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leads to some full-length Cav1.4 protein, and some cones surviving to drive photopic visual responses.
63.1 Introduction
The incomplete form of X-linked congenital stationary night blindness (iCSNB, CSNB2) is one of a heterogeneous group of human visual disorders due to defects in retinal neurotransmission, characterized by reduced function in rod and cone photoreceptor pathways (Miyake et al. 1986; Tremblay et al. 1995). The clinical features of CSNB2 are highly variable and may include reduced visual acuity, impaired night vision, refractive disorders, nystagmus and strabismus (Miyake et al. 1986; Boycott et al. 2000; Miyake 2002; Lodha et al. 2009). The full-field flash electroretinograms (ERGs) of CSNB2 patients reveal abnormal rodand cone-system responses, indicating impairment of synaptic transmission from photoreceptors to bipolar cells.
CSNB2 is an X-linked recessive condition due to mutations in CACNA1F, the gene for the pore-forming α1F-subunit of an L-type voltage-gated calcium channel, Cav1.4 (Bech-Hansen et al. 1998; Strom et al. 1998). Since Cav1.4 is the major mediator of transmitter release from rods and cones, it has been assumed that the impairment of synaptic transmission in CSNB2 is due to insufficiency of calcium currents through mutant Cav1.4 channels (Bech-Hansen et al. 1998; Strom et al. 1998; Baumann et al. 2004). To understand the pathophysiology of human CSNB2,
we have characterized two murine CSNB2 models: an engineered null mutant with a stop codon at amino acid residue 305 (G305X: Cacna1f G305X; (Mansergh et al.
2005; Orton et al. 2007; Raven et al. 2008)), and a spontaneous mutant in which the insertion of a retrovirus-like early transposon (ETn) causes the production of abnormal Cacna1f-encoded transcripts and proteins mutant (nob2: Cacna1f nob2; (Chang et al. 2006; Bayley and Morgans 2007)). Here we describe further investigations of these two models and discuss insights that we have gained from such studies.
63.2 Methods
Previous reports have described our methods for immunofluorescence (Mansergh et al. 2005; Morgans et al. 2005; Chang et al. 2006; Raven et al. 2008), optokinetic response (OKR: Prusky and Douglas 2004; Bonfield et al. 2007; Umino et al. 2008), and flash electroretinography (ERG: Mansergh et al. 2005; Orton et al. 2007; Doering et al. 2008; Raven et al. 2008). In G305X mice, differences between gene expression in adult wild-type and mutant retinas were characterized by a retina-specific microarray (Orton et al. 2007), and effects of mutations on retinal development and synaptogenesis were assessed by the TUNEL method and caspase immunocytochemistry (putatively indicating apotptosis) plus transmission electron microscopy (TEM) in P10-P28 mice (Orton et al. 2007; Raven et al. 2008). In adult nob2 mice, mRNA was cloned and sequenced, expressed protein was characterized
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by immunoblotting for Cav1.4/α1F and a pull-down assay for binding to filamin (a cytoskeletal protein), and the properties of wild-type and mutant Cav1.4 channels expressed in HEK cells were determined by patch-clamping (Doering et al. 2008).
63.3 Results
Although these models for CSNB2 are similar, they differ in several important
respects:
Cacna1fG305X: A loss-of-function mutation was created by inserting a selfexcising Cre-lox-neocassette into exon 7 of Cacna1f, the murine orthologue of CACNA1F (Mansergh et al. 2005). Its scotopic ERG had an a-wave of marginally reduced amplitude and no post-receptoral b-wave and oscillatory potentials (Fig. 63.1a and b), while its photopic ERG (Fig. 63.1c), as well as visual evoked
Fig. 63.1 Electroretinogram (ERG) findings in Cacna1f G305X mice. (a) Scotopic ERG: Intensity-
response series of scotopic electroretinograms in wild-type (Cacna1fWT) and mutant mice (Cacna1f G305X/G305X, Cacna1f G305X/Y). In the Cacna1f G305X mice, the ERG b-wave is absent, pro-
viding an electronegative configuration to the bright-flash responses. Oscillatory potentials (OPs)
are also absent in mutant mice. Numbers at the left of individual recordings are stimulus intensity in log cd s/m. (b) Comparison of ERG responses between a Cacna1f G305X mutant mouse
and a wild-type animal with intravitreal injection of CoCl2. The bright-flash response after CoCl2 injection in the wild-type is identical to the one in the mutant, suggesting that the Cacna1f-mutant
ERG response is mostly generated by photoreceptor activity. (c) Photopic ERG is undetectable in Cacna1f G305X mice (modified from Mansergh et al. 2005)
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potentials and multi-unit activities in the superior colliculus, were absent (Mansergh et al. 2005). Immunoreactive Cav1.4 protein was not detectable in the outer plexiform layer (OPL), dendrites of second-order neurons sprouted into the photoreceptor layer, and TEM showed a profound loss of photoreceptor synapses (Mansergh et al. 2005). Microarray of retina from affected males and females revealed marked reductions in expression of cone-specific genes (data not shown). Photopic spatial contrast-sensitivity (CS) functions for optokinetic responses (OptoMotryTM) of wild-type controls were as described previously ((Prusky and Douglas 2004; Umino et al. 2008); Bonfield 2009), with optimal drift speed 12 d/s, peak contrast sensitivity 15 (threshold contrast 6.5%) at spatial frequency (SF) 0.061–0.1
c/d, and acuity (highest SF for response at 100% contrast) >0.4 c/d. In contrast, affected G305X mice (Cacna1f G305X/y and Cacna1f G305X/G305X) gave no optokinetic response, while heterozygous females (Cacna1f G3O5X/+) responded robustly
Fig. 63.2 Electroretinogram (ERG) findings in Cacna1fnob2 mice. (a) Scotopic ERG (dark
adapted): Intensity-response series of scotopic electroretinograms in wild-type (Cacna1f WT) and mutant (Cacna1f nob2) mice. Scotopic ERG b-wave is nearly absent in mutant mice at low inten-
sity and appears at higher intensity, though with a reduced amplitude, providing an electronegative configuration to the bright-flash responses. The oscillatory potentials (OPs) are absent in mutant mice at low intensity, but are present at high intensity. (b, c) Details of scotopic ERG at intensities 1.89 log cd/m2 (b) and –0.81 log cd/m2 (c). Wild type and mutant ages nearly P52. The b-wave and OP can be elicited, especially at higher luminance and their amplitude increases with increasing stimulus intensity. (d, e) Photopic ERG (light-adapted). (d: high intensity of 1.89 log cd/m2; e: low intensity of 0.38 log cd/m2) a-wave, b-wave and oscillatory potentials are small but clearly evident in Cacna1f nob2 mice at both intensities of stimulus
Fig. 63.3 Photopic spatial (sine-wave grating) contrast sensitivity (CS) function. (a) G305X mice Spatial CS function of heterozygous females (Cacna1f +/G305X, 3 l, n = 9) showed reduced CS vs unaffected male (Cacna1f WT) littermates. Cacna1f G305X/Y: no response to any spatial frequency or contrast. (b) nob2 mice Spatial CS function of affected males (Cacna1f nob2/y, 3 l, n = 15). The CS was similar to that of WT (Cacna1f WT) in nob2 litters 2 and 3, but not to that of Cacna1f +/G305X in nob2 liter 1
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