Download Functional expression of P2 receptors in the inner ear of chicken

Neuroscience Letters 553 (2013) 24–28
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Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet
Functional expression of P2 receptors in the inner ear of chicken
embryo
Fabian Galindo a , Eduardo Monjaraz a , Salvador Galicia b , Jorge Cebada b ,
Celso Cortés c , Amira Flores a,∗
a
Instituto de Fisiología, Benemérita Universidad Autónoma de Puebla, Av. 14 Sur 6301 Colonia Jardines de San Manuel CP 72570, Puebla, Pue., Mexico
Escuela de Biología, BUAP, Blvd. Valsequillo y Av. San Claudio, Edif. 112-A C.U. Colonia Jardines de San Manuel CP 72570, Puebla, Pue., Mexico
c
Facultad de Medicina, BUAP, Av. 13 Sur 2702 Colonia Volcanes CP 72410, Puebla, Pue., Mexico
b
h i g h l i g h t s
•
•
•
•
ATP modulate the spontaneous afferent discharge in the inner ear during development.
P2X receptors have a significative effect on the vestibular afferent discharge.
P2Y receptors predominate in the basilar papilla of chicken.
The purinergic effects in both systems are more important in younger animals.
a r t i c l e
i n f o
Article history:
Received 9 January 2013
Received in revised form 18 June 2013
Accepted 5 August 2013
Keywords:
ATP
Inner ear
Development
Chicken
P2X receptor
P2Y receptor
a b s t r a c t
The purpose of the present study was to investigate the modulation of spontaneous afferent activity by
ATP during embryonic development in a preparation isolated chicken inner ear. This work was performed
using multiunit and single-unit extracellular recordings from the posterior semicircular canal nerve and
the basilar papilla nerve. ␣,␤-meATP, a P2X receptor agonist, notably increased the discharge frequency
of the vestibular afferents between E15 and E18, but not in the basilar papilla. In contrast, the P2Y receptor agonist UTP produced a slight increase in the discharge frequency of basilar papilla afferents, without
apparent changes in the vestibular afferent activity. 2-MeSATP, a P2Y agonist, increased the basal discharge of the primary afferents in a dose-age dependent way, but when we applied the antagonist of
P2Y receptor, Reactive Blue 2 (10−4 M), the effect of 2-MeSATP decreased significantly. This was observed
both in vestibule and basilar papilla. Using RT-PCR the presence of P2X3 , P2Y1 , P2Y2 and P2Y6 mRNA was
documented in the vestibular system with more important presence during the early stage (E15) than the
later stage (E21), however in the basilar papilla we found only the P2Y1 , P2Y2 and P2Y6 mRNA with the
same temporal course as in the vestibule. These results confirm our pharmacological findings. Together
this data suggests a role for P2X receptors-mediated purinergic signaling in vestibular synaptic organization. Temporal changes in P2Y receptors during development might be involved in the establishment
of the endolymphatic ion composition needed for normal vestibular and auditory transduction and/or
specific cellular differentiation.
© 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Extracellular
nucleotides,
particularly
adenosine
5 triphosphate (ATP), are important signaling molecules that
mediate diverse biological effects via cell surface receptors [1,4].
∗ Corresponding author at: Instituto de Fisiología, Laboratorio de Neurobiología,
Benemérita Universidad Autónoma de Puebla, Apartado Postal 406, Puebla, Pue., CP
72000, Mexico. Tel.: +52 222 2 29 55 00x7303; fax: +52 222 2 29 55 00x7323.
E-mail addresses: [email protected], amira.fl[email protected]
(A. Flores).
0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neulet.2013.08.010
ATP can act at both ionotropic (P2X) and metabotropic receptors (P2Y). Multiple isoforms of P2X and P2Y receptors, which
differ in molecular structure, kinetics and sensitivity for agonists
and antagonists have been cloned and characterized [4,10,15].
Important developmental roles including cell differentiation,
neurogenesis, gastrulation, survival and maturation of neurons,
have been suggested for these receptors [6].
Although ATP and their receptors have been localized in the
inner ear [18], little is known concerning the potential role of ATP in
the regulation of the hair cells response, neurotransmitter release,
discharge of the afferent neurons and their participation in the process of maturation during inner ear development. The present study
F. Galindo et al. / Neuroscience Letters 553 (2013) 24–28
25
Table 1
Primer pairs used in the RT–PCR reactions.
Target mRNA
P2X1
P2X3
P2Y1
P2Y2
P2Y6
␤actin
GenBank access no.
NM204519
XM426413
NM205333
XM425667
NM205195
NM 001017992
Upper and lower primer sequences
5 -CAGACACAGGACAGCATCG-3
5 -GTCACAGTTCCAGTCGATG-3
5 -GACACTGTCATCGAGTCCT-3
5 -GGACGCTGTTCTTGATGAAG-3
5 -CGTCATGTGCAAGCTGCAG-3
5 -TCAGAGCTTTCACGATCAGC-3
5 -CACCACGTACATGTTCAACC-3
5 -CATCACCGAACTGTATATGAC-3
5 -CGTTGTCATTGGGCAGATCT-3
5 -CAATCAGCTCGTCTTTCTGG-3
5 -CCTGCTTGCTGATCCACATCTGCTG-3
5 -ATCCTGCGTCTGGACCTGGCTG-3
was performed to examine a potential role for ATP in the excitability
of the avian inner ear. For this purpose, we investigated the effects
of P2X and P2Y agonists and antagonists in the firing rate of the
afferent fibers from the posterior semicircular canal and from the
basilar papilla of the chicken during embryonic development. In
addition, we studied the temporal course of P2X and P2Y receptor
expression in the vestibular periphery and the basilar papilla.
2. Materials and methods
2.1. Embryo preparations
All procedures described in this study were in accordance with
the technical guidelines for production, care, and use of animals in
the laboratory issued by SAGARPA México (NOM-062 ZOO-1999)
and by the National Institutes of Health Guide for the Care and
Use of Laboratory Animals. All efforts were made to minimize animal suffering and to reduce the number of animals used. We used
chicken embryos (E) (Gallus domesticus) of 15, 18 and 21 days supplied by ALPES (Tehuacán, Mexico). The age of the embryos given
in days was established by reference to the staging criteria of Hamburger and Hamilton (1951) [8]. Embryos were cooled, removed
from the egg, and decapitated. The inner ear was dissected out in
Ringer’s solution containing (in mM): NaCl 124; KCl 5; NaH2 PO4
2.2; CaCl2 2; NaHCO3 26; MgSO4 2 and glucose 10; with a pH of
7.3–7.4 after saturation with 95% O2 and 5% CO2 [25]. The isolated
preparation (vestibular end organs or basilar papilla) was transferred to a recording chamber with a constant temperature of 37 ◦ C
(TC-102 of Medical System Co).
2.2. Electrophysiological recording
Multiunit extracellular recordings were obtained from the posterior semicircular canal nerve using a suction electrode. Electrical
activity amplified by means of a conventional AC amplifier (Model
P511, GRASS) was fed to an oscilloscope (Tektronix 2201) and to a
window discriminator (121 WPI), the output of which was passed
to a microcomputer for on-line analysis of the neuronal spike discharge rate [27].
In the basilar papilla, extracellular single-unit recordings were
made with glass micropipettes filled with 2 M NaCl (30–50 M).
Signals were digitized and sampled at 100 kHz (Digidata 1320A;
Axon Instruments). The analyses of the spikes were made with the
pClamp 9.0 (Molecular Devices) software.
2.3. Drug application
All chemicals were purchased from Sigma–Aldrich. Drugs were
either bath substitution – or locally applied as indicated. Data are
presented as mean ± SEM, and statistical analysis performed using
Annealing temperature (◦ C)
RT-PCR product (bp)
58
621
58
384
64
370
58
402
60
544
65
578
Student’s paired or unpaired t test and analysis of variance (ANOVA)
followed by post hoc Newman–Keuls test.
2.4. Standard RT-PCR
Total RNA from crista ampullaris and basilar papilla was isolated
using Quick-RNATM MiniPrep (Zymo Research, USA) according to
the manufacturer’s directions. The concentration of total RNA for
each sample was determined by absorbance measurements at 260
and 280 nm (Biophotometer, Eppendorf, USA.). The integrity of the
RNA was assessed by electrophoresis on a denaturing agarose gel.
RNA samples were stored at −30 ◦ C until analyzed by RT–PCR. cDNA
was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). cDNA obtained was subjected to
the PCR protocol as follows: each reaction mixture (20 ␮l final volume) included 10 ␮l PyroStartTM Fast PCR Master Mix (Fermentas,
USA), 1 ␮l (1 ␮g/␮l) of each specific primer (Table 1), 6.5 ␮l of water
and 1.5 ␮l (50 ng/␮l) of cDNA and reaction mixtures were processed
in a Mastercycler gradient (Eppendorf, USA). For amplification an
initial denaturation step at 94 ◦ C for 5 min was followed by 35 cycles
of PCR. Each cycle consisted of three temperature steps: 30 s at
94 ◦ C, 30 s at the optimal annealing temperature (Table 1), and 60 s
at 72 ◦ C. Amplification was concluded with a final elongation step
at 72 ◦ C for 10 min. To verify their identity, amplification products
were resolved by electrophoresis in 1.5% agarose gels and visualized by ethidium bromide staining, and the intensities were then
measured by scanning the gel with the ChemiDoc Gel Documentation System (Bio-Rad, USA). Controls without reverse transcriptase
were used to demonstrate the absence of contaminating DNA.
PCR products were sequenced and were cleaned using QIAquick
PCR purification columns (Qiagen; USA). The suitability of the gelpurified PCR product was evaluated by automatic DNA sequencing
using an ABI PRISM 310 sequence analyser (Perkin–Elmer Applied
Biosystems, USA).
3. Results
3.1. Vestibule
We studied the effect of ATP, ␣␤-meATP, UTP and 2MeSATP
on the basal discharge of the vestibular afferents. Fig. 1A, shows
that ATP (10−8 –10−4 M) significantly enhanced the spontaneous
afferent electrical activity in a dose-age dependent manner. The
maximum effect was observed in E15 and E18. Likewise, application of the P2X receptor agonist ␣␤-meATP (10−9 –10−4 M), showed
an important excitatory effect and increased the activity in a doseage dependent manner. 10−4 M caused the main excitatory effect in
E15 (208 ± 4.4%), E18 (226 ± 6.7%), and E21 (197 ± 4.1%) compared
with basal discharge (Fig. 1B).
UTP, a P2Y agonist, (10−6 –10−3 M) did not change the electrical
activity at any age studied (not shown). However, the P2Y agonist
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F. Galindo et al. / Neuroscience Letters 553 (2013) 24–28
with age. The P2X3 receptor had a particular behavior (Fig. 3A);
its expression was different at the three ages, showing increased
expression at E18 over E15 and E21 (187% and 167%, respectively).
This results match with the electrophysiological findings where the
P2X agonist ␣,␤-meATP had a major effect at age E18. Expression of
P2X3 , P2Y1 and P2Y2 receptors was also dependent on age; reaching
its maximum levels at E21 (Fig. 3B and C). P2Y6 had an important expression, as P2Y2 , but there were not statistical differences
between the ages tested (Fig. 3D).
3.2. Basilar papilla
We explored the effect of ATP receptors activation on auditory afferents discharge. Fig. 4A shows that ATP 10−3 M increased
the basal discharge at the three tested ages (E15, 25.73 ± 3.83%;
E18, 30.57 ± 1.8% and E21, 7.77 ± 1.7%). We did not find significant
changes with ␣,␤-meATP (10−5 –10−3 M) (not shown), but when
UTP and 2-MeSATP were applied (10−3 M) we observed an increase
in discharge frequency of the afferents at E15 and E18, (Fig. 4B and
C).
Application of Reactive Blue 2 (100 ␮M) by bath substitution
decreased significantly the spontaneous activity (>50%), and the
excitatory effect of 2-MeSATP was completely abolished at all ages
(not shown).
Interestingly, the RT-PCR assays showed that the basilar papilla
only expressed mRNA encoding for P2Y receptor isoforms. P2Y2
receptor showed a significant increase in its level of expression with
increased age of the chick embryo (Fig. 3C).
4. Discussion
Fig. 1. ATP and ␣␤-meATP effects on the basal discharge of vestibular afferents at different developmental ages. (A) ATP dose-response-age curve
fitting with the Hill equation yielded, (E15) EC50 = 4.031 × 10−7 (pEC50
6.395 ± 0.3599); Hill coefficient (nH) = 0.65 ± 0.223); (E18) EC50 = 4.42 × 10−6
(pEC50 5.354 ± 0.05978); nH = 0.87 ± 0.0714; (E21) EC50 = 3.134 × 10−6 (pEC50
5.504 ± 0.4259) nH = 0.513 ± 0.333. The effect reached its maximum level at
the highest concentrations tested at E18 (181.0 ± 2.157%). (B) ␣␤-meATP doseresponse-age curve fitting. (E15) EC50 = 3.53 × 10−6 (pEC50 5.451 ± 0.2596)
nH = 0.64 ± 0.29; (E18) EC50 = 9.66 × 10−6 (pEC50 5.015 ± 0.030), nH = 0.72 ± 0.31;
(E21) EC50 = 6.029 × 10−6 (pEC50 5.220 ± 0.092), nH = 0.59 ± 0.06. The maximal
responses for E15 (205.6 ± 11.7%) and E18 (234.7 ± 21.21%) were always greater
than the changes produced in E21 (185 ± 3.804%). Points are the means ± SEM and
the line drawn is the best-fit curve obtained.
2-MeSATP, applied by microperfusion (10−5 –10−3 M), increased
the spontaneous electrical activity of the primary afferents in doseage dependent manner. The effect was more apparent in E15 than
in E21 (192.4 ± 18.9% vs 132.87 ± 6.81%) with 10−3 M (Fig. 2A).
When we applied the antagonist of P2Y receptors, Reactive Blue 2
(10−4 M), a decrease in afferent spontaneous activity was observed
at the three ages tested (Fig. 2B). Likewise, the stimulatory effect
of 2-MeSATP (10−3 M) on the basal discharge in E15 is reduced to
79.6 ± 1.98% in the presence of Reactive Blue 2, 10−4 M (Fig. 2C).
Using RT-PCR we detected the presence of P2X3 , P2Y1 , P2Y2
and P2Y6 receptors, however, their pattern of expression differed
P2 receptors have been found in different structures of the mammalian inner ear [2–4,9,16,17,22,26,28]; however this is the first
study where the presence and possible functional role of P2 receptors in the avian inner ear are demonstrated during embryonic
development.
Spontaneous activity in the auditory nerve fibers before the
onset of hearing is essential for the survival of target neurons in
the cochlear nuclei, accurate wiring of auditory pathways, and the
refinement of the tonotopic maps [14,20,30].
Tritsch et al. [29] demonstrated that supporting cells within
Kolliker’s organ in the rat cochlea initiates bursts of electrical activity in spiral ganglion neurons before the onset of hearing through
ATP-dependent excitation of hair cells. These results suggest that
peripheral, non-sensory cells are essential for the maturation of
auditory pathways in the brain. Recent results show that spontaneous impulses in inner hair cells are intrinsically generated
throughout pre-hearing stage of development and does not require
waves of depolarization linked to ATP release [11]. This intrinsic
activity disappears at the onset of hearing due to a change in the
ion channels expressed by the hair cells [12].
However, Jones et al. [13] showed that in the chicken embryo
the E12–E16-17 period of endogenous cochlear signaling is critical for neurotrophic support of central relay nuclei for guiding
normal developmental refinements in central binaural processing
pathways. After this period, the cochlear sensor acquires the ability
to perform ambient sound detection and encoding.
In our study, design of the oligonucleotides for the molecular identification of P2X and P2Y receptor subunits was made in
concordance with the results of the pharmacology used for electrophysiological recordings of the basal discharge from the afferent
neurons and according to the results obtained by other authors (3,
5, 9, 21, 22).
In the vestibular system of E15 and E18 we found an important
excitatory effect evoked with the P2X agonist ␣,␤-meATP. The RT-
F. Galindo et al. / Neuroscience Letters 553 (2013) 24–28
27
Fig. 2. Effect of a P2Y agonist (2MeSATP) and a P2Y antagonist (Reactive Blue 2) on the basal discharge of vestibular afferents at different developmental ages. (A)
2MeSATP dose-response-age curve fitting with the Hill equation yielded (E15) EC50 = 2.86 × 10−6 (pEC50 = 5.543 ± 0.1354); nH = 0.83 ± 0.09; (E18) EC50 = 161.8 × 10−6
(pEC50 = 3.791 ± 0.0789); nH = 1.03 ± 0.32; (E21) EC50 = 18.0 × 10−6 (pEC50 = 4.744 ± 0.08101); nH = 1.23 ± 0.23.The maximal responses for E15 (188.4 ± 3.301%) and E18
(182.9 ± 3.872%), were greater significantly than for E21 (134.2 ± 1.131%). On mature embryos (E18 and E21) only high concentrations of 2MeSATP (10−3 M) induced an
increase in basal discharge. Points are the means ± SEM and the line drawn is the best-fit curve obtained. (B) The effect of Reactive Blue 2 (10−4 M) is clearly observed at all
embryonic ages but particularly at E15 (*, p < 0.05). (C) Reactive Blue 2 (10−4 M) blockage of the excitatory effect of 2MeSATP (10−3 M; *, p < 0.05).
PCR assays showed the presence of mRNA encoding P2X3 , but not
for P2X1 and P2X2 (data no show). However, mRNA for P2Y receptors was also present. Consonant with this, incubation with a P2X
receptors agonist by microperfusion showed a stimulatory effect
on the discharge frequency.
The synaptogenesis process in the vestibular system has been
divided into three stages (presents at periphery and central level):
early synaptogenesis (E8–E9), mid-synaptogenesis (E11–E13), and
late synaptogenesis (E14–E17) [7,24]. Late synaptogenesis is
critical for the establishment of the final contacts between hair
cells and afferents as well as between the afferent neurons and
Fig. 3. Semi-quantitative analysis of P2X3 (384 bp), P2Y1 (370 bp), P2Y2 (402 bp)
and P2Y6 (544 bp) mRNA expression in the vestibular end organs and basilar papilla
of chick embryos at E15, E18 and E21. Each panel shows the amplified mRNA for
the corresponding receptor, bar graphs show the values of band intensity (arbitrary
units) normalized with respect to ␤-actin. Values are expressed as mean ± S.E.M.
(n = 4; * p < 0.05).
Fig. 4. Effect of ATP and P2Y agonists (2MeSATP and UTP) on the basal discharge of
auditory afferents at different developmental ages. ATP and both agonists induce (at
10−3 M concentration) an increase of the electrical activity at E15 and E18 (*, p < 0.05;
**, p < 0.01). All drugs were tested at lower concentrations but no significant effects
were observed.
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F. Galindo et al. / Neuroscience Letters 553 (2013) 24–28
the second order neurons into the vestibular nucleus, at central level. Possibly ATP is helping neurons to find and establish
the correct synapses at periphery and central level in the vestibular system. Consistent with this, our results in E21 showed
minimal changes, probably because the synaptic contacts are
established and the ATP role in this context may not be needed.
Similar findings were observed in the auditory system, where the
youngest animals responded better to the action of ATP. In the
basilar papilla, the most prominent increase in electrical activity
evoked by ATP and the P2X receptor and P2Y receptor agonists
was observed at E15, an important moment for the establishment of hearing function, and we did not observe any important
change in E18 and E21, periods where the hearing has started
already.
Regarding new possible roles for P2Y receptors, it is worth
mentioning that in supporting cells and epithelial cells some
experiments indicate that ATP, acting through a highly sensitive purinergic/IP3 -mediated signaling pathway, is the principal
paracrine mediator implicated in the propagation of Ca2+ waves
[23]. Cells of the outer sulcus participate in the absorption of
cations and it is possible that these cells are responsible for
maintaining the correct concentration of K+ ions [19]. The P2Y4
receptors are expressed in developing outer sulcus cells, and then
down-regulated in the adult [26]. Homeostasis of the luminal
fluid composition is also controlled by P2Y receptors in the apical membrane of strial marginal cells and vestibular dark cells
of rodents [20,22]. Moreover, P2Y1 receptor expression has been
demonstrated in several structures during chicken embryonic
development [5,21].
The present study revealed for the first time by RT-PCR the presence of P2X3 , P2Y1 , P2Y2 and P2Y6 – mRNA in the inner ear of
chicken embryos in three ages (E15, E18 and E21), with a temporal
variation in their expression patterns.
Our results suggest that ATP could to modulate the spontaneous
activity of the auditory and vestibular afferent fibers at different
embryonic developmental stages in the chicken inner ear. The P2X
receptors in the vestibule have a very important effect on the afferent discharge while P2Y receptors maintain a more discreet profile.
In the basilar papilla, only found the presence of P2Y receptors. In
both organs, the purinergic effects are more important in younger
animals probably because during defined moments in the embryonic development, ATP is able to help to establish synaptic contacts
or to maintain an early communication between hair cells and
primary afferents or may play an important role in signal transduction in the inner ear. Later, ATP may be substituted by other
substances like glutamate that probably persist during the adult
stage.
Acknowledgments
We thank Dr. Ricardo Félix for critical reading of the manuscript,
and Dr. Antonio Arias-Montaño for his help with data analysis.
This work was partly supported by PIFI-SEP 2012 and VIEP-BUAP
SAL/G/2013 grants to A.F.
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