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T. N. Sidorova, R. M. Sappington, S. D. Crish, W. S. Lambert, D. J. Calkins; Cell Specific Localization of BDNF mRNA in the Mouse Visual Pathway Using Fluorescent in situ Hybridization. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3173.
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© ARVO (1962-2015); The Authors (2016-present)
Brain-derived neurotrophic factor (BDNF) is a major trophic factor for retinal ganglion cells (RGCs). While it is commonly accepted that ganglion cell BDNF is obtained from target sites in the brain, specifics of the synthesis and distribution of BDNF remain unclear. Several studies have described BDNF immunoreactivity in retina, however there is little information about cell-specific BDNF mRNA expression. In the present study, we examined BDNF mRNA expression in the mouse visual system using fluorescent in situ hybridization (FISH) combined with immunolabeling with glial and neuronal markers.
Digoxigenin-labeled RNA (DIG-RNA) BDNF probes (450 bp length) were transcribed in anti-sense and sense orientation using SP6 and T7 PNA polymerases, respectively. ISH with tyramide signal amplification (TSA Plus Fluorescence System) in combination with peroxidase (POD)-based visualization was performed on wholemount retinas and superior colliculi (SC) from 3 month C57BL/6 mice to detect cell specific BDNF mRNA expression. Immunolabeling was then performed for the astrocyte marker glial fibrillary acidic protein (GFAP) and the neuronal marker phosphorylated neurofilament (SMI31) to identify specific cell populations expressing BDNF mRNA.
Robust BDNF mRNA signal was present in SMI31-positive RGC cell bodies. No colocalization of in situ signal with GFAP in wholemount retina was found. In the SC, BDNF mRNA was expressed in neuronal cell bodies, however, signal was also found in long, SMI31-positive processes in the gray and white matter layers of the superficial (retinorecipient) SC.
Intriguingly, BDNF mRNA is produced in the inner retina and is expressed exclusively by RGCs. Within the superficial SC, BDNF mRNA is produced by collicular neurons as expected. BDNF message in SMI31-positive processes in the collicular layers made up of RGC axons suggests that retinally expressed BDNF mRNA may be transported for use at distal sites.
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