Novel method to quantify conditioned fear-based neuronal activity in rat brain in vivo using high-resolution photoacoustic imaging

项目摘要/摘要 精神健康障碍，如创伤后应激障碍(PTSD)，是复杂的，留下了挑战 用于制定有效的干预措施。通过以下方式增加我们对这些疾病的详细了解 研究它们背后的微神经回路，可以帮助为这些复杂的问题提供个性化的解决方案 条件。这种微神经回路可以通过研究一小群高度活跃的神经元来研究， 统称为神经元集合，负责独特的行为反应，例如恐惧 学习，在创伤后应激障碍中。到目前为止，对神经元集合的行为研究主要是在体外进行的，使用 与神经元活动相关的即刻早期基因(例如c-Fos)。活体成像方法目前 有限(非常有侵入性或低分辨率)，通常测量间接神经元活动，如血液 氧依赖反应。为了解决这一知识鸿沟，我们提出了一种新的高分辨率成像 检测和量化体内恐惧相关神经元活动的方法，目的是发现更多 调节与创伤后应激障碍相关的恐惧学习的神经元群的详细作用。 具体地说，我们建议使用光声(PA)成像来映射Fos/Lacz中的激活神经元 转基因大鼠模型。由神经元活动诱导的Fos与LacZ基因融合产生活性(Fos) 细胞能够在酶作用下将亲显色底物，如X-Gal，切割成PA活性染料。我的 在大鼠大脑中进行的幻影和体外先导研究展示了使用这些染料和图像的高对比度PA图像 具有非常高的信噪比的重建。我们假设在恐惧习得的过程中， 将在内侧前额叶皮质(一个紧密的区域)激活并检测到神经元集合 与恐惧行为相关)使用新型Fos/Lacz PA成像系统，提供了一种跟踪手段 这些组合在活体内的纵向变化。为了验证这一假设，我们将使用男性/女性Fos/Lacz 转基因大鼠在受到足部电击(恐惧)时将给予皮质X-Gal。X-Gal产品将 进行PA成像(目标1)，这些图像将用传统的免疫组织化学方法进行验证 脑片Fos和β-半乳糖苷酶的检测(目标2)。意义：该项目将提供更深层次的 洞察与恐惧相关的神经元集合的作用，并提供一种新的、低成本、高分辨率的 纵向测量Fos表达的方法学途径(代表神经元群) 活着。这将广泛用于行为研究，不仅在创伤后应激障碍，而且在各种精神病学 科学，使我们能够更好地定义复杂的精神健康障碍背后的机制。 我的培训将集中在三个主要领域：分子神经科学和显微镜(由赞助商Dr。 CONTI)、行为/动物建模(由共同发起人、佩林博士领导)和PA成像的使用(由关键培训领导 团队成员，Avanaki医生)，帮助我迈向神经放射内科医生兼科学家的职业目标。

Project Summary/Abstract Mental health disorders, such as post-traumatic stress disorder (PTSD), are complex, leaving challenges for the development of effective interventions. Increasing our detailed understanding of these disorders, by studying the micro-neurocircuitry behind them, can help provide individualized solutions to these complicated conditions. This micro-neurocircuitry can be investigated by studying small groups of highly-active neurons, collectively known as neuronal ensembles, that are responsible for unique behavioral responses, e.g. fear learning, in PTSD. Behavioral studies examining neuronal ensembles have thus far been largely ex vivo, using immediate early genes associated with neuronal activity (e.g. c-Fos). In vivo imaging methods are currently limited (very invasive or low resolution), and generally measure indirect neuronal activity, such as the blood oxygen dependent response. To address this knowledge gap, we propose a new high-resolution imaging approach to detect and quantify fear-related neuronal activity in vivo, with the goal of discovering more detailed roles of the neuronal ensembles that mediate fear learning associated with PTSD. Specifically, we propose using photoacoustic (PA) imaging to map activated neurons in a Fos/LacZ transgenic rat model. Fusion of Fos, which is induced by neuronal activity, with the lacZ gene gives active (Fos+) cells the ability to cleave pro-chromogenic substrates, such as X-Gal, enzymatically, into PA active dyes. My phantom and ex vivo pilot studies in rat brains demonstrate high-contrast PA images using these dyes and image reconstructions with a very high signal-to-noise ratio. We hypothesize that during fear acquisition, unique neuronal ensembles will be activated and detectable in the medial prefrontal cortex (a region tightly associated with fear behavior) using a novel Fos/LacZ PA imaging system, providing a means to track longitudinal changes in these ensembles in vivo. To test this hypothesis, we will use male/female Fos/LacZ transgenic rats that will be administered cortical X-Gal upon exposure to footshock (fear). The X-Gal product will be PA imaged (Aim 1) and these images will be validated with traditional methods for immunohistochemical detection of Fos and β-galactosidase in brain slices (Aim 2). Significance: This project will provide deeper insight into the role of fear-related neuronal ensembles and provide a new, low cost, high-resolution methodological approach to measure Fos expression longitudinally (representative of neuronal ensembles) in vivo. This will be broadly useful for behavioral research, not only in PTSD, but also in a variety of psychiatric science by allowing us to better define mechanisms behind complex mental health disorders. My training will focus on three main areas: molecular neuroscience and microscopy (led by sponsor, Dr. Conti), behavioral/animal modeling (led by co-sponsor, Dr. Perrine), and use of PA imaging (led by key training team member, Dr. Avanaki), facilitating my path towards my neuroradiology physician-scientist career goal.