电从新生儿鼠害隔离脑干，脊髓准备允许呼吸中枢网络输出记录
Summary
The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.
Abstract
While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.
Introduction
呼吸是一个复杂和生命活动由大脑控制，允许双氧（O 2）的摄取和二氧化碳（CO 2）的消除。中枢性呼吸的驱动器是由一个复杂的网络，位于脑干的两个哺乳动物1，两栖类2类，爬行类3，4鸟类和鱼类5生成。即使呼吸的研究可在体内进行处理，精密机械研究需要直接访问的呼吸控制网络。为此，Adrian和Buytendijk开发出减少的金鱼制剂，其中电极放置在脑干表面记录用鳃通风5相关联的产生的节奏。这一做法后来被Suzue于1984年6适应新生老鼠用。这种制剂的出现，导致呼吸神经生物学显著的进步。由于它是比较简单的，该技术呈现ħERE是适合于各种有节奏的运动行为和他们的新生啮齿动物起源的基本调查。
该方法的总的目标是记录吸气活动的神经相关，一个呼吸状节律称为假想呼吸，通过呼吸网络产生。这种方法可以用在广泛的研究目标，目标吸气响应呼吸变化或药理学在野生型7和转基因动物的8。鉴于实验关于记录的信号的生理相关性在低温下进行的，没有感觉传入，并且其中葡萄糖和 O 2的脑脊液内的浓度高的条件下，问题已经被提出。虽然有在体内和体外条件差别明显（例如，吸气突发的频率）的事实仍然是存在呼吸网络 6的核心元素使得能够研究相关 的一个重要的体内平衡功能9,10一个健壮的节奏。
后面的发展和使用该技术的基本原理是，以促进直接进入呼吸网络的脑干元素，这是在体内难以进入，特别是在新生儿。脑干被置于严格控制的条件下：录制的节奏不被从肺部或颈动脉体周围神经传入调制，使研究专注于呼吸中枢驱动器本身11上。因此，该访问被用来施加激励和记录的输出信号。在对比体积描记法记录，呼吸节律是通过其所有组件整个身体调制（例如，肺腹胀，外围化学传感器），使之难以应用于精确的刺激。
在ewborn大鼠，该协议包括上记录的分离的脑干第四前根信号和截短脊髓，保持在人造脑脊髓液（ACSF）中。由脑干脊髓制剂产生的节奏由链接到吸气信号 9个体慢突发。隔离脑干，脊髓制剂很容易在大鼠0产后天可记录到4（P0 – P4）7。这种方法通常被用来评估呼吸道网络的低氧反应，并且还响应于高碳酸血症，酸中毒或药物。急性缺氧协议这里介绍。被撤销的学联O 2获得此刺激;这种方法通常用于评估耐受性和响应性缺氧的侮辱。该协议导致从第一分钟节奏抑郁直到缺氧暴露的端部（ 图1）12。这个凹陷反转在后低氧恢复12。关于实验设计，它必须注意到，脑桥，位于脑干延髓部分，对节律发生器8的抑制作用是重要的。因此，全脑干延髓和脊髓的筹备工作显示较低的节奏。用于记录分离的样品中的脑桥夹杂物根据实验13的目标确定;延髓网络上脑桥影响的研究将需要记录有和没有脑桥比较结果14。此外，这种技术的优点之一是延伸制剂的喙部，以包括中脑和/或间脑区域15,16，使得可以评估这些区域的脑桥延髓呼吸网络的影响的可能性。
Protocol
Representative Results
Discussion
呼吸活动的准确定量是很有挑战性的。的确，呼吸是一个函数，既可以是自动和自愿的，即根据环境，人体的需要，情绪状态和行为调制。这种技术的优点是负责产生呼吸命令神经元件的隔离。因此，脑干脊髓制剂和体积描记法电生理记录是互补的技术，研究了整个神经网络呼吸体外和体内分别。在脑干脊髓制备的膜片钳记录（例如，从腹面接近）也可以设想，并允许在一保存呼吸<…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors sincerely thank the Canadian Institutes of Health Research MOP 130258 and the Star Foundation for Children’s Health Research, along with the Molly Towell Foundation, for the provision of the research facility and financial support. The authors also sincerely thank Dr. Kinkead Richard for manuscript proofreading and advice.
Materials
| Sylgard | Sigma Aldrich | 761036-5EA | Use under hood |
| NaCl | Bioshop | SOD002 | |
| KCl | Bioshop | POC888 | |
| CaCl2 | Bioshop | CCL444 | |
| MgCl2 | Bioshop | MAG510 | |
| NaHCO3 | Bioshop | SOB999 | |
| NaH2PO4 | Bioshop | SPM306 | |
| D-glucose | Bioshop | GLU501 | |
| Carbogen | Linde | 343-02-0006 | |
| Temperature Controller | Warner Instruments, Hamden, CT, USA | TC-324B | |
| Suction electrode | A-M Systems, Everett, WA, USA | model 573000 | |
| Differential AC amplifier | A-M Systems, Everett, WA, USA | model 1700 | |
| Moving averager | CWE, Ardmore, PA, USA | model MA-821 | |
| Data acquisition system | Dataq Instruments, Akron, OH, USA | model DI-720 | |
| LabChart software | ADInstruments, Colorado Springs, CO, USA | ||
| Prism sofware | Graphpad, La Jolla, CA, USA | ||
| Dissection chamber | Plastic box (e.g. petri box) will do | ||
| Recording chamber | Home made | ||
| Base | Kanetec, Bensenville, IL, USA | MB | |
| Micromanipulator | World Precision Instrument Inc, Sarasota, FL, USA | KITE-R | |
| Base | Kanetec, Bensenville, IL, USA | MB | |
| Peristaltic pump | Gilson, Middleton, WI, USA | MINIPULS 3 | |
| Faraday Cage | Home made | ||
| Computer |
Referências
- Feldman, J. L., Del Negro, ., A, C., Gray, P. A. Understanding the rhythm of breathing: so near, yet so far. Annu Rev Physiol. 75, 423-452 (2013).
- Taylor, A. C., Kollros, J. J. Stages in the normal development of Rana pipiens larvae. Anat Rec (Hoboken). 94, 7-13 (1946).
- Takeda, R., Remmers, J. E., Baker, J. P., Madden, K. P., Farber, J. P. Postsynaptic potentials of bulbar respiratory neurons of the turtle. Respir Physiol. 64, 149-160 (1986).
- Bouverot, P. Control of breathing in birds compared with mammals. Physiol Rev. 58, 604-655 (1978).
- Adrian, E. D., Buytendijk, F. J. Potential changes in the isolated brain stem of the goldfish. J Physiol. 71, 121-135 (1931).
- Suzue, T. Respiratory rhythm generation in the in vitro brain stem-spinal cord preparation of the neonatal rat. J Physiol. 354, 173-183 (1984).
- Fournier, S., et al. Gestational stress promotes pathological apneas and sex-specific disruption of respiratory control development in newborn rat. J Neurosci. 33, 563-573 (2013).
- Caravagna, C., Kinkead, R., Soliz, J. Post-natal hypoxic activity of the central respiratory command is improved in transgenic mice overexpressing Epo in the brain. Respir Physiol Neurobiol. 200, 64-71 (2014).
- Onimaru, H., Arata, A., Homma, I. Neuronal mechanisms of respiratory rhythm generation: an approach using in vitro preparation. Jpn J Physiol. 47, 385-403 (1997).
- Onimaru, H. Studies of the respiratory center using isolated brainstem-spinal cord preparations. Neurosci Res. 21, 183-190 (1995).
- Ballanyi, K., Onimaru, H., Homma, I. Respiratory network function in the isolated brainstem-spinal cord of newborn rats. Prog Neurobiol. 59, 583-634 (1999).
- Viemari, J. C., Burnet, H., Bevengut, M., Hilaire, G. Perinatal maturation of the mouse respiratory rhythm-generator: in vivo and in vitro studies. Eur J Neurosci. 17, 1233-1244 (2003).
- Rybak, I. A., Abdala, A. P., Markin, S. N., Paton, J. F., Smith, J. C. Spatial organization and state-dependent mechanisms for respiratory rhythm and pattern generation. Prog Brain Res. , 165-201 (2007).
- Hilaire, G., Viemari, J. C., Coulon, P., Simonneau, M., Bevengut, M. Modulation of the respiratory rhythm generator by the pontine noradrenergic A5 and A6 groups in rodents. Respir Physiol Neurobiol. 143, 187-197 (2004).
- Okada, Y., Kawai, A., Muckenhoff, K., Scheid, P. Role of the pons in hypoxic respiratory depression in the neonatal rat. Respir Physiol. 111, 55-63 (1998).
- Voituron, N., Frugiere, A., Gros, F., Macron, J. M., Bodineau, L. Diencephalic and mesencephalic influences on ponto-medullary respiratory control in normoxic and hypoxic conditions: an in vitro study on central nervous system preparations from newborn rat. Neurociência. 132, 843-854 (2005).
- Somjen, G. G. Ion regulation in the brain: implications for pathophysiology. Neuroscientist. 8, 254-267 (2002).
- Danneman, P. J., Mandrell, T. D. Evaluation of five agents/methods for anesthesia of neonatal rats. Lab Anim Sci. 47, 386-395 (1997).
- Formenti, A., Zocchi, L. Error signals as powerful stimuli for the operant conditioning-like process of the fictive respiratory output in a brainstem-spinal cord preparation from rats. Behav Brain Res. 272, 8-15 (2014).
- Bierman, A. M., Tankersley, C. G., Wilson, C. G., Chavez-Valdez, R., Gauda, E. B. Perinatal hyperoxic exposure reconfigures the central respiratory network contributing to intolerance to anoxia in newborn rat pups. J App Physiol. 116, 47-53 (2014).
- Umezawa, N., et al. Orexin-B antagonized respiratory depression induced by sevoflurane, propofol, and remifentanil in isolated brainstem-spinal cords of neonatal rats. Respir Physiol Neurobiol. 205, 61-65 (2015).
- Ruangkittisakul, A., Secchia, L., Bornes, T. D., Palathinkal, D. M., Ballanyi, K. Dependence on extracellular Ca2+/K+ antagonism of inspiratory centre rhythms in slices and en bloc preparations of newborn rat brainstem. J Physiol. 584, 489-508 (2007).
- Cayetanot, F., Bodineau, L., Frugiere, A. 5-HT acting on 5-HT(1/2) receptors does not participate in the in vitro hypoxic respiratory depression. Neurosci Res. 41, 71-78 (2001).
- Onimaru, H., Homma, I. Whole cell recordings from respiratory neurons in the medulla of brainstem-spinal cord preparations isolated from newborn rats. Pflugers Archiv : European journal of physiology. 420, 399-406 (1992).
- Paton, J. F. Rhythmic bursting of pre- and post-inspiratory neurones during central apnoea in mature mice. J Physiol. 502 (Pt 3), 623-639 (1997).
- Morin-Surun, M. P., Boudinot, E., Kato, F., Foutz, A. S., Denavit-Saubie, M. Involvement of NMDA receptors in the respiratory phase transition is different in the adult guinea pig in vivo and in the isolated brain stem preparation. J Neurophysiol. 74, 770-778 (1995).
- Otsuka, H. Effects of volatile anesthetics on respiratory activity and chemosensitivity in the isolated brainstem-spinal cord of the newborn rat. Hokkaido Igaku Zasshi. 73, 117-136 (1998).
- Gestreau, C., et al. Task2 potassium channels set central respiratory CO2 and O2 sensitivity. PNAS. 107, 2325-2330 (2010).
- Caravagna, C., Soliz, J. PI3K and MEK molecular pathways are involved in the erythropoietin-mediated regulation of the central respiratory command. Respir Physiol Neurobiol. 206C, 36-40 (2014).
- Tree, K., Caravagna, C., Hilaire, G., Peyronnet, J., Cayetanot, F. Anandamide centrally depresses the respiratory rhythm generator of neonatal mice. Neurociência. 170, 1098-1109 (2010).
- Arata, A. Respiratory activity of the neonatal dorsolateral pons in vitro. Respir Physiol Neurobiol. 168, 144-152 (2009).
- Onimaru, H., Homma, I. A novel functional neuron group for respiratory rhythm generation in the ventral medulla. J Neurosci. 23, 1478-1486 (2003).
- St-John, W. M., Paton, J. F. Characterizations of eupnea, apneusis and gasping in a perfused rat preparation. Respir Physiol. 123, 201-213 (2000).
