32 Zhao, Zhijian Müller, Frank Spehr, Marc 163020 160000 Publikationsserver der RWTH Aachen University Aachen English Cyclic guanosine monophosphate (cGMP) is a second messenger that plays an important role in many cell types of the retina. It is involved in a variety of intracellular signaling processes mostly by activating protein kinases or by binding to ion channels. In photoreceptors, cGMP is crucially involved in visual signal transduction. In many neurons of the inner retina, cGMP is synthesized by soluble guanylate cyclases (sGC) upon activation by nitric oxide (NO). So far, there is no common view of the contribution of cGMP to retinal adaptation or to the modulation of signaling processes in other retinal cell types, although many attempts to investigate those processes have been made in the past.Using stimulation of the retina with the nitric oxide donor SNAP followed by immunohistochemistry with an antibody against cGMP, I could demonstrate cGMP synthesis controlled by NO in a variety of cell types of the intact mouse retina. However, this method does not enable to resolve the dynamics of this process. I, therefore, aimed at the expression of the FRET-based genetically encoded cGMP sensor Cygnet-2.1 in retinal cultures and in the intact retina to monitor the production of cGMP in real time using imaging techniques. In control experiments I expressed Cygnet-2.1 functionally using the lipofectamine transfection in FlpTS GC-A cells (derived from HEK-293 cells) that served as model system. In these cells the synthesis and degradation of intracellular cGMP could be monitored upon activation of membrane-bound guanylate cyclases with atrial natriuretic peptide (ANP). The next aim was to establish Cygnet-2.1 expression in retinal cells. To this end, retinal cultures were established and seven different AAV serotypes encoding GFP were screened for their transduction efficiency in this in vitro model. AAV2 was selected as the most promising vector based on its wide range of transduction patterns among the retinal cell types and the high transduction efficiency. In the next step, AAV-mediated transduction was established in vivo. AAVs were delivered via ocular injection to newborn mice. Again, AAV2 was determined as the most promising candidate for transduction of retinal neurons. Finally, Cygnet-2.1 sensors were expressed in dissociated retinal cultures by using lipofectamine transfection and AAV-mediated transduction. Using lipofectamine transfection, changes in Cygnet-2.1 fluorescence revealed a fast increase in the internal cGMP concentration upon NO stimulation in cultured retinal neurons that were immunoreactive for sGC. Upon AAV2-mediated transduction, no change in the Cygnet-2.1 fluorescence could be detected in retinal cells upon stimulation with NO donors or in the FlpTS GC-A cells upon stimulation with ANP. Most likely, AAV-mediated transduction did not yield functional Cygnet-2.1 sensors. Nevertheless, the proof of principle experiments clearly demonstrated that it is possible to investigate cGMP metabolism in the retina under physiological conditions using Cygnet-2.1. In future, the established methods in this thesis can drive the investigation of cGMP modulation in vivo and in vitro by means of optogenetic approaches and, thereby, foster our understanding of retinal adaptation and signal modulation. Aachen, Techn. Hochsch., Diss., 2015 ; PUB:(DE-HGF)11, ; 2, ; Dissertation / PhD Thesis Dissertation Aachen, Techn. Hochsch. 2015 RWTH-2015-00840 2015 https://publications.rwth-aachen.de/record/463047