MICROVESSELS ISOLATED FROM BRAIN - LOCALIZATION OF MUSCARINIC SITES BY RADIOLIGAND BINDING AND IMMUNOFLUORESCENT TECHNIQUES

The present investigation was carried out to determine the extent to w hich muscarinic acetylcholine receptors (mAChRs) in vascular and periv ascular structures were colocalized with glial fibrillary acidic prote in (GFAP)-positive structures. To this aim, an immunocytochemical appr oach on free-floating cryosections and isolated microvessels obtained from rat brain was performed to study the possible colocalization of i mmunostaining with the anti-mAChR protein antibody (M35) and an anti-G FAP antibody. Double-labeling experiments were carried out by fluoresc ent techniques. Confocal microscopic observations of GFAP and M35 immu noreactivities on free-floating sections showed a high degree of coloc alization on astrocyte processes associated with large vessels or capi llaries. This pattern suggests that muscarinic receptors are associate d with astrocytic endfeet. Confocal microscopic observations of immuno reactivity from isolated cerebral microvessels strengthen this conclus ion since double-labeling of M35 and GFAP showed that perivascular ast rocytic structures remained attached to the isolated microvessels and were present on vascular segments showing M35 immunoreactivity. In ano ther set of experiments, the specific binding of [H-3]quinuclidinylben zylate ([H-3]QNB) to isolated microvessel membrane preparations from c erebral cortex, caudate nucleus, thalamus, and cerebellum showed that a constant binding yield (20% in bovine and 40% in rat) was observed f or microvessels compared with the corresponding brain region. Accordin g to our immunocytochemical results, the astrocytic membrane remaining attached to microvessels may account for the majority of the muscarin ic binding to isolated microvessels. [H-3]QNB binding values found in isolated microvessels cannot therefore be considered as artifacts with out 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.