CENTRAL RESPIRATORY CHEMORECEPTION IN AMPHIBIANS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。A.具体目的本申请中的具体目的没有改变。实验已经按照特定目标1中的描述进行，具体地说，我们在体外蝌蚪脑干制备中使用荧光染料BCECF收集了细胞内的pH值比率。B.研究和结果我们已经进行了目标1中描述的实验，以研究蝌蚪脑干的细胞内pH(Phi)。我们已经在蝌蚪延髓的已知化学感受区和非化学敏感区的细胞中记录了Phi。我们目前正在收集高碳酸血症期间的数据，并计划很快收集氯化铵预脉冲期和等渗高碳酸血症期间的数据。此外，我们最近完成的一项研究表明，pH敏感的染料BCECF在体外不会改变自发呼吸节律或对中枢化学感受器刺激的呼吸反应。这项研究包括计算不同孵育模式下负载BCECF的细胞的百分比，记录染料孵育前后的整个神经呼吸活动，以及我们在高碳酸酸血症期间光学记录phi的能力。这些数据目前正在编入同行评议研究手稿，将在本RCMI预算年度内提交。Gdovin，M.J.，Costanzo，M.C.，Ortiz，B.J.，Zamora，D.A.，以及Jackson，V.V.体外蝌蚪脑干制备、呼吸生理学和神经生物学中细胞内pH和自发呼吸节律的光学记录。我们的荧光成像系统遇到了技术上的困难；特别是我们用来捕获、收集和分析数据的计算机停止了工作。我们使用的数据采集软件的支持人员环球成像技术支持告诉我们，我们需要升级到一台更新的计算机，有更多的可用硬盘空间。虽然我们暂时解决了这个问题，但我们预计很快就会收到他们的报价，并有资金购买这个较新的系统。C.意义我们成功地记录了蝌蚪Rana catesbeiana脑干中含有BCECF染料的呼吸运动神经活动。这项研究的一个非常重要的发现是，染料的存在不会改变自发的呼吸运动输出或对中枢CO2化学感受器刺激的呼吸反应。更重要的是，我们可以在体外记录细胞内pH的光学数据。正如最初的拨款建议中所述，我们很快就会第一个报告phi的变化确实是脊椎动物模型中呼吸马达输出的变化的原因。D.计划我们为第二年提出的研究计划与最初的拨款申请中所述的计划是一致的。具体地说，我们将继续进行目标1中描述的实验。此外，我们将开始使用BASH应用试剂进行实验，以确定依赖Na+/H+和氯的HCO3-交换器在调节细胞内pH中的作用，如目标2中所述。除了上述进展，我还指导了我实验室的三年级博士生黛博拉·A·萨莫拉(Debora A.Zamora)，她是我实验室的一名博士生，与助学金写作有关。她在2005年春天提交了一份关于她的博士研究的F31拨款提案。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A. Specific AimsThe specific aims in the application have not changed. Experiments have been conducted as described in Specific Aim 1, specifically; we have collected intracellular pH ratios using the fluorescent dye BCECF in the in vitro tadpole brainstem preparation. B. Studies and Results We have performed experiments as described in Aim 1 to investigate intracellular pH (pHi) in the tadpole brainstem. We have recorded pHi in cell in both known chemoreceptive regions and non-chemosensitive regions of the tadpole medulla. We are currently collecting data during hypercapnic acidosis, and plan to soon collect data during the ammonium chloride prepulse and isohydric hypercapnia. In addition, we recently completed a study that demonstrated that the pH sensitive dye, BCECF, does not alter the spontaneous respiratory rhythm or the respiratory response to central chemoreceptor stimulation in vitro. This study involved calculating the percentage of cells that were loaded with BCECF following various incubation paradigms, recording whole nerve respiratory activities prior to and after dye incubation, and our ability to optically record pHi during hypercapnic acidosis. These data are currently being incorporated into a peer review research manuscript to be submitted during the current RCMI budget year. Gdovin, M.J., Costanzo, M.C., Ortiz, B.J., Zamora, D.A., and Jackson, V.V. ¿Optical recordings of intracellular pH and spontaneously respiratory rhythmicity in the in vitro tadpole brainstem preparation¿ Respiration Physiology and Neurobiology. We experienced technical difficulties with our fluorescence imaging system; specifically the computer that we use to capture, collect, and analyze data stopped working. We were told by Universal Imaging technical support, the support staff for the data acquisition software we use, that we needed to upgrade to a newer computer with more available hard drive space. While we temporarily fixed the problem, we expect to receive a quote from them soon and have funds to purchase this newer system. C. Significance We have successfully recorded respiratory motor nerve activity in the BCECF dye loaded brainstem of the tadpole, Rana catesbeiana. A very significant finding of this study is that the presence of the dye does not alter the spontaneous respiratory motor output or the respiratory response to central CO2 chemoreceptor stimulation. More importantly, we can record optical data of intracellular pH in vitro. As stated in the original grant proposal, we will soon be the first to report that the changes in pHi are indeed responsible for changes in respiratory motor output in a vertebrate model.D. PlansOur proposed research plan for year 2 is consistent with those described in the original grant application. Specifically, we will continue performing experiments described in Aim 1. In addition, we will begin to conduct experiments using bath applied agents to determine the roles of the Na+/H+ and Cl- dependent HCO3- exchangers in the regulation of intracellular pH, as described in Aim 2. In addition to the above progress, I also mentored Debora A. Zamora, a third year Ph.D. student in my lab, pertaining to grant writing. She submitted an F31 grant proposal in the Spring of 2005 pertaining to her doctoral research.