The Circuit Mechanisms Underlying the Spatiotemporal Properties of Stage 1 Retinal Waves

Spontaneous activity is a hallmark of developing neural systems. In the developing visual system, patterned spontaneous activity begins in the retina, where waves of depolarizations, called retinal waves propagate across a network of neurons. Retinal waves propagate laterally across the retina, but also downstream in the visual circuit through the dorsal lateral geniculate nucleus, the superior colliculus (SC), and the primary visual cortex. Throughout the development of the mouse visual system, retinal waves are observed in three stages starting from embryonic day 16 (E16) to eye opening, corresponding to postnatal day 13 (P13). The circuits that mediate each stage of retinal waves change throughout development. Stage 2 waves are observed between P1-P10 and are initiated by the release of acetylcholine (ACh) from starburst amacrine cells (SACs), leading to the activation of nicotinic acetylcholine receptors (nAChRs). They propagate through a network of SACs that themselves express nAChRs during development. Stage 3 waves are observed between P10-P14 and are initiated by the release of glutamate from bipolar cells, leading to the activation of ionotropic glutamate receptors. Stage 3 waves propagate through a network of bipolar and amacrine cells via glutamate transmission and gap junction coupling. Stage 1 waves are observed between E16-P0 and the initiation and propagation mechanisms for Stage 1 waves have been thought to mainly rely on gap junction coupling between retinal ganglion cells (RGCs), unlike the other two stages the details of their initiation and propagation mechanisms are poorly understood./nThe focus of this dissertation is to describe the spatiotemporal properties and circuit mechanisms of Stage 1 waves, identify how distinct these waves are from Stage 2 waves, and then begin to probe their function in the development of the retina. In Chapter 1, I summarize the current understanding of the mechanisms shaping the spatiotemporal properties of all stages of retinal waves. In Chapter 2, I used a custom-built macroscope and a two-photon microscope to perform population calcium imaging in embryonic retinas. I first characterized the spatiotemporal properties of Stage 1 waves. Then, using genetic and pharmacological manipulations, I determined the relative role of gap junctions and nAChRs in mediating Stage 1 waves. Stage 1 waves initiate at several locations across the retina and propagate across finite regions of varying areas. Additionally, I found that waves that spanned a large retinal surface area were more sensitive to nAChRs and gap junction antagonists, as they were either abolished or significantly reduced in frequency in the presence of those pharmacological agents. I also found that waves in mice lacking the 2 subunit of nAChRs (β2-nAChR-KO) exhibited reduced propagation areas. The application of a general nAChR antagonist in the β2-nAChR-KO had no effect on wave properties. However, the application of a gap junction antagonist greatly reduced both the area and frequency of waves, suggesting a compensatory role for gap junctions in the β2-nAChR-KO./nThe disruption of Stage 1 and 2 waves in the β2-nAChR-KO allowed for further investigation into the role of retinal waves in regulating the maturation of RGCs. We specifically investigated a subpopulation of RGCs, called intrinsically photosensitive RGCs (ipRGCs). Using immunohistochemistry, we observed a significant reduction in ipRGC density between P1 and P7 retinas in both WT and β2-nAChR-KO mice, suggesting that ipRGC cell death observed in the first postnatal week of development occurs independently of normal retinal waves. Moreover, there was no significant change in density or spatial distribution between WT and β2-nAChR-KO mice at P1 and P7, also indicating this developmental step occurs independently of normal waves. In Chapter 3, I discuss the implications of these results on our current understanding of retinal waves. Specifically, I highlight newly found key similarities and differences between Stage 1 and 2 waves, and future experiments necessary to fully understand the distinction between them.

Advisor: Feller, Marla B