| Popis: |
We have paid close attention to the elegant work from Dr Cangiano's group, in particular to the pioneer work in obtaining electrical recordings from mouse photoreceptors using patch-clamp electrodes. As with any technique, perforated or whole cell patch-clamp techniques have limitations, due in part to the dialysis of the pipette content into the cell and/or of the cell content into the pipette. These limitations are evident in small cells such as mammalian photoreceptors. Intracellular access through patch electrodes in mouse photoreceptors introduces such artefacts as rundown of the light responses and spontaneous increase in rod–cone coupling. Dr Cangiano's group has reported with great detail these limitations in two recent publications (Cangiano et al. 2012; Asteriti et al. 2014). However, despite all the limitations, intracellular access yields critical information about mammalian photoreceptor physiology that cannot be obtained with other techniques, including important information about the state of electrical coupling between neighbouring photoreceptors. We agree with Asteriti and Cangiano that the kinetics of the rod light responses we recorded in intact mouse retinae during the daytime, as reported in Jin et al. (2015), are at the higher end of the range of published values. These relatively slow kinetics may possibly reflect rundown due to perforated patch recording. As indicated in the Methods section of our paper, we found differences between the protocol used by the Cangiano group and ours. In particular, we used pipettes of relatively high resistance (15–20 MΩ) and found that this was key to obtaining stable recordings and limited rundown. It was not our intention to infer that our recordings were perfectly physiological, but we now feel that a few additional words could have been added to prevent the reader from making the assumption that they were. Specifically, we should have clarified that we had optimized the patch-clamp procedure in order to minimize the rundown and coupling effects that may result from patch clamping. In response to ‘It further surprised us the statement of Jin et al. (2015) that the “kinetics of the current and voltage responses to light compared favourably with those measured with suction and patch electrodes in mouse retinal slices”’, this statement was referring to the overall sensitivity and kinetics, including the polarity of the responses, and was not intended to make a precise comparison. However, we disagree with Asteriti and Cangiano on their claim that the possible rundown/coupling effect of patch clamping significantly complicates the interpretation of our findings. Indeed, electrical recording of single photon responses, tracer coupling and receptor field measurements provide compelling evidence that rod coupling is minimal during the day/subjective day. This demonstrates that, under our conditions, patching of a rod per se has limited effect on the state of rod electrical coupling. It follows that the day/night difference in coupling reflected in the increase in the number of effectively coupled rods (Ne), tracer coupling and receptor field size at night is due to the action of a circadian clock, as there is no reason to believe that the coupling effect of the patch electrode would differ between day and night. Similarly, the rundown effect due to patching should be independent of the time of day, and hence the day/night difference in response kinetics essentially reflects the action of the clock. Finally, the rundown effect has been shown to have no impact on the rod response amplitude (Cangiano et al. 2012) and therefore is probably not responsible for the decrease in response amplitude at night. Thus, as the effects of rundown and coupling due to intracellular access should be the same day and night, and daytime measurements demonstrate that both have limited impact on rod electrical coupling and response kinetics, we conclude that the day/night difference in rod electrical coupling and light response properties we observed is real and if anything may have been slightly underestimated. Finally, we acknowledge Asteriti and Cangiano's last statement: ‘one must realize the limitations of patch recordings of photoreceptors and plan for appropriate controls, timing of the recordings and devise alternative approaches.’ We do realize the limitations of patch recording mouse photoreceptors, but we think that the advantages of the technique greatly outweigh its limitations. The mechanisms through which intracellular access of mouse photoreceptors induces rundown and spontaneous electrical coupling remain unknown, but it is legitimate to think that they will eventually be identified and counteracted. The Jin et al. (2015) paper establishes time of day as a key determinant of the rod light response and electrical coupling. So far, we have used single cell recordings and single photon response statistics, tracer coupling and receptive field size as proxies of rod electrical coupling. We are moving towards simultaneous patch-clamp recordings of pairs of adjacent rods in various mouse models. Direct measurements of the rod junctional conductance and the use of genetic tools will not only provide important clues about the biophysical mechanisms involved in the regulation of rod function but also help in the understanding of the perturbations triggered by accessing mammalian photoreceptors with perforated patch electrodes. This will move the field forward. |
Plný text