Regional distribution of receptors

Radioligand binding on membrane preparations provides information concerning the interaction between drugs and well-defined receptors, as well as about the occurrence of such receptors in certain tissues or organs. However, most tissues and organs are very complex and comprise a number of different cell types, each with their own receptor and response specificity. In this context, the brain is especially complex since it contains a great number of different neurones. Yet, neurones that are responsible for very specific brain functions are often confined to small regions (nuclei) of the brain. Information about the regional distribution of receptors in complex tissues such as the brain therefore contributes to our understanding of their physiological role and their implication in certain pathophysiological conditions. However, radioligand binding experiments on membrane preparations provide only a little information: the resolution is limited by the resolution of the dissection. Autoradiography of radiolig-and binding to thin sections of brain allows the localization of receptors with a much higher degree of resolution (i.e. to the light microscopic level) (Figure 48). However, the resolution is still insufficient to make a distinction between pre- and postsynaptic receptors.

Figure 48 Classical technique for autoradiographic detection of receptors on thin tissue section. Steps: 1) incubation with radioligand, 2) wash, 3) put in casette, 4) overlay film, 5) close cover and expose, 6) remove film and develop.

Figure 49 Autoradiography of [3H]idazoxan binding to imidazoline binding sites (I2-receptors) in a cross section of the human medulla: left: grey scale, right: adopted colours. Reprinted from Progress in Histochemistry and Cytochemistry 26, De Vos, H., De Backer, J.-P., Convents, A., De Keyser, J. and Vauquelin, G., Identification of alpha2 adrenoceptors in the human nucleus olivarius by radioligand binding, 259-265. Copyright (1992), with permission from Elsevier.

Figure 49 Autoradiography of [3H]idazoxan binding to imidazoline binding sites (I2-receptors) in a cross section of the human medulla: left: grey scale, right: adopted colours. Reprinted from Progress in Histochemistry and Cytochemistry 26, De Vos, H., De Backer, J.-P., Convents, A., De Keyser, J. and Vauquelin, G., Identification of alpha2 adrenoceptors in the human nucleus olivarius by radioligand binding, 259-265. Copyright (1992), with permission from Elsevier.

For this approach, tissues are cut in thin (usually not exceeding 10 ^M) slices and put on coverslips. The slices are subsequently incubated with radioligand and washed. The radioactivity on the slices can then be determined by apposition of a sensitive film or by dipping the slice in a photographic emulsion. After exposure (from a few hours for [125I]-labelled ligands to a few weeks for [3H]-labelled ligands), the film or emulsion is developed. Black corresponds to a high density of radioligand (Figure 49, left). Newer detection techniques are available for [3H]-labelled ligands.

High-density detectors (e.g. lead)

Figure 50 Principle of PET scan. Reprinted from Trends in Pharmacological Sciences, 22, Reader, A.J. and Zweit, J., Developments in whole-body molecular imaging of live subjects, 604-607, © (2001), with permission from Elsevier.

High-density detectors (e.g. lead)

Figure 50 Principle of PET scan. Reprinted from Trends in Pharmacological Sciences, 22, Reader, A.J. and Zweit, J., Developments in whole-body molecular imaging of live subjects, 604-607, © (2001), with permission from Elsevier.

They rely on the apposition of a solid scintillator sheet and real-time imaging of the emitted photons. This brings down the exposure time to hours instead of days and weeks. Commercially available radioactive standards can be exposed along with the tissue sections and this allows the conversion of the different grey levels into binding values. The amount of radioligand bound to each location of the slice can therefore be quantified by densitometry. Computer-assisted image analysis greatly facilitates this task and it also allows different grey levels to be replaced by specific colours (Figure 49).

Finally, the in vivo determination of the regional distribution of receptors in e.g. the brain can be obtained by the PET scan technique (Figure 50). 'PET' stands for positron emission tomography and involves the administration of a positron emitting radioligand to a patient. The radioligand will accumulate in receptor-rich areas and the emitted positrons will, upon encounter with electrons, annihilate to produce gamma rays, which can be detected. This technique is fairly safe for the patient since the radioactive half-life is very short (in the order of minutes) but, on average, the positrons will only meet electrons a few millimetres away from their point of departure so that the degree of resolution is rather poor (Figure 51).

Figure 51 Distribution of 5-HT2 receptors in the brain. Accumulation of positron emitting radioligand ([18F]-setoperone) in brain 'sections' in untreated (total) and Ziprosidone-treated (non-specific accumulation) humans. Reproduced from Fischman, A. J., Bonab, A. A., Babich, J. W., Alpert, N. M., Rauch, S. L., Elmaleh, D. R., Shoup, T. M., Williams, S. A. and Rubin, R. H. (1996) Positron emission tomographic analysis of central 5-hydroxytryptamine2 receptor occupancy in healthy volunteers treated with the novel antipsychotic agent, ziprasidone. Journal of Pharmacology and Experimental Therapeutics, 279, 939-947, with permission from the American Society for Pharmacology and Experimental Theraputics.

Figure 51 Distribution of 5-HT2 receptors in the brain. Accumulation of positron emitting radioligand ([18F]-setoperone) in brain 'sections' in untreated (total) and Ziprosidone-treated (non-specific accumulation) humans. Reproduced from Fischman, A. J., Bonab, A. A., Babich, J. W., Alpert, N. M., Rauch, S. L., Elmaleh, D. R., Shoup, T. M., Williams, S. A. and Rubin, R. H. (1996) Positron emission tomographic analysis of central 5-hydroxytryptamine2 receptor occupancy in healthy volunteers treated with the novel antipsychotic agent, ziprasidone. Journal of Pharmacology and Experimental Therapeutics, 279, 939-947, with permission from the American Society for Pharmacology and Experimental Theraputics.

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