Circuit mechanisms underlying embryonic retinal waves

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