Sense- and antisense-targeting of oxytocinergic systems in the rat hypothalamus

3' phosphorothioate modified sense or antisense oligonucleotides to oxytocin transcripts were used for in vivo targeting of oxytocin (OT) neurons in the rat hypothalamus. Intracerebroventricular injections of antisense probe resulted in a loss of systemic OT. However, abundant immunoreactive OT as well as oxytocin mRNA hybridization signal was visualized in the hypothalamo neurohypophysial system (HNS) of these animals. RT-PCR of hypothalamic homogenates revealed clearly detectable amounts of cytoplasmic OT mRNA in spite of sense or antisense treatment. Immunostaining with an antibody to DNA-RNA triple helix resulted in cytoplasmic reaction product in the HNS in the antisense group, which was not found when tissue sections had been pretreated with RNase. Animals injected with the sense probe showed a less pronounced but significant loss of systemic OT while immunoreactivity for this peptide in the posterior lobe seemed to be unaffected. RT-PCR of OT encoding mRNA extracted from sense injected rats indicated that these transcripts were of smaller size than samples from antisense treated animals or controls. Immunostaining with the triple helix antibody revealed distinct immunoreactive dots in cellular nuclei throughout the brain in the sense group. Our findings suggest that sense and antisense probes may not readily be employed as "functional antagonists" since peptidergic neurons are probably capable of responding in various ways to the treatment. RNase H may be less important in hypothalamic neurons as commonly suggested. Targeted transcripts are likely to form complexes which may somehow interact with secretion. Triple helix formation in the nucleus may not be able to induce an efficient transcriptional arrest. Although endocrine and behavioral changes observed in antisense treated animals seem to confirm the hypothesis that a selective translational "knock out" can be achieved with in vivo hybridization strategies, the actual underlying molecular events are far from being understood. On the other hand, sense or antisense strategies may provide valuable insights into molecular and cellular events associated with neurosecretion.