Division of Biology and Biological Engineering, Tianqiao and Chrissy Chen Institute for Neuroscience, California Institute of Technology, Pasadena, United States.
Howard Hughes Medical Institute, Chevy Chase, United States.
Elife. 2024 Nov 28;12:RP92380. doi: 10.7554/eLife.92380.
Monitoring neuronal activity at single-cell resolution in freely moving engaged in social behaviors is challenging because of their small size and lack of transparency. Extant methods, such as Flyception, are highly invasive. Whole-brain calcium imaging in head-fixed, walking flies is feasible but the animals cannot perform the consummatory phases of social behaviors like aggression or mating under these conditions. This has left open the fundamental question of whether neurons identified as functionally important for such behaviors using loss- or gain-of-function screens are actually active during the natural performance of such behaviors, and if so during which phase(s). Here, we perform brain-wide mapping of active cells expressing the Immediate Early Gene using a high-sensitivity/low background fluorescence in situ hybridization (FISH) amplification method called HCR-3.0. Using double-labeling for hr38 mRNA and for GFP, we describe the activity of several classes of aggression-promoting neurons during courtship and aggression, including P1 cells, an intensively studied population of male-specific interneurons. Using HI-FISH in combination with optogenetic activation of aggression-promoting neurons (opto-HI-FISH), we identify candidate downstream functional targets of these cells in a brain-wide, unbiased manner. Finally, we compare the activity of P1 neurons during sequential performance of courtship and aggression, using intronic vs. exonic probes to differentiate newly synthesized nuclear transcripts from cytoplasmic transcripts synthesized at an earlier time. These data provide evidence suggesting that different subsets of P1 neurons may be active during courtship vs. aggression. HI-FISH and associated methods may help to fill an important lacuna in the armamentarium of tools for neural circuit analysis in .
在自由活动的参与社交行为的中监测神经元的活动在单细胞分辨率下是具有挑战性的，因为它们的体积小而且缺乏透明度。现有的方法，如 Flyception，具有高度的侵入性。在头部固定、行走的苍蝇中进行全脑钙成像在技术上是可行的，但在这些条件下，动物无法完成像攻击或交配这样的社交行为的完成阶段。这就留下了一个基本问题，即使用功能丧失或功能获得筛选来识别对这些行为具有功能重要性的神经元，在自然表现这些行为时是否实际上是活跃的，如果是这样，在哪个阶段活跃。在这里，我们使用一种称为 HCR-3.0 的高灵敏度/低背景荧光原位杂交（FISH）扩增方法，对表达即时早期基因的活跃细胞进行全脑映射。使用针对 hr38 mRNA 和 GFP 的双重标记，我们描述了在求偶和攻击期间几种促进攻击的神经元的活性，包括 P1 细胞，这是一种经过深入研究的雄性特异性中间神经元群体。使用 HI-FISH 结合促进攻击神经元的光遗传学激活（optogenetic activation of aggression-promoting neurons，opto-HI-FISH），我们以一种全脑、无偏倚的方式识别这些细胞的候选下游功能靶点。最后，我们使用内含子与外显子探针来区分新合成的核转录物和更早时间合成的细胞质转录物，比较 P1 神经元在连续进行求偶和攻击时的活性。这些数据提供的证据表明，不同子集的 P1 神经元可能在求偶与攻击期间具有活性。HI-FISH 和相关方法可能有助于填补在 神经回路分析工具包中的一个重要空白。
