LRIT3 Differentially Affects Connectivity and Synaptic Transmission of Cones to ON- and OFF-Bipolar Cells

Abstract

Purpose: Mutations in LRIT3 lead to complete congenital stationary night blindness (cCSNB). Using a cCSNB mouse model lacking Lrit3 (nob6), we recently have shown that LRIT3 has a role in the correct localization of TRPM1 (transient receptor potential melastatin 1) to the dendritic tips of ON-bipolar cells (BCs), contacting both rod and cone photoreceptors. Furthermore, postsynaptic clustering of other mGluR6 cascade components is selectively eliminated at the dendritic tips of cone ON-BCs. The purpose of this study was to further define the role of LRIT3 in structural and functional organization of cone synapses.

Methods: Exhaustive electroretinogram analysis was performed in a patient with LRIT3 mutations. Multielectrode array recordings were performed at the level of retinal ganglion cells in nob6 mice. Targeting of GluR1 and GluR5 at the dendritic tips of OFF-BCs in nob6 retinas was assessed by immunostaining and confocal microscopy. The ultrastructure of photoreceptor synapses was evaluated by electron microscopy in nob6 mice.

Results: The patient with LRIT3 mutations had a selective ON-BC dysfunction with relatively preserved OFF-BC responses. In nob6 mice, complete lack of ON-pathway function with robust, yet altered signaling processing in OFF-pathways was detected. Consistent with these observations, molecules essential for the OFF-BC signaling were normally targeted to the synapse. Finally, synaptic contacts made by ON-BC but not OFF-BC neurons with the cone pedicles were disorganized without ultrastructural alterations in cone terminals, horizontal cell processes, or synaptic ribbons.

Conclusions: These results suggest that LRIT3 is likely involved in coordination of the transsynaptic communication between cones and ON-BCs during synapse formation and function.

Figure 1

Full-field ERG of the cCSNB patient with LRIT3 mutations. Electroretinogram traces of the patient affected with cCSNB due to LRIT3 mutations (right) as compared to a normal subject (left). No recordable responses for the dark-adapted 0.01 ERG recording were detected. Dark-adapted 3.0 and 10.0 ERGs showed an a-wave with normal implicit time and amplitude but a severely reduced b-wave, leading to an electronegative waveform. Light-adapted 3.0 ERGs showed amplitudes at the lower limit of normal but implicit time shift for both the a-wave and the b-wave. The a-wave had a broadened trough in the patient with LRIT3 mutations, and there was a sharply rising b-wave with no oscillatory potentials. Light-adapted 3.0 flicker ERGs showed amplitudes at the lower limit of normal but a broadened trough and a mildly delayed implicit time. ON- and OFF-BC recordings revealed a normal a-wave both in amplitude and implicit time. A severely reduced b-wave and a preserved d-wave, in keeping with ON-pathway dysfunction with OFF-pathway preservation, were detected.

Figure 2

ON- and OFF-responses of RGCs in Lrit3^nob6/nob6 retinas. Representative RGC activity recording from an Lrit3^+/+ retina (A) and an Lrit3^nob6/nob6 retina (B). The gray box corresponds to the light stimulus. Horizontal scale bar: 500 ms; vertical scale bar: 50 μV. Representative raster plot (up) and peristimulus time histogram (bottom) from an Lrit3^+/+ retina (C) and an Lrit3^nob6/nob6 retina (D). The gray box corresponds to the light stimulus. Horizontal scale bar: 1 second; vertical scale bar: 50 events/bin. Maximum firing rate of ON- (E) and OFF-responses (F) in Lrit3^nob6/nob6 retinas compared to Lrit3^+/+ retinas relative to the spontaneous activity. Bars represent median values. Asterisks represent results that are significantly different (P < 0.05). (G) Time for which the maximum firing rate is reached after the light stimulus onset for ON-responses in Lrit3^+/+ retinas and the light stimulus offset for OFF-responses in Lrit3^nob6/nob6 retinas compared to Lrit3^+/+ retinas. Bars represent median values. Asterisks represent results that are significantly different (P < 0.05).

Figure 3

Localization of ionotropic glutamate receptors GluR1 and GluR5 and pikachurin in Lrit3^nob6/nob6 retinas. Representative confocal images of cross-sections centered on OPL of Lrit3^+/+ and Lrit3^nob6/nob6 retinas stained with antibodies against (A) GluR1 (red) and PSD-95 (green) and merge, (B) GluR1 (red) and GluR5 (green) and merge, and (C) pikachurin (red) and PSD-95 (green) and merge. Arrowheads represent putative cone synapses. Scale bar: 10 μm.

Figure 4

Ultrastructure of rod and cone synapses in Lrit3^nob6/nob6 mice. Representative electron microscopy images of rod (A) and cone (B) ribbon synapses in Lrit3^nob6/nob6 retinas compared to Lrit3^+/+ retinas. Rod spherules and cone pedicles are shown in yellow, invaginating dendrites of both rod and cone ON-BCs are shown in red, horizontal cell processes are shown in blue, and flat-contacting dendrites of cone OFF-BCs are marked with asterisks. Scale bar: 1 μm. (C) Quantification of synaptic elements present in rod spherules. Synapses are classified as diads if they have identifiable ribbon with adjacent horizontal cell process. If spherules additionally have ON-BC process next to the synaptic ribbon, they are designated as triads. (D) Quantification of synaptic elements at the cone pedicles of Lrit3^nob6/nob6 retinas compared to Lrit3^+/+ retinas as a percentage of all counted synapses. In addition to diads and triads classified as described above, flat contacts at the base of the pedicle away from the ribbon were also distinguished and labeled as OFF type. Not-otherwise-classified contacts are labeled as NC (not classified). Error bars represent standard error of the mean values.

Figure 5

Serial ultrathin section images of a cone pedicle in an Lrit3^nob6/nob6 retina. Serial electron microscopy images of a cone pedicle in an Lrit3^nob6/nob6 retina. A gallery shows images of every two sections. Cone pedicle is shown in yellow, invaginating dendrite of cone ON-BC is shown in red, and horizontal cell processes are shown in blue. Scale bar: 1 μm.