Chen Kaiyun, Featherstone David E, Broadie Kendal
Department of Biological Sciences, University of Illinois at Chicago, USA.
J Vis Exp. 2009 May 21(27):1348. doi: 10.3791/1348.
Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of <8 hours (at 25 degrees C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.
果蝇是用于研究胚胎发育和功能神经科学的首要遗传模型。传统上，这些领域彼此相当孤立，有着各自独立的历史和科学社群。然而，这些通常截然不同的领域之间的交叉点在于神经回路形成最后阶段功能性电信号特性的获得以及功能性化学突触分化所依据的发育程序。这个交叉点是一个极其重要的研究领域。在果蝇中，这些功能发育阶段发生在胚胎发育最后三分之一时期的<8小时内（25摄氏度）。由于坚硬、不可渗透的表皮角质层的沉积，这个发育后期长期以来被认为难以进行研究。一项突破性进展是应用可局部涂抹在角质层上的水聚合手术胶水，从而能够对晚期胚胎进行可控解剖。通过背部纵向切口，胚胎可以被平铺，将腹侧神经索和体壁肌肉组织暴露于实验研究之下。然后可以采用全细胞膜片钳技术从可单独识别的神经元和体壁肌肉进行记录。这些记录配置已被用于追踪离子电流的出现和成熟以及动作电位在神经元和肌肉中的传播。影响这些电特性的基因变异体已被表征，以揭示离子通道和相关信号复合物的分子组成，并开始探索功能分化的分子机制。一个特别的重点是中枢神经系统和外周突触连接的组装。谷氨酸能神经肌肉接头（NMJ）最便于进行光学成像和电生理记录相结合的研究。使用玻璃吸引电极刺激外周神经，在电压钳制的肌肉中记录兴奋性接头电流（EJC）。这种记录配置已被用于描绘突触的功能分化，并追踪突触前谷氨酸释放特性的出现和成熟。此外，突触后特性可以通过将谷氨酸离子导入或压力施加到肌肉表面来独立测定，以测量谷氨酸受体场的出现和成熟。因此，在胚胎突触形成过程中，可以分别或联合监测突触前和突触后元件。这个系统已被大量用于分离和表征损害胚胎突触形成的基因变异体，从而揭示控制突触连接的特化和分化以及功能性突触信号特性的分子机制。
