autoradiographic localization - Europe PMC

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Dec 19, 1979 - the locus ceruleus and nucleus tractus solitarii, and parts of the spinal cord. .... (the periventricular nucleus, pars rotundocellularis) were also.
Proc. Natl. Acad. Sci. USA Vol. 77, No. 3, pp. 1696-1700, March 1980

Neurobiology

Noradrenergic al and a2 receptors: Light microscopic autoradiographic localization (adrenergic receptors/brain/autoradiography)

W. SCOTT YOUNG III AND MICHAEL J. KUHAR* Departments of Pharmacology and Experimental Therapeutics and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Communicated by Saul Roseman, December 19, 1979

ABSTRACT [3H]WB4101 and p43H]aminoclonidine were used for light microscopic autoradiographiclocalization of al and a2 adrenergic receptors, respectively, in the rat brain. The binding of these ligands to slide-mounted tissue sections had all of the characteristics associated with al and a2 receptors. It was saturable with appropriate kinetic constants and was blocked only by other a-adrenergic drugs with the appropriate potency. Autoradiographic studies revealed a distribution of a-adrenergic receptors throughout the nervous system. Certain areas had elevated levels. These included parts of the olfactory bulb and nucleus, parts of the cerebral cortex and dentate gyrus, the more medial portions of the hypothalamus and thalamus, the locus ceruleus and nucleus tractus solitarii, and parts of the spinal cord. In certain areas, the distribution of cal and a2 receptors was markedly different. These results provide some rational basis for the observed actions of a-adrenergic drugs on the central nervous system. For example, the finding of high densities of a2 receptors in the nucleus tractus solitarii is most likely related to its antihypertensive action. The observed codistribution of a2 receptors with opiate receptors would provide an explanation of the observation that a2 agonists block opiate withdrawal. The results are also discussed in relationship to the anatomy of catecholamine systems in the brain.

Norepinephrine and epinephrine are neurotransmitter and humoral substances in the brain and periphery. Pharmacological studies have indicated that there are different types of receptors for these compounds. Although the division of adrenergic receptors into a and f types has been accepted for some time, Langer (1) and Berthelsen and Pettinger (2) have proposed two distinct oa receptor populations, designated as a l and a2. This distinction has pharmacological importance; for example, centrally acting antihypertensive agents fall into the category of a2 agents. Like many other receptor sites, noradrenergic a receptors can be identified by binding methods. The radiolabeled drugs WB-4101 and p-aminoclonidine (PAC) are suitable ligands for selectively identifying al and a2 receptors, respectively, in brain membranes (3, 4). We have localized distinct populations of a l and a2 binding sites in intact tissue sections of rat brain by light microscopic autoradiographic methods. METHODS The procedure used is a general one for localizing drug and neurotransmitter binding sites (5). It involves labeling receptors in slide-mounted tissue sections with a high degree of specificity and generating autoradiograms by the subsequent apposition of emulsion-coated coverslips. The following procedure was used for routine autoradiThe 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.

ographic studies. Sections (6 ,um) of rat (male, Sprague-Dawley, 200 g) brain were thaw-mounted onto microscope slides. For labeling of al receptors, the mounted tissue sections were incubated in 1.1 nM [3H]WB-4101 (New England Nuclear, 25.4 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) for 70 min at ice-bath temperatures in 0.17 M Tris-HCl, pH 7.6/0.001% ascorbic acid. After consecutive 5- and 20-min washes in cold buffer without drug, the slide-mounted tissue sections were dried in a stream of cold, dry air. For localizing a2 receptors, sections were incubated in 2.5 nM [3H]PAC (synthesized by Bruno Rouot and tritiated by New England Nuclear to 47.5 Ci/mmol) for 60 min, washed twice for 5 and 10 min, and dried. Extensive preliminary experiments showed that these incubation conditions gave an optimal, reproducible labeling of receptors and provided specific-to-nonspecific ratios of 3-5:1 in the whole sections. The selected incubation times were chosen such that the maximal association of drug with receptor was attained. The washing conditions were selected so that the nonspecific binding was greatly reduced while the specific binding was reduced by no more than 10 or 15%. Sections (10 Atm) of rat forebrain containing the caudate nucleus and septum were routinely selected for these preliminary experiments. These biochemical measurements of receptor binding were carried out by scraping the tissue sections from the slides and measuring the radioactivity by scintillation spectrometry (5). Control slides were generated for both ligands by adding 100 AtM norepinephrine to the incubations. As in our experiments with several other receptors, this preparative procedure did not alter the binding characteristics of the a receptors in any measureable way (5-8). As described below, the binding was saturable, of a high affinity, and exhibited appropriate pharmacological characteristics. Autoradiograms were exposed for 3 months for [3H]WB-4101 and for 4 months for [3H]PAC and developed, fixed, and stained as described (5). The slides were viewed by both brightfield and darkfield microscopy. Under these conditions, the autoradiographic technique provides quantitative data because autoradiographic grain densities are proportional to time of exposure and tissue content of radioactivity (5). All of the observations reported here were reproducible and found in several sections from the same and three different animals. RESULTS Before beginning autoradiographic studies, it was necessary to identify the conditions under which the bulk of the binding of the tritiated drugs to the mounted tissue sections had the characteristics associated with relevant a receptors. Accordingly, we carried out various kinetic and pharmacologic studies to assess the significance of the binding. Abbreviation: PAC, p-aminoelonidine. * To whom all correspondence should be addressed. 1696

Proc. Natl. Acad. Sci. USA 77 (1980)

Neurobiology: Young and Kuhar Kinetic Analysis of [3HIWB4101 and [3H]PAC Binding. Sections of rat brain were prepared as described in Methods and incubated with various concentrations of the tritiated ligands. We observed saturation of the specific binding of the drugs in the nanomolar range. [3H]WB-4101 showed a dissociation constant of 0.47 i 0.03 nM (mean ± SEM, n = 3), whereas [3H]PAC showed a Kd of 1.61 ± 0.72 nM (n = 3). The Bmax values were 31.0 i 4.0 fmol/mg (n = 3) and 5.89 ± 1.21 fmol/mg (n = 3) of tissue wet weight for WB-4101 and PAC binding, respectively (Fig. 1). These values are in general agreement with the results found in other studies (3). Pharmacological Specificity. The binding of [3H]WB-4101 had the pharmacological characteristics associated with an al receptor. For example, prazosin was 130 times more potent than clonidine in displacing WB-4101, and both were more potent than the isomers of norepinephrine (Fig. 2A). On the other hand, the binding of [3HJPAC had the characteristics associated with an a2 receptor. Clonidine and (-)norepinephrine were 500 times more potent than prazosin and (+)norepinephrine in displacing [3H]PAC (Fig. 2B).

1697

'D c

.00 0 o-

10-9

1O

10-7

-106

10-5

Drug concentration, M

FIG. 2. (A) Pharmacological specificity of [3H]WB-4101 binding in mounted tissue sections. 0, Prazosin; 0, clonidine; X, (-)norepinephrine; 0, (+)norepinephrine. (B) Pharmacological specificity of [3H]PAC binding. *, Clonidine; X, (-)norepinephrine; 0, (+)nor-

epinephrine; @, prazosin.

8

B

3

223/ 1

E1 6

21 2334

z

PAC bountrdinn

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FIG.

1.

al1

(A) Saturation kinetics for

shown are from one experiment. bound/free in

(Inset)

(fmol-ml)/(pmol-mg).

Kd

receptor binding. Data Scatchard plot: B/F

= 0.45 nM; Bmax

fmol/mg tissue wet weight. (B) Saturation kinetics for

ax2

=

=

35.0

receptor

binding. Data shown are from one experiment. (Inset) Scatchard plot (see A). Kd = 0.92 nM;

B)

*, Total

bound;

BmaX

= 3.47 fmol/mg tissue wet weight. (A and

A, specific binding;

*,

nonspecific binding.

Autoradiographic Studies. Autoradiograms were prepared described in Methods. Both al and a2 receptors were found fairly widespread in the rat brain. Although the variation in receptor densities in the different areas was not as great as observed with other receptors (5-8), certain areas did have enriched levels of binding sites compared to others. Also, there were striking differences between the binding localizations of [3H]WB-4101 and [3H]PAC. White matter areas always showed a low level of autoradiographic grains comparable to the background level observed in controls. The olfactory bulb and its nucleus exhibited some of the highest levels of a-receptor binding in the brain. However, the distributions were markedly different for the two ligands. WB-4101 binding was highest in the external plexiform layer of the olfactory bulb, whereas PAC was very high in the external plexiform layer of the olfactory nucleus (Fig. 3). The al-receptor density in the olfactory bulb was the highest obas

served in the brain.

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Neurobiology: Young and Kuhar

*

DENSE

E

MODERATE

LOW

Proc. Natl. Acad. Sci. USA 77 (1980)

A12760im | LG LMO

LGI

FIG. 3. Diagram of a-adrenergic receptor distribution in the olfactory bulb and nucleus and frontal pole of the cerebral cortex. The distributions of al- and a2-receptor binding were different. Grain densities were as follows: Dense, >40 grains per 1000 MAm2; moderate, 20-40; low, 6-20; and background,