The contacts between the membranes of type I hair cells and the neuronal calyx appear in thin section electron micrographs as two conspicuously electron dense membranes separated by a very regular space (). In close up views of these calyceal contacts we observe periodic electron dense elements that project from the juxtaposed surfaces of the contacting plasma membranes into the intercellular space and often appear to form cross-bridges ( inset). The regular spacing, enhanced electron density of the membranes, and the periodic cross bridges, are all common structural features observed in intercellular junctions(Furuse and Tsukita, 2006
). Freeze-fracture images of the plasma membrane of vestibular type-I hair cells show IMPs organized in parallel linear arrays (, arrows). These parallel linear arrays of IMPs resemble the IMP organization of SJs (Kachar et al., 1986
) but an optimal alignment of the section with the orientation of this linear array of particles is necessary for visualization of the cross-bridges in thin section electron micrographs. We tested by immunofluorescence for the presence of Caspr. Caspr immunoreactivity was observed at the contacts between the calyx and type I hair cells in mouse, rat (), and guinea pig. The Caspr immunofluorescence at the calyx was even stronger than that at the hemiparanodes (). To quantify the cellular distribution of Caspr immunolabeling in the calyceal contacts with the hair cells, we counted gold particles along the membranes of both sides of the contacts and found that 86% of the total gold particles (n=398) was associated with the nerve membrane ().
Septate junction-like features and localization of Caspr at type I hair cell calyceal synapses
The structural features of the contact between the type I hair cells and the calyx and the presence of Caspr suggest that this contact is supported by a septate-like junction. Previous ultrastructural studies have shown disruption of paranodal SJs in Caspr-/-
mouse (Bhat et al., 2001
). To explore the role of Caspr in calyceal contacts, we performed thin section electron microscopy of utricle and ampullae of Caspr-/-
. When comparing with Caspr+/+
, we do not detect changes in the overall ultrastructural morphology of Caspr-/-
sensory epithelium, including the calyceal terminals and the hair cells (data not shown). However, in the Caspr-/-
, the membranes of the calyceal contact do not exhibit the characteristic electron dense appearance and the gap between the membranes is very irregular and often significantly enlarged (). This enlargement of the intercellular space was specific to the calyceal synapses, as we did not observe swellings at the contacts between the same calyx and the supporting cells (). We measured the gap between the membranes at the calyceal synapses; it averaged 28 ± 4 nm (n = 279) in Caspr+/+
, but this value increased to 64 ± 55 nm (n = 436) in Caspr-/-
. The box plot of the data () shows the increased variability in the separation between the membranes. These results indicate that Caspr is required for maintaining the regular separation between hair cells and the calyx membranes and for recruiting other proteins that produce the characteristic electron dense profile of these membranes.
Ablation of Caspr leads to disruption of the calyceal synaptic contact
KCNQ4 is a potassium channel enriched at the calyceal synapses. In the adult rat, its major expression is in the calyx, but early in development it is expressed in both hair cells and the calyx (Hurley et al., 2006
). In order to determine if KCNQ4 localization is related or dependent on Caspr we performed a co-immunofluorescence localization assay. Confocal images tangential to the calyceal junction show a remarkable colocalization of these two proteins in adult rat (), but clustering of Caspr at these junctions preceded that of KCNQ4 in the first postnatal days (data not shown). Previous studies have shown that ion channels at the paranodal membranes are mislocalized in Caspr-/-
(Bhat et al., 2001
). Using immunofluorescence, we analyzed the distribution of KCNQ4 in the vestibular system of adult Caspr-/-
. Whereas Caspr colocalizes with KCNQ4 at the calyceal synapses of Caspr+/+
(), Caspr was not detected in the Caspr-/-
with the same experimental conditions and the distribution of KCNQ4 in the calyx in Caspr-/-
was notably faint and with a more disperse pattern of localization (, ). We analyzed the distribution of KCNQ4 labeling in the calyceal contacts by quantifying the number of KCNQ4 immunogold particles at these contacts and determining the percentage of particles associated with the membranes of hair cells and the nerve calyx for both genotypes. In Caspr+/+
, the frequency of gold particles associated with the calyx inner face (0.54 particles/μm, n=100) was higher than that associated with the hair cell (0.11 particles/μm) or the calyx outer face (0.09 particles/μm). In contrast, in Caspr-/-
, the frequency of gold particles associated with the calyx outer face (0.69 particles/μm, n=90) was higher than that associated with the calyx inner face (0.47 particles/μm) and the hair cells (0.17 particles/μm). These data confirm that KCNQ4 is indeed redistributed to outer face of the calyx of Caspr-/-
(), as illustrated in the diagrams (). Together, these results demonstrate that Caspr is necessary for recruiting or retaining KCNQ4 at the calyceal synaptic contacts.
Localization of KCNQ4 at the calyceal membrane depends on Caspr
Ablation of Caspr leads to decreases in KCNQ4 expression and its redistribution in the calyx membranes
To characterize the redistribution of KCNQ4, we quantitatively compared the distribution of KCNQ4 immunofluorescence at the inner and outer faces of Caspr+/+ and Caspr-/- calyces. While we detected KCNQ4 immunofluorescence at the outer face of virtually all Caspr-/- calyces, KCNQ4 immunofluorescence was detected in only around 15% of Caspr+/+ calyces (n=82 cells). This result per se indicates a redistribution of KCNQ4 immunofluorescence in the calyx membranes. In order to evaluate the redistribution of KCNQ4 immunoreactivity, we compared profiles of fluorescence intensity spanning inner and outer faces of calyces in Caspr+/+ and Caspr-/-. For that we subtracted the fluorescence background and used as a parameter the ratio (R) between the maximum values for the fluorescence peaks corresponding to the inner (i) and outer (o) faces of each measured calyx (for each individual cell, R=[i+i']/[o+o']) and found that Caspr+/+ indeed displays much higher R values (Rwt=9.8±7.8, n=10) than Caspr-/- (Rko=1.3±0.4, n=28). A representative example of this comparative analysis is shown in . The profiles clearly illustrate that KCNQ4 immunofluorescence is highly enriched at the inner face of Caspr+/+ calyces while it is much reduced in the Caspr-/- calyces. The quantitative immunofluorescence and immunoEM data further support the model depicted in , in which ablation of Caspr leads to decreases in KCNQ4 expression accompanied by the redistribution of these channels at the membranes of the calyx.