GluA2-Containing AMPA Receptors Distinguish Ribbon-Associated from Ribbonless Afferent Contacts on Rat Cochlear Hair Cells

Abstract

Mechanosensory hair cells release glutamate at ribbon synapses to excite postsynaptic afferent neurons, via AMPA-type ionotropic glutamate receptors (AMPARs). However, type II afferent neurons contacting outer hair cells in the mammalian cochlea were thought to differ in this respect, failing to show GluA immunolabeling and with many "ribbonless" afferent contacts. Here it is shown that antibodies to the AMPAR subunit GluA2 labeled afferent contacts below inner and outer hair cells in the rat cochlea, and that synaptic currents in type II afferents had AMPAR-specific pharmacology. Only half the postsynaptic densities of type II afferents that labeled for PSD-95, Shank, or Homer were associated with GluA2 immunopuncta or presynaptic ribbons, the "empty slots" corresponding to ribbonless contacts described previously. These results extend the universality of AMPAergic transmission by hair cells, and support the existence of silent afferent contacts.

Keywords: AMPA receptors; cochlear afferents; outer hair cells; postsynaptic densities.

Figure 1.

Ribbons and AMPAR clusters in cochlear whole mounts, and maximum intensity projections of confocal z-stacks of the medial region of the organ of Corti from an adult rat viewed from the endolymphatic surface including 24 adjacent OHCs and 5 IHCs. A, OHCs: immunolabel for the presynaptic ribbon marker (CtBP2, red channel). Immunolabel for the postsynaptic marker GluA2 (green channel). Merged and magnified inserts: CtBP2 and GluA2 puncta overlapped in the x- to y-plane. Rotation to the z- to x-planes or z- to y-planes reveals displacement between presynaptic and postsynaptic markers. B, IHCs: presynaptic and postsynaptic immunolabels. CtBP2 (red) and GluA2 (green) immunopuncta were consistently juxtaposed at the IHCs. Magnified insert in the x- to y-plane shows clear separation of presynaptic and postsynaptic labels. The x- to z-labels and z- to y-labels were not as well segregated as those in OHCs. C, Quantification of the number and the percentage of juxtaposed CTBP2 and GluA2 puncta in OHCs. D, Quantification of the number and the percentage of juxtaposed CtBP2 and GluA2 puncta in the IHCs. n = 3-9 independent preparations; 50 IHCs, 72 OHCs for A–D. There were no statistically significant differences in number or correlation among the immunolabels (one-way ANOVA test, p > 0.05). Scale bars: A, B, 5 µm; magnified inserts, 2.5 µm.

Figure 2.

AMPA receptors mediate synaptic transmission from OHC to type II afferents in young (1- to 2-week-old) rat cochlea. A, Inward synaptic currents (small downward deflections) evoked by high-potassium saline solution were reduced, then eliminated by exposure to CP-465,022. B, Diary plot showing a partial block of EPSCs by 1 µm CP465,022 followed by complete block by 10 µm, then partial recovery after washout. Recordings [holding potential (Vhold), −80 mV] were made in 40 mm external potassium to increase EPSC frequency. C, Cumulative fraction plot of EPSCs from fiber in B. EPSC amplitudes decreased in the presence of 1 µm CP465,022 (red). D, Scaled EPSC waveforms before (black) and during exposure to 1 µm CP465,022 (red) showing identical kinetics.

Figure 3.

Calcium-impermeant glutamate receptors carry synaptic currents in type II afferents in young (1- to 2-week-old) rat cochlea. A, Averaged synaptic currents in type II fiber containing 100 µm spermine at indicated membrane potentials (not corrected for junction potential). The number of events in each average current range from 115 to 624. B, Current–voltage curve of synaptic currents for spermine-loaded type II fiber (red) compared with control data (black; panel is from the study by Weisz et al., 2009, used with permission). Average current amplitude with SDs shown for spermine data. C, Average synaptic currents in a type II fiber (averaged over 10 s bins) before (black) and during exposure to 20 µm philanthotoxin (mean with SD). D, Average synaptic currents in a type II fiber (averaged over 10 s bins) before (black) and during exposure to 10 µm Naspm (mean with SD).

Figure 4.

IHC synaptic immunopuncta. Confocal z-stacks of five IHCs in the middle turn of the organ of Corti from an adult rat viewed from the endolymphatic surface. A, B, Immunolabeling for postsynaptic density proteins PSD-95 (red channel) and Shank (green channel; A) or Homer (green channel; B) show closely coincident puncta of these postsynaptic density markers (magnified inserts in the x- to y-plane). C, Immunolabeling for the CtBP2 red channel, and the postsynaptic marker PSD-95. Magnified insert shows juxtaposition in the x- to y-plane. D, Immunolabel comparing CtBP2 and Shank distribution. E, Immunolabel comparing CtBP2 and Homer. F, Quantification of presynaptic and postsynaptic immunopuncta in IHCs. G, Quantification of percentage juxtaposition of postsynaptic density proteins. H, Quantification of percentage juxtaposition for presynaptic ribbon marker CtBP2 and the postsynaptic density proteins (PSD-95, Shank, and Homer). A–H: n = 40-60 IHCs from four to five independent preparations. There were no statistically significant differences in number or correlation among these immunopuncta (one way-ANOVA test; p > 0.05). Scale bars: low magnification, 5 µm; high magnification, 2.5 µm.

Figure 5.

OHC synaptic immunopuncta. Confocal z-stack of OHCs in the middle turn of the organ of Corti from an adult rat viewed from the endolymphatic surface. A, Immunolabeling with the postsynaptic density proteins PSD-95 (red channel) and Shank (green channel) show an interconnected series of puncta along the base of the OHCs. PSD-95 and Shank puncta are closely coincident (magnified insert, x- to y-plane). B, Immunolabeling with the presynaptic ribbon marker CtBP2 (red) and postsynaptic marker Shank (green). C, Immunolabel for CtBP2 (red) and PSD-95 (green). D, Immunolabel for CtPB2 (red) and Homer (green). Magnified inserts (x- to y-plane) in each case show more extensive postsynaptic density distribution than presynaptic ribbon label. E, Presynaptic and postsynaptic immunopuncta at the OHCs. There were significantly fewer CtBP2 or GluA2 puncta than postsynaptic density puncta (PSD-95, Shank, or Homer; one way-ANOVA, p = 0.01; Bonferroni’s multiple comparison test; n = 3-7; 72-168 OHCs). F, Thumbnails of the base of individual OHCs immunolabeled for CtBP2 (red channel) and Shank (green channel). Many Shank immunopuncta had no associated CtBP2 puncta. G, Thumbnails of the base of individual OHCs immunolabeled for GluA2 (red channel) and Shank (green channel). Many of the Shank immunopuncta had no associated GluA2 puncta. H, Percentage juxtaposition of the CtBP2 and PSDs. The ratio of PSD-95, Shank, or Homer puncta juxtaposed to CTBP2 was significantly smaller than the ratio of CTBP2 puncta juxtaposed to the postsynaptic density proteins (one way-ANOVA, p ≤ 0.01; Bonferroni’s multiple comparison test; i.e., many PSDs had no ribbon). Scale bars: wide view, 5 µm; magnified inserts and thumbnails, 2.5 µm. I, Schematic drawing of OHC and IHC synapses. At the IHC afferent synapse, CtBP2/GluA2 relates closely in number to postsynaptic density markers. At the OHC afferent synapse, only a subset of postsynaptic density proteins relates to CtBP2/GluA2 synaptic markers.

Figure 6.

Single type II fibers visualized by intracellular labeling. A, Apical turn of a young (P8) rat organ of Corti with biocytin-filled type II fiber after streptavidin–peroxidase reaction. Scale bar, 125 µm. B, Higher magnification of boxed areas in A, showing trajectory and terminal branches. C, Biocytin-filled type II fiber reacted with streptavidin-Alexa Fluor 488 (green). OHC nuclei labeled with DAPI (blue). Magnifications show en passant (red arrows) and terminal (white arrowheads) swellings of branches from boxed regions. D, Biocytin-streptavidin-Alexa Fluor 488-filled fiber combined with FM1-43-labeled OHCs (red). Magnifications show terminal branches enwrapping the base of outer hair cells. E, Biocytin-streptavidin-Alexa Fluor 488-filled fiber combined with a CtPB2 immunolabel (red). Magnifications show an approximation of some terminal branches to CtBP2 puncta. F, Combined immunolabel for PSD-95 (green) and CtBP2 (red) among OHCs of young rat cochlea. Magnification shows pearl chain pattern found in adult cochlea. Scale bars: B–G, 5 µm; magnifications, 2.5 µm.