Network mechanisms for respiratory rhythm generation

描述（由申请人提供）：新证据表明，两种机制不同、空间分离的网络可以独立产生呼吸节律。我们建议，这两个网络在音乐会上满足相互冲突的需求，自适应性和鲁棒性所需的血液气体稳态的维护。这一新发现强调了网络相互作用在节奏生成中的作用。因为目前可用的制备允许容易地接近神经元，但过于减少（横向切片制备），或保留感兴趣的网络，但不允许容易地接近它们（整块制备），我们已经开发了一种倾斜的矢状切片制备，保留整块存在的网络，但提供良好的访问感兴趣的网络。我们使用光学记录，使用膜渗透性Ca++指示剂来确定切割切片的最佳参数，并常规使用光学记录技术来监测局部网络活动，沿着暴露在矢状切片表面的延髓腹外侧区呼吸调制神经元柱。我们将使用光信号来识别2类内源性爆发呼吸神经元，并使用细胞内记录技术进行详细研究。这些内源性爆发的不同之处在于，一个群体改变其爆发频率作为膜电位的函数（电压依赖性内源性爆发; VDEB），而另一个则没有（电压非依赖性内源性爆发; VIEB）。由于这种差异，VDEB循环周期可以通过瞬时去极化或超极化脉冲来改变;而VIEB对此类输入不太敏感。这为我们的假设提供了一种机制;我们建议VDEBs介导对呼吸节律生成网络的周期到周期的适应性，而VIEBs通过快速恢复基线节律来保持网络稳定性。我们建议在网络水平上测试这一假设，通过选择性地调制VIEB和VDEB网络，并在单个神经元水平上，通过表征突触隔离VIEB和VDEB的扰动响应。

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.