Development of gaba binding sites in chick embryo optic lobe: Effect of triton X-100

The temporal course of the development of GABA receptor sites in chick optic lobe was studied as a parameter of neuronal differentiation in the central nervous system. At 10 days of incubation, specific [3H]GABA binding was of 0.08 pmol/optic lobe and increased 7-8 fold between 12 and 16 days of incubation, reaching at 16 days a value of 0.60 pmol/optic lobe. This coincides with the period of arrival of the retinal fibers to the optic lobe. After this stage, the number of GABA binding sites decreased to a value of 0.35 pmol/optic lobe at hatching. After hatching a new increase appeared which reached at 5 days post-hatching a value of 0.87 pmol/optic lobe. Scatchard analysis of the saturation binding data obtained at 16 days of incubation and at hatching revealed the presence of two binding sites: one with high affinity and the other with low affinity, while at 12 days of incubation, the earliest stage examined, only the low-affinity binding site appeared. The high-affinity binding site for [3H]GABA was inhibited by muscimol, GABA, and bicuculline (IC50: 0.006, 0.002 and 10 microM, respectively). These values correspond to the potencies shown by those compounds in the binding to the synaptic GABA receptor. Treatment of the synaptic membranes with Triton X-100 showed a marked increase in the amount of specific [3H]GABA binding after 16 days of incubation reaching a 3-fold increase at hatching. These results suggest that endogenous inhibitors of the higher affinity binding site, probably appear during this period.     

Effects of light- and dark-rearing on the postnatal development of GABA receptor sites in the chick optic lobe

To investigate the ability of GABA receptor sites to undergo environmental-dependent plastic changes, the postnatal developmental pattern of GABA receptors was studied under different levels of light stimulation, i.e. normal-, light- and dark-rearing. At hatching the specific binding of [3H]GABA was 1.74 +/- 0.36 pmol/optic lobe. In normally reared chicks the number of GABA binding sites showed a transient increase with the highest value at the 6th day (7.0 +/- 1.32 pmol/optic lobe). This value is higher than the one reached at the adult stage. Between the 3rd and 6th day, there was a 33.7% increase in specific [3H]GABA binding in light-reared compared with normally reared animals (P less than 0.05). In the dark-reared chicks, the specific binding was 36.4% lower than that found in normally reared (P less than 0.02). However, the changes in receptor density were transient since at the 17th day the number of GABA binding sites returned to adult levels. Scatchard analysis revealed that the differences observed in the high affinity GABA binding sites between the three groups were due to modifications in the total number of binding sites while the affinity remained unchanged. The maximal number of binding sites were: 2.71, 7.01 and 1.79 pmol/mg protein in normally, light- and dark-reared chicks, respectively; while the apparent dissociation constants were unaffected: 3.2, 3.4 and 3.6 nM, respectively. These results show that, during postnatal development, different conditions of visual experience produce synaptic changes at the molecular level. These changes probably occur within a period of high plasticity, prior to the end of a critical period.     

3H-muscimol binding in synaptosomal fractions from bovine and developing rabbit retinas

Bovine and rabbit retina synaptosomal fractions bind the potent and specific GABA agonist, [3H]-muscimol with high affinity and limited capacity. The high degree of pharmacological specificity and the subcellular distribution of these binding sites are similar to those reported for [3H]-GABA binding sites. These observations suggest that these sites represent the recognition sites for GABA receptors. The specific binding of [3H]-muscimol in retinal homogenates from different aged rabbits reveal a distinct developmental profile with a fivefold to six fold increase in binding between days 5 and 13. Thus, it appears that GABA receptor development continues after eye opening in rabbits (day 9-10) and that receptor maturation is delayed by at least two to three days with respect to published of GABA uptake and evoked release.     

Benzodiazepine ([3H]flunitrazepam) binding in cat visual cortex: ontogenesis of normal characteristics and the effects of dark rearing

[3H]Flunitrazepam (FNZ) binding sites were characterized in homogenates of cat visual cortex during normal postnatal development and following dark rearing from birth. In parallel experiments, the distribution and density of [3H]FNZ binding sites were examined by in vitro autoradiographic or 'scrape' methods. In homogenates, Bmax measurements showed low early values, rising to a peak in receptor density at about 60 days postnatal, followed by a decline in adulthood. At all ages, gamma-aminobutyric acid (GABA) altered the Kd, but not the Bmax of [3H]FNZ binding sites. Kd values showed a general increase with age, parallelled by an increased sensitivity to GABA. Receptor autoradiography revealed that the highest density of [3H]FNZ binding sites was in layer IV of cats of all ages. Deafferentation of extrinsic inputs to the visual cortex by surgical undercutting did not alter this pattern of laminar distribution, indicating that the receptors were associated with intrinsic cortical elements rather than subcortical inputs. Dark rearing had no effect on [3H]FNZ laminar distribution in the visual cortex. The Bmax was higher at 30 days postnatal, but did not differ significantly thereafter. Modulation by GABA was concomitantly higher at 30 days, but lower than normal in dark-reared animals at ages greater than 30 days postnatal. The results are discussed in relation to the normal and abnormal development of GABA receptors in the cat visual cortex.     

GABA(A) receptor sites in the developing human foetus

GABA(A) receptor sites were characterised in cerebral cortex tissue samples from deceased neurologically normal infants who had come to autopsy during the third trimester of pregnancy. Pharmacological parameters were obtained from homogenate binding studies which utilised the 'central-type' benzodiazepine ligands [3H]diazepam and [3H]flunitrazepam, and from the GABA activation of [3H]diazepam binding. It was found that the two radioligands behaved differently during development. The affinity of [3H]flunitrazepam for its binding site did not vary significantly between preparations, whereas the [3H]diazepam K(D) showed marked regional and developmental variations: infant tissues showed a distinctly lower affinity than adults for this ligand. The density of [3H]flunitrazepam binding sites increased approximately 35% during the third trimester to reach adult levels by term, whereas [3H]diazepam binding capacity declined slightly but steadily throughout development. The GABA activation of [3H]diazepam binding was less efficient early in the trimester, in that the affinity of the agonist was significantly lower, though it rose to adult levels by term. The strength of the enhancement response increased to adult levels over the same time-frame. The results strongly suggest that the subunit composition of cortical GABA(A) sites changes significantly during this important developmental stage.     

Some properties of solubilized GABA receptor

gamma-Aminobutyric acid (GABA) receptor was solubilized from synaptic membrane of the rat brain by various detergents. Nonidet P-40, a non-ionic detergent, was found to be an effective solubilizing agent, since it caused no interference on the receptor binding assay, yielded a [3H]muscimol binding protein with a high specific activity and no aggregation, and preserved good stability of the solubilized fraction. Ammonium sulfate precipitation of the solubilized supernatant significantly increased the binding of [3H]muscimol to GABA receptor, possibly by removing heat-stable and small molecular inhibitory substances. The specific [3H]muscimol binding to the soluble fraction obtained by Nonidet P-40 treatment and subsequent ammonium sulfate precipitation, was saturable with KD 13 and 64 nM, and Bmax 3.4 and 1.8 pmol/mg protein, respectively. The enhancement of the [3H]muscimol binding by diazepam as found in synaptic membrane was also detected in the soluble fraction. Molecular weight of the [3H]muscimol binding site was determined by gel filtration on Sephadex G-200 and was calculated to be 270,000 daltons. This value was identical with that of the [3H]flunitrazepam binding site which appeared in the same solubilized fraction. These results indicate that the properties of solubilized GABA receptor are identical to those of membrane-bound GABA receptor. Furthermore, the present results suggest that both GABA and benzodiazepine receptors may reside on the same macromolecule in synaptic membrane.     

In ovo chronic neurosteroid treatment affects the function and allosteric interactions of GABA(A) receptor modulatory sites

We investigated the effects of in ovo chronic administration of the endogenous neurosteroid epipregnanolone (5beta-pregnan-3beta-ol-20-one) on the GABA(A) receptor complex present in chick optic lobe synaptic membranes. Chronic epipregnanolone treatment failed to exert any effect on the chick optic lobe total protein content and wet weight at the different doses tested. [3H]Flunitrazepam control binding remained unaltered after neurosteroid exposure, however, the positive allosteric modulation of this ligand by 4 microM allopregnanolone was reduced in a dose-dependent manner by neurosteroid treatment. Embryo exposure to 30 microM epipregnanolone decreased allopregnanolone EC(50) and E(max) values. Analyses of saturation binding isotherms disclosed that such administration had no effect on K(d) and B(max) values for [3H]flunitrazepam and [3H]GABA binding. [3H]GABA binding modulation disclosed an increase in allopregnanolone EC(50) value with a decrease in its E(max) value. Diazepam EC(50) and E(max) values were enhanced, while low affinity sodium pentobarbital EC(50) value was reduced by epipregnanolone treatment. The investigation of the GABA(A) receptor function revealed that administration of this neurosteroid reduces the efficacy of GABA to induce 36Cl(-) influx into microsacs prepared from chick optic lobe. These results indicate that endogenous neurosteroid epipregnanolone chronically administered in ovo produces homologous uncoupling between steroid modulatory sites, and those corresponding to benzodiazepine and GABA receptors. Thus epipregnanolone is able to induce heterologous changes in the allosteric linkage between benzodiazepine and barbiturate modulatory sites, and the GABA receptor site. Taken jointly with results on epipregnanolone enhancing effects on [3H]flunitrazepam and [3H]GABA binding, in the context of its endogenous synthesis, our present findings support this neurosteroid as the endogenous modulator of GABA(A) receptor sites and function during chick optic lobe development.     

Development of gamma-aminobutyric acid (GABA)ergic neurons in cerebral cortical neurons in primary culture

The developmental patterns of gamma-aminobutyric acid (GABA)ergic neurons in primary culture obtained from the neopallium of 15-day-old fetus of mouse were investigated in terms of morphological features, GABA metabolism and GABA receptor binding. Morphological investigations revealed that these cells possessed typical features of neurons and the formation of synapses was detected at 10 days after the inoculation. During neuronal growth on polylysine surfaces, GABA contents and activity of GABA transaminase (GABA-T) showed a progressive increase in the time of culture. Similarly, L-glutamic acid decarboxylase (GAD) showed a progressive elevation during neuronal development in vitro, which corresponded well with the change in immunoreactivity to anti-GAD examined immunohistochemically. In addition, the high K+-evoked release of [3H]GABA also showed an enhancement during the growth in vitro. The numbers of binding sites (Bmax) for [3H]muscimol and [3H]flunitrazepam (FLN) also showed increases with the time of incubation, although affinity (Kd) to the labeled ligands did not show any noticeable changes. Moreover, it was observed that [3H]FLN binding was enhanced by GABA even in neurons cultured for 7 days. These results indicate that cerebral cortical neurons in primary culture possess GABA biosynthesizing and degrading systems including a high-affinity uptake mechanism for GABA. The present results also indicate that these cells possess synaptic contacts as well as GABAA receptors coupled with benzodiazepine receptor from a relatively early stage of cellular development.     

Muscimol binding in rat brain: Association with synaptic GABA receptors

[3H]Muscimol binding to crude synaptic membrane fractions of the rat central nervous is saturable with a high affinity dissociation constant of 2.2 nM. The regional distribution of this binding in rat brain and the effects of freezing, sodium and Triton X-100 are similar to those previously reported for [3H]GABA binding to the the synaptic GABA receptor site. Also, the substrate specificity of [3H]muscimol binding is identifical to that observed for the GABA receptor. Thus, [3H]muscimol is displaced, stereospecifically, only by those drugs and amino acids which are known to neurophysiologically interact with the synaptic GABA receptor but is unaffected by agents which activate or inhibit other neurotransmitter receptors. This suggests that the behavioral effects observed after the systemic administration of muscimol are probably the result of GABA receptor activation.     

Benzodiazepine receptors in human brain: characterization, subcellular localization and solubilization

1. Benzodiazepine receptors have been characterized in human brain mainly using [3H]-Ro 15-1788 and [3H]-flunitrazepam. Both ligands present a very high affinity for the receptor sites (Kd values of 0.56 and 1.53 nM respectively). 2. GABA enhanced the affinity of [3H]-flunitrazepam and [3H]-diazepam, but not that of [3H]-Ro 15-1788 and [3H]-methyl-beta-carboline 3-carboxylate for their specific binding sites as well in cerebral as in cerebellar human cortex. 3. Subcellular distribution of the benzodiazepine receptors revealed a main synaptosomal localization in human cerebral cortex, cerebellum and striatum. 4. Solubilized benzodiazepine receptors were obtained using 0.5% sodium deoxycholate and were characterized with [3H]-Ro 15-1788. The solubilized receptors are still coupled to GABA receptors since the [3H]-flunitrazepam specific binding was enhanced in the presence of micromolar concentrations of GABA.     

3H]MK-801 binding sites in neonate rat brain

[3H]MK-801 binding sites are present in neonate rat brain as early as 3 days after birth. Immature hippocampus and cortex contain approximately one sixth the concentration of binding sites of the adult, while brainstem concentration is twice as high as that of adult. [3H]MK-801 binding is stimulated by glutamate and glycine and blocked by phencyclidine and Mg2+ both in 7-day-old neonate and adult, indicating that as early as 7 days postnatally, the N-methyl-D-aspartate-type glutamate receptor and MK-801 binding site are functionally coupled.     

Developmental study of [3H]TCP and [3H]glycine binding sites in the rat hippocampus

The postnatal development of [3H]thienylphencyclidine ([3H]TCP) sites in rat hippocampus has been studied autoradiographically and with membrane preparations. [3H]TCP binding increased progressively from birth to adulthood; this is due to a change in the maximal number of sites (Bmax) but not in the affinity (Kd). A different developmental pattern was found for strychnine-insensitive [3H]glycine binding which also increased after birth, but reached adult levels earlier than [3H]TCP binding. The ontogenesis of TCP or glycine sites also differed from that previously described for N-methyl-D-aspartate (NMDA) sites in the hippocampus. In neonatal, as in adult hippocampus, [3H]TCP binding was enhanced by NMDA or glycine and reduced by Mg2+. We suggest that TCP sites are functionally coupled to the NMDA receptor-ion channel complex in developing as in mature hippocampus, but that there are developmental changes in the receptor channel complex.     

Differential ontogenesis of type I and II benzodiazepine receptors in mouse cerebellum

The ontogeny of type I and type II benzodiazepine binding sites was studied in mouse cerebellum by displacement of [3H]flunitrazepam binding by zolpidem, a ligand specific for the type I sites. Type I binding sites predominate throughout development and in the adult while type II sites account for 25% of total cerebellar benzodiazepine binding sites at birth and, during development, decrease to 10% or less in the adult. On a per cerebellum basis type II sites increase during the first postnatal week and then remain at a steady level while type I sites increase until adulthood. These results may indicate a specific localization of the type II sites (and of the corresponding alpha-protein subunits in the GABA/benzodiazepine receptor complex) in structures already present at birth and developing during a short early postnatal period. The affinity of zolpidem for its high affinity (type I) binding sites increases during cerebellar ontogeny, this increase possibly indicates an epigenetic (post-translational) 'maturation' process of the corresponding receptor molecule. Hill numbers indicate the existence of an additional binding site heterogeneity greater during development but still present in the adult; probably this is to be related to the simultaneous presence of different 'maturation' stages during development and with a certain variety of the final products.     

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.     

Characterization of benzodiazepine and gamma-aminobutyric recognition sites and their endogenous modulators

The solubilization and partial purification from rat brain cortex homogenates of [3H]gamma-aminobutyric acid (GABA) and [3H]diazepam recognition sites and of their endogenous modulators (GABA-modulin and an endogenous compound which specifically bound [3H]diazepam) are reported. A high percentage of GABA binding sites (virtually free of benzodiazepine binding sites) was solubilized from homogenates of rat brain cortex incubated at 0 degree C with 1% Triton X-100 and a mixture of protease inhibitors. A large proportion of benzodiazepine binding sites was solubilized in the absence of apparent GABA binding capacity by incubating crude synaptic membrane preparations at 37 degree C with 0.05% Triton X-100. The characteristics of these two solubilized binding sites resemble those of the membrane-bound binding sites. However, unlike the membrane-bound sites, solubilized GABA and benzodiazepine binding sites are insensitive to GABA stimulation, while solubilized GABA binding sites are no longer protected by the benzodiazepines against heat inactivation. These results indicate that GABA and benzodiazepine recognition sites reside in two different molecules which, when bound to membranes, can interact reciprocally and modulate their binding affinity for specific ligands.     

The ontogeny of [3H]muscimol binding sites in the C57BL/6J mouse cerebellum

The characteristics of [3H]muscimol binding were investigated in cerebellar sections from 7-day-old mice. The binding sites were found to possess the kinetic properties and pharmacological specificity characteristic of high-affinity GABAA receptors. [3H]Muscimol binding sites in the developing C57BL/6J mouse cerebellum were visualized by light microscopic autoradiography. A distinct band of labeling situated over the molecular layer was apparent from day 1 to day 7. The external granule cell layer remained unlabeled throughout development. Labeling over the internal granule cell layer gradually increased from birth; it became more dense and well defined until adult levels of grain density were reached at 35-42 days of age. The deep cerebellar nuclei were moderately labeled at birth and gradually decreased in density thereafter. The observed ontogeny of granule cell [3H]muscimol binding sites suggests that the synthesis of receptors is initiated at a time immediately after cessation of cell division, coinciding with the beginning of granule cell translocation across the molecular layer. Since, at this time, granule cells have not yet formed synapses with the GABAergic Golgi II cells, nor have they, in turn, formed the vast majority of synaptic contacts with Purkinje cells, it follows that receptor appearance precedes the formation of afferent connections, and may also precede efferent synaptic contacts. The timing of the appearance of [3H]muscimol binding sites raises the possibility that their initial acquisition may be related to developmental events other than the interaction of the granule cell with its pre- or postsynaptic neuronal partners.     

Quantitative autoradiographic study of the postnatal development of benzodiazepine binding sites and their coupling to GABA receptors in the rat brain.

The postnatal development of benzodiazepine binding sites in the rat brain was studied by quantitative receptor autoradiography using [3H]flunitrazepam. The coupling of these sites to GABA receptors was assessed in 43 cerebral structures by examining the effects of in vitro addition of GABA on flunitrazepam specific binding. Benzodiazepine-specific binding was relatively high at birth and exhibited an heterogeneous distribution pattern, anatomically different from the adult one. Data showed a sequential development of benzodiazepine receptors in relation to the time course of maturation of cerebral structures. A proliferation peak which paralleled rapid brain growth was noticed. High levels of benzodiazepine sites were transiently observed in some areas, e.g. thalamus and hypothalamus, and might be related to maturational events. In every brain structure examined, benzodiazepine binding sites were linked to GABA receptors. However, enhancement of flunitrazepam specific binding by exogenous GABA differed according to the structures studied and decreased during development, suggesting some changes in the control of GABA/benzodiazepine regulation during postnatal maturation.     

Ontogenesis of muscimol binding sites in cat visual cortex

In vitro receptor binding techniques were used to study the characteristics, distribution, and ontogenesis of muscimol binding sites in cat visual cortex. [3H] Muscimol, a GABA agonist, labelled a single population of binding sites with a KD of 18 nM in adult cats. Specific binding was saturable, reversible and was blocked by the addition of GABA or (+) bicuculline. Autoradiograms revealed that the highest density of [3H] muscimol binding occurred in cortical layer IV. Similar patterns of [3H] muscimol binding were observed at all ages examined, although the binding densities differed. The peak [3H] muscimol binding density, corrected for amount of protein, occurred at 3 months postnatally. In 3 day old and adult cats binding density was 41% and 69%, respectively, of the peak value.     

Decreased GAB A, Benzodiazepine, and Picrotoxinin Receptor Binding in Brains of Rats After Cobalt-Induced Epilepsy

In previous studies of experimental and human epilepsy, defects have been shown in the gamma-aminobutyric acid (GABA) receptors. We further investigated the role of the GABA/benzodiazepine/picrotoxinin receptor complex in the epileptic focus and also in other regions of the rat brain. The focus was induced by cobalt implantation to the right motor cortex, and the brains were dissected 16-19 days after the operation. Benzodiazepine (using [3H]flunitrazepam as a ligand; FLU), GABA [3H]muscimol; MUS), and picrotoxinin [( 35S]t-butylbicyclophosphorothionate; TBPS) receptor bindings were measured in different brain areas and the values were compared with glass-implanted controls. In the focal area, the specific receptor binding decreased in the order TBPS greater than FLU greater than MUS. In the perifocal area only TBPS binding decreased, and Scatchard analysis showed a decrease in the number of binding sites (p less than 0.05) without any effect on binding affinity. No change was seen in the binding characteristics of the other areas studied. According to our results, in cobalt-induced epilepsy the GABA/benzodiazepine/picrotoxinin receptor complex is modulated in the focal area; this may lead to a defect in chloride conductance, which in turn induces disturbed control of neuronal activity in the epileptic focus.     

GABA receptor binding in rat striatum: Localization and effects of denervation

Intrastriatal injection of 2 μg of kainic acid, a conformationally restricted analogue of glutamate, causes degeneration of GABAergic neurons intrinsic to the striatum while sparing axons of extrinsic neurons terminating in or passing through the region. From 2 to 15 days after striatal lesion with kainate, the sodium-independent binding of [³H]-γ-aminobutyric acid (GABA) to striatal membranes is increased 200% above that of control. Differences in the amount of endogenous GABA contaminating the membrane preparations do not account for the increased receptor binding. Scatchard analysis reveals an increased affinity of the GABA receptor in the kainate-lesioned striatum with no change in the number of binding sites. The subcellular distribution of the receptor as well as the sensitivity of the receptor to several agonists and antagonists is unchanged by the kainate lesion. Ablation of the nigrostriatal dopaminergic pathway by nigral injection of 8 μg of 6-hydroxydopamine reduces by 22% the specific binding of [³H]GABA in the ipsilateral striatum and attenuates by 33% the increase in GABA receptor binding produced by the striatal kainate lesion. These studies demonstrate that: (1) the sodium-independent binding sites for GABA in striatum are localized on axons of extrinsic neurons, and (2) the affinity of these receptors for GABA increases in response to GABAergic denervation.