The 5-HT1A Receptor and Brain Development

描述（由申请人提供）：5-羟色胺1A受体（5-HT1A-R）特异性配体治疗抑郁、焦虑和许多相关症状的疗效促使人们对该神经递质受体的生理意义和信号传导机制进行研究。这些研究表明，前脑中这种受体的缺失，特别是在小鼠出生后发育的早期，会导致焦虑水平升高。申请人实验室的研究表明，在海马神经元来源的细胞系中刺激这种受体可以保护这些细胞免受凋亡。这种保护机制涉及刺激丝裂原活化蛋白激酶（MAPK）途径，该途径反过来通过蛋白激酶C α (PKCc0)发出信号，引起促凋亡蛋白caspase-3的抑制。由于正常的情绪状态是前脑各区域（如海马、杏仁核和前额叶皮质）之间适当的神经元连接的产物，先前的观察表明5-HT1A-R在这些大脑区域神经元的分裂和成熟中起重要作用。目前的提议将通过使用培养的海马切片以及5-HT1A-R（+/+）和5-HT1A-R（-/-）小鼠来验证这一假设。在出生后5、10、15和20天培养的小鼠海马切片将用5- HT1A激动剂8-羟基-2（二正丙基氨基）四氢萘（8-OH-DPAT）和免疫组织化学技术处理，用于研究刺激关键蛋白，如PKC(x)、MAPK和CREB，促进神经元分裂、成熟和存活。进行此类信号研究的一个重要目标是阐明5-HT1A-R信号的年龄依赖性调节及其对神经元细胞分裂（通过BrdU掺入测量）和成熟（通过MAP-2和突触素染色测量）的影响。凋亡的调节将通过使用抗活性caspase-3抗体（记录caspase-3的激活）和脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）（监测凋亡相关的DNA片段）来测试。同时，MAPK、CREB、PKCcq和传递MAPK信号的下游转录因子（如elk - 1）的激活以及caspase-3的抑制将通过使用海马切片裂解物的Western blot分析来测量。从处理过的切片中制备的RNA将被反向转录以制备cDNA探针用于微阵列分析，以测试基因表达的调节，特别是细胞骨架相关蛋白Arc和即时早期蛋白如Fos和jun。类似的研究将通过将8-OH-DPAT注射到5-HT1A-R（+/+）和5-HT1A-R（-/-）小鼠幼鼠（d5, dl0, d15, d20）中进行，并制备大脑冷冻切片以监测MAPK， CREB，PKCcq以及这种5-HT1A-R信号对海马及相关区域神经元细胞分裂和成熟的影响。从5-HT1A-R（+/+）和5-HT_A-R（-/-）小鼠幼崽中获得的结果将有助于描述5-HT1A-R信号传导的具体作用，并为5-HT1A-R在大脑发育中的机制作用提供新的思路。

DESCRIPTION (provided by applicant): The efficacy of the serotonin 1A receptor (5-HT1A-R)-specific ligands in treating depression, anxiety, and many related symptoms has prompted studies into the physiological significance and signaling mechanisms of this neurotransmitter receptor. Such studies have shown that the absence of this receptor in the forebrain, specifically during early postnatal development of mice, results in elevated anxiety levels. Studies in the applicant's laboratory have shown that stimulation of this receptor in a hippocampal neuron-derived cell line causes protection of these cells against apoptosis. The mechanism responsible for this protection involves stimulation of the mitogen activated protein kinase (MAPK) pathway, which in turn signals through protein kinase C alpha (PKCc0 to cause inhibition of the proapoptotic protein caspase-3. Since the normal emotional state is a product of appropriate neuronal connections among the various regions of the fore brain, such as hippocampus, amygdala, and prefrontal cortex, preceding observations suggest that the 5-HT1A-R plays an important role in division and maturation of neurons in these brain regions. The current proposal will test this hypothesis by using both cultured hippocampal slices as well as 5-HT1A-R (+/+) and 5-HT1A-R (-/-) mice. Cultured hippocampal slices from mice at post-natal days 5, 10, 15, and 20 will be treated with the 5- HT1A agonist 8-hydroxy-2 (di-n-propylamino) tetralin (8-OH-DPAT) and immunohistochemical techniques used to study stimulation of key proteins, like PKC(x, MAPK and CREB, that promote neuronal division as well as maturation and survival. An important objective of performing such signaling studies will be to elucidate the profile of age-dependent regulation of 5-HT1A-R signaling and its effect on division (measured by BrdU incorporation) and maturation (measured by MAP-2 and synaptophysin staining) of neuronal cells. Regulation of apoptosis will be tested by using an anti-active caspase-3 antibody (to record activation of caspase-3) and deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (to monitor apoptosis-associated DNA fragmentation). In parallel, the activation of MAPK, CREB, PKCcq and downstream transcription factors that relay the MAPK signals (like Elk-l), as well as the inhibition of caspase-3 will be measured by Western blot analysis using lysates of the hippocampal slices. RNA prepared from the treated slices will be reverse transcribed to prepare cDNA probes for microarray analysis to test for the regulation of gene expression, especially of the cytoskeleton-associated protein Arc and the immediate early proteins like Fos and Jun. Similar studies will be carried out by injecting 8-OH-DPAT into 5-HT1A-R (+/+) and 5-HT1A-R (-/-) mouse pups (d5, dl0, d15, d20) and preparing cryosections of brains to monitor activation of MAPK, CREB, PKCcq and also the effect of such 5-HT1A-R signaling on division and maturation of neuronal cells in the hippocampus and associated areas. Results obtained from 5-HT1A-R (+/+) and 5-HT_A-R (-/-) mouse pups will help delineate the specific effects of 5-HTtA-R signaling and shed new light on the mechanistic role of the 5-HT1A-R in brain development.