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Localization and characterization of 35S-t-butylbicyclophosphorothionate binding in rat brain: an autoradiographic study.

Publication ,  Journal Article
Edgar, PP; Schwartz, RD
Published in: J Neurosci
February 1990

35S-t-butylbicyclophosphorothionate (TBPS) binding to slide-mounted rat brain sections was characterized for subsequent autoradiographic analysis. Cortical brain mash slices, preincubated with EDTA to remove endogenous GABA, were used for biochemical characterization. Steady state for 35S-TBPS binding was reached by 3 hr of incubation at 22 degrees C. The association rate constant (K1) and dissociation rate constant (K2) were 0.377 min-1 microM-1 and 0.011 min-1, respectively. Dissociation was monophasic and slow (t1/2 = 80 min). The kinetically derived KD was 29.4 nM. Scatchard analysis indicated a single population of binding sites with a KD of 21.0 +/- 2.2 nM and a Bmax of 1.59 +/- 0.13 pmol/mg protein. Both picrotoxin and muscimol inhibited 35S-TBPS binding completely with IC50s of 251 +/- 13 nM and 203 +/- 41 nM and nHs of 0.98 and 1.4, respectively. The distribution of 35S-TBPS binding sites in the rat brain resembles that of other ligands that bind to GABAA receptor complex with some regionally specific differences. Regions with a high degree of 35S-TBPS binding included the inferior colliculus, medial septal nucleus, central and paracentral nuclei of the thalamus, olfactory tubercle, zona incerta, dentate gyrus, and substantia nigra. 35S-TBPS preferentially bound to the molecular vs granular layer of the cerebellum. Omission of the preincubation markedly but variably decreased 35S-TBPS binding. The greatest regional decreases occurred in areas with a high degree of GABA synthesis. In addition, 35S-TBPS binding was inhibited to different degrees in the cell layers of the cerebellum. The addition of 1 microM GABA to the incubation medium of preincubated slices also produced variable decreases in 35S-TBPS binding to cerebellar layers. These findings support previous studies that demonstrate GABAA receptor heterogeneity. Our study confirms the suitability of 35S-TBPS for use as a ligand in autoradiography and demonstrates that the distribution of 35S-TBPS binding sites is significantly influenced by the preincubation-incubation conditions used.

Duke Scholars

Published In

J Neurosci

DOI

ISSN

0270-6474

Publication Date

February 1990

Volume

10

Issue

2

Start / End Page

603 / 612

Location

United States

Related Subject Headings

  • gamma-Aminobutyric Acid
  • Tissue Distribution
  • Sulfur Radioisotopes
  • Rats, Inbred Strains
  • Rats
  • Neurology & Neurosurgery
  • Male
  • Bridged-Ring Compounds
  • Bridged Bicyclo Compounds, Heterocyclic
  • Bridged Bicyclo Compounds
 

Citation

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MLA
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Edgar, P. P., & Schwartz, R. D. (1990). Localization and characterization of 35S-t-butylbicyclophosphorothionate binding in rat brain: an autoradiographic study. J Neurosci, 10(2), 603–612. https://doi.org/10.1523/JNEUROSCI.10-02-00603.1990
Edgar, P. P., and R. D. Schwartz. “Localization and characterization of 35S-t-butylbicyclophosphorothionate binding in rat brain: an autoradiographic study.J Neurosci 10, no. 2 (February 1990): 603–12. https://doi.org/10.1523/JNEUROSCI.10-02-00603.1990.
Edgar, P. P., and R. D. Schwartz. “Localization and characterization of 35S-t-butylbicyclophosphorothionate binding in rat brain: an autoradiographic study.J Neurosci, vol. 10, no. 2, Feb. 1990, pp. 603–12. Pubmed, doi:10.1523/JNEUROSCI.10-02-00603.1990.

Published In

J Neurosci

DOI

ISSN

0270-6474

Publication Date

February 1990

Volume

10

Issue

2

Start / End Page

603 / 612

Location

United States

Related Subject Headings

  • gamma-Aminobutyric Acid
  • Tissue Distribution
  • Sulfur Radioisotopes
  • Rats, Inbred Strains
  • Rats
  • Neurology & Neurosurgery
  • Male
  • Bridged-Ring Compounds
  • Bridged Bicyclo Compounds, Heterocyclic
  • Bridged Bicyclo Compounds