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Vesicular glutamate transporter 3 is strongly upregulated in cochlear inner hair cells and spiral ganglion cells of developing circling mice
Neuroscience Letters 584 (2015) 320–324
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Short communication
Vesicular glutamate transporter 3 is strongly upregulated in cochlear inner hair cells and spiral ganglion cells of developing circling mice Youngeun Lee a , Hak Rim Kim b , Seung Cheol Ahn c,∗ a
Department of Nanobio Medical Science, Dankook University, 119 Dandaero, Anseo-dong, Cheonan-si, Chungnam 330-714, Republic of Korea Department of Pharmacology, College of Medicine, Dankook University, 119 Dandaero, Anseo-dong, Cheonan-si, Chungnam 330-714, 119 Dandaero, Republic of Korea c Department of Physiology, College of Medicine, Dankook University,119 Dandaero, Anseo-dong, Cheonan-si, Chungnam 330-714, Republic of Korea b
h i g h l i g h t s • IHC VGLUT3 IR was higher in circling mice at P14. • SGC VGLUT3 IR was higher in circling mice at P14. • VGLUT3 expression in protein level was most prominent in circling mice at P14.
a r t i c l e
i n f o
Article history: Received 18 September 2014 Received in revised form 23 October 2014 Accepted 31 October 2014 Available online 3 November 2014 Keywords: VGLUT3 Cochlea Inner hair cell Spiral ganglion cell Circling mice
a b s t r a c t Vesicular glutamate transporter 3 (VGLUT3) plays a major role in hearing, and mice lacking the VGLUT3 are congenitally deaf due to absence of glutamate release at the inner hair cell afferent synapses. However, whether VGLUT3 is expressed normally in the cochleae of developing circling mice (homozygous (cir/cir) mice), the animal model for human deafness type DFNB6, has not been established. In this study, we investigated the developmental expression of VGLUT3 in cochlear inner hair cells (IHCs) and spiral ganglion cells (SGCs) of homozygous (cir/cir) mice from postnatal day (P)1 to P14 using immunofluorescence (IF) staining and Western blot. VGLUT3 immunoreactivity (IR) and protein expression increased progressively with age in homozygous (cir/cir) and control mice (heterozygous (+/cir) mice and ICR mice). The rank order of VGLUT3 IR in IHCs and SGCs in P14 mice was homozygous (cir/cir) mice = heterozygous (+/cir) mice > ICR mice. The rank order of total protein expression was homozygous (cir/cir) mice > heterozygous (+/cir) mice = ICR mice at P14. IF staining and Western blot analysis indicated that developmental VGLUT3 expression in cochleae was most prominent in homozygous (cir/cir) mice. The possible contribution of VGLUT3 upregulation in the cochlear degeneration is discussed. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Glutamatergic transmission requires transport of the excitatory amino acid into secretory vesicles by a family of vesicular glutamate transporters (VGLUTs) [1,7,21]. Three isoforms of VGLUTs, named VGLUT1–3, have been identified in the brain [5,6,20]. Among them, VGLUT3 is the most important isoform for hearing; the lack of VGUT3 leads to seizure and deafness in mice [19] and the absence of acoustic startle reflex and vestibular-ocular reflex in zebra fish [16]. It is also reported that DFNA25 type deafness is attributed to a mutation in the gene encoding VGLUT3 in human [18].
∗ Corresponding author. Tel.: +82 41 550 3852; fax: +82 41 565 6167. E-mail address: [email protected] (S.C. Ahn). http://dx.doi.org/10.1016/j.neulet.2014.10.053 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Auditory VGLUT3 expression is regulated developmentally or environmentally. In cochlea, inner hair cells (IHCs) express VGLUT3 by embryonic day 19 in mice [19], and its expression is gradually upregulated by P60 in both IHCs and SGCs in rats [17]. In brainstem auditory circuits, VGLUT3 is expressed transiently in different superior olivary complex nuclei between P0 and P12 [2], and its expression is upregulated by cochlear damage in the lateral superior olive [11] in rats. These reports raise a possibility that developmental VGLUT3 expression might be different in the auditory system of genetically abnormal animals, such as mouse models of human deafness. The circling mouse is a mouse model of human deafness (DFNB6 type). The main cause of deafness is the relatively early degeneration of the organ of Corti and the loss of SGCs [4,12,13]. The stereociliary defects in the cochlear hair cells can be observed as
Y. Lee et al. / Neuroscience Letters 584 (2015) 320–324
early as P10 and the earliest complete hair cell degeneration is reported at P21 [4]. It has not been reported what the expression pattern of VGLUT3 is like in hair cells or SGCs during this early degenerating period in circling mice. The aim of this study was to investigate the temporal expression pattern of VGLUT3 in the cochleae of developing circling mice aged under P14. 2. Material and methods 2.1. Cochlear preparations Female heterozygous (+/cir) mice were mated with male homozygous (cir/cir) mice, and their offspring (P1–P2 and P13–P14 mice) were used for this study. As homozygous (cir/cir) mice were discovered within a group of ICR out-bred mice, ICR mice of the same age were also used as control. At least three animals were included in each experiment. The genotype of homozygous (cir/cir) mice was assessed with polymerase chain reaction analysis according to our previous report [9]. The animals were maintained at the Animal Facility of Dankook University. The Dankook University Institutional Animal Care and Use Committee approved this study. After mice were deeply anesthetized with isoflurane, their cochleae were removed in ice-cold phosphate buffered saline (0.19 M PBS), fixed in 4% paraformaldehyde (PFA) for 2 h, and decalcified in 5% EDTA for 3 h. After serial treatment with cold sucrose solutions, cochleae were embedded in frozen section compounds, and kept frozen at −80 ◦ C until use. 2.2. Staining and analysis Immunofluorescence (IF) staining was conducted using antiVGLUT3 antibody (1:5000, AB5421; Millipore, Milford, MA, USA). Mid-modiolar cochlear sections of 10 m were prepared (Leica CM 1900; Nussloch, Germany) for IF staining. Sections were incubated overnight at 4 ◦ C with anti-VGLUT3 antibody in PBS-based
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blocking buffer containing 0.3% Triton X-100, and 1% normal goat serum. The next day, the sections were washed with PBS and incubated at room temperature with a secondary antibody (Alexa Fluor 555 goat anti-guinea pig IgG, A21435; Invitrogen, Carlsbad, CA, USA) diluted 1:250 in PBS containing 0.3% Triton X-100. Nuclei were stained with DAPI (46-diamidino-2-phenylindole). The stained sections were observed under a confocal laser scanning microscope (LSM700; Carl Zeiss Meditec, Jena, Germany). The same parameter setting used to scan the cells of higher fluorescence was used in scanning the cells of lower fluorescence. The staining intensities were measured with the LSM700 image program. The border of the hair cell to be analyzed was outlined by a graphic tool provided by LSM700 image program to produce mean intensity. To measure the VGLUT3 intensities of SGCs, images were taken using a higher magnification and the averaged intensities per field were used as mean intensities. The final intensity was obtained by subtracting the background intensity. The background intensity was the averaged value of the intensities of three different spots outside the region of interest. The modified images with a graphics program (Adobe Photoshop 7.0) are presented in this manuscript. Statistical analysis was performed with Origin 7.0 (Origin Lab). Data are expressed as mean ± standard error. The independent t-test was used for comparisons, and a p < 0.05 was considered significant. 2.3. Western blots Mice cochleae were mixed with T-PER® tissue protein extraction reagent (Thermo Scientific, Rockford, IL, USA). The homogenized samples were boiled in Laemmli sample buffer. The samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore). Protein transfer was confirmed by staining the membrane with Ponceau S red and the membrane was incubated in Odyssey® blocking buffer for 1 h at room temperature. The membrane was stained with anti-VGLUT3 antibody (1:1000, AB5421; Millipore, Milford, MA, USA) overnight at 4 ◦ C. After
Fig. 1. VGLUT3 IR confined to IHCs. At P2, VGLUT3 IR (red signal) was weak in IHCs of homozygous (cir/cir) mice (A: homo), heterozygous (+/cir) mice (B: hetero), and ICR mice (C), but became prominent at P14 in all mice tested (D: homozygous (cir/cir) mice, E: heterozygous (+/cir) mice, F: ICR mice). The intensity of VGLUT3 IR was weakest in IHCs of ICR mice (F). G is a low magnification view showing VGLUT3 IR confined to IHC in P14 heterozygous (+/cir) mice. *Indicates outer hair cells. Nuclei were stained with DAPI (blue signal). Graph above G shows the temporal expression pattern of VGLUT3 IR. Y axis indicates mean intensities of VGLUT3 IR and X axis indicates ages of mice tested (Hom: homozygous (cir/cir) mice, het: heterozygous (+/cir) mice). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. VGLUT3 IR in SGCs. At P2, VGLUT3 IR (red signal) was weak in IHCs of homozygous (cir/cir) mice (A: homo), heterozygous (+/cir) mice (B: hetero), and ICR mice (C), but became prominent at P14 in all mice tested (D: homozygous (cir/cir) mice, E: heterozygous (+/cir) mice, F: ICR mice). Nuclei were stained with DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
extensive washing with Tris-buffered saline–Tween 20, the membrane was stained with an appropriate IR-dye tagged secondary antibody. Densitometry of the bands was performed with software for an Odyssey infrared imaging system (LI-COR® Biosciences). 3. Results 3.1. VGLUT3 IR The temporal expression patterns of VGLUT3 were investigated in the cochlear hair cells of P1–P2 and P13–P14 mice (ICR mice, heterozygous (+/cir) mice, and homozygous (cir/cir) mice). VGLUT3 IR confined to IHCs was weak at P1–P2 but became stronger with age in all mice tested (Fig. 1). As the staining pattern was not different depending on location (apex, middle, or basal turn), all IR signals from stained IHCs were collected and analyzed. The mean intensities of IHC VGLUT3 IR at P1–P2 were as follows: 95.7 ± 16.0 (ICR mice, n = 12, five cochleae, three animals), 85.4 ± 26.8 (heterozygous (+/cir) mice, n = 13, five cochleae, three animals), and 66.2 ± 15.6 (homozygous (cir/cir) mice, n = 19, five cochleae, four animals). The three values were not significantly different. The mean intensities of IHC VGLUT3 IR at P13–P14 were as follows: 293.4 ± 49.7 (ICR mice, n = 15, five cochleae, three animals), 537.6 ± 78.6 (heterozygous (+/cir) mice, n = 18, six cochleae, three animals), and 703.2 ± 94.6 (homozygous (cir/cir) mice, n = 13, eight cochleae, four animals) (Fig. 1 inset). The mean intensities obtained from homozygous (cir/cir) mice and heterozygous (+/cir) mice were not significantly different, but the mean intensity of the ICR mice was significantly lower than those of the homozygous (cir/cir) mice and heterozygous (+/cir) mice (independent t-test, p < 0.05). An age-dependent increase in VGLUT3 IR was also observed in SGCs (Fig. 2). The mean intensities of VGLUT3 IR per field at P1-P2 were 116.2 ± 30.7 (ICR mice, 24 fields, eight cochleae, four animals), 118.2 ± 18.7 (heterozygous (+/cir) mice, 18 fields, eight cochleae,
four animals), and 173.8 ± 20.2 (homozygous (cir/cir) mice, 24 fields, 10 cochleae, five animals). The three values were not significantly different. At P13–P14, they were 237.6 ± 56.2 (ICR mice, 16 fields, five cochleae, three animals), 315.8 ± 41.5 (heterozygous (+/cir) mice, 14 fields, five cochleae, three animals), and 410.4 ± 36.4 (homozygous (cir/cir) mice, 26 fields, nine cochleae, five animals). Only the difference between the homozygous (cir/cir) mice and ICR mice was significant (independent t-test, p < 0.05). 3.2. Total VGLUT3 expression in cochleae As we found developmental increases in VGLUT3 IR in all mice tested, we quantified the total expression level of VGLUT3 by Western blot (Fig. 3). The basal expression levels of VGLUT3 at P1–P2 were similar in homozygous (cir/cir) mice, heterozygous (+/cir) mice, and ICR mice. The increases in total VGLUT3 expression at P13–P14 normalized to that of P1–P2 cochleae were three-fold (3.08 ± 0.40, three animals, five cochleae) in homozygous (cir/cir) mice, 1.5-fold in heterozygous (+/cir) mice (1.48 ± 0.15, three animals, five cochleae), and 1.5-fold in ICR mice (1.45 ± 0.15, three animals, five cochleae) (Fig. 3B). 4. Discussion Developmental VGLUT3 expression in cochlear IHCs and SGCs has been recently reported in rats [17]. In that report, VGLUT3 expression was low at P0 in IHCs and SGCs but increased progressively with age, which was very similar to our data. The unexpected finding was that VGLUT3 upregulation was strongest in homozygous (cir/cir) mice compared to ICR mice or heterozygous (+/cir) mice at P14. This might simply reflect the influence of the cir gene, but, as the IHCs and SGCs of homozygous (cir/cir) mice are in the process of deterioration, the possibility that VGLUT3 upregulation is affected by pathologic changes of cochleae should be considered.
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homozygous (cir/cir) mice [4,12,13]. In homozygous (cir/cir) mice, VGLUT3-positive puncta are mainly present in the cytoplasm and perinuclear region of SGCs. No matter what the cause of SGC destruction is, if SGCs start to degenerate, glutamate released from glutamate-overloaded SGCs will accelerate the degeneration of adjacent SGCs. Although the possible contribution of VGLUT3 upregulation to the degeneration of IHCs and SGCs is suggested, it is apparent that VGLUT3 overexpression itself cannot be a cause of the early deafness reported in P18 homozygous (cir/cir) mice [4]. To date, there is no evidence that excess VGLUT3 expression will harm the hearing function. The exact role of VGLUT3 overexpression at around P14 in homozygous (cir/cir) mice needs further investigation. Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology (2012R1A1A2040535). Fig. 3. Total expression of VGLUT3 measured by Western blot. Representative immunoblots of VGLUT 3 at P1–P2 and P13–P14 are shown in A. Quantitative analysis of VGLUT3 expression levels at P13–P14 normalized to those at P1–P2 levels are shown in B. -actin bands were used as loading controls (n = 5). *P < 0.05, **P < 0.01.
It has been reported that cochlear ablation leads to the VGLUT3 upregulation in the rat cochlear nucleus [8] and lateral superior olive [11]. As cochlear hair cells are reported to degenerate after P10 in homozygous (cir/cir) mice [4], VGLUT3 upregulation in SGCs, the primary relay neurons of the auditory pathway, might be explained in the same point of view. One thing to be considered is that our case is different with the cochlear ablation-induced VGLUT3 upregulation reported in rat lateral superior olive or cochlear nucleus because we did not find any organs of Corti showing complete degeneration in homozygous (cir/cir) mice at P14. Thus, if enhanced VGLUT3 expression in SGCs is to be explained in terms of cochlear pathology, it needs explanations other than complete degeneration of the organ of Corti. One of the possible explanations might be abnormal or absent neurotransmitter release from IHCs, which might produce a similar effect in that it cuts synaptic input just as complete degeneration of the organ of Corti does. The scanning electron microscopic level stereocilliary bundle defect reported in P10 homozygous (cir/cir) mice [4] supports this idea because the process of neurotransmitter release from IHCs starts with deflection of hair bundles by mechanical stimuli. The SGC degeneration reported in homozygous (cir/cir) mice [4,12,13] also supports the absence of or abnormal neurotransmitter release from IHCs because the absence of glutamate release from IHCs leads to the early degeneration of SGCs in VGLUT3 knock-out mice [19]. In contrast to VGLUT3 upregulation in SGCs, we have not found an appropriate explanation to VGLUT3 upregulation in IHCs yet. Though some VGLUT3 upregulation has been reported in the nervous system [3,11,22], it has never been reported at the peripheral receptor level. We speculate that VGLUT3 upregulation in IHCs may reflect an increased activity of VGLUT3 or glutamate overload due to the abnormal release of glutamate from the IHCs. If abnormal release of glutamate leads to glutamate overload, it will possibly contribute to cell destruction: glutamate released from deteriorating hair cells overloaded with glutamate will accelerate the destruction of nearby hair cells if the hair cells start to degenerate. There have been reports showing glutamate cytotoxicity in hair cells [10,14] and one report suggested the aggravating actions of glutamate released from deteriorating hair cells, which supports our hypothesis [15]. This mechanism will also contribute to SGC destruction, which has been reported in
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Short communication
Vesicular glutamate transporter 3 is strongly upregulated in cochlear inner hair cells and spiral ganglion cells of developing circling mice Youngeun Lee a , Hak Rim Kim b , Seung Cheol Ahn c,∗ a
Department of Nanobio Medical Science, Dankook University, 119 Dandaero, Anseo-dong, Cheonan-si, Chungnam 330-714, Republic of Korea Department of Pharmacology, College of Medicine, Dankook University, 119 Dandaero, Anseo-dong, Cheonan-si, Chungnam 330-714, 119 Dandaero, Republic of Korea c Department of Physiology, College of Medicine, Dankook University,119 Dandaero, Anseo-dong, Cheonan-si, Chungnam 330-714, Republic of Korea b
h i g h l i g h t s • IHC VGLUT3 IR was higher in circling mice at P14. • SGC VGLUT3 IR was higher in circling mice at P14. • VGLUT3 expression in protein level was most prominent in circling mice at P14.
a r t i c l e
i n f o
Article history: Received 18 September 2014 Received in revised form 23 October 2014 Accepted 31 October 2014 Available online 3 November 2014 Keywords: VGLUT3 Cochlea Inner hair cell Spiral ganglion cell Circling mice
a b s t r a c t Vesicular glutamate transporter 3 (VGLUT3) plays a major role in hearing, and mice lacking the VGLUT3 are congenitally deaf due to absence of glutamate release at the inner hair cell afferent synapses. However, whether VGLUT3 is expressed normally in the cochleae of developing circling mice (homozygous (cir/cir) mice), the animal model for human deafness type DFNB6, has not been established. In this study, we investigated the developmental expression of VGLUT3 in cochlear inner hair cells (IHCs) and spiral ganglion cells (SGCs) of homozygous (cir/cir) mice from postnatal day (P)1 to P14 using immunofluorescence (IF) staining and Western blot. VGLUT3 immunoreactivity (IR) and protein expression increased progressively with age in homozygous (cir/cir) and control mice (heterozygous (+/cir) mice and ICR mice). The rank order of VGLUT3 IR in IHCs and SGCs in P14 mice was homozygous (cir/cir) mice = heterozygous (+/cir) mice > ICR mice. The rank order of total protein expression was homozygous (cir/cir) mice > heterozygous (+/cir) mice = ICR mice at P14. IF staining and Western blot analysis indicated that developmental VGLUT3 expression in cochleae was most prominent in homozygous (cir/cir) mice. The possible contribution of VGLUT3 upregulation in the cochlear degeneration is discussed. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Glutamatergic transmission requires transport of the excitatory amino acid into secretory vesicles by a family of vesicular glutamate transporters (VGLUTs) [1,7,21]. Three isoforms of VGLUTs, named VGLUT1–3, have been identified in the brain [5,6,20]. Among them, VGLUT3 is the most important isoform for hearing; the lack of VGUT3 leads to seizure and deafness in mice [19] and the absence of acoustic startle reflex and vestibular-ocular reflex in zebra fish [16]. It is also reported that DFNA25 type deafness is attributed to a mutation in the gene encoding VGLUT3 in human [18].
∗ Corresponding author. Tel.: +82 41 550 3852; fax: +82 41 565 6167. E-mail address: [email protected] (S.C. Ahn). http://dx.doi.org/10.1016/j.neulet.2014.10.053 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Auditory VGLUT3 expression is regulated developmentally or environmentally. In cochlea, inner hair cells (IHCs) express VGLUT3 by embryonic day 19 in mice [19], and its expression is gradually upregulated by P60 in both IHCs and SGCs in rats [17]. In brainstem auditory circuits, VGLUT3 is expressed transiently in different superior olivary complex nuclei between P0 and P12 [2], and its expression is upregulated by cochlear damage in the lateral superior olive [11] in rats. These reports raise a possibility that developmental VGLUT3 expression might be different in the auditory system of genetically abnormal animals, such as mouse models of human deafness. The circling mouse is a mouse model of human deafness (DFNB6 type). The main cause of deafness is the relatively early degeneration of the organ of Corti and the loss of SGCs [4,12,13]. The stereociliary defects in the cochlear hair cells can be observed as
Y. Lee et al. / Neuroscience Letters 584 (2015) 320–324
early as P10 and the earliest complete hair cell degeneration is reported at P21 [4]. It has not been reported what the expression pattern of VGLUT3 is like in hair cells or SGCs during this early degenerating period in circling mice. The aim of this study was to investigate the temporal expression pattern of VGLUT3 in the cochleae of developing circling mice aged under P14. 2. Material and methods 2.1. Cochlear preparations Female heterozygous (+/cir) mice were mated with male homozygous (cir/cir) mice, and their offspring (P1–P2 and P13–P14 mice) were used for this study. As homozygous (cir/cir) mice were discovered within a group of ICR out-bred mice, ICR mice of the same age were also used as control. At least three animals were included in each experiment. The genotype of homozygous (cir/cir) mice was assessed with polymerase chain reaction analysis according to our previous report [9]. The animals were maintained at the Animal Facility of Dankook University. The Dankook University Institutional Animal Care and Use Committee approved this study. After mice were deeply anesthetized with isoflurane, their cochleae were removed in ice-cold phosphate buffered saline (0.19 M PBS), fixed in 4% paraformaldehyde (PFA) for 2 h, and decalcified in 5% EDTA for 3 h. After serial treatment with cold sucrose solutions, cochleae were embedded in frozen section compounds, and kept frozen at −80 ◦ C until use. 2.2. Staining and analysis Immunofluorescence (IF) staining was conducted using antiVGLUT3 antibody (1:5000, AB5421; Millipore, Milford, MA, USA). Mid-modiolar cochlear sections of 10 m were prepared (Leica CM 1900; Nussloch, Germany) for IF staining. Sections were incubated overnight at 4 ◦ C with anti-VGLUT3 antibody in PBS-based
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blocking buffer containing 0.3% Triton X-100, and 1% normal goat serum. The next day, the sections were washed with PBS and incubated at room temperature with a secondary antibody (Alexa Fluor 555 goat anti-guinea pig IgG, A21435; Invitrogen, Carlsbad, CA, USA) diluted 1:250 in PBS containing 0.3% Triton X-100. Nuclei were stained with DAPI (46-diamidino-2-phenylindole). The stained sections were observed under a confocal laser scanning microscope (LSM700; Carl Zeiss Meditec, Jena, Germany). The same parameter setting used to scan the cells of higher fluorescence was used in scanning the cells of lower fluorescence. The staining intensities were measured with the LSM700 image program. The border of the hair cell to be analyzed was outlined by a graphic tool provided by LSM700 image program to produce mean intensity. To measure the VGLUT3 intensities of SGCs, images were taken using a higher magnification and the averaged intensities per field were used as mean intensities. The final intensity was obtained by subtracting the background intensity. The background intensity was the averaged value of the intensities of three different spots outside the region of interest. The modified images with a graphics program (Adobe Photoshop 7.0) are presented in this manuscript. Statistical analysis was performed with Origin 7.0 (Origin Lab). Data are expressed as mean ± standard error. The independent t-test was used for comparisons, and a p < 0.05 was considered significant. 2.3. Western blots Mice cochleae were mixed with T-PER® tissue protein extraction reagent (Thermo Scientific, Rockford, IL, USA). The homogenized samples were boiled in Laemmli sample buffer. The samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore). Protein transfer was confirmed by staining the membrane with Ponceau S red and the membrane was incubated in Odyssey® blocking buffer for 1 h at room temperature. The membrane was stained with anti-VGLUT3 antibody (1:1000, AB5421; Millipore, Milford, MA, USA) overnight at 4 ◦ C. After
Fig. 1. VGLUT3 IR confined to IHCs. At P2, VGLUT3 IR (red signal) was weak in IHCs of homozygous (cir/cir) mice (A: homo), heterozygous (+/cir) mice (B: hetero), and ICR mice (C), but became prominent at P14 in all mice tested (D: homozygous (cir/cir) mice, E: heterozygous (+/cir) mice, F: ICR mice). The intensity of VGLUT3 IR was weakest in IHCs of ICR mice (F). G is a low magnification view showing VGLUT3 IR confined to IHC in P14 heterozygous (+/cir) mice. *Indicates outer hair cells. Nuclei were stained with DAPI (blue signal). Graph above G shows the temporal expression pattern of VGLUT3 IR. Y axis indicates mean intensities of VGLUT3 IR and X axis indicates ages of mice tested (Hom: homozygous (cir/cir) mice, het: heterozygous (+/cir) mice). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. VGLUT3 IR in SGCs. At P2, VGLUT3 IR (red signal) was weak in IHCs of homozygous (cir/cir) mice (A: homo), heterozygous (+/cir) mice (B: hetero), and ICR mice (C), but became prominent at P14 in all mice tested (D: homozygous (cir/cir) mice, E: heterozygous (+/cir) mice, F: ICR mice). Nuclei were stained with DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
extensive washing with Tris-buffered saline–Tween 20, the membrane was stained with an appropriate IR-dye tagged secondary antibody. Densitometry of the bands was performed with software for an Odyssey infrared imaging system (LI-COR® Biosciences). 3. Results 3.1. VGLUT3 IR The temporal expression patterns of VGLUT3 were investigated in the cochlear hair cells of P1–P2 and P13–P14 mice (ICR mice, heterozygous (+/cir) mice, and homozygous (cir/cir) mice). VGLUT3 IR confined to IHCs was weak at P1–P2 but became stronger with age in all mice tested (Fig. 1). As the staining pattern was not different depending on location (apex, middle, or basal turn), all IR signals from stained IHCs were collected and analyzed. The mean intensities of IHC VGLUT3 IR at P1–P2 were as follows: 95.7 ± 16.0 (ICR mice, n = 12, five cochleae, three animals), 85.4 ± 26.8 (heterozygous (+/cir) mice, n = 13, five cochleae, three animals), and 66.2 ± 15.6 (homozygous (cir/cir) mice, n = 19, five cochleae, four animals). The three values were not significantly different. The mean intensities of IHC VGLUT3 IR at P13–P14 were as follows: 293.4 ± 49.7 (ICR mice, n = 15, five cochleae, three animals), 537.6 ± 78.6 (heterozygous (+/cir) mice, n = 18, six cochleae, three animals), and 703.2 ± 94.6 (homozygous (cir/cir) mice, n = 13, eight cochleae, four animals) (Fig. 1 inset). The mean intensities obtained from homozygous (cir/cir) mice and heterozygous (+/cir) mice were not significantly different, but the mean intensity of the ICR mice was significantly lower than those of the homozygous (cir/cir) mice and heterozygous (+/cir) mice (independent t-test, p < 0.05). An age-dependent increase in VGLUT3 IR was also observed in SGCs (Fig. 2). The mean intensities of VGLUT3 IR per field at P1-P2 were 116.2 ± 30.7 (ICR mice, 24 fields, eight cochleae, four animals), 118.2 ± 18.7 (heterozygous (+/cir) mice, 18 fields, eight cochleae,
four animals), and 173.8 ± 20.2 (homozygous (cir/cir) mice, 24 fields, 10 cochleae, five animals). The three values were not significantly different. At P13–P14, they were 237.6 ± 56.2 (ICR mice, 16 fields, five cochleae, three animals), 315.8 ± 41.5 (heterozygous (+/cir) mice, 14 fields, five cochleae, three animals), and 410.4 ± 36.4 (homozygous (cir/cir) mice, 26 fields, nine cochleae, five animals). Only the difference between the homozygous (cir/cir) mice and ICR mice was significant (independent t-test, p < 0.05). 3.2. Total VGLUT3 expression in cochleae As we found developmental increases in VGLUT3 IR in all mice tested, we quantified the total expression level of VGLUT3 by Western blot (Fig. 3). The basal expression levels of VGLUT3 at P1–P2 were similar in homozygous (cir/cir) mice, heterozygous (+/cir) mice, and ICR mice. The increases in total VGLUT3 expression at P13–P14 normalized to that of P1–P2 cochleae were three-fold (3.08 ± 0.40, three animals, five cochleae) in homozygous (cir/cir) mice, 1.5-fold in heterozygous (+/cir) mice (1.48 ± 0.15, three animals, five cochleae), and 1.5-fold in ICR mice (1.45 ± 0.15, three animals, five cochleae) (Fig. 3B). 4. Discussion Developmental VGLUT3 expression in cochlear IHCs and SGCs has been recently reported in rats [17]. In that report, VGLUT3 expression was low at P0 in IHCs and SGCs but increased progressively with age, which was very similar to our data. The unexpected finding was that VGLUT3 upregulation was strongest in homozygous (cir/cir) mice compared to ICR mice or heterozygous (+/cir) mice at P14. This might simply reflect the influence of the cir gene, but, as the IHCs and SGCs of homozygous (cir/cir) mice are in the process of deterioration, the possibility that VGLUT3 upregulation is affected by pathologic changes of cochleae should be considered.
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homozygous (cir/cir) mice [4,12,13]. In homozygous (cir/cir) mice, VGLUT3-positive puncta are mainly present in the cytoplasm and perinuclear region of SGCs. No matter what the cause of SGC destruction is, if SGCs start to degenerate, glutamate released from glutamate-overloaded SGCs will accelerate the degeneration of adjacent SGCs. Although the possible contribution of VGLUT3 upregulation to the degeneration of IHCs and SGCs is suggested, it is apparent that VGLUT3 overexpression itself cannot be a cause of the early deafness reported in P18 homozygous (cir/cir) mice [4]. To date, there is no evidence that excess VGLUT3 expression will harm the hearing function. The exact role of VGLUT3 overexpression at around P14 in homozygous (cir/cir) mice needs further investigation. Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology (2012R1A1A2040535). Fig. 3. Total expression of VGLUT3 measured by Western blot. Representative immunoblots of VGLUT 3 at P1–P2 and P13–P14 are shown in A. Quantitative analysis of VGLUT3 expression levels at P13–P14 normalized to those at P1–P2 levels are shown in B. -actin bands were used as loading controls (n = 5). *P < 0.05, **P < 0.01.
It has been reported that cochlear ablation leads to the VGLUT3 upregulation in the rat cochlear nucleus [8] and lateral superior olive [11]. As cochlear hair cells are reported to degenerate after P10 in homozygous (cir/cir) mice [4], VGLUT3 upregulation in SGCs, the primary relay neurons of the auditory pathway, might be explained in the same point of view. One thing to be considered is that our case is different with the cochlear ablation-induced VGLUT3 upregulation reported in rat lateral superior olive or cochlear nucleus because we did not find any organs of Corti showing complete degeneration in homozygous (cir/cir) mice at P14. Thus, if enhanced VGLUT3 expression in SGCs is to be explained in terms of cochlear pathology, it needs explanations other than complete degeneration of the organ of Corti. One of the possible explanations might be abnormal or absent neurotransmitter release from IHCs, which might produce a similar effect in that it cuts synaptic input just as complete degeneration of the organ of Corti does. The scanning electron microscopic level stereocilliary bundle defect reported in P10 homozygous (cir/cir) mice [4] supports this idea because the process of neurotransmitter release from IHCs starts with deflection of hair bundles by mechanical stimuli. The SGC degeneration reported in homozygous (cir/cir) mice [4,12,13] also supports the absence of or abnormal neurotransmitter release from IHCs because the absence of glutamate release from IHCs leads to the early degeneration of SGCs in VGLUT3 knock-out mice [19]. In contrast to VGLUT3 upregulation in SGCs, we have not found an appropriate explanation to VGLUT3 upregulation in IHCs yet. Though some VGLUT3 upregulation has been reported in the nervous system [3,11,22], it has never been reported at the peripheral receptor level. We speculate that VGLUT3 upregulation in IHCs may reflect an increased activity of VGLUT3 or glutamate overload due to the abnormal release of glutamate from the IHCs. If abnormal release of glutamate leads to glutamate overload, it will possibly contribute to cell destruction: glutamate released from deteriorating hair cells overloaded with glutamate will accelerate the destruction of nearby hair cells if the hair cells start to degenerate. There have been reports showing glutamate cytotoxicity in hair cells [10,14] and one report suggested the aggravating actions of glutamate released from deteriorating hair cells, which supports our hypothesis [15]. This mechanism will also contribute to SGC destruction, which has been reported in
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