Sodium and calcium currents shape action potentials in immature mouse inner hair cells

Abstract

Journal of PhysiologyImmature IHCs from the mouse express, in addition to an inward L-type Ca 2+ current containing the a1D (Ca v 1.3) subunit (Platzer et al. 2000), a large Na + current (I Na ; Kros et al. 1993;Kros, 1996). Although Na + currents have been reported in vestibular and cochlear hair cells of other vertebrates including mammals (Evans & Fuchs 1987;Sugihara & Furukawa, 1989;Sokolowski et al. 1993; Witt et al. 1994;Oliver et al. 1997;Lennan et al. 1999;Masetto et al. 2003), little is known about whether and how they contribute to the physiological responses of the hair cells. To gain more insight into the biophysical basis of the action potential, we set out to investigate the relative contribution of both I Ca and I Na to action potentials at body temperature and using a physiological extracellular Ca 2+ concentration. METHODS Tissue preparationApical-coil IHCs (n = 325) of CD-1 mice (Swiss CD-1, Charles Rivers, Margate, UK) were studied in acutely dissected organs of Corti from E16.5 to P20, where the day of birth (P0) corresponds to E19.5. For embryonic experiments only, mice were paired overnight and checked for vaginal plugs the following morning. Assuming ovulation occurs midway through the dark cycle, the midpoint of the light cycle of the day following mating is considered to be E0.5. Adult and neonatal mice were killed by cervical dislocation and embryos by decapitation, in accordance with UK Home Office regulations. The cochleae were dissected in extracellular solution composed of (mM): 135 NaCl, 5.8 KCl, 1.3 CaCl 2 , 0.9 MgCl 2 , 0.7 NaH 2 PO 4 , 5.6 D-glucose, 10 Hepes-NaOH, 2 sodium pyruvate. Amino acids and vitamins for Eagle's minimum essential medium (MEM) were added from concentrates (Invitrogen, Paisley, UK). The pH was adjusted to 7.5 and the osmolality was about 308 mmol kg _1 . The organs of Corti were transferred to a microscope chamber containing extracellular solution, in which they were immobilized with a nylon mesh fixed to a stainless steel ring. The chamber (volume 2 ml) was perfused at a flow rate of about 10 ml h _1 from a peristaltic pump and mounted on the stage of an upright microscope (Zeiss ACM, Germany or Olympus, Japan). The organs of Corti were observed with Nomarski differential interference contrast optics (w 40 water-immersion objectives). To expose the basolateral surfaces of the cells, a small tear was made in the epithelium with a suction pipette (tip diameter about 2-4 mm) filled with extracellular solution. The cells were then cleaned by a stream of fluid from this pipette before patching. Only cells of healthy appearance were selected for electrophysiological recordings. Criteria included an intact hair bundle, cell membranes with a smooth surface, absence of vacuoles in the cytoplasm and lack of Brownian motion of mitochondria. The position of cells along the cochlea was recorded as fractional distance from the extreme apex. In the immature cochlea, cells were positioned at a fractional distance of between 0.16 and 0.24. Mature IHCs were positioned bet...

Distinct capacity for differentiation to inner ear cell types by progenitor cells of the cochlea and vestibular organs

“…These values are set in large measure by K + -selective inwardly rectifying and outwardly rectifying channels and are within the physiological range for postnatal HCs (Eatock and Hurley, 2003 within the normal range reported from postnatal HCs. We did not see the mixed Ca 2+ -Na + spikes that are typical of mouse IHCs in the first postnatal week (Marcotti et al, 2003b). All Atoh1 + cells lacked the voltage-gated Na + currents of immature HC subtypes (Chabbert et al, 2003;Eckrich et al, 2012;Géléoc et al, 2004;Li et al, 2010;Marcotti et al, 2003b;Oliver et al, 1997;Witt et al, 1994;Wooltorton et al, 2007).…”

“…We did not see the mixed Ca 2+ -Na + spikes that are typical of mouse IHCs in the first postnatal week (Marcotti et al, 2003b). All Atoh1 + cells lacked the voltage-gated Na + currents of immature HC subtypes (Chabbert et al, 2003;Eckrich et al, 2012;Géléoc et al, 2004;Li et al, 2010;Marcotti et al, 2003b;Oliver et al, 1997;Witt et al, 1994;Wooltorton et al, 2007). Again, these channels either were never expressed or had stopped being expressed by the earliest time point we examined, 12 days of in vitro differentiation.…”

Ion Channel Gene Expression in the Inner Ear

“…They are expressed in rat outer hair cells on postnatal days 0-9, but expressed barely by day 18 (Oliver et al 1997). In mouse inner hair cells, Na + current (potential candidate Nav1.7; SCN9A) is present as early as ED16.5 and disappears by the onset of hearing on postnatal day 12 (Marcotti et al 2003). In comparison, spiral ganglion cells of adult mouse are immunoreactive for Nav1.2 (SCN2A) and Nav1.6 (SCN8A) (Hossain et al 2005).…”

“…Ion channels play an important role in signal transmission, and their malfunction can lead to both hearing and vestibular dysfunction (Coucke et al 1999;Holt and Corey 1999;Kharkovets et al 2000;Hunter 2001;Nicolas et al 2001;Casimiro et al 2001;Ashmore 2002;Dumont and Gillespie 2003;Marcotti et al 2003;Rüttiger et al 2004;Wangemann et al 2004). Studies of voltage-gated ion channel expression in inner ear tissues (Adamson et al 2002;Davis 2003) and expression of ionic currents during the development of hearing (Fuchs and Sokolowski 1990;Mammano and Ashmore 1996;Kros et al 1998;Michna et al 2003) provide evidence for the fundamental importance of ion channels in defining cellular specialization.…”

Spatiotemporal pattern of action potential firing in developing inner hair cells of the mouse cochlea

“…At the onset of hearing, mammalian IHCs produce graded receptor potentials to open voltage-gated calcium channels, which in turn trigger synaptic vesicle fusion and glutamate exocytosis into the synaptic cleft (1). In contrast, immature IHCs fire spontaneous and evoked action potentials (APs) until the onset of hearing (postnatal day 10 to postnatal day 12, P10-P12, in the mouse) (2)(3)(4)(5). Calcium AP firing in hair cells may refine synaptic connections along the ascending auditory pathway (6)(7)(8)(9), as a single AP triggers synaptic vesicle exocytosis, and convey information arising from hair cells to the higher auditory centers (3,10,11).…”