Autoradiography Assessment of Muscarinic Receptors in the Central Nervous System with a Special Focus on the Selectivity for M1 and M2 Muscarinic Receptors: Specific Protocol for M1 Muscarinic Receptors Labeling

The detection of muscarinic receptor binding sites is a crucial step in many experimental conditions. Despite radioligand binding to cell membranes derived from the peripheral tissues as well as large portions of some central nervous system regions (see Chapter 1 ) allowing to obtain well-defined receptor characteristics, receptor autoradiography is the preferred choice in exploring receptor density and distribution in the brain as it provides high spatial resolution and capability to detect infinitesimally small receptor changes. The development of autoradiography has made important progress, and some different modes (phosphor imaging) are used nowadays. Here, we describe standard autoradiography protocols for labeling of brain muscarinic receptors with different radioligands: [3H]-QNB and [3H]-NMS, [3H]-pirenzepine and [3H]-AFDX-384. According to our previous data, we have shown that [3H]-pirenzepine is highly specific for M1 muscarinic receptors and here we provide the protocol for detection of this muscarinic receptor subtype. [3H]-AFDX-384, however, did not show sufficient specificity for M2 muscarinic receptors and represents binding to M2/M4. Specific attention is paid to the muscarinic subtype detection and the limits are emphasized.