Our study reveals a unique role for the L-type calcium channel CaV1.3a in regulating Ribeye assembly and maintaining juxtaposition of synaptic components in developing zebrafish hair cells. Modulation of ribbon-synapse morphology by CaV1.3a and a requirement for alignment of synaptic components is supported by our findings that (1) genetically disrupting or pharmacologically blocking CaV1.3a produces enlarged synaptic ribbons and less refined ribbon synapses (i.e., changes in synaptic ribbon shape and a greater number of synaptic ribbons per single PSD), (2) pharmacologically activating L-type calcium channels leads to smaller or absent synaptic ribbons, (3) cav1.3a mutants show a progressive loss of presynaptic and postsynaptic juxtaposition, and (4) mature hair-cell synapses are not susceptible to short-term pharmacological block of CaV1.3a.
These data, combined with our previous study, reveal an interplay between Ribeye and CaV
1.3 that is necessary for proper hair-cell ribbon synapse formation and maturation. We propose a model by which Ribeye-containing aggregates initially accumulate at the basolateral end of hair cells to form synaptic ribbons that stabilize afferent-nerve-fiber contacts and cluster CaV
1.3 channels (Sheets et al., 2011
1.3 channels then regulate synaptic-ribbon size during a critical period of development and contribute to the refinement and maintenance of synaptic contacts.
Several of our observations support a mechanism by which Ca2+ influx through CaV1.3 channels regulates the assembly of Ribeye protein, thereby affecting synaptic-ribbon size and morphology. Acute block of CaV1.3a during a critical time window of hair-cell maturation results in a rapid increase in presynaptic accumulations of both zebrafish isoforms of Ribeye, whereas activation of L-type calcium channels generally produces a decrease in presynaptic Ribeye. In contrast, we see no significant difference in the level of ribeye b transcripts in drug-treated larvae, suggesting that the changes in Ribeye intensity we observed in drug-exposed larvae were not attributable to regulation of ribeye at the transcriptional level. Additionally, in larvae with either mutant allele of cav1.3a, we observed a significantly greater number of Ribeye aggregates in the hair-cell body but no difference in the number of synaptic ribbons. These data suggest that Ca2+ influx though CaV1.3a may propagate Ca2+ signaling through-out the hair cell (e.g., Ca2+ induced Ca2+ release from ER stores) that regulates not only the accumulation of Ribeye at the synapse but also assembly of synaptic-ribbon precursors.
Previous studies of hair-cell synapses in other species have reported that synaptic ribbon size positively correlates with calcium influx (Martinez-Dunst et al., 1997
; Schnee et al., 2005
; Frank et al., 2009
). In relation to these studies, our results initially seem paradoxical; how is it that we observe enlarged ribbons when calcium influx is blocked? A key observation in our study is that Ca2+
-mediated changes in synaptic ribbon size occur during a critical window of hair-cell development. In our experiments, the plasticity of hair-cell ribbons was apparent only during early developmental stages, which were not examined in the previous studies referenced above. However, in agreement with the descriptions of mature synapses in other species, we observe that relatively mature hair-cell synapses at 5 dpf are not susceptible to acute pharmacological block of CaV
1.3a. The actual source of heterogeneity of the size of hair-cell ribbon bodies is not clear. We speculate that larger cytosolic aggregates of Ribeye or early attachment of Ribeye aggregates before calcium currents peak may generate larger ribbon bodies that are able to recruit additional calcium channels to the ribbon synapse (Frank et al., 2010
; Sheets et al., 2011
). Such a scenario could explain why larger ribbons showed greater calcium influx in mature hair cells (Frank et al., 2009
Considering that we observe a similar phenomenon in pinealocytes as we do in hair cells—namely, that pharmacological manipulation of L-type calcium channels modulates presynaptic Ribeye accumulation—we propose that Ca2+
influx through L-type calcium channels may regulate synaptic-ribbon morphology in other ribbon synapse-containing cell types. Previous ultra-structural studies of pineal organ and photoreceptor synapses have shown that synaptic ribbons are dynamic structures whose size and shape change in response to illumination (Vollrath and Spiwoks-Becker, 1996
; Spiwoks-Becker et al., 2004
) or diurnal cycle (Hull et al., 2006
; Spiwoks-Becker et al., 2008
). Moreover, recent studies report that manipulating internal Ca2+
levels with a chelator or ionophore also produces structural changes in photoreceptor synaptic ribbons (Spiwoks-Becker et al., 2004
; Regus-Leidig et al., 2010
), but the sources of intracellular Ca2+
were not identified. Our results point to presynaptic L-type Ca2+
channels as the initial source of Ca2+
that mediates dynamic changes in synaptic-ribbon morphology. Additional studies identifying downstream targets of Ca2+
influx may not only reveal essential signaling pathways for hair-cell synapse maturation but also uncover mechanisms of ribbon-synapse plasticity in other cell types.
Overall, both our genetic and pharmacological evidence support the idea that Ca2+
influx modulates the size, morphology, and, to some extent, the number of synaptic ribbons at active zones. How CaV
1.3a refines the synapse and maintains the juxta-position of presynaptic and postsynaptic components in hair cells is less clear. The function of CaV
1.3a in synaptic maintenance appears to be independent of its role in synaptic transmission, because vglut3
mutants do not show a similar phenotype. Because we observed a failure to maintain synaptic alignment in R284C
larvae, wherein nonconducting CaV
1.3a channels localize correctly to synaptic ribbons, we propose that the physical presence of CaV
1.3a is not sufficient to maintain postsynaptic juxtaposition. Instead, the phenotype indicates that Ca2+
influx through CaV
1.3a may be mediating yet-to-be identified intracellular processes required for synaptic maintenance. Accordingly, we tested whether long-term block would result in loss of juxtaposition by exposing WT larvae to isradipine overnight, but synaptic juxtaposition was unaffected (). This result suggests that either long-term block was not able to phenocopy the effects of congenital loss of CaV
1.3a or that CaV
1.3a may indeed play a structural role in maintaining ribbon synapses. Interestingly, the R284C
amino acid substitution is within an extracellular loop of CaV
1.3a (IS5–IS6), raising the possibility that this extracellular loop may interact with postsynaptic components. A similar interaction has been reported for the neuromuscular junction (Nishimune et al., 2004
; Chen et al., 2011
). At this type of synapse, the direct interaction of an extracellular loop of presynaptic P/Q-type and N-type voltage-gated calcium channels with muscle-derived laminin β
2 is required for proper active-zone organization. Additional investigation may address whether CaV
1.3a channel function or postsynaptic protein interaction with the IS5–IS6 extracellular loop is critical for maintaining synaptic alignment.
In conclusion, our results reveal several important roles for CaV1.3a in both the maturation and maintenance of hair-cell ribbon synapses. Future studies exploring the downstream mechanisms of the mediation of synaptic-ribbon size by CaV1.3 channel may shed light on not only hair-cell synaptic maturation but also reveal a general mechanism of ribbon-synapse plasticity relevant for synaptic function.