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Preferentially regulated expression of connexin 43 in the developing spiral ganglion neurons and afferent terminals in post-natal rat cochlea.

Liu WJ, Yang J - Eur J Histochem (2015)

Bottom Line: We further performed quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) to identify the presence of Cx43 mRNA in the modiolus (mainly containing SGNs).Cx43 mRNA was higher at P8, compared with P1, and subsequently decreased at P14.These results indicated that Cx43 correlated with cochlear synaptogenesis and establishment of auditory neurotransmission.

View Article: PubMed Central - PubMed

Affiliation: Xinhua Hospital, Shanghai Jiaotong University, Shanghai Jiaotong University Ear Institute. Liuwj38@163.com.

ABSTRACT
The expression pattern of connexin 43 (Cx43) in the cochlea is not determined and is controversial. Since the presence of Cx43 is essential for hearing, we re-examined its distribution during postnatal development of rat cochlea. Cx43 protein was expressed in spiral ganglion neurons (SGNs) and their neurite terminals innervating the inner and outer hair cells (IHCs and OHCs) as early as birth (post-natal day 0, P0), and persisted until P14. Double immunofluorescence staining, using two antibodies against Cx43 and TUJ1, a marker for all SGNs and afferent terminals, showed that immunoreactivity for Cx43 and TUJ1 was perfectly colocalized in SGNs and afferent terminals associated with the IHCs and OHCs. However, beyond P14, Cx43 immunostaining could no longer be detected in the region of the synaptic terminals at the bases of IHCs and OHCs (P17, adult). In contrast, Cx43 maintained its expression in SGNs into adulthood. We further performed quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) to identify the presence of Cx43 mRNA in the modiolus (mainly containing SGNs). Cx43 mRNA was higher at P8, compared with P1, and subsequently decreased at P14. These results indicated that Cx43 correlated with cochlear synaptogenesis and establishment of auditory neurotransmission.

No MeSH data available.


Related in: MedlinePlus

Cx43 and TUJ1 immunolabeling and double labeling for Cx43 and TUJ1 in the mid turn of the rat cochlea at P8, P14 and the merged image +DAPI. A,B,C) An overview of Cx43 (green) and TUJ1 (red) labeling in the mid turn of the rat cochlea at P8 and the merged image +DAPI; Note that Cx43 immunolabeling is first present in the interdental cells (idc) of the spiral limbus (sb), and disappears from the intra-ganglion spiral bundle (igsb), where only TUJ1 labeling is detected in the igsb, and no overlap between Cx43 and TUJ1 immunoreactivity is found; Cx43 immunoreactivity includes the spiral ganglion neurons (sgns), the inner spiral plexus (isp), and outer spiral bundles (osb), the idcs of the sb, and a few fibrocytes of the spiral ligament (sl). D,E,F) Details of Cx43 (green) and TUJ1 (red) expression in the organ of Corti in the mid turn of P8 rats and the merged image +DAPI; There is a substantial overlap (yellow) between the distribution of Cx43 and TUJ1 immunolabeling in the nerve terminals below the inner hair cells (ihcs) and the three rows of outer hair cells (ohcs), except for TUJ1-labeled tunnel crossing fibers (tcfs - shown in red), which cross the tunnel of Corti to contact the ohcs. G,H,I) An overview of Cx43 (green) and TUJ1 (red) labeling in the mid turn of the rat cochlea at P14; sgns and afferent terminals innervating the ihcs and ohcs (as identified with TUJ1 labeling) show pronounced Cx43 immunolabeling; Cx43 overlaps perfectly with TUJ1 in the sgns, the inner spiral plexus (isps) and outer spiral bundles (osbs). J,K,L) Details of Cx43 and TUJ1 expression in the organ of Corti in the mid turn of P14 rats and the merged image +DAPI; note that anti-Cx43- and anti-TUJ1 labeled three slender processes contacting both ohcs and Deiters’ cells which approach the basilar membrane; merged confocal images showing a substantial overlap (yellow) of the expression of Cx43 and TUJ1 labeling below the ihcs and beneath the three rows of ohcs. sv, stria vascularis; os, outer sulcus cells; igsb, intra-ganglion spiral bundle.
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fig006: Cx43 and TUJ1 immunolabeling and double labeling for Cx43 and TUJ1 in the mid turn of the rat cochlea at P8, P14 and the merged image +DAPI. A,B,C) An overview of Cx43 (green) and TUJ1 (red) labeling in the mid turn of the rat cochlea at P8 and the merged image +DAPI; Note that Cx43 immunolabeling is first present in the interdental cells (idc) of the spiral limbus (sb), and disappears from the intra-ganglion spiral bundle (igsb), where only TUJ1 labeling is detected in the igsb, and no overlap between Cx43 and TUJ1 immunoreactivity is found; Cx43 immunoreactivity includes the spiral ganglion neurons (sgns), the inner spiral plexus (isp), and outer spiral bundles (osb), the idcs of the sb, and a few fibrocytes of the spiral ligament (sl). D,E,F) Details of Cx43 (green) and TUJ1 (red) expression in the organ of Corti in the mid turn of P8 rats and the merged image +DAPI; There is a substantial overlap (yellow) between the distribution of Cx43 and TUJ1 immunolabeling in the nerve terminals below the inner hair cells (ihcs) and the three rows of outer hair cells (ohcs), except for TUJ1-labeled tunnel crossing fibers (tcfs - shown in red), which cross the tunnel of Corti to contact the ohcs. G,H,I) An overview of Cx43 (green) and TUJ1 (red) labeling in the mid turn of the rat cochlea at P14; sgns and afferent terminals innervating the ihcs and ohcs (as identified with TUJ1 labeling) show pronounced Cx43 immunolabeling; Cx43 overlaps perfectly with TUJ1 in the sgns, the inner spiral plexus (isps) and outer spiral bundles (osbs). J,K,L) Details of Cx43 and TUJ1 expression in the organ of Corti in the mid turn of P14 rats and the merged image +DAPI; note that anti-Cx43- and anti-TUJ1 labeled three slender processes contacting both ohcs and Deiters’ cells which approach the basilar membrane; merged confocal images showing a substantial overlap (yellow) of the expression of Cx43 and TUJ1 labeling below the ihcs and beneath the three rows of ohcs. sv, stria vascularis; os, outer sulcus cells; igsb, intra-ganglion spiral bundle.

Mentions: To verify our fixative protocol, anti-Cx26 antibody was used for staining. Punctate labeling of Cx26 was detected in the basal cells in the stria vascularis, all fibrocytes of the spiral ligament, and between supporting cells in the organ of Corti (data not shown), consistent with a previous study.11 This was the first evidence, based on immunofluorescent localization, that Cx43 was present in the neurite projections to the hair cell region, from birth to P14, where there was extensive colocalization between Cx43 and TUJ1 in double-labeling experiments. TUJ1, a specific marker of neural cells,21-23 provided an excellent tool for the visualization of immature and mature type I and type II SGNs and their neurite projections associated with both IHCs and OHCs.17, 24-26 The present study confirmed previous findings in the mammalian cochlea, that TUJ1 was strongly and exclusively expressed in the developing and mature SGNs and the afferent terminals below OHCs and IHCs. We extended these findings by demonstrating that the intra-ganglion spiral bundle (IGSB) in the rat, which represents the efferent system, showed positive immunoreactivity to TUJ1 throughout postnatal stages of development, while TUJ1 immunoreactivity had been observed in human intra-ganglion spiral bundle.27 In addition, immunoreactivity for Cx43 and TUJ1 was colocalized in the IGSB (from P0 to P5) within the spiral ganglion. At P0, the earliest stage that we studied, a gradient of Cx43 expression levels in different cochlear turns was evident. The projection of TUJ1-positive afferent fibers into the organ of Corti also followed the same kind of gradients. At the apical turn, prominent Cx43 immunolabeling (in green) was uniformly distributed in the almost all SGNs, their distal neurites projecting to the lesser epithelial ridge, and the region of future IHCs. Similarly, TUJ1 (in red) showed strong labeling in the SGNs and the neurite projections to the IHC region. Merged images showed that virtually complete overlap (in yellow) of both fluorescence signals in the area mentioned, indicating colocalization of Cx43 and TUJ1 (Figure 2 A-I). No positive immunostaining was detected in the negative control (Figure 2 J-L). In the middle turn, in addition to Cx43-positive nerve terminals beneath the IHC, Cx43 immunoreactivity was first detected in the OHC region, coinciding perfectly with the first appearance of TUJ1 expression in the OHC region. SGNs and IGSB were strongly positive to Cx43 and TUJ1, where colocalization of TUJ1 with Cx43 was also seen (Figure 3 A-I). In the basal turn, Cx43-immunopositive nerve terminals became well defined beneath IHCs and the three rows of OHCs, corresponding to the TUJ1-specific labeling of inner spiral plexus and outer spiral bundle, respectively. Merged images revealed Cx43 colocalized extensively with TUJ1 in the outer spiral bundle and inner spiral plexus, as well as most SGNs and IGSB (Figure 3 J-O). At P5, a gradient of expression vanished, both Cx43- and TUJ1-expressing outer spiral bundle at the base of OHCs became well defined with fibers reaching among the Deiters’ cell in the apical turn, accompanied by stronger expression in the inner spiral plexus (Figure 4 A-I). No positive immunostaining was detected in the negative control (Figure 4 J-L). In the mid and basal region, Cx43- and TUJ1-labeled inner spiral plexus formed a calyceal-type innervation to the IHCs, and outer spiral bundles beneath all three rows of OHCs appeared cup-like, with almost all SGNs and IGSB being Cx43-positive. TUJ1 was perfectly colocalized in these sites (Figure 5 A-O). Because the gradient of expression was not observed at P5, the processing of our sections precluded analysis of apical and basal turn tissue at subsequent developmental stages, and we focused on the expression pattern of Cx43 in the mid turn of the cochlea. In the mid turn of P8 rat, we observed the absence of Cx43 immunoreactivity in the IGSB, and Cx43 immunolabeling was first detected in the interdental cells of the spiral limbus. Punctate Cx43 and TUJ1 immunolabeling was detected at the base of the OHCs. The inner spiral plexus labeling was more restricted to the basolateral region of the IHCs. Merged images showed a substantial overlap between the distribution of Cx43-positive and TUJ1-positive nerve terminals below the IHCs and OHCs (Figure 6 A-F). By P14, three parallel rows of Cx43- and TUJ1-positive outer spiral bundles extended from the base of the OHCs to the basilar membrane. The strong staining of the inner spiral plexus indicated innervation of the basolateral region of the IHCs. Double immunofluorescent staining exhibited Cx43 staining colocalizing nearly completely with TUJ1 staining in the inner spiral plexus and outer spiral bundles, and SGNs (Figure 6 G-L). From P14 onward, as shown for P17, regulation of Cx43 expression occurred. The inner spiral plexus and outer spiral bundles did not show any immunoreactivity for Cx43, as shown by the absence of colocalization of immunolabeling for Cx43 and TUJ1 in the synaptic region associated with the organ of Corti, but robust Cx43 immunolabeling remained in the SGNs. Type II fibrocytes of the spiral ligament and interdental cells maintained strong expression of Cx43 (Figure 7 A-I). In the adult, the overall pattern of Cx43 immunostaining was similar to that found at P17, and Cx43 was still constrained to SGNs. Omission of primary antisera resulted in loss of these specific labeling profiles in adult cochlear tissues (Figure 8 E-F).


Preferentially regulated expression of connexin 43 in the developing spiral ganglion neurons and afferent terminals in post-natal rat cochlea.

Liu WJ, Yang J - Eur J Histochem (2015)

Cx43 and TUJ1 immunolabeling and double labeling for Cx43 and TUJ1 in the mid turn of the rat cochlea at P8, P14 and the merged image +DAPI. A,B,C) An overview of Cx43 (green) and TUJ1 (red) labeling in the mid turn of the rat cochlea at P8 and the merged image +DAPI; Note that Cx43 immunolabeling is first present in the interdental cells (idc) of the spiral limbus (sb), and disappears from the intra-ganglion spiral bundle (igsb), where only TUJ1 labeling is detected in the igsb, and no overlap between Cx43 and TUJ1 immunoreactivity is found; Cx43 immunoreactivity includes the spiral ganglion neurons (sgns), the inner spiral plexus (isp), and outer spiral bundles (osb), the idcs of the sb, and a few fibrocytes of the spiral ligament (sl). D,E,F) Details of Cx43 (green) and TUJ1 (red) expression in the organ of Corti in the mid turn of P8 rats and the merged image +DAPI; There is a substantial overlap (yellow) between the distribution of Cx43 and TUJ1 immunolabeling in the nerve terminals below the inner hair cells (ihcs) and the three rows of outer hair cells (ohcs), except for TUJ1-labeled tunnel crossing fibers (tcfs - shown in red), which cross the tunnel of Corti to contact the ohcs. G,H,I) An overview of Cx43 (green) and TUJ1 (red) labeling in the mid turn of the rat cochlea at P14; sgns and afferent terminals innervating the ihcs and ohcs (as identified with TUJ1 labeling) show pronounced Cx43 immunolabeling; Cx43 overlaps perfectly with TUJ1 in the sgns, the inner spiral plexus (isps) and outer spiral bundles (osbs). J,K,L) Details of Cx43 and TUJ1 expression in the organ of Corti in the mid turn of P14 rats and the merged image +DAPI; note that anti-Cx43- and anti-TUJ1 labeled three slender processes contacting both ohcs and Deiters’ cells which approach the basilar membrane; merged confocal images showing a substantial overlap (yellow) of the expression of Cx43 and TUJ1 labeling below the ihcs and beneath the three rows of ohcs. sv, stria vascularis; os, outer sulcus cells; igsb, intra-ganglion spiral bundle.
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fig006: Cx43 and TUJ1 immunolabeling and double labeling for Cx43 and TUJ1 in the mid turn of the rat cochlea at P8, P14 and the merged image +DAPI. A,B,C) An overview of Cx43 (green) and TUJ1 (red) labeling in the mid turn of the rat cochlea at P8 and the merged image +DAPI; Note that Cx43 immunolabeling is first present in the interdental cells (idc) of the spiral limbus (sb), and disappears from the intra-ganglion spiral bundle (igsb), where only TUJ1 labeling is detected in the igsb, and no overlap between Cx43 and TUJ1 immunoreactivity is found; Cx43 immunoreactivity includes the spiral ganglion neurons (sgns), the inner spiral plexus (isp), and outer spiral bundles (osb), the idcs of the sb, and a few fibrocytes of the spiral ligament (sl). D,E,F) Details of Cx43 (green) and TUJ1 (red) expression in the organ of Corti in the mid turn of P8 rats and the merged image +DAPI; There is a substantial overlap (yellow) between the distribution of Cx43 and TUJ1 immunolabeling in the nerve terminals below the inner hair cells (ihcs) and the three rows of outer hair cells (ohcs), except for TUJ1-labeled tunnel crossing fibers (tcfs - shown in red), which cross the tunnel of Corti to contact the ohcs. G,H,I) An overview of Cx43 (green) and TUJ1 (red) labeling in the mid turn of the rat cochlea at P14; sgns and afferent terminals innervating the ihcs and ohcs (as identified with TUJ1 labeling) show pronounced Cx43 immunolabeling; Cx43 overlaps perfectly with TUJ1 in the sgns, the inner spiral plexus (isps) and outer spiral bundles (osbs). J,K,L) Details of Cx43 and TUJ1 expression in the organ of Corti in the mid turn of P14 rats and the merged image +DAPI; note that anti-Cx43- and anti-TUJ1 labeled three slender processes contacting both ohcs and Deiters’ cells which approach the basilar membrane; merged confocal images showing a substantial overlap (yellow) of the expression of Cx43 and TUJ1 labeling below the ihcs and beneath the three rows of ohcs. sv, stria vascularis; os, outer sulcus cells; igsb, intra-ganglion spiral bundle.
Mentions: To verify our fixative protocol, anti-Cx26 antibody was used for staining. Punctate labeling of Cx26 was detected in the basal cells in the stria vascularis, all fibrocytes of the spiral ligament, and between supporting cells in the organ of Corti (data not shown), consistent with a previous study.11 This was the first evidence, based on immunofluorescent localization, that Cx43 was present in the neurite projections to the hair cell region, from birth to P14, where there was extensive colocalization between Cx43 and TUJ1 in double-labeling experiments. TUJ1, a specific marker of neural cells,21-23 provided an excellent tool for the visualization of immature and mature type I and type II SGNs and their neurite projections associated with both IHCs and OHCs.17, 24-26 The present study confirmed previous findings in the mammalian cochlea, that TUJ1 was strongly and exclusively expressed in the developing and mature SGNs and the afferent terminals below OHCs and IHCs. We extended these findings by demonstrating that the intra-ganglion spiral bundle (IGSB) in the rat, which represents the efferent system, showed positive immunoreactivity to TUJ1 throughout postnatal stages of development, while TUJ1 immunoreactivity had been observed in human intra-ganglion spiral bundle.27 In addition, immunoreactivity for Cx43 and TUJ1 was colocalized in the IGSB (from P0 to P5) within the spiral ganglion. At P0, the earliest stage that we studied, a gradient of Cx43 expression levels in different cochlear turns was evident. The projection of TUJ1-positive afferent fibers into the organ of Corti also followed the same kind of gradients. At the apical turn, prominent Cx43 immunolabeling (in green) was uniformly distributed in the almost all SGNs, their distal neurites projecting to the lesser epithelial ridge, and the region of future IHCs. Similarly, TUJ1 (in red) showed strong labeling in the SGNs and the neurite projections to the IHC region. Merged images showed that virtually complete overlap (in yellow) of both fluorescence signals in the area mentioned, indicating colocalization of Cx43 and TUJ1 (Figure 2 A-I). No positive immunostaining was detected in the negative control (Figure 2 J-L). In the middle turn, in addition to Cx43-positive nerve terminals beneath the IHC, Cx43 immunoreactivity was first detected in the OHC region, coinciding perfectly with the first appearance of TUJ1 expression in the OHC region. SGNs and IGSB were strongly positive to Cx43 and TUJ1, where colocalization of TUJ1 with Cx43 was also seen (Figure 3 A-I). In the basal turn, Cx43-immunopositive nerve terminals became well defined beneath IHCs and the three rows of OHCs, corresponding to the TUJ1-specific labeling of inner spiral plexus and outer spiral bundle, respectively. Merged images revealed Cx43 colocalized extensively with TUJ1 in the outer spiral bundle and inner spiral plexus, as well as most SGNs and IGSB (Figure 3 J-O). At P5, a gradient of expression vanished, both Cx43- and TUJ1-expressing outer spiral bundle at the base of OHCs became well defined with fibers reaching among the Deiters’ cell in the apical turn, accompanied by stronger expression in the inner spiral plexus (Figure 4 A-I). No positive immunostaining was detected in the negative control (Figure 4 J-L). In the mid and basal region, Cx43- and TUJ1-labeled inner spiral plexus formed a calyceal-type innervation to the IHCs, and outer spiral bundles beneath all three rows of OHCs appeared cup-like, with almost all SGNs and IGSB being Cx43-positive. TUJ1 was perfectly colocalized in these sites (Figure 5 A-O). Because the gradient of expression was not observed at P5, the processing of our sections precluded analysis of apical and basal turn tissue at subsequent developmental stages, and we focused on the expression pattern of Cx43 in the mid turn of the cochlea. In the mid turn of P8 rat, we observed the absence of Cx43 immunoreactivity in the IGSB, and Cx43 immunolabeling was first detected in the interdental cells of the spiral limbus. Punctate Cx43 and TUJ1 immunolabeling was detected at the base of the OHCs. The inner spiral plexus labeling was more restricted to the basolateral region of the IHCs. Merged images showed a substantial overlap between the distribution of Cx43-positive and TUJ1-positive nerve terminals below the IHCs and OHCs (Figure 6 A-F). By P14, three parallel rows of Cx43- and TUJ1-positive outer spiral bundles extended from the base of the OHCs to the basilar membrane. The strong staining of the inner spiral plexus indicated innervation of the basolateral region of the IHCs. Double immunofluorescent staining exhibited Cx43 staining colocalizing nearly completely with TUJ1 staining in the inner spiral plexus and outer spiral bundles, and SGNs (Figure 6 G-L). From P14 onward, as shown for P17, regulation of Cx43 expression occurred. The inner spiral plexus and outer spiral bundles did not show any immunoreactivity for Cx43, as shown by the absence of colocalization of immunolabeling for Cx43 and TUJ1 in the synaptic region associated with the organ of Corti, but robust Cx43 immunolabeling remained in the SGNs. Type II fibrocytes of the spiral ligament and interdental cells maintained strong expression of Cx43 (Figure 7 A-I). In the adult, the overall pattern of Cx43 immunostaining was similar to that found at P17, and Cx43 was still constrained to SGNs. Omission of primary antisera resulted in loss of these specific labeling profiles in adult cochlear tissues (Figure 8 E-F).

Bottom Line: We further performed quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) to identify the presence of Cx43 mRNA in the modiolus (mainly containing SGNs).Cx43 mRNA was higher at P8, compared with P1, and subsequently decreased at P14.These results indicated that Cx43 correlated with cochlear synaptogenesis and establishment of auditory neurotransmission.

View Article: PubMed Central - PubMed

Affiliation: Xinhua Hospital, Shanghai Jiaotong University, Shanghai Jiaotong University Ear Institute. Liuwj38@163.com.

ABSTRACT
The expression pattern of connexin 43 (Cx43) in the cochlea is not determined and is controversial. Since the presence of Cx43 is essential for hearing, we re-examined its distribution during postnatal development of rat cochlea. Cx43 protein was expressed in spiral ganglion neurons (SGNs) and their neurite terminals innervating the inner and outer hair cells (IHCs and OHCs) as early as birth (post-natal day 0, P0), and persisted until P14. Double immunofluorescence staining, using two antibodies against Cx43 and TUJ1, a marker for all SGNs and afferent terminals, showed that immunoreactivity for Cx43 and TUJ1 was perfectly colocalized in SGNs and afferent terminals associated with the IHCs and OHCs. However, beyond P14, Cx43 immunostaining could no longer be detected in the region of the synaptic terminals at the bases of IHCs and OHCs (P17, adult). In contrast, Cx43 maintained its expression in SGNs into adulthood. We further performed quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) to identify the presence of Cx43 mRNA in the modiolus (mainly containing SGNs). Cx43 mRNA was higher at P8, compared with P1, and subsequently decreased at P14. These results indicated that Cx43 correlated with cochlear synaptogenesis and establishment of auditory neurotransmission.

No MeSH data available.


Related in: MedlinePlus