Chronic GABA Research Articles

Chronic unilateral inhibition of GABA synthesis in the amygdala increases specificity of conditioned fear in a discriminative fear conditioning paradigm in rats

Neural activity in the amygdala is critical for fear learning. In anxiety disorder patients, bilateral hyperactivity of the amygdala can be observed. This hyperactivation is often associated with the facilitation of fear learning and/or over-generalization of conditioned fear. In contrast, hypoactivity of the amygdala, e.g. by pharmacological interventions, attenuates or blocks fear learning. To date, little is known about how neural excitability of the amygdala affects specificity or generalization of fear. Therefore, the present study utilized chronic inhibition of GABA synthesis in the amygdala to increase excitability and investigated the effect on the specificity of fear learning. In rats, unilateral cannulas aiming at the amygdala were implanted. The cannulas were connected to subcutaneously implanted osmotic mini pumps that delivered either the GABA synthesis inhibitor L-allylglycine or its inactive enantiomer D-allylglycine. Following one week of chronic GABA synthesis manipulation, the rats were submitted to a discriminative fear conditioning protocol. In addition, anxiety-like behavior in the light-dark box was measured. Our data show that chronic unilateral L-AG infusions into the amygdala improve the specificity of learned fear, support safety learning, and reduce fear generalization and anxiety. This data demonstrates that moderately increased amygdala excitability can be beneficial for the specificity of fear learning and highlights the potential application for therapeutic interventions.

Activation of arcuate nucleus GABA neurons promotes luteinizing hormone secretion and reproductive dysfunction: Implications for polycystic ovary syndrome

BackgroundEnhanced GABA activity in the brain and a hyperactive hypothalamic-pituitary-gonadal axis are associated with polycystic ovary syndrome (PCOS), the most common form of anovulatory infertility. Women with PCOS exhibit elevated cerebrospinal fluid GABA levels and preclinical models of PCOS exhibit increased GABAergic input to GnRH neurons, the central regulators of reproduction. The arcuate nucleus (ARN) is postulated as the anatomical origin of elevated GABAergic innervation; however, the functional role of this circuit is undefined. MethodsWe employed a combination of targeted optogenetic and chemogenetic approaches to assess the impact of acute and chronic ARN GABA neuron activation. Selective acute activation of ARN GABA neurons and their fiber projections was coupled with serial blood sampling for luteinizing hormone secretion in anesthetized male, female and prenatally androgenised (PNA) mice modelling PCOS. In addition, GnRH neuron responses to ARN GABA fiber stimulation were recorded in ex vivo brain slices. Chronic activation of ARN GABA neurons in healthy female mice was coupled with reproductive phenotyping for PCOS-like features. FindingsAcute stimulation of ARN GABA fibers adjacent to GnRH neurons resulted in a significant and long-lasting increase in LH secretion in male and female mice. The amplitude of this response was blunted in PNA mice, which also exhibited a blunted LH response to GnRH administration. Infrequent and variable GABAA-dependent changes in GnRH neuron firing were observed in brain slices. Chronic activation of ARN GABA neurons in healthy females impaired estrous cyclicity, decreased corpora lutea number and increased circulating testosterone levels. InterpretationARN GABA neurons can stimulate the hypothalamic-pituitary axis and chronic activation of ARN GABA neurons can mimic the reproductive deficits of PCOS in healthy females. Unexpectedly blunted HPG axis responses in PNA mice may reflect a history of high frequency GnRH/LH secretion and reduced LH stores, but also raise questions about impaired function within the ARN GABA population and the involvement of other circuits.

Polysomnographic abnormalities in succinic semialdehyde dehydrogenase (SSADH) deficiency.

Patients with SSADH deficiency, a disorder of chronically elevated endogenous GABA and GHB, were studied for sleep symptoms and polysomnography. We hypothesized that patients would have excessive daytime somnolence and decreased REM sleep. Polysomnography and MSLT were performed on patients enrolled for comprehensive clinical studies of SSADH deficiency. Sleep studies were obtained in the sleep laboratories at CNMC and NIH. Sleep recordings were obtained in 10 patients with confirmed SSADH deficiency. Thirteen overnight polysomnograms were obtained in 10 patients (7 male, 3 female, ages 11-27 y). Eleven MSLT studies were completed in 8 patients. Polysomnograms showed prolongation of REM stage latency (mean 272 +/- 89 min) and decreased percent stage REM (mean 8.9%, range 0.3% to 13.8%). Decreased mean sleep latency was present in 6 of 11 MSLTs. SSADH deficiency is associated with prolonged latency to stage REM and decreased percent stage REM. This disorder represents a model of chronic GABA and GHB accumulation associated with suppression of REM sleep.

Contribution of brainstem GABA A synaptic transmission to morphine analgesic tolerance

Chronic opioid-induced analgesic tolerance remains a major obstacle to improving clinical management of moderate to severe chronic pain. Our understanding of the underlying mechanisms for opioid tolerance is only partially understood at present. In this study, we investigated the effects of chronic morphine on GABA A receptor-mediated synaptic transmission, a major opioid target for pain inhibition, and the behavioral role of GABA synaptic transmission in the development of morphine tolerance. In the nucleus raphe magnus (NRM), a critical brainstem site for opioid analgesia, the GABA A receptor-mediated inhibitory postsynaptic current (IPSC) was significantly increased in NRM neurons kept in a morphine-tolerant state from chronic morphine-treated rats. The potency of cAMP analogs for enhancing the GABA IPSC was also enhanced. The protein kinase A (PKA) inhibitor H89 reversed the chronic morphine-induced synaptic adaptation in GABA IPSCs. Behaviorally, a low dose of GABA A receptor antagonist bicuculline microinjected into the NRM, ineffective alone, blocked morphine antinociception in control rats, but failed to do so in morphine-tolerant rats. With chronic treatment through daily NRM microinjections, bicuculline augmented the development of morphine tolerance, whereas the GABA A receptor agonist muscimol or H89 significantly attenuated the development of morphine tolerance. These results suggest that chronic morphine increases GABA synaptic activity through upregulation of the AMP system in morphine-tolerant NRM neurons and that while chronic GABA A receptor antagonism in the NRM augments morphine tolerance, chronic activation of NRM GABA A receptors or PKA inhibition reduces morphine tolerance with increased analgesic efficacy of chronic morphine.

Prolonged exposure to γ-aminobutyric acid up-regulates stably expressed recombinant α1β2γ2s GABA A receptors

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABA A receptors following their prolonged exposure to drugs. Exposure (48 and/or 96 h) of human embryonic kidney (HEK 293) cells stably expressing recombinant α1β2γ2s GABA A receptors for γ-aminobutyric (GABA, 1 mM) and muscimol (100 μM), but not for diazepam (1 μM), enhanced the maximum number ( B max) of [ 3H]flunitrazepam binding sites without affecting their affinity ( K d). The GABA-induced enhancement in B max was reduced by the GABA receptor antagonist, bicuculline (100 μM), and by cycloheximide (10 μl/ml), a protein synthesis inhibitor. GABA (100 μM) enhanced the affinity of [ 3H]flunitrazepam binding to vehicle- and GABA-pretreated, but not to diazepam-pretreated, HEK 293 cells. The results suggest that chronic GABA treatment up-regulates stably expressed GABA A receptors, presumably by stimulating their synthesis. Unlike chronic diazepam, which produced functional uncoupling of GABA and benzodiazepine binding sites, chronic GABA failed to produce this effect.

Anxiolytic-like effects of acute and chronic GABA transporter inhibition in rats.

Acute GABA transporter inhibition can induce anxiolytic-like behaviors. The present analysis addressed whether chronic treatment (23 days via drinking water) with a GABA transporter inhibitor affects rat behavior similar to acute treatment and interferes with additional benzodiazepine-receptor agonistic treatment. Seventy-one rats divided into seven groups were acutely treated with either vehicle, diazepam (2 mg/kg), zolpidem (0.05 mg/kg), tiagabine (19 mg/kg) or chronically with tiagabine with or without acute diazepam or zolpidem. Animals were behaviorally characterized in an elevated plus-maze. None of the treatments induced changes in the activity of the animals. Acute and chronic treatment with tiagabine induced anxiolytic-like effects, similar to acute doses of diazepam. Acute diazepam did not enhance chronic tiagabine effects, whereas acute zolpidem attenuated the anxiolytic-like effects of chronic tiagabine. It is concluded that anxiolytic effects of acute GABA-uptake inhibition by tiagabine persist under chronic treatment and are sensitive to concomitant use of benzodiazepine receptor ligands.

Sodium lactate elicits anxiety in rats after repeated GABA receptor blockade in the basolateral amygdala

Repeated administration of the GABA A receptor antagonist bicuculline methiodide into the basolateral nucleus of the amygdala at doses subthreshold to eliciting a full response will eventually produce long-term ‘priming’, such that heart rate, blood pressure as well as anxiety are increased at the lower doses. The present study was conducted in order to determine if the long-term priming of anxiety within the basolateral nucleus is producing a condition similar to that seen in human panic disorder by testing the response elicited by i.v. lactate infusions, since lactate infusions induce a panic attack in patients with panic disorder. Male Wistar rats were fitted with femoral arterial and venous catheters and chronic microinjection cannulae into the basolateral nucleus. Repeated daily injections of a subthreshold dose of bicuculline methiodide into the basolateral nucleus for 4–5 days elicited a primed response, while the same procedure with artificial cerebrospinal fluid vehicle (a-CSF; sham-primed) had no effect. Following priming, rats received both sodium lactate infusions (0.5 N, 10 ml/kg) or 0.9% saline in a random order separated by 48 h. Heart rate and blood pressure were monitored throughout the infusion and the animals were immediately placed in the social interaction test to assess their anxiety response. Only primed and not sham-primed rats responded to a lactate infusion with significant increases in heart rate, blood pressure and experimental anxiety. Thus, rats which are primed with chronic subthreshold GABA receptor blockade in the basolateral nucleus develop a sensitivity to sodium lactate, similar to human panic disorder patients.

The GABA-withdrawal syndrome: a model of local status epilepticus.

The GABA-withdrawal syndrome (GWS) is a model of local status epilepticus following the interruption of a chronic GABA infusion into the rat somatomotor cortex. GWS is characterized by focal epileptic electroencephalographic discharges and associated contralateral myoclonus. In neocorticai slices obtained from GWS rats, most neurons recorded in the GABA-infused area are pyramidal neurons presenting bursting properties. The bursts are induced by white-matter stimulation and/or intracellular depolarizing current injection and correlate with a decrease of cellular sensitivity to GABA, caused by its prolonged infusion. This effect is related to a calcium influx that may reduce the GABAA receptormediated inward current and is responsible for the bursting properties. Here we present evidence for the involvement of calcium- and NMDA-induced currents in burst genesis. We also report modulatory effects of noradrenaline appearing as changes on firing patterns of bursting and nonbursting cells. Complementary histochemical data reveal the existence of a local noradrenergic hyperinnervation and an ectopic expression of tyrosine hydroxylase mRNAs in the epileptic zone.

GABA and muscimol as reversible inactivation tools in learning and memory.

Reversible inactivation of brain areas is a useful method for inferring brain-behavior relationships. Infusion of GABA or of the GABA receptor agonist muscimol is considered one interesting reversible inactivation method because it may not affect fibers of passage and may therefore be compared to axon-sparing types of lesions. This article reviews the data obtained with this method in learning and memory experiments. A critical analysis of data, collected in collaboration with Simon Brailowsky, with chronic GABA infusion is presented, together with an illustration of data obtained with muscimol-induced inactivation.

GABA(B)R1a/R1b-type receptor antisense deoxynucleotide treatment of melanotropes blocks chronic GABA(B) receptor inhibition of high voltage-activated Ca2+ channels.

GABA(B) and dopamine D2 receptors, both of which acutely inhibit adenylyl cyclase and high voltage-activated Ca2+ channels (HVA-CCs), are found in high levels in the melanotrope cells of the pituitary intermediate lobe. Chronic D2 receptor agonist application in vitro has been reported to result in inhibition of HVA-CC activity by down-regulation. Here we report that chronic GABA(B), but not GABA(A), agonist treatment also resulted in HVA-CC inhibition. Two GABA(B) receptor variants have been cloned and shown to inhibit adenylyl cyclase in HEK-293 cells. We have constructed an antisense deoxynucleotide knockdown-type probe that is complementary to 18 bp from the point at which the two sequences first become homologous. Chronic coincubation with baclofen and GABA(B) antisense nucleotide completely eliminated the inhibition of the channels by baclofen alone but had no reversing effect on HVA-CC inhibition by the D2 agonist quinpirole. A scrambled, missense nucleotide also had no reversing effect. Incubation with a D2 antisense knockdown probe eliminated the ability of a D2 agonist to inhibit the channels but had no effect on baclofen blockade. These results show the existence an R1a/R1b type of GABA(B) receptor, which, like the D2 receptor, is coupled to chronic HVA-CC inhibition in melanotropes.

Function of GABA ρ1 receptors is up-regulated by chronic GABA treatment in Xenopus oocytes

This study was aimed to investigate the effect of chronic γ-aminobutyric acid (GABA) treatment on homomeric GABA ρ1 receptors expressed in Xenopus oocytes. The ρ1 mRNA-injected oocytes were incubated with 10 mM GABA for 2, 6, 24, 48 or 72 h, prior to the assessment of GABA-gated ion currents. The results showed that GABA exposure for more than 6 h dramatically enhanced the peak GABA-activated currents. In addition to current amplitude, the apparent GABA potency and cooperativity were significantly increased. The enhancement reached its plateau after a 24-h exposure. The up-regulation of GABA current amplitude was completely inhibited by cycloheximide, a translation inhibitor. This suggests that chronic GABA treatment may increase the translational activity of GABA ρ1 subunit in Xenopus oocytes.

γ-Aminobutyric acid A receptor regulation: heterologous uncoupling of modulatory site interactions induced by chronic steroid, barbiturate, benzodiazepine, or GABA treatment in culture

Prolonged administration of anxiolytic, sedative, and anticonvulsant drugs that act through the GABA A receptor (GAB AR) can evoke tolerance and dependence, suggesting the existence of an endogenous mechanism(s) for altering the ability of such agents to interact with the GABA AR. Uncoupling appears to be one such mechanism. This is a decrease in the allosteric interactions between the benzodiazepine (BZD) recognition site and other agonist or modulator sites on the GABA AR, as measured by potentiation of [ 3H]flunitrazepam ([ 3H]FNZ) binding. To investigate the mechanism(s) of uncoupling, neuronal cultures were treated chronically with 3α-hydroxy-5β-pregnan-20-one (pregnanolone), pentobarbital, flurazepam, or GABA, then tested for enhancement of [ 3H]FNZ binding by these substances. The results indicate that BZDs, barbiturates, and steroids, as well as GABA itself, are capable of inducing both heterologous and homologous uncoupling. Surprisingly, different chronic drug treatments produce different patterns of homologous and heterologous uncoupling. Chronic exposure to pregnanolone, GABA, flurazepam or pentobarbital induces complete uncoupling of barbiturate-BZD site interactions, partial uncoupling of GABA-BZD site interactions, but different amounts of uncoupling of steroid-BZD site interactions. In addition, the EC 50 for pregnanolone-induced homologous uncoupling (1.7 μM) is over an order of magnitude greater than that for heterologous uncoupling of GABA and BZD sites (82 nM). Moreover, heterologous uncoupling by pregnanolone is inhibited by the GABA site antagonist SR-95531, whereas homologous uncoupling by pregnanolone is resistant to SR-95531. Therefore, there are at least two distinct ways in which GABA AR modulatory site interactions can be regulated by chronic drug treatment.

Effects of GABA B receptor antagonists on two models of focal epileptogenesis

The acute effects of two GABA B receptor antagonists (phaclofen and CGP-35348) were studied in two types of epileptogenic activity: that produced by intracortical injections of baclofen and that appearing after withdrawal of chronic intracerebral GABA infusion (the GABA-withdrawal syndrome, GWS). Intracortical baclofen induced two types of electrographic paroxysmal discharges: one consisting of single spike-and-wave (pattern 1) and another of poly spike-and-wave patterns (pattern II). Both patterns showed similar latencies and temporal evolution of spike frequency discharges. Phaclofen, applied directly into the baclofen-induced epileptogenic focus, suppressed pattern 11 but was ineffective in modifying both pattern I and the GWS. CGP-35348, administered systemically, inhibited both patterns I and II. Intracortical microinjection of baclofen or phaclofen in rats showing a GWS had no effect, nor the systemically given CGP 35348. These results indicate a differential participation of GABA B receptors in GABA-related epileptic syndromes of cortical origin.

A potassium current controls burst termination in rat neocortical neurons after GABA withdrawal

Bursting activities were investigated under conditions of reduced outward K + currents in neocortical slices obtained from rats presenting the γ-aminobutyric acid (GABA)-withdrawal syndrome (GWS), a focal epilepsy consecutive to the interruption of a chronic intracortical GABA infusion into the somatomotor cortex. These bursts were induced by intracellular depolarizing current injection and/or by white matter stimulation. Tetraethylammonium (TEA) at doses which did not change input resistance, spike duration or first interspike time interval abolished the burst terminating process and induced plateau-like potentials (up to 500 ms) which were tetrodotoxin-resistant and blocked by Ca 2+ antagonists Cd 2+ and Co 2+. Therefore, it appears that bursts during GWS are generated by Ca 2+-dependent plateau potentials which are terminated by a K + current highly sensitive to TEA.

High expression of noradrenaline, choline acetyltransferase and glial fibrillary acidic protein in the epileptic focus consecutive to GABA withdrawal. An immunocytochemical study

Interruption of a chronic GABA infusion into the rat somatosensory cortex induces the appearance of focal epileptic manifestations, known as the ‘GABA withdrawal syndrome’ (GWS). The aim of the present study was to determine, by immunocytochemistry, if neurotransmitters other than GABA are involved in GWS, namely: noradrenaline (NA), serotonin, choline acetyltransferase (CAT), cholecystokinin, neuropeptide Y, somatostatin and glial fibrillary acid protein (GFAP). Immunocytochemical data were compared in three animal groups: GABA-, saline- and L-aspartate ( L-Asp)-infused rats. Only GABA-infused rats presented epileptic manifestations after interruption of the infusion. Saline- and L-Asp-infused rats served as controls. Observations were limited to the region surrounding the cortical infusion site. GABA-infused rats showed in the zone of the epileptic focus a number of cell bodies strongly immunoreactive to NA antibodies much larger than control rats. In addition, NA-immunoreactive fibers formed a dense plexus and some of them were observed around perikarya. In saline- and L-Asp-infused rats, the NA-immunolabelled fibers were sparse and NA immunolabelling was rarely observed in cell bodies. These results contrast to those obtained for the serotonergic system, where no significant difference was observed among the three groups of rats. CAT immunolabelling was observed in cell bodies, but not in nerve terminals in rats of the three groups. The number of CAT-immunoreactive cell bodies was much greater in GABA-infused rats than in the control animals. GFAP immunolabelling showed an important number of astrocytes throughout the cortex of the GABA-infused hemisphere, whereas, astrocytic reaction was limited to the infusion site in controls. Immunocytochemical data concerning peptides revealed cortical neuronal elements labelled similarly in the three groups of rats. Noradrenergic, cholinergic and glial modifications observed mainly in GABA-infused rats can result from lesion and from a specific action of GABA in chronic infusion. These modifications may contribute to the epileptogenesis of GWS, as recently demonstrated by electrophysiological recordings that show a modulating action of NA on firing activity of neurons involved in the epileptic focus.

Chronic GABA treatment downregulates the GABA A receptor α2 and α3 subunit mRNAS as well as polypeptide expression in primary cultured cerebral cortical neurons

Chronic GABA exposure of mammalian primary cultured cortical neurons results in a downregulation of the GABA-benzodiazepine receptor complex. In the present study, the mRNA levels, as well as polypeptide expression, for the GABA A receptor α 2 and α 3 subunits in cultured embryonic mouse cerebral cortical neurons (7 day old) were examined using northern analysis and immunoblotting techniques following chronic GABA treatment. The α 1 subunit mRNA or polypeptide could not be detected in these neurons. The steady state levels of mRNA for the GABA A receptor α 2 and α 3 subunits showed a decrease in comparison with untreated neurons. There was no change in the level of the β actin or poly(A) + RNA under the same experimental conditions. This agonist-induced reduction in the GABA A receptor α 2 and α 3 subunit mRNA was blocked by the concomitant exposure of neurons to R 5135, an antagonist of GABA A receptor. The polypeptide expression for the GABA A receptor α 2 and α 3 subunits in chronically GABA-treated neurons also showed a decline and this change was also blocked by the concomitant exposure of cells to GABA and R 5135. These results indicate that the chronic exposure of the GABA A receptor complex to agonist downregulates the expression of the α subunits of the receptor complex, which may be related to an observed decreases in the number of binding sites and GABA-induced 36Cl-influx in the cortical neurons.

Gamma-Aminobutyric acid-A (GABAA) agonists down-regulate GABAA/benzodiazepine receptor polypeptides from the surface of chick cortical neurons.

The impermeant SH-cleavable reagent, 125I-labeled 3,3'-dithiopropionyl 1-sulfosuccinimidyl 1'-glycyltyrosine (DPSgt) and specific immunoprecipitation were used to quantitate gamma-aminobutyric acid-A (GABAA) receptor polypeptides with domains exposed on the surface of chick cortical neurons. The 125I label incorporated into 50- and 53-kDa receptor subunits was completely removed by washing the cells with glutathione (GSH) buffer. A single dose of 100 microns GABA or GABA agonist was added to the neurons, and after 5 days in culture the cells were washed and labeled with 125I-DPSgt. This treatment with GABA or isoguvacine reduced the level of the receptor 125I-labeled subunits by 50-60%, while the reduction by 4,5,6,7-tetrahydroisoazolo[5,4-c]pyridin-3-ol was less than 17%. The subunit down-regulation by agonist was prevented by the GABAA antagonist 3 alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (R5135). Direct iodination of membranes isolated from treated cells revealed a similar loss of subunits, indicating that the down-regulated polypeptides are not retained intracellularly. Furthermore, the level of intracellular [3H]flunitrazepam binding (per mg of protein) did not change significantly during chronic GABA treatment, while the fraction of intracellular binding rose from 7% to 15% of the total, owing to a decline in surface binding. The fate of the surface subunits during acute agonist exposure was examined by labeling intact neurons with 125I-DPSgt at 0 degrees C, incubating with GABA for 2-4 h at 37 degrees C and then washing with GSH buffer. Of the 50-53-kDa receptor peptides originally on the surface, 16 +/- 2% became protected from GSH during the 2-h GABA treatment. This sequestration was not found in cells incubated without GABA, with GABA + R5135, or with GABA at 0 degree C. A consistently lower level of sequestered subunits was recovered after a 4- versus 2-h GABA treatment at 37 degrees C, suggesting polypeptide degradation. Overall, the results indicate that GABAA receptor sequestration and subsequent degradation play an important role in agonist-dependent down-regulation.

Chronic infusions of GABA into the medial prefrontal cortex induce spatial alternation deficits in aged rats

It has been proposed that functions associated with the prefrontal cortex could change as a consequence of aging. Previous experiments in young rats have demonstrated that anatomical lesions or chronic GABA infusions into this area produce deficits in spatial delayed alternation tasks. The present study examines the effect of chronic (7 days) GABA or saline infusion into the prefrontal cortex on the performance of delayed alternation task in old rats (24 months). The results suggested that aged rats needed more sessions to acquire the delayed alternation task. GABA infusions into the prefrontal cortex produced deficits in spatial alternation tasks similar to those previously observed in young rats. Performance rapidly recovered after the infusion period. Histological analysis showed similar lesion size in both groups. The results suggest that aged prefrontal cortex and/or related areas participating in the acquisition of the delayed alternation task are more sensitive to aging processes. Furthermore, the prefrontal cortex is important for the retention of a previously learned spatial delayed alternation task. The structures involved in functional recovery from these deficits appear to be fully functional in aged rats.

Chronic GABA exposure down-regulates GABA-benzodiazepine receptor-ionophore complex in cultured cerebral cortical neurons

Cerebral cortical cultured neurons were characterized for GABA-benzodiazepine (BZ) receptor complex, and the effect of chronic exposure of cortical neurons to GABA on GABA-BZ receptor system was investigated. In the intact cells, the [ 3H]flunitrazepam binding was rapid and saturable, with an apparent K d of 4.2±1.5 nM and B max of 776±54 fmol/mg protein. Specifically bound [ 3H]flunitrazepam was displaced in a concentration-dependent manner by various BZ receptor ligands such as Ro15-1788, DMCM, Ro15-4513, clonazepam, alprazolam, diazepam and zolpidem, and enhanced by GABA, muscimol and pentobarbital. GABA induced enhancement of 36Cl-influx in a concentration-dependent manner (EC 50 = 9±2 μM). Chronic exposure of the cultured neurons to GABA resulted in a reduced [ 3H]flunitrazepam, [ 3H]GABA, [ 3H]Ro15-1788, [ 3H]Ro15-4513 and [ 35S]TBPS binding, a reduced enhancement of [ 3H]flunitrazepam binding by GABA, and a reduced GABA-induced 36Cl-influx susceptible to reversal by concomitant exposure of the cultures to R 5135, a GABA A-receptor antagonist. These findings indicate that cerebral cortical cultured neurons provide an ideal model to study GABA-BZ receptor complex using binding and 36Cl-influx assays, and chronic exposure of cortical cultures to GABA leads to a down-regulation of GABA-BZ receptor system. It is a GABA A receptor-mediated slow process.

Cytophotometric analysis of magnocellular azure B-RNA and Fuelgen-DNA following chronic GABA infusion into the nucleus basalis of rats

This investigation was undertaken to examine possible cytopathic effects of GABA infusion on nucleus basalis (NBM) magnocellular neurons. Sixty-three male Long-Evans rats received unilateral, intra-NBM infusions of either GABA100 (100 μg/μl/h), GABA10 (10 μg/μl/h), or ultrafiltered saline (1 μl/h) for a period of 24 hours. Rats from each of these groups were sacrificed at either 24 hours, 48 hours or 8 days following initiation of infusions. The sham operated hemisphere of each rat served as a control for the infused hemisphere. After stoichiometric azure B-RNA and Feulgen-DNA staining of brain sections, scanning-integrating microdensitometry was used to quantify GABA-induced alterations in these well established indices of neuronal toxicity. These results provide evidence that the neurotoxic effects of 24 hours of 100μg/μl-h GABA infusion are manifested within 48 hours post-initiation of infusions. Although 24 hours of 10 μg/μl-h GABA infusion suppressed NBM neuronal metabolism, the lower magnitude and duration of this effect signified an impending recovery. GABA infusion resulted in little if any NBM neuronal chromatin template impairment (i.e., reduced Feulgen-DNA reactivity), irrespective of the dosage employed and the delay prior to sacrifice.