DOI: 10.6078/d12d9f
title: Excitatory neurotransmission activates compartmentalized calcium transients in Müller glia without affecting lateral process motility

description: Neural activity has been implicated in the motility and outgrowth of glial
 cell processes throughout the central nervous system. Here we explore this
 phenomenon in Müller glia, which are specialized radial astroglia that are
 the predominant glial type of the vertebrate retina. Müller glia extend
 fine filopodia-like processes into retinal synaptic layers, in similar
 fashion to brain astrocytes and radial glia which exhibit perisynaptic
 processes. Using two-photon volumetric imaging, we found that during the
 second postnatal week, Müller glial processes were highly dynamic, with
 rapid extensions and retractions that were mediated by cytoskeletal
 rearrangements. During this same stage of development, retinal waves led
 to increases in cytosolic calcium within Müller glial lateral processes
 and stalks. These comprised distinct calcium compartments, distinguished
 by variable participation in waves, timing, and sensitivity to an M1
 muscarinic acetylcholine receptor antagonist. However, we found that
 motility of lateral processes was unaffected by the presence of
 pharmacological agents that enhanced or blocked wave-associated calcium
 transients. Finally, we found that mice lacking normal cholinergic waves
 in the first postnatal week also exhibited normal Müller glial process
 morphology. Hence, outgrowth of Müller glial lateral processes into
 synaptic layers is determined by factors that are independent of neuronal
 activity.
description: This dataset includes Müller glial calcium imaging and
 morphological data underlying all figures for the referenced manuscript,
 in which details about data acquisition and processing can be found in the
 'Materials and Methods' section. For Figures
 1 and 5, morphological measurements were obtained from two-photon
 volumetric images of membrane-tagged Müller glial processes in live flat
 mount retina. All morphological images were band pass filtered in XY space
 to reduce noise, corrected for 3D drift and photobleaching, and time
 course movies were collapsed into a single temporally color-coded image
 stack for subsequent quantification. For each field of view (FOV),
 processes were counted and grouped into one of 7 categories depending on
 whether motility occurred. Process counts and their associated Z-planes
 were then imported into MATLAB, where Z-planes for all counts within each
 FOV were normalized from 0 to 1 to enable comparison across cells and
 experiments. This morphological dataset is found in
 "motilityDataWithZ.mat".  For Figures 2-4,
 two-photon calcium imaging was performed on live flat mount retina in
 which Müller glia were labeled with a genetically encoded or organic
 calcium indicator. Following motion correction, regions of interest (ROIs)
 were semiautomatically placed over glial stalks and lateral processes
 using a segmentation algorithm. Average fluorescence intensity within
 these ROIs was normalized and smoothed using a median filter. Traces were
 Z scored, and a threshold Z score of 3 was used to define calcium
 transients, which were then further defined as wave-associated if they
 occurred within 3 seconds of the peak of a wave-associated EPSC.
 Using MATLAB scripts, measurements including proportion of ROIs
 participating in retinal waves, calcium transient amplitudes, and calcium
 transient latencies between stalks and lateral processes were obtained and
 can be found in "caImagingData.mat". This dataset also contains
 data from simultaneous electrophysiological recording of retinal waves,
 including EPSC amplitude and inter-wave interval. For
 Figure 6, Müller glial morphology was visualized by filling individual
 cells with fluorescent dye and obtaining volumetric two-photon images of
 dye-filled cells. Cells were traced using FIJI's Simple Neurite
 Tracer plugin, and measurements including number of tips, total process
 length, total branch points, and primary branches from stalk were derived
 from traced paths. Paths were converted into binarized skeletons and
 registered to correct for XY displacement of the stalk between the GCL and
 INL. Sholl analysis was performed using concentric rings spaced 1 µm
 apart, in the XY, XZ, and YZ planes. For the XZ and YZ planes, the center
 of each Sholl radius was placed on the end of the stalk closest to the INL
 in orthogonal projections of traced cells. Sholl radii were normalized to
 the total IPL thickness. Convex hull area was defined as the area of the
 smallest convex polygon enclosing the entire skeleton in an XY maximum
 projection image. These measurements and Sholl analysis data are in the
 MATLAB structures 'fillData.mat', 'sholl_xy.mat',
 'sholl_xz.mat', and sholl_yz.mat'.
 MATLAB scripts were used to extract data points of interest from
 these datasets for subsequent computation of summary statistics. There is
 a separate script associated with each figure. For each figure, the
 underlying data to be included was compiled into a source data file (eg.
 "Figure 1-source data 1.csv"). R scripts referencing these
 source data files were used to apply statistical testing. 
description: See README.txt for more information about datasets provided.
 These should be loaded into MATLAB, and then to extract relevant data, run
 the MATLAB script(s) associated with each figure.
 Statistical analyses may be run using the enclosed R scripts.
 These R scripts reference the source data which was extracted from the
 main .mat morphology or calcium imaging datasets. This source data is also
 included.