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Ca 2þ is an important intracellular messenger and regulator in both physiological and pathophysiological mechanisms in the hearing organ. Investigation of cellular Ca 2þ homeostasis in the mature cochlea is hampered by the special anatomy and high vulnerability of the organ. A quick, straightforward and reliable Ca 2þ imaging method with high spatial and temporal resolution in the mature organ of Corti is missing. Cell cultures or isolated cells do not preserve the special microenvironment and intercellular communication, while cochlear explants are excised from only a restricted portion of the organ of Corti and usually from neonatal pre-hearing murines. The hemicochlea, prepared from hearing mice allows tonotopic experimental approach on the radial perspective in the basal, middle and apical turns of the organ. We used the preparation recently for functional imaging in supporting cells of the organ of Corti after bulk loading of the Ca 2þ indicator. However, bulk loading takes long time, is variable and nonselective, and causes the accumulation of the indicator in the extracellular space. In this study we show the improved labeling of supporting cells of the organ of Corti by targeted single-cell electroporation in mature mouse hemicochlea. Single-cell electroporation proved to be a reliable way of reducing the duration and variability of loading and allowed subcellular Ca 2þ imaging by increasing the signal-tonoise ratio, while cell viability was retained during the experiments. We demonstrated the applicability of the method by measuring the effect of purinergic, TRPA1, TRPV1 and ACh receptor stimulation on intracellular Ca 2þ concentration at the cellular and subcellular level. In agreement with previous results, ATP evoked reversible and repeatable Ca 2þ transients in Deiters', Hensen's and Claudius' cells. TRPA1 and TRPV1 stimulation by AITC and capsaicin, respectively, failed to induce any Ca 2þ response in the supporting cells, except in a single Hensen's cell in which AITC evoked transients with smaller amplitude. AITC also caused the displacement of the tissue. Carbachol, agonist of ACh receptors induced Ca 2þ transients in about a third of Deiters' and fifth of Hensen's cells. Here we have presented a fast and cellspecific indicator loading method allowing subcellular functional Ca 2þ imaging in supporting cells of the organ of Corti in the mature hemicochlea preparation, thus providing a straightforward tool for deciphering the poorly understood regulation of Ca 2þ homeostasis in these cells.

Hearing impairment is the most common sensory deficit, affecting more than 400 million people worldwide. Sensorineural hearing losses currently lack any specific or efficient pharmacotherapy largely due to the insufficient knowledge of the pathomechanism. Purinergic signaling plays a substantial role in cochlear (patho)physiology. P2 (ionotropic P2X and the metabotropic P2Y) as well as adenosine receptors expressed on cochlear sensory and non-sensory cells are involved mostly in protective mechanisms of the cochlea. They are implicated in the sensitivity adjustment of the receptor cells by a K+ shunt and can attenuate the cochlear amplification by modifying cochlear micromechanics. Cochlear blood flow is also regulated by purines. Here, we propose to comprehend this field with the purine-immune interactions in the cochlea. The role of harmful immune mechanisms in sensorineural hearing losses has been emerging in the horizon of cochlear pathologies. In addition to decreasing hearing sensitivity and increasing cochlear blood supply, influencing the immune system can be the additional avenue for pharmacological targeting of purinergic signaling in the cochlea. Elucidating this complexity of purinergic effects on cochlear functions is necessary and it can result in development of new therapeutic approaches in hearing disabilities, especially in the noise-induced ones.

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Exploring the development of the hearing organ helps in the understanding of hearing and hearing impairments and it promotes the development of the regenerative approaches-based therapeutic efforts. The role of supporting cells in the development of the organ of Corti is much less elucidated than that of the cochlear sensory receptor cells. The use of our recently published method of single-cell electroporation loading of a fluorescent Ca2+ probe in the mouse hemicochlea preparation provided an appropriate means to investigate the Deiters’ cells at the subcellular level in two different cochlear turns (apical, middle). Deiters’ cell’s soma and process elongated, and the process became slimmer by maturation without tonotopic preference. The tonotopically heterogeneous spontaneous Ca2+ activity less frequently occurred by maturation and implied subcellular difference. The exogenous ATP- and UTP-evoked Ca2+ responses were maturation-dependent and showed P2Y receptor dominance in the apical turn. By monitoring the basic structural dimensions of this supporting cell type as well as its spontaneous and evoked purinergic Ca2+ signaling in the hemicochlea preparation in different stages in the critical postnatal P5-25 developmental period for the first time, we showed that the soma and the phalangeal process of the Deiters’ cells go through age- and tonotopy-dependent changes in the morphometric parameters and purinergic signaling.

Background:Selegiline [(-)-deprenyl] is widely used for the treatment of Parkinson’s disease in humans.Objective:Time-dependence of tissue distribution of selegiline following per os administration to rats.Method:Oral administration of radiolabeled selegiline to rats resulted in a pattern of tissue distribution similar to that following intraperitoneal injection. Analyses were done using both reversed-phase HPLC and also by counting radioactivity in various body compartments of rats.Results:As a consequence of oral administration of 30 mg/kg of selegiline, its level in the stomach was extremely high (179.57 µg/g tissue through 54.67 µg/g at 15 min to 120 min), that is one magnitude higher than that in the serum level. High selegiline concentrations were also detected in the lacrimal glands (7.45 µg/g), kidneys (6.87 µg/g), livers (6.01 µg/g) and lungs (3.47 µg/g) after 30 minutes of application, which were higher than after intraperitoneal injections.Conclusion:The relatively high tissue levels remained for 120 min monitoring. Selegiline levels in the brain (1.69 µg/g) and in the testes (1.88 µg/g) were also considerably higher than following intraperitoneal administration during the entire period of observation (15 to 120 min).