K+-evoked Ca2+-dependent release of d-[3H]aspartate from terminals of the cortico-pontine pathway

Pontine nuclei disseted from rat brain slices released previously accumulated d-[³H]aspartate (d-Asp) and [¹⁴C]γ-aminobutyrate (GABA) Ca-depentlytly exposed to 50 mM K. These efflux rates were substantially increased by including 0.5 mg/ml bovine serum albumin in the superfusion fluid. Degeneration of the cortico-pontine fibres 5 days after cutting the crus cerebri caused an 80% reduction in the fractional rate of Ca-dependent d-Asp release and a 60% reduction in uptake. The fractional rate of GABA efflux was significantly less reduced than that of d-Asp efflux, and GABA uptake was nearly unchanged.     

Efflux mechanism of phenylalanine and tryptophan in rat cerebral cortex slices

Phenylalanine and tryptophan effluxes from rat cerebral cortex slices were studied in small oscillating superfusion cups. The slices superfused for 120 min gained sodium and water and lost potassium only slightly more than those only preincubated for 30 min. There was a linear correlation between the rates of spontaneous efflux of [³H]phenylalanine and [³H]tryptophan and the phenylalanine or tryptophan contents in the slices. No signs of saturation of the exit were discernible within the amino acid concentration range of 0.1 to 18 mmol per kg slice wet weight. Extracellular histidine, phenylalanine and tryptophan stimulated the efflux of [³H]phenylalanine and [³H]tryptophan by saturable exchange. Only histidine inhibited the efflux intracellularly, while intracellular phenylalanine and tryptophan seemed to enhance it.The results suggest that the efflux of phenylalanine and tryptophan from brain cells is carrier-mediated in the presence of other extracellular amino acids but that spontaneous efflux into amino acid-free medium may mainly occur via non-mediated physical diffusion.     

Evidence that prejunctional adenosine receptors regulating acetylcholine release from rat hippocampal slices are linked to an N-ethylmaleimide-sensitive G-protein,but not to adenylate cyclase or dihydropyridine-sensitive Ca2+-channeIs

Electrically evoked [³H]acetylcholine ([³H]ACh) release from slices of the rat hippocampus was reduced in a dose-dependent manner by the adenosine A,-receptor agonist R-phenylisopropyladenosine (R-PIA) in the concentration range 0.1–10 μM. The maximal effect was observed with I μM R-PIA. Treatment with N-ethylmaleimide (NEM, 100 μ M, 10 min), which inactivates nucleotide-binding proteins (G-proteins), caused a slight increase in the basal overflow (0.17 ± 0.01%v. 0.10 ± 0.003% in the control slices), but did not affect stimulated release (0.73 ± 0.05%vs. 0.74 ± 0.03% in the control slices). N-ethylmaleimide pretreatment significantly reduced the prejunctional inhibitory effect of R-PIA on [³H]ACh release in a non-competitive manner. The S₂/S₁ ratio was 0.92 ± 0.03 in controls and was reduced to 0.32 ± 0.02 by I μ Mm R-PIA in the control slices and to 0.57 ± 0.03 after NEM pretreatment. Stimulation of cyclic AMP-accumulation by forskolin (I μ M) and rolipram (30 μ M) before the second stimulation (S₂) enhanced the S₂/S₁ ratio by about 30% to 1.26 ± 0.12, but did not reduce the inhibitory effect of R-PIA (I μ MM). The Ca²⁺-channel agonist Bay K 8644 (I μ MM), a concentration that increases K⁺-evoked noradrenaline release, did not affect the basal or electrically evoked [³H]ACh overflow, or the prejunctional effects of R-PIA (0.1 and I μ MM) on [³H]ACh release. Our results suggest that the presynaptic inhibitory effects of A₁-receptor agonists on [³H]ACh release are exerted via a nucleotide-binding protein that can be inhibited by NEM. However, the inhibitory effect is apparently not caused by a change in adenylate cyclase activity or by affecting dihydropyridine-sensitive Ca²⁺-channels.     

Release of endogenous 5-hydroxytryptamine from resting and stimulated enteric neurons

Physiological and biochemical evidence has indicated that there may be serotoninergic neurons in the enteric nervous system. A critical step in the identification of a neurotransmitter is the demonstration of the release of the substance upon nerve stimulation. We now report the release of endogenous 5-hydroxytryptamine from enteric neurons. Segments of guinea-pig small intestine were everted and perfused in vitro through the newly created serosal lumen. Tests with [³H]5-hydroxytryptamine revealed the existence of a tissue barrier preventing diffusion of mucosal (enteroendocrine cell) 5-hydroxytryptamine into the perfusate; thus, all 5-hydroxytryptamine in the perfusate was of neural origin. The gut was stimulated electrically. 5-Hydroxytryptamine in the perfusate and in the myenteric plexus was assayed by a specific radioenzymatic method. 5-Hydroxytryptamine was present in the myenteric plexus; it was released into the perfusate spontaneously and the release was enhanced by electrical stimulation. The stimulated, but not the spontaneous, release of the amine was Ca²⁺-dependent. Comparison with the release of newly taken up [³H]5-hydroxytryptamine showed that the specific radioactivity of electrically released 5-hydroxytryptamine was higher than that of either the spontaneously released or tissue amine. Stimulation also increased the release of 5-hydroxytryptamine more than that of its metabolites in the perfusate.These results indicate that 5-hydroxytryptamine is an endogeneous constituent of the enteric nervous system, that it is released by electrical field stimulation of enteric nerves, and that newly taken up 5-hydroxytryptamine is released preferentially by these neurons.     

Modulation of GABA release by α-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-d-aspartate receptors in matrix-enriched areas of the rat striatum

Using a new in vitro superfusion device, the release of preloaded [³H]GABA was examined in microdiscs of tissues taken from sagittal slices in matrix-enriched areas of the rat striatum. Potassium (9 mM, 15 mM) stimulated the release of [³H]GABA in a concentration- and calcium-dependent manner and the veratridine (1 μM)-evoked release of [³H]GABA was completely abolished in the presence of tetrodotoxin (1 μM).The selective glutamatergic agonist α-amino-3-hydroxy-5-methylisoxazole-4-propionate (1 mM) enhanced the potassium-evoked release of [³H]GABA as well as the basal outflow of [³H]GABA. This latter effect was found to be calcium-dependent, partially diminished by tetrodotoxin (1 μM), completely blocked by 6,7-dinitro-quinoxaline-2,3-dione (0.1 mM), which is generally used as an antagonist of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors, but not affected by ( + )-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801, 10μM), a specific antagonist of N-methyl-d-aspartate receptors.Similarly, N-methyl-d-aspartate (1 mM) enhanced both the potassium (9mM) and the α-amino-3-hydroxy-5-methylisoxazole-4-propionate (1 mM )-evoked release of [³H]GABA but when used alone, due to the presence of magnesium in the superfusion medium, was ineffective on the basal efflux of [³H]GABA. A stimulatory effect of N-methyl-d-aspartate (1 mM) on the basal outflow of [³H]GABA was observed, however, when magnesium was omitted from the superfusion medium. The stimulatory effect of N-methyl-d-aspartate (1 mM) observed in the presence of α-amino-3-hydroxy-5-methylisoxazole-4-propionate was not potentiated by glycine (1 μM, in the presence of strychnine 1 gmM) and the N-methyl-d-aspartate-evoked response seen in the absence of magnesium was not enhanced byd-serine (1 mM). suggesting that endogenous glycine is already acting on N-methyl-d-aspartate receptors. In fact, in the absence of magnesium, 7-chloro-kynurenate (1 mM) completely abolished the stimulatory effect of N-methyl-d-aspartate on the release of [³H]GABA confirming that under our conditions, the glycine site of the N-methyl-d-aspartate receptor is saturated. N-methyl-d-aspartate-evoked responses were all blocked by MK801 (10 μM). Finally, the N-methyl-d-aspartate-evoked response seen in the absence of magnesium was markedly reduced in the presence of tetrodotoxin (1 μM). While potassium (9 mM) markedly stimulated the release of [³H]GABA from purified synaptosomes from the rat striatum, neither α-amino-3-hydroxy-5-methylisoxazole-4-propionate alone nor N-methyl-d-aspartate in the presence of α-amino-3-hydroxy-5-methylisoxazole-4-propionate or in the absence of magnesium enhanced the release of [³H]GABA. A slight but not significant stimulatory effect was observed with N-methyl-d-aspartateandd-serine using a magnesium-free superfusion medium.Altogether, these results strongly suggest that α-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-d-aspartate receptors are present on dendrites and/or cell bodies of efferent GABAergic neurons but not on their nerve terminals in matrix-enriched areas of the rat striatum.     

Perikaryal cell labeling in the subthalamic nucleus following the injection of 3h-γ-aminobutyric acid into the pallidal complex: An autoradiographic study in cat

Although the subthalamic nucleus is thought to exert a major influence on the corpus striatum output cells, there is little information available on the transmitter or transmitters involved.In a series of autoradiographic experiments in which various different radiolabeled putative transmitter substances were injected separately into the pallidal complex of cats, it was noted that ³H-gamma-aminobutyric acid injection consistently resulted in perikaryal labeling in the subthalamic nucleus. ³H-γ-aminobutyric acid injection in the lateral part of the external pallidal segment resulted in labeled cells situated laterally in the subthalamic nucleus, while ³H-aminobutyric acid injection in the internal pallidal segment (entopeduncular nucleus) resulted in cell labeling more medially in the subthalamic nucleus. Perikaryal cell labeling was also noted in the lateral putamen following ³H-γ-aminobutyric acid injection in the external pallidal segment in cats pretreated with systemic amino-oxyacetic acid. No cell groups other than the striatum and subthalamic nucleus could be made to label with ³H-γ-aminobutyric acid. Furthermore, no perikaryal cell labeling in the subthalamic nucleus was seen to follow injections of ³H-d-aspartate or ³H-serotonin into the pallidal complex. The findings suggest that ³H-γ-aminobutyric acid, but not aspartate or serotonin, undergoes high affinity uptake and retrograde transport by subthalamo-pallidal neurons. Bearing in mind the many reservations discussed, the observation implies that ³H-γ-aminobutyric acid may be a transmitter in the subthalamo-pallidal pathway.     

Dopamine release from the rat substantia nigra in vitro. Effect of raphe lesions and veratridine stimulation

In order to eliminate the 5-hydroxytryptaminergic input to the substantia nigra lesions were placed in the dorsal and medial raphe nuclei in a number of rats. The release of exogenously applied [³H]dopamine from the partially denervated substantia nigra was determined in vitro and found to be very similar to the release observed from slices of control substantia nigra. These results lend further support to the theory that the release of exogenously applied [³H]dopamine at the level of the substantia nigra occurs mainly from dopaminergic dendrites, rather than from terminals of 5-hydroxytryptamine-containing neurons.A veratridine-induced release of [³H]dopamine from the pars reticulata of the substantia nigra is also described. An almost complete blockade of veratridine (3.0 μM) stimulation was observed with 100 nM tetrodotoxin. Similar effects of veratridine and tetrodotoxin were also observed on [³H]dopamine release from slices of corpus striatum. These results suggest that dendrites of the dopaminergic neurones in the substantia nigra contain fast, tetrodotoxin-sensitive sodium channels.     

Choline uptake, acetylcholine synthesis and release by Limulus abdominal ganglia

Isolated ganglia from the ventral nerve cord of the horseshoe crab, Limulus polyphemus, were incubated in [³H]choline and subsequently analyzed for choline uptake, conversion of choline to acetylcholine and the release of the newly synthesized acetylcholine. The ganglia readily accumulated radioactivity when incubated in Chao's solution containing 2 μM [³H]choline. The rate of uptake was 0.08 pmols/min/mg tissue and 26% of the [³H]choline taken up during a 1 h period was converted to [³H]acetylcholine. A 15 min exposure of ganglia to 90 mM K⁺ prior to incubation in [³H]choline caused an 89% increase in choline uptake and a 150% increase in its conversion to [³H]acetylcholine. The presence of unlabeled acetylcholine in the uptake medium inhibited both uptake and conversion of [³H]choline.There was a 5-fold increase in the efflux of radioactivity when ganglia incubated in 2 μM [³H]choline were superperfused with 90 mM K⁺. The increased efflux of radioactivity was Ca²⁺-dependent and was inhibited by Mg²⁺ (44%) and by Co²⁺ (72%). Similarly, addition of veratridine caused a Ca²⁺ and Na⁺-dependent release of radioactivity from prelabeled ganglia. Analysis of the superperfusate revealed that virtually all of the released radioactivity was [³H]acetylcholine.The abdominal ganglia of Limulus take up choline at micromolar concentrations, convert substantial amounts of it to acetylcholine and possess a depolarization-triggered, Ca²⁺-requiring mechanism for the specific release of acetylcholine. These results give further support to the view that the abdominal ganglia of Limulus contain a population of cholinergic terminals.     

Morphological, neurochemical, and behavioral studies on serotonergic denervation and graft-induced reinnervation of the rat hippocampus.

A procedure was developed to conduct simultaneously immunocytochemical and neurochemical studies on the serotonergic system in adjacent 300-micron-thick slices of rat hippocampus. This procedure was applied to correlate morphological (innervation pattern and density), neurochemical (5-hydroxytryptamine and 5-hydroxyindolacetic acid levels and [3H]5-hydroxytryptamine uptake and release) and behavioral (spatial learning) effects of neurotoxin-induced denervation and reinnervation by grafting fetal mesencephalic raphe cells. Intracerebroventricular injections of a low dose of 5,7-dihydroxytryptamine caused a discrete serotonergic denervation of the hippocampus. Eleven months after lesioning, 5-hydroxytryptamine and 5-hydroxyindolacetic acid levels and [3H]5-hydroxytryptamine uptake capacity were decreased by 50-60%. By this time, the residual fibers displayed an enhanced vulnerability towards K(+)-induced depolarization. Grafting of a fetal raphe cell suspension resulted in a reinnervation of the host hippocampus. The pattern of reinnervation was comparable to control innervation and the density was supranormal at the level of the graft. As observed semiquantitatively, the innervation density decreased with distance from the core of the graft. Neurochemical studies showed that the fibers were capable of synthesizing, metabolizing and releasing 5-hydroxytryptamine. The turnover of 5-hydroxytryptamine in both the denervated and the reinnervated hippocampus was comparable to that in control tissue. Previous behavioral testing of the denervated and of the denervated and implanted animals did not reveal any effect on spatial learning, either in an individual or in a social test paradigm. The latter data substantiate the notion that interference with the hippocampal serotonergic innervation does not hamper adequate spatial learning.     

Radioautographic study of uptake and storage of indoleamines in the rabbit enterochromaffin cells

After in vitro intra-arterial injection of tritium-labeled 5-hydroxytryptamine or -hydroxytrytophan ([³H]5-HT or [³H]5-HTP) (5·10^?7 M) in the presence of cold noradrenaline (5·10^?6 m) into rabbit colon, a clear-cut labelling pattern was observed by radioautography. Labelled cells were observed within the mucosa. The labeling was less important after [³H]5-HTP than after [³H]5-HT injection. Ultrastructural study indicates that labeling is confined to the cytoplasm and coincides with polymorphic secretory granules.Hence these labeled cells display not only APUD cells characteristics but also true neuronal properties.     

Differential effects of 5,7-dihydroxytryptamine-induced serotoninergic degeneration on 5-HT1A receptors and 5-HT uptake sites in the rat brain

The time-course of 5,7-dihydroxytryptamine-induced lesions (2, 5 and 14 days after i.c.v. injection of 150 μg) and the effects of acute reserpine treatment (10 mg/kg, i.p., one or 5 days before scheduled death), were evaluated by autoradiography of [³H]paroxetine binding sites in the rat brain. Reserpine had no significant effect on [³H]paroxetine binding, indicating that the depletion of serotonin is not sufficient per se to alter the serotonin uptake sites in any region. Two days after the 5,7-dihydroxytryptamine lesion, [³H]paroxetine binding was already decreased in the majority of brain regions. In the caudate putamen these binding sites were significantly decreased only 14 days after the lesion, whereas the ventral tegmental area (or the enclosed median forebrain bundle), the dorsal raphe (mainly the ventral portion) and the median raphe maintained their high density of serotonin uptake sites even after 14 days. Results were similar using [³H]citalopram as ligand for the serotonin uptake sites, in the brains of rats lesioned 5 days before death; an exception was the ventral portion of the dorsal raphe, where there was a significant increase with [³H]paroxetine and a decrease with [³H]citalopram binding. In adjacent sections of the same brains we also measured [³H]8-OH-DPAT binding, confirming that it completely disappears in the dorsal raphe after the lesion. Thus, considering the extent of serotonin cell body degeneration, there appears to be a paradoxical mismatch between the excessive loss of [³H]8-OH-DPAT binding and the resistance of [³H]citalopram or [³H]paroxetine binding in the dorsal raphe, suggesting that the two binding sites may undergo adaptive regulation in surviving neurons.     

Comparative study of the release of glutamate and GABA, newly synthesized from glutamine, in various regions of the central nervous system

The in vitro release of [³H]glutamate and [³H]γ-aminobutyric acid, both newly synthesized from [³H]glutamine, were studied in various regions of the rat and pigeon brain. The amount of transmitter released from slices during stimulation by excess K⁺, as well as the ratio of the amounts of glutamate and γ-aminobutyrate released, varied considerably from region to region. Very high levels of release of glutamate were observed in rat striatum and in rat and pigeon hippocampus whereas only moderate release of glutamate was found in rat and pigeon cerebellum, rat cochlear nucleus and rat substantia nigra. The highest levels of release of γ-aminobutyrate were observed in the rat substantia nigra, hippocampus and striatum. Particularly low levels of release of glutamate was observed in the pigeon optic tectum. The release of γ-aminobutyrate and glutamate in all structures was largely calciumdependent.These results suggest that the release of glutamate and γ-aminobutyrate newly synthesized from glutamine reflects the neurotransmitter function of the two amino acids within various CNS regions. They further suggest that the glutamate synthesizing and releasing pool is different from the γ-aminobutyrate synthesizing and releasing pool. The similarity between the release of glutamate in rat and pigeon cerebellum and hippocampus suggest some structural homology in both species.     

Exchange and efflux of tryptophan from superfused cerebral cortex slices

Summary The efflux and exchange of L-tryptophan (Trp) from rat cerebral cortex slices were studied in a superfusion system. The substrate specificity of Trp exchange was assessed by measuring the stimulation of [³H]Trp exit provoked by other extracellular amino acids. Large neutral amino acids were the most potent, but also glutamic acid, lysine and glycine had some effect. The stimulation caused by extracellular Trp and phenylalanine persisted also at 0 ° C though severalfold attenuated. Only intracellular histidine provoked slight inhibition of [³H]Trp efflux and intracellular Trp, phenylalanine and lysine had a small stimulatory effect. The results suggest an involvement of carrier-mediated processes in the exchange and efflux of Trp. The substrate specificities of the exchange and efflux are not apparently identical.     

Effect of hydroxycitric acid on serotonin release from isolated rat brain cortex.

There is evidence that hydroxycitric acid (HCA), an extract of dried fruit rind of South Asian trees of the genus Garcinia cambogia, can reduce food intake in experimental animals. In the present study, we investigated the effect of HCA on basal and potassium-depolarization evoked increase in radiolabeled serotonin ([3H]-5-HT) release from rat brain cortex slices in vitro. HCA (10 microM-1 mM) altered the baseline of spontaneous tritium efflux but had no significant effect on potassium-evoked release of [3H]-5-HT. When applied on its own, HCA (10 microM-1 mM) elicited a concentration-dependent increase in efflux of [3H]-5-HT reaching a maximum at 300 microM. We conclude that HCA can increase the release of radiolabeled 5-HT from the isolated rat brain cortex.     

Presynaptic effect of L-glutamic acid on the release of dopamine in rat striatal slices

The release of [³H] dopamine ([³H] DA) was estimated in serial superfusate fractions of rat striatal slices continuously superfused with L-[3,5-³H]-tyrosine. L-glutamic acid (5 · 10^?5 M), but not the D-stereoisomer, increased the spontaneous release of [³H] DA (60%). The stimulating effect of L-glutamic acid was still observed in the presence of tetrodotoxin (5 · 10^?7 M), suggesting that the amino-acid acts at a presynaptic site. Moreover, atropine (10^?6 M) or pempidine (10^?5 M) which blocks the acetylcholine (ACh) evoked release of [³H] DA did not reduce the stimulatory effect of L-glutamic acid on [³H] DA release, thus excluding the possible intervention of striatal cholinergic neurons. The data obtained support the hypothesis of a direct control of DA release from nerve terminals by glutamatergic neurons.     

Inactivation of presynaptic 5-HT autoreceptors by lithium in rat hippocampus

The effect of lithium ion on the electrically stimulated 5-[3H]hydroxytryptamine (5-HT) release from the rat hippocampal slices preloaded with [3H]5-HT was studied. Electrically stimulated [3H]5-HT release decreased when the slices were exposed to 5-HT in a concentration-dependent manner. Lithium (2.5 mM) did not affect [3H]5-HT release when added alone to the superfusion medium. However, the inhibitory effect of 5-HT (1 microM) on [3H]5-HT release was abolished by lithium. The results suggest that lithium may inhibit the regulation of 5-HT release via presynaptic 5-HT autoreceptors in rat hippocampus.     

Sulphur-containing excitatory amino acid-stimulated inositol phosphate formation in primary cultures of cerebellar granule cells is mediated predominantly by N-methyl-d-aspartate receptors

The stimulatory effect of excitatory sulphur-containing amino acids on inositol phosphate formation was investigated in primary cultures of cerebellar granule cells. l-Cysteine sulphinate (CSA), l-cysteate (CA), l-homocysteine sulphinate (HCSA), l-homocysteate (HCA) and S-sulpho-l-cysteine (SSC) dose-dependently stimulated the formation of [^u3H]inositol phosphates exhibiting EC₅₀ values in the range 60–200 μM and maximal effects of six- to 17-fold that of basal [³H]inositol phosphate levels. Endogenous l-glutamate spontaneously released into the extracellular medium or following exposure of cells to HCSA, HCA or SSC did not contribute significantly to formation of [³H]inositol phosphates, whereas 10% of the total [³H]inositol phosphates accumulated following exposure to CSA and CA was due to released l-glutamate. The selective N-methyl-d-aspartate receptor antagonist, d,l-2-amino-5-phosphonopentanoic acid (APV, 500 μM) attenuated by 20% (HCSA) to between 80 and 100% (CSA, CA, SSC, HCA) the formation of [³H]inositol phosphates induced by 1 mM sulphur-containing amino acids. When, however, HCSA was used at 100 μM (a concentration near to its EC₅₀ for phosphoinositide hydrolysis), APV inhibited induced responses by 70%. Sulphur-containing amino acid-stimulated [³H]inositol phosphate formation was unaffected by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM). Inhibition of sulphur-containing amino acid-stimulated [³H]inositol phosphate formation by co-administration of APV and CNQX was similar to that obtained in the presence of APV alone. CSA-, CA-, SSC- and HCA-stimulated [³H]inositol phosphate formation was markedly reduced by removal of Ca²⁺ from the extracellular medium whereas that stimulated by HCSA was less affected. A similar inhibitory profile was observed when the levels of sulphur-containing amino acid-induced increases in intracellular free calcium ([Ca²⁺₁) were measured in the presence of 500 μM APV; 1 mM HCSA-induced responses being inhibited by only 30% whereas responses to the remaining sulphur-containing amino acid (also at 1 mM) were inhibited by >45%. When the sulphur-containing amino acids were used at concentrations approximating their EC₅₀ values for phosphoinositide hydrolysis, APV inhibited the induced increases in [Ca²⁺]_i by 70–100%. HCA and SSC co-administered with the less efficacious but selective metabotropic glutamate receptor agonist, ( ± )-1-aminocyclopentane-trans-1,3-dicar☐ylic acid (trans-ACPD) at maximally effective concentrations (1 mM) of each agonist stimulated [³H]inositol phosphate formation in an additive manner. In the case of CSA, CA and HCSA, however, co-administration with trans-ACPD caused no increase in level of [³H]inositol phosphate formation compared to that observed in the presence of either sulphur-containing amino acid alone. In cells pre-treated with 1 μg/ml pertussis toxin, HCA-stimulated [³H]inositol phosphate formation was not significantly affected, however CSA-, CA- and HCSA-stimulated [³H]inositol phosphate formation was significantly but only partially (by 40%) inhibited. Taken together, these results indicate that in cerebellar granule cells sulphur-containing amino acid-stimulated [³H]inositol phosphate formation is mediated predominantly by N-methyl-d-aspartate receptor activation. There is evidence however to support an additional role for CSA, CA and particularly HCSA as agonists of the metabotropic glutamate receptor.The data are consistent with the sulphur-containing amino acids possibly having a physiological role as endogenous activators of both ionotropic and metabotropic excitatory amino acid receptors. The present findings may, however, be of more significance in suggesting possible mechanisms by which the sulphur-containing amino acids or disturbances in sulphur-containing amino acid metabolism are linked to the aetiology of a number of neuropathologies.     

Potassium-evoked release of [14C]GABA and [3H]taurine from rat olfactory bulb slices

Rat olfactory bulb slices were preloaded with [³H] taurine or with [¹⁴C] GABA. Upon stimulation of the slices with increasing concentrations of KCl, we observed release of [³H] taurine or [¹⁴C] GABA. Superfusion of the slices with high concentrations of K⁺ in the absence of Ca²⁺ in the perfusion medium, led to a marked decrease in the stimulated release of both [³H] taurine and [¹⁴C] GABA.     

Effect of arginine on basal and high potassium-induced efflux of [3H]D-aspartate from rat striatal slices.

There are conflicting reports in the literature regarding the effects of nitric oxide as well as the involvement of the cyclic GMP pathway on the transmitter release. To study the influence of the availability of the nitric oxide precursor arginine on the glutamate transmission process, rat striatal slices preloaded with the tritiated glutamate analogue D-aspartate were used. L-Arginine stimulated in a concentration-dependent way (0.01-10.0 mM) the high potassium-induced efflux of [3H]D-aspartate. The basal release was increased only by 10 mM L-arginine. Neither the basal nor the depolarization-induced efflux of [3H]D-aspartate was affected by D-arginine. The L-arginine effect was abolished by the nitric oxide synthase inhibitor L-arginine methyl ester and was not modified by cyclic GMP. Only at high concentrations of L-arginine (10 mM) could an elevation of cyclic GMP level be demonstrated. The results are discussed in terms of direct presynaptic action of nitric oxide on [3H]D-aspartate efflux and a possible modulation of glutamate release by the availability of arginine.     

The distribution of serotonin binding sites in the hippocampal region of the rat brain. An autoradiographic study

The distribution of serotonin binding sites was studied in the rat hippocampal region by using contact-film autoradiography after in vitro incubations of brain sections with 5-[3H]hydroxytryptamine, [3H]spiperone, and [3H]ketanserin, respectively. Biochemical studies of the 5-[3H]hydroxytryptamine binding to sections cut through the hippocampal region showed that at saturating concentrations of 5-[3H]hydroxytryptamine (2-2.5 nM) the specific binding was at least 50% of the total. The 5-[3H]hydroxytryptamine binding sites were found to be heterogeneously distributed within the hippocampal region with the highest densities present in the following parts: layers I and II and layers IV through VI of the entorhinal area, the radial layer of the subiculum and subfield CA1 of the Ammon's horn and the molecular layer of the area dentata. Moderate to low densities of binding was observed in layer III of the entorhinal area, the pre- and parasubiculum, the stratum pyramidale of the Ammon's horn, and the granular cell layer of the area dentata. Removal of the 5-hydroxytryptamine nerve terminals by systemic injections of the 5-hydroxytryptamine neurotoxin parachloroamphetamine resulted in no detectable reductions of 5-[3H]hydroxytryptamine binding in any brain region. Lesions of hippocampal cell bodies by intrahippocampal injections of ibotenic acid prevented the binding of 5-[3H]hydroxytryptamine within the area of the cell loss. Comparisons between the distribution of 5-hydroxytryptamine immunoreactive nerve terminals and the 5-[3H]hydroxytryptamine binding sites showed that in some areas of sparse 5-hydroxytryptamine innervation the 5-[3H]hydroxytryptamine binding was close to background (e.g. the pyramidal cell layer, the stratum lucidum) whereas in areas with little 5-[3H]hydroxytryptamine binding (e.g. layer III of the lateral entorhinal area, the presubiculum) a very dense 5-hydroxytryptamine innervation was found. The hippocampal 5-[3H]hydroxytryptamine binding was displaced neither by ketanserin (1 microM) nor by spiperone (1 microM), two drugs that bind to cortical 5-hydroxytryptamine2 receptors in the rat brain. Furthermore, the pattern of hippocampal [3H]spiperone binding differed considerably from that of 5-[3H]hydroxytryptamine. The [3H]ketanserin binding in the hippocampal region did not exceed background levels, except in the hilus of area dentata in the ventral hippocampus and entorhinal layer VI at the same level, where moderate binding was found.(ABSTRACT TRUNCATED AT 400 WORDS)