Characterization of electrically evoked [3H]-D-aspartate release from hippocampal slices

Electrical stimulation has certain advantages over chemical stimulation methods for the study of neurotransmitter release in brain slices. However, measuring detectable quantities of electrically evoked release of endogenous or radiolabeled markers of excitatory amino acid neurotransmitters has required current intensities or frequencies much higher than those usually required to study other transmitter systems. We demonstrate here that [3H]-D-aspartate (D-ASP) release can be detected from hippocampal slices at lower stimulation intensities in the presence of a glutamate reuptake inhibitor. Subsequently, we optimized the electrical stimulus parameters for characterizing electrically evoked D-ASP release. Under the experimental conditions described, greater than 90% of electrically evoked D-ASP release is calcium-dependent. Evoked D-ASP release is markedly reduced by pre-treating slices with the synaptic vesicle toxin bafilomycin A1 (BAF A1) or in the presence of 10-mM magnesium. Evoked D-ASP release is also reduced to variable degrees by N- and P/Q type voltage-sensitive calcium channel antagonists. Neither spontaneous efflux nor evoked D-ASP release were affected by NMDA, AMPA or group I metabotropic glutamate receptor (mGluR) antagonists. Evoked D-ASP release was reduced in the presence of an adenosine A1 receptor agonist and potentiated by treatment with a group I mGluR5 agonist. Evoked [3H]-D-ASP release was similar in magnitude to evoked [3H]-L-glutamate (L-GLU) release. Finally, in separate experiments using the same electrical stimulus parameters, more than 90% of electrically evoked endogenous L-GLU release was calcium dependent, a pattern similar to that observed for evoked [3H]-D-ASP release. Taken together, these results indicate that electrically evoked [3H]-D-ASP release mimics evoked glutamate release in brain slices under the experimental conditions employed in these studies.     

Regional release of [3H]dopamine from rat brain in vitro: effects of opioids on release induced by potassium, nicotine, and L-glutamic acid

Previous studies have suggested that the release of dopamine (DA) in the rat brain may be sensitive to modulation by opioid agents, including the endogenous opioid peptides (enkephalins and endorphins). The present study examined the effects of morphine and the enkephalin analogue D-Ala2-Met5-enkephalinamide (DALA) on the release of radiolabeled DA from superfused slices of rat brain regions. The release of preloaded [3H]DA was evoked from slices of the caudate-putamen (CP) by application of potassium (K+), nicotine (NIC), or L-glutamic acid (L-GLU). The release of [3H]DA from slices of the nucleus accumbens (NA), olfactory tubercle (OT), and substantia nigra (SN) was evoked by L-GLU. Both K+ and NIC evoked a concentration-related release of [3H]DA from CP slices. K+-induced release was only partially dependent on calcium (Ca2+), while NIC-evoked release was completely Ca2+ independent. Neither morphine nor DALA influenced the release of [3H]DA evoked by K+ or NIC. L-GLU produced a concentration-dependent release of [3H]DA from slices of CP, NA, OT, and SN. In all four brain regions, this release was (a) Ca2+-dependent, (b) strongly inhibited by low concentrations of magnesium (Mg2+), (c) greater than the release evoked by D-GLU, (d) attenuated by the putative L-GLU receptor antagonist glutamic acid diethylester (GDEE), and (e) insensitive to tetrodotoxin (TTX) except in the SN. Morphine produced a significant inhibition of L-GLU-evoked [3H]DA release from all four regions. Naloxone, which by itself had no significant effect on the L-GLU-evoked release of [3H]DA, blocked the inhibitory effect of morphine on this release in the CP but not in the other regions. Levorphanol and dextrorphan were equipotent in reducing the glutamate-stimulated release of [3H]DA from CP slices. DALA had no effect on L-GLU-induced release in any of the brain regions examined. The results indicate that L-GLU provokes regional release of DA by acting at a Mg2+-sensitive glutamate receptor. This release is selectively modified by morphine through a mechanism which is insensitive to naloxone.     

Exocytotic release of dopamine in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice

The present study used voltammetry to ascertain whether electrically stimulated somatodendritic dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice was due to exocytosis or dopamine transporter reversal, as has been debated. The maximal concentration of electrically evoked dopamine release was similar between ventral tegmental area slices from dopamine transporter knockout and C57BL/6 mice. Dopamine transporter blockade (10 μM nomifensine) in slices from C57BL/6 mice inhibited dopamine uptake but did not alter peak evoked dopamine release. In addition, dopamine release and uptake kinetics in ventral tegmental area slices from dopamine transporter knockout mice were unaltered by the norepinephrine transporter inhibitor, desipramine (10 μM), or the serotonin transporter inhibitor, fluoxetine (10 μM). Furthermore, maximal dopamine release in ventral tegmental area slices from both C57BL/6 and dopamine transporter knockout mice was significantly decreased in response to Na⁺ channel blockade by 1 μM tetrototoxin, removal of Ca²⁺ from the perfusion media and neuronal vesicular monoamine transporter inhibition by RO-04-1284 (10 μM) or tetrabenazine (10 and 100 μM). Finally, the glutamate receptor antagonists AP-5 (50 and 100 μM) and CNQX (20 and 50 μM) had no effect on peak somatodendritic dopamine release in C57BL/6 mice. Overall, these data suggest that similar mechanisms, consistent with exocytosis, govern electrically evoked dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice.     

THE EFFECT OF BICUCULLINE, METRAZOL, PICROTOXIN AND STRYCHNINE ON THE RELEASE OF [3H]GABA FROM RAT BRAIN SLICES

Each of the four convulsants used significantly influenced the release of [³H]-GABA from brain slices, without affecting [³H]GABA uptake. Bicuculline (10^?5M, but not 10-fold higher or lower concentrations) potentiated the electrically evoked release of [³H]GABA but not the resting release, whereas metrazol (10^?4 to 10^?6 M) was found to inhibit resting but not electrically evoked release. Strychnine (10^?4 and 10^?5 M) and picro-toxin (10^?4 M) inhibited electrically evoked release.     

Thyrotropin-releasing hormone (TRH) and its analog (DN-1417): interaction with pentobarbital in choline uptake and acetylcholine synthesis of rat brain slices

TRH or its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-proliamide) given 15 min after intravenous (i.v.) administration of pentobarbital (30 mg/kg) markedly shortened the pentobarbital-induced sleeping time in rats. This effect was almost completely abolished by intracerebroventricular pretreatment with atropine methylbromide (20 micrograms/rat), thereby suggesting the involvement of cholinergic mechanism. The action mechanism was investigated using rat brain slices. TRH (10(-6)-10(-4)M) or DN-1417 (10(-7)-10(-5)M) caused significant increases in the uptake of [3H]-choline into striatal slices. TRH(10(-4)M) or DN-1417(10(-5)M) also stimulated the conversion of [3H]-choline to [3H]-acetylcholine in striatal slices. A 30% reduction of acetylcholine synthesis from [3H]-choline in hippocampal slices and a 40% reduction of [3H]-choline uptake in slices of cerebral cortex, hippocampus and hypothalamus were observed in rats pretreated with pentobarbital (60 mg/kg, i.v.). TRH or DN-1417 (20 mg/kg, i.v.) given 15 min after the administration of pentobarbital markedly reversed both of the pentobarbital effects. Direct application of pentobarbital (5 X 10(-4)M) to slices in vitro also caused a 20-40% reduction of [3H]-choline uptake of cerebral cortex, hippocampus and diencephalon. A concomitant application of TRH(10(-4)M) or DN-1417(10(-5)M) and pentobarbital abolished the pentobarbital effect. These results provide neurochemical evidence that the antagonistic effects of TRH and DN-1417 on pentobarbital-induced narcosis are closely related to alterations in the rat brain choline uptake and acetylcholine synthesis, which are considered to be measures of the activity of cholinergic neurons.     

Adenosine receptor mediated inhibition of noradrenaline release from slices of the rat hippocampus

Adenosine and adenosine analogues inhibited electrically evoked 3H-noradrenaline (3H-NA) release from slices of the rat hippocampus in vitro in a dose -dependent manner in the concentration range 0.01–100 M. L-phenylisopropyladenosine (L-PIA) was more potent than 5′-N-carboxamidoadenosine (NECA), which was more potent than adenosine. The adenosine uptake blocker dipyridamole (3 M) enhanced the effect of exogenous adenosine, and had a slight inhibitory effect per se. The effect of L-PIA on NA release was competitively antagonized by 8-phenyltheopylline; pA2=7.1. Enprophylline (300 M), theophylline (300 M) and 8-phenyltheophylline (1–10 M) enhanced the evoked 3H-NA release per se, while no such enhancement was seen with the non-xanthine phosphodiesterase inhibitor ZK 62.711 (Rolipram) (30 M).It is concluded that adenosine, at physiologically relevant concentrations, inhibits electrically evoked NA release from terminals in the central nervous system. Alkylxanthines increase evoked NA release from hippocampal terminals, wich probably not related to cyclic AMP but may partly involve inhibition of endogenous adenosine acting as a modulator of transmitter release in the hippocampal slice preparation.     

GABA augments basal and electrically stimulated H-norepinephrine release in hypothalamic, preoptic area and cortical slices of female rats

These studies examined the regulation by GABA of norepinephrine release from hypothalamus, preoptic area and frontal cortex. Using superfused brain slicesfrom female rats, we show that 100 μM GABA enhances both basal and electrically stimulated release of ³H-norepinephrine in all three brain regions. The GABA_A agonist muscimol (100 μM) significantly augments ³H-norepinephrine release, but it is somewhat less effective than GABA. The GABA_B agonist baclofen has little or no effect on basal ³H-norepinephrine efflux. GABA also augments both the magnitude and duration of electrically evoked ³H-norepinephrine release in slices from all three brain regions. GABA facilitation of electrically stimulated ³H-norepinephrine release is mediated through GABA_A receptors as evidenced by its blockad by 10 μM bicuculline, a GABA_A antagonist, but not by 200 μM 2-OH-saclofen, a GABA_B antagonist. These data show that the inhibitory amino acid neurotransmitter GABA enhances both basal and evoked release of ³H-norepinephrine in brain slices from female rats. These effects are predominantly mediated by GABA_A receptors. GABA modulation of hypothalamic norepinephrine release may play a role in the regulation of gonadotropin secretion and reproductive behaviors such as lordosis.     

Study of Differential Effects of Kainic Acid on Metabolic Rates, Utilizing Exogenous or Endogenous Substrates, in Rat Brain Slices

CO2 production from exogenous glucose of cortical, whole hippocampal, and CA3 region hippocampal slices, as well as O2 consumption of whole hippocampal slices, were measured in the presence of different concentrations of kainic acid. A moderate, significant increase of CO2 production was seen only in the CA3 region hippocampal preparation at kainic acid concentrations of 10(-4)-10(-2) M. The O2 consumption, at the expense of endogenous energy stores of whole hippocampal slices, was substantially increased by 10(-3) M kainic acid when the slices were incubated without exogenous glucose. The effect was partly paralleled by the use of high (50 mM) K+ concentration. Some of the possible factors involved in the differential metabolic responses of brain slices to the action of kainic acid are discussed briefly.     

Exocytotic release of dopamine in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice

The present study used voltammetry to ascertain whether electrically stimulated somatodendritic dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice was due to exocytosis or dopamine transporter reversal, as has been debated. The maximal concentration of electrically evoked dopamine release was similar between ventral tegmental area slices from dopamine transporter knockout and C57BL/6 mice. Dopamine transporter blockade (10 μM nomifensine) in slices from C57BL/6 mice inhibited dopamine uptake but did not alter peak evoked dopamine release. In addition, dopamine release and uptake kinetics in ventral tegmental area slices from dopamine transporter knockout mice were unaltered by the norepinephrine transporter inhibitor, desipramine (10 μM), or the serotonin transporter inhibitor, fluoxetine (10 μM). Furthermore, maximal dopamine release in ventral tegmental area slices from both C57BL/6 and dopamine transporter knockout mice was significantly decreased in response to Na⁺ channel blockade by 1 μM tetrototoxin, removal of Ca²⁺ from the perfusion media and neuronal vesicular monoamine transporter inhibition by RO-04-1284 (10 μM) or tetrabenazine (10 and 100 μM). Finally, the glutamate receptor antagonists AP-5 (50 and 100 μM) and CNQX (20 and 50 μM) had no effect on peak somatodendritic dopamine release in C57BL/6 mice. Overall, these data suggest that similar mechanisms, consistent with exocytosis, govern electrically evoked dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice.     

Pentobarbital Antagonizes the A1 Adenosine Receptor-Mediated Inhibition of Hippocampal Neurotransmitter Release

Barbiturates have been shown to be competitive antagonists at A1 adenosine receptors in radioligand binding studies. The present study investigates the effects of pentobarbital on the A1 receptor-mediated inhibition of neurotransmitter release from rabbit hippocampal slices. The inhibition of the electrically evoked release of [3H]noradrenaline by the A1 receptor agonist (R)-N6-phenylisopropyladenosine (R-PIA) was antagonized by pentobarbital with an apparent pA2 value of 3.5. Low concentrations of pentobarbital alone altered neither basal nor evoked release of [3H]noradrenaline, whereas 1,000 microM pentobarbital enhanced the basal and reduced the evoked release. In the presence of 8-phenyltheophylline, pentobarbital (200 microM and 1,000 microM) reduced the evoked noradrenaline release. Pentobarbital also antagonized the inhibition of [3H]acetylcholine release by R-PIA. In contrast to the noradrenaline release model, the evoked release of acetylcholine was enhanced by the presence of pentobarbital (50-500 microM), an effect that was lost in the presence of 8-phenyltheophylline. These results indicate that pentobarbital, in addition to a direct inhibitory action at higher concentrations, has a facilitatory effect on neurotransmitter release by blocking presynaptic A1 adenosine receptors. The possible relevance of these findings for the excitatory effects of barbiturates is discussed.     

Effects of Cholecystokinin Peptides and GV 150013, a Selective CholecystokininB Receptor Antagonist, on Electrically Evoked Endogenous GABA Release from Rat Cortical Slices

The effects of cholecystokinin (CCK) agonists and antagonists on spontaneous and electrically evoked endogenous GABA release from rat cerebral cortex slices were evaluated. Neither the nonselective and CCK(B)-selective receptor agonists CCK-8S (3-1,000 nM) and CCK-4 (3-1,000 nM), respectively, nor the selective CCK(B) and CCK(A) receptor antagonists GV 150013 (3-30 nM) and L-364,718 (10-100 nM), respectively, significantly affected spontaneous GABA release. CCK-8S (1-1,000 nM) and CCK-4 (1-1,000 nM) increased the electrically (5 and 10 Hz)-evoked GABA release. On the contrary, GV 150013 (10 and 30 nM) significantly decreased the electrically evoked GABA release only when the slices were stimulated at the higher 10 Hz frequency. The CCK-8S- and CCK-4-induced increases in electrically evoked GABA release were counteracted by GV 150013, but not by L-364,718. Furthermore, GV 150013 at 3 nM shifted to the right the CCK-4 concentration-response curve, whereas at the higher 10 nM concentration it dramatically flattened the curve. Finally, in cortical slices obtained from rats chronically treated with GV 150013, the concentration-response curve of CCK-4 was shifted to the left and the peak effect of the peptide was significantly higher than that observed in naive animals. These results suggest that CCK increases electrically evoked, but not spontaneous, endogenous GABA release from rat cortical slices, possibly by activating local CCK(B) receptors. In addition, chronic treatment with the novel CCK(B) receptor antagonist GV 150013 leads to an enhanced responsiveness of cortical slices to CCK-4 application.     

Effects of ammonia onl-glutamate uptake in cultured astrocytes

The effect of ammonia onl-glutamate (L-GLU) uptake was examined in cultured astrocytes. Acute ammonia treatment (5–10 mM) enhanced L-[³H]GLU uptake by 20–42% by increasing the V_max; this persisted for 2 days and then started to decline. Ammonia, however, did not affect the uptake ofd-[³H]aspartate (D-ASP), a non-metabolizable analog of L-GLU, that uses the same transport carrier as L-GLU. Also, L-GLU uptake was not affected during the first 2 min of the assay. Thus, ammonia did not have an acute effect on L-GLU transport (translocation); rather, ammonia enhanced the accumulation or “trapping” of L-GLU or its by-products. Chronic ammonia treatment, on the other hand, inhibited L-GLU transport in astrocytes by ∼30–45% and this was due to a decrease in V_max, suggesting that the number of L-GLU transporters was decreased. This inhibitory effect was observed after 1 day of treatment and persisted for at least 7 days. The inhibition of L-GLU transport was partially reversible following removal of ammonia. The effects of ammonia on L-GLU transport and uptake may explain the abnormal L-GLU neurotransmission observed in hyperammonemia/hepatic encephalopathy, and the brain swelling associated with fulminant hepatic failure.     

Inhibitory cholinergic control of endogenous GABA release from electrically stimulated cortical slices and K-depolarized synaptosomes

In the present study we characterize the optimal experimental conditions under which to investigate the cholinergic regulation of endogenous electrically evoked γ-aminobutyric acid (GABA) release from guinea pig cortical slices. Superfusion with the neuronal GABA reuptake inhibitor, SKF89976A (10 μM) caused cortical GABA release to be linearly correlated with the frequency of electrical stimulation (5, 10, 20 Hz). Electrically evoked GABA release (10 Hz) was tetrodotoxin-sensitive and Ca²⁺-dependent and was under GABA_B autoreceptor control. Under these experimental conditions, acetylcholine (0.1–10 μM) and physostigmine (30 μM) decreased the electrically evoked GABA release while the M₂ receptor antagonist AFDX-116 (0.01–0.1 μM) counteracted these effects. Similar results were also observed in a cortical synaptosomal preparation stimulated with K⁺ (10 mM). These findings demonstrate an inhibitory cholinergic regulation of electrically evoked GABA release via M₂ receptors located on cortical GABAergic terminals.     

Aspartate Aminotransferase for Synthesis of Transmitter Glutamate in the Medulla Oblongata: Effect of Aminooxyacetic Acid and 2-Oxoglutarate

The effects of aminooxyacetic acid (AOAA), a transaminase inhibitor, and 2-oxoglutarate, a precursor to glutamate by the activity of aspartate aminotransferase (AAT), on slices of rat medulla oblongata, cerebellum, cerebral cortex, and hippocampus were studied. The slices were superfused and electrically stimulated. There was a Ca2+-dependent stimulus-evoked release of endogenous glutamate, gamma-aminobutyric acid (GABA), and beta-alanine in all regions examined. AOAA (10(-4) and 10(-3) M) decreased the release of glutamate in the medulla oblongata and cerebellum but not in the hippocampus. L-Canaline, a specific inhibitor of ornithine aminotransferase, did not affect the glutamate release in the medulla. 2-Oxoglutarate (10(-3) M) increased the release of glutamate in the medulla oblongata and cerebellum but not in the cerebral cortex and hippocampus. Treatment with AOAA (10(-4) M) almost abolished the activities of AAT in all regions studied. AOAA (10(-4) and 10(-3) M) increased the stimulus-evoked release of GABA in the cerebellum, cerebral cortex, and hippocampus, whereas the stimulus-evoked release of beta-alanine was decreased by this agent in all regions studied. These results suggest the participation of AAT in the synthesis of the transmitter glutamate in the medulla oblongata and cerebellum of the rat.     

Influence of Cannabinoids on Electrically Evoked Dopamine Release and Cyclic AMP Generation in the Rat Striatum

Abstract: Using the endogenous cannabinoid receptor agonist anandamide, the synthetic agonist CP 55940 {[1α,2β( R )5α]-(−)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol}, and the specific antagonist SR 141716 [ N -(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide hydrochloride], second messenger activation of the central cannabinoid receptor (CB1) was examined in rat striatal and cortical slices. The effects of these cannabinoid ligands on electrically evoked dopamine (DA) release from [³H]dopamine-prelabelled striatal slices were also investigated. CP 55940 (1 µ M ) and anandamide (10 µ M ) caused significant reductions in forskolin-stimulated cyclic AMP accumulation in rat striatal slices, which were reversed in the presence of SR 141716 (1 µ M ). CP 55940 (1 µ M ) had no effect on either KCl- or neurotransmitter-stimulated ³H-inositol phosphate accumulation in rat cortical slices. CP 55940 and anandamide caused significant reductions in the release of dopamine after electrical stimulation of [³H]dopamine-prelabelled striatal slices, which were antagonised by SR 141716. SR 141716 alone had no effect on electrically evoked dopamine release from rat striatal slices. These data indicate that the CB1 receptors in rat striatum are negatively linked to adenylyl cyclase and dopamine release. That the CB1 receptor may influence dopamine release in the striatum suggests that cannabinoids play a modulatory role in dopaminergic neuronal pathways.     

Interaction of kainic acid with Na(+)-dependent glutamate binding and uptake in the cerebral cortex of the developing mouse.

The effect of kainic acid, a structural analogue and specific agonist of glutamate, was studied on the Na(+)-dependent binding and uptake of this amino acid in cerebral cortex preparations from 7-day-old and 30-day-old mice. The specific binding of glutamate to a crude synaptic membrane fraction and uptake into cortical slices increased several fold during this period. Kainic acid (0.5 mM or 5 mM) significantly reduced glutamate binding and this effect was more pronounced in membrane fractions from older animals. In contrast to this, the inhibitory action of kainic acid on glutamate uptake was twofold more potent in 7-day-old mice. The results are discussed from the viewpoint of the relationship between the Na(+)-dependent binding of glutamate and its uptake.     

Role of Adenylate Cyclase in Presynaptic α2-Adrenoceptor- and μ-Opioid Receptor-Mediated Inhibition of [3H]Noradrenaline Release from Rat Brain Cortex Slices

Rat brain cortex slices, prelabelled with [3H]noradrenaline, were superfused and exposed to electrical biphasic block pulses (1 Hz; 12 mA, 4 ms) or to the Ca2+ ionophore A 23187 (10 microM) in the presence of 1.2 mM Ca2+. Forskolin (10 microM), 8-bromo-cyclic AMP (300 microM), and dibutyryl-cyclic AMP (300 microM) facilitated both the electrically evoked and A 23187-induced [3H]noradrenaline release, whereas the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX, 300 microM) and 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62771, 30 microM) enhanced the electrically evoked release only. The inhibitory effects of clonidine (1 nM-1 microM) and the facilitatory effect of phentolamine (0.01-10 microM) on the electrically evoked [3H]noradrenaline release were strongly reduced in the presence of 8-bromo-cyclic AMP. Clonidine (1 microM) reduced and phentolamine (3 microM) enhanced A 23187-induced [3H]noradrenaline release, provided that the slices were simultaneously exposed to forskolin. The inhibitory effects of morphine (1 microM) and [D-Ala2-D-Leu5]enkephalin (DADLE, 0.3 microM), like that of the Ca2+ antagonist Cd2+ (15 microM), on the electrically evoked release of [3H]noradrenaline were not affected by 8-bromo-cyclic AMP. Moreover, morphine and DADLE did not inhibit A 23187-induced release in the absence or presence of forskolin. These data strongly suggest that in contrast to presynaptic mu-opioid receptors, alpha 2-adrenoceptors on noradrenergic nerve terminals are negatively coupled to adenylate cyclase and may thus reduce neurotransmitter release by inhibiting the feed-forward action of cyclic AMP on the secretion process.     

Effects of glutamic acid diethylester on synaptic transmission in the skate ampullae of Lorenzini

The effects on synaptic transmission of glutamic acid diethylester (GDEE), a glutamate receptor blocker, were investigated by recording spike activity from single nerve fibers in the electroreceptor cells of the skate (Raja clavata) ampullae of Lorenzini. It was found that adding GDEE to the bathing medium led to a concentration-dependent reduction in or complete blockade of background and evoked receptor activity; 10^–6 M GDEE was the minimum effective concentration. It was also shown that GDEE reversibly blocked postsynaptic response produced by excitatory amino acids: L-glutamate (L-GLU) and L-asparate (L-ASP). Findings suggest the involvement of L-GLU or a related substance in synaptic transmission in the ampullae of Lorenzini.I. P. Pavlov Institute of Physiology of the Academy of Science of the USSR, Leningrad, USSR. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 323–327, May–June, 1987.     

Selective Stimulation of Neurotransmitter Release from Chick Retina by Kainic and Glutamic Acids

The excitatory action of kainic and glutamic acids in chick whole retina was demonstrated as an immediate stimulation of the release of labeled gamma-aminobutyric acid (GABA) and glycine in a superfusion system. This stimulatory effect was 3-10 times greater than that produced by a depolarizing K+ concentration; in addition, it was independent of Ca2+ in the medium, but notably inhibited when Na+ was omitted from the medium. Under identical experimental conditions, neither kainic nor glutamic acid had any effect on the release of labeled dopamine or alpha-aminoisobutyric acid, thus indicating that their effect is not unspecific or due to cell damage. Similar although less marked stimulation of labeled GABA and glycine release by kainic acid was obtained in subcellular retinal fractions, particularly in fraction P1, which contained photoreceptor terminals and outer segments. This stimulation was also Ca2+ independent and greatly reduced when Na+ was omitted from the medium. It is suggested that the stimulation of GABA release by kainic and glutamic acids is probably due to a Na+-dependent, carrier-mediated mechanism that responds to the entry of Na+ produced by the interaction of glutamic and kainic acids with retinal membranes. In cortical or striatal slices from mouse brain, these acids had a negligible stimulatory effect on GABA and dopamine release.     

ACTION OF THE NEUROTOXIN KAINIC ACID ON HIGH AFFINITY UPTAKE OF l-GLUTAMIC ACID IN RAT BRAIN SLICES

Kainic acid is a linear competitive inhibitor (K_is 250 μm ) of the ‘high affinity’ uptake of l -glutamic acid into rat brain slices. Kainic acid inhibits the ‘high affinity’ uptake of l -glutamic, d -aspartic and l -aspartic acids to a similar extent. Kainic acid is not actively taken up into rat brain slices and is thus not a substrate for the ‘high affinity’ acidic amino acid transport system or any other transport system in rat brain slices. Kainic acid (300 μm ) does not influence the steady-state release or potassium-stimulated release of preloaded d -aspartic acid from rat brain slices. Kainic acid binds to rat brain membranes in the absence of sodium ions in a manner indicating binding to a population of receptor sites for l -glutamic acid. Only quisqualic and l -glutamic acid inhibit kainic acid binding in a potent manner. The affinity of kainic acid for these receptor sites appears to be some 4 orders of magnitude higher than for the ‘high affinity’l -glutamic acid transport carrier. Dihydrokainic acid is approximately twice as potent as kainic acid as an inhibitor of ‘high affinity’l -glutamic acid uptake but is some 500 times less potent as an inhibitor of kainic acid binding and at least 1000 times less potent as a convulsant of immature rats on intraperitoneal injection. Dihydrokainic acid might be useful as a ‘control uptake inhibitor’ for the effects of kainic acid on ‘high affinity’l -glutamic acid uptake since it appears to have little action on excitatory receptors. N-Methyl-d -aspartic acid is a potent convulsant of immature rats, but does not inhibit kainic acid binding or ‘high affinity’l -glutamic acid uptake. N-Methyl-d -aspartic acid might be useful as a ‘control excitant’ that activates different excitatory receptors to kainic acid and does not influence ‘high affinity’l -glutamic acid uptake.