Proc. Natl. Acad. Sci. USA Vol. 81, pp. 1585-1589, March 1984 Neurobiology Quantitative autoradiography of !,3- and j82-adrenergic receptors in rat brain (norepinephrine/epinephrine/'2,5-labeled pindolol/Ultrofflm) THOMAS C. RAINBOW, BRUCE PARSONS, AND BARRY B. WOLFE Department of Pharmacology/G3, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 Communicated by Louis B. Flexner, November 21, 1983 ABSTRACT We have used quantitative autoradiography dards, the optical density readings can be quantitatively con- to localize in rat brain /3i- and .82-adrenergic receptors. These verted to amount of radioligand bound per mg of protein receptors were labeled in vitro with 1251-labeled pindolol, an (11). Thus, in the present study we have combined the tech- antagonist of 13-adrenergic receptors that binds nonselectively niques of quantitative autoradiography and quantitative analy- to both .p2 and #2 subtypes. The selective inhibition of 1251_ sis of -adrenergic receptor subtypes to allow determination labeled pindolol binding with specific antagonists of .BI and 132 of subtype densities with a resolution of =100 Am. These receptors allowed the visualization of 18-adrenergic receptor studies, in addition to confirming the previous reports on the subtypes. High levels of ,B1 receptors were observed in the cin- distribution of subtypes in gross areas, such as the entire gulate cortex, layers I and II of the cerebral cortex, the hippo- cerebral cortex or cerebellum, have also disclosed that 381 campus, the Islands of Calleja, and the gelatinosus, mediodor- and 832 receptors are distributed heterogeneously among afa- sal, and ventral nuclei of the thalamus. High levels of #82 recep- tomically discrete nuclei and subregions of rat brain. This tors were found in the molecular layer of the cerebellum, over new technology will enable investigators to examine -ad- pia mater, and in the central, paraventricular, and caudal lat- renergic receptor subtypes in the brain at a much higher ana- eral posterior thalamic nuclei. Approximately equal levels of tomical resolution than was previously possible. #1 and #B2 receptors occurred in the substantia nigra, the olfac- tory tubercle, layer IV of the cerebral cortex, the medial preoptic nucleus, and all nuclei of the medulla. The pro- METHODS nounced differences in the ratio of #I to #82 receptors among Male Sprague-Dawley rats (200-250 g) were decapitated, brain regions suggests that the subtypes of13-adrenergic recep- the brains mounted on a cryostat chuck, and 32-gm sections tors may play different roles in neuronal function. were cut, thawed, and mounted onto microscope slides as described (10). The sections were stored overnight at -20'C ,1-Adrenergic receptors were originally subclassified by and then stored at -70'C for up to a week before use. The Lands et al. (1) into 181 and 82 receptors on the basis of the slides were allowed to warm to room temperature 20 min rank order of potency of a series of catecholamines in stimu- before use. They were then immersed in a Coplin slide jar lating responses in several tissue preparations. Thus, cate- containing 35 ml of a Tris-saline buffer (20 mM Tris'HCl/135 cholamine-stimulated responses of cardiac and adipose tis- mM NaCl, pH 7.4), 1251-labeled (1251I-pindolol) pindolol (pre- sues were shown to be mediated by ,1 receptors; these re- pared as described in ref. 12), and various competing drugs. sponses in bronchiolar and vascular smooth muscle were After incubation for 70 min at 230C, the slides were washed found to be mediated by /32 receptors. More recently, Minne- (3 x 20 min) in 40C Tris-saline buffer, rinsed quickly in cold man et al. (2) and Hancock et al. (3) characterized and quan- distilled water to remove buffer salts, and rapidly dried on a tified the subtypes of /3-adrenergic receptors using the bind- slide warmer at 60'C. We either removed labeled brain sec- ing of radioligands to tissue homogenates. Minneman et al. tions for scintillation counting or exposed them for either 4.5 (4) have also shown that it is likely that only two subtypes of or 24 hr against LKB Ultrofilm, as described (10), to gener- ,1-adrenergic receptors exist in mammalian species. In addi- ate autoradiograms. Autoradiograms were quantified using a tion, Stiles et al. (5) have shown that the primary protein microcomputer-assisted densitometer at an anatomical reso- structures of mammalian /,3 and 132 receptors are distinct, but lution of 100 Aum. The optical density readings were convert- the proteins of, for example, cardiac 1-adrenergic receptors ed into the amount of radioligand bound per mg of protein, were conserved over a wide range of species including hu- using 32-,um-thick 125I-labeled brain-mash standards (11, 14). mans, dogs, pigs, rabbits, and rats (6). Detailed inhibition curves constructed from optical density In the central nervous system, B-adrenergic receptors are readings were analyzed using a nonlinear least-squares asymmetrically distributed with /31 receptors being found in method via the computer program FITSITES on the NIH- highest concentrations in forebrain structures, such as the supported PROPHET network. The untransformed data cerebral cortex, caudate, and hippocampus (2). In the cere- were tested for homology with one, two, and three separate bellum, the P2 subtype predominates (2). Up to the present, binding-site models, and F-test analysis was used to deter- the measurement of these receptor subtypes has been done mine the most appropriate fit (15). This method yielded esti- using homogenates of crude membrane preparations from mates of the densities of the separate binding sites and of the gross brain structures, such as the cerebral cortex or the cer- affinities of each site for competing drugs. The linear least- ebellar cortex. Recently, however, a quantitative autoradio- squares method was used for regression analysis of Scat- graphic method has been devised to measure neurotransmitter chard (16) plots made from optical density measurements. receptors in brain (7-10). This method involves manual or For inhibition and saturation analysis, sections correspond- computerized densitometric measurement of autoradiograms ing to Paxinos and Watson (13) stereotaxic levels 10-14 and made with LKB Ultrofilm. Using a series of density stan- 35-40 were used for receptor measurements of the caudate putamen, and the molecular layer of cerebellum, respective- ly, as initial studies indicated little or no rostral-caudal vari- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: 1251-pindolol, 125I-labeled pindolol. 1585 Downloaded by guest on October 2, 2021 1586 Neurobiology: Rainbow et al. Proc. NatL Acad Sci. USA 81 (1984) ation in 125I-pindolol binding. Optical density measurements were averaged across the entire dorsoventral and mediolat- eral extent of these structures. RESULTS Z 0 Specificity of Binding to Brain Regions. Using ° ° 1251-Pindolol 2E ICI 118,551 a scintillation counter to determine the amount of 125I-pindo- 5a p2 selective lol bound to tissue sections, we determined that the binding 0 of the ligand was at equilibrium after a 70-min incubation at U- 50 room temperature and that optimum ratios of total to non- specific binding occurred after three separate washes of 20 0~~~~~~~~~ min each in 40C Tris-saline buffer. This washing procedure did not result in any loss of specific binding. All subsequent characterization of 125I-pindolol binding was made by analy- * CEREBELLUM 0j sis of optical density readings from autoradiograms of the caudate putamen and the molecular layer of the cerebellum. 8 6 Optical density measurements were averaged from the entire 10 4 rostral-caudal extent of these structures. Analysis of inhibi- -LOG (DRUG) tion curves indicated that 125I-pindolol binding to frozen FIG. 1. Inhibition of '25I-pindolol binding by ICI 89,406 and ICI brain sections was inhibited by isoproterenol with an affinity 118, 551. 125I-Pindolol (150 pM) was incubated with slide-mounted comparable to that observed in membrane studies (17, 18) cerebellar sections in the presence and absence of various concen- (Kd = 0.3 x 10-6 M) and was stereospecifically inhibited trations of the 813-selective antagonist ICI 89,406 (o) or the 32-selec- by propranolol withl-propranolol being 100 times more po- tive antagonist ICI 118,551 (0). Autoradiograms of the molecular tent than d-propranolol (data not shown). Based on these re- layer of the cerebellum were analyzed, and the data are plotted as a sults, we defined the nonspecific binding of 125I-pindolol as percent of the binding occurring in the absence of any competing the binding that occurs in the presence of 100,uM isoprotere- ligand. The lines are the computer-generated best fit to a two-site model. This is a representative experiment of 11 or 12 similar experi- nol. Scatchard analysis indicated that 1251-pindolol binding ments. was saturable [Bmax (caudate) = 153 ± 14 fmol per mg of protein] and of an affinity similar to that found in membrane studies (17) (Kd = 51 ± 5 x 10-12 M; n = 3). It was also Anatomical Distribution ofl13 and 132 Receptors. The distri- observed that 1,uM I-propranolol reproducibly inhibited button of total _251-pindolol binding to 8-adrenergic recep- more 125I-pindolol binding than 100,Ml-isoproterenol (data tors was similar to previous descriptions of the binding of not shown). Since there always appeared to be a plateau in [3H]dihydroalprenolol, a nonselectivePreceptor antagonist the inhibition curves ofl-propanolol andl-isoproterenol (22, 23). We observed high levels of total binding in the cau- around 5-20% of total binding, and since,M1 d-propranolol date putamen, the olfactory tubercle, the superficial layers inhibited125I-pindolol binding to a great extent, we felt that of the cerebral cortex, and the superior colliculus, the cingu- the extra inhibition by,M1 l-propranolol represented inhi- late cortex, the substantia nigra, the dorsal caudal subicu- bition of nonspecific (nonreceptor) binding.