A comparison of the effects of acute and one year's continuous neuroleptic treatment on the release of [3H]glutamate and [3H]acetylcholine from rat striatal slices

The effect of neuroleptic drugs administered acutely or continuously for 1 year on the release of [³H]glutamate and [³H]acetylcholine from striatal slices in vitro has been compared.Acute in vivo administration of haloperidol, trifluoperazine and clozapine increased the potassium-evoked release of [³H]acetylcholine from striatal slices in a dose-dependent fashion, whereas sulpiride was without effect. None of the neuroleptics given acutely had any effect on the potassium-evoked striatal release of [³H]glutamate.Potassium-evoked striatal release of [³H]acetylcholine in animals receiving 1 year's continuous administration of haloperidol, trifluoperazine or sulpiride was no different from that in age-matched control animals, but was less than controls in animals receiving clozapine for 1 year. All drugs caused a decrease in potassium-evoked striatal [³H]glutamate release following drug administration for 1 year compared to age-matched controls.The reversal of the acute action of neuroleptic drugs on striatal [³H]acetylcholine and [³H]glutamate release is consistent with a functional increase in striatal dopamine transmission following long-term neuroleptic treatment.     

Uptake and release of [3H]GABA by growth cones isolated from neonatal rat brain

A subcellular fraction highly enriched in neuronal growth cones was isolated from 5-day-old rat forebrain by a recently described method. The growth cone fraction was shown to have a sodium- and temperature-dependent, high-affinity (Km = 4.4 microM) uptake system for [3H]GABA. Electron microscopic autoradiography confirmed that this uptake was into growth cones since only these structures were heavily labelled with silver grains. High potassium induced the release of newly accumulated [3H]GABA from the growth cone fraction, about half of which was Ca2+-dependent. The presence of uptake and release systems for GABA in growth cones may simply reflect the development of growth cones into nerve terminals. Alternatively, these observations may indicate a role for neurotransmitter release in synaptogenesis.     

A possible neuronal release of [14C] GABA from the rat cerebellum in vivo

Release patterns for exogenously applied [¹⁴C] labelled -amino-n-butyric-acid (GABA) have been investigated in rat cerebellar cortex in vivo. An increase in [¹⁴C] GABA release could be evoked by stimulating with high (40 mM) K⁺ or veratridine (10^–4 M) but not with direct electrical stimulation. Biphasic patterns for high K⁺ and possibly veratridine stimulated release of GABA suggest the existence of two separate anatomical sources of isotope which are sensitive to these depolarising stimuli. Both K⁺ and veratridine-evoked GABA release are calcium dependent. Studies involving partial replacement of Na⁺ with HEPES, (N-2-hydroxyethyl-piperazine-N-2-ethane-sulphonic acid), sucrose or choline chloride also reveal a sodium dependency of [¹⁴C] GABA release. These studies collectively indicate a neuronal source for evoked GABA release, a criterion for transmitter identification not previously satisfied in the cerebellar cortex.Supported in part by a grant from S.R.C. to N.D. and C.G.D.Supported by CAPES and FAPESP (Brazil)Supported by CNPq and FAPESP (Brazil)     

gamma-Aminobutyric acid and taurine release in the striatum of the rat during hypoglycemic coma, studied by microdialysis

Extracellular levels of striatal gamma-aminobutyric acid (GABA) and taurine were monitored during insulin-induced hypoglycemia using microdialysis. At the onset of isoelectricity in the electroencephalogram (EEG), a transient 5-fold increase in the levels of GABA occurred. Taurine levels increased 5 min following the onset of isoelectricity and continued to increase during the entire isoelectric period. The results demonstrate that events associated with the onset of isoelectricity during hypoglycemia trigger an increase in extracellular concentrations of GABA and taurine. The discrepancy in time-course of these changes may reflect differences in compartmentation, function and metabolism of the two amino acids.     

Autoradiographic localization of binding sites for [3H]gamma-aminobutyrate, [3H]muscimol,(+)[3H]bicuculline methiodide and [3H] flunitrazepam in cultures of rat cerebellum and spinal cord

Cultures of rat cerebellum and spinal cord were used to visualize binding sites for [³Hγaminobutyrate, [³Hmuscimol, [³Hbicuculline methiodide and [³Hflunitrazepam by autoradiography. In cerebellar cultures, many large neurons (presumably Purkinje cells) and interneurons were labelled. In spinal cord cultures, these compounds were mainly bound to small and medium-sized neurons, whereas the majority of large neurons were unlabelled. No binding sites for these radioligands were found on glial cells. Binding of [³Hγ-aminobutyrate, [³Hmuscimol and [³Hbicuculline methiodide was markedly reduced or inhibited by adding unlabelled γ-aminobutyrate, muscimol and bicuculline (10^?3m) respectively to the incubation medium. Addition of a thienobenzazepine markedly reduced binding with [³Hflunitrazepam.It is concluded that tissue cultures are an excellent tool to visualize the cellular localization of binding sites for neurotransmitters and drugs using autoradiography.     

Effect of DN-1417, a synthetic thyrotropin-releasing hormone analogue, on [3H]GABA binding in the cerebellum of ataxic rats

The neonatal administration of cytocine arabinoside (40 mg/kg, s.c.) induced marked ataxia and hypoplastic cerebellum in male Sprague-Dawley rats. Specific [3H]GABA binding in the cerebellum was significantly reduced by cytosine arabinoside, whereas [3H]GABA binding in the cerebral cortex was not changed. The administration of DN-1417 (1 mg/100 g body wt., i.p.), a synthetic derivative of thyrotropin-releasing hormone (TRH), significantly increased [3H]GABA binding in the cerebellum of cytosine arabinoside-induced ataxic rats. These results suggest that TRH may play a role in regulating GABA receptors involved in the function of the rat cerebellum.     

Pharmacological and ionic features of gamma-aminobutyric acid receptors influencing electrical properties of melanotrophs isolated from the rat pars intermedia

Characteristics of the gamma-aminobutyric acid receptors on melanotrophs of the rat pars intermedia were studied by intracellular recording. Muscimol and 3-amino-1-propanesulfonic acid, but not baclofen or glycine, mimicked the depolarization and conductance increase produced by gamma-aminobutyric acid on the melanotrophs. These effects appeared to be due to an increase in chloride ion conductance since the null potentials for all three drugs were the same and were affected by changes in external or internal chloride ion concentration but not by changes in the concentrations of other ions present in the recording solution or by the addition of the calcium-channel blocker cobalt. Bicuculline abolished the effect of muscimol. Picrotoxin reduced the effect of gamma-aminobutyric acid; so too did furosemide. Muscimol mimicked the ability of gamma-aminobutyric acid to reduce the depolarization produced by excess potassium and this effect was also blocked by bicuculline. Rat melanotrophs thus appear to possess gamma-aminobutyric acid receptor-ionophore complexes similar to the classical sort found in neurons in the mammalian central nervous system. Furthermore, the parallels between the electrical responses observed and secretory effects previously noted, reinforce the view that electrical activity may participate in stimulus-secretion coupling in melanotrophs.     

Role of presynaptic dopamine receptors in regulation of the glutamatergic neurotransmission in rat neostriatum

In experiments with the use of a push-pull cannula and simultaneous recording of electrical activity at the site of perfusion, the release of L-[3H]glutamic acid from rat neostriatum induced by K+-depolarization (60 mM K+ in perfusate) has been shown to be inhibited by replacing Ca2+ in the perfusion medium by Co2+. In contrast, release of L-[3H]glutamate induced by electrical stimulation of frontal cortex is enhanced by replacement of these cations. Application of dopamine (10(-5)-10(-3) M). apomorphine (10(-4) M) or beta-phenylethylamine (10(-3) M) as well as stimulation of the substantia nigra enhanced the basal release of L-[3H]glutamate. Haloperidol (10(-4) M) completely abolished the effects of apomorphine and beta-phenylethylamine, and partially abolished the effect of dopamine. The enhancement induced by apomorphine is strongly dependent on the presence of Na+ in the perfusion medium. On the other hand, apomorphine (10(-4) M) and beta-phenylethylamine (10(-3) M) inhibited the release of glutamate induced by electrical stimulation of the frontal cortex and that by K+-depolarization (the latter was shown for apomorphine). This inhibition is also abolished by haloperidol. A possible functional role of endogenous dopamine in the regulation of glutamatergic neurotransmission in rat neostriatum is discussed.     

The effect of some putative neurotransmitters on the release of 5-hydroxytryptamine and gamma-aminobutyric acid from slices of the rat midbrain raphe area.

The effect of various putative transmitters has been studied on the efflux of [³H]5-hydroxytryptamine and [³H]γ-aminobutyric acid from small superfused slices of the midbrain raphe area of the rat. The slices apparently contained functionally intact terminals for these transmitters since a depolarizing stimulus (50 mM KCl) stimulated the rate of efflux of [³H]5-hydroxytryptamine and [³H]γ-aminobutyric acid in a calcium-dependent fashion. Furthermore, slices accumulated radioactivity with apparent high affinity mechanisms. γ-Aminobutyric acid (50 and 100 μ m) and substance P (100 and 500μ m) stimulated the efflux of [³H]5-hydroxytryptamine. The effect of γ-aminobutyrate was blocked by picrotoxin (50 μ m) but not by strychnine (1 μ m). Other inhibitory amino acids (β-alanine, taurine and glycine) were without effect on the release of [³H]5-hydroxytryptamine. l-Noradrenaline (0.2–1 mM) stimulated the efflux of [³H]γ-aminobutyric acid but not that of [3H]5-hydroxytryptamine. Phentolamine (10μ m) but not (±)-propranolol (10 μ m) abolished the effect of 1 mM noradrenaline. Neither dopamine nor 5-hydroxytryptamine influenced the efflux of [3H]γ-aminobutyric acid.It is possible that interactions in the midbrain raphe area proposed from other studies can be mediated through presynaptic influences on transmitter release.     

Further characterization of [3H]gamma-aminobutyric acid release from isolated neuronal growth cones: role of intracellular Ca2+ stores

We have recently shown that growth cones isolated from neonatal rat forebrain possess uptake and release mechanisms for the neurotransmitter gamma-aminobutyric acid. About half of the K+-induced release of [3H]gamma-aminobutyric acid from isolated growth cones is dependent on extracellular Ca2+. The remaining component of the [3H]gamma-aminobutyric acid release is unaffected by removal of extracellular Ca2+ and is resistant to blockade by the voltage-sensitive Ca2+-channel blocker methoxyverapamil. In the present series of experiments we have used caffeine to assess the possible role of intracellular stores of Ca2+ in supporting that component of the K+-induced release of [3H]gamma-aminobutyric acid from isolated growth cones that is independent of extracellular Ca2+. We have chosen caffeine because of its well established effect of releasing Ca2+ from smooth endoplasmic reticulum in muscle. We found that caffeine can release [3H]gamma-aminobutyric acid from isolated growth cones. This effect persists in Ca2+-free medium, in the presence of methoxyverapamil and in the absence of Na+. Furthermore, isobutylmethylxanthine could not substitute for caffeine suggesting that the caffeine effect is not due to phosphodiesterase inhibition and the subsequent rise in intracellular cyclic nucleotides. A combination of the mitochondrial poisons, Antimycin A and sodium azide had no effect on the release of [3H]gamma-aminobutyric acid induced either by caffeine or by high K+. We conclude that caffeine causes the release of Ca2+ from a non-mitochondrial store within the growth cone and that this Ca2+ store supports that component of the K+-induced release of [3H]gamma-aminobutyric acid that is independent of extracellular Ca2+.     

gamma-Aminobutyric acid decreases levels of messenger ribonucleic acid encoding prolactin in the rat pituitary

The effect of gamma-aminobutyric acid (GABA) on levels of messenger ribonucleic acid (mRNA) encoding prolactin (PRL) was studied in cultured anterior pituitary cells, in vitro and in intact rats, in vivo. As quantitated by hybridization to a 32P-labeled rat PRL complementary deoxyribonucleic acid (cDNA) probe, levels of PRL mRNA in cultured pituitary cells were decreased by about 50% following 3 days exposure to 10(-5) M GABA. This effect was mimicked by muscimol (10(-6) M) and antagonized by bicuculline (10(-5) M). An increase of endogenous GABA levels in vivo effected by injection of GABA transaminase blockers (aminooxyacetic acid, 20 mg/kg, twice daily; vinyl GABA, 800 mg/kg) into rats resulted in a similar decrease in rat PRL mRNA levels in the adenohypophysis 3-4 days following commencement of the drug treatment. These findings suggest that GABA might inhibit PRL gene expression by a direct action on lactotrophs of the adenohypophysis.     

Release of D-[3H]aspartate from the dorsolateral septum after electrical stimulation of the fimbria in vitro

A new in vitro preparation from guinea-pig brain is described. The preparation consists of a slice of dorsolateral septum with adhering fimbria. The viability of the preparation was demonstrated. Fibre volleys, single and multiple unit discharges and evoked postsynaptic field potentials could be detected essentially as in vivo. Electrical stimulation of the CA3 hippocampal-septal excitatory connections through the fimbria releasedd-[³H]aspartate, as a marker forl-glutamate, from the dorsolateral septum. The release was specific. The presence of presynaptic fibre volleys at both high (1.5 mm) and low (0.1 mm) concentrations of Ca²⁺, but efflux ofd-[³H]aspartate only at high Ca²⁺ concentrations strongly favour a transmitter-related release.The results strongly suggest that two axon collaterals from the same hippocampal CA3 pyramidal cells usel-glutamate as neurotransmitter.     

Cellular localization of the uptake of [3H]taurine and [3H]β-alanine in cultures of the rat central nervous system

The cellular localization of the uptake of [³H]taurine and [³H]β-alanine was studied in cultures of rat central nervous system using autoradiography. In brain stem and spinal cord cultures, both amino acids were taken up by neurones and glial cells. In cerebellar cultures, [³H]taurine was accumulated by all glial cells and by a small number of neurones, whereas [³H]β-alanine was only taken up by glial elements. The uptake of both amino acids was sodium and temperature dependent, indicating an active transport mechanism.The results provide further support for the hypothesis that taurine and β-alanine are neurotransmitters in the mammalian central nervous system.     

Antagonistic effect of delta-aminovaleric acid on bicuculline-insensitive gamma-aminobutyric acid B (GABA B) sites in the rat's brain

The effect of delta-aminovaleric acid (delta-AV) on bicuculline-insensitive gamma-aminobutyric acid B (GABA B) sites in the central nervous system (CNS) was investigated by binding studies and experiments on slices in vitro. delta-AV inhibited [3H]GABA (10 nM) binding to GABA B sites in a rat brain membrane preparation with an IC50 value of 10(-4) M. It also inhibited [3H]baclofen (20 nM) binding with an IC50 value of 10(-4) M. In preparations of hippocampal slices, (-)-baclofen (5 microM) reduced the population spikes evoked by stimulating the Schaffer collaterals in CA1 pyramidal cells in the presence of 100 microM bicuculline. delta-AV (1 mM) antagonized this inhibitory action of baclofen. Since baclofen is an agonist of GABAB sites, our results indicate that delta-AV has an antagonistic effect on GABAB sites in the CNS.     

Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors modulate release of inhibitory amino acids in rat spinal cord dorsal horn

Local inhibition within the spinal cord dorsal horn is mediated by the neurotransmitters GABA and glycine and strongly influences nociceptive and temperature signaling. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are expressed by inhibitory interneurons and have been shown to modulate GABA release in other regions of the CNS. In the spinal cord, there is morphological evidence for presynaptic AMPA receptor subunits in GABAergic dorsal horn neurons, but functional data are lacking. To determine if AMPA receptors are indeed functional at presynaptic terminals of inhibitory neurons, we recorded evoked and miniature inhibitory postsynaptic currents (mIPSPs) in the superficial dorsal horn of the rat spinal cord. We show that AMPA receptor activation enhances spontaneous release of inhibitory amino acids in the presence of tetrodotoxin onto both lamina II neurons and NK1 receptor-expressing (NK1R+) lamina I neurons. This effect is sensitive to the concentration of extracellular Ca2+, yet is not fully blocked in most neurons in the presence of Cd2+, suggesting possible Ca2+ entry through AMPA receptors. Postsynaptic Ca2+ elevation is not required for these changes. AMPA-induced increases in mIPSP frequency are also seen in more mature dorsal horn neurons, indicating that these receptors may play a role in nociceptive processing in the adult. In addition, we have observed AMPA-induced depression of evoked release of GABA and glycine onto lamina I NK1R+ neurons. Taken together these data support a role for presynaptic AMPA receptors in modulating release of GABA and glycine in the superficial dorsal horn. Because inhibition in the dorsal horn is important for controlling pain signaling, presynaptic AMPA receptors acting to modulate the inhibitory inputs onto dorsal horn neurons would be expected to impact upon pain signaling in the spinal cord dorsal horn.     

In vivo [14C]amino acid profiles in discrete hypothalamic regions during push-pull perfusion in the unrestrained rat.

In the unanesthetized rat, the differential release of [¹⁴C]amino acids into a perfusate from a push-pull cannula was analyzed for 20 circumscribed areas of the hypothalamus. To serve as an exogenous precursor for [¹⁴C]amino acid synthesis. [U-¹⁴C]glucose was injected in a vol of 0.5–1.0 μl into a discrete hypothalamic region 20 min prior to a push-pull perfusion. Then, at 15min intervals, the labeled site was perfused for 5 min with an artificial cerebrospinal fluid usually at a rate of 10μl per min. Each sample of perfusate was extracted and assayed by two-dimensional, thin-layer chromatography for the presence of eight amino acids and residual glucose.The pattern of recovery of ¹⁴C-labeled substances as well as the distribution of ¹⁴C between γ-aminobutyric acid, glutamate, aspartate, alanine, taurine and glycine depended solely upon the neuroanatomical region perfused. Near the lateral edge of the ventromedial nucleus, 67% of the amino acid activity was accounted for by [¹⁴C]GABA. Perfusion sites medial to or within this nucleus, or others near the anterior hypothalamus or paraventricular nucleus, yielded a recovery of labeled GABA that was equal to that of glutamate, together accounting for two-thirds of the total ¹⁴C recovery. Within many perfusion loci, small amounts of [¹⁴C]alanine and [¹⁴C]aspartate were detected, whereas within most sites the respective recovery of [¹⁴C]aspartate, [¹⁴C]alanine, [¹⁴C]taurine and [¹⁴C]glycine was negligible. Finally, the metabolism of [¹⁴C]amino acids in the rat's hypothalamus decreased exponentially over time with little identifiable content obtained 80 min following injection of the glucose precursor.These results demonstrate that putative amino acid transmitters can be recovered from the brain of a freely moving animal, and can be characterized in vivo within specific neuroanatomical regions.     

Neural elements containing glutamic acid decarboxylase (GAD) in the dorsal lateral geniculate nucleus of the rat; immunohistochemical studies by light and electron microscopy

Immunoreactive constituents of the dorsal lateral geniculate nucleus of adult albino rats were examined by light- and electron-microscopy, using the unlabelled antibody enzyme method, following treatment of brain slices with a purified antibody to glutamic acid decarboxylase. The neuropil of the dorsal lateral geniculate nucleus displayed a conspicuous granular immunoreactivity. In addition, the antibody was bound to a class of small neurons of characteristic morphology. These cells possessed few (commonly 2-4) sparsely branched, long dendrites from some of which immunoreactive appendages were traced. Many cells were bipolar in form, and the dendrites of some appeared to be preferentially orientated. The immunoreactive cells closely resembled intrinsic interneurons characterized in previous Golgi studies of this nucleus. By electron-microscopy, immunoreactive presynaptic elements were present both in the extraglomerular neuropil and in the synaptic glomeruli. The former were axon terminals containing flattened synaptic vesicles and making Gray type II axo-dendritic synaptic contact; they appeared to correspond to axon terminals whose origin in the thalamic reticular nucleus has been established in previous studies, but it is possible that some were axon terminals of intrinsic interneurons. The immunoreactive glomerular components also contained flattened vesicles, were presynaptic to presumptive projection cell dendrites, postsynaptic to retinal axon terminals, and participated in triplet (triadic) and other complex synaptic arrangements. They corresponded in all respects to the synaptic portions of the complex dendritic appendages of intrinsic interneurons, identified and characterized in previous studies. The finding that there are high levels of glutamic acid decarboxylase in the cell bodies, dendritic shafts and dendritic appendages of intrinsic interneurons in the dorsal lateral geniculate nucleus of the rat, and in the axon terminals of fibres projecting to this site from the thalamic reticular nucleus, allows us to conclude that the inhibitory inputs to the geniculo-cortical projection cells from both of these sources are probably mediated by gamma-aminobutyric acid.     

Inactivation by 6-hydroxydopamine of the cerebral factor(s) responsible for the inhibition of the autoxidation of norepinephrine in vitro

The effect of extracts from rat cerebral cortex was examined on the stability of norepinephrine-HC1 (NE) in 16 mM Na-phosphate buffer, pH 7.4, at 37 degrees C. The autoxidation products of NE were detected spectrophotometrically at 480 nm. Dialysed samples from a synaptosomal preparation and from the 100,000 g supernatant of a crude homogenate were tested. Aliquots from these preparations, in the range of 0.005-5.0 or 0.01-10.0 micrograms protein/ml, respectively, produced up to 80-85% inhibition of the autoxidation of 100 microM NE for a period of at least 3 h. Similar results were obtained with albumin and ovalbumin at 10- and 10(3)-times higher concentrations, respectively. After the preparations were exposed to 0.1-1.0 mg 6-hydroxydopamine-HC1/mg protein for 5 min at 25 degrees C followed by rapid dialysis, the maximal inhibitory effect was reduced to between 95% to less than 5% of control values. The percent inactivation by a given quantity of 6-hydroxydopamine (6-OHDA) was inversely related to the potency of the untreated sample. Additional observations are presented which suggest that the destruction of the antioxidant activity is caused by breakdown products of 6-OHDA reacting with nucleophilic sites of the preparation. Similar inactivating substances are expected to be formed from other autoxidizing catecholamines, although at a slower rate.     

Effects of somatostatin-14 on the in vitro release of [3H]GABA from slices of rat caudatoputamen

Slices (300 microns) of rat caudatoputamen were incubated in Krebs-Henseleit medium and loaded with [3H]glutamine, part of which was converted to [3H]GABA. This conversion takes place only in GABA-neurons most of which probably contribute to the striatonigral pathway. After a 24 min equilibration period, release of radioactivity was stimulated with veratridine (3.1-4 mumol/l) or K+ (15-25 mmol/l) in the absence or presence of somatostatin-14. From the radioactivity released [3H]GABA was separated by cationic exchange chromatography and measured. Somatostatin-14 affected the release of [3H]GABA in a manner which depended on its concentration as well as on the extent of stimulus-evoked release. Somatostatin-14 (1 nmol/l) enhanced the moderate release (2-4% of tissue content) elicited by veratridine (3.1 mumol/l) or K+ (20 mmol/l), but had no effect on the more pronounced release (5-8% of tissue content) elicited by veratridine (4 mumol/l) or K+ (25 mmol/l). Somatostatin-14 (10 nmol/l) had no effect on the moderate release of [3H]GABA, but diminished the pronounced one. Further experiments provided evidence that the somatostatin-14-induced enhancement was not brought about by a direct action on GABA-neurons but was probably indirect, i.e. mediated by other striatal neurons. In contrast, the diminution of the release of [3H]GABA caused by somatostatin-14 may be due to its direct action on releasing neurons. Two antisera against somatostatin lowered the pronounced release indicating that endogenous somatostatin may also enhance the release of [3H]GABA. In addition, endogenous somatostatin seems also to be able to diminish the release under certain experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)