Immunohistochemistry localises myosin-7a to cochlear efferent boutons.

Background: Myosin 7a is an actin-binding motor protein involved in the formation of hair-cell stereocilia both in the cochlea and in the vestibular system. Mutations in myosin 7a are linked to congenital hearing loss and are present in 50% of Type-1 Usher syndrome patients who suffer from progressive hearing loss and vestibular system dysfunction. Methods: Myosin 7a is often used to visualise sensory hair cells due to its well characterised and localised expression profile. We thus conducted myosin-7a immunostaining across all three turns of the adult rat organ of Corti to visualise hair cells. Results: As expected, we observed myosin 7a staining in both inner and outer hair cells. Unexpectedly, we also observed strong myosin 7a staining in the medial olivocochlear efferent synaptic boutons contacting the outer hair cells. Efferent bouton myosin-7a staining was present across all three turns of the cochlea. We verified this localisation by co-staining with a known efferent bouton marker, the vesicular acetylcholine transporter. Conclusions: In addition to its role in stereocilia formation and maintenance, myosin 7a or certain myosin-7a expression variants might play a role in efferent synaptic transmission in the cochlea and thus ultimately influence cochlear gain regulation. Our immunohistochemistry results should be validated with other methods to confirm these serendipitous findings.

Intercellular Ca2+ signalling in the adult mouse cochlea.

KEY POINTS: Intercellular Ca2+ waves are increases in cytoplasmic Ca2+ levels that propagate between cells. Periodic Ca2+ waves have been linked to gene regulation and are thought to play a crucial role in the development of our hearing epithelium, the organ of Corti and the acquisition of hearing. We observed regular periodic intercellular Ca2+ waves in supporting cells of an ex vivo preparation of the adult mouse organ of Corti, and these waves were found to propagate independently of extracellular ATP and were inhibited by the gap junction blockers 1-octanol and carbenoxolone. Our results establish that the existence of periodic Ca2+ waves in the organ of Corti is not restricted to the prehearing period. ABSTRACT: We have investigated wave-like cytoplasmic calcium (Ca2+ ) signalling in an ex vivo preparation of the adult mouse organ of Corti. Two types of intercellular Ca2+ waves that differ in propagation distance and speed were observed. One type was observed to travel up to 100 µm with an average velocity of 7 µm/s. Such waves were initiated by local tissue damage in the outer hair cell region. The propagation distance was decreased when the purinergic receptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 50 µm) or suramin (150 µm) were added to the extracellular buffer. Immunocytochemical analysis and experiments with calcium indicator dyes showed that both P2X and P2Y receptors were present in supporting cells. A second class of waves identified to travel longitudinally along the organ of Corti propagated at a lower velocity of 1-3 µm/s. These 'slow' Ca2+ waves were particularly evident in the inner sulcus and Deiters' cells. They travelled for distances of up to 500 µm. The slow Ca2+ signalling varied periodically (approximately one wave every 10 min) and was maintained for more than 3 h. The slow waves were not affected by apyrase, or by the P2 receptor agonists suramin (150 µm) or PPADS (50 µm) but were blocked by the connexin channel blockers octanol (1 mm) and carbenoxolone (100 µm). It is proposed that the observed Ca2+ waves might be a physiological response to a change in extracellular environment and may be involved in critical gene regulation activities in the supporting cells of the cochlea.

S-nitrosylation of HDAC2 regulates the expression of the chromatin-remodeling factor Brm during radial neuron migration.

Dynamic epigenetic modifications play a key role in mediating the expression of genes required for neuronal development. We previously identified nitric oxide (NO) as a signaling molecule that mediates S-nitrosylation of histone deacetylase 2 (HDAC2) and epigenetic changes in neurons. Here, we show that HDAC2 nitrosylation regulates neuronal radial migration during cortical development. Bead-array analysis performed in the developing cortex revealed that brahma (Brm), a subunit of the ATP-dependent chromatin-remodeling complex BRG/brahma-associated factor, is one of the genes regulated by S-nitrosylation of HDAC2. In the cortex, expression of a mutant form of HDAC2 that cannot be nitrosylated dramatically inhibits Brm expression. Our study identifies NO and HDAC2 nitrosylation as part of a signaling pathway that regulates cortical development and the expression of Brm in neurons.