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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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?HT_1A , 5?HT_1B , 5?HT_2A , and 5?HT_2C . 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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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 [³ H]ketanserin (5‐HT_2A receptors; NEN Life Sciences) or
70 to 85 Ci/mmol [³ H]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 [³ H]ketanserin (NEN Life Sciences; for 5‐HT_2A receptors) or
70 to 85 Ci/mmol [³ H]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 [³ H]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 [³ H]8‐OH‐DPAT (Amersham Pharmacia Biotech, 222 to 225 Ci/mmol) or [³ H]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)
[³ H]Hyperfilm autoradiography film (Amersham Pharmacia Biotech)
[³ H]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 [¹²⁵ I](‐)iodocyanopindolol ([¹²⁵ I]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)
[³ H]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 [¹²⁵ I] 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 [¹²⁵ I].

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

Figure 1.23.1 Saturation binding plot of [³ H]‐mesulergine binding to the 5‐HT_2C receptor expressed in CHO cells using data shown in Table . Solid circles represent total binding in fmol/mg protein and solid squares represent the nonspecific binding data. Nonspecific binding data were analyzed by fitting the data to a straight line with an origin of zero to obtain the slope of the line (dotted line). Total binding data were fit to the equation in , , using nonlinear regression analysis (as described above; solid line) to obtain estimates of receptor density (B_max ), K_D , and slope factor, which for this experiment were 249 fmol/mg protein, 0.56 nM, and 0.91, respectively. The dashed line without data points represents specific binding calculated using the values of parameters B_max , K_D , and slope factor.

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Figure 1.23.2 Competition binding plot of ketanserin competing for [³ H]‐mesulergine binding to the 5‐HT_2C receptor expressed in CHO cells using data shown in Table . Solid circles represent [³ H]‐mesulergine binding in fmol/mg protein. Data were fit to the equation in , , using nonlinear regression analysis (as described above; solid line) to obtain estimates of [³ H]mesulergine binding in the absence (B_o ) and presence of maximal concentration of ketanserin (B_i ) and for the concentration of ketanserin that reduces [³ H]mesulergine binding by 50%, which for this experiment were 324 fmol/mg protein, 85 fmol/mg protein and 360 nM, respectively. A K_i of 157 nM was obtained using the Cheng‐Prusoff correction.

View Image

Figure 1.23.3 Flow chart for quantitation of autoradiograms.

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Videos

Literature Cited

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

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited