Regulation of cyclic AMP level by progesterone in ovariectomized rat neocortex.

Exposure of neocortical slices to progesterone, without prior treatment with estrogen, augmented forskolin-induced cyclic AMP within 15 min. 30 nM progesterone produced approximately 1/2 the maximal effect but as little as 10 nM progesterone produced a detectable increase in cyclic AMP. When forskolin was replaced by dideoxyforskolin, an analog that does not directly stimulate adenylyl cyclase but shares many of its other actions, progesterone did not augment cyclic AMP. Progesterone also failed to affect increased cyclic AMP that followed exposure to norepinephrine or isoproterenol. The effect of progesterone upon cyclic AMP was also evident when tetrodotoxin was added to block voltage-dependent sodium channels, suggesting that intercellular communication that is dependent upon action potentials was not necessary. The effect of progesterone was at least partially blocked by antagonists of GABAA receptor action, suggesting the involvement of GABAA or GABAA-like receptors. The effect of progesterone was also not homogeneous over the neo cortex. While forskolin-stimulated cyclic AMP was augmented by progesterone in the parietal and occipital regions, it was suppressed in the frontal region. These results are envisioned as a progesterone action upon a small and perhaps compartmentalized component of the cellular cyclic AMP system, an effect that is made detectable in our whole-tissue assay by the well known ability of forskolin to potentiate many hormonal effects upon cyclic AMP.

Decreased expression of GABAA receptor alpha6 and beta3 subunits in stargazer mutant mice: a possible role for brain-derived neurotrophic factor in the regulation of cerebellar GABAA receptor expression?

The cerebellar granule cells of the spontaneous recessive mutant mouse strain, stargazer (stg/stg), fail to express brain-derived neurotrophic factor mRNA. This deficit is exclusive to these neurons and is believed to underlie the motor irregularities displayed by stg/stg, though the molecular basis for their phenotype has still to be resolved. Brain-derived neurotrophic factor has been shown to play a role in the postnatal maturation of cerebellar granule cells. Differentiation of these neurons, postnatally, is characterised by a switch in their GABAA receptor subunit expression profile. Notably, the GABAA receptor alpha6 subunit, which is specific to these neurons, becomes detectable at postnatal days 10-14 (P10-14). To determine whether cerebellar GABAA receptor expression has been compromised in stg/stg mice, the expression levels of GABAA receptor alpha1, alpha6, beta2 and beta3 subunits were compared between stg/stg mice and the appropriate wild-type background strain, C57BL/6J (+/+). By quantitative immunoblotting, it was found that the expression of the alpha6 and beta3 subunits was 23+/-8% and 38+/-12% (mean+/-S.E.M., n=6) of control (+/+) levels, respectively. In contrast, the expression of the alpha1 and beta2 subunits was not significantly different from controls, being 116+/-11% and 87+/-24% (mean+/-S.E.M., n=6) of +/+ levels, respectively. Total specific [3H]Ro15-4513 binding activity detected in cerebellar membranes prepared from stg/stg was not significantly different from +/+ mice. However, the benzodiazepine agonist-insensitive subtype of [3H]Ro15-4513 binding activity, a pharmacological motif of alpha6 subunit-containing GABAA receptors, was lower in stg/stg mice relative to the +/+ strain which correlated with the lowered level of alpha6 subunit expression. Thus, we have identified an abnormality in the GABAA receptor profile of stg/stg mutant mice that might underpin its irregular phenotype.

Sequestration of gamma-aminobutyric acidA receptors on clathrin-coated vesicles during chronic benzodiazepine administration in vivo.

Chronic administration of benzodiazepine agonists produces behavioral tolerance. For induction of tolerance, the use-dependent down-regulation of gamma-aminobutyric acidA (GABA[A])/ benzodiazepine receptors is a potential cellular mechanism. We previously identified GABA(A) receptors on clathrin-coated vesicles from rat brain, suggesting that surface receptors can be internalized via endocytosis. To examine a role for coated vesicles in GABA(A) receptor down-regulation in vivo, fractions were obtained from mouse brain microsomes through density centrifugation and treatment with 0.1% Triton X-100. This coated vesicle preparation was enriched in clathrin subunits and clathrin light-chain kinase and had twice the level of [3H]flunitrazepam binding as did vesicles not exposed to Triton. Adult mice were treated with lorazepam (2 mg/kg/day) for 7 days via osmotic minipump, achieving a serum level of 103 +/- 8.9 ng/ml. The level of flunitrazepam bound to coated vesicles was increased by 83 +/- 13% in the lorazepam-treated mice compared with vehicle-treated controls. The Bmax value for [3H]flunitrazepam binding to synaptic membranes from lorazepam-treated animals was 33 +/- 4% lower than that of controls. The amount of GABA(A) receptor alpha-1 subunits, as quantified by Western blotting, followed a similar pattern. Relative to controls, immunoreactivity for alpha-1 subunits in coated vesicles from lorazepam-treated mice was increased by 60.0 +/- 10.3%, whereas that in synaptic membranes declined by 12 +/- 6%. These results indicate that lorazepam-dependent GABA(A) receptor sequestration occurs in mouse brain. Furthermore, it is suggested that this sequestration may play a role in GABA(A) receptor down-regulation in vivo.

Aberrant expression of GABAA receptor subunits in the tottering mouse: an animal model for absence seizures.

The single-locus mutant mouse tottering (tg) is an established model for absence seizures. We have previously reported an impairment in GABA-induced chloride uptake in tg brain [Tehrani and Barnes, Epilepsy Res. 1995;22:13-21]. In order to determine if this alteration in GABAA receptor function can be related to specific receptor isoforms, we examined the radioligand binding properties of GABAA receptors and the expression of GABAA receptor subunit mRNAs in the cerebral cortex. Saturation binding of [3H]flunitrazepam revealed a significantly lower Kd value in tg cortical tissues (1.77 +/- 0.05 nM) in comparison to that for the background C57BL/6J strain (3.23 +/- 0.23 nM), while the Bmax values were indistinguishable. Biphasic displacement of [3H]flunitrazepam binding by 2-oxoquazepam showed that low affinity binding sites account for 36 +/- 7.6 and 51 +/- 7.5% of the total in control and tg, respectively. The level of [35S]-t-butylbicyclophosphorothionate (TBPS) binding to tg cortical membranes was 73.6 +/- 5.8% of that in controls. Paired measurements by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed no significant differences in the levels of GABAA receptor alpha 1, alpha 3, alpha 5, beta 2, beta 3, gamma 2 or gamma 3 subunit mRNAs between tg and control cortex. However, tg tissues showed elevated levels of alpha 2- and beta 1-subunit mRNAs, representing 256 and 177%, respectively, those of controls. For the tg cortex, the enhanced expression of GABAA receptor alpha 2 and beta 1 subunits correlates with recombinant subtypes known to have low affinity for 2-oxoquazepam and impaired binding of TBPS. These aberrant properties of GABAA receptors could influence the development or propagation of phenotypic seizures in the tottering mouse.

Clathrin-coated vesicles from bovine brain contain uncoupled GABAA receptors.

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABAA receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [3H]muscimol. a GABAA-specific agonist. [3H]flunitrazepam a benzodiazepine agonist, and [35S]t-butylbiocyclophosphorthionate (TBPS), a ligand for GABAA receptor channels. Under standard conditions, the binding level of [3H]muscimol, [3H]flunitrazepam, and [35S]TBPS to coated vesicles represented 12.3 +/- 1.8%, 7.9 +/- 1%, and 10.2 +/- 1.8%, respectively, of that in crude synaptic membranes. Coated vesicles showed a single [3H]flunitrazepam binding site with a KD value (12 nM) which was 9-fold that for synaptic membranes. The allosteric coupling between binding sites was measured by the addition of GABA to [3H]flunitrazepam and [35S]TBPS binding assays. For [3H]flunitrazepam binding to synaptic membranes, GABA gave an EC50 = 2.0 microM and at saturation (100 microM) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonist SR95531. In contrast, neither GABA nor SR95531 had a significant effect on [3H]flunitrazepam binding to CCVs, indicating that the allosteric interaction between GABA and benzodiazepine binding sites is abolished. Likewise, GABA displaced nearly all of the [35S]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, indicating that coupling between the GABA binding sites and chloride channel is also impaired. Thus GABAA receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABAA receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic membranes, CCVs contained 110 +/- 14% and 29.5 +/- 3.8%, respectively, of the immunoreactivity for GABAA receptor beta 2 and alpha 1 subunits. Thus, in comparison to GABAA receptors on synaptic membranes, those on CCVs have a reduced alpha 1/beta 2-subunit ratio. It may be suggested that a selective decline in the content of alpha 1 subunits in coated vesicles could in part account for GABAA receptor uncoupling.

Reduced function of gamma-aminobutyric acidA receptors in tottering mouse brain: role of cAMP-dependent protein kinase.

The single-locus mutant mouse tottering (tg) displays spontaneous seizures that resemble those in human petit-mal epilepsy. In order to examine alterations in GABAA receptor function which could arise as a result of this mutation, the influx of 36Cl- was determined using microsacs (membrane vesicles) isolated from the brain of tg/tg and coisogenic C57BL/6J (+/+) control mice. In microsacs from both tg/tg and +/+ strains, the maximum level of 36Cl- uptake induced by 50 microM GABA was observed during five seconds of incubation at 28 degrees C. Compared to +/+, the GABA-dependent 36Cl- uptake in tg/tg microsacs was significantly lower and faded rapidly during longer incubations. The levels of gated 36Cl- uptake in tg/tg microsacs were 45 +/- 6.3%, 65 +/- 9.9%, and 33 +/- 6.1% of control (+/+) values for 3-, 5-, and 10-s incubations, respectively. GABAA receptor-specific agonists (30 microM), muscimol, isoguvacine and THIP (4,5,6,7-tetrahydroisoazolo-[5,4-c]pyridin-3-ol) induced 36Cl- influx in the order muscimol > GABA > isoguvacine > THIP. This order was similar for both strains, but the agonist-dependent influx was always significantly lower in tg/tg compared to +/+. Treatment of the microsacs with 10 microM H-89, a membrane-permeant inhibitor of the cAMP-dependent protein kinase (protein kinase A, PKA), was without effect on GABA-gated 36Cl- uptake in +/+, but increased the gated uptake in tg/tg microsacs by 44 +/- 16%. PKA was assayed using [gamma-32]ATP and kemptide as the substrate. Triton X-100 (0.1%) increased both the basal and 8-Br-cAMP dependent PKA activity in microsacs by 3-4 four fold, showing that most of the enzyme was intravesicular. In the presence of Triton, the basal activity of PKA in the tg/tg preparations was twice that of +/+, while the strain difference was no longer apparent in assays containing 8-Br-cAMP. The data suggest that an abnormal elevation of protein kinase A activity in tottering mouse brain contributes to an impairment of GABAA receptor function. It is suggested that the resulting loss of inhibition could play a role in induction of the seizures which characterize the mutant phenotype.

Age-related levels of GABA/benzodiazepine binding sites in cerebrum of F-344 rats: effects of exercise.

We examined gamma-aminobutyric acid (GABA), benzodiazepine and convulsant sites of postsynaptic GABA/benzodiazepine receptors (GBZR) in cerebral membranes of inbred Fischer 344 male rats as a function of age. In aged rats (23 to 24 months), the benzodiazepine binding site as determined by [3H]flunitrazepam was 47% and 43% lower than corresponding values in young adult (3 to 4 months) and mature (10 to 12 months) rats, respectively. The decrease was due to the loss of binding density rather than a change in affinity. No statistically significant age-related changes in [3H]muscimol binding were observed when 5 nM or 40 nM labeled muscimol were used. GABA produced a dose-dependent stimulation of flunitrazepam binding in all age groups, but the maximum stimulation in aged animals was significantly higher (24%) than in young and mature animals. The [35S]TBPS binding site, the convulsant site of GABA/benzodiazepine receptors, was unaffected with age. We also studied the effects of exercise on GBZR binding sites of aged rats. The decline of flunitrazepam binding sites and the high sensitivity of flunitrazepam binding to regulation by GABA in aged animals were reversed by 8 to 10 weeks of endurance exercise. Endurance exercise did not have any significant effect on muscimol or TBPS binding sites. Results suggest that there are aged-related alterations of GBZR binding sites and that these modifications can be reversed by exercise.

GABAA receptors in mouse cortical homogenates are phosphorylated by endogenous protein kinase A.

Biochemical, molecular, and electrophysiological studies suggest that phosphorylation of beta subunits of the GABAA receptor (GaR) by exogenous protein kinase A inactivates the receptor channels. We have developed a method which for the first time allows the study of GaR phosphorylation in brain tissues by endogenous PKA. Desalted homogenates or crude synaptic membranes from mouse cerebral cortex were incubated with [gamma-32P]ATP and 8-Br-cAMP or chlorophenylthio-cAMP. Extracts from these incubations were immunoprecipitated by polyclonal antibodies against native GaR and analyzed by SDS-gel electrophoresis and autoradiography. In both homogenates and membranes, cAMP-dependent incorporation of 32P was observed for a 57-kDa peptide, and to a lesser extent 51- to 53-kDa peptides. Phosphorylation of affinity-purified GaR by the catalytic subunit of PKA also produced a major 57-kDa phosphopeptide and a minor 51-kDa phosphopeptide. Limited digestion by S. aureus V-8 protease of the 57-kDa phosphopeptide from the desalted homogenates or from purified receptors produced a major 32P-labeled fragment of 11 kDa, suggesting that the phosphorylation site is similar to that shown previously to reduce GaR function. The phosphorylation of GaRs in homogenates was time dependent and blocked by H-89 or protein kinase inhibitor 5-24, specific inhibitors of protein kinase A. Prolonged incubations resulted in dephosphorylation of the 57-kDa phosphoprotein by a microcystin-LR sensitive phosphatase. In cortical homogenates the level of cAMP-dependent phosphorylation of the 57-kDa GaR peptide was more than 5 times that obtained with washed synaptic membranes. However, assays of PKA using the heptamer kemptide as substrate showed that the specific activity in the particulate fraction was 57% that of the homogenate. This suggests that GaRs on synaptic membranes are preferentially phosphorylated by a cytoplasmic form of protein kinase A. By comparing the [3H]flunitrazepam-photolabeled 53-kDa GaR subunit with the 51-57 kDa [32P]peptides from cortical homogenates, the molar ratio of [32P]/[3H] was estimated at 0.43, suggesting that a substantial fraction of the GaR pool is phosphorylated under these conditions.

Regulation of gamma-aminobutyric acidB (GABAB) receptors in cerebral cortex during the estrous cycle.

We examined binding of the GABAB receptor agonist baclofen to brain synaptic membranes as a function of the natural variations in gonadal steroids that occur during the estrous cycle of the adult rat. We found that the binding of baclofen to neocortical membranes varied systematically as a function of the estrous cycle, with the lowest binding occurring during the estrus stage. Binding to archicortical (hippocampal) and hypothalamic preparations also varied with the estrous cycle, except that the lowest level of binding in these latter cases occurred during the diestrus stage. The variation of [3H]baclofen binding during the estrous cycle was different with respect to the binding of [3H]muscimol, an agonist for GABAA receptors, and [3H]8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), an agonist for serotonin 5-HT1A receptors that shares similar G proteins and effectors with GABAB receptors. Saturation binding studies of cortical GABAB receptors showed that apparent receptor density (Bmax) rather than affinity (Kd) best accounted for the change in binding during the estrous cycle in that Bmax, like total specific binding, was at a minimum during the estrus stage. The robust regulation of GABAB receptors in neocortex was unexpected and its functional significance is at present unknown. However, the correlation of the menstrual cycle with mood and other behavioral changes, and the correlations of the estrous and menstrual cycles with seizure susceptibility, may somehow depend upon hormonal regulation of transmitter systems such as the one we have observed here.

Identification of GABAA/benzodiazepine receptors on clathrin-coated vesicles from rat brain.

To investigate the subcellular compartments that are involved in the endocytosis and intracellular trafficking of GABAA/benzodiazepine receptors, we have studied the distribution and properties of clonazepam-displaceable binding of [3H]flunitrazepam to membrane fractions from rat brain. The microsomal fraction was subjected to density centrifugation and gel filtration to isolate clathrin-coated vesicles. Homogeneity of the coated-vesicle fraction was demonstrated by using electron microscopy and by analysis of clathrin subunits and clathrin light-chain kinase. Vesicles exhibiting specific binding of [3H]flunitrazepam eluted from the sieving gel as a separate peak, which was coincident with that for coated vesicles. Scatchard analysis of equilibrium binding of [3H]flunitrazepam to coated vesicles yielded a KD value of 21 +/- 4.7 nM and a Bmax value of 184 +/- 28 fmol/mg. The KD value for coated vesicles was 12-19-fold that found with microsomal or crude synaptic membranes. This low-affinity benzodiazepine receptor was not identified on any other subcellular fraction and thus appears to be a novel characteristic of coated vesicles. The Bmax value for coated vesicles, expressed per milligram of protein, corresponded to 16 and 115% of that found for crude synaptic and microsomal membrane fractions, respectively. Because the trafficking of neurotransmitter receptors via clathrin-coated vesicles is most likely to occur through endocytosis, the data suggest that an endocytotic pathway may be involved in the removal of GABAA/benzodiazepine receptors from the neuronal surfaces of the rat brain. This mechanism could play a role in receptor sequestration and down-regulation that is produced by exposure to GABA and benzodiazepine agonists.

Agonist-dependent internalization of gamma-aminobutyric acidA/benzodiazepine receptors in chick cortical neurons.

An impermeant benzodiazepine receptor ligand was prepared by derivatization of the aminobenzodiazepine 1012-S with 4-sulfophenylisothiocyanate. The resulting N-(4-sulfophenyl)-thiocarbamoyl derivative of 1012-S (SPTC-1012S) was purified by reverse-phase HPLC, and the predicted structure was verified by mass spectrometry. The apparent affinity of SPTC-1012S (IC50 = 9.8 +/- 2.9 nM) for displacement of [3H]flunitrazepam from intact chick cortical neurons was similar to that of 1012-S (IC50 = 4.0 +/- 0.3 nM). However, at concentrations from 0.1 to 10 microM, 1012-S was consistently more efficacious than SPTC-1012S, a finding indicating that 6-8% of the benzodiazepine receptor pool was not accessible to the impermeant compound. This inaccessible pool was eliminated by permeabilization of the cells with saponin or Triton X-100, a result suggesting that approximately 7% of neuronal benzodiazepine receptors are intracellular. Acute treatment (1-4 h at 37 degrees C) of neurons with 100 microM gamma-aminobutyric acid (GABA) or 100 nM clonazepam had little effect on the level of [3H]flunitrazepam binding but increased the proportion of intracellular receptors by 61 and 74%, respectively, compared with untreated controls. Similar treatment with 1 mM GABA increased the level of intracellular sites by 154-176%. The effect of GABA on receptor internalization was blocked by cotreatment with the GABAA receptor antagonist R 5135. The results suggest that SPTC-1012S can be used as a probe to study the internalization of the GABAA/benzodiazepine receptor complex under normal conditions or following acute or chronic treatment with agonists.

Sodium channel density in hypomyelinated brain increased by myelin basic protein gene deletion.

Trophic control over the expression and membrane distribution of voltage-dependent ion channels is one of the principal organizing events underlying the maturation of excitable cells. The myelin sheath is a major structural determinant of regional ion channel topography in central axons, but the exact molecular signals that mediate local interactions between the oligodendrocyte and axolemma are not known. We have found that large caliber fibre pathways in the brain of the mutant mouse shiverer (shi, gene on chromosome 18), whose developmental fate of myelination is averted by deletion of five exons in the myelin basic protein gene, have a striking excess of sodium channels. As cytoplasmic membranes of shiverer oligodendroglia still adhere to axons, the evidence indicates that myelin basic protein or a myelin basic protein-dependent glial transmembrane signal associated with compact myelin formation, rather than a simple glial-axon contact inhibition or an intrinsic genetic program of neuronal differentiation, could be critical in downregulating sodium channel density in axons. Here we use the shiverer mutant to show that mature central nervous system projection neurons with large caliber unmyelinated fibres sustain functional excitability by increasing sodium channel density. This axon plasticity, triggered by the absence of a single glial protein, contributes to the unexpectedly mild degree of neurological impairment in the mutant brain without myelin, and may be a potentially inducible mechanism determining the recovery of function from dysmyelinating disease.

Basal and drug-induced cAMP levels in cortical slices from the tottering mouse.

Basal and drug-induced levels of cAMP were determined in cortical slices from mice which were homozygous for the tottering (tg/tg) gene defect as well as from co-isogenic controls (+/+). Basal levels of cAMP were 77 +/- 16% higher in tg/tg slices compared to the controls. This difference was abolished by exposure of the slices to propranolol, a beta-adrenergic receptor antagonist. Both isoproterenol and veratridine stimulated cAMP formation, but only small differences were observed in the cAMP levels in tg/tg and +/+ slices after this treatment. Of the veratridine-dependent increase in cAMP, approximately 40% was blocked by propranolol treatment of slices from both strains. The results suggest that a higher level of endogenous norepinephrine release in tottering mice contributes to an elevation of basal cAMP levels.

Effect of 2-hydroxy-saclofen, an antagonist of GABAB action, upon the binding of baclofen and other receptor ligands in rat cerebrum.

2-Hydroxysaclofen (2-OH-saclofen), a newly available compound which blocks certain physiological actions of the gamma-aminobutyric acidB (GABAB) agonist, baclofen, was found to displace [3H]baclofen at least 10-fold more potently than did phaclofen, a previously available antagonist of GABAB action. 2-OH-Saclofen reduced both the affinity and apparent density of baclofen binding sites and displaced baclofen binding at least 60-fold more potently than it displaced the binding of ligands for 3 other transmitters present in the rat cerebral cortex.

Chronic exposure of developing cortical neurons to GABA down-regulates GABA/benzodiazepine receptors and GABA-gated chloride currents.

Cultures of cerebral neurons were prepared from chick embryos, 8.5 days in ovo, and maintained in vitro. Following chronic exposure of these cells to GABA, the levels of [3H]flunitrazepam binding in situ and electrophysiological responsiveness to gamma-aminobutyric acid (GABA) was examined. Treatment with 100 microM GABA for 7 days reduced [3H]flunitrazepam binding in situ by 70 +/- 8% compared to untreated controls. The binding of [3H]N-methylscopolamine was unaffected by this treatment. The reduction in [3H]flunitrazepam binding was prevented by concomitant exposure of developing neurons to the GABA antagonist R 5135, suggesting that GABAA receptor occupancy is required. The loss of bezodiazepine receptors was dependent on the GABA concentration in the culture medium and a half-saturation (IC50) value of 11.2 +/- 3.7 microM was estimated. Whole-cell patch-clamp recordings were obtained to assess the functional properties of the labile receptor pool observed in the binding studies. Neurons cultured with 100 microM GABA for 7 days showed a 60-70% reduction in the peak current amplitudes observed in response to application of 10-100 microM GABA. However, the rate of rapid desensitization, quantified by measuring changes in input conductance, was unchanged by chronic GABA exposure, yielding decay time constants of 27.1 +/- 2.1 and 34.7 +/- 4.7 s for control and treated cells, respectively. The results are consistent with a GABA modulation of the GABAA/benzodiazepine receptor complex by means of down-regulation.

cAMP increases the rate of GABAA receptor desensitization in chick cortical neurons.

During prolonged application of GABA to cultured neurons from the chick embryo cerebrum, whole-cell voltage-clamp recordings show a decline in GABA-gated chloride currents due to desensitization. At a holding potential of -60 mV, desensitization can normally be described as a single exponential process with a time constant (tau) of 7-10 sec at a GABA concentration of 100 microM. After exposure to 50 microM forskolin, the peak amplitude of the GABA-induced currents declined and a fast component of desensitization (tau = 0.92 sec) appeared, whereas the slow component was essentially unchanged. This effect of forskolin was reversible after washing. Similar, although less robust effects were produced by incubation with 8-Br-cAMP, but not by 1,9-dideoxyforskolin. The desensitization process could also be measured by the GABA-dependent uptake of 36Cl- into the cultured neurons. A 20-sec incubation with 10 microM GABA in physiological saline produced a 29% inhibition of subsequent GABA-gated 36Cl- uptake in the presence of 40 mM K+. This inhibition was increased to 64% by the addition of 100 microM forskolin to the preincubation medium. This effect of forskolin was prevented by 100 microM 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase. A similar acceleration of desensitization was produced by 0.5 mM 8-Br-cAMP or by 0.5 mM isobutylmethylxanthine, but these compounds themselves failed to produce desensitization in the absence of exogenous GABA. In the presence of GABA, cAMP analogs were effective in the order 8-(4-chlorophenylthio)-cAMP greater than 8-Br-cAMP greater than N6,O2'-dibutyryl-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

GABA down-regulates the GABA/benzodiazepine receptor complex in developing cerebral neurons.

Neuronal cultures of the chick embryo cerebrum were used to study the chronic effects of gamma-aminobutyric acid (GABA) on the expression of the GABA/benzodiazepine receptor complex. A 7 day exposure of developing neurons to 100 microM GABA produced a 70% reduction in the level of [3H]flunitrazepam binding to intact cells, when compared to untreated controls. The reduction was due to a decrease in receptor density (Bmax) rather than the affinity. The same treatment also caused a 75% reduction in the rates of GABA-gated 36Cl- uptake by intact cells, without an effect on the basal (GABA-independent) flux. Eight days after removal of GABA from the medium of treated cultures, the neurons recovered [3H]flunitrazepam binding to levels corresponding to 74% of unexposed, age-matched controls. The results are consistent with a GABA-induced down-regulation of the GABA/benzodiazepine receptor.

Interaction of t-butylbicyclophosphorothionate with gamma-aminobutyric acid-gated chloride channels in cultured cerebral neurons.

The role of t-butylbicyclophosphorothionate (TBPS) as an antagonist of gamma-aminobutyric acid (GABA) was studied with primary cultures of neurons from the chick embryo cerebrum. The addition of GABA stimulated the uptake of 36Cl- by neurons and the dose dependence of this effect followed hyperbolic kinetics with a K0.5 = 1.3 microM for GABA. TBPS proved to be a potent inhibitor of GABA-dependent Cl- uptake (IC50 = 0.30 microM). Analysis of the kinetics of this process revealed that TBPS is a noncompetitive inhibitor (Ki = 0.15 microM) with respect to GABA. Scatchard analysis of direct binding of [35S]TBPS to membranes isolated from neuronal cultures gave curvilinear plots. These could be resolved by nonlinear regression methods into two components with KD values of 3.1 nM and 270 nM. The TBPS binding constant for this lower affinity site agreed well with the IC50 and Ki values for inhibition of Cl- flux, suggesting that this site is physiologically relevant to GABA antagonism. GABA was a noncompetitive displacer of [35S]TBPS binding to the lower affinity site. The Ki value for this displacement by GABA (1.7 microM) was comparable to the value for GABA enhancement of Cl- flux. The binding of [35S]TBPS to its low-affinity site on neuronal membranes was ninefold higher in the presence of Cl- than with gluconate, an impermeant anion. The rank order for anion stimulation of [35S]TBPS binding was Br- greater than or equal to SCN- greater than Cl- greater than or equal to NO3- greater than I- greater than F- greater than gluconate.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of perinatal administration of isobutyl methylxanthine (IBMX) on [3H]-DA binding in the forebrain of the rat.

Rats of both sexes treated with the phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) during the critical, perinatal period when sexual differentiation is conferred upon the brain. Their gain in body weight during development, spontaneous locomotor activity, duration of the oestrous cycle and characteristics of forebrain dopamine binding were compared with control animals as they matured. Treatment with IBMX had no effect on weight gain in either sex. However it reduced spontaneous motor activity in both sexes and disrupted the oestrous cycle in females. Forebrain binding capacity for [3H]-DA was higher in males than in females. This difference was abolished by perinatal treatment with IBMX which reduced male values to those of females. The pattern of displacement of [3H]-DA by unlabelled inhibitors was sexually dimorphic, indicating qualitative differences in the nature of the recognition sites. Perinatal treatment with IBMX modified these displacement patterns in both sexes. The results are consistent with the hypothesis that cyclic AMP is an internal coupling factor which is involved in oestrogen-mediated sexual differentiation of the brain.

Ontogeny of the GABA receptor complex in chick brain: studies in vivo and in vitro.

The ontogeny of the gamma-aminobutyric acid-A (GABAA) receptor complex in the chick brain was studied by specific binding of [3H]muscimol, [3H]flunitrazepam (Flu) and [35S]t-butylbicyclophosphorothionate (TBPS) to isolated membranes. During development in ovo, the specific binding of muscimol and flunitrazepam increased from day 8 and reached 50% of adult levels of day 20, while a comparable level of TBPS binding was achieved by day 17. The increases in TBPS and Flu binding were reflected in Bmax rather than Kd changes. In embryonic brain, only a low-affinity site for muscimol (Kd = 23 nM) was observed while an additional high-affinity site (Kd = 0.4 nM), as well as the low-affinity site, was found in adult tissue. Similar studies were carried out with cultures of cerebral neurons prepared from 8-day embryos. The level of specific binding of muscimol, Flu and TBPS increased in culture, achieved one half of the maximum level by days 4-5, maximal levels by day 10 and decreased slowly thereafter. The maximal levels in culture corresponded, respectively, to 27%, 67% and 57% of these found in the 18-day embryo. The binding of Flu to membranes from neurons, embryos and adults was enhanced by addition of GABA while TBPS binding was inhibited. The EC50 and IC50 values for these effects corresponded to those for gating of chloride channels. These findings indicate a coordinated expression of receptors for GABA, benzodiazepines and convulsant/TBPS during neuronal maturation both in vivo and in vitro. The schedule for this postsynaptic ontogeny is very similar to that for presynaptic markers of GABAergic neurons (see companion paper).