THE SPREAD AND UPTAKE PATTERN OF INTRACEREBRALLY ADMINISTERED OLIGONUCLEOTIDES IN NERVE AND GLIAL-CELL POPULATIONS OF THE RAT-BRAIN

The fate of 15-mer phosphorothioate-modified antisense oligonucleotide s to c-fos was followed after their microinjection into rat brain. Usi ng radiolabeled oligonucleotides, it was demonstrated that the bulk of the material stays in the injected region but that a minor part is tr ansported with the projection pathways to regions far away from the si te of injection, Using tetramethylrhodamine-isothiocyanate (TRITC) lab eling as well as fluorescein isothiocyanate (FITC) labeling, it was fo und that the oligonucleotides were taken up by a great number of cells within 30 minutes after the injection. A diffuse cytoplasmic staining and also nuclear staining were observed in these cells, which could b e identified exclusively as neurons by double labeling for the neuron- specific protein NeuN, At later times (6, 24, and 48 hours), the appea rance of the oligonucleotides changed gradually to a punctate cytoplas mic staining, which by electron microscopic analysis was shown to be c aused by the presence of the oligonucleotides in intracellular vesicle s. The pattern of intracellular fluorescence was changed when the olig onucleotides were injected together with the cationic lipid 1,2-bis(ol eoyloxy)-3-(trimethylammonio)propane (DOTAP), A small number of astroc ytes and microglial cells were found to be labeled by the oligonucleot ides, but only at later times after the injection and exclusively in a punctate cytoplasmic manner. Thus, the uptake of oligonucleotides in the nerve and glial cell populations of the brain might involve differ ent mechanisms, the one in the neurons appearing to be very rapid and potent.