Electrophysiological Validation of Monosynaptic Connectivity between Premotor Interneurons and the aCC Motoneuron in the Drosophila Larval CNS
Electrophysiological Validation of Monosynaptic Connectivity between Premotor Interneurons and the aCC Motoneuron in the Drosophila Larval CNS
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DOI：
10.1523/jneurosci.2463-21.2022
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发表时间：
2022-08-31
影响因子：
5.3
通讯作者：
Baines, Richard A.
中科院分区：
文献类型：
--
作者：
Giachello, Carlo N. G.;Hunter, Iain;Baines, Richard A.
果蝇连接组项目旨在绘制整个幼虫和成年果蝇神经网络的突触连接，这对于理解神经系统的发育和功能至关重要。到目前为止，该项目已经产生了大量的电子显微镜数据，这些数据促进了特定突触的重建，包括许多幼虫运动回路中的突触。虽然这一突破代表了技术上的杰作，但数据仍然没有得到充分利用，部分原因是缺乏对重建的功能验证。尝试验证连接体项目提出的连接性，主要依赖于行为测定和/或跨突触伴侣的GFP重建（GRASP）或GCaMP成像。虽然这些技术是有用的，但它们具有有限的空间或时间分辨率。突触连接的电生理学测定克服了这些限制。在这里，我们结合联合收割机膜片钳记录与光遗传学刺激在男性和女性的幼虫，测试突触连接提出的连接体重建。具体来说，我们使用多个驱动线，以确认前运动中间神经元和前角细胞运动神经元之间的几个连接，连接组项目建议，单突触。相比之下，我们的研究结果还表明，基于GRASP成像的结论可能会提供关于细胞之间连接的假阳性结果。我们还提出了一种新的成像工具，基于与我们的电生理学相同的技术，作为GRASP成像的有利替代方案。最后，在测试的八个Gal 4系中，五个在它们所靶向的前运动中间神经元中可靠地表达。因此，我们的工作强调需要确认功能性突触连接，驱动线特异性，并使用适当的遗传工具来支持连接体项目。
The Drosophila connectome project aims to map the synaptic connectivity of entire larval and adult fly neural networks, which is essential for understanding nervous system development and function. So far, the project has produced an impressive amount of electron microscopy data that has facilitated reconstructions of specific synapses, including many in the larval locomotor circuit. While this breakthrough represents a technical tour de force, the data remain underutilized, partly because of a lack of functional validation of reconstructions. Attempts to validate connectivity posited by the connectome project, have mostly relied on behavioral assays and/or GFP reconstitution across synaptic partners (GRASP) or GCaMP imaging. While these techniques are useful, they have limited spatial or temporal resolution. Electrophysiological assays of synaptic connectivity overcome these limitations. Here, we combine patch-clamp recordings with optogenetic stimulation in male and female larvae, to test synaptic connectivity proposed by connectome reconstructions. Specifically, we use multiple driver lines to confirm that several connections between premotor interneurons and the anterior corner cell motoneuron are, as the connectome project suggests, monosynaptic. In contrast, our results also show that conclusions based on GRASP imaging may provide false-positive results regarding connectivity between cells. We also present a novel imaging tool, based on the same technology as our electrophysiology, as a favorable alternative to GRASP imaging. Finally, of eight Gal4 lines tested, five are reliably expressed in the premotor interneurons they are targeted to. Thus, our work highlights the need to confirm functional synaptic connectivity, driver line specificity, and use of appropriate genetic tools to support connectome projects.