SKF 89976A Research Articles

GABA induces norepinephrine exocytosis from hippocampal noradrenergic axon terminals by a dual mechanism involving different voltage-sensitive calcium channels

GABA can evoke norepinephrine (NE) release by activating GABAA receptors or GABA transporters on noradrenergic terminals. The heterocarrier-induced release occurs by conventional exocytosis. We here characterized the mechanism of the GABAA receptor-induced release and investigated what type(s) of voltage-sensitive Ca2+ channels (VSCCs) are involved in the GABA heterocarrier and GABA(A) receptor-evoked release. The effect of GABA in superfused rat hippocampal synaptosomes prelabeled with [(3)H]-NE was partially prevented by bicuculline or the GABA uptake inhibitor SKF 89976A and abolished by blocking both GABAA receptors and GABA transporters. The release elicited through GABAA receptors was Ca2+-dependent, prevented by Cd2+ or by botulinum toxin C, and modulated through alpha2 autoreceptors. The GABAA receptor-evoked release was insensitive to nifedipine and to omega-conotoxin MVIIC, but was inhibited ( approximately 50%) by omega-conotoxin GVIA. The heterocarrier-evoked release, nifedipine-insensitive, was inhibited approximately 30% either by omega-conotoxin GVIA or by omega-conotoxin MVIIC; the combined toxins produced approximately 60% inhibition. To conclude: a) the releases of NE evoked by activation of GABA(A) receptors and GABA heterocarriers are additive, although they both occur by conventional exocytosis; b) the heterocarrier-induced release requires activation of N and P/Q type channels, whereas the GABAA receptor-induced release only involves channels of the N type.

Effects of GABA-transporter (GAT) inhibitors on rat behaviour in open-field and elevated plus-maze

The behavioural consequences of inhibition of gamma-aminobutyric acid (GABA) uptake were studied. Two GABA uptake inhibitors, tiagabine and SKF 89976-A, were administered to rats, and behaviour was analysed 30 min later in a standard open field, an enriched open field, and an elevated plus-maze. Eight groups of animals received either saline (0.9%), tiagabine, or SKF 89976-A. At a dose of 18.5 mg/kg, tiagabine, an established antiseizure drug, impaired motor coordination, enhanced exploratory activity and reduced anxiety related behaviour. SKF 89976-A exhibited minimal effects over the dose range tested. These results indicate that inhibition of GABA uptake might be a pharmacological strategy to treat not only epilepsy, but also anxiety disorders.

GABA sensitivity of spectrally classified horizontal cells in goldfish retina.

We studied the GABA sensitivity of horizontal cells in the isolated goldfish retina. After the glutamatergic input to the horizontal cells was blocked with DNQX, GABA depolarized the monophasic and biphasic horizontal cells. The pharmacology of these GABA-induced depolarizations was tested with the GABA receptor antagonists bicuculline-methiodide and picrotoxin, the GABA transporter agonist nipecotic acid, and the GABA transporter antagonist SKF 89976-A. The GABA-induced responses of monophasic horizontal cells consisted of two components; one with characteristics of GABA-gated chloride channels, and one with characteristics of GABA transporters. In biphasic horizontal cells, we only found evidence for GABA-gated chloride channels. The results show that monophasic horizontal cells in goldfish contain the two components of a positive feedback loop (GABA transporters and GABA-gated chloride channels), as described in salamander. Furthermore, our results indicate that the monophasic horizontal cells may project directly to the biphasic horizontal cells, via an excitatory GABAergic pathway. We propose that the function of these GABAergic systems in horizontal cells is to abolish cone dominance in bipolar cells surround in the dark-adapted retina.

Activity at the GABA transporter contributes to acute cellular swelling produced by metabolic impairment in retina

The role of the GABA transporter in acute toxicity in chick retina due to metabolic inhibition was investigated by the use of several substrate (nipecotic acid, THPO) and nonsubstrate (SKF 89976A, NO711) GABA transport inhibitors. Metabolic stress-induced acute toxicity in the retina is characterized by swelling of distinct populations of retinal neurons and selective release of GABA into the medium. Inhibitor concentrations were based on that needed to attenuate 14C-GABA uptake at its approximate KM concentration by > or = 70%. Under basal conditions, substrate, but not nonsubstrate, inhibitors increased extracellular GABA, but did not cause histological swelling per se. Under conditions of glycolytic inhibition, nonsubstrate, but not substrate, inhibitors significantly attenuated acute toxicity. Metabolic stress-induced acute toxicity was not altered by the GABA agonist muscimol, nor did muscimol reverse the protective effects of nonsubstrate transport inhibitors, suggesting that an increase in extracellular GABA during metabolic stress was not a component of the acute phase of toxicity. The results indicate that during metabolic inhibition, activity at the GABA transporter contributes to acute cellular swelling.

Gp120 can revert antagonism at the glycine site of NMDA receptors mediating GABA release from cultured hippocampal neurons.

The effects of the human immunodeficiency virus type 1 envelope protein gp120 on the release of GABA elicited by N-methyl-D-aspartate (NMDA) from rat hippocampal neurons in primary culture has been investigated. NMDA (1-300 microM) increased in a concentration-dependent manner (EC50 =37.9+/-12 microM) the release of [3H]-GABA. The effect of 100 microM NMDA was prevented by 30 microM of the GABA transport inhibitor N-(4,4-diphenyl-3-butenyl)guvacine (SKF 100330A). Glycine (10 microM) or gp120 (0.01 microM) affected neither the basal nor the NMDA-evoked [3H]-GABA release. The NMDA (100 microM)-evoked release was prevented by 5,7-dichloro-kynurenic acid (5,7-DCKA), a selective antagonist at the glycine site of the NMDA receptor, in a concentration-dependent manner (IC50 approximately 0.3 microM). Glycine (3-10 microM) or gp120 (0.003-0.01 microM) produced reversal of the 5,7-DCKA antagonism in a way that suggested competition at a same site; gp120 was at least 3 orders of magnitude more potent than glycine. It is suggested that gp120 may mimic glycine at NMDA receptors.

Tyrosine 140 of the γ-Aminobutyric Acid Transporter GAT-1 Plays a Critical Role in Neurotransmitter Recognition

The gamma-aminobutyric acid (GABA) transporter GAT-1 is located in nerve terminals and catalyzes the electrogenic reuptake of the neurotransmitter with two sodium ions and one chloride. We now identify a single tyrosine residue that is critical for GABA recognition and transport. It is completely conserved throughout the superfamily, and even substitution to the other aromatic amino acids, phenylalanine (Y140F) and tryptophan (Y140W), results in completely inactive transporters. Electrophysiological characterization reveals that both mutant transporters exhibit the sodium-dependent transient currents associated with sodium binding as well as the chloride-dependent lithium leak currents characteristic of GAT-1. On the other hand, in both mutants GABA is neither able to induce a steady-state transport current nor to block their transient currents. The nontransportable analog SKF 100330A potently inhibits the sodium-dependent transient in the wild type GAT-1 but not in the Y140W transporter. It partly blocks the transient of Y140F. Thus, although sodium and chloride binding are unimpaired in the tyrosine mutants, they have a specific defect in the binding of GABA. The total conservation of the residue throughout the family suggests that tyrosine 140 may be involved in the liganding of the amino group, the moiety common to all of the neurotransmitters.

Electrical stimulation of the substantia nigra reticulata: detection of neuronal extracellular GABA in the ventromedial thalamus and its regulatory mechanism using microdialysis in awake rats.

A combination of electrical stimulation and microdialysis was used to study the nigrothalamic gamma aminobutyric acid (GABA)ergic system and its regulatory mechanisms in awake rats. Extracellular GABA levels in the ventromedial nucleus of the thalamus were detected in 3-min fractions collected before, during and after a 10-min stimulation period of the substantia nigra reticulata. Electrical stimulation of the substantia nigra reticulata increased the GABA levels to 155% of basal values in the ventromedial thalamus only during the first 3-min interval upon stimulation. The increase in GABA levels was tetrodotoxin-dependent, implicating an exocytotic origin. The basal levels of extracellular GABA in the ventromedial thalamus were of nonexocytotic origin. To study the mechanism underlying the fast compensatory response in neuronal GABA release after nigral stimulation, local infusions into the ventromedial thalamus of reuptake inhibitors and GABA antagonists were performed and the effect of nigral stimulation was examined under the various applications. Local infusion of the reuptake inhibitors nipecotic acid (500 microM) and SKF 89976-A (20 and 50 microM) increased extracellular GABA levels to 350%, 180% and 600%, respectively, of basal values in the ventromedial thalamus tetrodotoxin-independently. Under these conditions, the increase in extracellular GABA was absent (nipecotic acid) or suppressed (20% of basal values; SKF 89976-A for both doses), leaving it unsolved whether or not the uptake system was responsible for the fast compensation in neuronal GABA after stimulation. The GABA-A antagonist bicucilline (50 microM) was ineffective when infused locally in the ventromedial thalamus, but prolonged the increase in neuronal GABA release after nigral stimulation; the GABA levels were increased during two 3-min samples to approximately 165%, indicating a functional role for GABA-A receptors in regulating the release of GABA from nigrothalamic GABAergic neurons. The GABA-B receptor antagonist CGP 35348 (50 microM) did not affect GABA levels when infused locally in the ventromedial thalamus and neither affected the response in neuronal GABA after stimulation. This finding does not support a role for GABA-B receptors in controlling the release from the nigrothalamic neurons.

GABAergic drugs and sexual motivation, receptivity and exploratory behaviors in the female rat.

Female rats were allowed to pace sexual interactions in a bilevel chamber, where a sexually vigorous male was tethered to the bottom level. Exploratory behaviors (sniffing, rearing), locomotor activity (expressed as number of level changes and periods of inactivity) as well as items of sexual motivation (latency to descend to the male's level, approaches towards the male and genital exploration) were recorded. In addition, sexual receptivity was evaluated in a non-paced situation. A test for motor impairment was also performed. The GABA transaminase inhibitor gamma-acetylene GABA reduced exploratory behaviors at doses much lower than those needed to reduce receptivity. The GABA reuptake inhibitor SKF 100330A did not affect any behavior category at doses of 15 and 30 mg/kg, but had a sedative action at 60 mg/kg. This was shown as impaired motor coordination and an almost total absence of activity in the bilevel chamber. Receptivity was not impaired, however. The mixed GABAA/ GABAB agonist progabide reduced exploratory behaviors and receptivity without producing motor impairment at a dose of 400 mg/kg. The GABAA agonist THIP impaired motor coordination and reduced receptivity and exploratory behaviors at a dose of 10 mg/kg. A larger dose, 20 mg/kg, had a strong sedative action. Only a small proportion of the animals descended to the males level. The GABAB agonist baclofen reduced receptivity at a dose that had no effect on motor coordination or exploratory behaviors. None of the drugs had a specific effect on sexual motivation. Whenever behaviors reflecting motivation were reduced, there were also other behavioral effects indicative of sedation. These data show that GABA receptor agonists, particularly the GABAB agonist baclofen, reduce sexual receptivity at doses that have only slight effect on motor functions or exploratory behaviors. In contrast, non-specific enhancement of GABAergic activity by a transaminase or reuptake inhibitor have effects on motor functions and exploratory behaviors at doses much lower than those needed to reduce receptivity.

Attenuation of excitotoxic cell swelling and GABA release by the GABA transport inhibitor SKF 89976A.

Acute excitotoxicity in the chick retina is characterized by cellular swelling and the subsequent selective release of GABA. In order to understand the source of GABA release, embryonic day 15 retina were incubated with 1 mM glutamate for 30 min in the presence or absence of the GABA transport inhibitor SKF 89976A (1-100 microM). SKF 89976A dose-dependently attentuated glutamate-induced GABA release (IC50, 39 microM). Histological examination of retina showed that SKF 89976A greatly reduced cellular swelling caused by glutamate exposure. Interaction of SKF 89976A with glutamate receptors was ruled out as a possible reason for protection vs acute glutamate excitotoxicity, since SKF 89976A had no effect on glutamate receptor-induced 22Na+ influx. In contrast, the NMDA antagonist, MK-801, significantly blocked glutamate-evoked 22NA+ uptake. These studies indicate that reversal of the GABA transporter contributes to the bulk of GABA release during acute excitotoxicity in retina. Further, a net effect of the presence of SKF 89976A during glutamate exposure is reduction in cellular swelling. It is not clear at present if attenuation of swelling is mediated specifically by an interaction with the GABA transporter or by a nonspecific or indirect effect of SKF 89976A.

Inhibition of SKF 89976–A of the y–aminobutyric acid release from primary neuronal chick cultures

Neuronal cultures were made from the 8-d-old embryonic chick telencephalon. The primary culture model was further improved, the medium composition was modified, and the cells grown for 10 d, which allowed the development of relatively differentiated neurones. A superfusion protocol was developed and applied to study the release of [3H]-gamma-aminobutyric acid ([3H]GABA). High endogenous activity levels of glutamate decarboxylase (GAD) and of a Ca-dependent potassium stimulated [3H]GABA release were used as criteria for GABAergic differentiation. The influence of the non-substrate inhibitor of GABA transport, SKF 89976-A, on the GABA release, was studied using the primary neuronal culture. The release was found to be inhibited by SKF 89976-A at higher concentrations (= 400 microM).

Frequency-dependent enhancement of hippocampal inhibition by GABA uptake blockers

The effects of GABA uptake inhibitors, SKF 89976A and SKF 100330A, on recurrent inhibition were studied in the rat hippocampal slice preparation by the antidromic-orthodromic stimulation test. Population spikes evoked orthodromically by stimulation of the stratum radiatum and recorded in the CA1 pyramidal cell body layer were inhibited antidromically by stimulation of the alveus by a single pulse or by a train of pulses, either at low or at high frequency. Low frequency train conditioning produced less inhibition than a single pulse. The uptake blockers had no effect or slightly enhanced the inhibition produced by single stimuli or low frequency trains. High frequency train conditioning produced more and much longer inhibition than a single pulse. This inhibition was further substantially enhanced and prolonged by the drugs. Frequency-dependent enhancement of inhibition may be responsible for suppression of epileptiform discharges by GABA uptake blockers.

Depolarization by K + and glutamate activates different neurotransmitter release mechanisms in gabaergic neurons: Vesicular versus non-vesicular release of GABA

Neurotransmitter release and changes in the concentration of intracellular free calcium ([Ca ++] i) were studied in cultured GABAergic cerebral cortical neurons, from mice, upon depolarization with either an unphysiologicaLly high potassium concentration (55 mM) or the physiological excitatory neurotransmitter glutamate (100 μM). Both depolarizing stimuli exerted prompt increases in the release of preloaded [ 3H]GABA as well as in [Ca ++] i. However, the basic properties of transmitter release and the increase in [Ca ++] i, under a variety of conditions were different during stimulation with K + or glutamate. Potassium-evoked release of [ 3H]GABA consisted of two phases, a rapid, large and transient phase followed by a smaller, more persistent second phase. The rapid phase was inhibited (60%) by nocodazole which reduced the number of vesicles in the neuntes by 80%. This rapid phase of the GABA release was also reduced by organic (verapamil) and inorganic (Co ++) Ca ++ channel blockers but was insensitive to the GABA transport inhibitor SKF 89976A. In contrast, the second phase was less sensitive to nocodazole and Ca ++ channel antagonists but could be inhibited by SKF 89976A. The glutamateinduced [ 3H]GABA release, which was mainly mediated by N-methyl- d-aspartate receptors, consisted of a single, sustained phase. This was insensitive to nocodazole, partly inhibited by verapamil and could be blocked by Co ++ as well as SKF 89976A. The action of Co ++ could be attributed to a block of N-methyl- d-aspartate-associated ion channels. These findings strongly suggest that the majority of the K +-stimulated GABA release is dependent upon vesicles whereas the glutamate induced release is non-vesicular and mediated by a depolarization-dependent reversal of the direction of high-affinity GABA transport. The basic differences in the mode of action of the two depolarizing stimuli were reflected in the properties of the increase in [Ca ++] i elicited by 55 mM K + and 100μM glutamate, respectively. The K +-induced increase in [Ca ++] i was reduced by both verapamil and Ca ++-free media whereas the corresponding glutamate response was only sensitive to Ca ++-free conditions. Exposure of the cells to nocodazole or SKF 89976A had no effect on the ability of K + or glutamate to increase [Ca ++] i. Altogether, the results clearly demonstrate that K +-induced transmitter release from these GABAergic neurons is vesicular in nature whereas that induced by the neurotransmitter glutamate is not.

Pharmacological characterization of GABAB-mediated responses in the CA1 region of the rat hippocampal slice

It is generally accepted that the bicuculline-resistant responses to GABA are mediated through the activation of GABAB receptors that mediate a slow IPSP. However, a number of reported observations are difficult to reconcile with this model. Specifically, GABAB antagonists only partially block bicuculline-resistant GABA responses, and both 4-aminopyridine (4-AP) and carbachol have been reported to block responses to the selective GABAB agonist baclofen, but not GABA itself. Thus, it has been argued that baclofen and GABA increase potassium conductance through separate receptor mechanisms. This suggestion is not easily reconcilable with the postulated physiological role of GABAB receptors in mediating the slow IPSP. We have addressed these discrepancies by using the new GABAB antagonists 2-hydroxy-saclofen (2-OH-SAC) and CGP 35348 in the presence of the GABA uptake inhibitor SKF 89976A. The weak antagonism of 2-OH-SAC against the bicuculline-resistant GABA response was improved when the GABA uptake was inhibited with SKF 89976A, allowing for the application of lower GABA concentrations. Under these circumstances, 2-OH-SAC and CGP 35348 strongly antagonized GABA and baclofen responses, but did not have any effect on outward currents evoked by 5-HT. The slow IPSP evoked in the presence of glutamate antagonists was reversibly inhibited by CGP 35348 (IC50 = 14 microM), without affecting the fast IPSP. Carbachol (0.3-20 microM) had no effect on outward currents evoked by either baclofen or GABA. 4-AP (5 microM to 1 mM), despite causing a large increase in cell excitability, did not change baclofen responses. Higher concentrations of 4-AP (5 mM) induced inward current, and reduced both baclofen and GABA outward currents to a similar extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of transporter mediated ?-aminobutyric acid (GABA) release by SKF 89976-A, a GABA uptake inhibitor, studied in a primary neuronal culture from chicken

The effect of SKF 89976-A, a lipophilic non-substrate inhibitor of the gamma-aminobutyric acid (GABA) transporter, on the release of radioactive GABA and D-aspartate has been studied. Neuronal cultures from 8 day old chick embryos, grown for six days, served as a model. The cultures were incubated with [3H] D-aspartate and [14C] GABA with the subsequent addition of high or low concentrations of SKF 89976-A. Finally the cultures were exposed to differently composed media for either 30 or 300 seconds. The release was quantified, using liquid scintillation counting. The efflux of [3H] D-aspartate and [14C] GABA was increased by [K+] and time, and a minimum value was obtained at [Ca2+] 1.05 mM. The release of both [3H] D-aspartate and [14C] GABA was inhibited by SKF 89976-A. The obtained results indicate that transporter mediated processes are the major mechanisms of transmitter release in the investigated model.

On the activity of gamma-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes.

The uptake of radioactive gamma-aminobutyric acid (GABA) and D-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P2-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [3H]GABA and for D-[3H]aspartate with an increasing Vmax during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P2-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake of D-aspartate. We found no non-saturable uptake of D-aspartate.

GABAergic Influences on Somatostatin Secretion from Hypothalamic Neurons Cultured in Defined Medium

The effects of GABAergic influences intrinsic to the hypothalamus on the secretion of somatostatin were studied using cultured fetal rat hypothalamic neurons. The existence of GABAergic neurons within the cultures was confirmed by immunocytochemistry. These neurons appeared to be actively secreting GABA as antagonism of GABAA receptors with bicuculline and picrotoxinin caused a dose-dependent increase in the release of immunoreactive somatostatin (SRIF), which was Ca(2+)-dependent. Although exogenous GABA inhibited SRIF secretion at concentrations of 10(-6) M and greater, muscimol, a GABAA agonist, inhibited SRIF release at 10(-8) M, whereas baclofen, a GABAB agonist, required concentrations two orders of magnitude greater to produce an effect. Phaclofen, a GABAB antagonist, was inactive (10(-8)-10(-4) M). A GABA uptake inhibitor, SKF 89976A, produced a dose-dependent inhibition of SRIF release. These results, therefore, support a role for intrahypothalamic GABA neurons in the regulation of SRIF secretion in the neonatal rat, predominantly via a type A receptor, and provide further evidence for a neuroendocrine role for GABA in controlling growth hormone secretion.

Biochemical and behavioral studies following subchronic administration of GABA uptake inhibitors in mice

N-(4,4-Diphenyl-3-butenyl) nipecotic acid (SKF(R)-89976A) and N-(4,4-diphenyl-3-butenyl) guvacine (SKF 100330A) are potent inhibitors of the uptake of GABA and have anticonvulsant properties. In the present study, the effects of these compounds on several behavioral and biochemical measures were determined, following subchronic administration. Administration of SKF(R)-89976A (8.9 mg/kg) for 14 days caused a small but significant reduction in its potency to protect against pentylenetetrazole-induced seizures, whereas treatment with SKF 100330A (13.6 mg/kg) had no significant effect. The percentage of animals rendered cataleptic by administration of either GABA uptake inhibitor was reduced by treatment for as few as 4 days and treatment with SKF(R)-89976A for 14 days resulted in a 4-fold increase in the CD 50 for induction of catalepsy. The binding of [ 3H]GABA A and [ 3H]GABA B in membranes from the forebrain of the mouse were not influenced by treatment with drug nor was synaptosomal uptake of [ 3H]GABA. Likewise, the binding of [ 3H]sulpiride in striatal membranes of the mouse was unaffected by repeated exposure to SKF(R)-89976A. These results demonstrate that prolonged administration of GABA uptake inhibitors produced only a small reduction in anticonvulsant potency, whereas liability to side-effects, as demonstrated by the reduction in catalepsy, was substantially reduced.

On the inhibition of GABA release from a preparation of rat brain nerve-endings by SKF 89976-A, a GABA uptake inhibitor

Fractions enriched in nerve-terminals were made in order to investigate the release of γ-aminobutyric acid (GABA). A P 2-preparation made from whole rat brain was pre-labeled with [ 3H]GABA and thereafter incubated in high concentrations (1 mM) of the GABA uptake inhibitor SKF 89976-A. The nerve-terminals were thereafter incubated in depolarizing concentrations of KCl and finally the medium and nerve-terminals separated by a filtration procedure. The amount of [ 3H]GABA present on the filter and in the supernatant was measured by liquid scintillation counting and used to assess the release rate. SKF 89976-A reduced the KCl-induced efflux of [ 3H]GABA from the nerve-terminals. The inhibition was as effective against KCl-induced [ 3H]GABA efflux in the presence as well as in the absence of Ca 2+. Our finding may demonstrate an inhibition of the inside-out activity of the GABA transporter.

Interactions of GABA A antagonists with deltamethrin, diazepam, pentobarbital, and SKF100330A in the rat dentate gyrus

Activation of dentate gyrus granule cells leads to a period of depressed excitability mediated primarily by recurrent inhibition. This response can be increased in intensity and duration by drugs that enhance GABA A-mediated inhibition including GABA uptake blockers (SKF100330A), benzodiazepines (diazepam), and barbiturates (pentobarbital). It can be attenuated or abolished by administration of the GABA A antagonists picrotoxin and bicuculline. Type II pyrethroids, such as deltamethrin, produce effects in vitro which lead one to expect that they would decrease GABA-mediated inhibition. However, administration of deltamethrin to animals intensifies the inhibitory consequences of granule cell activation by many fold. To determine the role of GABA A-mediated mechanisms in the action of deltamethrin, we have looked at its antagonism by bicuculline and picrotoxin. Neither GABA A antagonist reduced the duration of the inhibition produced by deltamethrin. The only effect observed was a small but significant reduction in intensity of inhibition during the period of 10–40 msec after activation of the granule cells. For comparative purposes we have also examined the antagonism produced by bicuculline and picrotoxin on agents known to enhance GABA A-mediated inhibition. These antagonists were quite effective in antagonizing the enhancement of inhibition produced by diazepam a and SKF 100330A. Bicuculline was also effective in antagonizing pentobarbital while picrotoxin was not. These results suggest that the prolongation of inhibition produced by deltamethrin in dentate granule cells does not result simply from an extension of GABA A-mediated inhibition, but rather derives from a different source.

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

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