Cone photoreceptors (cones) are essential for high-resolution daylight vision and colour perception. Loss of cones in hereditary retinal diseases has a dramatic impact on human vision. The mechanisms underlying cone death are poorly understood, and consequently, there are no treatments available. Previous studies suggest a central role for calcium (Ca2+) homeostasis deficits in photoreceptor degeneration; however, direct evidence for this is scarce and physiological measurements of Ca2+ in degenerating mammalian cones are lacking.

Here, we took advantage of the transgenic HR2.1:TN-XL mouse line that expresses a genetically encoded Ca2+ biosensor exclusively in cones. We cross-bred this line with mouse models for primary (“cone photoreceptor function loss-1”, cpfl1) and secondary (“retinal degeneration-1”, rd1) cone degeneration, respectively, and assessed resting Ca2+ levels and light-evoked Ca2+ responses in cones using two-photon imaging. We found that Ca2+ dynamics were altered in cpfl1 cones, showing higher noise and variable Ca2+ levels, with significantly wider distribution than for wild-type and rd1 cones. Unexpectedly, up to 21% of cpfl1 cones still displayed light-evoked Ca2+ responses, which were larger and slower than wild-type responses. In contrast, genetically intact rd1 cones were characterized by lower noise and complete lack of visual function.

Our study demonstrates alterations in cone Ca2+ dynamics in both primary and secondary cone degeneration. Our results are consistent with the view that higher (fluctuating) cone Ca2+ levels are involved in photoreceptor cell death in primary (cpfl1) but not in secondary (rd1) cone degeneration. These findings may guide the future development of therapies targeting photoreceptor Ca2+ homeostasis.

Introduction

Human vision depends primarily on cone photoreceptors, which mediate high-resolution, day-light colour vision. Rod photoreceptors (rods), on the other hand, are responsible for vision under dim-light conditions, i.e. at night. Cone loss can occur as a consequence of a mutation in the cone itself (primary cone degeneration) or as a “side-effect” of rod photoreceptor loss (secondary cone degeneration). A rapid cone loss as a consequence of mutations in cone-specific Pde6c (cGMP-specific phosphodiesterase) is present in the cpfl1 mouse, an animal model for primary cone degeneration (1–3). Secondary cone degeneration, on the other hand, is observed as a consequence of primary rod photoreceptor cell death in diseases like Retinitis Pigmentosa (4,5). The rd1 mouse carries a rod-specific Pde6b mutation and is a well-known model for secondary cone degeneration (6). We used cpfl1 and rd1 mouse lines to investigate the role of Ca2+ homeostasis in primary and secondary cone degeneration, respectively.

In the intact photoreceptor, Ca2+ has many important functions (Fig. 1A and B) , including modulating the transduction cascade in the outer segment (OS), and triggering the transmitter release from the axon terminals (7–9). In the dark, high cGMP levels in the OS activate cyclic nucleotide-gated (CNG) channels and allow for Ca2+ influx (10–12). In the light, Ca2+ levels drop due to cGMP hydrolysis by phosphodiesterase 6 (PDE6) and the subsequent closure of CNG channels. Low Ca2+ levels allow dissociation of Ca2+ from guanylate cyclase-activating proteins (GCAPs), disinhibition of guanylate cyclase (GC), restoring cGMP levels. This close coupling between cGMP and Ca2+ levels, together with the finding that mutant photoreceptors show an abnormal cGMP accumulation (13–15), led to the hypothesis of cGMP-mediated Ca2+ “overload” triggering degeneration (16).
Photoreceptor Ca2+ signalling and experimental model. (A) The phototransduction cascade tightly regulates outer segment (OS) Ca2+ influx via cGMP-dependent cyclic nucleotide-gated (CNG) channels. Currents through CNG channels modulate the membrane potential and, thereby, voltage-gated Ca2+ channels (VGCCs) in the photoreceptor axon terminal and glutamate release. (B) Ca2+ is highly compartmentalized in healthy photoreceptors and diffusion is restricted between OS, inner segment (IS), soma, and axon terminal. (C) Vertical slice of a transgenic HR2.1:TN-XL mouse retina (outer retina shown), with cones selectively expressing the Ca2+ biosensor TN-XL (green; cf. green areas in (B); grey, photoreceptor nuclei stained with DAPI). (D) Ca2+ responses (bottom) to a 1-s light-flash recorded from a cone axon terminal (top; yellow circle) in a slice retinal preparation (mean, black trace; single trials, grey traces). Ca2+ signals shown as the change in ratio (ΔR = FA/FD) of the acceptor fluorescence (FA) to the donor fluorescence (FD) (Methods). PDE6, phosphodiesterase 6; cGMP, 3’-5’-cyclic guanosine monophosphate; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars: c, 20 µm; d, 5 µm.
Figure 1.

Photoreceptor Ca2+ signalling and experimental model. (A) The phototransduction cascade tightly regulates outer segment (OS) Ca2+ influx via cGMP-dependent cyclic nucleotide-gated (CNG) channels. Currents through CNG channels modulate the membrane potential and, thereby, voltage-gated Ca2+ channels (VGCCs) in the photoreceptor axon terminal and glutamate release. (B) Ca2+ is highly compartmentalized in healthy photoreceptors and diffusion is restricted between OS, inner segment (IS), soma, and axon terminal. (C) Vertical slice of a transgenic HR2.1:TN-XL mouse retina (outer retina shown), with cones selectively expressing the Ca2+ biosensor TN-XL (green; cf. green areas in (B); grey, photoreceptor nuclei stained with DAPI). (D) Ca2+ responses (bottom) to a 1-s light-flash recorded from a cone axon terminal (top; yellow circle) in a slice retinal preparation (mean, black trace; single trials, grey traces). Ca2+ signals shown as the change in ratio (ΔR = FA/FD) of the acceptor fluorescence (FA) to the donor fluorescence (FD) (Methods). PDE6, phosphodiesterase 6; cGMP, 3’-5’-cyclic guanosine monophosphate; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars: c, 20 µm; d, 5 µm.

Many studies support this “high Ca2+ hypothesis” (15,17–20), however, there are also several contradicting results: For instance, treatments with Ca2+ channel blockers, aimed at reducing Ca2+ overload and, thus, preventing or at least delaying photoreceptor degeneration, produced inconsistent results for primary rod degeneration (21–24). Furthermore, data from CNGA3-deficient mice show that in the absence of cone CNG channels (and CNG channel-mediated Ca2+ influx) cones still degenerate (25). Additionally, a functional study in Pde6cw59 mutant zebrafish found no increase in cone Ca2+ levels, but instead a suppression of (spontaneous) Ca2+ transients (26). Finally, also an alternative hypothesis has been promoted, which is based on the idea that photoreceptor death might be triggered by too low Ca2+ (i.e. the “low Ca2+ hypothesis”) (27).

Here, we set out to study Ca2+ dynamics in degenerating cones using a transgenic mouse line (HR2.1:TN-XL) that expresses a genetically encoded ratiometric Ca2+ biosensor selectively in cones and allows monitoring of light-evoked Ca2+ signals in individual cone axon terminals (28–31). In healthy photoreceptors, opening of CNG channels in the dark depolarizes the cone membrane. Voltage-gated Ca2+ channels (VGCCs) in the synaptic terminal translate this depolarization into a Ca2+ influx, which in turn triggers synaptic vesicle fusion with the membrane and glutamate release (32–35). Thus, the presynaptic Ca2+ level in photoreceptors can serve as a proxy for upstream CNG channel activation in the OS (28).

To study cone Ca2+ in primary and secondary cone degeneration, we crossbred the HR2.1:TN-XL mouse line with the cpfl1 and rd1 PDE6 mutant lines, respectively. Ca2+ signals were compared between these cpfl1 and rd1 mutant crossbreds and the “wild-type” retina of HR2.1:TN-XL mice. We observed differential changes in Ca2+ in the two mutant lines: In primary cone degeneration, Ca2+ levels were more variable, noisy, and tended to be higher than in wild-type (wt). Notably, a substantial fraction of these cones showed robust light-evoked Ca2+ responses. On the other hand, in secondary cone degeneration, cone Ca2+ levels appeared to be lower and less variable compared to wt and did not show light-evoked Ca2+ responses. Our data are consistent with the idea of higher Ca2+ levels being involved in primary (cpfl1), but not in secondary (rd1) cone degeneration.

Materials and Methods

Animals

The transgenic mouse line HR2.1:TN-XL (C57BL/6J background) expresses the Ca2+ biosensor TN-XL (36) under the control of the human red opsin promoter (HR2.1) selectively in cone photoreceptors (28) (Fig. 1C). To study primary and secondary cone degeneration, we crossbred the biosensor line with the original rd1 and cpfl1 mutant animals to generate the HR2.1:TN-XL x cpfl1 (C57BL/6J background) and HR2.1:TN-XL x rd1 (C57BL/6J x C3H background) lines. Using PCR amplification and NdeI digestion, we verified that these lines were free of the rd8 (Crb1) mutation (37). While a previous study on C3H rd1 and congenic C3H wt animals, the latter of which show normal electroretinographic responses, had in principle ruled out the presence of the Nob5 (Gpr179) mutation (38), we additionally performed a PCR-based analysis employed recently in a survey on C3H mouse lines (39). As shown in Supplementary Material, Figure S1, the Nob5 mutation is absent in C3H rd1 mice used in our study. For simplicity, in the later parts of the manuscript, we refer to the biosensor lines as wt, cpfl1, and rd1. For Ca2+ imaging experiments (see below), we used mice in two time windows (postnatal days 18-20 (P18-20) and P30-33), to which we refer to as P18+ and P30+, respectively. See results for a justification for the use of these time-frames. In general, we used mice irrespective of gender.

Prior to Ca2+ imaging, the mice were dark-adapted for approx. 2 hours and then anaesthetized using isoflurane (CP Pharma, Burgdorf, Germany). For immunostainings, we used P20 and P30 as analysis time points and the animals were anaesthetized with CO2. In both cases, anaesthetized mice were killed by decapitation. All procedures were performed in accordance with the law on animal protection issued by the German Federal Government (Tierschutzgesetz) and approved by the institutional animal welfare committee of the University of Tübingen.

Immunohistochemistry and fluorescence microscopy

Eyes were marked on the nasal side prior to enucleation. They were fixed in 4% PFA in 0.1 M phosphate buffer saline (PBS, pH 7.4) (∼1 hour) and cryo-protected in 30% sucrose in PBS at 4oC overnight. Then, eyes were embedded in Tissue-Tek OCT compound (Sakura Finetek Europe, Alphen aan Den Rijn, Netherlands) and stored at −20 oC until cryo-sectioning into 12 μm thick vertical sections. Sections were rehydrated with PBS, permeabilized in 0.1% Triton X-100 in PBS containing blocking solution (10% goat or donkey serum, 1% BSA). As primary antibodies, we used rabbit anti-GFP (1:600; Millipore, Darmstadt, Germany) or mouse anti-GFP (1:500; Abcam, Cambridge, UK); sheep anti-cGMP (1:500, from Harry W.M. Steinbusch, Maastricht University, Maastricht, Netherlands) and guinea pig anti-glycogen phosphorylase (1:1000, kind gift from B. Pfeiffer-Guglielmi, University of Tübingen, Tübingen, Germany). As secondary antibodies, we used Alexa Fluor 568 donkey anti-sheep (1:500; Invitrogen, Carlsbad, USA), Alexa Fluor 488 goat anti-rabbit (1:500; Molecular Probes, Eugene, USA), Alexa Fluor 488 goat anti-mouse (1:500; Molecular Probes) and FITC goat anti-guinea-pig (1:300; Sigma, Saint Louis, USA). The terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay (Roche Diagnostics, Mannheim, Germany) was performed using the TMR red kit as per manufacturer’s instruction. Z-stack and mosaic images were captured on an Imager Z1 ApoTome microscope using a 20x air objective (0.8 NA; cf. Fig. 2A, wt and cpfl1) or a 63x oil immersion objective (1.4 NA; cf. Fig. 2A, rd1), and AxioVision (v.4.8.1.0) software (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). Figures were assembled either in Adobe Photoshop CS5 (Adobe Systems, San Jose, USA) or Canvas 11 (ACD Systems International Inc., Seattle, USA).
cGMP accumulation in photoreceptors of Pde6 mutant mice expressing the HR2.1:TN-XL Ca2+-biosensor. (A) Cross-sections of wt (left), cpfl1 (centre), and rd1 (right) outer retinas immunostained for cGMP (magenta) at P20 (top) and P30 (bottom). The biosensor TN-XL is shown in green. (B) Cone density (in # cells/10 μm) in rd1 animals is significantly reduced at P20, while at P30 both cpfl1 and rd1 retinas show cone loss. (C) The percentage of cGMP-positive cones is strongly increased in cpfl1 retina at P20 and P30, while wt and rd1 cones are negative for cGMP. Scale bars: a, 10 µm. P20 data obtained from 9 sections from 3 wt mice (9/3); cpfl1, 11/3; rd1, 9/3; P30: wt, 14/4; cpfl1, 11/3; rd1, 9/3); *** =P < 0.001. OPL, outer plexiform layer, ONL, outer nuclear layer.
Figure 2.

cGMP accumulation in photoreceptors of Pde6 mutant mice expressing the HR2.1:TN-XL Ca2+-biosensor. (A) Cross-sections of wt (left), cpfl1 (centre), and rd1 (right) outer retinas immunostained for cGMP (magenta) at P20 (top) and P30 (bottom). The biosensor TN-XL is shown in green. (B) Cone density (in # cells/10 μm) in rd1 animals is significantly reduced at P20, while at P30 both cpfl1 and rd1 retinas show cone loss. (C) The percentage of cGMP-positive cones is strongly increased in cpfl1 retina at P20 and P30, while wt and rd1 cones are negative for cGMP. Scale bars: a, 10 µm. P20 data obtained from 9 sections from 3 wt mice (9/3); cpfl1, 11/3; rd1, 9/3; P30: wt, 14/4; cpfl1, 11/3; rd1, 9/3); *** =P < 0.001. OPL, outer plexiform layer, ONL, outer nuclear layer.

Analysis of immunodata

The data were obtained from at least three different animals and for each animal at least three immunostained vertical sections were quantified, using mosaic images acquired at 20× magnification. For analysis, the area of the outer nuclear layer (ONL) and the length of the retinal sections were determined using the Zeiss Axiovision software. cGMP positive cells were counted in both biosensor and non-biosensor lines and were expressed as percent positive cells in the ONL (rd1 retina), or as percent positive cones (wt, rd1 and cpfl1). To estimate cone density (number of somata per 10 μm) for P20, P24 and P30 in both biosensor and non-biosensor lines, cones were labelled using glycogen phosphorylase (40) and counted in defined areas. Cones were only included if at least the inner segment (IS) and the soma could be clearly identified. To quantify dying cells, the in situ TUNEL assay was also performed on vertical sections (Supplementary Material, Fig. S2). We measured the area of the ONL, divided it by the total number of cells (obtained from DAPI staining) in that area and determined the fraction of TUNEL-positive nuclei (Supplementary Material, Fig. S3). Statistical comparisons were made using the Wilcoxon rank-sum test using IGOR Pro (Wavemetrics, Lake Oswego, USA).

Two-photon Ca2+ imaging

The preparation of retinal slices for Ca2+ imaging was performed as described previously (28,31). In brief, eyes were enucleated and dissected in carboxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF) solution, which contained (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 0.5 l-glutamine, and 20 glucose; maintained at pH 7.4 using carboxygen. Consecutive vertical slices (∼200 µm thick) were cut using a tissue chopper (41) and then transferred to the recording chamber of a two-photon microscope, where they were constantly perfused with warmed (∼37 °C) ACSF. The microscope (for details, see (42)), consisted of a customized MOM (movable objective microscope; designed by W. Denk, MPI of Neurobiology, Martinsried; purchased from Science Products/Sutter Instruments, Novato, USA), equipped with a mode-locked Ti:Sapphire laser (MaiTai-HP DeepSee; Newport Spectra-Physics, Darmstadt, Germany) tuned to 860 nm, two fluorescence detection channels (483 band-pass (BP) 32; 535 BP 50; AHF, Tübingen, Germany), and a 20x water-immersion objective (XLUMPlanFL, 0.95 NA; Olympus, Hamburg, Germany). The system was controlled by the image acquisition software ScanM (by M. Müller, MPI of Neurobiology, Martinsried, Germany, and T.E.) running under IGOR Pro 6.3, or CfNT (M. Müller, MPI, Martinsried, Germany). A custom-built dichromatic light stimulator (29), equipped with two band-pass filtered LEDs (UV filter: 360 BP 12; green: 578 BP 10) and mounted below the recording chamber, was used to present temporally-modulated, full-field light stimuli (approx. 2 mm in diameter) to the retinal slices. Ca2+ recordings were performed with a constant background illumination of 104 P*s 1 (photo-isomerization rate) for at least 15 seconds. Light stimuli consisted of a series of 1-second bright flashes with 4-second intervals. Flashes evoked similar photo-isomerization rates in both medium (M-) and short (S-) wavelength-sensitive cones (green LED: 6.7; UV LED: 6.5). At these light levels, rods are expected to be saturated and, therefore, light-evoked responses should originate in cones and not in electrically coupled rods. To record Ca2+ levels and light-evoked Ca2+ responses in cone axon terminals, we captured image series (128 x 16 pixels at 31.25 Hz), aligned with the outer plexiform layer (OPL) and covering an area of ∼75 x 10 µm on the slice (Fig. 1D). We recorded both fluorescence channels, as TN-XL allows ratiometric measurements via FRET between its fluorophores enhanced cyan fluorescent protein (eCFP, donor) and citrine (acceptor).

In a subset of experiments, absolute Ca2+ concentrations were measured using an “ex-vivo slice calibration” procedure (31). We recorded the fluorescence ratio (see Analysis of Ca2+ imaging data) first in a standard ACSF solution (Rbase, see below and Fig. 1D), then in Ca2+-free ACSF supplemented with ionomycin (5 μM) and EGTA (10 mM) to determine the minimum ratio (Rmin), and finally in ACSF supplemented with ionomycin (5 μM) and high Ca2+ (2.5 mM) to determine the maximum (saturating) ratio (Rmax). The absolute Ca2+ concentration was estimated using Rmin, Rmax, the donor fluorescence with a saturating Ca2+ level (FD, Ca-bound) and under Ca2+-free conditions (FD, Ca-free), as well as the in-vivo dissociation constant (Kd =0.77, see (43)) for TN-XL:

Analysis of Ca2+ imaging data

All Ca2+ data were analysed using custom scripts for IGOR Pro 6.3 (Wavemetrics). To extract cone Ca2+ signals, regions of interest (ROIs) were drawn manually around the cone terminals (Fig. 1D) and the pixels within ROIs were averaged for each image frame. An ROI placed outside the OPL was used to determine the baseline fluorescence in both detection channels for background subtraction. The relative Ca2+ level was then determined as the ratio (R) between acceptor and donor fluorescence (FA/FD) signals after background subtraction. For each ROI, stimulus trials were averaged and the mean trace was filtered (box-car, 320 ms filter-width).

Relative resting Ca2+ level (Rbase) was defined as the mean prior to the light flash (cf. Fig. 3A). Further, we quantified the area between the base line and light response (“response area”, RA), response amplitude (ΔR), response rise (t2080). The latter was determined by fitting a sigmoid to the response rise to determine the duration between the time points when 20% and 80% ΔR were reached. As a measure for the “slowness” of a light response, we determined a “response lag” (tlag), which we defined as the duration between t20% and tdecay, the time point when the Ca2+ level started to recover (= to increase). In addition, we quantified response noise (Rnoise), defined as s.d. of the traces (trials) after subtracting the low pass-filtered mean response (cf. Fig. 6A).
Cone resting Ca2+ levels. (A) Resting Ca2+ level (Rbase) was determined as the mean trace prior to light flash. (B) Distribution of relative cone Rbase in the three mouse lines at P18+ (left; wt, 853 cells from 3 mice (853/3); cpfl1, 944/4; rd1, 878/5) and P30+ (right; wt, 1,825/8; cpfl1, 1,048/8; rd1, 818/5; thick outline, mean distribution across mice; grey outlines, distributions from individual mice). Distributions were statistically compared using the K-S test (5% alpha level; **P < 0.01; *** P < 0.001; n.s.  0.37). (C) Box-and-whisker plot of estimated absolute cone Ca2+ concentrations at P30+ for the three lines (wt, 16/2; cpfl1, 16/4; rd1, 16/3). Data points ≤ 10 nM were considered to represent unrealistically low Ca2+ levels (possibly due to a limitation of TN-XL, as discussed in (28)) and were thus excluded from the analysis.
Figure 3.

Cone resting Ca2+ levels. (A) Resting Ca2+ level (Rbase) was determined as the mean trace prior to light flash. (B) Distribution of relative cone Rbase in the three mouse lines at P18+ (left; wt, 853 cells from 3 mice (853/3); cpfl1, 944/4; rd1, 878/5) and P30+ (right; wt, 1,825/8; cpfl1, 1,048/8; rd1, 818/5; thick outline, mean distribution across mice; grey outlines, distributions from individual mice). Distributions were statistically compared using the K-S test (5% alpha level; **P < 0.01; *** P < 0.001; n.s.  0.37). (C) Box-and-whisker plot of estimated absolute cone Ca2+ concentrations at P30+ for the three lines (wt, 16/2; cpfl1, 16/4; rd1, 16/3). Data points ≤ 10 nM were considered to represent unrealistically low Ca2+ levels (possibly due to a limitation of TN-XL, as discussed in (28)) and were thus excluded from the analysis.

Previous studies showed that during slicing, cells close to the surface might be mechanically damaged and show abnormal Ca2+ ratios (28). We tried to address this issue by recording from cones at least 20-50 µm in the slice and by excluding cones with unstable baseline and/or extreme noise level. We used the noise distribution of the wt P30+ dataset and determined from the histogram the noise level (Rnoise,max) that excluded the 5% cones with the highest noise (95th percentile). Then, from all datasets we excluded cones with Rnoise >Rnoise,max unless they displayed clear light responses (RA > 10 RA,noise, with RA,noise defined as the s.d. over the whole 5-s trace times the trial duration).

To compare responsiveness to light stimuli between the different data sets, we categorized the cones into “responsive” and “non-responsive”. To include only cones with robust light responses into the “responsive” category, we again used the wt P30+ dataset: We determined the value (RA,50%) that divided the RA histogram (cf.Fig. 4C) into halves and defined the 50% cones with the larger RA values as responsive. This RA,50% was then applied to the other datasets. Note that cones for which the response curve could not be fitted (to determine timing parameters, see above) were not classified as “responsive”, even if they fulfilled the RA >RA,50% criterion.
Light-evoked cone Ca2+ responses. (A) Determining area under the response curve (RA) and relative resting Ca2+ level (Rbase). (B) Exemplary Ca2+ signals (mean of ≥ 10 trials) in response to a light flash recorded from individual wt (black), cpfl1 (blue), and rd1 (red) cones at P30+. (C) RA as function of Rbase at P18+ (left) and P30+ (right). Each dot represents a single cone (colours as in B; P18+: wt, 853 from 3 mice (853/3); cpfl1, 944/4; rd1, 878/5; P30+: wt, 1,825/8; cpfl1, 1,048/8; rd1, 818/5). (D) Distribution of RA in “responsive” cones (for definition, see Methods); only data points between the two dashed lines in (C) were included (colours as in b). (E) Box-and-whisker plot of normalized RA at P18+ and P30+. At P18+, wt: 0.65 (median) (black, 160/3), cpfl1: 0.62 (blue, 32/4). At P30+, wt: 0.84, (730/8), cpfl1: 1.13, (99/8). RA values were statistically compared using the Wilcoxon rank-sum test (5% alpha level; ***P < 0.001; n.s. P = 0.47).
Figure 4.

Light-evoked cone Ca2+ responses. (A) Determining area under the response curve (RA) and relative resting Ca2+ level (Rbase). (B) Exemplary Ca2+ signals (mean of ≥ 10 trials) in response to a light flash recorded from individual wt (black), cpfl1 (blue), and rd1 (red) cones at P30+. (C) RA as function of Rbase at P18+ (left) and P30+ (right). Each dot represents a single cone (colours as in B; P18+: wt, 853 from 3 mice (853/3); cpfl1, 944/4; rd1, 878/5; P30+: wt, 1,825/8; cpfl1, 1,048/8; rd1, 818/5). (D) Distribution of RA in “responsive” cones (for definition, see Methods); only data points between the two dashed lines in (C) were included (colours as in b). (E) Box-and-whisker plot of normalized RA at P18+ and P30+. At P18+, wt: 0.65 (median) (black, 160/3), cpfl1: 0.62 (blue, 32/4). At P30+, wt: 0.84, (730/8), cpfl1: 1.13, (99/8). RA values were statistically compared using the Wilcoxon rank-sum test (5% alpha level; ***P < 0.001; n.s. P = 0.47).

Statistics

In total, we analysed n = 2,678 (89% of 3,007 cells) wt, n = 1,992 (81% of 2,469 cells) cpfl1, and n = 1,696 (92% of 1,845 cells) rd1 cones. To compare Ca2+ level distributions statistically, we fitted the distribution of Rbase for each line and time point with a Gaussian that was convoluted with an exponential (ExpModGauss in IGOR Pro) and used Rbase at the distribution peak to centre the histograms for comparison between data sets (Fig. 3B). Rbase distributions were compared using the Kolmogorov-Smirnov test (K-S test, IGOR Pro) for an alpha level of 5%. To compare the light response sizes statistically, we first normalized RA of the responsive cells for all the datasets to the mean RA of wt cones at P30+. To compare noise levels, we normalized Rnoise measured in the mutant lines and at other time windows to Rnoise,max (see above). The Wilcoxon rank-sum test (as implemented in IGOR Pro; 5% alpha level) was used to compare RA, tlag, and Rnoise between mouse lines and time windows. These data are shown as box-and-whisker plots (top whisker: 90th percentile; bottom whisker: 10th percentile).

Results

Normal wt cones and Ca2+ biosensor-expressing wt cones were previously found to be functionally equivalent, showing no differences in electroretinographic (ERG) recordings and opsin expression (28–30). To ascertain that the expression of the genetically encoded Ca2+ sensor TN-XL in mutant photoreceptors would not alter the degeneration phenotype, we first assessed degeneration markers in HR2.1:TN-XL crossbred mutants. For our analysis, we focussed on two time-windows, postnatal days (P) 18-20 and P30-33, to which we refer to as P18+ and P30+, respectively. In both rd1 and cpfl1 genotypes, these two periods correspond to times just beyond the onset of cone degeneration (P18+) and when roughly 50% of all cones are gone (P30+) (3,44).

Presence of biosensor does not alter Pde6 mutant phenotype

Cone morphology in the wt HR2.1:TN-XL retina (control) appeared normal, whereas HR2.1:TN-XL x cpfl1 cones were characterized by misplaced somata (Fig. 2A), as described before for non-biosensor cpfl1 cones (3). Due to primary rod loss, HR2.1:TN-XL x rd1 retina showed a marked thinning of the outer nuclear layer (ONL) (45), accompanied by dramatic changes in cone morphology (rounded somata with strongly altered neurite morphology; Fig. 2A), virtually identical to what has been described in the (non-biosensor) rd1 mouse (5). We counted cones in the three mouse lines and found cone densities to be significantly reduced over time in HR2.1:TN-XL x cpfl1 and HR2.1:TN-XL x rd1 (Fig. 2B), indicating severe cone degeneration compared to wt HR2.1:TN-XL. Cone densities were within the ranges reported for non-biosensor cpfl1 and rd1 (Table 1). In addition, we performed TUNEL assays to evaluate the number of dying cells at P20 and P30 (Supplementary Material, Fig. S2) and found the fraction of TUNEL-positive cells in HR2.1:TN-XL x cpfl1 and HR2.1:TN-XL x rd1 lines to be similar to that reported for cpfl1 and rd1 before, respectively (Table 1) (3,38).

Table 1.

Cone density and percentage of TUNEL-positive cells in the studied mouse lines

cone density[(10 µm)−1]
TUNEL positive cells[%]
P20P30P20P30
“wild-type” HR2.1:TN-XL1.28 (1.19)1.31 (1.35)0.17 (0.14)0.03 (0.03)
HR2.1:TN-XL x cpfl11.09 (1.14)0.63 (0.69)0.32 (0.38)0.17 (0.17)
HR2.1:TN-XL x rd10.81 (0.80)0.64 (0.63)2.51 (2.77)0.76 (0.84)
wt1.4 [P21] (61)1.4 (61)0.08 (15)0.04 (15)
cpfl11.12 (1.13) [P24]0.7 (, 61)0.15 (15)0.02 (15)
rd10.75 (62)0.60 (61)1.64 (15)0.55 [P28] (15)
cone density[(10 µm)−1]
TUNEL positive cells[%]
P20P30P20P30
“wild-type” HR2.1:TN-XL1.28 (1.19)1.31 (1.35)0.17 (0.14)0.03 (0.03)
HR2.1:TN-XL x cpfl11.09 (1.14)0.63 (0.69)0.32 (0.38)0.17 (0.17)
HR2.1:TN-XL x rd10.81 (0.80)0.64 (0.63)2.51 (2.77)0.76 (0.84)
wt1.4 [P21] (61)1.4 (61)0.08 (15)0.04 (15)
cpfl11.12 (1.13) [P24]0.7 (, 61)0.15 (15)0.02 (15)
rd10.75 (62)0.60 (61)1.64 (15)0.55 [P28] (15)

For calculation of cone density and % TUNEL positive cells, see Methods. To allow for a comparison with earlier literature, values shown in this table are expressed as mean, with median (where available) in parenthesis. In cases where P20/P30 values were not available for comparison, the actual postnatal age is given in square brackets. cpfl1 at P24: median 1.13 ± 0.18 SD, 14 vertical sections from 5 mice.

value given (“50% of wt”) was estimated using data given in (61). References in superscript brackets.

Table 1.

Cone density and percentage of TUNEL-positive cells in the studied mouse lines

cone density[(10 µm)−1]
TUNEL positive cells[%]
P20P30P20P30
“wild-type” HR2.1:TN-XL1.28 (1.19)1.31 (1.35)0.17 (0.14)0.03 (0.03)
HR2.1:TN-XL x cpfl11.09 (1.14)0.63 (0.69)0.32 (0.38)0.17 (0.17)
HR2.1:TN-XL x rd10.81 (0.80)0.64 (0.63)2.51 (2.77)0.76 (0.84)
wt1.4 [P21] (61)1.4 (61)0.08 (15)0.04 (15)
cpfl11.12 (1.13) [P24]0.7 (, 61)0.15 (15)0.02 (15)
rd10.75 (62)0.60 (61)1.64 (15)0.55 [P28] (15)
cone density[(10 µm)−1]
TUNEL positive cells[%]
P20P30P20P30
“wild-type” HR2.1:TN-XL1.28 (1.19)1.31 (1.35)0.17 (0.14)0.03 (0.03)
HR2.1:TN-XL x cpfl11.09 (1.14)0.63 (0.69)0.32 (0.38)0.17 (0.17)
HR2.1:TN-XL x rd10.81 (0.80)0.64 (0.63)2.51 (2.77)0.76 (0.84)
wt1.4 [P21] (61)1.4 (61)0.08 (15)0.04 (15)
cpfl11.12 (1.13) [P24]0.7 (, 61)0.15 (15)0.02 (15)
rd10.75 (62)0.60 (61)1.64 (15)0.55 [P28] (15)

For calculation of cone density and % TUNEL positive cells, see Methods. To allow for a comparison with earlier literature, values shown in this table are expressed as mean, with median (where available) in parenthesis. In cases where P20/P30 values were not available for comparison, the actual postnatal age is given in square brackets. cpfl1 at P24: median 1.13 ± 0.18 SD, 14 vertical sections from 5 mice.

value given (“50% of wt”) was estimated using data given in (61). References in superscript brackets.

Next, we assessed cGMP accumulation in the outer retina (3,15): In wild-type retina, whether expressing the TN-XL biosensor or not, essentially no cGMP accumulation could be observed in the ONL, at P20 and P30 (Fig. 2A and C) (3). In rd1 retina, the overall percentage of cGMP positive cells in the ONL at P20 was similar for biosensor and non-biosensor expressing retina (rd1: 7.44 ± 1.09% SD, n = 9 vertical sections from 3 mice; HR2.1:TN-XL x rd1: 6.91 ± 0.91% SD, P =  0.5, n = 9 vertical sections from 3 mice) and all available evidence suggests that this cGMP accumulation is restricted to rods exclusively. Since degenerating cones may lose the expression of conventional markers during the final stages of cell death, in non-biosensor rd1 retina there is a degree of uncertainty as to whether rd1 cones do show cGMP accumulation or not. However, in HR2.1:TN-XL x rd1 retina, no cGMP positive cones could be detected, indicating that rd1 cGMP accumulation was indeed restricted to rods only (Fig. 2A and C). In non-biosensor cpfl1 retina, approximately 50% of the cones were positive for cGMP (3,15), similar to what was found in HR2.1:TN-XL x cpfl1 cones (Fig. 2C).

In summary, we did not find any significant differences between the cones of the TN-XL biosensor-expressing and “normal” rd1 and cpfl1 mutants, suggesting that the mutant lines crossbred with HR2.1:TN-XL strain are suitable models to study cone function in health and disease. In addition, we showed that cGMP levels are strongly elevated in cpfl1 but not in rd1 cones. In the following, we will be addressing results obtained from the TN-XL expressing animals and, for ease of use, refer to these simply as wt (wild-type), rd1, and cpfl1.

Resting Ca2+ is very heterogeneous within cone populations and differs between mutants

To evaluate potential differences in Ca2+ homeostasis between mutant and wt lines, we recorded Ca2+ levels from TN-XL-expressing cones in acute vertical retinal slices using two-photon Ca2+ imaging (Fig. 1C and D; see Methods). We first assessed relative resting Ca2+ (Rbase; Fig. 3A) and found that at the beginning of cone degeneration, at P18+, resting Ca2+ level distributions were comparable in wt and cpfl1, whereas the distribution for rd1 was more narrow (Fig. 3B, left column). At P30+, when cone degeneration had progressed, the Ca2+ level distribution in rd1 remained narrow, whereas the distributions for both wt and cpfl1 broadened (Fig. 3B, right column). As some broadening was also observed in the wt, it may partially reflect developmental changes. Despite this, the effect was more pronounced in cpfl1. Taken together, these data suggest that the mutations in cpfl1 and rd1 have opposite effects on cone Ca2+ resting levels: As the degeneration progressed, cpfl1 cone Ca2+ levels became increasingly heterogeneous, whereas rd1 cone Ca2+ levels were (and remained) more homogenous across the population.

In a subset of experiments (n = 9 slices from 9 mice), we estimated the absolute resting Ca2+ concentrations in cones using an ex-vivo calibration approach in retinal slices (31,46). Because the difference in relative Ca2+ level distribution between wt and the two mutants was more prominent at P30+, we performed the calibration experiments at this age window. In wt cones, we determined an absolute cone Ca2+ concentration of 200 nM (median; n = 15 cones in 2 slices from 2 mice), which is in agreement with the literature (35,47). In cpfl1 cones, the Ca2+ concentration was 339 nM (n = 14 cones in 4 slices from 4 mice), with a higher variability compared to wt (Fig. 3C). While these differences were not statistically significant, the higher variability in cpfl1 Ca2+ concentration was consistent with the results from the relative Ca2+ distributions (Fig. 3B). Because of the low number of remaining cones in the rd1 retina at P30+, we could only get a rough estimate for the absolute Ca2+ level in rd1 cones (180 nM; n = 4 cones in 3 slices from 3 mice; Fig. 3C). However, since most Ca2+ values in rd1 cones were below the detection limit of the TN-XL biosensor, the actual intracellular Ca2+ levels in rd1 cones are likely to be much lower, similar to those measured under Ca2+-free conditions.

Unexpected, robust light evoked Ca2+ responses in cpfl1 cones

Previous ERG studies have reported very weak responses of cpfl1 retina to bright-field light flashes but could not pinpoint the origin of these responses (2). To test whether cpfl1 cones respond to light, we recorded cone Ca2+ signals and presented 1-s full-field light flashes on a constant background (cf. Fig. 1D). In wt cones, a light-evoked a decrease in the fluorescence ratio (FA/FD) indicates a decrease in Ca2+ level due to a light-induced hyperpolarization of the cone (Fig. 4A). To quantify Ca2+ response sizes, we determined the response area (RA). Light-evoked Ca2+ responses were detected in wt, and to our surprise, also in cpfl1 cones but not in rd1 cones (Fig. 4B). In general, response size tended to increase as resting Ca2+ levels rose.

As expected, we found the highest percentage of cones that met our conservative “responsiveness” criterion (Methods) in wt retina. The percentage of responsive wt cones increased from ∼36% at P18+ to ∼50% at P30+, (Fig. 4C and D), possibly due to postnatal maturation of mouse retina (48,49). In cpfl1 retina, we found a substantial percentage of cones that displayed light responses comparable to those measured in wt. At P18+, ∼8% of the recorded cpfl1 cones were responsive; later this fraction increased to ∼21%. Moreover, the response size dramatically increased in cpfl1 cones at P30+ (Fig. 4E). Remarkably, at this stage of cpfl1 degeneration, about half of the cones were already lost. In comparison, in the secondary cone degeneration model rd1, where cones are genetically intact, less than 1% of the recorded cones were responsive.

Next, we wanted to see if the light-evoked Ca2+ response recorded in cpfl1 cones differed from those in wt cones. Here, we focused on response kinetics, as responses appeared “slower” in cpfl1 compared to wt (cf. Fig. 4B). First, we determined the “response lag” (Δtlag) defined as the time period between the time when 20% of the response amplitude was reached (t20%) and the time when the Ca2+ level started to recover for a 1-s stimulus (tdecay; Fig. 5A). We found that Δtlag was significantly longer in cpfl1 than in wt cones at both ages (Fig. 5B). Moreover, Δtlag in cpfl1 cone responses increased significantly between P18+ and P30+. Taken together, our data suggest that even at P30+, a substantial fraction of cpfl1 cones generate large, although sluggish light-evoked responses.
Kinetics of light-evoked Ca2+ responses in wt and cpfl1 cones. (A) Exemplary light-evoked Ca2+ responses (means of ≥ 10 trials; smoothed using box-car, 320 ms filter-width) at P18+ and P30+ from wt (black) and cpfl1 (blue). Response lag time (Δtlag) was determined as the time between 20% of response rise (t20%) and beginning of response recovery (tdecay). (B) Box-and-whisker plot for Δtlag for 1-s light flashes. At P18+, wt: 0.91 (median), (160 cones from 3 mice (160/3)); cpfl1: 0.99, (32/4). At P30+, wt: 0.93, (730/8), cpfl1: 1.03, (99/8). Δtlag were statistically compared using the Wilcoxon rank-sum test (5% alpha level; *** P < 0.001; n.s. P = 0.051).
Figure 5.

Kinetics of light-evoked Ca2+ responses in wt and cpfl1 cones. (A) Exemplary light-evoked Ca2+ responses (means of ≥ 10 trials; smoothed using box-car, 320 ms filter-width) at P18+ and P30+ from wt (black) and cpfl1 (blue). Response lag time (Δtlag) was determined as the time between 20% of response rise (t20%) and beginning of response recovery (tdecay). (B) Box-and-whisker plot for Δtlag for 1-s light flashes. At P18+, wt: 0.91 (median), (160 cones from 3 mice (160/3)); cpfl1: 0.99, (32/4). At P30+, wt: 0.93, (730/8), cpfl1: 1.03, (99/8). Δtlag were statistically compared using the Wilcoxon rank-sum test (5% alpha level; *** P < 0.001; n.s. P = 0.051).

Cpfl1 cones are noisier than wt and rd1 cones

We noticed that Ca2+ traces recorded in cpfl1 cones appeared noisier compared to those in wt cones (Fig. 6A and B). This noise may reflect higher (spontaneous) activity caused by fluctuations in cGMP levels, possibly related to the cGMP accumulation we detected in cpfl1 cones (50) (cf. Fig. 2A). To include rd1 cones in this comparison, we analysed light-responsive and non-responsive cones separately (Fig. 6C and D). At P18+, noise levels were not significantly different between responsive cpfl1 and wt cones, whereas non-responsive cpfl1 cones were noisier than their wt counterparts. At P30+, both responsive and non-responsive wt cones became noisier, but much less so than cpfl1. The dramatic increase in Ca2+ signal noise recorded in cpfl1 cones may indicate that cGMP fluctuations progressively increased in responsive cones. In contrast, rd1 cones showed significantly lower noise than wt in both time windows. The small increase in rd1 cone noise at P30+ may relate to the remodelling of the outer retina connectivity (5). In fact, this notion is supported by a study showing that the oscillatory activity that arises in the outer rd1 retina (P30 and later) at least partially depends on voltage-gated Ca2+ channel activity in cone terminals (45).
Ca2+ noise levels in cones. (A) Exemplary of Ca2+ response to a 1-s light flash (top, black trace). Noise was determined from traces with the low-pass filtered response (top, green trace; box-car, 320 ms filter-width) subtracted (bottom). (B) Exemplary noise traces at P18+ (top) and P30+ (bottom) from wt (black) and cpfl1 (blue) cones. (C) Box-and-whisker plot of normalized noise (Rnoise) for responsive wt and cpfl1 cones at P18+ and P30+. At P18+, wt: 0.31 (median) (160 cones from three mice, 160/3), cpfl1: 0.36 (32/4). At P30+, wt: 0.47 (730/8), cpfl1: 0.59 (99/8). (D) Box-and-whisker plot of normalized Rnoise for non-responsive wt, cpfl1, and rd1 cones at P18+ and P30+. At P18+, wt: 0.33 (597/3), cpfl1: 0.40 (870/4), rd1: 0.26 (877/5). At P30+, wt: 0.41 (894/8), cpfl1: 0.51 (841/8), rd1: 0.29 (816/5). Rnoise values were statistically compared using the Wilcoxon rank-sum test (5% alpha level; ***P ≤ 0.001; n.s. P = 0.10).
Figure 6.

Ca2+ noise levels in cones. (A) Exemplary of Ca2+ response to a 1-s light flash (top, black trace). Noise was determined from traces with the low-pass filtered response (top, green trace; box-car, 320 ms filter-width) subtracted (bottom). (B) Exemplary noise traces at P18+ (top) and P30+ (bottom) from wt (black) and cpfl1 (blue) cones. (C) Box-and-whisker plot of normalized noise (Rnoise) for responsive wt and cpfl1 cones at P18+ and P30+. At P18+, wt: 0.31 (median) (160 cones from three mice, 160/3), cpfl1: 0.36 (32/4). At P30+, wt: 0.47 (730/8), cpfl1: 0.59 (99/8). (D) Box-and-whisker plot of normalized Rnoise for non-responsive wt, cpfl1, and rd1 cones at P18+ and P30+. At P18+, wt: 0.33 (597/3), cpfl1: 0.40 (870/4), rd1: 0.26 (877/5). At P30+, wt: 0.41 (894/8), cpfl1: 0.51 (841/8), rd1: 0.29 (816/5). Rnoise values were statistically compared using the Wilcoxon rank-sum test (5% alpha level; ***P ≤ 0.001; n.s. P = 0.10).

Discussion

In many inherited retinal diseases, mutations that affect the photoreceptor OS lead to cell death, and the pathways associated with this are often linked to aberrant Ca2+ signalling. Here, we monitored Ca2+ dynamics directly during both primary and secondary cone degeneration, to show that Ca2+ signalling was indeed altered. Remarkably, although cpfl1 cones suffer from primary Pde6c mutations, a subpopulation was found to be functional, while genetically intact rd1 cones were entirely dysfunctional. Our results raise new questions as to the possible involvement of Ca2+ noise and random fluctuations, which could explain the stochastic nature of cone cell death (51–53). In addition, our study highlights the importance of using in vivo single-cell imaging techniques to improve our understanding of the intracellular processes leading to retinal degeneration.

Cone Ca2+ dynamics are altered in primary cone degeneration

Cpfl1 cones carry Pde6c mutations (1), exhibit cGMP accumulation, and are expected to show altered Ca2+ homeostasis (3,16). In line with previous studies, we found that the percentage of cones showing cGMP accumulation remained constant between P20 and P30. High cGMP is predicted to cause changes in Ca2+ homeostasis, as evidenced indirectly by the increased activity of Ca2+-dependent calpain-type proteases (3,15). Our study confirms this prediction: Compared to wt, we found that cpfl1 cones showed higher relative resting Ca2+ levels. In line with this, we found in our calibration experiments that cpfl1 cones tended to have elevated absolute Ca2+ concentrations. In addition, cpfl1 cones had larger light responses, their resting Ca2+ was much more heterogeneous, and they displayed increased Ca2+ noise levels. Overall, our results on primary cpfl1 cone degeneration support the high Ca2+ hypothesis.

These results are interesting in the context of previous studies aimed at developing treatments for hereditary retinal degeneration. The use of inhibitors of the synaptic L-type VGCCs induced a delay in rd1 photoreceptor degeneration (21), which implied that photoreceptor Ca2+ overload (16) would occur primarily via the synaptic axon terminal. We show that Ca2+ levels in the axon terminals of PDE6 mutant cones may indeed be increased; however, these changes are tightly linked to primary alterations of Ca2+ in the cone OS, as evidenced by our light stimulation experiments. Thus, when therapeutic strategies aimed at blocking either Ca2+-permeable channels in the synapse (i.e. VGCCs) or in the OS (i.e. CNG-gated channels) (38,54) are compared, the latter appear to be a more suitable target.

Cone Ca2+ dynamics in secondary cone degeneration

In contrast to cpfl1 cones, rd1 cones are genetically intact and did not show cGMP accumulation. Yet, major cone loss was evident already at P20, progressing rapidly at P30. In rd1, cone OS and overall morphology changed dramatically in the absence of supporting rods as the photoreceptor layer thinned out. In addition, virtually no rd1 cone responded to light, in line with previous ERG studies (23,38,54–56).

In this scenario, a modulation of cone terminal Ca2+ via the OS seems questionable. The resting Ca2+ distribution in the rd1 cone population was significantly different at both P18+ and P30+ compared to wt, and rd1 cone noise levels were significantly lower at both time points. When Ca2+ was washed out of rd1 cones using ionomycin, the resting Ca2+ levels hardly changed at all. These results altogether point at a very low resting Ca2+ in cones upon loss of rods and structural disruption of the outer retina, indicating that rd1 secondary cone cell death may be caused by exceedingly low Ca2+ levels ((57); discussed in (11,27)). This conclusion would have important implications for therapeutic developments, which often aim at lowering intracellular Ca2+. While such an approach may be viable for primary cone degeneration (see above) it could be detrimental in secondary cone degeneration. To better understand Ca2+ dynamics in secondary cone degeneration, it may be necessary to study further animal models that exhibit a slower cone loss, not overlapping with postnatal retinal development, such as the Pde6b-mutant rd10 mouse (4,15).

Cpfl1 cones show light evoked responses

To our surprise, a substantial number of cpfl1 cones responded to light stimulation. These Ca2+ responses were larger and slower compared with wt cones, possibly due to low remnant PDE activity. Interestingly, cones treated with the PDE6 inhibitor zaprinast exhibited similar changes in response time course (28). Zaprinast application also caused a (reversible) increase in resting Ca2+, consistent with our finding of higher absolute Ca2+ in cpfl1 cones. The cpfl1 allele carries two different mutations, one 116bp intronic insertion between exons 4 and 5 resulting in a splice defect, and one 1bp deletion resulting in a frameshift in exon 7 (1). These two mutations together are expected to abolish PDE6C function completely. Nevertheless, a small proportion of correctly spliced Pde6c transcript carrying only the 1bp deletion appears to be present in cpfl1 cones (1), suggesting that they express lower amounts of PDE6C protein. Due to the severity of the genetic damage, it is unlikely, however, that this protein is functional. On the other hand, cpfl1 mice do show weak responses in photopic ERG recordings (2). Here, a possible explanation is that cpfl1 cones recruit low levels of rod Pde6a, which would be expected to slow-down cone response kinetics (58), similar to what we observed. Irrespective of the underlying mechanism, our results show that cpfl1 mice do have a residual cone function at least until one month postnatal.

Degenerating cones show altered noise levels

In wt cones, Ca2+ noise levels became significantly higher from P18+ to P30+, suggesting changes in PDE/cGMP/Ca2+ signalling with time, as the development and maturation of cones and retinal circuitry progresses (48). Previous studies have found that cone noise depends on variations in CNG channel activity and fluctuations in cGMP levels in the OS (50,59). Moreover, cone noise persists in the downstream circuitry (60).

Our data show altered Ca2+ noise levels in both primary and secondary cone degeneration. At P30+, in both responsive and non-responsive cpfl1 cones, Ca2+ noise was significantly higher than in their wt counterparts, possibly due to the higher overall Ca2+ levels in cpfl1 cones. It is tempting to speculate that such variations in Ca2+ noise (or Ca2+ fluctuations above or below a critical threshold) over time could contribute to the stochastic nature of photoreceptor cell death. Although at the tissue level, photoreceptor degeneration appears as a well-timed process, the time point when cell death is initiated at the level of the individual photoreceptor is random; it is inherently impossible to predict when a specific photoreceptor is going to die (51–53).

The precise processes that may be triggered by Ca2+  fluctuations remain unknown, but could potentially be linked to activation of Ca2+-dependent kinases (61,62) with bearings on gene expression (63) and/or Ca2+-dependent calpain-type proteases (3,19). On the other hand, a recent in vivo Ca2+ imaging study followed degenerating cones over a time-course of several hours in intact zebrafish larvae carrying the cpfl1-like Pde6cw59 mutation and reported suppressed Ca2+ fluctuations when compared to wt (26). At present, it is unclear where these differences between mutant cones in zebrafish and mouse arise, but they could be related to developmental stage, experimental conditions, and perhaps species differences. Ca2+ fluctuations may have either beneficial or detrimental consequences. In rd1 cones, on the other hand, Ca2+ noise was significantly lower than in wt, most likely because of the lower Ca2+ baseline levels. An exciting topic for future studies will be to understand if and how increased Ca2+ noise or overall changes in Ca2+ signalling contribute to these degenerative events.

Concluding remarks and future aspects

Several investigations were based on the assumption that altered Ca2+ signalling is involved in or serves as a trigger for rod and cone degeneration (e.g. (21,54,64)). Our results indeed show alterations in the dynamics of Ca2+ signalling in both primary and secondary cone degeneration. For primary cone degeneration, our data is in line with the “high Ca2+ hypothesis”, suggesting that cell death may be triggered by Ca2+ overload, possibly via higher random fluctuations in Ca2+ levels. This may have a bearing on the stochastics of cone degeneration (as discussed above) but still leaves open the question whether changes in Ca2+ and/or Ca2+-dependent enzymes are cause or consequence of primary cone degeneration. Secondary cone degeneration, on the other hand, is very likely associated with low Ca2+ levels and is, thus, more consistent with the “low Ca2+ hypothesis”. This may also be true for primary photoreceptor degeneration triggered by mutations that cause low Ca2+ (e.g. CNG channel mutations). Taken together, these findings may have important ramifications for therapeutic approaches focusing on blocking Ca2+ influx in hereditary retinal degeneration. Here, our study may help to reconcile both the high and low Ca2+ hypotheses (16,27) and contribute to resolving a long-lasting controversy on the use of Ca2+ channel antagonists for the treatment of inherited retinal degeneration (21–23).

To gain insight into the precise timing and the role of Ca2+ signalling in the execution of cell death, Ca2+ imaging experiments will need to be combined with cellular activity assays for Ca2+ targets such as kinases, proteases, or histone deacetylases (65,66). On a different note, our study also showed that even genetically impaired cones may show considerable functional activity, highlighting the importance of Ca2+ measurements at single-cell resolution to obtain direct readouts on the functionality and health of cones.

Supplementary Material

Supplementary Material is available at HMG online.

Acknowledgements

We thank Gordon Eske, Norman Rieger, and Klaudija Masarini for technical assistance; Simone De Giovanni, Christian Behrens, and Philipp Berens for helpful discussions; Ayse Sahaboglu-Tekgoez, and Robin Kemmler for technical advice and discussions.

Conflict of Interest statement. None declared.

Funding

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) (Werner Reichardt Centre for Integrative Neuroscience Tübingen, EXC 307 to TE; EU 42/8-1 to TE; KFO 134 and PA 1751/7-1 to FPD; TR 1238/4-1 to DT), fortüne to TE, Neuro-Ophthalmologische Gesellschaft to TE, and the European Union (DRUGSFORD; HEALTH-F2-2012-304963 to FPD).

References

1

Chang
B.
Grau
T.
Dangel
S.
Hurd
R.
Jurklies
B.
Sener
E.C.
Andreasson
S.
Dollfus
H.
Baumann
B.
Bolz
S.
, et al. . (
2009
)
A homologous genetic basis of the murine cpfl1 mutant and human achromatopsia linked to mutations in the PDE6C gene
.
Proc. Natl. Acad. Sci. U. S. A
,
106
,
19581
19586.,

2

Schaeferhoff
K.
Michalakis
S.
Tanimoto
N.
Fischer
M.D.
Becirovic
E.
Beck
S.C.
Huber
G.
Rieger
N.
Riess
O.
Wissinger
B.
, et al. . (
2010
)
Induction of STAT3-related genes in fast degenerating cone photoreceptors of cpfl1 mice
.
Cell. Mol. Life Sci
.,
67
,
3173
3186
.

3

Trifunović
D.
Dengler
K.
Michalakis
S.
Zrenner
E.
Wissinger
B.
Paquet-Durand
F.
(
2010
)
cGMP-dependent cone photoreceptor degeneration in the cpfl1 mouse retina
.
J. Comp. Neurol
.,
518
,
3604
3617
.

4

Gargini
C.
Terzibasi
E.
Mazzoni
F.
Strettoi
E.
(
2007
)
Retinal organization in the retinal degeneration 10 (rd10) mutant mouse: a morphological and ERG study
.
J. Comp. Neurol
.,
500
,
222
238
.

5

Lin
B.
Masland
R.H.
Strettoi
E.
(
2009
)
Remodeling of cone photoreceptor cells after rod degeneration in rd mice
.
Exp. Eye Res
.,
88
,
589
599
.

6

Bowes
C.
Li
T.
Danciger
M.
Baxter
L.C.
Applebury
M.L.
Farber
D.B.
(
1990
)
Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMP-phosphodiesterase
.
Nature
,
347
,
677
680
.

7

Mercer
A.J.
Thoreson
W.B.
(
2011
)
The dynamic architecture of photoreceptor ribbon synapses: cytoskeletal, extracellular matrix, and intramembrane proteins
.
Vis. Neurosci
.,
28
,
453
471
.

8

Koch
K.W.
Dell’Orco
D.
(
2013
)
A calcium-relay mechanism in vertebrate phototransduction
.
ACS Chem. Neurosci
.,
4
,
909
917
.

9

Schmitz
F.
(
2014
)
Presynaptic [Ca(2+)] and GCAPs: aspects on the structure and function of photoreceptor ribbon synapses
.
Front. Mol. Neurosci
.,
7
,
3.

10

Koutalos
Y.
Yau
K.W.
(
1996
)
Regulation of sensitivity in vertebrate rod photoreceptors by calcium
.
Trends Neurosci
.,
19
,
73
81
.

11

Burns
M.E.
Arshavsky
V.Y.
(
2005
)
Beyond counting photons: trials and trends in vertebrate visual transduction
.
Neuron
,
48
,
387
401
.

12

Burns
M.E.
Pugh
E.N.
(
2010
)
Lessons from photoreceptors: turning off g-protein signaling in living cells
.
Physiology (Bethesda)
.,
25
,
72
84
.

13

Farber
D.B.
Lolley
R.N.
(
1974
)
Cyclic guanosine monophosphate: elevation in degenerating photoreceptor cells of the C3H mouse retina
.
Science
.,
186
,
449
451
.

14

Paquet-Durand
F.
Hauck
S.M.
van Veen
T.
Ueffing
M.
Ekström
P.
(
2009
)
PKG activity causes photoreceptor cell death in two retinitis pigmentosa models
.
J. Neurochem
.,
108
,
796
810
.

15

Arango-Gonzalez
B.
Trifunović
D.
Sahaboglu
A.
Kranz
K.
Michalakis
S.
Farinelli
P.
Koch
S.
Koch
F.
Cottet
S.
Janssen-Bienhold
U.
, et al. . (
2014
)
Identification of a common non-apoptotic cell death mechanism in hereditary retinal degeneration
.
PLoS One
,
9
,
e112142.

16

Fox
D.A.
Poblenz
A.T.
He
L.
(
1999
)
Calcium overload triggers rod photoreceptor apoptotic cell death in chemical-induced and inherited retinal degenerations
.
Ann. N. Y. Acad. Sci
.,
893
,
282
285
.

17

Olshevskaya
E.V.
Calvert
P.D.
Woodruff
M.L.
Peshenko
I.V.
Savchenko
A.B.
Makino
C.L.
Ho
Y.S.
Fain
G.L.
Dizhoor
A.M.
(
2004
)
The Y99C mutation in guanylyl cyclase-activating protein 1 increases intracellular Ca2+ and causes photoreceptor degeneration in transgenic mice
.
J. Neurosci
.,
24
,
6078
6085
.

18

Doonan
F.
Donovan
M.
Cotter
T.G.
(
2005
)
Activation of multiple pathways during photoreceptor apoptosis in the rd mouse
.
Invest. Ophthalmol. Vis. Sci
.,
46
,
3530
3538
.

19

Paquet-Durand
F.
Azadi
S.
Hauck
S.M.
Ueffing
M.
van Veen
T.
Ekström
P.
(
2006
)
Calpain is activated in degenerating photoreceptors in the rd1 mouse
.
J. Neurochem
.,
96
,
802
814
.

20

Sanges
D.
Comitato
A.
Tammaro
R.
Marigo
V.
(
2006
)
Apoptosis in retinal degeneration involves cross-talk between apoptosis-inducing factor (AIF) and caspase-12 and is blocked by calpain inhibitors
.
Proc. Natl. Acad. Sci
.,
103
,
17366
17371
.

21

Frasson
M.
Sahel
J.A.
Fabre
M.
Simonutti
M.
Dreyfus
H.
Picaud
S.
(
1999
)
Retinitis pigmentosa: rod photoreceptor rescue by a calcium-channel blocker in the rd mouse
.
Nat. Med
.,
5
,
1183
1187
.

22

Bush
R.A.
Kononen
L.
Machida
S.
Sieving
P.A.
(
2000
)
The effect of calcium channel blocker diltiazem on photoreceptor degeneration in the rhodopsin Pro213His rat
.
Invest. Ophthalmol. Vis. Sci
.,
41
,
2697
2701
.

23

Pawlyk
B.S.
Li
T.
Scimeca
M.S.
Sandberg
M.A.
Berson
E.L.
(
2002
)
Absence of photoreceptor rescue with D-cis-diltiazem in the rd mouse
.
Invest. Ophthalmol. Vis. Sci
.,
43
,
1912
1915
.

24

Barabas
P.
Cutler Peck
C.
Krizaj
D.
(
2010
)
Do calcium channel blockers rescue dying photoreceptors in the Pde6b (rd1) mouse?
Adv. Exp. Med. Biol
.,
664
,
491
499
.

25

Xu
J.
Morris
L.
Thapa
A.
Ma
H.
Michalakis
S.
Biel
M.
Baehr
W.
Peshenko
I.V.
Dizhoor
A.M.
Ding
X.Q.
(
2013
)
cGMP accumulation causes photoreceptor degeneration in CNG channel deficiency: evidence of cGMP cytotoxicity independently of enhanced CNG channel function
.
J. Neurosci
.,
33
,
14939
14948
.

26

Ma
E.Y.
Lewis
A.
Barabas
P.
Stearns
G.
Suzuki
S.
Krizaj
D.
Brockerhoff
S.E.
(
2013
)
Loss of Pde6 reduces cell body Ca(2+) transients within photoreceptors
.
Cell Death Dis
.,
4
,
e797.

27

Fain
G.L.
Lisman
J.E.
(
1999
)
Light, Ca2+, and photoreceptor death: new evidence for the equivalent-light hypothesis from arrestin knockout mice
.
Invest. Ophthalmol. Vis. Sci
.,
40
,
2770
2772
.

28

Wei
T.
Schubert
T.
Paquet-Durand
F.
Tanimoto
N.
Chang
L.L.
Koeppen
K.
Ott
T.
Griesbeck
O.
Seeliger
M.W.
Euler
T.
, et al. . (
2012
)
Light-driven calcium signals in mouse cone photoreceptors
.
J. Neurosci
.,
32
,
6981
6994
.

29

Baden
T.
Schubert
T.
Chang
L.
Wei
T.
Zaichuk
M.
Wissinger
B.
Euler
T.
(
2013
)
A Tale of Two Retinal Domains: Near-Optimal Sampling of Achromatic Contrasts in Natural Scenes through Asymmetric Photoreceptor Distribution
.
Neuron
,
80
,
1206
1217
.

30

Kemmler
R.
Schultz
K.
Dedek
K.
Euler
T.
Schubert
T.
(
2014
)
Differential regulation of cone calcium signals by different horizontal cell feedback mechanisms in the mouse retina
.
J. Neurosci
.,
34
,
11826
11843
.

31

Kulkarni
M.
Schubert
T.
Baden
T.
Wissinger
B.
Euler
T.
Paquet-Durand
F.
(
2015
)
Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina
.
J. Vis. Exp
., 10.3791/52588.

32

Heidelberger
R.
Heinemann
C.
Neher
E.
Matthews
G.
(
1994
)
Calcium dependence of the rate of exocytosis in a synaptic terminal
.
Nature
,
371
,
513
515
.

33

Rieke
F.
Schwartz
E.A.
(
1994
)
A cGMP-gated current can control exocytosis at cone synapses
.
Neuron
,
13
,
863
873
.

34

Choi
S.Y.
Borghuis
B.G.
Borghuis
B.
Rea
R.
Levitan
E.S.
Sterling
P.
Kramer
R.H.
(
2005
)
Encoding light intensity by the cone photoreceptor synapse
.
Neuron
,
48
,
555
562
.

35

Choi
S.Y.
Jackman
S.
Thoreson
W.B.
Kramer
R.H.
(
2008
)
Light regulation of Ca2+ in the cone photoreceptor synaptic terminal
.
Vis. Neurosci
.,
25
,
693
700
.

36

Mank
M.
Reiff
D.F.
Heim
N.
Friedrich
M.W.
Borst
A.
Griesbeck
O.
(
2006
)
A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change
.
Biophys. J
.,
90
,
1790
1796
.

37

Schubert
T.
Gleiser
C.
Heiduschka
P.
Franz
C.
Nagel-Wolfrum
K.
Sahaboglu
A.
Weisschuh
N.
Eske
G.
Rohbock
K.
Rieger
N.
, et al. . (
2015
)
Deletion of myosin VI causes slow retinal optic neuropathy and age-related macular degeneration (AMD)-relevant retinal phenotype
.
Cell. Mol. Life Sci
.,
72
,
3953
3969.,

38

Paquet-Durand
F.
Beck
S.
Michalakis
S.
Goldmann
T.
Huber
G.
Mühlfriedel
R.
Trifunović
D.
Fischer
M.D.
Fahl
E.
Duetsch
G.
, et al. . (
2011
)
A key role for cyclic nucleotide gated (CNG) channels in cGMP-related retinitis pigmentosa
.
Hum. Mol. Genet
.,
20
,
941
947
.

39

Chang
B.
(
2015
)
Survey of the nob5 mutation in C3H substrains
.
Mol. Vis
.,
21
,
1101
1105
.

40

Pfeiffer-Guglielmi
B.
Fleckenstein
B.
Jung
G.
Hamprecht
B.
(
2003
)
Immunocytochemical localization of glycogen phosphorylase isozymes in rat nervous tissues by using isozyme-specific antibodies
.
J. Neurochem
.,
85
,
73
81
.

41

Werblin
F.S.
(
1978
)
Transmission along and between rods in the Tiger Salamander Retina
.
J. Physiol
.,
280
,
449
470
.

42

Euler
T.
Hausselt
S.E.
Margolis
D.J.
Breuninger
T.
Castell
X.
Detwiler
P.B.
Denk
W.
(
2009
)
Eyecup scope–optical recordings of light stimulus-evoked fluorescence signals in the retina
.
PflüGers Arch. Eur. J. Physiol
.,
457
,
1393
1414
.

43

Hendel
T.
Mank
M.
Schnell
B.
Griesbeck
O.
Borst
A.
Reiff
D.F.
(
2008
)
Fluorescence changes of genetic calcium indicators and OGB-1 correlated with neural activity and calcium in vivo and in vitro
.
J. Neurosci
.,
28
,
7399
7411
.

44

Carter-Dawson
L.D.
LaVail
M.M.
Sidman
R.L.
(
1978
)
Differential effect of the rd mutation on rods and cones in the mouse retina
.
Investig. Ophthalmol. Vis. Sci
.,
17
,
489
498
.

45

Haq
W.
Arango-Gonzalez
B.
Zrenner
E.
Euler
T.
Schubert
T.
(
2014
)
Synaptic remodeling generates synchronous oscillations in the degenerated outer mouse retina
.
Front. Neural Circuits
,
8
,
108.

46

Sheng
Z.
Choi
S.Y.
Dharia
A.
Li
J.
Sterling
P.
Kramer
R.H.
(
2007
)
Synaptic Ca2+ in darkness is lower in rods than cones, causing slower tonic release of vesicles
.
J. Neurosci
.,
27
,
5033
5042
.

47

Johnson
J.E.
Perkins
G.A.
Giddabasappa
A.
Chaney
S.
Xiao
W.
White
A.D.
Brown
J.M.
Waggoner
J.
Ellisman
M.H.
Fox
D.A.
(
2007
)
Spatiotemporal regulation of ATP and Ca2+ dynamics in vertebrate rod and cone ribbon synapses
.
Mol. Vis
.,
13
,
887
919
.

48

Szél
A.
Röhlich
P.
Mieziewska
K.
Aguirre
G.
van Veen
T.
(
1993
)
Spatial and temporal differences between the expression of short- and middle-wave sensitive cone pigments in the mouse retina: a developmental study
.
J. Comp. Neurol
.,
331
,
564
577
.

49

Gibson
R.
Fletcher
E.L.
Vingrys
A.J.
Zhu
Y.
Vessey
K.A.
Kalloniatis
M.
(
2013
)
Functional and neurochemical development in the normal and degenerating mouse retina
.
J. Comp. Neurol
.,
521
,
1251
1267
.

50

Angueyra
J.M.
Rieke
F.
(
2013
)
Origin and effect of phototransduction noise in primate cone photoreceptors
.
Nat. Neurosci
.,
16
,
1692
1700
.

51

Clarke
G.
Collins
R.A.
Leavitt
B.R.
Andrews
D.F.
Hayden
M.R.
Lumsden
C.J.
McInnes
R.R.
(
2000
)
A one-hit model of cell death in inherited neuronal degenerations
.
Nature
,
406
,
195
199
.

52

Clarke
G.
Lumsden
C.J.
McInnes
R.R.
(
2001
)
Inherited neurodegenerative diseases: the one-hit model of neurodegeneration
.
Hum. Mol. Genet
.,
10
,
2269
2275
.

53

Sahaboglu
A.
Paquet-Durand
O.
Dietter
J.
Dengler
K.
Bernhard-Kurz
S.
Ekström
P.A.
Hitzmann
B.
Ueffing
M.
Paquet-Durand
F.
(
2013
)
Retinitis pigmentosa: rapid neurodegeneration is governed by slow cell death mechanisms
.
Cell Death Dis
.,
4
,
e488.

54

Read
D.S.
McCall
M.A.
Gregg
R.G.
(
2002
)
Absence of voltage-dependent calcium channels delays photoreceptor degeneration in rd mice
.
Exp. Eye Res
.,
75
,
415
420
.,

55

Bonaventure
N.
Wioland
N.
Karli
P.
(
1985
)
Enhanced sensory convergence to the visual cortex in the rodless (rd/rd) mouse
.
Doc. Ophthalmol
.,
61
,
97
103
.

56

Strettoi
E.
Pignatelli
V.
Rossi
C.
Porciatti
V.
Falsini
B.
(
2003
)
Remodeling of second-order neurons in the retina of rd/rd mutant mice
.
Vision Res
.,
43
,
867
877
.

57

Woodruff
M.L.
Wang
Z.
Chung
H.Y.
Redmond
T.M.
Fain
G.L.
Lem
J.
(
2003
)
Spontaneous activity of opsin apoprotein is a cause of Leber congenital amaurosis
.
Nat. Genet
.,
35
,
158
164
.

58

Kolandaivelu
S.
Chang
B.
Ramamurthy
V.
(
2011
)
Rod phosphodiesterase-6 (PDE6) catalytic subunits restore cone function in a mouse model lacking cone PDE6 catalytic subunit
.
J. Biol. Chem
.,
286
,
33252
33259
.

59

Holcman
D.
Korenbrot
J.I.
(
2005
)
The Limit of Photoreceptor Sensitivity: Molecular Mechanisms of Dark Noise in Retinal Cones
.
J. Gen. Physiol
.,
125
,
641
660
.

60

Ala-Laurila
P.
Greschner
M.
Chichilnisky
E.J.
Rieke
F.
(
2011
)
Cone photoreceptor contributions to noise and correlations in the retinal output
.
Nat. Neurosci
.,
14
,
1309
1316
.

61

Hauck
S.M.
Ekström
P.A.
Ahuja-Jensen
P.
Suppmann
S.
Paquet-Durand
F.
van Veen
T.
Ueffing
M.
(
2006
)
Differential modification of phosducin protein in degenerating rd1 retina is associated with constitutively active Ca2+/calmodulin kinase II in rod outer segments
.
Mol. Cell. Proteomics
,
5
,
324
336
.

62

Azadi
S.
Paquet-Durand
F.
Medstrand
P.
van Veen
T.
Ekström
P.A.
(
2006
)
Up-regulation and increased phosphorylation of protein kinase C (PKC) delta, mu and theta in the degenerating rd1 mouse retina
.
Mol. Cell. Neurosci
.,
31
,
759
773
.

63

Azadi
S.
Johnson
L.E.
Paquet-Durand
F.
Perez
M.T.R.
Zhang
Y.
Ekström
P.A.
van Veen
T.
(
2007
)
CNTF+BDNF treatment and neuroprotective pathways in the rd1 mouse retina
.
Brain Res
.,
1129
,
116
129
.

64

Takano
Y.
Ohguro
H.
Dezawa
M.
Ishikawa
H.
Yamazaki
H.
Ohguro
I.
Mamiya
K.
Metoki
T.
Ishikawa
F.
Nakazawa
M.
(
2004
)
Study of drug effects of calcium channel blockers on retinal degeneration of rd mouse
.
Biochem. Biophys. Res. Commun
.,
313
,
1015
1022
.

65

Qiu
Z.
Ghosh
A.
(
2008
)
A calcium-dependent switch in a CREST-BRG1 complex regulates activity-dependent gene expression
.
Neuron
,
60
,
775
787
.

66

Tian
X.
Gala
U.
Zhang
Y.
Shang
W.
Nagarkar Jaiswal
S.
di Ronza
A.
Jaiswal
M.
Yamamoto
S.
Sandoval
H.
Duraine
L.
, et al. . (
2015
)
A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis
.
PLoS Biol
.,
13
,
e1002103.

Supplementary data