The role of CaV3.2 T-type calcium channel-expressing sensory neurons in nociception and chronic pain

CaV3.2 T型钙通道表达感觉神经元在伤害感受和慢性疼痛中的作用

• 批准号: 9177698
• 负责人: Daniel M DuBreuil
• 金额: $ 2.92万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9177698
• 关键词: Action Potentials; Acute; Address; Adverse effects; Affect; Afferent Neurons; Alpha Cell; Analgesics; Anatomy; Behavior; Behavioral; Behavioral Mechanisms; Biological Process; Calcium; Calcium Signaling; Calcium ion; Cells; Characteristics; Chronic; Consensus; Data; Development; Exhibits; Fiber; Hyperalgesia; Hypersensitivity; Imagery; Impairment; In Vitro; Individual; Inflammatory; Injury; Ion Channel; Kinetics; Knock-in Mouse; Knockout Mice; Light; Measures; Mechanics; Membrane Potentials; Methods; Mibefradil; Modeling; Molecular; Molecular Profiling; Mouse Strains; Mus; Nerve; Neurons; Nociception; Nociceptors; Opiates; Opioid Receptor; Peripheral; Pharmacology; Physiology; Play; Population; Property; Rest; Rodent; Role; Sensory; Sensory Thresholds; Site; Slice; Spinal; Spinal Cord; Spinal Ganglia; Stimulus; Synapses; Synaptic Potentials; Synaptic Transmission; Syndrome; T-Type Calcium Channels; TRPV1 gene; Testing; Thermal Hyperalgesias; Tissues; Transgenic Organisms; Withdrawal; Work; allodynia; awake; chronic pain; deltorphin; dorsal horn; experimental study; in vivo; ineffective therapies; inhibitor/antagonist; mechanical allodynia; mouse model; neuronal excitability; neurotransmission; neurotransmitter release; novel; presynaptic; public health relevance; receptor; spinal nerve posterior root; therapeutic target; voltage; ziconotide

 DESCRIPTION (provided by applicant): Voltage-gated calcium ion (CaV) channels play important roles in sensory transduction and are important targets of non-opioid analgesics. CaV3.2 channels are the dominant T-type current in DRG and exhibit interesting properties, such as activation near the resting membrane potential and slow kinetics of closing. These properties allow them to control neuronal excitability, lower activation thresholds, and prolong Ca2+ entry to boost calcium signaling. CaV3.2-knockout mice have impaired nociception and CaV3.2 channel expression is increased in a cell specific manner in several chronic pain models. Although it is clear that CaV3.2 channels are critical for the development of hyperalgesia, the biological function of CaV3.2-expressing neurons is still debated. In this work, I seek to define CaV3.2-expressing sensory neurons using functional anatomy and pharmacology and elucidate the role of CaV3.2 channels in hyperalgesia. In order to accomplish this, I have expressed the light-activated ion channel channelrhodopsin-2 (ChR2) specifically in neurons that express CaV3.2 channels using transgenic knock-in mice. This allows for the identification and control of CaV3.2-expressing neurons in vitro and in vivo. I propose a strategy to answer two questions: 1) What anatomical and functional characteristics define CaV3.2-expressing inputs to spinal dorsal horn? 2) How do CaV3.2- containing and CaV3.2-lacking neurons contribute to hyperalgesia? To examine the neuronal subpopulations that express CaV3.2 channels, as well as the mechanism by which they contribute to chronic pain, I will use a combination of behavior and slice physiology. In awake behaving mice, nociceptor activation elicits a rapid paw withdrawal. Using acute spinal cord slices, I will examine the functional distribution of CaV3.2- expressing afferent fibers into the spinal cord and test their pharmacological sensitivity to opiate and non-opiate analgesics. I hypothesize that CaV3.2 expressing sensory neurons represent a heterogeneous group of high-threshold and low-threshold sensory neurons and co-express δ-opioid receptors, but not µ-opoid receptors. To induce hyperalgesia, a feature of many chronic pain syndromes, I will use prolonged activation of ChR2-expressing sensory neurons. Preliminary data suggests that prolonged stimulation of CaV3.2-expressing sensory neurons is sufficient to generate mechanical hyperalgesia. In this proposal, I propose experiments testing for the contribution of CaV3.2-expressing and CaV3.2-lacking sensory neurons to mechanical hyperalgesia and allodynia, as well as thermal hyperalgesia. Using acute spinal cord slices, I will examine the mechanism of these behavioral effects by recording fEPSPs in acute spinal cord sections. I will test for increased contribution o CaV3.2 channels to neurotransmission using the CaV3 inhibitor mibefradil. This study will be the first to examine the entire population of CaV3.2-expressing sensory neurons in vivo and in vitro and will expand our understanding of the peripheral mechanism underlying chronic pain.

 描述（由适用提供）：电压门控钙离子（CAV）通道在感觉传递中起着重要作用，并且是非阿片类镇痛药的重要目标。 CAV3.2通道是DRG中的主要T型电流，并且具有有趣的特性，例如在静息膜电位附近的激活和闭合的缓慢动力学。这些特性使它们可以控制神经元令人兴奋，较低的激活阈值，并延长Ca2+进入以增强钙信号传导。 Cav3.2敲除小鼠在几种慢性疼痛模型中以细胞特异性方式增加了诺科的障碍，而Cav3.2通道表达增加。尽管很明显，Cav3.2通道对于杀伤性的发展至关重要，但仍在争论表达Cav3.2的神经元的生物学功能。在这项工作中，我试图使用功能解剖学和药理学来定义表达CAV3.2的感觉神经元，并阐明Cav3.2通道在Hypergensia中的作用。为了实现这一目标，我表达了使用转基因敲入小鼠在神经元中特别表达CAV3.2通道的光激活的离子通道通道Ropopsin-2（ChR2）。这允许在体外和体内识别和控制CAV3.2表达神经元。我提出了一个回答两个问题的策略：1）哪些解剖学和功能特征定义了cav3.2表达脊柱背角的输入？ 2）含Cav3.2的含Cav3.2含量和cav3.2的神经元如何导致痛觉过敏？为了检查表达Cav3.2通道的神经元亚群，以及它们导致慢性疼痛的机制，我将使用行为和切片生理学的组合。在醒着的行为小鼠中，伤害感受器的激活会引起迅速的爪子戒断。使用急性脊髓切片，我将检查Cav3.2-表达传入纤维的功能分布到脊髓中，并测试其药物敏感性以优化和非兼容镇痛药。我假设表达感觉神经元的Cav3.2代表了一组高阈值和低阈值感觉神经元和共表达δ-阿片类受体，但不指氧受体。综合征，我将使用长期激活ChR2表达ChR2的感觉神经元。初步数据表明，表达CAV3.2表达的感觉神经元的长期刺激足以产生机械性痛觉过敏。在此提案中，我提出了对CAV3.2表达和CAV3.2磁性的感觉神经元对机械性痛觉过敏和异常性痛的贡献以及热痛觉过敏的贡献的实验测试。使用急性脊髓切片，我将通过在急性脊髓切片中记录FEPSP来检查这些行为效应的机制。我将使用CAV3抑制剂Mibefradil测试c cav3.2对神经传递的贡献的增加。这项研究将是第一个检查体内和体外表达CAV3.2的感觉神经元的全部人群的研究，并将扩展我们对慢性疼痛背后的外围机制的理解。