AII amacrine cells: quantitative reconstruction and morphometric analysis of electrophysiologically identified cells in live rat retinal slices imaged with multi-photon excitation microscopy

Abstract

AII amacrine cells have been found in all mammalian retinas examined and play an important role for visual processing under both scotopic and photopic conditions. Whereas ultrastructural investigations have provided a detailed understanding of synaptic connectivity, there is little information available with respect to quantitative properties and variation of cellular morphology. Here, we performed whole-cell recordings from AII amacrine cells in rat retinal slices and filled the cells with fluorescent dyes. Multi-photon excitation microscopy was used to acquire image stacks and after deconvolution, we performed quantitative morphological reconstruction by computer-aided manual tracing. We reconstructed and performed morphometric analysis on 43 AII amacrine cells, with a focus on branching pattern, dendritic lengths and diameters, surface area, and number and distribution of dendritic varicosities. Compared to previous descriptions, the most surprising result was the considerable extent of branching, with the maximum branch order ranging from approximately 10–40. We found that AII amacrine cells conform to a recently described general structural design principle for neural arbors, where arbor density decreases proportionally to increasing territory size. We confirmed and quantified the bi-stratified morphology of AII amacrine cells by analyzing the arborizations as a function of retinal localization or with Sholl spheres. Principal component and cluster analysis revealed no evidence for morphological subtypes of AII amacrines. These results establish a database of morphometric properties important for studies of development, regeneration, degeneration, and disease processes, as well as a workflow compatible with compartmental modeling.

Electrotonic signal processing in AII amacrine cells: compartmental models and passive membrane properties for a gap junction-coupled retinal neuron

“…Whereas the AII cells have relatively small dendritic trees, detailed quantitative analysis recently revealed that the extent of branching is considerably larger than suggested by earlier investigations (Zandt et al 2017). The dendritic tree is bi-stratified with arboreal and lobular dendrites in the proximal and distal part of the inner plexiform layer, respectively.…”

“…Various aspects of the methods have previously been described in detail (Oltedal et al 2009;Zandt et al 2017). The use of animals in this study was carried out under the approval of and in accordance with the regulations of the Animal Laboratory Facility at the Faculty of Medicine at the University of Bergen (accredited by AAALAC International).…”

“…During image acquisition, exposure to IR laser light was controlled by an electronic shutter (LS6ZM2; Vincent Associates, Rochester, NY, USA), thereby minimizing the total exposure time. An image stack was acquired as a series of optical sections (1024 × 1024 pixels) with XY pixel size ~70 to ~80 nm (depending on the magnitude of the digital zoom factor) and collected at a focal plane interval of 0.4 µm, sufficient to satisfy Nyquist rate sampling (for details, see Zandt et al 2017). For each image stack, we acquired two channels and at each focal plane two images were averaged on-line.…”

“…Processed image stacks were saved in 16-bit TIFF format, utilizing the whole dynamic range. For additional details, see Zandt et al (2017).…”

“…Quantitative morphological reconstruction of the fluorescently labeled cells was done with Neurolucida software (64 bit; MBF Bioscience, Williston, VT, USA) running under Windows 7 Pro 64-bit (for a detailed description, see Zandt et al 2017). The 3D reconstruction of the soma was made by tracing it with multiple contours at a series of different focal planes.…”