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Calcium-Induced Calcium Release Supports Recruitment of Synaptic Vesicles in Auditory Hair Cells

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Calcium-Induced Calcium Release Supports Recruitment of Synaptic Vesicles in Auditory Hair Cells J Neurophysiol 115: 226–239, 2016. First published October 28, 2015; doi:10.1152/jn.00559.2015. Calcium-induced calcium release supports recruitment of synaptic vesicles in auditory hair cells X Manuel Castellano-Muñoz,1 Michael E. Schnee,1 and Anthony J. Ricci1,2 1Department of Otolaryngology, Stanford University School of Medicine, Stanford, California; and 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California Submitted 8 June 2015; accepted in final form 23 October 2015 Castellano-Muñoz M, Schnee ME, Ricci AJ. Calcium-induced ronal functions such as neuronal excitability, gene expression, calcium release supports recruitment of synaptic vesicles in auditory and synaptic plasticity and release (Bouchard et al. 2003). In hair cells. J Neurophysiol 115: 226–239, 2016. First published Octo- central synapses both endoplasmic reticulum (ER) and mito- Downloaded from ber 28, 2015; doi:10.1152/jn.00559.2015.—Hair cells from auditory chondria are well-known intracellular Ca2ϩ stores, and their and vestibular systems transmit continuous sound and balance infor- Ca2ϩ homeostatic modulation alters synaptic transmission pre- mation to the central nervous system through the release of synaptic and postsynaptically (Bardo et al. 2006; Emptage et al. 2001; vesicles at ribbon synapses. The high activity experienced by hair Llano et al. 2000). CICR is also suggested to contribute to cells requires a unique mechanism to sustain recruitment and replen- synaptic transmission at ribbon synapses (Babai et al. 2010; ishment of synaptic vesicles for continuous release. Using pre- and Lelli et al. 2003). http://jn.physiology.org/ postsynaptic electrophysiological recordings, we explored the poten- tial contribution of calcium-induced calcium release (CICR) in mod- Calcium imaging identified CICR in turtle auditory papilla ulating the recruitment of vesicles to auditory hair cell ribbon syn- hair cells (Tucker and Fettiplace 1995), frog semicircular canal apses. Pharmacological manipulation of CICR with agents targeting (Lelli et al. 2003), P6–P11 mouse inner hair cells (Iosub et al. endoplasmic reticulum calcium stores reduced both spontaneous post- 2015; Kennedy and Meech 2002), and rat and guinea pig outer synaptic multiunit activity and the frequency of excitatory postsyn- hair cells (Evans et al. 2000; Mammano et al. 1999). In aptic currents (EPSCs). Pharmacological treatments had no effect on mammalian outer hair cells, CICR is functionally associated to hair cell resting potential or activation curves for calcium and potas- subsynaptic Ca2ϩ stores in close proximity to efferent termi- sium channels. However, these drugs exerted a reduction in vesicle nals (Lioudyno et al. 2004). In addition, Ca2ϩ can be released release measured by dual-sine capacitance methods. In addition, by inositol triphosphate-gated Ca2ϩ stores at the base of the by 10.220.33.6 on September 23, 2016 calcium substitution by barium reduced release efficacy by delaying outer hair cell hair bundle (Mammano et al. 1999). Although release onset and diminishing vesicle recruitment. Together these results demonstrate a role for calcium stores in hair cell ribbon pharmacological data demonstrate the presence of intracellular stores in hair cells, their physiological role is debatable. Intra- synaptic transmission and suggest a novel contribution of CICR in 2ϩ hair cell vesicle recruitment. We hypothesize that calcium entry via cellular Ca stores have been functionally associated with the calcium channels is tightly regulated to control timing of vesicle control of BK channel activity in inner hair cells (Beurg et al. fusion at the synapse, whereas CICR is used to maintain a tonic 2005; Marcotti et al. 2004), modulation of outer hair cell calcium signal to modulate vesicle trafficking. electromotility (Dallos et al. 1997), homeostatic control of 2ϩ hair cell; dual-sine capacitance; Ca2ϩ-induced Ca2ϩ release; intra- presynaptic Ca levels (Kennedy and Meech 2002; Tucker cellular stores; ribbon synapse; synaptic transmission and Fettiplace 1995), time-dependent segregation of afferent and efferent signaling (Im et al. 2014), and regulation of vesicular trafficking, exocytosis, and synaptic transmission HAIR CELLS, the sensory receptors in the auditory and vestibular (Hendricson and Guth 2002; Lelli et al. 2003). systems, convert mechanical information into synaptic activity Here we performed auditory nerve multiunit and single-unit through the release of neurotransmitter at ribbon synapses. recordings as well as hair cell dual-sine capacitance experi- Each hair cell contains tens of synaptic ribbons (Schnee et al. ments to study the potential contribution of CICR to hair cell 2005, 2011; Sneary 1988), presynaptic specializations sur- synaptic transmission. Pharmacological and divalent cation rounded by synaptic vesicles and associated to active zones and substitution results are consistent with a role for CICR in the ϩ L-type Ca2 channels (Issa and Hudspeth 1994; Roberts et al. recruitment of vesicles to support maintained release in audi- 1990; Tucker and Fettiplace 1995). Similar to other sensory tory hair cell ribbon synapses. synapses, hair cell ribbon synapses operate in a graded fashion, reaching high release rates and exhibiting little fatigue. Both of MATERIALS AND METHODS these properties require rapid vesicle replenishment by a mech- anism that is not well understood. Tissue preparation. The auditory papilla of red-eared sliders (Tra- 2ϩ 2ϩ chemys scripta elegans) was dissected as previously described (Sch- Ca -induced Ca release (CICR) is a mechanism by nee et al. 2005). All animal procedures were approved by the Stanford which the influx of Ca2ϩ through Ca2ϩ channels in the plasma 2ϩ Institutional Animal Care and Use Committee (IACUC) and are in membrane activates Ca release from intracellular stores accord with National Institutes of Health guidelines and standards. (Verkhratsky 2005). CICR is implicated in a number of neu- Turtle half-head preparations were used for multiunit activity mea- surements from the eighth cranial nerve. The turtle head was split in Address for reprint requests and other correspondence: M. Castellano- half and pinned in a Sylgard dissection chamber either with an Muñoz, Inst. of Bioengineering, Miguel Hernández Univ., Avenida de la external solution similar to that used in patch-clamp recordings or Universidad, s/n 03202 Elche, Alicante, Spain (e-mail: [email protected]). with bicarbonate-buffered perilymph containing (in mM) 126 NaCl, 226 0022-3077/16 Copyright © 2016 the American Physiological Society www.jn.org STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES 227 2.5 KCl, 13 NaHCO3, 1.7 NaH2PO4, 1.8 CaCl2, 1 MgCl2, and 5 preparation in the recording dish. A gravity-controlled perfusion glucose (continuously bubbled with 95% O2-5% CO2). The brain was pipette was located ϳ5 mm above the otic capsule and was connected removed and the auditory nerve exposed (Fig. 1A), cutting the con- to a perfusion system with a flow rate of roughly 1 ml/min. nections to posterior ampulla and saccule. The ventral otic membrane For intracellular hair cell recordings, the inner ear was dissected was trimmed to allow access to perfusion prior to mounting of the from the otic capsule in external solution containing (in mM) 128 NaCl, 0.5 KCl, 2.8 CaCl2, 2.2 MgCl2, 10 HEPES, 6 glucose, 2 A Drug Delivery Recording creatine monohydrate, 2 ascorbate, and 2 pyruvate and pH was Pipette adjusted to 7.6 and osmolality to 275 mosmol/kg. The external solution was supplemented with 20 ␮M curare to eliminate efferent activity, and 100 nM apamin was included in some of the experiments to block SK activity. We found no evidence of remaining efferent activity with incubation with curare in both pre- and postsynaptic recordings. To disrupt mechanotransduction channels, after extrane- ous tissue was trimmed and the otoconia removed the papilla was Neck Nose incubated for 15 min in external solution and perfused with 200 ␮lof Downloaded from B 5 mM BAPTA before and after removal of the tectorial membrane 0.4 with a fine insect pin. In some experiments where the tectorial membrane was left intact, cell visualization was impaired but no obvious electrophysiological effects were observed. The basilar pa- 0.2 Vm pilla was transferred to the recording chamber and secured with single strands of dental floss. Cells were imaged with an Axioskop 2 FS plus 0 (Zeiss, Thornwood, NY) with bright field optics using a ϫ60 0.9 NA water objective (LUMPlan Fl/IR, Olympus). Perfusion of bath and http://jn.physiology.org/ caffeine drugs was delivered with a Minipuls 3 pump (Gilson, Middleton, WI). -0.2 Electrophysiology. For multiunit activity we used the turtle half- 50 head preparation, in which the auditory nerve was inserted into an hourglass-shaped suction electrode with a micromanipulator (Na- rishige, East Meadow, NY) and compound action potentials were s/sekips recorded with a differential AC preamplifier (Grass, P55 Astro-Med, West Warwick, RI). One electrode was inserted into the borosilicate 25 * suction pipette, and the neutral electrode was in contact with the bath. The signal was band-pass filtered (1 Hz–1 kHz) and amplified 1,000 by 10.220.33.6 on September 23, 2016 times. Compound action potentials were collected through a data acquisition interface (CED Micro 1401 mkII, Cambridge Electronic 0 Design) and analyzed with Spike2 software (Cambridge Electronic 0 1000 2000 Design). Noise levels were identified by blocking afferent
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