Intrastriatal and intraventricular injections of oligodeoxynucleotides in the rat brain: tissue penetration, intracellular distribution and c-fos antisense effects.
We have determined the time course, the spatial spread in brain tissue, and the intracellular distribution of biotin- and fluorescein-labeled phosphorothioate oligodeoxynucleotides (ODNs) following single injections into the rat striatum or the lateral ventricle. These time and space parameters were correlated with the ability of c-fos phosphorothioate antisense ODNs to suppress the induction of Fos protein by cocaine. A rapid and dose-dependent tissue penetration of labeled ODNs was observed following either intrastriatal or intraventricular injections of a constant sample volume. Inspection of tissue sections by confocal microscopy uncovered a distinct change in the intracellular disposition of labeled ODNs during the 24 h post-injection period. At 1, 6 and 12 h, the vast majority of the fluorescent signal was confined to the interstitial spaces throughout the zone penetrated by ODNs. Neuronal nuclei displayed faint labeling along the outer portion of the nucleus at 1 and 6 h post-injection. At these time-points, ODNs were not detected in the cytoplasm. By 16 h, ODNs were barely detectable in the extracellular space and absent from neuronal nuclei. Instead, ODNs were seen in large cytoplasmic granules of neurons throughout the tissue zone penetrated by the ODNs. Experiments with intrastriatal injections of antisense ODNs to c-fos mRNA revealed Fos suppression between 3 and 12 h, but not at 16 and 24 h. This combined analysis has revealed that (1) restricted tissue penetration by ODNs limits their antisense effects on protein expression, and (2) depletion of extracellular ODNs and sequestration of c-fos antisense ODNs into large intracellular granules coincides with the loss of their biological activity.
Grzanna
R
R
The DuPont Pharmaceuticals Company, Central Nervous System Diseases Research, Experimental Station E-400-4430, Wilmington, DE 19880-0400, USA. reinhard.grzanna@dupontpharma.com
Dubin
J R
JR
Dent
G W
GW
Ji
Z
Z
Zhang
W
W
Ho
S P
SP
Hartig
P R
PR
D000818:Animals; D016375:Antisense Elements (Genetics) / Q000494:pharmacology; D001710:Biotin; D001923:Brain Chemistry / Q000502:physiology; D003342:Corpus Striatum / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000502:physiology; D018014:Gene Transfer Techniques; D007276:Injections, Intraventricular; D008297:Male; D018613:Microscopy, Confocal; D009474:Neurons / Q000737:chemistry / Q000502:physiology; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D016375:Antisense Elements (Genetics); D009838:Oligodeoxyribonucleotides; D016760:Proto-Oncogene Proteins c-fos; D001710:Biotin