Laser scanning and electron microscopic evidence for rapid and specific in vivo labelling of cholinergic neurons in the rat basal forebrain with fluorochromated antibodies

Abstract

Recently developed methods for the selective labelling of cholinergic basal forebrain neurons containing the low-affinity neurotrophin receptor p75 (p75 NTR) in vivo and in vitro are based on carbocyanine 3 (Cy3)-tagged antibodies directed against p75 NTR. The present study focuses on the maintenance of this neuronal label after injection of such fluorescent antibodies into the cerebral ventricle. One, 3, and 10 days after injection this marker exclusively stains neurons immunoreactive for the cholinergic markers choline acetyltransferase and vesicular acetylcholine transporter in the rat medial septum, diagonal band and nucleus basalis. Thirty days after injection the in vivo labelling was nearly abolished. Predominant labelling of lysosomes was shown by electron microscopic analysis following photoconversion of the Cy3-label to an electron-dense reaction product. The pre-labelling of cholinergic neurons might facilitate pharmacological and electrophysiological approaches in living slices and cell culture systems as well as detailed investigations focused on the transport of neurotrophins in vivo and in animals with experimentally altered p75 NTR expression.