Measuring neurotransmitter receptors in the brain

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 nonspecifically 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.The number of receptors in a tissue preparation may be determined by plotting the ratio of the bound to free radioligand against the total boundligand.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 and the other to which the ligand binds with low affinity and high capacity.
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 abinding 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 nervemembrane 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, antagonismof alpha 1 adrenoceptors, histamine1 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.