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. 1999 Jun 22;96(13):7553-7.
doi: 10.1073/pnas.96.13.7553.

Selective loss of cone function in mice lacking the cyclic nucleotide-gated channel CNG3

M Biel  1 , M Seeliger, A Pfeifer, K Kohler, A Gerstner, A Ludwig, G Jaissle, S Fauser, E Zrenner, F Hofmann
Affiliations

Selective loss of cone function in mice lacking the cyclic nucleotide-gated channel CNG3

M Biel et al. Proc Natl Acad Sci U S A. .

Abstract

Two types of photoreceptors, rods and cones, coexist in the vertebrate retina. An in-depth analysis of the retinal circuitry that transmits rod and cone signals has been hampered by the presence of intimate physical and functional connections between rod and cone pathways. By deleting the cyclic nucleotide-gated channel CNG3 we have generated a mouse lacking any cone-mediated photoresponse. In contrast, the rod pathway is completely intact in CNG3-deficient mice. The functional loss of cone function correlates with a progressive degeneration of cone photoreceptors but not of other retinal cell types. CNG3-deficient mice provide an animal model to dissect unequivocally the contribution of rod and cone pathways for normal retinal function.

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Figures

Figure 1
Figure 1
Targeted disruption of the CNG3 gene. (a Upper) Structure of CNG3 protein showing the location of the six transmembrane segments (–6), the ion-conducting pore (P), and the cyclic nucleotide-binding domain (CNBD). The portion of the protein encoded by exons (E) 5–7 is indicated. (Lower) Restriction maps of the CNG3 target locus, targeting vector, and mutant allele. Exons (E5–E7) and introns are indicated by solid boxes and lines, respectively. The 284-bp fragment amplified by RT-PCR is displayed as a solid box below E7. For gene targeting of the CNG3 locus a portion of E7 (shown in gray) encoding transmembrane segments 3–6 was replaced by the neomycin-resistance cassette (neo). The thymidine kinase (tk) was used for negative selection. Ec, EcoRV; Sp, SphI; St, StuI; Bs, BspEI; Sa, SacI; pBSK, pBluescript SK vector; wt, wild type; mt, mutant. (b) Identification of CNG3+/−, CNG3+/+, and CNG3−/− mice by Southern blot analysis. The wild-type and mutant alleles gave 18- and 6.5-kb fragments, respectively, after EcoRV digestion and hybridization with the external probe. (c) RT-PCR of RNA isolated from retinae of CNG3+/+ and CNG3−/− mice with primers that amplify CNG3, the α-subunit (CNG1), and the β-subunit (CNG4) of rod photoreceptor channel.
Figure 2
Figure 2
ERG responses from 2-month-old CNG3+/+ and CNG3−/− mice. (a) Dark-adapted, single-flash ERG series in a CNG3+/+ and in a CNG3−/− mouse. Note the similar appearance except for smaller amplitudes at higher flash intensities in the CNG3−/− mouse. (b) Superposition of the responses from a. Up to 0.01 cds/m2, the responses do not differ in b-wave amplitude (open arrowheads). With a further increase in stimulus luminance, the difference in b-wave but not a-wave amplitude becomes obvious (solid arrowheads). (c) a-Wave amplitude from four CNG3−/− and four CNG3+/+ littermates as a function of the logarithm of the flash intensity. Boxes indicate the 25–75% quantile range, the bars indicate the 5 and 95% quantiles, and the central bar indicates the median of the CNG3−/− data. The red lines delimit the normal range given by the 5 and 95% quantile of the CNG3+/+ littermates. No differences between control and knockout mice were detectable, pointing to a pure rod origin of the a-wave at the tested intensity range. (d) b-Wave amplitude from four CNG3−/− and four CNG3+/+ littermates as a function of the logarithm of the flash intensity. At intensities higher than 0.01 cds/m2, the b-wave of the CNG3−/− mice does not increase further in amplitude, presumably because of the lack of cone contribution. (e) Light-adapted single-flash ERG in a CNG3+/+ and a CNG3−/− mouse, a human control, and an achromat by using flashes of 3-cds/m2 intensity. No stimulus-related activity can be recorded in the CNG3−/− mouse as in the human achromat.
Figure 3
Figure 3
ERG responses from 2-month-old dark-adapted CNG3+/+ and CNG3−/− mice to trains of flashes (flicker) of a fixed intensity, but varying frequency. (a and b) Scotopic flicker ERG series in a CNG3+/+ (a) and in a CNG3−/− (b) mouse obtained with a flash intensity of 0.01 cds/m2. Note the similar appearance and the normal flicker fusion frequency in the CNG3−/− mouse. (c and d) Scotopic flicker ERG series in a CNG3+/+ (c) and in a CNG3−/− (d) mouse obtained with a flash intensity of 3 cds/m2. Because of the high flash intensity, the ERG response of the CNG3−/− mouse is smaller even at the lowest frequency, and the flicker fusion frequency is only about 10% of normal.
Figure 4
Figure 4
Retinal morphology of 2-month-old CNG3−/− and CNG3+/+ mice. (a) Cryosection of the retina of a CNG3+/+ mouse incubated with the CNG3 antibody. The antibody specifically labels the outer segments of cone photoreceptors (arrows). (b) A section of a CNG3−/− retina incubated with the CNG3 antibody reveals no labeling. (c and d) Detection of cone photoreceptors with the lectin peanut agglutinin in CNG3+/+ (c) and CNG3−/− (d) mice. Note the reduced number of cones in CNG3−/− mice (arrows). [Bar = 10 μm (a and b) and 20 μm (c and d).] The sections (a–d) were taken from the central region of the inferior-nasal retina. PhR, inner and outer segments of the photoreceptors; ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (e and f) Electron microscopy photomicrographs. (e) Ultrastructural localization of CNG3 on the membranous disks of a cone outer segment of a CNG3+/+ mouse (arrows). (f) Cone outer segment of a CNG3−/− mouse revealing partial degeneration of disk structure. A rod outer segment next to the cone outer segment is marked by an asterisk. [Bar = 0.2 μm (e and f).]
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