Age-Dependency of Neurite Outgrowth in Postnatal Mouse Cochlear Spiral Ganglion Explants

Background: The spatial gap between cochlear implants (CIs) and the auditory nerve limits frequency selectivity as large populations of spiral ganglion neurons (SGNs) are electrically stimulated synchronously. To improve CI performance, a possible strategy is to promote neurite outgrowth toward the CI, thereby allowing a discrete stimulation of small SGN subpopulations. Brain-derived neurotrophic factor (BDNF) is effective to stimulate neurite outgrowth from SGNs. Method: TrkB (tropomyosin receptor kinase B) agonists, BDNF, and five known small-molecule BDNF mimetics were tested for their efficacy in stimulating neurite outgrowth in postnatal SGN explants. To modulate Trk receptor-mediated effects, TrkB and TrkC ligands were scavenged by an excess of recombinant receptor proteins. The pan-Trk inhibitor K252a was used to block Trk receptor actions. Results: THF (7,8,3′-trihydroxyflavone) partly reproduced the BDNF effect in postnatal day 7 (P7) mouse cochlear spiral ganglion explants (SGEs), but failed to show effectiveness in P4 SGEs. During the same postnatal period, spontaneous and BDNF-stimulated neurite outgrowth increased. The increased neurite outgrowth in P7 SGEs was not caused by the TrkB/TrkC ligands, BDNF and neurotrophin-3 (NT-3). Conclusions: The age-dependency of induction of neurite outgrowth in SGEs was very likely dependent on presently unidentified factors and/or molecular mechanisms which may also be decisive for the age-dependent efficacy of the small-molecule TrkB receptor agonist THF.

Hybrid optogenetic and electrical stimulation for greater spatial resolution and temporal fidelity of cochlear activation

Compared to electrical stimulation, optogenetic stimulation has the potential to improve the spatial precision of neural activation in neuroprostheses, but it requires intense light and has relatively poor temporal kinetics. We tested the effect of hybrid stimulation, which is the combination of subthreshold optical and electrical stimuli, on spectral and temporal fidelity in the cochlea by recording multiunit activity in the inferior colliculus of channelrhodopsin (H134R variant) transgenic mice. Pulsed light or biphasic electrical pulses were delivered to cochlear spiral ganglion neurons of acutely deafened mice, either as individual stimuli or as hybrid stimuli for which the timing of the electrical pulse had a varied delay relative to the start of the optical pulse. Facilitation occurred when subthreshold electrical stimuli were applied at the end of, or up to 3.75 ms after subthreshold optical pulses. The spread of activation resulting from hybrid stimulation was significantly narrower than electrical-only and optical-only stimulation (p<0.01), measured at equivalent suprathreshold levels of loudness that are relevant to cochlear implant users. Furthermore, temporal fidelity, measured as maximum following rates to 300 ms pulse trains bursts up to 240 Hz, was 2.4-fold greater than optical-only stimulation (p<0.05). By significantly improving spectral resolution of electrical- and optical-only stimulation and the temporal fidelity of optical-only stimulation, hybrid stimulation has the potential to increase the number of perceptually independent stimulating channels in a cochlear implant.

Estrogen inhibits the apoptosis of spiral ganglion cells in C57BL/6J mice with presbycusis by downregulating AQP4.

BackgroundEstrogen has a protective effect on age-related hearing loss (ARHL), but the mechanism has not been elucidated. Cochlear spiral ganglion neurons (SGN) are an important link between the cochlear hair cells and the auditory center. This study used C57BL/6J mice as models of elderly individuals to observe the protective effect of estrogen against cochlear spiral ganglion cell apoptosis.MethodsFifty mice were divided into the following five groups (10 mice/group): the young group at 3 months of age (3 m), the elderly group at 12 months of age (12 m), the ovariectomized group at 12 months of age (12 m ovx), the ovariectomized group at 12 months of age + estrogen treatment for 1 month (E2 1 m), and the ovariectomized group at 12 months of age + estrogen treatment for 2 months (E2 2 m). The auditory brain stem response (ABR) was analyzed to detect changes in the hearing threshold, enzyme-linked immunosorbent assays (ELISAs) were used to determine the serum estrogen levels,Hematoxylin eosin (HE) staining and transmission electron microscopy (TEM) were used to observe the morphological changes in the cochlea spiral ganglion neurons (SGN), and TUNEL staining was used to observe the apoptosis of SGN. The expression of AQP4 was observed by immunofluorescence, and the mRNA expression levels of AQP4, Caspase-3, Bax and Bcl-2 in the cochlea spiral ganglion were determined by qRT-PCR. Cell experiments: Primary cultures of spiral ganglion cells were divided into a control group (SGC), DMSO group, D-gal group, D-gal + E2 group, D-gal + TGN020 group and D-gal + E2 + TGN020 group. Immunofluorescence was used to observe the AQP4 expression, qRT-PCR was used to observe the AQP4, Caspase-3, Bax and Bcl-2 mRNA expression levels, and flow cytometry was used to observe the apoptosis rate.ResultsAn increased hearing threshold was observed in the elderly mice (P < 0.001). After removal of the ovaries, the hearing threshold of the mice in the 12 m ovx group was higher than it was in the 12 m control group (P < 0.05), and this increased threshold was accompanied by an increased loss of spiral ganglion cells, increased apoptosis (P < 0.01), and increased AQP4 expression (P < 0.001). Treatment with exogenous estrogen reversed all these changes. Cell experiments: D-gal increased the apoptosis rate (P < 0.001) and AQP4 expression (P < 0.001). Estrogen and the AQP4 inhibitor TGN020 both reduced the apoptosis rate and AQP4 expression.ConclusionEstrogen inhibited apoptosis of cochlear spiral ganglion cells in aged C57BL/6J mice by downregulating AQP4, thus achieving a protective effect on ARHL.

Cisplatin Toxicology: The Role of Pro-inflammatory Cytokines and GABA Transporters in Cochlear Spiral Ganglion

Background: The current study was conducted to examine the specific activation of pro-inflammatory cytokines (PICs), namely IL-1β, IL-6 and TNF-α in the cochlear spiral ganglion of rats after ototoxicity induced by cisplatin. Since γ-aminobutyric acid (GABA) and its receptors are involved in pathophysiological processes of ototoxicity, we further examined the role played by PICs in regulating expression of GABA transporter type 1 and 3 (GAT-1 and GAT-3), as two essential subtypes of GATs responsible for the regulation of extracellular GABA levels in the neuronal tissues. Methods: ELISA and western blot analysis were employed to examine the levels of PICs and GATs; and auditory brainstem response was used to assess ototoxicity induced by cisplatin. Results: IL-1β, IL-6 and TNF-α as well as their receptors were significantly increased in the spiral ganglion of ototoxic rats as compared with sham control animals (P<0.05, ototoxic rats vs. control rats). Cisplatin-ototoxicity also induced upregulation of the protein levels of GAT-1 and GAT-3 in the spiral ganglion (P<0.05 vs. controls). In addition, administration of inhibitors to IL-1β, IL-6 and TNF-α attenuated amplification of GAT-1 and GAT-3 and improved hearing impairment induced by cisplatin. Conclusion: Our data indicate that PIC signals are activated in the spiral ganglion during cisplatin-ototoxicity which thereby leads to upregulation of GABA transporters. As a result, it is likely that de-inhibition of GABA system is enhanced in the cochlear spiral ganglion. This supports a role for PICs in engagement of the signal mechanisms associated with cisplatin-ototoxicity, and has pharmacological implications to target specific PICs for GABAergic dysfunction and vulnerability related to cisplatin-ototoxicity.

Comprehensive transcriptome analysis of cochlear spiral ganglion neurons at multiple ages

Inner ear cochlear spiral ganglion neurons (SGNs) transmit sound information to the brainstem. Recent single cell RNA-Seq studies have revealed heterogeneities within SGNs. Nonetheless, much remains unknown about the transcriptome of SGNs, especially which genes are specifically expressed in SGNs. To address these questions, we needed a deeper and broader gene coverage than that in previous studies. We performed bulk RNA-Seq on mouse SGNs at five ages, and on two reference cell types (hair cells and glia). Their transcriptome comparison identified genes previously unknown to be specifically expressed in SGNs. To validate our dataset and provide useful genetic tools for this research field, we generated two knockin mouse strains: Scrt2-P2A-tdTomato and Celf4-3xHA-P2A-iCreER-T2A-EGFP. Our comprehensive analysis confirmed the SGN-selective expression of the candidate genes, testifying to the quality of our transcriptome data. These two mouse strains can be used to temporally label SGNs or to sort them.