MECHANISMS AND LOCALIZATION OF CO2 SENSITIVE CNS NEURONS

CO2 敏感中枢神经元的机制和定位

• 批准号: 6184278
• 负责人: GEORGE B RICHERSON
• 金额: $ 29.43万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6184278
• 关键词: acetylcholine; alkalosis; carbon dioxide; chemoreceptors; cholinergic agents; hypercapnia; immunocytochemistry; laboratory rat; medulla oblongata; membrane potentials; muscarinic receptor; neuroanatomy; neurons; neuropharmacology; potassium channel; respiratory acidosis; tissue /cell culture; voltage /patch clamp

DESCRIPTION (Adapted from the applicant's abstract): The location of central respiratory chemoreceptors and their cellular mechanisms have not been defined. Understanding these mechanisms is important because blood CO2 level is the dominant drive for respiration under normal conditions, and because abnormalities of chemoreception occur in many common diseases, including chronic obstructive pulmonary disease, sleep apnea and possibly sudden infant death syndrome. Previous work in this laboratory has identified putative central respiratory chemoreceptor neurons within the medullary raphe, which are highly chemosensitive. These neurons have now been isolated in tissue culture, where they express the same electrophysiological properties, chemosensitivity and neurotransmitters as medullary raphe neurons in brain slices and in vivo. Together with ongoing work in brain slices, this tissue culture model of cellular chemosensitivity provides an opportunity to study the fundamental mechanisms of central respiratory chemoreception at a highly detailed cellular level. The proposed work will examine a number of basic unanswered questions about chemosensitivity of medullary neurons. 1) How does the degree of chemosensitivity of medullary raphe neurons compare to that of neurons within the ventrolateral medulla and other brainstem respiratory nuclei that are also putative sites for chemoreception? 2) What are the neurotransmitters contained in chemosensitive medullary neurons? 3) How does acetylcholine modulate chemosensitive neurons? 4) Does CO2 act through a change in intracellular pH alone, or does extracellular pH or CO2 also modulate neuronal activity? 5) What are the ionic mechanisms of chemosensitivity? One set of experiments will use perforated patch clamp recordings to quantitatively compare chemosensitivity of neurons within the medullary raphe with other putative chemoreceptor sites. Another set of experiments will first identify chemosensitive neurons of the medullary raphe, then immunohistochemistry will be used to determine which neurotransmitters they contain. A third set of experiments will use patch clamp recordings to determine how chemosensitive raphe neurons are affected by acetylcholine agonists. A combination of patch clamp recording and fluorescence measurement of intracellular pH will then be used to determine the primary stimulus to chemosensitive raphe neurons. Preliminary work has identified a novel calcium-activated nonselective cation current in stimulated raphe neurons. Whole-cell patch clamp recordings will be used to determine the properties of this current, and how respiratory acidosis modulates it. For the first set of experiments, brain slices will be used exclusively. For the remaining experiments, cultured neurons will be used along with brain slices to verify the observations seen in culture. The proposed experiments should define the neurotransmitter content, and mechanisms of chemosensitivity of the medullary raphe, a brainstem site that is an excellent candidate for central respiratory chemoreception. Disturbances of breathing are common in human diseases. Understanding the basic mechanisms involved in modulation of neuronal activity by CO2 and pH may help provide successful treatment for these diseases.

描述（改编自申请人的摘要）： 中央呼吸性化学感受器及其细胞机制尚未 已定义。 了解这些机制很重要，因为血液CO2 水平是正常条件下呼吸的主要驱动力，并且 由于化学感受的异常发生在许多常见疾病中，所以 包括慢性阻塞性肺部疾病，睡眠呼吸暂停以及可能 猝死综合症。 该实验室的先前工作 确定了推定的中枢呼吸性化学感受器神经元 髓质raphe，高度化学敏感。 这些神经元现在有 在组织培养中分离出来，它们表达相同 电生理特性，化学敏感性和神经递质作为 脑切片和体内的髓质raphe神经元。 以及持续的 在脑切片中工作，这种细胞化学敏感性的组织培养模型 提供了研究中央基本机制的机会 高度详细的细胞水平上的呼吸性化学感受。 这 拟议的工作将研究许多有关的基本未解决问题 髓质神经元的化学敏度。 1）如何 与神经元相比，髓质神经元的化学敏度 在腹外侧髓质和其他脑干呼吸核中 也推定了化学感受的地点吗？ 2）什么是 化学敏感性髓质神经元中包含的神经递质？ 3）如何 乙酰胆碱会调节化学敏感性神经元吗？ 4）二氧化碳通过 单独的细胞内pH变化，或者也会发生细胞外pH或CO2 调节神经元活动？ 5）什么是离子机制 化学敏感性？ 一组实验将使用穿孔的斑块夹 记录以定量比较神经元内神经元的化学敏度 带有其他推定的化学感受器位点的髓质raphe。 另一组 实验将首先鉴定髓质的化学敏感性神经元 raphe，然后将使用免疫组织化学来确定 它们包含的神经递质。 第三组实验将使用补丁 夹具记录以确定如何影响化学敏感的Raphe神经元 通过乙酰胆碱激动剂。 斑块夹记录和 然后将使用细胞内pH的荧光测量来确定 化学敏感性Raphe神经元的主要刺激。 初步工作 确定了一种新型的钙激活的非选择性阳离子电流 刺激的Raphe神经元。 全细胞贴片钳记录将用于 确定该电流的特性以及呼吸酸中毒 调节它。 对于第一组实验，将使用大脑切片 只。 对于其余的实验，将使用培养的神经元 与大脑切片一起验证文化中看到的观察结果。 这 提出的实验应定义神经递质含量，并且 髓质raphe的化学敏度机制，脑干部位 是中枢呼吸性化学感受的绝佳候选者。 呼吸障碍在人类疾病中很常见。 了解 二氧化碳和pH调节神经元活性的基本机制 可能有助于为这些疾病提供成功的治疗方法。