Effects of cocaine administration on receptor binding and subunits mRNA of GABA(A)-benzodiazepine receptor complexes

The effects of intermittent intraperitoneal (i.p.) administration of cocaine (20 mg/kg) on GABA(A)-benzodiazepine (BZD) receptors labeled by t-[(35)S]butylbicyclophosphorothionate (TBPS), and on several types of mRNA subunits were investigated in rat brain by in vitro quantitative receptor autoradiography and in situ hybridization. Phosphor screen imaging with high sensitivity and a wide linear range of response was utilized for imaging analysis. There was a significant decrease in the level of alpha 1, alpha 6, beta 2, beta 3, and gamma 2 subunits mRNA, with no alteration of [(35)S]TBPS binding in any regions in the brain of rats at 1 h following a single injection of cocaine. In chronically treated animals, the mean scores of stereotyped behavior were increased with the number of injections. The level of beta 3 subunit mRNA was decreased in the cortices and caudate putamen, at 24 h after a final injection of chronic administrations for 14 days. In the withdrawal from cocaine, the frontal cortex and hippocampal complexes showed a significant increase in [(35)S]TBPS binding and alpha1 and beta 3 subunit mRNA in the rats 1 week after a cessation of chronic administration of cocaine. These findings suggest that the disruption of GABA(A)-BZD receptor formation is closely involved in the development of cocaine-related behavioral disturbances. Further studies on the physiological functions on GABA(A)-BZD receptor complex will be necessary for an explanation of the precise mechanisms underlying the acute effects, development of hypersensitization, and withdrawal state of cocaine.     

Differential expression of GABA(A) receptor subunit mRNAs and ligand binding sites in rat brain following phencyclidine administration

Recent biochemical observations have suggested the abnormalities in the gamma-amino-butyric acid (GABA)ergic system in schizophrenic brains. In the present study, we investigated the subunits gene expressions and ligand binding of the GABA(A) receptor following acute and chronic administration of phencyclidine (PCP), which induces schizophrenia-like symptoms, in rats using in situ hybridization and in vitro quantitative autoradiography. PCP i.p. administration at a daily dose of 7.5 mg/kg resulted in a significant decrease in expression of alpha 1 subunit mRNA in cerebral cortices (cingulate (-13%) and temporal cortex (-6%)) and hippocampal formation (CA1 (-11%), CA2 (-10%), CA3 (-11%) and dentate gyrus (-12%)) 1 h after a single treatment. In the repeated PCP administrations for 14 days, the expression of beta 2 mRNA in the cerebellum (-10%) and of beta 3 mRNA in the cerebral cortices (cingulate (-12%), parietal (-16%) and temporal cortex (-16%), caudate putamen (-18%), inferior colliculus (-18%), and cerebellum (-15%) were significantly decreased. In addition, [(35)S]t-butylbicyclophosphorothionate (TBPS) binding was also reduced in layer IV of the frontoparietal cortex (-14%), inferior colliculus (-17%), and cerebellum (-12%) following chronic PCP treatment, while no changes were observed following acute PCP treatment. These results indicate that single and repeated administrations of PCP independently regulate the expression of GABA(A)/benzodiazepine (BZD) receptor subunits mRNA and its receptor binding in the brain.     

Changes of GABA(A) receptor binding and subunit mRNA level in rat brain by infusion of NOS inhibitor

In the present study, we have investigated the effects of prolonged inhibition of nitric oxide synthase (NOS) by infusion of NOS inhibitor, L-nitroarginine, to examine the pentobarbital-induced sleep, modulation of GABA(A) receptor binding, and GABA(A) receptor subunit mRNA level in rat brain. Pre-treatment with L-nitroarginine 30 min before pentobarbital treatment (60 mg/kg, i.p.) significantly increased the duration of sleep in rats. However, the duration of pentobarbital-induced sleep was shortened by the prolonged infusion of L-nitroarginine into ventricle. We have investigated the effect of NOS inhibitor on GABA(A) receptor binding characteristics in discrete areas of brain regions by using autoradiographic and in situ hybridization techniques. Rats were infused with L-nitroarginine (10, 100 pmol/10 microl/h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps. The levels of [(3)H]muscimol and [(3)H]flunitrazepam binding were markedly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, there was no change in the level of [(35)S]TBPS binding. The levels of beta2-subunit were elevated in the cortex, brainstem, and cerebellar granule layers. By contrast, the levels of beta3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in L-nitroarginine-infused rats. Following L-nitroarginine treatment, the levels of alpha6- and delta-subunits which were strictly localized to the cerebellum, were not changed in the cerebellar granule layer. These results show that the prolonged inhibition of NOS by L-nitroarginine-infusion markedly elevates [(3)H]muscimol and [(3)H]flunitrazepam binding throughout the brain, and alters GABA(A) receptor subunit mRNA levels in different directions. Chronic inhibition of NO generation has differential effects on the various expressions of GABA(A) receptor subunits. These suggest the involvement of different regulatory mechanisms for the NO-induced expression of GABA(A) receptor.     

Binding pattern of [35S]t-butylbicyclophosphorothionate is not altered following electroconvulsive shock treatment in rats

Single or repeated electroconvulsive shock (ECS) treatment-induced changes in [35S]t-butylbicyclophosphorothionate [( 35S]TBPS) binding patterns in specific regions, i.e., cerebral cortex, cerebellum, hippocampus, and striatum of rat brain were investigated. Specific [35S]TBPS binding in these brain regions was not altered following a single or repeated administration of ECS, nor was the inhibition of [35S]TBPS binding to GABA affected. These observations tend to suggest that the picrotoxin-site on the GABA receptor complex may not be directly involved in electroconvulsive shock.     

Alterations in GABA(A) receptor expression in neonatal ventral hippocampal lesioned rats: comparison of prepubertal and postpubertal periods

Rats with neonatal ventral hippocampal lesions (NVHL) have been studied as a neurodevelopmental animal model of schizophrenia. NVHL rats exhibit postpubertal emergence of hyperresponsiveness to stress, suggesting increased mesolimbic dopamine (DA) activity. However, previous studies have not yielded clear evidence of this. Disturbances in the gamma-amino-butyric acid (GABA)-ergic system as well as the dopaminergic system are thought to be present in schizophrenia. To determine whether GABA(A) receptors play a role in the abnormal postpubertal behavior in NVHL rats, we compared changes in expression of mRNA of GABA(A) receptor subunits and in [(35)S] t-butylbicyclophosphorothionate ([(35)S] TBPS) binding in the prepubertal and postpubertal periods. Male pups were lesioned with ibotenic acid at postnatal day 7 (PD 7), and in situ hybridization and quantitative autoradiography were then performed. In NVHL rats, alpha1 subunit mRNA expression in prefrontal cortex was decreased at PD 35 (prepubertal period; by 21.7%), but increased at PD 56 (postpubertal period; by 21.4%) when compared with sham controls. beta2 subunit mRNA expression was increased in PFC in the postpubertal period (by 24.3%). beta3 subunit mRNA expression was increased in the caudate-putamen in the postpubertal period (by 37.2%). [(35)S] TBPS binding was increased in PFC only in the postpubertal period (by 17.7%). These findings suggest that dysfunction of the GABAergic system exists in NVHL rats. Furthermore, developmental and regional changes in GABA(A) receptor expression appear to occur in compensation for the attenuation of GABAergic system activity in NVHL rats.     

Aging related alterations in GABAA receptor subunit mRNA levels in Fischer rats.

The influence of aging on the binding of ligands to picrotoxin binding sites as well as steady state levels of mRNA for various alpha subunits of gamma-aminobutyric acid (GABA) receptor complex was investigated in male Fischer F-344 rats. In aged rats, the binding of [35S]t-butyl-bicyclophosphorothionate (TBPS) was significantly reduced. This decrease in TBPS binding derived from a reduced density of binding sites, rather than from affinity changes, in both cerebral cortex and cerebellum. In aged rats, alpha 1 mRNA level decreased approximately 70% between age 6 months and 24 months in the cerebral cortex (P less than 0.005). In contrast, alpha 1 mRNA remained unchanged in the cerebellum of old rats. The association of a decrease in picrotoxin binding sites in the cerebral cortex with a decline in alpha 1 mRNA level in the cerebral cortex and in alpha 2 mRNA level in the cerebellum is indicated. alpha 6 mRNA level increased with age in the cerebellum. These findings indicate a selective age related modulation in the stoichiometry of GABAA receptor in aging.     

Altered atypical coupling of gamma-aminobutyrate type A receptor agonist and convulsant binding sites in subunit-deficient mouse lines

We searched for subunit correlations for GABA(A) receptor-associated atypically GABA-insensitive [35S]TBPS binding. The homomeric beta3 subunit receptors could be excluded, as GABA-insensitive [35S]TBPS binding was present in beta3-/- mice. Localization of GABA-insensitive [35S]TBPS binding correlated best with those of delta, alpha4 and alpha6 subunit mRNAs. The amounts of GABA-insensitive [35S]TBPS binding components were increased in delta-/- mice, but dramatically reduced in alpha6-/- mice, suggesting a role for alpha6 but excluding delta subunits.     

Electroconvulsive shock alters GABAA receptor subunit mRNAs: Use of quantitative PCR methodology

Electroconvulsive shock (ECS) may affect several neurotransmitter systems in brain, including the GABAergic inhibitory system. We used a quantitative PCR-based assay to evaluate mRNAs for five GABAa receptor subunits at 2 to 24 h after ECS. mRNAs for the alpha 1 and beta2 subunits were significantly increased in cerebellum at 4 and 8 h after ECS, and returned to control levels at 24 h. No changes were observed in alpha2, beta3, gamma1, or gamma2 subunits, and no changes in any subunit evaluated were observed in cortex or hippocampus. These data corroborate prior results obtained for the alpha 1 subunit using Northern hybridization, and illustrate the utility of the PCR assay in quantitating low-abundance mRNAs.     

"Nonhedonic" food motivation in humans involves dopamine in the dorsal striatum and methylphenidate amplifies this effect

The effects of a single and repeated administration of cocaine on GABA(B) receptor subunit mRNA was investigated in rat brain by in situ hybridization. Following a single administration of cocaine, no significant change was observed in any brain regions examined, neither 1 h nor 24 h after administration. During repeated administration of cocaine, behavioral sensitization with increased stereotyped behavior was observed. A significant increase in the level of GABA(B(1)) mRNA was observed in the nucleus accumbens (11.4%), CA1 field of the hippocampus (16.8%), and thalamus (16.5%) 1 day after repeated administrations of cocaine for 14 consecutive days. The level of mRNA returned to the basal level 1 week after the final injection of repeated cocaine treatment. The observed changes in the mRNA level after the repeated cocaine may imply changes of GABA(B(1)) subunit in molecular mechanisms which underlie development of behavioral sensitization.     

Prenatal alteration and distribution of the GABA(B1) and GABA(B2) receptor subunit mRNAs during rat central nervous system development

The prenatal developmental expression changes and distribution of the gamma-aminobutyric acid (GABA)(B1) and GABA(B2) receptor subunit were investigated using in situ hybridization and RNase protection assay (RPA). We defined a different expression pattern of GABA(B1) subunit mRNA to that of GABA(B2) subunit. GABA(B1) subunit mRNA signals were moderately expressed in the cerebral cortex neuroepithelium of discrete brain regions on gestational day (GD) 11.5 and 12.5 and were highly expressed in the brain and spinal cord on GD 13.5 and 15.5. However, GABA(B2) subunit mRNAs were not detected on GD 11.5 and 12.5 and were first weakly detected on GD 13.5. On GD 15.5, 17.5, and 19.5, these subunit mRNAs were found in the retina, hippocampus, cerebral cortex, spinal cord, and cerebellum area. On GD 19.5 and 21.5, these subunits mRNA signals increased in the cerebral cortex, hippocampus, thalamus, and cerebellum, but decreased in the spinal cord, spinal ganglion, and midbrain, reaching similar levels to that of the adult brain. On GD 21.5, these subunit mRNAs were similarly expressed in almost all brain areas with a higher expression level of GABA(B1) subunit mRNA than GABA(B2) subunit mRNA. Our results found that GABA(B1) and GABA(B2) subunit mRNAs showed different expression patterns, with the GABA(B1) subunit expressed earlier and higher. We suggest that GABA(B1) and GABA(B2) subunits might have a role in the fetal brain during the gestational period for pre- and post-synaptogenesis, proliferation, differentiation, and neuronal maturation, and GABA(B1) subunit may be more important than GABA(B2) subunit during rat prenatal development.     

Decreased expression of the GABAA receptor in fragile X syndrome

After our initial discovery of under expression of the GABA(A) receptor delta subunit in a genome wide screening for differentially expressed mRNAs in brain of fragile X mice, a validated model for fragile X mental retardation syndrome, we analyzed expression of the 17 remaining subunits of the GABA(A) receptor using real-time PCR. We confirmed nearly 50% under expression of the delta subunit and found a significant 35%-50% reduction in expression of 7 additional subunit mRNAs, namely alpha(1), alpha(3), and alpha(4), beta(1) and beta(2) and gamma(1) and gamma(2), in fragile X mice compared to wild-type littermates. In concordance with previous results, under expression was found in cortex, but not in cerebellum. Moreover, decreased expression of specific GABA(A) receptor subunits in fragile X syndrome seems to be an evolutionary conserved hallmark since in the fragile X fly (Drosophila melanogaster) model we also found almost 50% under expression of all 3 subunits which make up the invertebrate GABA receptor, namely Grd, Rdl and Lcch3. In addition, we demonstrated a direct correlation between the amount of dFmrp and the expression of the GABA receptor subunits Rdl and Grd. Our results add evidence to previous observations of an altered GABAergic system in fragile X syndrome. Because GABA(A) receptors are the major inhibitory receptors in brain, involved in anxiety, depression, insomnia, learning and memory and epilepsy, processes also disturbed in fragile X patients, the well described GABA(A) receptor pharmacology might open new powerful opportunities for treatment of the behavioral and epileptic phenotype associated with fragile X syndrome.     

Persistent elevation in GABAA receptor subunit mRNAs following social stress.

Stress is associated with alterations in GABA/benzodiazepine binding and function. We evaluated effects of social stress on GABAA receptor subunit (alpha 1 and gamma 2) mRNAs by Northern hybridization. In cortex, no change was observed in either subunit mRNA immediately after stress, but a 4 hours mRNAs for both subunits were increased. These changes persisted for 72 hours after stress, and returned to baseline levels at 7 days. No changes in mRNAs were observed in sham-treated mice. No changes in either subunit mRNA were observed in stressed or sham-treated mice in cerebellum or hippocampus. In undefeated resident mice, mRNAs for both subunits in cortex were unaffected at 24 hours after the stress episode. Social stress is associated with increases in GABAA receptor alpha 1 and gamma 2 subunit mRNAs in cortex.     

Stable GABA(A) receptor intermediates in SF-9 cells expressing alpha1, beta2 and gamma2 subunits

Spodoptera frugiperda insect cells (Sf-9 cells) were used to study GABA(A) receptor assembly. Time courses of the expression level of alpha1beta2 and alpha1beta2gamma2 receptor protein showed [(3)H]muscimol binding to appear 2 hr before [(3)H]flunitrazepam and [(35)S]TBPS binding. This indicates that muscimol may bind to pentamers with an immature conformation or to molecules smaller than the pentamer. Binding studies performed on fractions from sucrose gradients loaded with solubilized alpha1beta2 or alpha1beta2gamma2 containing membranes revealed no binding other than to the pentameric fractions. Western blotting on fractionated sucrose gradients, however, clearly revealed the existence of GABA(A) receptor intermediates. The alpha1 subunit was seen in fractions corresponding to molecules smaller than the pentamer only when co-expressed with gamma2, indicating that the gamma2 subunit is needed for the alpha1 to form relatively long lasting intermediates. Moreover, Western blots revealed multiple isoforms for each subunit. In general, it was primarily the lower molecular weight forms that were detected in the pentameric fractions. The exception being for the alpha1 and gamma2 forms in subunit combinations that did not contain both of these subunits (i.e., alpha1, gamma2, alpha1beta2, beta2gamma2), where higher molecular weight forms were strongly represented. These findings show that alpha1 and gamma2 prefer specific protein forms when expressed together.     

Effect of prenatal exposure to diazepam on brain GABA(A) receptor mRNA levels in rats examined at late fetal or adult ages

This study tested the hypotheses that in utero exposure to diazepam (DZ): (1) exerts long-lasting effects on GABA(A) receptor function by altering GABA(A) receptor subunit mRNA levels in specific brain regions of adult animals and/or (2) alters GABA(A) subunit mRNA expression in exposed fetuses. We assayed levels of mRNAs encoding several of the most predominant GABA(A) receptor subunits as well as cyclophilin mRNA. Analysis of mRNA levels in the cortex in adults showed that only gamma2S mRNA levels varied significantly with prenatal drug exposure, an effect unrelated to DZ action to the GABA(A) receptor. Analysis in fetuses indicated that mRNA levels varied as a function of both fetal sex and fetal drug environment. Irrespective of sex, DZ exposure increased both alpha1 and cyclophilin mRNAs in fetal brainstem whereas the mRNA levels of gamma2S were increased and decreased, respectively, in the telencephalon and hypothalamus of DZ-exposed fetuses.     

Antidepressant effects on GABA-stimulated 36Cl(-) influx in rat cerebral cortex are altered after treatment with GABA(A) receptor antisense oligodeoxynucleotides

Antidepressants act at the GABA(A) receptor to inhibit GABA-stimulated 36Cl(-) influx and GABA reduction of [35S]TBPS binding. This study examined how selective knock-down (via antisense oligodeoxynucleotides, aODNs) of GABA(A) receptor subunits modified antidepressant activity. The specific aODNs used were for the alpha1, beta1, beta2 or gamma2 subunits of the GABA(A) receptor. The aODN microinjections reduced corresponding GABA(A) receptor subunit mRNA levels by 30-40% as assessed by RT-PCR. The inhibitory effect of the antidepressants amitriptyline and mianserin on GABA-stimulated 36Cl(-) influx was decreased after microinjections of alpha1, beta1, or beta2 subunit aODNs but potentiated after microinjections of gamma2 subunit aODNs. This pattern of aODNs effect on amitriptyline and mianserin modulation of GABA-stimulated 36Cl(-) influx was the same for both antidepressants and similar to GABA but different than that of diazepam and bicuculline. We conclude that multiple subunits of the GABA(A) receptor regulate the effect of amitriptyline and mianserin on the GABA(A) receptor chloride ionophore complex. However, the exact identity of the subunit mediating the direct or allosteric modulation of the antidepressant effect on GABA-stimulated 36Cl(-) influx remains unclear.     

Visualization of alpha5 subunit of GABAA/benzodiazepine receptor by 11C Ro15-4513 using positron emission tomography

Although [(11)C]Ro15-4513 and [(11)C]flumazenil both bind to the central benzodiazepine (BZ) receptors, the distributions of the two ligands are not identical in vivo. Moreover, the in vivo pharmacological properties of [(11)C]Ro15-4513 have not been thoroughly examined. In the present study, we examined the pharmacological profile of [(11)C]Ro15-4513 binding in the monkey brain using positron emission tomography (PET). [(11)C]Ro15-4513 showed relatively high accumulation in the anterior cingulate cortex, hippocampus, and insular cortex, with the lowest uptake being observed in the pons. Accumulation in the cerebral cortex was significantly diminished by the BZ antagonist flumazenil (0.1 mg/kg, i.v.), but not that in the pons. Using the pons as a reference region, the specific binding of [(11)C]Ro15-4513 in most of the cerebral cortex including the limbic regions clearly revealed two different affinity sites. On the other hand, specific binding in the occipital cortex and cerebellum showed only a low affinity site. Zolpidem with affinity for alpha1, alpha2, and alpha3 subunits of GABA(A)/BZ receptor fully inhibited [(11)C]Ro15-4513 binding in the occipital cortex and cerebellum, while only about 23% of the binding was blocked in the anterior cingulate cortex. Diazepam with affinity for alpha1, alpha2, alpha3, and alpha5 subunits inhibited the binding in all brain regions. Since Ro15-4513 has relatively high affinity for the alpha5 subunit in vitro, these in vivo bindings of [(11)C]Ro15-4513 can be interpreted as the relatively high accumulation in the fronto-temporal limbic regions representing binding to the GABA(A)/BZ receptor alpha5 subunit.     

Synchronous postnatal increase in alpha1 and gamma2L GABA(A) receptor mRNAs and high affinity zolpidem binding across three regions of rat brain

The objective of this study was to correlate postnatal changes in levels of mRNAs encoding predominant GABA(A) receptor subunits with a functional index of receptor development. This study is the first to quantify the temporal relationship between postnatal changes in predominant GABA(A) receptor mRNAs and zolpidem-sensitive GABA(A) receptor subtypes. In Experiment 1, we measured zolpidem displacement of 3H-flunitrazepam from rat cerebral cortex, hippocampus, and cerebellum at 0, 6, 14, 21, 29, and 90 postnatal days. Three independent 3H-flunitrazepam sites with high (K(i)=2. 7+/-0.6 nM), low (K(i)=67+/-4.8 nM), and very low (K(i)=4.1+/-0.9 mM) affinities for zolpidem varied in regional and developmental expression. In Experiment 2, we used RNAse protection assays to quantify levels of alpha1, alpha2, beta1, beta2, gamma2S and gamma2L mRNAs in the above regions at the same postnatal ages. Although there was a high degree of regional variation in the developmental expression of zolpidem-sensitive GABA(A) receptors and subunit mRNAs, a dramatic increase in high affinity zolpidem binding sites and alpha1 mRNA levels occurred within all three regions during the second postnatal week. Furthermore, a temporal overlap was observed between the rise in alpha1 mRNA and high affinity zolpidem binding and a more prolonged increase in gamma2L in each region. These results point to the inclusion of the alpha1 and gamma2L subunits in a GABA(A) receptor subtype with a high zolpidem affinity and suggest that a global signal may influence the emergence of this subtype in early postnatal life.