Angel L. De Blas

Immunohistochemical localization of GABAA receptors in the retina of the new world primate Saimiri sciureus.

A large population of amacrine cells in the retina are thought to use GABA as an inhibitory neurotransmitter in their synaptic interactions within the inner plexiform layer. However, little is known about their synaptic targets; the neurons that express the receptors for GABA have not been clearly identified. Recently, the GABAA receptor has been isolated and antibodies have been raised against it. These antibodies have proven useful for the immunocytochemical localization of the receptor, and two brief reports describing the distribution of GABAA receptor immunoreactivity in the retina have appeared (Richards et al., 1987; Mariani et al., 1987). We used a monoclonal antibody (62-3G1) against the GABAA receptor to study the retina of the New World primate Saimiri sciureus. Labeled somata were found in the inner nuclear layer (INL) and ganglion cell layer (GCL). The staining was confined to what appeared to be the cells plasmalemma and small cytoplasmic granules. Most of the labeled neurons in the INL had small somata (5-7 microns in diameter) located at the vitreal edge of the layer. They arborized in two laminae (approximately 2 and 4) of inner plexiform layer (IPL). Ventral to the optic disc (2.5 mm) they comprised 29% of the cells present. A few of the labeled neurons appeared to be interplexiform cells or flat bipolar cells, with labeled processes that extended into both the IPL and the inner half of the outer plexiform layer. In the GCL, the labeled somata were among the largest present (13-20 microns in diameter), and 2.5 mm ventral to the optic disc they made up 15% of the cells present. Experiments in which immunoreactive somata were retrogradely labeled following the injection of fluorescent tracers into the optic tract provided a conclusive demonstration that some of the immunoreactive somata were ganglion cells. The antibody often labeled their axons in the optic fiber layer. This suggests that the GABAA receptors are transported anterogradely to the retinal terminal fields. The dendrites of the immunoreactive ganglion cells extended into the 2 laminae of labeled processes in the IPL, and their primary dendritic arbors were, at any given eccentricity, quite similar in appearance. This homogeneity suggests that they comprise a particular subset of the ganglion cells. Sections simultaneously labeled with the monoclonal antibody against the GABAA receptor and antisera against either L-glutamic acid decarboxylase (GAD) or GABA revealed that the GAD/GABA was distributed much more widely in the IPL than the GABAA receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Endogenous benzodiazepine-like molecules in the human, rat and bovine brains studied with a monoclonal antibody to benzodiazepines

The anti-benzodiazepine monoclonal antibody 21-7F9 has been used for the identification and study of endogenous benzodiazepine-like molecules in the human, rat and bovine brains. A sandwich radioimmunoassay has been designed for the quantification of the membrane-bound endogenous benzodiazepine-like molecules. The localization of these molecules is not restricted to the brain tissue. They are also present in kidney, liver and spleen as well as in the neuroblastoma X glioma NG108-15 hybrid cell line. Immunoblots show benzodiazepine-like immunoreactivity in the membrane proteins of all of these tissues. The membrane-bound benzodiazepine-like molecules are resistant to limited proteolysis of the membranes. Moreover, this treatment increases the binding of the monoclonal antibody 21-7F9 to the membranes, probably by exposing sites that normally are not accessible to the antibody. Immunocytochemistry experiments show that benzodiazepine-like molecules are also present in samples of human cerebella that have been stored in paraffin since 1940, 15 years before the first chemical synthesis of benzodiazepines. The results indicate that the cerebellar benzodiazepine-like molecules recognized by the antibody are the product of biological (not chemical) synthesis. Benzodiazepine-like immunoreactivity has also been detected in NG108-15 cells that have been cultured for 3 months in serum-free medium. These results suggest that the cells could biosynthesize benzodiazepine-like molecules.

Immunocytochemical localization of the GABAA/benzodiazepine receptor in the guinea pig cochlear nucleus: evidence for receptor localization heterogeneity.

Immunocytochemistry with a monoclonal antibody against the GABAA/benzodiazepine receptor showed labeled axo-dendritic synapses in the anteroventral cochlear nucleus. In the dorsal cochlear nucleus, label was seen apposing both axo-somatic and axo-dendritic terminals. The results suggest a heterogeneous distribution of GABA receptors, together with a possible segregation of receptor subtypes between somata and dendrites in certain neurons. The presence of cytoplasmic labeling in some neurons might reflect a higher receptor turnover rate in these neurons.

Synaptic and extrasynaptic GABAA receptor and gephyrin clusters.

Publisher Summary In the brain, most interneurons are GABAergic. Glycinergic neurons are mainly localized in the spinal cord. Apart from the neurotransmittermediated signaling that occurs between interneurons and between interneurons and principal neurons, interneurons can be electrically coupled to each other via gap junctions. Powerful tools are being used to reveal the molecular organization of excitatory and inhibitory synapses and the various signaling pathways involved in these synapses. Recent developments in genomics and proteomics, gene knockout animal models, protein-protein interaction assays (such as yeast two-hybrid), and the production and application of novel specific antibodies in combination with immunofluorescence microscopy (confocal and two-photon) and electron microscopy Immunogold techniques are advancing at a very rapid pace our knowledge of the organization of the molecular machinery of chemical synapses.

Solubilization of Brain Benzodiazepine Receptors with a Zwitterionic Detergent: Optimal Preservation of Their Functional Interaction with the GABA Receptors

Abstract: Rat brain benzodiazepine receptors have been solubilized by means of the zwitterionic detergent CHAPS under conditions in which the GABA stimulation of [3H]flunitrazepam binding to the benzodiazepine receptors is maintained intact. This stimulation is partially or totally abolished when using other conventional deter gents.

Differential Regulation of the Postsynaptic Clustering of γ-Aminobutyric Acid Type A (GABAA) Receptors by Collybistin Isoforms

Collybistin promotes submembrane clustering of gephyrin and is essential for the postsynaptic localization of gephyrin and γ-aminobutyric acid type A (GABAA) receptors at GABAergic synapses in hippocampus and amygdala. Four collybistin isoforms are expressed in brain neurons; CB2 and CB3 differ in the C terminus and occur with and without the Src homology 3 (SH3) domain. We have found that in transfected hippocampal neurons, all collybistin isoforms (CB2SH3+, CB2SH3−, CB3SH3+, and CB3SH3−) target to and concentrate at GABAergic postsynapses. Moreover, in non-transfected neurons, collybistin concentrates at GABAergic synapses. Hippocampal neurons co-transfected with CB2SH3− and gephyrin developed very large postsynaptic gephyrin and GABAA receptor clusters (superclusters). This effect was accompanied by a significant increase in the amplitude of miniature inhibitory postsynaptic currents. Co-transfection with CB2SH3+ and gephyrin induced the formation of many (supernumerary) non-synaptic clusters. Transfection with gephyrin alone did not affect cluster number or size, but gephyrin potentiated the clustering effect of CB2SH3− or CB2SH3+. Co-transfection with CB2SH3− or CB2SH3+ and gephyrin did not affect the density of presynaptic GABAergic terminals contacting the transfected cells, indicating that collybistin is not synaptogenic. Nevertheless, the synaptic superclusters induced by CB2SH3− and gephyrin were accompanied by enlarged presynaptic GABAergic terminals. The enhanced clustering of gephyrin and GABAA receptors induced by collybistin isoforms was not accompanied by enhanced clustering of neuroligin 2. Moreover, during the development of GABAergic synapses, the clustering of gephyrin and GABAA receptors preceded the clustering of neuroligin 2. We propose a model in which the SH3− isoforms play a major role in the postsynaptic accumulation of GABAA receptors and in GABAergic synaptic strength.

Monoclonal antibodies to benzodiazepines

Abstract: Four hybridoma lines secreting monoclonal antibodies to benzodiazepines were produced after BALB/c mice were immunized with a benzodiazepine‐bovine serum albumin conjugate. The monoclonal antibodies were purified from ascites fluids, and their binding affinities for benzodiazepines and other benzodiazepine receptor ligands were determined. These antibodies have very high binding affinities for diazepam, flunitrazepam, Ro5‐4864, Ro5‐3453, Ro11–6896, and Ro5–3438 (the KD values are in the 10−9M range). However, these antibodies have low affinities for the benzodiazepine receptor inverse agonists (β‐carbolines) and antagonists (Ro15–1788 and CGS‐8216).

Mammalian brain antigens defined by monoclonal antibodies

Seventy-two hybridoma lines that produce monoclonal antibodies to molecules of a rat synaptosomal plasma membrane fraction (SPM) were generated. The topographical distribution of the antigens in the cerebellum and other areas of the brain was studied by light microscopy immunocytochemistry. Some of the antibodies recognize exclusively neuronal antigens while others bind to specific glial molecules. Some of the antigens have a distribution limited to certain classes of neurons. There are antigens localized in both the cell bodies and processes while others are present only in the latter. Immunoblots of SPM proteins indicate that some antibodies react specifically with one or few of these proteins while other antibodies react with many. The latter antibodies also generally react with many brain cell types. Particularly interesting is the monoclonal antibody 8-6A2 which binds to many SPM proteins but only recognizes large neurons with long axons. A further characterization of the antigens was done by enzyme-linked immunosorbent assays and immunoblots of known purified proteins. The results indicate that antibody 8-2H5 binds specifically to clathrin, 8-7A5 to actin, 8-1E7 to the glial fibrillary acidic protein and both 8-3A5 and 7-2C12 to collagen. In contrast, the antibodies 4-4C3, 2-4H3, 4-4G7 and 6-6A8 bind to antigenic determinants present in many purified proteins.

Purification of the γ-Aminobutyric AcidA/Benzodiazepine Receptor Complex by Immunoaffinity Chromatography

Abstract: The bovine γ‐aminobutyric acidA/benzodiazepine receptor complex has been purified by a novel immunoaffinity chromatography method on immobilized monoclonal antibody 62‐3G1. Immunopurification of the complex was achieved in a single step with an improved yield over affinity chromatography on the benzodiazepine Ro 7‐1986/1. High‐resolution sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of the immunoaffinity‐purified receptor revealed three major peptide bands of 51,000, 55,000, and 57,000 Mr which were also present in the Ro 7‐1986/1 affinity‐purified receptor. Peptide mapping, immunoblotting with subunit specific antibodies, and photoaffinity labeling with [3H]flunitrazepam and [3H]muscimol have been used for the identification of receptor subunits, including several which comigrated in a single band in SDS‐PAGE.

I. Demonstration and purification of an endogenous benzodiazepine from the mammalian brain with a monoclonal antibody to benzodiazepines

Four hybridoma lines secreting monoclonal antibodies to benzodiazepines were produced after BALB/c mice were immunized with a benzodiazepine-bovine serum albumin conjugate. The monoclonal antibodies were purified from ascites fluids, and their binding affinities for benzodiazepines and other benzodiazepine receptor ligands were determined. These antibodies have very high binding affinities for diazepam, flunitrazepam, Ro5-4864, Ro5-3453, Ro11-6896, and Ro5-3438 (the Kd values are in the 10(-9) M range). However, these antibodies have very low affinities for the benzodiazepine receptor inverse agonists (beta-carbolines) and antagonists (Ro15-1788 and CGS-8216). One of the monoclonal antibodies (21-7F9) has been used to demonstrate the existence of benzodiazepine-like molecules in the brain and for the purification of these molecules. Immunocytochemical experiments show that these molecules are neuronal and not glial and that they are ubiquitously distributed throughout the brain. Immunoblots indicate the presence of benzodiazepine-like epitopes in several brain peptides. An endogenous substance that binds to the central-type benzodiazepine receptor with agonist properties has been purified to homogeneity from the bovine brain. The purification consisted on immunoaffinity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reverse phase HPLCs. The purified substance has a small molecular weight and its activity is protease resistant. The endogenous substance blocks the binding of agonists, inverse agonists and antagonists to the central-type benzodiazepine receptor but it does not inhibit the binding of Ro5-4864 to the peripheral-type benzodiazepine receptor. The neurotransmitter gamma-aminobutyric acid increases the affinity of the benzodiazepine receptor for the purified substance. Preliminary evidence indicates that the purified substance is a benzodiazepine with a molecular structure that is identical or very close to N-desmethyldiazepam.