Antinociception produced by receptor selective opioids: modulation of spinal antinociceptive effects by supraspinal opioids

The effect of intracerebroventricular administration of low-antinociceptive doses of selective μ-(DAMGO) or δ-(DPDPE) opioid agonists on the dose-dependent antinociceptive effects produced by intrathecal administration of sequentially increasing doses of selective μ-, δ-, or κ- (U50, 488H) opioid agonists was evaluated, in the rat, using the Randall-Selitto paw-withdrawal test. When DPDPE or U50,488H was administered intrathecally, the low doses of both intracerebroventricular DAMGO and intracerebroventricular DPDPE markedly enhanced the antinociceptive effects of both intrathecal opiods. In contrast, when DAMGO was administered intrathecally, both intracerebroventricular DAMGO and intracerebroventricular DPDPE, administered in low doses, markedly antagonized the antinociceptive effects of the intrathecal opioid. In addition, the intracerebroventricular administration of low-antinociceptive dose of a second μ-opioid agonist, morphiceptin, antagonized the antinociceptive effects of intrathecal morphiceptin. The antagonism of the antinociceptive effects observed with spinal administration of DAMGO is dose-dependent, with the effect observed only at low doses. Furthermore, the antagonism cannot be explained by a reduction in motor deficits produced by intrathecal administration of DAMGO, because there were no differences in motor deficits, measured with an accelerating Rotarod treadmill, between intrathecal DAMGO administered as a single agent or as part of a combination regimen. The differences in antinociceptive effects obtained with the various supraspinal and spinal combinations are discussed in terms of the interactions that may occur between brainstem and spinal opioid receptor sites.     

Analgesic synergy and improved motor function produced by combinations of μ-δ- and μ-κ-opioids

This study evaluated the effects of intrathecal administration of a low-analgesic dose of the selective μ-agonist DAMGO co-administered with sequentially increasing doses of either the selective δ-agonist DPDPE or the selective κ-agonist, U50,488H on mechanical nociceptive thresholds in the rat. Potent analgesic synergy was observed with both combinations. Since an elevation in nociceptive threshold can result from motor deficits, as well as true analgesia, we also evaluated the effects of the combination regimens on motor coordination using a rotarod apparatus. The combination regimens produced significantly less motor deficits than those observed when DPDPE and U50,488H were administered as single agents. These findings of enhanced analgesia with decreased motor side-effects associated with administration of fixed μ/δ or μ/κ combinations suggest that co-administration of opiates that act at different receptors may constitute a superior approach to the treatment of pain.     

Inhibition of spinal opioid analgesia by supraspinal administration of selective opioid antagonists

The effect of intracerebroventricular administration of a selective mu- (CTOP) or delta- (ICI 174,864) opioid receptor antagonist on the antinociceptive effects produced by intrathecal administration of selective mu- (DAMGO), delta- (DPDPE) and kappa- (U50-488H) opioid receptor agonists was evaluated using the Randall-Selitto paw-withdrawal test, in the rat. While the intracerebroventricular administration of CTOP or ICI 174,864, alone, had no effect on nociceptive thresholds, intracerebroventricular administration of CTOP and ICI 174,864 produced marked antagonism of the antinociceptive effects of intrathecal DAMGO. The antinociceptive effects of intrathecal administration of DPDPE or U50,488H were not antagonized by intracerebroventricular administration of CTOP or ICI 174,864. These data suggest that, in the rat, along with the established descending antinociceptive pathways, there is an ascending antinociceptive control mechanism projecting from the spinal cord to the brainstem. The ascending antinociceptive control involves mu- and delta-opioid agonism at supraspinal sites and appears to be mediated selectively by mu-, but not by delta- or kappa-opioid agonism at the spinal level.     

Streptozotocin-induced diabetes selectively alters the potency of analgesia produced by μ-opioid agonists, but not by δ- and κ-opioid agonists

To investigate the possible mechanisms of the alterations in morphine-induced analgesia observed in diabetic mice, we examined the influence of streptozotocin-induced (STZ-induced) diabetes on analgesia mediated by the different opioid receptors. The antinociceptive potency of morphine (10 mg/kg), administered s.c., as determined by both the tail-pinch and the tail-flick test, was significantly reduced in diabetic mice as compared to that in controls. Mice with STZ-induced diabetes had significantly decreased sensitivity to intracerebroventricularly (i.c.v.) administered μ-opioid agonists, such as morphine (10 μg) and [d-Ala², N-Me Phe⁴,Gly-ol⁵]enkephalin (DAMGO, 0.5 μg). However, i.c.v. administration of [d-Pen^2,5]enkephalin (DPDPE, 5 μg), a δ-opioid agonist, and U-50,488H (50 μg), a κ-opioid agonist, produced pronounced antinociception in both control and diabetic mice. Furthermore, there were no significant differences in antinociceptive potency between diabetic and control mice when morphine (1 μg), DAMGO (10 μg), DPDPE (0.5 μg) or U-50,488H (50 μg) was administered intrathecally. In conclusion, mice with STZ-induced diabetes are selectively hyporesponsive to supraspinal μ-opioid receptor-mediated antinociception, but they are normally responsive to activation of δ- and κ-opioid receptors.     

Dissociation of antinociceptive and motor effects of supraspinal opioid agonists in the rat

This study compared the antinociceptive and motor effects produced by intracerebroventricular administration of selective mu- (DAMGO) and delta- (DPDPE) opioid receptor agonists in the rat. Changes in nociceptive thresholds were measured using the Randall-Selitto paw-withdrawal test and changes in motor coordination were evaluated using the rotarod treadmill test. Both DAMGO and DPDPE produced statistically significant, dose-dependent increases in mechanical nociceptive thresholds compared to vehicle controls. However, in the motor coordination studies, neither opioid agonist produced statistically significant changes in rotarod performance scores. The dissociation of antinociceptive and motor effects at this supraspinal site differs from the strong association between antinociceptive and motor effects produced by intrathecal administration of the same opioid agonists.     

Analgesic synergy and improved motor function produced by combinations of mu-delta- and mu-kappa-opioids

This study evaluated the effects of intrathecal administration of a low-analgesic dose of the selective mu-agonist DAMGO co-administered with sequentially increasing doses of either the selective delta-agonist DPDPE or the selective kappa-agonist, U50,488H on mechanical nociceptive thresholds in the rat. Potent analgesic synergy was observed with both combinations. Since an elevation in nociceptive threshold can result from motor deficits, as well as true analgesia, we also evaluated the effects of the combination regimens on motor coordination using a rotarod apparatus. The combination regimens produced significantly less motor deficits than those observed when DPDPE and U50,488H were administered as single agents. These findings of enhanced analgesia with decreased motor side-effects associated with administration of fixed mu/delta or mu/kappa combinations suggest that co-administration of opiates that act at different receptors may constitute a superior approach to the treatment of pain.     

Selective opioid δ agonists elicit antinociceptive supraspinal/spinal synergy in the rat

A multiplicative antinociceptive interaction of morphine activity at supraspinal and spinal sites has been clearly established and is thought to be responsible, in part, for the clinical utility of this compound in normal dose-ranges. While synergistic actions of μ-opioid receptor agonists have been shown, it is unclear whether a similar interaction exists for opioid agonists acting via δ-opioid receptors. Responses to acute nociception were determined with the 52°C hot plate, 52°C warm-water tail-flick and the Hargreaves paw-withdrawal tests. The peptidic opioid δ₁ agonist [ -Pen², -Pen⁵]enkephalin (DPDPE) or δ₂ agonist [ -Ala²,Glu⁴]deltorphin (DELT) were given into the rostral–ventral medulla (RVM), intrathecally (i.th.) or simultaneously into both the RVM and i.th. (1:1 fixed ratio). Both of the opioid δ agonists produced dose-dependent antinociception in all tests. With the exception of DPDPE in the hot plate test, isobolographic analysis revealed that the supraspinal/spinal antinociceptive interaction for both DPDPE and DELT were synergistic in all nociceptive tests. These data suggest that opioid δ agonists exert a multiplicative antinociceptive interaction between supraspinal and spinal sites to acute noxious stimuli and suggest possibility that compounds acting through δ-opioid receptors may have sufficient potency for eventual clinical application.     

Comparison of G-protein activation in the brain by μ-, δ-, and κ-opioid receptor agonists in μ-opioid receptor knockout mice

Mice lacking the μ-opioid receptor gene have been developed by a gene knockout procedure. In this study, the activity of opioid receptor coupled G-proteins was examined to investigate whether there is a change in the extent of coupling for μ-, δ-, and κ-opioid receptors in μ-opioid receptor knockout mice. Selective agonists of μ- (DAMGO), δ- (DPDPE), and κ- (U-69,593) opioid receptors stimulated [³⁵S]GTPγS binding in the caudate putamen and cortex of wild-type mice. In contrast, only U-69,593 stimulated [³⁵S]GTPγS binding in these regions of μ-opioid receptor knockout mice. These results confirmed the absence of G-protein activation by a μ-opioid receptor agonist in μ-opioid receptor knockout mice, and demonstrated that coupling of the κ-opioid receptor to G-proteins is preserved in these mice. However, G-protein activation by the δ-opioid receptor agonist, DPDPE, was reduced in the μ-opioid receptor knockout mice, at least in the brain regions studied using autoradiography.     

Antisense oligodeoxynucleotides to the κ-1 receptor block the antinociceptive effects of Δ-THC in the spinal cord

Intrathecal pretreatment of mice with an antisence oligodeoxynucleotide directed against the κ-1 receptor significantly reduced the antinociceptive effects of the kappa receptor agonist U50,488 as well as Δ⁹-THC, the major psychoactive ingredient found in cannabis. A mismatched oligodeoxynucleotide which contained four switched bases did not block the antinociception produced by U50,488 orΔ⁹-THC. Furthermore, κ-1 antisense did not alter the antinociceptive effects of either the mu receptor-selective opioid DAMGO, or the delta receptor-selective opioid DPDPE. By using κ-1 antisense, we were able to demonstrate that an interaction occurs between the cannabinoids and opioids in the spinal cord.     

Differential effects of 3-isobutyl-1-methylxanthine injected intrathecally or intracerebroventricularly on antinociception induced by opioids administered intracerebroventricularly in the mouse

Various doses of 3-isobutyl-1-methylxanthine (IBMX), a cAMP phosphodiesterase inhibitor, injected intrathecally (i.t.) or intracerebroventricularly (i.c.v.) alone did not show any antinociceptive effect. IBMX (0.01 to 1 ng) pretreatment i.t. for 10 min dose-dependently attenuated the inhibition of the tail-flick response induced by i.c.v. administered morphine (2 μg), β-endorphin (1 μg), and U50, 488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzeocetamide), 60 μg. However, pretreatment with IBMX i.c.v. did not affect the inhibition of the tail-flick response induced by morphine, β-endorphin, and U50, 488H administered i.c.v. Neither i.c.v. nor i.t. pretreatment with IBMX attenuated the inhibition of the tail-flick response induced by D-Pen²-D-Pen⁵-enkephalin (DPDPE; 10 μg) administered i.c.v. Our results suggest that spinal but not supraspinal cAMP phosphodiesterases are involved in mediating antinociception induced by morphine, β-endorphin and U50, 488H administered supraspinally. However, neither spinal nor supraspinal cAMP phosphodiesterase is involved in mediating antinociception induced by DPDPE administered supraspinally.     

Up-regulation of spinal μ-opioid receptor function to activate G-protein by chronic naloxone treatment

The effects of repeated s.c. administrations of an μ-opioid receptor antagonist naloxone on the G-protein activation induced by μ-opioid receptor agonists [ -Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin (DAMGO), endomorphin-1 and endomorphin-2 in the mouse spinal cord was studied, monitoring guanosine-5′-o-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding. All μ-opioid receptor agonists concentration-dependently increased the [³⁵S]GTPγS binding. The increases of [³⁵S]GTPγS binding induced by agonists were significantly enhanced in mice pretreated with naloxone. Under the present condition, chronic treatment with naloxone significantly increased the density of [³H]DAMGO binding sites with an increase in K_d values in spinal cord membranes, indicating an increase in μ-opioid receptors on the membrane surface. These findings suggest that chronic treatment with an μ-opioid receptor antagonist naloxone leads to the supersensitivity to activate G-protein by μ-opioid receptor agonists with an increase in μ-opioid receptors in membranes of the mouse spinal cord.     

Studies on the intrathecal effect of β-endorphin in primate

The intrathecal administration of β-endorphin in the primate through an indwelling spinal catheter, produced a significant elevation in the nociceptive threshold as measured by the discrete trial shock titration task. The time of onset, duration of effect and magnitude of effect were all dose-dependent over a range of 150–750 μg. The effects were antagonized in a dose-dependent fashion by the systemic administration of naloxone. Aside from the elevations in the shock titration threshold produced by intrathecal β-endorphin, no untoward effects on the animal's motor function or behavioral reactivity was noted. Significantly, unlike morphine, intrathecal β-endorphin failed to produce any signs of scratching behavior at the doses used in these experiments. Once daily administration of intrathecal β-endorphin (500 μg) showed a significant progressive decline in the antinociceptive effect over an 8-day period. Animals made tolerant to β-endorphin in this fashion showed a significantly reduced response to an otherwise active dose of intrathecal morphine, indicating evidence for cross tolerance.     

Effects of neonatal cocaine treatment and gender on opioid agonist-stimulated [S]GTPγS binding in the striatum and nucleus accumbens

Prenatal cocaine exposure increases μ-opioid receptor binding in dopaminergic terminal areas and enhances behavioral responsiveness to μ-opioid agonists. We investigated the influence of early postnatal cocaine treatment on in vitro μ- and δ-opioid receptor activation in male and female weanling rats. Pups received subcutaneous injections of either 20 mg/kg cocaine HCl or saline once daily on postnatal days 1 through 5. On postnatal day 25, animals were decapitated and their brains were removed and frozen for later sectioning. Opioid receptor activation was assessed in the striatum and the shell of the nucleus accumbens by autoradiographic analysis of agonist-stimulated [³⁵S]GTPγS binding. Brain sections were incubated in the presence of [³⁵S]GTPγS, GDP, and either the μ-opioid agonist [ -Ala²-N-MePhe⁴-Gly⁵-ol]enkephalin (DAMGO) or the δ-opioid agonist -Pen²-D-Pen⁵-enkephalin (DPDPE). Baseline binding was assessed in the absence of agonist, and nonspecific binding was determined by the addition of unlabeled GTPγS. Film images were quantified using brain mash-calibrated [¹⁴C] standards. Neonatal cocaine treatment had no effect on either baseline or agonist-stimulated [³⁵S]GTPγS binding. However, males exhibited significantly greater activation than females of δ-opioid receptors in both striatum and accumbens shell, regardless of neonatal treatment. These findings indicate a gender difference in δ-opioid receptor function that could mediate behavioral differences in response to opioid agonists.     

Differential antagonism of endomorphin-1 and endomorphin-2 spinal antinociception by naloxonazine and 3-methoxynaltrexone

To determine the role of spinal mu-opioid receptor subtypes in antinociception induced by intrathecal (i.t.) injection of endomorphin-1 and -2, we assessed the effects of β-funaltrexamine (a selective mu-opioid receptor antagonist) naloxonazine (a selective antagonist at the mu₁-opioid receptor) and a novel receptor antagonist (3-methoxynaltrexone) using the paw-withdrawal test. Antinociception of i.t. endomorphins and [ -Ala², MePhe⁴, Gly(ol)⁵]enkephalin (DAMGO) was completely reversed by pretreatment with β-funaltrexamine (40 mg/kg s.c.). Pretreatment with a variety of doses of i.t. or s.c. naloxonazine 24 h before testing antagonized the antinociception of endomorphin-1, -2 and DAMGO. Judging from the ID₅₀ values of naloxonazine, the antinociceptive effect of endomorphin-2 was more sensitive to naloxonazine than that of endomorphin-1 or DAMGO. The selective morphine-6β-glucuronide antagonist, 3-methoxynaltrexone, which blocked endomorphin-2-induced antinociception at each dose (0.25 mg/kg s.c. or 2.5 ng i.t.) that was inactive against DAMGO, did not affect endomorphin-1-induced antinociception but shifted the dose–response curve of endomorphin-2 3-fold to the right. These findings may be interpreted as indicative of the existence of a novel mu-opioid receptor subtype in spinal sites, where antinociception of morphine-6β-glucuronide and endomorphin-2 are antagonized by 3-methoxynaltrexone. The present results suggest that endomorphin-1 and endomorphin-2 may produce antinociception through different subtypes of mu-opioid receptor.     

Induction of fos-like immunoreactivity by opioids in guinea-pig brain

In the present study the effects of intracerebroventricular (i.c.v.) administration of 100 nmol of morphine, the selective μ-receptor agonist DAMGO, the δ-receptor agonist DPDPE and the κ-receptor agonist U50,488H, on the induction of Fos-like immunoreactivity (Fos-LI) in the guinea-pig brain were investigated using immunohistochemical techniques. Guinea-pigs given i.c.v. injection of opioids showed marked increases in the number of Fos-LI nuclei within a large number of brain regions, several of which, including hypothalamic nuclei, paraventricular thalamic nucleus, the amygdala, periaqueductal gray, superior and inferior colliculi, the piriform and entorhinal cortices, have been shown to be activated under stressful or aversive conditions. Pretreatment with the opioid antagonist, naltrexone, before administration of morphine or U50,488H, inhibited Fos-LI induction indicating that the effects of the opioids were mediated by opioid receptors. U50,488H administration resulted in higher numbers of Fos-LI stained neurons compared to morphine in most regions other than the nucleus accumbens and interpeduncular nucleus. Morphine and DAMGO produced significantly higher numbers of Fos-LI neurons in the nucleus accumbens shell region than U50,488H, which may reflect the more powerful reinforcing/rewarding effects of μ-receptor agonists. Thus the present study supports a critical role for the nucleus accumbens shell region in the reinforcing/rewarding effects of opioids.     

Comparison of the antinociceptive and motor effects of intrathecal opioid agonists in the rat

This study compared the antinociceptive and motor effects produced by intrathecal administration of selective mu-, delta-, and kappa-opioid receptor agonists in the rat. Changes in nociceptive threshold were measured using the Randall-Selitto paw-withdrawal test and changes in motor coordination were evaluated using the rotarod treadmill test. Each opioid agonist produced statistically significant, dose-dependent increases in mechanical nociceptive thresholds compared to vehicle controls. In the motor coordination studies, DAMGO and DPDPE, but not U50,488H, produced statistically significant decreases in rotarod performance scores compared to vehicle controls. The results of these studies suggest that motor side-effects produced by opioid agonists need to be considered when interpreting the results of antinociceptive tests that are dependent on a normally functioning motor system.     

Opioid receptors on peripheral sensory axons

Opioid receptors have been demonstrated by light microscopic techniques in fine cutaneous nerves in naive animals. The present study extends these findings by showing that 29 and 38% of unmyelinated cutaneous sensory axons can be immunostained for μ- or δ-opioid receptors respectively. Local cutaneous injection of DAMGO, a μ-opioid ligand, ameliorates the nociceptive behaviors caused by local cutaneous injection of glutamate, a purely nociceptive chemical stimulus showing that the μ-receptors are functional. By contrast the δ-opioid ligand [2-

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-penicillamine, 5-

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-penicillamine]enkephalin (DPDPE) had no effect on these behaviors. These findings indicate a wider function for opioid receptors in naive animals than previously envisioned.     

The antinociceptive effect of the μ-opioid fentanyl is reduced in the presence of the α2-adrenergic antagonist idazoxan in inflammation

Interactions between α₂-adrenergic and μ-opioid systems play an important role in the modulation of hyperalgesic states. The antinociceptive effects of α₂-adrenergic agonists and μ-opioids are potentiated when co-administered; however, attempts to induce cross reversal of the antinociceptive effects of α₂-adrenergic and μ-opioid systems have produced contradictory results. We have studied the possible endogenous tonic control of the α₂-adrenergic systems in the modulation of pain in inflammation, and the interactions between the two antinociceptive systems in rat spinal cord nociceptive reflexes activated by both natural and electrical stimulation. The facilitatory actions of the α₂-adrenergic antagonist idazoxan were compared in control rats and in animals with carrageenan-induced paw inflammation. The antinociceptive effect of the μ-opioid fentanyl was tested alone and in the presence of idazoxan. In agreement with some previous observations, idazoxan i.v. produced no change in responses to natural and electrical stimulation in normal animals. In animals with inflammation, idazoxan only induced facilitation of responses evoked by noxious thermal stimulation but not by mechanical or electrical stimulation. Fentanyl reduced the responses to either stimuli with lower potency in the presence of idazoxan, but only in animals with inflammation. Its dose–response curve was shifted to the right between 1.8- and 3.5-fold depending on the stimulus used. It is concluded that the increase of thermal responses by idazoxan in animals with inflammation is probably due to changes in the peripheral blood flow. Nevertheless, since an interaction with μ-opioids is clear in inflammation, endogenous α₂-adrenergic systems play an important role in the modulation of the effectiveness of opioids during inflammation.     

Effects of intrathecal ACTH on opiate A algesia in the rat

The effects of administration of opiates and ACTH onto the lumbar spinal cord of rats were investigated. ACTH, morphine and β-endorphin were administered to the lumbar spinal cord via chronically implanted spinal catheters. Effects on nociceptive thresholds using the tail-flick test were observed. Intrathecal morphine and β-endorphin caused analgesia whereas effects of intrathecal ACTH were variable. About half the animals showed small increases in tail-flick latency with intrathecal ACTH, the other half showing either decreases in tail-flick latency or no effect. In contrast, intrathecal ACTH consistently reversed the analgesia caused by either intrathecal morphine or β-endorphin. The results are discussed in terms of interactions between ACTH and opiates.     

Role of opioid receptors (μ, δ1, δ2) in modulating responses of nociceptive neurons in the superficial and deeper dorsal horn of the medulla (trigeminal nucleus caudalis) in the rat

This report describes the effects of intravenously administered agonists and antagonists at μ-, δ₁- and δ₂-opioid receptors on the Aδ- and C-fiber-evoked responses of trigeminal nociceptive neurons in anesthetized rats. Extracellular single unit recordings were made from 61 nociceptive neurons (23 NS, 38 WDR) in the superficial and 37 nociceptive neurons (3 NS, 34 WDR) in the deeper dorsal horn of the medulla (trigeminal nucleus caudalis). Administration of either the δ₁-receptor agonist [

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-Pen^2,5]enkephalin (DPDPE; 0.05–2 mg/kg), the δ₂-receptor agonist [

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-Ala²,Glu⁴]deltorphin (DELT; 1–2 mg/kg) or the μ-receptor agonist [

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-Ala²,N-MePhe⁴,Gly⁵-ol]enkephalin (DAMGO; 0.05–1 mg/kg) inhibited the Aδ- and C-fiber-evoked responses of nociceptive neurons in the superficial and deeper dorsal horn. The inhibitory effect was more pronounced on the C-fiber-evoked responses than on the Aδ-fiber-evoked responses. In other neurons, DPDPE also produced facilitation, or inhibition followed by facilitation, or differential effects (inhibition of the C-fiber-evoked responses and facilitation of the Aδ-fiber-evoked responses) on the Aδ- and C-fiber-evoked responses. The effects of DPDPE were antagonized by 7-benzylidenenaltrexone (BNTX, 0.4–1 mg/kg), a δ₁-receptor antagonist, in 88% (7/8) of neurons. Naltriben (NTB, 0.7–1 mg/kg), a δ₂-receptor antagonist, antagonized the effects of both DELT and DPDPE. A smaller dose of NTB (0.3 mg/kg), which failed to reverse the effects of DPDPE in 100% (4/4) of neurons, effectively antagonized the effects of DELT in 100% (6/6) of neurons. The inhibitory action of DAMGO was completely antagonized by naloxone (0.2 mg/kg) in 100% (6/6) of neurons. The results of the present investigation suggest that: (1) μ-, δ₁- and δ₂-opioid receptors play an important role in the inhibitory modulation of the Aδ- and C-fiber-evoked responses of nociceptive neurons in the superficial and deeper dorsal horn of the medulla; (2) 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 (3) NTB, in a limited dose range, can discriminate between δ₁- and δ₂-opioid receptor subtypes.