Unraveling respiratory rhythmic integration from rhythm generation to motor outpu

描述（由申请人提供）：哺乳动物呼吸由三个阶段组成：吸气（I）、吸气后（E1）和主动呼气（E2）。众所周知，分离前B <$tzinger复合物（preB <$tC）的横向切片制备物自发地产生吸气活动。呼气群体活动的缺乏与理论一致，即需要更大的网络来产生三个不同的阶段。PreB？tC只是存在于脑干腹外侧延髓中的几个相互作用的网络之一，并在脑干腹外侧延髓中向吻尾方向延伸，统称为腹侧呼吸柱（VRC）。VRC网络之间的功能相互作用，更具体地说，呼气节律如何从呼吸网络中出现，在很大程度上是未知的。基于这些观察结果，我们假设VRC切片中较长的爆发持续时间是由于吸气（I）和吸气后（E1）阶段的部分分离，而不频繁的喙部爆发活动反映了主动呼气（E2）。我们进一步假设，不同的阶段是由突触抑制和突触相互作用形成的，当暴露于发作性缺氧时，突触相互作用解离，这在多种病理中很常见。迄今为止，这些相互作用仅在体内和原位制备中进行了研究。不幸的是，无论是体内还是原位制备都不能像切片制备那样经受相同程度的细胞僵硬。当试图理解呼吸相产生的细胞机制时，这成为一个主要的缺点。我们开发了一种新的水平切片（VRC切片，小鼠，p5-p7; 700 - 1100 μ m），将整个VRC从面神经核的吻侧边缘隔离到脊髓中的C3，并保留双侧连接。初步意见， VRC切片中的群体记录显示：与横向切片相比，爆发持续时间更宽，第二嘴期，存在加巴嗪时同步的preB ² tC和嘴期，以及加巴嗪后的频率不规则性 暴露于间歇性缺氧，类似于体内观察到的结果。因此，我们的首要目标是功能性地表征呼吸三个阶段产生的基础网络到网络的相互作用。我们结合联合收割机电生理学、光遗传学和药理学技术，采用三种方法实现这一目标：（1）绘制整个切片的群体节律，并将运动前活动与运动输出相关联，（2）研究突触相互作用在建立不同呼吸时相中的作用，（3）检查暴露于急性间歇性缺氧后前B tC和喙部节律之间的相互作用。这些实验将进一步加深我们对负责产生呼吸节律阶段的相互作用网络的理解， 在整个延髓VRC的间歇性缺氧的不稳定网络的影响。

DESCRIPTION (provided by applicant): Mammalian breathing is composed of three phases: inspiration (I), post-inspiration (E1), and active expiration (E2). It is well known that the transverse slice preparation isolating the preB¿tzinger complex (preB¿tC) spontaneously generates inspiratory activity. The absence of expiratory population activity is consistent with th theory that a larger network is required to generate the three different phases. The preB¿tC is only one of several interacting networks that exist bilaterally and extend rostrocaudally in the ventrolateral medulla of the brainstem and is collectively termed the ventral respiratory column (VRC). The functional interactions between VRC networks, and more specifically how expiratory rhythms emerge from the respiratory network are largely unknown Based on these observations, we hypothesize that the longer burst durations in the VRC slice are due to the partial separation of inspiratory (I) and post-inspiratory (E1) phases, and the infrequent rostral burst activity reflects active expiration (E2). We further hypothesize that the different phases ar shaped by synaptic inhibition and synaptic interactions dissociate when exposed to episodic hypoxia, a common occurrence in multiple pathologies. These interactions have so far only been studied from in vivo and in situ preparations. Unfortunately neither in vivo nor in situ preparations are amenable to the same degree of cellular rigor as a slice preparation. This becomes a major disadvantage when trying to understand the cellular mechanisms underlying the generation of respiratory phases. We have developed a novel horizontal slice (VRC slice, mice, p5-p7; 700-1100¿m) that isolates the entire VRC from the rostral edge of the facial nucleus to C3 in the spinal cord and that retains bilateral connectivity. Initial observations from population recordings in the VRC slice reveal: broader burst durations compared to the transverse slice, a second rostral phase, synchronized preB¿tC and rostral phases in the presence of gabazine, and frequency irregularities after exposure to episodic hypoxia, similar to results seen in vivo. Thus, our overarching goal is to functionally characterize network-to-network interactions that underlie the generation of the three phases of respiration. We combine electrophysiological, optogenetic, and pharmacological techniques in three approaches toward this goal: (1) mapping population rhythms across the slice and correlating pre-motor activity with motor output, (2) investigating the role of synaptic interactions in establishing different respiratory phases, (3) examining the interactions between the preB¿tC and rostral rhythms following exposure to acute intermittent hypoxia. These experiments will further our understanding into the interacting networks responsible for generating the phases of breathing rhythms and provide insight into the destabilizing network effects of episodic hypoxia throughout the medullary VRC.