Analyses of inner brain Opsins in the vertebrate CNS

Opsins are light receptive proteins expressed in vertebrate eyes mediating vision and circadian entrainment. Yet, Opsins are also present in many cells outside the eyes. Over recent years, work by multiple groups has shown that many- if not the majority- of these non-visual Opsins can also function as light receptors. Consistently, several studies established that measurable amounts of light penetrate vertebrates brains. Here, we focus on the functional investigation of a particularly slowly evolving group of ciliary- type Opsins, the encephalopsin/ tmt-opsin (ETO) group in zebrafish and medakafish. Our previous work revealed so far that vertebrate brains express these Opsins in specific subsets of interneurons and motorneurons. We also showed that light can change the membrane potential of neurons at the position of tmtopsin expressing cells in medaka fish tectal slices. This led us to hypothesize that environmental light impacts on the information processing in the vertebrate brain by modulating the membrane potential of Opsin-expressing neurons, and hence might lead to light-dependent behavioural alterations. We meanwhile generated mutations in medakafish tmtopsin1b and 2. First analyses of different tmtopsin1b mutant alleles revealed a consistent behavioral phenotype, as well as changes in the levels of a specific preprohormone. With this principle evidence of functional importance at hand, we now aim to further explore these mutants on additional behavioral, molecular and cellular levels. The behavioral analyses include assays on different behaviors, as well as circadian entrainment tests. The molecular analyses will expand on the already uncovered preprohormone target, but also include additional quantitative sequencing approaches. The cellular level aims at unraveling the neuronal projections of tmtopsin cells in vivo to obtain a better understanding of the input and output areas of these cells. The labeled tmtopsin+ neurons will also allow us to perform electrophysiological experiments under different light conditions, as well as in wildtype vs. mutant fish. This combination of approaches will allow us to gain a more comprehensive understanding of the function of Opsins outside the eyes. In order to discriminate between species-specific and more widely conserved functions, we also added an evolutionary dimension to our proposal. Zebrafish and medaka inhabit different habitats and are separated by about the same evolutionary distance like birds versus mammals. We aim to compare the function of a selected set of orthologous ETO family members between both species. With our work we likely gain a better understanding of the function of non-visual photoreception, a phenomenon present across the vertebrate phylum. This should also allow a better judgement of the impact of light pollution (artificial light at the wrong time and place) on animals. Last, but not least, the non-visual Opsins under study here might also be useful for new optogenetic applications in the future.