Network mechanisms for respiratory rhythm generation

描述（由申请人提供）：新的证据表明，两个机械上不同，空间上分离的网络可以独立地产生呼吸节律。我们提出，这两个网络一致满足维持血气稳态所需的适应性和鲁棒性的冲突需求。这一新发现强调了网络相互作用在节律产生中的作用。由于目前可用的准备可以很容易地进入神经元，但太过减少（横切片准备），或者保留感兴趣的网络，但不允许容易地进入它们（整体准备），我们已经开发了一种倾斜矢状切片准备，它保留了整体存在的网络，但提供了良好的访问感兴趣的网络。我们使用薄膜渗透性钙离子指示器进行光学记录，以确定切割切片的最佳参数，并使用光学记录技术常规监测暴露在矢状切片表面的腹外侧髓质呼吸调节神经元柱上的局部网络活动。我们将使用光信号识别两类内源性破裂呼吸神经元，并使用细胞内记录技术进行详细研究。这些内源性爆裂物的不同之处在于，一种种群改变其爆裂频率作为膜电位的函数（电压依赖性内源性爆裂物；VDEB），而另一种种群则不改变其爆裂频率（电压非依赖性内源性爆裂物；VIEB）。由于这种差异，VDEB周期可以通过瞬态去极化或超极化脉冲来改变；而vieb对这种输入则不那么敏感。这为我们的假设提供了一种机制；我们认为，vdeb介导了周期对周期的呼吸节律产生网络适应性，而vieb通过快速恢复基线节律来维持网络稳定性。我们建议在网络水平上通过选择性地调节VIEB和VDEB网络来验证这一假设，并在单个神经元水平上通过表征突触隔离的VIEB和VDEB的扰动反应来验证这一假设。

DESCRIPTION (provided by applicant): New evidence indicates that two mechanistically distinct, spatially separate networks can independently generate respiratory rhythm. We propose that the two networks in concert meet the conflicting demands of adaptive-ness and robustness required for the maintenance of blood-gas homeostasis. This new finding puts emphasis on the role of network interactions in rhythm generation. Because preparations currently available allow easy access to neurons but are too reduced (transverse slice preparation), or retain the networks of interest but do not allow easy access to them (en bloc preparation), we have developed a tilted saggital slice preparation that retains the networks present en bloc but affords good access to the networks of interest. We used optical recordings using a membrane permeable Ca++ indicator to determine the best parameters for cutting the slice, and use optical recording techniques routinely to monitor local network activity along the column of respiration modulated neurons in ventrolateral medulla that are exposed at the surface of the saggital slice. We will use optical signals to identify 2 classes of endogenously bursting respiratory neurons for detailed investigation using intracellular recording techniques. These endogenous bursters differ in that one population changes its bursting frequency as a function of membrane potential (voltage-dependent endogenous burster; VDEB), while the other does not (voltage-independent endogenous burster; VIEB). Due to this difference, VDEB cycle period can be altered by transient de- or hyperpolarizing impulses; while VIEBs will be less sensitive to such inputs. This provides a mechanism for our hypothesis; we propose that VDEBs mediate cycle-to-cycle adaptiveness to respiratory rhythm-generating networks, while VIEBs maintain network stability by rapidly restoring baseline rhythm. We propose to test this hypothesis at the network level, by selectively modulating VIEB and VDEB networks, and at the single neuron level, by characterizing perturbation responses in synaptically isolated VIEBs and VDEBs.