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

小行星9509168 该项目解决了以下假设：心脏亚型脂肪酸结合蛋白（H-FABP）的发育表达与大脑中神经递质系统（即GABA）的同时发育有关。该假设基于1）通过免疫组织化学的蛋白质的相似定位，2）当H-FABP也存在时，记录的脑中特定途径/区域的分化时间，3）显示游离脂肪酸对GABA转运蛋白和GABA-a受体两者的影响的数据，和4）体外研究显示当加入外源性H-FABP时，突触体对GABA的摄取增强。该假设将通过开发新生小鼠小脑的细胞培养系统来解决，使用已建立的协议。将使用针对合成肽制备的抗血清和使用合成寡核苷酸探针检查培养物的H-FABP表达。将通过测量培养物的3 H- GABA摄取来验证GABA转运蛋白的存在。接下来，将研究外源性添加的脂肪酸对H-FABP水平和GABA摄取速率的影响。最后，将反义寡核苷酸加入培养物中以抑制H-FABP的表达，并测定对GABA摄取的影响。本提案中所述的活动旨在获得足够的试点数据，以保证提交重大赠款提案。 ***

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. ***