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Abstract— The binding of [3H]muscimol, a potent GABA agonist, to crude synaptic membranes prepared from rat brain was studied using a filtration method to isolate membrane‐bound ligand. Specific binding was found to be saturable and occurred to two binding sites of Kd5 5 and 30 nm. Binding was Na+‐independent and enhanced by both freezing and Triton treatment. Regional and subcellular distribution studies and pharmacological characterization of specific [3H]muscimol binding are consistent with binding to the synaptic GABA receptor.

After pretreatment of rat brain synaptic membranes with adenosine deaminase to remove endogenous adenosine, 2-chloroeHladenosine, a stable analog of adenosine, binds to two sites with The concept that adenosine (Ado) and ATP can function as modulators or neurotransmitters in mammalian tissues has been developed by Burnstock (1) from pharmacological data generated in peripheral systems; it is postulated that the nucleoside produces its effects via specific P1 purinergic receptors and the nucleotide produces its effects via P2 type receptors. Biochemical, electrophysiological and, to a lesser extent, behavioral and autoradiographical data have suggested a similar neuromodulatory-neurotransmitter role for these purines in central nervous system function (2, 3).Ado is an effective regulator of cyclic AMP formation in brain slices (4, 5) and homogenates (6), acting via membranelocalized extracellular receptor mechanisms that are susceptible to blockade by alkylxanthines such as theophylline (7). It is also a potent inhibitor of neurotransmitter release, modulating the release of acetylcholine, norepinephrine, and dopamine in central and peripheral nervous tissue (8-10). It is especially active in striatal synaptosomal preparations, where the nucleoside inhibits dopamine release [50% inhibition concentration (IC5o) = 10 nM; 10].Both Ado and ATP depress cell firing in the mammalian central nervous system (3,11,12), acting via alkylxanthinesensitive receptor mechanisms. Conversely, theophylline and other adenosine antagonists increase cell firing (11). Although the effects of the purines on cell firing may be attributed to inhibition of excitatory neurotransmitter release, it is thought that a direct inhibitory effect via purinergic PI receptors may also occur; however, there is controversy about the evidence for the existence of P2 receptors in the central nervous system (3).Behaviorally, Ado has sedative, hypnotic, and anticonvulsant activity (13-16), in contrast to caffeine and theophylline, which are central stimulants (17).Evidence for depolarization-evoked, calcium-dependent release of Ado and ATP from brain slices (18,19) and synaptosomal preparations (12, 20) suggests a potential physiological role for purine-modulated alterations in cell firing, transmitter release and cyclic AMP metabolism, and their sequelae.Demonstration of high-affinity binding of a suitable radioligand to brain membranes has become an additional criterion for the identification of an endogenous substance as a putative neurotransmitter. For Ado and ATP, their involvement in nearly all facets of cell function (21) Subcellular fractions from rat brain were prepared by the method of Whittaker (27); the nuclear, crude synaptosomal, myelin, enriched synaptosomal, mitochondrial, and microsomal subfractions were homogenized by using a Polytron (Brinkmann PT 20-ST generator, setting 5.5 for 10 sec) in distilled water; centrifuged at 48,000 X g for 10 min; and stored at -80°C for at least 18 hr before use. After dissection of rat brain r...