Gamma-aminobutyric Acid Efflux Research Articles

Dopamine receptor cooperativity synergistically drives dyskinesia, motor behavior, and striatal GABA neurotransmission in hemiparkinsonian rats

The striatum undergoes significant neuroplasticity both in Parkinson's Disease (PD) and following dopamine (DA) replacement therapy with l-DOPA. Unfortunately, these changes also contribute to the emergence of l-DOPA-induced dyskinesia (LID). While convergent strategies have demonstrated independent roles for DA D1 -receptors (D1R) and D2-receptors (D2R) in LID, DA receptor cooperativity, either by cellular or circuit mechanisms, has also been implicated in the dyskinetic brain. How this cooperativity is substantiated is vitally important given that l-DOPA, once converted to DA, stimulates all DA receptors. The present experiments sought to characterize the effect of individual or collective stimulation of D1R and D2R-like receptors both systemically and intrastriatally. In experiment 1, hemiparkinsonian l-DOPA-primed rats received systemic doses of the D1R agonist SKF38393 and D2R-like agonist quinpirole. Dyskinesia and motor improvement were monitored using the abnormal involuntary movements scale (AIMs) and the forepaw adjustment steps test (FAS), respectively. In experiment 2, SKF38393 and quinpirole were administered intrastriatally via reverse-phase in vivo microdialysis while coincident changes in striatal glutamate and gamma-Aminobutyric acid (GABA) were monitored. SKF38393 and quinpirole dose-dependently increased AIMs. When threshold DA agonist doses were co-administered, AIMs and motor performance were synergistically enhanced. Like systemic experiments, striatal co-administration of threshold concentrations of DA agonists resulted in synergistic exacerbation of AIMs, and concurrent increases in GABA efflux. These data highlight the role of striatal DA receptor cooperativity in LID and suggest a central role for striatal GABA release in these effects.

Aluminum-Activated Malate Transporters Can Facilitate GABA Transport.

Plant aluminum-activated malate transporters (ALMTs) are currently classified as anion channels; they are also known to be regulated by diverse signals, leading to a range of physiological responses. Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires a specific amino acid motif in ALMTs that shares similarity with a GABA binding site in mammalian GABAA receptors. Here, we explore why TaALMT1 activation leads to a negative correlation between malate efflux and endogenous GABA concentrations ([GABA]i) in both wheat (Triticum aestivum) root tips and in heterologous expression systems. We show that TaALMT1 activation reduces [GABA]i because TaALMT1 facilitates GABA efflux but GABA does not complex Al3+ TaALMT1 also leads to GABA transport into cells, demonstrated by a yeast complementation assay and via 14C-GABA uptake into TaALMT1-expressing Xenopus laevis oocytes; this was found to be a general feature of all ALMTs we examined. Mutation of the GABA motif (TaALMT1F213C) prevented both GABA influx and efflux, and resulted in no correlation between malate efflux and [GABA]i We conclude that ALMTs are likely to act as both GABA and anion transporters in planta. GABA and malate appear to interact with ALMTs in a complex manner to regulate each other's transport, suggestive of a role for ALMTs in communicating metabolic status.

Neural basis for a heritable phenotype: differences in the effects of apomorphine on startle gating and ventral pallidal GABA efflux in male Sprague-Dawley and Long-Evans rats.

RationalePrepulse inhibition (PPI) of startle is a measure of sensorimotor gating that is heritable and deficient in certain psychiatric disorders, including schizophrenia. Sprague–Dawley (SD) rats are more sensitive to PPI disruptive effects of dopamine (DA) agonists at long interstimulus intervals (60–120 ms) and less sensitive to their PPI-enhancing effects at short (10–30 ms), compared with Long–Evans (LE) rats. These heritable strain differences in sensitivity to the PPI disruptive effects of DA agonists must ultimately reflect neural changes "downstream" from forebrain DA receptors.ObjectiveThe current study evaluated the effects of the DA agonist, apomorphine (APO), on ventral pallidal (VP) gamma-aminobutyric acid (GABA) and glutamate efflux and PPI in SD and LE rats.MethodsPPI was tested in SD and LE rats after vehicle or APO (0.5 mg/kg, subcutaneously (s.c.)) in a within-subject design. In different SD and LE rats, VP dialysate was collected every 10 min for 120 min after vehicle or APO (0.5 mg/kg, s.c.) and analyzed for GABA and glutamate content by capillary electrophoresis (CE) coupled with laser-induced fluorescence (LIF).ResultsAs predicted, SD rats exhibited greater APO-induced PPI deficits at long intervals and less APO-induced PPI enhancement at short intervals compared to LE rats. APO significantly reduced VP GABA efflux in SD but not in LE rats; glutamate efflux was unaffected in both strains.ConclusionHeritable strain differences in PPI APO sensitivity in SD vs LE rats parallel, and may be mediated by, strain differences in the VP GABA efflux.

Alterations in Extracellular Levels of Gamma-Aminobutyric Acid in the Rat Basolateral Amygdala and Periaqueductal Gray During Conditioned Fear, Persistent Pain and Fear-Conditioned Analgesia

Evidence suggests an important role for supraspinal gamma-aminobutyric acid (GABA) in conditioned fear and pain. Using dual probe microdialysis coupled to HPLC, we investigated alterations in extracellular levels of GABA simultaneously in the rat basolateral amygdala and dorsal periaqueductal gray during expression of conditioned fear, formalin-evoked nociception, and fear-conditioned analgesia. Re-exposure to a context previously paired with footshock significantly increased the duration of freezing and 22-kilohertz ultrasonic vocalization, and reduced formalin-evoked nociceptive behavior. Upon re-exposure to the context, GABA levels in the basolateral amygdala were significantly lower in fear-conditioned animals compared with non-fear-conditioned controls, irrespective of intraplantar formalin/saline injection. GABA levels in the dorsal periaqueductal gray were lower in rats receiving intraplantar injection of formalin, compared with saline-treated controls. GABA levels sampled were sensitive to nipecotic acid and calcium infusion. No specific fear-conditioned analgesia-related alterations in GABA efflux were observed in these regions despite the ability of rats undergoing dual probe microdialysis to express this important survival response. In conclusion, expression of contextually induced fear- and pain-related behavior are accompanied by suppression of GABA release in the basolateral amygdala and dorsal periaqueductal gray, respectively, compared with non-fear, non-pain controls. This study investigates alterations in levels of the neurotransmitter GABA simultaneously in the rat amygdala and periaqueductal grey during expression of pain- and fear-related behavior and fear-induced analgesia. The results enhance our understanding of the role of this neurotransmitter in pain, memory of pain and control of pain during fear.

GABAergic transmission in the rat ventral pallidum mediates a saccharin palatability shift in conditioned taste aversion

We previously found that the blockade of gamma-aminobutyric acid (GABA)(A) receptors in the ventral pallidum (VP) alters the taste palatability of a conditioned stimulus (CS) from aversive to ingestive after the establishment of conditioned taste aversion (CTA). Because these results suggest that GABAergic transmission in the VP mediates decreased palatability of the taste in CTA, the present study aimed to examine the effects of taste stimulation on the extracellular release of GABA in the VP using in vivo microdialysis. Initially, rats received a paired presentation of 5 mm saccharin or 0.3 mm quinine solution with an intraperitoneal injection of 0.15 m lithium chloride (S-CTA and Q-CTA groups) or saline (S-control and Q-control groups). After conditioning, microdialysis was carried out before, during and after the presentation of the CS via an intra-oral cannula. We measured the latency of the first aversive orofacial responses to the CS as behavioral indices. In the S-CTA group, which rapidly rejected the CS (within 100 s), the GABA efflux was significantly increased (147%) and was maintained for 2 h. On the other hand, the S-control group expressed no aversive responses and showed no significant alterations in GABA efflux. Although the Q-CTA group immediately expressed aversive responses to the CS (within 30 s), GABA release was not changed by presentation of the CS, which was similar in the Q-control group. These findings suggest that the palatability shift from ingestive to aversive in conditioned aversion to saccharin, but not quinine, is mediated by the change in GABAergic transmission in the VP.

GABA Transporter Preserving Ongoing Spontaneous Neuronal Activity at Firing Subthreshold

There has been compelling evidence that the GABA transporter is crucial not only for removing gamma-aminobutyric acid (GABA) from but also releasing it into extracellular space, thereby clamping ambient GABA (GABA in extracellular space) at a certain level. The ambient GABA is known to activate extrasynaptic GABA receptors and provide tonic inhibitory current into neurons. We investigated how the transporter regulates the level of ambient GABA, mediates tonic neuronal inhibition, and influences ongoing spontaneous neuronal activity. A cortical neural network model is proposed in which GABA transporters on lateral (L) and feedback (F) inhibitory (GABAergic) interneurons are functionally made. Principal (P) cell assemblies participate in expressing information about elemental sensory features. At membrane potentials below the reversal potential, there is net influx of GABA, whereas at membrane potentials above the reversal potential, there is net efflux of GABA. Through this transport mechanism, ambient GABA concentration is kept within a submicromolar range during an ongoing spontaneous neuronal activity time period. Here we show that the GABA transporter on L cells regulates the overall level of ambient GABA across cell assemblies, and that on F cells it does so within individual cell assemblies. This combinatorial regulation of ambient GABA allows P cells to oscillate near firing threshold during the ongoing time period, thereby reducing their reaction time to externally applied stimuli. We suggest that the GABA transporter, with its forward and reverse transport mechanism, could regulate the ambient GABA. This transporter-mediated ambient GABA regulation may contribute to establishing an ongoing subthreshold neuronal state by which the network can respond rapidly to subsequent sensory input.

Effects of acute and repeated restraint stress on gaba efflux in the rat basolateral and central amygdala

Stress can precipitate onset of multiple mood-related disorders, including depression. Examination of the neural basis of this phenomenon has highlighted the amygdala as a key component. Alterations in amygdalar activity and structure accompany various mood-related disorders, and interestingly, amygdalar morphology and behavior can be altered in animals subjected to repeated stress. Gamma-aminobutyric acid (GABA) transmission in the amygdala represents an important means by which information flow, activity, and function can be controlled; therefore, we determined the effects of acute and repeated restraint stress (RRS) on GABA efflux in the basolateral and central amygdalar complexes. In vivo microdialysis revealed that acute restraint stress increased GABA efflux in the basolateral amygdala, whereas central amygdala efflux remained unchanged. Animals subjected to prior repeated stress displayed no acute stress-mediated increases in GABA efflux in the basolateral amygdala, an event accompanied by no changes in basal GABA concentrations. Conversely, repeated restraint stress had no effect on GABA efflux or basal GABA levels in the CeA. Collectively, these data demonstrate that acute stress elicits unique and region-specific increases in GABA efflux in the rat amygdala, and that prior repeated stress differentially modifies this response.

GABAergic mechanism mediated via D1 receptors in the rat periaqueductal gray participates in the micturition reflex: an in vivo microdialysis study

The periaqueductal gray (PAG) is critically involved in the micturition reflex, but little is known about the neuronal mechanisms involved. The present study elucidated dynamic changes in dopamine (DA), glutamate and gamma-aminobutyric acid (GABA) in the rat PAG during the micturition reflex, with a focus on dopaminergic modulation using in vivo microdialysis combined with cystometrography. Extracellular levels of DA and glutamate increased, whereas levels of GABA decreased, in parallel with the micturition reflex. Application of a D(1) receptor antagonist into the PAG produced increases in maximal voiding pressure (MVP) and decreases in intercontraction interval (ICI), suggesting that the micturition reflex was facilitated by D(1) receptor blockade. The D(1) receptor antagonist prevented micturition-induced decreases in GABA efflux but had no effect on DA or glutamate. Neither a D(2) receptor antagonist nor a D(1)/D(2) receptor agonist affected these neurochemical and physiological parameters. Micturition-induced inhibition of GABA was not observed in 6-hydroxydopamine (6-OHDA)-lesioned rats, an animal model of Parkinson's disease. 6-OHDA-lesioned rats exhibited bladder hyperactivity evaluated by increases in MVP and decreases in ICI, mimicking facilitation of the micturition reflex induced by D(1) receptor blockade. These findings suggest that the micturition reflex is under tonic dopaminergic regulation through D(1) receptors, in which a GABAergic mechanism is involved. Bladder hyperactivity observed in 6-OHDA-lesioned rats may be caused by dysfunction of GABAergic regulation underlying the micturition reflex. The present findings contribute to our understanding not only of the neurophysiology of the micturition reflex but also of the pathophysiology of lower urinary tract dysfunction in patients with Parkinson's disease.

Excitatory and Inhibitory Actions of Isoflurane on the Cholinergic Ascending Arousal System of the Rat

The cholinergic arousal systems are known to critically regulate the state of consciousness. The aim of this study was to determine the effect of isoflurane on the inhibitory or excitatory neurotransmitters efflux in important nuclei within the cholinergic arousal system using in vivo intracerebral microdialysis. The efflux of glutamate, gamma-aminobutyric acid (GABA), or acetylcholine in the posterior hypothalamus (PH), the basal forebrain (BF), and the somatosensory cortex (S1BF) of rats was detected using intracerebral microdialysis under an awake condition and at 0.5-2.0 minimum alveolar concentration (MAC) isoflurane anesthesia. The intrabasalis perfusion of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate on the cortical acetylcholine effluxes was also examined under both conditions. Isoflurane had no influence on the glutamate and GABA efflux in the PH, whereas in the BF, it dose-dependently increased glutamate efflux and decreased GABA efflux. A transient increase in glutamate efflux at 1.0 MAC and a decrease in GABA at 0.5-1.5 MAC were observed in the S1BF. Isoflurane dose-dependently decreased acetylcholine efflux in the S1BF. Perfusion of the BF with AMPA increased acetylcholine efflux in the S1BF with electroencephalographic activation during 0.75 MAC isoflurane anesthesia, suggesting an inhibitory action of isoflurane on AMPA receptors in the BF. However, N-methyl-D-aspartate had no effect on these parameters. Isoflurane induces both excitatory and inhibitory actions in the cholinergic arousal system. The predominant inhibitory action of isoflurane over its excitatory action at the BF would result in the decrease in the acetylcholine efflux in the S1BF.

The role of glycineB binding site and glycine transporter (GlyT1) in the regulation of [3H]GABA and [3H]glycine release in the rat brain.

The effect of N-methyl-D-aspartic acid (NMDA), a selective glutamate receptor agonist, on the release of previously incorporated [3H]gamma-aminobutyric acid(GABA) was examined in superfused striatal slices of the rat. NMDA (0.01 to 1.0 mM) increased [3H]GABA overflow with an EC50 value of 0.09 mM. The [3H]GABA releasing effect of NMDA was an external Ca2+-dependent process and the GABA uptake inhibitor nipecotic acid (0.1 mM) potentiated this effect. These findings support the view that NMDA evokes GABA release from vesicular pool in striatal GABAergic neurons. Addition of glycine (1 mM), a cotransmitter for NMDA receptor, did not influence the NMDA-induced [3H]GABA overflow. Kynurenic acid (1 mM), an antagonist of glycineB site, decreased the [3H]GABA-releasing effect of NMDA and this reduction was suspended by addition of 1 mM glycine. Neither glycine nor kynurenic acid exerted effects on resting [3H]GABA outflow. These data suggest that glycineB binding site at NMDA receptor may be saturated by glycine released from neighboring cells. Glycyldodecylamide (GDA) and N-dodecylsarcosine, inhibitors of glycineT1 transporter, inhibited the uptake of [3H]glycine (IC50 33 and 16 microM) in synaptosomes prepared from rat hippocampus. When hippocampal slices were loaded with [3H]glycine, resting efflux was detected whereas electrical stimulation failed to evoke [3H]glycine overflow. Neither GDA (0.1 mM) nor N-dodecylsarcosine (0.3 mM) influenced [3H]glycine efflux. Using Krebs-bicarbonate buffer with reduced Na+ for superfusion of hippocampal slices produced an increased [3H]glycine outflow and electrical stimulation further enhanced this release. These experiments speak for glial and neuronal [3H]glycine release in hippocampus with a dominant role of the former one. GDA, however, did not influence resting or stimulated [3H]glycine efflux even when buffer with low Na+ concentration was applied.

The neuroprotective agent MS-153 stimulates glutamate uptake

We investigated the effect of ( R)-(−)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), a novel neuroprotective agent, on l-[ 3H]glutamate uptake through GLT-1, a Na +/K +-dependent glial glutamate transporter, expressed in COS-7 cells. MS-153 (1–100 μM) accelerated the l-[ 3H]glutamate uptake through GLT-1 in a concentration-dependent and time-dependent manner. Eadie–Hofstee analysis revealed that MS-153 significantly decreased the K m of the glutamate uptake by COS-7 cells expressing GLT-1. In contrast, [ 3H]gamma-aminobutyric acid (GABA) uptake through a glial GABA transporter was not affected. In addition, MS-153 increased Na + currents through GLT-1 expressed in Xenopus oocytes. We also investigated the effect of MS-153 on amino acid efflux from rat hippocampal slices. The increase in glutamate efflux induced by 50 mM KCl was significantly attenuated by the treatment with MS-153 at 10 μM, while MS-153 had no significant effect on the K +-evoked efflux of GABA. Furthermore, the increase in glutamate efflux by ischemia (hypoxia/aglycemia) was partially, but significantly inhibited by MS-153. These results suggest that the cerebroprotective effect of MS-153 in this ischemic model in vivo is due to the specific reduction of the glutamate concentration in the extracellular space, which can probably be attributed to the acceleration of glutamate uptake by the indirect modulation of the glutamate transporter activity.

Efflux of gamma-aminobutyric acid caused by changes in ion concentrations and cell swelling simulating the effect of cerebral ischaemia.

The relationships among ischaemic GABA efflux from brain tissue and extracellular and intracellular concentrations of sodium, chloride and potassium ions were investigated by means of 1) transverse hippocampal slices from rat and 2) functional expression of a high affinity GABA transporter in Xenopus oocytes. Brain slices were incubated for 20 min in medium where extracellular sodium and chloride were substituted with impermeant ions. Isethionate (Iseth) substitution for chloride generated a 7-fold increase in GABA efflux. Choline (Chol) but not N-methyl-D-glucamine (NMDG) substitution for sodium likewise increased GABA efflux. Reducing the osmolarity of the medium by decreasing both sodium and chloride concentrations (Hyp) increased GABA efflux 3-fold. This release was blocked by mannitol (Man). Blocking sodium channels with 1 microM of tetrodotoxin (TTX) also increased the release 3-fold. Energy deprivation (ED) increased the GABA release 50-fold. ED/Iseth left the release unchanged, ED/Chol increased the GABA efflux by 23%, whereas ED/NMDG reduced the release by 41%. Adding mannitol did not block the ED-evoked release, whereas TTX reduced it by 52%. Release of preloaded [3H]-GABA from oocytes expressing the GAT-1 GABA transporter was then examined. Depolarisation by current injection or 100 mM extracellular K+ did not increase GABA release. Sodium chloride injection, however, caused membrane depolarisation and a 100-fold increased GABA efflux from the oocytes. This release was blocked when the osmolarity was increased extracellularly by adding mannitol. These results show that 1) TTX releases GABA from brain tissue but blocks release during ED, 2) the high affinity GABA carrier must be altered in order to reverse, 3) ischaemic GABA release is sodium independent, and is modulated by large cations, 4) mannitol blocks the reversal of high affinity carriers in oocytes, but the release from brain slices during ED is unaffected. Taken together, the results suggest that ischaemic release of GABA from brain tissue does not occur by means of reversed high affinity carriers alone, but rather that it is controlled by more complex mechanisms.

Non-neuronal endogenous GABA efflux from the rat oviduct

The subcellular distribution of Gamma-aminobutyric acid (GABA) was studied in the rat oviduct. The highest content of GABA was found in the soluble fraction. The effect of chemical stimulation of the endogenous GABA efflux from the rat oviduct was examined. High K + concentrations could not induce elevation of the GABA efflux. Instead, a continuous spontaneous GABA efflux without change for long periods of time was observed. The total GABA content and GABA concentration were determined in the rat oviduct on days 1, 5, 10, 15, 30, 35 and 40 of the postnatal period and also during the estrous cycle. During postnatal development the GABA levels increase gradually with time reaching at prepuberal age a concentration similar to that found in diestrous rats. In the estrous cycle both GABA content and GABA concentration reached the highest value in the proestrous and the lowest value in the estrous phase. These findings support the hypothesis that GABA efflux may be modulated by the changes in oviductal fluid volume during the estrous cycle.

Inhibitory effects of the psychoactive drug modafinil on gamma-aminobutyric acid outflow from the cerebral cortex of the awake freely moving guinea-pig. Possible involvement of 5-hydroxytryptamine mechanisms.

The effects of modafinil on acetylcholine and GABA outflow from the cerebral cortex of awake freely moving guinea pigs provided with an epidural cup were studied. In the dose range of 3-30 mg/kg s.c. modafinil produced a dose dependent significant inhibition of GABA outflow without influencing cortical acetylcholine release. Methysergide (2 mg/kg, i.p.) and ketanserin (0.5 mg/kg, i.p.) but not prazosin (0.14 mg/kg, i.p.) counteracted the inhibitory action of modafinil on cortical GABA outflow. Modafinil both acutely and chronically in the same dose range increased striatal 5-HIAA levels and 5-HT utilization in the rat (acute) and mouse (chronic). The action on cortical GABA release may be dependent on activity at 5-HT2 receptors, since the action of modafinil in this respect is blocked by the non-selective 5-HT antagonist methysergide and the 5-HT2 antagonist ketanserin. The involvement of 5-HT mechanisms in the inhibitory action of modafinil on cortical GABA release is also suggested by the findings that 5-HT metabolism may become increased by modafinil at least in the striatum. The reduction of cortical GABA outflow via 5-HT2 receptors by modafinil is probably related to some of its actions on the central nervous system including behavioural effects.

Changes in the Release of Gamma‐Aminobutyric Acid and Catecholamines in the Preoptic/Septal Area Prior to and During the Preovulatory Surge of Luteinizing Hormone in the Ewe

Abstract The technique of intracranial microdialysis was used to monitor changes in the outflow of the catecholamines, noradrenaline, adrenaline and dopamine and the inhibitory amino-acid gamma-aminobutyric acid (GABA) in the preoptic/septal area of the conscious ewe during an oestradiol-induced surge of luteinizing hormone (LH). The same animals were sampled twice from an identical brain site, once in the presence of oestradiol and once in its absence, when no surge occurred and LH levels remained low. Changes in the outflow of GABA, noradrenaline and adrenaline (but not dopamine) were related to changes in LH secretion. Specifically, GABA outflow was maximal in the hours following oestradiol administration but began a sustained fall some 10 h before the surge began, to level off just before the first increment in LH secretion. Low GABA concentrations were maintained until after gonadotrophin levels had once more returned to baseline. The release of all three catecholamines was pulsatile. Noradrenergic activity was greater in the presence of oestradiol although activity did not alter over the 20 h of sampling. The pulse frequency of adrenaline was maximal in the hours immediately prior to the LH surge and minimal in the hours following its initiation. These data suggest that a decrease in GABAergic transmission in the vicinity of the LH-releasing hormone cell bodies is a necessary component of the neural mechanism by which the oestradiol-induced surge of LH is generated. A general increase in noradrenergic activity coupled with changes in the release of adrenaline at the time of the surge may be additional prerequisites for successful ovulation.

Releasable GABA in tubular epithelium of rat kidney

The distribution of gamma-aminobutyric acid (GABA) in the rat kidney was examined by immunocytochemical techniques. GABA-like immunoreactivity (GABA-LI) was predominantly confined to the renal tubules, including the ascending parts of the distal tubules, and the loops of Henle, the collecting tubules and ducts, and the connective parts of the convoluted tubules. In GABA-positive cortical tubules, about half of the epithelial cells were labelled. The labelled cell type showed the ultrastructural features of principal cells. Depolarizing stimulation by ouabain and high K+ concentration evoked the efflux of endogenous GABA from kidney slices. The present findings, along with previous results, suggest that GABA released from renal tubular epithelium, and transported with the urine, might be involved in the modulation of contractility in the urinary tract.

Endogenous Release of γ‐Aminobutyric Acid from the Medial Preoptic Area Measured by Microdialysis in the Anaesthetised Rat

The characteristics of gamma-aminobutyric acid (GABA) release as monitored by microdialysis have been investigated in the chloral hydrate anaesthetised rat. The high outflow of GABA following insertion of the microdialysis probe (membrane 2 mm in length, 0.5 mm in diameter) into the medial preoptic area was found to decline to a stable baseline level after 2 h. After this time, perfusion with a medium containing 100 mM potassium ions evoked a 56-fold increase in GABA outflow. The addition of the calcium channel blocker verapamil (100 microM) to the perfusion medium induced significant 25 and 50% reductions in basal and potassium-stimulated GABA outflow, respectively. In the same animals, verapamil caused an 80% decrease in potassium-stimulated noradrenaline outflow. The glutamic acid decarboxylase inhibitors 3-mercaptopropionic acid and L-allylglycine added to the perfusion medium at a concentration of 10 mM reduced basal GABA release by approximately 50% with different time-courses of action. Ethanolamine-O-sulfate, a GABA-transaminase inhibitor, induced significant increases in basal GABA outflow 90 min after inclusion in the perfusion medium. These results demonstrate that microdialysis is a suitable technique with which to monitor extracellular levels of GABA and provide in vivo data on GABA release and degradation mechanisms.

Mechanisms of [3H] γ‐aminobutyric acid release by chromaffin cells in primary culture

The basal and evoked [3H] gamma-aminobutyric acid (GABA) release from chromaffin cells in primary cultures was studied and compared with that of [3H]NA. [3H]GABA was found to be released, in a dose-dependent fashion, by different secretagogues known to induce noradrenaline (NA) release, that is, the cholinergic agonist nicotine, high-potassium chloride, veratridine, and calcium ionophores. In general, there was a parallelism between percentages of release of both [3H]GABA and [3H]NA, although in all circumstances the former were lower. The nicotine- and high-potassium-evoked [3H]GABA release was absolutely calcium dependent, thus indicating the existence of a exocytotic-like mechanism, whereas in the veratridine-induced release, a calcium-independent component was also detected. This latter component was sodium dependent, as it showed an absolute requirement for extracellular sodium and was enhanced by ouabain. Moreover, it was inhibited by known GABA uptake inhibitors, which indicate that this component of [3H]GABA release induced by veratridine could be due to GABA outflow through the membrane carrier. The above results, together with that obtained from studies about subcellular localization of [3H]GABA taken up by chromaffin cells, seem to support the existence of two mechanisms for [3H]GABA release by chromaffin cells: one calcium-dependent, exocytotic-like, and another calcium-independent and sodium-dependent, possibly mediated by the GABA carrier. Both processes could have a functional role on the regulation of extracellular GABA levels and so in the control of catecholamine release by chromaffin cells.

Changes in [3H]nitrendipine binding and gamma-aminobutyric acid release in rat hippocampus following repeated morphine administration.

An antagonistic effect of calcium on the action of morphine was studied in rat hippocampal slices. The effect of repeated administration of morphine on gamma-aminobutyric acid (GABA) release and binding of [3H]nitrendipine, a calcium antagonist, was also examined. (1) In rat brain hippocampal slices, morphine enlarged the amplitude of the field potentials evoked in pyramidal neurons, disinhibiting them through basket cells. When the calcium concentration was elevated, potentiation of the field potentials by morphine was reduced. Decrease of the calcium concentration, on the other hand, enhanced the potentiating effect of morphine. Following repeated administration of morphine, its enhancing effect on the field potentials in slices was not observed. (2) In hippocampal membrane fractions obtained from rats repeatedly treated with morphine, enhancement of [3H]nitrendipine binding was observed. (3) In hippocampal slice preparations from rats receiving morphine repeatedly, K+ (45 mM)-stimulated [3H]GABA efflux was enhanced. The above results indicate that morphine antagonizes calcium, thereby reducing the release of transmitters. Furthermore, increase in calcium channels following repeated treatment of rats with morphine may explain the mechanism underlying development of tolerance.

Low-affinity gamma-aminobutyric acid transport in rat brain.

The low-affinity (Km = 100-200 microM) gamma-aminobutyric acid (GABA) transporter from membrane vesicles from rat brain has been characterized and found to be in many aspects similar to the well-known sodium- and chloride-coupled high-affinity gamma-aminobutyric acid transporter (Km = 2-4 microM). Influx by this system is sodium and chloride dependent and stimulated by an interior negative membrane potential. Steady-state levels obtained by both systems are lowered by the sodium channel openers veratridine and aconitine. However, while the channel blocker tetrodotoxin fully reverses this inhibition with the high-affinity system, this is not the case for its low-affinity counterpart. Furthermore, the toxin from the scorpion Androctonus australis Hector inhibited high-affinity transport only. Efflux of gamma-aminobutyric acid taken up by the high-affinity system displayed a Km of about 100 microM. Exchange catalyzed by the low-affinity system was observed in the absence of external sodium and chloride. Furthermore, both activities copurified in the fractionation procedure developed to purify the high-affinity transporter. All these observations are consistent with the idea that both activities are manifestations of only one gamma-aminobutyric acid transporter. The high-affinity binding site represents the extracellular and the low-affinity site the cytosolic aspect of the transporter. In addition, it was found that right-side-out synaptosomes also contain a low-affinity GABA transporter. This apparently represents a different transport protein.