Characterization of 5‐HT1A,B and 5‐HT2A,C Serotonin Receptor Binding

William P. Clarke1, Kelly A. Berg1, Georgianna Gould1, Alan Frazer2

1 The University of Texas Health Science Center, San Antonio, Texas, 2 The Audie L. Murphy Memorial Hospital Division, University of Texas Health Science Center, San Antonio, Texas
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.23
DOI:  10.1002/0471141755.ph0123s12
Online Posting Date:  May, 2001
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Abstract

This unit describes assays for measuring the binding of radioligands to two major types of receptors for 5‐hydroxytryptamine (5‐HT or serotonin), 5‐HT1 and 5‐HT2 receptors, in homogenates of brain tissue or cloned into cells in culture. The specific receptor subtypes covered are 5‐HT1A, 5‐HT1B, 5‐HT2A, and 5‐HT2C. In addition, methodology for using quantitative autoradiography to measure radioligand binding to serotonin receptors in brain slices is described. Protocols are provided for characterization of both saturation and competition binding assays, and instructions for data analysis of these assays is also described. In addition, methodology is provided for the quantification (image analysis) of radioligand binding in brain tissue sections to determine receptor density, preparation of rat brain sections for quantitative autoradiography, and thionin staining of thaw‐mounted tissue sections to define certain brain regions.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Binding Properties to Cloned 5‐HT2A and 5‐HT2C Receptors Expressed in Cells—Saturation Binding
  • Basic Protocol 2: Measurement of Ligand Affinity to Cloned 5‐HT2A AND 5‐HT2C Receptors Expressed in Cells—Competition Binding
  • Support Protocol 1: Data Analysis for Saturation and Competition Assays
  • Basic Protocol 3: Measurement of 5‐HT1A Receptor Binding in Tissue Membrane Homogenates
  • Basic Protocol 4: Quantitative Autoradiography of 5‐HT1A Binding to Rat Brain
  • Support Protocol 2: Preparation of Brain Tissue Sections for Quantitative Autoradiography
  • Support Protocol 3: Quantification of Radioligand Binding in Brain Tissue Sections by Image Analysis
  • Basic Protocol 5: Quantitative Autoradiography of 5‐HT1B Binding to Rat Brain
  • Support Protocol 4: Thionin Staining of Thaw Mounted Tissue Sections
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Binding Properties to Cloned 5‐HT2A and 5‐HT2C Receptors Expressed in Cells—Saturation Binding

  Materials
  • CHO cell line expressing 5‐HT 2A or 5‐HT 2C serotonin receptors in 15‐cm plates
  • Hanks Basic Salt Solution (HBSS; Life Technologies), ice‐cold
  • HEPES homogenization buffer, pH 7.4 (see recipe), 37° and 4°C
  • HEPES assay buffer, pH 7.4, 4°C
  • 60 to 90 Ci/mmol [3H]ketanserin (5‐HT 2A receptors; NEN Life Sciences) or
  • 70 to 85 Ci/mmol [3H]mesulergine (5‐HT 2C receptors; Amersham)
  • Masking ligand: methysergide (5‐HT 2A receptors) or mianserin (5‐HT 2C receptors)
  • 0.5% (v/v) polyethyleneimine (Sigma)
  • Scintillation fluid (e.g., Beckman Ready‐Solv)
  • Rubber cell scraper (Gilco)
  • 1‐ml disposable transfer pipets (Fisher)
  • 50‐ml round plastic centrifuge tubes (Nalgene)
  • 2‐ml cryotube
  • Polytron homogenizer (Brinkmann Instruments)
  • Refrigerated Sorvall RC‐5 centrifuge and SA‐600 rotor
  • Potter‐Elvehjem glass‐Teflon homogenizer (VWR) or equivalent, prechilled
  • 7‐ml scintillation vials (Fisher)
  • GF/C filter strips (Whatman)
  • 24‐channel cell harvester (Brandel)
  • Scintillation counter (Beckman)
  • Additional equipment and reagents for Lowry protein assay ( appendix 3A).

Basic Protocol 2: Measurement of Ligand Affinity to Cloned 5‐HT2A AND 5‐HT2C Receptors Expressed in Cells—Competition Binding

  Materials
  • Test (competitor) compounds
  • HEPES assay buffer, pH 7.4 at 4°C (see recipe)
  • 60 to 90 Ci/mmol [3H]ketanserin (NEN Life Sciences; for 5‐HT 2A receptors) or
  • 70 to 85 Ci/mmol [3H]mesulergine (Amersham; for 5‐HT 2C receptors)
  • Competitor ligand
  • Additional reagents and equipment for preparing membranes (see protocol 1).

Support Protocol 1: Data Analysis for Saturation and Competition Assays

  Materials
  • Spreadsheet program (e.g., Microsoft Excel)
  • Graphing program (e.g., GraphPad Prism, KaleidaGraph)

Basic Protocol 3: Measurement of 5‐HT1A Receptor Binding in Tissue Membrane Homogenates

  Materials
  • 5‐HT 1A homogenate wash buffer, ice‐cold (see recipe)
  • 225‐ to 300‐g male Sprague‐Dawley rat (Harlan)
  • 222 to 225 Ci/mmol [3H]8‐OH‐DPAT (Amersham Pharmacia Biotech)
  • 5‐HT 1A homogenate assay buffer (see recipe)
  • 10 mM 5‐HT in 0.02% ascorbic acid (see recipe)
  • Ecolume scintillation fluid (Fisher)
  • Polytron homogenizer (Brinkmann), chilled
  • 50‐ml round plastic centrifuge tubes (Nalgene)
  • Sorvall RC‐5 centrifuge and SS‐34 rotor
  • Glass homogenizer and pestle (e.g., Potter‐Elvehjem, VWR)
  • Scintillation vials (e.g., Fisher)
  • Scintillation counter (e.g., Packard Instruments)
  • 12 × 75–mm glass culture tubes (e.g., VWR)
  • Brandel cell harvester
  • 2 × 12–in. #25 glass fiber filter papers (Schleicher & Schuell) for tissue harvester (VWR)
  • Additional materials for Bradford protein assay ( appendix 3A)

Basic Protocol 4: Quantitative Autoradiography of 5‐HT1A Binding to Rat Brain

  Materials
  • 69 Ci/mmol [3H]8‐OH‐DPAT (Amersham Pharmacia Biotech, 222 to 225 Ci/mmol) or [3H]p‐MPPF (NEN)
  • 170 mM Tris⋅Cl buffer, pH 7.6 ( appendix 2A), room temperature and 4°C (adjust pH at desired temperature)
  • Brain tissue sections mounted on gelatin‐coated slides (see protocol 6)
  • 1 mM WAY 100635 or 5 mM p‐MPPF (RBI); 1000× stock solution
  • Plastic Coplin staining jars (PGC Scientifics) or slide mailers (VWR Scientific Products)
  • Large glass staining dishes and glass slide racks (PGC Scientifics)
  • Slide warmer (VWR Scientific Products; optional)
  • Film cassettes for autoradiography (VWR Scientific Products)
  • [3H]Hyperfilm autoradiography film (Amersham Pharmacia Biotech)
  • [3H]standard slides, low‐activity (American Radiolabeled Chemicals e.g., ART 123, see also Amersham RPA 506 and 507)
  • Powder‐free gloves

Support Protocol 2: Preparation of Brain Tissue Sections for Quantitative Autoradiography

  Materials
  • 225‐ to 300‐g male Sprague‐Dawley rat
  • 0.9% (w/v) saline solution at 4°C in a beaker on ice
  • Tissue mounting media (e.g., Tissue Tek, VWR Scientific Products)
  • Rat guillotine (e.g., Braintree Scientific)
  • Dissection instruments: bone rongeurs, curved iris scissors, dissecting knife, and spatula
  • Dry ice, powdered
  • Small (∼8 × 12–cm) resealable plastic bags for storage (e.g., Whirl‐Pak; VWR Scientific Products)
  • Cryostat or microtome set between −20° and 16°C (e.g., Leica)
  • Gelatin‐coated microscope slides (see recipe), chilled
  • Plastic slide boxes (e.g., VWR Scientific Products)
NOTE: The use of powder‐free gloves is required for this protocol.

Support Protocol 3: Quantification of Radioligand Binding in Brain Tissue Sections by Image Analysis

  Materials
  • Light box (e.g., Northern Lights; Imaging Research)
  • Macintosh personal computer (PowerMac or G3 recommended) with frame grabber card (e.g., Scion VG‐5 NuBus) and NIH Image software
  • Copy stand (e.g., Kaiser)
  • 50‐mm camera lens (e.g., Schneider)
  • CCD black and white camera with resolution TV lines 580 horizontal, 350 vertical (e.g., COHU; http://www.cohu‐cameras.com)

Basic Protocol 5: Quantitative Autoradiography of 5‐HT1B Binding to Rat Brain

  Materials
  • 2200 Ci/mmol specific activity [125I](‐)iodocyanopindolol ([125I]ICYP) and filter kit (NEN Life Science)
  • 5 mM (‐)isoproterenol (see recipe)
  • 170 mM Tris⋅Cl/150 mM NaCl/0.01% ascorbic acid buffer, pH 7.4 (see recipe), room temperature
  • Brain tissue sections mounted on gelatin‐coated slides (see protocol 6)
  • 170 mM Tris⋅Cl buffer, pH 7.4 ( appendix 2A), room temperature and 4°C (adjust pH at desired temperature)
  • 5 mM 5‐HT (e.g., RBI, Sigma, Tocris)
  • [3H]Hyperfilm autoradiography film (Amersham Pharmacia Biotech)
  • Plastic Coplin staining jars (PGC Scientifics) or slide mailers (VWR Scientific Products)
  • Gamma counter (Pakcard Instruments)
  • Large glass staining dishes and glass slide racks (PGC Scientifics)
  • Slide warmer (VWR Scientific Products; optional)
  • Film cassettes for autoradiography (VWR Scientific Products)
  • Tritium standards, high activity (American Radiolabeled Chemicals, ART 123A)
  • Powder free‐gloves
  • Lead foil shielding and plastic β shield
CAUTION: Since [125I] emits β and γ nuclear particles, it is advisable to conduct the experiment behind a shield (e.g., lead foil shielding wrapped around a plastic β shield; PGC Scientifics) to minimize personal exposure. Also, conduct the binding assay under a hood, if possible, to reduce exposure to volatile [125I].

Support Protocol 4: Thionin Staining of Thaw Mounted Tissue Sections

  Materials
  • 100% (200 proof), 95%, 70%, and 50% ethanol, low grade
  • Hemo‐De tissue clearing agent (Fisher)
  • Thionin stain (see recipe)
  • Permount (Fisher)
  • Tissue sections thaw‐mounted on slides in slide trays (see protocol 6)
  • Plastic Coplin jars
  • Coverslips (e.g., 22 × 50–mm, Fisher)
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Figures

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Literature Cited

Literature Cited
   Barnes, N.M. and Sharp, T. 1999. A review of central 5‐HT receptors and their function. Neuropharmacol. 38:1083‐1152.
   Berg, K.A., Clarke, W.P., Sailstad, C., Saltzman, A., and Maayani, S. 1994. Signal transduction differences between 5‐hydroxytryptamine type 2A and type 2C receptor systems. Mol. Pharmacol 46:477‐484.
   Boess, F.G. and Martin, I.L. 1994. Molecular biology of 5‐HT receptors. Neuropharmacol. 33:275‐317.
   Chamberlain, J., Offord, S.J., Wolfe, B.B., Tyau, L.S., Wang, H.L., and Frazer, A. 1993. Potency of 5‐hydroxytryptamine1a agonists to inhibit adenylyl cyclase activity is a function of affinity for the “low‐affinity” state of [3H]8‐hydroxy‐N,N‐dipropylaminotetralin ([3H]8‐OH‐DPAT) binding. J. Pharmacol. Exp. Ther 266:618‐25.
   Cheng, Y.‐C. and Prusoff, W.H. 1973. Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22:3099‐3103.
   Frazer, A. and Hensler, J.G. 1990. 5‐HT1A mediated responses and 5‐HT1A receptors: Effects of treatments that modify serotonergic neurotransmission. Ann. N.Y. Acad. Sci 600:460‐475.
   Gaddum, J.H. and Picarelli, Z.P. 1957. Two kinds of tryptamine receptor. Br. J. Pharmacol. 12:323‐328.
   Geary, W.A. II, Toga, A.W., and Wooten, G.F. 1985. Quantitative film autoradiography for tritium: Methodological considerations. Brain Res. 337:99‐108.
   Gozlan, H., Thibault, S., Laporte, A.M., Lima, L., and Hamon, M. 1995. The selective 5‐HT1A antagonist radioligand [3H]WAY 100635 labels both G‐protein‐coupled and free 5‐HT1A receptors in rat brain membranes. Eur. J. Pharmacol 288:173‐86.
   Hoyer, D. and Schoeffter, P. 1991. 5‐HT receptors: Subtypes and second messengers. J. Recept. Res. 11:197‐214.
   Hoyer, D., Clarke, D.E., Fozard, J.R., Hartig, P.R., Martin, G.R., Mylecharane, E.J., Saxena, P.R., and Humphrey, P.P. 1994. International Union of Pharmacology classification of receptors for 5‐ hydroxytryptamine (Serotonin). Pharmacol. Rev 46:157‐203.
   Kehne, J.H., Baron, B.M., Carr, A.A., Chaney, S.F., Elands, J., Feldman, D.J., Frank, R.A., van Giersbergen, P.L., McCloskey, T.C., Johnson, M.P., McCarty, D.R., Poirot, M., Senyah, Y., Siegel, B.W., and Widmaier, C. 1996. Preclinical characterization of the potential of the putative atypical antipsychotic MDL 100,907 as a potent 5‐HT2A antagonist with a favorable CNS safety profile. J. Pharmacol. Exp. Ther. 277:968‐981.
   Kenakin, T. 1997. Pharmacologic analysis of drug‐receptor interaction. 3rd edition. Raven Press, New York.
   Kennett, G.A., Wood, M.D., Bright, F., Trail, B., Riley, G., Holland, V., Avenell, K.Y., Stean, T., Upton, N., Bromidge, S., Forbes, I.T., Brown, A.M., Middlemiss, D.N., and Blackburn, T.P. 1997. SB 242084, a selective and brain penetrant 5‐HT2C receptor antagonist. Neuropharmacol. 36:609‐620.
   Khawaja, X. 1995. Quantitative autoradiographic characterization of the binding of [3H]WAY‐100635, a selective 5‐HT1A receptor antagonist. Brain Res. 673:217‐225.
   Khawaja, X., Evans, N., Reilly, Y., Ennis, C., and Minchin, M.C. 1995. Characterization of the binding of [3H]WAY‐100635, a novel 5‐hydroxytryptamine‐1A receptor antagonist, to rat brain. J. Neurochem. 64:2716‐2726.
   Limbird, L.E. 1996. Cell Surface Receptors: A short course on theory and methods. Kluwer Academic Publishers, Boston, Mass. xs
   Martin, G.R. and Humphrey, P.P. 1994. Receptors for 5‐hydroxytryptamine: Current perspectives on classification and nomenclature. Neuropharmacol 33:261‐273.
   Meltzer, H.Y. 1995. Atypical antipsychotic drugs. In Psychopharmacology: The Fourth Generation of Progress (F.E. Bloom and D.J. Kupfer, eds.) pp. 1277‐1286. Raven Press, New York.
   Nelson, D.L., Monroe, P.J., Lambert, G., and Yamamura, H.I. 1987. [3H]spiroxatrine lables a serotonin1a‐like site in the rat hippocampus. Life Sci. 41:1567‐76.
   Nichols, D.E. 1997 Role of serotoninergic neurons and 5‐HT receptors in the action of hallucinogens. In Serotonergic Neurons and 5‐HT Receptors in the CNS. (H.G. Baumgarten and M. Gothert) pp. 563‐585. Springer Verlag, New York.
   Offord, S.J., Ordway, G.A., and Frazer, A. 1988. Application of [125I]iodocyanopindolol to measure 5‐hydroxytryptamine1B receptors in the brain of the rat. Pharmacol. Exp. Ther. 244:144‐53.
   Paxinos, G. and Watson, C. 1986. The rat brain in stereotaxic coordinates. 2nd ed Academic Press, San Diego.
   Peroutka, S.J. and Snyder, S.H. 1979. Multiple serotonin receptors: Differential binding of [3H]5‐hydroxytrptamine. [3H]lysergic acid diethylamide and [3H]spiroperidol. Mol. Pharmacol 16:687‐99.
   Rapport, M.M., Green, A.A., and Page, I.H. 1948. Serum vasoconstrictor (serotonin). IV. Isolation and characterization. J. Biol. Chem 176:1243‐1251.
   Rosen, R.C., Lane, R.M., and Menza, M. 1999. Effects of SSRIs on sexual function: A critical review. J. Clin. Psychopharmacol 19:67‐85.
   Roth, B.L., Willins, D.L., Kristiansen, K., and Kroeze, W.K. 1998. 5‐Hydroxytryptamine2‐family receptors (5‐hydroxytryptamine2A, 5‐ hydroxytryptamine2B, 5‐hydroxytryptamine2C): Where structure meets function. Pharmacol. Ther. 79:231‐257.
   Saltzman, A. G., Morse, B., Whitman, M.M., Ivanshchenko, Y., Jaye, M., and Felder, S. 1991. Cloning of the human serotonin 5‐HT2 and 5‐HT1C receptor subtypes. Biochem. Biophys. Res. Commun. . 181:1469‐1478.
   Segraves, R.T. 1998. Antidepressant‐induced sexual dysfunction. J. Clin. Psychiatry 59:48‐54.
   Thielen, R.J., Fangon, N.B., and Frazer, A. 1996. 4‐(2'‐Methoxyphenyl)‐1‐[2'‐[N‐(2″‐pyridinyl)‐p‐iodobenzamido]ethyl] piperazine and 4‐(2'‐methoxyphenyl)‐1‐[2'‐[N‐(2″‐pyridinyl)‐p‐fluorobenzamido]ethyl]piperazine, two new antagonists at pre‐and postsynaptic serotonin‐1A receptors. J. Pharmacol. Exp. Ther. 277:661‐70.
   Verge, D., Daval, G., Marcinkiewicz, M., Patey, A., el Mestikawy, S., Gozlan, H., and Hamon, M. 1986. Quantitative autoradiography of multiple 5‐HT1 receptor subtypes in the brain of control or 5,7‐dihydroxytryptamine‐treated rats. J. Neurosci 6:3474‐82
   Zifa, E. and Fillion, G. 1992 5‐Hydroxytryptamine receptors. Pharmacol. Rev. 44:401‐458.
Key References
   Gozlan et al., 1995. See above.
  Information about the antagonist [3H]WAY 100635 and quantitative autoradiography.
   Hensler, J.G., Kovachich, G.B., and Frazer, A. 1991. A quantitative autoradiographic study of serotonin1A receptor regulation. Effect of 5,7‐dihydroxytryptamine and antidepressant treatments. Neuropsychopharmacology 4:131‐44.
  Uses [3H] 8‐Hydroxy‐DPAT in quantitative autoradiography.
   Khawaja, X. 1995. Quantitative autoradiographic characterization of the binding of [3H]WAY‐100635, a selective 5‐HT1A receptor antagonist. Brain Res. 673:217‐225.
  Information about the antagonist [3H]WAY 100635 and quantitative autoradiography.
   Kung, H.F., Stevenson, D.A., Zhuang, Z.P., Kung, M.P., Frederick, D., and Hurt, S.D. 1996. New 5‐HT1A receptor antagonist: [3H]p‐MPPF. Synapse 23:344‐346.
  Information about binding of the antagonist [3H]p‐MPPF.
   Limbird, L.E. 1996. See above
  General information about quantitative autoradiography and homogenate binding, formulas, and curve fits to obtain KD and IC50 values.
   Martial, J., Lal, S., Dalpe, M., Olivier, A., de Montigny, C., and Quirion, R. 1989. Apparent absence of serotonin1B receptors in biopsied and post‐mortem human brain. Synapse 4:203‐209.
  Basic protocol for 5‐HT1B autoradiography was derived from this reference.
   Pazos, A. and Palacios, J.M. 1985. Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. Serotonin‐1 receptors. Brain Res. 346:205‐230.
  Overview of serotonin 1 receptors in rat brain by quantitative autoradiography.
   Pazos, A., Engel, G., and Palacios, J.M. 1985. Beta‐Adrenoceptor blocking agents recognize a subpopulation of serotonin receptors in brain. Brain Res. 343:403‐408.
  Basic protocol for 5‐HT1B autoradiography was derived from this reference.
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