Action Of Kainate Research Articles

Furthering pharmacological and physiological assessment of the glutamatergic receptors at the Drosophila neuromuscular junction

Drosophila melanogaster larval neuromuscular junctions (NMJs) serve as a model for synaptic physiology. The molecular sequences of the postsynaptic glutamate receptors have been described; however, the pharmacological profile has not been fully elucidated. The postsynaptic molecular sequence suggests a novel glutamate receptor subtype. Kainate does not depolarize the muscle, but dampens evoked EPSP amplitudes. Quantal responses show a decreased amplitude and area under the voltage curve indicative of reduced postsynaptic receptor sensitivity to glutamate transmission. ATPA, a kainate receptor agonist, did not mimic kainate's action. The metabotropic glutamate receptor agonist t-ACPD had no effect. Domoic acid, a kainate/AMPA receptor agonist, blocks the postsynaptic receptors without depolarizing the muscle. However, SYM 2081, a kainate receptor agonist, did depolarize the muscle and reduce the EPSP amplitude at 1 mM but not at 0.1 mM. This supports the notion that these are generally a quisqualate subtype receptors with some oddities in the pharmacological profile. The results suggest a direct postsynaptic action of kainate due to partial antagonist action on the quisqualate receptors. There does not appear to be presynaptic auto-regulation via a kainate receptor subtype or a metabotropic auto-receptor. This study aids in furthering the pharmokinetic profiling and specificity of the receptor subtypes.

Presynaptic AMPA and kainate receptors increase the size of GABAergic terminals and enhance GABA release

In the developing cerebellum, NMDA receptors promote the maturation of axonal terminals of inhibitory interneurons. We compared the effects of AMPA/kainate receptor agonists in cultured cerebellar cells from GAD65-eGFP mice. Both AMPA and kainate augmented granule cell survival without affecting interneurons. The action of kainate was blocked by an AMPA but not by a NMDA receptor antagonist, suggesting AMPA receptor involvement. AMPA and kainate increased the size of the GABAergic terminals and the action of kainate was insensitive to NMDA blockers. Whole cell recordings in granule neurons revealed that chronic treatment for 5 days with kainate as well as NMDA decreased AMPA receptor expression while interneuronal kainate receptors were depressed by kainate treatment. Acute kainate application increased mIPSCs frequency in both granule neurons and interneurons and this effect was only partially blocked by an AMPA receptor antagonist. In contrast to what was reported for NMDA, chronic treatment with kainate induced a significant decrease of the basal mIPSCs frequency but increased the acute action of kainate on mIPSCs. Direct recordings from presynaptic GABAergic terminals suggest that AMPA and kainate receptors are present in developing GABAergic terminals and their activation affects the size of GABAergic terminals and spontaneous GABA release.

Non‐targeted profiling of lipids during kainate‐induced neuronal injury

Kainate is a glutamate analog that has been widely used in pharmacological studies of neuronal injury related to ischemic conditions and epilepsy. While altered lipid metabolism has been implicated in kainate action, no study has yet investigated the associated changes in lipid metabolites on a systems scale. Here we describe a mass spectrometry-based approach for profiling of lipid mixtures in a nontargeted fashion. Combined with tandem mass spectrometry, this method aims to identify lipids that are altered between two conditions, the kainate-treated and the control hippocampal tissues. In addition to reductions in major phospholipids with mainly polyunsaturated fatty acyl chains, we find elevated levels of ions that correspond to acylated forms of phosphatidylethanolamines and ceramides. Acylated phosphatidylethanolamines are neuroprotective lipids and precursors for anandamide, which signals via cannabinoid receptors. Quantitative analysis of ceramides shows that many molecular species with different acyl compositions are increased during kainate treatment. This increase is mainly restricted to neurons rather than other brain cells in the hippocampus as revealed by immunohistochemistry of brain slices.

A pharmacological investigation of the role of GLU K5-containing receptors in kainate-driven hippocampal gamma band oscillations

Low concentrations of kainate can induce gamma frequency (25–80 Hz) oscillations in hippocampal slices as well as other brain structures in vitro. Little is known, however, about the kainate receptor (KAR) subtypes that underlie this type of rhythmic neuronal network activity. In this study, the role of GLU K5 subunit-containing KARs in kainate-induced hippocampal gamma frequency oscillations was assessed using GLU K5-selective pharmacological ligands. Activation of GLU K5-containing subunits using the selective agonists ( RS)-2-amino-3-(3-hydroxy-5- tert-butylisoxazol-4-yl)propanoic acid (ATPA; 0.1–1 μM) or iodowillardiine (0.1–1 μM) failed to induce gamma frequency oscillations in area CA3 of the rat hippocampal slice. Likewise, preincubation with a selective GLU K5 antagonist, ( RS)-3-(2-carboxybenzyl)willardiine (UBP296), did not prevent the appearance of gamma oscillations induced by 150 nM kainate. However, addition of UBP296 (10 μM) to hippocampal slices in which kainate-driven gamma oscillations were pre-established resulted in an approximately 50% reduction in gamma frequency power. These effects occurred in the absence of any effect on AMPA receptor-mediated synaptic transmission. Furthermore, carbachol-induced gamma oscillations were also unaffected by application of UBP296. These results suggest that GLU K5-containing KARs are not alone sufficient to generate gamma frequency oscillations, but are involved in maintaining neuronal network activity induced by the actions of kainate at other KARs such as GLU K6.

Enhanced taurine release in cultured cerebellar granule cells in cell-damaging conditions.

The release of taurine from cultured cerebellar granule neurons was studied in different cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and in the presence of free radicals. The effects of both ionotropic and metabotropic glutamate receptor agonists on the release were likewise investigated. The release of [3H]taurine from the glutamatergic granule cells was increased by K+ (50 mM) and veratridine (0.1 mM), the effect of veratridine being the greater. Hypoxia and ischemia produced an initial increase in release compared to normoxia but resulted in a diminished response to K+. Hypoglycemia, oxidative stress and free radicals enhanced taurine release, and subsequent K+ treatment exhibited a correspondingly greater stimulation. A common feature of taurine release in all the above conditions was a slow response to the stimulus evoked by K+ and particularly to that evoked by veratridine. All ionotropic glutamate receptor agonists potentiated taurine release, but only the action of kainate seemed to be receptor-mediated. Metabotropic receptor agonists of group I slightly stimulated the release. The prolonged taurine release seen in both normoxia and cell-damaging conditions may be of importance in maintaining homeostasis in the cerebellum and reducing excitability for a longer period than other neuroprotective mechanisms.

Actions of kainate and AMPA selective glutamate receptor ligands on nociceptive processing in the spinal cord

Kainate receptors expressing the GluR5 subunit of glutamate receptor are present at high levels on small diameter primary afferent neurones that are considered to mediate nociceptive inputs. This suggests that GluR5 selective ligands could be novel analgesic agents. The role of kainate receptors on C fibre primary afferents has therefore been probed using three compounds that are selective for homomeric GluR5 receptors. The agonist, ATPA, and the antagonists, LY294486 and LY382884, have been tested in four models of nociception: responses evoked by noxious stimulation of the periphery have been recorded electrophysiologically (1) from hemisected spinal cords from neonatal rats in vitro, (2) from single motor units in adult rats in vivo, (3) from dorsal horn neurones in adult rats in vivo, and (4) in hotplate tests with conscious mice. In some protocols comparisons were made with the AMPA selective antagonist GYKI 53655. The agonist ATPA reduced nociceptive reflexes in vitro, but failed to have effects in vivo. In all tests, the GluR5 antagonists reduced nociceptive responses but only at doses that also affected responses to exogenous AMPA. The AMPA antagonist reduced nociceptive responses at doses causing relatively greater reductions of responses to exogenous AMPA. The results indicate that GluR5 selective ligands do reduce spinal nociceptive responses, but they are not strongly analgesic under these conditions of acute nociception.

Metabotropic and ionotropic glutamate receptors regulate calcium channel currents in salamander retinal ganglion cells.

1. Glutamate suppressed high-voltage-activated barium currents (IBa, HVA) in tiger salamander retinal ganglion cells. Both ionotropic (iGluR) and metabotropic (mGluR) receptors contributed to this calcium channel inhibition. 2. Trans-ACPD (1-aminocyclopentane-trans-1S,3R-dicarboxylic acid), a broad-spectrum metabotropic glutamate receptor agonist, suppressed a dihydropyridine-sensitive barium current. Kainate, an ionotropic glutamate receptor agonist, reduced an omega-conotoxin GVIA-sensitive current. 3. The relative effectiveness of selective agonists indicated that the predominant metabotropic receptor was the L-2-amino-4-phosphonobutyrate (L-AP4)-sensitive, group III receptor. This receptor reversed the action of forskolin, but this was not responsible for calcium channel suppression. l-AP4 raised internal calcium concentration. Antagonists of phospholipase C, inositol trisphosphate (IP3) receptors and ryanodine receptors inhibited the action of metabotropic agonists, indicating that group III receptor transduction was linked to this pathway. 4. The action of kainate was partially suppressed by BAPTA, by calmodulin antagonists and by blockers of calmodulin-dependent phosphatase. Suppression by kainate of the calcium channel current was more rapid when calcium was the charge carrier, instead of barium. The results indicate that calcium influx through kainate-sensitive glutamate receptors can activate calmodulin, which stimulates phosphatases that may directly suppress voltage-sensitive calcium channels. 5. Thus, ionotropic and metabotropic glutamate receptors inhibit distinct calcium channels. They could act synergistically, since both increase internal calcium. These pathways provide negative feedback that can reduce calcium influx when ganglion cells are depolarized.

Differential expression of fibronectin, tenascin-C and NCAMs in cultured hippocampal astrocytes activated by kainate, bacterial lipopolysaccharide or basic fibroblast growth factor

Different reports demonstrated that reactive glial cells express increased amounts of adhesion and matrix molecules. Despite a wealth of information on the expression of these molecules during development and after lesion, very little is known of how this expression is regulated. In the present report we used Western blots and immunocytochemistry to investigate the expression of neural cell adhesion molecule (NCAM), fibronectin and tenascin-C in cultured astrocytes from rat hippocampus. The effects of three different extracellular signals were analyzed: the glutamatergic receptor agonist kainic acid, the basic fibroblast growth factor (bFGF) and the bacterial lipopolysaccharide. Each treatment had a specific pattern of glial activation and differentially modified the expression of these proteins. Treatment of astrocytes with kainic acid resulted in an increase of tenascin-C, a decrease of fibronectin and a shift of NCAMs isoforms: NCAM 140 and PSA-NCAM (polysialic acid-rich NCAMs) were increased while NCAM 120 was decreased. bFGF increased fibronectin, tenascin-C and NCAM 120, while decreasing PSA-NCAM. Finally, the treatment of astrocytes with lipopolysaccharide induced a significant increase of fibronectin, tenascin-C and NCAM 120 but did not modify the expression of NCAM 140 and PSA-NCAM. These data suggest different mechanisms for modulation of cell surface interactions. They suggest that glial activation by bFGF and lipopolysaccharide are associated with an increase of the adhesive properties, while kainate action is rather associated with a decrease of the adhesiveness of astrocytes.

The effect of high pressure on glycine- and kainate-sensitive receptor channels expressed in Xenopus oocytes.

The effect of high pressure on the response to glycine or kainate of voltage-clamped Xenopus oocytes micro-injected with messenger-RNA derived from either rat spinal cord or whole brain, respectively, has been investigated. Current responses were measured at 1 bar (= 10(5) Pa), 50 bar, 100 bar and 150 bar, with PO2 fixed at 1 bar and the balance helium. Glycine elicited a depolarizing current response which was antagonized by nanomolar concentrations of strychnine. The responses reversibly desensitized, with a decay constant of 0.01 s-1, when glycine concentrations greater than 250 microM were used. The decay constant was insensitive to both glycine concentration and pressure. Resensitization was complete within 4 min. Kainate elicited a depolarizing current which was non-desensitizing. The response was slightly sensitive to glutamate diethyl ester (50 microM), which increased the EC50 by 25%. The action of glycine was highly pressure sensitive. The dose-response curves established at 50 bar, 100 bar and 150 bar were shifted progressively to the right, with no effect on the maximal current. The EC50 increased from 216 microM to 296 microM at 50 bar, to 345 microM at 100 bar, and to 425 microM at 150 bar. The action of kainate was unaffected by pressure. No shift in the dose-response curves was established, nor was there any effect on the maximum current. The EC50 was 113 microM at 1 bar, and 111 microM at both 50 bar and 100 bar.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of potassium and N-methyl-D-aspartate on the aspartate aminotransferase activity in cultured cerebellar granule cells.

The effect of potassium depolarization and N-methyl-D-aspartate (NMDA) on the activity of aspartate aminotransferase (AAT; EC 2.6.1.1), an enzyme suggested to be involved in neurotransmitter glutamate synthesis, was studied in cultured cerebellar granule neurons. Both KCl and NMDA increased AAT activity in a dose-dependent manner. When cells were treated 48-72 hr with 40 mM KCl or 150 microM NMDA the AAT was enhanced about 65-75%. The EC50 for NMDA and KCl were 25 microM and 17 mM, respectively. The effect of NMDA and KCl was specific for AAT without affecting the activity of other enzymes like lactate dehydrogenase or protein content and it was observed only in granule cells but not in astrocytes or cortical neurons. The effect of KCl was not mediated by an activation of excitatory amino acid receptors and was Ca(++)-dependent. The effect of NMDA was completely blocked by Mg++ and NMDA antagonists. The increase of AAT induced by AAT and KCl was blocked by cycloheximide and actinomycin D, suggesting an involvement of de novo synthesis of proteins and RNA. Kainic acid and quinolinic acid were also effective in increasing the AAT activity. The action of kainate was less effective than that of NMDA and it was observed only at relatively low concentrations (10 microM). Quinolinic acid raised the activity of AAT about 45% at a concentration of 500 microM. Other non-NMDA agonists did not modify the AAT activity. From these findings we can conclude that NMDA and KCl exert a trophic action on cerebellar granular neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulatory and inhibitory actions of excitatory amino acids on inositol phospholipid metabolism in rabbit retina. Evidence for a specific quisqualate receptor subtype associated with neurones

The effects of excitatory amino acid agonists on [ 3H]inositol phosphates (InsPs) levels have been examined in rabbit retinal tissues under basal conditions and after agonist stimulation. Quisqualate (QA) is the most effective excitatory amino acid agonist at stimulating InsPs accumulation with an EC 50 value of 0·1 μ m. The responses for maximally effective concentrations of QA with either ibotenate or kainate were not additive, which suggested that all the excitatory amino acid agonists which stimulate InsPs accumulation (quisqualate, kainate, NMDA, glutamate, ibotenate, aspartate) have a common site of action. None of the following antagonists: dl-2-amino-5-phosphonovalerate (APV), dl-2-amino-4-phosphonobutyrate (APB) and glutamate dimethyl ester (GDEE), prazosin, ketanserin or atropine influenced the excitatory amino agonist stimulation of InsPs. These data suggest the presence of a specific QA-receptor subtype in the retina. QA, and to a lesser extent other excitatory amino acid agonists, were also effective in stimulating InsPs accumulation and the mobilization of internal calcium levels in 3–5-day-old retinal cultures but not in the older cultures (25–30 days old), which lack neurones but contain Müller cells. The QA receptor subtypes linked to InsPs accumulation in the retina are therefore present on neurones. Kainate and NMDA had a weak inhibitory action on the effect of the carbachol-induced stimulation of InsPs at 50 μ m. The NMDA action was abolished by APV, whereas this antagonist had no effect on the action of kainate. Experiments with tetrodotoxin, cadmium and verapamil indicate that the kainate and NMDA action on the carbachol-induced stimulation of InsPs does not take place through an indirect release of substances from neighbouring neurones. The mode of action of NMDA and kainate in reducing agonist-mediated InsPs formation in the retina still requires elucidation.

Kainate evokes the release of endogenous glycine from striatal neurons in primary culture

The actions of 56 mM KCl and excitatory amino acid (EAA) agonists on the release of endogenous glycine (Gly) from striatal neurons in primary culture was examined. During a 3 min period, 2 × 10 6 striatal neurons released 743 ± 51 pmol of Gly. In the presence of 56 mM KCl, an additional 492 ± 52 pmol of Gly (+ 66%) were released, 75% of which was dependent upon the presence of extracellular calcium. When striatal neurons were exposed to 1 mM N- methyl- d-aspartate (NMDA) or quisqualate (QA), endogenous Gly released was increased by 370 ± 71 (+ 50%) or 120 ± 31 (+ 16%) pmol, respectively. In the presence of 1 mM kainate (KA), however, the release of endogenous Gly increased by 994 ± 82 pmol (+ 135%). Interestingly, while KA (1 mM) was twice as effective as KCl (56 mM) in evoking the release of endogenous Gly, KCl was 5 times more effective than KA in evoking the release of endogenous γ-aminobutyric acid (GABA). KA-induced increases of endogenously released Gly were dose-dependent (EC 50, 100 μM), saturable and not significantly reduced in the absence of extracellular calcium. The actions of KA were blocked by coincubation with 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX), a competitive antagonist at the KA receptor. These data suggest that the release of endogenous Gly from striatal neurons in primary culture is regulated principally by EAA actions at the KA receptor system.

Effects of CNQX, APB, PDA, and kynurenate on horizontal cells of the tiger salamander retina.

Effects of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-4-phosphonobutyrate (APB), cis-2,3-piperidine dicarboxylic acid (PDA), and kynurenate (KYN) on the depolarizing actions of glutamate and kainate on horizontal cells (HCs) were studied in the larval tiger salamander retina. APB, PDA, and KYN hyperpolarized the HCs, but they failed to block either the actions of glutamate and kainate, or the HC light responses. APB and PDA did not cause membrane polarizations in either rods or cones, suggesting that the HC hyperpolarizations were not mediated by presynaptic actions of these compounds. CNQX, the newly synthesized non-NMDA (N-Methyl-D-Aspartate) receptor antagonist, blocked the HC light responses and the action of kainate, but not that of glutamate. These results suggest that the synaptic receptors in the tiger salamander HCs are probably non-NMDA although extra-synaptic NMDA receptors may exist in these cells.

Glutamate and kainate increase intracellular sodium activity in leech neuropile glial cells

Na+-selective, double-barrelled microelectrodes were used to measure intracellular Na+ activity (aiNa) and membrane potential (Em) in neuropile glial cells of isolated segmental ganglia in the leech Hirudo medicinalis. Bath application of glutamate (10(-3) M) resulted in membrane depolarizations of about 5 mV and a concomitant increase of aiNa by between 2 and 10 mM. Kainate (10(-4) M) elicited depolarizations of up to 40 mV amplitude followed by a prominent after hyperpolarization. During kainate, aiNa increased by 7 to 25 mM. In contrast to glutamate, an initial decrease of aiNa was detected during the action of kainate. N-methyl-D-aspartate (NMDA, 10(-5)-10(-3) M) had no effect of Em and aiNa. The results indicate that leech glial cells have a kainate-preferring non-NMDA glutamate receptor.

Potentiation by Kainate of Excitatory Amino Acid Release in Striatum: Complementary In Vivo and In Vitro Experiments

The effect of kainate on extracellular levels of amino acids in corpus striatum was investigated in vitro and in vivo, to elucidate the mechanism underlying its neurotoxicity. Kainate increased extracellular glutamate and aspartate in both striatal slices in vitro and intact striatum in vivo, as previously reported. Both in vitro and in vivo, DL-threo-3-hydroxyaspartate increased extracellular glutamate and aspartate levels (to between 150 and 200% of basal), and also enhanced their kainate-evoked release. The action of kainate in vivo was reduced by prior frontal decortication, whereas in vitro the kainate-evoked responses were only slightly reduced by tetrodotoxin, and remained above control values. These results confirm that kainate increases extracellular glutamate and aspartate, and provide evidence that this is due to synaptic release evoked by an action on receptors on glutamatergic neurone terminals. These findings may be relevant to the understanding of epilepsy.

Excitatory amino acid receptors coupled with guanylate cyclase in primary cultures of cerebellar granule cells

Primary cultures of cerebellar granule cells have been used in pharmacologically and functionally characterizing excitatory amino acid recognition sites coupled with guanylate cyclase. When granule cells were incubated in physiological culture conditions (Locke's solution, pH 7.4), only kainate and, to a lesser extent, L-glutamate increased cyclic GMP (cGMP) levels. Under these conditions, L-aspartate, N-methyl-D-aspartate (NMDA), and quisqualate were inactive. When granule cells were incubated in the absence of extracellular Mg2+ or in the presence of the depolarizing agent veratrine, L-glutamate, L-aspartate, and NMDA became as effective as kainate in enhancing cGMP formation. The action of kainate was preferentially antagonized by 2,3-cis-piperidindicarboxylate, whereas the action of L-glutamate was preferentially antagonized by (+/-)2-amino-5-phosphonovalerate. These data suggest that 2 different excitatory amino acid recognition sites (activated by kainate or by L-glutamate, L-aspartate, and NMDA, respectively) are coupled with guanylate cyclase in primary cultures of cerebellar granule cells: While the coupling of the recognition site for kainate with guanylate cyclase operates under resting conditions and in the presence of Mg2+, the coupling of the recognition site for L-glutamate, L-aspartate, and NMDA with guanylate cyclase requires depolarizing conditions or the absence of extracellular Mg2+.

Reduction of brain taurine: Effects on neurotoxic and metabolic actions of kainate

The effects of chronic administration of 2-guanidinoethane sulfonic acid on the levels of intra- and extracellular amino acids in the rat hippocampus were studied. The tissue content of taurine was selectively reduced by almost one third after 9 days of peroral administration of 1% 2-guanidinoethane sulfonate. Extracellular levels of amino acids were monitored with the brain microdialysis method. The taurine concentration in the extracellular fluid was depressed in relation to the decrease in intracellular taurine. Unexpectedly, extracellular (but not intracellular) glutamate was doubled in 2-guanidinoethane sulfonate treated animals. The kainic acid evoked release of taurine was suppressed in the 2-guanidinoethane sulfonate group, whereas the kainate stimulated efflux of glutamate was elevated after 2-guanidinoethane sulfonate administration. The acute metabolic effects of kainate were studied by measuring the efflux of the adenosine triphosphate breakdown products hypoxanthine, xanthine, inosine and adenosine. No differences were found between control and 2-guanidinoethane sulfonate treated rats with respect to basal or kainic acid evoked release of purine catabolites. Also, the neuronal loss caused by kainate injection into the hippocampus was not modified by 2-guanidinoethane sulfonate treatment, suggesting that endogenous taurine does not affect these responses. We conclude that chronic administration of 2-guanidinoethane sulfonate does not sensitize central neurons to the metabolic and toxic actions of kainate.

Effect of excitatory amino acids and analogues on [3H]acetylcholine release from amacrine cells of the rabbit retina.

The pharmacology of cholinergic amacrine cells has been further studied by examining the effects of excitatory amino acids and antagonists on [3H]acetylcholine (ACh) release from the retinas of anaesthetized rabbits. Exposure of the retina to glutamate (5 mM), aspartate (5 mM), kainate (8 microM) and quisqualate (8 microM) abolished the light-evoked release of [3H]ACh but increased the spontaneous resting release four- to fivefold. N-methyl-D-aspartate (NMDA) (5 mM) in normal Krebs bicarbonate medium abolished the light-evoked release of [3H]ACh but did not affect the resting release. However, in Mg-free medium, NMDA (0.5 mM) abolished the light-evoked release of [3H]ACh and increased the resting release fivefold. The effects of other agonists were not altered in Mg-free medium. The amplitude of the electroretinogram (e.r.g.) b-wave was not significantly reduced by glutamate, aspartate or NMDA (in normal or Mg-free medium). Kainate and quisqualate reduced the b-wave amplitude to approximately 50 and 30% of controls respectively. The general excitatory amino acid antagonist, cis-2,3-piperidine dicarboxylic acid (PDA) (2 mM) blocked the light-evoked release of [3H]ACh, but had no significant effect on the e.r.g. b-wave amplitude or on the resting release of [3H]ACh. L(+)-2-amino-4-phosphonobutyrate (L(+)-APB) decreased the light-evoked release of [3H]ACh and the amplitude of the e.r.g. b-wave in parallel (correlation coefficient 0.995). D(-)-APB had similar effects but was fifteen times less potent. Since the L(+)-compound is known to mimic the photoreceptor transmitter on the depolarizing, but not hyperpolarizing, bipolar cells, these results strongly suggest that the [3H]ACh released in response to light originates mainly from the 'on' (displaced) cholinergic amacrine cells. Our experiments give no information on the origin of the spontaneously released [3H]ACh. PDA (2-5 mM) blocked the effects of glutamate, aspartate, kainate, quisqualate and NMDA on the resting release of [3H]ACh. In contrast, D(-)-APB (5 mM), which is a relatively non-specific excitatory amino acid antagonist in the spinal cord, blocked only the actions of kainate and had no blocking effect on the actions of glutamate or aspartate (the putative bipolar cell transmitters) or NMDA. D(-)-2-amino-5-phosphonovalerate (APV) which is a relatively selective NMDA antagonist in the spinal cord failed to discriminate between the effects of kainate and NMDA on the resting release of [3H]ACh. D-alpha-aminoadipate at concentrations up to 5 mM had no effect on any of the agonists.(ABSTRACT TRUNCATED AT 400 WORDS)

Excitatory amino acid receptors and guanosine 3',5'-cyclic monophosphate in incubated slices of immature and adult rat cerebellum

Addition of the excitatory amino acids l-glutamate, l-aspartate and their analogues kainate and N-methyl- d-aspartate to incubated slices of adult rat cerebellum led to large increases in cyclic GMP levels. The order of apparent potencies was kainate > N-methyl- d-aspartate > glutamate and aspartate. d-α-aminoadipate and Mg 2+ inhibited responses to N-methyl- d-aspartate while glutamic acid diethyl ester was most effective against those to glutamate; responses to kainate were least affected by the antagonists. The exicitant amino acids also elicited large elevations of cyclic GMP levels in slices of immature (8 day) cerebellum. Kainate was less effective than in the adult but induced two responses distinguishable by their different time courses, concentration dependencies and sensitivity to antagonists. N-methyl- d-aspartate, glutamate and aspartate were 5 to 10-fold more potent than in the adult. Responses to N-methyl- d-aspartate were similarly inhibited by d-α-aminoadipate and Mg 2+ but those to glutamate were more resistant to glutamic acid diethyl ester than in the adult. It is concluded that the accumulation of cyclic GMP in response to excitant amino acids in the adult cerebellum is mediated via the operation of receptor types showing pharmacological characteristics expected of excitatory amino acid receptors. The actions of kainate in the immature cerebellum appear to be mediated by receptors different from those on which it acts primarily in the adult.

Distribution and pharmacological properties of synaptic and extrasynaptic glutamate receptors on crayfish muscle.

1. The distribution of glutamate sensitivity was measured in the opener muscle in the walking legs of the crayfish (Cambarus clarkii). L-Glutamate was ionophoretically applied under visual control. 2. Bundles of a few muscle fibres were isolated and viewed with Nomarski optics. Two axons, presumably excitatory and inhibitory, branched widely over the surface of individual muscle fibres, forming numerous clusters of boutons or varicosities. 3. Glutamate sensitivity was measured from the slope of the dose-response curves obtained by ionophoretic application of L-glutamate and expressed as mV/nC. The highest sensitivities were about 100 mV/nC, obtained at the edge of synaptic boutons. The sensitivity declined to less than 5% about 2 micrometer away from the synaptic terminal. The time course of glutamate potentials was approximately the same as that of spontaneous synaptic potentials. 4. Glutamate depolarization started within 300 microsec after ionophoretic release of glutamate. This time lag was shorter than the synaptic delay of the nerve-evoked synaptic potential measured with an extracellular micro-electrode. This indicates that glutamate depolarization results from a direct action on the post-synaptic receptor. 5. Application of L-alpha-kainic acid decreased the amplitude of the glutamate potential produced at the synaptic region, whereas kainate increased the amplitude of the glutamate potential at the extrasynaptic region. It is suggested that the pharmacological properties of the extrasynaptic receptor differ from those of the synaptic receptor. Possible mechanisms for the different actions of kainate are discussed.