Functional diversity of GABAA receptor ligand-gated chloride channels in rat synaptoneurosomes

Experiments were performed to examine neurochemically the functional diversity of GABA_A receptors as measured by muscimol-, 5α-pregnane-3α,21-diol-20-one (THDOC)-, and pentobarbital-stimulated ³⁶C1^? uptake, and region-specific changes in muscimol THDOC- and THDOC-induced potentiation of muscimol-stimulated ³⁶CI– uptake in rats treated acutely or subacutely with a subconvulsive dose of bicuculline. The data, for stimulation of ³⁶C1– uptake by muscimol showed a single binding site interaction in the cerebral cortex, hippocampus and cerebellum. The concentration-response curves for muscimol in the cerebral cortex and hippocampus were steep and indicated an increase of, approximately 130% at the maximum concentration. In contrast, the curve for the cerebellum was shallow and, exhibited a smaller maximal response (∽60%). Apparent affinity for muscimol also differed among these brain regions. The regional differences in ³⁶CI– uptake induced by THDOC and pentobarbital were not as apparent as those induced by muscimol; however, the maximal modulatory effect of pentobarbital, in the hippocampus was significantly higher than that in the cerebellum. In rats treated subacutely with a subconvulsive dose of bicuculline, a significant increase in muscimol-stimulated ³⁶C1- uptake was observed in the cerebellum but not in the frontal cortex or hippocampus. Analysis of the concentration-response curves for muscimol-stimulated ³⁶CI^? uptake in the cerebellum revealed that the Vmax for muscimol in the subacutely treated group was significantly higher than those for muscimol in the control and acutely treated groups without any differences in the KD value. In addition, THDOC-induced potentiation of muscimol-stimulated ³⁶CI^? uptake in the subacutely treated group was significantly higher than those in the control and acutely treated groups when a lower concentration (10 nM) of THDOC was used. These results suggest that regional variation of ³⁶C1^? uptake stimulated by muscimol is more apparent than those stimulated by THDOC and pentobarbital. The present results, along with our previous findings, also indicate that region-specific bicuculline up-regulation of GABA_A receptors in the cerebellum involves changes not only in binding characteristics but also in function of GABA receptor-gated chloride channel. © 1995 Wiley-Liss, Inc.     

Biochemical and behavioral effects of steroids on GABAA receptor function in long- and short-sleep mice

The in vitro and in vivo effects of alphaxalone, a steroid anesthetic, and two physiological steroids, tetrahydrodeoxycorticosterone (THDOC) and pregnenolone sulfate (PS), on GABA_A receptor function were evaluated in long-sleep (LS) and short-sleep (SS) mice. In vitro, both alphaxalone and THDOC enhanced GABAergic inhibition as measured by [³H]FNZ binding and GABA-stimulated ³⁶Cl⁻ flux. However, with the exception of alphaxalone potentiation of [³H]FNZ binding, which was greater in SS brain regions, LS and SS mice did not differ in their degree of enhancement. Pregnenolone sulfate produced mixed agonistic and antagonistic effects on GABAergic function, dependent upon brain region, with few differences between the lines of mice. In vivo effects of these steroids on sleep time indicated that, like other anesthetic agents, THDOC and alphaxalone induced longer sleep times in LS mice. Antagonism by PS of ethanol-induced sleep time was observed in LS mice only; however, this effect was dependent upon the dose of ethanol used and on the vehicle used to prepare the steroid. Pentobarbital-induced sleep time was not reduced by PS treatment in either line of mouse. These results demonstrate that few differences in sensitivity of the GABAergic receptor to these steroids exist between LS and SS mice. Thus, unlike the differences between LS and SS mice in GABAergic mediation of responses to ethanol and benzodiazepines, there is little genetic variability in subtypes of GABA_A receptors capable of modulation by steroids in these lines of mice.     

Glucocorticoids are modulators of GABAA receptors in brain

Modulation of binding of [³H]muscimol, a GABA_A receptor agonist, by natural and synthetic glucocorticoids was investigated in crude synaptosomal membranes and in brain sections of rat. In adrenalectomized (Adx) rats, muscimol binding was reduced by 30–50% in cerebral cortex, cerebellum, thalamus and hippocampus, as compared to sham-operated controls. This decrease was due to reduced binding affinities of GABA receptors for muscimol. In contrast muscimol binding was increased by 38% in the hypothalamus and did not change in the pons-medulla after Adx. Nanomolar concentrations of corticosterone and pregnenolone-sulfate, but not dexamethasone, enhanced muscimol binding in brain regions that were characterized by reduced binding following Adx. This steroid-induced increase in muscimol binding was due to enhanced affinities of GABA receptors.     

Changes of GABAA receptor binding and subunit mRNA level in rat brain by infusion of subtoxic dose of MK-801

In the present study, we have investigated the effects of prolonged inhibition of NMDA receptor by infusion of subtoxic dose of MK-801 to examine the modulation of GABA_A receptor binding and GABA_A receptor subunit mRNA level in rat brain. It has been reported that NMDA-selective glutamate receptor stimulation alters GABA_A receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunit. However, we have investigated the effect of NMDA antagonist, MK-801, on GABA_A receptor binding characteristics in discrete brain regions by using autoradiographic and in situ hybridization techniques. The GABA_A receptor bindings were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS in rat brain slices. Rats were infused with MK-801 (1 pmol/10 μl per h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]muscimol binding were highly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, the [³H]flunitrazepam binding and [³⁵S]TBPS binding were increased only in specific regions; the former level was increased in parts of the cortex, thalamus, and hippocampus, while the latter binding sites were only slightly elevated in parts of thalamus. The levels of β2-subunit were elevated in the frontal cortex, thalamus, hippocampus, brainstem, and cerebellar granule layers while the levels of β3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in MK-801-infused rats. The levels of α6- and δ-subunits, which are highly localized in the cerebellum, were increased in the cerebellar granule layer after MK-801 treatment. These results show that the prolonged suppression of NMDA receptor function by MK-801-infusion strongly elevates [³H]muscimol binding throughout the brain, increases regional [³H]flunitrazepam and [³⁵S]TBPS binding, and alters GABA_A receptor subunit mRNA levels in different directions. The chronic MK-801 treatment has differential effect on various GABA_A receptor subunits, which suggests involvement of differential regulatory mechanisms in interaction of NMDA receptor with the GABA receptors.     

Distribution of GABA binding site subtypes in rat pituitary gland

GABA_A and GABA_B binding sites in rat pituitary gland were investigated using equilibrium binding assays in vitro. Specific binding of both [³H]GABA and [³H]muscimol could be detected in both anterior and neurointermediate lobes, with a relative concentration in the anterior lobe. [³H]GABA binding was discriminated into GABA_A and GABA_B receptor type binding using baclofen. GABA_B sites were detectable in the anterior but not in the neurointermediate lobe. Saturation analysis of [³H]muscimol binding to whole pituitary gland membranes demonstrated that the pituitary contains two classes of GABA_A sites differing in affinity, as found in the CNS, although the number of sites is considerably lower than in the CNS.     

Pregnancy-induced alterations of GABAA receptor sensitivity in maternal brain: an antecedent of post-partum ‘blues’?

Pregnancy increases affinity of [³H]muscimol binding to GABA_A receptors in the rat forebrain. Post-partum, the receptor affinity is further increased concomitantly with a reduction of the receptor density. These changes may result from an action of endogenous placental and adrenal steroids, tetrahydroprogesterone and tetrahydrodeoxycorticosterone, which in vitro behave as allosteric agonists of GABA_A receptors. The alterations may contribute to the psycholigical phenomena associated with pregnancy and the puerperium.     

Biochemical and behavioral effects of steroids on GABAA receptor function in long- and short-sleep mice.

The in vitro and in vivo effects of alphaxalone, a steroid anesthetic, and two physiological steroids, tetrahydrodeoxycorticosterone (THDOC) and pregnenolone sulfate (PS), on GABAA receptor function were evaluated in long-sleep (LS) and short-sleep (SS) mice. In vitro, both alphaxalone and THDOC enhanced GABAergic inhibition as measured by [3H]FNZ binding and GABA-stimulated 36Cl- flux. However, with the exception of alphaxalone potentiation of [3H]FNZ binding, which was greater in SS brain regions, LS and SS mice did not differ in their degree of enhancement. Pregnenolone sulfate produced mixed agonistic and antagonistic effects on GABAergic function, dependent upon brain region, with few differences between the lines of mice. In vivo effects of these steroids on sleep time indicated that, like other anesthetic agents, THDOC and alphaxalone induced longer sleep times in LS mice. Antagonism by PS of ethanol-induced sleep time was observed in LS mice only; however, this effect was dependent upon the dose of ethanol used and on the vehicle used to prepare the steroid. Pentobarbital-induced sleep time was not reduced by PS treatment in either line of mouse. These results demonstrate that few differences in sensitivity of the GABAergic receptor to these steroids exist between LS and SS mice. Thus, unlike the differences between LS and SS mice in GABAergic mediation of responses to ethanol and benzodiazepines, there is little genetic variability in subtypes of GABAA receptors capable of modulation by steroids in these lines of mice.     

Studies on picrotoxin binding sites of GABAA receptors in rat cortical synaptoneurosomes

Experiments were performed to characterize [³⁵S]TBPS binding in rat cortical Synaptoneurosomes, which have vesicular structures containing both pre- and postsynaptic elements. Scatchard analysis revealed a single component of [³⁵S]TBPS binding sites with K_D and B_max values of 76.1 nM and 1.97 pmoles/mg protein, respectively, under physiological conditions. GABA and muscimol inhibited [³⁵S]TBPS binding in a concentration-dependent manner. IC₅₀ values of these GABA_A agonists in displacing synaptoneurosomal [³⁵S]TBPS binding are comparable to previously reported EC₅₀ values of the agonist-stimulated ³⁶C1⁻ uptake in Synaptoneurosomes by these agents. Furthermore, the IC₅₀ values of these GABA_A agonists were better correspondence to those determined by [³H]muscimol binding in synaptoneurosomal preparations as reported by Delorey and Brown (3) than those determined in membrane preparations. Although bicuculline increased [³⁵S]TBPS binding in a concentration dependent manner in cortical membranes, it did not affect synaptoneurosomal [³⁵S]TBPS binding. Benzodiazepine agonists and inverse agonists (0.1 to 10 μM) did not show any effects on the binding in the absence of muscimol. However, benzodiazepine agonists potentiated and inverse agonists antagonized muscimol-induced inhibition of synaptoneurosomal [³⁵S]TBPS binding. In addition, an anesthetic steroid, THDOC, and pentobarbital inhibited synaptoneurosomal [³⁵S]TBPS binding in a concentration dependent manner. These results suggest that allosteric modulation of [³⁵S]TBPS binding by various ligands which interact with GABA_A supramolecular complexes remain intact in Synaptoneurosomes. It appears that this preparation is useful for investigating correlation between functional ³⁶Cl⁻ uptake and individual binding studies of each of the GABA_A receptor complex.     

Effect of the pregnane-related GABA-active steroid alphaxalone on mice performance in the staircase test

We evaluated the modulatory effect of the GABA-active neurosteroid alphaxalone on the staircase test behavior of mice. Results were compared with the benzodiazepine alprazolam, the GABA_A agonist muscimol and the peripheral steroids corticosterone and progesterone. Alphaxalone and alprazolam reduced rearing activity in a dose-dependent manner, at doses that did not suppress climbing. The rearing-suppression effect of alprazolam, but not of alphaxalone, was blocked by the benzodiazepine antagonist flumazenil. No such dissociation between the effect on rearing and climbing was obtained with muscimol, and both activities were suppressed, in a flumazenil-insensitive pattern, at high doses. Corticosterone and progesterone did not affect the behavior of the mice. The lack of sensitivity of both phenobarbital and alphaxalone to flumazenil indicates that neither agents act via the benzodiazepine recognition site at the GABA_A receptor complex.     

Neuroactive steroids induce GABAA receptor‐mediated depolarizing postsynaptic potentials in hippocampal CA1 pyramidal cells of the rat

Intracellular recordings were performed in area CA1 pyramidal cells of rat hippocampal slices to determine the effects of certain steroids on inhibitory postsynaptic potentials/currents (IPSP/Cs) mediated by GABA_A receptors. Following application of the steroids 5α-pregnan-3α,21-diol-20-one (5α-THDOC), alphaxalone and 5β-pregnan-3α-ol-20-one (pregnanolone) hyperpolarizing PSPs developed into biphasic responses consisting of an early hyperpolarizing and a late depolarizing PSP sequence. Steroid-induced depolarizing PSPs could be elicited in the presence of antagonists to non-NMDA, NMDA, and GABA_B receptors, indicating that these receptor types do not contribute significantly to the initiation of these responses. Depolarizing PSPs were completely blocked by both GABA_A receptor antagonists bicuculline and t-butylbicyclophosphorothionat (TBPS) providing evidence for their mediation by GABA_A receptors. The reversal potential of steroid-induced late inward PSCs, measured in single-electrode voltage clamp, was ?29.9 ± 5.3 mV, whereas the early outward current, which corresponded to the early hyperpolarizing component of PSPs, reversed at ?68.2 ± 1.5 mV. Depolarizing PSPs and late inward PSCs were sensitive to reduction of extracellular [HCO₃^–] and block of carbonic anhydrase by application of acetazolamide. The results suggest that certain neuroactive steroids can induce GABA_A receptor-mediated depolarizing PSPs, which are dependent on HCO₃^–.     

Characterization of neuronal migration disorders in neocortical structures: quantitative receptor autoradiography of ionotropic glutamate, GABAA and GABAB receptors

Epileptiform activity was previously described [ Luhmann et al. (1998 ) Eur.J. Neurosci., 10, 3085–3094] in the neocortex of the adult rat following freeze lesioning of the newborn neocortex. After a survival time of 3 months, a small area of dysplastic cortex surrounded by histologically normal (exofocal) neocortex was observed. The dysplastic cortex is characterized by the formation of a small sulcus and a three- to four-layered architecture. Two questions are addressed here: (i) is the hyperexcitability associated with changes in binding to major excitatory and inhibitory transmitter receptors in the dysplastic cortex?; and (ii) do such changes also occur in the exofocal cortex? Alterations in binding to glutamatergic N-methyl-d -aspartate (NMDA), (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), kainate and GABA_A and GABA_B (γ-aminobutyric acid) receptors are demonstrated with quantitative in vitro receptor autoradiography by using the ligands [³H]MK-801, [³H]AMPA, [³H]kainate, [³H]muscimol and [³H]baclofen, respectively. In the dysplastic cortex, the binding to NMDA, AMPA and kainate receptors is significantly increased, whereas the binding to GABA_A and GABA_B receptors is reduced. Exofocal areas of the lesioned hemisphere show an imbalance between excitatory and inhibitory receptor binding with an up-regulation of the binding to AMPA and kainate, and a down-regulation to GABA_A receptors. The binding to GABA_B and NMDA receptors is not significantly changed in the exofocal areas. The imbalance between excitatory and inhibitory receptors may cause the hyperexcitability, as previously found in the identical experimental model, and may also induce epileptiform activity in the human cortex with migration disorders.     

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.     

Regional distribution of GABAA receptor binding sites in cat somatosensory and motor cortex

Inhibition in primary sensory cortex plays a role in neuronal response to periperal stimuli. For many neurons in cat primary somatosensory cortex, blockade of GABA_A receptors by bicuculline results in receptive field enlargement. The magnitude of this effect varies with the neuron's adaption characteristics and its location in particular laminae and submodality regions. To test whether these variation are correlated with the distribution of GABA_A receptors, we analyzed [³H] muscimol binding in cat primary somatosensory and motor cortical areas. The highest levels of binding were in layers IV was distinguished by highest levels were in layers V–VI. In somatosensory cortical areas, layer IV, levels of binding were significantly higher in posterior area 3b than in anterior area 3b. These difference may correspond to the rapidly adaption and slowly adapting submodality regions which have been described in this area. The laminar distribution of [³H] muscimol binding differed from that of [³H]flunitrazepam, and neither resembled the distribution of magnitude of bicuculline's effects on receptive field size. The laminar distribution of [³H] muscimol binding was highly correlated with the area density of GABA-immunoreactive neurons described in a companion study. © 1994 Wiley-Liss, Inc.     

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam and [³⁵S]TBPS in rat brain slices. The density of [³H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [³H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [³H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [³H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [³⁵S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg²⁺. Elevated [³⁵S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg²⁺. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.     

Age-related modifications on the GABAA 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.     

The Neuroactive Steroid 5α-Tetrahydrodeoxycorticosterone Increases GABAergic Postsynaptic Inhibition in Rat Neocortical Neurons in vitro

The neuroactive steroid 5α-pregnane-3α,21-diol-20-one (5α-tetrahydrodeoxycorticosterone; 5α-THDOC) has been shown to potentiate GABA-induced chloride currents in cell cultures and subcellular preparations. In this study, we recorded from pyramidal neurons in an in vitro slice preparation of the adult rat frontal neocortex using intracellular microelectrodes. 5α-THDOC (10 μM) increased and prolonged the inhibitory postsynaptic potential (IPSP). The mean maximal synaptic conductance of the early, GABA_A receptor-mediated, IPSP was enhanced to more than 700%, the one at the maximum of the late, partially GABA_A receptor-mediated, IPSP to approximately 400%. The progesterone/glucocorticoid receptor antagonist RU 38486 did not prevent the IPSP increase. At a concentration of 1 μM 5α-THDOC increased only the early IPSP to about 125%. Responses to the iontophoretically applied specific GABA_A receptor agonist muscimol but not to the specific GABA_B receptor agonist L-baclofen were enhanced by 5α-THDOC (10 μM). In the giga-seal whole-cell configuration when the GABA_B receptor-mediated IPSP component was absent due to intracellular perfusion, 5α-THDOC (10 μM) increased IPSPs to a similar extent as in the conventional microelectrode recordings. Excitatory postsynaptic potentials, resting membrane potential, input resistance and action potential amplitude were not affected by 5α-THDOC (10 μM). These data demonstrate that in neocortical tissue of the rat 5α-THDOC enhances GABAergic inhibition by interacting with postsynaptic GABA_A receptors while synaptic excitation and parameters of electric excitability remain unchanged.     

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.     

Modulation of the GABA receptor complex by a steroid anaesthetic

The interactions of a steroid anaesthetic, alphaxalone, with the GABA receptor-ionophore complex were investigated by two different experimental approaches. In the rat cuneate nucleus slice, alphaxalone (0.1-10 microM) potentiated depolarizing responses to superfused GABA and muscimol, but not those to glycine. The potentiating effect of alphaxalone was unaltered by the benzodiazepine antagonist Ro 15-1788. Alphaxalone (0.1-30 microM) also enhanced [3H]muscimol binding to rat brain membranes in the presence of Cl-ions; the enhancing effect on [3H]muscimol binding was abolished by Triton X-100. Analysis of binding curves for [3H]muscimol indicated that the steroid anaesthetic increases the affinity for [3H]muscimol of low affinity binding sites; this property is shared by pentobarbitone. The physiologically inactive beta-hydroxy isomer of the steroid was without activity in either of the experimental situations at 30 microM. It is suggested that alphaxalone and pentobarbitone share a common mode of action on the GABA system, which may be relevant to the mechanisms by which these drugs produce anaesthesia.     

Zinc modulates GABAB binding in rat brain

The effects of ZnCl₂ on [³H]GABA binding to GABA_A and GABA_B binding sites were investigated using receptor autoradiography. At concentrations exceeding 100 μM, zinc non-competitively inhibited GABA_B binding in a dose dependent fashion. GABA_A binding was not inhibited significantly by zinc eliminating the possibility of a non-specific effect of zinc. Increased calcium concentrations up to 10 MM enhanced total GABA_B binding but did not prevent zinc induced inhibition of GABA_B binding, indicating a separate site of action for these cations at the GABA_B binding site. In some regions, zinc modulates GABA_B binding in a biphasic manner as concentrations of 10–100 μM zinc significantly enhanced GABA_B binding in the hippocampus and the molecular layer of the cerebellum but not in the thalamus. These results provide further evidence for a neuromodulatory role for zinc in the central nervous system.     

Characterization and regional distribution of serotonin S2-receptors in human brain

[³H]Ketanserin binding was characterized in vitro in human brain homogenates and the regional distribution of the sites was determined.In human brain, [³H]ketanserin was found to bind on serotonin (5-HT) S₂-receptors; only 5-HT antagonists competed with the labelled ligand at nanomolar concentrations; other drugs were much less active or inactive. Special attention was paid to the choice of a displacer, here methysergide, to determine the blank value (non-displaceable binding). [³H]Ketanserin binding in human brain displayed similar binding characteristics to the S₂-receptor in the rat frontal cortex, high affinity (K_d 0.69 nM) and relatively slow dissociation rate.The regional distribution of serotonin S₂-receptors labelled with [³H]ketanserin was studied in 30 different regions of human brain. The highest number of receptors was measured in the cortex. However, within the cortex the distribution was also inhomogeneous, a much lower number of sites being found in the pre- and post-central gyri. In the dopaminergic areas and the cerebellum the number of sites was quite low, and only few binding sites were detected in the corpus callosum, the medulla and the hypophysis.The large number of serotonin S₂-receptors in the human cortex suggests that serotonin has an important role in this brain region.