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Frans Vinberg, Hanna Heikkinen, Ari Koskelainen; Modulation of Dim Flash Responses by Lowered Extracellular Calcium in GCAP-/- Mouse Rods. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5264.
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Reducing extracellular [Ca2+] leads to a large transient increase of the photoreceptor outer segment current together with persistent deceleration and desensitization of dim flash responses (Yau et al., Nature 292:502-5; Bastian & Fain, J. Physiol. 330:307-329). These effects are probably caused either directly or indirectly by highly increased [cGMP] during low Ca2+ treatment in darkness (Matthews, J. Physiol. 484(2): 267-286), thus limiting the use of low [Ca2+]out in investigating Ca2+ dependent light adaptation mechanisms. Here GCAP-/- mouse rods are used to investigate how lowered [Ca2+]out modulates dim flash responses when the Ca2+-dependent acceleration of cGMP synthesis is absent.
Rod photoreceptor flash (2 - 20 ms) responses were recorded with transretinal ERG from WT (C57Bl/6) and GCAP-/- mouse retinas in 1 mM [Ca2+]out and in EGTA buffered ~30 nM [Ca2+]out. HEPES buffered Ringer solution with 2 mM aspartate was used to remove b-wave and higher-order neuron components. The glial component (slow PIII) was removed with BaCl2. Stability of the responses, especially under low [Ca2+]out, was facilitated by conducting the experiments at 25oC.
The saturated photoresponse amplitude of WT rods was 135 ± 20 µV in 1 mM [Ca2+]out and it transiently exceeded 1 mV, reaching a steady state value of 340 ± 40 µV when the retinas were exposed to low [Ca2+]out (n = 10). The fractional dim flash response sensitivity decreased from 3.1 ± 0.6 %/R* to 1.0 ± 0.2 %/R* and the time for the dim flash response to reach the peak amplitude (tp) increased from 310 ± 20 to 560 ± 70 ms as the retinas were switched from 1 mM Ca2+ to low 30 nM Ca2+ perfusion. In GCAP-/- rods the low calcium treatment did not increase the saturated photoresponse amplitude significantly. Moreover, on the contrary to the WT retinas, the tp of the GCAP-/- rod responses decreased from 670 ± 40 to 470 ± 10 ms when switched from 1 mM to 30 nM [Ca2+]out (n = 2). The accelerated dim flash response kinetics in GCAP-/- rods was accompanied by ~4 -fold decrease in fractional sensitivity. In preliminary simulations, these changes in GCAP-/- rods under low [Ca2+]out could be accounted for with ~4-5-fold reduction in R* lifetime.
The GCAP-/- rods can be used to study how [Ca2+]out modulates phototransduction in mammalian rods. Our results indicate that dim flash responses are significantly shaped by lowered extracellular [Ca2+] in the absence of GCAPs, most probably through shortening of R* lifetime.
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