Monoclonal Antibodies Recognizing the Benzodiazepine Receptor of Chick Embryo Brain

Abstract

The benzodiazepine receptor (BZDR) of the embryonic chick brain contained three subunit proteins with molecular weights of 48-kilodalton (KD), 50-KD and 51-KD at a pI of 5.6, as demonstrated by two-dimensional gel electrophoresis and fluorography of the 3H-flunitrazepam (FNZ)-photolabeled receptor. Monoclonal antibodies (mAB) against the receptor were produced by using the spleen cells of one mouse immunized with the three subunit proteins extracted from SDS-PAGE gels. When the radioligand-labeled membranes were subjected to two-dimensional gel electrophoresis followed by immunoblotting using the mAB 2C3, both 50-KD and 51-KD bands with a pI of 5.6 were immunoreactive and radioactive. Thus, the mAB 2C3 recognized a common epitope on the 50-KD and 51-KD subunits of the BZDR. In addition, the mAB 2C3 was used with immunocytochemistry to determine the distribution of the receptor in the chick embryo brain. The BZDR immunoreactivity was observed among various brain areas, including hippocampus, optic tectum and cerebellum. The reaction product was localized in the neuronal membranes and cytoplasm. Certain neurons in the culture derived from embryonic chick brains were also immunoreactive as detected by immunocytochemical staining.

Distributions of GABAA, GABAB, and benzodiazepine receptors in the forebrain and midbrain of pigeons

“…Dietl et al (1988) also only found modest ligand binding for BDZ sites in the visceral basal ganglia in pigeon. In chicks, the abundance of GABAA/ BDZ receptors in the visceral basal ganglia has also been found to be modest with ligand binding, immunohistochemistry, and in situ hybridization histochemistry (Stewart et al, 1988;Yin and Fan, 1990;Bateson et al, 1991;Glencorse et al, 1991). Our immunohistochemical data, however, showed that the visceral basal ganglia indeed contained a mat of dendrites labeled for GABAA~BDZ receptors, resembling our previously observed GABAergic terminals (which surround dendrites).…”

“…The septum is lightly (GABAAIBDZ) to moderately (GABAB) labeled for the receptors, but dense in GABAergic terminals. Other authors have also shown a relatively low abundance of GABA receptor labeling in the avian septum (Dietl et al, 1988;Stewart et al, 1988;Yin and Fan, 1990;Bateson et al, 1991;Canonaco et al, 1991Canonaco et al, , 1992Glencorse et al, 1991).…”

Downregulation and subcellular redistribution of the γ-aminobutyric acidA receptor induced by tunicamycin in cultured brain neurons

“…The percentage of cells containing ␣ 1 mRNA is lower than that immunostained by mAb 2C3. The difference probably results from that mAb 2C3 recognizes two putative ␣-subunits [Yin and Fan, 1990].…”

“…Fluorography was carried out according to previous procedures [Yin and Fan, 1990]. In brief, cultured cell homogenates were incubated with 5 nM 3 H-FNZ in PBSS for 60 min on ice followed by irradiation with long-wavelength ultraviolet light at a distance of 3 cm for 45 min at 4°C.…”

“…The blots were incubated with 5% nonfat milk in PBS for 1 h and then a monoclonal antibody (mAb) 2C3 against chicken GABA A R (ascites fluid: 1:200 dilution) [Yin and Fan, 1990] overnight at 4°C. They were then incubated with a peroxidase-conjugated goat anti-mouse IgG antibody (Sigma) for 1 h at RT.…”