Cellular and subcellular localization of alpha-1 adrenoceptors in the rat visual cortex

Neuroscience 141 (2006) 1783–1792

CELLULAR AND SUBCELLULAR LOCALIZATION OF ALPHA-1 ADRENOCEPTORS IN THE RAT VISUAL CORTEX K. NAKADATE,a,b K. IMAMURAb,c* AND Y. WATANABEb,d

over, a small number of immunoreaction products were also detected in axons and presynaptic sites. These findings provide the first quantitative evidence regarding the cellular and subcellular localization of alpha-1 adrenoceptor immunoreactivity in visual cortex. Moreover, the ultrastructural distribution of alpha-1 adrenoceptor immunoreactivity suggests that alpha-1 adrenoceptors are transported mainly into dendrites and that they exert effects at postsynaptic sites of neurons. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

a Department of Histology and Neurobiology, Dokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu-machi, Tochigi 3210293, Japan b Department of Neuroscience, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita-shi, Osaka 565-0874, Japan c

Laboratory of Visual Neurocomputing, Brain Research Institute, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan

d Department of Physiology, Osaka City University Graduate School of Medicine, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan

Key words: noradrenaline, receptor, immunohistochemistry, immunoelectron microscopy, synapse, transport.

Abstract—Noradrenaline is thought to play modulatory roles in a number of physiological, behavioral, and cellular processes. Although many of these modulatory effects are mediated through alpha-1 adrenoceptors, basic knowledge of the cellular and subcellular distributions of these receptors is limited. We investigated the laminar distribution pattern of alpha-1 adrenoceptors in rat visual cortex, using immunohistochemistry at both light and electron microscopic levels. Affinity-purified antialpha-1 antibody was confirmed to react only with a single band of about 70 – 80 kDa in total proteins prepared from rat visual cortex. Alpha-1 adrenoceptors were widely distributed though all cortical layers, but relatively high in density in layers I, II/III, and V. Immunoreactivity was observed in both neuronal perikarya and processes including apical dendrites. In doublelabeling experiments with anti-microtubule-associated protein 2, anti-neurofilament, anti-glial fibrillary acidic protein, anti-glutamic acid decarboxylase 65/67, anti-2-3-cyclic nucleotide 3-phosphodiesterase, and anti-tyrosine hydroxylase antibodies, alpha-1 adrenoceptors were found mainly in dendrites and somata of microtubule-associated protein 2-immunopositive neurons. About 20% of alpha-1 adrenoceptors were in GABAergic neurons. A small number of alpha-1 adrenoceptors were also distributed in axons of excitatory neurons, astrocytes, oligodendrocytes and noradrenergic fibers. Using an immunoelectron microscopic technique, numerous regions of alpha-1 adrenoceptor immunoreactivity were found in cell somata, on membranes of dendrites, and in postsynaptic regions. More-

Biogenic amines in the brain, including those of the noradrenergic (NA) system, play important roles in cellular, physiological, and behavioral modulation. The majority of NA fibers in the brain arise from a single source of NAcontaining neurons in the locus coeruleus (LC; A6) (Dahlstrom and Fuxe, 1964). NA fiber projections are widespread throughout the cerebral cortex (Ungerstedt, 1971; Maeda and Shimizu, 1972) and play important roles in the regulation of cortical function (Sara and Segal, 1991). Evoked firing patterns of neurons in the locus coeruleus are highly stereotypical; these neurons respond to a variety of afferent inputs (Nakamura, 1977; Foote et al., 1980, 1983; Aston-Jones and Bloom, 1981). The multitude of physiological effects resulting from NA activation are thus likely a result of different types of postsynaptic responses to NA released at different loci. In fact, it is possible that the various functional effects attributed to NA are the result of differential patterns of activation of adrenoceptor subtypes. NA mediates effects in the brain via several different G-protein-linked receptors, broadly classified as alpha and beta adrenoceptors (Berthelsen and Pettinger, 1977; Raymond et al., 1990). Alpha adrenoceptors have been divided into alpha-1 and alpha-2 subtypes based on their differences in affinity for a variety of agonists and antagonists (Morrow et al., 1985; Morrow and Creese, 1986; Minneman, 1988; Han and Minneman, 1991). Ligand binding studies have demonstrated the existence of high-affinity alpha-1 adrenoceptor binding sites in the rat brain (Morrow and Creese, 1986). The distribution of alpha-1 adrenoceptors in the brain has been determined in radioligand binding studies, which have typically employed 3H-prazosin or 125I-2-{[b(4-hydroxyphenyl)ethyl]aminomethyl}-1-tetralone (HEAT) as a ligand (Young and Kuhar, 1980; Jones et al., 1985). The NA projections in visual cortex have been shown to play important roles in the formation of synapses (Blue and Parnavelas, 1982) and synaptic plasticity during develop-

*Correspondence to: K. Imamura, Laboratory of Visual Neurocomputing, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Tel: ⫹81-48-462-1111x7154; fax: ⫹81-48-4679685. E-mail address: [email protected] (K. Imamura). Abbreviations: ANOVA, analysis of variance; BSA, bovine serum albumin; CNPase, 2-3-cyclic nucleotide 3-phosphodiesterase; DAB, 3-3-diaminobenzidine tetrahydrochloride; EDTA, ethylenediamineN,N,N=,N=-tetraacetic acid; GAD65/67, glutamic acid decarboxylase 65/67; GFAP, glial fibrillary acidic protein; HEAT, 2-{[b-(4-hydroxyphenyl)ethyl]aminomethyl}-1-tetralone; IP3, inositol 1,4,5-triphosphate; MAP2, microtubule-associated protein 2; NA, noradrenaline or noradrenergic; PB, phosphate buffer; PBS, phosphate-buffered saline; PBS-T, phosphate-buffered saline containing 0.3% Triton X-100; ROI, region of interest; RT, room temperature; SDS, sodium dodecyl sulfate; TH, tyrosine hydroxylase; TPBS, phosphate-buffered saline containing 0.1% Tween 20; Tris–HCl, 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride.

0306-4522/06$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.05.031

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ment (Komatsu, 1996; Kirkwood et al., 1999). It has been reported that alpha-1 adrenoceptor play a major role in NAinduced intracellular Ca2⫹ concentration responses in visual cortex (Kobayashi et al., 2000). Alpha-1 adrenoceptors are required for the formation of excitatory synapses in rat visual cortex (Nakadate et al., 2006). Moreover, it has been reported that activation of alpha-1 adrenoceptors selectively suppressed the horizontal propagation of excitation in the supragranular layers of rat visual cortex (Kobayashi et al., 2000). This alpha-1 adrenoceptor-dependent suppression may play important roles in visual information processing. Full understanding of these various aspects of NA-activated alpha-1 adrenoceptor activity in visual cortex requires determination in detail of the pattern of distribution of alpha-1 adrenoceptors in the visual cortex. In the present study, using immunohistochemical and immunoelectron microscopic methods, we for the first time examined the subcellular distribution of alpha-1 adrenoceptors in rat visual cortex.

EXPERIMENTAL PROCEDURES Animals In this study, a total of eight Long-Evans strain male rats were used at postnatal day 56. All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and approved by the Animal Research Committee of Osaka Bioscience Institute (No. 98-30). All efforts were made to minimize the suffering of animals and to reduce the number of animals used in the present study.

Alpha-1 adrenoceptor antibody Polyclonal antibody to alpha-1 adrenoceptor (PA1-047) was purchased from Affinity BioReagents Inc. (Golden, CO, USA). It had been raised against the sequence of amino acids 339 –349 of the 3rd intracellular loop of the human alpha-1 adrenoceptor sequence (FSREKKAAK). This sequence is specific to all alpha-1 adrenoceptor subtypes (alpha-1A/C, Stewart et al., 1994; 1B, Strausberg et al., 2002; 1D, Schwinn et al., 1995), being conserved in neither other adrenoceptor subtypes (alpha-2a, b; beta-1, 2, 3) nor other G-protein-coupled receptors (NCBI, BLAST program). To check the specificity of immunostaining, the antibodies were preadsorbed with synthetic peptides based on the findings of epitope mapping. Briefly, for alpha-1 adrenoceptor polyclonal antibody, 100 ␮g or 1 mg of synthetic peptide (FSREKKAAK) was incubated with 10 ␮g of antibody at 4 °C for 24 h on a rotation vortex. The supernatant solution containing blocked antibody was then tested by immunoblotting and immunohistochemical analysis, as described below.

Protein preparation for Western blot analysis The rats were perfused through the left ventricle with ice-cold 0.1 M phosphate-buffered saline (PBS, pH 7.4). The brain was rapidly removed and homogenized in 10 volumes of ice-cold homogenate buffer (20 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride [Tris–HCl] at pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, and 150 mM NaCl) containing protease inhibitors (one tablet/10 ml homogenate buffer, CompleteTM Mini, Roche Diagnostics, Basel, Switzerland). Homogenates were then centrifuged at 500⫻g for 5 min at 4 °C, and the supernatants were dissolved in sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris–HCl, pH 6.8, containing 3% SDS, 5% glycerol, and 2% 2-mercaptoethanol) and then boiled for 5 min. Protein concentrations

were measured using a protein assay kit (Bio-Rad, Hercules, CA, USA) and determined with bovine serum albumin (BSA) as a reference protein as described (Bradford, 1976).

Immunoblot analysis SDS-PAGE was performed as described (Laemmli, 1970), and Western blotting was performed using the ECL-Plus immunoblotting detection system (Amersham Life Science, Buckinghamshire, UK) according to the manufacturer’s instructions. Proteins were separated by SDS–polyacrylamide gel electrophoresis (8% gels) and electrophoretically transferred at 50 V for 60 min onto a polyvinylidene difluoride membrane (ImmobilonTM-P, Millipore Co., Bedford, MA, USA). After a blocking step of incubation with 5% (w/v) skim milk (Becton Dickinson Microbiology System, Sparks, MD, USA) and Block-Ace (Yukijirushi Nyugyo Co., Sapporo, Japan) in phosphate-buffered saline containing 0.1% Tween 20 (TPBS, pH 7.5) for 1 h at room temperature (RT), the membrane was washed and incubated with primary antibody (1.0 ␮g/ml in TPBS containing 1% BSA and 5% skim milk) for 1 h at RT. Then, after incubation with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2000 dilution in TPBS containing 1% BSA and 5% skim milk) for 45 min at RT, the immunoreactive bands were detected with an ECL-Plus kit (Amersham Life Science).

Tissue preparation for immunohistochemical and immunoelectron microscopic analysis Animals were deeply anesthetized with an overdose of sodium pentobarbiturate (50 mg/kg, i.p.; Nembutal®; Abbott Laboratory, IL, USA) and perfused through the left ventricle with ice-cold 0.9% saline. For immunohistochemical and immunofluorescence analysis, fixative containing 4% paraformaldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (PB, pH 7.4) was perfused. The brain was quickly removed, post-fixed overnight at 4 °C in the same fixative, cryoprotected in graded concentrations of sucrose (final 30%) in 0.1 M PB, frozen on dry ice, and cut into 50 ␮m coronal sections with a freezing microtome. For immunoelectron microscopic study, animals were perfused with physiological saline, followed by ice-cold fixative containing 4% paraformaldehyde, 15% saturated picric acid, and 0.1% glutaraldehyde in 0.1 M PB (pH 7.4). The brains were removed from the skulls, and then post-fixed with the same fixative for 24 h at 4°C. Brains were washed in 0.1 M PB, and then sections were cut on a microslicer (DTK-1000, Dosaka EM, Kyoto, Japan) at 50 ␮m thickness. Some of these sections were processed for electron microscopic analysis as described below.

Immunohistochemistry: light microscopic analysis Immunohistochemistry was performed using the free-floating method. Fifty micrometer sections were washed in phosphatebuffered saline containing 0.3% Triton X-100 (PBS-T) and incubated at RT for 90 min in PSB-T containing 1% hydrogen peroxide. After several washes, sections were incubated with blocking solution [5% normal goat serum (Vector Laboratories, Burlingame, CA, USA), 2% BSA (Sigma Chemical Co., MO, USA), and 10% Block-Ace (Dainihon Seiyaku Co., Tokyo, Japan) in PBS-T] at RT for 2 h, and then incubated for 2 days at 4 °C with the antibody to alpha-1-adrenoceptors (2.0 ␮g/ml in PBS). After washes in PBS, the sections were incubated with biotinylated goat anti-rabbit antibody (Vector Laboratories) at RT for 2 h. The sections were then washed and reacted with avidin– biotin peroxidase complex (ABC kit, Vector Laboratories) at RT for 2 h. The sections were subsequently incubated in 50 mM Tris–HCl (pH 7.3) containing 0.05% 3-3-diaminobenzidine tetrahydrochloride (DAB; Dojindo, Kumamoto, Japan) and 0.003% hydrogen peroxide. All sections were mounted on gelatin-coated slides, dehydrated through graded

K. Nakadate et al. / Neuroscience 141 (2006) 1783–1792 concentrations of ethanol, cleared in xylene, and then mounted with Mount-Quick (Daido Sangyo Co., Ltd. Japan). In addition, as controls, some sections were incubated in solution without the primary antibody, with preimmune serum, or with primary antibody pre-adsorbed using the synthetic peptide.

Double-immunofluorescence staining Sections were incubated with a blocking solution, and then overnight at 4 °C with conjugated primary antibodies. One primary antibody was rabbit anti-alpha-1 adrenoceptor antibody (2.0 ␮g/ml in PBS-T), and the other was one of the following mouse monoclonal antibodies: anti-MAP2 (microtubule-associated protein 2; 1:100, Boehringer Mannheim Biochemica, Germany) antibody, anti-neurofilament antibody (1:1000, ICN Biomedicals, Inc, CA, USA), anti-GFAP antibody (glial fibrillary acidic protein, 1:1000, Boehringer Mannheim Biochemica), anti-GAD 65/67 antibody (1:1000, Stresgen Bioreagents, BC, Canada), anti-2-3-cyclic nucleotide 3-phosphodiesterase (CNPase) antibody (1:1000, Sigma), or anti-TH antibody (tyrosine hydroxylase, 1:1000, DiaSorin, MN, USA). Sections were rinsed with PBS-T and incubated with conjugated secondary antibodies, FITCconjugated goat anti-rabbit IgG (1:400 in PBS, Jackson ImmunoResearch, West Grove, PA, USA), and TRITC-conjugated goat antimouse IgG (1:400, Jackson ImmunoResearch, West Grove, PA, USA). Immunofluorescence images were obtained under a confocal laser scanning microscope (Carl Zeiss LSM510) and analyzed using Adobe Photoshop 5.5.

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visual cortex (in nine randomly chosen 100⫻200 ␮m rectangular regions) using image-analysis software (NIH Image, National Institutes of Health). For quantification of double labeling, the intensity of yellow color, indicating double labeling, was extracted from the original image using software (Photoshop, Adobe Systems Inc., CA, USA). The threshold for extraction was set at twice the baseline value. The sampled signal was quantitatively measured using NIH Image.

RESULTS Specificity of alpha-1 adrenoceptor antibody The specificity of affinity-purified antibodies to alpha-1 adrenoceptor was examined with immunoblot analysis of membrane fractions obtained from postnatal day (PND) 56 rat brain (Fig. 1). The alpha-1 adrenoceptor antibody labeled a single band with a molecular weight of about 70 – 80 kDa (Fig. 1A, Alpha-1 lane). The immunoreactivity was completely adsorbed by preincubation of the primary antibodies with excess amounts of the respective epitope peptides (Fig. 1, Pre-adsorbed lane).

Immunoelectron microscopic analysis Sections were cryoprotected in solutions containing 15 and 30% sucrose in 0.1 M PB. The sections were freeze-thawed and incubated in a blocking solution containing 10% normal goat serum in 0.1 M PBS for 2 h, followed by incubation with primary antibodies (2.0 ␮g/ml) diluted in PBS containing 3% normal goat serum overnight at 4 °C. After three brief rinses, the sections were incubated with biotinylated secondary antibody (diluted 1:100 in PBS) for immunoperoxidase reaction, or with 1.4 nm gold-coupled secondary antibody (diluted 1:100 in PBS; Nanogold, Nanoprobes, Stony Brook, NY, USA) for immunogold reaction, and then reacted with the ABC kit or HQ Silver kit (Nanoprobes), respectively. After treatment with OsO4, sections were stained with uranyl acetate, dehydrated, and embedded in Epon-812 resin (TAAB, Switzerland). Ultrathin sections were cut on an ultramicrotome (Ultracut S; Reichert-Nissei, Tokyo, Japan) and examined with an H-7100 electron microscope (Hitachi Co., Ltd., Tokyo, Japan). Synaptic profiles were classified according to their membrane thickenings as either asymmetrical (Gray’s type I) or symmetrical (Gray’s type II) (Gray, 1959).

Image analysis Photo-images of HRP-DAB-reacted sections and Nissl-stained sections were captured using a CCD camera system (Fujix Digital Camera HC-2500 3CCD®, Fujifilm Co., Tokyo, Japan) and processed using software (Photograb-2500®, Fujifilm Co., Tokyo, Japan). Anatomic structures were identified by direct observation of Nissl-stained sections, using the atlases of (Paxinos and Watson, 1986) and (Paxinos, 1995) as basic references. For quantification of alpha-1 immunoreactivity in each section, the intensity of immunoreactivity was measured [in regions of interest (ROI) 80⫻200 ␮m, 11 ROIs in each of three sections] using image-analysis software (NIH Image version 1.63, National Institutes of Health, USA). Statistical comparisons were performed using one-way analysis of variance (ANOVA). Fisher’s post hoc test was then used for comparison when significance was established by ANOVA. Findings of P⬍0.05 were considered significant. After immunofluorescence sections were captured, we measured the optical densities of alpha-1 adrenoceptor signals in the

Fig. 1. Specificity of the polyclonal anti-alpha-1 adrenoceptor antibody as determined by immunoblot analysis. The anti-alpha-1 adrenoceptor antibody recognizes a single band of molecular weight 70 – 80 kDa (arrow) within rat visual cortical membrane fraction (Alpha-1 lane). No immunoreactivity is found in Western blot using serum pre-adsorbed with synthetic peptide of the epitope (Pre-adsorbed lane).

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Fig. 2. Distribution of alpha-1 adrenoceptors in rat visual cortex. These photographs of immunostaining were taken from the primary visual cortex (area 17, Oc1). (A) A Nissl-stained section shows the anatomic structures of primary visual cortex. Layers (I to VI) of visual cortex are represented in Fig. 1A as I, II/III, IV, and V/VI. The laminar pattern of alpha-1 adrenoceptor staining is shown in B. Higher densities of alpha-1 adrenoceptor appear in layers I to III and the upper portion of V/VI (B). White rectangular areas in B are enlarged in C and D. In layers I to III (C) and the upper portion of V/VI (D), alpha-1 adrenoceptors are distributed in neuronal perikarya and fibers. (E) Incubation in antiserum preadsorbed with immunizing peptide. (F) Optical density of immunoreactivity for alpha-1 adrenoceptor is plotted against cortical layer. Closed circles and closed squares indicate alpha-1 adrenoceptor staining and preadsorbed staining, respectively. * P⬍0.05, ** P⬍0.01. Scale bars⫽50 ␮m in D and E⫽200 ␮m.

Lack of cross-reactivity on immunohistochemistry was confirmed as follows. Alpha-1 adrenoceptor immunoreactivity was completely absent after omission of the primary antibody (data not shown), and immunoreactivity was completely adsorbed by pre-incubation of the primary antibodies with an excess amount of the antigen synthetic peptide (Fig. 2E). This antibody thus appeared to bind specifically to the alpha-1 adrenoceptor protein. Laminar distribution of alpha-1 adrenoceptors in rat visual cortex The laminar pattern of immunolabeling of alpha-1 adrenoceptors was examined (Fig. 2). Although immunoreactivity was found throughout the layers of visual cortex, it was relatively low in layers IV and VI. Optical density was significantly lower in these layers than in layer I (ANOVA, Fisher post hoc test, P⬍0.05, Fig. 2B and F). The immunoreactivity of alpha-1 adrenoceptors was small and dotlike in appearance. In layers I to III (Fig. 2C), large numbers of immunoreactive somata and fibers were present, diffusely in neuronal perikarya as well as along apical dendrites and in relatively low density in the nucleus. Reaction product was also widely distributed in layers V/VI (Fig. 2D). No staining was detected in tissue either incubated in antisera preadsorbed with a synthetic peptide (Fig. 2E) or with omission of primary antisera (data not shown). These findings together indicate that alpha-1 im-

munoreactivity is lower in cortical layers IV and VI, where thalamic afferents terminate. Alpha-1 adrenoceptors are mainly expressed in the dendrites of excitatory neurons in rat visual cortex To determine the cell types and cellular compartments of alpha-1 adrenoceptors, we used double-immunostaining with fluorescently labeled antibodies to alpha-1 adrenoceptors and many subcellular markers (Fig. 3). Doublestaining using anti-alpha-1 antibody (green) and antiMAP2 antibody (red) revealed alpha-1 adrenoceptors on the dendrites of cortical neurons (Fig. 3A). Fig. 3B is a higher-magnification photograph of the white rectangular area in Fig. 3A, in which 60 –70% of the total alpha-1 adrenoceptor signal overlapped with that of MAP2 (Fig. 3B and Table 1). Co-expression of alpha-1 adrenoceptor (green) and glutamic acid decarboxylase 65/67 (GAD65/ 67) (red) was detected for about 20% of total alpha-1 adrenoceptor signal (Fig. 3C and D, Table 1). The results obtained with an antibody (red) to an axonal marker, neurofilament, revealed a small fraction of alpha-1 adrenoceptors (green) distributed in neuronal axons (Fig. 3E and F), with 5% co-localization (Table 1). Results of double-staining using anti-alpha-1 antibody (green) and anti-CNPase antibody (red, Fig. 3G), anti-GFAP antibody (red, Fig. 3H), or anti-tyrosine hydroxylase antibody (red, Fig. 3I) demonstrated small numbers of alpha-1 adrenoceptors localized

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Fig. 3. Laser scanning microscopic images of double-immunofluorescence of alpha-1 adreoceptor (green fluorescence) with MAP2 (A and B, red fluorescence), GAD65/67 (C and D, red fluorescence), Neurofilament (E and F, red fluorescence), CNPase (G, red fluorescence), GFAP (H, red fluorescence) and TH (I, red fluorescence). All images are from layer II/III. B, D, and F are higher-magnification views of white rectangular areas in figures A, C, and E, respectively. Scale bar⫽10 ␮m.

in oligodendrocytes, astrocytes, and catecholaminergic fibers within rat visual cortex (Table 1). Because there may be GFAP-immunonegative astrocytes and/or GFAP-immunopositive non-astrocytic cells in rat visual cortex, the ratio of co-localization of immunoreactivity provides an approximation for the amount of alpha-1 adrenoceptors on astrocytes. Table 1. Ratios of co-localization of immunoreactivity for alpha-1 adrenoceptors with those for several cellular marker proteins Marker

Mean⫾SD (%)

Dendritic marker (MAP2) Axonal marker (neurofilament) GABAergic cell marker (GAD65/67) Astrocyte marker (GFAP) Oligodendrocyte marker (CNPase) Catecholaminergic fiber marker (TH)

66.7⫾10.1 4.7⫾2.1 22.4⫾6.5 5.3⫾1.9 3.9⫾1.0 8.4⫾6.2

Subcellular co-localization values are the mean⫾standard deviation (n⫽9). Each value obtained as the ratio of co-localization area/total area of alpha-1 adrenoceptor expression.

Observation of alpha-1 adrenoceptors in both pre- and postsynaptic membranes To determine the subcellular components of alpha-1 adrenoceptors in rat visual cortex, we next performed immunoelectron microscopic analysis using the anti-alpha-1 adrenoceptor antibody (Fig. 4). The immunoperoxidase products within neuronal perikarya were associated with the Golgi apparatus and the endoplasmic reticulum, particularly those positioned near the plasma membrane (Fig. 4A). Alpha-1 adrenoceptors were distributed both near and far from the plasma membrane of dendrites (Fig. 4B). Alpha-1 adrenoceptor immunoreactivity was also located in the spinous portion of the dendrites and near the synapses that apposed dendrites (Fig. 4B and 4C). There was alpha-1 adrenoceptor immunoreactivity in the postsynaptic as well as presynaptic regions of both excitatory and inhibitory types of synapses (Fig. 4D and E). Because immunoelectron microscopy with the HRPDAB method used in the above experiment could not precisely determine the sites of immunoreactivity, we next

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Fig. 4. Electron microscopy reveals alpha-1 adrenoceptors within a perikaryon, dendrite, and synapse in rat visual cortex. These pictures were taken from ultrathin sections in which lead citrate counterstain was omitted to facilitate visualization of immunolabeling. (A) Arrows point to immunoreactivity near the plasma membrane. Arrowhead points to immunoreactivity somewhat distant from the plasma membrane. The presence of alpha-1 immunoreactivity in the vicinity of endoplasmic reticulum (ER) indicates that these sites of immunoreactivity are localized to the perikaryal cytoplasm. Figures B and C show sites of immunopositivity on dendrites. In figure B, the arrow points to immunoreactivity near the plasma membrane, and arrowheads point to immunoreactivity far from the plasma membrane. Discrete regions of immunoreactivity are associated with the spinous portion of the dendrite (open arrow). In C, the arrow points to immunoreactivity near the region of synaptic contact (arrowhead). (D) The arrow shows the immunoreactive postsynaptic site of an excitatory neuron (asymmetric synapse, Gray’s type I synapse). (E) The arrow points to immunoreactive postsynaptic sites and arrowhead points to immunoreactive presynaptic sites. T in figures C–E indicates the presynaptic terminal. Scale bars⫽200 nm.

used immunogold-silver enhanced immunoelectron microscopy (Fig. 5). Immunogold particles associated with alpha-1 adrenoceptors were distributed both near and far from the plasma membrane of dendrites (Fig. 5A). Alpha-1 adrenoceptors were localized in the postsynaptic region both near and far from the postsynaptic density (Fig. 5B), and were also distributed in the presynaptic region both near and far from the site of synaptic contact (Fig. 5C and D). Immunoreactivity was found near the postsynaptic membrane in inhibitory synapses, as well (Fig. 5E).

DISCUSSION Specificity of alpha-1 adrenoceptor antibody In the present study, we examined the distribution of alpha-1 adrenoceptors in rat visual cortex using a commercially available antibody. Because no data were available on the specificity of this anti-alpha-1 adrenoceptor antibody for rat brain tissue, including the visual cortex, we first assessed specificity with immunoblot analysis and immunohistochemical analysis. In immunoblot analysis, the antialpha-1 adrenoceptor antibody labeled a single band with a molecular weight of 70 – 80 kDa, and a specific pattern of immunoreactivity for alpha-1 adrenoceptor was observed in immunohistochemical analysis. Shen et al. (2000) determined using immunoblot analysis that the distribution of alpha-1 adrenoceptor subtype proteins (alpha-1A, B, and D) varied in different tissues of neonatal and adult rats: However, the molecular weight of each alpha-1 adreno-

ceptor subtype protein was identical, at about 80 kDa. Earlier studies using purified and characterized alpha-1 adrenoceptor subtype proteins demonstrated that each alpha-1 adrenoceptor subtype protein had no difference in their molecular weights (Leeb-Lundberg et al., 1984; Venter et al., 1984; Lomasney et al., 1986; Sawutz et al., 1987; Graham et al., 1996). Although, the sequence of antigenic peptide was conserved in neither other subtype of alpha adrenoceptors nor other G-protein-coupled receptors, it is likely that the present anti-alpha-1 adrenoceptor antibody recognized all alpha-1A, B, and D adrenoceptor proteins in rat visual cortex. Determination by immunohistochemical analysis of detailed laminar and subcellular distribution of alpha-1 adrenoceptors Numerous studies employing the ligand-binding autoradiography technique for tissue sections developed by (Young and Kuhar, 1979a) reported a characteristic pattern of distribution of alpha-1 adrenoceptors in the CNS (Young and Kuhar, 1979b, 1980; Dashwood, 1983; Rainbow and Biegon, 1983; Jones et al., 1985; Palacios et al., 1987; Chamba et al., 1991). Three ligands were often used in these studies: 3H-WB4101, 3H-prazosin, and 125I-HEAT. In rat and mouse visual cortex, alpha-1 adrenoceptor binding sites were observed through all cortical layers, and strong signals were observed in deeper layers. On the other hand, one study found that the density of alpha-1 adrenoceptors was highest in layer I, with no prominent

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Fig. 5. Immunogold labeling of alpha-1 adrenoceptors in dendrites and synapses in rat visual cortex. These pictures were taken from counterstained sections. (A) The arrow points to immunoreactivity near the plasma membrane of a dendrite, and arrowheads point to immunoreactivity far from the plasma membrane. (B) Alpha-1 adrenoceptors are localized postsynaptically; the arrows and arrowhead point to alpha-1 adrenoceptors near and far from the post-synaptic density, respectively. Figures C and D show pictures of presynaptic alpha-1 adrenoceptor distribution. The arrow in C points to immunogold staining of alpha-1 adrenoceptors near the presynaptic plasma membrane. The arrow and arrowhead in D point to the immunogold staining of alpha-1 adrenoceptors near and far from the site of synaptic contact, respectively. (E) There is immunoreactivity near the post-synaptic membrane of the inhibitory synapse. T in figures B–E indicates the presynaptic terminal. Scale bars⫽200 nm.

differences in density among other layers (Schliebs and Godicke, 1988). The region of highest density of alpha-1 adrenoceptors corresponds well to the pattern of termination of NA fibers, with the highest density of such terminations in layer I of rat visual cortex (Levitt and Moore, 1978; Parnavelas et al., 1985). Autoradiographic studies are compromised by instability when multiple ligands are used, interfering with clear detection of the tissue distribution of receptor molecules. In addition, the low cellular resolution of the autoradiographic ligand binding technique does not permit accurate identification of ligand-bound neurons in the brain that express a specific alpha-1 adrenoceptor subtype. After the first cloning of an alpha-1 adrenoreceptor by Lefkowitz and collaborators (Cotecchia et al., 1988), it has become possible to utilize the in situ hybridization technique to examine the distribution of neurons in the brain that synthesize mRNA to these various adrenoceptors (Nicholas et al., 1991). In a study using the in situ hybridization technique, intense staining of mRNA of both alpha1A/D and alpha-1B adrenoceptors was found through layers II–VI of rat brain (Pieribone et al., 1994). However, the in situ hybridization technique reveals only the somata, in which the receptors are synthesized, and not the distribu-

tion of receptor protein in the dendritic and axonal ramifications. Thus, findings obtained with the two techniques are complementary and together provide important clues to the functional roles of receptors. In contrast, we studied alpha-1 adrenoceptors immunohistochemically and found intense immunoreactivity for alpha-1 adrenoceptors in layers I, II/III, and V. Moreover, immunolabeling for alpha-1 adrenoceptors examined at high cellular resolution revealed signals mainly in the dendrites and postsynaptic regions of excitatory, and to a lesser extent, inhibitory neurons. In cultured visuocortical neurons, localization of alpha-1 adrenoceptors was reported in the pyramidal celllike somata and dendrites using the fluorescently labeled alpha-1 adrenoceptor-selective antagonist BODIPY FL prazosin (Wang et al., 1997). Moreover, in that study, alpha-1 receptor binding sites were visualized as fluorescent “hot spots” on the somata and dendrites. These hot spots were described as circular or roughly oval and were fairly homogeneous in size, with most diameters varying from 0.5–1.5 ␮m. This range of spot diameters is similar to that of synapses described previously (Shkolnik-Yarros, 1971; Gabbott and Stewart, 1987; Harris and Stevens, 1989; Chicurel and Harris, 1992; Harris and Kater, 1994;

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Trommald and Hulleberg, 1997) and is much larger than the expected diameter of single receptors. In the present study, subcellular localization of alpha-1 adrenoceptors was observed in the neuronal perikarya, along dendritic membranes, and at postsynaptic sites. It is possible that localization of functional alpha-1 adrenoceptors revealed by fluorescently labeled antagonist corresponded to a part of receptors that identified in the synaptic membrane in the present experiments. Functional considerations Alpha-1 adrenoceptors are known to be involved in physiological responses to NA and in the formation of synapses during postnatal development. For example, a pharmacological study has shown that alpha-1 adrenoceptors play a major role in NA-induced intracellular Ca2⫹ responses in rat visual cortex (Kobayashi et al., 1999). In addition, after the destruction of terminals by treatment with the NA neurotoxin DSP-4, the intracellular Ca2⫹ response to NA via alpha-1 adrenoceptors was selectively reduced (Yamamoto et al., 2001). Although the present study revealed postsynaptic localization of alpha-1 adrenoceptors, the significant reduction of Ca2⫹ response to alpha-1 adrenoceptor activation following pharmacological elimination of adrenergic fibers suggests that a small fraction of presynaptic alpha-1 adrenoceptors still play an important role in increasing intracellular Ca2⫹ responses. In addition, activation of alpha-1 adrenoceptors in postsynaptic membrane coupled with that of phospholipase C via an inhibitory G protein, produces inositol 1,4,5triphosphate (IP3) (Minneman and Esbenshade, 1994). IP3 can in turn activate its specific receptor on the endoplasmic reticulum to induce the release of stored Ca2⫹ (Berridge, 1993). Thus, alpha-1 adrenoceptor activation by NA plays an important role in the rapid elevation of intracellular Ca2⫹ in both pre- and postsynaptic compartments. In addition to alpha-1 adrenoceptors, serotonin 5HT2 receptors are known to couple with phospholipase C via an inhibitory G protein (Roth and Chuang, 1987; Minneman and Esbenshade, 1994; Weng et al., 1994). Blockade of alpha-1 adrenoceptors and serotonin 5HT2 receptors was reported to prevent induction of LTP at inhibitory synapses in the rat visual cortex (Komatsu, 1996). The study by Komatsu (1996) also suggested that LTP induction required activation of postsynaptic GABAB receptors and that the effects of LTP induction were mediated by facilitation of induction of IP3 formation by alpha-1 adrenoceptor and 5HT2 receptors, which then caused rapid Ca2⫹ release from the internal stores in postsynaptic cells. A recent study showed that alpha-1 adrenoceptor also related the changes in the density of excitatory synapses in rat visual cortex (Nakadate et al., 2006). In that study, synaptic density was significantly altered by treatment with alpha-1 adrenoceptor antagonists or agonist. The alpha-1 adrenoceptor antagonists prazosin and HEAT dose-dependently reduced the density of synapses. Conversely, an alpha-1 adrenoceptor agonist, methoxamine, increased synaptic density. Methoxamine also competitively inhibited the effect of alpha-1 adrenoceptor antagonist on the density of synapses, and reduced the effects of a NA neuro-

toxin. These findings suggest that the facilitatory effects of the NAergic projection system on the formation of excitatory synapses and their maintenance are mediated primarily by an alpha-1 adrenoceptor subtype. This may be related to the fact that brain-derived neurotrophic factor is synthesized in NAergic neurons and anterogradely transported to the neocortex for modulation of neural circuitry (Fawcett et al., 1998).

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(Accepted 12 May 2006) (Available online 22 June 2006)