Michigan Neuroscience Institute, University of Michigan, 205 Zina Pitcher pl, Ann Arbor, MI, 48109, USA.
Brain Struct Funct. 2021 Mar;226(2):481-499. doi: 10.1007/s00429-020-02194-4. Epub 2021 Jan 2.
Transcript labeling in intact tissues using in situ hybridization chain reaction has potential to provide vital spatiotemporal information for molecular characterization of heterogeneous neuronal populations. However, large tissue labeling in non-perfused or fresh-frozen rodent and postmortem human samples, which provide more flexible utilization than perfused tissues, is largely unexplored. In the present study, we optimized the combination of in situ hybridization chain reaction in fresh-frozen rodent brains and then evaluated the uniformity of neuronal labeling between two clearing methods, CLARITY and iDISCO. We found that CLARITY yielded higher signal-to-noise ratios but more limited imaging depth and required longer clearing times, whereas, iDISCO resulted in better tissue clearing, greater imaging depth and a more uniform labeling of larger samples. Based on these results, we used iDISCO-cleared fresh-frozen rodent brains to further validate this combination and map the expression of a few genes of interest pertaining to mood disorders. We then examined the potential of in situ hybridization chain reaction to label transcripts in cleared postmortem human brain tissues. The combination failed to produce adequate mRNA labeling in postmortem human cortical slices but produced visually adequate labeling in the cerebellum tissues. We next, investigated the multiplexing ability of in situ hybridization chain reaction in cleared tissues which revealed inconsistent fluorescence output depending upon the fluorophore conjugated to the hairpins. Finally, we applied our optimized protocol to assess the effect of glucocorticoid receptor overexpression on basal somatostatin expression in the mouse cortex. The constitutive glucocorticoid receptor overexpression resulted in lower number density of somatostatin-expressing neurons compared to wild type. Overall, the combination of in situ hybridization chain reaction with clearing methods, especially iDISCO, may find broad application in the transcript analysis in rodent studies, but its limited use in postmortem human tissues can be improved by further optimizations.
原位杂交链反应在完整组织中的转录本标记有可能为异质神经元群体的分子特征提供重要的时空信息。然而，在非灌注或新鲜冷冻的啮齿动物和尸检人类样本中进行大规模组织标记，提供了比灌注组织更灵活的利用方式，但在很大程度上尚未得到探索。在本研究中，我们优化了原位杂交链反应在新鲜冷冻的啮齿动物大脑中的组合，然后评估了两种透明化方法（CLARITY 和 iDISCO）之间神经元标记的均匀性。我们发现 CLARITY 产生了更高的信噪比，但成像深度更有限，需要更长的透明化时间，而 iDISCO 则导致更好的组织透明化、更大的成像深度和更大样本的更均匀标记。基于这些结果，我们使用 iDISCO 透明化的新鲜冷冻啮齿动物大脑进一步验证了这种组合，并绘制了与情绪障碍相关的几个感兴趣基因的表达图谱。然后，我们检查了原位杂交链反应在清除后的人类大脑组织中标记转录本的潜力。该组合未能在尸检人类皮质切片中产生足够的 mRNA 标记，但在小脑组织中产生了视觉上足够的标记。接下来，我们研究了原位杂交链反应在透明化组织中的多重标记能力，结果显示，根据与发夹连接的荧光团的不同，荧光输出不一致。最后，我们应用我们优化的方案来评估糖皮质激素受体过表达对小鼠皮质中基础生长抑素表达的影响。与野生型相比，糖皮质激素受体的组成型过表达导致生长抑素表达神经元的数量密度降低。总的来说，原位杂交链反应与透明化方法的组合，特别是 iDISCO，可能在啮齿动物研究中的转录本分析中得到广泛应用，但在尸检人类组织中的有限应用可以通过进一步优化得到改善。
