In this study, we have analyzed cone visual functions without interference from rod responses. This experimental design has allowed us to demonstrate that CaBP4 is crucial for synaptic transmission from cone photoreceptors to second-order neurons. To evaluate pure cone function, it is essential to block rod responses. Cone-mediated light responses can be isolated using rod-saturating conditions or flicker ERG at high frequency. However, residual rod responses might still be detected in photopic responses,
15 and the use of a rod-saturating background might also affect cone responses. The drawback of flicker ERG in mice is the low signal-to-noise ratio at frequencies that are thought to be greater than rod temporal resolution.
17 To characterize cone responses under photopic and scotopic conditions, we used knockout mice that have no functional rods. Diverse knockout models of rod dysfunction have been developed, including rhodopsin-deficient mice and rod α-transducin–deficient mice.
11 18 19 The advantage of the rod α-transducin–deficient model over the rhodopsin-deficient mice is that the structure of rods is normal for the first 3 months, whereas that of rhodopsin-deficient mice degenerates rapidly.
18 19
In this study, to investigate the role of CaBP4 for cone vision, we crossbred
Cabp4 –/– with
Gnat1 –/– mice. These
Cabp4 –/– Gnat1 –/– double-knockout mice allowed us to study the effect of CaBP4-deficiency without interference from rod-mediated responses. No detectable changes in retinal morphology between double-knockout mice and single-knockout mice were observed, and the cones were as well preserved in double-knockout mice as in single-knockout mice. These results indicate that the absence of photoexcitation in rods does not affect the CaBP4-deficient phenotype morphologically, confirming the suitability of this model to study the effect of CaBP4 deficiency on cone signaling function. These observations are in accordance with other findings that demonstrate that the absence of functional cones and rods does not affect the structural development of the photoreceptor synapses after eye opening.
20 Synaptic contacts between photoreceptor terminals, horizontal cell processes, and bipolar cell dendrites were also reported to be relatively normal in double cone cyclic nucleotide-gated cation channel /rhodopsin knockout mice up to postnatal week 4.
20
The absence of morphologic changes between these Cabp4 –/– and Cabp4 –/– Gnat1 –/– mice also suggests that changes in the photoreceptor terminals associated with rod-mediated light responses, including light-dependent changes of the calcium concentration at the synapse, do not contribute to the mechanisms underlying the phenotype of Cabp4 –/– mice. The light-independent phenotype corroborates the putative role of CaBP4 in photoreceptor synaptogenesis.
Electroretinograms were recorded to investigate the effect of CaBP4 deficiency on cone function. Single-flash ERG responses of the Cabp4 –/– Gnat1 –/– mice were severely reduced in scotopic conditions, whereas there was no significant difference in photopic conditions compared with Cabp4 –/– mice. Reduced-flicker ERG demonstrates that amplitudes and sensitivities of Cabp4 –/– Gnat1 –/– mice and Cabp4 –/– mice were significantly attenuated compared with those of Gnat1 –/– mice and wild-type mice, suggesting that few neural connections between cone cells and bipolar cells are functional in Cabp4 –/– and Cabp4 –/– Gnat1 –/– mice, in agreement with the results obtained using single-flash ERG recordings. These data demonstrate that in the absence of CaBP4, the synaptic transmission of the light response from cones to second-order neurons is severely affected. Unexpectedly, flicker ERG of Gnat1 –/– mice under scotopic and photopic conditions was attenuated compared with that of wild-type mice. This difference might be attributed to the different genetic backgrounds of wild-type mice and knockout mice.
Gresh et al.
21 reported that a reduced amplitude of flicker ERG correlates with increasing flicker frequency more noticeably in Balb/c mice than in C57Bl/6 mice. To confirm the genetic background effects of mice for flicker ERG, double-heterozygous mice that comprise mixed C57BL/6, 129SvEv, and BALB/c backgrounds were prepared and analyzed with flicker ERG under the same conditions used in this study. However, no significant differences were observed between C57BL/6 wild-type mice and double-heterozygous mice (data not shown).
We have previously shown that CaBP4 interacts with expressed α1F (Ca
v1.4) L-type, voltage-dependent calcium channels and modulates their functional properties.
2 Although the molecular identity of the cone Ca
2+ channels is not fully elucidated, some cone channels contain the Ca
v1.3 subunit.
22 23 24 Moreover, immunostaining of additional aggregates to typical horseshoe-shaped structures with an anti-Ca
v1.4 α1F antibody suggests that Ca
v1.4 α1F is also present in cone terminals.
25 The severely reduced cone signaling in CaBP4-deficient mice indicates that CaBP4 is also critical for the release of neurotransmitter from cone synaptic terminals. These results suggest that cones express the Ca
v1.4 subunit or a subunit highly homologous to it. The gene encoding the α-subunit of Ca
v1.4 appears to have diverged more recently from that of the human Ca
v1.3.
26 Their amino acid sequences share 70% overall similarity and 84% similarity between transmembrane segments. Furthermore, in humans, mutations in the gene encoding the Ca
v1.4 α-subunit cause CSNB2. ERG responses of CSNB2 patients show reduced b-wave amplitude, but their color vision is almost, if not completely, normal, suggesting that cones express a calcium channel other than Ca
v1.4.
Ca
v1.4-deficient mice also show ERG responses with an electronegative configuration and ectopic photoreceptor synapses (Orton NC, et al.
IOVS 2004;45:ARVO Abstract 2507). Cav1.4 and Cav1.3 share biophysical properties; both activate at more negative voltages and more rapidly than Cav1.2, and both inactivate more slowly than Cav1.2. However, Ca
v1.3 α-deficient mice do not exhibit electroretinogram changes.
27 The phenotypes of CSNB2 patients and Ca
v1.4-deficient and Ca
v1.3-deficient mice can be reconciled if cones can be shown to coexpress two types of L-type voltage-dependent calcium channels. This would also be consistent with the critical role of CaBP4 in modulating rod and cone neurotransmitter release.
The authors thank Daniel Possin, supported by Vision Core Grant EY01730, for his great expertise with the EM experiments and Rebecca Birdsong for her help during manuscript preparation.