Regional Selectivity of a γ-Aminobutyric Acid-Induced [3H]Acetylcholine Release Sensitive to Inhibitors of γ-Aminobutyric Acid Uptake

The effects of gamma-aminobutyric acid (GABA) on the release of [3H]acetylcholine ([3H]ACh) were studied in synaptosomes prepared from rat hippocampus, cerebral cortex, hypothalamus, and striatum and prelabelled with [3H]choline. When synaptosomes were exposed in superfusion to exogenous GABA (0.01-0.3 mM) the basal release of newly synthesized [3H]ACh was increased in a concentration-dependent way in hippocampus, cortex, and hypothalamus nerve endings. In contrast, the release of [3H]ACh was not significantly affected by GABA in striatal synaptosomes. The effect of GABA was not antagonized significantly by bicuculline or picrotoxin. Muscimol caused only a slight not significant increase of [3H]ACh release when tested at 0.3 mM whereas, at this concentration, (-)-baclofen was totally inactive. The GABA-induced release of [3H]ACh was counteracted by SKF 89976A, SKF 100561, and SKF 100330A, three strong and selective GABA uptake inhibitors. The data suggest that, in selective areas of the rat brain, GABA causes release of [3H]ACh following penetration into cholinergic nerve terminals through a GABA transport system.     

Physiological presynaptic facilitation of dopamine release in the cat caudate nucleus

Using halothane-anaesthetized cats implanted with push-pull cannulae, we investigated the effects of GABA application into the VM/VL on the release of [3H] DA continuously synthetized from [3H] tyrosine in the ipsilateral CN and SN and on single unit activity of nigral DA cells. GABA was applied (30 min) at a concentration of 10(-3) or 10(-5) M since the higher concentration reduces the local multi-unit activity in the VM/VL while the opposite response is observed with the lower one. The application of GABA into the VM/VL at a concentration of 10(-3) M resulted in an increase in nigral [3H] DA release, an inhibition of DA cell firing and a decrease in [3H] DA release in the CN. The latter effect is likely due to the inhibition of DA neuron activity which is triggered through DA autoreceptors by DA released from dendrites. In contrast, when applied at a concentration of 10(-5) M into the VM/VL, GABA stimulated [3H] DA release in the CN despite its inhibitory effect on single unit activity of DA cells. Furthermore, the nigral release of [3H] DA was no longer affected. These results indicated that DA release from nerve terminals was not dependent on nerve activity and they favour the existence of a potent facilitatory presynaptic regulation of DA release. The intervention of a presynaptic mechanism was further established by examining the effect of GABA (10(-5) M) application into the VM/VL on [3H] DA release in the CN shortly after a complete ipsilateral hemisection of the brain made at the meso-diencephalic level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of Protein Kinase C in γ-Aminobutyric Acid Release from Xenopus Oocytes Injected with Rat Brain mRNA

Abstract: Involvement of protein kinase C (PKC) in the release of γ-aminobutyric acid (GABA) was examined in Xenopus laevis oocytes injected with mRNA from rat cerebellum, as compared with findings in slices of rat cerebellum. The mRNA-injected oocytes preloaded with [³H]GABA showed spontaneous release of [³H]GABA, ∼0.5% of GABA content per 1 min. Stimulation with either Ca²⁺ ionophore (A23187) or a high K⁺ concentration increased the release of [³H]GABA from slices of rat deep cerebellar nucleus and mRNA-injected oocytes but not from noninjected and water-injected oocytes. 12- O -Tetradecanoylphorbol 13-acetate (10–300 n M ) but not 4α-phorbol 12,13-didecanoate (300 n M ) potentiated the A23187-stimulated release of [³H]GABA from slices and from mRNA-injected oocytes, in a concentration-dependent manner. Thus, machinery associated with release processes of GABA can be expressed in oocytes by injecting rat cerebellar mRNA, and PKC participates in GABA release from the functionally expressed GABAergic nerve terminals.     

Posttranslational Protein Modification by Polyamines in Intact and Regenerating Nerves

A 150,000-g supernatant from axoplasm of the giant axon of the stellate nerve of the squid and from rat sciatic and goldfish optic nerves was found to be able to incorporate covalently [3H]putrescine and [3H]spermidine into an exogenous protein (N,N'-dimethylcasein). Incorporation of radioactivity was inhibited by CuSO4, a specific inhibitor of transglutaminases, the enzymes mediating these reactions in other tissues. Analysis of pH and temperature range and enzyme kinetics displayed characteristics predicted for transglutaminase-mediated reactions. Transglutaminase activity increased during regeneration of both vertebrate nerves, but greater activity was found in segments of nerve containing no intact axons than in either intact segments or in segments containing regenerating axons. Polyacrylamide gel electrophoresis of endogenous modified proteins (in the absence of N,N'-dimethylcasein) showed labeling of 18-, 46- and 200-kilodalton proteins by both [3H]putrescine and [3H]spermidine. Analysis of the protein-bound radioactivity from intact and regenerating rat sciatic nerves demonstrated it to be predominantly in the form of the parent radioactive polyamine. These experiments demonstrate the covalent modification of proteins by polyamines at low levels in squid axoplasm and at relatively higher levels in rat sciatic and goldfish optic nerves. In the latter two cases, the activity of these modification reactions may be due in part to the modification of axonal proteins, but the majority of the activity occurs in nonneuronal cells of the nerve.     

Presynaptic Mechanisms Underlying the γ-Aminobutyric Acid-Evoked Receptor-Independent Release of [3H]Norepinephrine in Rat Hippocampus

The effects of gamma-aminobutyric acid (GABA) on the spontaneous efflux of [3H]norepinephrine ([3H]NE) were studied in synaptosomes prepared from rat hippocampus and prelabelled with [3H]NE. It had been observed previously that, when synaptosomes were exposed in superfusion to GABA, the basal release of the tritiated catecholamine was enhanced, apparently with no involvement of the known GABA receptors. The mechanisms underlying this effect have now been investigated. The potency of GABA as a releaser of [3H]NE was decreased by lowering the Na+ content of the superfusion medium, and its effect disappeared at 23 mM Na+. The GABA-induced [3H]NE release was counteracted by the GABA uptake inhibitor N-(4,4-diphenyl-3-butenyl)nipecotic acid (SKF 89976A), but it was unaffected by the NE uptake blockers desmethylimipramine and nisoxetine. The GABA-induced release of [3H]NE was Ca2+-dependent and tetrodotoxin-sensitive. The data support the hypothesis that GABA provoked [3H]NE release by a novel mechanism which involves penetration into the noradrenergic nerve terminals through a GABA carrier located on the NE terminals themselves. This uptake process might be electrogenic and provoke depolarization of the nerve terminals, causing an exocytotic release of [3H]NE.     

Effect of Taurine on Neurotransmitter Release from Insect Synaptosomes

The effect of taurine on the release of [3H]acetylcholine ([3H]ACH) and [3H]gamma-aminobutyric acid ([3H]GABA) from preloaded locust synaptosomes has been studied. Veratridine (100 microM) and K+ (100 mM) both evoked [3H]ACh release and this was reduced in a concentration-dependent manner by taurine (5, 10, and 20 mM). In contrast to this, veratridine induced no observable release of [3H]GABA, and the response to K+ was slight. In the presence of taurine, however, a concentration-dependent enhancement of [3H]GABA release was observed. Since nipecotic acid (1 mM), an inhibitor of neuronal GABA uptake, also revealed [3H]GABA release induced by veratridine, it is suggested that both this effect and that of taurine are due to prevention of GABA reuptake. These results suggest that taurine may act as a neuromodulator in insects.     

GABAergic Inhibition on Dopamine Cells of the Fish Retina: A [3H]Dopamine Release Study with Isolated Cell Fractions

Inner retinal cells including dopamine (DA) cells were isolated and fractionated from the carp (Cyprinus carpio) retina by an enzyme cell dissociation and metrizamide gradient centrifugation method. When gamma-aminobutyric acid (GABA) antagonists (bicuculline and picrotoxin) were added into the perfusate over such a cell fraction, they stimulated the release of [3H]DA which had been preloaded in the cell fraction. The action of GABA antagonists was dose and Ca2+ dependent. Their minimal effective concentration was very low (0.5 microM). A similar action was elicited by high K+. In the presence of excess GABA, this stimulatory action of GABA antagonists and high K+ on [3H]DA release was completely abolished. To interpret the action of GABA antagonists on DA cells, isolated cell fractions were preincubated with GABAse. After such a treatment, the stimulatory effects of GABA antagonists and high K+ on [3H]DA release were differentiated from each other; the former disappeared whereas the latter remained unchanged. The data strongly suggest that GABA inhibits the DA release from retinal DA cells and thus the GABA antagonists affect [3H]DA release from cell fractions not by a direct membrane action but by a disinhibition mechanism via GABA receptors on the DA cell bodies.     

Ca2+-Dependent and Ca2+-Independent [3H]GABA Release from Rat Brain Synaptosomes: the Effect of Temperature

The main inhibitory neurotransmitter GABA in the mammalian brain is distributed in the nerve terminals between two pools, vesicular (synaptic vesicles) and cytosolic. GABA is released from these pools by different mechanisms; there are calcium-activated exocytotic release and calcium-independent sodium-dependent release from the cytosolic pool (resulting from the membrane GABA transporter reversal). We investigated the influence of temperature on [³H]GABA release from rat brain synaptosomes, which was induced by stimulation of both these processes. In addition, we used -latrotoxin as a stimulant of [³H]GABA release. Synaptosomes from the rat brain were used in the experiments. 4-Aminopyridine (4-AP) and high [KCl] were applied to stimulate calcium-activated and calcium-independent [³H]GABA release, respectively. 4-AP-evoked [³H]GABA release was of the same intensity at 37 and 25°C (10.1 ± 1.2 and 10.1 ± 0.8% of total [³H]GABA incorporated into the synaptosomes, respectively). The effect of 4-AP on the ⁴⁵Ca²⁺ influx into synaptosomes was also temperature-independent: 0.775 ± 0.075 and 0.725 ± 0.100 nmol/min/mg of protein at 37 and 25°C, respectively. A drop in the effect of 4-AP was observed only at 15°C. When synaptosomes were depolarized with 50 mM KCl, a temperature decrease from 37°C to 25°C resulted in a twofold drop in the [³H]GABA release, from 20.5 ± 1.4 to 10.3 ± 0.7%; at 15°C [³H]GABA release dropped to less than one-third of the norm (6.0 ± 0.5%). -Latrotoxin-stimulated [³H]GABA release was diminished from 32.5 ± 2.5 at 37°C to 17.2 ± 1.3 at 25°C and 5.9 ± 0.4% at 15°C and was not affected by the presence or absence of calcium in the medium. It seems likely that the observed effect of temperature can be interpreted as based on the temperature dependence of the -latrotoxin insertion into the membrane. It is suggested that the pattern of the temperature sensitivity of GABA release from the synaptosomes can be used as a criterion for identification of the mode of neurotransmitter release.     

Retrograde Axonal Transport of Locally Synthesized Phosphoinositides in the Rat Sciatic Nerve

Although autoradiography has demonstrated local incorporation of [3H]inositol into axonal phospholipids after intraneural injection, retrograde axonal transport of phosphatidylinositol has only been demonstrated after injection of lipid precursor into the cell body regions (L4 and L5 dorsal root ganglia) of the sciatic nerve. We now report the retrograde axonal transport of inositol phospholipids synthesized locally in the axons. Following microinjection of myo-[3H]inositol into the rat sciatic nerve (50-55 mm distal to L4 and L5 dorsal root ganglia), a time-dependent accumulation of 3H label occurred in the dorsal root ganglia ipsilateral to the injection site. The ratio of dpm present in the ipsilateral dorsal root ganglia to that in the contralateral dorsal root ganglia was not significantly different from unity between 2 and 8 h following isotope injection but increased to 10-12-fold between 24 and 72 h following precursor injection. By 24 h following precursor injection, the ipsilateral/contralateral ratio of the water-soluble label in the dorsal root ganglia still remained approximately 1.0, whereas the corresponding ratio in the chloroform/methanol-soluble fraction was approximately 20. The time course of appearance of labeled lipids in the ipsilateral dorsal root ganglia after injection of precursor into the nerve at various distances from the dorsal root ganglia indicated a transport rate of at least 5 mm/h. Accumulation of label in the dorsal root ganglia could be prevented by intraneural injection of colchicine or ligation of the sciatic nerve between the dorsal root ganglia and the isotope injection site. These results demonstrate that inositol phospholipids synthesized locally in the sciatic nerve are retrogradely transported back to the nerve cell bodies located in the dorsal root ganglia.     

RELEASE OF [3H]GABA FROM RAT CORTICAL SLICES: NEURONAL VS GLIAL ORIGIN 1

Abstract— The effect of L-2,4 diaminobutyric acid (DABA) and β-alanine on the K⁺ stimulated release of [³H]GABA was examined using a continuous superfusion system in which a carrier mediated exchange diffusion could be demonstrated between [³H]GABA in preloaded rat cortical slices and unlabeled DABA and β-alanine in the superfusion medium. These structurally related amino acids were chosen to investigate the source of releasable [³H]GABA because of evidence suggesting they may have differing affinities for the GABA carrier transport system that are specific for neurons and glia, DABA having a greater affinity for the neuronal GABA system and β-alanine for the glial. Five millimolars-DABA in the superfusion medium nearly abolished the K⁺ stimulated release of [³H]GABA whereas β-alanine had little effect. The results and conclusions are discussed in terms of a postulated carrier mediated exchange of unlabeled DABA with a specific neuronal pool of [³H]GABA interfering with the K⁺ stimulated release of the radiolabeled GABA. The results provide indirect evidence in favor of a neuronal pool as the source of releasable [³H]GABA in this system.     

Uptake,exchange, and release of GABA by cerebellar glomeruli

Glomerular particles were isolated from the bovine cerebellar vermis and studied in vitro to further assess the possibility that -aminobutyric acid (GABA) is utilized as a neurotransmitter in this synaptic complex. Cerebellar glomeruli accumulated [³H]GABA at two different high affinity sites, with affinities (K _T) of 2.2×10^–6M and 3.0×10^–5M. These uptake sites could not be distinguished on the basis of their temperature sensitivities, sodium dependence, substrate specificities or responses to metabolic inhibitors. Although an exchange process contributed to the uptake measured in these experiments, a considerable amount of the [³H]GABA accumulated by glomerular particles was stored in an osmotically-sensitive, nonexchangeable pool. Glomerular particles preloaded with [³H]GABA exhibited a Ca²⁺-independent release of this amino acid in response to membrane depolarization. However, when preloaded glomerular particles were exposed to unlabeled GABA, which presumably displaced [³H]GABA from the exchangeable pool, a K^–-evoked and Ca²⁺-dependent release of the remaining [³H]GABA occurred. The observed net uptake, together with the depolarization-induced and Ca²⁺-dependent release, of [³H]GABA from glomerular particles supports the suggestion that functionally active GABAergic synapses are present in these structures.     

Calcium-Independent γ-Aminobutyric Acid Release from Growth Cones: Role of γ-Aminobutyric Acid Transport

Neuronal growth cones isolated in bulk from neonatal rat forebrain have uptake and K(+)-stimulated release mechanisms for gamma-aminobutyric acid (GABA). Up to and including postnatal day 5, the K(+)-stimulated release of [3H]GABA and endogenous GABA is Ca2+ independent. At these ages, isolated growth cones neither contain synaptic vesicles nor stain for synaptic vesicle antigens. Here we examined the possibility that the release mechanism underlying Ca2(+)-independent GABA release from isolated growth cones is by reversal of the plasma membrane GABA transporter. The effects of two GABA transporter inhibitors, nipecotic acid and an analogue of nipecotic acid, SKF 89976-A, on K(+)-stimulated release of [3H]GABA from superfused growth cones were examined. Nipecotic acid both stimulated basal [3H]GABA release and enhanced K(+)-stimulated release of [3H]GABA, which indicates that this agent can stimulate GABA release and is, therefore, not a useful inhibitor with which to test the role of the GABA transporter in K(+)-stimulated GABA release from growth cones. In contrast, SKF 89976-A profoundly depressed both basal and K(+)-stimulated [3H]GABA release. This occurred at similar concentrations at which uptake was blocked. These observations provide evidence for a major role of the GABA transporter in GABA release from neuronal growth cones.     

Biosynthesis of Neutral Glucocerebroside Homologues in the Absence of Myelin Assembly After Nerve Transection

The biosynthesis of myelin-associated glycolipids during various stages of myelination was studied by in vitro incorporation of [3H]Gal, [3H]Glc, or [35S]sulfate into the endoneurium of rat sciatic nerve. In the normal adult nerve, where the level of myelin assembly is substantially reduced and Schwann cells are principally involved in maintaining the existing myelin membrane, [3H]Gal was primarily incorporated into monogalactosyl diacylglycerol (MGDG) and the galactocerebrosides (GalCe) with lower levels of incorporation into the sulfatides. Such incorporation was enhanced 35 days after crush injury of the adult rat sciatic nerve, which is characterized by active myelin assembly. In contrast, at 35 days after permanent nerve transection where there is no axonal regeneration or myelin assembly, the incorporation of [3H]Gal or [3H]Glc into GalCe was nearly undetected whereas the incorporation of [3H]Gal into MGDG was completely inhibited. Instead, the 3H-labeled glycolipids in transected nerve were identified as the glucocerebrosides (GlcCe) and oligohexosylceramide derivatives with tetrahexosylceramide being a major product. In contrast, [35S]sulfate was incorporated into endoneurial sulfatides in the transected nerve, which suggests that endogenous GalCe rather than newly synthesized GalCe served as the substrate for the sulfotransferase reaction. The GlcCe homologues are not considered as constituents of the myelin membrane but are likely plasma membrane components synthesized in the absence of myelin assembly. It is likely that the cells responsible for GlcCe biosynthesis are Schwann cells, since they comprise 90% of the total endoneurial cell area in the distal nerve segment at 35 days after transection.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of GABA and beta-alanine transport and GABA membrane binding in the rat brain

It was found that rat brain nerve endings contain a high affinity and Na^- dependent transport system for [³H]β-alanine ([³H]β-ala). As determined from Michaelis-Menten plots, the [³H]β-ala K_m was 2.8 × 10^-5 M and the V_max was 0.29 nmol/mg protein/5 min. Under similar incubation conditions the [³H]GABA K_m was 3.8 x 10^-6M and the V_max was 6.3 nmol/mg protein/5 min. The [³H]β-ala and [³H]GABA transport systems were further characterized by determining the IC₅₀ values for a number of compounds. The compounds tested were GABA, β-ala, l -2,4-diaminobutyric acid. DL-3-hyd-roxy-GABA, β-guanidopropionic acid, strychnine, γ-guanidobutyric acid, imidazole-4-acetic acid, DL-proline, bicuculline, L-serine, glycine, l -α-ala and taurine. DABA, dl -3-hydroxy-GABA, β-guanidopro-pionic acid and γ-guanidobutyric acid were more potent inhibitors of [³H]GABA than [³H]β-ala transport. Strychnine, imidazole-4-acetic acid, proline and glycine were between 2 and 6 times more potent inhibitors of [³H]β-ala than [³H]GABA transport. β-Ala, bicuculline, serine, α-alanine and taurine were all markedly more potent (12–150 times) inhibitors of [³H]β-ala than [³H]GABA transport. IC₅₀ values were also determined for the above compounds for the sodium-dependent and the sodium-independent binding of [³H]GABA to both fresh and frozen brain membranes. In general, the potency of these compounds to inhibit either sodium-independent or sodium-dependent binding was greater in fresh tissue. It was also observed that the neurophysiologically‘glycine-like’amino acids were more potent inhibitors in the presence of NaCl. No significant correlations were found between [³H]GABA binding under any condition and [³H]GABA or [³H]β-ala transport into nerve endings.     

N-Terminal Arginylation of Sciatic Nerve and Brain Proteins Following Injury

N-terminal protein arginylation has been demonstrated in vitro and in situ and has been reported to increase following injury to sciatic nerves of rats. The present study attempts to demonstrate these reactions in vivo by applying [³H]Arg to the cut end of sciatic nerves in anesthetized rats and assaying for N-terminal arginylation using Edman chemistry and acid precipitation of labeled proteins in the proximal nerve segment. No evidence was found for arginylation in an aqueous soluble fraction. However, N-terminal arginylation was detected in a urea soluble fraction at 2 hours after nerve crush. The data show that arginylation of rat sciatic nerve proteins occurs in vivo and suggest that the arginylated proteins formed an aqueous insoluble/urea soluble aggregate after arginylation. In other experiments, rat brains were injured and assayed for arginylation in vitro to test the hypothesis that injury causes an up-regulation of these reactions. Results showed an activation of the reaction at 2 hours post crush and indicate that increases in N-terminal arginylation are likely to be a general response to injury in nervous tissue.     

Preferential release of newly synthesized [3H]GABA from striatal slices to preloaded [3H]GABA

The release of [³H]GABA which is newly synthesized from [³H]l-glutamic acid (GLU) has been examined using striatal slices obtained from the rat brain. It was found that 8–10% of [³H]GLU transported was converted to [³H]GABA during the incubation of striatal slices in the presence of nipecotic acid (5 × 10^?5 M). Nipecotic acid was added to the medium in order to prevent possible reuptake of [³H]GABA released during its synthesis, and it was found to have no significant effect on the formation of [³H]GABA from [³H]GLU as well as on the uptake of [³H]GLU. The application of high potassium (60 mM) stimulation exhibited a significant enhancement of the release of this newly synthesized [³H]GABA in a Ca²⁺ dependent manner. Kinetic analysis revealed that the evoked release of newly synthesized [³H]GABA was approximately two times greater than that of previously-loaded [³H]GABA, whereas no significant difference was observed in the spontaneous release. An immobilization stress in water failed to affect the release of newly synthesized [³H]GABA from striatal slices despite the occurrence of a significant enhancement of GABA formation in this structure.These results suggest that newly synthesized GABA may be preferentially released from its nerve terminals in response to the excitation of neurons at least in the striatum as compared with previously accumulated GABA.     

Efflux of Putative Transmitters from Superfused Rat Brain Slices Induced by Low Chloride Ion Concentrations

Slices of rat cerebral cortex, preloaded with [14C]gamma-aminobutyric acid (GABA) and either [3H]5-hydroxytryptamine (5-HT) or [3H]noradrenaline, were superfused with media in which varying concentrations of Cl- had been replaced with other monovalent anions. Rapid reduction of [Cl-], by superfusion with media containing instead the impermeant anions propionate, isethionate, gluconate, or methyl sulphate, caused increases in the efflux of tritiated biogenic amines, but the increase in that of [14C]-GABA was not significant. The increased efflux of [3H]5-HT evoked by superfusion with low Cl- levels when propionate was the replacement anion, was transient and was linearly related to the log[Cl-]-1. It was not affected by removal of Ca2+ or by addition of 10 mM Mg2+ and was delayed but not abolished by tetrodotoxin. The low Cl(-)-evoked efflux of [3H]5-HT was not affected by pretreatment with neuronal reuptake blockers but was inhibited by picrotoxin, strychnine, and 4-acetamido-4-isothiocyanostilbene-2,2-disulphonic acid and was enhanced by glycine. Muscimol and GABA were without effect. These observations are taken to indicate that the efflux of biogenic amines is brought about by terminal depolarisation due to outward movement of Cl- in low chloride-containing media. They are of relevance to other physiological and pharmacological studies in which anion concentrations are manipulated and suggest that the anion-evoked release phenomenon may provide a model for the analysis of Cl(-)-dependent mechanisms in nerve terminals.     

The Picrotoxinin Binding Site and Its Relationship to the GABA Receptor Complex

The binding characteristics of [3H] alpha-dihydropicrotoxinin to the picrotoxinin binding site were investigated in membrane preparations of adult rat forebrain and living cultures of rat cerebral cortex. The binding of [3H]alpha-dihydropicrotoxinin to rat forebrain was decreased by lysing, treating with Triton X-100, and heating. Coincubation with gamma-aminobutyric acid (GABA), benzodiazepines, or alterations in the Na+ or Cl- composition of the media had no effect on the binding to the rat brain preparation. However, in the living neurons in tissue culture both GABA and diazepam significantly decreased the binding of [3H]alpha-dihydropicrotoxinin. The dose-response relationships for GABA antagonism of [3H]alpha-dihydropicrotoxinin binding and for picrotoxinin antagonism of the GABA enhancement of [3H]flunitrazepam binding in cultured cortical neurons were also investigated. The Hill coefficients for these actions were reciprocal, suggesting that they result from complementary interactions between the binding sites for GABA and picrotoxinin. These data support the association of the picrotoxinin binding site with the postsynaptic GABA receptor complex.     

Pregnenolone sulfate induces NMDA receptor dependent release of dopamine from synaptic terminals in the striatum

Neuromodulators that alter the balance between lower-frequency glutamate-mediated excitatory and higher-frequency GABA-mediated inhibitory synaptic transmission are likely to participate in core mechanisms for CNS function and may contribute to the pathophysiology of neurological disorders such as schizophrenia and Alzheimer's disease. Pregnenolone sulfate (PS) modulates both ionotropic glutamate and GABA(A) receptor mediated synaptic transmission. The enzymes necessary for PS synthesis and degradation are found in brain tissue of several species including human and rat, and up to 5 nM PS has been detected in extracts of postmortem human brain. Here, we ask whether PS could modulate transmitter release from nerve terminals located in the striatum. Superfusion of a preparation of striatal nerve terminals comprised of mixed synaptosomes and synaptoneurosomes with brief-duration (2 min) pulses of 25 nM PS demonstrates that PS increases the release of newly accumulated [3H]dopamine ([3H]DA), but not [14C]glutamate or [3H]GABA, whereas pregnenolone is without effect. PS does not affect dopamine transporter (DAT) mediated uptake of [3H]DA, demonstrating that it specifically affects the transmitter release mechanism. The PS-induced [3H]DA release occurs via an NMDA receptor (NMDAR) dependent mechanism as it is blocked by D-2-amino-5-phosphonovaleric acid. PS modulates DA release with very high potency, significantly increasing [3H]DA release at PS concentrations as low as 25 pM. This first report of a selective direct enhancement of synaptosomal dopamine release by PS at picomolar concentrations via an NMDAR dependent mechanism raises the possibility that dopaminergic axon terminals may be a site of action for this neurosteroid.     

Activation of presynaptic ionotropic glutamate receptors stimulates GABA release from hippocampal and cortical nerve terminals in rats

One of the pathways implicated in a fine-tuning control of neurosecretory process is the activation of presynaptic receptors. The present study was focused on the role of presynaptic glutamate receptor activation in the regulation of inhibitory synaptic transmission in the rat hippocampus and cortex. We aimed to clarify what types of ionotropic glutamate receptors are involved in the modulation of GABA secretion, and what mechanism underlies this modulation. We have revealed that specific agonists of kainate and NMDA receptors, kainate and NMDA, like glutamate, induced the release of [3H]GABA from hippocampal and cortical nerve terminals suggesting the involvement of both types in the regulation of GABAergic transmission. Our results indicate preferential involvement of vesicular, but not cytosolic, pool in response to glutamate receptor activation. This is based on the finding that NO-711 (a specific inhibitor of plasma membrane GABA transporters), fails to attenuate [3H]GABA release. We have concluded that presynaptic glutamate receptor-induced modulation of the strength of synaptic response is due to increasing the release probability of synaptic vesicles.