Muscimol Binding Sites Research Articles

Honokiol and magnolol increase the number of [3H] muscimol binding sites three-fold in rat forebrain membranes in vitro using a filtration assay, by allosterically increasing the affinities of low-affinity sites.

1. The bark of the root and stem of various Magnolia species has been used in Traditional Chinese Medicine to treat a variety of disorders including anxiety and nervous disturbances. The biphenolic compounds honokiol (H) and magnolol (M), the main components of the Chinese medicinal plant Magnolia officinalis, interact with GABA(A) receptors in rat brain in vitro. We compared the effects of H and M on [3H]muscimol (MUS) and [3H]flunitrazepam (FNM) binding using EDTA/water dialyzed rat brain membranes in a buffer containing 150 mM NaCl plus 5 mM Tris-HCl, pH 7.5 as well as [35S]t-butylbicyclophosphorothionate (TBPS) in 200 mM KBr plus 5 mM Tris-HCl, pH 7.5. H and M had similar enhancing effects on [3H]MUS as well as on [3H]FNM binding to rat brain membrane preparations, but H was 2.5 to 5.2 times more potent than M. 2. [3H]FNM binding. GABA alone almost doubled [3H]FNM binding with EC50 = 450 nM and 200 nM using forebrain and cerebellar membranes, respectively. In the presence of 5 microM H or M the EC50 values for GABA were decreased to 79 and 89 nM, respectively, using forebrain, and 39 and 78 nM, using cerebellar membranes. H and M potently enhanced the potentiating effect of 200 nM GABA on [3H]FNM binding with EC50 values of 0.61 microM and 1.6 microM using forebrain membranes, with maximal enhancements of 33 and 47%, respectively. Using cerebellar membranes, the corresponding values were 0.25 and 1.1 microM, and 22 and 34%. 3. [3H]MUS binding. H and M increased [3H]MUS binding to whole forebrain membranes about 3-fold with EC50 values of 6.0 and 15 microM. Using cerebellar membranes, H and M increased [3H]MUS binding approximately 68% with EC50 values of 2.3 and 12 microM, respectively. Scatchard analysis revealed that the enhancements of [3H]MUS binding were due primarily to increases in the number of binding sites (Bmax values) with no effect on the high affinity binding constants (Kd values). The enhancing effect of H and M were not additive. 4. [35S]TBPS binding. H and M displaced [35S]TBPS binding from sites on whole rat forebrain membranes with IC50 values of 7.8 and 6.0 microM, respectively. Using cerebellar membranes, the corresponding IC50 values were 5.3 and 4.8 microM. These inhibitory effects were reversed by the potent GABA(A) receptor blocker R5135 (10 nM), suggesting that H and M allosterically increase the affinity of GABA(A) receptors for GABA and MUS by binding to sites in GABA(A) receptor complexes. 5. Two monophenols, the anesthetic propofol (2,6-diisopropylphenol, P) and the anti-inflammatory diflunisal (2',4'-difluoro-4-hydroxy-3-biphenyl carboxylic acid, D) also enhanced [3H]MUS binding, decreased the EC50 values for GABA in enhancing [3H]FNM binding and potentiated the enhancing effect of 200 nM GABA on [3H]FNM binding, although enhancements of [3H]MUS binding for these monophenols were smaller than those for H and M, using forebrain and cerebellar membranes. The enhancing effect of P and D on [3H]MUS binding were almost completely additive. 2,2'-biphenol was inactive on [3H]MUS and [3H]FNM binding. These, and other preliminary experiments, suggest that appropriate ortho (C2) and para (C4) substitution increases the GABA-potentiating activity of phenols. 6. The potentiation of GABAergic neurotransmission by H and M is probably involved in their previously reported anxiolytic and central depressant effects.

Bihemispheric reduction of GABA A 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 [ 3 H ]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.

Rat and human hippocampal alpha5 subunit-containing gamma-aminobutyric AcidA receptors have alpha5 beta3 gamma2 pharmacological characteristics.

The gamma-aminobutyric acid (GABA)A receptor is a hetero-oligomer consisting of five subunits, the combination of which confers unique pharmacological properties to the receptor. To understand the physiological role of native GABAA receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human alpha5 beta3 gamma2 and alpha5beta3gamma3 GABAA receptors stably expressed in L(tk-) cells were characterized with the alpha5-selective ligand [3H]L-655,708 and compared with the pharmacological characteristics of [3H]L-655,708 binding sites from rat and human hippocampus. Saturation analyses revealed a 9-fold selective affinity of [3H]L-655,708 for alpha5 beta3 gamma2 receptors (Kd = 1.7 +/- 0.4 nM), compared with alpha5 beta3 gamma3 receptors (Kd = 15 +/- 3 nM). Rat and human hippocampal [3H]L-655,708 binding sites had affinities of 2.2 +/- 0.6 and 1.0 +/- 0.2 nM, respectively, comparable to the affinity of alpha5 beta3 gamma2 receptors. Pharmacological analysis of [3H]L-655,708 binding sites in rat and human hippocampi revealed a strong correlation with the affinities of seven benzodiazepine site ligands for alpha5 beta3 gamma2 but not alpha5 beta3 gamma3 receptors. Immunoprecipitation of [3H]L-655,708 binding sites from rat hippocampus with a gamma2-selective antibody yielded 19 +/- 4% of total benzodiazepine binding sites measured using [3H]Ro15-1788, whereas no specific binding was measured after immunoprecipitation with an anti-gamma3 antibody. Combinatorial immunoprecipitations of [3H]muscimol binding sites with anti-alpha5 and anti-gamma2 or anti-alpha5 and anti-gamma3 antibodies established the preferential expression of alpha5 gamma2 receptors, accounting for 22 +/- 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of alpha5 beta3 gamma2 GABAA receptors in rat hippocampus, despite the clustering of alpha5 and gamma3 loci on the same chromosome.

Gamma-aminobutyric acidA receptor function is desensitised in rat cultured cerebellar granule cells following chronic flunitrazepam treatment.

This study examined gamma-aminobutyric acidA (GABA(A)) receptor function in cultured rat cerebellar granule cells by using microphysiometry following chronic flunitrazepam exposure, and correlated the findings with the alpha1 and beta2/3 subunit protein expression and [3H]muscimol binding after the same treatment paradigm. Flunitrazepam treatment reduced (p < 0.05) the maximal GABA-stimulated increase in extracellular acidification rate (Emax) (16.5 +/- 1.2% and 11.3 +/- 1.0%, 2-day control and treated cells, respectively; 17.4 +/- 1.0% and 9.9 +/- 0.7%, 7-day control and treated cells, respectively; best-fit Emax +/- SEM, n = 7), without affecting the GABA concentration required to elicit 50% of maximal response (EC50) (1.2 +/- 1.7 and 2.3 +/- 1.8 microM, 2-day control and treated cells, respectively; 1.7 +/- 1.5 and 1.5 +/- 1.5 microM, 7-day control and treated cells, respectively; best-fit EC50 +/- SEM, n = 7). Flunitrazepam exposure also abolished the flunitrazepam potentiation of the GABA response, caused a transient reduction of the GABA(A) receptor alpha1 and beta2/3 subunit proteins over the initial 2 days, but did not alter [3H]muscimol binding compared with vehicle-treated cells. The results suggest that changes in GABA(A) receptor subunit protein expression, rather than loss of [3H]muscimol binding sites, underlie the chronic flunitrazepam-mediated desensitisation of GABA(A) receptor function.

Long-term changes of ionotropic glutamate and GABA receptors after unilateral permanent focal cerebral ischemia in the mouse brain

Long-term hyperexcitability was found after unilateral, permanent middle cerebral artery occlusion in exofocal neocortical areas of the adult mouse [Mittmann et al. (1998) Neuroscience 85, 15–27]. The aim of the present study was to test the hypothesis in an identical paradigm of ischemia, whether alterations in the densities of both excitatory and inhibitory amino acid receptors may underlie these pathophysiological changes. Alterations in densities of [ 3H]dizocilpine, [ 3H] d, l- α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, [ 3H]kainate and [ 3H]muscimol binding sites were demonstrated with quantitative in vitro receptor autoradiography. All binding sites were severely reduced in the core of the ischemic lesion. A completely different reaction was found in the exofocal, histologically inconspicous parts of the somatosensory cortex and the more remote neocortical areas of both hemispheres. The [ 3H]muscimol binding sites were significantly reduced four weeks after ischemia in the motor cortex, hindlimb representation area and exofocal parts of the primary and secondary somatosensory cortices of both hemispheres. The focus of the reduction in [ 3H]muscimol binding sites was found in lower layer V and upper layer VI. Contrastingly, the densities of [ 3H]dizocilpine binding sites were found to be increased in these areas, whereas those of [ 3H] d, l- α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and [ 3H]kainate binding sites did not show significant changes. The [ 3H]dizocilpine binding site density increased predominantly in layers III and IV. All binding sites were also reduced in the retrogradely reacting, gliotic part of the ipsilateral ventroposterior thalamic nucleus, whereas the [ 3H] d, l- α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid binding sites were increased in the surround of the ipsilateral nucleus and no changes in binding sites were seen in the whole contralateral nucleus. We conclude that permanent local ischemia leads to a long-term and widespread impairment of the normal balance between binding sites of excitatory and inhibitory neurotransmitter receptors in neocortical areas far away from the focus of the post-ischemic tissue damage. The imbalance comprizes an up-regulation of the [ 3H]dizocilpine binding sites in the ion channels of N-methyl- d-aspartate receptors and a down-regulation of [ 3H]muscimol binding sites of the GABA A receptors in the ipsi- and contralateral neocortex. These changes at the receptor level explain the previously observed hyperexcitability with the appearance of epileptiform field potentials and the long duration of excitatory postsynaptic potentials four weeks after ischemia.

Development of MK-801, kainate, AMPA, and muscimol binding sites and the effect of dark rearing in rat visual cortex.

We used quantitative autoradiography to determine whether the development of glutamate receptors correlates with the plastic period for monocular deprivation in rat visual cortex. To study glutamate receptors, we incubated sections of rat visual cortex with tritiated (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10imin e maleate (MK-801), tritiated kainate, and tritiated amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA). [3H]MK-801 is a noncompetitive ligand for the N-methyl-D-aspartate (NMDA) receptor. [3H]kainate and [3H]AMPA are competitive ligands for non-NMDA receptors. To compare glutamate binding sites with a nonglutamate binding site, we studied [3H]muscimol, which binds to gamma-aminobutyric acid (GABA)A receptors. [3H]MK-801 binding was maximal at postnatal day 26 (P26) and decreased in adulthood. [3H]AMPA binding was maximal at P18. [3H]kainate binding and [3H]muscimol binding were not age dependent. Dark rearing partially prevented the age-dependent decrease in [3H]MK-801 binding but had no effect on [3H]kainate or [3H]AMPA binding. Dark rearing decreased muscimol binding in adult animals. These results suggest that NMDA receptors, but not other glutamate receptors or GABAA receptors, are likely to be critical for developmental plasticity in rat visual cortex.

Regional differences in NaCl-induced increase of the potency of bicuculline to displace [ 3H]muscimol binding

It has been shown that the potency of bicuculline to displace [ 3H]muscimol binding to crude brain membranes can be enhanced markedly by different anions. This study shows that although bicuculline alone was a more potent displacer of [ 3H]muscimol binding in cortical than in cerebellar membranes, the NaCl (250 mM)-induced leftward shift of the bicuculline inhibition curve of [ 3H]muscimol binding was considerably higher in cerebellum than in cortex. The same concentration of NaCl failed to affect either the affinity or the density of cortical and cerebellar [ 3H]muscimol binding sites. The results suggest that sodium chloride is able to reveal regional differences in bicuculline potency.

Age-related modifications on the GABA A receptor binding properties from Wistar rat prefrontal cortex

In the present communication we have investigated the pharmacological properties of the GABA A receptor from adult (3 months old) and aged (24 months old) Wistar rat prefrontal cortex. The prefrontal cortex is implicated in cognitive functions and stress and both processes seem to be altered during aging. These changes could be mediated by modifications in the GABA A receptor properties. Our results indicated the absence of generalized age-related modifications on the pharmacological properties of the GABA A receptor from prefrontal cortical membranes. Saturation experiments using the non-selective benzodiazepine [ 3H]flunitrazepam revealed that neither the K d values or the B max were modified during aging. Moreover, Cl 218 872 displacement of [ 3H]flunitrazepam showed no age-related modifications on either the K is or the relative proportion between the Type I and Type II benzodiazepine binding sites. Therefore, the benzodiazepine binding sites are well preserved in aged prefrontal cortex. On the other hand, saturation experiments using the GABA agonist [ 3H]muscimol demonstrated a decrease in the B max of the low affinity [ 3H]muscimol binding sites in aged rats (4.3 ± 0.8 pmol/mg protein vs. 2.3 ± 0.2 pmol/mg protein in adult and aged rats, respectively). However, no age-dependent modifications were observed in the allosteric interaction between GABA and benzodiazepine binding sites. These results demonstrate that the benzodiazepine binding sites and the GABA binding sites of the GABA A receptor complex from rat prefrontal cortical membranes are differentially affected by the aging process.

Diazepam-insensitive GABA A receptors in rat cerebellum and thalamus

There major populations of GABA A receptor binding sites are present in cerebellar membranes: diazepam-sensitive [ 3H]Ro15-4513 binding sites, diazepam-insensitive [ 3H]Ro15-4513 binding sites and high-affinity [ 3H]muscimol binding sites. All three populations contain a β subunit as shown by immunoprecipitation with antibodies that recognize all β subunits. The β3 subtype of β subunit is contained in all three populations, but only a similar low fraction (< 20%) in each. Thus, the majority contain β subunits other than β3 (β2 and β1) and β3 subunits are not selectively associated with nor lacking in any of the three binding populations. Antibodies to the γ2 subunit precipitated similar fractions of [ 3H]Ro15-4513, [ 3H]flunitrazepam and [ 3H]muscimol binding sites, showing that γ2 subunits are present in high-affinity muscimol binding isoforms, as well as a significant fraction of the diazepam-insensitive [ 3H]Ro15-4513 binding sites. Under conditions that identify the 56 kDa α6 subunit on SDS-PAGE as the diazepam-insensitive site of [ 3H]Ro15-4513 binding in cerebellum, no polypeptide showing diazepam-insensitive binding of [ 3H]Ro15-4513 could be photoaffinity-labeled in rat thalamus. These results suggest that α4 subunits in the thalamus participate primarily in subunit combinations which bind muscimol but not any benzodiazepine site ligands.

Interaction of allosteric ligands with GABA A receptors containing one, two, or three different subunits

The presence of allosteric binding sites on recombinant GABA A receptors formed after transfection of human embryonic kidney (HEK) 293 cells with α 1-, β 3-, γ 2-subunits, or with various combinations of these subunits, was systematically investigated. From all possible subunit combinations, high affinity [ 3H]muscimol binding sites were induced in cells transfected with α 1 β 3- or α 1 β 3 γ 2-subunits only. GABA A receptor associated [ 3H]flunitrazepam binding sites were induced in cells after transfection with α 1 γ 2- or α 1 β 3 γ 2-subunits, and [ 35S] t-butylbicyclophosphorothionate (TBPS) binding sites were found in cells transfected with β 3-, β 3 γ 2-, α 1 β 3-, or α 1 β 3 γ 2-subunits. Binding of [ 35S]TBPS could be inhibited by pentobarbital, etazolate, (+)-etomidate, alphaxalone, propofol, chlormethiazole, and 4′-chlorodiazepam (Ro 5-4864) with a potency which differed in cells transfected with β 3-, β 3 γ 2-, α 1 β 3-, or α 1 β 3 γ 2-subunits. Results obtained indicate that receptors with different subunit composition actually can be formed in HEK cells and exhibit distinct pharmacological properties.

Laminar distribution of MK-801, kainate, AMPA, and muscimol binding sites in cat visual cortex: A developmental study

We used quantitative autoradiography to determine whether the development of glutamate receptors correlates with the sensitive period for monocular deprivation in the visual cortex. To study glutamate receptors, we incubated sections of cat visual cortex with tritiated (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10imin e-maleate (MK-801), tritiated kainate, and tritiated amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA). [3H]MK-801 is a noncompetitive ligand for the N-methyl-D-aspartate (NMDA) receptor. [3H]kainate and [3H]AMPA are competitive ligands for non-NMDA receptors. We used [3H]muscimol, which binds to GABAA receptors, so that we would have one control ligand that binds to a nonglutamate receptor. When all layers were combined, the results confirmed our previous studies with homogenate binding. [3H]MK-801 and [3H]kainate binding were significantly greater at 42 days than at earlier or later times. [3H]AMPA and [3H]muscimol binding did not show such a peak. This suggests that MK-801 and kainate binding sites are more likely to be involved in plasticity than are AMPA and muscimol binding sites. In layers 2/3, MK-801 had the greatest age-dependent changes; in layers 5 and 6, kainate binding changed most with age. This suggests that the mechanisms of plasticity may vary with cortical layer.

Laminar distribution of MK‐801, kainate, AMPA, and muscimol binding sites in cat visual cortex: A developmental study

Laminar distribution of MK‐801, kainate, AMPA, and muscimol binding sites in cat visual cortex: A developmental study

Endogenous Phosphorylation of Distinct γ-Aminobutyric Acid Type A Receptor Polypeptides by Ser/Thr and Tyr Kinase Activities Associated with the Purified Receptor

We have investigated the phosphorylation of gamma-aminobutyric acid type A (GABAA) receptor purified from bovine cerebral cortex in the absence of added kinases. Incubation of the affinity-purified receptor with [gamma-32P]ATP and 500 microM MnCl2 yielded incorporation of 0.45 mol of 32P/mol of muscimol binding sites within 2 h at 30 degrees C. Mn2+ was much more effective than Mg2+ as activator. Phosphorylation of the receptor was observed on at least three different polypeptides of 51, 53, and 55 kDa. It was predominant on 51- and 53-kDa polypeptides that co-migrate with the [3H]flunitrazepam photoaffinity-labeled bands, suggesting that 32P incorporation mainly occurs on alpha-subunits. A monoclonal antibody specific for alpha-subunits adsorbed the endogenously phosphorylated GABAA receptor with a stoichiometry close to 1 mol of phosphate/mol of muscimol. The phosphorylation of the 51-kDa polypeptide, corresponding to alpha 1-subunit, exhibited a micromolar affinity for ATP and sigmoid kinetics (nH = 2). Major incorporation of phosphate occurred on serine and threonine residues in roughly equimolar ratio. By enzyme-linked immunosorbent assay and immunoblotting studies we also detected a minor incorporation on tyrosine residues; this was specific for a 55-kDa polypeptide. Comparison with molecular data suggests that at least alpha 1- and alpha 2-subunits (Ser and Thr residues) and possibly gamma 2-subunits (Tyr residue) are endogenously phosphorylated by multiple kinases, with a clear preference for alpha 1-subunit. The beta-subunits were not phosphorylated in our experimental conditions. The corresponding kinase activities are closely associated to the receptor protein, indicating a new complexity in the regulation of the GABAA receptor.

Characterisation of δ-subunit containing GABA A receptors from rat brain

Polyclonal antibodies have been raised in rabbits against the predicted cytoplasmic loop region of the δ-subunit of the GABA A receptor. These specifically identify the expressed fragment by Western blot but do not cross react with analogous polypeptides from the γ 1, γ 2 or γ 3-subunits. Polyclonal antisera immunoprecipitated [ 3H]muscimol binding sites from several brain regions consistent with the reported distribution of δ-subunit mRNA and also detected the δ-subunit by Western blot, identifying a polypeptide of 55KDa. Receptors immunoprecipitated from rat brain with the δ-antisera exhibited an atypical profile with respect to their radioligand binding properties. Receptors immunoprecipitated from all regions tested bound [ 3H]muscimol, but did not bind benzodiazepine site ligands [ 3H]Ro 15,1788 or [ 3H]flunitrazepam with high affinity. Receptors containing a δ-subunit accounted for 10.7 ± 2% of all GABA A receptors ([ 3H]muscimol binding sites) in the rat central nervous system as deduced from quantitative immunoprecipitation experiments, the largest population being in the cerebellum where approximately 27% of all receptors contained a δ-subunit. The pharmacology of the GABA (γ-aminonutyric acid) binding site on receptors immunoprecipitated from cerebellum with γ 2 and δ-antisera was compared. The rank order of potency of a series of 6 compounds to compete for [ 3H]muscimol binding sites was similar in these two populations, but muscimol had a significantly higher affinity for receptors containing the δ-subunit. These receptors therefore comprise a novel population of GABA A receptors which do not bind benzodiazepines but have a 5-fold higher affinity for muscimol. Coexistence of the δ-subunit with γ-subunits was investigated by purifying receptor on a γ 1, γ 2 or γ 3-immunoaffinity resins, separating the subunit components by polyacrylamide gel electrophoresis, transferring to nitrocellulose and probing with δ-subunit specific antibodies. The δ-subunit could not be detected in combination with any γ-subunit.

Changes in GABA-benzodiazepine receptor complex and in peripheral benzodiazepine receptors in male mice after copulation

We studied the effect of copulation on GABA and benzodiazepine (BZD) receptors in the male mouse. After copulation, there was an 18% increase in the in vitro number of [ 3H]muscimol binding sites in frontal cortex. No changes were observed in central BZD binding sites labelled either in vivo by [ 3H]flunitrazepam or in vitro (in olfactory bulbs and in frontal cortex) by [ 3H]flumazenil, but further in vitro studies demonstrated that the GABA-stimulated [ 3H]flunitrazepam binding was reduced in both frontal cortex and olfactory bulbs. Copulation increased the number of peripheral BZD binding sites labelled by 3H-Ro 5-4864 in olfactory bulbs by 22% and in heart by 36%, but not in frontal cortex or in testes. The changes of GABA/BZD and peripheral BZD receptors in mouse suggest that the GABAergic system may be affected by copulation.

Thiomuscimol, a new photoaffinity label for the GABA A receptor

Thiomuscimol inhibits [ 3H]muscimol binding to brain GABA A receptors. Exposure of Ag +-treated membrane preparations to UV radiation at 254 nm for 40 min in the presence of thiomuscimol (10 −5 M) produced a 20–30% irreversible decrease in high-affinity [ 3H]muscimol binding sites. The photoaffinity labeling of thiomuscimol was inhibited by GABA (10 −4 M) added prior to exposure to UV light. The data show that thiomuscimol can label the GABA A receptor site and that the ligand can be used as a photoaffinity label for purification and identification of GABA binding sites within the GABA A receptor complex.

Possible intermolecular interaction between quinolones and biphenylacetic acid inhibits gamma-aminobutyric acid receptor sites.

The combination of some new quinolone antibacterial agents with 4-biphenylacetic acid (BPAA), a metabolite of fenbufen, is known to specifically induce functional blockade of the gamma-aminobutyric acid (GABA) receptors. The mechanisms of these drug interactions were further examined. Scatchard analysis of [3H]muscimol binding to rat brain plasma membranes in the presence of enoxacin and BPAA revealed that a significant decrease in the number of muscimol binding sites was produced without affecting the affinity of binding to the receptors. In the presence of norfloxacin, BPAA inhibited muscimol binding the most potently of the six BPAA-related compounds tested. Fenbufen and 9,10-dihydro-gamma-oxo-2-phenanthrenebutyric acid also inhibited the binding, and 4-biphenylcarboxylic acid and methyl 4-biphenylacetate inhibited it slightly, but 3-benzoylpropionic acid exhibited no competitive inhibition. Accordingly, hybrid molecules of norfloxacin and BPAA were synthesized for stereochemical analysis of these drug interactions. A hybrid with a -CONH(CH2)3- chain between norfloxacin and BPAA (flexible structure) inhibited muscimol binding, and intracisternal injection of this hybrid caused clonic convulsions in mice more potently than the combination of norfloxacin and BPAA did. In contrast, a hybrid linked by -CONH- (stretched structure) showed almost no such inhibitory effect. 1H NMR analysis indicated the presence of intramolecular attraction at the quinoline ring of the hybrid exhibiting the antagonistic activity. These results suggest the possibility that quinolones and BPAA interact with the GABA receptor at nearby sites and that the binding affinity of quinolones to the GABA receptors is largely enhanced by the intermolecular interaction with BPAA.

Sexual differences in the effect of the GABAergic system on LH secretion and in the hypothalamic ontogenesis of GABA A receptors in prepubertal rats

The administration of aminooxyacetic acid (AOAA), which increases hypothalamic GABA concentrations, induced a significant increase in LH levels in female rats of 12, 16 and 18 days of age. This stimulatory effect of AOAA on serum LH levels was not observed at 21 and 25 days of age whereas a significant decrease in the LH concentrations by AOAA was found at 30 days of age. The neonatal androgenization of female rats abolished the effects of AOAA and no effects of AOAA were found in male rats at the different ages studied. The muscimol binding sites in medial basal and preoptic anterior hypothalamic areas showed a significant different sexual ontogenic pattern. Males rats castrated at birth showed as a very similar ontogenic pattern of [ 3H]muscimol binding sites to female rats and significantly different to male control rats. It is concluded that there are sexual differences in the effect of GABAergic system on LHRH-LH secretion and in the development of hypothalamic GABA A receptors during sexual maturation. These differences are probably connected with the neonatal exposure to androgens that would induced changes in the composition and probably in the properties of GABA A receptor that in turn modified its effects on LHRH neurons.

Region-specific changes of GABA A receptors by tolerance to and dependence upon pentobarbital

An experimental model for inducing tolerance to and dependence upon pentobarbital was characterized. Rats were infused with pentobarbital, 300 μg/10 μ1 per h i.c.v. for 6 or 7 days, through pre-implanted cannulas by osmotic minipumps. Measurement of brain and serum levels showed that pentobarbital remained mainly inside of the brain. Measurements of sleeping time and susceptibility to convulsant-induced seizures indicated a substantial degree of tolerance and dependence (24 h after termination of infusion). Results of GABA A receptor binding assays showed marked regional variations. While [ 35S]t-butylbicyclophosphorothionate (TBPS) binding sites were increased in frontal cortices and striata of pentobarbital-dependent animals, K D was increased in striata of tolerant animals. Dependence upon pentobarbital was correlated with increased [ 3H]flunitrazepam binding sites in all three regions examined. Both high and low affinity [ 3H]muscimol binding sites were increased in dependent animals, but low affinity sites were decreased in frontal cortices of tolerant animals. K Ds of [ 3H]muscimol high affinity sites were increased in cerebellum after animals developed dependence upon pentobarbital. K Ds of [ 3H]muscimol low affinity sites were decreased in striata of pentobarbital tolerant animals. These findings further support the hypothesis that GABA A receptors in discrete areas of the brain have different subunit compositions and are regulated differentially by pharmacological modulators.

Amino acid levels and gamma-aminobutyric acidA receptors in rat neostriatum, cortex, and thalamus after neonatal 6-hydroxydopamine lesion.

The amino acid gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in brain, and GABAergic neurons have been proposed to play a major role in basal ganglia physiology. In the neostriatum (caudate putamen), medium-sized aspiny interneurons, as well as neostriatal output neurons that project to several brain regions, use GABA as their neurotransmitter. Dopamine fibers arising from the substantia nigra represent a major input to the neostriatum where, besides their classic neurotransmitter role, they are seemingly involved in the regulation of amino acid neurotransmitter release. To further characterize the nature of some of the amino acid/dopamine interactions, selective dopaminergic deafferentations were produced in neonatal rats (3 days postnatal) by intraventricular administration of the neurotoxin 6-hydroxydopamine (6-OHDA); the noradrenergic neurons were protected by prior administration of desmethylimipramine. After a 3-month survival, levels of catecholamines, indoleamines, and amino acids were determined in cingulate cortex, thalamus, and neostriatum. In addition, GABAA receptors were measured in membrane preparations from these three regions, using the specific agonist [3H]muscimol. In the 6-hydroxydopamine-lesioned rats, levels of dopamine and its metabolites homovanillic acid, 3,4-dihydroxyphenylacetic acid, and 3-methoxytyramine were decreased, as expected, in cortex and neostriatum, but remained unmodified in thalamus. In all three regions, serotonin content was increased; its metabolite, 5-hydroxyindole-3-acetic acid, was also elevated, but only in cortex and neostriatum. The levels of GABA were increased in neostriatum and thalamus, but remained unmodified in cortex. Glycine was increased in all three regions examined. There were also increases of phosphatidylethanolamine and serine in thalamus, and of aspartic acid and alanine in neostriatum. The density of GABAA binding sites was increased in neostriatum, but remained unchanged in cortex and thalamus. The changes in amino acid levels and [3H]muscimol binding sites induced by a neonatal 6-hydroxydopamine treatment differ from those found after similar lesions in adult animals, possibly because of the plastic and synaptic rearrangements that can still occur during early postnatal development. The present results also demonstrate that adaptations occur in response to a dopaminergic deafferentation at an early age and that these exhibit a regional specificity.