Activity-dependent expression and distribution of M1 muscarinic ACh receptors in visual cortex neuronal cultures

Abstract

The fluorescently labeled muscarinic M1 receptor-selective antagonist BODIPY® FL pirenzepine has been employed to study the activity-dependent distribution and expression of muscarinic M1 ACh receptors (M1AChRs) in cultured neurons derived from rat visual cortex. Displacement experiments showed that like pirenzepine, binding of BODIPY® FL pirenzepine was specific to M1 receptors and its K(i) was similar to that of unlabeled pirenzepine. Using confocal laser scanning microscopy, M1 receptors were predominantly localized to cell bodies early in development in the culture environment. After 2 weeks in culture, the receptors showed labeling not only in cell bodies but also in neuritic processes, especially on the initial segments of the processes. Chronic membrane depolarization with 40 mM potassium chloride caused a dramatic increase in M1 receptor expression on these neurons. Conversely, blockade of neural activity with 0.1 μM TTX decreased expression of the receptors. Receptor expression increased after cells were treated chronically with 50 nM pirenzepine, whereas it decreased after exposure to 10 μM carbachol. The results demonstrate for the first time the exact location of muscarinic receptors in living cultured neurons and also the activity- dependent expression of M1 receptors on these neurons. Both chronic membrane depolarization and antagonist application upregulate receptor expression, whereas blocking bioelectrical activity or chronic agonist application downregulates expression.