Synaptic profiles during neurite extension, refinement and retraction in the developing cochlea

Abstract

Background: During development, excess synapses form between the central and peripheral nervous systems that are then eliminated to achieve correct connectivity. In the peripheral auditory system, the developing type I spiral ganglion afferent fibres undergo a dramatic re-organisation, initially forming connections with both sensory inner hair cells (IHCs) and outer hair cells (OHCs). The OHC connections are then selectively eliminated, leaving sparse innervation by type II afferent fibres, whilst the type I afferent synapses with IHCs are consolidated.

Results: We examined the molecular makeup of the synaptic contacts formed onto the IHCs and OHCs during this period of afferent fibre remodelling. We observed that presynaptic ribbons initially form at all the afferent neurite contacts, i.e. not only at the expected developing IHC-type I fibre synapses but also at OHCs where type I fibres temporarily contact. Moreover, the transient contacts forming onto OHCs possess a broad set of pre- and postsynaptic proteins, suggesting that functional synaptic connections are formed prior to the removal of type I fibre innervation. AMPA-type glutamate receptor subunits were transiently observed at the base of the OHCs, with their downregulation occurring in parallel with the withdrawal of type I fibres, dispersal of presynaptic ribbons, and downregulation of the anchoring proteins Bassoon and Shank. Conversely, at developing type I afferent IHC synapses, the presence of pre- and postsynaptic scaffold proteins was maintained, with differential plasticity in AMPA receptor subunits observed and AMPA receptor subunit composition changing around hearing onset.

Conclusions: Overall our data show a differential balance in the patterns of synaptic proteins at developing afferent IHC versus OHC synapses that likely reflect their stable versus transient fates.

Figure 1

Visualization of synaptic ribbons during the development of the innervation of IHCs and OHCs by type I and type II afferent fibres respectively. All images are from the mid-turn of the cochlea. Triple labelling showing CtBP2/RIBEYE puncta to mark synaptic ribbons (red), type I nerve fibres labelled with TMRD (blue; A-F) and type II fibres labelled with anti-peripherin (green; G-I) from P0–P6. A-C TMRD-positive type I fibres innervating the IHCs, initially extending to the apical cell regions before consolidating at the basolateral region where the synaptic ribbons and localised. D-F TMRD-positive type I fibres also temporarily innervate the OHCs, localising at the basal region of the OHCs where synaptic ribbons are localised. At P6, the ribbons disperse in parallel with retraction of type I fibres. G-I Peripherin-positive type II fibres innervating the OHCs. The formation of the outer spiral bundles proceeds despite dispersal of the presynaptic ribbons. Scale bar 5 mm.

Figure 2

Developmental changes in CtBP2/RIBEYE labelling in IHCs and OHCs between E18 and adult.A-F Maximal projection confocal images of the Organ of Corti in cross section illustrating the immunofluorescent localisation of CtBP2/RIBEYE-positive puncta (red) superimposed on transmitted light images for anatomical reference. Scale bar: 10 mm. G-L Fluorescence images of CtBP2/RIBEYE immunostaining, without the transmitted light images, to highlight the changes in CtBP2/RIBEYE over development. M,N Quantification of the changes in the number of CtBP2/RIBEYE puncta per (M) IHCs and (N) OHCs at P0, P3, P6, P12 and adult. *p < 0.05, ***p < 0.001.

Figure 3

3D reconstruction of the ribbons in OHCs during cochlear development.A-C Confocal images of one row of whole mount cochlear preparations immunostained against CtBP2/RIBEYE at (A) P3, (B) P6, and (C) P12 were deconvolved prior to 3D reconstruction (Image Pro Plus with 3D suite). Numbers on the X and Y axis represent mm in the Y and Z direction respectively. The 3D reconstructions reveal the spatial distribution of the CtBP2/RIBEYE puncta in the OHCs, which were highly concentrated in the basal synaptic region of the OHCs at P3, before dispersing at P6 and then significantly downregulated by P12. The lateral and medial sides of the OHCs are at the left and right sides of each image respectively. The first row of OHCs is on the right side and the third row of OHCs on the left side of each image.

Figure 4

Developmental changes in presynaptic Bassoon in IHCs and OHCs.A-E Maximal projection confocal images of the Organ of Corti in cross section illustrating the immunofluorescent localisation of Bassoon (green) and CtBP2/RIBEYE positive puncta (red) superimposed on transmitted light images for anatomical reference from P0 to adult. Scale bar 10 mm. F-J Fluorescence images of Bassoon and CtBP2/RIBEYE immunostaining, without the transmitted light images, to highlight the developmental changes. K-P Quantification of the developmental changes in the total number of Bassoon puncta (K,N), the percentage of synaptic Bassoon puncta as measured by co-localisation with CtBP2 puncta (L,O), and the intensity changes in synaptic Bassoon puncta (M,P), in IHCs (K,L,M) and OHCs (N,O,P) respectively. In all cases n = 6 animals. *p < 0.05, **p < 0.005, ***p < 0.001.

Figure 5

Changes in GluA2/3 subunits in IHCs and OHCs during development.A-E Example fluorescent whole-mount images of GluA2/3 (green) and CtBP2/RIBEYE labelling (red). Scale bar 5 mm. Grey dotted lines delineate the OHC and IHC regions. Boxed regions from both the IHC (left) and OHC (right) regions are shown below each image for each age. F Quantification of the total number of GluA2/3 puncta in IHCs between P0 and adult. G Quantification of the developmental changes in synaptic GluA2/3 in IHCs, as assessed by per cent co-localisation with CtBP2/RIBEYE puncta. H Quantification of the total number of GluA2/3 puncta in OHCs between P0 and adult. I Quantification of the developmental changes in synaptic GluA2/3 in OHCs, as assessed by per cent co-localisation with CtBP2/RIBEYE puncta. In all graphs, data are represented as mean ± SEM; n ≥ 5 in all age groups. *p < 0.05, ***p < 0.001.

Figure 6

Changes in GluA4 subunit in IHCs and OHCs during development.A-E Example maximal projection whole-mount confocal images of GluA4 (green) and CtBP2 (red) immunolabelling from P0 to adult. Scale bar 10 mm. Grey dotted lines delineate the OHC and IHC regions. F Quantification of the average total number of GluA4 puncta per IHC from P0 to adult. G Quantification of the developmental changes in synaptic GluA4, as assessed by per cent co-localisation with CtBP2/RIBEYE puncta, in IHCs. H Average total number of GluA4 puncta per OHC from P0 to adult. I Percent of synaptic GluA4, as measured by per cent co-localisation with CtBP2/RIBEYE puncta, from P0 to adult. In all graphs, data are represented as mean ± SEM; n ≥ 5 in all age groups. **p < 0.005, ***p < 0.001.

Figure 7

Developmental profile of Shank1 at IHCs and OHCs in the organ of Corti.A-D Example fluorescent whole-mount images revealing immunolabelling of Shank1 (green) and CtBP2/RIBEYE (red). Shank1 is evident at the base of IHCs from P0 through to adulthood. Shank1 is significantly weaker in the OHCs. The strongest Shank1 expression is seen in OHCs at P3 followed by downregulation such that in the adult no Shank1 was observed. Scale bar 10 mm. Grey dotted lines delineate the OHC and IHC regions. In each image, the area denoted by the white square is shown in the magnified form below: Shank1 alone (green, LHS) and overlay of Shank1 and CtBP2 (red, RHS). E-J Quantification of total Shank1 puncta number (E,H), Shank1 puncta volume (F,I) and per cent Shank1 co-localised with CtBP2/RIBEYE (G,J) in IHCs and OHCs from P0 to adult (n = 6 for all age groups). *p < 0.05, **p < 0.01, **p < 0.005.