Spontaneous activity is a hallmark of developing neural systems. In the retina, spontaneous activity comes in the form of retinal waves, comprised of three stages persisting from embryonic day 16 (E16) to eye opening at postnatal day 14 (P14). Though postnatal retinal waves have been well characterized, little is known about the spatiotemporal properties or the mechanisms mediating embryonic retinal waves, designated Stage 1 waves. Using a custom-built macroscope to record spontaneous calcium transients from whole embryonic retinas, we show that Stage 1 waves are initiated at several locations across the retina and propagate across a broad range of areas. A gap junction antagonist, meclofenamic acid, reduced the frequency and size of Stage 1 waves, nearly abolishing them. The general nAChR antagonist, hexamethonium similarly nearly abolished Stage 1 waves. Application of the α4β2 nAChR antagonist dihydro-ß-erythroidine only slightly reduced the frequency of waves but significantly reduced the number of cells that participated in waves. Thus, Stage 1 waves are mediated by a complex circuitry involving subtypes of nAChRs and gap junctions. Stage 1 waves in mice lacking the β2 subunit of the nAChRs (β2-nAChR-KO) were reduced, but in contrast to WT mice, they persisted in the hexamethonium and were completely blocked by meclofenamic acid. To assay the impact of Stage 1 waves on retinal development, we compared the spatial distribution of a subtype of retinal ganglion cells, intrinsically photosensitive retinal ganglion cells (ipRGCs), which undergo a significant amount of cell death, in WT and β2-nAChR-KO mice. We found that the developmental decrease of ipRGC density is preserved between WT and β2-nAChR-KO mice, indicating that processes regulating ipRGC distribution are not influenced by spontaneous activity.

This is data for Figure 5. Refer to methods for data that were collected and processed.

From the methods section:

Epifluorescent macroscope calcium imaging

Epifluorescent calcium imaging was obtained on a custom-built macroscope with an Olympus XLFLUOR4X/340 4X 0.28 NA objective and a Teledyne Kinetix camera.  Collectively, this macroscope has 4.7 mm x 4.7 mm FOV, and 1.5 µm/pixel. All movies were taken at a 1 Hz frequency and pixels were binned 4 x 4 bringing the resolution down to 5.9 µm/pixel, still maintaining single-cell resolution. Cal520 and GCaMP6s excitation was evoked with a 474 nm LED. A full description and building instructions can be found at: https://github.com/Llamero/DIY_Epifluorescence_Macroscope

Analysis of ipRGC densities

To image the number of ipRGCs in fixed retinas, dissected retinas from P1 and P7 mice were fixed in 4% PFA for 30 minutes. The fixed retinas were subsequently mounted on a slide with vectashield and a cover slip, then imaged within an hour of mounting on the macroscope. For P1 retinas, Z-stacks were acquired by manually turning the focus knob.

We first identified the centroid of each ipRGCs. For P7 retinas, where there is more space between cells, we employed the following automatic segmentation. Images were bandpass filtered and somata automatically segmented using the Morpholibj (Legland et al., 2016) classic watershed tool to obtain 8-bit binarized masks. The masks were then processed in MATLAB in order to obtain the centroid locations and nearest neighbor distances for each soma. For P1 retina, where there is less space between cells and in fact cells seem to form clusters, automatic segmentation was not possible. Therefore, cells were manually marked using the ImageJ multipoint tool, and soma locations were exported as a CSV file. For all ages, the centroid data was imported to MATLAB for further analyses.

Density was quantified by dividing the microscope field of view up into 200×200 μm squares, manually excluding ones that did not cover the retina or covered partial or damaged parts of the retinas. Out of the resulting squares (~150 per P7 retina, ~100 per P1 retinas) 75 squares (for P7 retinas) and 50 squares (for P1 retinas) were randomly selected and the average density of TdTom+ cells in those squares were calculated.

Tif images can be opened using FIJI (Fiji is just imageJ).