The 5-HT1A Receptor and Brain Development

5-HT1A 受体和大脑发育

• 批准号: 7445477
• 负责人: PROBAL BANERJEE
• 金额: $ 1.08万
• 依托单位: COLLEGE OF STATEN ISLAND
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-18 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7445477
• 关键词: Affect; Agonist; Alcoholism; Alzheimer' s Disease; Antibodies; Anxiety; Apoptotic; Autoreceptors; Behavior; Binding; Biological Assay; Brain; Brain Diseases; Bromodeoxyuridine; CREB1 gene; Cell Death Inhibition; Cell Line; Cell Proliferation; Cell division; Cells; Cocaine; Cocaine Abuse; Cyclin-Dependent Kinase 4 Inhibitor B; DNA Nucleotidylexotransferase; Data; Development; Differentiation Antigens; Disease; Employee Strikes; Endopeptidases; Hippocampus (Brain); Immunohistochemistry; In Vitro; Isoenzymes; Knowledge; Label; Ligands; Light; Link; Measurement; Measures; Mediating; Mental Depression; Mitogen-Activated Protein Kinases; Mitotic; Molecular; Monitor; Moods; Mus; Neuronal Differentiation; Neurons; Neurotransmitter Receptor; Pathway interactions; Peptide Hydrolases; Phosphorylation; Play; Polymerase Chain Reaction; Principal Investigator; Proliferating Cell Nuclear Antigen; Prosencephalon; Protein Kinase C Alpha; Proteins; Regulation; Relative (related person); Role; Schizophrenia; Serotonin Receptor 5-HT1A; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Slice; Specificity; Staging; Staining method; Stains; Symptoms; Synapses; Synaptic Potentials; Synaptophysin; Testing; Threonine; Time; Western Blotting; age related; caspase-3; day; design; emotion regulation; in vitro Assay; neurogenesis; neuronal survival; neuroprotection; novel; novel therapeutics; postnatal; receptor; synaptogenesis

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.

描述(由申请人提供)：5-HT1A受体(5-HT1A-R)特异性配体在治疗抑郁、焦虑和许多相关症状方面的有效性促使人们对这种神经递质受体的生理意义和信号机制进行研究。这类研究表明，前脑中缺乏这种受体，特别是在小鼠出生后的早期发育过程中，会导致焦虑水平上升。申请人实验室的研究表明，刺激海马神经元来源的细胞系中的这种受体可以保护这些细胞免受凋亡的影响。这种保护的机制涉及刺激丝裂原激活的蛋白激酶(MAPK)途径，进而通过蛋白激酶Cα(PKCc0)发出信号，导致促凋亡蛋白caspase-3的抑制。由于正常的情绪状态是前脑不同区域(如海马体、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在这些脑区神经元的分裂和成熟过程中发挥着重要作用。目前的建议将通过使用培养的海马片以及5-HT1A-R(+/+)和5-HT1A-R(-/-)小鼠来验证这一假设。来自出生后5、10、15和20天的小鼠的海马片将被5-HT1A激动剂8-羟基-2(二正丙氨基)四氢萘(8-OH-DPAT)和免疫组织化学技术处理，用于研究对关键蛋白的刺激，如PKC(x，MAPK和CREB)，促进神经元分裂以及成熟和存活。进行这类信号研究的一个重要目标将是阐明5-HT1A-R信号的年龄依赖性调节及其对神经细胞分裂(通过BrdU掺入)和成熟(通过MAP-2和突触素染色)的影响。通过使用抗caspase-3抗体(用于记录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)(用于监测与凋亡相关的DNA片断)来测试对细胞凋亡的调节。同时，MAPK、CREB、PKCcq和传递MAPK信号的下游转录因子(如ELK-L)的激活以及对Caspase-3的抑制将通过使用海马片的裂解物进行Western印迹分析来测量。从处理过的切片中制备的RNA将被逆转录成cDNA探针，用于微阵列分析，以测试基因表达的调控，特别是细胞骨架相关蛋白Arc和即刻早期蛋白质如Fos和Jun.类似的研究将通过向5-HT1A-R(+/+)和5-HT1A-R(-/-)小鼠(d5，d0，d15，d20)注射8-OH-DPAT和制备大脑冰冻切片来监测MAPK，CREB，PKCcq的激活，以及这些5-HT1A-R信号对海马区和相关区域神经细胞分裂和成熟的影响。从5-HT1A-R(+/+)和5-HTA-R(-/-)小鼠身上获得的结果将有助于描述5-HTTA-R信号的具体作用，并为5-HT1A-R在脑发育中的机制作用提供新的线索。