BIOCHEMICAL CORRELATES OF 5-HT NEURONAL ACTIVITY

这项延续提案的重点是对 5-羟色胺能神经元中色氨酸羟化酶的活性 大鼠大脑的神经元。因为这种酶是限速的 合成递质5-羟色胺(5-羟色胺)其活性是 对于确定这一重要信息的可用性至关重要 物质，现在被牵连为广泛的调解人 各种功能，包括情绪或情感，焦虑， 抗伤害性感觉、睡眠、体温调节和中枢 控制血压。5-羟色胺的合成在 阿司匹林对5-羟色胺神经元冲动流量增加的反应 可能涉及色氨酸H因此酶的激活的机制 从刺激的大脑皮层5-羟色胺投射准备的显示 依赖于频率的激活，并被碱性逆转 磷酸酶。另一方面，抑制5-羟色胺的产生 降低酶活性，同样是通过碱性磷酸酶 可逆过程。现在计划进行实验，以进一步 描述上清液动力学行为的变化 酶制剂对不同浓度5-羟色胺的响应 水合氯醛麻醉雄性SD大鼠的羟色胺能神经元放电 Dawley大鼠在中枢不同区域，具有天然的 辅因子，6R-L-红血球-5，6，7，8-四氢生物蝶呤；2)至 研究候选传递物质的作用， 中缝背核立体定向微量注射 5-羟色胺上行投射中酶活性的调节 该核团对清醒大鼠的影响；3)检测其作用 色氨酸上的各种递质或推定的递质 切片孵育后的羟基酶活性；4) 为了确定体外活性和动力学的变化 上清液酶对这些条件的响应特性 操纵在体内表达；5)启动有目的的研究 在确定酶的激活是否对 生理刺激是由直接磷酸化的 酵素。这些后一项研究将需要一种抗体 色氨酸羟基酶。产生5-羟色胺代谢物和5-羟色胺 在Trp H分析中，被分离并用高效液相色谱定量 电化学检测。应该获得重要的见解 从这项关于细胞和分子机制的工作中 维持5-羟色胺水平，从而维持5-羟色胺的调节功能。

This continuation proposal focusses on the regulation of tryptophan hydroxylase (Trp H) activity in the serotonergic neurons of the rat brain. Since this enzyme is rate limiting in the synthesis of the transmitter, serotonin, (5-HT) its activity is central for determining the availability of this important substance, which is now implicated as a mediator in a wide variety of functions including mood or affect, anxiety, antinociception, sleep, temperature regulation and central control of blood pressure. Synthesis of 5-HT is enhanced in response to increased 5-HT neuronal impulse flow by a mechanism that may involve activation of Trp H. Thus enzyme prepared from stimulated cortical 5-HT projections shows an activation that is frequency dependent, and reversed by alkaline phosphatase. Inhibition of 5-HT firing on the other hand decreases enzyme activity, again by an alkaline phosphatase reversible process. Experiments are now planned 1) to further characterize the changes in kinetic behaviour of supernatant preparations of the enzyme in response to different levels of 5- HT neuronal firing in chloral hydrate anaesthetized male Sprague Dawley rats in different regions of CNS, with the natural cofactor, 6R-L-erythro-5,6,7,8-tetrahydrobiopterin; 2) to examine the role of candidate transmitter substances, microinfused stereotaxically into the dorsal raphe nucleus on the regulation of enzyme activity in ascending 5-HT projections supplied by this nucleus in awake rats; 3) to examine the effects of various transmitters or putative transmitters on tryptophan hydroxylase activity after incubation with slice preparations; 4) to establish whether in vitro changes in activity and kinetic properties of supernatant enzyme in response to these manipulations are expressed in vivo; 5) to initiate studies aimed at determining whether the activation of enzyme in response to physiological stimuli results from a direct phosphorylation of the enzyme. These latter studies will require an antibody to tryptophan hydroxylase. 5-HT metabolites and 5-HTP, generated in the Trp H assay, are isolated and quantitated by HPLC with electrochemical detection. Important insights should be gained from this work regarding cellular and molecular mechanisms for maintenance of 5-HT levels and hence 5-HT mediated function.