Phillips Wiktor S, Herly Mikkel, Del Negro Christopher A, Rekling Jens C
Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark; and Department of Applied Science, The College of William and Mary, Williamsburg, Virginia.
Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark; and.
J Neurophysiol. 2016 Feb 1;115(2):1063-70. doi: 10.1152/jn.00904.2015. Epub 2015 Dec 9.
Study of acute brain stem slice preparations in vitro has advanced our understanding of the cellular and synaptic mechanisms of respiratory rhythm generation, but their inherent limitations preclude long-term manipulation and recording experiments. In the current study, we have developed an organotypic slice culture preparation containing the preBötzinger complex (preBötC), the core inspiratory rhythm generator of the ventrolateral brain stem. We measured bilateral synchronous network oscillations, using calcium-sensitive fluorescent dyes, in both ventrolateral (presumably the preBötC) and dorsomedial regions of slice cultures at 7-43 days in vitro. These calcium oscillations appear to be driven by periodic bursts of inspiratory neuronal activity, because whole cell recordings from ventrolateral neurons in culture revealed inspiratory-like drive potentials, and no oscillatory activity was detected from glial fibrillary associated protein-expressing astrocytes in cultures. Acute slices showed a burst frequency of 10.9 ± 4.2 bursts/min, which was not different from that of brain stem slice cultures (13.7 ± 10.6 bursts/min). However, slice cocultures that include two cerebellar explants placed along the dorsolateral border of the brainstem displayed up to 193% faster burst frequency (22.4 ± 8.3 bursts/min) and higher signal amplitude (340%) compared with acute slices. We conclude that preBötC-containing slice cultures retain inspiratory-like rhythmic function and therefore may facilitate lines of experimentation that involve extended incubation (e.g., genetic transfection or chronic drug exposure) while simultaneously being amenable to imaging and electrophysiology at cellular, synaptic, and network levels.
对急性脑干细胞体外切片制剂的研究推进了我们对呼吸节律产生的细胞和突触机制的理解，但它们固有的局限性妨碍了长期操作和记录实验。在当前的研究中，我们开发了一种包含前包钦格复合体（preBötC）的器官型切片培养制剂，前包钦格复合体是腹外侧脑干的核心吸气节律发生器。我们使用钙敏荧光染料，在体外培养7至43天的切片培养物的腹外侧（可能是前包钦格复合体）和背内侧区域测量了双侧同步网络振荡。这些钙振荡似乎由吸气神经元活动的周期性爆发驱动，因为对培养的腹外侧神经元的全细胞记录显示出类似吸气的驱动电位，并且在培养物中未检测到来自表达胶质纤维酸性蛋白的星形胶质细胞的振荡活动。急性切片显示爆发频率为10.9±4.2次/分钟，与脑干切片培养物的爆发频率（13.7±10.6次/分钟）没有差异。然而，与急性切片相比，包含沿脑干背外侧边界放置的两个小脑外植体的切片共培养物显示爆发频率快达193%（22.4±8.3次/分钟），信号幅度高340%。我们得出结论，包含前包钦格复合体的切片培养物保留了类似吸气的节律功能，因此可能有助于涉及延长孵育时间的实验（例如基因转染或长期药物暴露），同时便于在细胞、突触和网络水平进行成像和电生理研究。
