■已经报道了视网膜神经节细胞(RGC)的生理变化,但这还没有在灵长类动物中进行调查。通过在猕猴的中央凹RGC中同时表达钙指示剂(GCaMP6s)和光遗传学致动器(ChrimsonR),我们在体内重新激活RGC,并评估其在PR丢失后数周和数年的反应.
■我们使用了体内钙成像方法来记录灵长类中央凹中光遗传诱发的RGC活动。在PR消融后的10周时间内纵向进行细胞尺度记录,并与2年前失去PR输入的RGC的反应进行比较。
■三只眼睛接受了PR消融,雄性猕猴(M1)的右眼,雌性猕猴(M2)的左眼,和雄性猕猴(M3)的右眼。两只动物用于体内记录,1用于组织学评估。
■用通过自适应光学扫描光检眼镜(AOSLO)递送的超快激光烧蚀视锥。25Hz660nm光的0.5秒脉冲光遗传刺激RGC,并且使用AOSLO记录得到的GCaMP荧光信号。PR消融后立即重复测量10周,在2.3年和控制RGC。
■钙上升时间,衰变常数,光遗传学介导的RGC的敏感性指数来自体内221个RGC(动物M1)和218个RGC(动物M2)的GCaMP荧光记录。
■PR消融后,在受试者1的10周观察期内,RGC中钙反应的平均衰减常数下降了1.5倍(标准偏差1.6±0.5秒至0.6±0.3秒),受试者2的8周内下降了2.1倍(标准偏差2.5±0.5秒至1.2±0.2秒).钙上升时间和敏感性指数稳定。PR消融后2.3年,光遗传学再激活仍有可能。
■在PR消融术后的几周内,灵长类中央凹RGC出现了改变的钙动力学。光遗传学介导的钙反应的平均衰减常数降低了1.5至两倍。这是灵长类动物视网膜中这种现象的首次报道,需要进一步的工作来了解这些变化在细胞存活和活动中的作用。
■专有或商业披露可在本文末尾的脚注和披露中找到。
UNASSIGNED: Physiological changes in retinal ganglion cells (RGCs) have been reported in rodent models of photoreceptor (PR) loss, but this has not been investigated in primates. By expressing both a calcium indicator (GCaMP6s) and an optogenetic actuator (ChrimsonR) in foveal RGCs of the macaque, we reactivated RGCs in vivo and assessed their response in the weeks and years after PR loss.
UNASSIGNED: We used an in vivo calcium imaging approach to record optogenetically evoked activity in deafferented RGCs in primate fovea. Cellular scale recordings were made longitudinally over a 10-week period after PR ablation and compared with responses from RGCs that had lost PR input >2 years prior.
UNASSIGNED: Three eyes received PR ablation, the right eye of a male Macaca mulatta (M1), the left eye of a female Macaca fascicularis (M2), and the right eye of a male Macaca fascicularis (M3). Two animals were used for in vivo recording, 1 for histological assessment.
UNASSIGNED: Cones were ablated with an ultrafast laser delivered through an adaptive optics scanning light ophthalmoscope (AOSLO). A 0.5 second pulse of 25 Hz 660 nm light optogenetically stimulated RGCs, and the resulting GCaMP fluorescence signal was recorded using an AOSLO. Measurements were repeated over 10 weeks immediately after PR ablation, at 2.3 years and in control RGCs.
UNASSIGNED: The calcium rise time, decay constant, and sensitivity index of optogenetic-mediated RGC were derived from GCaMP fluorescence recordings from 221 RGCs (animal M1) and 218 RGCs (animal M2) in vivo.
UNASSIGNED: After PR ablation, the mean decay constant of the calcium response in RGCs decreased 1.5-fold (standard deviation 1.6 ± 0.5 seconds to 0.6 ± 0.3 seconds) over the 10-week observation period in subject 1 and 2.1-fold (standard deviation 2.5 ± 0.5 seconds to 1.2 ± 0.2 seconds) within 8 weeks in subject 2. Calcium rise time and sensitivity index were stable. Optogenetic reactivation remained possible 2.3 years after PR ablation.
UNASSIGNED: Altered calcium dynamics developed in primate foveal RGCs in the weeks after PR ablation. The mean decay constant of optogenetic-mediated calcium responses decreased 1.5- to twofold. This is the first report of this phenomenon in primate retina and further work is required to understand the role these changes play in cell survival and activity.
UNASSIGNED: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.