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P2 receptors modulate respiratory rhythm but do not contribute to central CO2 sensitivity in vitro A.R. Lorier1 , K. Peebles, T. Brosenitsch, D.M. Robinson, G.D. Housley, G.D. Funk∗ Department of Physiology, Faculty of Medicine and Health Science, University of Auckland, Private Bag 92019, Auckland, New Zealand Accepted 14 April 2004
Abstract Multiple brainstem sites are proposed to contribute to central respiratory chemosensitivity, however, the underlying molecular mechanisms remain unknown. P2X2 subunit-containing ATP receptors, which mediate pH-sensitive currents, appear to contribute to central chemosensitivity in vivo [J. Physiol. 523 (2000) 441]. However, recent data from P2X2 knockout mice [J. Neurosci. 23 (2003) 11315] indicate that they are not essential. To further explore the role of P2 receptors in central chemosensitivity, we examined the effects of P2 receptor agonists/antagonists on respiratory-related activity and CO2 -sensitivity of rhythmically-active in vitro preparations from neonatal rat. Our main findings: (i) that putative chemosensitive regions of the ventrolateral medulla are immunoreactive for the P2X2 subunit; (ii) that ATP potentiates respiratory frequency in a dose-dependent, and PPADS-sensitive (P2 receptor antagonist), manner; and (iii) that the increase in burst frequency produced by increasing CO2 is unaffected by PPADS, indicate that ATP is a potent modulator of respiratory activity, but that P2 receptors do not contribute to central chemosensitivity in vitro. © 2004 Elsevier B.V. All rights reserved. Keywords: Chemosensitivity, central; CO2 , chemosensitivity; Mammals, rat; Pharmacological agents, P2 receptor antagonist PPADS; Receptors, P2
1. Introduction One of the most important tonic modulatory inputs to the central respiratory network is from central chemoreceptors. Their activity not only maintains ∗ Corresponding author. Present address: Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, 7-50 Medical Sciences Building, Edmonton, Alta., Canada T6G 2H7. Tel.: +1-780-492-8330; fax: +1-780-492-8915. E-mail address:
[email protected] (G.D. Funk). 1 Present address: Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, 7-50 Medical Sciences Building, Edmonton, Alta., Canada T6G 2H7.
respiratory activity at rest, increased activation of central chemoreceptors by elevations in PCO2 and the associated decrease in extracellular pH elicits rapid increases in ventilation that are crucial for the homeostatic regulation of breathing (Cherniack, 1993). The type and location of chemosensitive neurons mediating this response, however, are not known. Historically, chemosensitivity has been attributed to regions along the ventral surface of the medulla. However, recent data suggest a more generalized distribution of chemosensitivity within the brainstem to include not only regions along the ventral medulla such as the retrotrapezoid nucleus (Li and Nattie, 1997), but also
1569-9048/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2004.04.007
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the region of the nucleus tractus soliatarius (NTS) (Dean et al., 1989), locus coeruleus (Oyamada et al., 1998), rostral aspect of the ventral respiratory group (VRG) (Kawai et al., 1996) and the medullary raphe (Richerson, 1995). The intriguing possibility that respiratory neurons themselves contribute to central chemosensitivity has also been raised (Kawai et al., 1996). The adequate stimulus for central chemosensitive neurons is believed to be the local changes in H+ concentration that result from changes in PCO2 (Cherniack, 1993; Ritucci et al., 1998). However, the molecular mechanisms underlying central chemosensitivity remain unknown. The recent suggestion that ionotropic, ATP-gated ion channels, P2X receptors, may contribute (Spyer and Thomas, 2000) is controversial. P2X receptor involvement is supported by several observations. (i) Currents mediated by P2X receptors containing the P2X2 subunit, are significantly potentiated by physiologically-relevant reductions in pH (King et al., 1996). (ii) P2X2 receptor and mRNA labeling are apparent throughout respiratory regions of the medulla in adult rat, including a number of putative chemosensitive regions (Kanjhan et al., 1999; Yao et al., 2000; Thomas et al., 2001; Gourine et al., 2003). (iii) Eighty-five percent of the inspiratory neurons in the preBötzinger complex (preBötC) are chemosensitive and this chemosensitivity is blocked by P2 receptor antagonists (Thomas and Spyer, 2000). (iv) The gain of the CO2 response curve in anaesthetized rat is reduced by local injections into the ventral–lateral medulla (VLM) of the general P2 receptor antagonist suramin and the desensitizing agonist ␣,-meATP (Thomas et al., 1999). On the other hand, it has recently been demonstrated that central respiratory CO2 sensitivity is unaffected in mice lacking P2X2 receptors (Rong et al., 2003). Thus, either P2X receptors are not involved in central CO2 sensitivity, or they contribute (in a non-essential manner), and non-P2X receptor-dependent mechanisms compensate for the loss of the P2X receptor component during development in the knockout animals. The main objective of this study was to further explore the contribution of P2 receptors in mediating central respiratory chemosensitivity. To this end, we explored P2X2 receptor subunit immunolabeling in putative chemosensitive sites in the medulla of neonatal rat, measured the effects of ATP on
respiratory-related rhythm in vitro and assessed the effects of the P2 receptor antagonist, PPADS, on the CO2 sensitivity of rhythmically-active brainstem–spinal cord preparations. We selected the in vitro approach because it allowed us to avoid the two main technical limitations associated with exploring this question in vivo. First, at the concentrations used in vivo, most ATP receptor antagonists are non-selective. Suramin, the most commonly used general P2 antagonist, and to a lesser degree PPADS, antagonize glutamatergic transmission, inhibit ectoATPase activity, inhibit IP3 induced Ca2+ release, and have a variety of other non-specific effects when used at high concentrations (>100 M for suramin and >10 M PPADs) (Motin and Bennett, 1995; Nakazawa et al., 1995; Lambrecht, 2000). Since glutamate is essential in both the generation of respiratory rhythm and transmission of respiratory inputs to motoneurons (Funk et al., 1993), reductions in respiratory activity and CO2 sensitivity associated with high concentrations of suramin or PPADs (Thomas et al., 1999) cannot, in most cases, be directly attributed to endogenous activation of ATP receptors. Second, central chemosensitivity most likely reflects activity of heterogeneous neurons located at multiple sites within the brainstem (Feldman et al., 2003), with neurons in different regions possibly employing different chemosensing mechanisms (Ballantyne and Scheid, 2001). As a result, to definitively assess the contribution of medullary ATP receptors to central chemosensitivity requires that ATP receptors be simultaneously blocked throughout the brainstem, which is extremely difficult to accomplish in vivo. The in vitro approach allowed us to control drug concentration to exploit the limited specificity of PPADS, and simultaneously affect all medullary chemosensitive sites. These data have been presented in abstract form (Lorier et al., 2003).
2. Methods 2.1. Electrophysiology 2.1.1. Rhythmically active in vitro preparations Brainstem–spinal cord preparations were prepared from neonatal Wistar rats ranging in age from postnatal day 0–3 (P0–P3) using methods
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previously described (Miles et al., 2002). Briefly, animals were anesthetized with ether and decerebrated. The brainstem–spinal cord was isolated in cold artificial cerebrospinal fluid (aCSF) containing (in mM): 120 NaCl, 3 KCl, 1.0 CaCl2 , 2.0 MgSO4 , 26 NaHCO3 , 1.25 NaH2 PO4 , 20 d-glucose, equilibrated with 95% O2 /5% CO2 . Preparations extended from the caudal-pontine level rostrally to approximately the seventh cervical segment caudally. Once dissected free, the preparation was pinned down with the ventral surface up on SylgardTM resin in a recording chamber perfused with aCSF. Medullary slice preparations were also prepared from neonatal (P0–3) Wistar rats, as described previously (Funk et al., 1993, 1997). In brief, once the brainstem–spinal cord was isolated as described above, it was pinned to a wax chuck and sectioned using a vibratome (Pelco-101, Ted Pella, CA, USA or Leica VT1000S). A series of 100–200 m sections were cut and examined for landmarks. When the compact division of nucleus ambiguus (cNA) was no longer evident and the rostral margin of the inferior olive first appeared in these thin sections, a single 700 m section was cut. This slice extended from the caudal margin of the cNA to obex and contained the preBötC, rostral ventral respiratory group, XII motor nuclei and rostral XII nerve rootlets. Slices were fixed rostral surface up in a recording chamber perfused with aCSF. For all experiments with the brainstem–spinal cord preparation and the majority of experiments with the medullary slice (see below), we used a recording chamber with a volume of 10 ml. The total volume of recirculating solution was 100 ml and flow rate was maintained at ∼5 ml/min. Solution was aerated in a heated reservoir and bath temperature was gradually increased over 30 min from room temperature to 27–28 ◦ C before recording. Thirty minutes prior to the start of data collection, slice preparations were perfused with solution containing 9 mM K+ , as required for long-term maintenance of respiratory network activity (Smith et al., 1991; Funk et al., 1993). While the reasons that maintained activity requires elevated K+ are not known, the assumption is that isolation of the slice from the brainstem–spinal cord preparation reduces the excitability of a critical population of neurons. The elevated K+ presumably depolarizes neurons, restoring excitability to levels consistent
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with rhythm generation (for additional discussion see Funk et al., 1993). In an initial set of experiments using the medullary slice to explore the effects of ATP on rhythmic activity, preparations were placed in a 2 ml recording chamber, and perfused, without recirculation, at a flow rate of 2 ml/min. All experiments and procedures were approved by the local animal ethics committee and performed in accordance with New Zealand guidelines for the care, handling and treatment of experimental animals. 2.1.2. Nerve root recordings Inspiratory motoneuron activity was recorded from severed ends of C4 or C1 cervical (brainstem–spinal cord), or XII cranial nerve roots (medullary slice) using suction electrodes. Signals were amplified, band-pass filtered (100 Hz–3 kHz), full-wave rectified, integrated using a leaky integrator (τ = 25 ms), and displayed on a chart recorder and an oscilloscope. They were also recorded on videotape via pulse code modulation (Vetter Model 402 or 3000A) for storage and off-line analysis. 2.1.3. Drugs and method of application Drugs used included: adenosine triphosphate disodium salt (ATP-Na+ , 0.25–1.0 mM, Sigma), adenosine triphosphate Mg2+ salt (ATP-Mg2+ , 0.1–1.0 mM, Sigma), and pyridoxal-phosphate-6-azophenyl-2 ,4 disulphonic acid 4-sodium (PPADS, P2 receptor antagonist (Lambrecht, 2000), 10–100 M, Sigma). All drugs were dissolved in standard extracellular solution and applied directly to the perfusion solution. Note that since ATP is rapidly hydrolyzed to produce adenosine, which has modulatory actions of its own through P1 receptors (Herlenius et al., 1997; Ralevic and Burnstock, 1998), all experiments involving exogenous application of ATP were performed with the P1 receptor antagonist, theophylline (200 M, Sigma) (Ralevic and Burnstock, 1998), added to the aCSF. 2.1.4. Measurement of central respiratory chemosensitivity in vitro To change the pH of the aCSF, the solution flask and dissection bath were bubbled with either a low CO2 mixture (2% CO2 and 98% O2 , pH = 7.88 ± 0.03) or a high CO2 mixture (8% CO2 and 92% O2 ,
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pH = 7.38 ± 0.03). These gas mixtures and the 95% O2 /5% CO2 were prepared by an industrial supplier and their composition checked using a Datex digital gas analyzer. To maintain a consistent rate of gas bubbling in the flask and bath throughout the experiment, the flow rates from each gas cylinder were matched using regulators. Central chemosensitivity of the brainstem–spinal cord preparation was assessed using a protocol similar to that described previously (Kawai et al., 1996). Following dissection, preparations were equilibrated for 60 min in aCSF bubbled with 95%O2 and 5% CO2 . The pH of the aCSF was raised by bubbling the solution with a 2% CO2 mixture. Preliminary experiments indicated that it took ∼10 min for the rhythmic activity to stabilize following this transition. The preparations were maintained at 2% CO2 for 24 min, pH was then lowered by bubbling with the 8% CO2 mixture for 24 min before returning to the 2% CO2 mixture for 24 min. Involvement of ATP receptors was assessed by adding PPADS (10–100 M) to the perfusion solution. An equilibration period of 30 min was allowed and the 2, 8, 2% CO2 protocol repeated. PPADS was then washed out for 30 min and the recovery response recorded. The pH of the superfusate was measured throughout the experiment with a macroelectrode (pH Meter CG 840) that was placed in one end of the bath and calibrated prior to each experiment using standard calibration buffers. 2.1.5. Data analysis The effects of the stimuli (changing CO2 levels or bath-application of drugs) on frequency and peak amplitude of integrated XII and C4 inspiratory bursts were assessed using custom-written LabVIEWTM acquisition and analysis protocols (Funk et al., 1997; Miles et al., 2002). Response time-course was calculated by averaging inspiratory burst frequency and amplitude in 1-min bins for 10 min prior to stimulus onset, throughout stimulus application and for 30 min during recovery. These values obtained from individual preparations were then averaged to obtain the response time-course. Maximum effects of ATP or CO2 on inspiratory frequency or burst amplitude were calculated by comparing values averaged over the last 5 min of control, the last 5 min
of treatment and the last 5 min of recovery. Efficacy of PPADS in blocking the ATP- or CO2 -mediated changes in frequency or amplitude was assessed by comparing the magnitude of the increase induced in the absence and then presence of antagonist. Parameters are reported in absolute terms, or relative to control (prestimulus) levels, as mean ± S.E. All statistical analyses were performed on raw data and differences between means identified using Student t-test with Bonferroni correction for multiple comparisons. Values of P < 0.05 were assumed significant. 2.1.6. Immunohistochemistry Neonatal (P3) animals were anesthetized deeply with sodium pentobarbital (6 g/kg, i.p.) and perfused through the heart with 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4). Following perfusion, brainstems were removed and post-fixed overnight in 4% formaldehyde. Subsequently, brainstems were infiltrated with 30% sucrose for ∼12 h, placed in a 1:1 mixture of 30% sucrose and Tissue-Tek® for an additional 24 h, then embedded and frozen in Tissue-Tek® at −80 ◦ C until use. Prior to immunochemical labeling, frozen transverse sections (14 m) were cut in a series of 6 and thaw-mounted onto microscope slides. Immunolabeling was performed using polyclonal rabbit P2X2 (1:4000, Roche Biosciences) and goat anti-rabbit IgG-biotin (1:200 Vector). The specificity of the P2X2 antibody has previously been characterized (Xiang et al., 1998; Bo et al., 1999). Slide mounted sections were initially washed in PBS before being incubated in 0.5% H2 O2 in methanol for 20 min. After further PBS washes, sections were blocked with 10% goat serum diluted in PBS containing 0.3% Triton-X (PBS-Tx) for 2 h and incubated for 40 h at 4 ◦ C in rabbit anti-P2X2 antibody diluted in PBS-Tx. Sections were then rinsed in PBS-Tx three times for 30 min each, incubated in goat anti-rabbit biotin for 1 h, and then washed in PBS three times for 10 min each, before being incubated in ABC Elite reagent (Vector) diluted 1:100 in 0.5 M NaCl-PBS for 1 h. Finally, after washes in both 0.5 M NaCl-PBS and PBS, sections were allowed to react in 0.3 mg/ml diaminobenzidine diluted in PBS containing 0.03% NiCl2 and 0.008% H2 O2 , washed in PBS, and coverslipped with glycerol gel.
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3. Results 3.1. P2X2 R immunolabeling is distributed throughout putative chemosensitive regions of the medulla The hypothesis that purinergic transmission is involved in central chemosensitivity implies that chemosensitive neurons are tonically or phasically modulated by ATP, and that pH-sensitivity of the P2X2 containing P2X receptors enhances the strength of this purinergic modulatory input. A necessary condition of this hypothesis is that chemosensitive brainstem neurons express P2X receptors that contain the P2X2 subunit. We explored this question using immunohistochemistry. P2X2 immunolabeling was found throughout many brainstem nuclei in the neonatal rat (Fig. 1). Large, darkly-labeled neurons (presumptive motoneurons) were prominent in many motor nuclei, including XII, NA, VII and V. Labeling was also strong within the inferior olive, and putative chemosensitive regions of the brainstem including the VLM, NTS, medullary raphe, retrotrapezoid nucleus and locus coeruleus. P2X2 immunolabeled neurons were found scattered throughout the VLM ventral to the compact formation of NA (Fig. 1A and B and D–F), including at the level of the preBötC, the proposed site of respiratory rhythm-generation. P2X2 immunolabeling was also strong in neurons of the NTS (Fig. 1A and C) and retrotrapezoid nucleus (Fig. 1G and I). Labeling of locus coeruleus was more moderate (Fig. 1J and K). In the medullary raphe, P2X2 immunolabeling was detected in both neurons and fibre tracts (Fig. 1G and H). 3.2. ATP modulates respiratory network activity A second prediction following from the hypothesis that P2X receptors contribute to central chemosensitivity is that ATP receptor activation will potentiate respiratory activity. In a preliminary set of experiments designed to test this hypothesis we established, using medullary slices, the concentration range over which ATP modulated respiratory-related activity. Respiratory activity was not significantly altered by 0.1 mM ATP. At 1 mM, ATP caused a 91 ± 18% increase in frequency (10.1 ± 1.3 in control to a peak of 18.2 ± 1.1 cycles/min, n = 7) (Fig. 2A). Effects were sustained throughout the period of drug exposure and
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fully reversible, with frequency returning to 10.4 ± 1.4 cycles/min following washout of ATP (Fig. 2C). At 10 mM, frequency increased as drug washed into the bath, but burst amplitude decreased to the point that rhythmic inspiratory-related XII nerve output ceased (Fig. 2B). We then examined the effects on respiratory output of applying ATP at two submaximal concentrations (250 and 500 M) to the aCSF bathing rhythmically active in vitro preparations. These concentrations were selected to facilitate use of the lowest possible PPADS concentrations in subsequent antagonist experiments (see Fig. 3). ATP caused similar, dose-dependent increases in respiratory frequency in brainstem–spinal cord and medullary slice preparations. In brainstem–spinal cord preparations (n = 7), ATP at 250 and 500 M increased frequency by 18 ± 6 and 32 ± 7%, respectively, from a control level of 6.8 ± 0.7 to 7.7 ± 0.6 cycles/min (250 M) and 8.4 ± 0.7 cycles/min (500 M) (Fig. 3A and C). Frequency also returned toward control levels (7.6 ± 0.8 cycles/min) following ATP washout. In slices, ATP increased frequency by 21±4 and 38±5% at 250 and 500 M, respectively (Fig. 3D and F) from a control level of 11.1 ± 0.7 to 13.4 ± 0.7 cycles/min (250 M) and 15.3 ± 0.8 cycles/min (500 M) (n = 4). Frequency returned to control levels (10.8 ± 1.2 cycles/min) following washout of ATP from the slice. ATP caused a minor decrease in C4 burst amplitude (8.7±3% at 250; 18±3% at 500 M) in the brainstem–spinal cord preparation, but had no significant effect on XII burst amplitude in slice preparations. To confirm that these actions of ATP were mediated through activation of P2 receptors, we tested in the same preparations whether the P2 receptor antagonist, PPADS, altered the ATP dose response. After the control responses to 250 and 500 M ATP were established (see above), PPADS was applied to the bath at a concentration of 10 M. This concentration was chosen to avoid the non-specific actions of PPADS. Following a 30 min equilibration period, the ATP dose response was repeated. Alone, PPADS was without significant effect on frequency or burst amplitude in either preparation. In slices, frequency was 10.8 ± 1.2 cycles/min in control and 12 ± 0.6 cycles/min in PPADS. In the brainstem–spinal cord preparation, frequency was 7.8 ± 0.6 cycles/min in control and
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Fig. 1. Chemosensitive regions of the brainstem show P2X2 receptor subunit immunolabeling. Photomicrographs of transverse sections at various rostrocaudal levels of neonatal rat brainstem ((A) is most caudal, and (J) is most rostral). Small boxes in (A, D, G and J) indicate the region shown at a higher magnification in adjacent images. At the level of obex (A), P2X2 immunolabeling was detected in the VLM (B) and NTS (C). In more rostral sections (D, G, and J) labeling was observed in the rostral VLM (E and F), the medullary raphe (H), retrotrapezoid nucleus (I), and locus coeruleus (K). No labeling was detected when primary antibody was omitted (L). Abbreviations: NTS, nucleus of the solitary tract; NA, nucleus ambiguus; XII, hypoglossal nucleus; IO, inferior olive; SpV, spinal trigeminal nucleus; VII, facial nucleus; moV; trigeminal motor nucleus; meV, mesencephalic trigeminal sensory nucleus; and LC, locus coeruleus. Scale bars: 100 m.
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Fig. 2. Integrated XII nerve recordings ( XII) from a medullary slice preparation illustrating the effects on inspiratory-related output of bath applying (A) 1 mM and (B) 10 mM ATP. Arrows indicate onset of drug application. Group data for the 1 mM response are included (C) (n = 7).
Fig. 3. ATP potentiates inspiratory frequency in vitro. Integrated recordings from (A and B) C4 ( C4) and (D and E) XII ( XII) nerves illustrate that bath application of ATP increases respiratory frequency in (A) brainstem–spinal cord and (D) medullary slice preparations. This potentiation was reduced by (B and E) PPADS (10 M). Group data are shown in (C) (n = 7) and (F) (n = 4) (∗ P < 0.05).
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7.2 ± 0.6 cycles/min in PPADS. PPADS did, however, significantly attenuate the effects of ATP on respiratory frequency by up to 51% (Fig. 3C and F). In the presence of PPADS, frequency increased by only 8±4 and 16 ± 6% (brainstem–spinal cord, Fig. 3B and C) and 11 ± 2 and 24 ± 6% (slice, Fig. 3E and F) in response to 250 and 500 M ATP, respectively. The inhibitory actions of PPADS were partially reversible in the slice (Fig. 3F) with frequency increasing by 16 ± 2 and 27 ± 5% with 250 and 500 M ATP, respectively, following washout. In the brainstem–spinal cord the effect of PPADS could not be reversed (Fig. 3C).
5.75 ± 0.9 cycles/min while burst amplitude fell by only 7%. PPADS was also without effect on the CO2 response (Fig. 4B, C and E). With the transition from 2 to 8% CO2 , frequency increased by 91 ± 30%, while burst amplitude did not change. Frequency returned to baseline levels with the transition back to 2% CO2 (Fig. 4C and E). The CO2 response was repeated once more following a 30 min washout period. The CO2 responses measured under control, in the presence of PPADS and following PPADS washout were indistinguishable.
3.3. Central CO2 sensitivity in vitro is not sensitive to PPADS
4. Discussion
We next tested the hypothesis that P2X receptors contribute to central chemosensitivity in the neonatal rat brainstem–spinal cord preparation. Increasing the CO2 concentration of the gas used to bubble the aCSF from 2 to 8% caused pH of the extracellular solution to decrease steadily from 7.88 ± 0.03 to 7.38 ± 0.03 over 10 min. With the switch back to 2% CO2 , pH gradually increased, attaining initial control levels within ∼12 min. The time course of changes in respiratory-related activity followed changes in bath pH. Starting from a baseline of 6.54 ± 0.53 cycles/min in 2% CO2 , frequency increased steadily over ∼10 min with the switch to 8% CO2 to plateau at a level 74 ± 13% higher than in 2% CO2 (Fig. 4A, C and E). Frequency remained elevated throughout the high CO2 exposure. It returned to control levels (6.54 ± 0.8 cycles/min) within 10 min of switching back to 2% CO2 (Fig. 4A, C and E). Inspiratory burst amplitude was not significantly affected by the changes in CO2 , decreasing only minor amounts during the last half of the 8% CO2 treatment (Fig. 4D and F). In a preliminary set of experiments, PPADS was applied at a concentration of 10 M. As PPADS was without effect on the CO2 response of the brainstem–spinal cord preparations at this minimal dose, we increased the concentration of PPADS to 100 M to ensure block of P2 receptors. At this higher concentration, PPADS (100 M) did not significantly affect baseline respiratory activity measured at 2% CO2 . Frequency decreased 12% from 6.54 ± 0.8 to
In spite of significant advances in our understanding of the regions and neurons that mediate central respiratory CO2 sensitivity, the molecular mechanism(s) that transduces increases in PCO2 (or the resulting decreases in pH) into increased respiratory output remains unknown. In this study we explored the controversial hypothesis that the pH-sensitivity of currents mediated by P2X2 subunit-containing ATP receptors is an important molecular mechanism contributing to central CO2 chemosensitivity. Experiments were performed in vitro using rhythmically-active brainstem–spinal cord and medullary slice preparations in which non-specific actions of ATP receptor antagonists could be minimized and P2X2 receptors could be antagonized throughout the medulla, not just within respiratory regions of the brainstem (Thomas et al., 1999; Thomas and Spyer, 2000). There were three main findings. (i) P2X2 receptors are widely expressed in the brainstem of neonatal rat, suggesting that purinergic transmission subserves a variety of functions in the neonatal brainstem. (ii) P2X2 immunolabeling in respiratory regions of the VLM, combined with the potentiation of respiratory frequency by P2 receptor activation, supports a role for purinergic signaling in modulating the activity of central respiratory networks. (iii) Despite P2X2 immunolabeling within putative medullary chemosensitive sites, the inability of the P2X receptor antagonist PPADS to alter central chemosensitivity indicates that P2 receptors do not contribute to central chemosensitivity in the neonate in vitro. The hypothesis that the pH-sensitivity of P2X2 receptor-mediated currents provides the molecular
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Fig. 4. PPADS does not alter the effects of CO2 on inspiratory-related activity. Integrated C1 nerve recordings ( C1) from (A and B) a single brainstem–spinal cord preparation and (C–F) group data (n = 6) illustrate that the time course and magnitude of the increase in respiratory frequency associated with increasing CO2 levels from 2 to 8% is the same in (A) control and (B) after bath application of PPADS (100 M).
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basis of central respiratory chemosensitivity derives from the characterization, in expression systems, that P2X2 -containing P2 receptors are markedly potentiated by reductions in pH (King et al., 1996), and the widespread distribution of P2X2 receptors in the brainstem of adult rat (Kanjhan et al., 1999; Yao et al., 2000). The premise is that non-rhythmic chemoreceptive neurons, which provide tonic drive to respiratory networks, or respiratory neurons, express P2X2 subunit-containing P2 receptors and receive tonic ATP mediated input. As PCO2 increases and extracellular pH decreases, these tonic ATP inputs are potentiated and respiratory output increases. On the basis of this hypothesis we tested the following predictions: (1) P2X2 receptors are expressed in chemosensitive regions of the brainstem; (2) activation of ATP receptors potentiates respiratory output and; (3) block of pH-sensitive ATP receptors alters central respiratory chemosensitivity. 4.1. Chemosensitive regions of the brainstem are immunoreactive for the P2X2 receptor subunit Historically, regions near the ventral surface of the medulla have been considered the primary sites mediating central chemosensitivity (Nattie, 1999). However, increases in ventilation following local acidification of multiple areas through the brainstem (Coates et al., 1993; Bernard et al., 1996; Nattie and Li, 1996; Li and Nattie, 1997), c-fos expression in numerous regions following hypercapnia (Teppema et al., 1997; Okada et al., 2002), attenuation (but not block) of CO2 sensitivity following lesion or inhibition of neuronal activity in multiple areas (Berger and Cooney, 1982; Dreshaj et al., 1998; Gray et al., 2001; Nattie and Li, 2002) and the presence of pH-sensitive neurons in many of these same locations (Okada et al., 1993b; Kawai et al., 1996; Oyamada et al., 1998; Wang et al., 2001) underlie the emerging view that CO2 sensitivity is not mediated by a single locus, but results through combined action of sites distributed through the brainstem (Feldman et al., 2003). If P2X2 receptors contribute to CO2 sensitivity, a necessary condition is that they are expressed by neurons in chemosensitive regions of the brainstem or directly on respiratory neurons. This appears to be the case in adult rat. P2X2 mRNA or protein is present in many of the putative chemosensory regions including
the VLM, VRG (including the preBötC), medullary raphe, locus coeruleus, and NTS (Kanjhan et al., 1999; Yao et al., 2000; Thomas et al., 2001; Gourine et al., 2003). The pattern of P2X2 labeling in the neonatal rat brainstem was similar to that of the adult (Funk et al., 1997; Kanjhan et al., 1999; Yao et al., 2000; Thomas et al., 2001; Miles et al., 2002). Immunolabeling was present in many regions of the brainstem, with labeling particularly evident in motor nuclei, as described previously in neonatal (Funk et al., 1997; Miles et al., 2002) and adult motoneurons (Funk et al., 1997; Kanjhan et al., 1999; Yao et al., 2000). Our focus on putative chemosensitive regions revealed moderate to strong P2X2 immunolabeling in the VLM (caudal and rostral VRG), medullary raphe, retrotrapezoid nucleus, locus coeruleus, and NTS. The widespread expression of P2X2 receptor subunit throughout the neonatal brainstem is consistent with the involvement of this receptor family in developmental processes or intercellular signaling functions. Of particular relevance to this study is that P2X2 immunolabeling in the VLM supports modulation of cardio-respiratory activities by purinergic signaling systems. 4.2. P2 receptor activation increases respiratory frequency The role of ATP as a neurotransmitter in the CNS is best characterized within sensory systems (North, 2002). An understanding of ATP’s role in motor systems is limited. Involvement has been most convincingly demonstrated in Xenopus tadpole where ATP-mediated inhibition of a K+ conductance in spinal locomotor neurons initiates swimming episodes (Dale and Gilday, 1996). In mammalian systems, a role for purinergic transmission in motor control is implicated by the ubiquitous expression of P2 receptors on motoneurons (in neonatal and adult animals) as well as the ATP-mediated excitation of motoneurons (Funk et al., 1997; Miles et al., 2002). Involvement of purinergic signaling in the efferent limb of the respiratory motor control system in vitro is supported by the ATP-mediated, and PPADS-sensitive, potentiation of both respiratory frequency (present study) and XII and phrenic motoneuron burst amplitude (Funk et al., 1997; Miles et al., 2002). This
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view is also supported by: expression of P2X subunits (P2X1 , P2X2 , P2X5 and P2X6 ) in the rostral VLM (Thomas et al., 2001) and P2X2 subunits in a subset of functionally identified VLM respiratory neurons (Gourine et al., 2003) and ATP-mediated excitation of inspiratory neurons in vitro (Lorier et al., 2002) and inspiratory and expiratory neurons in vivo (Thomas and Spyer, 2000; Gourine et al., 2003). Given these observations, the finding that microinjection of ATP into the VLM markedly reduces or abolishes phrenic nerve output (Thomas et al., 2001) is surprising. However, the potentiation of respiratory frequency by 1 mM ATP and loss of respiratory rhythm with 10 mM ATP in vitro (Fig. 2C) suggests that the interruption of rhythm following application of 20 mM ATP in vivo may reflect depolarization block. 4.2.1. Neurons, ionic mechanisms and receptor subtypes mediating actions of ATP Identifying the brainstem regions or neurons responsible for the ATP-mediated increase in frequency was not an objective of this study. However, the fact that respiratory frequency was potentiated to a similar degree in both the brainstem–spinal cord and medullary slice preparations suggests that the necessary neurons, or at least a significant portion of these neurons, are contained within the slice. Indeed P2 receptors are expressed by neurons in many regions contained within the slice (Kanjhan et al., 1999; Yao et al., 2000; Thomas et al., 2001), including respiratory neurons (Gourine et al., 2003). Whether the frequency potentiation reflects activation of respiratory or non-respiratory neurons, or both, is not clear. ATP excitation of respiratory motoneurons (Funk et al., 1997; Miles et al., 2002) is unlikely to contribute since inspiratory burst frequency, rather than burst amplitude, was affected. Direct activation of respiratory neurons is likely to be involved since they are excited by ATP, both in vivo (Thomas and Spyer, 2000) and in vitro (Lorier et al., 2002). However, it is equally possible that the frequency potentiation results from excitation of non-respiratory neurons providing excitatory input to the respiratory rhythm-generating network. Establishing the ionic mechanisms underlying the actions of ATP on respiratory rhythm will first require the identification of the neurons responding to ATP. In other systems, ATP excites neurons through
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a number of different pathways including activation of non-selective cationic currents (Sugasawa et al., 1996; Funk et al., 1997; Khakh et al., 1997; Miles et al., 2002), inhibition of K+ conductances (Harms et al., 1992; Dale and Gilday, 1996) or by modulating the activity of other neurotransmitter systems, including glutamatergic transmission (Li and Perl, 1995; Khakh and Henderson, 1998; Knight et al., 2002), which is essential for both the generation and transmission of respiratory activity (Funk et al., 1993). The purinergic receptor subtypes responsible for the ATP-mediated potentiation of respiratory frequency also remain to be fully elucidated. Extensive P2X2 subunit immunolabeling throughout the neonatal medulla, including the VLM, presented in this study suggests that receptors containing P2X2 subunits contribute to the ATP response. Expression of other P2X subunits in the neonatal brainstem is unknown, but the expression of P2X1 –P2X6 subunits in the adult rat brainstem (Yao et al., 2000) suggests that multiple receptor subtypes or heteromeric receptors could also be involved. PPADS is not selective for one particular P2 subtype, but its attenuation of ATP’s action on respiratory rhythm suggests involvement of P2X1 , P2X2 , P2X3 , P2X5 , P2X2/3 , P2X1/5 or P2Y1 receptors (Lambrecht, 2000). That the block of ATP responses by PPADS was only partial may suggest involvement of P2X4 and P2X6 receptor subunits, since these are not sensitive to PPADS (Lambrecht, 2000). However, it is as likely that the incomplete block reflects the low concentration of PPADS used. Additional information regarding the composition of the P2X receptors involved may be derived from the observation that there was incomplete recovery of the ATP response following PPADS block. The kinetics of PPADS inhibition varies at different receptor subtypes. PPADS inhibition at P2X3 receptors has a rapid onset and recovery. However, at P2X1 , P2X2 and P2X5 receptors, and as seen here, inhibition is slower and often only partially reversible (Buell et al., 1996). Under normal physiological conditions, the effects of adenosine at P1 receptors must also be considered since ATP is rapidly hydrolyzed to adenosine (Zimmermann, 2000), and adenosine is a potent inhibitor of respiratory rhythm (Herlenius et al., 1997). However, involvement of adenosinergic signaling in the present results can be discounted because ex-
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periments were performed in the presence of a P1 receptor antagonist. 4.3. Central respiratory chemosensitivity in vitro is not dependent on P2X receptor activation Interest in the potential involvement of P2 receptors in central chemosensitivity arose with the description that ATP currents are modulated by extracellular pH (King et al., 1996). The P2X2 subunit appears to be critical in this pH sensitivity. Currents through homomeric P2X2 receptors are potentiated more than 20-fold with reductions in pH from 7.4 to 6.3 (King et al., 1996), whereas homomeric P2X1 , P2X3 and P2X4 receptor currents are inhibited (Stoop et al., 1997). P2X2 /P2X3 and P2X2 /P2X6 heteromeric receptors are also potentiated by acidic shifts in pH (King et al., 2000), although the response occurs over a narrower pH range, and the potentiation is smaller than for homomeric P2X2 receptors. Co-expression of P2X1 and P2X2 produces biphasic currents with the two components displaying differing pH sensitivities (Brown et al., 2002). Examination of P2 receptor involvement in central chemosensitivity in vivo has been confounded by the lack of adequate P2 receptor antagonists. Indirect electrophysiological measurements conducted in vivo on anesthetized adult rats support a contribution of P2X receptors. In particular, the gain of the CO2 response curve is reduced by local injections into the VLM of the general P2 receptor antagonist suramin and the desensitizing agonist ␣,-meATP (Thomas et al., 1999). In addition, antagonizing P2 receptors with suramin attenuates the excitatory effects of CO2 on respiratory neurons (Thomas and Spyer, 2000). A prominent role for P2X2 receptors in the CO2 response, however, is questioned by three pieces of evidence from this same group. First, high concentrations of antagonist (suramin) are required in vivo (Thomas and Spyer, 2000). Second, only 20% of inspiratory neurons of the VLM express the P2X2 subunit (Gourine et al., 2003). Most importantly, the CO2 ventilatory response of wild-type mice and knockout mice lacking the P2X2 subunit are indistinguishable (Rong et al., 2003). Thus, either P2X2 receptors are not involved in central chemoreception, or during development non-P2X receptor-mediated mechanisms of central chemosensitivity compensate for the lack of
the P2X receptor component. Indeed there are examples in knockout animals where a secondary mechanism can compensate during development for the lack of a gene or gene product (Drago et al., 2003). To distinguish between these two alternatives, we employed an in vitro approach, primarily because this allowed us to apply the P2 antagonist, PPADS, globally to all putative medullary chemosensitive sites at concentrations where specificity is maintained. The in vitro brainstem–spinal cord preparation has been utilized in numerous studies to advance our understanding of central chemosensitivity (Suzue, 1984; Harada et al., 1985; Voipio and Ballanyi, 1997; Ballanyi et al., 1999; Ballantyne and Scheid, 2001). As seen in the present study, it produces a robust, repeatable response to elevations of CO2 in the perfusion solution. However, the fact that the in vitro response is blunted relative to in vivo conditions and is primarily mediated by increases in frequency rather than tidal volume, has led some to question whether the responses in vitro are relevant to understanding central chemoreception in vivo. A variety of factors have been proposed to account for the altered response profiles. For example, the blunted response in vitro may in part reflect reduced temperature (Forster et al., 1997). It may also reflect altered state. The ventilatory response to CO2 is state-dependent. Compared to wakefulness it is reduced during sleep and anesthesia (Nattie, 1999). The state of the in vitro preparation is unknown, but differences in state between in vitro and in vivo conditions may contribute to the blunted CO2 response in vitro. Developmental factors may also contribute. While CO2 sensitivity in intact rat either increases developmentally (Matsuoka and Mortola, 1995) or shows a biphasic time course with high responsiveness in the neonate (Stunden et al., 2001), CO2 sensitivity of peripherally chemodenervated neonatal rats is reduced relative to adults (Serra et al., 2001). The frequency versus tidal volume response, on the other hand, may reflect the effects of vagotomy. In neonatal rat in vivo, bilateral vagotomy produces an immediate increase in phrenic amplitude and tidal volume. Increased phrenic burst amplitude in vitro would limit the range over which phrenic nerve burst amplitude could increase in response to CO2 (Ballanyi et al., 1999). It is unlikely that this is the only factor, however, since vagotomized adult animals still respond to hypercapnia with an increased
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burst amplitude (Xu et al., 1994; Golder et al., 2001). Despite differences in response profiles in vitro and in vivo, and debate regarding underlying mechanism, there is a growing consensus that the brainstem–spinal cord preparation produces a response to CO2 that can be useful in resolving mechanisms of central chemosensitivity (Ballanyi et al., 1999; Ballantyne and Scheid, 2001). In this context, our finding that the brainstem–spinal cord preparations produced a CO2 response typical of in vitro preparations and that this response is not affected by PPADS, combined with recent data from P2X2 knockout mice in which CO2 sensitivity is normal (Rong et al., 2003), strongly suggest that P2X2 receptors do not contribute to central chemosensitivity. We cannot exclude the possibility that the source of tonic ATP input to respiratory networks has been removed in vitro, in which case ATP receptor block would have no effect on CO2 sensitivity even if ATP receptors normally contribute. However, it is clear from both our own work and that of others (Suzue, 1984; Harada et al., 1985; Okada et al., 1993a,b; Kawai et al., 1996; Voipio and Ballanyi, 1997; Oyamada et al., 1998; Ballanyi et al., 1999; Ballantyne and Scheid, 2001) that the brainstem–spinal cord preparation does respond to pH in vitro. Thus, we can conclude that the brainstem–spinal cord preparation contains functional chemoreceptors and that transduction of the CO2 stimulus into increased respiratory output does not require ATP receptors. The possibility that differences in responses obtained in wild type neonatal animals in vitro and adults in vivo reflects a developmental change in the mechanisms mediating central respiratory chemosensitivity also remains, as newborn rats are very immature animals. However, recent data from adult P2X2 knockout mice in vivo indicate that CO2 sensitivity is unaffected in the complete absence of P2X2 receptors (Rong et al., 2003). In addition, while suramin reduces the slope of the CO2 response in adult rats by approximately 50% (Thomas et al., 1999), suramin alone significantly depresses respiratory neuronal (Thomas and Spyer, 2000), and network activity (Thomas et al., 1999) when used at the high concentrations required in vivo. Thus, CO2 response curves were not generated from the same baseline conditions. While relevant controls were performed in the in vivo studies (Thomas et al., 1999; Thomas and Spyer, 2000), de-
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termining whether the depressive action of suramin reflects action at P2 receptors or nonspecific actions at glutamate receptors or via other pathways can only be resolved by single cell analysis. When in vitro data and in vivo data from wild type and P2X2 knockout animals are considered in total, we find the data suggesting that P2X2 receptors are not involved in central chemosensitivity most compelling. Immunohistochemical data also support the conclusion that P2X2 receptors do not contribute to central chemosensitivity. P2X2 receptors are expressed by a variety of neuron types throughout the brainstem and spinal cord of neonatal and adult rat (Kanjhan et al., 1999; Yao et al., 2000; Thomas et al., 2001) including inferior olivary neurons that are not chemosensitive (Ritucci et al., 1998). If P2X2 receptors have a special role in mediating the CO2 sensitivity of the respiratory system, one might expect a certain degree of specificity for these receptors on chemosensitive neurons. Data suggesting that the purinergic system is not involved in central chemosensitivity highlights the potential importance of other transmitter systems in mediating this reflex. Monoaminergic neurons, including noradrenergic locus coeruleus neurons and serotonergic neurons of the medullary raphe are proposed to have an important role in central CO2 sensitivity. Neurons located within the locus coeruleus receive a respiratory-related modulation and are CO2 sensitive (Oyamada et al., 1998). Serotonergic medullary raphe neurons are also excited by acidosis, project to respiratory nuclei and have dendritic projections in close proximity to blood vessels projecting from the basilar artery (Wang et al., 2001; Bradley et al., 2002), an anatomical arrangement making them ideally suited for CO2 sensing. The parallel developmental increase in the number of acid-stimulated raphe neurons and the central CO2 response in vivo (Wang and Richerson, 1999) also support involvement of the serotonergic system in CO2 sensing. In conclusion, the widespread expression of P2X2 receptor protein in the neonatal rat brainstem, including areas important for respiratory control, and the potentiation of respiratory frequency by ATP suggests that purinergic signaling systems form an important element of the respiratory control system. Constant CO2 sensitivity before and during P2 receptor block also suggests that purinergic mechanisms are not involved in central respiratory chemoreception.
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Acknowledgements This work was supported by the Health Research Council of New Zealand, Auckland Medical Research Foundation, Canadian Institutes of Health Research, the Alberta Heritage Foundation for Medical Research and the Canadian Foundation for Innovation.
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