From January to December, 1984, 3 sets of experiments will be performed. The first set comprises completion of the melatonin uptake, thin-layer chromatography (TLC) and ultrastructural studies done during the first 9 months of the TIDA award. To complete the uptake studies, the data will be reduced in the anatomic regions not yet analyzed, including levels of spinal cord other than lumbar. Any differences in uptake depending on time of injection within the photoperiod will be determined. Besides %whole blood and dpm/mg, results will also be expressed as %dose administered, and statistical significance will be obtained. To complete the TLC experiments, the two-dimensional chromatography method will be used, since this technique should provide better detection and separation of melatonin metabolites than the one-dimensional procedure we have used. Points other than the 10 minute time point will be assessed, to try to detect a higher % of metabolites. The assessment of thin sections of lumbar cord, sciatic nerve, midbrain, hypothalamus, pituitary, and pineal in the 4 main treatment categories will be completed for the ultrastructural experiments. The second set of experiments will be performed in vitro to determine the direct effects of melatonin on fetal mouse brain and spinal cord explants. Timed pregnancies will be generated in our colony, and tissue obtained at the 13-14th day of gestation. In the first group of manipulations, various dilutions of melatonin will be added to the culture media for 15, 30, 60 or 120 minutes (acute group). In the second group, cultures will be chronically treated with these same dilutions daily for 2 weeks to 2 months. Protein and RNA synthesis will be measured, and any ultrastructural changes assessed. The third set of experiments will be to determine the effects of melatonin on axonal transport. The gastrocnemius muscle of 3 to 6 month old mice will be injected with horseradish peroxidase (HRP), and the mice killed by intravascular perfusion at 6, 24 and 48 hours post-injection. The distribution of reaction product in distal and proximal sciatic nerve segments and anterior horn cells of the sciatic motor nucleus will be assessed at these time intervals by light and electron microscopy. In the acute experiments, mice will be injected intraperitioneally (ip) with 50 ug melatonin 5, 10 or 20 minutes prior to the HRP injection. In the chronic experiments, mice will be injected (ip) with 50 ug melatonin daily for up to 7 weeks, prior to the HRP injection.
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