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Acid-sensing ionic-channel functional expression in the vestibular endorgans
Neuroscience Letters 463 (2009) 199–202
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Acid-sensing ionic-channel functional expression in the vestibular endorgans Rosario Vega ∗ , Uxmal Rodríguez, Enrique Soto Instituto de Fisiología, Benemérita Universidad Autónoma de Puebla, Apartado Postal 406, Puebla, Pue. 72000, Mexico
a r t i c l e
i n f o
Article history: Received 13 June 2009 Received in revised form 29 July 2009 Accepted 31 July 2009 Keywords: Proton-gated currents ASIC Amiloride Acetylsalicylic acid FMRF-amide Afferent neurons Inner ear
a b s t r a c t In the vestibular system, the electrical discharge of the afferent neurons has been found to be highly sensitive to external pH changes, and acid-sensing ionic-channels (ASIC) have been found to be functionally expressed in afferent neurons. No previous attempt to assay the ASIC function in vestibular afferent neurons has been done. In our work we studied the electrical discharge of the afferent neuron of the isolated inner ear of the axolotl (Ambystoma tigrinum) to determine the participation of proton-gated currents in the postransductional information processing in the vestibular system. Microperfusion of FMRF-amide significantly increased the resting activity of the afferent neurons of the semicircular canal indicating that ASIC currents are tonically active in the resting condition. The use of ASIC antagonists, amiloride and acetylsalicylic acid (ASA), significantly reduced the vestibular-nerve discharge, corroborating the idea that the afferent neurons of the vestibular system express ASICs that are sensitive to amiloride, ASA, and to FMRF-amide. The sensitivity of the vestibular afferent-resting discharge to the microperfusion of ASIC acting agents indicates the participation of these currents in the establishment of the afferent-resting discharge. © 2009 Elsevier Ireland Ltd. All rights reserved.
The acid-sensing ion-channel (ASIC) has been shown to participate in the tissue response to acidity, inflammation, and ischemia. The ASICs also seem to participate in the sensory coding of various sensory modalities, such as the sensation of sour taste, pain, and touch. ASICs have also been shown to participate in synaptic transmission and plasticity [15,8,7], and their activation has a significant role in the physiopathology of ischemic brain injury, the damage to the nervous system produced by inflammatory processes such as in multiple sclerosis, and also in epilepsy [4,33,17,10]. The ASICs have been found to be sensitive to amiloride, to the nonsteroid antiinflammatory drugs, to low concentrations of Zn2+ , to the lanthanide Gd3+ , and to the peptidic toxins psalmotoxin1 and APTx2 that selectively block the ASIC1a and the ASIC3 subunits. The ASICs are coactivated by high concentrations of Zn2+ (>100 M) and by the snail neuropeptide FMRF-amide that inhibits their desensitization (reviewed by Xiong et al. [35]). Recently an antagonistic action of aminoglycosides on the ASIC currents in dorsal root-ganglion neurons has also been shown [30]. In the vestibular system, the electrical discharge of the afferent neurons has been found to be highly sensitive to external pH changes [31]. The ASIC1b and ASIC4 subunits have been cloned and identified by RT-PCR from the rat vestibular apparatus [6,11]. Voltage clamp of vestibular afferent neurons from rats showed
∗ Corresponding author. E-mail addresses: [email protected], [email protected], [email protected], axolotl [email protected] (R. Vega). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.07.086
expression of an ASIC current sensitive to amiloride, gadolinium, lead, and acetylsalicylic acid. The ASIC current in vestibular afferent neurons was enhanced by FMRF-amide and zinc, and the functional expression of the current was correlated with smaller-capacitance neurons [22]. Acidification of the extracellular pH generated action potentials in vestibular neurons, suggesting a functional role of ASICs in their excitability. Immunoreactivity to ASIC1a and ASIC2a subunits was found in small vestibular ganglion neurons and afferent fibers that run throughout the macula utricle and crista stroma. The ASIC2b, ASIC3, and ASIC4 were expressed to a lesser degree in vestibular ganglion neurons. The ASIC1b subunit was not detected in the vestibular endorgans and no acid pH-sensitive currents or ASIC immunoreactivity was found in hair cells [22]. No previous attempt to assay the ASIC function in vestibular afferent neurons has been done. In our work we studied the action of FMRF-amide, acetylsalicylic acid (ASA) amiloride, and Gd3+ on the electrical activity of vestibular afferent neurons in the isolated inner ear of the axolotl (Ambystoma tigrinum) to determine the participation of proton-gated currents in the postransductional information processing in the vestibular system. Animal care and procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Experiments were made on wild larval axolotl (A. tigrinum, 30–60-g body weight). The animals were anesthetized by immersion in 3aminobenzoic acid ethyl ester (0.1% in water) and subsequently decapitated. The otic capsule was opened ventrally and the inner ear structures identified as previously reported [27]. Briefly, the nerve fibers originating from the lateral canal were dissected and
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R. Vega et al. / Neuroscience Letters 463 (2009) 199–202
the cartilaginous otic capsule, including the whole vestibular system, was cut out and isolated from the skull. The isolated vestibule was transferred to a recording chamber and continuously perfused with Ringer solution of the following composition (in mM): KCl 2.5, NaCl 111, CaCl2 1.8, MgCl2 1, glucose 10, HEPES 5, pH 7.4. Multiunit extracellular recordings were obtained from the lateral semicircular canal nerve using a suction electrode. Electrical activity, amplified by means of a conventional AC amplifier (P-15, Grass Inst., Boston, MA), was filtered at cut-off frequencies of 100 and 3000 Hz and monitored in an oscilloscope (Tektronix, Beaverton, OR). The signal was also led to a window discriminator (Model 121, WPI, Sarasota, FL), the output of which was connected to a microcomputer for on-line analysis of the discharge rate [28]. Drugs were applied by bath perfusion or by microperfusion as indicated in each case. For bath application, the desired drug concentration was added to the perfusion Ringer solution and the recording chamber bath was completely replaced using a rapid exchange suction-perfusion system. For microperfusion 20 L of the drug were pressure ejected in 2 s from a micropipette (100m tip diameter, flow rate 10 L/s) positioned near (>500 m) the origin of the afferent fibers. Concentrations given herein are those originally in the pipette. Because the bath volume was 2 mL, the drug concentration decays exponentially to about 1% of its original value in a few milliseconds [32]. No drug was tested unless the basal discharge was stable. When more than one drug was used, sufficient time (at least 10 min) was allowed to wash off the previous drug, and no drug was used until the basal discharge level returned to the control level. The data are expressed as the mean ± the standard error. Recordings of the electrical discharge of the semicircular canal neurons were reliably obtained from the isolated inner ear of the axolotl. To corroborate the potential role of ASICs in the generation of the basal discharge in the vestibular afferent neurons, we used FMRF-amide, which has been shown to decrease the desensitization rate of ASIC currents in vestibular afferent neurons [22]. Microperfusion of 1 mM FMRF-amide (n = 58) significantly increased the resting activity of the semicircular canal afferent
neurons 57 ± 7% (paired Student’s t-test P = 0.00004) (Fig. 1). To determine whether the FMRF-amide action was produced on already active ASICs, the influence of pH on the response to FMRFamide was studied. The preparations were bathed in extracellular saline solution with pHs 8.8 and 6.0. Microperfusion of 1 mM FMRFamide (20 L) at pH 6.0 (n = 11) produced a significant 65 ± 32% excitatory effect in 5 out of 11 experiments (Fig. 1). In contrast, the microapplication of 1 mM FMRF-amide while the preparation was being bathed with a pH 8.8 Ringer solution (n = 10) produced an increase of 36 ± 10% of the resting discharge of the semicircular canal afferent neurons (paired Student’s t-test P = 0.0008), thus indicating that most probably the action of the FMRF-amide is dependent on the state of the ASIC channel. To further corroborate the potential role of ASICs in the vestibular afferent neurons, the action of the ASIC antagonists Gd3+ , amiloride, and acetylsalicylic acid were studied. The bath perfusion of 100 M amiloride (n = 7) significantly decreased the resting discharge of the afferent neurons of the semicircular canal to 84 ± 5% of the control value after a 5-min perfusion (paired Student’s t-test P = 0.034) (Fig. 2A). Bath perfusion of 1 mM acetylsalicylic acid (n = 7) produced a significant slowly developing inhibition of the resting discharge of the afferent neurons of the semicircular canal after a 10-min bath perfusion, a time at which discharge was 69 ± 11% of the control value (paired Student’s t-test P = 0.035) (Fig. 2B). The GdCl3 (100 M) was perfused in the bath solution (n = 4) during 30 min. The resting discharge of the afferent neurons was 99 ± 6% after 5-min bathing with Gd3+ , 109 ± 6% at 10 min, and 72 ± 12% after 20 min. Although there was an inhibitory effect, this was not significant (one-way ANOVA test; data not shown). Our results corroborate the idea that the afferent neurons of the vestibular system express ASICs that are sensitive to amiloride, ASA, and to FMRF-amide. The sensitivity of the vestibular afferentresting discharge to the microperfusion of FMRF-amide is relevant. It is known that FMRF-amide by itself cannot activate the ASIC currents, but FMRF-amide has been shown to modify the ASIC current desensitization [3,34]. Thus, the action of FMRF-amide shows that
Fig. 1. Influence of the pH on the action FMRF-amide. In (A–C) frequency versus time graphs of the vestibular afferent neuron discharge rate. After 2 min of control record, 1 mM FMRF-amide microperfusion produced an increase of the afferent neuron basal discharge. The amplitude of FMRF-amide action was modulated by the extracellular pH (indicated in each graph). In (D) bar graph showing the pH dependence of FMRF-amide excitatory effect on the vestibular afferent basal discharge rate. Results are expressed as mean ± SE.
R. Vega et al. / Neuroscience Letters 463 (2009) 199–202
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Fig. 2. Actions of amiloride and ASA. In (A) frequency versus time plot showing a typical record. After 2 min of control activity, 100 M amiloride was perfused into the bath. In this experiment, afferent discharge decreased from a mean of 83 impulses per second (ips) to 51 ips after 10 min. In (B) a typical record of the 1 mM ASA action. In this experiment, afferent discharge decreased from a mean of 23 ips to 13 ips after 10 min. Results are expressed as mean ± SE.
ASIC channels are tonically active in the vestibular afferent neurons and participate in the generation of its basal discharge. The advantage of using the amphibian preparation for the recording of the electrical discharge of the vestibular afferent neurons, resides in the long time and stable recordings of the afferent discharge that may be obtained [27,29,31], allowing for extensive manipulation such as stable pH changes in the bathing media. It is worth to note that amphibians lack type-I hair cells, and in consequence data obtained refer exclusively to type-II hair cell systems. The reduced action of the FMRF-amide at pH 8.8 is in agreement with the idea of a tonic activation of the ASIC channels that would be reduced at this pH. It should be noted that the binding of the FMRF-amide to the ASIC channels has a pH dependence, reducing its affinity at lower pHs [4,25]. The lack of effect of FMRF-amide at pH 6.0 in some experiments may be due to a desensitization of ASICs due to the sustained acid perfusion. The most probable source of activity for the ASIC channels is an acidification of intersynaptic space by the sustained release of glutamate from the hair cells. Although there is no direct demonstration of an acidification of the synaptic space in the hair cell basal pole, it is known that some homomeric ASIC 3 and 1a and heteromeric combinations of ASICs mostly of ASIC3, 2a and b, and 1a, have activation thresholds above pH 7 [14]. The possibility that the FMRF-amide can interact with receptors of the RF-amide-peptides cannot be discarded [13,21]. This family of receptors has been described all along the evolutionary scale [9]. In the Ambystoma mexicanum it has been shown that FMRF-amide modulates olfactory responses [26], which implies that FMRF-amide or a similar RF-amide peptide is released from the hair cells. Thus the expression of RF-amide receptors in the afferent neurons mediating the observed actions of FMRF-amide in our system should be further investigated. Amiloride has been shown to block the channels of the Deg-EnaC family, Ca channels, Na+ –H+ , Na+ –Ca2+ , and diverse membrane ATPases [24]. Although the inhibitory action of amiloride on the afferent discharge coincides with a potential blockade of ASIC channels, it is not possible to discard other actions producing this effect, such as a presynaptic calcium-channel block leading to a reduction in neurotransmitter release. Thus amiloride actions should only be considered as indicative but not direct evidence of ASIC participation in the afferent activity. The inhibitory action of ASA on the discharge rate of the vestibular afferent neurons is also indicative of the ASIC participation on the basal discharge, although the ASA inhibits the cyclooxigenase that can lead to an increase in the arachidonic acid concentration and subsequent increase in the ASIC current [1]. It also has been shown to increase the NMDA receptor activity [12]. These actions tend to increase the afferent discharge, not to decrease it as we found. Salicylates can also block L-type calcium channels,
and sodium and potassium channels, producing a decrease in the afferent activity [18–20]. In consequence the ASA actions should also be considered as another indication of ASIC participation in the afferent activity. The non-significant inhibitory effect of gadolinium could be due to the characteristics of the ASIC subunits expressed in the axolotl inner ear and to the complex biological actions of gadolinium. Gadolinium ions inhibited ASIC3 and ASIC2a + ASIC3 responses with much higher potency IC50 approximately 40 M than the ASIC2a response IC50 ≥ 1 mM [5]. Also it is worth to note that gadolinium has additional actions on other membrane proteins such as the AMPA receptors, upon which gadolinium exerts an excitatory action [16]. The AMPA receptors have been shown to mediate an excitatory input to the afferent neurons [29], thus gadolinium may exert both excitatory and inhibitory actions on the discharge of the vestibular afferent neurons. The ASIC current may contribute to postsynaptic depolarization and facilitate NMDA receptor activation [33]. It has been shown that hair cell afferent transmission is glutamatergic [29] and that NMDA receptors participate in the activation of the afferent neurons [27]. Glutamatergic synaptic vesicles have been shown to have an acidic pH of about 5.8 [23]. Thus protons are coreleased with glutamate and may activate ASICs, depolarizing the afferent neurons and facilitating the NMDA receptor activation. At the same time, protons may limit the activation of glutamate receptors [31] and limit the presynaptic calcium current in the hair cells [2]. The precise action of protons in the afferent-receptoneural junction in hair cells is complex. The data suggest that it is essentially a boosting mechanism that increases the amplitude of the synaptic response but limits its duration. Acknowledgments The authors wish to thank Dr. Ellis Glazier for editing this English-language manuscript. This research was partially supported by National Council of Science and Technology of México (CONACyT grant 46511) to E.S., and Vicerrectoria de Investigación (VIEP-BUAP)-CONACyT grants 20/SAL06-G and 20/SAL06-I to RV and ES. References [1] N.J. Allen, D. Attwell, Modulation of ASIC channel in rat cerebellar Purkinje neurons by ischaemia-related signals, J. Physiol. 543 (2002) 521–529. [2] A. Almanza, F. Mercado, R. Vega, E. Soto, Extracellular pH modulates the voltagedependent Ca2+ current and low threshold K+ current in hair cells, Neurochem. Res. 33 (2007) 1435–1441. [3] C.C. Askwith, C. Cheng, M. Ikuma, C. Benson, M.P. Price, M.J. Welsh, Neuropeptide FF and FMRFamide potentiate acid-evoked currents from sensory neurons and proton-gated DEG/ENaC channels, Neuron 26 (2000) 133–141.
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[4] C.C. Askwith, C.J. Benson, M. Welsh, P.M. Snyder, DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 6459–6463. [5] K. Babinski, S. Catarsi, G. Biagini, P. Séguéla, Mammalian ASIC2a and ASIC3 subunits co-assemble into heteromeric proton-gated channels sensitive to Gd3+ , J. Biol. Chem. 275 (2000) 28519–28525. [6] E.L. Bässler, T.J. Ngo-Anh, H.S. Geisler, J.P. Ruppersberg, S. Gründer, Molecular and functional characterization of acid-sensing ion channel (ASIC) 1b, J. Biol. Chem. 276 (2001) 33782–33787. [7] A.A. Beg, G.G. Ernstrom, P. Nix, M.W. Davis, E.M. Jorgensen, Protons act as a transmitter for muscle contraction in C. elegans, Cell 132 (2008) 149–160. [8] L. Bianchi, M. Driscoll, Protons at the gate: DEG/ENaC ion channels help us feel and remember, Neuron 34 (2002) 337–340. [9] G.J. Dockray, The expanding family of RFamide peptides and their effects on feeding behaviour, Exp. Physiol. 89 (2004) 229–235. [10] M.A. Friese, M.J. Craner, R. Etzensperger, S. Vergo, J.A. Wemmie, M.J. Welsh, A. Vincent, L. Fugger, Acid-sensing ion channel-1 contributes to axonal degeneration in autoimmune inflammation of the central nervous system, Nat. Med. 13 (12) (2007) 1483–1489. [11] S. Gründer, H.S. Geissler, E.L. Bässler, J.P. Ruppersberg, A new member of acidsensing ion channels from pituitary gland, Neuroreport 11 (2000) 1607–1611. [12] M.J. Guitton, J. Caston, J. Ruel, R.M. Johnson, R. Pujol, J.L. Puel, Salicylates induce tinnitus through activation of cochlear NMDA receptor, J. Neurosci. 23 (2003) 3944–3952. [13] S.K. Han, X. Dong, J.I. Hwang, M.J. Zylka, D.J. Anderson, M.I. Simon, Orphan G protein-coupled receptors MrgA1 and MrgC11 are distinctively by RFamiderelated peptides through the G␣q/11 pathway, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 14740–14745. [14] M. Hesselager, D.B. Timmermann, P.K. Ahring, pH dependency and desensitization kinetics of heterologously expressed combinations of acid-sensing ion channel subunits, J. Biol. Chem. 279 (2004) 11006–11015. [15] S. Kellenberger, L. Schild, Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure, Physiol. Rev. 82 (2002) 735–767. [16] S. Lei, J.F. Macdonald, Gadolinium reduces AMPA receptor desensitization and deactivation in hippocampal neurons, J. Neurophysiol. 86 (2001) 173–182. [17] E. Lingueglia, Acid-sensing ion channels in sensory perception, J. Biol. Chem. 282 (2007) 17325–17329. [18] Y. Liu, X. Li, Effects of salicylate on voltage-gated sodium channels in rat inferior colliculus neurons, Hear. Res. 193 (2004) 68–74. [19] Y. Liu, X. Li, Effects of salicylate on transient outward and delayed rectifier potassium channels in rat inferior colliculus neurons, Neurosci. Lett. 369 (2004) 115–120. [20] Y. Liu, X. Li, C. Ma, J. Liu, H. Lu, Salicylate blocks L-type calcium channels in rat inferior colliculus neurons, Hear. Res. 205 (2005) 271–276.
[21] T. Meeusen, I. Mertens, E. Clyden, G. Baggerman, R. Nichols, R.J. Nachman, R. Huybrechts, A. De Loof, L. Schoofs, Identification in Drosophila melanogaster of the invertebrate G protein-coupled FMRFamide receptor, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 15363–15368. [22] F. Mercado, I.A. Lopez, D. Acuna, R. Vega, E. Soto, Acid-sensing ionic channels in the rat vestibular endorgans and ganglia, J. Neurophysiol. 96 (2006) 1615–1624. [23] G. Miesenbock, D.A. De Angelis, J.E. Rothman, Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins, Nature 394 (1998) 192–195. [24] Y. Murata, K. Harada, F. Nakajima, J. Maruo, T. Morita, Non-selective effects of the amiloride and its analogues on ion transport systems and their cytotoxicities in cardiac myocitos, Jpn. J. Pharmacol. 68 (1995) 279–285. [25] O. Ostrovskaya, L. Moroz, O. Krishtal, Modulatory action of RFamide-related peptides on acid-sensing ionic channels is pH dependent: the role of arginine, J. Neurochem. 91 (2004) 252–255. [26] D. Park, S.R. Zawacki, H.L. Eisthen, Olfatory signal by molluscan tetrapeptide (FMRFamide) in axolotls (Ambystoma mexicanum), Chem. Senses 28 (2003) 339–348. [27] E. Soto, A. Flores, C. Erostegui, R. Vega, Evidence for NMDA receptor in the afferent synaptic transmission of the vestibular system, Brain Res. 633 (1994) 289–296. [28] E. Soto, E. Manjarrez, R. Vega, A microcomputer based expert system for neuronal spike train data analysis, Int. J. Med. Informat. 44 (1997) 203–212. [29] E. Soto, R. Vega, Actions of excitatory aminoacid agonists and antagonists on the primary afferents of the vestibular system of the axolotl (Ambystoma mexicanum), Brain Res. 462 (1988) 104–111. [30] E. Soto, A. Garza, O. Ramirez, R. Vega, Aminoglycosides block of the acid-sensing ionic-channel (ASIC) in dorsal-root ganglion neurons from the rat, Soc. Nsci. Abstr. 39 (2009) (abstract 7127). [31] R. Vega, F. Mercado, H. Chávez, A. Limón, A. Almanza, A. Ortega, M.E. Perez, E. Soto, pH modulate the vestibular afferent discharge and its response to excitatory amino acids, Neuroreport 14 (2003) 1327–1328. [32] D.R. Waud, On diffusion from a point source, J. Pharmacol. Exp. Ther. 159 (1968) 123–128. [33] J.A. Wemmie, J. Chen, C.C. Askwith, A.M. Hruska-Hageman, M.P. Price, B.C. Nolan, P.G. Yoder, E. Lamani, T. Hoshi, J.H. Freeman Jr., M.J. Welsh, The acidactivated ion channel ASIC contributes to synaptic plasticity, learning, and memory, Neuron 34 (2002) 463–477. [34] J. Xie, M.P. Price, J.A. Wemmie, C.C. Askwith, M.J. Welsh, ASIC3 and ASIC1 mediate FMRFamide-related peptide enhancement of H+ -gated currents in cultured dorsal root ganglion neurons, J. Neurophysiol. 89 (2003) 2459–2465. [35] Z.G. Xiong, G. Pignataro, M. Li, S.Y. Chang, R.P. Simon, Acid-sensing ion channels (ASICs) as pharmacological targets for neurodegenerative diseases, Curr. Opin. Pharmacol. 8 (2008) 25–32.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Acid-sensing ionic-channel functional expression in the vestibular endorgans Rosario Vega ∗ , Uxmal Rodríguez, Enrique Soto Instituto de Fisiología, Benemérita Universidad Autónoma de Puebla, Apartado Postal 406, Puebla, Pue. 72000, Mexico
a r t i c l e
i n f o
Article history: Received 13 June 2009 Received in revised form 29 July 2009 Accepted 31 July 2009 Keywords: Proton-gated currents ASIC Amiloride Acetylsalicylic acid FMRF-amide Afferent neurons Inner ear
a b s t r a c t In the vestibular system, the electrical discharge of the afferent neurons has been found to be highly sensitive to external pH changes, and acid-sensing ionic-channels (ASIC) have been found to be functionally expressed in afferent neurons. No previous attempt to assay the ASIC function in vestibular afferent neurons has been done. In our work we studied the electrical discharge of the afferent neuron of the isolated inner ear of the axolotl (Ambystoma tigrinum) to determine the participation of proton-gated currents in the postransductional information processing in the vestibular system. Microperfusion of FMRF-amide significantly increased the resting activity of the afferent neurons of the semicircular canal indicating that ASIC currents are tonically active in the resting condition. The use of ASIC antagonists, amiloride and acetylsalicylic acid (ASA), significantly reduced the vestibular-nerve discharge, corroborating the idea that the afferent neurons of the vestibular system express ASICs that are sensitive to amiloride, ASA, and to FMRF-amide. The sensitivity of the vestibular afferent-resting discharge to the microperfusion of ASIC acting agents indicates the participation of these currents in the establishment of the afferent-resting discharge. © 2009 Elsevier Ireland Ltd. All rights reserved.
The acid-sensing ion-channel (ASIC) has been shown to participate in the tissue response to acidity, inflammation, and ischemia. The ASICs also seem to participate in the sensory coding of various sensory modalities, such as the sensation of sour taste, pain, and touch. ASICs have also been shown to participate in synaptic transmission and plasticity [15,8,7], and their activation has a significant role in the physiopathology of ischemic brain injury, the damage to the nervous system produced by inflammatory processes such as in multiple sclerosis, and also in epilepsy [4,33,17,10]. The ASICs have been found to be sensitive to amiloride, to the nonsteroid antiinflammatory drugs, to low concentrations of Zn2+ , to the lanthanide Gd3+ , and to the peptidic toxins psalmotoxin1 and APTx2 that selectively block the ASIC1a and the ASIC3 subunits. The ASICs are coactivated by high concentrations of Zn2+ (>100 M) and by the snail neuropeptide FMRF-amide that inhibits their desensitization (reviewed by Xiong et al. [35]). Recently an antagonistic action of aminoglycosides on the ASIC currents in dorsal root-ganglion neurons has also been shown [30]. In the vestibular system, the electrical discharge of the afferent neurons has been found to be highly sensitive to external pH changes [31]. The ASIC1b and ASIC4 subunits have been cloned and identified by RT-PCR from the rat vestibular apparatus [6,11]. Voltage clamp of vestibular afferent neurons from rats showed
∗ Corresponding author. E-mail addresses: [email protected], [email protected], [email protected], axolotl [email protected] (R. Vega). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.07.086
expression of an ASIC current sensitive to amiloride, gadolinium, lead, and acetylsalicylic acid. The ASIC current in vestibular afferent neurons was enhanced by FMRF-amide and zinc, and the functional expression of the current was correlated with smaller-capacitance neurons [22]. Acidification of the extracellular pH generated action potentials in vestibular neurons, suggesting a functional role of ASICs in their excitability. Immunoreactivity to ASIC1a and ASIC2a subunits was found in small vestibular ganglion neurons and afferent fibers that run throughout the macula utricle and crista stroma. The ASIC2b, ASIC3, and ASIC4 were expressed to a lesser degree in vestibular ganglion neurons. The ASIC1b subunit was not detected in the vestibular endorgans and no acid pH-sensitive currents or ASIC immunoreactivity was found in hair cells [22]. No previous attempt to assay the ASIC function in vestibular afferent neurons has been done. In our work we studied the action of FMRF-amide, acetylsalicylic acid (ASA) amiloride, and Gd3+ on the electrical activity of vestibular afferent neurons in the isolated inner ear of the axolotl (Ambystoma tigrinum) to determine the participation of proton-gated currents in the postransductional information processing in the vestibular system. Animal care and procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Experiments were made on wild larval axolotl (A. tigrinum, 30–60-g body weight). The animals were anesthetized by immersion in 3aminobenzoic acid ethyl ester (0.1% in water) and subsequently decapitated. The otic capsule was opened ventrally and the inner ear structures identified as previously reported [27]. Briefly, the nerve fibers originating from the lateral canal were dissected and
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the cartilaginous otic capsule, including the whole vestibular system, was cut out and isolated from the skull. The isolated vestibule was transferred to a recording chamber and continuously perfused with Ringer solution of the following composition (in mM): KCl 2.5, NaCl 111, CaCl2 1.8, MgCl2 1, glucose 10, HEPES 5, pH 7.4. Multiunit extracellular recordings were obtained from the lateral semicircular canal nerve using a suction electrode. Electrical activity, amplified by means of a conventional AC amplifier (P-15, Grass Inst., Boston, MA), was filtered at cut-off frequencies of 100 and 3000 Hz and monitored in an oscilloscope (Tektronix, Beaverton, OR). The signal was also led to a window discriminator (Model 121, WPI, Sarasota, FL), the output of which was connected to a microcomputer for on-line analysis of the discharge rate [28]. Drugs were applied by bath perfusion or by microperfusion as indicated in each case. For bath application, the desired drug concentration was added to the perfusion Ringer solution and the recording chamber bath was completely replaced using a rapid exchange suction-perfusion system. For microperfusion 20 L of the drug were pressure ejected in 2 s from a micropipette (100m tip diameter, flow rate 10 L/s) positioned near (>500 m) the origin of the afferent fibers. Concentrations given herein are those originally in the pipette. Because the bath volume was 2 mL, the drug concentration decays exponentially to about 1% of its original value in a few milliseconds [32]. No drug was tested unless the basal discharge was stable. When more than one drug was used, sufficient time (at least 10 min) was allowed to wash off the previous drug, and no drug was used until the basal discharge level returned to the control level. The data are expressed as the mean ± the standard error. Recordings of the electrical discharge of the semicircular canal neurons were reliably obtained from the isolated inner ear of the axolotl. To corroborate the potential role of ASICs in the generation of the basal discharge in the vestibular afferent neurons, we used FMRF-amide, which has been shown to decrease the desensitization rate of ASIC currents in vestibular afferent neurons [22]. Microperfusion of 1 mM FMRF-amide (n = 58) significantly increased the resting activity of the semicircular canal afferent
neurons 57 ± 7% (paired Student’s t-test P = 0.00004) (Fig. 1). To determine whether the FMRF-amide action was produced on already active ASICs, the influence of pH on the response to FMRFamide was studied. The preparations were bathed in extracellular saline solution with pHs 8.8 and 6.0. Microperfusion of 1 mM FMRFamide (20 L) at pH 6.0 (n = 11) produced a significant 65 ± 32% excitatory effect in 5 out of 11 experiments (Fig. 1). In contrast, the microapplication of 1 mM FMRF-amide while the preparation was being bathed with a pH 8.8 Ringer solution (n = 10) produced an increase of 36 ± 10% of the resting discharge of the semicircular canal afferent neurons (paired Student’s t-test P = 0.0008), thus indicating that most probably the action of the FMRF-amide is dependent on the state of the ASIC channel. To further corroborate the potential role of ASICs in the vestibular afferent neurons, the action of the ASIC antagonists Gd3+ , amiloride, and acetylsalicylic acid were studied. The bath perfusion of 100 M amiloride (n = 7) significantly decreased the resting discharge of the afferent neurons of the semicircular canal to 84 ± 5% of the control value after a 5-min perfusion (paired Student’s t-test P = 0.034) (Fig. 2A). Bath perfusion of 1 mM acetylsalicylic acid (n = 7) produced a significant slowly developing inhibition of the resting discharge of the afferent neurons of the semicircular canal after a 10-min bath perfusion, a time at which discharge was 69 ± 11% of the control value (paired Student’s t-test P = 0.035) (Fig. 2B). The GdCl3 (100 M) was perfused in the bath solution (n = 4) during 30 min. The resting discharge of the afferent neurons was 99 ± 6% after 5-min bathing with Gd3+ , 109 ± 6% at 10 min, and 72 ± 12% after 20 min. Although there was an inhibitory effect, this was not significant (one-way ANOVA test; data not shown). Our results corroborate the idea that the afferent neurons of the vestibular system express ASICs that are sensitive to amiloride, ASA, and to FMRF-amide. The sensitivity of the vestibular afferentresting discharge to the microperfusion of FMRF-amide is relevant. It is known that FMRF-amide by itself cannot activate the ASIC currents, but FMRF-amide has been shown to modify the ASIC current desensitization [3,34]. Thus, the action of FMRF-amide shows that
Fig. 1. Influence of the pH on the action FMRF-amide. In (A–C) frequency versus time graphs of the vestibular afferent neuron discharge rate. After 2 min of control record, 1 mM FMRF-amide microperfusion produced an increase of the afferent neuron basal discharge. The amplitude of FMRF-amide action was modulated by the extracellular pH (indicated in each graph). In (D) bar graph showing the pH dependence of FMRF-amide excitatory effect on the vestibular afferent basal discharge rate. Results are expressed as mean ± SE.
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Fig. 2. Actions of amiloride and ASA. In (A) frequency versus time plot showing a typical record. After 2 min of control activity, 100 M amiloride was perfused into the bath. In this experiment, afferent discharge decreased from a mean of 83 impulses per second (ips) to 51 ips after 10 min. In (B) a typical record of the 1 mM ASA action. In this experiment, afferent discharge decreased from a mean of 23 ips to 13 ips after 10 min. Results are expressed as mean ± SE.
ASIC channels are tonically active in the vestibular afferent neurons and participate in the generation of its basal discharge. The advantage of using the amphibian preparation for the recording of the electrical discharge of the vestibular afferent neurons, resides in the long time and stable recordings of the afferent discharge that may be obtained [27,29,31], allowing for extensive manipulation such as stable pH changes in the bathing media. It is worth to note that amphibians lack type-I hair cells, and in consequence data obtained refer exclusively to type-II hair cell systems. The reduced action of the FMRF-amide at pH 8.8 is in agreement with the idea of a tonic activation of the ASIC channels that would be reduced at this pH. It should be noted that the binding of the FMRF-amide to the ASIC channels has a pH dependence, reducing its affinity at lower pHs [4,25]. The lack of effect of FMRF-amide at pH 6.0 in some experiments may be due to a desensitization of ASICs due to the sustained acid perfusion. The most probable source of activity for the ASIC channels is an acidification of intersynaptic space by the sustained release of glutamate from the hair cells. Although there is no direct demonstration of an acidification of the synaptic space in the hair cell basal pole, it is known that some homomeric ASIC 3 and 1a and heteromeric combinations of ASICs mostly of ASIC3, 2a and b, and 1a, have activation thresholds above pH 7 [14]. The possibility that the FMRF-amide can interact with receptors of the RF-amide-peptides cannot be discarded [13,21]. This family of receptors has been described all along the evolutionary scale [9]. In the Ambystoma mexicanum it has been shown that FMRF-amide modulates olfactory responses [26], which implies that FMRF-amide or a similar RF-amide peptide is released from the hair cells. Thus the expression of RF-amide receptors in the afferent neurons mediating the observed actions of FMRF-amide in our system should be further investigated. Amiloride has been shown to block the channels of the Deg-EnaC family, Ca channels, Na+ –H+ , Na+ –Ca2+ , and diverse membrane ATPases [24]. Although the inhibitory action of amiloride on the afferent discharge coincides with a potential blockade of ASIC channels, it is not possible to discard other actions producing this effect, such as a presynaptic calcium-channel block leading to a reduction in neurotransmitter release. Thus amiloride actions should only be considered as indicative but not direct evidence of ASIC participation in the afferent activity. The inhibitory action of ASA on the discharge rate of the vestibular afferent neurons is also indicative of the ASIC participation on the basal discharge, although the ASA inhibits the cyclooxigenase that can lead to an increase in the arachidonic acid concentration and subsequent increase in the ASIC current [1]. It also has been shown to increase the NMDA receptor activity [12]. These actions tend to increase the afferent discharge, not to decrease it as we found. Salicylates can also block L-type calcium channels,
and sodium and potassium channels, producing a decrease in the afferent activity [18–20]. In consequence the ASA actions should also be considered as another indication of ASIC participation in the afferent activity. The non-significant inhibitory effect of gadolinium could be due to the characteristics of the ASIC subunits expressed in the axolotl inner ear and to the complex biological actions of gadolinium. Gadolinium ions inhibited ASIC3 and ASIC2a + ASIC3 responses with much higher potency IC50 approximately 40 M than the ASIC2a response IC50 ≥ 1 mM [5]. Also it is worth to note that gadolinium has additional actions on other membrane proteins such as the AMPA receptors, upon which gadolinium exerts an excitatory action [16]. The AMPA receptors have been shown to mediate an excitatory input to the afferent neurons [29], thus gadolinium may exert both excitatory and inhibitory actions on the discharge of the vestibular afferent neurons. The ASIC current may contribute to postsynaptic depolarization and facilitate NMDA receptor activation [33]. It has been shown that hair cell afferent transmission is glutamatergic [29] and that NMDA receptors participate in the activation of the afferent neurons [27]. Glutamatergic synaptic vesicles have been shown to have an acidic pH of about 5.8 [23]. Thus protons are coreleased with glutamate and may activate ASICs, depolarizing the afferent neurons and facilitating the NMDA receptor activation. At the same time, protons may limit the activation of glutamate receptors [31] and limit the presynaptic calcium current in the hair cells [2]. The precise action of protons in the afferent-receptoneural junction in hair cells is complex. The data suggest that it is essentially a boosting mechanism that increases the amplitude of the synaptic response but limits its duration. Acknowledgments The authors wish to thank Dr. Ellis Glazier for editing this English-language manuscript. This research was partially supported by National Council of Science and Technology of México (CONACyT grant 46511) to E.S., and Vicerrectoria de Investigación (VIEP-BUAP)-CONACyT grants 20/SAL06-G and 20/SAL06-I to RV and ES. References [1] N.J. Allen, D. Attwell, Modulation of ASIC channel in rat cerebellar Purkinje neurons by ischaemia-related signals, J. Physiol. 543 (2002) 521–529. [2] A. Almanza, F. Mercado, R. Vega, E. Soto, Extracellular pH modulates the voltagedependent Ca2+ current and low threshold K+ current in hair cells, Neurochem. Res. 33 (2007) 1435–1441. [3] C.C. Askwith, C. Cheng, M. Ikuma, C. Benson, M.P. Price, M.J. Welsh, Neuropeptide FF and FMRFamide potentiate acid-evoked currents from sensory neurons and proton-gated DEG/ENaC channels, Neuron 26 (2000) 133–141.
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