As expected from the cCSNB phenotype,
Lrit3nob6/nob6 retinas showed no cone-driven ON-responses at the level of ganglion cells. A similar phenotype resulting in the absence of short-latency ON-responses is also observed in other mouse models of cCSNB where components of the mGluR6 cascade are mutated or absent.
18,19,42–44 Thus, our results confirm that the mGluR6 signaling cascade is nonfunctional in mice lacking
Lrit3. In contrast, the OFF-responses are present and have a normal firing rate in
Lrit3nob6/nob6 mice. Similarly, in other mouse models with cCSNB, the major defect is characterized by the absence of ON-responses and relatively unaffected OFF-responses. However,
Lrit3nob6/nob6 mice also showed OFF-responses, which were significantly delayed in onset latencies. The distribution of latencies clearly indicates a delay in the OFF-responses for most if not all recorded RGCs (
Fig. 2G), which may suggest synaptic changes at the photoreceptor to OFF-BC synapse. However, a follow-up study is needed to define if the different types of ganglion cells are differently affected by this latency delay, bearing in mind previous and recent findings showing the existence of at least 32 different types of ganglion cells.
45–53 Of note, in other mouse models with cCSNB, variability in minor altered OFF-responses has been reported.
16,18,19,42–44 These findings show that although the main defect in cCSNB arises from a lack of ON-pathway signaling, the OFF-pathway may also be affected. This situation seems to be strikingly different for mutations in presynaptic proteins present at the photoreceptor terminals and implicated in the incomplete type of CSNB. For example, a mouse model lacking the α-subunit of the calcium channel Cav1.4
54,55 exhibits lack of both ON- and OFF-responses when evaluated at either bipolar or ganglion cell levels.
56,57 Therefore, we think it is likely that the OFF-response deficits observed in the
Lrit3 knockout mouse model represent a secondary effect resulting from the primary alterations in ON-pathways. Indeed, recent studies
42,58 at the level of bipolar or ganglion cells, using different species, contradict the notion that ON- and OFF- signals are truly selective for a single pathway. In addition, crosstalk between ON- and OFF-pathways mediated by amacrine cells has been described in mammalian retinas. Multistratified amacrine cells receive ON-signals via chemical synapses with ON-bipolar cell axon terminals in the inner plexiform layer and then deliver these signals to OFF ganglion cells.
59 This may explain minor altered OFF-responses observed in mouse models with cCSNB. Furthermore, we confirmed that full-field ERG recordings performed according to ISCEV standards in a patient with
LRIT3 mutations display characteristic responses of cCSNB.
24 Namely, there was absence of detectable responses to a dim flash as well as electronegative waveform to a bright flash under scotopic conditions, keeping with rod ON-pathway dysfunction.
60,61 Moreover, photopic ERG responses indicate a selective ON-bipolar pathway dysfunction with OFF-bipolar pathway preservation.
62 We further documented these abnormalities by performing long-duration stimulations, which revealed a normal a-wave but a severely reduced ON–b-wave and a preserved OFF–d-wave. However, long-duration stimulations allow only a qualitative rather than quantitative analysis owing to the high variability for OFF-responses,
63 and we were not able to conclude if these OFF-responses are delayed in the patient as shown for the mouse model. It has been previously demonstrated that photoreceptors release glutamate at synaptic ribbons and that the neurotransmitter subsequently diffuses toward the dendritic tips of OFF-BCs.
64–66 So, the displacement of ON-bipolar dendrites and concomitant increase in the number of flat contacts that we observed in
Lrit3nob6/nob6 mice may affect transmission to OFF-BCs through steric occlusion. Despite the fact that the exact mechanisms underlying deficits in OFF-pathways induced by the loss of LRIT3 function remain to be elucidated, some recent techniques allowing imaging of glutamate release dynamics in the retina
67,68 would be useful to investigate this hypothesis in the future. However, species differences may also account for the observed different onset of the OFF-responses observed in mice lacking LRIT3 and the patient harboring
LRIT3 mutations. Indeed, in general the cone system shows considerable variation across species.
69 This is also highlighted by the different cone ERG waveforms always seen in mouse models for cCSNB compared to patients with the same gene defect. The photopic b-wave seems to be more severley reduced in mouse models than in patients.
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