RETROGRADE SIGNALING IN LTP

描述：本项目的总体目的是研究一氧化氮（NO）、cGMP和cGMP依赖性蛋白激酶（PKG）在海马Schaffer侧侧突触的长期增强（LTP）中的作用。虽然人们普遍认为该突触的LTP是由突触后钙通过NMDA受体或在某些情况下通过电压门控钙通道进入引起的，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，表达机制被认为是突触后或突触前的，很少有人认为两者都是。这个中心的PI和他的合作者显然是处于突触前阵营。突触前表达机制的含义是，在突触后感知Ca升高和突触前递质释放变化之间一定存在某种形式的通信。一些所谓的逆行信使参与了这个过程。这些包括NO， CO， AA，血小板活化因子和神经营养因子。PI和合作者已经积累了大量证据，表明NO作为逆行信使发挥作用，该资助旨在进一步探索这一作用，并确定NO的下游靶标，如cGMP和PKG，在介导递质释放的突触前变化中。该组还确定了LTP表达的不同阶段。晚期（L-LTP）在诱导后约90分钟开始，需要3或4组100 Hz， 1秒的刺激，依赖于蛋白质和RNA的合成，而早期（E-LTP）在诱导后约60分钟发生，由1组刺激诱导，而不是。还有一个最近描述的中间阶段，它依赖于PKA而不是蛋白质合成，尽管这个项目没有具体解决这个中间阶段。本项目的两个具体目标是进一步探索NO、cGMP和PKG在海马Schaffer侧支突触早期和晚期LTP中的作用。对于早期LTP， PI和他的同事们提出：1)在NO合成酶或可溶性guanylyl环化酶靶向缺失或过表达的小鼠中检测LTP；2)利用细胞内注射方法在海马切片上检测这些分子在LTP过程中的作用部位；3)利用免疫细胞化学检测这些分子的细胞定位；4)利用电生理和成像技术在培养的海马神经元中检测这些分子在LTP诱导和表达中的作用，其中突触前和突触后神经元可用于研究。对于晚期LTP，他们提出：1)研究NO在晚期LTP和早期LTP中是否有不同的作用位点；2)确定NO对晚期LTP的一些下游靶点，重点关注他们有初步证据的两个候选靶点，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.