Effects of d-aspartate on excitatory amino acid-induced release of [H]GABA from goldfish retina

The rate of release of [³H]GABA from isolated intact goldfish retinas was studied. Release of [³H]GABA is markedly stimulated by the inclusion in the incubation medium of the photoreceptor neurotransmitter candidates l-glutamate (l-Glu) and l-aspartate (l-Asp), and the glutamate analogs, kainate and quisqualate. At micromolar concentrations, kainate and quisqualate are effective releasers of [³H]GABA, whereas millimolar concentrations of l-Glu and l-Asp are required to release comparable amounts of [³H]GABA. The d-isomers of aspartate (d-Asp) and glutamate (d-Glu) are able to release [³H]GABA, but only when applied at high concentrations (3–30 mM). In the presence of 5 mM d-Asp, the effect of l-Glu in releasing [³H]GABA was markedly potentiated. This dose-response curve of l-Glu was shifted to the left in the presence of d-Asp, although the maximal amount of release was unchanged. d-Asp at 5 mM only slightly increased the GABA release induced by quisqualate, and it did not increase the GABA release induced by kainate. Finally, low concentrations of l-Asp were potentiated by d-Asp, but higher concentrations of l-Asp (3–10 mM) were clearly inhibited by this agent. This biphasic effect of d-Asp on l-Asp-induced release of [³H]GABA is a possible explanation for previously conflicting reports of d-Asp's effect on l-Asp action^2,8,29. Our data suggest that d-Asp has both pre- and postsynaptic sites of action.     

Effect of oxidative stress on the release of [H]GABA in cultured chick retina cells

The effect of ascorbate (1.5 mM)/Fe²⁺ (7.5 μM)-induced oxidative stress on the release of pre-accumulated [³H]γ-aminobutyric acid ([³H]GABA) from cultured chick retina cells was studied. Depolarization of control cells with 50 mM K⁺ increased the release of [³H]GABA by 1.01 ± 0.16% and 2.5 ± 0.3% of the total, in the absence and in the presence of Ca²⁺, respectively. Lipid peroxidation increased the release of [³H]GABA to 2.07 ± 0.31% and 3.6 ± 0.39% of the total, in Ca²⁺-free or in Ca²⁺-containing media, respectively. The inhibitor of the GABA carrier, 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711) blocked almost completely the release of [³H]GABA due to K⁺-depolarization in the absence of Ca²⁺, but only 65% of the release occurring in the presence of Ca²⁺ in control and peroxidized cells. Under oxidative stress retina cells release more [³H]GABA than control cells, being the Ca²⁺-independent mechanism, mediated by the reversal of the Na⁺/GABA carrier, the most affected. MK-801 (1 μM), a non-competitive antagonist of the NMDA receptor-channel complex, blocked by 80% the release of [³H]GABA in peroxidized cells, whereas in control cells the inhibitory effect was of 40%. The non-selective blocker of the non-NMDA glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), inhibited the release of [³H]GABA by 30% and 70% in control and peroxidized cells, respectively. Glycine (5 μM) stimulated [³H]GABA release evoked by 50 mM K⁺-depolarization in control but not in peroxidized cells. The release of -[³H]aspartate (a non-metabolized analog of -glutamate) evoked by 50 mM K⁺, in the absence of Ca²⁺, was significantly higher in peroxidized cells (6.76 ± 0.64% of the total) than in control cells (3.79 ± 0.27% of the total). The results suggest that oxidative stress induced by ascorbate/Fe²⁺ causes an excessive release of endogenous excitatory amino acids upon K⁺-depolarization. The glutamate released may activate NMDA and non-NMDA receptors, raising the intracellular Na⁺ concentration and consequently stimulating the release of [³H]GABA by reversal of the Na⁺/GABA carrier.     

Glutamate increases the [Ca]i but stimulates Ca-independent release of [H]GABA in cultured chick retina cells

The effect of glutamate of [Ca²⁺]_i and on [³H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca²⁺]_i by Ca²⁺ influx through Ca²⁺ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca²⁺ channel blocker. Mobilization of Ca²⁺ by glutamate required the presence of external Na⁺, suggesting that Na⁺ mobilization through the ionotropic glutamate receptors is necessary for the Ca²⁺ channels to open. The increase in [Ca²⁺]_i was not related to the release of [³H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca²⁺ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [³H]GABA was significantly depressed by Ca_o²⁺, but it was dependent on Na_o⁺, just as was observed for the [³H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [³H]GABA release. Both veratridine- and glutamate-induced [³H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [³H]GABA evoked by glutamate. Continuous depolarization with 50 mM K⁺ induced maximal release of [³H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [³H]GABA release induced by 50 mM K⁺, and this effect was blocked by MK-801, suggesting that the effect of K⁺ on [³H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K⁺ depolarization. We conclude that glutamate induces Ca²⁺-independent release of [³H]GABA through reversal of the GABA carrier due to Na⁺ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca²⁺ entering by the pathways open by glutamate. This Ca²⁺ does not lead to exocytosis, probably because the Ca²⁺ channels used are located at sites far from the active zones.     

Characterization of the excitatory amino acid receptor-mediated release of [H]acetylcholine from rat striatal slices

The pharmacological nature of the interaction of excitatory amino acids with striatal cholinergic neurons was investigated in vitro. Agonists of excitatory amino acid receptors evoked the release of [³H]acetylcholine from slices of rat striatum, in the presence of magnesium (1.2 mM). Removal of magnesium from the medium markedly increased the release of [³H]acetylcholine evoked by all excitatory amino acid receptor agonists tested, with the exception of kainate. In the absence but not the presence of magnesium, a clear rank order of potency was found: N-methyl-dl-aspartate = ibotenate >l-glutamate >l-aspartate cysteate > kainate = quisqualate.The excitatory amino acid receptor mediating [³H]acetylcholine release resembles the N-methyl-d-aspartate preferring (N-type) receptor, as previously characterized electrophysiologically, according to 3 criteria: (1) rank order of potency of agonists; (2) magnesium-sensitivity; and (3) antagonism by 2-amino-5-phosphonovalerate.The release of [³H]acetylcholine evoked by N-methyl-dl-aspartate was blocked by tetrodotoxin (0.5 μM). Moreover, N-methyl-dl-aspartate failed to evoke [³H]acetylcholine release from slices of hippocampus, where cholinergic afferents, rather than interneurons, are found. These results suggest that excitatory amino acids act at receptors on the dendrites of striatal cholinergic interneurons, giving rise to action potentials and release of acetylcholine from cholinergic nerve terminals.     

Evoked efflux of [H]GABA from goldfish retina in the dark

H1 horizontal cells of goldfish retina probably are GABAergic and receive synaptic excitation from red cone photoreceptors in the dark. This study was designed to detect efflux of [³H]GABA from H1 cells by a physiological stimulus in order to obtain information regarding the identity of the red cone transmitter and obtain information on the role of dopaminergic interplexiform cells (DA-IPCs), the other synaptic input to H1 cells. Efflux of [³H]GABA was studied by biochemical analysis of perfused isolated retinas. Retinas were incubated in dim red light for 15 min in 0.72 μM [³H]GABA, rinsed for 30 min in red light and subjected to darkness under a variety of conditions. Radioactivity in the perfusate was determined by liquid scintillation spectroscopy. The findings are: 1. both l-glutamate and l-aspartate cause a dose-dependent efflux of [³H]GABA from H1 cells, 2. inclusion of 3.2 mM d-aspartate in the perfusion medium potentiates l-glutamate and totally inhibits l-aspartate, 3. retinas perfused in the standard Ringer do not show increased [³H]GABA efflux when placed in the dark, 4. when 3.2 mM d-aspartate is in the perfusion medium, there is significant dark-induced [³H]GABA efflux which is reduced with light onset, 5. 100 μM dopamine inhibits the dark-induced efflux of [³H]GABA. These results show that efflux of [³H]GABA from H1 cells can be detected under physiological conditions strongly suggesting that the H1 cell is GABAergic and, in addition, is subjected to antagonistic inputs from red cones and DA-IPCs. Furthermore, since d-aspartate potentiates l-glutamate and inhibits l-aspartate, and is required for the detection of dark-induced efflux of [³H]GABA, it is unlikely that the transmitter for red cones is l-aspartate but more likely is l-glutamate.     

GABA and benzodiazepine-induced modification of [C]l-glutamic acid release from rat spinal cord slices

The spontaneous and potassium-evoked release of [¹⁴C]-label from rat spinal cord slices preloaded with [¹⁴C]l-glutamic acid and its modification by GABA and related drugs, such as flurazepam, was studied as a possible indirect measure of presynaptic inhibition and of the ability of benzodiazepines to augment it. GABA (100 μM) reduced the spontaneous release of [¹⁴C]-label (glutamate) provided that GABA metabolism was blocked by amino-oxyacetic acid (AOAA), but failed to reduce the potassium-evoked release of glutamate, although muscimol (10 μM) had some effect. In contrast, flurazepam (1–100 μM) did not affect spontaneous release but produced some inhibition of the evoked release (through a system insensitive to 10 μM bicuculline). This inhibition became more marked in the presence of both GABA and AOAA, and was then overcome by bicuculline. It is concluded that either some benzodiazeophine receptors must be occupied for GABA to produce an effect on evoked release and/or, that the benzodiazepines can only augment GABA function once a certain amount has been released. Studies of the rapid distribution of [¹⁴C]-label from glutamate, to GABA, glutamine and other amino acids, using high voltage electrophoresis, showed the importance of blocking metabolic pathways in studies of this kind.     

Forskolin attenuates the evoked release of [H]GABA from striatal neurons in primary culture

The actions of the diterpene forskolin, and cyclic AMP analogues, on the evoked release of [³H]GABA (γ-aminobutyric acid) was examined in intact striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to forskolin (100 μM) resulted in a 40–55% attenuation of [³H]GABA release evoked by either KCl (30 mM) or veratrine (2 μg/ml), while baseline levels of release were unaffected. The dose-dependence for forskolin in striatal neurons. Exposure of striatal neurons to membrane-permeable identical to the dose-dependent elevation of cyclic AMP levels by forskolin in striatal neurons. Exposure of striatal neurons to membrane-permeable analogues of cyclic AMP, such as p-chlorophenylthio cyclic AMP (0.5 mM) and dibutyryl cyclic AMP (1 mM), resulted in a 25 and 26% attenuation of [³H]GABA release, respectively; dibutyryl cyclic GMP (1 mM) was without effect. The similarity between the actions of forskolin and the cyclic AMP analogues suggests that, in striatal neurons in primary culture, the elevation of cyclic AMP levels results in the attenuation of the evoked release of [³H]GABA. The greater effectiveness of forskolin, compared to the cyclic AMP analogues, may be related to the recently reported, additional direct actions of forskolin on neuronal membrane ion channels.     

Effects of glycine and GABA on the ganglion cells of the retina of the skate Raja erinacea

Application of glycine and GABA caused inhibition of light-evoked activity from different populations of ‘ON’ center ganglion cells in the retina of the skate (Raja erinacea). Cells affected by glycine were not affected by GABA and vice-versa. Addition of 100 μM strychnine to the glycine-containing perfusate caused a resumption in light-evoked responses of the cells. The action of GABA was antagonized by 100 μM bicuculline methochloride, but not by picrotoxin. Autoradiography revealed the presence of [³H]glycine accumulating amacrine cells.     

l-Glutamate evoked release of GABA from cultured avian retina cells does not require glutamate receptor activation

γ-Aminobutyric acid (GABA) and l-glutamate are the major inhibitory and excitatory transmitters in the central nervous system. Recent evidence has indicated that l-glutamate may stimulate GABA release by a novel exchange mechanism (Nascimento and De Mello, J. Neurochem., 1985, 45: 1820–1827). Here we provide strong support for this hypothesis by showing that the l-glutamate-evoked release of [³H]GABA from cultured avian retina cells is not dependent on the activation of excitatory amino acid receptors. Retina cells were found to incorporate [³H]GABA into a pool that was released when cultures were treated with l-glutamate (100 μM). This release was unaffected when calcium ions were removed, but was prevented when NaCl was replaced by LiCl. d-Aspartate, which in tracer experiments was shown to be taken into cells by the same carrier as l-glutamate, was also able to evoke release of [³H]GABA, with the same requirement for NaCl. In addition, l-glutamate and d-aspartate uptake by retina cells was inhibited in more then 80% when the uptake was measured in the presence of LiCl. As opposed to GABA, the release of acetylcholine (ACh) promoted by l-glutamate showed characteristics of classical mechanisms of neurotransmitter release. Glutamate-induced efflux of ACh was Ca²⁺-dependent and was not affected when NaCl was replaced by LiCl. Also, d-aspartate was ineffective in eliciting the release of ACh. Even at high concentrations, antagonists of excitatory amino acid receptors were unable to diminish the glutamate-evoked release of [³H]GABA. The antagonists tested were: 2-amino-5-phosphonovalerate and d-α-aminoadipate, which are relatively selective for N-methyl-d-aspartate receptors; l-glutamate diethyl ester, which selectively blocks quisqualate sites, and cis-2,3-piperidine dicarboxylate, w which non-selectively antagonizes all types of excitatory amino acid receptors. The data show that excitatory amino acid receptors are not involved in the Na-dependent l-glutamate-evoked release of [³H]GABA and support the concept that a glutamate-GABA exchange mechanism operates in the central nervous system. Since glutamate and GABA exert antagonistic effects on the electrophysiology of nerve cells, this mechanism might be important in regulating neuronal excitability.     

Nigroamygdaloid dopamine neurons: Nigral modulation of their activity

In order to study the mechanisms regulating the dopaminergic nigroamygdaloid cells, the release of dopamine was observed in the central nucleus of the amygdaloid complex. Halothane anesthetized rats were implanted, according to the experiment, with one or two push-pull cannulae in the central nuclei of the amygdala (ACE), the substantia nigra (SN) and/or the caudate nucleus (CN). Cannulae were supplied with artificial cerebrospinal fluid (CSF) containing tritiated tyrosine, and labeled dopamine [³H]DA was evaluated in successive superfusate fractions. Electrical stimulation of the medial forebrain bundle with an implanted bipolar electrode induced an increase of the [³H]DA release in the ipsi- and contralateral ACE. Electrical stimulation of the SN produced only a very delayed effect in the ipsilateral ACE but an immediate and large increase of [³H]DA release in the contralateral structure. Superfusion of unlabeled DA and α-methyl-p-tyrosine in the SN remained ineffective on the [³H]DA release in the ipsilateral ACE. In this structure the release of [³H]DA was, however, decreased by nigral superfusion with γ-amino-butyric acid (GABA).d-(+)-Amphetamine (1 μM), when superfused in the CN, induced a large enhancement of the [³H]DA release in the ipsilateral ACE simultaneously with the local increase of [³H]DA release. The results presented here are in agreement with the previous studies concerning the anatomical organization of the dopaminergic nigroamygdaloid pathway. The DA cell bodies located in the SN appear insensitive to a local action of DA, perhaps due to a lack of autoreceptors. They are, however, powerfully inhibited by GABA and the relation observed between the [³H]DA release in the CN and ACE support the hypothesis that the SN can act as a relay between the extrapyramidal and limbic systems.     

Muscarinic receptors on dopamine terminals in the cat caudate nucleus: Neuromodulation of [H]dopamine release in vitro by endogenous acetylcholine

The directly acting muscarinic receptor agonist oxotremorine (1.8–10 μM) produced an increase in electrically evoked [³H]dopamine release from slices of the cat caudate. The maximal increase caused by oxotremorine was 40%, and was antagonized by the muscarinic receptor blocking agent atropine (0.1 μM). Exposure to the acetylcholinesterase (AChE) inhibitor physostigmine (1 μM) resulted in a 50% increase in electrically evoked [³H]dopamine release. The increase caused by physostigmine was also antagonized by atropine (0.1 μM).Atropine did not, however, alter the modulations in [³H]dopamine release mediated by the dopamine autoreceptor: the increase in electrically evoked [³H]dopamine release caused by the dopamine receptor antagonist S-sulpiride (0.1 μM) and the decrease caused by the dopamine receptor agonist pergolide (30 nM) were unaffected by atropine (0.1 μM). These results indicate that the muscarinic receptor-mediated and dopamine autoreceptor-mediated presynaptic effects on [³H]dopamine release are independent.The present results suggest that in the electrically depolarized caudate slice in vitro, released endogenous acetylcholine may interact with muscarinic receptors faciliting depolarization-evoked [³H]dopamine release,ifAChE is inhibited. These muscarinic receptors may be located on dopamine nerve terminals. In the context of present neuroanatomical knowledge, the action of released endogenous acetylcholine on dopamine terminals may be a non-synaptic neuromodulation.     

Presynaptic effect of 7-OH-DPAT on evoked [H]-acetylcholine release in rat striatal synaptosomes

The objective of the present experiments was to study the presynaptic effect of 7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7-OH-DPAT, a D₂-like dopamine receptor agonist) on [³H]-acetylcholine ([³H]-ACh) release induced by potassium (15 mM, 25 mM and 60 mM), potassium channel-blockers (4-aminopyridine, 4-AP; tetraethylammonium, TEA and quinine) and veratridine to gain insight into the mechanisms involved in the activation of the D₂ dopamine-receptor subtype located at striatal cholinergic nerve terminals. 7-OH-DPAT (1 μM) inhibited the evoked [³H]-ACh release induced by K⁺ 15 mM in a similar percentage than that obtained during basal conditions (30% and 27%, respectively). Nevertheless, in the presence of 25 mM and 60 mM of K⁺ the inhibitory effect of 7-OH-DPAT was completely abolished. 4-AP (1–100 μM) and TEA (1 and 5 mM) significantly enhanced [³H]-ACh release, showing 69.32%±7.60% (P<0.001) and 52.27%±5.64% (P<0.001), respectively, at the highest concentrations tested. In these conditions, 7-OH-DPAT (1 μM) inhibited the release induced by potassium channel-blockers 25–27%. Quinine (0.1–1 μM) did not alter [³H]-ACh release either in the presence or absence of 7-OH-DPAT. Veratridine 10 μM evoked [³H]-ACh release in the presence of a low-calcium medium, but in such conditions 7-OH-DPAT (1 μM) did not modify the neurotransmitter release in the absence or presence of veratridine. Present data indicate that activation of the presynaptic D₂ dopamine receptor inhibits the [³H]-ACh release by increasing K⁺ conductance, as high K⁺ concentrations abolished the inhibitory control of 7-OH-DPAT on [³H]-ACh release. This effect could be mediated by potassium channels different from those sensitive to 4-AP, TEA and quinine. In addition, the presynaptic D₂ dopamine-receptor activation seems to not involve changes in intracellular Ca²⁺.     

Modulation of noradrenaline release from hippocampal slices by hexachlorocyclohexane isomers. Effects of GABAergic compounds

The effects of hexachlorocyclohexane (HCH) isomers and some GABAergic compounds on [³H]noradrenaline (NA) release from rat hippocampal slices prelabelled with 80 nM [³H]NA were determined. The convulsant γ-HCH isomer facilitated (EC₅₀ = 21 μM) and the depressant δ-HCH isomer reduced (EC₅₀ = 48 μM) the Ca²⁺-dependent K⁺-evoked release of [³H]NA, whereas α- and β-HCH isomers did not show any effect. Moreover, α- and δ-HCH isomers antagonized the facilitation of evoked [³H]NA release induced by the γ-HCH isomer. The GABAergic convulsant drugs, bicuculline, picrotoxin and pentylenetetrazol, did not cause any modification of the evoked [³H]NA release even at high concentrations. Neither bicuculline nor picrotoxin blocked the effects of HCH isomers on K⁺-evoked release of [³H]NA. Exposure of slices to diazepam reduced the K⁺-evoked release of [³H]NA (EC₅₀ = 33 μM) in a manner similar to that of the δ-HCH isomer. In addition, diazepam (50 μM) blocked the γ-HCH effect and caused an additive inhibitory response with the δ-HCH isomer. On the other hand, diazepam and δ-HCH induced a time-dependent Ca²⁺-independent enhancement of basal [³H]NA release. The results suggest that modulation of [³H]NA release in the hippocampus by HCH isomers may be involved in the central actions of these compounds, and that sites other than the classic GABA_A receptor may underlie their presynaptic mechanisms of action.     

NMDA regulation of dopamine release from proximal and distal dendrites in the cat substantia nigra

The NMDA regulation of the dendritic release of [³H]dopamine ([³H]DA) synthesized from [³H]tyrosine was investigated in vitro using a microsuperfusion procedure in the pars compacta (SNC) and the pars reticulata (SNR) of the cat substantia nigra. The spontaneous release of [³H]DA was threefold higher in the SNC than in the SNR and amphetamine (1 μM) enhanced similarly [³H]DA release in both nigral areas. In the absence of magnesium, NMDA (50 μM) stimulated markedly the release of [³H]DA in the SNC and SNR, these effects being completely prevented by MK 801 (1 μM), the NMDA receptor antagonist. The DA uptake inhibitor, nomifensine (5 μM), increased the amount of [³H]DA recovered in SNC (×2) and SNR (×3) superfusates but did not significantly modify the NMDA-evoked responses. The effects of NMDA seen in the absence or presence of nomifensine persisted when the two nigral areas were continuously superfused with tetrodotoxin (1 μM). These results are in favor of the presence of NMDA receptors on dopaminergic dendritic arborizations and indicate that the stimulation of these receptors facilitates in a similar way the release of DA from proximal and distal dendrites.     

Effect of chronic desipramine treatment on adrenoceptor modulation of [H]dopamine release from rat nucleus accumbens slices

The effects of α₂- and β-adrenoceptor agonists on the 25 mM K⁺-induced release of [³H]dopamine ([³H]DA from nucleus accumbens slices of chronic desipramine (DMI)- and saline-treated rats were investigated using a superfusion technique. The K⁺-induced release of [³H]DA from nucleus accumbens slices was shown to be Ca²⁺-dependent by ascorbic acid. In experiments with isoproterenol, ascorbic acid was added to the superfusion media in order to prevent the otherwise rapid oxidation of the drug. The K⁺-induced release of [³H]DA from nucleus accumbens slices of saline-treated rats was significantly decreased by the α₂-adrenoceptor agonist, clonidine(10 μM; 89 ± 2.4%of control values; P < 0.002), and significantly enhanced by the β-adrenoceptor agonist, isoproterenol(1 and 10 μM; 122 ± 4.3and171 ± 2.9%of control values, P < 0.002andP < 0.001, respectively). The basal release of [³H]DA was strongly enhanced by 10 μM but not 1 μM isoproterenol. Chronic DMI pretreatment (10 mg/kg i.p. for 28 days) did not significantly alter the K⁺-induced release of [³H]DA. Chronic DMI treatment attenuated the α-adrenoceptor-mediated inhibition of [³H]DA release, while the β-adrenoceptor-mediated stimulation remained unchanged. The net effect of chronic DMI treatment therefore would appear to be a facilitation of dopaminergic neurotransmission in the mesolimbic system. This is consistent with behavioural evidence which suggests that the function of the mesolimbic dopaminergic reward system is facilitated by chronic treatment with antidepressant drugs.     

Somatostatin and cholecystokinin octapeptide differentially modulate the release of [H]acetylcholine from caudate nucleus but not cerebral cortex: Role of dopamine receptor activation

The effects of somatostatin (SOM) and cholecystokinin octapeptide (CCK-8) on basal and potassium-induced release of acetylcholine (ACh) were investigated in slices of rat caudate nucleus (CN) and, for comparison, cerebral cortex (CX). Potassium (5–55 mM) produced a concentration-dependent increase in the release of [³H]ACh in the presence of extracellular Ca²⁺. SOM (1 μ), CCK-8 (1 μM) and the dopamine (DA) receptor agonist, apomorphine (APO, 30 μM) inhibited the K⁺-induced (35 mM) release of [³H]ACh by 26–32% from CN, but did not affect ACh release from CX. Other peptides (1 μM), such as Met-enkephalin, vasoactive intestinal peptide, thyrotropin-releasing hormone and substance P, had no effect on release of [³H]ACh in CN or CX. Sulpiride (SULP), a dopamine receptor antagonist, prevented the effects of APO and SOM, but not CCK-8, to inhibit [³H]ACh release. The results indicate that: (1) SOM and CCK-8 inhibit the release of [³H]ACh in CN, but not CX; and (2) the inhibitory effect of SOM, but not CCK-8, on [³H]ACh release is mediated by dopaminergic mechanisms.     

Effect of pentobarbitone on the spontaneous efflux of γ-amino acids from rabbit retina

The effect of pentobarbitone on the spontaneous release of radioactivity from rabbit retinas preloaded with [³H]GABA, [³H]DABA, [³H]β-alanine and [³H]glycine was studied. In high concentration of pentobarbitone (10⁻³ M) the rate of spontaneous efflux of neuronal [³H]GABA, [³H]DABA and [³H]β-alanine was reduced but not that of [³H]glycine. Glial release of [³H]GABA was much less influenced. At lower concentrations (10⁻⁴-10⁻⁵ M) there was an initial increase in the release of [³H]GABA, [³H]DABA and [³H]β-alanine followed by a decrease, indicating several components in the release systems for the amino acids.     

Acute effects of γ-vinyl GABA (vigabatrin) on hippocampal GABAergic inhibition in vitro

The acute effects of γ-vinyl-GABA (GVG) on GABAergic inhibition were investigated in the hippocampal slice preparation using the paired-pulse test of inhibition during extracellular recordings. Superfusion of GVG (100–500 μM) for 60 min resulted in a concentration-dependent decrease in GABAergic inhibition. Slices superfused with higher concentrations of GVG (0.5–1 mM) were hyperexcitable as demonstrated by the appearance of multiple spikes. Binding studies showed that GVG (1 mM) had no effect on the binding of [³H]flunitrazepam or [³H]TBOB and displaced no more than 15% of specific [³H]GABA binding, which indicates that GVG-induced disinhibition is not mediated through an action at the GABA_A receptor complex. Consistent with this suggestion is the finding that GVG (500 μM) had little effect on the inhibition of the orthodromically evoked CA1 population spike produced by the GABA_A receptor agonist muscimol (10 μM), whereas this inhibition was considerably attenuated by the GABA_A receptor antagonist, bicuculline methiodide (5 μM). The results of this study suggest that the acute actions of GVG on the GABAergic neurotransmitter system are not involved in its anticonvulsant effect.     

Downregulation of muscarinic- and 5-HT1B-mediated modulation of [H]acetylcholine release in hippocampal slices of rats with fimbria-fornix lesions and intrahippocampal grafts of septal origin

Adult Long-Evans female rats sustained electrolytic fimbria-fornix lesions and, two weeks later, received intrahippocampal suspension grafts of fetal septal tissue. Sham-operated and lesion-only rats served as controls. Between 6.5 and 8 months after grafting, both the [³H]choline accumulation and the electrically evoked [³H]acetylcholine ([³H]ACh) release were assessed in hippocampal slices. The release of [³H]ACh was measured in presence of atropine (muscarinic antagonist, 1 μM), physostigmine (acetylcholinesterase inhibitor, 0.1 μM), oxotremorine (muscarinic agonist, 0.01 μM–10 μM), mecamylamine (nicotinic antagonist, 10 μM), methiothepin (mixed 5-HT₁/5-HT₂ antagonist, 10 μM), 8-OH-DPAT (5-HT_1A agonist, 1 μM), 2-methyl-serotonin (5-HT₃ agonist, 1 μM) and CP 93129 (5-HT_1B agonist, 0.1 μM–100 μM), or without any drug application as a control. In lesion-only rats, the specific accumulation of [³H]choline was reduced to 46% of normal and the release of [³H]ACh to 32% (nCi) and 43% (% of tissue tritium content). In the grafted rats, these parameters were significantly increased to 63%, 98% and 116% of control, respectively. Physostigmine reduced the evoked [³H]ACh release and was significantly more effective in grafted (−70%) than in sham-operated (−56%) or lesion-only (−54%) rats. When physostigmine was superfused throughout, mecamylamine had no effect. Conversely, atropine induced a significant increase of [³H]ACh release in all groups, but this increase was significantly larger in sham-operated rats (+209%) than in the other groups (lesioned: +80%; grafted: +117%). Oxotremorine dose-dependently decreased the ([³H]ACh) release, but in lesion-only rats, this effect was significantly lower than in sham-operated rats. Whatever group was considered, 8-OH-DPAT, methiothepin and 2-methyl-serotonin failed to induce any significant effect on [³H]ACh release. In contrast, CP 93129 dose-dependently decreased [³H]ACh release. This effect was significantly weaker in grafted rats than in the rats of the two other groups. Our data confirm that cholinergic terminals in the intact hippocampus possess inhibitory muscarinic autoreceptors and serotonin heteroreceptors of the 5-HT_1B subtype. They also show that both types of receptors are still operative in the cholinergic terminals which survived the lesions and in the grafted cholinergic neurons. However, the muscarinic receptors in both lesioned and grafted rats, as well as the 5-HT_1B receptors in grafted rats show a sensitivity which seems to be downregulated in comparison to that found in sham-operated rats. In the grafted rats, both types of downregulations might contribute to (or reflect) an increased cholinergic function that results from a reduction of the inhibitory tonus which ACh and serotonin exert at the level of the cholinergic terminal.     

Clathrin-coated vesicles from bovine brain contain uncoupled GABAA receptors

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABA_A receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [³H]muscimol, a GABA_A-specific agonist, [³H]flunitrazepam, a benzodiazepine agonist, and [³⁵S]t-butylbiocyclophosphorthionate (TBPS), a ligand for GABA_A receptor channels. Under standard conditions, the binding level of [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS to coated vesicles represented 12.3±1.8%, 7.9±1%, and 10.2±1.8%, respectively, of that in crude synaptic membranes. Coated vesicles showed a single [³H]flunitrazepam binding site with a K_D value (12 nM) which was 9-fold that for synaptic membranes. The allosteric coupling between binding sites was measured by the addition of GABA to [³H]flunitrazepam and [³⁵S]TBPS binding assays. For [³H]flunitrazepam binding to synaptic membranes, GABA gave an EC₅₀=2.0 μM and at saturation (100 μM) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonist SR95531. In contrast, neither GABA nor SR95531 had a significant effect on [³H]flunitrazepam binding to CCVs, indicating that the allosteric interaction between GABA and benzodiazepine binding sites is abolished. Likewise, GABA displaced nearly all of the [³⁵S]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, indicating that coupling between the GABA binding sites and chloride channel is also impaired. Thus GABA_A receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABA_A receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic membranes, CCVs contained 110±14% and 29.5±3.8%, respectively, of the immunoreactivity for GABA_A receptor β2 and α1 subunits. Thus, in comparison to GABA_A receptors on synaptic membranes, those on CCVs have a reduced α1/β2-subunit ratio. It may be suggested that a selective decline in the content of α1 subunits in coated vesicles could in part account for GABA_A receptor uncoupling.