Furthermore, events of similar magnitude could often have different designs. afferents requires the sequential activation of alpha9-comprising nicotinic ACh receptors (9*nAChRs) and small-conductance, calcium-dependent potassium channels (SK) in type II hair cells. Gradations in the strength of efferent-mediated inhibition across the crista likely reflect variations in 9*nAChRs and/or SK activation in type II hair cells from those different areas. However, in turtle cristae, neither inference has been confirmed with direct recordings from type II hair cells. To address these gaps, we performed whole-cell, patch-clamp recordings from type II hair cells within a split-epithelial preparation of the turtle posterior crista. Here, we can very easily visualize and record hair cells while keeping their native location within the neuroepithelium. Consistent with 9*nAChR/SK activation, ACh-sensitive currents in type II hair cells were inward at hyperpolarizing potentials but reversed near ?90 mV to produce outward currents that typically peaked around ?20 mV. ACh-sensitive currents were largest in torus hair Nepsilon-Acetyl-L-lysine cells but absent from hair cells near the planum. In current clamp recordings under zero-current conditions, ACh robustly hyperpolarized type II hair cells. ACh-sensitive reactions were Nepsilon-Acetyl-L-lysine reversibly clogged from the 9nAChR antagonists ICS, strychnine, and methyllycaconitine as well as the SK antagonists apamin and UCL1684. Intact efferent terminals in the split-epithelial preparation spontaneously released ACh that also triggered 9*nAChRs/SK in type II hair cells. These launch events were accelerated with high-potassium external solution and all events were clogged by strychnine, ICS, methyllycaconitine, and apamin. These findings provide direct evidence that activation of 9*nAChR/SK in turtle type II hair cells underlies efferent-mediated inhibition of bouton afferents. =?is the concentration Nepsilon-Acetyl-L-lysine of ACh, is the response to ACh at concentration is the Hill coefficient. Results For orientation, the cellular organization of the neuroepithelium in our split-epithelial preparation is best illustrated using an immunohistochemical image taken from longitudinal sections of the posterior crista (Number ?(Figure1D).1D). Here, hair cells, calyx-bearing afferents, and efferent terminals are stained with myosin 7A (magenta), calretinin (white), and synapsin (green), respectively. Type II hair cells and efferent terminals are distributed throughout the crista while type I hair cells are limited to the central zone (CZ). Type I hair cells in the CZ are distinguished by the presence of calyx-bearing afferents which can be very easily visualized during patch-clamp recordings using DIC optics. For this study, we exclusively recorded from type II hair cells located in one of three regions of the crista designated as Torus, Central Zone, or Planum (Number ?(Figure1D).1D). The bulk of the recordings were made in type II hair cells from your torus region. All type II hair cells were recognized by their crista location, characteristic shape, and lack of calyx ending, all of which was confirmed in many Nepsilon-Acetyl-L-lysine recordings by visualizing fluorescent fills with Alexa594-hydrazide after going whole cell (Numbers 1E,F). During patch-clamp recordings, the lack of the signature type I hair cell potassium current IKL offered further confirmation that we were recording from type II hair cells (Rennie and Correia, 1994; Rsch and Eatock, 1996; Brichta et al., 2002). A total of 240 cristae from 165 turtles were collected for this study from which 323 type II hair cells from your three regions were recorded. Cells were deemed healthy offered the cell membrane appeared intact, there was no obvious swelling, and the resting membrane potential was stable at ?40 mV or reduce. Standard recordings from type II hair cells near the torus: acetylcholine-sensitive inward and outward current in type II hair cells To enhance conditions for observing 9*nAChR-mediated reactions in turtle posterior crista hair cells, we 1st recorded the current response of torus type II hair cells near ?20 mV before and during the application of 100 M acetylcholine (ACh). This approach was used since: (1) Bouton afferents innervating type II hair cells near the torus (BT) showed the most strong inhibitory reactions during efferent activation (Brichta and Goldberg, 2000b; Holt et al., 2006, 2015a); and (2) Maximum 9nAChR-mediated activation of SK potassium Rabbit Polyclonal to CDC25B (phospho-Ser323) currents in additional hair cell systems ranges Nepsilon-Acetyl-L-lysine from ?40 to ?10 mV (Fuchs and Murrow, 1992; Nenov et al., 1996a; Glowatzki.