Name: autoradiography as a simple and powerful method for visualization and characterization of pharmacological targets
Uploaded: 2019-03-12T15:00:00
Description: Watch this Scientific Journal Video about Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets at JoVE.com

The work was supported by the Lundbeck Foundation (Grant R133-A12270) and the Novo Nordisk Foundation (Grant NNF0C0028664). The authors thank Dr. Ales&#780; Marek for the supply of [3H]radioligand.

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...

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...

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 (Kd) and density of binding sites (Bmax), as well as for determining the inhibition constant (Ki) of competing ligands1,2. 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 &#947;-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 rat8,9,10, 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 technique9,11. 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.

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

autoradiography as a simple and powerful method for visualization and characterization of pharmacological targets

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 (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) 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 &#947;-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.

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...

Watch this Scientific Journal Video about Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets at JoVE.com

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

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

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

Research

JoVE Journal

Neuroscience

A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously ...

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...