CAA: Relationship Between Fatty Acid-binding Proteins and Neurotransmitter Systems in Developing Brain

这个项目提出了一个假设，即脂肪酸结合蛋白（H- FABP）心脏亚型的发育表达与大脑中神经递质系统，即GABA的同时发育有关。该假说基于以下几个方面：1)免疫组织化学对蛋白质的相似定位；2)记录的大脑中特定通路/区域在H-FABP存在时的分化时间；3)显示游离脂肪酸对GABA转运体和GABA-a受体均有影响的数据；4)体外研究显示，当外源性H-FABP加入时，突触体对GABA的摄取增强。这一假设将通过开发一种来自新生小鼠小脑的细胞培养系统来解决，使用已建立的协议。将使用合成肽制备的抗血清和合成寡核苷酸探针检测培养物H-FABP的表达。GABA转运体的存在将通过测量培养物的3H- GABA摄取来验证。接下来，将研究外源添加脂肪酸对H-FABP水平和GABA摄取速率的影响。最后，将反义寡核苷酸添加到培养物中以抑制H-FABP的表达，并确定对GABA摄取的影响。本提案中描述的活动旨在获得足够的试点数据，以保证提交一份主要的赠款提案。＊＊＊

9509168 Sellner This project addresses the hypothesis that the developmental expression of the heart subtype of fatty acid-binding protein (H- FABP) is related to the simultaneous development of neurotransmitter systems in the brain, namely GABA. The hypothesis is based on 1) similar localization of the proteins by immunohistochemistry, 2) the documented timing of differentiation of particular pathways/regions in the brain at a time when H-FABP is also present, 3) data showing effects of free fatty acids on both the GABA transporter and the GABA-a receptor, and 4) in vitro studies showing an enhanced uptake of GABA by synaptosomes when exogenous H-FABP is added. The hypothesis will be addressed by developing a cell culture system from neonatal mouse cerebellum, using established protocols. The cultures will be examined for their expression of H-FABP using antiserum prepared against synthetic peptides and using synthetic oligonucleotide probes. The presence of the GABA transporter will be verified by measuring 3H- GABA uptake by the cultures. Next, the efdects of exogenously added fatty acids on the levels of H-FABP and on rates of GABA uptake will be studied. Finally, antisense oligonucleotides will be added to the cultures to inhibit the expression of H-FABP, and effects on GABA uptake will be determined. The activities described in this proposal are designed to obtain sufficient pilot data to warrant submission of a major grant proposal. ***