- Email: [email protected]
GABA-mediated modulation of ATP-induced intracellular calcium responses in nodose ganglion neurons of the rat
Neuroscience Letters 584 (2015) 168–172
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
GABA-mediated modulation of ATP-induced intracellular calcium responses in nodose ganglion neurons of the rat Takuya Yokoyama a,b , Shou Fukuzumi a , Hitomi Hayashi a , Nobuaki Nakamuta a,b , Yoshio Yamamoto a,b,∗ a b
Laboratory of Veterinary Anatomy and Cell Biology, Faculty of Agriculture, Iwate University, Morioka, Japan Department of Basic Veterinary Science, United Graduate School of Veterinary Science, Gifu University, Gifu, Japan
h i g h l i g h t s • • • • •
ATP increased intracellular Ca2+ levels in nodose ganglion neurons via P2X receptor. ATP increased intracellular Ca2+ levels in satellite cells via P2Y receptor. GABA inhibited ATP-induced intracellular Ca2+ responses in isolated neurons. Bicuculline enhanced ATP-induced Ca2+ responses in neurons with satellite cells. Neuronal excitability may be modulated by GABA from satellite cells in nodose ganglion.
a r t i c l e
i n f o
Article history: Received 16 September 2014 Received in revised form 6 October 2014 Accepted 7 October 2014 Available online 17 October 2014 Keywords: Nodose ganglion Sensory neuron Satellite cell ATP GABA
a b s t r a c t We examined ATP-induced intracellular Ca2+ ([Ca2+ ]i ) responses in the neurons and satellite cells from one of the viscerosensory ganglia, the nodose ganglion (NG), as well as the GABA-mediated modulation of ATPinduced neuronal [Ca2+ ]i responses using intracellular calcium imaging. In neurons with satellite cells, ATP induced [Ca2+ ]i increases in both the neurons and satellite cells. The P2X receptor agonist, ␣,-meATP, induced [Ca2+ ]i increases in neurons and this response was inhibited by the P2X receptor antagonist, PPADS. On the other hand, the P2Y receptor agonist, ADP, induced [Ca2+ ]i increases in satellite cells, and this response was inhibited by the P2Y receptor antagonist, MRS2179. RT-PCR detected the expression of P2X2, P2X3, P2Y1, and P2Y2 receptor mRNAs in NG extracts. Immunohistochemistry revealed that NG neurons and satellite cells were immunoreactive to P2X2 and P2X3, and P2Y1 and P2Y2 receptors, respectively. In isolated neurons, the ATP-evoked [Ca2+ ]i increase was inhibited by GABA. However, in neurons with satellite cells, the GABAA receptor antagonist, bicuculline, enhanced the ATP-induced [Ca2+ ]i increase in neurons. These results suggest that viscerosensory neuronal excitability may be modulated by GABA from satellite cells in NG. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The nodose ganglion (NG) is the distal sensory ganglion of vagus nerve and contains viscerosensory neurons related to the vagus reflex for gastrointestinal, respiratory, and cardiovascular functions [1–3]. NG contains the cell bodies of pseudounipolar neurons, which transmit sensory information from visceral organs to the medulla oblongata. NG neurons are surrounded by satellite cells, a
∗ Corresponding author at: Laboratory of Veterinary Anatomy and Cell Biology, Faculty of Agriculture, Iwate University, 18-8 Ueda 3-chome, Morioka, Iwate 020-8550, Japan. Tel.: +81 19 621 6273; fax: +81 19 621 6273. E-mail address: [email protected] (Y. Yamamoto). http://dx.doi.org/10.1016/j.neulet.2014.10.008 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
type of glial cell in the peripheral nervous system. Although neurons in the sensory ganglia are devoid of synaptic contacts, neurons and satellite cells possess receptors for various neurotransmitters, and previous studies have suggested that neurotransmitters nonsynaptically act within sensory ganglia [4,5]. Adenosine 5 -triphosphate (ATP) has been shown to mediate interactions between neurons and satellite cells in sensory ganglia. ATP induced an increase in intracellular Ca2+ ([Ca2+ ]i ) in both the neurons and satellite cells of the trigeminal ganglion (TG) in the mouse [6,7]. Furthermore, electrical stimulation evoked the release of ATP from the neuronal cell bodies of the dorsal root ganglion (DRG), which, in turn, induced [Ca2+ ]i increases in the adjacent satellite cells [8]. ATP is known to act on two families of purinoreceptors, ionotropic P2X and G protein-coupled P2Y receptors,
T. Yokoyama et al. / Neuroscience Letters 584 (2015) 168–172
which consist of seven (P2X1-7) and eight (P2Y1, 2, 4, 6, 11–14) individual receptor subunits, respectively [9,10]. In the sensory ganglia, functional P2X2 and P2X3 receptors were expressed in the neurons of the DRG and TG [11,12], and ATP evoked [Ca2+ ]i increases in TG neurons in the mouse via P2X3 receptor [7]. On the other hand, ATP induced [Ca2+ ]i increases in the satellite cells of the TG via multiple P2Y receptors [6]. Although P2X2 and P2X3 receptor immunoreactivities were detected in NG neurons of the rat [13], the presence and function of P2 purinoreceptors in NG neurons and satellite cells remain unclear. In addition to ATP, immunoreactivity to the major inhibitory neurotransmitter gamma-amino butyric acid (GABA) was detected in the neuronal cell bodies of DRG, TG, and NG in the rat [14]. GABAA receptor immunoreactivity was also localized in the DRG neuronal cell bodies of the rat [15]. In the TG of the rat, functional GABAA receptors were expressed in neuronal cell bodies, and the surrounding satellite cells were immunoreactive for GABA [16]. These findings suggested that an endogenous GABAergic modulatory system may exist in the sensory ganglia. Furthermore, GABA was shown to inhibit the excitatory effects of ATP on the DRG neurons of the rat via GABAA receptors [17]. Therefore, ATP-induced neuronal excitability is expected to be modulated by endogenous GABA in the sensory ganglia. In the present study, we investigated [Ca2+ ]i changes in NG neurons and satellite cells using NG neurons with satellite cells as well as isolated neurons to determine whether ATP- and GABAmediated neuron-satellite cell interactions existed in NG. We measured the [Ca2+ ]i responses of neurons and satellite cells to ATP and the agonists of P2X and P2Y receptors to examine the expression and function of those receptors. We also examined the mRNA expression and immunohistochemical localization of P2X2, P2X3, P2Y1, and P2Y2 receptors in NG by RT-PCR and immunohistochemistry. We evaluated [Ca2+ ]i changes in NG neurons and satellite cells following the application of ATP and GABA or their antagonists. 2. Materials and methods 2.1. Animals Eighteen male Wistar rats (8–10 weeks old; 180–200 g) were purchased from Japan SLC (Hamamatsu, Japan). All animal experiments in the present study were approved by the Local Animal Ethics Committee of Iwate University (accession number #A201325).
169
phosphate-6-azophenyl-2,4-disulfonic acid (PPADS; P178; Sigma), adenosine 5 -diphosphate (ADP; A2754; Sigma), 2 -deoxy-N6 methyladenosine-3 ,5 -diphosphate (MRS 2179; M3808; Sigma), and GABA (A2129; Sigma) were prepared from stocks at the desired concentrations in HR. (+) Bicuculline (14340; Fluka) was dissolved in DMSO and diluted to the final concentration in HR. 2.3. RT-PCR RT-PCR analysis was performed to investigate the mRNA expression of the P2X2, P2X3, P2Y1, and P2Y2 receptors in NG. For RT-PCR analysis, rats were anesthetized using diethyl ether and euthanized by exsanguination from the abdominal aorta. NG was removed and frozen in liquid nitrogen. Total RNA from NG was extracted using a magnetic bead method (MELT total RNA extraction kit, Ambion, Austin, TX, USA). RT-PCR was performed using a QIAGEN One Step RT-PCR Kit (Qiagen, Tokyo, Japan) with gene-specific primers for P2X2, P2X3, P2Y1, P2Y2, and -actin as internal controls. Details of the primers used in the present study are shown in Table 1. Reverse transcription was performed for 30 min at 50 ◦ C and initial PCR activation was incubated for 15 min at 95 ◦ C. Following reverse transcription, PCR amplification was performed 40 times as follows: 30 s at 94 ◦ C for denaturation, 30 s at 60 ◦ C for annealing, and 1 min at 72 ◦ C for extension. After PCR amplification, a final extension was performed for 10 min at 72 ◦ C. PCR end products were visualized on 2% agarose gels using ethidium bromide. The mRNA templates were omitted for a negative control. 2.4. Immunohistochemistry The methods of immunohistochemistry have been described previously [18]. The following antibodies were used: polyclonal guinea pig antibody against P2X2 (1:100; GP14106, Neuromics, Edina, MN, USA), and polyclonal rabbit antibody against P2X3 (1:1000; RA10109, Neuromics), P2Y1 (1:100; APR-009, Alomone Labs, Jerusalem, Israel), or P2Y2 (1:100; APR-010, Alomone Labs). As secondary antibodies, Alexa Fluor 488 conjugated donkey antibody against either guinea pig IgG (1:200; 706-545-148, Jackson ImmunoResearch, West Grove, PA, USA), or rabbit IgG (1:200; A21206, Invitrogen, Carlsbad, CA, USA) were used. After incubation with secondary antibodies, sections were then counterstained with DAPI and coverslipped with mounting medium (Fluoromount, Diagnostic Biosystems, Pleasanton, CA, USA). Sections were examined with a confocal laser scanning microscope (C1; Nikon, Tokyo, Japan).
2.2. Intracellular calcium imaging
3. Results
The methods of intracellular calcium imaging were performed as previously reported [18], with some modifications. To prepare the specimens of NG neurons with satellite cells, NGs were placed in Dulbecco’s modified Eagle’s medium-F-12 (DMEM/F12; GIBCO, Tokyo) containing 0.4 mg/ml collagenase P (1213857; Roche Applied Science, Mannheim, Germany), and incubated for 25 min at 37 ◦ C. Partially digested ganglia were dropped onto coverslips, and then incubated at 37 ◦ C in a humidified atmosphere of 95% air–5% CO2 . For the preparations of isolated NG neurons, several pieces of NG were placed in DMEM/F12 containing 2.0 mg/ml collagenase P and 0.5 mg/ml trypsin (Difco, Detroit, MI, USA), and incubated for 1 h at 37 ◦ C. The suspension was subjected to centrifugation at 1500 rpm for 5 min, and the supernatant was aspirated. The pellet of ganglion neurons was plated onto coverslips. Cells were cultured under the same conditions as neurons with satellite cells. KCl solution was prepared by simply adding KCl to HEPES-buffered Ringer’s solution (HR). The drug ATP (A2383; Sigma, St. Louis, MO, USA), ␣,-methylene ATP (␣,-meATP; M6517; Sigma), pyridoxal
3.1. ATP- and P2 purinoreceptors agonist-induced [Ca2+ ]i responses In NG neurons with satellite cells, the application of ATP (2.5 M) for 10 s induced transient [Ca2+ ]i increases in both the neurons and satellite cells (n = 8; Fig. 1A–E). KCl (60 mM) for 30 s evoked a [Ca2+ ]i increase in NG neurons, but did not change [Ca2+ ]i in the satellite cells (Fig. 1D and E). The P2X receptor agonist, ␣,-meATP (200 M), for 5 s evoked a transient [Ca2+ ]i increase in the neurons with satellite cells (Fig. 2A–E). However, the ␣,-meATP-induced [Ca2+ ]i response in the same neurons was reversibly inhibited by preincubating with the P2X receptor antagonist, PPADS (10 M), for 30 s (n = 4; Fig. 2C–E). The P2Y receptor agonist, ADP (10 M), for 5 s did not cause any [Ca2+ ]i response in NG neurons, whereas an increase in [Ca2+ ]i was observed in the same neurons with the application of KCl (60 mM) for 30 s (Fig. 2E). On the other hand, ADP (10 M) for 5 s induced a transient [Ca2+ ]i increase in the satellite cells surrounding NG neurons (Fig. 2F–J). The ADP-evoked
170
T. Yokoyama et al. / Neuroscience Letters 584 (2015) 168–172
Table 1 Primers for RT-PCR. mRNA (Accession #)
Primer sequences
Position
Product length
P2X2 (NM 053656)
5 -CAAGACCTGCGAGGTGTCAG-3 (sense) 5 -AGTTCGAGTGGTGGTAGTGC-3 (antisense) 5 -AGTCGGTGGTTGTGAAGAGC-3 (sense) 5 -CTGGACAGAATCCTTGCATTTGAT-3 (antisense) 5 -AGTTCAAGCAGAACGGAGACA-3 (sense) 5 -CTCAGTGGTCACATCACGGTT-3 (antisense) 5 -TCCTGCCTCAGTCCCAGTAG-3 (sense) 5 -AGTCCTCGTTGAAGCGACAT-3 (antisense) 5 -CCCTGAAGTACCCCATTGAA-3 (sense) 5 -ACCAGAGGCATACAGGGACA-3 (antisense)
501–520 932–951 222–241 483–506 1710–1730 2122–2142 347–366 555–574 275–294 497–516
451 bp
P2X3 (NM 031075) P2Y1 (NM 012800) P2Y2 (NM 017255) -Actin (NM 031144)
285 bp 433 bp 228 bp 242 bp
Fig. 1. ATP-induced [Ca2+ ]i responses recorded from NG neurons with satellite cells. (A) Phase contrast image of a neuron (N) with satellite cells (arrowhead). (B–D) Pseudocolor images of the fluorescence change in the neuron. (E) Time course of [Ca2+ ]i responses to ATP in the neuron and satellite glial cells (SGC). ATP induces [Ca2+ ]i increase in both the neurons and SGC. An increase in [Ca2+ ]i is observed in the neuron with the application of KCl, whereas SGC does not respond to KCl.
[Ca2+ ]i response in the satellite cells was abolished by preincubating with the P2Y receptor antagonist, MRS2179 (10 M), for 30 s (n = 4; Fig. 2H–J). The magnitude of the ADP-evoked [Ca2+ ]i increase in the satellite cells was partially returned by the removal of MRS2179. The satellite cells did not show any [Ca2+ ]i response to KCl (60 mM) for 30 s (Fig. 2J). 3.2. Gene expression and immunohistochemical localization of P2X2, P2X3, P2Y1, and P2Y2 receptors in NG The RT-PCR analysis detected mRNA amplification products for P2X2, P2X3, P2Y1, and P2Y2 receptors in NG extracts (Fig. 3A). A PCR-amplified product for -actin was also detected. No PCR product was detected in samples without mRNA. In sections stained with P2X2, P2X2 immunoreactivity was observed in the perinuclear cytoplasm of NG neurons, but not in satellite cells (Fig. 3B). Intense P2X3 immunoreactivity was observed in the perinuclear cytoplasm of small-sized neurons (diameter <40 m), whereas large-sized neurons (diameter >40 m) were weakly positive (Fig. 3C). No P2X3 immunoreactivity was observed in the satellite cells surrounding NG neurons. On the other hand, P2Y1 immunoreactivity was localized in the perinuclear cytoplasm and fine processes of the satellite cells, whereas NG neurons were negative (Fig. 3D). P2Y2 immunoreactivity was also observed in the perinuclear cytoplasm and fine processes of the satellite cells, but not in NG neurons (Fig. 3E). 3.3. Effects of GABA and bicuculline on ATP-induced [Ca2+ ]i responses In isolated neurons, ATP (2 M) for 30 s evoked a transient [Ca2+ ]i increase (Fig. 4A–E). The ATP-induced [Ca2+ ]i increase in the same neurons was partially inhibited by preincubating with GABA
Fig. 2. P2 purinoreceptors agonist-induced [Ca2+ ]i responses recorded from neurons with satellite cells. (A–E) [Ca2+ ]i responses to the P2X receptor agonist and antagonist in a neuron with satellite cells. (A) Phase contrast image of a neuron (N) with satellite cells (arrowhead). (B–D) Pseudocolor images of the fluorescence change in the neuron. (E) Time course of the [Ca2+ ]i responses to the P2X receptor agonist and its antagonist in the neuron. The P2X receptor agonist, ␣,-meATP, induces a [Ca2+ ]i increase in the neuron, and this response is inhibited by the P2X receptor antagonist, PPADS. The neuron does not respond to the P2Y receptor agonist, ADP. (F–J) [Ca2+ ]i responses to the P2Y receptor agonist and antagonist in satellite cells. (F) Phase contrast image of the satellite cells (arrowhead) surrounding NG neuron (N). (G–I) Pseudocolor images of the fluorescence change in satellite cells. (J) Time course of the [Ca2+ ]i responses to the P2Y receptor agonist and its antagonist in satellite cells. The P2Y receptor agonist, ADP, induces a [Ca2+ ]i increase, while this response is inhibited by the P2Y receptor antagonist, MRS2179.
(100 M) for 60 s and 120 s, respectively (n = 4; Fig. 4D and E). The magnitude of the ATP-induced [Ca2+ ]i increase in the neuron was partially returned by the removal of GABA. On the other hand, in NG neurons with satellite cells, ATP (10 M) for 5 s evoked a [Ca2+ ]i increase in both the neurons and satellite cells (Fig. 5). However,
T. Yokoyama et al. / Neuroscience Letters 584 (2015) 168–172
171
Fig. 4. ATP-induced [Ca2+ ]i responses in the presence of GABA recorded from isolated neurons. (A) Phase contrast image of isolated neurons (N). (B–D) Pseudocolor images of the fluorescence change in the neurons. (E) Time course of ATP-induced [Ca2+ ]i responses in the presence of GABA in the neurons. ATP induces a transient [Ca2+ ]i increase, while this response is partially inhibited in the presence of GABA.
Fig. 3. Expression of P2X and P2Y receptors in NG. (A) RT-PCR for the expression of P2X and P2Y receptor mRNAs in NG. PCR-amplified products for P2X2, P2X3, P2Y1, and P2Y2 receptors are detected in NG. (B–E) Immunoreactivity for P2X2 (B), P2X3 (C), P2Y1 (D), and P2Y2 (E) in NG. Nuclei are stained with DAPI (blue). (B) P2X2 immunoreactivity is observed in NG neurons, but not in the surrounding satellite cells. (C) Intense P2X3 immunoreactivity is observed in the small-sized neurons, while large-sized neurons are weakly positive. No P2X3 immunoreactivity is observed in satellite cells. (D) P2Y1 immunoreactivity is observed in satellite cells, but not in NG neurons. (E) Satellite cells are also immunoreactive to P2Y2, while NG neurons are negative.
preincubation with the GABAA receptor antagonist, bicuculline (100 M), for 30 s enhanced the magnitude of the ATP-induced [Ca2+ ]i increase in the neurons (n = 4). The ATP-induced [Ca2+ ]i increase in the satellite cells was not altered in the presence of bicuculline. DMSO, the vehicle for bicuculline, did not cause any responses (data not shown). 4. Discussion In the present study, ATP induced [Ca2+ ]i responses in NG neurons, and the P2X receptor agonist, ␣,-meATP induced [Ca2+ ]i increases that were inhibited by PPADS. Therefore, [Ca2+ ]i increases in NG neurons may be mediated by ATP via P2X receptors. Furthermore, RT-PCR and immunohistochemistry revealed that P2X2
Fig. 5. ATP-induced [Ca2+ ]i responses in the presence of the GABAA receptor antagonist recorded from neurons (N) with satellite glial cells (SGC). ATP evokes [Ca2+ ]i increases in neurons, and this response is enhanced in the presence of the GABAA receptor antagonist, bicuculline. SGC also exhibits [Ca2+ ]i increases with the application of ATP, whereas this response is not altered in the presence of bicuculline.
and P2X3 receptors were expressed in NG neurons. In the sensory ganglia, the neuronal cell bodies of NG were previously shown to express functional P2X2 and P2X3 receptors [19]. Furthermore, previous electrophysiological studies have shown that the excitatory effects of ATP on NG neurons were predominantly mediated by homomeric P2X2 and heteromeric P2X2/3 receptors [20]. Thus, ATP may activate NG neurons via P2X2, P2X3, and P2X2/3 receptors. A previous study reported that the P2Y receptor agonist, adenosine 5 -O-(2-thiodiphosphate), induced [Ca2+ ]i increases in the satellite cells of the TG in the mouse [6]. In the present study, ATP induced [Ca2+ ]i responses in the satellite cells of the NG, and the P2Y receptor agonist, ADP, induced [Ca2+ ]i increases that were abolished by MRS2179. Thus, [Ca2+ ]i increases in the satellite cells of the NG may be mediated by ATP via P2Y receptors. RT-PCR and immunohistochemistry revealed the expression of P2Y1 and P2Y2 receptors in satellite cells. These results suggest that ATP activates satellite cells via P2Y1 and P2Y2 receptors. Because MRS2179 has been described as a selective P2Y1 receptor antagonist based on its
172
T. Yokoyama et al. / Neuroscience Letters 584 (2015) 168–172
pharmacological characteristics [21], P2Y1 receptors may mainly mediate ATP-induced responses in satellite cells. The reduction observed in the ATP-induced [Ca2+ ]i increase in isolated NG neurons by GABA in the present study suggests the presence of inhibitory interactions between extrinsic GABA and ATP for neuronal excitability. Furthermore, ATP-induced responses in NG neurons with satellite cells were enhanced in the presence of bicuculline. These results suggest the existence of an intrinsic GABAergic modulatory system based on a close relationship between neurons and satellite cells, and also that GABA inhibits ATP-induced responses in NG neurons via GABAA receptors. A previous calcium imaging study has shown that bicuculline induces [Ca2+ ]i increases in NG neurons with satellite cells, but not in isolated neurons [18]. This finding suggests that GABA is released from satellite cells. Satellite cells may inhibit ATP-evoked neuronal excitability by releasing GABA in NG. In the present study, we showed that ATP induces [Ca2+ ]i increases in NG neurons and satellite cells via P2X and P2Y receptors, respectively. We also demonstrated that an ATP- and GABA-mediated modulatory system for NG neuronal excitability exists between neurons and satellite cells. Viscerosensory neuronal excitability may be modulated by GABA from satellite cells in NG. References [1] K.N. Browning, D. Mendelowitz, Musings on the wanderer: what’s new in our understanding of vago-vagal reflexes? II. Integration of afferent signaling from the viscera by the nodose ganglia, Am. J. Physiol. Gastrointest. Liver Physiol. 284 (2003) 8–14. [2] B. Canning, N. Mori, S.B. Mazzone, Vagal afferent nerves regulating the cough reflex, Respir. Physiol. 152 (2006) 223–242. [3] R.D. Foreman, C. Qin, Neuromodulation of cardiac pain and cerebral vasculature: neural mechanisms, Cleve. Clin. J. Med. 76 (2009) 75–79. [4] D. Julius, A.I. Basbaum, Molecular mechanisms of nociception, Nature 413 (2001) 203–210. [5] M. Hanani, Satellite glial cells in sensory ganglia: from form to function, Brain Res. Rev. 48 (2005) 457–476. [6] M. Weick, P.S. Cherkas, W. Härtig, T. Pannicke, O. Uckermann, A. Bringmann, M. Tal, A. Reichenbach, M. Hanani, P2 receptors in satellite glial cells in trigeminal ganglia of mice, Neuroscience 120 (2003) 969–977.
[7] S. Ceruti, M. Fumagalli, G. Villa, C. Verderio, M.P. Abbracchio, Purinoceptormediated calcium signaling in primary neuron-glia trigeminal cultures, Cell Calcium 43 (2008) 576–590. [8] X. Zhang, Y. Chen, C. Wang, L.Y. Huang, Neuronal somatic ATP release triggers neuron-satellite glial cell communication in dorsal root ganglia, Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 9864–9869. [9] B.B. Fredholm, M.P. Abbracchio, G. Burnstock, J.W. Daly, T.K. Harden, K.A. Jacobson, P. Leff, M. Williams, Nomenclature and classification of purinoceptors, Pharmacol. Rev. 46 (1994) 143–156. [10] V. Ralevic, G. Burnstock, Receptors for purines and pyrimidines, Pharmacol. Rev. 50 (1998) 413–492. [11] E.C. Burgard, W. Niforatos, T. van Biesen, K.J. Lynch, E. Touma, R.E. Metzger, E.A. Kowaluk, M.F. Jarvis, P2X receptor-mediated ionic currents in dorsal root ganglion neurons, J. Neurophysiol. 82 (1999) 1590–1598. [12] P.M. Dunn, M. Liu, Y. Zhong, B.F. King, G. Burnstock, Diinosine pentaphosphate: an antagonist which discriminates between recombinant P2X3 and P2X2/3 receptors and between two P2X receptors in rat sensory neurones, Br. J. Pharmacol. 130 (2000) 1378–1384. [13] Z. Xiang, X. Bo, G. Burnstock, Localization of ATP-gated P2X receptor immunoreactivity in rat sensory and sympathetic ganglia, Neurosci. Lett. 256 (1998) 105–108. [14] E. Szabat, S. Soinila, O. Happola, A. Linnala, I. Virtanen, A new monoclonal antibody against the GABA-protein conjugate shows immunoreactivity in sensory neurons of the rat, Neuroscience 47 (1992) 409–420. [15] F.J. Alvarez, B. Taylor-Blake, R.E.W. Fyffe, A.L. De Blas, A.R. Light, Distribution of immunoreactivity for the beta 2 and beta 3 subunits of the GABAA receptor in the mammalian spinal cord, J. Comp. Neurol. 365 (1996) 392–412. [16] H. Hayasaki, Y. Sohma, K. Kanbara, K. Maemura, T. Kubota, M. Watanabe, A local GABAergic system within rat trigeminal ganglion cells, Eur. J. Neurosci. 23 (2006) 745–757. [17] E. Sokolova, A. Nistri, R. Giniatullin, Negative cross talk between anionic GABAA and cationic P2X ionotropic receptors of rat dorsal root ganglion neurons, J. Neurosci. 21 (2001) 4958–4968. [18] Y. Shoji, M. Yamaguchi-Yamada, Y. Yamamoto, Glutamate- and GABA-mediated neuron-satellite cell interaction in nodose ganglia as revealed by intracellular calcium imaging, Histochem. Cell Biol. 134 (2010) 13–22. [19] D.A. Cockayne, P.M. Dunn, Y. Zhong, W. Rong, S.G. Hamilton, G.E. Knight, H.Z. Ruan, B. Ma, P. Yip, P. Nunn, S.B. McMahon, G. Burnstock, A.P. Ford, P2X2 knockout mice and P2X2/P2X3 double knockout mice reveal a role for the P2X2 receptor subunit in mediating multiple sensory effects of ATP, J. Physiol. 567 (2005) 621–639. [20] C. Virginio, R.A. North, A. Surprenant, Calcium permeability and block at homomeric and heteromeric P2X2 and P2X3 receptors, and P2X receptors in rat nodose neurons, J. Physiol. 510 (1998) 27–35. [21] E. Camaioni, J.L. Boyer, A. Mohanram, T.K. Harden, K.A. Jacobson, Deoxyadenosine bisphosphate derivatives as potent antagonists at P2Y1 receptors, J. Med. Chem. 41 (1998) 183–190.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
GABA-mediated modulation of ATP-induced intracellular calcium responses in nodose ganglion neurons of the rat Takuya Yokoyama a,b , Shou Fukuzumi a , Hitomi Hayashi a , Nobuaki Nakamuta a,b , Yoshio Yamamoto a,b,∗ a b
Laboratory of Veterinary Anatomy and Cell Biology, Faculty of Agriculture, Iwate University, Morioka, Japan Department of Basic Veterinary Science, United Graduate School of Veterinary Science, Gifu University, Gifu, Japan
h i g h l i g h t s • • • • •
ATP increased intracellular Ca2+ levels in nodose ganglion neurons via P2X receptor. ATP increased intracellular Ca2+ levels in satellite cells via P2Y receptor. GABA inhibited ATP-induced intracellular Ca2+ responses in isolated neurons. Bicuculline enhanced ATP-induced Ca2+ responses in neurons with satellite cells. Neuronal excitability may be modulated by GABA from satellite cells in nodose ganglion.
a r t i c l e
i n f o
Article history: Received 16 September 2014 Received in revised form 6 October 2014 Accepted 7 October 2014 Available online 17 October 2014 Keywords: Nodose ganglion Sensory neuron Satellite cell ATP GABA
a b s t r a c t We examined ATP-induced intracellular Ca2+ ([Ca2+ ]i ) responses in the neurons and satellite cells from one of the viscerosensory ganglia, the nodose ganglion (NG), as well as the GABA-mediated modulation of ATPinduced neuronal [Ca2+ ]i responses using intracellular calcium imaging. In neurons with satellite cells, ATP induced [Ca2+ ]i increases in both the neurons and satellite cells. The P2X receptor agonist, ␣,-meATP, induced [Ca2+ ]i increases in neurons and this response was inhibited by the P2X receptor antagonist, PPADS. On the other hand, the P2Y receptor agonist, ADP, induced [Ca2+ ]i increases in satellite cells, and this response was inhibited by the P2Y receptor antagonist, MRS2179. RT-PCR detected the expression of P2X2, P2X3, P2Y1, and P2Y2 receptor mRNAs in NG extracts. Immunohistochemistry revealed that NG neurons and satellite cells were immunoreactive to P2X2 and P2X3, and P2Y1 and P2Y2 receptors, respectively. In isolated neurons, the ATP-evoked [Ca2+ ]i increase was inhibited by GABA. However, in neurons with satellite cells, the GABAA receptor antagonist, bicuculline, enhanced the ATP-induced [Ca2+ ]i increase in neurons. These results suggest that viscerosensory neuronal excitability may be modulated by GABA from satellite cells in NG. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The nodose ganglion (NG) is the distal sensory ganglion of vagus nerve and contains viscerosensory neurons related to the vagus reflex for gastrointestinal, respiratory, and cardiovascular functions [1–3]. NG contains the cell bodies of pseudounipolar neurons, which transmit sensory information from visceral organs to the medulla oblongata. NG neurons are surrounded by satellite cells, a
∗ Corresponding author at: Laboratory of Veterinary Anatomy and Cell Biology, Faculty of Agriculture, Iwate University, 18-8 Ueda 3-chome, Morioka, Iwate 020-8550, Japan. Tel.: +81 19 621 6273; fax: +81 19 621 6273. E-mail address: [email protected] (Y. Yamamoto). http://dx.doi.org/10.1016/j.neulet.2014.10.008 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
type of glial cell in the peripheral nervous system. Although neurons in the sensory ganglia are devoid of synaptic contacts, neurons and satellite cells possess receptors for various neurotransmitters, and previous studies have suggested that neurotransmitters nonsynaptically act within sensory ganglia [4,5]. Adenosine 5 -triphosphate (ATP) has been shown to mediate interactions between neurons and satellite cells in sensory ganglia. ATP induced an increase in intracellular Ca2+ ([Ca2+ ]i ) in both the neurons and satellite cells of the trigeminal ganglion (TG) in the mouse [6,7]. Furthermore, electrical stimulation evoked the release of ATP from the neuronal cell bodies of the dorsal root ganglion (DRG), which, in turn, induced [Ca2+ ]i increases in the adjacent satellite cells [8]. ATP is known to act on two families of purinoreceptors, ionotropic P2X and G protein-coupled P2Y receptors,
T. Yokoyama et al. / Neuroscience Letters 584 (2015) 168–172
which consist of seven (P2X1-7) and eight (P2Y1, 2, 4, 6, 11–14) individual receptor subunits, respectively [9,10]. In the sensory ganglia, functional P2X2 and P2X3 receptors were expressed in the neurons of the DRG and TG [11,12], and ATP evoked [Ca2+ ]i increases in TG neurons in the mouse via P2X3 receptor [7]. On the other hand, ATP induced [Ca2+ ]i increases in the satellite cells of the TG via multiple P2Y receptors [6]. Although P2X2 and P2X3 receptor immunoreactivities were detected in NG neurons of the rat [13], the presence and function of P2 purinoreceptors in NG neurons and satellite cells remain unclear. In addition to ATP, immunoreactivity to the major inhibitory neurotransmitter gamma-amino butyric acid (GABA) was detected in the neuronal cell bodies of DRG, TG, and NG in the rat [14]. GABAA receptor immunoreactivity was also localized in the DRG neuronal cell bodies of the rat [15]. In the TG of the rat, functional GABAA receptors were expressed in neuronal cell bodies, and the surrounding satellite cells were immunoreactive for GABA [16]. These findings suggested that an endogenous GABAergic modulatory system may exist in the sensory ganglia. Furthermore, GABA was shown to inhibit the excitatory effects of ATP on the DRG neurons of the rat via GABAA receptors [17]. Therefore, ATP-induced neuronal excitability is expected to be modulated by endogenous GABA in the sensory ganglia. In the present study, we investigated [Ca2+ ]i changes in NG neurons and satellite cells using NG neurons with satellite cells as well as isolated neurons to determine whether ATP- and GABAmediated neuron-satellite cell interactions existed in NG. We measured the [Ca2+ ]i responses of neurons and satellite cells to ATP and the agonists of P2X and P2Y receptors to examine the expression and function of those receptors. We also examined the mRNA expression and immunohistochemical localization of P2X2, P2X3, P2Y1, and P2Y2 receptors in NG by RT-PCR and immunohistochemistry. We evaluated [Ca2+ ]i changes in NG neurons and satellite cells following the application of ATP and GABA or their antagonists. 2. Materials and methods 2.1. Animals Eighteen male Wistar rats (8–10 weeks old; 180–200 g) were purchased from Japan SLC (Hamamatsu, Japan). All animal experiments in the present study were approved by the Local Animal Ethics Committee of Iwate University (accession number #A201325).
169
phosphate-6-azophenyl-2,4-disulfonic acid (PPADS; P178; Sigma), adenosine 5 -diphosphate (ADP; A2754; Sigma), 2 -deoxy-N6 methyladenosine-3 ,5 -diphosphate (MRS 2179; M3808; Sigma), and GABA (A2129; Sigma) were prepared from stocks at the desired concentrations in HR. (+) Bicuculline (14340; Fluka) was dissolved in DMSO and diluted to the final concentration in HR. 2.3. RT-PCR RT-PCR analysis was performed to investigate the mRNA expression of the P2X2, P2X3, P2Y1, and P2Y2 receptors in NG. For RT-PCR analysis, rats were anesthetized using diethyl ether and euthanized by exsanguination from the abdominal aorta. NG was removed and frozen in liquid nitrogen. Total RNA from NG was extracted using a magnetic bead method (MELT total RNA extraction kit, Ambion, Austin, TX, USA). RT-PCR was performed using a QIAGEN One Step RT-PCR Kit (Qiagen, Tokyo, Japan) with gene-specific primers for P2X2, P2X3, P2Y1, P2Y2, and -actin as internal controls. Details of the primers used in the present study are shown in Table 1. Reverse transcription was performed for 30 min at 50 ◦ C and initial PCR activation was incubated for 15 min at 95 ◦ C. Following reverse transcription, PCR amplification was performed 40 times as follows: 30 s at 94 ◦ C for denaturation, 30 s at 60 ◦ C for annealing, and 1 min at 72 ◦ C for extension. After PCR amplification, a final extension was performed for 10 min at 72 ◦ C. PCR end products were visualized on 2% agarose gels using ethidium bromide. The mRNA templates were omitted for a negative control. 2.4. Immunohistochemistry The methods of immunohistochemistry have been described previously [18]. The following antibodies were used: polyclonal guinea pig antibody against P2X2 (1:100; GP14106, Neuromics, Edina, MN, USA), and polyclonal rabbit antibody against P2X3 (1:1000; RA10109, Neuromics), P2Y1 (1:100; APR-009, Alomone Labs, Jerusalem, Israel), or P2Y2 (1:100; APR-010, Alomone Labs). As secondary antibodies, Alexa Fluor 488 conjugated donkey antibody against either guinea pig IgG (1:200; 706-545-148, Jackson ImmunoResearch, West Grove, PA, USA), or rabbit IgG (1:200; A21206, Invitrogen, Carlsbad, CA, USA) were used. After incubation with secondary antibodies, sections were then counterstained with DAPI and coverslipped with mounting medium (Fluoromount, Diagnostic Biosystems, Pleasanton, CA, USA). Sections were examined with a confocal laser scanning microscope (C1; Nikon, Tokyo, Japan).
2.2. Intracellular calcium imaging
3. Results
The methods of intracellular calcium imaging were performed as previously reported [18], with some modifications. To prepare the specimens of NG neurons with satellite cells, NGs were placed in Dulbecco’s modified Eagle’s medium-F-12 (DMEM/F12; GIBCO, Tokyo) containing 0.4 mg/ml collagenase P (1213857; Roche Applied Science, Mannheim, Germany), and incubated for 25 min at 37 ◦ C. Partially digested ganglia were dropped onto coverslips, and then incubated at 37 ◦ C in a humidified atmosphere of 95% air–5% CO2 . For the preparations of isolated NG neurons, several pieces of NG were placed in DMEM/F12 containing 2.0 mg/ml collagenase P and 0.5 mg/ml trypsin (Difco, Detroit, MI, USA), and incubated for 1 h at 37 ◦ C. The suspension was subjected to centrifugation at 1500 rpm for 5 min, and the supernatant was aspirated. The pellet of ganglion neurons was plated onto coverslips. Cells were cultured under the same conditions as neurons with satellite cells. KCl solution was prepared by simply adding KCl to HEPES-buffered Ringer’s solution (HR). The drug ATP (A2383; Sigma, St. Louis, MO, USA), ␣,-methylene ATP (␣,-meATP; M6517; Sigma), pyridoxal
3.1. ATP- and P2 purinoreceptors agonist-induced [Ca2+ ]i responses In NG neurons with satellite cells, the application of ATP (2.5 M) for 10 s induced transient [Ca2+ ]i increases in both the neurons and satellite cells (n = 8; Fig. 1A–E). KCl (60 mM) for 30 s evoked a [Ca2+ ]i increase in NG neurons, but did not change [Ca2+ ]i in the satellite cells (Fig. 1D and E). The P2X receptor agonist, ␣,-meATP (200 M), for 5 s evoked a transient [Ca2+ ]i increase in the neurons with satellite cells (Fig. 2A–E). However, the ␣,-meATP-induced [Ca2+ ]i response in the same neurons was reversibly inhibited by preincubating with the P2X receptor antagonist, PPADS (10 M), for 30 s (n = 4; Fig. 2C–E). The P2Y receptor agonist, ADP (10 M), for 5 s did not cause any [Ca2+ ]i response in NG neurons, whereas an increase in [Ca2+ ]i was observed in the same neurons with the application of KCl (60 mM) for 30 s (Fig. 2E). On the other hand, ADP (10 M) for 5 s induced a transient [Ca2+ ]i increase in the satellite cells surrounding NG neurons (Fig. 2F–J). The ADP-evoked
170
T. Yokoyama et al. / Neuroscience Letters 584 (2015) 168–172
Table 1 Primers for RT-PCR. mRNA (Accession #)
Primer sequences
Position
Product length
P2X2 (NM 053656)
5 -CAAGACCTGCGAGGTGTCAG-3 (sense) 5 -AGTTCGAGTGGTGGTAGTGC-3 (antisense) 5 -AGTCGGTGGTTGTGAAGAGC-3 (sense) 5 -CTGGACAGAATCCTTGCATTTGAT-3 (antisense) 5 -AGTTCAAGCAGAACGGAGACA-3 (sense) 5 -CTCAGTGGTCACATCACGGTT-3 (antisense) 5 -TCCTGCCTCAGTCCCAGTAG-3 (sense) 5 -AGTCCTCGTTGAAGCGACAT-3 (antisense) 5 -CCCTGAAGTACCCCATTGAA-3 (sense) 5 -ACCAGAGGCATACAGGGACA-3 (antisense)
501–520 932–951 222–241 483–506 1710–1730 2122–2142 347–366 555–574 275–294 497–516
451 bp
P2X3 (NM 031075) P2Y1 (NM 012800) P2Y2 (NM 017255) -Actin (NM 031144)
285 bp 433 bp 228 bp 242 bp
Fig. 1. ATP-induced [Ca2+ ]i responses recorded from NG neurons with satellite cells. (A) Phase contrast image of a neuron (N) with satellite cells (arrowhead). (B–D) Pseudocolor images of the fluorescence change in the neuron. (E) Time course of [Ca2+ ]i responses to ATP in the neuron and satellite glial cells (SGC). ATP induces [Ca2+ ]i increase in both the neurons and SGC. An increase in [Ca2+ ]i is observed in the neuron with the application of KCl, whereas SGC does not respond to KCl.
[Ca2+ ]i response in the satellite cells was abolished by preincubating with the P2Y receptor antagonist, MRS2179 (10 M), for 30 s (n = 4; Fig. 2H–J). The magnitude of the ADP-evoked [Ca2+ ]i increase in the satellite cells was partially returned by the removal of MRS2179. The satellite cells did not show any [Ca2+ ]i response to KCl (60 mM) for 30 s (Fig. 2J). 3.2. Gene expression and immunohistochemical localization of P2X2, P2X3, P2Y1, and P2Y2 receptors in NG The RT-PCR analysis detected mRNA amplification products for P2X2, P2X3, P2Y1, and P2Y2 receptors in NG extracts (Fig. 3A). A PCR-amplified product for -actin was also detected. No PCR product was detected in samples without mRNA. In sections stained with P2X2, P2X2 immunoreactivity was observed in the perinuclear cytoplasm of NG neurons, but not in satellite cells (Fig. 3B). Intense P2X3 immunoreactivity was observed in the perinuclear cytoplasm of small-sized neurons (diameter <40 m), whereas large-sized neurons (diameter >40 m) were weakly positive (Fig. 3C). No P2X3 immunoreactivity was observed in the satellite cells surrounding NG neurons. On the other hand, P2Y1 immunoreactivity was localized in the perinuclear cytoplasm and fine processes of the satellite cells, whereas NG neurons were negative (Fig. 3D). P2Y2 immunoreactivity was also observed in the perinuclear cytoplasm and fine processes of the satellite cells, but not in NG neurons (Fig. 3E). 3.3. Effects of GABA and bicuculline on ATP-induced [Ca2+ ]i responses In isolated neurons, ATP (2 M) for 30 s evoked a transient [Ca2+ ]i increase (Fig. 4A–E). The ATP-induced [Ca2+ ]i increase in the same neurons was partially inhibited by preincubating with GABA
Fig. 2. P2 purinoreceptors agonist-induced [Ca2+ ]i responses recorded from neurons with satellite cells. (A–E) [Ca2+ ]i responses to the P2X receptor agonist and antagonist in a neuron with satellite cells. (A) Phase contrast image of a neuron (N) with satellite cells (arrowhead). (B–D) Pseudocolor images of the fluorescence change in the neuron. (E) Time course of the [Ca2+ ]i responses to the P2X receptor agonist and its antagonist in the neuron. The P2X receptor agonist, ␣,-meATP, induces a [Ca2+ ]i increase in the neuron, and this response is inhibited by the P2X receptor antagonist, PPADS. The neuron does not respond to the P2Y receptor agonist, ADP. (F–J) [Ca2+ ]i responses to the P2Y receptor agonist and antagonist in satellite cells. (F) Phase contrast image of the satellite cells (arrowhead) surrounding NG neuron (N). (G–I) Pseudocolor images of the fluorescence change in satellite cells. (J) Time course of the [Ca2+ ]i responses to the P2Y receptor agonist and its antagonist in satellite cells. The P2Y receptor agonist, ADP, induces a [Ca2+ ]i increase, while this response is inhibited by the P2Y receptor antagonist, MRS2179.
(100 M) for 60 s and 120 s, respectively (n = 4; Fig. 4D and E). The magnitude of the ATP-induced [Ca2+ ]i increase in the neuron was partially returned by the removal of GABA. On the other hand, in NG neurons with satellite cells, ATP (10 M) for 5 s evoked a [Ca2+ ]i increase in both the neurons and satellite cells (Fig. 5). However,
T. Yokoyama et al. / Neuroscience Letters 584 (2015) 168–172
171
Fig. 4. ATP-induced [Ca2+ ]i responses in the presence of GABA recorded from isolated neurons. (A) Phase contrast image of isolated neurons (N). (B–D) Pseudocolor images of the fluorescence change in the neurons. (E) Time course of ATP-induced [Ca2+ ]i responses in the presence of GABA in the neurons. ATP induces a transient [Ca2+ ]i increase, while this response is partially inhibited in the presence of GABA.
Fig. 3. Expression of P2X and P2Y receptors in NG. (A) RT-PCR for the expression of P2X and P2Y receptor mRNAs in NG. PCR-amplified products for P2X2, P2X3, P2Y1, and P2Y2 receptors are detected in NG. (B–E) Immunoreactivity for P2X2 (B), P2X3 (C), P2Y1 (D), and P2Y2 (E) in NG. Nuclei are stained with DAPI (blue). (B) P2X2 immunoreactivity is observed in NG neurons, but not in the surrounding satellite cells. (C) Intense P2X3 immunoreactivity is observed in the small-sized neurons, while large-sized neurons are weakly positive. No P2X3 immunoreactivity is observed in satellite cells. (D) P2Y1 immunoreactivity is observed in satellite cells, but not in NG neurons. (E) Satellite cells are also immunoreactive to P2Y2, while NG neurons are negative.
preincubation with the GABAA receptor antagonist, bicuculline (100 M), for 30 s enhanced the magnitude of the ATP-induced [Ca2+ ]i increase in the neurons (n = 4). The ATP-induced [Ca2+ ]i increase in the satellite cells was not altered in the presence of bicuculline. DMSO, the vehicle for bicuculline, did not cause any responses (data not shown). 4. Discussion In the present study, ATP induced [Ca2+ ]i responses in NG neurons, and the P2X receptor agonist, ␣,-meATP induced [Ca2+ ]i increases that were inhibited by PPADS. Therefore, [Ca2+ ]i increases in NG neurons may be mediated by ATP via P2X receptors. Furthermore, RT-PCR and immunohistochemistry revealed that P2X2
Fig. 5. ATP-induced [Ca2+ ]i responses in the presence of the GABAA receptor antagonist recorded from neurons (N) with satellite glial cells (SGC). ATP evokes [Ca2+ ]i increases in neurons, and this response is enhanced in the presence of the GABAA receptor antagonist, bicuculline. SGC also exhibits [Ca2+ ]i increases with the application of ATP, whereas this response is not altered in the presence of bicuculline.
and P2X3 receptors were expressed in NG neurons. In the sensory ganglia, the neuronal cell bodies of NG were previously shown to express functional P2X2 and P2X3 receptors [19]. Furthermore, previous electrophysiological studies have shown that the excitatory effects of ATP on NG neurons were predominantly mediated by homomeric P2X2 and heteromeric P2X2/3 receptors [20]. Thus, ATP may activate NG neurons via P2X2, P2X3, and P2X2/3 receptors. A previous study reported that the P2Y receptor agonist, adenosine 5 -O-(2-thiodiphosphate), induced [Ca2+ ]i increases in the satellite cells of the TG in the mouse [6]. In the present study, ATP induced [Ca2+ ]i responses in the satellite cells of the NG, and the P2Y receptor agonist, ADP, induced [Ca2+ ]i increases that were abolished by MRS2179. Thus, [Ca2+ ]i increases in the satellite cells of the NG may be mediated by ATP via P2Y receptors. RT-PCR and immunohistochemistry revealed the expression of P2Y1 and P2Y2 receptors in satellite cells. These results suggest that ATP activates satellite cells via P2Y1 and P2Y2 receptors. Because MRS2179 has been described as a selective P2Y1 receptor antagonist based on its
172
T. Yokoyama et al. / Neuroscience Letters 584 (2015) 168–172
pharmacological characteristics [21], P2Y1 receptors may mainly mediate ATP-induced responses in satellite cells. The reduction observed in the ATP-induced [Ca2+ ]i increase in isolated NG neurons by GABA in the present study suggests the presence of inhibitory interactions between extrinsic GABA and ATP for neuronal excitability. Furthermore, ATP-induced responses in NG neurons with satellite cells were enhanced in the presence of bicuculline. These results suggest the existence of an intrinsic GABAergic modulatory system based on a close relationship between neurons and satellite cells, and also that GABA inhibits ATP-induced responses in NG neurons via GABAA receptors. A previous calcium imaging study has shown that bicuculline induces [Ca2+ ]i increases in NG neurons with satellite cells, but not in isolated neurons [18]. This finding suggests that GABA is released from satellite cells. Satellite cells may inhibit ATP-evoked neuronal excitability by releasing GABA in NG. In the present study, we showed that ATP induces [Ca2+ ]i increases in NG neurons and satellite cells via P2X and P2Y receptors, respectively. We also demonstrated that an ATP- and GABA-mediated modulatory system for NG neuronal excitability exists between neurons and satellite cells. Viscerosensory neuronal excitability may be modulated by GABA from satellite cells in NG. References [1] K.N. Browning, D. Mendelowitz, Musings on the wanderer: what’s new in our understanding of vago-vagal reflexes? II. Integration of afferent signaling from the viscera by the nodose ganglia, Am. J. Physiol. Gastrointest. Liver Physiol. 284 (2003) 8–14. [2] B. Canning, N. Mori, S.B. Mazzone, Vagal afferent nerves regulating the cough reflex, Respir. Physiol. 152 (2006) 223–242. [3] R.D. Foreman, C. Qin, Neuromodulation of cardiac pain and cerebral vasculature: neural mechanisms, Cleve. Clin. J. Med. 76 (2009) 75–79. [4] D. Julius, A.I. Basbaum, Molecular mechanisms of nociception, Nature 413 (2001) 203–210. [5] M. Hanani, Satellite glial cells in sensory ganglia: from form to function, Brain Res. Rev. 48 (2005) 457–476. [6] M. Weick, P.S. Cherkas, W. Härtig, T. Pannicke, O. Uckermann, A. Bringmann, M. Tal, A. Reichenbach, M. Hanani, P2 receptors in satellite glial cells in trigeminal ganglia of mice, Neuroscience 120 (2003) 969–977.
[7] S. Ceruti, M. Fumagalli, G. Villa, C. Verderio, M.P. Abbracchio, Purinoceptormediated calcium signaling in primary neuron-glia trigeminal cultures, Cell Calcium 43 (2008) 576–590. [8] X. Zhang, Y. Chen, C. Wang, L.Y. Huang, Neuronal somatic ATP release triggers neuron-satellite glial cell communication in dorsal root ganglia, Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 9864–9869. [9] B.B. Fredholm, M.P. Abbracchio, G. Burnstock, J.W. Daly, T.K. Harden, K.A. Jacobson, P. Leff, M. Williams, Nomenclature and classification of purinoceptors, Pharmacol. Rev. 46 (1994) 143–156. [10] V. Ralevic, G. Burnstock, Receptors for purines and pyrimidines, Pharmacol. Rev. 50 (1998) 413–492. [11] E.C. Burgard, W. Niforatos, T. van Biesen, K.J. Lynch, E. Touma, R.E. Metzger, E.A. Kowaluk, M.F. Jarvis, P2X receptor-mediated ionic currents in dorsal root ganglion neurons, J. Neurophysiol. 82 (1999) 1590–1598. [12] P.M. Dunn, M. Liu, Y. Zhong, B.F. King, G. Burnstock, Diinosine pentaphosphate: an antagonist which discriminates between recombinant P2X3 and P2X2/3 receptors and between two P2X receptors in rat sensory neurones, Br. J. Pharmacol. 130 (2000) 1378–1384. [13] Z. Xiang, X. Bo, G. Burnstock, Localization of ATP-gated P2X receptor immunoreactivity in rat sensory and sympathetic ganglia, Neurosci. Lett. 256 (1998) 105–108. [14] E. Szabat, S. Soinila, O. Happola, A. Linnala, I. Virtanen, A new monoclonal antibody against the GABA-protein conjugate shows immunoreactivity in sensory neurons of the rat, Neuroscience 47 (1992) 409–420. [15] F.J. Alvarez, B. Taylor-Blake, R.E.W. Fyffe, A.L. De Blas, A.R. Light, Distribution of immunoreactivity for the beta 2 and beta 3 subunits of the GABAA receptor in the mammalian spinal cord, J. Comp. Neurol. 365 (1996) 392–412. [16] H. Hayasaki, Y. Sohma, K. Kanbara, K. Maemura, T. Kubota, M. Watanabe, A local GABAergic system within rat trigeminal ganglion cells, Eur. J. Neurosci. 23 (2006) 745–757. [17] E. Sokolova, A. Nistri, R. Giniatullin, Negative cross talk between anionic GABAA and cationic P2X ionotropic receptors of rat dorsal root ganglion neurons, J. Neurosci. 21 (2001) 4958–4968. [18] Y. Shoji, M. Yamaguchi-Yamada, Y. Yamamoto, Glutamate- and GABA-mediated neuron-satellite cell interaction in nodose ganglia as revealed by intracellular calcium imaging, Histochem. Cell Biol. 134 (2010) 13–22. [19] D.A. Cockayne, P.M. Dunn, Y. Zhong, W. Rong, S.G. Hamilton, G.E. Knight, H.Z. Ruan, B. Ma, P. Yip, P. Nunn, S.B. McMahon, G. Burnstock, A.P. Ford, P2X2 knockout mice and P2X2/P2X3 double knockout mice reveal a role for the P2X2 receptor subunit in mediating multiple sensory effects of ATP, J. Physiol. 567 (2005) 621–639. [20] C. Virginio, R.A. North, A. Surprenant, Calcium permeability and block at homomeric and heteromeric P2X2 and P2X3 receptors, and P2X receptors in rat nodose neurons, J. Physiol. 510 (1998) 27–35. [21] E. Camaioni, J.L. Boyer, A. Mohanram, T.K. Harden, K.A. Jacobson, Deoxyadenosine bisphosphate derivatives as potent antagonists at P2Y1 receptors, J. Med. Chem. 41 (1998) 183–190.