Brain Research Research report. Xiao-Min Wang, Jian-Qun Yan, Kai-Ming Zhang, Sukhbir S. Mokha )

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1 Brain Research Research report Role of opioid receptors Ž m, d, d. 1 in modulating responses of nociceptive neurons in the superficial and deeper dorsal horn of the medulla Žtrigeminal nucleus caudalis. in the rat Xiao-Min Wang, Jian-Qun Yan, Kai-Ming Zhang, Sukhbir S. Mokha ) Department of Physiology, Meharry Medical College, 1005 D.B. Todd BouleÕard, NashÕille, TN 3708, USA Accepted 5 June 1996 Abstract This report describes the effects of intravenously administered agonists and antagonists at m-, d1- and d-opioid receptors on the Adand C-fiber-evoked responses of trigeminal nociceptive neurons in anesthetized rats. Extracellular single unit recordings ere made from 61 nociceptive neurons Ž 3 NS, 38 WDR. in the superficial and 37 nociceptive neurons Ž 3 NS, 34 WDR. in the deeper dorsal horn of the,5 medulla trigeminal nucleus caudalis. Administration of either the d -receptor agonist D-Pen xenkephalin Ž DPDPE; 0.05 mgrkg. 1, 4 x 4 5 the d -receptor agonist D-Ala,Glu deltorphin DELT; 1 mgrkg or the m-receptor agonist D-Ala, N-MePhe,Gly -olx enkephalin Ž DAMGO; mgrkg. inhibited the Ad- and C-fiber-evoked responses of nociceptive neurons in the superficial and deeper dorsal horn. The inhibitory effect as more pronounced on the C-fiber-evoked responses than on the Ad-fiber-evoked responses. In other neurons, DPDPE also produced facilitation, or inhibition folloed by facilitation, or differential effects Žinhibition of the C-fiber-evoked responses and facilitation of the Ad-fiber-evoked responses. on the Ad- and C-fiber-evoked responses. The effects of DPDPE ere antagonized by 7-benzylidenenaltrexone Ž BNTX, mgrkg., a d -receptor antagonist, in 88% Ž 7r8. of neurons. Naltriben Ž 1 NTB, mgrkg., a d -receptor antagonist, antagonized the effects of both DELT and DPDPE. A smaller dose of NTB Ž 0.3 mgrkg., hich failed to reverse the effects of DPDPE in 100% Ž 4r4. of neurons, effectively antagonized the effects of DELT in 100% Ž 6r6. of neurons. The inhibitory action of DAMGO as completely antagonized by naloxone Ž 0. mgrkg. in 100% Ž 6r6. of neurons. The results of the present investigation suggest that: Ž. 1 m-, d1- and d-opioid receptors play an important role in the inhibitory modulation of the Ad- and C-fiber-evoked responses of nociceptive neurons in the superficial and deeper dorsal horn of the medulla; Ž. selective inhibition of the C-fiber-evoked responses by activation of opioid receptors may account for the opioid-mediated selective suppression of second or persistent pain as compared to first pain; and Ž. 3 NTB, in a limited dose range, can discriminate beteen d1- and d-opioid receptor subtypes. Keyords: Pain; Medullary dorsal horn; Trigeminal nucleus caudalis; Opioid receptors; Antinociception; Nociception 1. Introduction The dorsal horn of the medulla Žtrigeminal nucleus caudalis. plays an important role in processing nociceptive and thermosensory information originating from the orofa4,40 x. It contains high densities of m-, d- and cial region k-opioid receptors and receptor subtypes Žm1 and m, d1 and d. particularly in the superficial laminae 1,3 5,7,17,43 x. These receptors are located on neurons, small diameter primary afferent fibers 14x and on the terminals ) Corresponding author. Fax: q ; mokhas1@ccvax.mmc.edu of ascending and descending pathays. The effects of opiates and opioid peptides have been extensively investi,6,13,51 x, ith a major action being an inhibition gated of nociceptive transmission in the dorsal horn. With a fe exceptions 9,0,3,33,39,50 x, previous studies focused on neurons in the deeper dorsal horn of the spinal cord 11 13,15,19,50 x. There is relatively little information available regarding the opioid-mediated modulation of neuronal responses in the dorsal horn of the medulla x. Therefore, e recently examined the role of multiple opioid receptors in modulating thermal stimuli-evoked responses of nociceptive neurons in the superficial and deeper dorsal horn 30,31,33x and the activity of cold-receptive neurons in the superficial dorsal horn of the medulla 3 x r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 转载

2 36 X.-M. Wang et al.rbrain Research Recent behavioral 6,7,4,44 46x and in vitro electro16x support the importance of d1- and d-opioid receptor subtypes in antinociception. Ho- physiological studies ever, the role that d-opioid receptor subtypes Ž d, d. 1 might play in modulating the responses of nociceptive neurons has not been examined previously in in vivo electrophysiological studies. The present study as, therefore, designed to investigate the effects of opioid receptor Ž m, d, d. 1 activation on the Ad- and C-fiber-evoked responses of physiologically characterized nociceptive neurons in the superficial and deeper dorsal horn of the medulla. Some of the results have been presented in an abstract form 34 x.. Materials and methods Experiments ere performed on 7 male Sprague Daley rats Ž g. anesthetized ith urethane Ž1.5 grkg, i.p... Tracheal and jugular cannulations ere performed. Some animals ere paralyzed ith gallamine triethiodide Ž 50 mgrkg, i.v.. and artificially respired. The electrical activity of the heart, end-tidal CO and rectal core temperature ere monitored continuously. To improve the stability of recordings from the superficial and deeper dorsal horn of the medulla, a small metallic brass plate as fixed to the skull and the head as ventroflexed. The medulla as exposed by clearing the overlying musculature, cutting through and deflecting the dura and arachnoid matter. The exposed surface of the medulla as covered ith agar Ž4% agar in normal saline at about 408C. to further improve the stability of recording from neurons in the superficial dorsal horn of the medulla. Extracellular, single-unit recordings ere made ith glass-coated, platinum-plated, tungsten microelectrodes 8x advanced ith a stepping motor-driven micromanipulator. Recordings ere made caudal to the obex Žy0.5 to y.0 mm. ith particular attention paid to the medial to-thirds of the trigeminal nucleus caudalis, a region that receives terminations of the maxillary and mandibular divisions. Neuronal activity as amplified and recorded on an oscilloscope to allo for discrimination of single units. Spontaneous and evoked activity as fed to a indo discriminator and to a microcomputer to allo further Ž analysis of the data on-line and off-line MI softare, Southeastern, PA.. Stimulus-evoked neural activity as continuously monitored on-line by constructing peristimulus time histograms and dot-raster displays. Because spike amplitude sometimes varied ith respiration and discharge rate, spike shape, receptive field characteristics, spontaneous activity and latency ere closely monitored to ensure that the same unit as studied throughout the recording period. Fig. 1A shos the spike shape of a selectively nocireceptive neuron located in the superficial dorsal horn. The location of recordings in the superficial versus the deeper dorsal horn of the medulla based on depth, spike shapes, receptive fields has been described and discussed in detail in previous studies 9,31,3 x. To limit the recordings from neurons in the superficial dorsal horn of the medulla, the electrode penetrations did not exceed 00 mm from the pial surface. For recordings from neurons in the deeper dorsal horn, penetrations did not exceed 500 mm. Recording sites ere marked in some experiments by making discrete electrolytic lesions Ž 8 0 ma for 0 s., and reconstructed from 50 mm serial, transverse sections stained ith Cresyl violet. The location of a selectively nocireceptive Ž nociceptive-specific, NS. neuron in the superficial dorsal horn is illustrated in Fig. 1B. The face as carefully shaved for adequate stimulation and mapping of receptive fields. The responses of neurons to stimulation of their receptive fields ere characterized by using brush, pressure, pinch, squeeze and radiant heat. Neurons ere classified as selectively nocireceptive Ž nociceptive-specific, NS. and multireceptive Žide dynamic range, WDR.. NS neurons are driven exclusively by noxious stimuli, hereas WDR neurons receive non-noxi- Fig. 1. Spike shape and recording site of selectively nocireceptive neurons in the superficial dorsal horn of the medulla. A: spike shape of a selectively nocireceptive neuron in the superficial dorsal horn Žrecording depth: 90 mm. of the medulla. Scale bars0.1 ms. B: recording site of a selectively nocireceptive neuron in the superficial dorsal horn Žrecording depths100 mm. of the medulla. The arro points to the electrolytic lesion. Scale bars00 mm.

3 X.-M. Wang et al.rbrain Research Fig.. Time course of the inhibitory effect produced by DELT Ž1 mgrkg. on the Ad- and C-fiber-evoked responses of a multireceptive neuron in the superficial dorsal horn Ž 50 mm. of the medulla. Each point in this and subsequent figures represents total number of spikes-evoked per stimulation trial Ž 10 stimuli, -ms pulse repeated at 0.3 Hz.. Top: peristimulus time histograms of the neural activity evoked by Ad- and C-fiber Ž 10 consecutive stimulus trials.. The latency for the Ad-fiberevoked responses is 7 ms and for the C-fiber-evoked responses is 58 ms. Arro indicates the time of the stimulus. Bottom: time course of the inhibitory effect of DELT. The effects of one agonist or agonistrantagonist combination ere tested on only one neuron in 46 out of 7 experiments as described in our previous studies x. In the remaining 6 experiments, the effects of drugs ere tested on to neurons per animal. Hoever, sufficient time Ž 3 5 h. as alloed to elapse before testing the effect of a drug on the second neuron. In a fe instances, the effects of several drugs ere tested on one neuron. Hoever, the effect of a subsequent drug as tested only after recovery from the previously administered drug. The total number of spikes evoked by the Ad- or C-fiber stimulation as counted in each trial Ž 10 stimuli. to determine the effects of agonists and antagonists at m-, d1- and d-opioid receptors. The responses during drug administration ere compared ith the control Ž baseline. responses Ž4 6 determinations prior to each drug administration.. Neurons shoing more than 10% variability in control responses ere eliminated from the analysis. The effect induced by the application of a drug as defined as inhibitory or excitatory only hen the responses ere reduced or enhanced by ) 0% relative to the control responses. An antagonist as considered to be effective if the action of the agonist as reduced by 50%. The data are expressed as the mean" S.E.M. Statistical comparisons ere made by the use of the Student s t-test and x -test. P as considered as statistically significant. ous inputs as ell. Nociceptive neurons ere activated by electrical stimulation of the receptive field ith needle electrodes. Each stimulation trial consisted of a series of 10 stimuli Ž ms single pulse at a repetition rate of 0.3 Hz. at a stimulus intensity of.5 times the threshold of the neuron for a C-fiber response. A clear separation Žbased on latencies. of the Ad- and C-fiber-evoked responses as evident Ž as shon in the histogram of Fig... Responses evoked consistently at a latency greater than 30 ms ere attributed to the C-fiber excitatory inputs, hereas those evoked at latencies ranging from 3 30 ms ere attributed to the Ad-fiber excitatory inputs Žon the basis of a 0 30 mm conduction distance.. Responses evoked at a latency of -3 ms ere excluded because these ere too close to the stimulus artifact to be counted accurately.,5 D-Pen xenkephalin Ž DPDPE; mol.t.s , D- Ala,Glu 4 xdeltorphin II Ž DELT; mol.t.s 78.9., D- 4 5 Ala, N-MePhe,Gly -olxenkephalin ŽDAMGO; mol.t.s and naloxone hydrochloride Ž mol.t.s ere obtained from Sigma Chemical Co. Ž St. Louis, MO.. Naltriben methanesulfonate Ž NTB; mol.t.s and 7- Benzylidenenaltrexone hydrochloride ŽBNTX; mol.t.s ere obtained from Research Biochemicals Int. Ž Natick, MA.. Drugs ere dissolved either in saline Ž0.9% NaCl. or in distilled ater immediately prior to use. All drugs ere injected intravenously Ž i.v.. in a volume of ml over s folloed by 150 ml of normal saline to flush the cannula. 3. Results Extracellular single unit recordings ere made from 61 nociceptive neurons Ž 3 NS, 38 WDR. in the superficial dorsal horn Ž SDH. and 37 nociceptive neurons Ž3 NS, 34 WDR. in the deeper dorsal horn Ž DDH.. All neurons had ipsilateral orofacial mechanoreceptive fields and most ere not spontaneously active. Latencies for the C-fiber-evoked responses ranged from 34 to 84 ms, and for the Ad-fiberevoked responses these latencies ranged from 3 to ms Effects of DELT on the Ad- and C-fiber-eÕoked responses The effects of DELT Ž 1 mgrkg. ere tested on 5 superficial dorsal horn neurons Ž 6 NS, 19 WDR, Table 1. and 6 deeper dorsal horn neurons Ž NS, 4 WDR, Table.. Administration of DELT Ž 1 mgrkg. produced a reversible inhibition of the Ad-fiber-evoked responses in 45% Ž 14r31. of neurons Ž 4 NS, 10 WDR, Tables 1 and. and of the C-fiber-evoked responses in 87% Ž 7r31. of neurons Ž 7 NS, 0 WDR, Tables 1 and.. A typical example demonstrating the effect of DELT is illustrated in Fig.. The effect as significant ithin 3 6 min, peaked ithin 6 9 min, and persisted for 0 40 min. The maximal inhibitions Žexpressed as percentage of preinjection control values. ere 39" 6% Ž n s 14. on the Ad-fiber-evoked

4 38 X.-M. Wang et al.rbrain Research Table 1 Effects of DELT, DPDPE and DAMGO on the Ad- and C-fiber-evoked responses of nociceptive-specific Ž NS. and ide dynamic range Ž WDR. neurons in the superficial dorsal horn of the medulla DELT DPDPE DAMGO NS WDR NS WDR NS WDR Ad C Ad C Ad C Ad C Ad C Ad C Inhibition 4r6 5r6 8r19 17r19 8r14 8r14 11r16 13r16 5r5 5r5 5r8 7r8 Excitation 0r6 1r6 0r19 0r19 1r14 3r14 1r16 r16 0r5 0r5 0r8 0r8 No effect r6 0r6 11r19 r19 5r14 3r14 4r16 1r16 0r5 0r5 3r8 1r8 Total responses and 66" 5% Ž n s 7. on the C-fiber-evoked responses, respectively. The maximal inhibition as more pronounced on the C-fiber-evoked responses as compared to the Ad-fiber-evoked responses Žgrouped-sample t-test, P An excitatory effect of DELT as observed on the C-fiber-evoked responses in only one neuron Ž 1 NS.. DELT did not alter the Ad-fiber-evoked responses in 55% Ž 17r31. of neurons Ž 4 NS, 13 WDR., nor the C-fiberevoked responses in 10% Ž 3r31. of neurons Ž 3 WDR Effects of DPDPE on the Ad- and C-fiber-eÕoked responses The effects of DPDPE ere tested on 34 superficial dorsal horn neurons Ž 17 NS, 17 WDR, Table 1. and 4 deeper dorsal horn neurons Ž 3 NS, 1 WDR, Table.. Administration of DPDPE Ž 0.05 mgrkg. produced a reversible reduction in the Ad-fiber-evoked responses in 41% Ž 4r58. of neurons Ž 9 NS, 15 WDR, Tables 1 and. and the C-fiber-evoked responses in 5% Ž 30r58. of neurons Ž 9 NS, 1 WDR, Tables 1 and.. In most of these neurons, the inhibitory effect as significant ithin 3 6 min, peaked ithin 9 1 min and persisted for 0 60 min after the injection of DPDPE. The inhibitory effect produced by small doses Ž mgrkg. of DPDPE is illustrated in Fig. 3. Similar to the effect of DELT, the maximal inhibition as more pronounced on the C-fiberevoked responses as compared to the Ad-fiber-evoked responses. The maximal inhibitory effects ere 36" 5% Ž ns4. on the Ad-fiber-evoked responses and 63" 6% Ž ns30. on the C-fiber-evoked responses, respectively Ž grouped-sample t-test, P DPDPE also produced excitation, or inhibition folloed by excitation, on the Ad-fiber-evoked responses in 5% Ž 3r58. of neurons Ž1 NS, WDR. and on the C-fiber-evoked responses in 16% Ž 9r58. of neurons ŽŽ 4 NS, 5 WDR., Tables 1 and.. DPDPE enhanced the Ad-fiber-evoked responses by 39" 7% Ž ns3. and the C-fiber-evoked responses by 67"9% Ž ns9.. In addition, DPDPE differentially affected the Ad- and C-fiber-evoked responses in 4 Ž 3 NS, 1 WDR. superficial dorsal horn neurons Ž Fig. 4., producing facilitation of the Ad-fiber-evoked responses and inhibition of the C-fiber-evoked responses. Effects of DPDPE, inhibition or facilitation or biphasic changes, ere not dose-related, i.e., lo doses produce facilitation, hereas high doses produce inhibition or vice versa. There as no significant effect of DPDPE on the Ad-fiber-evoked responses in 47% Ž 7r58. of neurons Ž 7 NS, 0 WDR. and on the C-fiber-evoked responses in 6% Ž 15r58. of neurons ŽŽ 4 NS, 11 WDR., Tables 1 and.. DPDPE inhibited the Ad-fiber-evoked responses of a significantly large percentage of WDR neurons in the superficial dorsal horn as compared to the Ž deeper dorsal horn x -test, P s Furthermore, the percentage of neurons unaffected by DPDPE as significantly smaller in the superficial dorsal horn as compared to the deeper dorsal horn Ž Tables 1 and.. Table Effects of DELT, DPDPE and DAMGO on the Ad- and C-fiber-evoked responses of nociceptive-specific Ž NS. and ide dynamic range Ž WDR. neurons in the deeper dorsal horn of the medulla DELT DPDPE DAMGO NS WDR NS WDR NS WDR Ad C Ad C Ad C Ad C Ad C Ad C Inhibition 0r r r4 3r4 1r3 1r3 4r1 8r1 0r0 0r0 6r11 11r11 Excitation 0r 0r 0r4 0r4 0r3 1r3 1r1 3r1 0r0 0r0 0r11 0r11 No effect r 0r r4 1r4 r3 1r3 16r1 10r1 0r0 0r0 5r11 0r11 Total

5 X.-M. Wang et al.rbrain Research Fig. 3. Effects of small doses of DPDPE mgrkg on the Adand C-fiber-evoked responses of a multireceptive neuron in the deeper dorsal horn of the medulla. Fig. 5. Effects of small doses of DAMGO 0.05 mgrkg on the Ad- and C-fiber-evoked responses of a multireceptive neuron in the deeper dorsal horn of the medulla Effects of DAMGO on the Ad- and C-fiber-eÕoked responses The effects of DAMGO Ž mgrkg. ere tested on 13 superficial dorsal horn neurons Ž5 NS, 8 WDR, Table 1. and 11 deeper dorsal horn neurons Ž11 WDR, Table.. Compared to the effects of DPDPE and DELT, DAMGO produced a more consistent inhibitory effect on the Ad- and C-fiber-evoked responses Ž Tables 1 and.. DAMGO inhibited the Ad-fiber-evoked responses in 67% Ž 16r4. of neurons Ž 5 NS, 11 WDR., and the C-fiberevoked responses in 96% Ž 3r4. of neurons Ž5 NS, 18 WDR.. A typical example is shon in Fig. 5. The C- fiber-evoked responses ere predominantly inhibited by the administration of DAMGO Žthe maximal inhibition as 93"3%, ns3., hereas the Ad-fiber-evoked responses ere less affected Žthe maximal inhibition as 49"7%, ns16, grouped-sample t-test, P Sometimes, a smaller dose of DAMGO Ž 0.5 mgrkg. did not affect the Ad-fiber-evoked responses, but did affect the C-fiber-evoked responses Ž Fig. 6B.. The effect of DAMGO appeared ithin 3 min, peaked ithin 9 min and persisted for at least 60 min. DAMGO did not produce any effects on the Ad-fiber-evoked responses in 33% Ž 8r4. of neurons Ž 8 WDR. nor on the C-fiber-evoked responses in 4% Ž 1r4. of neurons ŽŽ 1 WDR., Tables 1 and The antagonism of the effects of DELT, DPDPE and DAMGO by NTB, BNTX and naloxone The inhibitory effect of DELT on the C-fiber-evoked responses as antagonized by NTB Ž mgrkg. in 100% Ž 14r14. of neurons Ž 3 NS, 11 WDR. hile antagonism of the inhibitory effect of DELT on the Ad-fiber- Fig. 4. Differential effects of DPDPE Ž 1 mgrkg. on the Ad- and C-fiber-evoked responses of a multireceptive neuron in the superficial dorsal horn Ž 60 mm. of the medulla. Fig. 6. Effects Ž lack of effects. of DELT Ž 1 mgrkg., DPDPE Ž1 mgrkg. and DAMGO Ž 0.5 mgrkg. on the Ad- and C-fiber-evoked responses of a multireceptive neuron in the superficial dorsal horn Ž80 mm. of the medulla. A: smaller dose of NTB Ž 0.3 mgrkg. blocked the inhibitory effect of DELT. B: DPDPE did not alter the responses, hereas DAMGO produced a naloxone-reversible inhibitory effect on the C-fiber-evoked responses.

6 40 X.-M. Wang et al.rbrain Research evoked responses as observed in 79% Ž 11r14. of neurons Ž 3 NS, 8 WDR.. The smallest dose of NTB Ž0.3 mgrkg., hich did not alter the effects of DPDPE in 100% Ž 4r4. of neurons Ž NS, WDR, as illustrated in Fig. 7A. antagonized the effects produced by DELT in 100% Ž 6r6, 1 NS, 5 WDR. of neurons Ž Fig. 6A.. Hoever, at higher doses Ž mgrkg. NTB also antagonized the effects of DPDPE in 100% Ž 4r4. of neurons Ž4 WDR. as illustrated in Fig. 7B. The antagonism appeared 5 min after administration of NTB and often persisted for min. BNTX Ž mgrkg. antagonized the effects of DPDPE in 88% Ž 7r8. of neurons Ž 3 NS, 4 WDR. as illustrated in Fig. 7C. The reversal by BNTX appeared Fig. 8. Lack of effects of saline Ž 0.3 ml. and NTB Ž 1 mgrkg. on the Adand C-fiber-evoked responses of neurons in the superficial dorsal horn of the medulla. A: lack of effect of saline on a selectively nocireceptive neuron in the superficial dorsal horn Ž 170 mm.. B: lack of effect of NTB on a multireceptive neuron in the superficial dorsal horn Ž 176 mm.. ithin 8 10 min and persisted for nearly 40 min. The inhibitory action of DAMGO as completely antagonized by naloxone Ž 0. mgrkg. in 100% Ž 6r6. of neurons Ž3 NS, 3 WDR, Fig. 6B.. The antagonism produced by naloxone as immediate, and a short-lasting facilitation of responses as also observed in some neurons. The effect of saline as examined on neurons in the present study, since the activity of some neurons in the superficial dorsal horn has been reported to be susceptible to saline x. Injection of 0.3 ml of saline had no effect on the C-fiber-evoked responses of r neurons Ž Fig. 8A.. In order to test if the antagonists affected the activity of these neurons per se, effects of NTB Ž ns4. or BNTX Ž ns3. administered alone ere examined. In general, neither NTB nor BNTX altered the Ad- and C-fiber-evoked responses Ž Fig. 8B.. 4. Discussion Fig. 7. Reversal of DPDPE effects by NTB and BNTX on three different neurons. A: smaller dose of NTB Ž 0.3 mgrkg. failed to antagonize the effect of DPDPE on the Ad- and C-fiber-evoked responses of a selectively nocireceptive neuron in the superficial dorsal horn Ž 86 mm. of the medulla. B: larger dose of NTB Ž 0.7 mgrkg. antagonized the effect of DPDPE on the Ad- and C-fiber-evoked responses of another selectively nocireceptive neuron in the superficial dorsal horn Ž 150 mm. of the medulla. C: BNTX Ž 1 mgrkg. antagonized the effect of DPDPE on the Ad- and C-fiber-evoked responses of a multireceptive neuron in the superficial dorsal horn Ž 00 mm. of the medulla Contribution of m- and d-opioid receptor actiõation to the modulation of Ad- and C-fiber-eÕoked responses in the medulla The present investigation provides the first in vivo electrophysiological evidence demonstrating opioid-receptor selective Ž m, d and d. 1 modulation of the Ad- and C-fiber-evoked responses of physiologically characterized nociceptive neurons in the superficial and deeper dorsal horn of the medulla. Our results suggest that inhibition of

7 X.-M. Wang et al.rbrain Research the Ad- and C-fiber-evoked responses by DAMGO, DPDPE and DELT is mediated by activation of m- and d-opioid receptors. Specifically, DAMGO, a m-opioid receptor-selective agonist, inhibited the Ad- and C-fiberevoked responses. This inhibitory effect as antagonized by lo doses of naloxone Ž 0. mgrkg.. Identical doses of naloxone have been shon previously to block the effects of m-opioid agonists 30,33 x, but not the effects of d-opioid receptor agonists 30 x. Furthermore, the Ad- and C-fiberevoked responses ere inhibited by DPDPE and DELT, to highly selective d-opioid receptor agonists 10,1,,7,41,44,45 x. Importantly, the inhibition by DPDPE and DELT as antagonized, respectively, by BNTX and NTB, highly selective d-opioid receptor antag37,41,4 x. Finally, neither NTB or BNTX alone, onists nor vehicle, altered the responses of nociceptive neurons. Although naloxone is knon to differentially modulate nociceptive input in the medullary dorsal horn 31 x, its ability to antagonize the inhibitory action of DAMGO is not compromised. The peak effects of naloxone, in gen31 x, hereas the antagonism of the inhibitory effect of DAMGO eral, ere reported to develop ithin 15 1 min appeared immediately Ž Fig. 6B.. We conclude that activation of m- as ell d-opioid receptors inhibits the Ad- and C-fiber-evoked responses in the dorsal horn of the medulla. Selective opioid agonists at m- and d-opioid receptors produced a more marked suppression of the C-fiber-evoked responses than of the Ad-fiber-evoked responses. Furthermore, opioids inhibited the C-fiber-evoked responses in a large percentage of neurons, hereas the Ad-fiber-evoked responses ere inhibited in a small percentage of neurons. These observations provide an explanation for the opioidmediated selective suppression of second or persistent pain as compared to first pain x 8. Signals for second pain are carried by C-fibers hereas Ad-fibers mediate first pain. Multiple factors may have contributed to the selectivity of opioid action in the present investigation. It is possible that opioid effects may depend on the firing frequency generated by Ad- and C-fibers. It has been reported that the effects of opioid agonists are dependent upon the frequency of firing generated by noxious mechanical and thermal stimuli, i.e., the higher the firing frequency the smaller the inhibitory effect of the opioid agonist 35 x. Based on our results, the inhibitory effect of opioids did not seem to depend on the response magnitude Žtotal spikesrstimulus., otherise there ould have been a stronger inhibitory effect on the Ad-fiber-evoked responses because of a smaller response magnitude as compared to the response magnitude of the C-fiber-evoked excitation. Another factor that may have contributed to the selectivity of opioid action is the stimulus intensity. The peripheral stimulus used in the present investigation as.5 times the threshold of the neuron for a C-fiber response. This ill excite the Ad-fiber more strongly than the C-fiber, and consequently generate a stronger Adfiber-evoked response hich may, therefore, be less sensitive to the effects of opioids. Recently it has been reported that opioids reduce the calcium current of small neurons, but not of large neurons in the trigeminal ganglion 47 x. It is, therefore, possible that opioids modulate the C-fiber nociceptive input in the medullary dorsal horn at both the primary afferent level and at the level of postsynaptic neurons, hereas the Ad-fiber nociceptive input is only modulated at the level of postsynaptic neurons. 4.. d - and d -opioid receptors modulate the Ad- and 1 C-fiber-eÕoked responses, but produce differential effects Recent behavioral studies provide strong evidence for the importance of d-opioid receptor subtypes Ž d, d. 1 in antinociception,41,44,45,49 x. Several findings in the present study indicate that activation of d1- and d-opioid receptors modulates the Ad- and C-fiber-evoked responses in the dorsal horn of the medulla. First, the d1-opioid receptor-selective agonist, DPDPE, inhibited the Ad- and C-fiber-evoked responses. The inhibition as antagonized by BNTX, a d -opioid receptor-selective antagonist 4 x. 1 Second, the Ad- and C-fiber-evoked responses ere also inhibited by DELT, a d-opioid receptor-selective agonist. This inhibitory effect of DELT as antagonized selectively by lo doses of NTB, a d-opioid receptor-selective antagonist 10,41,44,45 x. Interestingly, smaller doses of NTB Ž 0.3 mgrkg. hich antagonized the inhibitory action of DELT failed to alter the effects produced by DPDPE. These results are consistent ith the recent behavioral findings 1,,41,44,45 x. Hoever, NTB is apparently non-selective at higher doses since at a higher dose Ž0.7 mgrkg. it reversed the effects of both DPDPE and DELT. This finding is also consistent ith previous observations 46x here subcutaneous Ž 1 mgrkg. NTB antagonized the antinociceptive effect produced by both DELT and DPDPE. Although both in vitro 36,37x and in vivo studies in the mouse and in the rat 44,45x provide strong evidence that NTB is a selective d-opioid receptor antagonist, one has to be cautious considering the fact that only lo doses of NTB discriminate beteen d - and d -opioid receptor 1 subtypes. We suggest that the modulation of the Ad- and C-fiber-evoked responses by DPDPE and DELT is mediated by the activation of d1- and d-opioid receptors, respectively. The effects of DPDPE appear more complicated. In addition to an inhibitory action, DPDPE produced facilitation, or inhibition folloed by facilitation, hen adminis- tered systemically or iontophoretically 53 x. DPDPE also produced differential effects on the Ad- and C-fiber-evoked responses in the superficial dorsal horn. The facilitation produced by DPDPE may result from inhibition of g- aminobutyric acid Ž GABA. or glycine containing inhibitory interneurons present in the superficial dorsal horn 18,5,38,48 x. Glycine and GABA have been shon to mediate spontaneous inhibitory postsynaptic currents in neurons in the substantia gelatinosa 5 x.

8 4 X.-M. Wang et al.rbrain Research In summary, these results provide the first in vivo electrophysiological evidence that activation of d-opioid receptor subtypes Ž d, d. 1 inhibits the Ad- and C-fiberevoked responses of physiologically characterized nociceptive neurons in the superficial and deeper dorsal horn of the medulla in the rat. The inhibitory action produced by activation of opioid receptors Ž m, d, d. 1 as more pronounced on the C-fiber-evoked excitation than Ad-fiberevoked excitation. These observations provide a putative neural correlate for the antinociceptive effects of DPDPE and DELT observed in behavioral studies. Furthermore, our observations provide an explanation for the opioidmediated selective suppression of second or persistent pain as compared to first pain. Finally, these results demonstrate that in a limited dose range, NTB can discriminate beteen d1- and d-opioid receptor subtypes. 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