Department of Neurosciences and Addiction Studies, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Horm Behav. 2024 Aug;164:105599. doi: 10.1016/j.yhbeh.2024.105599. Epub 2024 Jul 3.
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 F-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.
褪黑素是一种多功能神经激素，在松果体以外的组织如海马中合成。海马褪黑素合成的关键酶是芳基烷基胺-N-乙酰转移酶（AANAT）。海马褪黑素合成的重要性尚未确定。我们使用基因沉默小干扰 RNA（siRNA）技术研究了海马 AANAT 在认知功能中的作用。通过 AANAT-siRNA 注射抑制海马局部褪黑素合成。通过 RT-PCR 技术获得 AANAT-siRNA 的时间基因沉默谱。MTT 测定法在 B65 神经细胞上测定 siRNA 剂量的细胞毒性。动物接受选定剂量的 AANAT-siRNA。然后，评估空间工作记忆（Y 迷宫）、物体识别记忆和空间参照记忆（Morris 水迷宫，MWM）。通过高架十字迷宫评估焦虑样行为。一周后，在 MWM 探针测试后，处死大鼠进行组织学分析。使用液相色谱-质谱技术测量海马褪黑素水平。AANAT-siRNA 组的海马褪黑素水平降低。接受 AANAT-siRNA 的动物在空间学习和工作记忆方面表现出缺陷，这分别通过逃逸潜伏期增加和自发交替减少得到验证。AANAT-siRNA 组的焦虑样行为增加和识别记忆缺陷。尼氏染色和活化 caspase-3 的免疫组织化学显示 AANAT-siRNA 组海马组织中的神经元丢失和细胞凋亡。F-FDG-PET 成像显示 AANAT mRNA 减少后葡萄糖代谢降低。数据表明，AANAT mRNA 和海马褪黑素合成可能是学习、记忆和认知某些方面以及海马细胞内稳态的重要因素。
