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PubMed1989

Developmental changes in the calcium dependency of gamma-aminobutyric acid release from isolated growth cones: correlation with growth cone morphology.

We have investigated the development of Ca2+-dependent gamma-[3H]aminobutyric acid [( 3H]GABA) release in superfused growth cone fractions isolated from rats between the postnatal ages of 1 and 11 days. We have compared this release with the overall morphology of the subcellular fractions, and identified those structures taking up [3H]GABA by electron microscopical autoradiography. In fractions isolated from rats between 1 and 5 days, K+-evoked [3H]GABA release was completely independent of extracellular Ca2+. After 5 days a Ca2+ dependency appeared, which increased with age, such that by 10 days approximately 50% of the K+-evoked release was Ca2+ dependent. Electron microscopical analysis showed that, at all ages, large numbers of GABAergic growth cones were present in the subcellular fractions. Up to postnatal day 5, the growth cones were synaptic vesicle sparse but, after this age, increasing numbers of synaptic vesicle-containing growth cones were seen. These results suggest that during maturation of GABAergic growth cones into synapses there is, initially, a mechanism for release that is independent of extracellular Ca2+ and that the appearance of a Ca2+-dependent [3H]GABA release from growth cones correlates with the appearance of synaptic vesicles.

Aging↗
PubMed1995

Novel properties of homomeric beta 1 gamma-aminobutyric acid type A receptors: actions of the anesthetics propofol and pentobarbital.

In this study we determined the influence of gamma-aminobutyric acid (GABA)A receptor subunit composition on the direct effects of the general anesthetics propofol, pentobarbital, and alphaxalone, using recombinant receptors expressed in Xenopus oocytes. cDNAs coding for human beta 1, alpha 1 beta 1, or alpha 1 beta 1 gamma 2S GABAA receptor subunits were injected into Xenopus oocytes, and responses induced by either GABA or anesthetics were measured by two-electrode voltage-clamp recording. Expression of homomeric beta 1 receptors resulted in the formation of a Cl- channel that was sensitive to picrotoxin and strychnine and could be activated, albeit with relatively low potency, by GABA. However, GABA-induced currents of homomeric beta 1 receptors were completely insensitive to the GABAA receptor antagonist bicuculline. Homomeric beta 1 receptors showed marked direct activation by propofol or pentobarbital, but not by alphaxalone. In contrast, these three anesthetics induced much weaker direct activation of Cl- currents in oocytes expressing alpha 1 beta 1 or alpha 1 beta 1 gamma 2S receptors. These data indicate that the beta 1 subunit of the GABAA receptor forms a functional Cl- channel that contains sites for the direct activating effects of GABA, propofol, and pentobarbital and this GABA site is not blocked by bicuculline.

Animals↗
PubMed1999

Regulation of the gamma-aminobutyric acid transporter activity by protein phosphatases in synaptic plasma membranes.

The influence of the phosphorylation dephosphorylation states on the gamma-aminobutyric acid (GABA) transporter activity of synaptic plasma membranes (SPM) was studied by using either specific phosphatase inhibitors or activators. Calyculin A and okadaic acid (phosphatase 1 and phosphatase 2A inhibitors) inhibited the GABA uptake by isolated SPM vesicles, whereas cyclosporin A (phosphatase 2B inhibitor) had a stimulatory effect (approximately 10%) which was higher (approximately 38%) when all these drugs were present in the reaction medium. On the other hand, intravesicular Ca2+, up to about 10 microM, inhibited the GABA uptake (approximately 50%) in a manner which appeared to be facilitated in the presence of PP1 and PP2A inhibitors and this inhibition was relieved by the calmodulin antagonist W-7. We also observed that isolated SPM vesicles contain both Ca(2+)-independent phosphatase activity that is significantly inhibited by PP1 and PP2A inhibitors, and Ca(2+)-dependent phosphatase activity that is abolished in the presence of the PP2B inhibitor, cyclosporin A. These results indicate that regulation of the SPM GABA transporter is determined by the internally localized Ca-calmodulin-dependent phosphatase activity (calcineurin), and that other phosphorylated sites, sensitive to PP1 and PP2A inhibitors, potentiate either the positive or negative effects exerted by those internal sites when they are in their phosphorylated or dephosphorylated states, respectively.

Animals↗
PubMed1986

Development of gamma-aminobutyric acid immunoreactivity in chick hyperstriatum ventrale and cerebellum: light and electron microscopical observations.

The presence of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the avian hyperstriatum ventrale and dorsal cerebeller vermis was investigated immunocytochemically using a recently characterized antiserum raised against GABA. Tissue from domestic chicks aged from 19 days in ovo to 28 days posthatch was studied with both light and electron microscopy using pre-embedding immunocytochemistry. Basket, stellate and Golgi cells in the cerebellum, considered to be GABA-ergic, exhibited specific GABA-like immunolabelling in perikarya and in axonal and dendritic processes throughout the developmental period investigated. Purkinje cells also exhibited specific GABA-like immunoreactivity in both pre- and posthatch birds but the distribution and intensity of the immunolabelling varied with age and also in its location within the Purkinje neuron. In prehatch birds Purkinje perikarya exhibited heavy immunostaining which was substantially reduced posthatch, whereas the Purkinje primary dendrites remained immunopositive throughout the developmental period. A small population of cells in the medial hyperstriatum ventrale (mHV) were GABA-positive prehatch, but no immunopositive perikarya were evident in any posthatch samples. Small GABA-positive punctate profiles, representing boutons and transversely sectioned axons or dendrites, were present in the neuropil of all age groups studied. Possible reasons for these findings are discussed and it is suggested that the loss of perikaryal immunostaining, both in the cerebellar Purkinje cells and in those of the mHV, may be governed by maturational processes.

Animals↗
PubMed1989

Shapes and projections of neurons with immunoreactivity for gamma-aminobutyric acid in the guinea-pig small intestine.

The distribution of nerve cell bodies and fibres with immunoreactivity for gamma-aminobutyric acid (GABA) has been studied in the guinea-pig small intestine. Cell bodies were common in myenteric ganglia but were extremely rare in the submucosa. Reactive fibres were numerous in the tertiary component of the myenteric plexus and in the circular muscle but they were rare in both myenteric and submucous ganglia. Reactive nerve fibres were absent from the mucosa. This distribution conforms to previous descriptions. Exposure to exogenous GABA, in vitro, was used to supplement endogenous stores of GABA. The morphology of cell bodies was better defined after this treatment. Nearly all cell bodies had type-I morphology, i.e., the cells had numerous short lamellar dendrites and one axon. Most axons ran anally. Some could be traced to the tertiary component of the myenteric plexus, others to the circular muscle. Removal of the myenteric plexus from a short length of intestine caused a loss of nerve fibres from the circular muscle beneath the site of operation and a decrease in fibre density in the circular muscle that extended anally from the lesion for about 1 mm. The nerve lesions caused no significant changes in the tertiary plexus. It is concluded that GABA is contained in motor neurons supplying the longitudinal and circular muscle, and that the neurons supplying the circular muscle may be inhibitory.

Animals↗
PubMed1992

Modulation of extracellular gamma-aminobutyric acid in the ventral pallidum using in vivo microdialysis.

Intracranial microdialysis was used to investigate the origin of extracellular gamma-aminobutyric acid (GABA) in the ventral pallidum. Changes in basal GABA levels in response to membrane depolarizers, ion-channel blockers, and receptor agonists were determined. Antagonism of Ca2+ fluxes with high Mg2+ in a Ca(2+)-free perfusion buffer decreased GABA levels by up to 30%. Inhibition of voltage-dependent Na+ channels by the addition of tetrodotoxin also significantly decreased basal extracellular GABA concentrations by up to 45%, and blockade of Ca2+ and Na+ channels with verapamil reduced extracellular GABA by as much as 30%. The addition of either the GABAA agonist, muscimol, or the GABAB agonist, baclofen, produced a 40% reduction in extracellular GABA. GABA release was stimulated by high K+ and the addition of veratridine to increase Na+ influx. High K(+)-induced release was predominantly Ca(2+)-dependent, whereas the effect of veratridine was potentiated in the absence of extracellular Ca2+. Both high K(+)- and veratridine-induced elevations in extracellular GABA were inhibited by baclofen, whereas only veratridine-induced release was antagonized by muscimol. These results demonstrate that at least 50% of basal extracellular GABA in the ventral pallidum is derived from Ca(2+)- or Na(+)-dependent mechanisms. They also suggest that Na(+)-dependent release of GABA via reversal of the uptake carrier can be shown in vivo.

Animals↗
PubMed1992

Neither amino nor carboxyl termini are required for function of the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain.

Antibodies were raised against synthetic peptides corresponding to several regions of the rat brain gamma-aminobutyric acid (GABA) transporter. According to our model, this glycoprotein has 12 transmembrane alpha-helices with both amino and carboxyl termini located in the cytoplasm. The antibodies recognized the intact transporter on Western blots. Upon papain treatment, a reconstitutively active transporter can be isolated upon lectin chromatography (Kanner, B. I., Keynan, S., and Radian, R. (1989) Biochemistry 28, 3722-3728). The papainized transporter runs on sodium dodecyl sulfate-polyacrylamide gels as a broad band with an apparent molecular mass between about 58 and 68 kDa as compared to 80 kDa for the untreated transporter. The transporter fragment was recognized by all the antibodies, except for that raised against the amino terminus. Pronase cleaves the transporter to a relatively sharp 60-kDa band, which reacts with the antibodies against the internal loops but not with either the amino- or the carboxyl-terminal. This pronase-treated transporter, upon isolation by lectin chromatography, was reconstituted. It exhibits full GABA transport activity. This activity exhibits the same features as the intact system including an absolute dependence on sodium and chloride as well as electrogenicity. We conclude that the amino- and carboxyl-terminal parts of the transporter, possibly including transmembrane alpha-helices 1, 2, and 12, are not required for the transport function.

Animals↗
PubMed1985

Excitatory transmitter release induced by high concentrations of gamma-aminobutyric acid (GABA) in crayfish neuromuscular junctions.

At the neuromuscular junction of very small crayfish (0.4-2 g) addition of gamma-aminobutyric acid (GABA) to the superfusing solution at concentrations exceeding 100 mmol/l elicited high frequency release of excitatory transmitter quanta. In seven experiments single application of 500 mmol/l GABA gave rise to instantaneous release of 70,000 to 130,000 quanta. These stores of transmitter were released by GABA in a first order process with time constants, tau q, of between 9 s and 20 s, the maximum rate of release, ñ0, reaching 10,000 quanta/s in some cases. After release had ceased in the presence of GABA, the preparation was allowed to recover for five minutes in normal solution. Subsequently, a second trial evoked about 50% of the release induced during the first application of GABA. Pretreatment of the preparation with 2 mumol/l serotonin (5-HT) facilitated GABA-induced transmitter release resulting in larger rates of release and consequently in a larger output of transmitter by a factor of about 3. The largest amount of transmitter released on a single application of GABA in the presence of serotonin comprised about 220,000 quanta with a maximum rate of release ñ0 approximately equal to 25,000 quanta/s. The release evoked by high GABA-concentrations did not depend markedly on extracellular Ca2+ or Mg2+, but required extracellular Na+. The effects induced by high concentrations of GABA on release of excitatory transmitter quanta were quantitatively similar to the effects of high glycine-concentrations on release of quanta from the inhibitory terminals (Finger 1983a, b).

Animals↗
PubMed1987

gamma-Aminobutyric acid opens Cl-channels in cultured astrocytes.

Cultured astrocytes from cerebral hemispheres of early postnatal rats responded to gamma-aminobutyric acid (GABA) with membrane depolarization. This depolarization was affected by changes in extracellular [Cl-] and depended on the membrane potential. The reversal potential of the GABA-induced depolarization was determined by double electrode voltage clamp or depolarization by elevated [K+]o and ranged between -38 and -53 mV. Cell input resistance decreased after addition of GABA with the same time course as the membrane depolarization. GABA responses were temperature dependent yielding a peak at about 14 degrees C. At higher temperatures a decrease in the GABA-induced depolarization was seen indicating that the depolarization may not be mediated by an enzyme-coupled carrier system. Addition of ouabain at different temperatures did not change the size of the GABA depolarization. This excludes the possibility that an electrogenic component of the temperature-sensitive Na+,K+-ATPase activity causes the decrease in GABA-dependent depolarization at higher temperatures. Intracellular [Cl-] was measured with Cl- sensitive microelectrodes and found to be higher than the value calculated for a free distribution according to the Nernst equation (-40 mV). Addition of furosemide did not alter the reversal potential, but reduced the size of the GABA-induced membrane depolarization. From these observations and previous experiments on the pharmacological properties of the membrane response we conclude that the ionic mechanism underlying the GABA-dependent membrane depolarization of astrocytes results from a transient increase in Cl- -conductance similar to that of the neuronal GABAA-receptor.

Animals↗
PubMed1988

Effect of gamma-aminobutyric acid (GABA) on vasodilation in resistance-sized arteries isolated from the monkey, rabbit, and rat.

The effect of gamma-aminobutyric acid (GABA) and papaverine on cerebral arteries of rat, rabbit, and monkey and the small mesenteric arteries of the rat were studied in vitro with a microvessel apparatus. GABA (1 x 10(-7) to 1 x 10(-3) M) did not affect the basal tension of arteries of rats at rest. In PGF2 alpha-contracted monkey basilar artery and middle cerebral artery and rat basilar artery, cumulative addition of GABA (1 x 10(-7) to 1 x 10(-3) M) did not produce any relaxation. Also in K+-contracted rat basilar artery and small mesenteric artery, cumulative additions of GABA, muscimol, or bicuculline did not result in relaxation. In K+-contracted rabbit basilar artery, GABA did not produce relaxation. However, the addition of papaverine (1 x 10(-7) to 1 x 10(-4) M) in either PGF2 alpha- or K+-contracted arteries, produced a concentration-dependent relaxation in all arteries tested. These results suggest that the failure of GABA or muscimol to induce relaxation is not due to a defect of the arterial smooth muscle relaxant mechanism, but rather is due to the inability of GABA or muscimol to directly relax the artery in this in vitro preparation. Therefore, the hypotensive effect of GABA seen in the rat is probably not due to direct vasodilation of mesenteric or cerebral arteries. These findings lend further support to the idea that GABA mediates its hypotensive effect through its action as an inhibitory neurotransmitter, as previously suggested by others.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Osmotic shock: a method to eliminate endogenous gamma-aminobutyric acid and account for the influence on benzodiazepine binding affinity in autoradiographic studies.

Inasmuch as the presence of endogenous gamma-aminobutyric acid (GABA) may affect benzodiazepine binding to tissue sections in autoradiographic studies, a protocol designed to check for this influence has been investigated. [3H]Flunitrazepam (1 nM) was used to label benzodiazepine receptors for autoradiographic localization. Bicuculline was added to the incubation medium of an additional set of tissue sections to antagonize any potential effect of endogenous GABA. Binding in these sections was compared to that occurring in another set in which excess GABA was added to "create" further GABA enhancement. Binding also was compared to adjacent sections which were treated similarly but also preincubated in distilled-deionized water to burst the cells by osmotic shock and eliminate endogenous GABA, thereby preventing any effect on benzodiazepine binding. The results indicated that endogenous GABA is indeed present in the slide-mounted tissue sections and is affecting benzodiazepine receptor binding differentially in various regions of the brain depending on the density of GABAergic innervation. Scatchard analysis of saturation data demonstrated that the alteration in benzodiazepine binding due to GABA was a result of a change in the affinity rather than number of receptors present. These experiments have been compared to the binding of the imidazodiazepine, [3H] Ro15-1788. We also show that the treatments affect endogenous GABA and not the receptors themselves. This suggests strongly that, when using a single nonsaturating concentration of radiolabeled benzodiazepine antagonist, autoradiographic studies to date may have been subject to erroneous interpretation due to the differential effects of endogenous GABA on benzodiazepine binding (increased affinity).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1974

Depolarizing actions of gamma-aminobutyric acid and related compounds on rat superior cervical ganglia in vitro.

1 Potential changes in rat superior cervical ganglia were recorded in vitro with surface electrodes.2 gamma-aminobutyric acid (GABA) produced a transient, low-amplitude ganglion depolarization at rest, and a transient hyperpolarization in ganglia depolarized by carbachol. Depolarization was not prevented by preganglionic denervation. The log dose-response curve for depolarization was sigmoid with a mean ED(50) of 12.5 muM.3 The ganglion was depolarized in similar manner by the following compounds (mean molar potencies relative to GABA (=1) in brackets): 3-aminopropane sulphonic acid (3.4), gamma-amino-beta-hydroxybutyric acid (0.27), beta-guanidino-propionic acid (0.12), guanidinoacetic acid (0.057), delta-aminovaleric acid (0.048), beta-alanine (0.01), 2,4-diaminobutyric acid, gamma-guanidinobutyric acid, taurine and N-methyl-GABA (all <0.01). The following compounds did not depolarize the ganglion at 10 mM concentrations: alpha- and beta-amino-n-butyric acids, alpha-amino-iso-butyric acid, glycine and glutamic acid.4 Depolarization declined in the continued presence of GABA. Ganglia thus ;desensitized' to GABA showed a diminished response to other amino acids but not to carbachol.5 The effect of GABA was not antagonized by hyoscine and hexamethonium in combination, in concentrations sufficient to block responses to carbachol.6 Responses to GABA were blocked more readily than those to carbachol by bicuculline (IC(50), 14 muM) and picrotoxin (IC(50), 37 muM). Strychnine (IC(50), 73 muM) was a relatively weak and less selective GABA-antagonist.7 It is concluded that sympathetic ganglion cells possess receptors for GABA and related amino acids which are (a) different from the acetylcholine receptors and (b) similar to GABA receptors in the central nervous system.

Alanine↗
PubMed2006

Alkylene tether-length dependent gamma-aminobutyric acid type A receptor competitive antagonism by tacrine dimers.

Bis(7)-tacrine was previously demonstrated as an antagonist of gamma-aminobutyric acid type A (GABA(A)) receptors. In this study, the effects of a series of alkylene-linked tacrine dimers on GABA(A) receptors were examined. In radioligand binding assay, the analogues differed in binding affinity for GABA(A) receptors, and potency monotonically increased as the tether was shortened from nine to two methylenes. Bis(2)-tacrine, the shortest tacrine dimer, could displace [(3)H]muscimol from rat brain membranes with an IC(50) of 0.48 microM, which was 11, 13 and 525 times more potent than the GABA(A) receptor antagonist (+)-bicuculline, bis(7)-tacrine and tacrine, respectively. In whole-cell patch-clamp recordings, these dimeric tacrine analogues competitively antagonized GABA-induced inward current with a rank order of potency of bis(2)-tacrine>bicuculline>bis(7)-tacrine>bis(9)-tacrine>tacrine, and the potency of bis(2)-tacrine was 11, 18 and 487 times higher than that of (+)-bicuculline, bis(7)-tacrine and tacrine, respectively. Bis(2)-tacrine shifted the GABA concentration-response curve to the right in a parallel manner, and the inhibition was voltage-independent between -80 and +20 mV. It can be concluded that the shorter the alkylene linkage in tacrine dimers the stronger the binding affinity and higher the antagonistic effect on the GABA(A) receptor will be.

Alkylation↗
PubMed1991

Regulation of cardiovascular sympathetic neurons by substance P and gamma-aminobutyric acid in the rat spinal cord.

The spinal regulation of cardiovascular sympathetic preganglionic neurons by substance P (SP) and gamma-aminobutyric acid (GABA) was investigated in conscious rats. Intrathecal injection at the T-9 spinal level of bicuculline, a GABAA receptor antagonist, evoked increases in mean arterial pressure (MAP) and heart rate (HR) which were maximal at 5.0 and 0.5 nmol, respectively. Phaclofen, a GABAB receptor antagonist, produced no cardiovascular changes up to 2 mumol while 10 mumol evoked a rise in MAP and HR. Muscimol, a GABAA receptor agonist, produced a decrease in MAP which was maximal at 5.0 nmol and had no effect on HR. Baclofen, a GABAB receptor agonist, was without cardiovascular effects up to 5.0 nmol, while 50 and 100 nmol evoked a fall in MAP and HR. The pressor response to SP (16.25 nmol, T-9) was antagonised by 0.5-50 nmol muscimol or baclofen in a dose-related manner and the pressor response to SP was still inhibited by 40 nmol GABA in capsaicin-treated animals. However, when SP was injected at T-2, the rise in both MAP and HR was blocked by 50 nmol baclofen. Similarly, 50 nmol muscimol blocked the rise in both MAP and HR induced by 15 nmol thyrotropin-releasing hormone. In contrast, 50 nmol glycine failed to alter the cardiovascular response to SP co-injected either at T-9 or T-2. Baclofen was found to reduce significantly the basal release of epinephrine when injected at the T-9 level. These results provide pharmacological evidence for a possible tonic GABAergic inhibitory input onto cardiovascular sympathetic preganglionic neurons mediated by GABAA and GABAB receptors.

Animals↗
PubMed1988

[Characteristics of the effect of glycine and gamma-aminobutyric acid on the spinal cord neurons in the lamprey].

Responses of isolated spinal cord neurons of lamprey on glycine and gamma-aminobutyric acid (GABA) were investigated by means of intracellular perfusion and concentration clamp techniques. Responses on both amino acids exhibited fast but not full desensitization. Preincubation of neurons in the solution of one mediator led to full disappearance of the response to other mediator. It is suggested that these amino acids act on the same channel-receptor complex.

Animals↗
PubMed1999

Determination of striatal extracellular gamma-aminobutyric acid in non-hibernating and hibernating arctic ground squirrels using quantitative microdialysis.

This study determined extracellular concentrations of gamma-aminobutyric acid ([GABA](ecf)) in striatum of non-hibernating and hibernating arctic ground squirrels to test the hypothesis that an increase in [GABA](ecf) was associated with profound CNS depression during hibernation. Quantitative microdialysis procedures were employed to circumvent the effects of low temperature on the relative recovery of the analyte across the dialysis membrane and yielded for the first time quantitative in vivo estimates of [GABA](ecf) in any brain region or any species. Laboratory housed, wild caught Arctic ground squirrels (Spermophilus parryii) were implanted intraperitoneally with radio transmitters that enabled the telemetric monitoring of activity and core body temperature (T(b)) and bilaterally implanted with cranial guide tubes that enabled the implantation of microdialysis probes into the striatum. Striatal [GABA](ecf) was determined in unrestrained, non-hibernating ground squirrels (T(b) range 34.7-38.9 degrees C) and hibernating ground squirrels (T(b) range 2.9-3.9 degrees C) using extrapolation to zero flow and very slow flow microdialysis techniques. The results show that [GABA](ecf) in non-hibernating squirrels was 73 nM and this level was decreased by approximately 50% during hibernation thereby suggesting that an increase in [GABA](ecf) does not play a major role in CNS depression during hibernation. The reduction of [GABA](ecf) parallels a decrease in plasma and CSF [glucose] and may be related to a decrease in GABA synthesis or reduced voltage dependent release. This paper demonstrates that measurement of extracellular concentrations of neurotransmitters in animals with vastly different body temperatures is possible using microdialysis techniques of extrapolation to zero flow or very slow flow rates that enable 100% recovery. Such quantitative techniques may prove valuable in the study of the neurochemistry of the cerebral mechanisms of hibernation and tolerance to cerebral ischemia exhibited by hibernating animals.

Adaptation, Physiological↗
PubMed1988

Gamma-aminobutyric acid in the medial rat nucleus accumbens: ultrastructural localization in neurons receiving monosynaptic input from catecholaminergic afferents.

Neurons containing gamma-aminobutyric acid (GABA) in the medial portion of the adult rat nucleus accumbens were characterized with respect to their ultrastructure, sites of termination, and catecholaminergic input. Antisera against GABA-conjugates and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), were localized within single sections by means of peroxidase-antiperoxidase (PAP) and immunoautoradiographic labeling methods. Peroxidase reaction product indicating GABA-like immunoreactivity (GABA-LI) was seen in medium-size (15-20 microns) perikarya containing either round and unindented or invaginated nuclear membranes. The cells with invaginated nuclei were few in number and usually exhibited more intense peroxidase reaction product in sections collected at the same distance from the surface of the tissue. Reaction product for GABA was also detected in proximal (1.5-3.0 microns) dendrites, axons, and terminals. Terminals with GABA-LI formed symmetric junctions on perikarya, proximal dendrites, and dendritic spines of neurons that usually lacked detectable immunoreactivity. Many of the GABAergic terminals also were apposed directly to other unlabeled terminals and to terminals exhibiting either peroxidase labeling for GABA or immunoautoradiographic labeling for TH. Many of the unlabeled terminals associated with the GABAergic axons formed asymmetric junctions on dendritic spines. From 138 TH-labeled, principally dopaminergic terminals that were examined in the medial nucleus accumbens, 4% were associated with the somata of GABAergic neurons and another 14% formed symmetric junctions with proximal dendrite showing GABA-LI. The remaining TH-immuno-reactive terminals either lacked recognizable densities or formed symmetric synapses on unlabeled dendrites and spines. A few of the unlabeled dendrites, as well as those containing GABA-LI, received symmetric synapses from both catecholaminergic and GABAergic terminals. We conclude that in the medial portion of the rat nucleus accumbens, GABA is localized to two morphologically distinct types of neurons, one or both of which receive monosynaptic input from catecholaminergic afferents, and that GABAergic terminals form symmetric synapses on other principally non-GABAergic neurons. The results also support earlier physiological evidence showing that GABA may modulate the output of other GABAergic and non-GABAergic neurons through presynaptic associations.

Animals↗
PubMed1994

Stable expression of a neuronal gamma-aminobutyric acid transporter, GAT-3, in mammalian cells demonstrates unique pharmacological properties and ion dependence.

The transport proteins that mediate gamma-aminobutyric acid (GABA) reuptake have been major targets for the development of agents to treat neurological diseases such as epilepsy, where augmentation of GABAergic function is indicated. The recent isolation of cDNAs for four distinct brain GABA carriers has provided an avenue for creating more specific and selective antagonists of GABA transport. An LLC-PK1 cell line stably expressing GABA transporter type 3 (GAT-3), a beta-alanine-sensitive neuronal GABA transporter, has been generated and used to examine the kinetics, ion dependence, and pharmacological properties of the transporter. In this cell line, the GAT-3 carrier transports GABA with an apparent Km of 4 microM and a Vmax of 1.25 x 10(-16) mol/cell/min. beta-Alanine is a relatively potent inhibitor of GAT-3 GABA transport, with a K(i) value of 34 microM. beta-Alanine also serves as a substrate for the carrier (Km = 29 microM, Vmax = 1.82 x 10(-16) mol/cell/min) and appears to interact with the transporter at the same or a similar site as GABA. Other experimental GABA transport antagonists developed as anticonvulsant agents, including tiagabine, Cl-966, SKF-100330-A, SKF-89976-A, and NO-711, are weak inhibitors of GAT-3 GABA transport, suggesting that their therapeutic effects may be more related to their ability to block GABA transporters other than GAT-3. GAT-3 exhibits a sigmoidal dependence on Na+ concentration, with a Hill coefficient of 1.65, suggesting that more than one Na+ ion is involved in the transport mechanism. In contrast, the transport activity shows a hyperbolic Cl- dependence, with a Hill coefficient of 1.05. The Km for Cl- is 78 mM, a value severalfold higher than has been noted for another cloned GABA carrier, GABA transporter type 1. Interestingly, for GAT-3 a reduction of the Cl- concentration results in a small but consistent increase in the apparent Km for GABA, suggesting that the interaction of chloride with the transporter may be an important initial event in the mechanism of transport. These results underscore the unique properties of GAT-3 and distinguish this transporter as a new target for the development of GABA-mimetic agents.

Animals↗