Visualization of 5‐HT Receptors Using Radioligand‐Binding Autoradiography

Roser Cortés1, M. Teresa Vilaró1, Guadalupe Mengod1

1 IIBB‐CSIC, IDIBAPS, CIBERNED, Barcelona
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 8.3
DOI:  10.1002/cpph.17
Online Posting Date:  December, 2016
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Abstract

Described in this unit are techniques to visualize the majority of serotonin (5‐hydroxytryptamine, 5‐HT) receptor subtypes in sections of frozen brain tissue using receptor autoradiography. Protocols for brain extraction and sectioning, radioligand exposure, autoradiogram generation, and data quantification are provided, as are the optimal incubation conditions for the autoradiographic visualization of receptors using agonist and antagonist radioligands. © 2016 by John Wiley & Sons, Inc.

Keywords: receptor autoradiography; radioligand binding; brain cryosections; brain tissue; 5‐HT receptor subtypes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparing and Pretreating Brain Tissue for Autoradiography
  • Basic Protocol 2: Radioligand Labeling of Receptors
  • Basic Protocol 3: Generation of Autoradiograms
  • Basic Protocol 4: Autoradiogram Analysis
  • Support Protocol 1: APTS (3‐Aminopropyltriethoxysilane)‐Coated Slides
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparing and Pretreating Brain Tissue for Autoradiography

  Materials
  • Rats, mice, guinea pigs, other species of laboratory animals or human postmortem brain tissue
  • 0.9% (v/v) NaCl saline solution
  • Isopentane (2‐methylbutane)
  • Dry ice pellets for freezing
  • Tissue freezing medium (embedding medium for cryosectioning): Jung Tissue Freezing Medium (Leica); other media are commercially available: e.g., Tissue‐Tek O.C.T. Compound (Sakura) or TBS TFM (Fisher Scientific)
  • Gloves
  • Mask if cutting human brain tissue
  • Dissection instruments including:
    • Bone cutter (cartilage cutter with round blades)
    • Small and large scissors
    • Forceps
    • Spatula
  • 100‐ml plastic beakers
  • Razor blades
  • Brain slicer (Brain Matrix, Ted Pella)
  • Thermometer for measuring −40°C or lower
  • Aluminum foil
  • Zipper freezer bags for tissue storage
  • Freezer (−80°C)
  • Microtome‐cryostat (−20°C) (e.g., Leica CM3050S, Microm, Thermo Fisher Scientific, cat. no. HM525)
  • Specimen stage (e.g., Leica 14037008587; Thermo Fisher Scientific, cat. no. 715710), antiroller (e.g., Leica 14041933816; Thermo Fisher Scientific, cat. no 449980 or 449990) and knife (e.g., Leica 14021604206; Thermo Fisher Scientific, cat. no. 152020) or blade (e.g., Leica 14035838383; Thermo Scientific MX35 Ultra Blades) compatible with microtome‐cryostat brand
  • Coated microscope‐glass slides (see protocol 5Support Protocol)
  • Slide boxes

Basic Protocol 2: Radioligand Labeling of Receptors

  Materials
  • Slide boxes
  • Incubation solution (radioligand solution; see composition in Tables 8.3.1 8.3.3)
  • [3H]‐ or [125I]‐ labeled ligands (see Tables 8.3.1 8.3.3 for different receptor subtypes)
  • Unlabeled ligands (competitor compounds) predissolved as concentrated stock solutions
  • Ice bath
  • Scintillation fluid, OptiphaseHisafe 2 (Perkin Elmer)
  • Slide‐mounted tissue sections (from protocol 1)
  • Wash buffer (see composition in Tables 8.3.1, 8.3.2, and 8.3.3)
  • Distilled water
  • Slide boxes
  • Powder‐free gloves
  • X‐ray protective glasses
  • Incubation jars: The number of slides to be incubated will determine the type of jars to be used: Hellendahl jars, Coplin jars, slide mailing boxes (VWR Scientific Products; Ted Pella) or slide staining racks and dishes (black boxes) from Kartell (see Fig.  A)
  • Radiation shielding leaded glass, when using 125I‐ligands
  • Leaded apron, when using 125I‐ligands
  • Leaded shield, when using 125I‐ligands
  • Lead container for discarded material
  • Scintillation counter (β or γ)
  • Air stream apparatus (e.g., hair dryer)
  • Cold dry air system (e.g., refrigerator with fan)
  • Timer
  • Forceps
NOTE: See Tables 8.3.1 to 8.3.3 for buffer compositions for the different 5‐HT receptor radioligands. Note that when 5‐HT is used as radioligand or as displacer, an MAO inhibitor (pargyline) and an antioxidant (ascorbic acid) are usually included in the incubation and washing buffers to prevent degradation of the transmitter. Pre‐incubation buffer should not contain pargyline or ascorbic acid to allow for catabolism of endogenous 5‐HT.CAUTION: When working with radioactive compounds it is important to strictly observe all safety rules for protection from contamination and radiation. Additional lead protection is needed when using 125I‐labeled agents.

Basic Protocol 3: Generation of Autoradiograms

  Materials
  • Incubated glass slides (from protocol 2)
  • Silica gel packets, 5 g (Silicagelpackets.co.uk)
  • Developer: 200 ml LX24 (X‐Ray developer Carestream Kodak 5070933) and 800 ml H 2O
  • Fixer: 200 ml AL4 (X‐Ray fixer Carestream Kodak 5071071) and 800 ml H 2O
  • Support to fix the slides (e.g., one‐side self‐adhesive plastic foil, by roll, to be cut to the size of the autoradiographic film; Aironfix, Eon Paper S.L., or other brands)
  • X‐ray cassettes (Hypercassette, Amersham)
  • X‐ray film (Carestream Kodak Biomax MR films, Sigma Aldrich)
  • Safelight (with 7.5‐ or 15‐W bulb and red Kodak GBX‐2 filter)
  • Zippered plastic bags
  • Developing trays or tanks (Richards, UK; model TX3)
  • Plastic radiolabeled standard microscales for calibration and quantification (American Radiolabeled Chemicals)

Basic Protocol 4: Autoradiogram Analysis

  Materials
  • Developed film(s) (from protocol 3)
  • Light box: Precision white light illuminator with adjustable intensity output (e.g., Northern Light desktop illuminator, Imaging Research, Interfocus)
  • Copy stand (e.g., Kaiser RS1, Kaiser Fototechnik) to hold the camera
  • Digital video camera (Q‐Imaging) equipped with an appropriate objective (e.g., Nikkor 55 mm, 1:2.6, Japan) via a C‐mount thread
  • Computerized image analysis system and software (MCID Core image analysis system, InterFocus)

Support Protocol 1: APTS (3‐Aminopropyltriethoxysilane)‐Coated Slides

  Materials
  • APTS (3‐Aminopropyltriethoxysilane 99%; Sigma‐Aldrich, cat. no. A3648)
  • Acetone puriss (Panreac, cat. no. 141007)
  • Distilled water
  • Microscope glass slides
  • Tissue paper
  • Stainless steel slide staining racks (Lipshaw)
  • Glass beakers or crystallizers
  • Heating oven
  • Storage boxes (e.g., plastic Tupperware‐type containers)
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Figures

Videos

Literature Cited

Literature Cited
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