Function and Expression of Connexins in the pre-Botzinger Complex

描述（由申请人提供）：间隙连接是跨越相邻细胞膜的孔，因此提供了这些细胞之间的电和细胞质连续性的手段。这些跨细胞通道由连接的半通道组成，每个半通道是称为连接蛋白（Cx）的整合膜蛋白亚基的六聚体。作为神经元之间的电连接的一种形式，缝隙连接已经涉及各种神经元网络的功能，包括甲壳动物口胃神经节、脊椎动物视网膜、哺乳动物的下橄榄复合体和延髓的自主网络（包括前Btzinger复合体（PBC））内的一些神经元网络。主要研究者的实验室专注于阐明神经元网络机制，产生和模式的神经元群体活动的节奏爆发。这种节律性网络的活动是从行走到信息处理等基本功能的基础。在PBC的情况下，这种活动与呼吸的吸气运动的产生有关。了解PBC功能的机制最终将是至关重要的了解中枢呼吸系统疾病，如中枢换气不足综合征和Rett综合征。研究结果a）啮齿类PBC内的神经元表达至少一些Cx，可能包括Cx 26、Cx 32和Cx 36，和B）假定的间隙连接阻断剂（解偶联剂）影响PBC爆发的产生，表明间隙连接在PBC节律发生中的作用。然而，在其他神经元群体中，解偶联剂对膜性质产生非特异性影响。此外，由于所采用的方法可能缺乏特异性，PBC内某些Cx的检测受到质疑。因此，缝隙连接作为细胞间通讯的一种形式对PBC功能是否重要的问题仍然没有得到解决。因此，本文提出的研究将通过实现两个具体目标来进一步研究这个问题。第一个目的是确定推定的间隙连接解偶联剂改变PBC输出的机制。为此，PBC神经元之间的细胞质连接性，以及解偶联剂（甘珀酸或CBX）和对照剂（烟酸或GZA）对这种连续性的潜在改变，将通过光漂白后荧光恢复（FRAP）显微镜技术进行评估。沿着FRAP研究，将检查CBX和GZA对膜特性（包括输入电阻和钙电流）的影响。本研究的第二个目的是进一步阐明PBC神经元内Cx表达的个体发育模式。这一目标将通过原位杂交检测从胚胎第12天到出生后第21天的小鼠组织中PBC神经元中Cx26、Cx32、Cx36、Cx45、Cx47和Cx59的转录物来实现。即使与感觉或下行输入隔离的神经元网络也会产生活动的爆发（即，中枢模式发生器）是诸如呼吸和运动的各种基本功能的基础。因此，研究这种节律性网络功能的机制将提供一个概念框架，在其中检查影响重复行为的障碍。关于本文提出的研究，更好地理解前B "tzinger复合体功能的机制将最终对理解中枢呼吸系统疾病如中枢换气不足综合征和Rett综合征至关重要。

DESCRIPTION (provided by applicant): Gap junctions are pores that span the membranes of adjacent cells, and as such provide a means of electrical and cytoplasmic continuity between those cells. These trans-cellular channels are made up of joined hemichannels, each a hexamer of integral membrane protein subunits termed connexins (Cx). As a form of electrical connectivity between neurons, gap junctions have been implicated in the functioning of various neuronal networks including some within the crustacean stomatogastric ganglion, the vertebrate retina, the inferior olivary complex of mammals, and autonomic networks of the medulla oblongata, including the pre-B"tzinger complex (PBC). The principle investigator's laboratory is focused on clarifying neuronal network mechanisms that generate and pattern rhythmic bursts of neuronal population activity. The activity of such rhythmogenic networks is fundamental to essential functions ranging from walking to information processing. In the case of the PBC, such activity is related to the generation of inspiratory movements of breathing. Understanding the mechanisms of PBC function will ultimately be vital to understanding central respiratory disorders such as central hypoventilation syndrome, and Rett Syndrome. The findings a) that neurons within the rodent PBC express at least some Cx, possibly including Cx26, Cx32, and Cx36, and b) that putative gap junction blockers (uncouplers) affect the generation of bursting by the PBC suggest a role for gap junctions in PBC rhythmogenesis. However, in other neuronal populations, uncouplers exert non-specific effects on membrane properties. Moreover, the detection of certain Cx within the PBC has been questioned due to a potential lack of specificity by the method employed. Accordingly the issue of whether gap junctions as a form of intercellular communication are important to PBC function remains unresolved. Thus, the research proposed herein will further examine this issue by accomplishing two specific aims. The first aim is to determine the mechanisms by which putative gap junction uncouplers alter PBC output. To this end the cytoplasmic connectivity between PBC neurons, and the potential alteration of this continuity by an uncoupler (carbenoxolone, or CBX) and a control agent (glycyrrhizic acid or GZA), will be evaluated by a fluorescence recovery after photobleaching (FRAP) microscopy technique. Along with the FRAP study, the effects of CBX and GZA on membrane properties including input resistance and calcium currents will be examined. The second aim of this research is to further elucidate ontogenetic patterns of Cx expression within PBC neurons. This aim will be accomplished using in situ hybridization to detect transcripts for Cx26, Cx32, Cx36, Cx45, Cx47, and Cx59 in PBC neurons in tissue from mice from embryonic day 12 through postnatal day 21. Neuronal networks that even in isolation from sensory or descending inputs generate bursts of activity (i.e., central pattern generators) underlie various essential functions such as breathing and locomotion. Accordingly, studies examining the mechanisms of such rhythmogenic network function will provide a conceptual framework within which to examine disorders affecting repetitive behaviors. In relation to the research proposed herein, better understanding the mechanisms of pre-B"tzinger Complex function will ultimately be vital to understanding central respiratory disorders such as central hypoventilation syndrome, and Rett Syndrome.