68.2.3 In Vivo Imaging

Confocal scanning laser ophthalmoscopy (cSLO) was performed as previously described (Seeliger et al. 2005). Briefly, cSLO imaging was performed using the HRA I (Heidelberg Engineering, Heidelberg, Germany) featuring two argon wavelengths (488 and 514 nm) in the short wavelength range and two infrared diode lasers (795 and 830 nm) in the long wavelength range. The laser wavelength of 795 nm was used for indocyanine-green (ICG) angiography with a barrier filter at 800 nm. The 488 nm wavelength was used for fundus autofluorescence analysis and detection of GFP expression using a barrier filter at 500 nm. Mouse eyes were subjected to spectral domain OCT (SD-OCT) using the SpectralisTM device (Heidelberg Engineering) featuring a broadband superluminescent diode at λ = 880 nm as low coherent light source. Each high resolution two-dimensional B-Scan recorded at 30◦ field of view consists of 1536 A-Scans, which are acquired at a speed of 40,000 scans per second. Optical depth resolution is ca. 7 μm with digital resolution reaching 3.5 μm (Wolf-Schnurrbusch et al. 2008). Resulting data were exported as 8 bit color image files and processed in Adobe Photoshop CS2 (Adobe Systems, San Jose, CA).

68.3 Results

68.3.1 Function

To investigate functional properties in cpfl1 mice, flash ERGs were recorded from 4-week-old wild type and cpfl1 mice under dark-adapted (Fig. 68.1a) and lightadapted (Fig. 68.1b and e) conditions. The ERG results were generally matching those in Cnga3–/– mice (deficient of the cone cyclic nucleotide-gated channel α subunit, Biel et al. 1999), suggesting a strongly reduced cone system but regular rod system function. While dark-adapted ERG responses up to approximately –2 log cd s/m2 intensity are evoked exclusively by the rod system (Tanimoto et al. 2009), the cone system begins to respond to stimuli brighter than –2 log cd s/m2 (Jaissle et al. 2001), and contributes to the trailing part of the scotopic b-wave (Biel et al. 1999). Consequently, the scotopic a-wave and the leading part of the scotopic b-wave do not change significantly in mice with selective loss of cone function (Biel et al. 1999). Indeed, we found a normal b-wave up to –2 log cd s/m2 intensity and a normal a-wave at all light intensities (Fig. 68.1a), but a reduction of the trailing edge of the b-wave especially at high intensities (arrow in Fig. 68.1c).

As the cpfl1 mouse line was named after its deficient cone system function (Chang et al. 2002), the photopic ERGs were strongly reduced as anticipated. However, unlike in Cnga3–/– mice (Biel et al. 1999), photopic responses were not completely absent in cpfl1 mice (arrows in Fig. 68.1b). To confirm that these changes

Fig. 68.1 Electroretinographic data from wild type and cpfl1 mice at 4 weeks of age. (a) Scotopic (dark-adapted) single flash ERG intensity series of a wild type (left) and a cpfl1 mouse (right). Vertical line crossing each trace shows the timing of the light flash. (b) Photopic (light-adapted) single flash ERG intensity series of a wild type (left) and a cpfl1 mouse (right). The photopic responses are strongly reduced but not completely absent (arrows in b). (c) Overlay of selected scotopic (top) and photopic (bottom) waveforms from the intensity series. The alteration of the trailing edge of the scotopic b-wave at high stimulus intensities is illustrated (arrow in c). (d) Scotopic (SC) and photopic (PH) b-wave amplitudes from wild-type and cpfl1 mice as a function of the logarithm of the flash intensity. Boxes indicate the 25–75% quantile range, whiskers indicate the 5 and 95% quantiles, and the asterisk indicates the median of the data. (e) Photopic bright flash ERG responses obtained from a wild-type and a cpfl1 mouse, and also from a functionally all-rod mouse (Cnga3–/–, cone cyclic nucleotide-gated channel deficient). Small response was detected in cpfl1 mice (arrow in e), indicating some remaining cone system function at 4 weeks of age

truly reflect residual cone system function, we performed a photopic bright flash ERG using a light stimulus of 4.1 log cd s/m2 intensity (Fig. 68.1e). Although response amplitudes are initially masked by a flash artefact, a small but distinct light-evoked response could be demonstrated in 4-week-old cpfl1 mice (arrow in Fig. 68.1e).