Calcium imaging in the insect nervous system using an innovative dye loading technique

神经科学的目标是了解神经系统的组织和功能，重点是脊椎动物和无脊椎动物模型动物。记录神经元的活动通常是通过细胞外或细胞内电极来实现的，这种电极测量具有高时间和高幅度分辨率的电脉冲和突触活动。在过去的几十年里，已经开发了光学记录，例如基于钙敏感染料，这些染料被加载到神经元或神经元群体中。一旦与钙离子结合，这些染料就会改变它们的荧光强度或波长，并产生光学信号，这些信号可以用灵敏的冷却CCD相机或共焦显微镜进行监测。由于钙是神经元功能和突触处理的必要分子，光学信号报告标记的神经元的兴奋活动-尽管不是以电生理测量的精度。然而，光学成像允许在细胞和网络水平上分析神经元处理的空间维度，并对我们对神经系统的理解做出了重大贡献。在一些模型系统中，如果蝇或斑马鱼，甚至可以获得遗传编码的钙指示剂，它可以在神经系统的特定细胞系中表达。相应的分子遗传学工具在其他生物体中还不可用，尽管如此，它们仍然是研究特定行为的模型系统。其中一个系统是像蟋蟀或丛林蟋蟀这样的声学交流昆虫，几十年来，它们一直是神经科学研究的重点，旨在了解听力、听觉模式识别和声音模式生成的神经机制。分析这些系统的进展主要基于电生理学和神经解剖学技术，因为用钙敏感染料标记中枢或传入神经元是困难的，需要通过微电极给这些神经元注入细胞内染料。受通过完整人体皮肤的离子导入药物的启发，我们最近开发了一种通过完整的神经鞘或神经节传递染料的方法。顶端直径为40-80微米的玻璃毛细管充满了钙敏感示踪剂，并附着在神经鞘上，例如昆虫的听神经。最初，它们被用作记录电极，一旦建立了良好的信号，电路就切换到离子导入染料输送模式。钙敏感染料进入神经轴突，然后从注射部位向两个方向移动，标记神经系统中的外周感觉器官和中央传入轴突树枝。没有其他的标签方法可以做到这一点。该方法还可以用来特异性地标记蟋蟀大脑中的听觉神经纤维区。我们的初步成像实验证明了这一原理，并证明了声音诱发的光信号报告了标记的听觉传入和中枢神经元的特定活动。我们现在的目标是进一步利用这一技术来系统地研究(1)。蟋蟀大脑中听觉处理的空间组织；(2)听觉器官中听觉传入的激活；(3)雄性蟋蟀鸣叫运动活动的时空活动模式。除了使用我们灵敏的CCD摄像系统外，我们还将使用更先进的共焦和双光子成像系统来提高数据的分辨率。所选择的目标代表了昆虫神经科学的中心问题，应该有助于进一步了解昆虫的声学通信。此外，计划中的实验将展示我们的方法作为昆虫神经科学中的一种新工具的多功能性，并将与更广泛的研究人员社区相关。

Neuroscience aims at understanding the organisation and function of nervous systems focussing on vertebrate and invertebrate model animals. Recording the activity of neurons is classically achieved with extra- or intracellular electrodes which measure the electrical spike and synaptic activity with high temporal and amplitude resolution. Over the last decades optical recordings have been developed, based e.g. on calcium sensitive dyes, which are loaded into neurons or populations of neurons. Upon binding to Calcium ions these dyes change their fluorescence intensity or wavelength and generate optical signals that can be monitored with sensitive cooled CCD cameras or confocal microscopes. As calcium is an essential molecule for neuronal functioning and synaptic processing the optical signals report the excitatory activity of the labelled neurons - although not with the precision of electrophysiological measurements. Optical imaging however, allows to analyse the spatial dimensions of neuronal processing at the cellular and network level and has substantially contributed to our understanding of nervous systems. In some model systems, like the fruit fly Drosophila or the zebrafish, even genetically encoded calcium indicators are available, which can be expressed in specific cell lines of the nervous system. The corresponding molecular-genetic tools are not (yet) available in other organisms, which nonetheless are model systems to study a specific behaviour. One of these system are acoustically communicating insects like crickets or bush-crickets, which for decades have been in the focus of neuroscience research aiming to understand the neuronal mechanisms underlying hearing, auditory pattern recognition and sound pattern generation. Progress in analysing these systems was mainly based on electrophysiological and neuroanatomical techniques, as loading central or afferent neurons with calcium sensitive dyes, is difficult and required loading these neurons with intracellular dye injection via microelectrodes. Inspired by the iontophoretic application of drugs through the intact human skin, we recently developed a dye delivery method through the intact sheath of nerves or ganglia. Glass capillaries with 40-80 micro-meter tip diameter are filled with the calcium sensitive tracer and are attached to the neuronal sheath e.g. of an insect auditory nerve. Initially they are used as recording electrodes, and once a good signal has been established the circuit is switched into iontophoretic dye delivery mode. The calcium sensitive dye moves into the axons of the nerve and then travels in both directions from the injection site, labelling the peripheral sensory organ and the central afferent axonal arborisations in the nervous system. No other labelling method can achieve this. The method can also be used to specifically label auditory neuropil regions in the brain of crickets. Our preliminary imaging experiments prove the principle and demonstrate that sound evoked optical signals report the specific activity of the labelled auditory afferents and central neurons. We now aim to further take advantage of this technique to systematically study (1.) the spatial organisation of auditory processing in the cricket brain; (2) the activation of auditory afferents in the hearing organ and (3) the spatiotemporal activity patterns underlying the singing motor activity in male crickets. Besides using our sensitive CCD camera system, we will use more advanced confocal and two-photon imaging systems to enhance the resolution of the data. The chosen objectives represent central questions in insect neuroscience and should contribute to further our understanding of insect acoustic communication. Moreover the planned experiments will demonstrate the versatility of our method as a new tool in insect neuroscience and will be relevant to the wider community of researchers.