Role of astroglia in cocaine-induced neurotoxicity

描述(申请人提供)：这项建议集中于开发一种新的原型光学扩散荧光(ODF)探针的技术，用于体内研究星形胶质细胞在可卡因诱导的血流动力学、新陈代谢和脑功能影响中的作用。这种光学探头是独一无二的，因为它使用多个波长在体内以高时间分辨率同时跟踪脑血容量(CBV)、氧合血红蛋白(HBO2)、脱氧血红蛋白(HBr)和细胞内钙([Ca++])i。我们已经在活体啮齿动物身上建立了新探针设计的原则证据，最近获得的结果表明，静脉注射可卡因和局麻药利多卡因后，皮质[Ca++]i显著增加，但不使用哌醋甲酯，这表明可卡因的局部麻醉特性，而不是其儿茶酚胺能作用，是这种作用的基础。我们假设可卡因诱导的[Ca++]i升高是潜在的有害的，因为[Ca++]i的增加会使组织更容易受到缺血的影响，继而是脑血流量的潜在下降，否则不会引起缺血和/或细胞损伤。我们建议进一步发展ODF技术，以分离可卡因引起的[Ca++]i升高的细胞来源。我们的主要假设是，可卡因诱导的[Ca++]i反应来自星形胶质细胞，而不是神经元。我们进一步假设谷氨酸在一定程度上介导了在大脑皮层观察到的星形细胞[Ca++]i的增加。为了验证我们的假设，我们提出了以下具体目标：i)证明ODF探针结合钙敏感荧光染料Rhod-2(AM)和细胞特异性组织加载技术可以用于区分活体神经元和星形胶质细胞网络中的[Ca++]i动态，同时跟踪CBV、HBO2和HBr的变化；ii)证明ODF探针测量的可卡因诱导的[Ca++]i反应部分源于星形胶质细胞；iii)选择性代谢型谷氨酸受体1(MGluR1)拮抗剂可以阻断可卡因诱导的[Ca++]i升高。这项研究对药物成瘾领域的意义将在于：1)首次实现一种新的实验方法，能够在活体内跟踪中枢神经系统特定和大型细胞群(星形胶质细胞)的[Ca++]i瞬变；2)直接研究星形胶质细胞在可卡因诱导的活体动物脑功能变化中的作用；3)首次获得与正常和暴露于可卡因的大脑中的CBV、HBO2和Hbr同步的[Ca++]i动态信息。我们的长期目标是进一步巩固和简化ODF探针技术，以评估大脑功能和氧气含量，从而使广大神经科学界能够获得在药物成瘾研究领域的进一步研究。有关星形胶质细胞在成瘾过程中的作用和/或滥用药物的神经毒性的新信息可能会导致确定新的治疗目标和新的治疗策略，这将有利于公众健康。这项建议的重点是开发一种新的原型光学扩散荧光(ODF)探针用于药物成瘾研究的技术。这项研究与公共卫生的相关性在于，与目前的技术(共焦显微镜和双光子显微镜)相比，新的探测器负担得起，因此研究人员可以广泛获得，从而有助于加强我们对导致药物成瘾的过程以及最终治疗和预防的了解。

DESCRIPTION (provided by applicant): This proposal focuses on developing technology for a new prototype optical diffusion fluorescence (ODF) probe to be used in vivo to study the role of astroglia cells in cocaine-induced effects on hemodynamics, metabolism and function in the brain. The optical probe in question is unique because it uses multiple wave- lengths to track cerebral blood volume (CBV), oxyhemoglobin (HbO2), deoxyhemoglobin (HbR) and intracellular calcium ([Ca++])i simultaneously at high temporal resolution in vivo. We have established proof-of- principle of the new probe design in the living rodent and recently obtained results demonstrating significant increases in cortical [Ca++]i following intravenous administration of cocaine and the local anesthetic lidocaine but not with methylphenidate indicating that cocaine's local anesthetic properties but not its catecholaminergic actions underlie this effect. We hypothesized that the cocaine-induced [Ca++]i increases are potentially harmful because the [Ca++]i increases would make the tissue more vulnerable to ischemia secondary to potential decreases in cerebral blood flow which would otherwise not induce ischemia and/or cell damage. We are proposing to further develop the ODF technology to isolate the cellular origin of the [Ca++]i increases induced by cocaine. Our main hypothesis states that the cocaine-induced [Ca++]I response originates from astroglia cells and not from neurons. We further hypothesize that glutamate in part mediates the increases in astrocytic [Ca++]i observed in cortex. To test our hypothesis we propose the following specific aims: i) To demonstrate that the ODF probe in combination with the calcium-sensitive fluorescence dye Rhod-2 (AM) and cell-specific tissue loading technology can be used to differentiate [Ca++]i dynamics in neuronal and astroglia cellular networks in vivo while simultaneously tracking changes in CBV, HbO2 and HbR; ii) To demonstrate that the cocaine-induced [Ca++]i response measured by the ODF probe originate in part from astroglia cells; iii) that the cocaine-induced [Ca++]i increases can be blocked by a selective metabotrobic glutamate receptor 1 (mGluR1) antagonist. The significance of this research to the field of drug addiction will be to 1) implement for the first time a new experimental methodology which is capable of tracking [Ca++]i transients from specific and large cell populations (astroglia cells) in the central nervous system in vivo, 2) directly address the role of astroglia cells in cocaine-induced changes in brain function in the living animal and 3) for the first time obtain information on [Ca++]i dynamics simultaneously with CBV, HbO2 and HbR in the normal and cocaine-exposed brain in vivo. Our long-term goal is to further solidify and simplify the ODF probe technology to assess brain function and oxygen content, thereby, allowing broad neuroscience community access to further studies in the area of drug addiction research. New information on the role of astroglia cells in the addiction process and/or neurotoxicity of drugs of abuse will potentially lead to identification of new treatment targets and new therapeutic strategies which will benefit public health. This proposal focuses on developing technology for a new prototype optical diffusion fluorescence (ODF) probe to be used in drug addiction research. The relevance of this research to public health is that the new probe in comparison to current technology (confocal and two-photon microscopes) is affordable and therefore can become widely accessible to researchers and thus help enhance our understanding of processes leading to addiction to drugs of abuse and ultimately to its treatment and prevention.