Brain Research, 302 (1984) 9-18 9 Elsevier BRE 10035 Quantitative~Analysis of [3H]Spiroperidol Binding to Rat Forebrain Sections: Plasticity of Neostriatal Dopamine Receptors after Nigrostriatal Injury KIM A. NEVE, C. ANTHONY ALTAR, CATHERINE A. WONG and JOHN F. MARSHALL Department of Psychobiology, Universityof California, Irvine, CA 92717 (U.S.A.) (Accepted October 18th, 1983) Key words: dopamine -- receptor proliferation -- supersensitivity-- autoradiography -- neostriatum - - dopamine receptors --[3H]spiroperidol The binding of [3H]spiroperidol to rat coronal sections in vitro was investigated using two procedures: swabbing studies, in which the tissue sections are wiped from the microscope slides after incubation in the presence of [3H]spiroperidol, and autoradiographic stud- ies, in which the autoradiographic negatives are analyzed using computer-assisted densitometry. In the swabbing studies, the pharma- cological and kinetic properties of butaclamol-displaceablebinding were investigated, and the following results suggest that [3H]spiro- peridol binds specifically to only a single site within the basal forebrain of tissue sections and that the site is the dopamine D-2 receptor. (1) The pseudo-first order and first order plots for the rate of association to and dissociation from tissue sections appeared to be linear. (2) Dopamine antagonists, such as haloperidol and butaclamol, were much more effective than dopamine agonists or the serotonin S-2 ligand, ketanserin, in inhibiting [3H]spiroperidol binding. (3) The ability of dopamine agonists to inhibit [3H]spiroperidoi binding was markedly reduced by the guanine nucleotide, Gpp(NH)p. (4) Saturation analysis of specific [3H]spiroperidol binding revealed a Ka and Bma x of 0.93 nM and 447 fmol/mg protein, and a Hill coefficient of 1.05. The findings are also compatible with the possibility that [3H]spiroperidol binds to several sites that have identical affinities for this ligand. Densitometric studies were used to assess the effect of lesions on [3H]spiroperidol binding in the neostriatum. Intrastriatal injection of kainic acid substantially reduced 1 #M (+)--butaclamol-displaceablebinding, indicating that the receptors are in large part on in- trinsic striatal neurons. Neostriatal [3H]spiroperidol binding was investigated 7 days after destruction of the mesotelencephalic dopa- mine system by the ventral tegmental injection of 6-hydroxydopamine. As determined by saturation analysis, the average values for Kd and Bm~ were 0.66 nM and 1212 fmol/mg protein in the intact striatum, and 0.82 nM and 1504 fmol/mg in the denervated striatum. The finding of a significant 23.8% increase in receptor density by the end 9 f the first postoperative week, a period during which behav- ioral supersensitivity to apomorphine increases rapidly, supports the hypothesis that a proliferation of D-2 receptors underlies the be- havioral manifestations of denervation supersensitivity. INTRODUCTION Receptor autoradiography is a versatile method for the localization and quantification of neurotrans- mitter binding sites in the brain. Using in vivo autora- diography, saturable [3H]spiroperidol binding that is displaced by other dopaminergic ligands has been seen in all areas of the rat brain that receive a signifi- cant dopaminergic innervationll, 13. In vitro receptor autoradiography using tritium-sensitive film permits the quantitative analysis of regional [3H]spiroperidol binding3L Autoradiographic studies are frequently supported by experiments in which tissue sections are swabbed from microscope slides for liquid scintilla- tion counting after incubation in the presence of the radioactive ligand. Such swabbing Studies have been used to determine the K d and Bmax for [3H]spiroperi- dol binding to forebrain tissue sections, and some progress has been made in separating serotonergic and dopaminergic components of this binding19. Still, quantitative autoradiography of [3H]spirope- ridol binding has not been fully exploited. There is a need for region-by-region microdensitometric analy- ses of the pharmacological and kinetic properties of [3H]spiroperidol binding, and a more complete anal- ysis of the characteristics of binding to the high-affini- ty dopamine antagonist (D-2) receptor in tissue sec- tions. Quantitative autoradiography also provides a Correspondence: J. F. Marshall, Department of Psychobiology, University of California, Irvine, CA 92717, U.S.A. 0006-8993/84/$03.00 (~ 1984 Elsevier Science Publishers B.V.