ARTICLE |
Correspondence to: Sujay K. Singh, Imgenex, 11494 Sorrento Valley Road, Suite B, San Diego, CA 92121.
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Summary |
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Serotonin (5-hydroxytryptamine, 5-HT) mediates many functions of the central and peripheral nervous systems by its interaction with specific neuronal and glial receptors. Fourteen serotonin receptors belonging to seven families have been identified through physiological, pharmacological, and molecular cloning studies. Monoclonal antibodies (MAbs) specific for each of these receptor subtypes are needed to characterize their expression, distribution, and function in embryonic, adult, and pathological tissues. In this article we report the development and characterization of MAbs specific to the serotonin 5-HT2A receptor. To generate MAbs against 5-HT2AR, mice were immunized with the N-terminal domain of the receptor. The antigens were produced as glutathionine S-transferase (GST) fusion proteins in insect cells using a Baculovirus expression system. The hybridomas were initially screened by ELISA against the GST5-HT2AR recombinant proteins and subsequently against GST control proteins to eliminate clones with unwanted reactivity. They were further tested by Western blotting against recombinant GST5-HT2AR, rat and human brain lysate, and lysate from cell lines transfected with 5-HT2AR cDNA. One of the MAbs G186-1117, which recognizes a portion of the 5-HT2AR N-terminus, was selected for further characterization. G186-1117 reacted with a band of molecular size 55 kD corresponding to the predicted size of 5-HT2AR in lysates from rat brain and a 5-HT2AR-transfected cell line. Its specificity was further confirmed by adsorption of immunoreactivity with recombinant 5-HT2AR but not with recombinant 5-HT2BR and 5-HT2CR. Rat brain sections and Schwann cell cultures were immunohistochemically labeled with this MAb. G186-1117 showed differential staining in various regions of the rat brain, varying from regions with no staining to regions of intense reactivity. In particular, staining of cell bodies and dendrites of the pyramidal neurons in the cortex was observed, which is in agreement with observations of electrophysiological studies. (J Histochem Cytochem 46:811824, 1998)
Key Words: serotonin 5-HT2A receptor, monoclonal antibody, human, rat, brain, immunohistochemistry, Western blotting
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Introduction |
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Serotonin (5-hydroxytryptamine, 5-HT), originally discovered as a blood serum factor, plays important roles in regulating diverse biological processes in the cardiovascular and gastrointestinal systems and in the central and peripheral nervous systems (
The 5-HT2 receptors (5-HT2Rs) are implicated in various normal physiological responses, e.g., smooth muscle contraction (
Localization of these receptors is generally done using radioligand binding followed by autoradiography (
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Materials and Methods |
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Generation and Purification of Recombinant GST Fusion Proteins as Immunogens
For the generation of immunogens, the cDNA fragments encoding the entire first extracellular domain in the N-terminus (amino acid residues 176 of 5-HT2AR, 156 of 5-HT2BR, and 155 of 5-HT2CR) were amplified from a human retina and human brain cDNA library by the polymerase chain reaction (PCR) using the THERMALASE Tbr Kit (Amresco; Solon, OH) and Biometra Trio-Thermoblock (Biometra; Tampa, FL). The forward primers (5-HT2AR, 5'-ccgggatccatggatattctttgtgaagaaaatact-3'; 5-HT2BR, 5'-cgcggatccaaaatggctctctcttacagagtgtctg-3'; and 5-HT2CR, 5'-cgcggatccaaaatggtgaacctgaggaatgcggtgc-3') and reverse primers (5-HT2AR, 5'-cagcccgggtcaccagtttttttcctggagatgaag-3'; 5-HT2BR, 5'-ccggaattctgcccagtgcagtttatttcc-3'; and 5-HT2CR, 5'-ccggaattcccagttttgtaccccgtctgg-3') were chosen from published human 5-HT2 receptor cDNA sequences (
The purification of GST fusion proteins was carried out according to published protocols (
Development of Monoclonal Antibodies to 5-HT2AR
Balb/c mice were immunized four times with 50 µg of GST5-HT2AR fusion protein at 1-week intervals. Four days after the last immunization, the mice were sacrificed and the splenocytes were fused with HGPRT(-) F0 myeloma cells (
Western Blot Analysis
Rat brain synaptic membrane was a kind gift of Dr. Doug Vetter (Salk Institute; La Jolla, CA). Human brain lysate (a brain medley from a 68-year-old white male who had died of cardiac arrest) was purchased from Clontech Laboratories (Palo Alto, CA). Lysate from NIH-3T3 cell stably transfected with rat 5-HT2AR (
Absorption of Antibodies with Recombinant 5-HT2R Proteins
To determine the specificity of the MAbs, the antibodies were preadsorbed with the recombinant 5-HT2R proteins. Briefly, 200 µg of either GST5-HT2AR fusion protein or negative control GST5-HT2BR or GST5-HT2CR fusion protein was incubated with 100 µg of MAb and 200 µl of glutathioneagarose beads for 2 hr at 4C. The beads were pelleted at 1000 x g for 3 min. The supernatant containing adsorbed or unadsorbed antibodies was tested by Western blotting and immunocytochemical analysis.
Immunostaining of Cultured Schwann Cells
Schwann cells were isolated from neonatal SpragueDawley rat sciatic nerves and purified by depletion of fibroblasts using complement protein, according to a modified protocol (
Immunohistochemical Staining of Rat Brain Sections
Adult SpragueDawley rats were anesthetized with sodium pentobarbital and perfused transcardially with 50 ml PBS and 250 ml 2% PFA plus 0.2% parabenzoquinone in 0.075 M phosphate buffer. The rat brain tissues were removed, postfixed for 2 hr in the same fixation buffer, and then cryoprotected in 30% sucrose in 0.1 M phosphate buffer overnight. Forty-µm sections were cut with a sliding microtome and stored in PBS until required for immunohistochemistry. Free-floating sections were washed for 10 min in Tris-buffered saline (TBS), incubated for 20 min in TBS containing 0.25% Triton X-100, and blocked for 60 min in TBS + 2% BSA. Staining was performed by incubating sections in 2 µg/ml anti-5-HT2A receptor antibody, G186-1117, in TBS + 0.25% Triton X-100 + 2% BSA for 40 hr at 4C. An IgG1 isotype-matched negative control antibody (PharMingen) was also used. After washing unbound antibodies with TBS + 0.25% Triton X-100, the sections were incubated with 1.5 µg/ml biotinylated donkey anti-mouse antibodies (Jackson ImmunoResearch Labs) in TBS + 0.25% Triton X-100 x 2% BSA for 2 hr at RT. The sections were then washed and incubated with an avidinbiotinperoxidase reagent (1:200 dilution; DAKO, Carpinteria, CA) for 60 min at RT. After washing, the sections were incubated with freshly prepared diaminobenzidine (DAB) solution (DAB kit; Vector Labs, Burlingame, CA) for 5 min. DAB-stained sections were rinsed three times in PBS and mounted on microscope slides, dehydrated in four changes of 100% alcohol, cleared with four changes of xylene, and mounted with a coverslip.
Immunofluorescence Staining for Confocal Microscopy of Rat Brain Sections
Adult rat brain sections were perfused and cut as described above. G186-1117, an antibody that was directly conjugated with FITC (Molecular Probes) was used for the staining. After several washes and blocking with 2% BSA in TBS + 0.25% Triton X-100, the sections were incubated with 2 µg/ml of the FITCG186-1117 antibodies for 40 hr at 4C. The sections were then washed extensively in TBS + 0.25% Triton X-100 and mounted in a glycerol/PBS mounting medium. A confocal laser scanning microscope was used to examine the staining, utilizing an immersion lens (x40) and a numerical aperture. For each tissue, focal series of 24 sections each, 0.5 mm apart, were scanned by the confocal laser microscope. The images were reconstructed and modified in Adobe Photoshop software (Adobe Systems; San Jose, CA) and printed on Fujifilm Pictro paper.
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Results |
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Cloning and Expression of Recombinant Proteins
The cDNA fragments corresponding to the N-terminal extracellular domain of 5-HT2A, 5-HT2B, and 5-HT2C receptors were amplified by PCR from a human retina (5-HT2AR) or brain cDNA library (5-HT2BR and 5-HT2CR) and cloned into pAcG2T Baculovirus expression vector. The authenticity of the PCR products was confirmed by nucleotide sequencing (using Sequenase Version 2.0 DNA Sequencing Kit; USB, Cleveland, OH). They were also confirmed to be in frame with the GST coding sequence. The fusion proteins from the insect cell lysate were purified using glutathioneSepharose beads. The purified proteins were analyzed on SDS-PAGE followed by Coomassie Blue staining to confirm the correct molecular size. These proteins were used to immunize mice, to screen the hybridomas, and to test the specificity of anti-5-HT2AR antibodies by Western blot analysis.
Generation of Hybridomas and Selection of Monoclonal Antibodies
One thousand hybridoma culture supernatants were tested for antibodies to recombinant 5-HT2AR fusion protein (GST5-HT2AR) by ELISA. Twenty of 200 ELISA-positive hybridomas were further tested by Western blotting using recombinant GST5-HT2AR protein. Seven of these hybridoma wells gave strong staining with the immunogen. These hybridomas were cloned by limiting dilution and subsequently subcloned to select stable antibody-secreting clones. Four clones that reacted with the N-terminal extracellular portion of the 5-HT2A receptor were selected for further characterization. On the basis of the specificity and intensity of reaction by immunohistochemistry and Western blotting, only one of these four clones, G186-1117, was selected for further characterization.
Characterization of the Specificity of MAb G186-1117 by Western Blot Analyses
We have tested the crossreactivity of the G186-1117 antibody against recombinant 5-HT2B and 5-HT2C receptor proteins. The 5-HT2AR shares approximately 27% sequence homology with the N-terminal extracellular domain of the 5-HT2B and 5-HT2C receptors. To test the crossreactivity of G186-1117 with 5-HT2BR and 5-HT2CR, equal amounts of recombinant GST5-HT2AR, GST5-HT2BR, and GST5-HT2CR were separated on SDS-PAGE gels and analyzed by Western blot. As shown in Figure 1, Figure G186-1117 reacted only with recombinant 5-HT2AR and not with 5-HT2BR and 5-HT2CR.
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Specificity of G186-1117 to the full-length 5-HT2A receptor was also tested by Western blot analysis using protein preparations from rat brain, human brain, and a 5-HT2AR cDNA-transfected cell line. G186-1117 detected an immunoreactive band of about 55 kD, the predicted size, in rat brain lysate (Figure 2A). A smear of immunoreactive proteins of sizes ranging from 40 to 55 kD was observed in human brain lysate (Figure 2B). G186-1117 detected an immunoreactive band of about 55 kD in lysates from NIH-3T3 cells that were transfected with 5-HT2AR cDNA (Figure 2C). GST5-HT2AR recombinant protein entirely depleted the immunoreactivity of G186-1117 against the positive bands in the purified rat brain synaptic membrane protein preparation, in the human brain lysate as well as in the 5-HT2AR-transfected cell lysates, whereas recombinant GST5-HT2BR and GST5-HT2CR failed to adsorb anti-5-HT2AR activity (Figure 2AC).
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Immunocytochemical Staining of Cultured Schwann Cells
Schwann cells that had been isolated from neonatal SpragueDawley rat sciatic nerves (
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Immunohistochemical Staining of Adult Rat Brain Sections with MAb G186-1117
Immunostaining observed with this antibody appeared to be specific (Figure 4B) and could not be reproduced when similar sections were processed with an isotype control (Figure 4A) or with G186-1117 preadsorbed with recombinant protein GST5-HT2AR (data not shown). The immunoreactivity of G186-1117 could not be blocked by the GST5-HT2BR and GST5-HT2CR recombinant proteins (data not shown). An immunohistochemical evaluation was carried out on brain sections from four different animals using G186-1117. Our analysis was not intended to serve as a thorough description of the immunohistochemical labeling seen with this antibody throughout the entire CNS but was aimed at comparing our data with the previously published data of in situ hybridization (
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In the main olfactory bulb, intense 5-HT2AR immunoreactivity was observed in cells in the mitral cell layer and in fibers in the external plexiform layer and glomerular layer (Figure 4D). Moderate to strong staining was also seen in most cortical regions. Cortical labeling appeared to be primarily associated with neurons in Layer V and with their proximal dendrites that ramified in cortical Layers II and III (Figure 4C). Staining was almost absent from cortical Layer I and was much less intense in Layers IV and VI. Robust 5-HT2AR immunoreactivity was noted in the piriform cortex (Figure 4E), olfactory tubercle, and ventral pallidum (Figure 4F), but was absent from the claustrum and endopiriform nucleus (Figure 4E). In agreement with previous in situ hybridization studies, staining for the 5-HT2AR was also undetectable throughout the septum (data not shown). Within the hippocampal formation, moderate 5-HT2AR immunostaining was seen in CA1 pyramidal cells and their dendrites in stratum raditum (Figure 4H) and in CA3 pyramidal cells and their dendrites in stratum lucidum (Figure 4G). Some staining was also noted in the dentate gyrus, just above the granule cell layer, although it was not possible to specifically determine the cell structure that stained.
In the medial mammillary nucleus, where intense mRNA for the 5-HT2AR has been reported, very little immunostaining was observed (Figure 5A). Similarly, in the pontine nuclei, where high levels of mRNA for the 5-HT2AR have been found, 5-HT2AR immunoreactivity was absent (Figure 5B). We cannot explain the discrepancy between the 5-HT2AR mRNA and the protein level in the mammilary nucleus. However, the absence of 5-HT2AR immunoreactivity in the pontine nuclei is in agreement with radioligand binding data (
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Immunofluorescence Staining of 5-HT2A Receptors on the Dendrites of Pyramidal Cells with MAb G186-1117
A typical subcellular localization of 5-HT2A receptor with G186-1117 was seen in the dendrites of pyramidal cells in the frontal cortex of rat brain (Figure 6AC). A series of optical sections through the apical dendrites of several neurons showed that most of the immunofluorescence staining was associated with the plasma membrane but some with cytoplasm. Optical sections through the dendrites showed that the staining of the dendrites was on the surface (Figure 6D). Therefore, staining appeared as a hollow tube (Figure 6E and Figure 6F and Figure 6GI, arrows). This type of plasma membrane staining was also shown in fusing myoblasts by
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Discussion |
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A large number of studies have been published on the distribution and localization of the 5-HT receptor by using radioligand binding followed by autoradiography, in situ hybridization, or electrophysiology (
We chose to use the N-terminal domain of the 5-HT2A receptor as immunogen because at this region, 5-HT2A, 5-HT2B, and 5-HT2C receptors have low homology (less than 30%) at the amino acid sequence level. In addition, there is 75% homology among human, rat, and mouse 5-HT2A receptors at this same region. This approach had a greater chance of generating 5-HT2AR-specific MAbs that would crossreact with human, rat, and mouse 5-HT2A receptors.
The specificity of G186-1117 to 5-HT2AR was examined by Western blot analysis. G186-1117 reacted with GST5-HT2AR but not with GST5-HT2BR or 5-HT2CR (Figure 1). The size of the band on Western blots of rat brain lysate (Figure 2A) and lysates from the NIH-3T3 cells transfected with 5-HT2AR cDNA (Figure 2C) was about 55 kD, which is similar to the value of 56 kD that was detected by the anti-idiotypic polyclonal antibody (
The specificity of G186-1117 was further examined by immunocytochemistry on cells expressing transfected and endogenous 5-HT2A receptors. Cultured rat Schwann cells that naturally express functional 5-HT2A receptors were also labeled with G186-1117 (Figure 3). These cells had previously been shown to label with an anti-idiotypic antibody (TH8) that recognizes 5-HT1B, 5-HT2A, and 5-HT2C receptors (
G186-1117 was found to stain brain tissues that were processed by different preservation protocols including frozen tissue, paraffin-embedded tissue, and perfused tissue (data not shown). For the perfused tissue, we have also used two different fixation buffers, including 2% paraformaldehyde plus 0.2% parabenzoquinone in 0.075 M phosphate buffer and 4% paraformaldehyde alone in 0.075 M phosphate buffer. These fixation protocols gave different staining patterns with different tissue preservation. In the present study, fixation with 2% paraformaldehyde plus 0.2% parabenzoquinone in 0.075 M phosphate buffer by perfusion gave the best results, which are shown in Figure 4 and Figure 5. Although the distribution of 5-HT2AR protein that we observed was largely in agreement with the distribution of mRNA reported in previous in situ hybridization studies (
The subcellular localization of 5-HT2AR with G186- 1117 suggests that most of the labeling is associated with the plasma membrane (Figure 6). In the present study, rat brain sections were stained for G186-1117 and images were analyzed by confocal laser scanning microscopy. These staining results are consistent with observations made by
In conclusion, we have developed and extensively characterized monoclonal antibodies to the 5-HT2A receptor. We have selected one MAb, G186-1117, that is specific for the 5-HT2A receptor, as defined by its immunoreactivity pattern in various tissues, its reactivity with a protein of expected molecular size, and by selective adsorption using recombinant 5-HT2AR but not 5-HT2BR and 5-HT2CR proteins. We anticipate that G186-1117 will be a useful tool for studying the anatomic distribution, regulation, and function of the 5-HT2A receptor.
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Acknowledgments |
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Supported in part by an SBIR grant from NIMH (Grant #1R43MH54437-1 to SKS) and grants from the NIH: NS14718, NS24635, RR04050 (to MHE), and an Overseas Research Student Award from the CVCP, UK (#ORS/96027038 to CW). SK is supported by the Wellcome Trust, UK.
We thank Dr Chun-Ming Huang for his support of this project. We are grateful to Dr Maryann E. Martone for valuble assistance with the confocal laser microscopy and for her critical reading of the manuscript. We thank Dr L. Descarries and Virginia CorneaHebert for their helpful discussions. We would also like to thank Ling-Pu Dong for his help in rat brain section preparation.
Received for publication October 28, 1997; accepted February 11, 1998.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abramowski D, Rigo M, Duc D, Hoyer D, Staufenbiel M (1995) Localization of the 5-hydroxytryptamine 2C receptor protein in human and rat brain using specific antisera. Neuropharmacology 34:1635-1645[Medline]
Andrade R, Nicoll RA (1987) Pharmacologically distinct actions of serotonin on single pyramidal neurones of the rat hippocampus recorded in vitro. J Physiol 394:99-124[Abstract]
Araneda R, Andrade R (1991) 5-hydroxytryptamine 2 and 5-hydroxytryptamine 1A receptors mediate opposing responses on membrane excitability in rat association cortex. Neuroscience 40:399-3412[Medline]
Azmitia EC, Yu I, Akbari HM, Kheck N, WhitakerAzmitia PM, Mrshak DR (1992) Antipeptide antibodies against the 5-HT1A receptor. J Chem Neuroanat 5:289-298[Medline]
Backstrom JR, Westphal RS, Canton H, SandersBush E (1995) Identification of rat serotonin 5-HT2C receptors as glycoproteins containing N-linked oligosaccharides. Brain Res Mol Brain Res 33:311-318[Medline]
Brockes JP, Fields KL, Raff MC (1979) Studies on cultured rat Schwann cells I. Establishment of purified populations from cultures of peripheral nerve. Biochem Pharmacol 165:105-118
Burnet PWJ, Eastwood SL, Lacey K, Harrison PJ (1995) The distribution of 5-HT1A and 5-HT2A receptor mRNA in human brain. Brain Res 676:157-168[Medline]
Choi DS, Maroteaux L (1996) Immunohistochemical localisation of the serotonin 5-HT2B receptor in mouse gut cardiovascular system and brain. FEBS Lett 391:45-51[Medline]
Choi DS, Ward SJ, Messaddeq N, Launay JM, Maroteaux L (1997) 5-HT2B receptor-mediated serotonin morphogenetic functions in mouse cranial neural crest and myocardiac cells. Development 124:1745-1755
Cohen ML, Wittenauer LA (1987) Serotonin receptor activation of phosphoinositide turnover in uterine fundal vascular and tracheal smooth muscle. J Cardiovasc Pharmacol 10:176-181[Medline]
el Mestikawy S, Riad M, Laporte AM, Verge D, Daval G, Gozlan H, Hamon M (1990) Production of specific anti-rat 5-HT1A receptor antibodies in rabbits injected with a synthetic peptide. Neurosci Lett 118:189-192[Medline]
Foguet M, Hoyer D, Pardo A, Parekh A, Kluxen FW, Kalkman HO, Stühmer W, Lübbert H (1992) Cloning and functional characterization of the rat stomach fundus serotonin receptor. EMBO J 11:3481-3487[Abstract]
Galfre G, Howe SC, Milstein C, Butcher GW, Howard JC (1977) Antibodies to major histocompatibility antigens produced by hybrid cell line. Nature 266:550-552[Medline]
Garlow SJ, Morilak DA, Dean R, Roth BL, Ciaranello RD (1993) Production and characterization of a specific 5-HT2 receptor antibody. Brain Res 615:113-120[Medline]
Gerard C, Langlois X, Gingrich J, Doucet E, Verge D, Kia HK, Raisman R, Gozlan H, el Mestikawy S, Hamon M (1994) Production and characterization of polyclonal antibodies recognizing the intracytoplasmic third loop of the 5-hydroxytryptamine 1A receptor. Neuroscience 62:721-739[Medline]
Gerard C, Martres MP, Lefevre K, Miquel MC, Verge D, Lanfumey L, Doucet E, Hamon M, el Mestikawy S (1997) Immunolocalization of serotonin 5-HT6 receptor-like material in the rat central nervous system. Brain Res 746:207-219[Medline]
Gerhardt CC, van Heerikhuizen H (1997) Functional charateristics of heterologously expressed 5-HT receptors. Eur J Pharmacol 334:1-23[Medline]
Gould RM, Jessen KR, Mirsky R, Tennekoon G (1992) The cell of Schwann: an update. In Martenson RE, ed. Myelin: Biology and Chemistry. Boca Raton, FL, CRC Press, 123-171
Grimaldi B, CloezTayarani I, Fillion MP, Mazie JC, Hen R, Fillion G (1995) Production and characterization of an antibody directed against the mouse 5-HT1B receptor. Neurosci Res 24:97-101[Medline]
GuilletDeniau I, Burnol AF, Girard J (1997) Identification and localization of a skeletal muscle serotonin 5-HT2A receptor coupled to Jak/STAT pathway. J Biol Chem 272:14825-14829
Hillion J, Catelon J, Raid M, Hamon M, De Vitry F (1994) Neuronal localization of 5-HT1A receptor mRNA and protein in rat embryonic brain stem cultures. Brain Res Dev Brain Res 79:195-202[Medline]
Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, Saxena PR, Humphrey PPA (1994) VII International Union of Pharmacology classification for 5-hydroxytryptamine (serotonin). Phamacol Rev 46:157-203
Hoyer D, Martin G (1997) 5-HT receptor clasification and nomenclature: towards a harmonization with human genome. Neuropharmacology 36:419-428[Medline]
Humphrey PPA, Hartig P, Hoyer D (1993) A proposed new nomenclature for 5-HT receptors. Trends Pharmacol Sci 6:233-236
Julius D, Huang KN, Livelli TJ, Axel R, Jessell TM (1990) The 5-HT2 receptor defines a family of structurally distinct but functionally conserved serotonin receptors. Proc Natl Acad Sci USA 87:928-932[Abstract]
Kalkman HO, Fozard JR (1991) 5-HT receptor types and their role in disease. Curr Opin Neurolol Neurosurg 4:560-565
Kharazia VN, Wenthold RJ, Weinberg RJ (1996) GluR1-immunopositive interneurons in rat neocortex. J Comp Neurol 368:399-412[Medline]
Kia HK, Brisorgueil MJ, Hamon M, Calas A, Verge D (1996a) Ultrastructural localization of 5-hydroxytryptamine 1A receptors in the rat brain. J Neurosci Res 46:697-708[Medline]
Kia HK, Miquel MC, Brisorgueil MJ, Daval G, Riad M, el Mestikawy S, Hamon M, Verge D (1996b) Immunocytochemical localization of serotonin 1A receptors in the rat central nervous system. J Comp Neurol 365:289-305[Medline]
Kia HK, Miquel MC, McKernan RM, Laporte AM, Lombard MC, Bourgoin S, Hamon M, Verge D (1995) Localization of 5-HT3 receptors in the rat spinal cord: immunohistochemistry and in situ hybridization. Neuroreport 6:257-261[Medline]
Kursar ND, Nelson DL, Wainscott DB, Cohen ML, Betz M (1992) Molecular cloning functional expression and pharmacological characterization of a novel serotonin receptor (5-hydroxytryptamine 2F) from rat stomach fundus. Mol Pharmacol 42:549-557[Abstract]
Langlois X, Gerard C, Darmon M, Chauveau J, Hamon M, el Mestikawy S (1995) Immunolabeling of central serotonin 5-HT1D-beta receptors in the rat mouse and guinea pig with a specific anti-peptide antiserum. J Neurochem 65:2671-2681[Medline]
Laubeya MK, Maloteaux JM, De Roe C, Trouet A, Laduron PM (1986) Different subcellular localization of muscarinic and serotonin (S2) receptors in human, dog, and rat brain. J Neurochem 46:405-412[Medline]
Lauder JM (1990) Ontogeny of the serotogenic system in the rat: serotonin as a developmental signal. Ann NY Acad Sci 600:297-313[Abstract]
Lauder JM (1993) Neurotransmitters as growth regulatory signals: role of receptors and second messengers. Trends Neurosci 16:233-240[Medline]
Launay JM, Birraux G, Bondoux D, Callert J, Choi DS, Loric S, Maroteaux L (1997) Ras involvment in signal transduction by the serotonin 5-HT2B receptor. J Biol Chem 271:3141-3147
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275
Maura D, Roccatagliata E, Ulivi M, Raiteri M (1988) Serotonin-glutamate interaction in rat cerebellum: involvement of 5-HT1 and 5-HT2 receptors. Eur J Pharmacol 145:31-38[Medline]
Matsouka H, Ishii M, Goto A, Sugimoto T (1985) Role of serotonin type 2 receptors in regulation of aldosterone production. Am J Physiol 234:234-238
Matthiessen L, Kia HK, Daval G, Riad M, Hamon M, Verge D (1993) Immunocytochemical localization of 5-HT1A receptors in the rat immature cerebellum. Neuroreport 4:763-766[Medline]
McBride PA, Mann JJ, Polley MJ, Wiley AJ, Sweeny JA (1987) Assessment of binding indices and physiological responsivenes of the 5-HT2 receptor on human platelets. Life Sci 40:1799-1809[Medline]
Mengod G, Pompeiano M, MartinezMir I, Palacios JM (1990) Localization of the mRNA for the 5-HT2 receptor by in situ hybridization histochemistry. Correlation with the distribution of receptor. Brain Res 524:139-143[Medline]
Miquel C, Doucet E, Riad M, Adrien J, Verge D, Hamon M (1992) Effect of the selective lesion of serotoninergic neurons on the regional distribution of 5-HT1A receptor mRNA in the rat brain. Brain Res Mol Brain Res 14:357-362[Medline]
Mirsky R, Jessen KR (1990) Schwann cell development and regulation of myelination. Semin Neurosci 2:423-435
Molnár E, Baude A, Richmond SA, Patel PB, Somogyi P, McIlhinney RAJ (1993) Biochemical and immunocytochemical characterization of antipeptide antibodies to a cloned GluR1 glutamate receptor subunit: cellular and subcellular distribution in the rat forebrain. Neuroscience 53:307-326[Medline]
Morales M, Battenberg E, de Lecea L, Sanna PP, Bloom FE (1996) Cellular and subcellular immunolocalization of the type 3 serotonin receptor in the rat central nervous system. Brain Res Mol Brain Res 36:251-260[Medline]
Morilak DA, Garlow SJ, Ciaranello RD (1993) Immunocytochemical localization and description of neurons expressing serotonin 2 receptors in the rat brain. Neuroscience 54:701-717[Medline]
Morilak DA, Somogyi P, LujanMiras R, Ciaraneloo D (1994) Neurons expressing 5-HT2 receptors in the rat brain: neurochemical identification of cell types by immunocytochemistry. Neuropsychopharmacology 11:157-166[Medline]
Mukerji J, Haghighi A, Seguela P (1996) Immunological characterization and transmembrane topology of 5-hydroxytryptamine 3 receptors by functional epitope tagging. J Neurochem 66:1027-1032[Medline]
Nemecek GM, Coughlin SR, Handley DA, Moskowitz MA (1978) Stimulation of aortic smooth muscle cell mitogenesis by serotonin. Proc Natl Acad Sci USA 83:674-678
Osborne NN (1982) Assay distribution and functions of serotonin in nervous tissues. In Osborne NN, ed. Biology of Serotonergic Transmission. Chichester, Wiley, 7-27
Patton JG, Alley MC, Mao SJ (1982) Evaluation of monoclonal antibodies to human plasma low density lipoproteins. A requirement for lipids to maintain antigenic structure. J Immunol Methods 55:193-203[Medline]
Pazos A, Cortes R, Palacios JM (1985) Quantitative autoradiographic mapping of serotonin receptors in rat brain II Serotonin-2 receptors. Brain Res 346:231-249[Medline]
Peroutka SJ (1988) 5-Hydroxytryptamine receptor subtypes. Annu Rev Neurosci 11:45-60[Medline]
Petralia RS, Wang YX, Singh S, Wu C, Shi LR, Wei J, Wenthold R (1997) A monoclonal antibody shows discrete cellular and subcellular localizations of mGluR1a metabotropic glutamate receptors. J Chem Neuroanat 13:79-93
Pomperiano M, Palacios JM, Mengod G (1994) Distribution of serotonin 5-HT2 receptor family mRNAs: comparison between 5-HT2A and 5-HT2C receptors. Mol Brain Res 23:163-178[Medline]
Raymond JR, Kim J, Beach RE, Tisher CC (1993) Immunohistochemical mapping of cellular and subcellular distribution of 5-HT1A receptors in rat and human kidneys. Am J Physiol 264:F9-19
Ridet JL, Tamir H, Privat A (1994) Direct immunocytochemical localization of 5-hydroxytryptamine receptors in the adult rat spinal cord: a light and electron microscopic study using an anti-idiotypic antiserum. J Neurosci Res 38:109-121[Medline]
Roth BL, Palvimaki EP, Berry S, Kahn N, Sachs N, Uler A, Choudhary MS (1995) 5-hydroxytryptamine 2A (5-HT2A) receptor desensitization can occur without down-regulation. J Pharmacol Exp Ther 275:1638-1646[Abstract]
Saltzman AG, Morse B, Whitman MM, Ivanshchenko Y, Jaye M, Felder S (1991) Cloning of the human serotonin 5-HT2 and 5-HT1C receptor subtypes. Biochem Biophys Res Commun 181:1469-1478[Medline]
Saxena PR (1995) Serotonin receptors: subtypes functional responses and therapeutic activity. Pharmacol Ther 66:339-368[Medline]
Schmuck K, Ullmer C, Engels P, Lubbert H (1994) Cloning and functional characterization of the human 5-HT2B serotonin receptor. FEBS Lett 342:85-90[Medline]
Shih JC, Chen K (1990) Molecular studies of 5-HT receptors. Ann NY Acad Sci 600:206-211[Medline]
Smith DB, Johnson KS (1988) Single-step purification of purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31-40[Medline]
Sugihara I, Lang EJ, Llinas R (1995) Serotonin modulation of inferior olivary oscillations and synchronicity: a multiple-electrode study in the rat cerebellum. Eur J Neurosci 7:521-534[Medline]
Sundaram H, Strange PG (1994) Characterisation of the human brain serotonin 5-HT1A receptor expressed in Chinese hamster ovary cells. Biochem Soc Trans 22:75S[Medline]
Tamir H, Liu K-P, Hsiung S-C, Yu P, Kirchgessner AL, Gershon MD (1991) Identification of serotonin receptors recognized by anti-idiotypic antibodies. J Neurochem 57:930-942[Medline]
Verdot L, Bertin B, Guilloteau D, Strosberg AD, Hoebke J (1995) Characterization and pharmacologically active anti-peptide antibodies directed against the first and second extracellular loops of the serotonin 5-HT1A receptor. J Neurochem 65:319-328[Medline]
Verdot L, Ferrer-di-Martino M, Bertin B, Strosberg AD, Hoebeke J (1994) Production of anti-peptide antibodies directed against the first and the second extracellular loop of the human serotonin 5-HT1A receptor. Biochimie 76:165-170[Medline]
Wang CD, Gallaher TK, Shih JC (1993) Site-directed mutagenesis of the serotonin 5-hydroxytrypamine 2 receptor: identification of amino acids necessary for ligand binding and receptor activation. Mol Pharmacol 43:931-940[Abstract]
WhitakerAzmitia PM (1991) Role of serotonin and other neurotransmitter receptors in brain development: basis for developmental pharmacology. Pharmacol Rev 43:553-561[Medline]
Willins DL, Deutch AY, Roth BL (1997) Serotonin 5-HT2A receptors are expressed on pyramidal cells and interneurons in the rat cortex. Synapse 27:70-82
Wright DE, Seroogy KB, Lundgren KH, Davis BM, Jennes L (1995) Comparative localization of serotonin 1A, 1C, and 2 receptor subtype mRNAs in rat brain. J Comp Neurol 351:357-373[Medline]
Yoder EJ, Lee B, Ellisman MH (1997a) The expression of serotonin receptors by cultured rat Schwann cells is a function of their differentiation: correlation with a quiescent myelinating phenotype. Mol Cell Neurosci 8:303-310[Medline]
Yoder EJ, Tamir H, Ellisman MH (1996) 5-hydroxytryptamine 2A receptors on cultured rat Schwann cells. Glia 17:15-27[Medline]
Yoder EJ, Tamir H, Ellisman MH (1997b) Serotonin receptors expressed by myelinating Schwann cells in rat sciatic nerve. Brain Res 753:299-308[Medline]