Preliminary Characterization of Voltage-Activated Whole-Cell Currents in Developing Human Vestibular Hair Cells and Calyx Afferent Terminals
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We present preliminary functional data from human vestibular hair cells and primary afferent calyx terminals during fetal development. Whole-cell recordings were obtained from hair cells or calyx terminals in semi-intact cristae prepared from human fetuses aged between 11 and 18 weeks gestation (WG). During early fetal development (11–14 WG), hair cells expressed whole-cell conductances that were qualitatively similar but quantitatively smaller than those observed previously in mature rodent type II hair cells. As development progressed (15–18 WG), peak outward conductances increased in putative type II hair cells but did not reach amplitudes observed in adult human hair cells. Type I hair cells express a specific low-voltage activating conductance, G K,L. A similar current was first observed at 15 WG but remained relatively small, even at 18 WG. The presence of a “collapsing” tail current indicates a maturing type I hair cell phenotype and suggests the presence of a surrounding calyx afferent terminal. We were also able to record from calyx afferent terminals in 15–18 WG cristae. In voltage clamp, these terminals exhibited fast inactivating inward as well as slower outward conductances, and in current clamp, discharged a single action potential during depolarizing steps. Together, these data suggest the major functional characteristics of type I and type II hair cells and calyx terminals are present by 18 WG. Our study also describes a new preparation for the functional investigation of key events that occur during maturation of human vestibular organs.
Keywordselectrophysiology human hair cells vestibular development
Most of our understanding about the cellular development of human peripheral vestibular organs comes from anatomical studies, which have documented their early growth and maturation (Sans and Dechesne 1985, 1987). Anatomical differentiation of human vestibular hair cells and supporting cells begins at the end of the embryonic period when fetal crown-rump length (CRL) is between 16.5 and 26 mm or approximately 8–9 weeks gestation (WG, Dechesne et al. 1987). At this stage, hair cells have short, polarized hair bundles and exhibit anatomical features, including synaptic bodies, which are consistent with synapse development (Sans and Dechesne 1985). Concomitantly, invading primary afferent fibers are juxtaposed with these nascent hair cells and exhibit postsynaptic densities (Sans and Dechesne 1985). Therefore, innervation by primary afferent fibers precedes full hair cell differentiation. However, synapse development occurs in tandem with morphological differentiation as reported in mouse vestibular organs (Rüsch et al. 1998) and mouse cochlea (Mbiene et al. 1988). Limited anatomical data are available for human tissue beyond this time.
We know that in amniotes (reptiles, birds, and mammals), further anatomical and physiological differentiation results in the emergence of type I and type II hair cells. In mature vestibular epithelia, these hair cell types can be distinguished by three features: (1) shape, (2) their primary afferent contacts, and (3) whole-cell conductances. The ubiquitous type II hair cell, which is present in all vertebrates, is cylindrically shaped, contacted by conventional bouton-like afferent terminals, and expresses an assortment of voltage- and ligand-gated conductances including: outward and inward rectifiers, A-like conductances, and calcium activated K+ conductances. Type I hair cells, in contrast, have a constricted neck, are contacted by an enveloping “cup-like” or calyx afferent terminal, and express a unique low-voltage activated K+ conductance called G K,L. In rodents, the emergence of G K,L marks the physiological differentiation of type I hair cells (Rüsch et al. 1998; Geleoc et al. 2004).
Although we know that mature human vestibular hair cells express type I and type II specific whole-cell conductances (Oghalai et al. 1998) similar to those seen in rodents, we have no data on the physiological differentiation of human hair cells. Even basic information about when each hair cell type begins to express characteristic whole-cell conductances is not known. From a broader perspective it means we do not know how data from rodents with rapid fetal development measured in days and weeks, translates to the relatively long human gestational period, measured in months. To address these significant shortcomings, we have established a semi-intact preparation of fetal human vestibular organs and have begun to examine the spatiotemporal expression of whole-cell conductances in hair cells and associated calyx primary afferent terminals during an important phase (11–18 WG) of peripheral vestibular development.
MATERIALS AND METHODS
The University of Newcastle Human Ethics Committee approved all procedures. Written consent was obtained from all tissue donors. Apart from gestational age, no other identifying information was supplied. Gestational age was determined by three criteria: (1) the date of the last menstrual period, (2) ultrasound measurement of CRL, and (3) foot length. Tissue was obtained from electively terminated fetal material. There were no known instances of abnormalities. Tissue specimens were collected in cold glycerol-based artificial cerebrospinal fluid (ACSF; see below) and transported to The University of Newcastle. Time between the tissue collection and the dissection of epithelium was less than 1 h.
Properties and characteristics of type I and II vestibular hair cells aged 11–14 WG and 15–18 WG
n = 13
n = 17
n = 13
821.0 ± 150.0*
776.6 ± 139.3#
158.1 ± 37.4*#
12.0 ± 1.0
12.3 ± 1.2
13.03 ± 34.0
11.5 ± 4.0
7.7 ± 1.6
7.3 ± 2.4
G max (nS)
3.5 ± 0.2*
11.9 ± 1.5*
V ½ (mV)
−22.4 ± 2.6
−25.6 ± 1.9
7.9 ± 0.7
7.2 ± 0.4
Whole-cell patch clamp recordings were obtained from hair cells and calyx afferent terminals in 31 semi-intact preparations of human fetal vestibular cristae (aged 11–18 WG).
Hair Cell Recordings
Hair cells lacking G K,L conductance exhibit an outward delayed rectifier conductance, GDR, which is similar to that expressed in type II hair cells throughout embryonic and postnatal development in rodent utricle and crista (Rüsch et al. 1998; Geleoc et al. 2004). The peak current–voltage (I–V) relationship of putative human type II hair cells increases with fetal age. For example, the 12 WG type II hair cell had a maximum peak current at +11 mV of ∼1 nA, which was substantially smaller than the peak current of ∼4.5 nA observed in the 17 WG type II hair cell (Fig. 2C). Similarly, group data shows that the peak current at +11 mV was significantly smaller at 11–14 WG than at 15–18 WG (1.0 ± 0.1 nA; n = 13 versus 2.5 ± 0.2 nA; n = 17, t (28) = 2.05, p = 0.00003).
Activation curves were obtained from the instantaneous tail currents immediately following a step to −39 mV (asterisks, Fig. 2A, B). These values were plotted against the holding potential prior to the step (Fig. 2D). Putative type II hair cells exhibit typical sigmoidal tail current activation curves. Boltzmann curves were fitted to calculate G MAX, V ½, and S values. For the 12 WG type II hair cell, G MAX was calculated as 3.9 nS versus 24.8 nS for the 17 WG type II hair cell. For group data, G MAX was significantly smaller in putative type II hair cells aged 11–14 WG (3.5 ± 0.3 nS, n = 13) compared to those aged 15–18 WG (11.9 ± 1.5 nS, n = 17, t (28) = 2.05, p = 0.00008). Notably, even at this later developmental stage, hair cells exhibit only ∼60 % of the G MAX values obtained in isolated adult human hair cells (11. 9 nS, n = 17 versus 19.3 nS calculated from Oghalai et al. 1998). These data support the notion that G MAX values continue to increase with gestational age.
Although G MAX values were smaller, V ½ and S values were not significantly different (t (28) = 2.05, p = 0.32 and t (28) = 2.05, p = 0.43, respectively) between the two age groups (see Table 1). Furthermore, our data show that the calculated V ½ and S values were similar to those obtained for embryonic mouse hair cells (Geleoc et al. 2004) and for G DR1 in postnatal rat hair cells (Rüsch et al. 1998). However, compared to isolated adult human hair cells (Oghalai et al. 1998), type II human fetal hair cells have a more depolarized V ½ and larger S values or a less steep Boltzmann slope (human adult–estimated V ½ = −47 mV, S = 6.1; human fetus V ½ = −34.5 mV, S = 7.9).
In addition to outward conductances, at hyperpolarized membrane potentials (more negative than −70 mV), we observed small inward rectifying conductances (∼2 nS) at all age groups examined. Examples of limited inward rectification can be observed in Figure 2. However, there was no significant difference in the conductance of inward rectifying conductances between the two age groups (2.00 nS, n = 13 versus 1.73 nS, n = 17 for 11–14 WG and 15–18 WG, respectively; t (28) = 2.05, p = 0.64).
In addition to the type II hair cells described above, we made recordings from a total of 13 presumed type I hair cells. These were classified as type I hair cells on the basis of their low input resistance (158.1 ± 37.4 MΩ, n = 13) and the presence of a small putative G K,L conductance. Although low input resistance is characteristically associated with type I hair cells (due to the presence of activated G K,L at resting membrane potentials), this may also reflect a “leaky” cell. Therefore, to satisfy the type I designation, the hair cell had to show evidence of deactivation at more hyperpolarized potentials. In other words, membrane potentials more negative than -90 mV, where G K,L is in a closed state, the hair cell’s input resistance was higher than at -69 mV when G K,L is open (158 MΩ at −69 mV versus 306 MΩ at −99 mV, n = 5). The input resistance values obtained for these presumptive type I hair cells at −69 mV are comparable to that reported for embryonic mouse hair cells (158 versus 55 MΩ, Geleoc et al. 2004). The difference in input resistance between the hair cell types was not related to hair cell size, as measurements of membrane capacitance were similar in all hair cells (see Table 1).
We recently reported similar collapsing tail currents in type I hair cells from a semi-intact preparation of the mouse crista. This feature is not present in acutely isolated hair cells (Lim et al. 2011). In mice, we attributed the collapse of tail currents to the close apposition of the calyx terminal. These cup-like terminals surround type I hair cells early in fetal development (Sans et al. 1994) and restrict potassium (K+) diffusion away from the type I hair cell. This results in K+ accumulation between hair cell and calyx thereby reducing the driving force and attenuating tail currents (Lim et al. 2011). The collapsing tail currents in the putative type I hair cell at 15 WG suggests a similar situation exists in the human fetal hair cells; i.e., the presence of a developing partial or full calyx is sufficient to influence the ionic microenvironment around the type I hair cell. Importantly, while putative G K,L is smaller in semi-intact human fetal type I hair cells, the presence of “collapsing” activation curves at 15 WG is indicative of an emerging type I hair cell.
As a consequence of collapsing activation curves, calculations of G MAX, V ½, and S (which assume stable K+ concentrations surrounding the hair cell and fixed K+ reversal potentials) are not valid when analyzing type I hair cells and are therefore not presented.
In current clamp, the average resting membrane potential of developing human calyx terminals was −64 ± 4 mV (n = 3). Current clamp recordings from rodent calyceal terminals typically show that a single action potential (AP) is discharged during depolarization (Rennie and Streeter 2006; Dhawan et al. 2010). Similarly, the human calyx recording shown in Figure 5C, with a resting membrane potential of −56 mV, also discharged a single AP in response to a depolarizing current step of 20 pA (Fig. 5C). We could not elicit multiple AP’s with increasing level of current injection. The AP had a 10 mV overshoot and showed a small after hyperpolarization, similar to that observed in mature gerbil calyces (Meredith et al. 2011). Our data, therefore, indicate that calyx terminals are not only present in the developing neuroepithelium but may also be functionally active as early as 15 WG. We have recordings from three calyceal terminals from human neuroepithelium aged 15, 17, and 18 WG, although only the calyx aged 18 WG discharged action potentials. However, we did not see evidence of synaptic quantal events in any of our voltage clamp recordings from calyceal terminals. Thus, it is unclear if the calyx synapse is fully functional at this early stage of development.
In this study, we established a semi-intact preparation of the human fetal vestibular organs and obtained the first whole-cell current recordings from developing human vestibular hair cells and the only recordings from human calyx primary afferent terminals. Our major finding is that the gestational period examined (11–18 WG) represents a crucial transitional phase where the mature functional characteristics of type I and type II hair cells emerge.
Recordings from Hair Cells
From our data, 11 to 14 WG marks the end of a nascent phase where type II vestibular hair cells express whole-cell conductances similar to, albeit smaller than, more mature fetal human hair cells (15–18 WG). Our results indicate that there is a significant increase in G MAX of type II hair cells during development. This increase in G MAX was not associated with a change in V ½ or S, the slope of the activation curve. This suggests the expression of a greater number of voltage-activated currents in type II hair cells with age rather than a change or an upregulation of different channel types. However, both fetal (this study) and adult (Oghalai et al. 1998) G MAX values recorded in human hair cells were smaller than those in rodents (Rüsch et al. 1998). These reduced G max values would have the effect of increasing hair cell input resistance. Therefore, for a given stimulus, the voltage gain would be enhanced, presumably resulting in more neurotransmitter release.
Increased input per human hair cell onto afferent terminals is amplified still further by the significantly increased convergence of human hair cells onto primary afferent terminals compared to rodents. A recent study estimated there were 36,000 hair cells in both fetal (16 WG) and adult human utricle (Severinsen et al. 2010). This is almost an order of magnitude greater (36,000 versus. 3,800) than values reported for the same structure in mice (Desai et al. 2005b). There are approximately 3,400 utricular afferent fibers in humans (Bergstrom 1973) and ∼680 in mice (Desai et al. 2005b) resulting in an increased ratio of hair cells per afferent (∼10:1 versus ∼5:1). A similar ratio also exists for cristae between human and mouse. In adult human cristae, the ratio of hair cells to afferent fibers is 5:1 (∼8,000 hair cells; ∼1,400 afferents; Lopez et al. 2005a; Lopez et al. 2005b), while in mouse cristae, the ratio is 1:2 (∼1,420 hair cells; ∼680 afferents; Desai et al. 2005a). Precisely, why there is potentially more hair cell transmitter release and greater convergence onto afferent terminals in humans than rodents is unclear, but these results suggest that human afferent discharge thresholds may be higher than those in rodents.
During the next phase of development (15–18 WG), there is continued maturation where adult-like features of the vestibular neuroepithelium begin to emerge. At this stage, whole-cell voltage-activated currents were more diverse and conductances were larger than earlier stages of development but still smaller than those observed in adult human hair cells (Oghalai et al. 1998). Indeed, 15 WG appears to be a milestone in hair cell development where an apparent small G K,L begins to emerge, resulting in the functional segregation of two hair cell types. Similar to other outward conductances described above, initially, G K,L is small and likely to increase in amplitude during development as has been observed in developing mouse vestibular hair cells (Geleoc et al. 2004). Our results also suggest that this physiological differentiation, as determined by the expression of G K,L , precedes morphological differentiation, as a recent study could not unambiguously distinguish type I and type II hair cells at 16 WG (Severinsen et al. 2010).
While future pharmacological studies will include antagonists such as linopirdine and XE-991 to establish the presence of G K,L in putative type I hair cells, there are several other features that suggest the increasing expression of G K,L. For example, low input resistance, a direct consequence of G K,L expression marks the physiological differentiation of type I hair cells. In mice, this lowered input resistance is first observed at E18 (Geleoc et al. 2004) and becomes more prevalent in both mice and rats during the first postnatal week (Rüsch et al. 1998; Geleoc et al. 2004). Our data show that putative human type I vestibular cells also exhibit significantly lower input resistances at ∼15 WG compared to type II hair cells. It has also been noted that G K,L activates at more positive membrane potentials in neonatal rats (average V ½ = 34 mV more depolarized in the first compared to the second postnatal week, Hurley et al. 2006). Our data are also consistent with these observations, as fetal G K,L is activated at more depolarized potentials (∼−59 mV; see Fig. 4) than reported in adult human vestibular hair cells that have an activation range of approximately −90 mV (Oghalai et al. 1998). Rodent data also show that the magnitude of G K,L in hair cells increases with age as does G MAX in cultured and acutely isolated epithelial preparations (Rüsch et al. 1998). Due to the collapse of tail currents at potentials more depolarized than ∼−29 mV, we could not determine G MAX in fetal human type I hair cells. As mentioned above, collapse of the activation curve is only observed in preparations that maintain the cellular microarchitecture around type I hair cells (Lim et al. 2011) and is consistent with the presence of partial or complete calyceal primary afferents surrounding presumed type I hair cells (see Fig. 4C). The presence of small but distinct G K,L as well as collapsing tail currents supports the notion that physiological differentiation of type I hair cells occurs at approximately 15 WG in human fetal hair cells.
Our data also shows that there is a transient expression of Na+ channels in human hair cells up to 14 WG. In rodent hair cells, Na+ conductances have markedly differing cellular, regional, and developmental expression profiles (Rüsch et al. 1998; Geleoc et al. 2004; Wooltorton et al. 2007; Li et al. 2010). Two types of Na+ currents have been identified in rats; I na,1 is found in all type I hair cells throughout development, whereas I Na,2, was present only in subsets of hair cells until P7 (Wooltorton et al. 2007). Our data show that V 1/2 act and V 1/2 deact values for presumptive Na+ currents in human hair cells are similar to those described for I na,1 in developing rat hair cells (Wooltorton et al. 2007). Pharmacological characterization of presumptive Na+ currents in fetal human hair cells is required to determine which subtypes are expressed during the developmental period examined.
The transient expression of Na+ channels during development is thought to result in AP generation and maybe important for release of trophic factors necessary for synaptic maturation (Chabbert et al. 2003). We observed Na+ conductances in hair cells over a period where the anatomical substrates of synapse formation appear (Sans et al. 1994) but prior to clear morphological differentiation of type I hair cells. Taken together, our data suggests the decline in Na+ conductances from human hair cells coincides with the increased expression of G K,L and the time when definitive calyceal recordings are first obtained.
Our data suggests a discrepancy between anatomical and physiological maturation of vestibular primary afferent terminals. Morphological studies show that penetration of the human vestibular neuroepithelium by primary afferents begins at ∼6.5 WG (Yokoh 1974), whereas contact with hair cells occurs by 9 WG (Sans and Dechesne 1985). Synaptic specializations including pre- and postsynaptic densities are also evident at this developmental stage (Sans and Dechesne 1985). This advanced development of synaptic elements suggests that calyceal terminals maybe functional very early during human development. The physiological readout of a “functional synapse” would be the presence of quantal synaptic events, appearing as brief miniature postsynaptic currents in calyceal voltage clamp recordings. This would indicate the activation of calyx postsynaptic receptors by neurotransmitter release from hair cells. Since we did not observe any quantal events in our calyceal recordings, this suggests three possibilities: (1) the synapses may not be mature (Songer and Eatock 2013); (2) damaged or immature hair bundles and consequently transduction apparatus may result in hyperpolarized hair cell membranes thereby reducing transmitter release; or (3) quantal release was compromised in our preparations. Although our current experimental setup could not distinguish between these three possibilities, it should be noted that calyx afferent terminals possess whole-cell conductances necessary for AP discharge by 18 WG. This would suggest that if the calyx/hair cell synapses were functional, then these signals could be transmitted by the afferent fiber to the CNS. Nevertheless, it still remains to be determined exactly when the human hair cell/calyx synapse becomes functionally active.
In short, while morphological data suggest that development of the unique hair cell/calyx synapse is around 8–9 WG, this appears to be well in advance of physiological maturation of G K,L in type I hair cells and the AP-generating conductances in afferents that have emerged by ∼15 WG. The chronology of afferent innervation and hair cell physiological differentiation in humans is therefore consistent with findings in rats where the encapsulation of presumed type I hair cells by calyces occurs independently and in advance of hair cell differentiation and the acquisition of G K,L (Rüsch et al. 1998). It should be noted that although G K,L is typically associated with type I vestibular hair cells, it has also been described in rat and gerbil calyceal terminals (Hurley et al. 2006; Rennie and Streeter 2006; Dhawan et al. 2010). Our recordings from human calyx terminals provide similar evidence for nascent G K,L .
Anatomical evidence suggests that the human vestibular neuroepithelium is well developed by 14 WG (Rosenhall 1972); however, prior to our study, functional data were not available. Although we know that hair cell innervation begins early in fetal development (9WG, Sans et al. 1994; Sans and Scarfone 1996), our data suggests that at this stage of development, hair cell conductances are not mature compared to adult human hair cells (Oghalai et al. 1998). By 15 WG, however, we observed early signs of G K,L in some hair cells. The presence of hair-cell-specific conductances, together with AP generation in calyx terminals, (as well as prior studies on afferent myelination at 8–9 WG, Sanchez-Fernandez and Rivera-Pomar 1983) implies that the vestibular neuroepithelium has the necessary machinery to transmit sensory signals to the CNS early in the second trimester of pregnancy. This prediction would be dependent on the mechanosensory transduction channels (associated with the hair bundles of hair cells) being functionally operational. While this has yet to be confirmed in human tissue, it appears to be a reasonable assumption since transduction signals can be evoked early in mouse vestibular and auditory hair cell development (E17–P0; Geleoc and Holt 2003; Lelli et al. 2009).
Understanding the timing of morphological and physiological development of human hair cells and their primary afferent terminals is critical if we are to apply regenerative technologies to human inner ears. Studies have shown that the processes involved in regeneration and recovery from injury often recapitulate those observed during development (Levic et al. 2007). Given the precise spatiotemporal patterning of ion channel expression and coincident primary afferent innervation, future human experiments will need to target the concomitant molecular signals that are necessary for cell survival, specification, differentiation, ion channel expression, and synaptogenesis. Some of these signals, whose appearance is fleeting during the compressed embryogenesis in rodents, presumably persist longer in the extended gestational period of humans. We propose that our semi-intact preparation presents a new opportunity for combined functional, anatomical, and molecular investigation of fetal hair cell development in humans.
This research was funded by The Garnett Passe and Rodney Williams Memorial Foundation and the National Health and Medical Research Council of Australia (Grants 1022717, 1048232). We would also like to thank Em. Prof Peter Barry for assisting with liquid junction potential calculations.
Conflict of Interest
The authors declare that they have no conflict of interest.
- Chabbert C, Mechaly I, Sieso V, Giraud P, Brugeaud A, Lehouelleur J, Couraud F, Valmier J, Sans A (2003) Voltage-gated Na + channel activation induces both action potentials in utricular hair cells and brain-derived neurotrophic factor release in the rat utricle during a restricted period of development. J Physiol 553:113–123PubMedCrossRefPubMedCentralGoogle Scholar
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