Quattrocolo Giulia, Isaac Maria, Zhang Yajun, Petros Timothy J
Kavli Institute for Systems Neuroscience and Centre for Neural Computation, Norwegian University of Science and Technology.
Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.
J Vis Exp. 2018 Jun 8(136):57723. doi: 10.3791/57723.
Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.
神经元命运的决定和成熟需要遗传程序与环境信号之间复杂的相互作用。然而，厘清调节这种分化过程的内在机制与外在机制的作用，对所有发育神经生物学家来说都是一个难题。对于GABA能中间神经元而言，这个问题更加突出，GABA能中间神经元是一个极其异质的细胞群体，它们起源于短暂的胚胎结构，并经历漫长的迁移阶段以分散至整个端脑。为了探究不同的脑环境如何影响中间神经元的命运和成熟，我们开发了一种从新生小鼠（P0 - P2）特定脑区收集荧光标记的未成熟中间神经元前体的方法。在这个年龄段，中间神经元迁移基本完成，这些细胞处于它们最终的静止环境中，突触整合相对较少。通过流式细胞术收集单细胞溶液后，将这些中间神经元前体移植到P0 - P2野生型新生幼崽体内。通过进行同位移植（例如，皮层到皮层）或异位移植（例如，皮层到海马体），人们可以评估在新脑环境中挑战未成熟中间神经元如何影响它们的命运、成熟和回路整合。可以在成年小鼠中获取大脑，并对移植细胞进行包括免疫组织化学、电生理和转录谱分析在内的多种事后分析。这种通用方法为研究人员提供了一种策略，以测定不同的脑环境如何影响神经元发育的多个方面，并确定特定的神经元特征主要是由固有遗传程序还是环境线索驱动。
