Tonotopic Distribution of Short-Term Adaptation Properties in the Cochlear Nerve of Normal and Acoustically Overexposed Chicks

Cochlear nerve adaptation is thought to result, at least partially, from the depletion of neurotransmitter stores in hair cells. Recently, neurotransmitter vesicle pools have been identified in chick tall hair cells that might play a role in adaptation. In order to understand better the relationship between adaptation and neurotransmitter release dynamics, short-term adaptation was characterized by using peristimulus time histograms of single-unit activity in the chick cochlear nerve. The adaptation function resulting from 100-ms pure tone stimuli presented at the characteristic frequency, +20 dB relative to threshold, was well described as a single exponential decay process with an average time constant of 18.6 ± 0.8 ms (mean ± SEM). The number of spikes contributed by the adapting part of the response increased tonotopically for characteristic frequencies up to ∼0.8 kHz. Comparison of the adaptation data with known physiological and anatomical hair cell properties suggests that depletion of the readily releasable pool is the basis of short-term adaptation in the chick. With this idea in mind, short-term adaptation was used as a proxy for assessing tall hair cell synaptic function following intense acoustic stimulation. After 48 h of exposure to an intense pure tone, the time constant of short-term adaptation was unaltered, whereas the number of spikes in the adapting component was increased at characteristic frequencies at and above the exposure frequency. These data suggest that the rate of readily releasable pool emptying is unaltered, but the neurotransmitter content of the pool is increased, by exposure to intense sound. The results imply that an increase in readily releasable pool size might be a compensatory mechanism ensuring the strength of the hair cell afferent synapse in the face of ongoing acoustic stress

FM Dye Photo-Oxidation as a Tool for Monitoring Membrane Recycling in Inner Hair Cells

Styryl (FM) dyes have been used for more than two decades to investigate exo- and endocytosis in conventional synapses. However, they are difficult to use in the inner hair cells of the auditory pathway (IHCs), as FM dyes appear to penetrate through mechanotransducer channels into the cytosol of IHCs, masking endocytotic uptake. To solve this problem we applied to IHCs the FM dye photo-oxidation technique, which renders the dyes into electron microscopy markers. Photo-oxidation allowed the unambiguous identification of labeled organelles, despite the presence of FM dye in the cytosol. This enabled us to describe the morphologies of several organelles that take up membrane in IHCs, both at rest and during stimulation. At rest, endosome-like organelles were detected in the region of the cuticular plate. Larger tubulo-cisternal organelles dominated the top and nuclear regions. Finally, the basal region, where the IHC active zones are located, contained few labeled organelles. Stimulation increased significantly membrane trafficking in the basal region, inducing the appearance of labeled vesicles and cistern-like organelles. The latter were replaced by small, synaptic-like vesicles during recovery after stimulation. In contrast, no changes in membrane trafficking were induced by stimulation in the cuticular plate region or in the top and nuclear regions. We conclude that synaptic vesicle recycling takes place mostly in the basal region of the IHCs. Other organelles participate in abundant constitutive membrane trafficking throughout the rest of the IHC volume.Open-Access-Publikationsfonds 2014peerReviewe

Linear Cyclodextrin Polymer Prodrugs as Novel Therapeutics for Niemann-Pick Type C1 Disorder

Niemann-Pick Type C1 disorder (NPC) is a rare lysosomal storage disease characterized by the accumulation of cholesterol in lysosomes. NPC has no FDA approved treatments yet, however 2-hydroxypropyl-β-cyclodextrin (HPβCD) has shown efficacy for treating the disease in both mouse and feline NPC models and is currently being investigated in late stage clinical trials. Despite promising results, therapeutic use of HPβCD is limited by the need for high doses, ototoxicity and intrathecal administration. These limitations can be attributed to its poor pharmacokinetic profile. In the attempt to overcome these limitations, we have designed a β-cyclodextrin (βCD) based polymer prodrugs (ORX-301) for an enhanced pharmacokinetic and biodistribution profile, which in turn can potentially provide an improved efficacy at lower doses. We demonstrated that subcutaneously injected ORX-301 extended the mean lifespan of NPC mice at a dosage 5-fold lower (800 mg/kg, body weight) the HPβCD dose proven efficacious (4000 mg/kg). We also show that ORX-301 penetrates the blood brain barrier and counteracts neurological impairment. These properties represent a substantial improvement and appear to overcome major limitations of presently available βCD-based therapy, demonstrating that this novel prodrug is a valuable alternative/complement for existing therapies