Trek channels and opioid signaling in the ventral tegmental area

描述（由申请人提供）：阿片类药物是疼痛管理的支柱，尽管它们有显著的副作用和成瘾倾向。滥用海洛因等阿片类药物与许多严重的健康问题有关，包括致命的过量服用、自然流产、肝炎和艾滋病毒/艾滋病等传染病以及心血管和肺部问题。鉴于其临床意义和对公众健康的不利影响，我们必须了解阿片类药物生理和行为影响的机制。本文提出的工作集中在阿片奖励的关键神经基质-腹侧被盖区（VTA）-并挑战有关介导阿片诱导的多巴胺能（DA）神经元去抑制的信号通路的传统智慧，阿片奖励的关键机制。阿片类药物引起的腹侧被盖区DA能神经元的去抑制涉及腹侧被盖区GABA能神经元的直接超极化。G蛋白门控内向整流K+（GIRK/KIR 3）通道被广泛认为介导阿片类药物诱导的GABA神经元超极化，这主要是由于它们在许多神经元群体中的代谢性突触后抑制中的作用。然而，我们最近试图验证这种范式失败了，揭示了GIRK通道不介导VTA GABA神经元的抑制，VTA DA神经元的去抑制，或阿片类药物的奖励相关行为效应。相反，我们的研究结果表明，阿片类药物对腹侧被盖区GABA神经元的急性抑制作用是由腺苷酸环化酶的抑制和随后的离子通道激活介导的，该离子通道表现出Trek亚家族2孔（K2 P）K+通道的独特调控和生物物理特征。本研究的目的是验证阿片类药物通过激活腹侧被盖区GABA神经元中的Trek通道间接刺激腹侧被盖区多巴胺神经元并诱发奖赏相关行为的假设。目前，关于Trek在腹侧被盖区表达的数据很少，也没有Trek通道参与阿片信号传导的报道。因此，我们将在AIM 1中开始，确定是Trek 1还是Trek 2在腹侧被盖区GABA神经元中携带莫尔激活的K+电流。针对Trek 1和Trek 2的功能阻断抗体以及单细胞RT-PCR将应用于涉及VTA GABA神经元切片的电生理研究。在AIM 2中，我们将使用可用的Trek基因敲除小鼠来测量Trek消融对VTA中阿片样物质信号传导的影响，以及对吗啡的运动刺激和强化作用的影响。在AIM 3中，我们将寻求更好地理解新的观察，即在Girk基因敲除小鼠中，VTA GABA神经元中MOR激活的K+电流显著增强。电生理学和行为学方法将被用来探测VTA GABA神经元中MOR激活的K+电流与慢性给药相关的复杂适应之间的关系。拟议的工作将重新构建我们对阿片受体下游信号传导的理解，因此，可能对疼痛管理和成瘾相关的诊断或治疗策略产生重大影响。 公共卫生相关性：阿片类药物靶向对疼痛处理和奖励很重要的神经回路，这些作用解释了它们的有益（镇痛）和有害（成瘾）作用。这一提议挑战了传统智慧，即在涉及奖赏的关键神经元群体中阿片信号传导的分子细节。清楚地了解阿片类药物奖赏的分子机制对于我们理解成瘾和设计更具选择性和有效的疼痛管理治疗方法至关重要。

DESCRIPTION (provided by applicant): Opioid-based drugs are mainstays for pain management despite their significant side effects and addictive liability. Abuse of opioid drugs such as heroin is linked to many serious health problems, including fatal overdose, spontaneous abortion, infectious disease such as hepatitis and HIV/AIDS, and cardiovascular and pulmonary problems. Given both their clinical significance and adverse impact on public health, it is imperative that we understand mechanisms underlying the physiological and behavioral effects of opioids. The work proposed herein centers on a key neural substrate of opioid reward - the ventral tegmental area (VTA) - and challenges conventional wisdom concerning the signaling pathway mediating the opioid-induced disinhibition of dopaminergic (DA) neurons, a key mechanism of opioid reward. The opioid-induced disinhibition of DA neurons in the VTA involves the direct hyperpolarization of VTA GABA neurons. G protein-gated inwardly- rectifying K+ (GIRK/KIR3) channels are widely-considered to mediate the opioid-induced hyperpolarization of GABA neurons, due largely to their documented roles in metabotropic postsynaptic inhibition in many neuron populations. Our recent attempt to validate this paradigm failed, however, revealing that GIRK channels do not mediate the inhibition of VTA GABA neurons, the disinhibition of VTA DA neurons, or reward-related behavioral effects of opioids. Instead, our findings suggest that the acute inhibitory actions of opioids on VTA GABA neurons are mediated by the inhibition of adenylyl cyclase and consequent activation of an ion channel exhibiting the unique regulatory and biophysical signature of the Trek subfamily of 2-pore (K2P) K+ channels. The goal of this study is to test the hypothesis that opioids indirectly stimulate VTA dopamine neurons, and evoke reward-relevant behaviors, by activating Trek channels in VTA GABA neurons. At present, there are scant data concerning Trek expression in the VTA and no reports of Trek channel involvement in opioid signaling. As such, we will begin in AIM 1 by determining whether it is Trek1 or Trek2 that carries the MOR- activated K+ current in VTA GABA neurons. Well-characterized function-blocking antibodies directed against Trek1 and Trek2, as well as single-cell RT-PCR, will be applied to electrophysiological studies involving VTA GABA neurons in slices. In AIM 2, we will use available Trek knockout mice to measure the impact of Trek ablation on opioid signaling in the VTA, and on the motor-stimulatory and reinforcing effects of morphine. In AIM 3, we will seek a better understanding of the novel observation that the MOR-activated K+ current in VTA GABA neurons is significantly enhanced in Girk knockout mice. Electrophysiological and behavioral approaches will be used to probe the relationship between the MOR-activated K+ current in VTA GABA neurons and the complex adaptations linked to chronic drug administration. The proposed work will reframe our understanding of signaling downstream from opioid receptors and as such, may have significant implications for diagnostic or therapeutic strategies relevant to pain management and addiction. PUBLIC HEALTH RELEVANCE: Opioid-based drugs target neural circuitry important for pain processing and reward, actions that explain both their beneficial (analgesic) and untoward (addictive) effects. This proposal challenges conventional wisdom concerning the molecular details of opioid signaling in a key neuron population involved in reward. A clear understanding of the molecular mechanisms of opioid reward is crucial to our understanding of addiction and to the design of more selective and effective therapeutic approaches to pain management.