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Division of Animal Physiology, School of Biosciences and Institute of Neuroscience, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK
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(Received 18 August 2003;
accepted after revision 15 December 2003)
Corresponding author R. W. Clarke: Division of Animal Physiology, School of Biosciences and Institute of Neuroscience, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK. Email: robert.clarke{at}nottingham.ac.uk
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Introduction |
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Glutamate is known to be a key transmitter in the sensitization of nociceptive circuits (Baranauskas & Nistri, 1998; Clarke, 2000), and N-methyl-D-aspartate (NMDA) receptors in particular have been shown to have a key role. NMDA receptor antagonists effectively block central sensitization of reflexes in rats (Woolf & Thompson, 1991) and human trials of clinically available antagonists such as ketamine and dextromethorphan have demonstrated good efficacy in all types of chronic pain states, albeit with a predictable range of side-effects including cognitive deficits (Sang, 2000; Fisher et al. 2000; Eide, 2000). One way of avoiding the side-effect issue may be to target subtypes of NMDA receptor, and selective antagonists for receptors containing the NR2B subunit have shown promise in this respect (Taniguchi et al. 1997; Boyce et al. 1999; Chizh et al. 2001). An alternative strategy is to focus on the less ubiquitous metabotropic glutamate receptors. The group I receptors (mGlu1 and mGlu5) have been consistently implicated in chronic pain mechanisms (Boxall et al. 1996; Fisher & Coderre, 1996; Budai & Larson, 1998; Baranauskas & Nistri, 1998; Hudson et al. 2002; Neugebauer, 2002; Fisher et al. 2002) and appear to offer a promising lead to antihyperalgesic therapies.
There is considerable evidence to support a role for tachykinins in the development of chronic pain (Clarke, 2000; Salter, 2002). Most attention has focused on the NK1 receptor, which has been implicated in most rodent-based models of chronic pain (Luo & Wiesenfeld-Hallin, 1995; Parsons et al. 1996; Liu & Sandkuhler, 1997; Ma & Woolf, 1997; De Felipe et al. 1998; Herrero et al. 2000; Gonzalez et al. 2000; Cahill & Coderre, 2002). However, blockade of this site in isolation has only a small impact on central sensitization in the spinalized rabbit (Houghton & Clarke, 1995) and has proved ineffective as an analgesic strategy in human patients (Hill, 2000). On the other hand, blockade of the NK3 receptor in the spinalized rabbit reduced the duration of sensitization generated by repetitive electrical stimulation of the sural nerve (Houghton et al. 2000), and a number of studies in rodents have implicated the NK3 receptor as a mediator of hyperalgesia (Linden et al. 1999; Zaratin et al. 2000; Barbieri & Nistri, 2001).
The object of the present study was to investigate the role of glutamate group I metabotropic, NR2B subunit-containing NMDA receptors, as well as NK1 and NK3 tachykinin receptors, in mediating central sensitization of hind limb withdrawal reflexes in the decerebrated, non-spinalized rabbit. For a positive control, the effects of non-selective blockade of spinal NMDA receptors has also been studied. Finally, as sensitization involves parallel activation of many different receptors (Hill, 2000; Clarke, 2000), we have also studied the effects of combined blockade of all tachykinin receptors. Some of these data have been published as abstracts (Harris et al. 2002; Harris et al. 2003).
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Methods |
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The spinal cord was exposed at L1 and a polythene cannula (O.D.;0.63 mm) inserted under the dura so that its tip lay near the lumbar enlargement. All animals were then decerebrated by suction to the precollicular level. The nerves to tibialis anterior (TA), semitendinosus (ST) and medial gastrocnemius (MG) were exposed in the left popliteal fossa, cut, and their desheathed central ends applied to paired silver recording electrodes. Anaesthesia was then discontinued and the animals paralysed with pancuronium bromide (Fort Dodge Animal Health, Southampton, UK), infused at 0.5 mg h–1 from a solution of 100 µg/ml in 100 mmol l–1 D-glucose, 100 mmol l–1 NaHCO3. Ventilation was maintained artificially on room air supplemented with oxygen. End tidal CO2 was monitored at intervals and maintained between 3.5 and 4.5%. Core temperature was held at 38 ± 0.5°C by the action of a thermostatically controlled heating blanket. Heart rate was derived from an ECG signal recorded from an intraoesophageal probe. Experiments were terminated by intravenous injection of saturated KCl solution.
Reflexes were evoked by electrical stimulation of the plantar skin of the foot at the heel and at the metatarsophalangeal joints of the middle two toes using paired, stainless steel 23 g needle electrodes separated by 4 mm. Electrical stimuli were constant current pulses of 1 ms duration. The stimulus was set to a multiple (between 1.5 and 4 times) of the threshold value for evoking reflexes. The need to record a measurable reflex response with some latitude to increase in size was the determinant of the multiple used in each experiment. It was often not possible to evoke reflexes in the flexor muscles with single shocks up to 10 mA. Therefore in 36 preparations, the toes were stimulated with triple pulses delivered at 250 Hz. To allow for comparisons with experiments in which single shocks were given, the threshold for evoking reflexes in animals requiring triple shocks was recorded as 10 mA (i.e. the highest possible single stimulus). Stimuli were delivered in blocks of 8 at 1 Hz, applied alternately to the heel and the toes at 2 min intervals.
Reflex responses were recorded as full-wave rectified compound action potentials from the appropriate muscle nerves. On the basis of previous studies (Clarke et al. 1989; Clarke et al. 1992a), only MG reflexes were recorded in response to heel stimulation and only TA and ST reflexes were recorded after stimulation at the toes. Neurogram signals were amplified 1000–10 000 times, filtered between 1 Hz and 6 kHz, and digitized at 20 kHz. The responses to each 8 stimulus block were averaged and integrated with respect to time. A second computer was used to make continuous records of blood pressure and heart rate.
Mustard oil conditioning stimuli, consisting of 2 x 50 µl aliquots of 20% mustard oil in liquid paraffin, were applied topically to either the two lateral or two medial toes of the left hind limb. No attempt was made to remove the oil after application. An initial conditioning stimulus was applied once reflexes had been stable (i.e. varying by <10% between each block of electrical stimuli) for at least 24 min. A minimum of 1 h after the conditioning stimulus, the test drug was given intrathecally or, in the case of SR 142,801, intravenously (see below). Reflexes were allowed to stabilize (i.e. not varying by > 10%) for 20 min before a second conditioning stimulus was applied to the pair of toes that had not been exposed to mustard oil at the first stimulus. Care was taken to ensure that each pair of toes (i.e. lateral or medial) was stimulated first an equal number of times within each group of animals. This design adds power by allowing the use of paired statistical tests, but restricts the experiment to the use of just a single dose of each drug in each animal. For this reason, high doses have been used in an attempt to ensure that most, if not all, relevant receptors were blocked.
CPCCOEt, L-733,060 and ZD-6021 were dissolved in 100% DMSO, so the initial stimulus in the animals receiving these compounds was performed after intrathecal injection of 60–75 µl DMSO to control for effects of the vehicle.
Table 1 shows the drugs used and the dose and route of administration for each. Dizocilpine was a gift of Merck, Sharp and Dohme Neuroscience Research (Harlow, UK) and was dissolved to a concentration of 10 mg ml–1 in Ringer's solution; CP-101,606 was a gift of Pfizer Central Research (Groton, CT, USA), and was dissolved in 1% DMSO in Ringer's solution to a strength of 10 or 30 mg ml–1; CPCCOEt (Tocris, Bristol, UK) was dissolved to concentrations of 10 or 30 mg ml–1 in 100% DMSO; and MPEP (Tocris) was dissolved to concentrations of 2 or 10 mg ml–1 in Ringer's solution. L-733,060 (Tocris) and ZD-6021 (a gift of AstraZeneca) were each dissolved in 100% DMSO to a concentration of 4 mg ml–1, whereas SR 142,801 (Sanofi Recherche, Montpelier, France) was solubilized in DMSO and diluted in 5% D-glucose solution to give a final concentration of 2 mg ml–1 (1% DMSO).
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DMSO as a drug vehicle
CPCCOEt, L-733,060 and ZD-6021 were dissolved in 100% DMSO as attempts to dilute solutions with aqueous media invariably resulted in the precipitation of the drugs. Given intrathecally, 60–75 µl DMSO induced an increase in blood pressure (peak increase of 24 ± 3 mmHg over preinjection values 2 min after injection) and significant (Friedman's ANOVA, P < 0.03) but transient (5 min) decreases in all three reflex responses. The change in blood pressure was sustained, so that 20 min after DMSO, it was a mean of 4 ± 1 mmHg greater than the mean pre-DMSO level of 91 ± 3 mmHg (paired t test, P < 0.05, n= 33). After DMSO, application of mustard oil to the toes induced changes in reflexes and increases in blood pressure that were not significantly different from those obtained from untreated controls (Fig. 1).
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There were no significant differences between absolute values for any reflex or blood pressure between drug treatment groups before mustard oil was applied (Kruskal–Wallis ANOVA or one-way ANOVA, P > 0.05), nor were there any differences in the effects of mustard oil on reflexes (peak effect or duration of action) or blood pressure (peak increase, Kruskal–Wallis ANOVA or one-way ANOVA, P > 0.1).
Metabotropic glutamate receptor antagonists
There were no obvious differences between the effects of the two doses of CPCCOEt so data have been pooled. Intrathecal CPCCOEt had no effects on baseline reflexes over and above those that were obtained with the vehicle (Wilcoxon tests, P > 0.8, n= 8). Blood pressure tended to decrease after the mGlu1 antagonist, with the mean pressure moving from 81 ± 4 mmHg to 76 ± 3 mmHg, but the change was not significant. However, this change was significantly different from the increase in pressure seen when DMSO was given alone (P < 0.0001, t test), i.e. the presence of the antagonist blunted the pressor response to DMSO. The two doses of MPEP were equally ineffective and data have been pooled. MPEP failed to alter baseline reflexes in any muscle nerve when given intrathecally at either 0.2 or 1 mg (Wilcoxon tests, P > 0.1). It also failed to have any significant or consistent effect on mean arterial pressure (predrug value 84 ± 5 mmHg, postdrug 78 ± 2 mmHg, paired t test, P > 0.1).
In the control state mustard oil induced significant increases in both flexor reflexes; a short-lived inhibition of the heel–MG response; and an increase in arterial blood pressure. None of these effects was significantly affected by CPCCOEt or MPEP (Fig. 2).
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Dizocilpine (1 mg I.TH.) significantly reduced the MG response to heel stimulation from a median of 342 µV ms (IQR 220–669 µV ms) to 125 µV ms (IQR 59–186 µV ms, Wilcoxon test, P < 0.04) but had no statistically distinguishable effects on either of the flexor responses per se (Wilcoxon tests, P > 0.8, n= 8). This drug also significantly (paired t test, P < 0.03) reduced mean arterial blood pressure to an average of 62 ± 6 mmHg, from a mean predrug value of 85 ± 6 mmHg. CP-101 606 (1–3 mg I.TH.) caused no significant changes in reflexes per se (Wilcoxon tests, P > 0.1, n= 10) but reduced arterial blood pressure from a predrug mean of 88 ± 4 to 76 ± 5 mmHg (paired t test, P < 0.02) 10 min after administration. The average decrease in blood pressure in this group of animals was not significantly different from that observed with dizocilpine (unpaired t test, P > 0.05).
Application of mustard oil to the toe tips gave rise to significant facilitation of both flexor (TA and ST) reflexes in the control state in both groups of animals receiving NMDA receptor antagonists (Figs 2 and 3). However, mustard oil induced significant changes in the heel–MG reflex and blood pressure only in the group of animals that were to receive CP-101,606 (Fig. 2).
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In the presence of CP-101,606, mustard oil potentiated both flexor reflexes but to significantly lower levels than in the control state (Figs 2 and 3). The median duration of increase for either reflex was unaffected by CP-101,606 (Fig. 2). The effects of mustard oil on the heel–MG reflex were unchanged by CP-101,606, but the increase in blood pressure induced by the conditioning stimulus was significantly greater in the first minute after the stimulus (paired t test, P < 0.04, Figs 2 and 3).
Tachykinin receptor antagonists
Intrathecal administration of L-733,060 (n= 6) or ZD-6021 (n= 10) at 300 µg had no effects on any reflex over and above those obtained with DMSO alone (Wilcoxon or paired t tests, P > 0.1, see above and Fig. 2). Blood pressure tended to decline to levels below control after both antagonists, but only in the case of ZD-6021 was this effect statistically significant. Ten minutes after ZD-6021, mean arterial pressure was 5 ± 3 mmHg below predrug controls, significantly different from the effect seen at the same time after DMSO given alone (paired t test, P < 0.01). SR 142,801 (n= 7) had no effects on any baseline variable (Wilcoxon or paired t tests, P > 0.1).
Prior to administration of L-733,060, mustard oil induced significant increases in the toes–TA and toes–ST reflexes but in these animals, the heel–MG reflex was not significantly affected by the conditioning stimulus (Fig. 2). There was a non-significant increase in mean arterial pressure of 5 ± 2 mmHg over premustard oil values of 103 ± 6 mmHg. After L-733,060, the effects of mustard oil on reflexes were statistically indistinguishable from those obtained in the absence of the drug (Wilcoxon tests, P > 0.1, Fig. 2). However, mustard oil-induced changes in blood pressure were statistically significant in the presence of L-733,060 (Fig. 2).
Prior to administration of SR 142,801, the conditioning stimulus increased both flexor reflexes, reduced the heel–MG reflex and increased blood pressure (Fig. 2). There were no significant differences between the effects of mustard oil on reflex responses or blood pressure recorded before and after application of SR 142,801 (Fig. 2).
In those animals that were to receive ZD-6021, mustard oil significantly enhanced both TA and ST reflexes, had no significant effect on the heel–MG reflex and increased arterial blood pressure (Fig. 2). After ZD-6021, mustard oil induced a significantly smaller peak increase in both toes–TA and toes–ST reflexes (Wilcoxon tests, P < 0.05, Fig. 2). All other effects of the conditioning stimulus, including the duration of enhancement of the flexor reflexes, were not significantly different from the pre-ZD-6021 controls (Fig. 2).
Combined administration of CP-101,606 and ZD-6021
Co-administration of CP-101,606 (3 mg I.TH.) with ZD-6021 (0.3 mg I.TH., n= 9) had no effects on any of the three reflexes per se (Wilcoxon tests, P > 0.05). However, this drug combination reduced arterial pressure by a mean of 48 ± 9 mmHg compared to the mean predrug control value of 93 ± 6 mmHg, 10 min after administration. This was a significantly greater effect than that obtained at the same time with either CP-101,606 or ZD-6021 given alone (ANOVA, P < 0.01, followed by Bonferroni test, P < 0.01). Prior to the drug combination, mustard oil induced significant increases in the TA and ST responses to toe stimulation (Figs 2 and 3), had no effect on the heel–MG reflex and increased blood pressure (Fig. 2). After the drug combination, potentiation of both flexor reflexes by mustard oil was significantly reduced compared to the predrug state (Wilcoxon tests, P < 0.05, Figs 2 and 3). The duration of effect was also decreased (Wilcoxon tests, P < 0.01). The increase in blood pressure induced by mustard oil was significantly larger than in the control state (Figs 2 and 3), but no other effects of mustard oil were altered by the two antagonists given together (Wilcoxon or paired t tests, P > 0.05).
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Discussion |
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In keeping with many animal and human studies (Woolf & Thompson, 1991; Ma & Woolf, 1995; Stubhaug et al. 1997; Baranauskas & Nistri, 1998; Boyce et al. 1999; Eide, 2000) the present results show that blockade of NMDA receptors inhibits the development of central sensitization. This effect is most likely to be mediated in the dorsal horn as a mustard oil conditioning stimulus has no effect on the excitability of 
-motoneurones per se (Cook et al. 1986). Although our finding is congruent with many previous observations, the high dose of antagonist used in the present study requires a little caution in interpretation of the data. The dose was selected on the grounds that it was found to reduce temporal summation of spinal reflexes without influencing the basal level of excitability in reflex circuits (Clarke et al. 2002). Dizocilpine reduced arterial blood pressure, but this effect is entirely predictable on the basis of what is known of NMDA receptor involvement in driving activity in sympathetic preganglionic neurones (Bazil & Gordon, 1993; Huang et al. 1997). It may be argued that the fall in blood pressure and/or changes in spinal blood flow are responsible for the effects of dizocilpine. However, mustard oil induces robust sensitization of reflexes in rabbits with a low thoracic spinal section which have blood pressure that is as low as the post-dizocilpine values observed in the present study (Clarke et al. 1992b; Clarke & Harris, 2001; Harris & Clarke, 2003). Furthermore, despite having clear cardiovascular effects, DMSO had no influence at all on the development of sensitization. The failure of dizocilpine to alter two of the three test reflexes shows that it could not have been acting at AMPA type glutamate receptors, as blockade of these sites would be expected to reduce all sensory inflow to the spinal cord (Cumberbatch et al. 1994). Finally, dizocilpine is known to block the nicotinic receptor channel (Yamakura et al. 2000), so it is conceivable that an antagonist action at these receptors may have contributed to the effect of the drug. However, nicotinic receptor activation is associated with depression rather than enhancement of spinal reflexes (Clarke et al. 1989; Ibrahim & Goldstein, 1989) rendering it unlikely that blockade of these receptors could account for the actions of dizocilpine. In view of this body of evidence, we can conclude that the effects of dizocilpine in the present study were indeed due to blockade of NMDA receptors in the spinal cord.
The involvement of NMDA receptors in sensitization was confirmed by the action of the NR2B-selective NMDA receptor antagonist CP-101,606, which has no known action at nicotinic receptors. This highly selective compound (Chenard et al. 1995) failed to induce motor discoordination in rats at doses up to 100 mg kg–1I.V. (Boyce et al. 1999), and is therefore likely to have remained selective for NR2B-subtype containing receptors at the doses used in the present study. The dose selected was based on the relative potencies of dizocilpine and CP-101,606 in reducing wind-up in spinal reflexes in rabbit (Boyce et al. 1999). In common with dizocilpine, CP-101,606 effectively suppressed the amplitude of facilitation of reflexes after application of mustard oil to the toes, but unlike the non-selective antagonist it had no effect on the duration of sensitization. This suggests that the overall effect of dizocilpine results from blockade of more than one type of NMDA receptor in the spinal cord (Karlsson et al. 2002). The effectiveness of CP-101,606 in reducing sensitization cannot be attributed to its depressor action (see above). As noted by others, antagonists selective for NR2B-subunit bearing NMDA receptors could provide a viable target for antihyperalgesic drugs that would be free from some of the more debilitating side-effects of the complete antagonists (Boyce et al. 1999; Merchant et al. 1999; Clarke, 2000; Chizh et al. 2001).
Non-involvement of metabotropic glutamate receptors in central sensitization
Blockade of group I metabotropic receptors by CPCCOEt (mGlu1) or MPEP (mGlu5) failed to alter any of the effects of mustard oil application. The very high doses used strongly suggest that this is a true negative result and indicates that these receptors do not make a substantial contribution to sensitization of spinal circuits in the rabbit after an acute noxious stimulus. Indeed, there was sufficient CPCCOEt present to reduce the pressor response to DMSO, indicating that it had reached effective concentrations at some receptors. MPEP has nanomolar affinity at mGlu5 receptors (Gasparini et al. 1999). Where they have an effect, drugs with this order of affinity are maximally effective when given at 100–300 µg by the intrathecal route in the present preparation (Harris & Clarke, 1993; Clarke et al. 1996). Thus, it is very unlikely that insufficient MPEP was applied to block mGlu5 receptors. Although clear cut, these findings appear to be at odds with the reports of the involvement of mGlu receptors in sensitized spinal processing in rodents (Boxall et al. 1996; Fisher & Coderre, 1996; Budai & Larson, 1998; Baranauskas & Nistri, 1998; Hudson et al. 2002; Neugebauer, 2002; Fisher et al. 2002). It has been suggested that the contribution of mGlu5 receptors in sensitized processing is mediated at a peripheral site that would not be readily accessed from the intrathecal injections used in the present study (Walker et al. 2001), explaining the lack of effect of MPEP. It is possible that substantial activation of mGluRs occurs only after very intense stimuli and that the relatively short-lived nociceptive input used in the present study was insufficient to ensure their activation, or that the effect of mGlu antagonists in other studies is a particular feature of nociceptive processing in rodents.
Tachykinin receptors in sensitization
Previous studies from this laboratory indicated that sensitization of withdrawal reflexes in spinalized preparations, evoked by electrical stimulation of peripheral nerves, is sensitive to blockade of NK3 receptors with minor involvement of NK1 receptors (Houghton & Clarke, 1995; Houghton et al. 2000). In the present study (with non-spinalized animals and using a ‘natural’ sensitizing stimulus) selective blockade of either of these receptors had no effect at all on sensitization, or any other effect of mustard oil, but simultaneous blockade of all tachykinin receptors gave rise to a slight reduction in enhancement of flexor reflexes. It would appear that either the integrity of descending fibre systems or the use of adequate stimulation of nociceptors (we cannot determine which) induced parallel activation of NK1 and NK3 receptors to contribute to the process of sensitization. NK2 receptors are not present on neurones in the spinal cord (Zerari et al. 1998) and presumably did not contribute to the effects of ZD-6021 in the present study. Although selective NK1 antagonists block central sensitization in the rat (Thompson et al. 1994; Ma & Woolf, 1997; Laird et al. 2001) and primates (Dougherty et al. 1994), they have proved ineffective in human clinical pain studies (Hill, 2000).
One of the suggestions put forward to explain this failure has been that inactivation of just one component of the many transmitter systems activated in parallel by noxious stimuli is insufficient to produce a clear antihyperalgesic action (Hill, 2000; Clarke, 2000). The present data support this view, which lay behind the final experiment in which blockade of NK1 and NK3 receptors was combined with antagonism at NR2B subunit containing NMDA receptors. Concomitant administration of ZD-6021 and CP-101,606 reduced both peak facilitation and the duration of enhancement of flexor reflexes, whereas each drug individually reduced only the amplitude of the effect. This suggests an additive effect of the two antagonists and indicates that blockade of multiple receptors may be the best way forward in terms of generating new and effective antihyperalgesic drugs. Our interpretation of the large and prolonged increases in blood pressure seen when mustard oil was given in the presence of CP-101,606 (especially when coadministered with ZD-6021) is that they reflect inactivation of the relevant drugs.
Inhibition of the heel–MG reflex
In previous studies we have noted weak or absent effects of mustard oil stimulation of the toe tips on the MG response to heel stimulation in non-spinal rabbits (Harris & Clarke, 2003). Where it was observed in the present study (in the CPCCOEt, MPEP, CP-101,606 and SR 142,801 groups), the effect proved resistant to any of the antagonists used and does not appear to involve activation of metabotropic glutamate, NR2B-NMDA or NK3 receptors.
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References |
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) and after (•) intrathecal administration of DMSO 60–75 µl. Each point is the median from 35 (untreated) or 33 (DMSO-treated) experiments and vertical lines indicate 1st or 3rd quartiles. Blood pressure data are shown as means ± SEM. Mustard oil was applied at time 0. There were no significant differences between the two conditions (Wilcoxon's and paired t tests, P > 0.05).
). * indicates a significant inhibition of the heel–MG reflex (Friedman's ANOVA, P < 0.05); 
indicates a significant difference between pre- and postdrug values (Wilcoxon or paired t tests, P < 0.05). For the reflex data, the height of the bars indicate median values from 8 (CPCCOEt and dizocilpine); 10 (MPEP, CP-101,606 alone and ZD-6021 alone); 6 (L-733,060); 7 (SR 142,801) or 9 (CP-101,606 + ZD-6021) experiments, and the vertical bars indicate the third quartile. For blood pressure data the bars and lines indicate means with standard error.