Abstract:
High resolution retinal imaging paired with intravitreal injection of a viral vector coding for the calcium indicator GCaMP has enabled visualization of activity dependent calcium changes in retinal ganglion cells (RGCs) at single cell resolution in the living eye. The inner limiting membrane (ILM) is a barrier for viral vectors, restricting transduction to a ring of RGCs serving the fovea in both humans and non-human primates (NHP). We evaluate peeling the ILM prior to intravitreal injection as a strategy to expand calcium imaging beyond the fovea in the NHP eye in vivo. Five Macaca fascicularis eyes (age 3-10y; n=3 individuals; 2M, 1F) underwent vitrectomy and 5 to 6-disc diameter ILM peel centered on the fovea prior to intravitreal delivery of 7m8:SNCG:GCaMP8s. Calcium responses from RGCs were recorded using a fluorescence adaptive optics scanning laser ophthalmoscope. In all eyes GCaMP was expressed throughout the peeled area, representing a mean 8-fold enlargement in area of expression relative to a control eye. Calcium recordings were obtained up to 11 degrees from the foveal center. RGC responses were comparable to the fellow control eye and showed no significant decrease over the 6 months post ILM peel, suggesting that RGC function was not compromised by the surgical procedure. In addition, we demonstrate that activity can be recorded directly from the retinal nerve fiber layer. This approach will be valuable for a range of applications in visual neuroscience including pre-clinical evaluation of retinal function, detecting vision loss, and assessing the impact of therapeutic interventions.
摘要:
高分辨率视网膜成像与编码钙指示剂GCaMP的病毒载体的玻璃体内注射配对,可以在活体眼中以单细胞分辨率可视化视网膜神经节细胞(RGC)中的活性依赖性钙变化。内界膜(ILM)是病毒载体的屏障,在人类和非人类灵长类动物(NHP)中,限制对服务于中央凹的RGC环的转导。我们评估了在玻璃体内注射之前剥离ILM作为在体内将钙成像扩展到NHP眼中的中央凹之外的策略。五个猕猴的眼睛(3-10岁;n=3个人;2M,1F)进行了玻璃体切除术,在玻璃体内递送7m8:SNCG:GCaMP8s之前,直径为5至6盘的ILM剥离位于中央凹中心。使用荧光自适应光学扫描激光检眼镜记录来自RGC的钙反应。在所有的眼睛中,GCaMP在整个剥皮区域表达,表示相对于对照眼的表达面积的平均8倍扩大。从中央凹中心获得高达11度的钙记录。RGC反应与对照眼相当,在ILM剥离后6个月内没有显着下降,提示RGC功能并未因外科手术而受损。此外,我们证明,活动可以直接从视网膜神经纤维层记录。这种方法对于视觉神经科学的一系列应用将是有价值的,包括视网膜功能的临床前评估。检测视力丧失,并评估治疗干预措施的影响。
■这项研究通过开发一种涉及剥离内界膜(ILM)并结合玻璃体内注射以扩展功能记录能力的新型技术,在视觉神经科学中取得了突破性进展。通过利用高分辨率视网膜成像与病毒载体介导的钙指示剂GCaMP的表达,该研究以单细胞分辨率实现了前所未有的视网膜神经节细胞(RGC)活性的可视化和评估。重要的是,该技术可以从以前无法进入的视网膜区域进行记录,显着扩大钙成像区域超出了中央凹。结果表明,RGC功能稳定,提示对视网膜生理学影响最小。这种创新的方法为视觉神经科学的各种应用提供了重要的前景。包括视网膜功能的临床前评估,视力丧失的检测,和治疗干预措施的评估。总的来说,这项研究代表了在理解和治疗视网膜退行性疾病方面迈出的重要一步,为视力恢复的研究和开发提供了新的途径。