Glutamate regulation of non-quantal release of acetylcholine in the rat neuromuscular junction

Glutamate, previously demonstrated to participate in regulation of the resting membrane potential in skeletal muscles, also regulates non-quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non-quantal ACh secretion was estimated by the amplitude of endplate hyperpolarization (H-effect) following blockade of skeletal muscle post-synaptic nicotinic receptors by (+)-tubocurarine and cholinesterase by armin (diethoxy-p-nitrophenyl phosphate). Glutamate was shown to inhibit non-quantal release but not spontaneous and evoked quantal secretion of ACh. Glutamate-induced decrease of the H-effect was enhanced by glycine. Glycine alone also lowered the H-effect, probably due to potentiation of the effect of endogenous glutamate present in the synaptic cleft. Inhibition of N-methyl-d-aspartate (NMDA) receptors with (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801), dl-2-amino-5-phosphopentanoic acid (AP5) and 7-chlorokynurenic acid or the elimination of Ca2+ from the bathing solution prevented the glutamate-induced decrease of the H-effect with or without glycine. Inhibition of muscle nitric oxide synthase by NG-nitro-l-arginine methyl ester (l-NAME), soluble guanylyl cyclase by 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and binding and inactivation of extracellular nitric oxide (NO) by haemoglobin removed the action of glutamate and glycine on the H-effect. The results suggest that glutamate, acting on post-synaptic NMDA receptors to induce sarcoplasmic synthesis and release of NO, selectively inhibits non-quantal secretion of ACh from motor nerve terminals. Non-quantal ACh is known to modulate the resting membrane potential of muscle membrane via control of activity of chloride transport and a decrease in secretion of non-quantal transmitter following muscle denervation triggers the early post-denervation depolarization of muscle fibres.     

Participation of receptors of the NMDA type in regulation by glutamate of alimentary motor program of the freshwater mollusc <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis>

A possible participation of receptors of the NMDA type in regulation by glutamate of the Lymnaea stagnalis alimentary program was studied in electrophysiological experiments. The specific antagonist of receptors of the N-methyl-D-aspartate (NMDA) type MK-801 has been shown to turn off the endogenous generation of the standard three-phase rhythm or the two-phase rhythm. Stimulation of receptors of this type by their specific agonist, NMDA, on the contrary, increased frequency of the alimentary rhythm and transformed it to the two-phase one. All NMDA effects are eliminated by MK-801. Apart from action on generation of central alimentary rhythms, ligands of receptors of the NMDA type change the tonical level of depolarization and activity of the alimentary circuit motoneurons. MK-801 decreased the initial level of the motoneuron B4 activity and inhibited the excitatory effect both of NMDA and of glutamate itself. There are also obtained data in favor of that earlier reported effect of transformation of the inhibitory response of neurons B4 to glutamate to the excitatory one at action of nitric oxide (NO) donors can be mediated by the specific NO effect on the activity of receptors of the NMDA type. The blocker of NMDA receptors MK-801 has been shown to inhibit the effect of transformation of the response to glutamate. The NO donor nitroprusside enhanced essentially the NMDA excitatory action, while the NO acceptor PTlO decreased it. The results obtained with use of ODQ, the blocker of NO-sensitive guanylyl cyclase (GC), allow thinking that effect of NO on activity of the NMDA receptors of the pond snail alimentary program can be realized through the metabolic pathway GC-cGMP. On the whole, the obtained results show the pond snail receptors of the NMDA type to participate in generation and rearrangements of rhythmical alimentary programs in the tonical excitatory effect on the alimentary program motoneurons in the NO-dependent transformation of the glutamate response.     

Generation of inositol phosphates, cytosolic Ca and secretion of noradrenaline in PC12 cells treated with glutamate

Glutamate transiently stimulated rat pheochromocytoma PC12 cells and caused an inositol trisphosphate formation and an increase in levels of Ca⁺ in the cytosol. The rank order of potency of glutamate> N-methyl-D-aspartate (NMDA) > KAINATE = quisqualate is characteristic of an interaction with NMDA receptors. The effect of glutamate on inositol trisphosphate formation disappeared in a low Mg²⁺ buffer and was not blocked by DL-2-amino-5-phosphonovalerate, an antagonist for NMDA receptors coupled to ion channels. Although glutamate failed to stimulate noradrenaline secretion, glutamate enhanced the effect of bradykinin, but not of Ca ionophore A23187, or KC1. These results suggest the existence of metabotropic glutamate receptors, different from previously reported receptors, in PC12 cells.     

Glutamate and nitric oxide modulate ERK and CREB phosphorylation in the avian retina: evidence for direct signaling from neurons to Müller glial cells

Glutamate signaling in the mature retinal tissue is very important for accurate sensory decoding by retinal neurons and orchestrates the fine-tuned output from the retina to higher-order centers at the cerebral cortex. In this study, we show that glutamate induces a rapid extracellular-regulated kinase and cAMP-responsive element binding protein (CREB) phosphorylation in cultured developing retinal neurons. This process is reliant on α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors and nitric oxide (NO) signaling and independent of NMDA receptors activation, as it is blocked by α-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate antagonists as well as inhibiting NO synthase with NG-nitro- l -arginine methyl ester but not by the NMDA channel blocker dizocilpine maleate. The effect of NO on extracellular-regulated kinase and CREB is mediated by the classical NO/soluble guanylyl cyclase/protein kinase G pathways as it is inhibited by the soluble guanylyl cyclase blocker 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one and the protein kinase G inhibitor KT5823, respectively. Immunocytochemical data suggest that increased CREB phosphorylation in response to glutamate occurs in glial cell nuclei. We also have supporting evidence suggesting that neuronally produced NO directly reaches the glial cells and stimulates CREB phosphorylation. Hence, the results indicate the importance of neuronal–glial communication and glutamate/NO/CREB linkage during retinal development.     

Modeling the effect of glutamate diffusion and uptake on NMDA and non-NMDA receptor saturation.

One- and two-dimensional models of glutamate diffusion, uptake, and binding in the synaptic cleft were developed to determine if the release of single vesicles of glutamate would saturate NMDA and non-NMDA receptors. Ranges of parameter values were used in the simulations to determine the conditions when saturation could occur. Single vesicles of glutamate did not saturate NMDA receptors unless diffusion was very slow and the number of glutamate molecules in a vesicle was large. However, the release of eight vesicles at 400 Hz caused NMDA receptor saturation for all parameter values tested. Glutamate uptake was found to reduce NMDA receptor saturation, but the effect was smaller than that of changes in the diffusion coefficient or in the number of glutamate molecules in a vesicle. Non-NMDA receptors were not saturated unless diffusion was very slow and the number of glutamate molecules in a vesicle was large. The release of eight vesicles at 400 Hz caused significant non-NMDA receptor desensitization. The results suggest that NMDA and non-NMDA receptors are not saturated by single vesicles of glutamate under usual conditions, and that tetanic input, of the type typically used to induce long-term potentiation, will increase calcium influx by increasing receptor binding as well as by reducing voltage-dependent block of NMDA receptors.     

Selective potentiation of N-methyl-D-aspartate-induced current by protein kinase C in Xenopus oocytes injected with rat brain RNA.

Glutamate receptors and protein kinase C (PKC) may play significant roles in long-term potentiation in hippocampus. To clarify the regulatory involvement of PKC in the functions of glutamate receptors, we examined the effects of PKC activation on current response induced by the activation of each subtype of glutamate receptor in Xenopus oocytes injected with rat brain RNA. Treatment with the PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), potentiated N-methyl-D-aspartate (NMDA)-induced current by about 2.5-fold, although it did not affect kainate-induced current at all. Quisqualate-mediated oscillatory current was almost abolished by this treatment. The TPA-induced potentiation of NMDA current was suppressed by staurosporine, an inhibitor of protein kinases. Pretreatment with 4-O-methyl-TPA, an inactive phorbol ester, had no effect on NMDA current. Current response mediated by NMDA receptors would thus appear to be modulated by PKC.     

Different receptors mediate stimulation of nitric oxide-dependent cyclic GMP formation in neurons and astrocytes in culture.

The ability of various compounds to stimulate cyclic GMP accumulation was studied in neuronal and astrocyte-enriched primary cultures from rat cerebrum. Glutamate was the only agonist eliciting a response in neurons whereas several agonists had an effect in astrocytes but only those due to norepinephrine and glutamate were of considerable magnitude. The responses were markedly inhibited by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine. The effect of glutamate appears to be mediated predominantly by NMDA receptors in neurons and by quisqualate AMPA-insensitive receptors in astrocytes.     

N-Methyl-<Emphasis Type="SmallCaps">d</Emphasis>-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors involved in the induction of sedative effects under an acute stress in neonatal chicks

Glutamate, an excitatory amino acid, acts at several glutamate receptor subtypes. Recently, we reported that central administration of glutathione induced hypnosis under stressful conditions in neonatal chicks. Glutathione appears to bind to the N-methyl-d-aspartate (NMDA) receptor. To clarify the involvement of each glutamate receptor subtype during stressful conditions, intracerebroventricular (i.c.v.) injection of several glutamate receptor agonists was given to chicks under social separation stress. Glutamate dose-dependently induced a hypnotic effect. NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate are characterized as ionotropic glutamate receptors (iGluRs). Although NMDA also induced sleep-like behavior or sedative effects, the potency of NMDA was less than that of glutamate. AMPA tended to decrease distress vocalizations induced by acute stress and brought about a sedative effect. Kainate and (S)-3, 5-dehydroxyphenylglycine, which is a metabotropic glutamate receptor agonist, had no influence on chick behavior. Thus, it is suggested that the iGluRs, NMDA and AMPA, are important in inducing hypnosis and sedation under acute stress in chicks.     

NMDA and non-NMDA receptors stimulation causes differential oxidative stress in rat cortical slices

Glutamate receptor activated neuronal cell death is attributed to a massive influx of Ca(2+) and subsequent formation of reactive oxygen species (ROS) but the relative contribution of NMDA and non-NMDA sub-types of glutamate receptors in excitotoxicity is not known. In the present study, we have examined the role of NMDA and non-NMDA receptors in glutamate-induced neuronal injury in cortical slices from young (20+/-2 day) and adult (80+/-5 day) rats. Treatment of slices with glutamate receptor agonists NMDA, AMPA and KA elicited the formation of reactive oxygen species (ROS) and neuronal cell death. In young slices, NMDA receptor stimulation caused a higher ROS formation and neurotoxicity, but KA was more effective in producing ROS and cell death in adult slices. AMPA exhibited an intermediate effect on ROS formation and toxicity in both the age groups. A significant protection in glutamate mediated ROS formation and neurotoxicity was observed in presence of NMDA or/and non-NMDA receptors antagonists APV and NBQX, respectively. This further confirms the involvement of both NMDA and non-NMDA receptors in glutamate mediated neurotoxicity. In adult slices, we did not find positive correlation between ligand induced neurotoxicity and mitochondrial depolarization. Though, NMDA and KA stimulation produced differential effect on ROS formation and neurotoxicity in young and adult slices, the mitochondrial depolarization was higher and comparable on NMDA stimulation in both the age groups as compared to KA, suggesting that the mitochondrial depolarization may not be a good indicator for neurotoxicity. Our results demonstrate that both NMDA and non-NMDA sub-types of glutamate receptors are involved in glutamate mediated neurotoxicity but their relative contribution is highly dependent on the age of the animal.     

N-Methyl-d-aspartate and α-amino-3-hydroxy-5-methyl-4- isoxazolepropionate receptors involved in the induction of sedative effects under an acute stress in neonatal chicks

Glutamate, an excitatory amino acid, acts at several glutamate receptor subtypes. Recently, we reported that central administration of glutathione induced hypnosis under stressful conditions in neonatal chicks. Glutathione appears to bind to the N-methyl-d-aspartate (NMDA) receptor. To clarify the involvement of each glutamate receptor subtype during stressful conditions, intracerebroventricular (i.c.v.) injection of several glutamate receptor agonists was given to chicks under social separation stress. Glutamate dose-dependently induced a hypnotic effect. NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate are characterized as ionotropic glutamate receptors (iGluRs). Although NMDA also induced sleep-like behavior or sedative effects, the potency of NMDA was less than that of glutamate. AMPA tended to decrease distress vocalizations induced by acute stress and brought about a sedative effect. Kainate and (S)-3, 5-dehydroxyphenylglycine, which is a metabotropic glutamate receptor agonist, had no influence on chick behavior. Thus, it is suggested that the iGluRs, NMDA and AMPA, are important in inducing hypnosis and sedation under acute stress in chicks.     

谷氨酸性突触在痛觉和记忆中的突触和分子机制

谷氨酸是哺乳动物脑中的兴奋性递质。中枢神经系统的谷氨酸性突触广泛参与痛觉传递,突触可塑性和递质的调节。谷氨酸的NMDA受体参与前脑相关的学习及功能。在这篇综述中,我们提出前脑的NMDA受体通过增强谷氨酸性突触传递导致长期性的炎痛。具有增强NMDA受体功能的小鼠会产生更多的慢性痛。NMDA NR2B受体抑制剂在未来可能被用来控制人类的慢性痛。     

Glutamate release is involved in PAF-increased cyclic GMP levels in hippocampus

The involvement of glutamate in PAF-increased cyclic GMP levels was studied. Glutamate treatment caused a dose-response increase of cyclic GMP levels in hippocampal slices. The presence of 1 mM glutamate did not modify the effect caused by 10(-7)M PAF. To elucidate the involvement of glutamate in this action, slices were treated with PAF in the presence of MK-801, a NMDA receptor antagonist. Results indicate that PAF-increased cyclic GMP levels were obtained by NMDA receptors activation. Finally, results obtained from the experiments performed with PAF in the presence of riluzole, to inhibit the glutamate release, demonstrated that glutamate release is a stage in the PAF-induced increase of cyclic GMP levels in hippocampus.     

NMDA and non-NMDA receptor-mediated differential Ca2+ load and greater vulnerability of motor neurons in spinal cord cultures

Glutamate receptor activated neuronal cell death has been implicated in the pathogenesis of motor neuron disease but the molecular mechanism responsible for neuronal dysfunction needs to be elucidated. In the present study, we examined the contribution of NMDA and non-NMDA sub-types of glutamate receptors in selective vulnerability of motor neurons. Glutamate receptor activated Ca2+ signaling, mitochondrial functions and neurotoxicity in motor neurons and other spinal neurons were studied in mixed spinal cord primary cultures. Exposure of cells to glutamate receptor agonists glutamate, NMDA and AMPA elevated the intracellular Ca2+, mitochondrial Ca2+ and caused mitochondrial depolarization and cytotoxicity in both motor neurons and other spinal neurons but a striking difference was observed in the magnitude and temporal patterns of the [Ca2+]i responses between the two neuronal cell types. The motor neurons elicited higher Ca2+ load than the other spinal neurons and the [Ca2+]i levels were elevated for a longer duration in motor neurons. AMPA receptor stimulation was more effective than NMDA. Both the NMDA and non-NMDA receptor antagonists APV and NBQX inhibited the Ca2+ entry and decreased the cell death significantly; however, NBQX was more potent than APV. Our results demonstrate that both NMDA and non-NMDA sub-types of glutamate receptors contribute to glutamate-mediated motor neuron damage but AMPA receptors play the major role. AMPA receptor-mediated excessive Ca2+ load and differential handling/regulation of Ca2+ buffering by mitochondria in motor neurons could be central in their selective vulnerability to excitotoxicity.     

Metabotropic glutamate receptor 5 modulates the nitric oxide-cGMP pathway in cerebellum in vivo through activation of AMPA receptors

Metabotropic glutamate receptors (mGluRs) modulate important processes in cerebellum including long-term depression, which also requires formation of nitric oxide (NO) and cGMP. Some reports suggest that mGluRs could modulate the NO-cGMP pathway in cerebellum. However this modulation has not been studied in detail. The aim of this work was to assess by microdialysis in freely moving rats whether activation of mGluR5 modulates the NO-cGMP pathway in cerebellum in vivo and to analyze the underlying mechanisms. We show that mGluR5 activation increases extracellular glutamate, citrulline and cGMP in cerebellum. Blocking NMDA receptors with MK-801 does not prevent any of these effects, indicating that NMDA receptors activation is not required. However in the presence of MK-801 the effects are more transient, returning faster to basal levels. Blocking AMPA receptors prevents the increase in citrulline and cGMP induced by mGluR5 activation, but not the increase in glutamate. The release of glutamate is prevented by tetrodotoxin but not by fluoroacetate, indicating that glutamate is released from neurons and not from astrocytes. Activation of AMPA receptors increases citrulline and cGMP. These data indicate that activation of mGluR5 induces an increase of extracellular glutamate which activates AMPA receptors, leading to activation of nitric oxide synthase and increased NO, which activates guanylate cyclase, increasing cGMP. The response mediated by AMPA receptors desensitize rapidly. Activation of AMPA receptors also induces a mild depolarization, allowing activation of NMDA receptors which prolongs the duration of the effect initiated by activation of AMPA receptors. These data support that the three types of glutamate receptors: mGluR5, AMPA and NMDA cooperate in the modulation of the grade and duration of activation of the NO-cGMP pathway in cerebellum in vivo. This pathway would modulate cerebellar processes such as long-term depression.     

Glutamate induces formation of free radicals in rat brain synaptosomes

The influence of glutamate and agonists of its ionotropic receptors on free radical formation in rat brain synaptosomes was investigated using the fluorescent dye DCFDA. Glutamate at concentrations of 100 μM and 1 mM increased the production of reactive oxygen species. This phenomenon was eliminated by removing calcium from the incubation medium. Addition of NMDA (100 μM) or kainate (100 μM) to a suspension of synaptosomes also led to free radical formation. The influence of glutamate receptor agonists was blocked by the specific antagonists MK-801 and NBQX. Thus, activation of NMDA and AMPA/kainate receptors can lead to oxidative stress in neuronal presynaptic endings.     

Glutamatergic transmission between antagonistic motor neurones in the locust

The pharmacology of the direct central connections between the fast extensor and flexor motor neurones of a locust (Schistocerca gregaria) hind leg was studied. A spike in the fast extensor produces an EPSP in the flexor motor neurones. Glutamate depolarized the flexor motor neurones when injected into the neuropil. Quisqualate, but not by kainate or NMDA, also depolarized the flexor motor neurones. The fast extensor was also depolarized by glutamate, and also by kainate, but not by quisqualate, AMPA or NMDA. The glutamate response in the flexor motor neurones and the EPSP evoked by a spike in FETi both had similar reversal potentials. The FETi-evoked EPSP was blocked by bath application of the glutamate antagonist glutamic acid diethyl ester. The responses of extrasynaptic somata receptors to glutamate were compared to the neuropil responses. Glutamate usually hyperpolarized the somata of FETi and the flexor motor neurones. The response of a flexor motor neurone to glutamate was abolished at potentials less negative than -90 mV. The results provide evidence for glutamate transmission at central synapses in the locust, and show that presumed synaptic receptors in the neuropil differ to the extrasynaptic soma response     

Activation of Mitogen-Activated Protein Kinase in Cultured Rat Hippocampal Neurons by Stimulation of Glutamate Receptors

Abstract: Mitogen-activated protein kinase (MAP kinase) was activated by stimulation of glutamate receptors in cultured rat hippocampal neurons. Ten micromolar glutamate maximally stimulated MAP kinase activity, which peaked during 10 min and decreased to the basal level within 30 min. Experiments using glutamate receptor agonists and antagonists revealed that glutamate stimulated MAP kinase through NMDA and metabotropic glutamate receptors but not through non-NMDA receptors. Glutamate and its receptor agonists had no apparent effect on MAP kinase activation in cultured cortical astrocytes. Addition of calphostin C, a protein kinase C (PKC) inhibitor, or down-regulation of PKC activity partly abolished the stimulatory effect by glutamate, but the MAP kinase activation by treatment with ionomycin, a Ca²⁺ ionophore, remained intact. Lavendustin A, a tyrosine kinase inhibitor, was without effect. In experiments with ³²P-labeled hippocampal neurons, MAP kinase activation by glutamate was associated with phosphorylation of the tyrosine residue located on MAP kinase. However, phosphorylation of Raf-1, the c- raf protooncogene product, was not stimulated by treatment with glutamate. Our observations suggest that MAP kinase activation through glutamate receptors in hippocampal neurons is mediated by both the PKC-dependent and the Ca²⁺-dependent pathways and that the activation of Raf-1 is not involved.     

Dizocilpine Inhibits Amphetamine-Induced Formation of Nitric Oxide and Amphetamine-Induced Release of Amino Acids and Acetylcholine in the Rat Brain

Glutamate receptor activation participates in mediation of neurotoxic effects in the striatum induced by the psychomotor stimulant amphetamine. The effects of the non-competitive NMDA receptor antagonist dizocilpine (MK-801) on amphetamine-induced toxicity and formation of nitric oxide (NO) in both striatum and cortex and on induced transmitter release in the nucleus accumbens were investigated. Repeated, systemic application of amphetamine elevated striatal and cortical lipid peroxidation and NO production. Moreover, amphetamine caused an immediate release of acetylcholine and aspartate and a delayed release of GABA in the nucleus accumbens. Surprisingly, glutamate release was not affected. Dizocilpine abolished the amphetamine-induced lipid peroxidation and NO production in striatum and cortex and diminished the elevation of neurotransmitter release. These findings suggest that amphetamine evokes neurotoxic effects in both striatal and cortical brain areas that are prevented by inhibiting NMDA receptor activation. The amphetamine-induced acetylcholine, aspartate and GABA release in the nucleus accumbens is also mediated through NMDA receptor-dependent mechanisms. Interestingly, the enhanced aspartate release might contribute to NMDA receptor activation in the nucleus accumbens, while glutamate does not seem to mediate amphetamine-evoked transmitter release in this striatal brain area.     

Oligodendrocyte differentiation from adult multipotent stem cells is modulated by glutamate

We used multipotent stem cells (MSCs) derived from the young rat subventricular zone (SVZ) to study the effects of glutamate in oligodendrocyte maturation. Glutamate stimulated oligodendrocyte differentiation from SVZ-derived MSCs through the activation of specific N-methyl--aspartate (NMDA) receptor subunits. The effect of glutamate and NMDA on oligodendrocyte differentiation was evident in both the number of newly generated oligodendrocytes and their morphology. In addition, the levels of NMDAR1 and NMDAR2A protein increased during differentiation, whereas NMDAR2B and NMDAR3 protein levels decreased, suggesting differential expression of NMDA receptor subunits during maturation. Microfluorimetry showed that the activation of NMDA receptors during oligodendrocyte differentiation elevated cytosolic calcium levels and promoted myelination in cocultures with neurons. Moreover, we observed that stimulation of MSCs by NMDA receptors induced the generation of reactive oxygen species (ROS), which were negatively modulated by the NADPH inhibitor apocynin, and that the levels of ROS correlated with the degree of differentiation. Taken together, these findings suggest that ROS generated by NADPH oxidase by the activation of NMDA receptors promotes the maturation of oligodendrocytes and favors myelination.     

Excitatory Amino Acid Receptors Coupled to the Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum During Development

The coupling of excitatory amino acid receptors to the formation of nitric oxide (NO) from arginine during the postnatal development of rat cerebellum was assayed in slice preparations by measuring cyclic GMP accumulation. In the immature tissue, N-methyl-D-aspartate (NMDA) and glutamate were highly efficacious agonists, whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate evoked only small responses. The effect of glutamate at all concentrations tested (up to 10 mM) was abolished by the NMDA antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801). In adult slices, AMPA and quisqualate were much more effective and their effects were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist for ionotropic non-NMDA receptors, whereas the apparent efficacy of NMDA was greatly reduced. The major changes took place between 8 and 14 days postnatum and, in the case of NMDA, part of the loss of sensitivity appeared to reflect a decline in the ambient levels of glycine with age. Moreover, a component of the response to glutamate in the adult was resistant to MK-801. Cyclic GMP accumulations induced by NMDA and non-NMDA agonists alike were Ca(2+)-dependent and could be antagonized by competitive NO synthase inhibitors in an arginine-sensitive manner, indicating that they are all mediated by NO formation. With one of the inhibitors, L-NG-nitroarginine, a highly potent component (IC50 = 6 nM) evident in slices from rats of up to 8 days old was lost during maturation, indicating that there may be a NO synthase isoform which is prominent only in the immature tissue. Cyclic GMP levels in adult slices under "basal" conditions were reduced markedly by blocking NMDA receptors, by inhibiting action potentials with tetrodotoxin, or by NO synthase inhibition, suggesting that the endogenous transmitter released during spontaneous synaptic activity acts mainly through NMDA receptors to trigger NO formation.