College of Engineering, Science and Environment, University of Newcastle, Callaghan, NSW, Australia.
PLoS One. 2022 Jul 19;17(7):e0271744. doi: 10.1371/journal.pone.0271744. eCollection 2022.
Electrical coupling between retinal neurons contributes to the functional complexity of visual circuits. "Cut-loading" methods allow simultaneous assessment of cell-coupling between multiple retinal cell-types, but existing analysis methods impede direct comparison with gold standard direct dye injection techniques. In the current study, we both improved an existing method and developed two new approaches to address observed limitations. Each method of analysis was applied to cut-loaded dark-adapted Guinea pig retinae (n = 29) to assess coupling strength in the axonless horizontal cell type ('a-type', aHCs). Method 1 was an improved version of the standard protocol and described the distance of dye-diffusion (space constant). Method 2 adjusted for the geometric path of dye-transfer through cut-loaded cells and extracted the rate of dye-transfer across gap-junctions in terms of the coupling coefficient (kj). Method 3 measured the diffusion coefficient (De) perpendicular to the cut-axis. Dye transfer was measured after one of five diffusion times (1-20 mins), or with a coupling inhibitor, meclofenamic acid (MFA) (50-500μM after 20 mins diffusion). The standard protocol fits an exponential decay function to the fluorescence profile of a specified retina layer but includes non-specific background fluorescence. This was improved by measuring the fluorescence of individual cell soma and excluding from the fit non-horizontal cells located at the cut-edge (p<0.001) (Method 1). The space constant (Method 1) increased with diffusion time (p<0.01), whereas Methods 2 (p = 0.54) and 3 (p = 0.63) produced consistent results across all diffusion times. Adjusting distance by the mean cell-cell spacing within each tissue reduced the incidence of outliers across all three methods. Method 1 was less sensitive to detecting changes induced by MFA than Methods 2 (p<0.01) and 3 (p<0.01). Although the standard protocol was easily improved (Method 1), Methods 2 and 3 proved more sensitive and generalisable; allowing for detailed assessment of the tracer kinetics between different populations of gap-junction linked cell networks and direct comparison to dye-injection techniques.
视网膜神经元的电耦合有助于视觉电路的功能复杂性。“切割加载”方法允许同时评估多种视网膜细胞类型之间的细胞耦合，但现有的分析方法阻碍了与金标准直接染料注射技术的直接比较。在本研究中，我们改进了一种现有的方法，并开发了两种新方法来解决观察到的局限性。分析的每种方法都应用于暗适应的豚鼠视网膜（n = 29），以评估无轴突水平细胞类型（“a 型”，aHCs）中的耦合强度。方法 1 是标准方案的改进版本，描述了染料扩散的距离（空间常数）。方法 2 调整了通过切割加载细胞的染料转移的几何路径，并根据耦合系数（kj）提取间隙连接中的染料转移速率。方法 3 测量垂直于切割轴的扩散系数（De）。在五种扩散时间（1-20 分钟）之一后或用耦合抑制剂甲氯芬酸（MFA）（20 分钟扩散后 50-500μM）测量染料转移。标准方案拟合指定视网膜层的荧光轮廓的指数衰减函数，但包括非特异性背景荧光。通过测量单个细胞体的荧光并从拟合中排除位于切割边缘的非水平细胞（p<0.001）（方法 1）来改进这一点。空间常数（方法 1）随扩散时间的增加而增加（p<0.01），而方法 2（p = 0.54）和 3（p = 0.63）在所有扩散时间内产生一致的结果。通过在每个组织内调整细胞-细胞间距来调整距离，可以减少所有三种方法的异常值的发生率。与方法 2（p<0.01）和 3（p<0.01）相比，方法 1 对 MFA 诱导的变化的检测灵敏度较低。尽管标准方案很容易改进（方法 1），但方法 2 和 3 更敏感且更具通用性；允许对不同群体的间隙连接连接细胞网络之间的示踪剂动力学进行详细评估，并与染料注射技术直接比较。
