Proc. Natl. Acad. Sci. USAVol. 77, No. 3, pp. 1696-1700, March 1980Neurobiology

Noradrenergic al and a2 receptors: Light microscopicautoradiographic 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 wereused for light microscopic autoradiographiclocalization of aland a2 adrenergic receptors, respectively, in the rat brain. Thebinding of these ligands to slide-mounted tissue sections hadall of the characteristics associated with al and a2 receptors.It was saturable with appropriate kinetic constants and wasblocked only by other a-adrenergic drugs with the appropriatepotency. Autoradiographic studies revealed a distribution ofa-adrenergic receptors throughout the nervous system. Certainareas had elevated levels. These included parts of the olfactorybulb 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 thespinal cord. In certain areas, the distribution of cal and a2 re-ceptors was markedly different. These results provide somerational basis for the observed actions of a-adrenergic drugs onthe central nervous system. For example, the finding of highdensities of a2 receptors in the nucleus tractus solitarii is mostlikely related to its antihypertensive action. The observed co-distribution of a2 receptors with opiate receptors would providean explanation of the observation that a2 agonists block opiatewithdrawal. The results are also discussed in relationship to theanatomy of catecholamine systems in the brain.

Norepinephrine and epinephrine are neurotransmitter andhumoral substances in the brain and periphery. Pharmaco-logical studies have indicated that there are different types ofreceptors for these compounds. Although the division of ad-renergic receptors into a and f types has been accepted forsome time, Langer (1) and Berthelsen and Pettinger (2) haveproposed two distinct oa receptor populations, designated as aland a2. This distinction has pharmacological importance; forexample, centrally acting antihypertensive agents fall into thecategory of a2 agents.

Like many other receptor sites, noradrenergic a receptorscan be identified by binding methods. The radiolabeled drugsWB-4101 and p-aminoclonidine (PAC) are suitable ligands forselectively identifying al and a2 receptors, respectively, inbrain membranes (3, 4). We have localized distinct populationsof al and a2 binding sites in intact tissue sections of rat brainby light microscopic autoradiographic methods.

METHODSThe procedure used is a general one for localizing drug andneurotransmitter binding sites (5). It involves labeling receptorsin slide-mounted tissue sections with a high degree of specificityand generating autoradiograms by the subsequent appositionof emulsion-coated coverslips.The following procedure was used for routine autoradi-

ographic studies. Sections (6 ,um) of rat (male, Sprague-Dawley,200 g) brain were thaw-mounted onto microscope slides. Forlabeling of al receptors, the mounted tissue sections were in-cubated in 1.1 nM [3H]WB-4101 (New England Nuclear, 25.4Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) for 70 min at ice-bathtemperatures in 0.17 M Tris-HCl, pH 7.6/0.001% ascorbic acid.After consecutive 5- and 20-min washes in cold buffer withoutdrug, the slide-mounted tissue sections were dried in a streamof cold, dry air. For localizing a2 receptors, sections were in-cubated in 2.5 nM [3H]PAC (synthesized by Bruno Rouot andtritiated by New England Nuclear to 47.5 Ci/mmol) for 60 min,washed twice for 5 and 10 min, and dried. Extensive prelimi-nary experiments showed that these incubation conditions gavean optimal, reproducible labeling of receptors and providedspecific-to-nonspecific ratios of 3-5:1 in the whole sections. Theselected incubation times were chosen such that the maximalassociation of drug with receptor was attained. The washingconditions were selected so that the nonspecific binding wasgreatly reduced while the specific binding was reduced by nomore than 10 or 15%. Sections (10 Atm) of rat forebrain con-taining the caudate nucleus and septum were routinely selectedfor these preliminary experiments. These biochemical mea-surements of receptor binding were carried out by scraping thetissue sections from the slides and measuring the radioactivityby scintillation spectrometry (5). Control slides were generatedfor both ligands by adding 100 AtM norepinephrine to the in-cubations. As in our experiments with several other receptors,this preparative procedure did not alter the binding charac-teristics of the a receptors in any measureable way (5-8). Asdescribed below, the binding was saturable, of a high affinity,and exhibited appropriate pharmacological characteristics.

Autoradiograms were exposed for 3 months for [3H]WB-4101and for 4 months for [3H]PAC and developed, fixed, and stainedas described (5). The slides were viewed by both brightfield anddarkfield microscopy. Under these conditions, the autoradi-ographic technique provides quantitative data because auto-radiographic grain densities are proportional to time of exposureand tissue content of radioactivity (5). All of the observationsreported here were reproducible and found in several sectionsfrom the same and three different animals.

RESULTSBefore beginning autoradiographic studies, it was necessary toidentify the conditions under which the bulk of the binding ofthe tritiated drugs to the mounted tissue sections had thecharacteristics associated with relevant a receptors. Accord-ingly, we carried out various kinetic and pharmacologic studiesto assess the significance of the binding.

Abbreviation: PAC, p-aminoelonidine.* To whom all correspondence should be addressed.

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Proc. Natl. Acad. Sci. USA 77 (1980) 1697

Kinetic Analysis of [3HIWB4101 and [3H]PAC Binding.Sections of rat brain were prepared as described in Methodsand incubated with various concentrations of the tritiated li-gands. We observed saturation of the specific binding of thedrugs in the nanomolar range. [3H]WB-4101 showed a disso-ciation 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). TheBmax values were 31.0 i 4.0 fmol/mg (n = 3) and 5.89 ± 1.21fmol/mg (n = 3) of tissue wet weight for WB-4101 and PACbinding, respectively (Fig. 1). These values are in generalagreement with the results found in other studies (3).

Pharmacological Specificity. The binding of [3H]WB-4101had the pharmacological characteristics associated with an alreceptor. For example, prazosin was 130 times more potent thanclonidine in displacing WB-4101, and both were more potentthan the isomers of norepinephrine (Fig. 2A). On the otherhand, the binding of [3HJPAC had the characteristics associatedwith an a2 receptor. Clonidine and (-)norepinephrine were500 times more potent than prazosin and (+)norepinephrinein displacing [3H]PAC (Fig. 2B).

8 3 B

E1 1223/

6

z 212334!-- PACbountrdinn

FIG. 1. (A) Saturation kinetics for al1 receptor binding. Data

shown are from one experiment. (Inset) Scatchard plot: B/F =

bound/free in (fmol-ml)/(pmol-mg). Kd = 0.45 nM; Bmax = 35.0

fmol/mg tissue wet weight. (B) Saturation kinetics for ax2 receptor

binding. Data shown are from one experiment. (Inset) Scatchard plot

(see A). Kd = 0.92 nM; BmaX = 3.47 fmol/mg tissue wet weight. (A and

B) *, Total bound; A, specific binding; *, nonspecific binding.

10-9 1O 10-7 -106 10-5Drug concentration, M

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

Autoradiographic Studies. Autoradiograms were preparedas described in Methods. Both al and a2 receptors were foundfairly widespread in the rat brain. Although the variation inreceptor densities in the different areas was not as great as ob-

served with other receptors (5-8), certain areas did have en-

riched levels of binding sites compared to others. Also, therewere striking differences between the binding localizations of[3H]WB-4101 and [3H]PAC. White matter areas always showeda low level of autoradiographic grains comparable to thebackground level observed in controls.The olfactory bulb and its nucleus exhibited some of the

highest levels of a-receptor binding in the brain. However, thedistributions were markedly different for the two ligands.WB-4101 binding was highest in the external plexiform layerof the olfactory bulb, whereas PAC was very high in the ex-

ternal plexiform layer of the olfactory nucleus (Fig. 3). Theal-receptor density in the olfactory bulb was the highest ob-served in the brain.

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0.00

o-

Neurobiology: Young and Kuhar

1698 Neurobiology: Young and Kuhar

* DENSE

E MODERATE A12760imLOW | LGI

LG LMOFIG. 3. Diagram of a-adrenergic receptor distribution in the ol-

factory bulb and nucleus and frontal pole of the cerebral cortex. Thedistributions of al- and a2-receptor binding were different. Graindensities were as follows: Dense, >40 grains per 1000 MAm2; moderate,20-40; low, 6-20; and background, <6. Note the high level of [3H]-WB-4101 binding in the lamina cellularium mitralium bulbi olfactoriiaccessoii (LMCA) and lamina molecularis bulbi olfactorii (LMO) and[3H]PAC binding in the external plexiform layer of the olfactorynucleus (LPE). Other abbreviations and the drawing are from Konigand Klippel (25).

In the cerebral cortex, there were moderate to low levelswidely distributed for both ligands. However, in general, PACshowed higher levels in the more peripheral layers. The anteriorpyriform cortex showed high levels of a2 receptors, as did a

medial portion of the very anterior part of the cerebral cortex.Both ligands exhibited a low to moderate binding in the caud-ate-putamen whereas the lateral septum showed high levels ofPAC binding. High levels of PAC binding were also found inthe basal forebrain anterior to the optic chiasm. The high levelsof a2 receptors in the pyriform cortex and lateral septumcontinued caudally, and the dorsal interstitial nucleus of thestria terminalis also showed high levels of a2 receptors.

In the hypothalamus and thalamus, receptors were widelydistributed. In general, receptors seemed to be higher in medialareas for both ligands although a2 receptors showed substantialenrichments in certain areas, including the supraoptic nucleus,the arcuate nucleus, the dorsomedial nucleus of the hypothal-amus, and some periventricular areas (Figs. 4 and 5). In theseareas the distributions of al and a2 receptors were often quitedifferent. For example, a2 receptors were concentrated in thearcuate and periventricular areas whereas al receptors weredistributed more laterally (Fig. 4). In the same coronal sections,elevated densities in the amygdala and dorsomedial thalamus(the periventricular nucleus, pars rotundocellularis) were alsoobserved for a2 receptors. In the hippocampal formation, themolecular layer of the dentate gyrus showed elevated levels ofal receptors compared to the remainder of the structure al-though the overall levels were still moderate to low. Caudally,however, the dentate gyrus showed fairly high levels. In mid-brain areas, the central gray seemed to be slightly higher thanthe remainder of midbrain areas for a1 receptors. PAC bindingin these areas was low, but somewhat elevated in the superiorcolliculus.

In the hindbrain, as in other parts of the brain, a l-receptordistributions were relatively low. However, we observed someslight elevations along the floor of the fourth ventricle as wellas in some areas along the base of the medulla, including thefacial and vagal nuclei and the superior olive. Areas containingthe catecholamine cell groups Al and A5 (23) are probablyincluded here. a2-Receptor distributions in the hindbrain weremarkedly elevated in certain areas. These areas included thelocus ceruleus (Fig. 6), nucleus tractus solitarii, nucleus com-missuralis, and nucleus raphe pallidus. Other areas that werelower but still elevated included the cochlear nuclei, parts ofthe floor of the fourth ventricle, and the substantia gelatinosaof the spinal trigeminal nucleus. Most other parts of the brainstem had a still lower level of receptors. In the cervical spinalcord, al-receptor distributions were low and quite evenlydistributed over gray matter. a2-Receptor distributions werealso low and quite evenly distributed except that lamina II hadmoderate levels.

B

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9, .,a.--.1 ..

t. e 'k .. , .:

rninFIG. 4. Distribution of a1 and a2 receptors in the hypothalamus. (B) Brightfield micrograph shows the medial and lateral hypothalamus

at about the level of A4890 Am according to Konig and Klippel (25). The optic tract (TO), the ventromedial nucleus of the hypothalamus (hvm),the third ventricle (double arrows), and the infundibular recess (one arrow) are shown. Bar, 500 ,gm. (A) Darkfield micrograph shows the dis-tribution of al receptors. Placement of the arrows is the same as in B. (C) a2 receptors are shown in the darkfield micrograph. Note the moremedial localization of a2 receptors and the more lateral localization of al receptors. Also, a2 receptors are present in the internal part of themedian eminence (above arrow) and the arcuate nucleus (at base of third ventricle). Adjacent control sections did not show any such patternsbut, rather, a low, even level of grains.

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

Proc. Natl. Acad. Sc. USA 77 (1980) 1699

FIG. 5. Darkfield micrographs showing a2-receptor distribution in a coronal section of rat brain. The level shown is approximately the sameas A3990 Am of Konig and Klippel (25). Bar, 1000,gm. Note the high densities of a2 receptors in the medial hypothalamus (the distributionat this level is different from the distribution at A4890gm shown in Fig. 4), the amygdala, and the periventricular nucleus (pars rotundocellularis)in the dorsomedial thalamus (large arrowhead). The bilateral smaller arrows touch the dorsomedial nucleus of the hypothalamus, pars dorsalis.An adjacent control section did not have any of these grain elevations. Abbreviations: ar, nucleus arcuatus; am, nucleus amygdaloideus medialis;abm, nucleus amygdaloideus basalis, pars medialis; ac, nucleus amygdaloideus centralis; H, hippocampal formation.~~~~~~~~~~~~-1 _.

b~~~~~

- 9~~~~~~~~~~~~~~~~~~~~~~~~~~~~,

FIG. 6. Distribution of al1 and ca2 receptors in the area of the locus ceruleus. (B) Brightfield micrograph show's the cerebellum (CB), themesencephalic nucleus of the trigeminal nerve (NTM), and locus ceruleus (asterisk is placed immediately to its right). Bar, 100 ,um. (A) Theaz2-receptor distribution is shown. Note the strikingly high level of ca2 receptors in the locus ceruleus. (D) Darkfield micrograph shows the locusceruleus in an adjacent section prepared as a control; there is a reduced grain density of about that found in the cerebellum in A. (C) The al1distribution is shown in the darkfield micrograph. Note that there is no enrichment in the locus ceruleus and a uniform low level of binding onlyslightly higher than the control values shown in E.

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1700 Neurobiology: Young and Kuhar

DISCUSSIONIn the central nervous system, many experiments indicate theseparate existence of the so-called al- and ac2-noradrenergicreceptors. In the periphery, it was originally proposed that a2receptors were presynaptically located on noradrenergic neu-rons because of their inhibition of norepinephrine release (1,2, 9). In the brain, some of these receptors may be locatedpresynaptically also. For example, several workers have foundthat a2 receptors appear to block norepinephrine release inbrain slices (9, 10). However, lesion experiments suggest thatnot all a2 receptors in brain are located presynaptically. Forexample, administration of 6-hydroxydopamine to depletenoradrenergic nerve terminals in the forebrain resulted in onlya slight decrease of a2 receptors in the septum and the amyg-dala whereas the remainder of the brain showed an increase inreceptors (11). Light microscopic studies do not have the reso-lution to settle this issue. Thus, in the brain, the location of thea2 receptors is not completely understood.

In electrophysiologic experiments, some areas of the brainappeared to be highly selective for a2 receptors. For example,Cedarbaum and Aghajanian (12) found that the neurons of thelocus ceruleus responded preferentially to iontophoresis of a2drugs rather than al drugs. Interestingly, our results are inagreement with their observations; we observed high densitiesof a2 receptors in the locus ceruleus whereas al-receptordensities were very low.Our observed localization of a receptors provides some ra-

tional explanation for many pharmacological and physiologicaleffects of these drugs. For example, clonidine, an a2 agonist,is a centrally acting antihypertensive agent. Many experimentssuggest that a primary site of its action is in the lower medulla,perhaps in the nucleus tractus solitarii (13, 14). In agreementwith this, we observed very high densities of receptors in thisnucleus. Although there was also a slight enrichment of al re-ceptors there, the density of a2 receptors was much greater bycomparison. Another example is the relationship betweena2-receptor stimulation and the blockade of opiate withdrawaleffects. Administration of clonidine blocks the appearance ofthe withdrawal syndrome in animals (15) and humans (16), andAghajanian (17) found that the firing rate of neurons in the locusceruleus increases during opiate withdrawal and that this isattenuated by administration of clonidine. Related to this, wefound elevated densities of a2 receptors in several areas pre-viously found to have elevated densities of opiate receptors(18-20). These areas include the substantiae gelatinosae of thespinal cord and the spinal trigeminal nucleus, the vagal nuclei,the locus ceruleus and adjacent areas of the fourth ventricle,the dorsomedial thalamus (nucleus periventricularis rotundo-cellularis), and parts of the amygdala. Thus, administration ofclonidine would affect many of the same brain areas as opiates.Curiously, this correlation is even found in parts of the olfactorynucleus. Another example of the correlation between locationof receptors and physiological effects of drugs was our obser-vation of high densities of a-adrenergic receptors in differentparts of the hypothalamus. a-Adrenergic agents have signifi-cant effects on hormonal levels (21, 22).A knowledge of the location of noradrenergic receptors in

brain is critical for understanding the functional anatomy ofthe adrenergic pathways. Only where there is a co-existenceof neurotransmitter and receptor can one have a functionalpathway of noradrenergic transmission. Related to this weobserved high densities of receptors in many areas of the brainpreviously shown to have high densities of catecholaminecontaining nerve terminals and cell bodies (for review, see ref.

23). However, one must keep in mind that we are only exam-ining, in this communication, high-affinity al- and a2-bindingsites. There are clearly other receptors such as the 3-adrenergicreceptors that are not labeled by [3H]WB-4101 or [3H]PAC, andthere may be even other a-adrenergic sites of a lower affinitythat were not labeled by these procedures. Nevertheless, we didfind high densities of receptors in many related areas. For ex-ample, the locus ceruleus and the nucleus tractus solitarii, areascontaining both catecholamine cell bodies and nerve terminals(23), have elevated receptor levels. Neurons containing epi-nephrine (23, 24) also terminate in these areas as well as areasin the diencephalon enriched in a2 receptors. The developmentof a method for simultaneously localizing receptors and cate-cholamines would be useful for exploring this further. A pre-liminary report of these findings has been published (26).

We acknowledge the technical assistance of Mrs. Naomi Taylor, Ms.Mary Conrad, and Ms. Shelley Young and the clerical assistance of Ms.Darlene Weimer and Mrs. Mary Flutka. We thank Dr. Bruno Rouotand Prof. Solomon Snyder for supplying several of the drugs. This re-search was supported by U.S. Public Health Service Grant MH25951.M.J.K. is the recipient of a Research Career Development Award(MH0053). W.S.Y. is the recipient of a postdoctoral fellowship(MH07624-02).

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