| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Cell Physiology and Pharmacology, University of Leicester, PO Box 138, University Road, Leicester, LE1 9HN, UK
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
S (100 µM) had no effect on holding current, but facilitated spontaneous excitatory postsynaptic current (sEPSC) frequency in 41% of recordings and facilitated spontaneous inhibitory postsynaptic currents (sIPSCs) in 20% of recordings. These were blocked by the P2 receptor antagonist suramin (100 µM). 
,ß-meATP also facilitated sEPSC and sIPSC frequency, while L-ß,-meATP facilitated only sIPSCs. The sEPSC facilitation by ATPS was blocked by TTX (but did not block facilitation of sIPSCs). sEPSC facilitation was blocked by PPADS (30 µM) and the selective P2X3 receptor antagonist A-317491 (3 µM), suggesting that modulation of sEPSCs involves P2X3 receptor subunits. ,ß-meATP-facilitated sIPSCs were also recorded in wild-type mouse MNTB neurones, but were absent in the MNTB from P2X1 receptor-deficient mice demonstrating a functional role for P2X1 receptors in the CNS.
(Received 21 April 2004;
accepted after revision 31 May 2004;
first published online 4 June 2004)
Corresponding author R. J. Evans: Department of Cell Physiology and Pharmacology, University of Leicester, PO Box 138, University Road, Leicester, LE1 9HN, UK. Email: RJE6{at}le.ac.uk
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
P2 receptors mediate a broad range of effects in the nervous system. For example, presynaptic P2X receptors regulate transmitter release (von Kugelgen et al. 1999; Nakatsuka & Gu, 2001; Smith et al. 2001) both through their depolarizing action and through direct calcium influx (Lalo & Kostyuk, 1998; Shibuya et al. 1999; Khakh & Henderson, 2000). Postsynaptic P2X receptors mediate fast excitatory transmission as well as having roles in sensory transduction and neuronal excitability (Jang et al. 2001; Vlaskovska et al. 2001). P2Y receptors exert both excitatory and inhibitory influences, regulating ion channels and transmitter release and mediating calcium waves in glial cells (Boehm et al. 1995; Harden et al. 1995; Ikeuchi & Nishizaki, 1995; Fam et al. 2000; Filippov et al. 2000).
The medial nucleus of the trapezoid body (MNTB) forms an inverting relay in the binaural auditory pathway (Barnes-Davies & Forsythe, 1995; Forsythe et al. 1998). It receives an excitatory glutamatergic input via the calyx of Held (Forsythe, 1994) and provides an inhibitory projection (Smith et al. 2000) to ipsilateral medial and lateral superior olives (MSO and LSO, respectively). MNTB neurones also receive excitatory glutamatergic inputs from non-calyceal terminals and glycinergic/GABAergic inhibitory inputs (Forsythe & Barnes-Davies, 1993; Hamann et al. 2003). Immunohistochemical, in situ and electrophysiological studies show that P2X receptors are expressed in the auditory system (Nikolic et al. 2001; Housley et al. 2002), hair cells (Glowatzki et al. 1997; Raybould & Housley, 1997; Housley et al. 1998), spiral ganglion (Salih et al. 1999) and brainstem, including the trapezoid nucleus and cochlear nucleus (Yao et al. 2000). There is also evidence for cochlea expression of P2Y receptors (Housley et al. 2002).
There are many regions in the CNS where P2 receptors have been localized but comparatively few specific functional roles have been identified. In this study, we have explored the role of P2 receptors in rat auditory brainstem. We have shown that ATP enhances excitatory and inhibitory transmission in the MNTB via distinct P2X receptor-ion channels and demonstrated a functional role for P2X1 receptor subunits in the CNS.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Transverse brainstem slices including the MNTB, were prepared as previously described (Barnes-Davies & Forsythe, 1995; Smith et al. 2000). In brief, 9- to 13-day-old Lister Hooded rats or mice (wild-type or P2X1 receptor deficient as previously described; Mulryan et al. 2000) were killed by decapitation and the brainstem removed into cooled (0–4°C) low-Na+, high-sucrose artificial cerebrospinal fluid (aCSF; see below). Transverse slices (120 µm thick) were cut sequentially in the rostral direction from the level of the 7th nerve. The slices were then incubated for 1 h at 37°C in normal aCSF (see below) bubbled with 95% O2–5% CO2, giving a pH of 7.4. Following incubation, the slice maintenance chamber was allowed to cool to room temperature.
For recording, one slice was transferred to a Peltier controlled environmental chamber mounted on the stage of an upright Axioskop microscope (Zeiss, Germany). The microscope was fitted with differential interference contrast (DIC) optics and individual cells were visualized with a x 40 water-immersion objective (Zeiss, NA 0.75). The environmental chamber (300–400 µl volume) was continuously superfused with normal aCSF (bubbled with 95% O2–5% CO2) at a rate of 0.7–1.0 ml min–1 using a peristaltic pump (Gilson, Minipuls 3), at a temperature of 27°C. Drugs were applied by switching between one of four perfusion lines, all of which entered directly into the recording chamber so as to minimize dead space.
Cell culture
Human embryonic kidney 293 (HEK-293) cells stably expressing the recombinant rat glycosilated P2X6 receptor were a gift from Dr I. P. Chessell (GlaxoSmithKline, UK). Cells were maintained in Eagle's medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids and 0.6 mg ml–1 Geneticine (Gibco BRL, UK) at 37°C in a humidified atmosphere of 5% CO2 and 95% air. When required for study, cells were attached to glass coverslips (13 mm) and used for experiment the next day.
Electrophysiological study
Recordings were made using the whole-cell patch-clamp technique as previously described (Barnes-Davies & Forsythe, 1995; Smith et al. 2000). Patch pipettes were made with a two stage vertical pipette puller (PP-83, Narishige, Japan) from standard walled filamented borosilicate glass (Clark Electromedical, GC150F-7.5) Pipette resistances were around 5 M
when filled with intracellular solution (see below). Recordings were made from visually identified MNTB principal neurones. An Axopatch 200B patch-clamp amplifier (Axon Instruments, CA, USA) was used. Series resistances were under 20 M and 70–80% compensation was used with 10 µs lag. Membrane currents were acquired by Digidata 1322A interface (Axon Instruments) with a PC computer using pCLAMP8 software (Axon Instruments). Data were filtered at 5 kHz with a low-pass Bessel filter and digitized at between 5 and 20 kHz. Spontaneous currents (sEPSCs and sIPSCs) were recorded at a holding potential of –70 mV. Analysis of sEPSCs and sIPSCs was conducted using the whole-cell analysis programs WinEDR and WinWCP (John Dempster, University of Strathclyde) with a detection amplitude threshold of 29.3 pA. For analysis of spontaneous currents we measured the number of events in 30 s intervals; control currents were measured 60–30 s before drug application, a test period was measured 30 s after the commencement of agonist perfusion and recovery was measured 5 min after drug washout. For experiments with the antagonist suramin the frequency of spontaneous events was measured 150 s after the start of suramin application. Pharmacological studies were conducted only on those MNTB neurones showing spontaneous IPSCs or EPSCs under control conditions. Resting average spontaneous rates could vary by up to 20%, hence only enhancements of over 2-fold and where the rate on washout returned to control levels were analysed further. Recordings from HEK-293 cells expressing recombinant rat P2X6 receptors were made using whole-cell patch-clamp and agonists were applied using a U-tube (Evans & Kennedy, 1994). Cells were perfused with an Etotal solution (see below). Whole-cell currents were recorded at holding potential of –60 mV.
Solution and drugs
The low-Na+, high-sucrose aCSF used for slice preparation contained (mM): 250 sucrose, 2.5 KCl, 10 glucose, 1.25 NaH2PO4, 26 NaHCO3, 2 sodium pyruvate, 3 myo-inositol, 0.5 ascorbic acid, 4 MgCl2 and 0.1 CaCl2. The normal aCSF used for incubation and control perfusion media for slices contained (mM): 125 NaCl, 2.5 KCl, 10 glucose, 1.25 NaH2PO4, 2 sodium pyruvate, 3 myo-inositol, 0.5 ascorbic acid, 2 CaCl2 and 1 MgCl2. The normal aCSF solutions were bubbled with 95% O2–5% CO2, giving a pH of 7.4. The patch solution for slices, contained (mM): 110 CsCl, 40 Hepes, 10 TEA-Cl, 1 EGTA (pH 7.3 adjusted by CsOH). The external solution for the HEK-293 cell line contained (mM): 150 NaCl, 2.5 KCl, 10 Hepes, 2.5 CaCl2 and 1 MgCl2 (pH 7.3 adjusted by NaOH). The patch solution for the HEK-293 cell line contained (mM): 140 potassium gluconate, 5 NaCl, 10 Hepes, 10 EGTA (pH 7.3 adjusted by KOH).
ATP, UTP, adenosine-5'-O-(3-thiotriphosphate) (ATPS), ,ß-methyleneATP (,ß-meATP), adenosine, suramin, pyridoxal-phosphate-6-azophenyl-2',4'-di-sulphonate (iso-PPADS) and tetrodotoxin (TTX) were purchased from Sigma (UK). 6-Cyano-7-nitro-quinoxaline-2,3-dione (CNQX), strychnine and bicuculline were purchased from Tocris Cookson (St Louis, MO, USA). L-ß,-Methylene ATP (L-ß,-meATP) was purchased from RBI (UK). A-317491 was a gift from Abbott Laboratories, USA.
Statistical analyses
Statistical analyses were performed with the Dunnett multiple comparisons or Chi-squared tests, with P < 0.05 considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Two distinct types of spontaneous synaptic transient inward currents were recorded from MNTB neurones in the brain slice; (i) fast currents with a half-decay time of 
3 ms and (ii) slow currents that decayed with a half-time of > 3 ms. Using a CsCl2-based internal solution and holding potential of –70 mV, both excitatory and inhibitory synaptic inputs gave rise to inward currents. The MNTB expresses AMPA receptors of the GluRD ‘flop’ spliced-variant that have rapid kinetics and are calcium permeable (Geiger et al. 1995), giving fast EPSC decay time constants (Barnes-Davies & Forsythe, 1995; Taschenberger & von Gersdorff, 2000). AMPA receptor-mediated currents were blocked by the glutamate receptor antagonist CNQX (10 µM) in all cells tested (5/5) (Fig. 1A, C and D). The slow currents mediated by corelease of GABA and glycine (Jonas et al. 1998; Smith et al. 2000) were abolished by combined application of the glycine receptor antagonist strychnine (1 µM) and the GABAA receptor antagonist bicuculline (10 µM) in all cells tested (5/5) (Fig. 1B, C and D). TTX (0.5 µM) had no effect on basal rates of spontaneous IPSCs or EPSCs in all cells tested (17/17). Co-application of all three antagonists blocked all spontaneous synaptic currents (Fig. 1A and B). Therefore spontaneous synaptic currents in the MNTB arise from glutamatergic ‘fast’ sEPSCs and mixed glycine–GABAergic ‘slow’ sIPSCs.
|
S evoked transient facilitation of sEPSC and sIPSC frequency
We applied ATPS, the metabolically stable ATP analogue, to examine the effects of P2 receptor activation on auditory brainstem activity. ATPS (100 µM) had no effect on the holding current of MNTB neurones (n=169), indicating that functional P2X receptors are not expressed on the cell body or dendrites of MNTB neurones. However, in 53% of neurones ATPS showed a dramatic potentiation of spontaneous synaptic current frequency (see Table 1; 47% of neurones showed no change in spontaneous synaptic currents). The effect was transient and decayed during the continued application of ATPS (over a time course of around 1–2 min). Analysis of this increased activity revealed three different patterns (Figs 2 and 3); (1) the most common response (34%, 57/169) was a transient increase in sEPSC frequency (Fig. 2); (2) 12% (20/169) of MNTB neurones showed a large increase in sIPSC frequency (Fig. 3A), and (3) 8% of neurones (13/169) showed a large increase of both sEPSC and sIPSC frequency (Fig. 3B). Overall, 41% of MNTB neurones showed increased sEPSC frequency and 20% showed increased sIPSC frequency (Table 1, Fig. 3C), however, ATPS had no effect on the mean current amplitude of sEPSCs (101 ± 8% of control) or sIPSCs (94 ± 7% of control). Following 3 min pre-application of suramin, co-application with ATPS had no effect on either sIPSC or sEPSC frequency (11/11 neurones) (Fig. 4). As a positive control following washout of suramin, ATPS increased sIPSC and sEPSC frequency in 27% and 55% of neurones (3/11 and 6/11), respectively. These results indicate that ATPS mediates the increase in spontaneous synaptic transmission through the activation of P2 receptors.
|
|
|
|
ATPS is an effective agonist at P2X receptors and at some P2Y receptors. To investigate which P2 receptors are expressed in the auditory brainstem we used a range of nucleotide agonists showing some P2Y subtype specificity and with action at P2X receptors. P2Y1, P2Y11, P2Y12 and P2Y13 are ADP sensitive, P2Y2 and P2Y4 receptors are UTP sensitive, and the P2Y6 receptor is UDP sensitive (Webb et al. 1993; Chen et al. 1996; Communi et al. 1996; Nicholas et al. 1996; Bogdanov et al. 1998; Hollopeter et al. 2001; Zhang et al. 2002).
UTP, UDP and ADP (100 µM) preferentially activate specific P2Y receptors but had no effect on the holding current of MNTB neurones or on the frequency of spontaneous synaptic currents (Fig. 5A, Table 1). As a positive control following the washout of ADP, UTP or UDP, ATPS increased the frequency of spontaneous synaptic events (Fig. 5A). ATP is metabolically unstable and is degraded to ADP, AMP and adenosine by ecto-nucleotidases (Kegel et al. 1997; Cunha et al. 1998; Ohkubo et al. 2000). For example in the caudal regions of the rat nucleus tractus solitarii, ATP mediates excitatory transmission indirectly through breakdown to adenosine and activation of A1 receptors (Cunha et al. 1998; Kato & Shigetomi, 2001). In the present study adenosine (10 µM) had no effect on resting frequency or ATPS-induced facilitation (Table 1). ATP (100 µM) had similar effects to ATPS on sEPSC and sIPSC frequency (Table 1), indicating that it was acting directly through P2X receptors (ATP had no significant effect on the mean current amplitude of sEPSCs 92 ± 7% (n= 9) and sIPSCs 97 ± 3% of control (n= 4)). These results indicate that P2Y or P1 adenosine receptors do not regulate MNTB spontaneous activity and that ATPS effects are most likely to be mediated through P2X receptors. The lack of expression of P2X receptors on acutely dissociated astrocytes suggests that ATPS is acting directly at neuronal P2X receptors to modulate release of excitatory mediators, e.g. glutamate (Jeremic et al. 2001).
|
To further characterize the P2X receptor subtypes underlying the ATPS-mediated responses we used metabolically stable and subtype-selective agonists. ,ß-meATP is an agonist at recombinant P2X receptors containing P2X1, P2X3 or P2X6 receptor subunits (e.g. homomeric P2X3 and heteromeric P2X2/3 receptors; North, 2002; Jones et al. 2004 and current study) and evoked increases in sIPSC and sEPSC frequency similar to ATPS (Table 1, Fig. 5B–D) with no effect on the mean current amplitudes (sEPSCs 100 ± 10% of control (n= 7) and sIPSCs 102 ± 13% control (n= 4)). The percentages of neurones that responded to ATPS, ATP and ,ß-meATP were similar, indicating no additional expression of ATPS-sensitive but ,ß-meATP-insensitive P2X receptors (Table 1). The relative contribution of individual subunits was investigated using L-ß,-meATP, which acts at P2X1 receptors, but not at P2X3 receptors (Evans et al. 1995; Lewis et al. 1995; Grubb & Evans, 1999). The properties of recombinant P2X6 homomeric receptors have been described recently; this receptor only forms functional channels when correctly glycosylated (Jones et al. 2004). ATP (100 µM), ,ß-meATP (100 µM) and L-ß,-meATP (100 µM) all evoked inward currents at P2X6 receptors (9.9 ± 1.2, 12.0 ± 0.9 and 4.5 ± 0.4 pA pF–1, respectively, Fig. 6A) expressed in HEK-293 cells.
|
-meATP (100 µM) evoked increases in sIPSC frequency (with no effect on the mean current amplitude 104 ± 9% of control (n= 5) but did not change sEPSC frequency (Fig. 6B–D, Table 1). The similar percentage of inhibitory neurones responding to ATPS, ATP, ,ß-meATP and L-ß,-meATP indicates that there are no additional inhibitory neurones expressing ATPS-sensitive but L-ß,-meATP-insensitive P2X receptors. To determine whether the ,ß-meATP-sensitive, but L-ß,-meATP-insensitive increase in sEPSCs was mediated by receptors containing P2X3 receptor subunits we used the selective P2X3 receptor antagonist A-317491 (Jarvis et al. 2002). ATPS-evoked increases in sEPSCs were abolished by A-317491 (3 µM) and iso-PPADS (30 µM) (Fig. 7).
|
,ß-meATP and L-ß,-meATP of the sIPSCs suggested the involvement of either P2X1 or P2X6 receptor subunits and is similar to that described recently in the somatosensory cortex (Pankratov et al. 2003). To determine the contribution of P2X1 receptors we compared responses in MNTB neurones from wild-type and P2X1 receptor-deficient mice (Mulryan et al. 2000). The sIPSC frequency was potentiated by ,ß-meATP in 8/8 MNTB neurones from wild-type mice, but ,ß-meATP failed to evoke any change in sIPSC frequency recorded from P2X1 receptor-deficient mouse MNTB neurones (0/5) (
2P < 0.01). These results demonstrate that in the mouse and probably in the rat, P2X1 receptors are expressed on the inhibitory projections to the MNTB.
Is action potential firing required for the modulatory actions of ATPS on sEPSC and sIPSC frequency in MNTB neurones?
The P2X receptor effects may depend on the cellular localization of the receptors; for example in intrinsic sensory neurones P2X receptors are present on the cell body and their activation leads to depolarization and firing of action potentials (Bertrand & Bornstein, 2002). P2X receptors may also be present at the presynaptic nerve terminal and directly regulate transmitter release via calcium influx through the receptor ion channel (Rhee et al. 2000; Kato & Shigetomi, 2001; Nakatsuka & Gu, 2001). We used the voltage-gated sodium channel blocker tetrodotoxin (TTX) to determine whether action potential propagation was required for P2X receptor-mediated facilitation of spontaneous synaptic transmission. Figure 8 shows the typical effect of TTX (0.5 µM) on spontaneous currents and the ATPS-evoked facilitation. TTX did not change the frequency of spontaneous currents in any neurones (17/17) and in the presence of TTX, ATPS no longer facilitated sEPSC frequency (Table 1, Fig. 8C). However the proportion of cells responding to ATPS with an increase in sIPSCs was unchanged (Table 1, Fig. 8C). These results suggest that P2X receptors are present predominantly on the cell body or fibre tracts in excitatory pathways, while for inhibitory pathways they are located on the presynaptic nerve terminals.
|
In order to determine if the facilitation of sIPSC frequency was Ca2+ dependent we tested the effect of ATPS in low external Ca2+ solution (low [Ca2+]o) and in the presence of TTX. As a positive control in 0.5 µM TTX, ATPS facilitated sIPSC frequency in 24% (4/17) of cells (Table 1, Fig. 8). After 5 min perfusion of low [Ca2+]o, ATPS no longer facilitated sIPSC frequency in any of the 16 cells tested (Table 1, Fig. 8), indicating that calcium influx via presynaptic P2X receptors mediates the increase in sIPSCs frequency.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
|
S (or indeed any of a number of purinergic agonists) had no effect on the holding current of MNTB neurones, suggesting that functional P2X receptor-ion channels are not present on MNTB somas (or in their short dendritic tree). P2X receptors could be present on the axonal projections of the MNTB, but this remains to be determined. In contrast, ATPS gave a profound increase in spontaneous transmitter release from both excitatory (11.5-fold) and inhibitory inputs (3.6-fold), indicating a high degree of purinergic regulation within the superior olivary complex. However not all inputs responded with an increase in activity (41% of excitatory and 20% of inhibitory inputs showed potentiation), suggesting that there is a heterogeneity in the neuronal input to the MNTB. ATPS is an agonist at all P2X receptors and many P2Y receptors. We have shown that only P2X receptors were involved in the reported effects since a range of P2Y receptor agonists (selective for all currently identified P2Y receptor subtypes) had no effect on spontaneous synaptic currents in the MNTB. Using subtype-selective purinergic agonists we have demonstrated the presence of molecularly distinct P2X receptors on excitatory and inhibitory inputs. MNTB neurones receive excitatory input from a single giant synapse (calyx of Held), which covers around half of the somatic surface area, as well as conventional glutamatergic synaptic terminals (Hamann et al. 2003). P2X receptor-mediated potentiation of sEPSCs was sensitive to TTX, indicating that the receptors were located predominantly on cell bodies or axon tracts of excitatory fibres innervating the MNTB (it is unlikely that a polysynaptic pathway is involved due to the orientation during cutting of the brain slice). The giant calyceal input is unlikely to be involved in P2X receptor stimulation as the increases in sEPSCs were blocked by TTX, hence they must require action potential propagation. Such spontaneous action potentials would generate giant sEPSCs at the calyx of Held, which were never observed. However, presynaptic P2X receptors have been observed at other giant synapses (Sun & Stanley, 1996). Previous reports have demonstrated a modest depression of the evoked EPSC at the calyx of Held/MNTB synapse by adenosine (Barnes-Davies & Forsythe, 1995) and in the present study adenosine had no effect on the frequency of sEPSCs. Thus the potentiation of spontaneous excitatory events is probably via non-calyceal high threshold excitatory inputs innervating the MNTB, as previously described (Hamann et al. 2003).
In addition to ATPS stimulation, transmitter release from the excitatory terminals was potentiated by ,ß-meATP (an agonist at recombinant receptors containing P2X1, P2X3 and P2X6 receptor subunits). The potentiation of sEPSCs was abolished by the P2X3 receptor-selective antagonist A-317491 (Jarvis et al. 2002) and insensitive to the P2X1 and P2X6 receptor subunit agonist L-ß,-meATP (Evans et al. 1995; Buell et al. 1996). These results indicate that the high threshold excitatory neurones express P2X3 receptor subunits. P2X3 receptor subunits are thought to be expressed predominantly, if not exclusively, on sensory nerves, suggesting that either the MNTB receives a sensory afferent input or more likely, given the anatomy of the brainstem, that P2X3 receptors are not expressed exclusively on sensory fibres. The incorporation of P2X3 receptor subunits into heteromeric channels can confer ,ß-meATP sensitivity to the resulting channel, e.g. P2X2/3 (where P2X2 receptor homomeric channels are essentially insensitive to ,ß-meATP) (Lewis et al. 1995; Le et al. 1998). The desensitizing nature of the response (1–2 min) is considerably slower than that for recombinant P2X3 receptors (1–2 s) and suggests that the P2X3 receptor subunit contributes ,ß-meATP sensitivity to a heteromeric P2X receptor on excitatory neurones.
The MNTB receives inhibitory drive from recurrent inputs and from other brainstem nuclei of the superior olivary complex (Guinan & Stankovic, 1996; Kopp-Scheinpflug et al. 2002). The mixed GABA–glycine-mediated sIPSCs reported are similar to those characterized previously in the medial superior olive (Smith et al. 2000). The sensitivity of P2X receptors located on inhibitory inputs to L-ß,-meATP (P2X1 or P2X6 receptor subunit selective) and the abolition of responses in P2X1 receptor-deficient mice suggest that the P2X channel on these neurones expresses P2X1 receptor subunits. This is consistent with in situ hybridization (Kidd et al. 1995; Collo et al. 1996) and immunohistochemical studies (Xiang et al. 1998; Yao et al. 2000; Rubio & Soto, 2001) that have shown these subunits to be expressed in the CNS and brainstem (Yao et al. 2000). However, it is unlikely that the response corresponds to homomeric P2X1 receptors as these show rapid desensitization (1–2 s) (Evans et al. 1995). It seems more likely that P2X1 receptor subunits contribute ,ß-meATP and L-ß,-meATP sensitivity to a heteromeric P2X receptor on the terminals of inhibitory neurones similar, for example, to the P2X1/2 heteromeric channel in superior cervical ganglion neurones (Calvert & Evans, 2004).
The TTX insensitivity of the P2X agonist effects on inhibitory pathways demonstrates that action potential propagation is not involved and suggests that the P2X receptors are present on the presynaptic nerve terminal. This is consistent with previous studies demonstrating presynaptic P2X receptors regulating transmitter release (Sun & Stanley, 1996; Nakatsuka & Gu, 2001; Smith et al. 2001) and including inhibitory glycinergic (Rhee et al. 2000; Jang et al. 2001) and GABAergic neurones (Hugel & Schlichter, 2000).
In summary we have shown that ATP can regulate both excitatory and inhibitory inputs to MNTB neurones by the discrete localization of functional P2X receptor subtypes in the brainstem; one mechanism is via P2X3-containing receptors located on excitatory neuronal cell bodies and a second is via presynaptic P2X1-containing receptors located on inhibitory neurones. P2X receptors have been described in the primary sensory apparatus of the cochlea and this study demonstrates that these receptors can also play a functional role in the regulation of auditory processing at the level of the brainstem. This is the first time that a functional role of P2X1 receptors has been demonstrated in the central nervous system.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Bertrand PP & Bornstein JC (2002). ATP as a putative sensory mediator: activation of intrinsic sensory neurons of the myenteric plexus via P2X receptors. J Neurosci 22, 4767–4775.
Boehm S, Huck S & Illes P (1995). UTP- and ATP-triggered transmitter release from rat sympathetic neurones via separate receptors. Br J Pharmacol 116, 2341–2343.[Medline]
Bogdanov YD, Wildman SS, Clements MP, King BF & Burnstock G (1998). Molecular cloning and characterization of rat P2Y4 nucleotide receptor. Br J Pharmacol 124, 428–430.[CrossRef][Medline]
Buell G, Lewis C, Collo G, North RA & Surprenant A (1996). An antagonist-insensitive P2X receptor expressed in epithelia and brain. EMBO J 15, 55–62.[Medline]
Calvert JA & Evans RJ (2004). Heterogeneity of P2X receptors in sympathetic neurons: contribution of neuronal P2X1 receptors revealed using knockout mice. Mol Pharmacol 65, 139–148.
Chang K, Hanaoka K, Kumada M & Takuwa Y (1995). Molecular cloning and functional analysis of a novel P2 nucleotide receptor. J Biol Chem 270, 26152–26158.
Chen ZP, Krull N, Xu S, Levy A & Lightman SL (1996). Molecular cloning and functional characterization of a rat pituitary G protein-coupled adenosine triphosphate (ATP) receptor. Endocrinol 137, 1833–1840.[Abstract]
Collo G, North RA, Kawashima E, Merlo-Pich E, Neidhart S, Surprenant A & Buell G (1996). Cloning OF P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels. J Neurosci 16, 2495–2507.
Communi D, Govaerts C, Parmentier M & Boeynaems JM (1997). Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase. J Biol Chem 272, 31969–31973.
Communi D, Parmentier M & Boeynaems JM (1996). Cloning, functional expression and tissue distribution of the human P2Y6 receptor. Biochem Biophys Res Commun 222, 303–308.[CrossRef][Medline]
Cunha RA, Sebastiao AM & Ribeiro JA (1998). Inhibition by ATP of hippocampal synaptic transmission requires localized extracellular catabolism by ecto-nucleotidases into adenosine and channeling to adenosine A1 receptors. J Neurosci 18, 1987–1995.
Evans RJ & Kennedy C (1994). Characterization of P2-purinoceptors in the smooth muscle of the rat tail artery: a comparison between contractile and electrophysiological responses. Br J Pharmacol 113, 853–860.[Medline]
Evans R, Lewis C, Buell G, Valera S, North R & Surprenant A (1995). Pharmacological characterization of heterologously expressed ATP-gated cation channels (P2x purinoceptors). Mol Pharmacol 48, 178–183.[Abstract]
Evans R, Lewis C, Virginio C, Lundstrom K, Buell G, Surprenant A & North R (1996). Ionic permeability of, and divalent cation effects on, two ATP-gated cation channels (P2X receptors) expressed in mammalian cells. J Physiol 497, 413–422.
Fam SR, Gallagher CJ & Salter MW (2000). P2Y1 purinoceptor-mediated Ca2+ signaling and Ca2+ wave propagation in dorsal spinal cord astrocytes. J Neurosci 20, 2800–2808.
Filippov AK, Brown DA & Barnard EA (2000). The P2Y1 receptor closes the N-type Ca2+ channel in neurones, with both adenosine triphosphates and diphosphates as potent agonists. Br J Pharmacol 129, 1063–1066.[CrossRef][Medline]
Forsythe ID (1994). Direct patch recording from identified presynaptic terminals mediating glutamatergic EPSCs in the rat CNS, in vitro. J Physiol 479, 381–387.
Forsythe ID & Barnes-Davies M (1993). The binaural auditory pathway: excitatory amino acid receptors mediate dual timecourse excitatory postsynaptic currents in the rat medial nucleus of the trapezoid body. Proc R Soc Lond B Biol Sci 251, 151–157.[Medline]
Forsythe ID, Tsujimoto T, Barnes-Davies M, Cuttle MF & Takahashi T (1998). Inactivation of presynaptic calcium current contributes to synaptic depression at a fast central synapse. Neuron 20, 797–807.[CrossRef][Medline]
Garcia-Guzman M, Soto F, Gomez-Hernandez JM, Lund PE & Stuhmer W (1997). Characterization of recombinant human P2X4 receptor reveals pharmacological differences to the rat homologue. Mol Pharmacol 51, 109–118.
Geiger JR, Melcher T, Koh DS, Sakmann B, Seeburg PH, Jonas P & Monyer H (1995). Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS. Neuron 15, 193–204.[CrossRef][Medline]
Glowatzki E, Ruppersberg JP, Zenner HP & Rusch A (1997). Mechanically and ATP-induced currents of mouse outer hair cells are independent and differentially blocked by d-tubocurarine. Neuropharmacology 36, 1269–1275.[CrossRef][Medline]
Grubb BD & Evans RJ (1999). Characterization of cultured dorsal root ganglion neuron P2X receptors. Eur J Neurosci 11, 149–154.[CrossRef][Medline]
Guinan JJ Jr & Stankovic KM (1996). Medial efferent inhibition produces the largest equivalent attenuations at moderate to high sound levels in cat auditory-nerve fibers. J Acoust Soc Am 100, 1680–1690.[CrossRef][Medline]
Hamann M, Billups B & Forsythe ID (2003). Non-calyceal excitatory inputs mediate low fidelity synaptic transmission in rat auditory brainstem slices. Eur J Neurosci 18, 2899–2902.[CrossRef][Medline]
Harden TK, Boyer JL & Nicholas RA (1995). P2-purinergic receptors: subtype-associated signaling responses and structure. Annu Rev Pharmacol Toxicol 35, 541–579.[CrossRef][Medline]
Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V, Yang RB, Nurden P, Nurden A, Julius D & Conley PB (2001). Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 409, 202–207.[CrossRef][Medline]
Housley GD, Jagger DJ, Greenwood D, Raybould NP, Salih SG, Jarlebark LE, Vlajkovic SM, Kanjhan R, Nikolic P, Munoz DJ & Thorne PR (2002). Purinergic regulation of sound transduction and auditory neurotransmission. Audiol Neurootol 7, 55–61.[CrossRef][Medline]
Housley GD, Luo L & Ryan AF (1998). Localization of mRNA encoding the P2X2 receptor subunit of the adenosine 5'-triphosphate-gated ion channel in the adult and developing rat inner ear by in situ hybridization. J Comp Neurol 393, 403–414.[CrossRef][Medline]
Hugel S & Schlichter R (2000). Presynaptic P2X receptors facilitate inhibitory GABAergic transmission between cultured rat spinal cord dorsal horn neurons. J Neurosci 20, 2121–2130.
Ikeuchi Y & Nishizaki T (1995). The P2Y purinoceptor-operated potassium channel is possibly regulated by the beta gamma subunits of a pertussis toxin-insensitive G-protein in cultured rat inferior colliculus neurons. Biochem Biophys Res Commun 214, 589–596.[CrossRef][Medline]
Inoue K (1998). ATP receptors for the protection of hippocampal functions. Jpn J Pharmacol 78, 405–410.[CrossRef][Medline]
Jang IS, Rhee JS, Kubota H & Akaike N (2001). Developmental changes in P2X purinoceptors on glycinergic presynaptic nerve terminals projecting to rat substantia gelatinosa neurones. J Physiol 536, 505–519.
Jarvis MF, Burgard EC, McGaraughty S, Honore P, Lynch K, Brennan TJ, Subieta A, Van Biesen T, Cartmell J, Bianchi B, Niforatos W & Kage K., Yu, H, Mikusa J, Wismer CT, Zhu CZ, Chu K, Lee CH, Stewart AO, Polakowski J, Cox BF, Kowaluk E, Williams M, Sullivan J & Faltynek C (2002). A-317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat. Proc Natl Acad Sci U S A 99, 17179–17184.
Jeremic A, Jeftinija K, Stevanovic J, Glavaski A & Jeftinija S (2001). ATP stimulates calcium-dependent glutamate release from cultured astrocytes. J Neurochem 77, 664–675.[CrossRef][Medline]
Jo YH & Schlichter R (1999). Synaptic corelease of ATP and GABA in cultured spinal neurons. Nat Neurosci 2, 241–245.[CrossRef][Medline]
Jonas P, Bischofberger J & Sandkuhler J (1998). Corelease of two fast neurotransmitters at a central synapse. Science 281, 419–424.
Jones CA, Vial C, Sellers LA, Humphrey PP, Evans RJ & Chessell IP (2004). Functional regulation of P2X6 receptors by N-linked glycosylation: identification of a novel ,ß-methylene ATP-sensitive phenotype. Mol Pharmacol 65, 979–985.
Kato F & Shigetomi E (2001). Distinct modulation of evoked and spontaneous EPSCs by purinoceptors in the nucleus tractus solitarii of the rat. J Physiol 530, 469–486.
Kegel B, Braun N, Heine P, Maliszewski CR & Zimmermann H (1997). An ecto-ATPase and an ecto-ATP diphosphohydrolase are expressed in rat brain. Neuropharmacol 36, 1189–1200.[CrossRef][Medline]
Khakh BS & Henderson G (2000). Modulation of fast synaptic transmission by presynaptic ligand-gated cation channels. J Auton Nerv Syst 81, 110–121.[CrossRef][Medline]
Kidd EJ, Grahames CB, Simon J, Michel AD, Barnard EA & Humphrey PP (1995). Localization of P2X purinoceptor transcripts in the rat nervous system. Mol Pharmacol 48, 569–573.[Abstract]
Kopp-Scheinpflug C, Lippe WR, Dorrscheidt GJ & Rubsamen R (2002). The medial nucleus of the trapezoid body in the gerbil is more than a relay: comparison of pre- and postsynaptic activity. J Assoc Res Otolaryngol 8, 8.
Lalo U & Kostyuk P (1998). Developmental changes in purinergic calcium signalling in rat neocortical neurones. Brain Res Dev Brain Res 111, 43–50.[CrossRef][Medline]
Le KT, Babinski K & Seguela P (1998). Central P2X4 and P2X6 channel subunits coassemble into a novel heteromeric ATP receptor. J Neurosci 18, 7152–7159.
Lewis C, Neidhart S, Holy C, North RA, Buell G & Surprenant A (1995). Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons. Nature 377, 432–435.[CrossRef][Medline]
Mulryan K, Gitterman DP, Lewis CJ, Vial C, Leckie BJ, Cobb AL, Brown JE, Conley EC, Buell G, Pritchard CA & Evans RJ (2000). Reduced vas deferens contraction and male infertility in mice lacking P2X1 receptors. Nature 403, 86–89.[CrossRef][Medline]
Nakatsuka T & Gu JG (2001). ATP P2X receptor-mediated enhancement of glutamate release and evoked EPSCs in dorsal horn neurons of the rat spinal cord. J Neurosci 21, 6522–6531.
Nicholas RA, Watt WC, Lazarowski ER, Li Q & Harden K (1996). Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: identification of a UDP-selective, a UTP-selective, and an ATP- and UTP-specific receptor. Mol Pharmacol 50, 224–229.[Abstract]
Nikolic P, Housley GD, Luo L, Ryan AF & Thorne PR (2001). Transient expression of P2X1 receptor subunits of ATP-gated ion channels in the developing rat cochlea. Brain Res Dev Brain Res 126, 173–182.[CrossRef][Medline]
North RA (2002). Molecular physiology of P2X receptors. Physiol Rev 82, 1013–1067.
Ohkubo S, Kimura J & Matsuoka I (2000). Ecto-alkaline phosphatase in NG108-15 cells: a key enzyme mediating P1 antagonist-sensitive ATP response. Br J Pharmacol 131, 1667–1672.[CrossRef][Medline]
Pankratov Y, Lalo U, Krishtal O & Verkhratsky A (2003). P2X receptor-mediated excitatory synaptic currents in somatosensory cortex. Mol Cell Neurosci 24, 842–849.[CrossRef][Medline]
Queiroz G, Meyer DK, Meyer A, Starke K & von Kugelgen I (1999). A study of the mechanism of the release of ATP from rat cortical astroglial cells evoked by activation of glutamate receptors. Neuroscience 91, 1171–1181.[CrossRef][Medline]
Raybould NP & Housley GD (1997). Variation in expression of the outer hair cell P2X receptor conductance along the guinea-pig cochlea. J Physiol 498, 717–727.
Rhee JS, Wang ZM, Nabekura J, Inoue K & Akaike N (2000). ATP facilitates spontaneous glycinergic IPSC frequency at dissociated rat dorsal horn interneuron synapses. J Physiol 524, 471–483.
Rice WR, Burton FM & Fiedeldey DT (1995). Cloning and expression of the alveolar type II cell P2u-purinergic receptor. Am J Respir Cell Mol Biol 12, 27–32.[Abstract]
Richardson PJ & Brown SJ (1987). ATP release from affinity-purified rat cholinergic nerve terminals. J Neurochem 48, 622–630.[CrossRef][Medline]
Rubio ME & Soto F (2001). Distinct localization of P2X receptors at excitatory postsynaptic specializations. J Neurosci 21, 641–653.
Salih SG, Housley GD, Raybould NP & Thorne PR (1999). ATP-gated ion channel expression in primary auditory neurones. Neuroreport 10, 2579–2586.[Medline]
Shibuya I, Tanaka K, Hattori Y, Uezono Y, Harayama N, Noguchi J, Ueta Y, Izumi F & Yamashita H (1999). Evidence that multiple P2X purinoceptors are functionally expressed in rat supraoptic neurones. J Physiol 514, 351–367.
Smith AB, Hansen MA, Liu DM & Adams DJ (2001). Pre- and postsynaptic actions of ATP on neurotransmission in rat submandibular ganglia. Neuroscience 107, 283–291.[CrossRef][Medline]
Smith AJ, Owens S & Forsythe ID (2000). Characterisation of inhibitory and excitatory postsynaptic currents of the rat medial superior olive. J Physiol 529, 681–698.
Sun XP & Stanley EF (1996). An ATP-activated, ligand-gated ion channel on a cholinergic presynaptic nerve terminal. Proc Natl Acad Sci U S A 93, 1859–1863.
Surprenant A, Schneider DA, Wilson HL, Galligan JJ & North RA (2000). Functional properties of heteromeric P2X1/5 receptors expressed in HEK cells and excitatory junction potentials in guinea-pig submucosal arterioles. J Auton Nerv Syst 81, 249–263.[CrossRef][Medline]
Taschenberger H & von Gersdorff H (2000). Fine-tuning an auditory synapse for speed and fidelity: developmental changes in presynaptic waveform, EPSC kinetics, and synaptic plasticity. J Neurosci 20, 9162–9173.
Tokuyama Y, Hara M, Jones EM, Fan Z & Bell GI (1995). Cloning of rat and mouse P2Y purinoceptors. Biochem Biophys Res Commun 211, 211–218.[CrossRef][Medline]
Virginio C, North RA & Surprenant A (1998a). Calcium permeability and block at homomeric and heteromeric P2X2 and P2X3 receptors, and P2X receptors in rat nodose neurones. J Physiol 510, 27–35.
Vlaskovska M, Kasakov L, Rong W, Bodin P, Bardini M, Cockayne DA, Ford AP & Burnstock G (2001). P2X3 knock-out mice reveal a major sensory role for urothelially released ATP. J Neurosci 21, 5670–5677.
von Kugelgen I, Goncalves J, Driessen B & Starke K (1998). Corelease of noradrenaline and adenosine triphosphate from sympathetic neurones. Adv Pharmacol 42, 120–125.[Medline]
von Kugelgen I, Norenberg W, Meyer A, Illes P & Starke K (1999). Role of action potentials and calcium influx in ATP- and UDP-induced noradrenaline release from rat cultured sympathetic neurones. Naunyn Schmiedebergs Arch Pharmacol 359, 360–369.[CrossRef][Medline]
Webb TE, Simon J, Krishek BJ, Bateson AN, Smart TG, King BF, Burnstock G & Barnard EA (1993). Cloning and functional expression of a brain G-protein-coupled ATP receptor. FEBS Lett 324, 219–225.[CrossRef][Medline]
Xiang Z, Bo X & Burnstock G (1998). Localization of ATP-gated P2X receptor immunoreactivity in rat sensory and sympathetic ganglia. Neurosci Lett 256, 105–108.[CrossRef][Medline]
Yao ST, Barden JA, Finkelstein DI, Bennett MR & Lawrence AJ (2000). Comparative study on the distribution patterns of P2X1-P2X6 receptor immunoreactivity in the brainstem of the rat and the common marmoset (Callithrix jacchus): association with catecholamine cell groups. J Comp Neurol 427, 485–507.[CrossRef][Medline]
Zhang FL, Luo L, Gustafson E, Palmer K, Qiao X, Fan X, Yang S, Laz TM, Bayne M & Monsma F Jr (2002). P2Y13: identification and characterization of a novel Galphai-coupled ADP receptor from human and mouse. J Pharmacol Exp Ther 301, 705–713.
|
Acknowledgements |
|---|
This article has been cited by other articles:
|
|
 |
|
  L. S. Harrington, R. J. Evans, J. Wray, L. Norling, K. E. Swales, C. Vial, F. Ali, M. J. Carrier, and J. A. Mitchell Purinergic 2X1 Receptors Mediate Endothelial Dependent Vasodilation to ATP Mol. Pharmacol., November 1, 2007; 72(5): 1132 - |
