Leikvoll Austin, Kara Prakash
Department of Neuroscience, University of Minnesota, Minneapolis, MN, United States.
Front Neurosci. 2023 May 12;17:1181828. doi: 10.3389/fnins.2023.1181828. eCollection 2023.
Two-photon imaging of genetically-encoded calcium indicators (GECIs) has traditionally relied on intracranial injections of adeno-associated virus (AAV) or transgenic animals to achieve expression. Intracranial injections require an invasive surgery and result in a relatively small volume of tissue labeling. Transgenic animals, although they can have brain-wide GECI expression, often express GECIs in only a small subset of neurons, may have abnormal behavioral phenotypes, and are currently limited to older generations of GECIs. Inspired by recent developments in the synthesis of AAVs that readily cross the blood brain barrier, we tested whether an alternative strategy of intravenously injecting AAV-PHP.eB is suitable for two-photon calcium imaging of neurons over many months after injection. We injected C57BL/6 J mice with AAV-PHP.eB-Synapsin-jGCaMP7s via the retro-orbital sinus. After allowing 5 to 34 weeks for expression, we performed conventional and widefield two-photon imaging of layers 2/3, 4 and 5 of the primary visual cortex. We found reproducible trial-by-trial neural responses and tuning properties consistent with known feature selectivity in the visual cortex. Thus, intravenous injection of AAV-PHP.eB does not interfere with the normal processing in neural circuits. and histological images show no nuclear expression of jGCaMP7s for at least 34 weeks post-injection.
传统上，对基因编码钙指示剂（GECIs）进行双光子成像依赖于向颅内注射腺相关病毒（AAV）或使用转基因动物来实现表达。颅内注射需要进行侵入性手术，且组织标记的体积相对较小。转基因动物虽然可以在全脑表达GECIs，但通常仅在一小部分神经元中表达，可能具有异常的行为表型，并且目前仅限于较老一代的GECIs。受最近易于穿过血脑屏障的AAV合成技术发展的启发，我们测试了静脉注射AAV-PHP.eB这种替代策略是否适用于注射后数月对神经元进行双光子钙成像。我们通过眶后窦向C57BL/6 J小鼠注射AAV-PHP.eB-Synapsin-jGCaMP7s。在经过5至34周的表达期后，我们对初级视觉皮层的第2/3层、第4层和第5层进行了传统和广角双光子成像。我们发现了可重复的逐次试验神经反应以及与视觉皮层中已知特征选择性一致的调谐特性。因此，静脉注射AAV-PHP.eB不会干扰神经回路的正常处理。组织学图像显示，注射后至少34周内jGCaMP7s无核表达。
