Monitoring Neural Activity in Freely Behaving Zebrafish Larvae with Bioluminescen

描述（由申请人提供）：现有的监测清醒、行为脊椎动物神经活动的技术是侵入性的，通常需要约束动物。在这里，我们建议使用生物发光来非侵入性地监测自由行为的斑马鱼遗传特定神经元的活动。光蛋白GFP-apoAequorin （Ga）将在斑马鱼幼体神经元中表达，并与其底物coelenterazine （CLZN）在体内构成Ca2+敏感生物发光传感器GFP-Aequorin （Ga）。然后，发光的闪光将报告自发和诱发Ca2+信号在目标神经元。这些“神经发光”反应可以用大面积光子计数探测器记录下来，同时用红外敏感摄像机监测行为。初步研究表明，转基因的泛神经元遗传鱼产生了大而快速的神经发光信号，可以连续记录许多天。这些光信号与神经元潜在电活动的关系将通过靶向膜片钳记录到泛神经元启动子下表达蛋白质的单个神经元来探索。为了探索该技术的局限性和敏感性，GA将专门针对下丘脑的下丘脑分泌素阳性神经元，中隔核的血清素能神经元和下丘脑腹侧的多巴胺能神经元。为了克服现有生物发光监测策略的主要限制，该策略需要一个完全黑暗的环境，我们提出了一种扩展该方法的快速时间门控，能够在正常照明条件下计数单个光子。因此，这种分析将使我们能够以高时间分辨率和稳定性监测一小部分神经元在许多天内不受约束的视觉行为中的活动。我们相信，快速、稳定的遗传报告神经活动的特性以及非成像检测策略可以提供一个有用的、容易实现的工具，用于监测遗传指定细胞类型在自然行为中的活动；一种有吸引力的替代方案，目前正在寻求其他更具技术挑战性的成像方法。

DESCRIPTION (provided by applicant): Existing techniques for monitoring neural activity in awake, behaving vertebrates are invasive and often require restraining the animal. Here we propose the use of bioluminescence to non- invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. The photoprotein GFP-apoAequorin (Ga) will be expressed in neurons of larval zebrafish and constituted in vivo with its substrate coelenterazine (CLZN) to form the Ca2+-sensitive bioluminescent sensor GFP-Aequorin (GA). Flashes of luminescence will then report spontaneous and evoked Ca2+ signals in the targeted neurons. These 'neuroluminescence' responses can be recorded with a large-area photon-counting detector while simultaneously monitoring behavior with an infrared-sensitive camera. Pilot studies have shown that transgenic, pan-neuronal GA fish produced large and fast neuroluminescent signals that could be recorded continuously for many days. The relationship of these light signals with the neurons underlying electrical activity will be explored by targeting patch-clamp recordings to individual neurons expressing the protein under pan-neuronal promoters. To explore the limits and the sensitivity of this technique, GA will specifically be targeted to the hypocretin-positive neurons of the hypothalamus, the serotonergic neurons of the raphe nuclei and the dopaminergic neurons of the ventral hypothalamus. To overcome a major limitation of existing bioluminescence monitoring strategies, which require a completely dark environment, we propose an extension of this method for fast temporal gating that is able to count single photons during normal lighting conditions. Thus, this assay will allow us to monitor, with high temporal resolution and stability, the activity of small subsets of neurons during unrestrained, visual behavior over a time period of many days. We believe that the fast, stable properties of GA's report of neural activity along with non- imaging detection strategies can provide a useful, easily implemented tool for monitoring the activity of genetically specified cell types during natural behavior; an attractive alternative to other more technically challenging imaging approaches currently being pursued.