Inhibition of hippocampal melatonin synthesis by siRNA induced learning and memory deficits in male rats.

ABSTRACT

Melatonin, the multi-functional neurohormone, is synthesized in the extra-pineal tissues such as the hippocampus. The key enzyme in hippocampal melatonin synthesis is arylalkylamine-N-acetyltransferase (AANAT). The importance of melatonin synthesis in the hippocampus has not yet been determined. We investigated hippocampal AANAT role in cognitive function using gene silencing small interference RNA (siRNA) technology. The hippocampal local melatonin synthesis was inhibited by AANAT-siRNA injection. The time-gene silencing profile of AANAT-siRNA was obtained by RT-PCR technique. The cytotoxicity of siRNA dose was determined by MTT assay on the B65 neural cells. Animals received the selected dosage of AANAT-siRNA. Then, the spatial working memory (Y maze), object recognition memory and spatial reference memory (Morris's water maze, MWM) were evaluated. The anxiety-like behaviors were evaluated by the elevated plus maze. After one week, following the probe test of MWM, the rats were sacrificed for histological analysis. The hippocampal melatonin levels were measured using the liquid chromatography-mass spectrometry technique. The hippocampal melatonin levels in the AANAT-siRNA group decreased. Animals receiving the AANAT-siRNA showed deficits in spatial learning and working memory which were verified by increased escape latency and reduced spontaneous alternations, respectively. There was an increase in anxiety-like behaviors as well as a deficit in recognition memory in the AANAT-siRNA group. The Nissl staining and immunohistochemistry of activated caspase-3 showed the neuronal loss and cell apoptosis in hippocampal tissue of the AANAT-siRNA group. The 18F-FDG-PET imaging displayed lower glucose metabolism following the reduction in AANAT mRNA. Data suggest that the AANAT mRNA and hippocampal melatonin synthesis might be an essential factor for learning, memory and some aspects of cognition, as well as homeostasis of hippocampal cells.