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Total bound ligand

Figure 2.6. Scatchard plot of the binding of a radioligand to a membrane preparation containing two binding sites. In this diagram, Bmax1 represents a high affinity (Kd1) low capacity site, whereas Bmax2 represents a low affinity (Kd2) high capacity site.

binding site as a receptor it is essential to show that the binding site is linked to an ion channel or secondary messenger system or that an electrophysiological response occurs as a direct consequence of the activation of the binding site. It is possible, for example, that the ligand binds to a portion of the nerve membrane that is not involved in neurotransmission. Following the discovery that tritiated benzodiazepines bind with high specificity to nerve membranes it took several years of further research to show that occupation of the benzodiazepine-binding site could lead to the enhanced sensitivity of the GABA-A receptor to the effects of GABA. Only when the functional activity of the benzodiazepine receptor was established could the binding site be justifiably called a receptor site.

The application of ligand binding techniques to the quantification of receptor sites in the brain has had important implications for psychopharmacology. It has now been possible to correlate the therapeutic potencies of some drugs with their receptor occupancy. Neuroleptics are known to block dopamine receptors in the brain. By studying the binding of a series of neuroleptics to the different types of dopamine receptor in nerve membrane preparations, it has been found that there is a good correlation between the occupancy of the D2 receptor subtype and the therapeutic potency. There does not appear to be a direct correlation between the binding of these drugs to adrenoceptors, histamine, 5-HT or acetylcholine receptors and their therapeutic potency. However, by considering the interaction of psychotropic drugs with these various receptors, it is possible to predict their side effects. For example, antagonism

Figure 2.7. Method used to determine the receptor number and the ligand affinity for the receptor by the Scatchard plot.

of alphaj adrenoceptors, histaminei and muscarinic receptors is associated with postural hypotension, sedation and anticholinergic side effects respectively. Thus by using this relatively simple technique it is possible to gain an insight into the site(s) of action of most classes of psychotropic drug and to predict with reasonable accuracy what their side effects will be. Whether the receptor affinity of a drug in vitro necessarily provides information about its mode of action in the brain is quite another issue.

Quantitative autoradiography

In this method, thin tissue sections of the brain are incubated with a specific radioligand, the unbound ligand removed by washing and the resulting tissue section placed on a sensitive photographic film. The sites where the radioligand binds to the tissue fog the film, and following its development grain counts, densitometric analysis and photometric techniques can be used to quantify the extent of the binding of the radioligand to specific cell structures. More recently, the application of computerized image analysis has simplified the problem of visualization and quantification. The system most widely used consists of a television camera linked to an IBM personal computer. This system can colour code images to enhance contrast, modify images, subtract one image from another and average densities in specific regions of the visual field. The ability of this system to include and process autoradiographic standards enables the density of the radioligand binding to be quantified. Finally, the system can tabulate, store and calculate the Bmax and Kd values of the radioligand.

One of the most important uses of autoradiography in psychopharma-cology lies in enabling the sites in the brain where a drug acts to be identified. For example, the brain region or specific neuronal circuit that is affected by a drug may be visualized. Since most psychotropic drugs have multiple effects in the brain, autoradiographic methods have helped to explain the complexity of the effects by identifying the receptors and their distribution in the brain. Changes in receptor density may reflect neuronal function. Receptor mapping has therefore been applied to biopsy and autopsy samples from patients with Parkinsonism, Alzheimer's disease, schizophrenia and depression. The results of such studies have already begun to throw light on the possible biochemical changes that underlie such diseases. There are practical limitations to the autoradiographic technique however. The resolution using the light microscope is limited and it is seldom possible to readily identify specific cell structures that contain the receptors or to distinguish between functional and non-functional receptors to which the ligand is bound. It should be emphasized that this is also a problem with conventional radioligand techniques as applied to membrane preparations or to tissue homogenates. To overcome the problem of resolution, electron microscopic autoradiography may eventually prove to be of value, particularly when this is combined with immunohistochemical techniques to improve the resolution.

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