Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

Podsumowanie

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

Streszczenie

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (K_d), inhibition constants (K_i) as well as the density of binding sites (B_max) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity γ-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

Wprowadzenie

Autoradiography is a method which provides images of radioactive decay. The technique is routinely used to study the tissue distribution of a protein of interest in vitro based on a specific pharmacological interaction between a radiolabelled compound and its target. This provides direct information about the selectivity of the ligand for the target. In vitro autoradiography may also be used for quantitative determination of pharmacological binding parameters of radioligands, such as the dissociation constant (K_d) and density of binding sites (B_max), as well as for determining the inhibition constant (K_i) of competing ligands^1,2. Compared to traditional homogenate radioligand binding, autoradiography has the advantage of being able to visualize spatial anatomy and giving succinct details of regional expression patterns³. The method of autoradiography is therefore a relevant alternative to immunocytochemistry, especially in the absence of a validated antibody. Autoradiography is easily implemented in a standard radioisotope laboratory given the availability of a suitable radioligand with the required pharmacological specificity, access to a microtome cryostat for preparing tissue sections, and a suitable imaging device that is able to analyze the distribution of radioactivity in the respective tissue sections. Notably, an important selection criterion for the radioligand is a limited amount of binding to non-target sites. This can be to other proteins, membranes or materials such as plastic or filters, and is collectively referred to as non-specific binding. Usually, non-specific binding is non-saturable but can be saturable if it involves a specific off-target protein. The best way of validating true specific binding is to compare to tissues lacking the target, e.g., genetically engineered (knock-out) tissue⁴.

Here, the methodology is illustrated with the autoradiographic characterization of the high-affinity binding site for γ-hydroxybutyric acid (GHB) in the mammalian brain. Understanding the pharmacological interaction between GHB and its binding site is of relevance as GHB is both a clinically useful drug in the treatment of narcolepsy and alcoholism⁵, but also a natural constituent of the mammalian brain and a recreational drug⁶. High-affinity GHB binding sites were first described using [³H]GHB binding to rat brain homogenate⁷. Over the years, further autoradiography studies with [³H]GHB and the analogue [³H]NCS-382 has showed a high density of binding sites in forebrain regions of rat^8,9,10, mouse⁹, pig¹¹and monkey/human brain¹². However, the molecular identity and exact functional relevance of these binding sites have remained elusive.

With the intention to further characterize the binding sites, and to facilitate studies on the physiological role of GHB, multiple radioligands incorporating different isotopes endowed with different affinities have been developed ([³H]GHB, [³H]NCS-382, [³H]HOCPCA and [¹²⁵I]BnOPh-GHB)^13,14,15,16(reviewed in¹⁷) (Figure 1). The combination of selective high-affinity radioligands and a very high tissue density of the binding sites have allowed for the production of high-quality images using the phosphor imaging technique^9,11. Along with an outline of the practical points in setting up an autoradiographic experiment and an illustration to exemplify details, the discussion section will emphasize i) the choice of radionuclide, ii) the choice of assay conditions, and iii) the use of phosphor imaging plates versus X-ray film. The overall goal of this paper is to provide technical, methodological and scientific details on the autoradiography technique for informing about tissue distribution and pharmacological analysis of protein targets.

Protokół

All animal handling was performed in compliance with the guidelines from The Danish Animal Experimentation Inspectorate.

NOTE: The protocol described here covers tissue preparation (i.e., mouse brain tissue), the in vitro autoradiographic assay in sufficient detail for setting up the method in a new lab, the exposure to phosphor imaging plates as well as subsequent densitometric analysis of autoradiograms (Figure 2) with the aim of localizing and quantifying radioligand binding in distinct anatomical structures. For histological comparison, a protocol for cresyl violet staining is included. Moreover, the determination of non-specific binding with a competing ligand is included within the protocol. For a detailed description on how to determine K_d, B_max or K_i, see previous publication⁴.

1. Tissue Preparation by Cryosectioning

1. Euthanize the mouse by cervical dislocation and immediately dissect out the brain using scissors and forceps. Directly proceed to the next step to avoid tissue damage.
2. Snap-freeze the tissue by submersion in powdered dry ice, gaseous CO₂ or isopentane. Directly transfer the frozen tissue to a cryostat with the temperature set to -20 °C. Alternatively, store the tissue at -80 °C until processing.
   NOTE: Avoid repeated thawing/freezing to reduce tissue damage.
3. Let the tissue acclimate to -20 °C in the cryostat for 20 min before further processing to avoid tissue shattering.
4. Cover the tissue holder with embedding medium outside the cryostat and quickly place the frozen tissue specimen in the desired orientation while the embedding medium is still liquid. For instance, place the mouse brain vertically onto cerebellum in order to achieve rostral coronal sections. Transfer the tissue holder back to the cryostat and expose the embedding medium to temperatures below -10 °C for hardening.
   NOTE: Fragile tissue specimen should be coated in embedding medium within the tissue molds prior to mounting.
5. Position the tissue holder in the microtome of the cryostat. Adjust the orientation of the tissue to avoid sloped sections.
6. Cut the tissue with the guidance of a stereotaxic atlas¹⁸ in sections of desired thickness (12 µm recommended for [³H] labelled ligands). Carefully straighten and unfold the section with a brush of small size if necessary and thaw-mount the section onto a microscope slide. Sequentially collect the sections from the region of interest for desired technical replication (e.g., 4 sections per slide).
7. Allow the sections on the slides to air-dry for 1 h before further handling.
   NOTE: Addition of desiccant material to slide boxes minimizes moisture build up on the tissue sections. Procotol can be paused here by storing sections long-term in slide boxes at -80 °C.

2. In vitro Autoradiography

CAUTION: Radioactivity. Work in a certified laboratory according to local regulations. Wear protective clothing. Dispose in accordance with radioactive decay or outsource to a certified company.

1. Thaw the sections for at least 30 min at room temperature (RT). Label the slides with experimental conditions. Use a pencil because the slides will be bathed in ethanol during subsequent staining.
2. Place the slides horizontally in plastic trays.
   NOTE: Positioning slides on an elevated platform within plastic trays facilitates their handling.
3. Pre-incubate the sections mounted on the slides in assay buffer adjusted to target in question (for GHB protocol, 50 mM KHPO₄ buffer pH 6.0 is used) by carefully applying an appropriate volume onto the slide (700 µL for 3-4 rodent coronal sections).
   NOTE: Make sure that every section is covered completely with liquid.
   1. Cover the plastic trays with a lid in order to avoid evaporation and pre-incubate at relevant temperature (for GHB protocol pre-incubate for 30 min at RT) under constant gentle (20 rpm) shaking on a plate shaker.
   2. For the determination of non-specific binding, supplement assay buffer with relevant concentration of unlabelled compound (for GHB protocol, 1 mM GHB).
      NOTE: Pre-incubation may not be necessary.
4. Pour off pre-incubation liquid from each slide and transfer the slides back to the plastic tray.
5. To avoid dehydration, immediately incubate the sections with relevant concentration of radioligand in assay buffer under desired conditions (for GHB protocol, 1 nM [³H]HOCPCA for 1 h at RT) by covering the sections completely with the radioligand solution (700 µL for 3-4 rodent coronal sections).
   1. Incubate under under constant gentle (20 rpm) shaking of plastic trays with closed lid.
      NOTE: The radioligand concentration can be validated by counting an aliquot in a liquid scintillation counter.
6. Remove the incubation solution by pouring off the liquid and transfer the slides into a microscope slide rack. Immediately proceed to the next step to avoid section dehydration.
7. Wash the slides. For the GHB protocol, wash with ice-cold assay buffer twice for 20 s and then rinse twice by dipping the slide rack into the trays filled with ice-cold distilled water to remove salts. Position the slides vertically in racks for air-drying for at least 1 h at RT or dry the slides for 5 min with a blower set to cold temperature.
   NOTE: Washing must be optimized, e.g., extensive washing may be useful for decreasing non-specific binding.
8. Transfer the slides to a fixator containing paraformaldehyde (PFA) powder for overnight fixation with PFA vapours at RT in order to protect the integrity of the ligand-target complex.
   CAUTION: PFA is toxic. Positionthe fixator in fume hood and avoid skin/eye contact with PFA.
9. The following day, transfer the slides to a desiccator box containing silica gel for 3 h at RT to eliminate moisture.

3. Exposure to Phosphor Imaging Plates and Scanning

1. Place the sections in a radiation-shielded imaging plate cassette with the tissue facing up. For subsequent quantification of radioligand binding, include a [³H]microscale in every cassette. Arrange the sections randomly and expose the sections for direct comparison in the same cassette.
2. Erase the tritium-sensitive phosphor imaging plate immediately before usage in order to remove accumulated signals from storage and to eliminate background signals. Therefore, load the plate into phosphor imaging machine and expose it to visible/infrared light according to the instructions of the manufacturer.
3. Remove the plate from phosphor imaging machine and immediately place it onto the sections in the cassette. Make sure that the cassette is closed completely. Expose the sections to the phosphor imaging plate for 3 days at RT shielded from light.
4. Because light erases signal from the imaging plate, carefully open the cassette in the dark and immediately transfer the imaging plate into the dark box of a phosphor imager or place the phosphor imager in a dark room.
   NOTE: Make sure to notate the spatial arrangement of the slides during exposure in order to identify individual specimen on the digital image after analysis. Therefore, phosphor imaging plates also display one corner cut in a distinct angle in order to identify the correct orientation of the plate on the digital picture.
5. Scan the plate at the highest resolution possible to obtain a digital image.

4. Optional: Cresyl Violet Staining of Tissue Sections

1. Prepare 1% cresyl violet solution by mixing 5 g of cresyl violet acetate in 500 mL of deionized water (dH₂O) until dissolved (approximately 2 h). Filter through a filter paper using a funnel into a new 500 mL bottle. Adjust pH to 3.5-3.8.
2. Position the slide staining set under fume hood. Prepare trays with the following solutions in white polypropylene trays (except for xylene):
   a. 50% ethanol/50% dH₂O
   b. 70% ethanol/30% dH₂O
   c. 100% ethanol
   d. 100% ethanol
   e. 100% dH₂O
   f. 1% cresyl violet
   g. 0.07% acetic acid (add 175 µL of acetic acid to 250 mL of dH₂O).
   h. 100% xylene in green solvent-resistant trays
   i. 100% xylene in green solvent-resistant trays
3. Transfer the slides to the fume hood and place them in a slide rack.
4. Dissolve the lipids through increasing graded series of ethanol in dH₂O into 100% ethanol (tray a-d) by dipping the slides for 1 min.
5. Rehydrate the specimens to dH₂O through descending concentrations of ethanol (tray a-d in reverse order, followed by tray e) by dipping the slides for 1 min.
6. Immerse the specimens in cresyl violet solution for 10 min.
7. Rinse the specimens in 0.07% acetic acid by lifting the slides up and down gently for 4-8 s. Wash the slides by dipping in dH₂O for 1 min.
8. Dehydrate the specimens by immersion of the slides for 30 s in ascending concentrations of ethanol (tray a-d).
9. Transfer the specimens through two trays of 100% xylene (tray h and i) to quench the ethanol.
10. Rehydrate the specimens to dH₂O through descending concentrations of ethanol (tray a-d in reverse order, followed by tray e) by dipping the slides for 1 min.
11. Remove the slides from saline with forceps. Add a few drops of organic solvent mounting media per slide and add a 24 x 60 mm coverslip on top to protect specimens. Remove air bubbles between the specimen and coverslip by gently pressing onto the coverslip.
    NOTE: Keep the remaining slides in xylene during mounting to prevent drying.
12. Dry the slides overnight in a fume hood at RT.
13. Obtain a picture of specimen with a microscope and 1.25X objective.

5. Densitometric Analysis of Digital Image

1. Measure relative optical densities (RODs) of each calibration standard from the [³H]microscale with an image analysis software.
   1. Select an area of equal size for each point of the [³H]microscale using a tool for Region creation from the menu item Region determination. Assign a number to each selected area by clicking on Number under the menu item Label.
   2. Export OD values for each point of the calibration standard by clicking File | Export | 2D region report. Transfer ROD values to a spreadsheet and normalize by the size of the selected area. Perform linear regression to obtain a standard curve for further densitometric analysis.
      NOTE: Make sure that the selected areas are labelled in order to identify matching ROD values and samples.
2. Perform quantification of autoradiograms using the proprietary imaging software by selecting the region of interest (ROI) using a Region creation tool in every section and measuring its ODs. Select the same region in every section by creating a template for the region of interest, which is copied and manually adjusted to minor variations in brain anatomy for each autoradiogram. Identify the anatomy of the ROI by comparison of autoradiograms with a brain atlas¹⁸. When multiple treatments are compared, perform the analysis blinded and randomized in order to avoid biased selection of ROIs.
3. Export ROD values and sizes of selected areas into a spreadsheet by clicking File | Export | 2D region report.
4. Divide the measured ROD of the selected ROI by its area to obtain the density per specific area.
5. Measure the ROD of the background of the plate and export corresponding ROD values and area size into a spreadsheet. Subtract the average background signal from every ROD value of each ROI.
6. Average the RODs of technical replicates, i.e., section replicates using tissue from the same animal.
7. Use the standard curve to convert RODs into units of radioligand binding, i.e., nCi/mg tissue equivalents (TE).
   NOTE: The term TE is used because standards are generated with materials simulating tissue.
8. Express binding by conversion of nCi/mg to nmol/mg TE according to the specific activity of the radioligand (Equation 1).
   (1)
9. To obtain specific binding values, subtract non-specific binding from total binding.
10. Average the binding of every biological replicate by using the average of the technical replicates of each animal (obtained in Step 5.6).

Wyniki

Using the described protocol, the anatomical distribution of the high-affinity GHB binding sites was visualized with the radiolabelled GHB analogue [³H]HOCPCA in mouse brain, which was cut into coronal, sagittal and horizontal sections (Figure 3). High levels of binding were observed in hippocampus and cortex, lower binding in striatum and no binding was detected in cerebellum, corresponding to previous reported expression patterns of the high-affi...

Dyskusje

The quality of an autoradiographic assay is most often determined by the sensitivity of the radioligand. A major contributing factor is the selected radioisotope, which is given by the availability of known ligands or by the feasibility of specific labelling techniques to yield ligands with appropriate specific activity (i.e., the amount of radioactivity per unit mole of a radioligand)²³and with limited amounts of chemical degradation. A large number of radioligands of known ligands are l...

Materiały

Name	Company	Catalog Number	Comments
Absolute ethanol	Merck Millipore	107017
Acetic acid	Sigma-Aldrich	A6283
BAS-TR2040 Imaging Plate	GE Healthcare Life Science	28956481	20x40 cm - Sensitive to tritium
Cresyl violet acetate	Sigma-Aldrich	C5042-10G
DPX (non-aqueous mounting medium for microscopy)	Merck Millipore	100579
O.C.T. Compound, 12 x 125 mL	Sakura	4583	Tissue-Tek
Paraformaldehyde	Sigma-Aldrich	16005-1KG-R
Superfrost Plus slides	VWR	631-9483	microscope slides
Tissue-Tek Manual Slide Staining Set	Sakura Finetek Denmark ApS	4451
Tritium Standard on Glas	American Radiolabeld Chemicals, Inc.	ART 0123
Xylene substitute	Sigma-Aldrich	A5597