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Characterization of P2 receptors modulating neural activity in rat rostral ventrolateral medulla
P2 receptors in the rostral ventrolateral medulla
Pergamon PII: S0306-4522(99)00376-0
Neuroscience Vol. 94, No. 3, pp. 867–878, 1999 867 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00
CHARACTERIZATION OF P2 RECEPTORS MODULATING NEURAL ACTIVITY IN RAT ROSTRAL VENTROLATERAL MEDULLA V. RALEVIC,* T. THOMAS, G. BURNSTOCK and K. M. SPYER Autonomic Neuroscience Institute, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, U.K.
Abstract—This study investigated the effects of ATP, and related compounds, on the activity of neurons within the rostral ventrolateral medulla, an area of fundamental importance in reflex control of the cardiovascular system. Extracellular recordings were made from single neurons in anaesthetized, paralysed and artificially ventilated rats. Ionophoretic application of a,b-methylene-ATP, adenosine 5 0 -O-(2-thiodiphosphate), UTP, 2-methylthio-ATP and ATP altered the ongoing activity in the majority of neurons (.74% of neurons), generally causing increases in the firing rate. Nine of 11 cells with presumed spinal projection were excited by ATP and/or the P2X-selective agonist a,b-methylene-ATP. Desensitization of the excitatory responses to a,b-methylene-ATP was observed in four of 20 rostral ventrolateral medulla neurons. For the remainder of the rostral ventrolateral medulla neurons, the increase in firing rate evoked by a,b-methylene-ATP, and by the other purine compounds tested, did not undergo desensitization. Suramin, a P2 receptor antagonist, blocked excitatory responses to adenosine 5 0 -O-(2-thiodiphosphate) or a,bmethylene-ATP in five of 16 neurons. These results indicate that ATP can modulate the activity of neurons in the rostral ventrolateral medulla via actions at P2 purine receptors. The data suggest that both P2X and P2Y receptors are involved, and that the functional expression of these receptors within the rostral ventrolateral medulla is not uniform. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: ATP, P2 receptors, rostral ventrolateral medulla.
arterial pressure. 27 Since that study was conducted, there have been significant advances in purine receptor research, and using this information and newly available purine receptor ligands, we aimed to determine the subtype identity of P2 receptors in the RVLM. Seven different mammalian P2X receptors (P2X1–7) and five P2Y receptors (P2Y1,2,4,6,11) have been cloned, characterized and formally recognized as members of the P2 receptor family. 2,8 It should be noted that, whereas P2Y receptors are single proteins, co-expressed P2X receptor proteins may combine to form heteromeric receptors. It is not yet known how the properties of the different P2X subunits influence the phenotype of the heteromeric receptor. This, together with co-expression of different types of endogenous P2 receptors, has contributed to the complex pharmacological response profiles exhibited by P2 receptors to many biological tissues, that do not match with those of any of the cloned receptors. The general lack of subtype-selective agonists and antagonists has proved a major handicap in P2 receptor characterization. Suramin and pyridoxalphosphate-6-azophenyl-2 0 ,4 0 disulphonic acid (PPADS) are P2 receptor antagonists, but do not block P2X4 and P2X6 receptors and a subpopulation of P2Y1 and P2Y2 receptors. a,b-meATP is a non-hydrolysable agonist and desensitizing agent selective for P2X1 and P2X3 receptors, 2-methylthio-ATP (2MeSATP) acts at both P2X and P2Y receptors, and adenosine 5 0 -O-(2-thiodiphosphate) (ADPbS) and UTP are selective for P2Y receptors. The different rates of desensitization of P2X receptors have also proved useful in their characterization: P2X1 and P2X3 receptors desensitize rapidly, whereas P2X2, P2X4, P2X6 and P2X7 receptors desensitize slowly or not at all. 7 P2Y receptors do not desensitize rapidly. The present study investigates the actions of ATP and its analogues on the ongoing activity of individual neurons throughout the RVLM in anaesthetized rats, with the aim of characterizing the P2 purine receptor subtypes involved. We
Extracellular ATP mediates its effects via cell surface P2 receptors that have been divided into two families according to whether they are ligand-gated cation channels (P2X receptors) or are coupled to G-proteins (P2Y receptors). 2,8,20 Electrophysiological studies conducted mainly on dissociated or cultured neurons and on brain slices have shown that ATP evokes rapid inward currents and modulates the activity of neurons via P2 receptors throughout the CNS, and evidence for an involvement of both P2X and P2Y receptors has been presented. 12,33 A physiological correlate is provided in the medial habenula and locus coeruleus, where ATP has been shown to mediate fast synaptic currents via P2X receptors; the currents are blocked by suramin and a,b-methylene-ATP (a,b-meATP) desensitization, and are mimicked by ATP and a,b-meATP. 5,18 Complementary immunohistochemical evidence has identified specific patterns of P2X subtype labelling in different regions of the brain, with P2X2, P2X4 and P2X6 being the most abundantly expressed subtypes. 4,30 We have obtained evidence for a role of purines in cardiovascular control primarily at the level of the medulla oblongata, including the rostral ventrolateral medulla (RVLM), 28 an area considered to be of fundamental importance in the mediation of reflex control of the cardiovascular system. 24,26 The present study arose from experimental studies indicating that ionophoretic application of ATP and its stable analogue, a,b-meATP, in anaesthetized rats, excites RVLM neurons in a suramin-sensitive manner and mediates an increase in mean *To whom correspondence should be addressed at: School of Biomedical Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham NG7 2UH, U.K. (present address). Tel.: +44-115-970-9480; fax: +44-115-970-9259. E-mail address: [email protected] (V. Ralevic) Abbreviations: ADPbS, adenosine 5 0 -O-(2-thiodiphosphate); HDA, hypothalamic defence area; a,b-meATP, a,b-methylene-ATP; 2MeSATP, 2-methylthio-ATP; PPADS, pyridoxalphosphate-6-azophenyl-2 0 ,4 0 -disulphonic acid; RVLM, rostral ventrolateral medulla. 867
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were particularly interested in neurons receiving input from the hypothalamic defence area (HDA), since studies from our laboratory have indicated a role for purines in the cardiovascular components of the response to stimulation of that region. 25 A preliminary account of our results was presented at the inaugural meeting of the International Society for Autonomic Neuroscience. 21 EXPERIMENTAL PROCEDURES
General methods Experiments were carried out on male Sprague–Dawley rats (300– 350 g) anaesthetized initially with pentobarbitone sodium (60 mg/kg, i.p., Sagatale) and supplemented as necessary with a-chloralose (40 mg/kg, i.v.). The depth of anaesthesia was assessed by monitoring the stability of blood pressure and heart rate, and the minimal effects on cardiovascular variables to a pinch to the paw. Following paralysis (see below), the stability of these variables alone was used to assess the level of anaesthesia. 28 All of the following procedures and protocols were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986. Surgical procedure The temperature of the animal was maintained at 37–37.58C with a servo-controlled heating pad. The left femoral artery and vein were cannulated for measurement of arterial blood pressure (Neurolog System) and administration of supplemental anaesthetic, respectively. The trachea was cannulated for artificial ventilation. The left aortic nerve was dissected and prepared for stimulation with bipolar silver electrodes. Using high-frequency stimulation (100–300 mA, 1 ms, 100 Hz), we tested that aortic nerve stimulation evoked only a vasodepressor response; a significant fall in blood pressure was always observed. Unfortunately, because of difficulties with dissection of the aortic nerve in some cases, this test was not applied in all animals. The rats were fixed in a stereotaxic frame, paralysed with gallamine triethiodide (Flaxedile, 3 mg/kg/h) and ventilated artificially with oxygen-enriched air (Harvard ventilator, model 683). Partial laminectomy at the T2–T3 level was performed and a concentric bipolar stimulating electrode (SNE 100, Rhodes Medical Electrodes; shaft diameter 0.5 mm, tip exposure 0.5 mm × 0.2 mm) placed in the T2 region of the spinal cord for antidromic identification of cells with spinal projection (50–200 mA, 0.2 ms, 1 Hz). The electrode was placed on the left side with its tip 1.0–2.0 mm below the dorsolateral sulcus. Neurons responding to electrical stimulation in T2–T3 at a constant latency (.five sweeps) were presumed to have spinal projection. The collision test was not used routinely. Muscles were removed from the back of the neck, an occipital craniotomy performed and the cerebellum removed by suction to allow access of microelectrodes to the rostral region of the brainstem. A hole (diameter 3 mm) was drilled in the skull 2 mm caudal and 1 mm lateral of bregma to allow a stimulating electrode to be placed 7 mm from the dorsal surface, in the HDA, 25 for identification of neurons with HDA input. The HDA was identified on the basis of the characteristic cardiorespiratory response observed on stimulation in this region. 25 Recordings The surface coordinates used for positioning of microelectrodes in the region of the RVLM were 1.5–2.0 mm rostral and 2.0–2.5 mm lateral of obex, and 2.5–3.6 mm below the dorsal surface of the medulla oblongata. Extracellular recordings were made from RVLM neurons with five-barrelled glass microelectrodes (tip diameter 1– 2 mm, 7–10 V). The recording barrel contained 4 M sodium chloride. The others contained the excitatory amino acid l-glutamate (0.2 M, pH 8.0), Pontamine Sky Blue dye (2%), and the remaining barrels were filled with purine receptor agonists and/or antagonists (0.02–0.2 M, pH 8.0). Drugs were applied by ionophoresis (Neurophore, Medical Systems) at 20–80 nA; a retaining current of 20 nA was applied to each drug barrel between drug ejection periods. Firing was induced in neurons with no ongoing activity by ionophoretic application of glutamate. Automatic current balancing on the Neurophore limited current artifacts. This was confirmed by the fact that ionophoretic
application of saline as a current control did not elicit or modify neuronal firing, and there was also a lack of response of some neurons to selected purine compounds. Neuronal activity was recorded on video tape via a digital interface (Instrutech, VR-100B), together with records of arterial blood pressure for off-line analysis.
Data analysis Data analysis was carried out off-line after analogue-to-digital data conversion (CED 1401-Plus data acquisition system). The commercially available software packages CED Spike 2 and Signal Averager were used to analyse data. Single-unit activity is represented throughout as rate histograms after discrimination of neuronal activity using a window discriminator (Digitimer D130).
Histology After recording from a neuron, Pontamine Sky Blue (2%) was injected with 2430 nA current for 5 min. At the end of the experiment the brain was removed, fixed in a 10% solution of formal saline, frozen and sectioned serially (80 mm), and counterstained with Neutral Red. The sections were viewed using a light microscope, and the location of marked injection sites mapped with the aid of the atlas of Paxinos and Watson. 19
Materials ADPbS (trilithium salt; 0.01 M), ATP (disodium salt; 0.2 M), lglutamate (0.2 M), UTP (sodium salt; 0.2 M), a,b-meATP (lithium salt; 0.02 M) and Pontamine Sky Blue were purchased from Sigma (Poole, U.K.). 2-MeSATP (tetrasodium salt; 0.02 M) was from Research Biochemicals. PPADS (0.001 and 0.01 M) was from Tocris Cookson (Bristol, U.K.). Suramin (0.02 and 0.2 M) was from Bayer. All drugs were dissolved in distilled water, except for a,b-meATP, which was dissolved in saline, and Pontamine Sky Blue, which was dissolved in 0.5 M sodium acetate. The pH of drugs for ionophoresis was adjusted to 8.0.
RESULTS
General properties of recorded neurons Extracellular recordings were made from a total of 105 neurons in 28 rats. Of these, 86 were excited by stimulation of the spinal cord, 75 of these synaptically only (see Figs 1–3 as examples) and 11 antidromically. In the case of cells with presumed spinal projection, the mean response latency was 5.12 ^ 1.0 ms (range 1.87–13.60 ms, n 11), suggesting that they had small myelinated axons. In addition, the activity of a large proportion (56 of 96 neurons) of the neurons studied was increased and/or inhibited by electrical stimulation in the HDA (50–200 mA, 1-ms pulse, 1 Hz). Of 45 neurons, 53% (24 of 45) of neurons showed an excitatory response only, 4% (two of 45) of neurons were inhibited only and 16% (seven of 45) of neurons had excitation but also late inhibition. In a further 11 neurons, where ongoing discharge rate was low, an excitatory response was observed but accompanying inhibitory responses could not be effectively analysed. In the case of the cells with presumed spinal projection, seven of 11 showed an excitatory response to HDA stimulation; the remaining four neurons were not excited by stimulation in the HDA. Thirty per cent (21 of 69) of RVLM neurons were excited by aortic nerve stimulation (100–300 mA, 1 ms, 1 Hz), as described previously. 31 In the case of 22% (five of 23) of the RVLM neurons, a continuous train of stimuli resulted in a prolonged inhibition of ongoing activity.
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Fig. 1. Effect of ionophoretic application of ATP and a,b-meATP on the ongoing firing rate of an RVLM neuron. This neuron was excited by electrical stimulation in the T2 region of the spinal cord (spinal; top; three superimposed sweeps), but not by stimulation in the HDA (not shown). (A) The neuron was excited in an effective dose-related manner (20–80 nA) by ionophoretic application of ATP. (B) By contrast, a,b-meATP at the same currents did not elicit an increase in the ongoing rate of firing. Bin size: 4 s.
Effects of the P2 receptor agonists ATP, a ,b -methylene-ATP, 2-methylthio-ATP, adenosine 5 0 -O-(2-thiodiphosphate) and UTP The actions of P2 receptor agonists on the firing properties of RVLM neurons without reference to their aortic nerve and HDA inputs will be described initially. Ionophoretic application of ATP (20–80 nA) elicited dose-related increases in the firing rate in 40 of 48 RVLM neurons (Fig. 1). Responses were maintained in the continuous presence of ATP (Fig. 1). ATP elicited a decrease in firing in a small number of neurons (n 4) and was ineffective in a further four neurons. In general, ionophoretic application of the P2X-selective agonist a,b-meATP (20–80 nA) elicited increases in neuronal discharge in a dose-related manner (35 of 50). Where ATP and a,b-meATP (20–80 nA) were applied ionophoretically to the same cell (19 neurons), nine of these were excited by both ATP and a,b-meATP (Figs 2, 3). In six neurons ATP mediated excitation, but a,b-meATP was ineffective (Fig. 1). Typically, in those neurons in which a,b-meATP elicited
an increase in firing, the excitatory response did not desensitize, either during repetitive or continuous application (Figs 4, 5). Of the 20 neurons in which this was studied, autodesensitization of the excitatory response to a,b-meATP was observed in only four neurons (see Figs 2 and 3 as examples). In three of these desensitization was complete, with firing decreasing to, or below, baseline levels in the continuous presence of a,b-meATP (Fig. 3). In two of these neurons, the excitatory response to ATP was partially blocked by a,b-meATP desensitization (Figs 2, 3). Ionophoretic application of 2MeSATP (Fig. 6), ADPbS (Fig. 7) and UTP (20–80 nA) elicited an effective doserelated excitation in the majority of cells in which their actions were tested (27 of 36 neurons), with inhibition in three of 36. In general, the increase in firing evoked by 2MeSATP did not desensitize on continuous (Fig. 6) or repeated application (five of six neurons). Desensitization of the response was not observed for excitation evoked by ADPbS or UTP. The P2Y-selective agonist ADPbS and the P2X-selective
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Fig. 2. Effect of repetitive application of a,b-meATP on the effect of subsequent application of ATP on the ongoing firing rate of an RVLM neuron. This neuron was excited by electrical stimulation in both the T2 region of the spinal cord (spinal; upper left; three superimposed sweeps) and in the HDA (upper right; three superimposed sweeps). (A) Ionophoretic application of ATP elicited an increase in the rate of firing. (B) Ionophoretic application of a,b-meATP elicited an increase in firing, which decreased with repeated application. (C) Following desensitization to a,b-meATP, ionophoretic application of ATP decreased the rate of neuronal firing. Bin size: 5 s.
agonist a,b-meATP were applied ionophoretically (20– 80 nA) to the same cell in five cases; three of these were excited by both agonists and in two of these excitation resulting from co-application of ADPbS and a,b-meATP was shown to be additive. In two cells, excitation was mediated by either ADPbS (Fig. 7) or a,b-meATP only. ATP and UTP
were applied ionophoretically (20–80 nA) to the same cell in six cells; all were excited by both agonists. Effects of co-application of purines with glutamate In four neurons, a,b-meATP and glutamate were applied
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Fig. 3. Effects of ionophoretic application of ATP and a,b-meATP on the firing rate of an RVLM neuron. This neuron was excited by electrical stimulation in the T2 region of the spinal cord (spinal; three superimposed sweeps), but not in the HDA (three superimposed sweeps), nor by repetitive stimulation of the aortic nerve (not shown). (A) Ionophoretic application of ATP elicited an increase in the rate of firing which was maintained in the presence of ATP. Bin size: 3 s. (B) Ionophoretic application of a,b-meATP elicited an increase followed by a decrease in firing; firing rate was reduced to below that of the baseline in the presence, and after offset of application, of a,b-meATP. Bin size: 1.5 s. (C) In the continuous presence of a,b-meATP, the excitatory response to ATP was only partially blocked. Bin size: 2 s.
separately initially and then simultaneously (each at both 20 and 40 nA). Co-application of a,b-meATP and glutamate mediated an excitation which was additive of the responses evoked by these agents applied individually (Fig. 5). Effects of the P2 receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2 0 ,4 0 -disulphonic acid The effect of ionophoretic application of suramin was studied using barrel concentrations of 0.02 M (25 neurons) and 0.2 M (five neurons). Of these 30 neurons, suramin was tested against agonist-evoked responses in 20 cases by ionophoresis for 2 min prior to and during ionophoresis of ATP, a,b-meATP or 2MeSATP. Suramin (0.02 M) had no effect on baseline firing in 15 of 25 neurons; it increased firing in seven neurons (Fig. 5) and reduced firing in three neurons (Fig. 8). In those neurons in which suramin increased firing, its effects as a potential antagonist of purine-mediated excitation were not assessed.
Suramin (0.02 M) blocked excitatory responses to 2MeSATP in two of seven neurons (Fig. 6). The selectivity of suramin at this concentration was confirmed in one neuron where excitation to 2MeSATP, but not that to glutamate, was blocked by suramin (Fig. 6). Suramin (0.02 M) blocked responses to a,bmeATP in four of nine neurons (Fig. 4), and did not block excitation to a,b-meATP in the remaining five neurons (Figs 5, 8). Suramin, used at a higher barrel concentration of 0.2 M, had no effect on baseline firing in three of five neurons; it increased firing in one neuron and reduced firing in one neuron. Suramin (0.2 M) blocked excitation mediated by ATP in three of four neurons; in one of these, an inhibitory effect of ATP was revealed. However, glutamate-mediated excitation was also blocked by suramin (n 2), suggesting that at this barrel concentration suramin acts non-selectively. Ionophoretic application of PPADS at a barrel concentration of 0.01 M (20–40 nA) produced excitation in 12 of 16 cells. Of the four cells in which it had no direct effects,
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Fig. 4. Effect of a,b-meATP on the ongoing firing rate of an RVLM neuron in the absence (B) and presence (C) of suramin. (A) This neuron was excited by electrical stimulation in the T2 region of the spinal cord (spinal; left; three superimposed sweeps) and in the HDA region (right; four superimposed sweeps). (B) The neuron was excited by a,b-meATP in a manner which did not desensitize either during the response or with repeated application of a,b-meATP. Bin size: 3 s. (C) In the presence of suramin, there was a small decrease in baseline firing and the response to a,b-meATP was blocked. Bin size: 2 s.
PPADS (0.01 M) blocked excitation to a,b-meATP in one cell and had no effect on excitation to a,b-meATP in another. In the remaining two cells, a,b-meATP was ineffective in altering firing rate. Ionophoretic application of PPADS at the lower barrel concentration of 0.001 M (20–40 nA) elicited an increase in firing in three of four cells. Physiological inputs of purine-sensitive rostral ventrolateral medulla neurons The physiological inputs of RVLM neurons excited by purine compounds are summarized in Table 1. In general, cells with presumed spinal projection were excited by ionophoretic application of ATP (four of six neurons) or a,b-meATP (five of seven neurons). In two cells with presumed spinal projection in which ATP and a,b-meATP were co-applied, ATP increased the firing, whilst a,b-meATP induced little, if any, change in firing. Cells with presumed spinal projection that were excited by stimulation in the HDA were generally excited by ATP (three of four
neurons) and a,b-meATP (four of five neurons). These effects of ATP and a,b-meATP on cells with presumed spinal projection do not appear to differ from the effects of these agents on the total population of neurons. Interestingly, all RVLM neurons that received an aortic input, whether excitatory or inhibitory, were excited by ATP, whereas only 81% of those RVLM neurons with no aortic input were excited by ATP (Table 1). a,b-meATP increased the firing rate of all neurons with an inhibitory aortic input, compared with an excitation of only 75% and 68% of neurons excited by and not responding to stimulation of the aortic nerve, respectively (Table 1). All neurons that were inhibited by stimulation in the HDA were excited by ATP, compared to ATP-mediated excitation of 79% of those with an excitatory input and 88% of those with no HDA input (Table 1). Localization of recording sites Twenty-three recording sites were marked by ionophoresis
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Fig. 5. Effect of a,b-meATP on the firing rate of an RVLM neuron in the absence and presence of suramin (A), during continuous application for .1 min (B) and during co-application of glutamate (C). The neuron was excited by electrical stimulation in the T2 region of the spinal cord (spinal; three superimposed sweeps), but not by stimulation of the HDA (not shown). (A) The neuron was excited by a,b-meATP in an effective dose-related manner, which was not blocked by suramin. (B) The excitatory response to a,b-meATP did not desensitize during continuous ionophoresis for greater than 2 min. (C) Ionophoresis of a,b-meATP simultaneously with glutamate (Glu) elicited an increase in firing which was additive of the individual increases in firing produced by these agents. Bin size: 2 s.
of Pontamine Sky Blue in eight animals. Examination under the light microscope revealed that the recording sites were located within the area of the RVLM, and these were mapped using a stereotaxic atlas 19 (Fig. 9). DISCUSSION
This study is the first to show that both P2X and P2Y receptors for ATP can modulate the ongoing activity of neurons with different inputs in the RVLM. This has implications for our understanding of central purinergic control of the cardiovascular system as, amongst other functions, the RVLM is critically involved in integration of cardiovascular reflexes. Physiological implications of P2 purine receptor modulation of rostral ventrolateral medulla neurons It is well established that a population of neurons within the ventrolateral medulla has a crucial role in maintaining the activity of vasomotor sympathetic preganglionic neurons and is important in reflex control of the cardiovascular
system. 9,24,26 The current study, showing that P2X and P2Y purine receptor ligands modulate the activity of RVLM neurons, extends the findings of an earlier study showing an increase in neuronal firing of bulbospinal vasomotor neurons following ionophoretic application of ATP and a,b-meATP into the rat RVLM. 27 It should be noted that the RVLM contains a variety of populations of neurons with different functions. 26 Those investigated in the present study included neurons with characteristics of sympathoexcitatory neurons. Modulation of their activity by P2 receptors is consistent with a role for ATP in the RVLM-mediated control of sympathetic activity and of the defence response, since many were also affected by HDA stimulation. The predominantly excitatory responses of the neurons recorded in the RVLM following ionophoretic application of P2 receptor agonists are consistent with the increase in arterial blood pressure observed when ATP and a,bmeATP are microinjected into the rat RVLM. 22,27 This contrasts with the hypotension and bradycardia that occur on microinjection of a,b-meATP and 2MeSATP into the nucleus tractus solitarius of the rat, 6 the site of termination of baroreceptor afferents, with neurons functionally linked
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Fig. 6. Effect of 2-MeSATP on the firing rate of an RVLM neuron in the absence (A–C) and presence (D) of suramin. This neuron was excited by electrical stimulation in both the T2 region of the spinal cord (spinal; upper left; three superimposed sweeps) and the HDA (upper right; two superimposed sweeps), but not by repetitive electrical stimulation of the aortic nerve (not shown). (A–C) 2MeSATP elicited an effective dose-related increase in firing of this neuron. Glutamate (Glut; A) was also shown to elicit an increase in firing, but suramin applied at the same currents had no effect on neuronal firing. (D) In the presence of suramin, excitatory responses to 2MeSATP were selectively blocked, those to glutamate being unaffected. Bin size: 2 s.
to presympathetic neurons in the RVLM by multisynaptic pathways. 14,24 Neurons within the RVLM with identified spinal, HDA and/or aortic nerve inputs were examined for their patterns of responses to P2 receptor agonists. Interestingly, all RVLM
neurons that received aortic nerve input, whether excitatory or inhibitory, were excited by ATP, compared with an ATPmediated excitation of 81% of neurons having no aortic input. All neurons with an inhibitory aortic nerve input were excited by a,b-meATP, but a proportion of those
Table 1. Percentage of rostral ventrolateral medulla neurons with different physiological inputs that respond with excitation by purines Purine
Excitatory spinal input No spinal input Excitatory HDA input Inhibitory HDA input No HDA input Excitatory aortic input Inhibitory aortic input No aortic input
ATP
a,b-meATP
2MeSATP
ADPbS
UTP
78 (28/36) 100 (11/11) 79 (19/24) 100 (6/6) 88 (21/24) 100 (11/11) 100 (4/4) 81 (21/26)
71 (35/49) 60 (3/5) 67 (18/27) 80 (4/5) 77 (17/22) 75 (9/12) 100 (4/4) 68 (13/19)
73 (11/15) — (1/1) 77 (10/13) — 67 (2/3) 60 (3/5) — 71 (5/7)
55 (6/11) — (1/1) — (1/2) — — (0/2) — (0/1) — 56 (5/9)
100 (5/5) 100 (2/2) 100 (3/3) — 100 (4/4) — — 100 (4/4)
Values are percentages, with numbers of cells given in parentheses.
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Fig. 7. Functional characterization and pharmacological responses to a,b-meATP and ADPbS of an RVLM neuron. The neuron was excited by electrical stimulation in the T2 region of the spinal cord (spinal; five superimposed sweeps; upper left), but not by repetitive stimulation of the aortic nerve (not shown). It fired with cardiac-related activity (CRA; upper right); R-wave-triggered spike interval histogram (lower) and arterial blood pressure (AP; upper) (average of 700 sweeps) are also shown. The neuron was excited by ionophoretic application of ADPbS, but not by a,b-meATP. Bin size: 5 s.
receiving an excitatory aortic nerve input was insensitive to a,b-meATP. This indicates differential expression of P2 receptors within the RVLM, but the exact pattern of P2X subtype selectivity detected by functional assays is more sophisticated than the P2 receptor ligands currently available can detect. The widespread distribution of P2 receptors throughout the RVLM suggests that the specificity of cell– cell communication for a neuron will rely on its neuronal connections, as well as the expression of its cell surface P2 receptors. Characterization of P2 purine receptors modulating activity of rostral ventrolateral medulla neurons An increase in neuronal firing rate evoked by the P2Xselective agonist a,b-meATP, and the P2Y-selective agonists ADPbS and UTP, suggests that both P2X and P2Y receptors can modulate the activity of neurons in the RVLM. Furthermore, the fact that single neurons responded to both a,bmeATP and ADPbS suggests that P2X and P2Y receptors may coexist on individual neurons in the RVLM. However, we cannot exclude the possibility that for one, or both, of these agonists modulation of neuronal firing was indirect following P2 receptor-mediated release of transmitters from adjacent neurons or from glial cells. 13 Our findings are in line with functional, immunohistochemical and ligand binding studies that have provided overwhelming evidence for the widespread distribution of ionotropic P2X receptors and
G-protein-coupled P2Y receptors throughout the CNS (see Introduction 10,11,32). Actions of ATP as a neurotransmitter in the CNS via fast ionotropic and slow metabotropic receptors brings ATP in line with most other neurotransmitters, such as acetylcholine, GABA, glutamate and 5-hydroxytryptamine, for which ligand-gated and G-protein-mediated receptor subclassification has already been established. 1 Specific effects at P2 receptors of the purine compounds were confirmed by antagonism of the excitatory responses with suramin (barrel concentration 0.02 M). However, at a higher concentration of suramin (0.2 M), non-selective effects were apparent as excitation evoked by glutamate was also blocked. This indicates that it is critical to evaluate the effects of suramin with care, as suggested previously. 17 Suramin is also able to inhibit ectonucleotidase activity, 20 which may contribute to its excitatory effect on ongoing neuronal activity. The responses of some neurons to a,b-meATP were not blocked by suramin, which may imply actions at P2X4 and P2X6 receptor proteins, which are suramin insensitive. As suramin does not block all subtypes of P2 receptors, the absence of functional antagonism cannot be regarded as evidence against an involvement of P2 receptors. The general lack of effect of suramin as an inhibitor of baseline firing may not be surprising given that suramin typically did not block excitation to exogenously applied purines. In those RVLM neurons in which suramin did block responses to ATP, and its analogues, there was not always a corresponding inhibition of spontaneous firing, suggesting that ATP is not released
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Fig. 8. Functional characterization and pharmacological responses of an RVLM neuron to a,b-meATP in the absence (A) and presence (B) of suramin. The neuron was excited by electrical stimulation in both the T2 region of the spinal cord (spinal; upper left) and the HDA; upper right; three superimposed sweeps). The neuron fired with minimal cardiac-related activity (CRA); R-wave-triggered spike interval histogram (lower right) and arterial blood pressure (AP; upper right) (averages of 1200 sweeps) are also shown. (A) Effective dose-related increase in firing rate to a,b-meATP. (B) Suramin did not block the effective doserelated increase in firing by a,b-meATP. Note that suramin decreased the baseline firing rate of this neuron. Bin size: 3 s.
tonically on to these neurons, at least not under the conditions of the present study. Sun et al. 27 have also reported that suramin (used at a barrel concentration of 0.1 M) is ineffective at altering the baseline firing of RVLM neurons, although, in contrast to the present study, suramin consistently blocked excitation of neuronal firing by ionophoretically applied ATP. This difference between the two studies may reflect the fact that the populations of RVLM neurons studied were significantly different in terms of their spinal projections and baroreceptor inputs. The effective dose of suramin used is also likely to be critical for P2 receptor blockade. The P2 receptor antagonist PPADS generally excited RVLM neurons, even when used at relatively low barrel concentrations, perhaps due to membrane depolarization, and thus could not be examined as an antagonist of responses to purines. Sensitivity of RVLM neurons to a,b-meATP implies actions at P2X1 or P2X3 receptor subunits. Recombinant and native P2X1 and P2X3 receptors have been shown to desensitize rapidly (,100 ms), whereas the other P2X subtypes desensitize slowly or not at all. 7 In the current study, desensitization to a,b-meATP observed in some neurons is consistent with an involvement of P2X1 and/or P2X3 receptor proteins. In two neurons, desensitization to
a,b-meATP reduced the firing rate to below baseline, suggesting that ATP is released on to a subpopulation of neurons in the RVLM. Sun 26 showed that, during application of a,bmeATP, the firing rate of reticulospinal neurons gradually declined, and this abolished subsequent responses to ATP. In the current study, the lack of desensitization of responses to ATP, 2MeSATP and a,b-meATP, which was observed for the majority of neurons, is unequivocal. For those neurons activated by a,b-meATP, this may indicate that a heteromeric receptor is formed from an a,b-meATP-sensitive P2X receptor subunit in combination with a non-desensitizing P2X subunit, as suggested for non-desensitizing a,b-meATP-sensitive P2X2 and P2X3 receptors in adult rat sensory ganglia. 15,16 Non-desensitizing P2X2, P2X4 and P2X6 proteins are highly expressed throughout the adult rat brain, 4,30 and are possible candidates for the non-desensitizing component of a heteromeric a,b-meATP-sensitive RVLM P2X receptor. Since the P2 receptors in the RVLM, as in most other brain regions, appear to be mainly non-desensitizing, rapid degradation of ATP by extracellular and neuronally released ectonucleotidases 29 may be more important for termination of ATP actions. The subtype identity of the P2Y receptors mediating excitation of RVLM neurons by ADPbS and UTP is unclear. UTP
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of a plexus of nerves in the nucleus tractus solitarius disappears after nodose ganglionectomy, consistent with a presynaptic location of these receptors. 30 a,b-meATP and ADPbS are non-hydrolysable agonists, which indicates that ATP does not have to be metabolized to adenosine for a functional response. In contrast, 2MeSATP, ATP and UTP are rapidly degraded, and actions at P1 receptors following degradation to adenosine have to be considered; both excitation and inhibition by adenosine of neuronal firing in the RVLM have been reported. 28 At least a part of the inhibitory responses to purines reported in the present study may be due to direct or indirect effects at P1 receptors, and in some cases this may be masked by excitatory effects mediated at P2 receptors. Co-application of a ,b -methylene-ATP with glutamate Ionophoretic co-application of the P2X-selective agonist a,b-meATP with the excitatory amino acid glutamate elicited excitatory responses that were additive. Thus, if ATP is coreleased as an excitatory transmitter with glutamate from neurons in the RVLM, there appears to be no functional synergism between these neurotransmitters, at least under the conditions of the present study. CONCLUSIONS
Fig. 9. Distribution of recording sites in the RVLM. The location of 23 recording sites was identified by ionophoretic deposition of Pontamine Sky Blue dye, and the positions mapped to coronal sections of the medulla oblongata. 19 Numbers indicate distance from the interaural line.
acts at P2Y2 (or P2U) receptors activated equipotently by ATP and UTP, and at P2Y4, P2Y6 and endogenous uridine nucleotide-specific receptors, which are activated by UTP and UDP, but not by ATP. However, UTP has also been shown to be a weak agonist at the P2X3 receptor. 3,23 An issue which could not be addressed in the present study is whether P2 receptors in the RVLM have a pre- or a postsynaptic location. Both pre- and postsynaptic expression of P2X receptors has been described in other brain regions. P2X2 immunoreactivity has been localized on neurons and their processes in defined brain nuclei; the P2X2 immunoreactivity
We have shown that P2 receptors can modulate the activity of neurons in the RVLM in vivo. This involves both P2X and P2Y receptors, although further characterization of the specific subtypes within each of these families awaits the development and use of selective agonists and antagonists. These findings support the concept of purinergic signalling in the CNS, and more specifically in control of cardiovascular function via reflex and central pathways involving the RVLM. Whilst these data do not imply the existence of specific purinergic pathways, they do support the notion of the co-transmission of purines with conventional excitatory and inhibitory transmitters. This adds to the potential of integrative actions at the level of the RVLM. Acknowledgements—We are grateful to the Royal Society (V.R.) and the British Heart Foundation (T.T.) for their generous support of this study. The study was supported by a British Heart Foundation programme grant (to K.M.S.). Linda Hutchinson is thanked for excellent technical assistance.
REFERENCES
1. Burnstock G. (1996) Development and perspectives of the purinoceptor concept. J. auton. Pharmac. 16, 295–302. 2. Burnstock G. and King B. F. (1996) Numbering of cloned P2 purinoceptors. Drug Dev. Res. 38, 67–71. 3. Chen C.-C., Akopian A. N., Sivilotti L., Colquhoun D., Burnstock G. and Wood J. N. (1995) A P2X purinoceptor expressed by a subset of sensory neurones. Nature 377, 428–431. 4. Collo G., North R. A., Kawashima E., Merlo-Pich E., Neidhart S., Surprenant A. and 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. 5. Edwards F. A., Gibb A. J. and Colquhoun D. (1992) ATP receptor mediated synaptic currents in the central nervous system. Nature 359, 144–146. 6. Ergene E., Dunbar J. C., O’Leary D. S. and Barraco R. A. (1994) Activation of P2-purinoceptors in the nucleus tractus solitarius mediate depressor responses. Neurosci. Lett. 174, 188–192. 7. Evans R. J. and Surprenant A. (1996) P2X receptors in autonomic and sensory neurones. Semin. Neurosci. 8, 217–223. 8. Fredholm B. B., Burnstock G., Harden K. T. and Spedding M. (1996) Receptor nomenclature. Drug Dev. Res. 39, 461–466. 9. Guyenet P. G. (1990) Role of the ventral medulla oblongata in blood pressure regulation. In Central Regulation of Autonomic Functions (eds Loewy A. D. and Spyer K. M.), pp. 145–167. Oxford University Press, Oxford. 10. Ikeuchi Y. and Nishizaki T. (1995) The P2Y purinoceptor-operated potassium channel is possibly regulated by the bg subunits of a pertussis toxininsensitive G-protein in cultured rat inferior colliculus neurones. Biochem. biophys. Res. Commun. 214, 589–596. 11. Ikeuchi Y. and Nishizaki T. (1996) P2-purinoceptor-operated potassium channel in rat cerebellar neurones. Biochem. biophys. Res. Commun. 218, 67–71. 12. Illes P., Nieber K. and No¨renberg W. (1996) Electrophysiological effects of ATP on brain neurones. J. auton. Pharmac. 16, 407–411.
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13. Inoue K., Nakazawa K., Fujimori K., Watano T. and Takanaka A. (1992) Extracellular adenosine-5 0 -triphosphate-evoked glutamate release in cultured hippocampal neurones. Neurosci. Lett. 134, 215–218. 14. Jordan D. (1995) Central nervous integration of cardiovascular control. In Cardiovascular Regulation (eds Jordan D. and Marshall J.), pp. 1–14. Cambridge University Press, Cambridge. 15. Khakh B. S., Humphrey P. P. and Surprenant A. (1995) Electrophysiological properties of P2X-purinoceptors in rat superior cervical, nodose and guineapig coeliac neurones. J. Physiol., Lond. 484, 385–395. 16. Lewis C., Neidhart S., Holy C., North R. A., Buell G. and Surprenant A. (1995) Coexpression of P2X2 and P2X3 receptor subunits can account for ATPgated currents in sensory neurones. Nature 377, 432–435. 17. Nakazawa K., Inoue K., Ito K., Koizumi S. and Inoue K. (1995) Inhibition by suramin and reactive blue 2 of GABA and glutamate receptor channels in rat hippocampal neurones. Naunyn-Schmiedeberg’s Arch. Pharmac. 351, 202–208. 18. Nieber K., Poelchen W. and Illes P. (1997) Role of ATP in fast excitatory synaptic potentials in locus coeruleus neurones of the rat. Br. J. Pharmac. 122, 423–430. 19. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates, 2nd edn. Academic, New York. 20. Ralevic V. and Burnstock G. (1998) Receptors for purines and pyrimidines. Pharmac. Rev. 50, 413–492. 21. Ralevic V., Thomas T., Burnstock G. and Spyer K. M. (1997) P2 receptor-mediated changes in activity of neurones in the rostral ventrolateral medulla of the rat: an in vivo ionophoretic study. J. auton. nerv. Syst. 65, 104. 22. Ralevic V., Thomas T. and Spyer K. M. (1998) Effects of P2 purine receptor agonists microinjected into the rostral ventrolateral medulla on the cardiovascular and respiratory systems of the anaesthetised rat. J. Physiol., Lond. 509P, 127P. 23. Robertson S. J., Rae M. G., Rowan E. G. and Kennedy C. (1996) Characterization of a P2X-purinoceptor in cultured neurones of the rat dorsal root ganglia. Br. J. Pharmac. 118, 951–956. 24. Spyer K. M. (1994) Central nervous mechanisms contributing to cardiovascular control. J. Physiol., Lond. 474, 1–19. 25. St Lambert J. H., Thomas T., Burnstock G. and Spyer K. M. (1997) A source of adenosine involved in cardiovascular responses to defense area stimulation. Am. J. Physiol. 272, R195–R200. 26. Sun M. K. (1996) Pharmacology of reticulospinal vasomotor neurones in cardiovascular regulation. Pharmac. Rev. 48, 465–494. 27. Sun M. K., Wahlestedt C. and Reis D. J. (1992) Action of externally applied ATP on rat reticulospinal vasomotor neurones. Eur. J. Pharmac. 224, 93–96. 28. Thomas T. and Spyer K. M. (1999) A novel influence of adenosine on ongoing activity in rat rostral ventrolateral medulla. Neuroscience 88, 1213–1223. 29. Todorov L., Mihaylova-Todorova S., Westfall T. D., Sneddon P., Kennedy C., Bjur R. A. and Westfall D. P. (1997) Neuronal release of soluble nucleotidases and their role in neurotransmitter inactivation. Nature 387, 76–79. 30. Vulchanova L., Arvidsson U., Riedl M., Wang J., Buell G., Surprenant A., North R. A. and Elde R. (1996) Differential distribution of two ATP-gated channels (P2X receptors) determined by immunocytochemistry. Proc. natn. Acad. Sci. U.S.A. 93, 8063–8067. 31. Zagon A. and Spyer K. M. (1996) Stimulation of aortic nerve evokes three different response patterns in neurones of rostral VLM of the rat. Am. J. Physiol. 271, R1720–R1728. 32. Zhang Y. X., Yamashita H., Oshita T., Sawamoto N. and Nakamura S. (1995) ATP increases extracellular dopamine level through stimulation of P2Y purinoceptors in the rat striatum. Brain Res. 691, 205–212. 33. Zimmermann H. (1994) Signalling via ATP in the nervous system. Trends Neurosci. 17, 420–426. (Accepted 25 June 1999)
Pergamon PII: S0306-4522(99)00376-0
Neuroscience Vol. 94, No. 3, pp. 867–878, 1999 867 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00
CHARACTERIZATION OF P2 RECEPTORS MODULATING NEURAL ACTIVITY IN RAT ROSTRAL VENTROLATERAL MEDULLA V. RALEVIC,* T. THOMAS, G. BURNSTOCK and K. M. SPYER Autonomic Neuroscience Institute, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, U.K.
Abstract—This study investigated the effects of ATP, and related compounds, on the activity of neurons within the rostral ventrolateral medulla, an area of fundamental importance in reflex control of the cardiovascular system. Extracellular recordings were made from single neurons in anaesthetized, paralysed and artificially ventilated rats. Ionophoretic application of a,b-methylene-ATP, adenosine 5 0 -O-(2-thiodiphosphate), UTP, 2-methylthio-ATP and ATP altered the ongoing activity in the majority of neurons (.74% of neurons), generally causing increases in the firing rate. Nine of 11 cells with presumed spinal projection were excited by ATP and/or the P2X-selective agonist a,b-methylene-ATP. Desensitization of the excitatory responses to a,b-methylene-ATP was observed in four of 20 rostral ventrolateral medulla neurons. For the remainder of the rostral ventrolateral medulla neurons, the increase in firing rate evoked by a,b-methylene-ATP, and by the other purine compounds tested, did not undergo desensitization. Suramin, a P2 receptor antagonist, blocked excitatory responses to adenosine 5 0 -O-(2-thiodiphosphate) or a,bmethylene-ATP in five of 16 neurons. These results indicate that ATP can modulate the activity of neurons in the rostral ventrolateral medulla via actions at P2 purine receptors. The data suggest that both P2X and P2Y receptors are involved, and that the functional expression of these receptors within the rostral ventrolateral medulla is not uniform. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: ATP, P2 receptors, rostral ventrolateral medulla.
arterial pressure. 27 Since that study was conducted, there have been significant advances in purine receptor research, and using this information and newly available purine receptor ligands, we aimed to determine the subtype identity of P2 receptors in the RVLM. Seven different mammalian P2X receptors (P2X1–7) and five P2Y receptors (P2Y1,2,4,6,11) have been cloned, characterized and formally recognized as members of the P2 receptor family. 2,8 It should be noted that, whereas P2Y receptors are single proteins, co-expressed P2X receptor proteins may combine to form heteromeric receptors. It is not yet known how the properties of the different P2X subunits influence the phenotype of the heteromeric receptor. This, together with co-expression of different types of endogenous P2 receptors, has contributed to the complex pharmacological response profiles exhibited by P2 receptors to many biological tissues, that do not match with those of any of the cloned receptors. The general lack of subtype-selective agonists and antagonists has proved a major handicap in P2 receptor characterization. Suramin and pyridoxalphosphate-6-azophenyl-2 0 ,4 0 disulphonic acid (PPADS) are P2 receptor antagonists, but do not block P2X4 and P2X6 receptors and a subpopulation of P2Y1 and P2Y2 receptors. a,b-meATP is a non-hydrolysable agonist and desensitizing agent selective for P2X1 and P2X3 receptors, 2-methylthio-ATP (2MeSATP) acts at both P2X and P2Y receptors, and adenosine 5 0 -O-(2-thiodiphosphate) (ADPbS) and UTP are selective for P2Y receptors. The different rates of desensitization of P2X receptors have also proved useful in their characterization: P2X1 and P2X3 receptors desensitize rapidly, whereas P2X2, P2X4, P2X6 and P2X7 receptors desensitize slowly or not at all. 7 P2Y receptors do not desensitize rapidly. The present study investigates the actions of ATP and its analogues on the ongoing activity of individual neurons throughout the RVLM in anaesthetized rats, with the aim of characterizing the P2 purine receptor subtypes involved. We
Extracellular ATP mediates its effects via cell surface P2 receptors that have been divided into two families according to whether they are ligand-gated cation channels (P2X receptors) or are coupled to G-proteins (P2Y receptors). 2,8,20 Electrophysiological studies conducted mainly on dissociated or cultured neurons and on brain slices have shown that ATP evokes rapid inward currents and modulates the activity of neurons via P2 receptors throughout the CNS, and evidence for an involvement of both P2X and P2Y receptors has been presented. 12,33 A physiological correlate is provided in the medial habenula and locus coeruleus, where ATP has been shown to mediate fast synaptic currents via P2X receptors; the currents are blocked by suramin and a,b-methylene-ATP (a,b-meATP) desensitization, and are mimicked by ATP and a,b-meATP. 5,18 Complementary immunohistochemical evidence has identified specific patterns of P2X subtype labelling in different regions of the brain, with P2X2, P2X4 and P2X6 being the most abundantly expressed subtypes. 4,30 We have obtained evidence for a role of purines in cardiovascular control primarily at the level of the medulla oblongata, including the rostral ventrolateral medulla (RVLM), 28 an area considered to be of fundamental importance in the mediation of reflex control of the cardiovascular system. 24,26 The present study arose from experimental studies indicating that ionophoretic application of ATP and its stable analogue, a,b-meATP, in anaesthetized rats, excites RVLM neurons in a suramin-sensitive manner and mediates an increase in mean *To whom correspondence should be addressed at: School of Biomedical Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham NG7 2UH, U.K. (present address). Tel.: +44-115-970-9480; fax: +44-115-970-9259. E-mail address: [email protected] (V. Ralevic) Abbreviations: ADPbS, adenosine 5 0 -O-(2-thiodiphosphate); HDA, hypothalamic defence area; a,b-meATP, a,b-methylene-ATP; 2MeSATP, 2-methylthio-ATP; PPADS, pyridoxalphosphate-6-azophenyl-2 0 ,4 0 -disulphonic acid; RVLM, rostral ventrolateral medulla. 867
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were particularly interested in neurons receiving input from the hypothalamic defence area (HDA), since studies from our laboratory have indicated a role for purines in the cardiovascular components of the response to stimulation of that region. 25 A preliminary account of our results was presented at the inaugural meeting of the International Society for Autonomic Neuroscience. 21 EXPERIMENTAL PROCEDURES
General methods Experiments were carried out on male Sprague–Dawley rats (300– 350 g) anaesthetized initially with pentobarbitone sodium (60 mg/kg, i.p., Sagatale) and supplemented as necessary with a-chloralose (40 mg/kg, i.v.). The depth of anaesthesia was assessed by monitoring the stability of blood pressure and heart rate, and the minimal effects on cardiovascular variables to a pinch to the paw. Following paralysis (see below), the stability of these variables alone was used to assess the level of anaesthesia. 28 All of the following procedures and protocols were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986. Surgical procedure The temperature of the animal was maintained at 37–37.58C with a servo-controlled heating pad. The left femoral artery and vein were cannulated for measurement of arterial blood pressure (Neurolog System) and administration of supplemental anaesthetic, respectively. The trachea was cannulated for artificial ventilation. The left aortic nerve was dissected and prepared for stimulation with bipolar silver electrodes. Using high-frequency stimulation (100–300 mA, 1 ms, 100 Hz), we tested that aortic nerve stimulation evoked only a vasodepressor response; a significant fall in blood pressure was always observed. Unfortunately, because of difficulties with dissection of the aortic nerve in some cases, this test was not applied in all animals. The rats were fixed in a stereotaxic frame, paralysed with gallamine triethiodide (Flaxedile, 3 mg/kg/h) and ventilated artificially with oxygen-enriched air (Harvard ventilator, model 683). Partial laminectomy at the T2–T3 level was performed and a concentric bipolar stimulating electrode (SNE 100, Rhodes Medical Electrodes; shaft diameter 0.5 mm, tip exposure 0.5 mm × 0.2 mm) placed in the T2 region of the spinal cord for antidromic identification of cells with spinal projection (50–200 mA, 0.2 ms, 1 Hz). The electrode was placed on the left side with its tip 1.0–2.0 mm below the dorsolateral sulcus. Neurons responding to electrical stimulation in T2–T3 at a constant latency (.five sweeps) were presumed to have spinal projection. The collision test was not used routinely. Muscles were removed from the back of the neck, an occipital craniotomy performed and the cerebellum removed by suction to allow access of microelectrodes to the rostral region of the brainstem. A hole (diameter 3 mm) was drilled in the skull 2 mm caudal and 1 mm lateral of bregma to allow a stimulating electrode to be placed 7 mm from the dorsal surface, in the HDA, 25 for identification of neurons with HDA input. The HDA was identified on the basis of the characteristic cardiorespiratory response observed on stimulation in this region. 25 Recordings The surface coordinates used for positioning of microelectrodes in the region of the RVLM were 1.5–2.0 mm rostral and 2.0–2.5 mm lateral of obex, and 2.5–3.6 mm below the dorsal surface of the medulla oblongata. Extracellular recordings were made from RVLM neurons with five-barrelled glass microelectrodes (tip diameter 1– 2 mm, 7–10 V). The recording barrel contained 4 M sodium chloride. The others contained the excitatory amino acid l-glutamate (0.2 M, pH 8.0), Pontamine Sky Blue dye (2%), and the remaining barrels were filled with purine receptor agonists and/or antagonists (0.02–0.2 M, pH 8.0). Drugs were applied by ionophoresis (Neurophore, Medical Systems) at 20–80 nA; a retaining current of 20 nA was applied to each drug barrel between drug ejection periods. Firing was induced in neurons with no ongoing activity by ionophoretic application of glutamate. Automatic current balancing on the Neurophore limited current artifacts. This was confirmed by the fact that ionophoretic
application of saline as a current control did not elicit or modify neuronal firing, and there was also a lack of response of some neurons to selected purine compounds. Neuronal activity was recorded on video tape via a digital interface (Instrutech, VR-100B), together with records of arterial blood pressure for off-line analysis.
Data analysis Data analysis was carried out off-line after analogue-to-digital data conversion (CED 1401-Plus data acquisition system). The commercially available software packages CED Spike 2 and Signal Averager were used to analyse data. Single-unit activity is represented throughout as rate histograms after discrimination of neuronal activity using a window discriminator (Digitimer D130).
Histology After recording from a neuron, Pontamine Sky Blue (2%) was injected with 2430 nA current for 5 min. At the end of the experiment the brain was removed, fixed in a 10% solution of formal saline, frozen and sectioned serially (80 mm), and counterstained with Neutral Red. The sections were viewed using a light microscope, and the location of marked injection sites mapped with the aid of the atlas of Paxinos and Watson. 19
Materials ADPbS (trilithium salt; 0.01 M), ATP (disodium salt; 0.2 M), lglutamate (0.2 M), UTP (sodium salt; 0.2 M), a,b-meATP (lithium salt; 0.02 M) and Pontamine Sky Blue were purchased from Sigma (Poole, U.K.). 2-MeSATP (tetrasodium salt; 0.02 M) was from Research Biochemicals. PPADS (0.001 and 0.01 M) was from Tocris Cookson (Bristol, U.K.). Suramin (0.02 and 0.2 M) was from Bayer. All drugs were dissolved in distilled water, except for a,b-meATP, which was dissolved in saline, and Pontamine Sky Blue, which was dissolved in 0.5 M sodium acetate. The pH of drugs for ionophoresis was adjusted to 8.0.
RESULTS
General properties of recorded neurons Extracellular recordings were made from a total of 105 neurons in 28 rats. Of these, 86 were excited by stimulation of the spinal cord, 75 of these synaptically only (see Figs 1–3 as examples) and 11 antidromically. In the case of cells with presumed spinal projection, the mean response latency was 5.12 ^ 1.0 ms (range 1.87–13.60 ms, n 11), suggesting that they had small myelinated axons. In addition, the activity of a large proportion (56 of 96 neurons) of the neurons studied was increased and/or inhibited by electrical stimulation in the HDA (50–200 mA, 1-ms pulse, 1 Hz). Of 45 neurons, 53% (24 of 45) of neurons showed an excitatory response only, 4% (two of 45) of neurons were inhibited only and 16% (seven of 45) of neurons had excitation but also late inhibition. In a further 11 neurons, where ongoing discharge rate was low, an excitatory response was observed but accompanying inhibitory responses could not be effectively analysed. In the case of the cells with presumed spinal projection, seven of 11 showed an excitatory response to HDA stimulation; the remaining four neurons were not excited by stimulation in the HDA. Thirty per cent (21 of 69) of RVLM neurons were excited by aortic nerve stimulation (100–300 mA, 1 ms, 1 Hz), as described previously. 31 In the case of 22% (five of 23) of the RVLM neurons, a continuous train of stimuli resulted in a prolonged inhibition of ongoing activity.
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Fig. 1. Effect of ionophoretic application of ATP and a,b-meATP on the ongoing firing rate of an RVLM neuron. This neuron was excited by electrical stimulation in the T2 region of the spinal cord (spinal; top; three superimposed sweeps), but not by stimulation in the HDA (not shown). (A) The neuron was excited in an effective dose-related manner (20–80 nA) by ionophoretic application of ATP. (B) By contrast, a,b-meATP at the same currents did not elicit an increase in the ongoing rate of firing. Bin size: 4 s.
Effects of the P2 receptor agonists ATP, a ,b -methylene-ATP, 2-methylthio-ATP, adenosine 5 0 -O-(2-thiodiphosphate) and UTP The actions of P2 receptor agonists on the firing properties of RVLM neurons without reference to their aortic nerve and HDA inputs will be described initially. Ionophoretic application of ATP (20–80 nA) elicited dose-related increases in the firing rate in 40 of 48 RVLM neurons (Fig. 1). Responses were maintained in the continuous presence of ATP (Fig. 1). ATP elicited a decrease in firing in a small number of neurons (n 4) and was ineffective in a further four neurons. In general, ionophoretic application of the P2X-selective agonist a,b-meATP (20–80 nA) elicited increases in neuronal discharge in a dose-related manner (35 of 50). Where ATP and a,b-meATP (20–80 nA) were applied ionophoretically to the same cell (19 neurons), nine of these were excited by both ATP and a,b-meATP (Figs 2, 3). In six neurons ATP mediated excitation, but a,b-meATP was ineffective (Fig. 1). Typically, in those neurons in which a,b-meATP elicited
an increase in firing, the excitatory response did not desensitize, either during repetitive or continuous application (Figs 4, 5). Of the 20 neurons in which this was studied, autodesensitization of the excitatory response to a,b-meATP was observed in only four neurons (see Figs 2 and 3 as examples). In three of these desensitization was complete, with firing decreasing to, or below, baseline levels in the continuous presence of a,b-meATP (Fig. 3). In two of these neurons, the excitatory response to ATP was partially blocked by a,b-meATP desensitization (Figs 2, 3). Ionophoretic application of 2MeSATP (Fig. 6), ADPbS (Fig. 7) and UTP (20–80 nA) elicited an effective doserelated excitation in the majority of cells in which their actions were tested (27 of 36 neurons), with inhibition in three of 36. In general, the increase in firing evoked by 2MeSATP did not desensitize on continuous (Fig. 6) or repeated application (five of six neurons). Desensitization of the response was not observed for excitation evoked by ADPbS or UTP. The P2Y-selective agonist ADPbS and the P2X-selective
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Fig. 2. Effect of repetitive application of a,b-meATP on the effect of subsequent application of ATP on the ongoing firing rate of an RVLM neuron. This neuron was excited by electrical stimulation in both the T2 region of the spinal cord (spinal; upper left; three superimposed sweeps) and in the HDA (upper right; three superimposed sweeps). (A) Ionophoretic application of ATP elicited an increase in the rate of firing. (B) Ionophoretic application of a,b-meATP elicited an increase in firing, which decreased with repeated application. (C) Following desensitization to a,b-meATP, ionophoretic application of ATP decreased the rate of neuronal firing. Bin size: 5 s.
agonist a,b-meATP were applied ionophoretically (20– 80 nA) to the same cell in five cases; three of these were excited by both agonists and in two of these excitation resulting from co-application of ADPbS and a,b-meATP was shown to be additive. In two cells, excitation was mediated by either ADPbS (Fig. 7) or a,b-meATP only. ATP and UTP
were applied ionophoretically (20–80 nA) to the same cell in six cells; all were excited by both agonists. Effects of co-application of purines with glutamate In four neurons, a,b-meATP and glutamate were applied
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Fig. 3. Effects of ionophoretic application of ATP and a,b-meATP on the firing rate of an RVLM neuron. This neuron was excited by electrical stimulation in the T2 region of the spinal cord (spinal; three superimposed sweeps), but not in the HDA (three superimposed sweeps), nor by repetitive stimulation of the aortic nerve (not shown). (A) Ionophoretic application of ATP elicited an increase in the rate of firing which was maintained in the presence of ATP. Bin size: 3 s. (B) Ionophoretic application of a,b-meATP elicited an increase followed by a decrease in firing; firing rate was reduced to below that of the baseline in the presence, and after offset of application, of a,b-meATP. Bin size: 1.5 s. (C) In the continuous presence of a,b-meATP, the excitatory response to ATP was only partially blocked. Bin size: 2 s.
separately initially and then simultaneously (each at both 20 and 40 nA). Co-application of a,b-meATP and glutamate mediated an excitation which was additive of the responses evoked by these agents applied individually (Fig. 5). Effects of the P2 receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2 0 ,4 0 -disulphonic acid The effect of ionophoretic application of suramin was studied using barrel concentrations of 0.02 M (25 neurons) and 0.2 M (five neurons). Of these 30 neurons, suramin was tested against agonist-evoked responses in 20 cases by ionophoresis for 2 min prior to and during ionophoresis of ATP, a,b-meATP or 2MeSATP. Suramin (0.02 M) had no effect on baseline firing in 15 of 25 neurons; it increased firing in seven neurons (Fig. 5) and reduced firing in three neurons (Fig. 8). In those neurons in which suramin increased firing, its effects as a potential antagonist of purine-mediated excitation were not assessed.
Suramin (0.02 M) blocked excitatory responses to 2MeSATP in two of seven neurons (Fig. 6). The selectivity of suramin at this concentration was confirmed in one neuron where excitation to 2MeSATP, but not that to glutamate, was blocked by suramin (Fig. 6). Suramin (0.02 M) blocked responses to a,bmeATP in four of nine neurons (Fig. 4), and did not block excitation to a,b-meATP in the remaining five neurons (Figs 5, 8). Suramin, used at a higher barrel concentration of 0.2 M, had no effect on baseline firing in three of five neurons; it increased firing in one neuron and reduced firing in one neuron. Suramin (0.2 M) blocked excitation mediated by ATP in three of four neurons; in one of these, an inhibitory effect of ATP was revealed. However, glutamate-mediated excitation was also blocked by suramin (n 2), suggesting that at this barrel concentration suramin acts non-selectively. Ionophoretic application of PPADS at a barrel concentration of 0.01 M (20–40 nA) produced excitation in 12 of 16 cells. Of the four cells in which it had no direct effects,
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Fig. 4. Effect of a,b-meATP on the ongoing firing rate of an RVLM neuron in the absence (B) and presence (C) of suramin. (A) This neuron was excited by electrical stimulation in the T2 region of the spinal cord (spinal; left; three superimposed sweeps) and in the HDA region (right; four superimposed sweeps). (B) The neuron was excited by a,b-meATP in a manner which did not desensitize either during the response or with repeated application of a,b-meATP. Bin size: 3 s. (C) In the presence of suramin, there was a small decrease in baseline firing and the response to a,b-meATP was blocked. Bin size: 2 s.
PPADS (0.01 M) blocked excitation to a,b-meATP in one cell and had no effect on excitation to a,b-meATP in another. In the remaining two cells, a,b-meATP was ineffective in altering firing rate. Ionophoretic application of PPADS at the lower barrel concentration of 0.001 M (20–40 nA) elicited an increase in firing in three of four cells. Physiological inputs of purine-sensitive rostral ventrolateral medulla neurons The physiological inputs of RVLM neurons excited by purine compounds are summarized in Table 1. In general, cells with presumed spinal projection were excited by ionophoretic application of ATP (four of six neurons) or a,b-meATP (five of seven neurons). In two cells with presumed spinal projection in which ATP and a,b-meATP were co-applied, ATP increased the firing, whilst a,b-meATP induced little, if any, change in firing. Cells with presumed spinal projection that were excited by stimulation in the HDA were generally excited by ATP (three of four
neurons) and a,b-meATP (four of five neurons). These effects of ATP and a,b-meATP on cells with presumed spinal projection do not appear to differ from the effects of these agents on the total population of neurons. Interestingly, all RVLM neurons that received an aortic input, whether excitatory or inhibitory, were excited by ATP, whereas only 81% of those RVLM neurons with no aortic input were excited by ATP (Table 1). a,b-meATP increased the firing rate of all neurons with an inhibitory aortic input, compared with an excitation of only 75% and 68% of neurons excited by and not responding to stimulation of the aortic nerve, respectively (Table 1). All neurons that were inhibited by stimulation in the HDA were excited by ATP, compared to ATP-mediated excitation of 79% of those with an excitatory input and 88% of those with no HDA input (Table 1). Localization of recording sites Twenty-three recording sites were marked by ionophoresis
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Fig. 5. Effect of a,b-meATP on the firing rate of an RVLM neuron in the absence and presence of suramin (A), during continuous application for .1 min (B) and during co-application of glutamate (C). The neuron was excited by electrical stimulation in the T2 region of the spinal cord (spinal; three superimposed sweeps), but not by stimulation of the HDA (not shown). (A) The neuron was excited by a,b-meATP in an effective dose-related manner, which was not blocked by suramin. (B) The excitatory response to a,b-meATP did not desensitize during continuous ionophoresis for greater than 2 min. (C) Ionophoresis of a,b-meATP simultaneously with glutamate (Glu) elicited an increase in firing which was additive of the individual increases in firing produced by these agents. Bin size: 2 s.
of Pontamine Sky Blue in eight animals. Examination under the light microscope revealed that the recording sites were located within the area of the RVLM, and these were mapped using a stereotaxic atlas 19 (Fig. 9). DISCUSSION
This study is the first to show that both P2X and P2Y receptors for ATP can modulate the ongoing activity of neurons with different inputs in the RVLM. This has implications for our understanding of central purinergic control of the cardiovascular system as, amongst other functions, the RVLM is critically involved in integration of cardiovascular reflexes. Physiological implications of P2 purine receptor modulation of rostral ventrolateral medulla neurons It is well established that a population of neurons within the ventrolateral medulla has a crucial role in maintaining the activity of vasomotor sympathetic preganglionic neurons and is important in reflex control of the cardiovascular
system. 9,24,26 The current study, showing that P2X and P2Y purine receptor ligands modulate the activity of RVLM neurons, extends the findings of an earlier study showing an increase in neuronal firing of bulbospinal vasomotor neurons following ionophoretic application of ATP and a,b-meATP into the rat RVLM. 27 It should be noted that the RVLM contains a variety of populations of neurons with different functions. 26 Those investigated in the present study included neurons with characteristics of sympathoexcitatory neurons. Modulation of their activity by P2 receptors is consistent with a role for ATP in the RVLM-mediated control of sympathetic activity and of the defence response, since many were also affected by HDA stimulation. The predominantly excitatory responses of the neurons recorded in the RVLM following ionophoretic application of P2 receptor agonists are consistent with the increase in arterial blood pressure observed when ATP and a,bmeATP are microinjected into the rat RVLM. 22,27 This contrasts with the hypotension and bradycardia that occur on microinjection of a,b-meATP and 2MeSATP into the nucleus tractus solitarius of the rat, 6 the site of termination of baroreceptor afferents, with neurons functionally linked
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Fig. 6. Effect of 2-MeSATP on the firing rate of an RVLM neuron in the absence (A–C) and presence (D) of suramin. This neuron was excited by electrical stimulation in both the T2 region of the spinal cord (spinal; upper left; three superimposed sweeps) and the HDA (upper right; two superimposed sweeps), but not by repetitive electrical stimulation of the aortic nerve (not shown). (A–C) 2MeSATP elicited an effective dose-related increase in firing of this neuron. Glutamate (Glut; A) was also shown to elicit an increase in firing, but suramin applied at the same currents had no effect on neuronal firing. (D) In the presence of suramin, excitatory responses to 2MeSATP were selectively blocked, those to glutamate being unaffected. Bin size: 2 s.
to presympathetic neurons in the RVLM by multisynaptic pathways. 14,24 Neurons within the RVLM with identified spinal, HDA and/or aortic nerve inputs were examined for their patterns of responses to P2 receptor agonists. Interestingly, all RVLM
neurons that received aortic nerve input, whether excitatory or inhibitory, were excited by ATP, compared with an ATPmediated excitation of 81% of neurons having no aortic input. All neurons with an inhibitory aortic nerve input were excited by a,b-meATP, but a proportion of those
Table 1. Percentage of rostral ventrolateral medulla neurons with different physiological inputs that respond with excitation by purines Purine
Excitatory spinal input No spinal input Excitatory HDA input Inhibitory HDA input No HDA input Excitatory aortic input Inhibitory aortic input No aortic input
ATP
a,b-meATP
2MeSATP
ADPbS
UTP
78 (28/36) 100 (11/11) 79 (19/24) 100 (6/6) 88 (21/24) 100 (11/11) 100 (4/4) 81 (21/26)
71 (35/49) 60 (3/5) 67 (18/27) 80 (4/5) 77 (17/22) 75 (9/12) 100 (4/4) 68 (13/19)
73 (11/15) — (1/1) 77 (10/13) — 67 (2/3) 60 (3/5) — 71 (5/7)
55 (6/11) — (1/1) — (1/2) — — (0/2) — (0/1) — 56 (5/9)
100 (5/5) 100 (2/2) 100 (3/3) — 100 (4/4) — — 100 (4/4)
Values are percentages, with numbers of cells given in parentheses.
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Fig. 7. Functional characterization and pharmacological responses to a,b-meATP and ADPbS of an RVLM neuron. The neuron was excited by electrical stimulation in the T2 region of the spinal cord (spinal; five superimposed sweeps; upper left), but not by repetitive stimulation of the aortic nerve (not shown). It fired with cardiac-related activity (CRA; upper right); R-wave-triggered spike interval histogram (lower) and arterial blood pressure (AP; upper) (average of 700 sweeps) are also shown. The neuron was excited by ionophoretic application of ADPbS, but not by a,b-meATP. Bin size: 5 s.
receiving an excitatory aortic nerve input was insensitive to a,b-meATP. This indicates differential expression of P2 receptors within the RVLM, but the exact pattern of P2X subtype selectivity detected by functional assays is more sophisticated than the P2 receptor ligands currently available can detect. The widespread distribution of P2 receptors throughout the RVLM suggests that the specificity of cell– cell communication for a neuron will rely on its neuronal connections, as well as the expression of its cell surface P2 receptors. Characterization of P2 purine receptors modulating activity of rostral ventrolateral medulla neurons An increase in neuronal firing rate evoked by the P2Xselective agonist a,b-meATP, and the P2Y-selective agonists ADPbS and UTP, suggests that both P2X and P2Y receptors can modulate the activity of neurons in the RVLM. Furthermore, the fact that single neurons responded to both a,bmeATP and ADPbS suggests that P2X and P2Y receptors may coexist on individual neurons in the RVLM. However, we cannot exclude the possibility that for one, or both, of these agonists modulation of neuronal firing was indirect following P2 receptor-mediated release of transmitters from adjacent neurons or from glial cells. 13 Our findings are in line with functional, immunohistochemical and ligand binding studies that have provided overwhelming evidence for the widespread distribution of ionotropic P2X receptors and
G-protein-coupled P2Y receptors throughout the CNS (see Introduction 10,11,32). Actions of ATP as a neurotransmitter in the CNS via fast ionotropic and slow metabotropic receptors brings ATP in line with most other neurotransmitters, such as acetylcholine, GABA, glutamate and 5-hydroxytryptamine, for which ligand-gated and G-protein-mediated receptor subclassification has already been established. 1 Specific effects at P2 receptors of the purine compounds were confirmed by antagonism of the excitatory responses with suramin (barrel concentration 0.02 M). However, at a higher concentration of suramin (0.2 M), non-selective effects were apparent as excitation evoked by glutamate was also blocked. This indicates that it is critical to evaluate the effects of suramin with care, as suggested previously. 17 Suramin is also able to inhibit ectonucleotidase activity, 20 which may contribute to its excitatory effect on ongoing neuronal activity. The responses of some neurons to a,b-meATP were not blocked by suramin, which may imply actions at P2X4 and P2X6 receptor proteins, which are suramin insensitive. As suramin does not block all subtypes of P2 receptors, the absence of functional antagonism cannot be regarded as evidence against an involvement of P2 receptors. The general lack of effect of suramin as an inhibitor of baseline firing may not be surprising given that suramin typically did not block excitation to exogenously applied purines. In those RVLM neurons in which suramin did block responses to ATP, and its analogues, there was not always a corresponding inhibition of spontaneous firing, suggesting that ATP is not released
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Fig. 8. Functional characterization and pharmacological responses of an RVLM neuron to a,b-meATP in the absence (A) and presence (B) of suramin. The neuron was excited by electrical stimulation in both the T2 region of the spinal cord (spinal; upper left) and the HDA; upper right; three superimposed sweeps). The neuron fired with minimal cardiac-related activity (CRA); R-wave-triggered spike interval histogram (lower right) and arterial blood pressure (AP; upper right) (averages of 1200 sweeps) are also shown. (A) Effective dose-related increase in firing rate to a,b-meATP. (B) Suramin did not block the effective doserelated increase in firing by a,b-meATP. Note that suramin decreased the baseline firing rate of this neuron. Bin size: 3 s.
tonically on to these neurons, at least not under the conditions of the present study. Sun et al. 27 have also reported that suramin (used at a barrel concentration of 0.1 M) is ineffective at altering the baseline firing of RVLM neurons, although, in contrast to the present study, suramin consistently blocked excitation of neuronal firing by ionophoretically applied ATP. This difference between the two studies may reflect the fact that the populations of RVLM neurons studied were significantly different in terms of their spinal projections and baroreceptor inputs. The effective dose of suramin used is also likely to be critical for P2 receptor blockade. The P2 receptor antagonist PPADS generally excited RVLM neurons, even when used at relatively low barrel concentrations, perhaps due to membrane depolarization, and thus could not be examined as an antagonist of responses to purines. Sensitivity of RVLM neurons to a,b-meATP implies actions at P2X1 or P2X3 receptor subunits. Recombinant and native P2X1 and P2X3 receptors have been shown to desensitize rapidly (,100 ms), whereas the other P2X subtypes desensitize slowly or not at all. 7 In the current study, desensitization to a,b-meATP observed in some neurons is consistent with an involvement of P2X1 and/or P2X3 receptor proteins. In two neurons, desensitization to
a,b-meATP reduced the firing rate to below baseline, suggesting that ATP is released on to a subpopulation of neurons in the RVLM. Sun 26 showed that, during application of a,bmeATP, the firing rate of reticulospinal neurons gradually declined, and this abolished subsequent responses to ATP. In the current study, the lack of desensitization of responses to ATP, 2MeSATP and a,b-meATP, which was observed for the majority of neurons, is unequivocal. For those neurons activated by a,b-meATP, this may indicate that a heteromeric receptor is formed from an a,b-meATP-sensitive P2X receptor subunit in combination with a non-desensitizing P2X subunit, as suggested for non-desensitizing a,b-meATP-sensitive P2X2 and P2X3 receptors in adult rat sensory ganglia. 15,16 Non-desensitizing P2X2, P2X4 and P2X6 proteins are highly expressed throughout the adult rat brain, 4,30 and are possible candidates for the non-desensitizing component of a heteromeric a,b-meATP-sensitive RVLM P2X receptor. Since the P2 receptors in the RVLM, as in most other brain regions, appear to be mainly non-desensitizing, rapid degradation of ATP by extracellular and neuronally released ectonucleotidases 29 may be more important for termination of ATP actions. The subtype identity of the P2Y receptors mediating excitation of RVLM neurons by ADPbS and UTP is unclear. UTP
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of a plexus of nerves in the nucleus tractus solitarius disappears after nodose ganglionectomy, consistent with a presynaptic location of these receptors. 30 a,b-meATP and ADPbS are non-hydrolysable agonists, which indicates that ATP does not have to be metabolized to adenosine for a functional response. In contrast, 2MeSATP, ATP and UTP are rapidly degraded, and actions at P1 receptors following degradation to adenosine have to be considered; both excitation and inhibition by adenosine of neuronal firing in the RVLM have been reported. 28 At least a part of the inhibitory responses to purines reported in the present study may be due to direct or indirect effects at P1 receptors, and in some cases this may be masked by excitatory effects mediated at P2 receptors. Co-application of a ,b -methylene-ATP with glutamate Ionophoretic co-application of the P2X-selective agonist a,b-meATP with the excitatory amino acid glutamate elicited excitatory responses that were additive. Thus, if ATP is coreleased as an excitatory transmitter with glutamate from neurons in the RVLM, there appears to be no functional synergism between these neurotransmitters, at least under the conditions of the present study. CONCLUSIONS
Fig. 9. Distribution of recording sites in the RVLM. The location of 23 recording sites was identified by ionophoretic deposition of Pontamine Sky Blue dye, and the positions mapped to coronal sections of the medulla oblongata. 19 Numbers indicate distance from the interaural line.
acts at P2Y2 (or P2U) receptors activated equipotently by ATP and UTP, and at P2Y4, P2Y6 and endogenous uridine nucleotide-specific receptors, which are activated by UTP and UDP, but not by ATP. However, UTP has also been shown to be a weak agonist at the P2X3 receptor. 3,23 An issue which could not be addressed in the present study is whether P2 receptors in the RVLM have a pre- or a postsynaptic location. Both pre- and postsynaptic expression of P2X receptors has been described in other brain regions. P2X2 immunoreactivity has been localized on neurons and their processes in defined brain nuclei; the P2X2 immunoreactivity
We have shown that P2 receptors can modulate the activity of neurons in the RVLM in vivo. This involves both P2X and P2Y receptors, although further characterization of the specific subtypes within each of these families awaits the development and use of selective agonists and antagonists. These findings support the concept of purinergic signalling in the CNS, and more specifically in control of cardiovascular function via reflex and central pathways involving the RVLM. Whilst these data do not imply the existence of specific purinergic pathways, they do support the notion of the co-transmission of purines with conventional excitatory and inhibitory transmitters. This adds to the potential of integrative actions at the level of the RVLM. Acknowledgements—We are grateful to the Royal Society (V.R.) and the British Heart Foundation (T.T.) for their generous support of this study. The study was supported by a British Heart Foundation programme grant (to K.M.S.). Linda Hutchinson is thanked for excellent technical assistance.
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