A growing body of research is showing that chronic circadian rhythm disruption, as experienced by rotational shift workers and patients suffering from sleep phase disorders, neurodegenerative diseases, and psychiatric disorders, can lead to behavioral and physiological problems such as insulin insensitivity, depressed or agitated mood, impulse control issues, and cognitive deficits. To regulate circadian rhythms, light information reaches the brain via efferent projections of intrinsically photosensitive melanopsin-expressing retinal ganglion cells (mRGCs) to the suprachiasmatic nucleus (SCN). In order to develop effective treatments for circadian rhythm disruption, it is imperative to understand and separate out the specific pathways through which light information reaches the SCN from other mRGC mediated effects, such as the pupillary light reflex which is mediated by mRGC projections to the olivary pretectal nucleus (OPN). This proposal aims to identify distinct retinal cell- mRGC-brain region circuits for different non-image forming (NIF) visual responses and molecular players that underlie those circuits. In order to identify mRGCs that specifically project to SCN o to OPN, TVA, an avian receptor not found in mammalian cells, will be expressed in the mRGCs of Opn4Cre (cre-recombinase driven by melanopsin promoter) mice via intravitreal injections of cre-dependent AAV. Stereotaxic injections of TVA dependent red glycoprotein-deleted rabies virus (EnvA-SAD?G-mCherry) into the SCN and blue glycoprotein- deleted rabies virus (EnvA-SAD?G-BFP) into the OPN will allow SAD?G to be taken up in mRGC axon terminals ending at the injection site. Glycoprotein deleted rabies virus is unable to spread transsynaptically but continues to replicate within the infected cells- effectively filling all processes with fluorescen protein and allowing for identification of mRGCs that specifically project to SCN (will express mCherry), those that specifically project to OPN (will express BFP), and subtypes of mRGCs. In a separate experiment, rabies virus glycoprotein (RV-G) will be targeted to mRGCs. Infection of RV-G-expressing mRGCs with SAD?G will allow the fluorescent rabies virus to spread one synapse in the retrograde direction. This will allow SCN or OPN specific mRGCs to label upstream retinal cells with the same colored SAD?G and may reveal exclusive retinal cell-mRGC-brain region di-synaptic circuits. Lastly, next generation sequencing will be applied to mRGC populations that are fluorescently labeled based on their central target projections to generate gene transcription profiles of target- specific mRGC populations. Comparisons between gene expression profiles of different target-defined mRGC populations will reveal genes that are preferentially expressed in mRGC populations that specifically project to SCN or OPN. All together, these experiments will identify brain region-specific mRGC projections and retinal cell inputs, uncover molecular players that underlie target-specific mRGC projections, and inform about potential targets for treating circadian rhythm disruption.
My proposed research will identify distinct cell-cell interactions that carry light information to the brain to synchronize circadian rhythms. Results from this research will shed light on how a very small population of cells in the eye mediate many different unconscious responses and will allow us to develop novel approaches to diagnose and treat circadian rhythm disruption prevalent in shift workers, jet lag, and disorders of the eye, metabolism and mood.
