the cultures and increases with their maturation. The tissue GABA concentration in mature cultures is similar to that of adult rat cortex in vivo. The synthetic enzyme, glutamate decar☐ylase, also increases with age as does high affinity GABA uptake. GABA uptake was blocked by l-2,4-diaminobutyrate (DABA) and had the properties of neuronal GABA uptake. Specific release by depolarizing media of both exogenous [³H]GABA and GABA synthesized from d-[U-¹⁴C]glucose was demonstrated. The GABA released by high potassium media had higher specific activity and a greater contribution from glucose (as compared to acetate) than GABA found in the medium in the absence of depolarization. Calcium dependency of evoked GABA release could be shown only after pretreatment of cultures with ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid or EGTA. Synaptosomes may exhibit greater calcium dependence of evoked transmitter release than intact cells in culture because their intracellular calcium stores are depleted during preparation. Glycine uptake by the cultures was much less in amount than was GABA uptake, and specific release of glycine could not be demonstrated.Specific binding of both a GABA agonist ([³H]muscimol) and an antagonist ([³H]bicuculline) was shown by membranes prepared from the cultures. By contrast, when [³H]muscimol binding to intact cells was studied, essentially all binding was sodium dependent and had the properties of GABA uptake binding. We conclude that the use of [³H]muscimol for receptor studies is valid only after the elimination of GABA uptake systems.Biochemical data from these studies support the concept that GABA is the transmitter for many cortical synapses. Glycine and taurine are not likely to be transmitters in these cortical cultures. When considered together with physiological data from the preceding paper, we have satisfied Werman's criteria (see ref. 36) for accepting GABA as the major inhibitory transmitter in the cortical culture system.     

Development of sensitivity to GABA and glycine in cultured cerebellar neurons

The effects of iontophoretically applied gamma-aminobutyric acid (GABA) and glycine on developing cerebellar neurons cultured for 7-40 days were intracellularly investigated. All neurons tested dose-dependently responded to both GABA and glycine. In mature neurons (after 25 days in culture) these amino acids inhibited spontaneous spikes, decreased the membrane input resistance and induced either hyperpolarization or depolarization of membrane potential. The mean reversal potential was -47 mV for GABA and -43 mV for glycine. Immature neurons, 7-12 days in culture, which were not spontaneously firing, also behaved in a similar manner as the mature ones, though the membrane resistance was not so largely changed by GABA or glycine and the reversal potential was more positive (-39 mV for GABA, -37 mV for glycine). These reversal potentials were shifted toward 0 mV by lowering the external Cl- concentration in either mature or immature neurons. The effects of GABA and glycine on mature or immature neurons were more or less inhibited by all of picrotoxin, bicuculline and strychnine. The effective concentrations of these antagonists, however, were lower in general in immature neurons. In mature neurons, picrotoxin and bicuculline became more selective to GABA than glycine and strychnine became more selective to glycine than GABA. These results suggest that sensitivities to GABA and glycine differentiate into selective types in the course of maturing of cerebellar cultured neurons.