The localization of fl-adrenergic receptors (fl-AdR) in the rat brain was examined immunocytochemically with antibody against affinity-purified fl2-receptors.
Brain Research, 485 (1989) 125-140 Elsevier
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Immunocytochemical localization of fl-adrenergic receptors in the rat brain A. Wanaka 1, H. Kiyama 1, T. Murakami 1, M. Matsumoto 2, T. Kamada 2, C.C. Malbon 3 and M. Tohyama ~ 1Department of Anatomy II and 2First Department of Internal Medicine, Osaka University Medical School, Osaka (Japan) and 3Department of Pharmacological Sciences, School of Medicine-Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY (U.S.A.) (Accepted 20 September 1988) Key words: Adrenergic receptor; Immunocytochemistry, Rat brain
The localization of fl-adrenergic receptors (fl-AdR) in the rat brain was examined immunocytochemically with antibody against affinity-purified fl2-receptors. This antibody was confirmed to cross-react with fll-AdR, fl-AdR were widely but unevenly distributed in the rat brain. In the forebrain and diencephalon, cells with fl-AdR were abundant in the cerebral cortex, hippocampus, and various hypothalamic nuclei other than the ventromedial hypothalamic nucleus and the suprachiasmatic nucleus. Few or no cells with fl-AdR were found in the thalamus except in the lateral and medial geniculate nucleus, habenular nucleus and paraventricular thalamic nucleus. The striatum, globus pallidus, and accumbens nucleus also contained few fl-AdR. In the lower brainstem, areas related to auditory function, such as the inferior colliculus, the nucleus of the lateral lemniscus and the cochlear nuclei, as well as areas related to cerebellar function, such as the reticulotegmental nucleus of the pons, the pontine nuclei, the lateral reticular nucleus and the inferior olive, were rich in fl-AdR. Considerable immunoreactivity was also found in the parabrachial nucleus and the nucleus of the solitary tract. INTRODUCTION The localization of catecholamine (CA) in the brain has been examined morphologically by histofluorescence 4"6'8-1°'2° and immunocytochemistry with antisera against various CA-synthesizing enzymes, such as tyrosine hydroxylase (TH) 13"21, dopamine-fl-hydroxylase ( D B H ) 36, and phenyl-Nmethyltransferase (PNMT) 32. C A receptors have been detected pharmacologically and electrophysiologically with the use of C A antagonists or agonists 19'42. The binding of C A and C A agonists to C A receptors has been examined morphologically by autoradiography 21. Recent advances in molecular biology and biochemical techniques have made it possible to explore the molecular profiles of receptors 3'16~18"23, enabling us to produce antibodies against purified receptors 22'3s. For example, the
location of receptors for v-aminobutyric acid ( G A B A ) 31 or glycine 2'37'38 has been determined with anti-receptor antisera by both light and electron microscopy. Previously, /Strader et al. showed immunocytochemically that Purkinje cells contain adrenergic receptors ( A d R ) 3s, and Ventimiglia et al. examined cultured hypothalamic neurons using anti-fl-adrenoreceptor antiserum 43. In addition, Aoki et al. demonstrated the presence of neurons containing fl-AdR-like immunoreactivity in the cerebral cortex and neostriatum of the rat 1. However, no information was available on the overall distribution of the fl-AdR in the brain. In the present study we employed antiserum against purified fl2-adrenergic receptors from guinea pig lung to examine immunohistochemically the precise location of fl-adrenergic receptors in the rat brain.
Correspondence: A. Wanaka, Department of Anatomy II, Osaka University Medical School, 4-3-57 Nakanoshima, Kitaku, Osaka, 530, Japan. 0006-8993J89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
126 MATERIALS AND METHODS
Tissue preparation Eight male Wistar rats, each weighing about 100 g, were perfused transcardially with 50 ml of ice-cold saline followed by 500 ml of modified Zamboni's fixative48 (0.1 M phosphate buffer containing 4% paraformaldehyde and 0.2% picric acid) under sodium pentobarbital anesthesia (60 mg/kg, i.p.). The brains were removed, immersed in the same fixative overnight at 4 °C, and soaked for one more night in phosphate-buffered saline (PBS, pH 7.4) containing 30% sucrose at 4 °C. Frozen sections 5 /~m thick were cut with a cryostat and prepared for immunocytochemical examination 5.
Immunocytochemical procedures T h e sections were divided into two groups, one for the demonstration of cells and fibers with AdR-like immunoreactivity (AdR-IR), the other for the identification of regions of the brain by Cresyl violet staining. All samples for immunocytochemical analysis were first rinsed in PBS for 20 min, then incubated in a humid atmosphere overnight at 15 °C with primary antiserum diluted to 1:50 with PBS. After three 10 min rinsings in PBS, the sections were incubated overnight at 15 °C in a humid atmosphere with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin (IgG) (Miles). The sections were washed in PBS 3 times for 10 min each time and cover-slipped in a PBS-glycerine mixture (1:1). Some of the samples of the first group were subjected to absorption tests to determine the specificity of the antiserum, these were first incubated with control serum (see below) instead of the primary antiserum, after which they were processed as described above.
Preparation and characterization of the antiserum Characterization of the antiserum has been reported elsewhere 22. Briefly, fl2-AdR was purified by affinity chromatography and ion-exchange chromatography from guinea pig lung in 0.1 ml of 200 mM NaC1 containing 100 mM KHzHPO 4 (pH 7.0) and 0.05% digitonin. The specific activity of the purified receptors was found to be greater than 0.5 nmol/mg protein. Separately, a washed cell pellet of 1 × 10 7 cells of sp2/o myeloma was suspended in 0.4 ml of
glutaraldehyde in PBS and incubated at 4 °C for 4 h. The cells were collected by centrifugation and washed with PBS; purified receptors were added, and the mixture was vortexed and incubated overnight at 4 °C. New Zealand white rabbits were immunized with 3 /~g of purified fl2-AdR coupled with the myeloma cells as an antigen. The antibody titer was checked by solid-phase enzyme-linked immunosorbent assay (ELISA) 22. An absorption control test was used to check the specificity of the immunostaining. First, 5 /~g of purified flz-AdR, which corresponded to more than 2.5 pmol/mg protein of receptor activity, was added to 500 ktl of the diluted (1:50) primary antiserum and incubated at 4 °C overnight. The mixture was centrifuged at 10,000 g for 30 min, and the supernatant was used for the test. The overall predicted structures of G-proteincoupled receptors such as al-, a2-, ill-, and fl2-AdR, and rhodopsin receptors are homologous 7"16'as. Therefore, when the cross-reactivity of anti-fl2-AdR antiserum with fll-AdR of rat adipose cells was tested by immunoprecipitation, between 60% and 84% of fll-AdR was immunoprecipitated 22. Accordingly, the antiserum used in this study appears to recognize both ill- and fl2-AdR. The cross-reactivity with rhodopsin receptors of the antiserum used in this study was considered to be low, because no immunostaining was seen in the outer segment or outer nuclear layer of the retina, which contains many rhodopsin receptors (Ishimoto et al., in preparation). In the cranial motor nuclei, immunostaining was slight to relatively moderate (for data, see Results) compared with the high density of quinuclidinyl benzilate binding sites 44, suggesting that there was little or no cross-reactivity of the antiserum with muscarinic cholinergic receptors. The possibility that the antiserum cross-reacted with dopamine receptors seems slight, because no immunostaining was noted in the striatum (data presented in Results) where the density of [3H]spiperone binding was high a6. Areas containing a high density of [3H]clonidine binding sites such as the nucleus of the solitary tract, locus coeruleus, and lateral parabrachial nucleus (data presented in Results) displayed high levels of immunoreactivity39'41. A rich plexus in the thalamus, which was strongly and specifically labeled with [ 125I][2-/3- (4 - hydroxyphenyl) - ethylaminomethyl- te-
127 tralone] 1415, did not exhibit immunoreactivity. Thus, the antiserum has little cross-reactivity with al-AdR, but may cross-react to some extent with a2-AdR. The likelihood of cross-reaction occurring between the antiserum used in this study and a2-AdR or dopamine receptors was examined in additional control experiments. The antiserum employed in this study was shown to inhibit the binding of ligand 2~. So that we examined whether or not the immunoreaction was displaced with excess amount of ligands. Details are as follows; anti-fl2-AdR antiserum was diluted with buffers containing either 1 mM alprenolol, 1 mM clonidine, or 1 mM spiperone. Sections were preincubated with one of these solutions before being examined immunocytochemically.
Nomenclature The terminology is based on the atlas of Paxinos and Watson 2s. RESULTS
Control experiments Absorption tests. Immunostaining was not detected when the sections were incubated with antiserum absorbed with purified fl2-AdR. Moreover, no immunostaining was seen in the sections that were incubated with normal rabbit serum instead of the primary antiserum, or incubated only with the secondary antiserum. Additional control experiments. Immunostaining was markedly reduced when the sections were incubated with the solution containing alprenolol. However, no reduction of the immunoreaction was seen in the sections incubated with the solution containing clonidine or spiperone. These findings strongly suggest that the antiserum against fl2-AdR used in these experiments recognized both subtypes of fl-AdR but had little cross-reactivity with a2-AdR and dopamine receptors.
Distribution of fl-AdR-like immunoreactivity (flAdR-IR) in the brain fl-AdR-IR was widely but unevenly distributed in a punctate pattern throughout the brain. The distribution of fl-AdR-IR structures in the brain is presented schematically in Fig. 1. fl-AdR-IR was
found mainly on the surface of the neurons, on the proximal parts of their processes, or within the cytoplasm, although the intensity of the immunoreaction differed among the regions of the brain (Fig. 2). Immunoreaction intensity was classified as strong, moderate, or weak. In some instances, the receptors had a varicose fiber-like appearance (Fig. 3A,B) or were distributed in the neuropil as fine dots (Figs. 3C and 5A,B), suggesting that receptors were localized on thin neuronal elements such as the distal dendrites and axon terminals. In this study, these structures will be called IR fibers.
(1) Telencephalon Weak immunostaining was observed in the internal granular layer and anterior olfactory nucleus of the olfactory bulb, but no fl-AdR-IR was detected in other parts (Fig. 1A). The olfactory tubercle contained many moderately labeled cells, mostly in the pyramidal layer, but with a few in the polymorphic and plexiform layers (Figs. 1B,C and 2A,B). No fl-AdR-IR were detected in the island of Calleja. Labeled cells extended laterally to occupy the pyramidal layer of the piriform cortex. Both the horizontal and vertical limbs of the nucleus of the diagonal band (DB) contained weakly labeled large cells (about 30 ttm in diameter) (Fig. 1C). The border between the DB and the accumbens nucleus contained a group of weakly stained oval cells of medium size (about 20 ktm in diameter). However, no fl-AdR-IR was noted in the accumbens nucleus itself (Fig. 1B,C). In the medial and lateral septal nuclei, only IR fibers were present, and they became more numerous in the dorsolateral direction. The bed nucleus of the stria terminalis contained fibers with fl-AdR-IR, but was devoid of neurons with fl-AdR, which were found elsewhere in the nucleus, especially in the infracommissural portion (Fig. 1D,E). Pallidal structures, such as the globus pallidus, ventral pallidum, and caudate putamen, had few fl-AdR (Figs. 1 B - G and 2F). In the cerebral cortex a number of cells showed moderate immunofluorescence (Figs. 1 and 2C,D), the density being highest in the cingulate cortex and frontal cortex including the prefrontal cortex. Labeled neurons were found in all layers except layer
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