Measuring neurotransmitter receptors in the brain

Little was known about the identity of neurotransmitter receptors in the brain until the early 1970s when several laboratories independently reported that a potent snake venom, alpha-bungarotoxin, could bind with high affinity to nicotinic receptors that occurred in the electric organs of certain species of fish. This laid the basis for ligand receptor binding studies. Such studies rely on the use of radiolabelled (generally with tritium or carbon-14) drugs or chemicals which have a high affinity for a specific receptor. Such ligands may be either agonists or antagonists that bind to the receptor, thereby enabling the number of receptors on a tissue to be determined by measuring the quantity of radioactive ligand that has been specifically bound.

In practice, pieces of brain tissue (e.g. membrane preparations or crude tissue homogenates) are incubated with the radioligand in a physiological buffer solution. The tissue is then filtered or centrifuged to separate the tissue from the incubation medium. The quantity of ligand bound to the tissue can then be estimated by solubilizing the tissue and counting the radioactivity in a liquid scintillation counter. As most radioligands also bind non-specifically to brain tissue, to the walls of the incubation tube and even to the filters used to separate the tissue from the incubation medium, it is essential to determine the amount of radioligand bound specifically to the receptor. This is done by incubating the tissue preparation in tubes containing the specific radioligand alone and also with the ligand together with another drug or compound which is not radioactively labelled but which also binds with high affinity to the same receptor. For example, to study the number of beta adrenoceptors in a brain or lymphocyte preparation, tritiated dihydroalprenolol ([3H]DHA) is used as the radioligand and DL-propranolol as the non-radioactive displacing agent. Thus propranolol will tend to displace all of the [3H]DHA bound to the beta receptor but is less effective in displacing any radioligand that is bound non-specifically, and possibly irreversibly, to other types of receptor or to non-receptor sites. The amount of radioactivity present in the tissue preparation that has been incubated with the radioligand alone and that remaining after the specifically bound ligand has been displaced by propranolol is then counted. The difference between the total (i.e. the radioactivity in the tube containing the radioligand alone) and the non-specifically bound activity (i.e. the radioactivity in the tube containing the radioligand and the displacing agent) gives a measure of the amount of radioactivity specifically bound to the beta adrenoceptor. This is illustrated in Figure 2.5.

The number of receptors in a tissue preparation may be determined by plotting the ratio of the bound to free radioligand against the total bound ligand. For one population of receptors in a tissue, this plot yields a straight line, the number of binding sites (termed the Bmax) being determined from the point of intersection of the y axis. This is illustrated in Figure 2.6.

This method of expressing the results of radioligand binding assays is known as a Scatchard plot, after the chemist who used it to study the binding of small molecules to proteins. A non-linear Scatchard plot often implies that there are two or more binding sites, one of these sites to which the ligand binds with high affinity and low capacity (shown as Bmax1 in Figure 2.7) and the other to which the ligand binds with low affinity and high capacity (shown as Bmax2 in Figure 2.7).

The affinity of a ligand for a receptor can be calculated from the slope of the plot, 1/slope being known as the Kd value or the binding affinity.

Thus by means of this relatively simple technique it is possible to determine the number of binding sites in a piece of brain tissue, their homogeneity and the affinity of the ligand for these sites. This enables changes in the density of specific receptors to be determined following drug treatment or as a result of disease. However, it must be emphasized that a binding site for a radioligand is not necessarily a receptor. To classify a

Figure 2.5. Diagrammatic representation of the binding of a radioactive ligand to a membrane preparation.

Figure 2.5. Diagrammatic representation of the binding of a radioactive ligand to a membrane preparation.


Total bound Total free


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