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K. Cusato, M. Srinivas, A. Bosco, D.C. Spray; Gap Junctions Mediate Cell Death in the Developing Retina . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1617.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have recently demonstrated that gap junctions mediate bystander cell death in the developing retina. If gap junctions spread naturally-occurring cell death during normal retinal development, inhibition of gap junctions should decrease developmental cell death. In this study we describe the effects of mefloquine (MFQ), a connexin-selective gap junction inhibitor, on cell death and gap junctional coupling in the developing retina. Further, we examine the efficacy of MFQ as an inhibitor of connexins expressed in the retina. Methods: Postnatal day 5 (P5) and P8 mice were anesthetized and decapitated. Retinas were dissected free, flatmounted on culture inserts, and incubated in medium containing 3 µM MFQ or control medium without MFQ for 1-2 hours. Gap junctional coupling was examined by scrapeloading. Retinas were fixed in 4% paraformaldehyde, rinsed, cryosectioned at 20 µm, and stained with DAPI. Pyknotic cells in the INL were quantified in 16 fields (400 µm in length) per retina (n=6 for P5 and n=3 for P8) and compared by paired t- test. Scrapeloaded retinas were labeled with Streptavidin cy-2. Neuroblastoma cells (N2A) were transfected with connexin36 (Cx36) or Cx43 and coupling was evaluated by the dual whole cell patch clamp method. Results: 3 µM MFQ significantly reduced cell death in P5 mouse retina (from 4.18 to 2.73 cells/field) and in P8 retina (from 17.85 to 12.35 cells/field). Gap junctional coupling was evidenced by dye-spread in scrapeloaded retinas and was also reduced by MFQ treatment. Further, 3 µM MFQ decreased coupling of cells transfected with Cx36 by 95%, while coupling in Cx43 transfectants was reduced by only 10%. Conclusions: Mefloquine decreases both cell death and gap junctional coupling in the developing retina. The concentration of MFQ used in this study inhibits Cx36 but not Cx43 suggesting that MFQ acts on neurons rather than glia. Taken together, these results suggest that gap junctions mediate a naturally occurring bystander effect of neuronal death in the developing retina.
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