Regenerated synapses between postnatal hair cells and auditory neurons

Abstract

Regeneration of synaptic connections between hair cells and spiral ganglion neurons would be required to restore hearing after neural loss. Here we demonstrate by immunohistochemistry the appearance of afferent-like cochlear synapses in vitro after co-culture of de-afferented organ of Corti with spiral ganglion neurons from newborn mice. The glutamatergic synaptic complexes at the ribbon synapse of the inner hair cell contain markers for presynaptic ribbons and postsynaptic densities. We found postsynaptic density protein PSD-95 at the contacts between hair cells and spiral ganglion neurons in newly formed synapses in vitro. The postsynaptic proteins were directly facing the CtBP2-positive presynaptic ribbons of the hair cells. BDNF and NT-3 promoted afferent synaptogenesis in vitro. Direct juxtaposition of the postsynaptic densities with the components of the preexisting ribbon synapse indicated that growing fibers recognized components of the presynaptic sites. Initiation of cochlear synaptogenesis appeared to be influenced by glutamate release from the hair cell ribbons at the presynaptic site since the synaptic regeneration was impaired in glutamate vesicular transporter 3 mutant mice. These insights into cochlear synaptogenesis could be relevant to regenerative approaches for neural loss in the cochlea.

FIG. 1

Components of afferent synapses in mouse cochlea. A HC ribbon synaspes from a P5 mouse were detected using antibodies against PSD-95 (red), NF (green) and CtBP2 (blue). Ribbons of both IHCs and OHCs were positive for CtBP2 and appeared as puncta. IHC nuclei were also positive for CtBP2. Postsynaptic densities were observed at the endings of SGNs in the IHC region with the PSD-95 antibody. B Representative confocal projections of the basal membranes of two IHCs are shown from a P30 mouse. The ribbon synapses were immunostained with antibodies to PSD-95 (red), GluR2/3, against AMPA receptors (green) and CtBP2 (blue). GluR2/3 and PSD-95 were co-localized, and apposed to the HC ribbons. Scale bars: 10 μm.

FIG. 2

Co-culture of de-afferented organ of Corti with SGNs. A An organ of Corti from a P5 mouse was immunostained with antibodies against NF (green) and myosin VIIa (blue) to label the SGNs and HCs. B A cross-sectional view shows the lines of separation followed with a micro-blade to mechanically axotomize the SGN fibers at the GER region close to IHCs and at the outer sulcus (dashed lines). C A de-afferented organ of Corti (P5) was co-cultured with isolated SGNs for 1 day. D, E De-afferented organs of Corti (P6) were co-cultured with isolated SGNs for 6 days. The fibers of SGNs extended to both IHCs (arrow, D) and OHCs (E). F SGN fibers innervated HCs at their basal surface after 6 days. Nuclei were stained with DAPI (red). Scale bars in A–E: 50 μm, F: 10 μm.

FIG. 3

Newly generated afferent-like cochlear synapses in vitro. A A de-afferented organ of Corti (P5) was co-cultured with isolated SGNs for 6 days. PSD-95-positive puncta were detected at the sites of innervation of the IHCs (arrows). An orthogonal view shows PSD-95 apposed to the basal surface of the IHC, presumably the synaptic zone (a′, dashed line in A). B After 6 days, immunostaining with an antibody against CtBP2 showed afferent-like synapses at the sites of IHC innervation. Enlarged views demonstrate that PSD-95 puncta were directly apposed to the CtBP2-positive presynaptic ribbons of the HCs (B′ and B″). C After 6 days, HC ribbons in the section of the de-afferented organ of Corti that was not innervated by SGNs were preserved, whereas postsynaptic densities were no longer present at synaptic zones.

FIG. 4

Neurotrophins promoted cochlear synaptogenesis. A De-afferented organs of Corti were co-cultured with SGNs. In one group of explants, NT-3 (10 ng/ml) and BDNF (10 ng/ml) were added to the culture. After 6 days, the explants were immunostained with antibodies to PSD-95, NF and CtBP2. Insets show enlarged views of synaptic zones in the yellow boxes. B After 6 days, addition of NTs significantly increased the percentage of IHCs innervated by SGNs in the co-cultured explant (Control, n = 8 explants; NTs, n = 11 explants). C After 6 days, addition of NTs significantly increased the average numbers of new synapses per IHC in the co-cultured explant (Control, n = 59 IHCs; NTs, n = 209 IHCs). *P < 0.05, Student’s t-test. Scale bar: 10 μm.

FIG. 5

Glutamate was not required for cochlear synaptogenesis. A–B De-afferented organs of Corti dissected from wild type (WT) and VGLUT3 knockout (VGLUT3^−/−) littermates were co-cultured with isolated SGNs from C57BL/6J mice for 6 days. PSD-95-positive puncta were detected in both groups. Enlarged views of the synaptic zones demonstrated that postsynaptic densities were formed along the SGN fibers and juxtaposed to the IHC ribbons. C The average number of new synapses generated per IHC was decreased in the explants of VGLUT3^−/− organs of Corti (WT, n = 23; VGLUT3^−/−, n = 23). *P < 0.05, Student’s t-test. Scale bars: 10 μm.