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Background: Loudness recruitment is commonly experienced by patients with putative endolymphatic hydrops. Loudness recruitment is abnormal loudness growth with high-level sounds being perceived as having normal loudness even though hearing thresholds are elevated. The traditional interpretation of recruitment is that cochlear amplification has been reduced. Since the cochlear amplifier acts primarily at low sound levels, an ear with elevated thresholds from reduced cochlear amplification can have normal processing at high sound levels. In humans, recruitment can be studied using perceptual loudness but in animals physiological measurements are used. Recruitment in animal auditory-nerve responses has never been unequivocally demonstrated because the animals used had damage to sensory and neural cells, not solely a reduction of cochlear amplification. Investigators have thus looked for, and found, evidence of recruitment in the auditory central nervous system (CNS). While studies on CNS recruitment are informative, they cannot rule out the traditional interpretation of recruitment originating in the cochlea.Design: We used techniques that could assess hearing function throughout entire frequency- and dynamic-range of hearing. Measurements were made from two animal models: guinea-pig ears with endolymphatic-sac-ablation surgery to produce endolymphatic hydrops, and naïve guinea-pig ears with cochlear perfusions of 13 mM 2-Hydroxypropyl-Beta-Cyclodextrin (HPBCD) in artificial perilymph. Endolymphatic sac ablation caused low-frequency loss. Animals treated with HPBCD had hearing loss at all frequencies. None of these animals had loss of hair cells or synapses on auditory nerve fibers.Results: In ears with endolymphatic hydrops and those perfused with HPBCD, auditory-nerve based measurements at low frequencies showed recruitment compared to controls. Recruitment was not found at high frequencies (> 4 kHz) where hearing thresholds were normal in ears with endolymphatic hydrops and elevated in ears treated with HPBCD.Conclusions: We found compelling evidence of recruitment in auditory-nerve data. Such clear evidence has never been shown before. Our findings suggest that, in patients suspected of having endolymphatic hydrops, loudness recruitment may be a good indication that the associated low-frequency hearing loss originates from a reduction of cochlear amplification, and that measurements of recruitment could be used in differential diagnosis and treatment monitoring of Ménière's disease.

Producing hair cells of the inner ear is the major goal of ongoing research that combines advances in developmental and stem cell biology. The recent advent of an inner ear organoid protocol-resulting in three-dimensional stem cell-derived tissues resembling vestibular sensory epithelia-has sparked interest in applications such as regeneration, drug discovery, and disease modeling. In this study, we adapted this protocol for a novel mouse embryonic stem cell line with a fluorescent reporter for Pax2 expression. We used Pax2EGFP/+ organoid formation to model otic induction, the pivotal developmental event when preplacodal tissue adopts otic fate. We found upregulation of Pax2 and activation of ERK downstream of fibroblast growth factor signaling in organoid formation as in embryonic inner ear development. Pax2 expression was evident from the EGFP reporter beginning at the vesicle formation stage and persisting through generation of the sensory epithelium. The native ventralizing signal sonic hedgehog was largely absent from the cell aggregates as otic vesicles began to form, confirming the dorsal vestibular organoid fate. Nonetheless, cochlear- or vestibular-like neurons appeared to delaminate from the derived otic vesicles and formed synaptic contacts with hair cells in the organoids. Cell lines with transcriptional reporters such as Pax2EGFP/+ facilitate direct evaluation of morphological changes during organoid production, a major asset when establishing and validating the culture protocol.

Stem cell therapy holds great promise for treating neurodegenerative disease, but major barriers to effective therapeutic strategies remain. A complete understanding of the derived phenotype is required for predicting cell response once introduced into the host tissue. We sought to identify major axonal guidance cues present in neurons derived from the transient overexpression of neurogenin-1 (Neurog1) in mouse embryonic stem cells (ESCs). Neurog1 upregulated the netrin-1 axon guidance receptors DCC (deleted in colorectal cancer) and neogenin (NEO1). Quantitative polymerase chain reaction results showed a 2-fold increase in NEO1 mRNA and a 36-fold increase in DCC mRNA in Neurog1-induced compared with control ESCs. Immunohistochemistry indicated that DCC was primarily expressed on cells positive for the neuronal marker TUJ1. DCC was preferentially localized to the cell soma and growth-cones of induced neurons. In contrast, NEO1 expression showed less specificity, labeling both TUJ1-positive and TUJ1-negative cells as well as uninduced control cells. Axonal outgrowth was directed preferentially toward aggregates of HEK293 cells secreting a recombinant active fragment of netrin-1. These data indicate that DCC and NEO1 are downstream products of Neurog1 and may guide the integration of Neurog1-induced ESCs with target cells secreting netrin-1. Differential expression profiles for netrin receptors could indicate different roles for this guidance cue on neuronal and non-neuronal cells.