RETROGRADE SIGNALING IN LTP

描述：本项目的总体目标是研究一氧化氮(NO)、cGMP和cGMP依赖的蛋白激酶(PKG)在海马区Schaffer侧支突触的长时程增强(LTP)中的作用。虽然人们普遍认为LTP是由突触后钙通过NMDA受体或在某些条件下通过电压门控性钙通道进入的增加而启动的，但LTP的表达机制仍存在很大争议。有证据表明，LTP的表达涉及突触后谷氨酸受体的变化，而其他数据表明，LTP涉及突触前递质释放的增加。因此，表达机制被认为要么是突触后的，要么是突触前的，很少有人同时对这两种机制提出异议。PI和他在这个中心的合作者显然属于突触前阵营。突触前表达机制的含义是，突触后感觉钙升高和突触前递质释放的变化之间肯定存在某种形式的沟通。一些所谓的逆行信使卷入了这一过程。这些物质包括NO、CO、AA、血小板激活因子和神经营养因子。PI及其合作者已经积累了大量的证据，表明NO作为逆行信使发挥了作用，本研究旨在进一步探索这一作用，并确定NO的下游靶点，如cGMP和PKG，它们在介导突触前递质释放的变化中。该小组还确定了LTP表达的不同阶段。晚期(L-LTP)在诱导后90min左右开始，需要3-4次100 Hz，1s的刺激，依赖于蛋白质和核糖核酸的合成；而早期(E-LTP)，发生在诱导后约60min，只需一次刺激，则不依赖于蛋白质和RNA的合成。还有一个最近描述的中间阶段，它依赖于PKA，但不依赖于蛋白质合成，尽管这个项目没有具体涉及这个中间阶段。本项目的两个具体目标是进一步探讨NO、cGMP和PKG在海马Schaffer侧支突触LTP早期和晚期中的作用。对于早期的LTP，Pi及其同事建议：1)检测LTP靶向缺失或过表达的小鼠海马神经元；2)使用海马片细胞内注射的方法测试LTP期间这些分子的作用部位；3)使用免疫细胞化学检测这些分子在细胞内的定位；4)利用电生理学和成像技术研究这些分子在LTP诱导和表达中的作用；3)使用电生理学和成像技术对突触前和突触后神经元进行研究。对于晚期LTP，他们建议：1)调查NO在晚期LTP和早期LTP中是否有不同的作用部位；2)确定晚期LTP中NO的一些下游靶点，重点放在他们有初步证据的两个候选者，ryanodine受体和CREB。

DESCRIPTION: The overall aim of this project is to investigate the roles of nitric oxide (NO), cGMP, and cGMP dependent protein kinase (PKG) in long-term potentiation (LTP) at the Schaffer collateral synapse in the hippocampus. While there is general agreement that LTP at this synapse is initiated by a rise in postsynaptic Ca entering through either NMDA receptors or, under some conditions, voltage-gated Ca channels, the mechanisms for expression of the LTP are highly controversial. There is evidence to suggest that expression involves changes in postsynaptic glutamate receptors, while other data suggest that LTP involves increases in presynaptic transmitter release. Expression mechanisms therefore are argued to be either postsynaptic or presynaptic with few people arguing both. The PI and his collaborators in this center are clearly in the presynaptic camp. The implication for a presynaptic expression mechanism is that there must be some form of communication between the postsynaptic sensing of a rise in Ca and the presynaptic change in transmitter release. A number of so-called retrograde messengers have been implicated in this process. These include NO, CO, AA, platelet activating factor, and neurotrophins. The PI and collaborators have amassed a considerable body of evidence to suggest that NO plays a role as a retrograde messenger, and this grant is intended to further explore this role and to identify the downstream targets of NO such as cGMP and PKG in mediating the presynaptic changes in transmitter release. This group has also identified various phases to the expression of LTP. The late phase (L-LTP), which begins about 90 min after induction and requires 3 or 4 trains of 100 Hz, 1 sec stimulation, is protein and RNA synthesis dependent while the early phase (E-LTP), which occurs during approximately the first 60 min after induction and is induced with 1 train of stimulation, is not. There is also a recently described intermediate phase, which is dependent on PKA but not protein synthesis, although this project does not specifically address this intermediate phase. The two specific aims for this project are to explore further the role of NO, cGMP, and PKG in early and late phase LTP at the Schaffer collateral synapse in hippocampus. For early LTP, the PI and his colleagues propose: 1) to examine LTP in mice with targeted deletions or overexpression of NO synthase or soluble guanylyl cyclase; 2) to test the site of action of these molecules during LTP using intracellular injection methods in hippocampal slices; 3) to examine the cellular localization of these molecules using immunocytochemistry; 4) to examine the roles of these molecules in the induction and expression of LTP using electrophysiological and imaging techniques in cultured hippocampal neurons where the pre- and postsynaptic neurons are accessible for study. For late LTP they propose: 1) to investigate whether NO has different sites of action for late vs. early LTP; 2) to determine some of the downstream targets of NO for late LTP, focusing on two candidate for which they have preliminary evidence, ryanodine receptors and CREB.