GluA3 subunits are required for appropriate assembly of AMPAR GluA2 and GluA4 subunits on cochlear afferent synapses and for presynaptic ribbon modiolar–pillar morphology
- Department of Otolaryngology, Washington University School of Medicine, United States
- Department of Neurobiology, University of Pittsburgh School of Medicine, United States
- Department of Otolaryngology, University of Pittsburgh School of Medicine, United States
Figures
Figure 1
ABRs, GluA1 and GluA2 immunolabeling and qRT-PCR in GluA3WT and GluA3KO.
(A) Mean ABR thresholds ( ± standard deviation [SD]) were similar between male GluA3WT and GluA3KO mice (F(1, 168) = 2.659, p = 0.11; two-way analysis of variance (ANOVA); GluA3WT n = 13; GluA3KO n …
-
Figure 1—source data 1
Data and statistical analysis for the ABR, PCR gels and qRT-PCR for GluA3WT and GluA3KO mice.
- https://cdn.elifesciences.org/articles/80950/elife-80950-fig1-data1-v2.zip
-
Figure 1—source data 2
Raw unedited PCR acrylamide gels for Gria2 and Gria4 flip/flop in GluA3WT and GluA3KO.
- https://cdn.elifesciences.org/articles/80950/elife-80950-fig1-data2-v2.zip
-
Figure 1—source data 3
Figures of the uncropped PCR acrylamide gels for Gria2 and Gria4 flip/flop in GluA3WT, and GluA3KO with relevant bands and lanes clearly labeled.
- https://cdn.elifesciences.org/articles/80950/elife-80950-fig1-data3-v2.zip
Figure 2
Immunohistofluorescence of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) pore-forming subunits GluA2, 3, and 4 on spiral ganglion neuron postsynaptic terminals in the organ of Corti.
Confocal microscope immunofluorescence images of afferent ribbon synapses in organ of Corti whole-mount samples from GluA3WT (left) and GluA3KO mice (right) in the mid-cochlea. Anti-GluA2 (green), …
Figure 3 with 1 supplement
Ultrastructural features of GluA3WT IHC-ribbon mid-cochlear synapses.
Transmission electron microscopy (TEM) micrographs of IHC synapses on the modiolar (A) and pillar sides (B). Aff.: afferent; IHC: inner hair cell; Eff.: efferent terminal. Scale bar: 0.5 µm. (A’, B’)…
-
Figure 3—source data 1
Data and statistical analysis for the ultrastuctural analysis of GluA3WT mice.
- https://cdn.elifesciences.org/articles/80950/elife-80950-fig3-data1-v2.zip
Figure 3—figure supplement 1
Representative serial electron micrographs and corresponding three-dimensional (3D) reconstructions of modiolar- or pillar-side inner hair cell (IHC)-ribbon synapses of the GluA3WT mice.
Synapses #10 and #24 correspond to the synapses shown in Figure 3. Scale bar: 0.5 μm.
Figure 4 with 1 supplement
Ultrastructural features of GluA3KO IHC-ribbon mid-cochlear synapses.
Transmission electron microscopy (TEM) micrographs of IHC synapses on the modiolar (A) and pillar sides (B) of GluA3KO mice. Aff.: afferent; IHC: inner hair cell. Scale bar: 0.5 µm. (A’, B’) …
-
Figure 4—source data 1
Data and statistical analysis for the ultrastuctural analysis of GluA3KO mice.
- https://cdn.elifesciences.org/articles/80950/elife-80950-fig4-data1-v2.zip
Figure 4—figure supplement 1
Representative serial electron micrographs and corresponding three-dimensional (3D) reconstructions of modiolar- or pillar-side inner hair cell (IHC)-ribbon synapses of the GluA3KO mice.
Synapses #15 and #24 correspond to the synapses shown in Figure 4. Representative serial electron micrographs of one pillar-side synapse with two ribbons; this synapse was not included in our …
Figure 5
Inner hair cell (IHC) modiolar–pillar structural differences in presynaptic ribbon size, ribbon shape, and vesicle size seen in GluA3WT were diminished or reversed in GluA 3KO.
(A) Whisker plots show the quantitative data of the surface area and volume of the postsynaptic density (PSD) and ribbon volume of GluA3WT (black) and GluA3KO (gray) mice. The error bar corresponds …
-
Figure 5—source data 1
Data and statistical analysis for the comparison of the ultrastructural analysis of WT vs. GluA3KO mice.
- https://cdn.elifesciences.org/articles/80950/elife-80950-fig5-data1-v2.zip
Figure 6
Inner hair cell (IHC)-ribbon synapse counts in 5-week-old male GluA3WT and GluA3KO mice.
(A) Confocal microscope immunofluorescence images of afferent ribbon synapses in organ of Corti whole-mount samples from GluA3WT (upper) and GluA3KO mice (lower) in the apical, middle, and basal …
-
Figure 6—source data 1
Confocal data and statistics of synapse counts for GluA3WT and GluA3KO.
- https://cdn.elifesciences.org/articles/80950/elife-80950-fig6-data1-v2.zip
Figure 7
Alteration of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit expression in GluA3KO mice.
(A) From images like in Figure 2: Summed pixel intensity per synapse (raw values, a.u.) for GluA2 (green), GluA4 (blue), and GluASum (black). In each subpanel, GluA3WT is on left and GluA3KO is on …
Figure 8 with 1 supplement
Modiolar- and pillar-side volume, intensity, and density of presynaptic ribbon and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunits.
(A) Quantification of CtBP2, GluA2, or GluA4 mean volume per image for GluA3WT (black, n = 2990 synapses from 14 images) compared to GluA3KO (gray, n = 2814 synapses from 14 images). Each point …
-
Figure 8—source data 1
Confocal data and statistics of synapse volumes and intensities for GluA3WT and GluA3KO.
- https://cdn.elifesciences.org/articles/80950/elife-80950-fig8-data1-v2.zip
Figure 8—figure supplement 1
Modiolar-, pillar-side groupings and example synapses from GluA3WT and GluA3KO.
(A) Schematic of an inner hair cell (IHC) when mounted with long axis parallel to the glass coverslip, such that the modiolar–pillar dimension of the cell is orthogonal to the coverslip. Per image, …
Figure 9
Spatial trends of synapse sphericity, volume, and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit relative abundance in the organ of Corti.
(A) Volume and sphericity per synapse vs. Z-axis position for an exemplar GluA3WT image from Figure 7 showing spatial oscillations in CtBP2, GluA2, and GluA4. Right: Inverse relationship between …
Author response image 1
