UC Berkeley

The retina offers unique insight into synaptic activity in the central nervous system. It is the only part of the central nervous system that can be visualized without advanced imaging or invasive methods. Its highly organized structure has turned it into a main target for neuroscientific studies, and while it has a simplistic anatomical organization its synaptic functions have only been partially elucidated. Zebrafish have been utilized to study vision since they have a cone-rich retina, high acuity vision and they are a convenient model to work with and manipulate genetically. Furthermore, zebrafish can absorb drugs and small molecules directly from their liquid environment thereby facilitating chemical manipulation of their nervous system. For these reasons we have chosen to use zebrafish to generate tools that improve our understanding of retinal synaptic activity in the outer plexiform layer of the retina, and in horizontal cells (HCs) in particular. Five genetically modified strains of zebrafish (4 of which were developed at the Kramer lab) are presented in this dissertation. Two strains have been modified using the introduction of foreign receptors, FaNaC and PSAM-GlyR, allowing controlled and reversible depolarization and hyperpolarization of HCs, respectively. Using these modified strains, we show that by changing the membrane potential of HCs we can manipulate neuronal function upstream, and downstream to the HC synapse. Using three additional strains, we explore the change in pH at different locations in the HC to cone synapse during lateral inhibition. Those locations include the AMPA receptor on HCs, and synaptophysin and the voltage gated calcium channel on the photoreceptor side. By combining our chemogenic tool FaNaC to depolarize HCs, and a genetically introduced pHluorin to detect pH changes, we reaffirm the hypothesis that pH is the signal which mediates lateral inhibition in the outer retina. Using the three pH probes, we show that the change in pH is highly localized nearby photoreceptor voltage-gated calcium channels. In conclusion, while HCs form a wide interconnected network that spans the entire retina, their communication with individual photoreceptors through acidification of the synaptic cleft is spatially constrained. Moreover, slight deviations in HC membrane potential greatly alter their effects on other retinal neurons including photoreceptors, bipolar cells and retinal ganglion cells.