Journal of Neuroscience, Vol 10, 603-612, Copyright © 1990 by Society for Neuroscience
Localization and characterization of 35S-t- butylbicyclophosphorothionate binding in rat brain: an autoradiographic study
PP Edgar and RD Schwartz
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710.
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.
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