Glen MacDonald

Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio).

Mechanoreceptive hair cells are extremely sensitive to aminoglycoside antibiotics, including neomycin. Hair cell survival was assessed in larval wild-type zebrafish lateral line neuromasts 4 h after initial exposure to a range of neomycin concentrations for 1 h. Each of the lateral line neuromasts was scored in live fish for the presence or absence of hair cells using the fluorescent vital dye DASPEI to selectively label hair cells. All neuromasts were devoid of DASPEI-labeled hair cells 4 h after 500 µM neomycin exposure. Vital DASPEI staining was proportional to the number of hair cells per neuromast identified in fixed larvae using immunocytochemistry for acetylated tubulin and phalloidin labeling. The time course of hair cell regeneration in the lateral line neuromasts was also analyzed following neomycin-induced damage. Regenerated hair cells were first observed using live DASPEI staining 12 and 24 h following neomycin treatment. The potential role of proliferation in regenerating hair cells was analyzed. A 1 h pulse-fix protocol using bromodeoxyuridine (BrdU) incorporation was used to identify S-phase cells in neuromasts. BrdU incorporation in neomycin-damaged neuromasts did not differ from control neuromasts 4 h after drug exposure but was dramatically upregulated after 12 h. The proliferative cells identified during a 1 h period at 12 h after neomycin treatment were able to give rise to new hair cells by 24–48 h after drug treatment. The results presented here provide a standardized preparation for studying and identifying genes that influence vertebrate hair cell death, survival, and regeneration following ototoxic insults.

Lateral line hair cell maturation is a determinant of aminoglycoside susceptibility in zebrafish (Danio rerio)

Developmental differences in hair cell susceptibility to aminoglycoside-induced cell death has been observed in multiple species. Increased sensitivity to aminoglycosides has been temporally correlated with the onset of mechanotransduction-dependent activity. We have used in vivo fluorescent vital dye markers to further investigate the determinants of aminoglycoside induced hair cell death in the lateral line of zebrafish (Danio rerio). Labeling hair cells of the lateral line in vivo with the dyes FM 1-43, To-Pro-3, and Yo-Pro-1 served as reliable indicators of hair cell viability. Results indicate that hair cell maturation is a determinant of developmental differences in susceptibility. The age dependent differences in susceptibility to aminoglycosides are independent of the onset of mechanotransduction-dependent activity as measured by FM 1-43 uptake and independent of hair cell ability to take up fluorescently conjugated aminoglycosides.

Ultrastructural analysis of aminoglycoside‐induced hair cell death in the zebrafish lateral line reveals an early mitochondrial response

Loss of the mechanosensory hair cells in the auditory and vestibular organs leads to hearing and balance deficits. To investigate initial, in vivo events in aminoglycoside‐induced hair cell damage, we examined hair cells from the lateral line of the zebrafish, Danio rerio. The mechanosensory lateral line is located externally on the animal and therefore allows direct manipulation and observation of hair cells. Labeling with vital dyes revealed a rapid response of hair cells to the aminoglycoside neomycin. Similarly, ultrastructural analysis revealed structural alteration among hair cells within 15 minutes of neomycin exposure. Animals exposed to a low, 25‐μM concentration of neomycin exhibited hair cells with swollen mitochondria, but little other damage. Animals treated with higher concentrations of neomycin (50–200 μM) had more severe and heterogeneous cellular changes, as well as fewer hair cells. Both necrotic‐like and apoptotic‐like cellular damage were observed. Quantitation of the types of alterations observed indicated that mitochondrial defects appear earlier and more predominantly than other structural alterations. In vivo monitoring demonstrated that mitochondrial potential decreased following neomycin treatment. These results indicate that perturbation of the mitochondrion is an early, central event in aminoglycoside‐induced damage. J. Comp. Neurol. 502:522–543, 2007.

Three-dimensional imaging of the intact mouse cochlea by fluorescent laser scanning confocal microscopy

The complex anatomy of the mammalian cochlea is most readily understood by representation in three-dimensions. However, the cochlea is often sectioned to minimize the effects of its anatomic complexity and optical properties on image acquisition by light microscopy. We have found that optical aberrations present in the decalcified cochlea can be greatly reduced by dehydration through graded ethanols followed by clearing with a mixture of five parts methyl salicylate and three parts benzyl benzoate (MSBB). Clearing the cochlea with MSBB enables acquisition of high-resolution images with multiple fluorescent labels, through the full volume of the cochlea by laser scanning confocal microscopy. The resulting images are readily applicable to three-dimensional morphometric analysis and volumetric visualizations. This method promises to be particularly useful for three-dimensional characterization of anatomy, innervation and expression of genes or proteins in the many new animal models of hearing and balance generated by genetic manipulation. Furthermore, the MSBB is compatible with most non-protein fluorophores used for histological labeling, and may be removed with traditional transitional solvents to allow subsequent epoxy embedding for sectioning.

Deafness and retinal degeneration in a novel USH1C knock-in mouse model.

Usher syndrome is the leading cause of combined deaf–blindness, but the molecular mechanisms underlying the auditory and visual impairment are poorly understood. Usher I is characterized by profound congenital hearing loss, vestibular dysfunction, and progressive retinitis pigmentosa beginning in early adolescence. Using the c.216G>A cryptic splice site mutation in Exon 3 of the USH1C gene found in Acadian Usher I patients in Louisiana, we constructed the first mouse model that develops both deafness and retinal degeneration. The same truncated mRNA transcript found in Usher 1C patients is found in the cochleae and retinas of these knock‐in mice. Absent auditory‐evoked brainstem responses indicated that the mutant mice are deaf at 1 month of age. Cochlear histology showed disorganized hair cell rows, abnormal bundles, and loss of both inner and outer hair cells in the middle turns and at the base. Retinal dysfunction as evident by an abnormal electroretinogram was seen as early as 1 month of age, with progressive loss of rod photoreceptors between 6 and 12 months of age. This knock‐in mouse reproduces the dual sensory loss of human Usher I, providing a novel resource to study the disease mechanism and the development of therapies.

A new method for imaging and 3D reconstruction of mammalian cochlea by fluorescent confocal microscopy

Traditional methods for anatomical and morphometric studies of cochlear tissues have relied upon either microdissection of the organ of Corti or the generation of serial sections of the cochlea. Such methods are time-consuming, disruptive to three-dimensional relationships and often restrict sampling to very limited numbers of cells. We have found that cells and tissue components of the cochlear duct may be labelled by fluorescent markers within intact cochleae, which are then embedded in epoxy resin for subsequent viewing by fluorescent microscopy methods. This approach allows imaging through thick optical volumes with preservation of three-dimensional relationships. Unlike sectioned tissue, alignment of the sample relative to the focal axis may be easily corrected by re-orientation of the optical volume with common image processing software. Fluorescently labelled cochleae embedded in epoxy can be viewed by most fluorescent microscopy methods including laser scanning confocal microscopy, multi-photon confocal microscopy and widefield epi-fluorescence microscopy with deconvolution. Furthermore, semi-thin sections made from these preparations are compatible with traditional histological stains, as well as allowing brightly labelled epi-fluorescent images.

Evidence that TRPC4 supports the calcium selective ICRAC-like current in human gingival keratinocytes

We previously demonstrated that high external [Ca2+] activated two Ca2+ currents in human gingival keratinocytes (HGKs): an initial small ICRAC-like current and a second large nonspecific cation current (Fatherazi S, Belton CM, Cai S, Zarif S, Goodwin PC, Lamont RJ, Izutsu KT; Pflugers Arch 448:93–104, 2004). It was recently shown that TRPC1, a member of the transient receptor potential protein family, is a component of the store-operated calcium entry mechanism in keratinocytes. To further elucidate the molecular identity of these channels, we investigated the expression of TRPC4 in gingival tissue and in cultured keratinocytes, and the effect of knockdown of TRPC4 expression on the Ca2+ currents and influx. Immunohistochemistry showed TRPC4 was present in gingival epithelium as well as in HGKs cultured in different [Ca2+]s. Results from tissue and cultured HGKs demonstrated TRPC4 expression decreased with differentiation. Knockdown of TRPC4 in proliferating HGKs with antisense oligonucleotides significantly reduced the intracellular [Ca2+] increase obtained upon exposure to high external [Ca2+]. Antisense knockdown of TRPC4 expression was confirmed by reverse transcriptase polymerase chain reaction, Western blot, and immunofluorescence microscopy of transfected HGKs. Immunofluorescence microscopy and patch clamp measurements in Lucifer-yellow-tagged, antisense-treated HGKs showed attenuation of TRPC4 expression levels as well as attenuation of the ICRAC-like current in the same cell, whereas the large nonspecific cation current was unchanged but significantly delayed. Cells transfected with a scrambled TRPC4 oligonucleotide showed no change in either the ICRAC-like or nonspecific currents. The results indicate that TRPC4 is an important component of the ICRAC-like channel in HGKs.

Practical Confocal Microscopy

The other chapters in this book give the reader an in-depth description of every important aspect of biological confocal microscopy that we could think of. This chapter is to provide the novice user of this instrument with a basic understanding of the practical information needed to use it effectively. Because the computer interfaces of the various commercial instruments vary greatly, this chapter will stress the important features of microscopical optics and the basics of sampling that are common to all instruments.

Three-dimensional confocal microscopy of the mammalian inner ear

Abstract Objective: Control the refractive index in fixed specimens of mammalian inner ear to reduce spherical aberrations that limit our ability to obtain 3-dimensional images of fluorescently labeled inner ear specimens by conventional laser scanning confocal microscopy. Study Design: Mouse inner ear specimens were fixed with minimal dissection, rapidly decalcified and fluorescently labeled by immunohistochemistry then impregnated by epoxy resin or a clearing agent composed of 5 parts methyl salicylate:3 parts benzyl benzoate. The specimens were imaged by both confocal microscopy and by widefield epi-fluorescent microscopy, with additional processing by deconvolution. Results: Rapid decalcification preserved tissue morphology and antigenicity for the antibodies tested. Although the epoxy allowed some reduction of spherical aberration, the clearing agent enabled optical volumes of high quality and resolution to be collected through the inner ear. The conditions for immunolabeling are important to ensure adequate perfusion of the immuno-labeling reagents throughout the specimen. Conclusion: Spherical aberration reduces signal intensity, contrast and resolution in optical microscopy. Creating a homogeneous refractive index throughout the inner ear to reduce spherical aberration allowed optical volumes to be collected through an intact, fluorescently labeled cochlea in a manner limited by the working distance of the objective lens rather than by spherical aberration. Optical volumes collected by this method from the mammalian inner ear promise to be useful for applications such as tracing innervation patterns, counting sensory cells or other structures over large regions of the sensory epithelium, and characterization of the inner ear in animal models of human deafness disorders.

Cardiomyocytes can induce rhythmic contraction of skeletal muscle cells. Potential use for infarct repair

Skeletal myoblasts form viable grafts of mature muscle in injured hearts, and these grafts contract when exogenously stimulated. It is not known, however, whether cardiac muscle can form electromechanical junctions with skeletal muscle and induce its synchronous contraction. Here we report that undifferentiated skeletal myoblasts expressed N-cadherin and connexin43, major adhesion and gap junction proteins of the intercalated disk. Both proteins were markedly down-regulated after the cells differentiated into myotubes. Similarly, differentiated skeletal muscle grafts in injured hearts had no detectable N-cadherin or connexin43; hence, electromechanical coupling did not occur after in vivo grafting. Surprisingly, however, skeletal myotubes contracted in synchrony with cardiomyocytes in coculture. Isoproterenol increased myotube contraction rates by 25% in coculture without affecting myotubes in monoculture, indicating that cardiomyocytes were the pacemakers. The gap junction inhibitor heptanol aborted myotube contractions but left spontaneous contractions of individual cardiomyocytes intact, suggesting gap junction-dependent myotube activation. Confocal microscopy revealed the expression of cadherin and connexin43 at junctions between myotubes and cardiomyocytes in vitro. Microinjection studies showed that myotubes were capable of dye transfer via gap junctions. Calcium imaging revealed that the contraction of cardiomyocytes induced synchronous, calcium-dependent contractions in myotubes. Thus, cardiomyocytes form electromechanical junctions with skeletal myotubes in coculture, and induce their synchronous contraction via gap junctions. The induction of similar junctions in vivo may be sufficient to make skeletal muscle grafts beat synchronously with the host myocardium.