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N. C. Orton, W. K. Stell, N. T. Bech-Hansen; The Cacna1f-Mutant Retina Shows Evidence of Failed Synaptogenesis, Abnormal Synaptic Ribbon Structure and Increased Photoreceptor Cell Death. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4466.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in CACNA1F, which encodes the Cav1.4 subunit of a voltage-gated L-type calcium channel, cause incomplete congenital stationary night blindness (CSNB2). We have previously shown significant neuronal rearrangements and a loss of photoreceptor to 2nd-order neuron synapses in the adult retina of the Cacna1f-mutant mouse. In order to establish whether these changes were due to failure of synapses to develop, or to failed maintenance of mature synapses in the OPL, we analyzed mutant retina in the postnatal period immediately before and after completion of synaptogenesis.
We assessed synaptic development in WT and Cacna1f-mutant retinas (P10, P14) by TEM and immunocytochemistry, and apoptosis (P10 to adult) by TUNEL. We counted rod and cone terminals in TEM images (about 11µm retinal width/field; n=3 animals) and TUNEL-positive nuclei (480µm retinal length/field; n=3-5 animals). Student's t-test was used to determine significance.
TEM at P14 revealed 40% fewer rod and 63% fewer cone terminals in mutant retina. The invagination of 2nd-order neurons into terminals, and typical synaptic ribbons, were never observed in the mutants (n=60 micrographs). We did see small, free-floating electron-dense objects in the residual terminals of mutants, but even if they represent immature/abnormal synaptic ribbons, they were 52% less numerous than ribbons in the WT. In contrast, TEM at P10 showed no significant difference between the absolute numbers of terminals in WT and mutant retinas, although the structure of synaptic ribbons in the mutants was abnormal. Immunocytochemical analysis at P14 revealed strong co-localization of presynaptic (ribeye) and postsynaptic (mGluR6) markers in the WT retinas, but not in the mutant retinas, confirming TEM observations. TUNEL labeling showed peak apoptosis in the ONL at P14, in WT as well as mutant retinas; however, the frequency of TUNEL-positive nuclei in the ONL at P14, P21 and P28 was higher in the mutant retinas, indicating both more frequent and more prolonged cell death in the mutant ONL.
This work points strongly to a critical role for Cav1.4 in photoreceptor synaptogenesis. Without Cav1.4, the formation of synaptic ribbons and invaginations of second-order neurons is abnormal. The absence of Cav1.4 also causes increased and prolonged loss of cells in the photoreceptor layer. Whether these changes are due to loss of calcium channel function, or to physical absence of this channel protein, is yet to be determined.
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