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J Physiol 592.21 (2014) pp 4611–4612
JOURNAL CLUB
Zebrafish in auditory research: are fish better than mice? Michael G. Leitner Department of Neurophysiology, Institute of Physiology and Pathophysiology, Philipps-University Marburg, 35037 Marburg, Germany
The Journal of Physiology
Email: [email protected]
Sensory inner and outer hair cells (IHCs and OHCs) in the mammalian organ of Corti mediate the perception of sound, and hair cells (HCs) in the vestibular system detect head movements. Their characteristic feature is the ‘hair bundle’ of actin-based stereocilia that are arranged in three rows, with increasing length toward the apical cell pole. Sound- or movement-induced deflection towards the longest row opens mechanoelectrical transduction (MET) channels, whereas movement in the opposite direction closes these ion channels. Accordingly, opening and closing of the MET channels transduce sound information and movement of the head into receptor potentials in auditory and vestibular hair cells, respectively. Depolarization induces calcium-dependent neurotransmitter release from specialized presynaptic structures in IHCs and vestibular HCs; the resultant EPSP modulates action potential firing in afferent nerves. In OHCs, receptor potentials produce somatic length changes that comprise the cochlear amplifier and underlie the high sensitivity and frequency selectivity of mammals. The response of the HC membrane potential to MET channel activation is tuned and characterized by a distinct profile of K+ channels in the basolateral membrane (Schwander et al. 2010). The use of rodents and guinea-pigs in auditory research has yielded significant insight into normal and pathophysiological hearing as well as HC function. The animals are easy to handle, exhibit short breeding times, and the anatomy of the inner ear is comparable to that of humans. Their hearing range overlaps with that of humans, but importantly, hair cell physiology appears, as far as one can determine, to be virtually identical in these animals and humans. The many available
genetic markers and genetically modified mouse strains that recapitulate human deafness and balance disorders highlight these similarities and demonstrate the applicability of rodents for studies of human hearing and deafness. However, rodents do not allow for large-scale mutagenesis and preclude high-throughput screening for drugs, because the sensory epithelia are embedded in a bony capsule (Schacht & Leitner, 2014). In recent years, the zebrafish (Danio rerio) has come to be appreciated as an alternative model system for auditory physiology (and other research fields), because these fish offer significant advantages over higher vertebrates. Zebrafish exhibit relative ease of genetic manipulation with fast procreation, and because the sensory organs are not coiled or encapsulated, HCs are readily accessible. Given these advantages, zebrafish might be suited to study HC physiology, but it has not yet been determined whether zebrafish and mammalian HCs are (electro)physiologically similar. One problem has been that the zebrafish cells are much smaller than mammalian HCs, which makes satisfactory experimental assessment more difficult. In a recent article in The Journal of Physiology, Olt, Johnson and Marcotti refined previously developed electrophysiological protocols to provide a detailed analysis of the biophysical properties of zebrafish HCs (Olt et al. 2014). These results allowed for the evaluation of zebrafish as models for mammalian HCs. Hair cells in zebrafish localize to the lateral line and to the inner ear structures the sacculus, utriculus and lagena. The lateral line is located along the body axis and detects directional movement and water flow. The zebrafish HCs are surrounded by a gelatinous and inertial cupula similar to the mammalian vestibular system. Given that zebrafish larvae are transparent and non-pigmented mutants are available, HCs in the lateral line can be labelled and visualized with fluorescent proteins and monitored with standard light microscopy. Accordingly, Olt and colleagues could assess HCs directly in the living fish, i.e. in an in vivo-like setting in controlled in vitro conditions. In the inner ear of zebrafish, the sacculus is the hearing organ (up to 4 kHz), the utriculus is required for balance function, and the lagena is believed to
C 2014 The Author. The Journal of Physiology C 2014 The Physiological Society
implement hearing and balance functions. Hair cells in the inner ear appear later in development, and the structures needed to be dissected for experimental analysis of the HCs (Olt et al. 2014). With whole-cell patch clamp, the authors characterized HCs during development (dpf/wpf, days/weeks post fertilization; larvae, 3 dpf to 2 wpf; juvenile, 2 wpf to sexual maturation; and adult, >6 months old) and at different relative positions within the organ (centre or edge). Hair cells in each sensory epithelium were characterized by a distinct profile of K+ currents that differed between developmental stages and the position within the organ. Hair cells in the centre of neuromasts in the lateral line developed a mature K+ current phenotype during the third week post fertilization (voltage-dependent delayed-rectifier K+ current, IK,D ; and large voltage-dependent and inactivating K+ current, IA ). In contrast, cells at the edge of the neuromast retained immature properties (IK,D ; small IA ; and calcium-activated K+ current, IK,Ca ). The persistently immature phenotype might represent differentiating HCs that are derived typically from the edge of the organ before they migrate to the centre. This ability of zebrafish to regenerate HCs remarkably dissociates fish from mammals and points to their extraordinary features. The characteristics of HCs along the lateral line were essentially the same, but were quite different from HCs of higher vertebrates. In the zebrafish inner ear, HCs displayed variable K+ current phenotypes early in development that converged on characteristic groups of K+ currents in adult zebrafish. Murine HCs also showed developmental changes (Marcotti et al. 2003), but the characteristics of immature zebrafish HCs appeared more variable and were different from those of higher vertebrates. In adult zebrafish, certain HCs displayed biophysical properties reminiscent of mammalian HCs; HCs at the edge of the lagena and throughout the utriculus were comparable to vestibular HCs in mice and birds. Hair cells in the centre of lagena and sacculus showed characteristics of immature rodent IHCs, such as a delayed rectifier (IK,D ) and an inwardly rectifying K+ current (IK1 ; Marcotti et al. 2003). In the centre of the lagena, HCs even generated action
DOI: 10.1113/jphysiol.2014.280438
Downloaded from J Physiol (jp.physoc.org) at UNIV OF NORTH CAROLINA on November 7, 2014
4612
Journal Club
potentials similar to spiking immature rodent IHCs (Marcotti et al. 2003). Accordingly, zebrafish HCs from the edge of the lagena or from the utriculus might constitute biophysical models for mammalian vestibular hair cells and those from the centre of the lagena and sacculus for developing IHCs, but not for mammalian cochlear HCs. Olt et al. (2014) identified voltage-dependent Ca2+ currents in HCs of the lateral line that were similar to presynaptic Cav 1.3 channels of mammalian auditory and vestibular HCs. These currents together with Ca2+ -dependent vesicle release suggested neuromasts as a potential model for the mammalian HC synapse. This was already proposed several decades ago, but detailed analysis of the underlying mechanisms is still required to evaluate zebrafish in comparison to other animal models. The study reveals variable and inhomogeneous biophysical properties of HCs in the sensory organs of zebrafish. These multiple HC types, the absence of OHC-based amplification and the physiological similarities between the lateral line and the vestibular system (rather than the cochlea) render extrapolations to mammals generally difficult. A confounding factor might be the evolutionary distance to humans that is also depicted by biophysical similarities of HCs in the lagena and the sacculus to those of goldfish and frogs (Olt et al. 2014). The recordings demonstrated comparable biophysical properties of certain HCs in zebrafish and mammals, but it is not clear whether the HC physiology itself is also similar. It remains to be elucidated whether the HC currents were mediated by protein orthologues and whether the orthologues are functionally similar. Of special note, strikingly different properties have been reported from zebrafish orthologues related to hearing in mammals (e.g. prestin, see
Schaechinger & Oliver, 2007). Of course, the similarity of protein expression and function determines the applicability of zebrafish as model system. On the positive side, large-scale mutagenesis screens can be performed in zebrafish to identify underlying proteins, but due to the stated differences from mammals, promising candidates will require more elaborate animal models in order to assess the applicability of zebrafish further. An additional important finding of the study concerns the widely used zebrafish anaesthetic, tricane methanesulfonate (MS-222). MS-222 reversibly inhibited K+ currents (IA and IK,Ca ) in juvenile HCs of the lateral line. Accordingly, the membrane potential of the HCs, the activity of the MET channels and the biophysical membrane properties in an anaesthetized fish might be altered significantly. This has the potential to produce misleading results and should be kept in mind when the analysis of HCs is the experimental goal. The study by Olt et al. (2014) raised several points worth considering prior to work with zebrafish HCs, as follows: (i) immature and mature HCs coexist throughout life, with different biophysical properties and probably with different protein components; (ii) HCs in the centre of adult sensory organs display a mature phenotype; (iii) grouping of findings into developmental stages and the HC location might increase the significance of the data; (iv) some HCs resemble mammalian vestibular or immature auditory HCs (similar K+ and Ca2+ currents), but the molecular components in zebrafish remain unknown; and (v) zebrafish offer the applicability of genetic and pharmacological high-throughput screenings, but due to the evolutionary distance from mammals the candidate mechanisms should be evaluated in additional model systems.
J Physiol 592.21
In summary, the biophysical results shown in this paper indicate that zebrafish provide promising models of mammalian HCs. Even so, the molecular identity of the involved proteins needs to be established, and zebrafish HC physiology requires further analysis to evaluate whether zebrafish HCs are genuinely similar and indeed recapitulate mammalian vestibular and auditory physiology. References Marcotti W, Johnson SL, Holley MC & Kros CJ (2003). Developmental changes in the expression of potassium currents of embryonic, neonatal and mature mouse inner hair cells. J Physiol 548, 383–400. Olt J, Johnson SL & Marcotti W (2014). In vivo and in vitro biophysical properties of hair cells from the lateral line and inner ear of developing and adult zebrafish. J Physiol 592, 2041–2058. Schacht J & Leitner MG (2014). In vitro models for otoxoic research. In Methods in Pharmacology and Toxicology: In Vitro Toxicology Systems, ed. Jennings P & Bal-Price A, Part IV. Chapter 9, pp. 199–224. Humana Press, New York. Schaechinger TJ & Oliver D (2007). Nonmammalian orthologs of prestin (SLC26A5) are electrogenic divalent/chloride anion exchangers. Proc Natl Acad Sci U S A 104, 7693–7698. Schwander M, Kachar B & M¨uller U (2010). Review series: the cell biology of hearing. J Cell Biol 190, 9–20.
Additional information Competing interests
None declared. Funding
M.G.L. is supported by a Research Grant of the University Medical Center Giessen und Marburg (UKGM 17/2013 MR)
C 2014 The Author. The Journal of Physiology C 2014 The Physiological Society
Downloaded from J Physiol (jp.physoc.org) at UNIV OF NORTH CAROLINA on November 7, 2014
J Physiol 592.21 (2014) pp 4611–4612
JOURNAL CLUB
Zebrafish in auditory research: are fish better than mice? Michael G. Leitner Department of Neurophysiology, Institute of Physiology and Pathophysiology, Philipps-University Marburg, 35037 Marburg, Germany
The Journal of Physiology
Email: [email protected]
Sensory inner and outer hair cells (IHCs and OHCs) in the mammalian organ of Corti mediate the perception of sound, and hair cells (HCs) in the vestibular system detect head movements. Their characteristic feature is the ‘hair bundle’ of actin-based stereocilia that are arranged in three rows, with increasing length toward the apical cell pole. Sound- or movement-induced deflection towards the longest row opens mechanoelectrical transduction (MET) channels, whereas movement in the opposite direction closes these ion channels. Accordingly, opening and closing of the MET channels transduce sound information and movement of the head into receptor potentials in auditory and vestibular hair cells, respectively. Depolarization induces calcium-dependent neurotransmitter release from specialized presynaptic structures in IHCs and vestibular HCs; the resultant EPSP modulates action potential firing in afferent nerves. In OHCs, receptor potentials produce somatic length changes that comprise the cochlear amplifier and underlie the high sensitivity and frequency selectivity of mammals. The response of the HC membrane potential to MET channel activation is tuned and characterized by a distinct profile of K+ channels in the basolateral membrane (Schwander et al. 2010). The use of rodents and guinea-pigs in auditory research has yielded significant insight into normal and pathophysiological hearing as well as HC function. The animals are easy to handle, exhibit short breeding times, and the anatomy of the inner ear is comparable to that of humans. Their hearing range overlaps with that of humans, but importantly, hair cell physiology appears, as far as one can determine, to be virtually identical in these animals and humans. The many available
genetic markers and genetically modified mouse strains that recapitulate human deafness and balance disorders highlight these similarities and demonstrate the applicability of rodents for studies of human hearing and deafness. However, rodents do not allow for large-scale mutagenesis and preclude high-throughput screening for drugs, because the sensory epithelia are embedded in a bony capsule (Schacht & Leitner, 2014). In recent years, the zebrafish (Danio rerio) has come to be appreciated as an alternative model system for auditory physiology (and other research fields), because these fish offer significant advantages over higher vertebrates. Zebrafish exhibit relative ease of genetic manipulation with fast procreation, and because the sensory organs are not coiled or encapsulated, HCs are readily accessible. Given these advantages, zebrafish might be suited to study HC physiology, but it has not yet been determined whether zebrafish and mammalian HCs are (electro)physiologically similar. One problem has been that the zebrafish cells are much smaller than mammalian HCs, which makes satisfactory experimental assessment more difficult. In a recent article in The Journal of Physiology, Olt, Johnson and Marcotti refined previously developed electrophysiological protocols to provide a detailed analysis of the biophysical properties of zebrafish HCs (Olt et al. 2014). These results allowed for the evaluation of zebrafish as models for mammalian HCs. Hair cells in zebrafish localize to the lateral line and to the inner ear structures the sacculus, utriculus and lagena. The lateral line is located along the body axis and detects directional movement and water flow. The zebrafish HCs are surrounded by a gelatinous and inertial cupula similar to the mammalian vestibular system. Given that zebrafish larvae are transparent and non-pigmented mutants are available, HCs in the lateral line can be labelled and visualized with fluorescent proteins and monitored with standard light microscopy. Accordingly, Olt and colleagues could assess HCs directly in the living fish, i.e. in an in vivo-like setting in controlled in vitro conditions. In the inner ear of zebrafish, the sacculus is the hearing organ (up to 4 kHz), the utriculus is required for balance function, and the lagena is believed to
C 2014 The Author. The Journal of Physiology C 2014 The Physiological Society
implement hearing and balance functions. Hair cells in the inner ear appear later in development, and the structures needed to be dissected for experimental analysis of the HCs (Olt et al. 2014). With whole-cell patch clamp, the authors characterized HCs during development (dpf/wpf, days/weeks post fertilization; larvae, 3 dpf to 2 wpf; juvenile, 2 wpf to sexual maturation; and adult, >6 months old) and at different relative positions within the organ (centre or edge). Hair cells in each sensory epithelium were characterized by a distinct profile of K+ currents that differed between developmental stages and the position within the organ. Hair cells in the centre of neuromasts in the lateral line developed a mature K+ current phenotype during the third week post fertilization (voltage-dependent delayed-rectifier K+ current, IK,D ; and large voltage-dependent and inactivating K+ current, IA ). In contrast, cells at the edge of the neuromast retained immature properties (IK,D ; small IA ; and calcium-activated K+ current, IK,Ca ). The persistently immature phenotype might represent differentiating HCs that are derived typically from the edge of the organ before they migrate to the centre. This ability of zebrafish to regenerate HCs remarkably dissociates fish from mammals and points to their extraordinary features. The characteristics of HCs along the lateral line were essentially the same, but were quite different from HCs of higher vertebrates. In the zebrafish inner ear, HCs displayed variable K+ current phenotypes early in development that converged on characteristic groups of K+ currents in adult zebrafish. Murine HCs also showed developmental changes (Marcotti et al. 2003), but the characteristics of immature zebrafish HCs appeared more variable and were different from those of higher vertebrates. In adult zebrafish, certain HCs displayed biophysical properties reminiscent of mammalian HCs; HCs at the edge of the lagena and throughout the utriculus were comparable to vestibular HCs in mice and birds. Hair cells in the centre of lagena and sacculus showed characteristics of immature rodent IHCs, such as a delayed rectifier (IK,D ) and an inwardly rectifying K+ current (IK1 ; Marcotti et al. 2003). In the centre of the lagena, HCs even generated action
DOI: 10.1113/jphysiol.2014.280438
Downloaded from J Physiol (jp.physoc.org) at UNIV OF NORTH CAROLINA on November 7, 2014
4612
Journal Club
potentials similar to spiking immature rodent IHCs (Marcotti et al. 2003). Accordingly, zebrafish HCs from the edge of the lagena or from the utriculus might constitute biophysical models for mammalian vestibular hair cells and those from the centre of the lagena and sacculus for developing IHCs, but not for mammalian cochlear HCs. Olt et al. (2014) identified voltage-dependent Ca2+ currents in HCs of the lateral line that were similar to presynaptic Cav 1.3 channels of mammalian auditory and vestibular HCs. These currents together with Ca2+ -dependent vesicle release suggested neuromasts as a potential model for the mammalian HC synapse. This was already proposed several decades ago, but detailed analysis of the underlying mechanisms is still required to evaluate zebrafish in comparison to other animal models. The study reveals variable and inhomogeneous biophysical properties of HCs in the sensory organs of zebrafish. These multiple HC types, the absence of OHC-based amplification and the physiological similarities between the lateral line and the vestibular system (rather than the cochlea) render extrapolations to mammals generally difficult. A confounding factor might be the evolutionary distance to humans that is also depicted by biophysical similarities of HCs in the lagena and the sacculus to those of goldfish and frogs (Olt et al. 2014). The recordings demonstrated comparable biophysical properties of certain HCs in zebrafish and mammals, but it is not clear whether the HC physiology itself is also similar. It remains to be elucidated whether the HC currents were mediated by protein orthologues and whether the orthologues are functionally similar. Of special note, strikingly different properties have been reported from zebrafish orthologues related to hearing in mammals (e.g. prestin, see
Schaechinger & Oliver, 2007). Of course, the similarity of protein expression and function determines the applicability of zebrafish as model system. On the positive side, large-scale mutagenesis screens can be performed in zebrafish to identify underlying proteins, but due to the stated differences from mammals, promising candidates will require more elaborate animal models in order to assess the applicability of zebrafish further. An additional important finding of the study concerns the widely used zebrafish anaesthetic, tricane methanesulfonate (MS-222). MS-222 reversibly inhibited K+ currents (IA and IK,Ca ) in juvenile HCs of the lateral line. Accordingly, the membrane potential of the HCs, the activity of the MET channels and the biophysical membrane properties in an anaesthetized fish might be altered significantly. This has the potential to produce misleading results and should be kept in mind when the analysis of HCs is the experimental goal. The study by Olt et al. (2014) raised several points worth considering prior to work with zebrafish HCs, as follows: (i) immature and mature HCs coexist throughout life, with different biophysical properties and probably with different protein components; (ii) HCs in the centre of adult sensory organs display a mature phenotype; (iii) grouping of findings into developmental stages and the HC location might increase the significance of the data; (iv) some HCs resemble mammalian vestibular or immature auditory HCs (similar K+ and Ca2+ currents), but the molecular components in zebrafish remain unknown; and (v) zebrafish offer the applicability of genetic and pharmacological high-throughput screenings, but due to the evolutionary distance from mammals the candidate mechanisms should be evaluated in additional model systems.
J Physiol 592.21
In summary, the biophysical results shown in this paper indicate that zebrafish provide promising models of mammalian HCs. Even so, the molecular identity of the involved proteins needs to be established, and zebrafish HC physiology requires further analysis to evaluate whether zebrafish HCs are genuinely similar and indeed recapitulate mammalian vestibular and auditory physiology. References Marcotti W, Johnson SL, Holley MC & Kros CJ (2003). Developmental changes in the expression of potassium currents of embryonic, neonatal and mature mouse inner hair cells. J Physiol 548, 383–400. Olt J, Johnson SL & Marcotti W (2014). In vivo and in vitro biophysical properties of hair cells from the lateral line and inner ear of developing and adult zebrafish. J Physiol 592, 2041–2058. Schacht J & Leitner MG (2014). In vitro models for otoxoic research. In Methods in Pharmacology and Toxicology: In Vitro Toxicology Systems, ed. Jennings P & Bal-Price A, Part IV. Chapter 9, pp. 199–224. Humana Press, New York. Schaechinger TJ & Oliver D (2007). Nonmammalian orthologs of prestin (SLC26A5) are electrogenic divalent/chloride anion exchangers. Proc Natl Acad Sci U S A 104, 7693–7698. Schwander M, Kachar B & M¨uller U (2010). Review series: the cell biology of hearing. J Cell Biol 190, 9–20.
Additional information Competing interests
None declared. Funding
M.G.L. is supported by a Research Grant of the University Medical Center Giessen und Marburg (UKGM 17/2013 MR)
C 2014 The Author. The Journal of Physiology C 2014 The Physiological Society
Downloaded from J Physiol (jp.physoc.org) at UNIV OF NORTH CAROLINA on November 7, 2014
