Introduction

Abstract Introduction Materials Results Discussion References

Two subtypes of the delta opioid receptor, termed delta-1 and delta-2, have been proposed on the basis of considerable pharmacological, behavioral and biochemical evidence (Hammond, 1993; Porreca and Burks, 1993; Zaki et al., 1996). The role of these receptor subtypes in the production of antinociception at the level of the spinal cord has been extensively studied in the mouse (Heyman et al., 1987; Mattia et al., 1992; Sofuoglu et al., 1991; Takemori and Portoghese, 1993), as well as the rat (Hammond et al., 1995; Malmberg and Yaksh, 1992; Stewart and Hammond, 1993, 1994). In contrast, comparatively little is known about the role of supraspinal delta opioid receptors in the production of antinociception, the specific sites at which delta opioid receptor agonists act or the relative involvement of the two different subtypes of delta opioid receptor in the supraspinal modulation of nociception. Most studies of supraspinal delta opioid receptors in the mouse (Jiang et al., 1991; Mattia et al., 1991; Roerig and Fujimoto, 1989; Sofuoglu et al., 1991) and the rat (Adams et al., 1993; Miaskowski et al., 1991; Negri et al., 1991) administered the agonists intracerebroventricularly. Unfortunately, such studies provide little information about the specific supraspinal sites at which delta opioid receptor agonists act to produce antinociception. With the recent development of antibodies to the delta opioid receptor, the neuroanatomic distribution of these receptors can be visualized with greater resolution than previously afforded by in vitro autoradiography. These studies have localized delta opioid receptors to fibers and varicosities in the NRM and NGCp (Arvidsson et al., 1995a; Kalyuzhny et al., 1996), two nuclei in the VMM that are implicated in the bulbospinal modulation of nociceptive transmission (Gebhart, 1982; Hammond, 1986; Jones, 1992). Although several investigators have reported that microinjection of delta opioid receptor agonists in the VMM produces antinociception (Jensen and Yaksh, 1986; Ossipov et al., 1995; Rossi et al., 1994), the role of the different subtypes of delta opioid receptor in the production of antinociception is not well understood. Also, despite the high concentration of enkephalin (Zamir et al., 1985), the relatively high densities of enkephalinergic terminals (Finley et al., 1981; Khachaturian et al., 1983) and existence of opioid receptors in the VMM (Arvidsson et al., 1995a, 1995b; Mansour et al., 1988, 1994), it is not known whether neurons in this region are tonically modulated by enkephalinergic inputs. The present study was therefore undertaken to (1) characterize the antinociception produced by microinjection of the prototypic delta-1 opioid receptor agonist DPDPE or the delta-2 opioid receptor agonist DELT into the NRM and NGCp using the radiant heat tail-flick and 55°C hot-plate tests in the rat and (2) determine the pharmacological specificity of this antinociception by challenging these agonists with the delta-1 opioid receptor antagonist BNTX and the delta-2 opioid receptor antagonist NTB. A final aim of this study was to determine whether neurons of the NRM and NGCp receive a tonic inhibitory enkephalinergic input mediated by delta opioid receptors by assessing the effects of the antagonists themselves on nociceptive threshold. Preliminary reports of this work have been published in abstract form (Thorat and Hammond, 1996, 1997).

	Methods and Materials

Abstract Introduction Materials Results Discussion References

These experiments were approved by the Institutional Animal Care and Use Committee of the University of Chicago. All procedures were conducted in accordance with the "Guide for Care and Use of Laboratory Animals" as published by the National Institutes of Health and the ethical guidelines of the International Association for the Study of Pain.

Animals. Male Sprague-Dawley rats (Sasco, Madison, WI) weighing 300 to 350g were housed three per cage and maintained on a 12-hr light/dark cycle with lights on at 6:00 a.m. Animals had free access to food and water. Rats were used only once in this study.

Surgical procedures. After induction of anesthesia with a mixture of ketamine hydrochloride (85 mg/kg i.p.) and xylazine (13 mg/kg i.p.), rats were stereotaxically prepared with a stainless steel guide cannula (26-gauge; Plastics One, Roanoke, VA) aimed either at NRM (interaural coordinates: AP, 1.6 to 2.5 mm; ML, 0.0 mm; DV, 4.0 to 5.0 mm) or NGCp (interaural coordinates: AP, 1.6 to 2.5 mm; ML, ±0.7 mm; DV, 4.0 to 5.0 mm). The incisor bar was fixed at 2.5 mm. The guide cannula was secured to the skull with stainless steel screws and dental acrylic cement. A 33-gauge stainless steel stylette was placed in each guide cannula to maintain its patency. After surgery, the rats were housed individually and allowed at least 7 days to recover from surgery.

Behavioral procedures. Nociceptive threshold was assessed by using the radiant heat tail-flick and 55°C hot-plate tests. Briefly, in the tail-flick test, a high-intensity light beam was focused on the blackened tail of the rat (IITC, Woodlawn Hills, CA). The tail-flick reflex latency was measured to the nearest tenth of a sec. Two successive determinations of response latency were made at different sites on the distal two thirds of the tail, and the average was recorded as the tail-flick latency. In the hot-plate test, the rat was placed on a enclosed copper hot-plate maintained at 55°C and the time between placement of the rat on the hot-plate and the occurrence of either a hind paw lick or a jump off the surface was recorded as the hot-plate latency. Rats with base-line tail-flick latencies of >5.2 sec or base-line hot-plate latencies of >15 sec were eliminated from the study. Mean base-line latencies for 20 treatment groups ranged from 3.5 to 4.5 sec in the tail-flick test and from 8.1 to 11.4 sec in the hot-plate test. Motor function was evaluated using the inclined plane test (Rivlin and Tator, 1977), which determined the largest angle at which the rat was able to maintain its position on the inclined plane. Mean base-line inclined plane angle varied from 40° to 50°. The tail-flick, inclined plane and hot-plate tests were conducted in succession in that order. All experiments were done between 10:00 a.m. and 4:00 p.m.

Experimental design. The first series of experiments determined the time course and dose dependence of the effects of DPDPE or DELT on nociceptive threshold. After determination of base-line response latencies and motor competency, rats were microinjected with either DPDPE (0.2-8.8 nmol), DELT (0.03-3.0 nmol) or their respective vehicle. Tail-flick and hot-plate latencies were redetermined 15, 30, 45 and 60 min later. Rats that did not respond within 14 sec on the tail-flick test or 40 sec on the hot-plate test after drug administration were removed to prevent tissue damage and assigned the cutoff latency. Dose-response lines for DPDPE or DELT were constructed using the individual tail-flick and hot-plate latencies obtained at the time of peak effect (45 and 30 min, respectively). The ED₅₀ was defined as the dose that produced 50% of the maximum possible increase in tail-flick or hot-plate latency (i.e., to 7 sec in the tail-flick test and to 25 sec in the hot-plate test). Fieller's theorem was used to determine the 95% CL (Finney, 1964). A two-way analysis of variance for repeated measures was used to compare the effects of the delta opioid receptor agonists to those of their respective vehicle. The Newman-Keuls test was used for post hoc comparisons among the individual group means.

Histology. At the end of the experiment, each rat was killed by inhalation of CO₂. The brain was removed and fixed by immersion in 10% formalin containing 30% sucrose. Twenty five-µm transverse sections were cut through the region traversed by the guide cannula using a cryostat microtome, collected on gelatinized slides and stained with Cresyl violet. The location of the injection sites was verified by two individuals without reference to the behavioral data and plotted on appropriate coronal sections from an atlas of the rat brainstem modified from that provided by Neurographics (Kanata, Ontario, Canada).

Drugs and microinjections. All drug and vehicle solutions were microinjected over a period of 60 sec in a volume of 0.4 µl using a 33-gauge injection cannula that protruded an additional 3 mm beyond the guide cannula. Delivery of the drug solution was monitored by following movement of a bubble in the calibrated tubing that connected the injector to a syringe mounted on a microinfusion pump. The drugs and their vehicle solutions were freshly prepared. Naltriben (lot no. XXI-146.3; molecular weight, 465.5) was a gift from G. D. Searle & Co. (Skokie, IL). BNTX hydrochloride (lot no. WY-III-69B; molecular weight, 466.0) was obtained from Research Biochemicals (Natick, MA) through the Research Technology Branch of the National Institute of Drug Abuse. Naltriben and BNTX were dissolved in 45% (w/v) HBC (lot no. UCD-695A; Research Biochemicals). DELT (Sigma Chemical, St. Louis, MO; lot no. 44H08641; molecular weight, 782.9) was dissolved in either saline for the dose-response studies or 45% (w/v) HBC for the antagonism studies. DPDPE (Sigma Chemical; lot no. 85H58551; molecular weight, 654.8, or Research Biochemicals; lot no. FRY-296F, molecular weight, 647.8) was dissolved in distilled water for the dose-response studies or saline for the antagonism studies.

	Results

Abstract Introduction Materials Results Discussion References

Distribution of microinjection sites in the VMM. Histological analysis revealed that the microinjection sites were distributed throughout the rostrocaudal extent of the NRM and NGCp. The very large number of rats and different treatment groups in this study precluded presentation of all the injection sites for each treatment group. Therefore, as there were no major differences in the distribution of microinjection sites among the various treatment groups, only the distribution of microinjection sites for the 1.2-nmol dose of DELT is presented (fig. 1). A number of sites located dorsal to the NGCp and ventral to the dorsal border of the facial nucleus, as well as a few sites in the most rostral aspect of the nucleus reticularis paragigantocellularis lateralis, were included within the NGCp for purposes of analysis. Microinjection sites located outside the NRM and NGCp included sites in the dorsal, lateral or caudal aspects of the nucleus reticularis gigantocellularis, as well as the facial nucleus, the medial longitudinal fasciculus, the pyramids, inferior olive or nucleus raphe obscurus.

View larger version (58K): [in this window] [in a new window]   Fig. 1.   Representative rostrocaudal distribution of the sites in the VMM at which 1.2 nmol DELT was microinjected in the rat. Numbers refer to distance caudal to the interaural line. , Sites in the NRM and NGCp. , Sites outside these two nuclei. 4th vent, fourth ventricle; 7, facial motor nucleus; 7g, genu of the seventh cranial nerve; 7t, tract of the seventh cranial nerve; dcn, dorsal cochlear nucleus; icp, inferior cerebellar peduncle; mlf, medial longitudinal fasciculus; ngc, nucleus reticularis gigantocellularis; P, pyramid; tr, trapezoid body; V, spinal trigeminal tract.

Effects of DPDPE in the VMM. Microinjection of 0.2 to 8.8 nmol of DPDPE in either the NRM or NGCp increased tail-flick latency in a dose-dependent manner, with the peak effect occurring between 30 and 45 min (fig. 2A). There was no difference between these nuclei in either the magnitude or the onset to effect of DPDPE (P > .27 for each treatment group). For example, microinjection of 8.8 nmol of DPDPE at seven sites in the NRM increased tail-flick latency to 6.4 ± 1.2, 9.0 ± 0.9 and 10.5 ± 1.1 sec at 15, 30 and 45 min, respectively. This same dose in the NGCp increased tail-flick latency to 5.8 ± 1.0, 8.2 ± 1.3 and 9.3 ± 1.1 sec at 15, 30 and 45 min, respectively. Therefore, the sites were grouped together as the VMM for subsequent analysis. Linear regression analysis estimated the ED₅₀ (95% CL) of DPDPE in the tail-flick test to be 0.66 (0.07-1.5) nmol (fig. 3A). Doses of DPDPE of >8.8 nmol could not be tested due to its limited solubility.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Time course of the change in response latencies in the (A) tail-flick and (B) hot-plate tests after microinjection of either distilled water () or 0.2 (), 1.0 (), 2.9 () or 8.8 () nmol of the delta-1 opioid receptor agonist DPDPE in the VMM. Symbols represent the mean ± S.E.M. of determinations in 12 to 17 rats. *, P < .05; **, P < .01 compared with vehicle at the corresponding time point.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Dose-response relationships for DPDPE () and DELT () in the (A) tail-flick and (B) hot-plate tests. Dose-response curves were generated by linear regression of data at time of peak effect, which corresponded to 45 min for DPDPE and 30 min for DELT in the tail-flick test. Dose-response curves in the hot-plate test were generated at 30 min for DPDPE and 15 min for DELT; these were not significant as indicated by the dashed line. Symbols represent the mean ± S.E.M. of determinations in 9 to 21 rats.

Effects of DELT in the VMM. Microinjection of 0.03 to 3.0 nmol of DELT in either the NRM or NGCp produced a dose-dependent increase in tail-flick latency, with the peak effect occurring within 30 min (fig. 4A). These sites were grouped together as the VMM for subsequent analysis because they did not differ with respect to either the magnitude or onset to effect of DELT (P > .2 for all treatment groups). For example, tail-flick latency determined 15, 30 and 45 min after microinjection of 1.2 nmol of DELT at seven sites in the NRM was 6.3 ± 0.9, 8.6 ± 1.2 and 8.9 ± 1.1 sec, respectively. Tail-flick latency determined 15, 30 and 45 min after microinjection of this same dose at seven sites in the NGCp was 5.8 ± 0.7, 9.2 ± 0.8 and 9.5 ± 0.8 sec, respectively. The ED₅₀ (95% CL) of DELT in the tail-flick test was 0.1 (0.03-0.21) nmol (fig. 3A). Microinjection of either 1.2 or 3.0 nmol of DELT at five sites in the nucleus reticularis gigantocellularis increased tail-flick latency with the same onset and to the same extent as did microinjection of these doses in the NRM and NGCp. Doses of DELT of >3.0 nmol could not be tested due to limited solubility.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Time course of the change in response latencies in the (A) tail-flick and (B) hot-plate tests after microinjection of either saline () or 0.03 (), 0.12 (), 0.3 (), 1.2 () or 3.0 () nmol of the delta-2 opioid receptor agonist DELT in the VMM. Symbols represent the mean ± S.E.M. of determinations in 9 to 21 rats. *, P < .05; **, P < .01 compared with vehicle at the corresponding time point.

Effects of BNTX or NTB in the VMM. Figure 5 illustrates the time course of the change in response latencies produced by microinjection of BNTX or NTB in the VMM. Neither tail-flick nor hot-plate latencies were altered after microinjection of 45 pmol of BNTX in the VMM. Similarly, microinjection of 0.15 nmol of NTB in the VMM did not alter either tail-flick or hot-plate latencies compared with vehicle. However, tail-flick and hot-plate latencies were significantly increased after microinjection of a 10-fold higher dose of NTB, 1.5 nmol, in the VMM (fig. 5, A and B). No consistent effects were noted on the inclined plane test after microinjection of BNTX or NTB (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Time course of the change in response latencies in the (A) tail-flick and (B) hot-plate tests after microinjection of either 45% (w/v) HBC (), 45 pmol of the delta-1 opioid receptor antagonist BNTX () or 0.15 () or 1.5 () nmol of the delta-2 opioid receptor antagonist NTB in the VMM. Symbols represent the mean ± S.E.M. of determinations in 12 to 17 rats. *, P < .05; **, P < .01 compared with HBC at the corresponding time point.

Effects of BNTX or NTB on the antinociception produced by microinjection of DPDPE or DELT in the VMM. Microinjection of the delta-1 opioid receptor antagonist BNTX in the VMM selectively antagonized the antinociception produced by microinjection of DPDPE at the same sites. However, the selectivity of this antagonism was dependent on the dose of BNTX. Pretreatment with 1.5 pmol of BNTX significantly attenuated the increase in tail-flick latency produced by microinjection of 5.3 nmol of DPDPE (fig. 6A) and produced a only marginal and transient antagonism of the increase in tail-flick latency produced by microinjection of DELT (fig. 6B). Increasing the dose of BNTX by 10-fold to 15 pmol completely antagonized the antinociceptive effect of DPDPE (fig. 6A). However, pretreatment with this higher dose of BNTX also antagonized the antinociceptive effect of DELT to a greater extent and for a longer duration (fig. 6B). Microinjection of 45 pmol of BNTX did not produce any greater antagonism of the antinociceptive effect of DELT than did the 15-pmol dose (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Time course of the antagonism of the increase in tail-flick latency produced by microinjection of (A) 5.3 nmol of DPDPE or (B) 1.2 nmol of DELT in the VMM of rats pretreated 5 min earlier by microinjection of either HBC (), 0.15 nmol of the delta-2 opioid receptor antagonist NTB () or 1.5 pmol () or 15 pmol () of the delta-1 opioid receptor antagonist BNTX. Symbols represent the mean ± S.E.M. of determinations in 7 to 13 rats. *, P < .05; **, P < .01 compared with the rats pretreated with HBC at the corresponding time point.

Influence of solvent on drug efficacy. In the antagonism study, 1.2 nmol of DELT increased tail-flick latency to a significantly greater extent than in the dose-response analysis (compare figs. 4A and 6B). This difference in effect could arise from (1) pretreatment with the HBC vehicle or (2) the use of HBC, rather than saline, as the solvent for DELT in the antagonism study. Additional animals were tested to examine this discrepancy. Microinjection of 1.2 nmol of DELT dissolved in saline in five rats that were pretreated 5 min earlier with HBC increased tail-flick latency to 8.8 ± 1.6 sec. These values were not different from the 8.9 ± 0.7 sec latency determined for this dose of DELT in the dose-response study in which it was also dissolved in saline (P > .8; fig. 3A). When 1.2 nmol of DELT was dissolved in HBC and injected by itself, it increased tail-flick latency to 11.6 ± 1.1 sec (n = 7). These values were significantly greater than the effect of this dose of DELT dissolved in saline (P < .05) but did not differ from the increase produced by microinjection of this formulation in rats that were pretreated with HBC (12.3 ± 0.7 sec). A similar enhancement occurred in the hot-plate test, in which response latency was increased to a greater extent and for a longer duration when DELT was dissolved in HBC compared with saline (data not shown). The enhanced efficacy of DELT in this set of experiments is therefore most likely due to the use of HBC as the solvent for DELT, and not to prior microinjection of HBC. The antinociceptive potency of intrathecally administered morphine, [D-Ala², D-Leu⁵]enkephalin and other peptidic analogs of enkephalin is similarly enhanced when HBC is used as the vehicle (Jang et al., 1992; Yaksh et al., 1991).

	Discussion

Abstract Introduction Materials Results Discussion References

Medullary delta-1 opioid receptors modulate nociception. The first finding of this study was that microinjection of DPDPE, a delta-1 opioid receptor agonist, in the NRM and NGCp increased tail-flick latency in a dose-dependent manner. This increase in tail-flick latency was antagonized by microinjection of as little as 1.5 pmol of the delta-1 opioid receptor antagonist BNTX at the same site. However, it was not attenuated by pretreatment with 0.15 nmol of the delta-2 opioid receptor antagonist NTB. The selective antagonism of the antinociceptive effect of DPDPE by BNTX but not NTB is strong evidence that the increase in tail-flick latency is specific and is mediated by a delta-1 opioid receptor. The finding that microinjection of DPDPE in the VMM increased tail-flick latency is contrary to previous reports that doses as high as 40 nmol of DPDPE were ineffective (Ossipov et al., 1995; Rossi et al., 1994). However, this study differs from previous reports in two important respects. First, DPDPE was administered in a much smaller volume in the present study (0.4 compared with 1.0 µl). Second, comparison of base-line tail-flick latencies suggests that the thermal stimulus in this study was less intense than that used by Rossi et al. (1994). Studies with intrathecally administered mu opioid receptor agonists indicate that antinociceptive potency is inversely related to the intensity of the noxious stimulus (Saeki and Yaksh, 1993). Even with the moderate intensity stimulus used in this study, the highest dose of DPDPE that could be tested did not increase tail-flick latency beyond 10 sec. Therefore, the antinociceptive effect of DPDPE may not have been observed in previous studies simply because the thermal stimulus was too intense.

Medullary delta-2 opioid receptors modulate nociception. Another finding of this study was that microinjection of DELT, a delta-2 opioid receptor agonist, in the NRM and NGCp also produced a dose-dependent increase in tail-flick latency, as well as a modest increase in hot-plate latency. The increase in tail-flick and hot-plate latency was antagonized by microinjection of 0.15 nmol of NTB at the same site but was not attenuated by 1.5 pmol of BNTX. The selective antagonism of the antinociceptive effect of DELT by NTB, but not BNTX, is strong evidence that the increase in tail-flick latency is specific and is mediated by a delta-2 opioid receptor. These observations are consistent with previous reports that microinjection of DELT in the VMM increases response latency in the radiant heat and warm-water tail-flick tests, as well as the hot-plate test (Ossipov et al., 1995; Rossi et al., 1994), and that the antinociceptive effect of DELT is antagonized by the nonequilibrium delta-2 opioid receptor antagonist [D-Ala²,Cys⁴]deltorphin but not the delta-1 opioid receptor antagonist [D-Ala², Leu⁵,Cys⁶]enkephalin (Ossipov et al., 1995). The doses of DELT that increased tail-flick latency in this study were 10-fold lower than those used in prior reports. The greater potency of DELT is likely due to our use of a less intense stimulus in the tail-flick test. Consistent with this interpretation, microinjection of 20 nmol of DELT in the VMM increased response latency to 47% of the maximum possible effect in the 52°C warm water tail-flick test but was inactive in the 55°C warm water tail-flick test (Ossipov et al., 1995).

Mechanisms by which delta-1 and delta-2 opioid receptors may produce antinociception. Immunocytochemical studies indicate that delta opioid receptor immunoreactivity in the NRM and NGCp is localized to fibers and varicosities and that immunoreactive soma are not present (Arvidsson et al., 1995a; Kalyuzhny et al., 1996). In fact, half of all spinally projecting neurons in the NRM are apposed by varicosities immunoreactive for the delta opioid receptor (Kalyuzhny et al., 1996). These observations suggest that delta opioid receptors are predominantly situated presynaptically in the NRM and NGCp. Because no somal staining for the delta opioid receptor was observed in this region, DELT or DPDPE probably do not produce antinociception by inhibiting the pain-facilitatory pathways that also originate in this region (Gebhart, 1993; Zhuo and Gebhart, 1990, 1991). Rather, microinjection of DELT or DPDPE in the VMM may produce antinociception by presynaptically inhibiting inhibitory inputs to these neurons. Candidate inhibitory inputs include those mediated by GABA, norepinephrine or enkephalin. Definition of the mechanism of action will require additional immunocytochemical studies of the relationship of delta opioid receptors to immunocytochemically defined inputs in this region, as well as neurochemical studies of the effects of delta opioid receptor agonists on the release of GABA, norepinephrine and enkephalin.

Selectivity of BNTX and NTB as antagonists of the delta-1 and delta-2 opioid receptor. Microinjection of 1.5 pmol of BNTX selectively antagonized the antinociception produced by DPDPE without affecting that produced by DELT. Conversely, microinjection of 0.15 nmol of NTB almost completely blocked the antinociception produced by DELT without attenuating the antinociception produced by microinjection of DPDPE. These findings are consistent the characteristics of the delta-1 and delta-2 subtypes of the delta opioid receptor (Hammond, 1993; Zaki et al., 1996). However, the selectivity of BNTX and NTB is maintained only in a limited dose range and the selection of dose requires careful attention. For example, a 10-fold increase in the dose of BNTX to 15 pmol resulted in a complete antagonism of the effects of DPDPE but also caused a significant, prolonged antagonism of the effects of DELT. Similarly, a 10-fold higher dose of NTB (1.5 nmol) by itself significantly increased tail-flick and hot-plate latencies. This effect probably arises from nonspecific effects of this high dose of NTB. Previous studies from this and other laboratories indicate that NTB has a narrow window of selectivity (Stewart and Hammond, 1993, 1994), and at high doses it can, like naltrindole, exert agonist-like effects (Stapelfeld et al., 1992; Stewart et al., 1994; Takemori et al., 1990, 1992).

Summary. The present results provide strong evidence that activation of delta-1, as well as delta-2, opioid receptors in the NRM and NGCp results in antinociception. The differential antagonism of the antinociceptive effects of DPDPE and DELT by the subtype-selective antagonists BNTX and NTB complements earlier studies of spinal delta opioid receptors and supports the existence of delta-1 and delta-2 opioid receptors at supraspinal, as well as spinal loci. These findings implicate both subtypes of the delta opioid receptor in the supraspinal modulation of nociception. However, neurons of the VMM that modulate nociception do not appear to receive a tonically active, inhibitory enkephalinergic input mediated by either delta-1 or delta-2 opioid receptors.