Purchase this article with an account.
Mark E. Pennesi, John W. Stoddard, Keith V. Michaels, Emily D. Blum, Anastasiya Maricle, Peter J. Francis; Expression and Localization of Serotonin Receptors in the Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6547.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We previously reported the presence of serotonin (5-HT) receptor and serotonin transporter (5HTT) mRNA transcripts in both mouse and rat retina. Their existence in the retina raises the possibility that modulation of 5-HT levels by selective serotonin reuptake inhibitors (SSRIs) may directly affect retinal physiology. In the brain, increased levels of serotonin induced by SSRIs, have been shown to elevate levels of growth factors including brain-derived neurotrophic factor (BDNF). BDNF has been demonstrated to attenuate photoreceptor death in animal models of retinal degeneration. The purpose of this study is to determine whether the previously described mRNA transcripts are translated into proteins and identify their location within the rodent retina.
Western blot analysis was performed using the Odyssey® Infrared Imaging System protocol (LI-COR). Total protein and total cell membrane protein isolates from mouse brain and retina were blotted on 15% Tris-Glycine gels. Nitrocellulose membranes were probed with commercially available anti-5-HT receptor polyclonal antibodies. Histological analysis was performed using mouse retinal sections cut at 10µm using a cryostat from eye cups fixed in 2% paraformaldehyde and embedded into O.C.T. Sections were stained with commercially available 5HT receptor antibodies and fluorescent images were acquired with an Olympus Fluoview 1000 confocal microscope.
Western blot analysis revealed a strong band near the predicted size for 5HT1a, 5HT3a and 5HTT in mouse brain and retina total protein samples. Total membrane isolations demonstrated a reduction in non-specific binding while retaining the identical bands for 5HT1a, 5HT3a and 5HTT. Furthermore, the respective bands were eliminated in Western blots performed with pre-incubated antibody and antigen. In mouse retina, staining for 5HT1a was observed in photoreceptors, ganglions cells and a population of cells in the inner aspect of the inner nuclear layer, while 5HT5a only localized to ganglion cells. In contrast, 5HT1b staining was strong in the outer plexiform layer, which appeared to be post-synaptic and 5HTT exhibited an intracellular space pattern stain consistent with glial cell localization.
Our initial Western blot and histological analysis provides evidence supporting the diverse expression of 5-HT receptors and the 5HT transporter in the mouse retina. Experiments are underway to further detect and co-localize the remaining 5-HT receptors to specific retinal cell types. These findings may establish mouse models as valuable tools in evaluating the efficacy of therapies designed to treat retinal disorders via modulation of serotonin physiology.
This PDF is available to Subscribers Only