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Neurons of the medial olivary complex inhibit cochlear hair cells through the activation of α9α10-containing nicotinic acetylcholine receptors (nAChRs). Efforts to study the localization of these proteins have been hampered by the absence of reliable antibodies. To overcome this obstacle, CRISPR-Cas9 gene editing was used to generate mice in which a hemagglutinin tag (HA) was attached to the C-terminus of either α9 or α10 proteins. Immunodetection of the HA tag on either subunit in the organ of Corti of adult mice revealed immunopuncta clustered at the synaptic pole of outer hair cells. These puncta were juxtaposed to immunolabeled presynaptic efferent terminals. HA immunopuncta also occurred in inner hair cells of pre-hearing (P7) but not in adult mice. These immunolabeling patterns were similar for both homozygous and heterozygous mice. All HA-tagged genotypes had auditory brainstem responses not significantly different from those of wild type littermates. The activation of efferent neurons in heterozygous mice evoked biphasic postsynaptic currents not significantly different from those of wild type hair cells. However, efferent synaptic responses were significantly smaller and less frequent in the homozygous mice. We show that HA-tagged nAChRs introduced in the mouse by a CRISPR knock-in are regulated and expressed like the native protein, and in the heterozygous condition mediate normal synaptic function. The animals thus generated have clear advantages for localization studies.

Auditory stimuli travel from the cochlea to the brainstem through type I and type II cochlear afferents. While type I afferents convey information about the frequency, intensity, and timing of sounds, the role of type II afferents remains unresolved. Limited recordings of type II afferents from cochlear apex of pre-hearing rats reveal they are activated by widespread outer hair cell stimulation, ATP, and by the rupture of nearby outer hair cells. Altogether, these lines of evidence suggest that type II afferents sense loud, potentially damaging levels of sound. To explore this hypothesis further, calcium imaging was used to determine the impact of acoustic trauma on the activity of type II cochlear afferents of young adult mice of both sexes. Two known marker genes (Th, Drd2) and one new marker gene (Tac1), expressed in type II afferents and some other cochlear cell types, drove GCaMP6f expression to reveal calcium transients in response to focal damage in the organ of Corti in all turns of the cochlea. Mature type II afferents responded to acute photoablation damage less often but at greater length compared to pre-hearing neurons. In addition, days after acoustic trauma, acute photoablation triggered a novel response pattern in type II afferents and surrounding epithelial cells, delayed bursts of activity occurring minutes after the initial response subsided. Overall, calcium imaging can report type II afferent responses to damage even in mature and noise-exposed animals and reveals previously unknown tissue hyperactivity subsequent to acoustic trauma. SIGNIFICANCE STATEMENT The function of type II cochlear afferents is currently unknown. The prevailing hypothesis is that these neurons detect excessively loud sound and tissue damage within the cochlea. However, this hypothesis has not been directly investigated in fully hearing, mature animals. To this end, we show that type II afferents in mature mice experience prolonged calcium transients in response to focal tissue damage as compared to young, pre-hearing mice. Previous traumatic noise exposure caused novel delayed response patterns. Together, our data support the role of type II cochlear afferents as tissue damage detectors in the cochlea and suggest that changes in type II afferent activity may contribute to pathologies resulting from traumatic noise exposure.

BackgroundObese children with asthma are more vulnerable to air pollution, especially fine particulate matter (PM2.5), but reasons are poorly understood. We hypothesised that differences in breathing patterns (tidal volume, respiratory rate and minute ventilation) due to elevated body mass index (BMI) may contribute to this finding.ObjectiveTo investigate the association of BMI with breathing patterns and deposition of inhaled PM2.5.MethodsBaseline data from a prospective study of children with asthma were analysed (n=174). Tidal breathing was measured by a pitot-tube flowmeter, from which tidal volume, respiratory rate and minute ventilation were obtained. The association of BMI z-score with breathing patterns was estimated in a multivariable model adjusted for age, height, race, sex and asthma severity. A particle dosimetry model simulated PM2.5 lung deposition based on BMI-associated changes in breathing patterns.ResultsHigher BMI was associated with higher tidal volume (adjusted mean difference (aMD) between obese and normal-range BMI of 25 mL, 95% CI 5–45 mL) and minute ventilation (aMD 453 mL·min−1, 95% CI 123–784 mL·min−1). Higher tidal volumes caused higher fractional deposition of PM2.5 in the lung, driven by greater alveolar deposition. This translated into obese participants having greater per-breath retention of inhaled PM2.5 (aMD in alveolar deposition fraction of 3.4%, 95% CI 1.3–5.5%), leading to worse PM2.5 deposition rates.ConclusionsObese children with asthma breathe at higher tidal volumes that may increase the efficiency of PM2.5 deposition in the lung. This finding may partially explain why obese children with asthma exhibit greater sensitivity to air pollution.

Outer hair cells (OHCs) in the mouse cochlea are contacted by up to three type II afferent boutons. On average, only half of these are postsynaptic to presynaptic ribbons. Mice of both sexes were subjected to acoustic trauma that produced a threshold shift of 44.2 ± 9.1 dB 7 days after exposure. Ribbon synapses of OHCs were quantified in post-trauma and littermate controls using immunolabeling of CtBP2. Visualization with virtual reality was used to determine 3-D cytoplasmic localization of CtBP2 puncta to the synaptic pole of OHCs. Acoustic trauma was associated with a statistically significant increase in the number of synaptic ribbons per OHC. Serial section TEM was carried out on similarly treated mice. This also showed a significant increase in the number of ribbons in post-trauma OHCs, as well as a significant increase in ribbon volume compared to ribbons in control OHCs. An increase in OHC ribbon synapses after acoustic trauma is a novel observation that has implications for OHC:type II afferent signaling. A mathematical model showed that the observed increase in OHC ribbons considered alone could produce a significant increase in action potentials among type II afferent neurons during strong acoustic stimulation. Supplementary Information The online version contains supplementary material available at 10.1007/s10162-020-00777-w.

Microfold (M) cells are phagocytic intestinal epithelial cells in the follicle-associated epithelium of Peyer's patches that transport particulate antigens from the gut lumen into the subepithelial dome. Differentiation of M cells from epithelial stem cells in intestinal crypts requires the cytokine receptor activator of NF-κB ligand (RANKL) and the transcription factor Spi-B. We used three-dimensional enteroid cultures established with small intestinal crypts from mice as a model system to investigate signaling pathways involved in M cell differentiation and the influence of other cytokines on RANKL-induced M cell differentiation. Addition of RANKL to enteroids induced expression of multiple M cell-associated genes, including Spib, Ccl9 [chemokine (C-C motif) ligand 9], Tnfaip2 (TNF-α-induced protein 2), Anxa5 (annexin A5), and Marcksl1 (myristoylated alanine-rich protein kinase C substrate) in 1 day. The mature M cell marker glycoprotein 2 ( Gp2) was strongly induced by 3 days and expressed by 11% of cells in enteroids. The noncanonical NF-κB pathway was required for RANKL-induced M cell differentiation in enteroids, as addition of RANKL to enteroids from mice with a null mutation in the mitogen-activated protein kinase kinase kinase 14 ( Map3k14) gene encoding NF-κB-inducing kinase failed to induce M cell-associated genes. While the cytokine TNF-α alone had little, if any, effect on expression of M cell-associated genes, addition of TNF-α to RANKL consistently resulted in three- to sixfold higher levels of multiple M cell-associated genes than RANKL alone. One contributing mechanism is the rapid induction by TNF-α of Relb and Nfkb2 (NF-κB subunit 2), genes encoding the two subunits of the noncanonical NF-κB heterodimer. We conclude that endogenous activators of canonical NF-κB signaling present in the gut-associated lymphoid tissue microenvironment, including TNF-α, can play a supportive role in the RANKL-dependent differentiation of M cells in the follicle-associated epithelium.