Microvessels Isolated from Brain: Localization of Muscarinic Sites by Radioligand Binding and Immunofluorescent Techniques

Abstract

The present investigation was carried out to determine the extent to which muscarinic acetylcholine receptors (mAChRs) in vascular and perivascular structures were colocalized with glial fibrillary acidic protein (GFAP)–positive structures. To this aim, an immunocytochemical approach on free-floating cryosections and isolated microvessels obtained from rat brain was performed to study the possible colocalization of immunostaining with the anti-mAChR protein antibody (M35) and an anti-GFAP antibody. Double-labeling experiments were carried out by fluorescent techniques. Confocal microscopic observations of GFAP and M35 immunoreactivities on free-floating sections showed a high degree of colocalization on astrocyte processes associated with large vessels or capillaries. This pattern suggests that muscarinic receptors are associated with astrocytic endfeet. Confocal microscopic observations of immunoreactivity from isolated cerebral microvessels strengthen this conclusion since double-labeling of M35 and GFAP showed that perivascular astrocytic structures remained attached to the isolated microvessels and were present on vascular segments showing M35 immunoreactivity. In another set of experiments, the specific binding of [3H]quinuclidinylbenzylate ([3H]QNB) to isolated microvessel membrane preparations from cerebral cortex, caudate nucleus, thalamus, and cerebellum showed that a constant binding yield (20% in bovine and 40% in rat) was observed for microvessels compared with the corresponding brain region. According to our immunocytochemical results, the astrocytic membrane remaining attached to microvessels may account for the majority of the muscarinic binding to isolated microvessels. [3H]QNB binding values found in isolated microvessels cannot therefore be considered as artifacts without any link with vascular function. Taken together, the present study strengthens the idea that the muscarinic receptors may be implicated in the functional relationship between glial and vascular structures.