SELECTIVE IN-VIVO FLUORESCENCE LABELING OF CHOLINERGIC NEURONS CONTAINING P75(NTR) IN THE RAT BASAL FOREBRAIN

The cholinergic system of the rat basal forebrain is used as a model f or the homologous region in humans which is highly susceptible to neur opathological alterations as in Alzheimer's disease. Cholinergic cells in the basal forebrain express the low-affinity neurotrophin receptor p75(NTR). This has been utilized for selective immunolesioning of cho linergic neurons after internalization of an immunotoxin composed of a nti-p75(NTR) and the ribosome-inactivating toxin saporin. However, the goal of many studies may be not the lesion, but the identification of cholinergic cells after other experimentally induced alterations in t he basal forebrain. Therefore, a novel cholinergic marker was prepared by conjugating the monoclonal antibody 192IgG directed against p75(NT R) with the bright red fluorochrome carbocyanine 3 (Cy3). Three days a fter intraventricular injection of Cy3-192IgG the fluorescence microsc opic analysis revealed a pattern of Cy3-labelled cells matching the di stribution of cholinergic neurons. Apparently the marker was internali zed within complexes of p75(NTR) and Cy3-192IgG which were then retrog radely transported to the cholinergic perikarya of the basal forebrain . In addition to the even labelling of somata, a strong punctate-like Cy3-immunofluorescence was seen in structures resembling lysosomes. Th e specificity of the in vivo staining was proven by subsequent immunol abelling of choline acetyltransferase (ChAT) with green fluorescent Cy 2-tagged secondary antibodies. In the medial septum, the diagonal band and the nucleus basalis only cholinergic neurons were marked by Cy3-1 92IgG. In parallel experiments, digoxigenylated 192IgG was not detecta ble within cholinergic basal forebrain neurons after intraventricular injection. Presumably, this modified antibody could not be internalize d. On the other hand, digoxigenylated 192IgC was found to be an excell ent immunocytochemical marker for p75(NTR) as shown by double labellin g including highly sensitive mouse antibodies directed against ChAT, B ased on the present findings, future applications of the apparently no n-toxic Cy3-192IgG and other antibodies for fluorescent in vivo and in vitro labelling are discussed. (C) 1998 Elsevier Science B.V. All rig hts reserved.