Characterization and differential regulation of GABAA and benzodiazepine receptors in rat neocortex.

We have characterized the gamma-aminobutyric acid-A (GABAA) and benzodiazepine (BZ) receptors in in vitro living slices of adult rat neocortex using [3H]SR95531, a GABAA antagonist, and [3H]flunitrazepam (FNZ), a BZ ligand. [3H]SR95531 labelled a single population of GABAA receptors with a Bmax of 1030.7 fmol/mg protein and a Kd of 43.5 nM. [3H]FNZ also labelled a single binding site with a Bmax of 4239 fmol/mg protein and a Kd of 22 nM. The GABAA receptor labelled using [3H]SR95531 could be down-regulated by 2 h preincubations in GABA and the GABAA agonist muscimol (8% and 11%, respectively). Increases in cellular electrical activity induced by a combination of veratridine and glutamate led to an average increase in GABAA receptor number of 58%. The BZ binding site labelled with [3H]FNZ was down-regulated by clonazepam (-55%), increased by GABA (+17%), but not altered by changes in electrical activity. The present results demonstrate the rapid differential regulation of a ligand-gated receptor by agonist stimulation or increases in bioelectric activity. Such regulation may provide clues to the nature of the modifications which occur following changes in cellular activity in the cortex.

[1]  M. Cynader,et al.  Ontogenesis of muscimol binding sites in cat visual cortex , 1984, Brain Research Bulletin.

[2]  F. Stephenson.  Understanding the GABAA receptor: a chemically gated ion channel. , 1988, The Biochemical journal.

[3]  M. Simmonds.  Evidence that bicuculline and picrotoxin act at separate sites to antagonize γ-aminobutyric acid in rat cuneate nucleus , 1980, Neuropharmacology.

[4]  T. Narahashi.  Chemicals as tools in the study of excitable membranes. , 1974, Physiological reviews.

[5]  J A Zivin,et al.  How to analyze binding, enzyme and uptake data: the simplest case, a single phase. , 1982, Life sciences.

[6]  M. Wilkinson,et al.  Benzodiazepine Receptors in Fish Brain: [3H]-Flunitrazepam binding and modulatory effects of GABA in rainbow trout , 1983, Brain Research Bulletin.

[7]  M. Wilkinson,et al.  Benzodiazepine ([3H])-flunitrazepam) binding sites in cerebellar and cerebral cortical slices of mouse brain , 1984, Brain Research Bulletin.

[8]  T. Nabeshima,et al.  Effects of Picrotoxin Treatment on GABAA Receptor Supramolecular Complexes in Rat Brain , 1989, Journal of neurochemistry.

[9]  S. Paul,et al.  Benzodiazepine Receptor Binding of Triazolobenzodiazepines In Vivo: Increased Receptor Number with Low‐Dose Alprazolam , 1987, Journal of neurochemistry.

[10]  M. Cynader,et al.  Phorbol 12,13-dibutyrate regulates muscarinic receptors in rat cerebral cortical slices by activating protein kinase C. , 1989, Brain research. Molecular brain research.

[11]  M. Caron,et al.  Removal of phosphorylation sites from the β2-adrenergic receptor delays onset of agonist-promoted desensitization , 1988, Nature.

[12]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[13]  M. Caron,et al.  Phosphorylation sites on two domains of the beta 2-adrenergic receptor are involved in distinct pathways of receptor desensitization. , 1989, The Journal of biological chemistry.

[14]  M. Cynader,et al.  Characteristics and distribution of muscimol binding sites in cat visual cortex , 1984, Brain Research.

[15]  W. Krieg.  Connections of the cerebral cortex. I. The albino rat. A. Topography of the cortical areas , 1946 .

[16]  M. Cynader,et al.  Benzodiazepine ([3H]flunitrazepam) binding in cat visual cortex: ontogenesis of normal characteristics and the effects of dark rearing. , 1987, Brain research.

[17]  M. Hollenberg.  Control of Receptor Function by Homologous and Heterologous Ligands , 1985 .

[18]  M. Hollenberg.  Biochemical mechanisms of receptor regulation , 1985 .

[19]  M. Cynader,et al.  The laminar distributions and postnatal development of neurotransmitter and neuromodulator receptors in cat visual cortex , 1986, Brain Research Bulletin.

[20]  M. Cynader,et al.  Pre- and postnatal development of GABA receptors in Macaca monkey visual cortex , 1991, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[21]  K. Subbarao,et al.  Different effects of pentobarbital on two gamma-aminobutyrate receptors from rat brain: channel opening, desensitization, and an additional conformational change. , 1988, Biochemistry.

[22]  W. Sieghart.  Multiplicity of GABAA--benzodiazepine receptors. , 1989, Trends in pharmacological sciences.

[23]  G. Paxinos,et al.  The Rat Brain in Stereotaxic Coordinates , 1983 .

[24]  W. Catterall.  Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes. , 1980, Annual review of pharmacology and toxicology.

[25]  M. Wilkinson,et al.  Tissue slices in radioligand binding assays: studies in brain, pineal and muscle. , 1986, Life sciences.

[26]  M. Cynader,et al.  A role for potassium channels in the regulation of cortical muscarinic acetylcholine receptors in an in vitro slice preparation. , 1989, Brain research. Molecular brain research.

[27]  M. Cynader,et al.  Regulation of Cortical Muscarinic Acetylcholine Receptors: Involvement of Potassium Channels , 1989 .

[28]  C. Laterre,et al.  GABA induces down-regulation of the benzodiazepine-GABA receptor complex in the rat cultured neurons. , 1987, European journal of pharmacology.

[29]  E. Sigel,et al.  Allosteric modulation by benzodiazepine receptor ligands of the GABAA receptor channel expressed in Xenopus oocytes , 1988, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[30]  J. Hablitz,et al.  Chronic exposure of developing cortical neurons to GABA down-regulates GABA/benzodiazepine receptors and GABA-gated chloride currents , 1989, Brain Research.

[31]  M. Cynader,et al.  Characterization of muscarinic acetylcholine receptors in rat cerebral cortex slices with concomitant morphological and physiological assessment of tissue viability. , 1989, Brain research. Molecular brain research.