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

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 labelled with tritium^{9,10,24,25,26}, which infers several advantages. Firstly, tritium (³H) is characterized by a long half-life (12.43 years) promoting long-term storage of individual batches. Secondly, the ligand-target interaction is not distorted by the radionuclide as ³H-ligands are biologically indistinguishable from their parent compounds. Tritium emits low energy β^–-particles, which travel only short distances in tissue resulting in high spatial resolution and, subsequently, greater distinction between anatomical structures⁴. Nonetheless, tritium labelling can only be produced to yield moderately high specific activities. This is due to the fact that available ³H-sources are contaminated with non-radioactive hydrogen. Generally, the higher the specific activity, the less radioligand is needed to yield sensitive detection²³. Moreover, it should be considered that ³H-ligands have the possibility to undergo hydrogen exchange with water depending on the stability of the ³H-label. To compensate for low expression or too low specific activity, iodine-125 can be used as the radionuclide¹³. The maximal specific activity of iodine-125 is approx. 100-fold higher than that of tritium. However, several additional considerations have to be made when working with iodine-125. For instance, the addition of iodine-125 normally induces structural alterations in the molecule which may impact the ligand-target interactions. As iodine-125 has a half-life of 60 days, correction for daily decay should be considered for specific activity and quantification of ligand-target interactions²³. ¹²⁵I-ligands emit γ-photons and in spite of much higher sensitivity, produce lower-resolution images. This is due to the inverse relationship between resolution and the maximum energy of the isotope (as discussed below). Finally, compared to tritium, increased care should be taken when handling iodine-125 due to the higher energy of the radionuclide.

Depending on the nuclide, tissue section thickness may influence resolution. The low-energy β^–-radiation of tritium limits its tissue reach to approx. 5 µm due to self-absorption. As a consequence, quantification is not influenced by tissue thickness when the section thickness exceeds 5 µm^27,28. By contrast, radioactive decay of high-energy isotopes has a greater tissue penetration, resulting in lower image resolution because ligands with a greater distance to the detection medium also contribute to image formation. Consequently, thinner sections promote higher resolution for high-energy radionuclides¹.

The establishment of an autoradiographic protocol requires knowledge of optimal binding conditions (e.g., buffer, pH and temperature) and pharmacological parameters of the radioligand in terms of affinity and kinetics. If the radioligand has not been characterized before, exploratory studies are necessary²⁹. Choosing an optimal radioligand concentration is guided both by the affinity of the radioligand and the abundance of binding sites. Normally, a concentration reflecting 5-6 times K_d is used to make sure that all binding sites are saturated³⁰. Another approach aims to yield the highest ratio of total-to-non-specific binding by selecting radioligand concentrations below K_d³¹ and saving radioligand solution at the same time. This approach is particularly useful when the binding site is highly abundant in the examined tissue since high radioligand concentrations would increase the chance of oversaturating autoradiograms, as the case for [³H]HOCPCA and the high-affinity GHB binding sites⁹. Furthermore, in order to efficiently label the whole population of the targeted protein, the radioligand should ideally bind to all possible target conformations. Especially in receptor autoradiography, the use of agonists may only reveal a partial number of total receptors since some might be present in low-affinity agonist states. In contrast, neutral antagonists most often display affinity for all receptor states^26,29.

Moreover, binding experiments are generally performed under equilibrium binding conditions. Therefore, the time needed to reach equilibrium binding under the desired experimental conditions (fixed radioligand concentration, buffer and temperature) should be determined in association experiments to ensure equilibrium binding within the scope of the experiment^4,23,30. After radioligand incubation, unbound radioligand is washed off by several incubations with assay buffer and typically followed by rinsing in distilled H₂O. Signal-to-noise ratios can be optimized by adjusting temperature and time depending on the dissociation rate of the radioligand^26,30,31.

Radioligands may display binding to non-biological materials, i.e., non-specific binding. Radioligand binding to cellular components other than the intended target is defined as unspecific binding. The contribution of unspecific binding to the total amount of radioligand binding is assessed in the presence of a competing unlabelled ligand that targets the same binding site as the radioligand. As the non-radioactive compound (displacer) is supplied in 10,000-fold excess, it occupies the binding site and the radioligand can only bind to off-target sites^26,31,32. Crucially, the unlabelled compound should be of a different chemical structure than the radioligand since this lowers the risk of displacing both specific as well as non-specific binding²³. Non-specific binding arising from binding to membranes can be a major problem especially in the case of fairly lipophilic radioligands. In some cases, extensive washing procedures may remove non-receptor bound radioligand and therefore improve the specific binding ratios²⁹.

When choosing an assay buffer, it is crucial to consider the effect of ionic strength and pH on the ligand-target interaction. Especially electrostatic interactions between polar ligands and hydrophilic components of binding sites are influenced by the ionic strength of the buffer. Therefore, supplementation with monovalent or divalent ions may impact the effective affinity constant^23,33. If the optimal buffer composition for the studied ligand-target interaction is unknown, different common buffers should be explored in pilot experiments. Buffers may also be supplemented with anti-oxidants such as ascorbic acid and inhibitors of degrading enzymes^29,34. Moreover, the ionization of specific groups within the binding site or on the ligand itself is influenced by pH and has critical effects on the equilibrium binding constant, the kinetic rate constants and non-specific binding^23,33. For example, probing the high-affinity GHB binding site with different GHB radioligands nicely illustrates the importance of pH on this binding target (Figure 6). Characterizing the optimal pH for the ligand-receptor interaction may also give clues about the importance of the target in relation to its biological relevance.

Another critical factor in digital autoradiography is the exposure time, i.e., the time needed to achieve quantifiable autoradiograms by exposing the radiolabelled tissue sections to phosphor imaging plates. Estimates are based on the amount of radioactivity in the sample, the energy and half-life of the isotope as well as the desired signal-to-noise ratio. In particular, prolonged exposure time results in saturated images and high background signal. Elevated background signal can be reduced by storing cassettes within lead-shielded environments to avoid cosmic radiation^1,4. Nevertheless, if suboptimal autoradiograms are achieved, the specimen can be exposed multiple times, provided that the ligand-target complex is fixed and the half-life of the radionuclide allows it.

Phosphor imaging plates can be reused and have a long lifespan when handled correctly, i.e., bending should be avoided and plates should be stored in a dry environment. The handling of phosphor imaging plates is guided by their sensitivity to light and cosmic radiation. Thus, it is important to erase the plate prior to every use in order to minimize background signal. Exposure of radiolabelled tissue sections to the imaging plates is done in radiation-shielded cassettes completely devoid of light. When transferring the plate to the scanner at the end of the exposure time, any ambient light should also be avoided as even short contact with white light reduces accumulated signal. Furthermore, plates should be scanned immediately after the removal of the specimen to avoid signal fading. A drawback of using phosphor imaging plates is the potential appearance of artefacts and residual 'ghost images' after repeated usage of the plates¹.

Working with tritium requires imaging plates without a protective coating to allow the low energy radiation to reach the phosphor crystals. Tritium-sensitive phosphor imaging plates are therefore more sensitive to contamination or damage due to incorrect handling. Once contaminated, tritium-sensitive plates cannot be cleaned and need to be replaced. Fixation of the ligand-target complex with PFA vapour reduces potential contamination of the plate, lengthening its lifespan. Moreover, dehydration of the tissue subsequent to fixation is a crucial step since tritium sensitive plates are sensitive to moisture. Due to its delicate nature, tritium-sensitive plates should not be used for other isotopes^1,4. In contrast, the plates for high-energy isotopes such as iodine-125 are more robust and their surface can even be cleaned by wiping with 70% ethanol.

Traditionally, radiosensitive film has been used for the spatial recording of radioactive decay. While images with high resolution can be obtained, film autoradiography has several limitations. Besides the necessity of hazardous chemicals and a darkroom for development, X-ray film is characterized by a narrow dynamic range. Therefore, it may be necessary to expose radiolabelled sections repeatedly with different exposure times in order to achieve quantifiable non-saturated images³⁵. Moreover, X-ray film exhibits limited sensitivity resulting in sustained exposure times for specimen labelled with low energy isotopes, i.e. tritium decay may require several months of exposure. Low sensitivity combined with small linear range makes the technique extremely time consuming, especially when optimal assay conditions have to be determined first^1,35.

With the development of phosphor imaging plates, several of these limitations have been addressed^35,36,37. The imaging plates serve to temporarily store images of radioactive decay, representing the spatial arrangement of radioligand in the tissue specimen. Photostimulable BaFBr:Eu²⁺ phosphor crystals are used to capture the radioactive energy emitted by the sample, as high energy radiation (e.g., X-rays, gamma rays or beta particles) results in the excitation of Eu²⁺ to Eu³⁺ and consequent trapping of the released electron in the phosphor lattice^4,37. Exposing the imaging plate to visible or infrared light reverses the reaction, i.e., the trapped electron is released and during the transformation of Eu³⁺ to Eu²⁺ luminescence is emitted. The emitted light is proportional to the amount of radioactivity and its detection by a photomultiplier enables the creation of a digital autoradiogram³⁷. This system provides an increase in sensitivity accompanied by a marked reduction in exposure time from month to days³. On top of that, the linear dynamic range is considerably increased, which reduces the chance of oversaturated images. Linearity is given within four to five orders of magnitude and has been validated repeatedly^{3,35,36,37,38}. Although film autoradiography still provides superior spatial resolution, efforts in scanning technology resulted in the improvement of resolution from 300 to 25 µm (pixel size), allowing the detailed differentiation between anatomical regions³. Overall, phosphor imaging plates are facilitating the acquirement of digital autoradiograms both due to an increased linear range and sensitivity. Reduced exposure time and a simplified development technique significantly lead to decreased time for data analysis allowing higher throughput.

Compared to autoradiography, useful pharmacological parameters such as affinity and density are also commonly characterized by the application of radioligands in tissue homogenate binding assays. This method has the advantage of producing results by measuring β-emitted radioactive decay with a liquid scintillation detector in an efficient manner². Being performed in a multi-well approach, e.g., in 96-well microtiter plates, such assays are useful for screening of libraries of compounds and for a larger number of concentration-dependent relationships. On top of that, conducting saturation analysis with this setup is often more feasible compared to autoradiography, which displays a risk of oversaturated images with high radioligand concentrations. However, performing homologous displacement of a fixed low radioligand concentration with non-radioactive ligands in order to obtain K_d and B_max circumvents the problem of oversaturated images (Figure 6)²². Homogenate binding and autoradiography produce similar estimates for ligand affinity constants whereas tissue density of the protein of interest may be underestimated in homogenate binding. Thus, it has been proposed that cell membrane disruption concomitant with tissue homogenisation might result in receptor loss or altered binding conditions^3,33. Moreover, errors in tissue dissection might produce homogenates contaminated with tissue from adjacent brain regions. In comparison, even complex binding patterns in small nuclei are visualized and differentiable due to the spatial anatomical resolution in autoradiography^3,33.

Immunohistochemistry also visualizes the distribution of a protein of interest anatomically. The method is capable of producing images with high anatomical resolution, as discrete tissue components can be identified at cellular level and even subcellular levels using electron microscopy. Expression levels are assessed based on the intensity of the staining. Nonetheless, absolute quantification of expression levels is difficult due to the lack of appropriate reference standards³⁹. Moreover, immunohistochemistry is dependent on the availability of a selective, well-validated antibody which is often a problem in receptor research.

Before deciding on performing in vitro autoradiography, several considerations need to be made. First of all, post-mortem tissue preparation including sectioning as well as repeated freezing and thawing might influence the preservation of binding sites². Furthermore, the method depends on the availability of an adequate radioligand, which displays high affinity and selectivity for the target in question². The radioligand should not display significant binding to off-target sites, and it should also demonstrate a favourable kinetic profile. This is necessary because the ligand-target complex must stay intact during the scope of the experiment. Moreover, when suitable non-radioactive compounds exist, the introduction of the radionuclide might become a critical factor. Thus, the molecule of interest should be equipped with suitable functional groups for efficient radiolabelling, which enables production of radioligands with sufficiently high specific activity and chemical stability⁴⁰. Another disadvantage of in vitro autoradiography is that the method only allows the use of an animal once. More elegantly is the extension to in vivo imaging methods such as position emission tomography (PET), which enables repeated scanning of the same animal and determination of occupancy and dynamic binding characteristics. PET is especially valuable for the study of higher mammals⁴¹ and for dose optimization in pre-clinical studies ^42,43,44.

Several modifications of the autoradiographic technique extend its application both in terms of the characterization of pharmacological targets in healthy and diseased states as well as in drug discovery and development. First of all, recent advances in imaging technology have led to the development of real-time autoradiography. Gas detectors of α/β-particles obviate the need for imaging plates or film by the direct measurement of disintegrations, thereby producing fast digital autoradiograms⁴⁵.

Moreover, in vitro autoradiography enables studies of the functionality of G protein-coupled receptors (GPCRs) on top of information about their anatomical distribution in post-mortem tissues. This variant of the method involves incubation of tissue sections with a radioactively labelled analogue of guanosine triphosphate (GTP), i.e., [³⁵S]guanosine 5'-γ-thiotriphosphate ([³⁵S]GTPγS), together with a non-radioactive agonist of the GPCR. When the agonist binds and elicits a response of the GPCR, the incorporation of [³⁵S]GTPγS can be localized and quantified via autoradiography, which reflects only the activated receptor population^2,46,47.

Ex vivo autoradiography represents another version of the technique, which assesses the regional binding of a radioligand after administration to a live experimental animal. Following the sacrifice of the animal, cryosectioning of the tissue in question, and autoradiographic exposure result in autoradiograms which reflect the radioligand binding in vivo². Ex vivo autoradiography is commonly employed within drug discovery and development programs in order to gain information on the pharmacokinetic profile of a lead compound, i.e., its absorption, distribution, metabolism and excretion (ADME). Particularly whole-body autoradiography provides insight about drug distribution to all organs and tissues. However, determination of non-specific binding and quantification is more difficult compared to in vitro autoradiography due to possible metabolism and degradation of the radioligand and no means for washing away unbound ligand⁴⁸.

Autoradiography is also used for preliminary testing and characterization of PET ligands⁴. The high-energy radionuclides carbon-11 and fluorine-18 are most often used for PET ligands. PET is a prominent, non-invasive application for radioligands because quantifiable 3D images of the radioligand binding in a living animal can be obtained^11,40,49.

In vitro autoradiography using phosphor imaging plates represents a valuable assay method for the pharmacological characterization of ligand-target interactions. The method produces reproducible results by the employment of a relatively fast and simple protocol once optimal assay conditions have been established. Anatomical distribution of a protein of interest is determined within its native microenvironment, which allows the study of its physiological, pharmacological and pathological role in healthy and diseased post-mortem tissue of experimental animals as well as humans^2,47.