CENTRAL RESPIRATORY CHEMORECEPTION IN AMPHIBIANS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。A.具体目标应用程序中的具体目标没有改变。实验已按照具体目标1中所述进行，具体而言；我们使用荧光染料BCECF收集蝌蚪脑干体外制备的细胞内pH值。B.研究和结果我们进行了Aim 1中描述的实验来研究蝌蚪脑干的细胞内pH （pHi）。我们记录了蝌蚪髓中已知的化学接受区和非化学敏感区细胞中的pHi。我们目前正在收集高碳酸血症酸中毒期间的数据，并计划很快收集氯化铵预脉冲和等水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敏感染料BCECF不会改变体外自发呼吸节律或对中枢化学受体刺激的呼吸反应。本研究包括计算在不同孵育模式下携带BCECF的细胞百分比，记录染料孵育前后的整个神经呼吸活动，以及我们在高糖酸中毒期间光学记录pHi的能力。这些数据目前正被纳入一份同行审查研究手稿，将在当前RCMI预算年度提交。Gdovin, m.j., Costanzo, m.c., Ortiz, b.j., Zamora, d.a.和Jackson， V.V.¿体外蝌蚪脑干制备中细胞内pH值和自发呼吸节律性的光学记录¿呼吸生理学和神经生物学。我们的荧光成像系统遇到了技术上的困难；特别是我们用来捕捉、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技术支持人员Universal Imaging的技术支持人员告诉我们，我们需要升级到一台有更多可用硬盘空间的新电脑。虽然我们暂时解决了这个问题，但我们希望很快收到他们的报价，并有资金购买这个更新的系统。C.意义我们成功地记录了蝌蚪蛙（Rana catesbeiana）携带BCECF染料的脑干的呼吸运动神经活动。本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呼吸对中央CO2化学受体刺激的反应。更重要的是，我们可以在体外记录细胞内pH的光学数据。正如最初的拨款提案中所述，我们将很快成为第一个报告pHi的变化确实是脊椎动物模型中呼吸运动输出变化的原因。我们建议的第二年研究计划与原始拨款申请中描述的计划一致。具体来说，我们将继续进行Aim 1中描述的实验。此外，我们将开始使用浴液应用剂进行实验，以确定Na+/H+和Cl-依赖的HCO3-交换剂在调节细胞内pH中的作用，如Aim 2所述。除了上述进展，我还指导了我实验室的三年级博士生Debora a . Zamora，关于拨款写作。2005年春，她提交了一份关于她博士研究的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.