Unraveling respiratory rhythmic integration from rhythm generation to motor outpu

描述（由申请人提供）：哺乳动物的呼吸由吸气(I)、吸气后（E1）和主动呼气（E2）三个阶段组成。众所周知，分离preB¿tzinger复合物（preB¿tC）的横片制备可以自发地产生吸气活性。呼气种群活动的缺失与理论一致，即需要更大的网络来产生三个不同的阶段。preB¿tC只是脑干腹外侧髓质中存在的几个相互作用网络中的一个，并向后侧延伸，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，呼气节律是如何从呼吸网络中产生的，在很大程度上是未知的。基于这些观察，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，而吻侧爆发活动的不频繁反映了主动呼气（E2）。我们进一步假设，不同的阶段是由突触抑制形成的，当暴露于间歇性缺氧时，突触相互作用会分离，这在多种疾病中很常见。到目前为止，这些相互作用仅从体内和原位制剂中进行了研究。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具有相同程度的细胞严谨性。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¿m），将整个VRC从面核的吻侧边缘分离到脊髓的C3，并保持双侧连通性。从

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.