Effects of repeated amphetamine treatment on regional GABAA receptor binding

The present study examined the effects of repeated exposure to amphetamine on GABA_A receptor binding in cortical and subcortical areas. The goal of the study was to determine whether changes in specific binding were related to behavioral sensitization. Animals were exposed to either saline (0.3 ml, s.c.; n=12) or -amphetamine (2.5 mg/kg, s.c.; n=12) for 6 consecutive days and sacrificed after a 14-day withdrawal period. Differences in GABA_A receptor binding in these two groups of animals were assessed using the GABA_A receptor antagonist [³H]SR 95531. To verify that the preceding treatment regimen led to the development of behavioral sensitization, a separate set of animals (n=8/group) was exposed to the same regimen and challenged with -amphetamine (2.5 mg/kg, s.c.) after the 14-day withdrawal period. As expected, preexposure to amphetamine led to the development of amphetamine sensitization. There were no differences in GABA_A receptor binding in animals preexposed to saline and amphetamine in the prefrontal cortex, caudate-putamen, hypothalamus, or cerebellum. These findings do not provide support for the idea that changes in GABA_A receptor binding in the medial prefrontal cortex or various subcortical areas are related to the development of behavioral sensitization.     

GABAA and GABAB receptors modulating basal and footshock-induced nitric oxide releases in rat prefrontal cortex

Using an in vivo brain microdialysis technique, we measured extracellular levels of nitric oxide (NO) metabolites (NO_x⁻) in the medial prefrontal cortex (mPFC) upon perfusion of γ-aminobutyric acid (GABA) receptor antagonists as well as agonists, and also examined the effects of GABA receptor agonists on mild intermittent footshock-induced NO releases in the mPFC in conscious rats. Perfusion of either bicuculline methiodide, a GABA_A receptor antagonist, or saclofen, a GABA_B receptor antagonist, through a microdialysis probe resulted in dose-dependent increases in NO_x⁻ levels. Higher-dose perfusion of either muscimol (50 μM), a GABA_A receptor agonist, or baclofen (250 μM), a GABA_B receptor agonist resulted in a significant decrease in NO_x⁻ levels. The elevated levels of NO_x⁻ after mild intermittent footshock were attenuated by perfusion of either muscimol (10 μM) or baclofen (50 μM), either of which alone did not affect basal NO_x⁻ levels. These findings are likely to provide helpful clues to our understanding of the inhibitory modulation of basal and footshock-induced NO metabolites releases by GABA_A and GABA_B receptors in the mPFC.     

Autoradiographic localization of α5 subunit-containing GABAA receptors in rat brain

Multiple subtypes of GABA_A receptors are expressed in the rat central nervous system (CNS). To determine the distribution and proportion of α5 subunit containing receptors, quantitative autoradiographic analyses were performed with both [³H]L-655,708 and [³H]Ro15-1788, an α5 selective and a non selective benzodiazepine binding site ligand, respectively. High densities of [³H]L-655,708 binding sites were observed in hippocampus and olfactory bulb, where α5 receptors accounted for 20–35% of total [³H]Ro15-1788 binding sites. Low levels of [³H]L-655,708 sites were associated with the cortex as well as amygdala, thalamic, hypothalamic and midbrain nuclei. These observations indicate that although [³H]L-655,708 binding sites have an overall low expression in rat CNS, they may contribute significantly to GABAergic inhibition in specific brain regions.     

Lower GABAA receptor binding in the amygdala and hypothalamus of spontaneously hypertensive rats

The central GABAergic system is associated with normal blood pressure regulation, but the role of GAGA receptors in genetic hypertension remains unclear. This study was conducted to investigate GABA_A receptor binding in several brain regions of spontaneously hypertensive (SHR) rats during development of hypertension. GAGA_A receptor binding was labeled with [³⁵S]TBPS and was assessed by quantitative autoradiography with the aid of a computer-assisted image analysis system. Densities of GABA_A receptor binding sites were significantly lower in all hypothalamic and amygdaloid nuclei evaluated in 4-week-old SHR rats, when compared with their age-matched normotensive Wistar-Kyoto rats. At 12 weeks of age, GABA_A receptor binding remained significantly lower in the central amygdaloid nucleus and paraventricular hypothalamic nucleus of SHR rats. Collectively, the results suggest that GABA_A receptors in these nuclei are likely to be involved in the initiation and maintenance of hypertension. In conclusion, this study supports a notion that downregulation of GABA_A receptor binding occurs in the hypothalamus and amygdala of SHR rats and may play a role in genetic hypertension.     

Localization of GABAA and GABAB receptor subtypes on serotonergic neurons

The effect of selective destruction of serotonin (5-HT)-containing neurons with 5,7-dihydroxytryptamine (5,7-DHT) on [3H] muscimol and (-)-[3H]baclofen binding was investigated in various rat brain regions. Ten days after intracerebroventricular 5,7-DHT, serotonin levels and [3H]imipramine binding were markedly decreased. 5,7-DHT reduced [3H]muscimol binding only in the mesencephalon, and (-)-[3H]baclofen binding was unmodified in all the areas considered. These results suggest that except in the mesencephalon GABA receptors may not be localized on serotonergic nerve terminals.     

Temperature-dependent effect of zolpidem on the GABAA receptor-mediated response at recombinant human GABAA receptor subtypes

The effects of zolpidem on the two forms of recombinant human GABA_A receptors (α1β2γ2s and α3β2γ2s) at different temperatures were functionally investigated, using the whole-cell patch recording configuration. In both forms, zolpidem potentiated the response to GABA in a concentration-dependent manner. At 16°C, the apparent dissociation constant (K_D) values for the α1β2γ2s and α3β2γ2s forms were 3.7×10⁻⁸ and 5.6×10⁻⁷ M, respectively. When the temperature was increased to 36°C, the K_D values for the α1β2γ2s and α3β2γ2s forms were 2.1×10⁻⁷ and 1.5×10⁻⁶ M, respectively. Although the affinity ratio was reduced from 15.1 to 7.1-fold the selectivity of zolpidem for the α1β2γ2s still remained at 36°C.     

Pharmacological characterisation of the histamine H3 receptor in the rat hippocampus

The purpose of this report was to pharmacologically characterise the histamine H₃ in the rat hippocampus using radioligand binding studies with the H₃ receptor antagonist [¹²⁵I]iodophenpropit and the H₃ receptor mediated inhibition of [³H]noradrenaline release. A dissociation constant of 0.33 nM and a maximal number of binding sites of 125 fmol/mg protein were found for [¹²⁵I]iodophenpropit. Competition studies showed stereoselectivity for the (R) and (S) enantiomers of α-methylhistamine and 10 μM of GTP_γS shifted the curve of (R)-α-methylhistamine rightwards. Up to 1 μM, (R)-α-methylhistamine displaced only 30% whereas the tested H₃-antagonists displaced 50–60% of the total [¹²⁵I]iodophenpropit bound. This indicates the presence of an additional non-H₃ receptor binding site(s) for [¹²⁵I]iodophenpropit in the rat hippocampus. This secondary site shows low affinity for H₃ agonists, but high affinity for the tested H₃ antagonists. Electrically evoked [³H]acetylcholine release was shown in slices of rat hippocampus. No H₃ receptor modulation of [³H]acetylcholine release from hippocampal slices was detectable. However, H₃ receptor activation inhibited 42% of the electrically-evoked [³H]noradrenaline release in rat hippocampal slices. The inhibition of [³H]noradrenaline release was effectively antagonized by the H₃ antagonists thioperamide and burimamide. We describe the pharmacological identification of the histamine H₃ receptor in the rat hippocampus and its similarities and differences from the cortical H₃ receptor. These studies enable us to investigate changes in density and functionality of the hippocampal H₃ receptor under (patho)physiological conditions.     

Autoradiographic localization of the GABAA receptor agonist [H]muscimol in rat cerebral vessels

By the use of combined in vitro radioreceptor binding and autoradiographic techniques with [³H]muscimol as a ligand, we analyzed the distribution of GABA_A receptor sites in the arteries of the circle of Willis as well as in the arteries and arterioles of the pial-arachnoid membrane in the rat. [³H]Muscimol was bound by sections of rat cerebral vessels in a manner consistent with the existence of GABA_A receptors, with K_d and B_max values of 46 nM and 0.60 pmol/mg tissue respectively. [³H]Muscimol was bound by the medial layer of cerebral arteries, while no specific binding was observed in the intima, the adventitia and the adventitial-medial border. These findings suggest that the vasodilatory action of GABA on in vitro preparations of cerebral vessels is mediated by muscular receptor sites. The posterior cerebral arteries are richer in [³H]muscimol binding sites than the anterior ones. Pial-arachnoid arterioles, which are of critical importance in controlling local cerebral blood flow, did not exhibit any significant binding of [³H]muscimol. These results may explain the difficulty in manipulating pharmacologically the cerebral tissue perfusion in intact animals using GABAergic agonists.     

Antidepressant effects on GABA-stimulated Cl influx in rat cerebral cortex are altered after treatment with GABAA 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 α1, β1, β2 or γ2 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 α1, β1, or β2 subunit aODNs but potentiated after microinjections of γ2 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.     

Benzodiazepine- and barbiturate-interactions with GABAA receptor responses on lactotrophes

Modulation of the biphasic effect of muscimol on prolactin secretion by benzodiazepines and secobarbital was investigated, using an in vitro superfusion system. The stimulatory effect of low concentrations of muscimol was potentiated by both classes of drugs, and the effect of benzodiazepines appeared to be mediated by central-type benzodiazepine receptors. Neither benzodiazepines nor secobarbital affected the inhibitory response to muscimol. Clonazepam reduced the potency of bicuculline methiodide as an antagonist of the stimulatory effect, but did not alter the potency of picrotoxinin. These results demonstrate a selective potentiation of one component of the GABAA receptor effect on lactotrophs by benzodiazepines and barbiturates and provide evidence for a functional effect of these drugs at a site without the CNS.     

Prolonged exposure to hypobaric hypoxia transiently reduces GABAA receptor number in mice cerebral cortex

The central nervous system is severely affected by hypoxic conditions, which produce alterations in neural cytoarchitecture and neurotransmission, resulting in a variety of neuropathological conditions such as convulsive states, neurobehavioral impairment and motor CNS alterations. Some of the neuropathologies observed in hypobaric hypoxia, corresponding to high altitude conditions, have been correlated with a loss of balance between excitatory and inhibitory neurotransmission, produced by alterations in glutamatergic and GABAergic receptors. In the present work, we have studied the effect of chronic hypobaric hypoxia (506 hPa, 18 h/day×21 days) applied to adult male mice on GABA_A receptors from cerebral cortex, to determine whether hypoxic exposure may irreversibly affect central inhibitory neurotransmission. Saturation curves for [³H]GABA specifically bound to GABA_A receptors in isolated synaptic membranes showed a 30% decrease in maximal binding capacity after hypoxic exposure (B_max control, 4.70±0.19, hypoxic, 3.33±0.10 pmol/mg protein), with no effect on GABA binding sites affinity (K_d control: 159.3±13.3 nM, hypoxic: 164.2±15.1 nM). Decreased B_max values were observed up to the 10th post-hypoxic day, returning to control values by the 15th post-hypoxic day. Pharmacological properties of GABA_A receptor were also affected by hypoxic exposure, with a 45 to 51% increase in the maximal effect by positive allosteric modulators (pentobarbital and 5α-pregnan-3α-ol-20-one). We conclude that long-term hypoxia produces a significant but reversible reduction on GABA binding to GABA_A receptor sites in cerebral cortex, which may reflect an adaptive response to this sustained pathophysiological state.     

Bihemispheric reduction of GABAA receptor binding following focal cortical photothrombotic lesions in the rat brain

Focal brain lesions may lead to neuronal dysfunctions in remote (exofocal) brain regions. In the present study, focal lesions were induced in the hindlimb representation area of the parietal cerebral cortex in rats using the technique of photothrombosis. Photothrombosis occurs after illumination of the brain through the intact skull following intravenous injection of the photosensitive dye Rose Bengal. This resulted in cortical lesions with a diameter of about 2 mm. Quantitative receptor autoradiography was used to study alterations in the density of []muscimol binding sites to GABA_A receptors seven days after lesion induction. A reduced GABA_A receptor binding (−13 to −27% of the control value) was found in layers II and III of remote exofocal regions in the ipsi- and contralateral cortex. The reduction was consistently more intense in the ipsilateral cortical areas than in those of the contralateral hemisphere. Using extracellular recordings, significant correlations between GABA_A receptor binding and paired pulse inhibition could be demonstrated. The present investigation demonstrates that focal brain lesions cause a widespread, functionally effective down-regulation of GABA_A receptors. These postlesional changes may result from lesion-induced alterations in cortical connectivity.     

Glucocorticoid binding sites in human temporal cortex

This work describes the presence of glucocorticoid binding sites in human temporal cortex obtained following partial lobectomy in two epileptic patients. Using [³H]dexamethasone as radioligand and cold cortisol or RU 28362 as competitor we found an apparent K_d of 2.8 nM with a B_max of approximately 34 fmol/mg protein. The order of potency of various unlabeled steroids to compete for [³H]dexamethasone binding was as follows: RU 28362 = RU 38486 = cortisol = dexamethasone > progesterone > spironolactone > estradiol. These data provide evidence for an intracellular mechanism by which circulating glucocorticoids might regulate neuronal function in the human cortex.     

Clathrin-coated vesicles from bovine brain contain uncoupled GABAA receptors

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABA_A receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [³H]muscimol, a GABA_A-specific agonist, [³H]flunitrazepam, a benzodiazepine agonist, and [³⁵S]t-butylbiocyclophosphorthionate (TBPS), a ligand for GABA_A 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 K_D 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 μM and at saturation (100 μM) 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 GABA_A receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABA_A 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 GABA_A receptor β2 and α1 subunits. Thus, in comparison to GABA_A receptors on synaptic membranes, those on CCVs have a reduced α1/β2-subunit ratio. It may be suggested that a selective decline in the content of α1 subunits in coated vesicles could in part account for GABA_A receptor uncoupling.     

Sexual dimorphism of GABAA receptor levels in subcortical brain regions of a woodland rodent (Apodemus sylvaticus)

This is the first report of quantitative autoradiography results showing sex differences of GABA_A receptor levels in brain regions of a wild rodent (wood mouse, Apodemus sylvaticus) living in its natural habitat. The labeling of this GABAergic site with its specific high affinity radioligand [³H] muscimol provided a heterogeneous and dimorphic binding pattern in some of the neural centers. In the female, higher ( ≥ 50 ≥ 65%) to moderately higher (<50%) binding levels than in the male, even after correction of the specific binding values using the calculated quenching coefficients, were observed in the substantia nigra pars reticulata and ventral lateral thalamic nucleus, brain centers that are relays of motor circuits. In the male, on the other hand, a higher level was only obtained in the caudateputamen. Relays of the stria terminalis-hypothalamic-central gray pathway such as the bed nucleus of the stria terminalis, the pontine central gray and the ventromedial hypothalamic nucleus, were among the other female brain areas with an extremely higher (>65%) to higher and moderately higher binding activity than in the male. From the saturation analyses, it appeared that the binding differences were mainly due to B_max variations, although closer examinations revealed that changes in the K_D might have also accounted for [³H] muscimol binding differences, as shown by the high K_D and B_max values in the bed nucleus of the stria terminalis, the substantia nigra pars reticulata and the pontine central gray of the female wood mouse. These findings suggest that the diamorphic binding activity of GABA_A receptors in the above brain regions might be involved in neuronal circuitry mechanisms related to sex-specific social behaviors in rodents living in their natural environmental conditions.     

Calcineurin-mediated GABAA receptor dephosphorylation in rats after kainic acid-induced status epilepticus

Calcineurin (CaN) is a neuronally enriched, calcium-dependent phosphatase, which plays an important role in a number of neuronal processes including development of learning and memory, and modulation of receptor's function and neuronal excitability as well as induction of apoptosis. It has been established in kindling model that the status epilepticus (SE)-induced increase in CaN activity is involved in the development of seizures through down-regulation of γ-aminobutyric acid A receptor (GABA_AR) activation. However, the mechanism by which CaN mediates GABA_A receptor dephosphorylation in SE is not fully understood. Here, using a model of kainic acid (KA)-induced SE and CaN inhibitor FK506, we observed the behaviors induced by KA and levels of CaN activity and CaN expression in hippocampus by immunobloting. The results showed that the SE-induced CaN activity was time-dependent, with a peak at 2 h and a return to basal level at 24 h, whereas a significant increase in CaN expression was seen at 24 h after SE. It is proposed that the rapid elevation in CaN activity after KA-induced SE is not likely due to an increase in CaN expression but rather an increase in CaN activation state or kinetics. In addition, we also demonstrated that pre-treatment with FK506 remarkably suppressed the SE-induced CaN activity and its expression, and reversed the SE-induced dephosphorylation of GABA_AR 2/3 subunits. Taken together, our data suggest that down-regulation in inhibition of GABA_AR 2/3 by CaN activity contributes to an elevation in neuronal excitability of hippocampus, which may be involved in development of chronic processes of seizures.     

Developmental changes of GABAA receptor-chloride channels in rat Meynert neurons

The developmental changes of GABA_A receptors were investigated in Meynert neurons freshly dissociated from day 0, 2 week-, and 6 month-old rats using both nystatin and gramicidin perforated patch recording modes under voltage-clamp conditions. The age-related changes in the current amplitude and threshold concentration in the concentration–response relationships for GABA indicated the developmental alteration of the GABA_A receptor subunits and the channel density. The GABA-induced

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measured by the gramicidin perforated patch mode shifted to more negative with development. The decay time constant of GABAergic inhibitory postsynaptic spontaneous currents (sIPSCs) in the synaptic active zone accelerated with aging. The GABA-induced currents were potentiated in a concentration dependent manner in the presence of benzodiazepine (BZP) agonists, diazepam (DZP) and zolpidem (ZPM). The potentiation rate of DZP on the GABA_A response decreased with aging, but not in the case of ZPM, which demonstrated a stronger action in the aging rat neurons. These results suggested that the GABA_A receptor·Cl⁻ channel complexes may thus change both the assembly and interaction of subunits as well as their functional roles with aging.     

Modulation of acetylcholine release via GABAA and GABAB receptors in rat striatum

In order to investigate whether changes in acetylcholine (ACh) release induced by GABA receptors are due to a direct or indirect effect on cholinergic neurons in the striatum, GABA_A and GABA_B receptor bindings were assayed in the striatum microinjected with ethylcholine mustard aziridinium ion (AF64A), a cholinergic neurotoxin. Intra-striatal injection of a selective concentration of AF64A (10 nmol) reduced GABA_A receptor binding without significantly altering GABA_B receptor binding. Treatment with a higher, less selective concentration of AF64A (20 nmol) reduced all markers examined. These results suggest that GABA_A, but not GABA_B receptors, are located on cholinergic neurons in the striatum, and that GABA can directly modulate ACh release through stimulation of GABA_A receptors. Findings further suggest that GABA can also indirectly modulate ACh release through stimulation of GABA_B receptors located on non-cholinergic neuronal elements in the striatum.     

Localization of specific neurotensin binding sites in the rat adrenal gland

Specific tritiated neurotensin binding sites were localized in the rat adrenal gland by receptor autoradiography and characterized using a tissue homogenate receptor binding assay. High levels of specific neurotensin binding sites were found in the inner layer of the adrenal cortex and lower amounts in the adrenal medulla with only background labeling in the outer cortical layers. The structure-activity profile of the specific neurotensin binding was consistent with binding to a physiological neurotensin receptor.