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Introduction
The peripheral vestibular system consists of 2 sack-like structures: the sacculus and utricle, and 3 tubular structures
known as the semicircular canals. Vestibular hair cells (VHC) were characterized as type I or type II based on their
characteristic innervation pattern[1]. Type I vestibular hair cells (VHC I), which are shaped like bottles or flasks with rounded bases and
short necks, are present in reptiles, birds and mammals, but not in fish or amphibians. VHC I is innervated by an afferent nerve
calyx engulfing the entire cell body. Type II hair cells (VHC II) have cylindrical shapes and are innervated by both afferent
and efferent nerve branches, mostly synapsing with the basal part of the
cell[2,3]. The vestibular endorgans detect both
angular velocity and linear acceleration, including gravity; the information from this system is conveyed to the central
nervous system via afferent nerve fibers. Meanwhile, the efferent terminals arising in the brainstem form direct synaptic
contacts with VHC II and form postsynaptic connections with the afferent calyx surrounding the VHC I.
There is a general consensus that acetylcholine (ACh) is the major transmitter released at efferent synapses in the
peripheral vestibular system[4_6]. By using histochemical techniques, acetylcholinesterase (AChE), the ACh inactivating
enzyme, was localized to the vestibular efferent neurons and
endings[7_10]. The choline acetyltransferase (ChAT) like
immunoreactivity was also traced to the efferent endings in vestibular
endorgans[11_13]. ChAT and AChE activity were found in
efferent terminals contacting the basal area of VHC II and the afferent chalices surrounding VHC I. Cholinergic receptors
belong to 2 different classes: muscarine cholinergic receptors and nicotinic cholinergic receptors (nAChR). By using various
nucleic acid and ligand-binding methods, nAChR were also localized in vestibular
endorgans[14_19].
A total of 17 nAChR subunits
(α1_α10, β1_β4, γ, δ, and ε) have been identified in vertebrate species which can
co-assemble to generate a wide variety of
nAChR[20]. The a9 and a10 subunits are localized predominantly in neuroepithelial
auditory and VHC[21_25], although their presence has also been found in other
tissues[26]. Recent studies have suggested the
localization of mRNA in VHC of mammals. Hiel et
al localized α9 AChR mRNA in VHC I and VHC II of rats using
in situ hybridization with 35S-labeled ribo-
probes[16]. Using immunohistochemistry techniques, Luebke
et al reported that in chinchillas, rats and guinea pigs,
immunoreactivity to the α9 AChR was confined to calyces around VHC I and the synaptic pole of VHC
II[19]. The single cell reverse transcription polymerase chain reaction (RT-PCR) technique has higher sensitivity than the
in situ hybridization technique and immunohistochemistry techniques, particularly in studies aimed to localize the cell specific expression of receptors in an
individual cell[27]. With the single cell RT-PCR technique, the cell specific expression of
α9 AChR in auditory hair cells has been
reported[27]. With the single cell RT-PCR technique, Lustig
et al demonstrated the expression of α9 AChR mRNA in both
the VHC I and VHC II of chicks[18]. However, expression of
α9 AChR has not been detected in the VHC of mammals by using
the single cell RT-PCR technique. It was reported that the innervation pattern of the ChAT-like immunostained efferents in
humans is similar to that of other mammals, including
rats[13]; there were many differences between the efferent vestibular
system of birds and that of mammals[6]. Further investigations on the ACh-ergic innervation pattern of the peripheral
vestibular system of rats will help us to understand the efferent innervation pattern of the human vestibule.
In the present study, in order to explore the cell specific expression of
α9 AChR in the VHC II of rats, we used the RT-PCR technique with gene-specific primers to detect the expression of
α9 AChR in the vestibular endorgans of rats, then used
single cell RT-PCR with the same primers to detect the expression of
α9 AChR in single rat VHC II.
Materials and methods
RT-PCR An adult Sprague-Dawley rat
(200_250 g) was anesthetized and decapitated in 1 run of the experiment. The
temporal bones were quickly removed and placed in ribonuclease (RNase)-free phosphate-buffered saline (PBS) at 4 °C and
pH 7.4, mimicking extracellular conditions containing (in mmol/L) NaCl (129.4), KCl (5),
MgSO4 (1.6),
Na2HPO4 (0.3),
CaCl2 (1.2),
KH2PO4 (0.4) and D-Glucose (16.7). The osmolarity of the PBS was adjusted to 295
mOsm/L by NaCl. The experiments were repeated 5 times. A total of 5 rats were used in the experiments. The vestibular organs, including the sacculus, utricle and
superior and horizontal ampulla, were dissected under Stereo Microscope
(JSZ, Jiangnan Novel Optics Co Ltd, Nanjing, China) and immediately homogenized in 1 mL Trizol reagent (Life Technologies Inc,
Gaithersburg, MD, USA). The total RNA of the vestibular endorgans was extracted from the tissue according to the manufacturer's instruction. The total RNA was
dissolved in 20 µL RNase-free water. The PBS was used as negative control at each run of the experiment.
Gene specific primers for α9 AChR
were used as outlined by Glowatzki[28]. The predicted fragment length for
α9 AChR was 576 bp. The primers were designed on different exons; the sense primer (nucleotide
664_689): 5'-CTCCTCATCCC-TTGCGTCCTCATAT-3' and the anti-sense primer (nucleotide
1239_1210): 5'-GAGGCACTTGGCAATGTATTCGATA-TTTT-3'. The gene specific primers for the
β-actin protein based on the rat β-actin mRNA sequence (NM_031144) were used as
internal control. The predicted fragment length for
β-actin cDNA was 213 bp; the sense primer (nucleotide
303_322 bp): 5'-CATTGTAACCAACT-GGGACG-3' and the anti-sense primer (nucleotide
497_515 bp): 5'-GAGGCATACA-GGGACAACA-3'. The primers were designed with the assistance of Primer Premier 5.0 Software (Premier Biosoft International, Palo Alto, CA,
USA) to amplify a fragment of the sequence, including an intron.
RT and PCR were performed in the same tube according to the manufacturer's instructions (SuperScript™ III, One-Step
RT-PCR System with Platinum®
Taq DNA Polymerase, Invitrogene, Life Technologies, Gaithersburg, MD, USA) in a
T-gradient thermal cycle (Biometra, Whatman Biometra, Goettingen, Germany). A 20 µL RT-PCR reaction system containing:
2×RT buffer (10 µL), 1 µL (10 pmol) gene specific primers, 1 µL (20 U) RNase Inhibitor (Promega, Madison, WI, USA), 6 µL
RNase-free water, 1 µL (1 U)
RT® Platinum Taq and a 1 µL total of RNA. The RT step was performed at 45 °C for 60 min. The
PCR had 40 cycles; the amplification conditions for
α9 AChR were as follows: 95 °C, 20 s; 58 °C, 30
s; 72 °C, 40 s. The amplification conditions for
β-actin were as follows: 95 °C, 20 s; 54 °C, 30 s; 72 °C, 30 s. After cycling, the reaction was
extended at 72 °C for 10 min. The products of the reaction were stored at 4 °C. Five microliters from the 20 µL products were
run on a 1.5% agarose gel containing ethidium bromide. Then remnant PCR products were given to the sequencing company
(BGI LifeTech Co Ltd, Beijing, China) for direct sequencing using an ABI377 sequencer (PE Applied Biosystems, Foster City,
CA, USA).
Cell preparation One rat was used for 1 run of the experiment. A total of 5 runs of experiments were done as part of this
study. The tissue of rat vestibular endorgans was dissected and immediately placed into calcium-free PBS supplemented
with collagenase IA (0.25 mg/mL) (Sigma, St Louis, MO, USA). After the endorgans were incubated in the PBS solution for
5 min at 4 °C, PBS without collagenase I A was used in exchange to the solution 3 times. Subsequ-ently, single hair cells were
mechanically dissociated from the endorgans by cutting through the epithelium with a sharpened needle under Stereo
Microscope (JSZ, Jiangnan Novel Optics Co, Ltd, Nanjing, China) at 4 °C. The single hair cells were prepared for single cell
RT-PCR.
Single cell harvest Single hair cells were moved to an inverted microscope (IX71, Olympus Corp, Tokyo, Japan). At 200×
total magnification, an individual VHC II was identified according to criteria outlined by Ricci
et al[29]. A glass pipette with a
tip diameter of 50 µm was attached to the micromanipulator (Narashige, Tokyo, Japan). The pipette tip was advanced with the
micromanipulator until contact with a VHC II, then gentle suction was used to aspirate the cell into the pipette. Subsequently,
the pipette with the cell was immerged into a fresh PBS solution at room temperature. The pipette went down with the
micromanipulator arm until its tip nearly made contact with the bottom of the glassy utensil and the VHC II were expelled by
gentle positive pressure. Finally, another glass pipette with a tip diameter of 10_20 µm, filled with about 2 µL RNase-free water
and 0.2 µL (2 U) RNase Inhibitor was used to aspirate the VHC II into the pipette again. The contents of the pipette were then
immediately expelled into a 200 µL microfuge PCR tube containing 4 µL RNase-free water with 0.4 µL (4 U) RNase Inhibitor
using positive pressure; the tip, which was about 2 mm long, was broken off. Samples of surrounding fluid and cell debris
were also aspirated and used as negative controls. The PCR tube was used in the following reaction or stored at -75 °C.
Single cell RT-PCR RT and PCR were performed in the same tube according to the manufacturer's instructions
(SuperScript™ III One-Step RT-PCR System with
Platinum® Taq DNA Polymerase, Life Technology, Gaithersburg, MD, USA)
in a thermal cycler (Biometro, Germany). A 20 µL RT-PCR reaction system containing: 2×RT buffer (10 µL), 1 µL (10 pmol) gene
specific primers, 1 µL (20 U) RNase Inhibitor, 6 µL RNase-free water, 1 µL (1 U)
RT® Platinum Taq and PBS with single VHC II
or PBS with cell debris or surrounding fluid (1 µL). These components were added and mixed by flicking the tube, followed
by brief centrifugation. The RT-PCR reaction conditions were the same as earlier described. After the first round of RT-PCR,
the second round of PCR was carried out. In the second round of PCR, 1 µL of the products yielded in the first round of PCR
was used as template, using the same primers. A 50 µL PCR reaction system containing:
10×Mg2+-free PCR buffer (5 µL), 3
µL(25 mmol/L) MgCl2 ,4 µL (2.5 mmol/L) dNTP, 1 µL (10 pmol) gene specific primers, 35 µL RNase-free water, 1 µL
Taq enzyme (Fermentas International Inc, Vilnius, Lithuania) and 1 µL template. The second round PCR had 35 cycles and reaction
conditions of amplification were the same as the the first round of PCR. Five microliters from the 50-µL products of the second
round reaction were run on a 1.5% agarose gel containing ethidium bromide. The remnant PCR products were then given to
the sequencing company (BGI Life Tech Co Ltd, Beijing, China) for directly sequencing.
Results
Expression of α9 AChR mRNA in the vestibular endorgans
The PCR products of the RT-PCR reaction using the total
RNA extracted from the vestibular endorgans as the template and the primers for
α9 AChR and β-actin, respectively, are shown in their predicted sizes, approximately 576 bp for
α9 AChR (Figure 1) and 213 bp for β-actin (Figure 2). The possible
amplification of the nuclear DNA would have resulted in a larger PCR fragment. Negative controls using the PBS as a template
for each run of the experiment were all negative. The messenger RNA encoding the
α9 AChR and β-actin was detected from the vestibular endo-rgans. The PCR products were directly sequenced; sequence analysis of the band confirmed identity to
rat β-actin and α9 AChR cDNA sequence in the predicted region.
Expression of α9 AChR mRNA in single VHC II
Messenger RNA encoding β-actin and α9 AChR was also detected in
a few of the VHC II. Each lane demonstrated a single band at the predicted size, approximately 213 bp for
β-actin (Figure 2) and 576 bp for α9 AChR (Figure 3). Seven single cells were examined for the expression of
β-actin and all 7 cells were positive. Twenty VHC II were examined for the expression of
α9 AChR and 4 VHC II were positive (Table 1). It is crucial to transfer the
single VHC II from the solution which contains the dissociated individual cells to fresh PBS to avoid false positive results. In
the negative control, the reaction that ran on the surrounding fluid bathing the single cell and cell debris failed to detect
b-
actin or α9 AChR mRNA. Seven and 18 negative controls for
β-actin and α9 AChR respectively were done and all were
negative. The PCR products have also been directly sequenced. Sequence analysis of the band confirmed identity to rat
β-actin and α9 AChR cDNA sequence in the predicted region.
Discussion
ACh has long been considered the efferent transmitter of the peripheral vestibular
system[5,30]. It was suggested that the
nAChR subunit a9 is one of the important mediators of efferent cholinergic signaling in the peripheral vestibular system.
α9 AChR was shown to be present in the vestibular sensory epithelium of rats in in situ hybridization and
immunohistochemistry studies. Hiel et al localized
α9 AChR mRNA in type I and type II VHC of rats using in situ hybridization
with 35S-labeled
riboprobes[16]. Luebke et al reported that in rats, immunoreactivity to the
α9 AChR was confined to calyces around type I hair cells and the synaptic pole of type II hair
cells[19]. Furthermore, Zuo et
al demonstrated the expression of α9 AChR mRNA in VHC utilizing green fluorescent protein coexpressed with
α9 AChR mRNA[31]. In this study, by using the
RT-PCR technology, the expressions of α9 AChR mRNA was detected in the vestibular endorgans of rat. By using the single
cell RT-PCR technique, it was shown that there was expression of
α9 AChR mRNA in single VHC II of rats. It also indicates
that VHC II of rats might contain α9 AChR. The present study confirmed the expression of
α9 AChR in vestibular endorgans of rats and the cell specific expression of
α9 AChR mRNA in individual VHC II of rats. Single cell RT-PCR is a highly sensitive
method which can detect mRNA molecule coding for a specific protein in an individual cell. One of the drawbacks of this
technique arises from high sensitivity: there is a high risk of false positive results due to
contamination[27]. In this study, it is crucial to transfer the single VHC II from the solution which contains the dissociated individual cells to fresh PBS to avoid
the false positive results. All negative controls being negative in this study indicate that this method could effectively avoid
the possible contamination and successfully detect the cell specific expression of mRNA in single VHC II.
VHC II are directly innervated by efferent nerve terminals and ACh is the major efferent transmitter in the peripheral
vestibular system of rats[4]. In addition,
α9 AChR have been localized to VHC II by using immunohistochemistry
techniques[19] and the in situ hybridization
technique[16]. Furthermore, ACh can induce modifications of VHC II membrane
potential[4]. In this experiment, we further demonstrated the localization of
α9 AChR mRNA in VHC II. We indicate that VHC II may possess
α9 AChR. The results indicate that VHC II may be directly modulated by ACh-ergic efferent terminals and
α9 AChR may be one of the important mediators of efferent cholinergic signaling in the peripheral vestibular system of rats.
The α9 AChR is specifically expressed
in hair cells of the inner ear and is believed to be involved in synaptic transmission
between efferent nerves and hair
cells[31]. It was also reported that
a10 AChR was a determinant of nAChR function in mammalian vestibular and cochlear mechanosensory hair cells, and efferent modulation of hair cell function occurs, at least
in part, through heteromeric nAChR assembled from both
a9 and a10 subunits[22,23]. Coinjection of Xenopus oocytes with
a9 and a10 cRNA results in the appearance of ACh-gated channels possessing properties that are distinct from
a9-injected oocytes. In particular, a9 and a10-injected oocytes show ~100-fold larger currents, have a unique current-voltage relationship,
exhibit more rapid and complete desensitization kinetics, and show a biphasic response to changes in extracellular
Ca2+ ions[22]. The single cell RT-PCR technique can be used to detect the coexpression of
a9 and a10 subunits in a VHC because it can detect a set of mRNA sequences in an individual
cell[27].
In summary, the results of this experiment provide the direct evidence to confirm that VHC II of rats might express mRNA
of α9 AChR. It indicates that VHC II may be directly modulated by ACh-ergic efferent terminals. However, the nature of the
expression of α9 AChR in rat VHC is still unclear. Further morphological and electrophysiological studies of VHC are needed
to address this issue.
References
1 Wersall J. Studies on the structure and innervation of the sensory epithelium of the cristae ampulares in the guinea pig; a light and electron
microscopic investigation. Acta Otolaryngol Suppl 1956; 126: 1_85.
2 Wersall J, Bagger-Sjoback D. Morphology of the vestibular sense organ. In: Kornhuber HH, editor. Handbook of Sensory Physio-logy.
Berlin: Springer-Verlag; 1974. p 123_70.
3 Lysakowski A. Synaptic organization of the crista ampullaris in vertebrates. Ann N Y Acad Sci 1996; 781: 164_82.
4 Guth PS, Norris CH. The hair cell acetylcholine receptors: a synthesis. Hear Res 1996; 98: 1_8.
5 Guth PS, Perin P, Norris CH, Valli P. The vestibular hair cells: post-transductional signal processing. Prog Neurobiol 1998; 54: 193_247.
6 Lysakowski A, Goldberg JM. Morphophysiology of the vestibular periphery. In: Highstein SM, Fay RR, Popper AN, editors. The
Vestibular System. New York: Springer; 2004. p 57_152.
7 Hilding D, Wersall J. Cholinesterase and its relation to the nerve endings in the inner ear. Acta Otolaryngol 1962; 55: 205_17.
8 Nomura Y, Gacek RR, Balogh K. Efferent innervation of vestibular labyrinth: Histochemical demonstration of Acetylcholinesterase
activity in the guinea pig inner ear. Arch Otolaryngol 1965; 81: 335_9.
9 Iurato S, Luciano L, Pannese E, Reale E. Histochemical localization of acetylcholinesterase (AChE) activity in the inner ear. Acta
Otolaryngol 1971; 279 (Suppl): 1_50.
10 Cohen GM. Acetylcholinesterase activity in the embryonic chick's inner ear. Hear Res 1987; 28: 57_63.
11 Gonzalez A, Meredith GE, Roberts BL. Choline acetyltransferase immunoreactive neurons innervating labyrinthine and lateral line sense
organs in amphibians. J Comp Neurol 1993; 332: 258_68.
12 Kong WJ, Egg G, Hussl B, Spoendlin H, Schrott-Fischer A. Localization of chat-like immunoreactivity in the vestibular endorgans of the
rat. Hear Res 1994; 75: 191_200.
13 Kong WJ, Hussl B, Thumfart WF, Schrott-Fischer A. Ultrastructural localization of ChAT-like immunoreactivity in the human vestibular
periphery. Hear Res 1998; 119: 96_103.
14 Ishiyama A, Lopez I, Wackym PA. Distribution of efferent cholinergic terminals and alpha-bungarotoxin binding to putative nicotinic
acetylcholine receptors in the human vestibular end-organs. Laryngoscope 1995; 105: 1167_72.
15 Wackym PA, Popper P, Lopez I, Ishiyama A, Micevych PE. Expression of alpha 4 and beta 2 nicotinic acetylcholine receptor subunit
mRNA and localization of alpha-bungarotoxin binding proteins in the rat vestibular periphery. Cell Biol Int 1995; 19: 291_300.
16 Hiel H, Elgoyhen AB, Drescher DG, Morley BJ. Expression of nicotinic acetylcholine receptor mRNA in the adult rat peripheral vestibular
system. Brain Res 1996; 738: 347_52.
17 Luo L, Bennett T, Jung HH, Ryan AF. Developmental expression of alpha 9 acetylcholine receptor mRNA in the rat cochlea and
vestibular inner ear. J Comp Neurol 1998; 393: 320_31.
18 Lustig LR, Hiel H, Fuchs PA. Vestibular hair cells of the chick express the nicotinic acetylcholine receptor subunit alpha 9. J Vestib Res
1999; 9: 359_67.
19 Luebke AE, Maroni PD, Guth SM, Lysakowski A. Alpha-9 nicotinic acetylcholine receptor immunoreactivity in the rodent vestibular
labyrinth. J Comp Neurol 2005; 492: 323_33.
20 Millar NS. Assembly and subunit diversity of nicotinic acetylcholine receptors. Biochem Soc Trans 2003; 31: 869_74.
21 Elgoyhen AB, Johnson DS, Boulter J, Vetter DE, Heinemann S. Alpha 9: an acetylcholine receptor with novel pharmacological properties
expressed in rat cochlear hair cells. Cell 1994; 79: 705_15.
22 Elgoyhen AB, Vetter DE, Katz E, Rothlin CV, Heinemann SF, Boulter J. alpha10: a determinant of nicotinic cholinergic receptor function
in mammalian vestibular and cochlear mechano-sensory hair cells. Proc Natl Acad Sci USA 2001; 98: 3501_6.
23 Morley BJ, Simmons DD. Developmental mRNA expression of the alpha10 nicotinic acetylcholine receptor subunit in the rat cochlea.
Brain Res Dev Brain Res 2002; 139: 87_96.
24 Simmons DD, Morley BJ. Differential expression of the alpha 9 nicotinic acetylcholine receptor subunit in neonatal and adult cochlear
hair cells. Brain Res Mol Brain Res 1998; 56: 287_92.
25 Drescher DG, Ramakrishnan NA, Drescher MJ, Chun W, Wang X, Myers SF,
et al. Cloning and characterization of alpha9 subunits of the
nicotinic acetylcholine receptor expressed by saccular hair cells of the rainbow trout (Oncorhynchus mykiss). Neuroscience 2004; 127:
737_52.
26 Sgard F, Charpantier E, Bertrand S, Walker N, Caput D, Graham D,
et al. A novel human nicotinic receptor subunit, alpha10, that confers
functionality to the alpha9-subunit. Mol Pharmacol 2002; 61: 150_9.
27 Glowatzki E, Wild K, Brandle U, Fakler G, Fakler B, Zenner HP,
et al. Cell-specific expression of the alpha 9 n-ACh receptor subunit in
auditory hair cells revealed by single-cell RT-PCR. Proc Biol Sci 1995; 262: 141_7.
28 Glowatzki E. Analysis of gene expression in the organ of Corti
revealed by single-cell RT-PCR. Audiol Neurootol 1997; 2: 71_8.
29 Ricci AJ, Rennie KJ, Cochran SL, Kevetter GA, Correia MJ. Vestibular type I and type II hair cells. 1: Morphometric identification in the
pigeon and gerbil. J Vestib Res 1997; 7: 393_406.
30 Highstein SM. The central nervous system efferent control of the organs of balance and equilibrium. Neurosci Res 1991; 12: 13_30.
31 Zuo J, Treadaway J, Buckner TW, Fritzsch B. Visualization of alpha9 acetylcholine receptor expression in hair cells of transgenic mice
containing a modified bacterial artificial chromosome. Proc Natl Acad Sci USA 1999; 96: 14100_5.
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