The antinociceptive action of an α2-adrenoceptor agonist in the spinal dorsal horn is due to a direct spinal action and not to activation of Descending Inhibition

In this electrophysiological study we tried to find out whether the spinal antinociceptive effect of a supraspinaly administered α₂-adrenoceptor agonist is due to a direct spinal effect or to activation of descending inhibition. The responses to wide-dynamic range (WDR) neurons of the spinal dorsal horn were studied following application of medetomidine, a selective α₂-adrenergic agonist, into the rostroventromedial medulla (RVM) or directly onto the spinal cord of the Intact and in spinal rats. The noxious electrical stimuli were applied to the ipsilateral receptive field in the plantar region of the hind paw, and responses mediated by A- and C-fibers to WDR neurons were separately evaluated. The reversal of medetomidine-induced effects was attempted by a systemic administration of atipamezole, a selective α₂-adronoceptor antagonist. Medetomldine injection into the RVM produced a dose-dependent, atipamezole-reversible attenuation of the C-fiber-mediated responses to WDR neurons of the spinal dorsal horn in both intact and spinal rats. Paradoxically, the spinal aMFnociceptive effect of supraspinally administered medstomidine was stronger in spinal rats. The A-fiber-mediated responses were significantly less attenuated by medetomidine than the C-fiber-mediated responses to the WDR neurons. Also a direct application of medetomidine onto the spinal cord produced a dose-dependent, atipamezole-reversible attenuation of the C-fiber-mediated responses, and this effect was identical in intact and in spinal rats. The medetomidins doses producing spinal antinociception were considerably lower with a direct spinal application than with a supraspinal application. These results indicate that spinal antinocicsption following spinal or supraspinal application of an α₂-adrenergic agonist is due to a direct activation of spinal α₂-adrenoceptors and not to descending inhibition. Activation of supraspinal α₂-adrenoceptors counteracts the spinal antinociceptive effect.     

Effect of α2 adrenergic agents upon central etorphine antinociception in the cat

Systemic (s.c.) administration of α₂ agonists clonidine (25–100 μg/kg) or guanfacine (50–400 μg/kg) elicited antinociception as assessed by the cat tail-flick model and potentiated in a dose-dependent manner the antinociceptive effect of etorphine (2.5 μg) administered directly into the periaqueductal gray. Conversely, systemic yohimbine (1 mg/kg) attenuated the effects of central etorphine, and diminished potentiation of etorphine by theα₂ agonists. Prior microinjection of clonidine (5μg) or guanfacine (5 μg) into the locus coeruleus (LC) reduced the intensity of central etorphine antinociception whereas central yohimbine (20 μg) pretreatment increased peak antinociceptive activity and prolonged the duration of etorphine. Thus, systemicα₂ agonists are inherently antinociceptive and potentiate central narcotic antinociception; however, the site of interaction betweenα₂ agonists and opiates does not appear to be the LC inasmuch asα₂ agonists attenuate the antinociceptive effect of etorphine when administered directly into the LC. A spinal site of action is suggested based upon known LC-spinal projections and our experimental observations.     

Antinociception produced by microinjection of morphine in the rat periaqueductal gray is enhanced in the foot, but not the tail, by intrathecal injection of α1-adrenoceptor antagonists

Antinociception produced by microinjection of morphine in the ventrolateral periaqueductal gray is mediated in part by α₂-adrenoceptors in the spinal cord dorsal horn. However, several recent reports demonstrate that microinjection of morphine in the ventrolateral periaqueductal gray inhibits nociceptive responses to noxious heating of the tail by activating descending neuronal systems that are different from those that inhibit the nociceptive responses to noxious heating of the feet. More specifically, α₂-adrenoceptors appear to mediate the antinociception produced by morphine using the tail-flick test, but not that using the foot-withdrawal or hot-plate tests. The present study extended these findings and determined the role of α₁-adrenoceptors in mediating the antinociceptive effects of morphine microinjected into the ventrolateral periaqueductal gray using both the foot-withdrawal and the tail-flick responses to noxious radiant heating in lightly anesthetized rats. Intrathecal injection of selective antagonists was used to determine whether the antinociceptive effects of morphine were modulated by α₁-adrenoceptors. Injection of the selective α₁-adrenoceptor antagonists prazosin or WB4101 potentiated the increase in the foot-withdrawal response latency produced by microinjection of morphine in the ventrolateral periaqueductal gray. In contrast, either prazosin or WB4101 partially reversed the increase in the tail-flick response latency produced by morphine. These results indicate that microinjection of morphine in the ventrolateral periaqueductal gray modulates nociceptive responses to noxious heating of the feet by activating descending neuronal systems that are different from those that inhibit the nociceptive responses to noxious heating of the tail. More specifically, α₁-adrenoceptors mediate a pro-nociceptive action of morphine using the foot-withdrawal response, but in contrast, α₁-adrenoceptors appear to mediate part of the antinociceptive effect of morphine determined using the tail-flick test.     

Antinociception produced by systemic R(+)-baclofen hydrochloride is attenuated by CGP 35348 administered to the spinal cord or ventromedial medulla of rats

This study examined the sites in the central nervous system at which subcutaneously-administered R(+) - baclofen hydrochloride (baclofen), the most active isomer of this prototypic γ-aminobutyric acid (GABAB receptor agonist, acts to produce antinociception in the rat. To determine whether baclofen acts in the spinal cord, either saline or the GABAB receptor antagonist CGP 35348 was injected intrathecally in rats pretreated 24 min earlier with 1 or 3 mg/kg s.c. baclofen. Intrathecal (i.t.) injection of 3 or 10 μg of CGP 35348 antagonized the increase in tail-flick and hot-plate latency produced by either dose of baclofen. To determine whether baclofen acts at sites in the ventromedial medulla (VMM), either saline or CGP 35348 was microinjected in the nucleus raphe magnus or nucleus reticularis gigantocellularis pars a of rats pretreated 24 min earlier with 1 or 3 mg/kg s.c. baclofen. Microinjection of 0.5 or 3 μg of CGP 35348 at sites in the VMM produced at best only a very modest attenuation of the antinociceptive effects of baclofen. These data suggest that systemically-administered baclofen acts at sites in both the spinal cord and the VMM, but that its antinociceptive effects are likely to be mediated to a greater extent by a spinal, rather than medullary site of action. However, a definitive comparison of the relative contribution of GABAB receptors in these two regions is precluded by differences in the diffusion and concentrations of the antagonist in the spinal cord and brainstem. Finally, microinjection of 0.5 or 3.0 μg of CGP 35348 in the nucleus raphe magnus or nucleus reticularis gigantocellularis pars a of saline-pretreated rats did not alter tail-flick or hot-plate latency. This finding suggests that, unlike GABA_A receptors, GABAB receptors do not mediate the tonic GABAergic input to neurons in these nuclei.     

Microinjection of carbachol in the lateral hypothalamus produces opposing actions on nociception mediated by α1- and α2-adrenoceptors

Electrical stimulation of the lateral hypothalamus (LH) produces antinociception partially blocked by intrathecal α-adrenergic antagonists, but the mechanism underlying this effect is not clear. Evidence from immunological studies demonstrates that substance P-immunoreactive neurons in the LH project near the A7 catecholamine cell group, a group of noradrenergic neurons in the pons known to effect antinociception in the spinal cord dorsal horn. Such evidence suggests that LH neurons may activate A7 neurons to produce antinociception. To test this hypothesis, the cholinergic agonist carbachol was microinjected into the LH at doses of 63, 125 and 250 nmol and the resulting effects on tail-flick and nociceptive foot-withdrawal latencies were measured. All three doses significantly increased response latencies on both tests, with the 125-nmol dose providing the optimal effect. Intrathecal injection of the opioid antagonist naltrexone (97 nmol) partially reversed antinociception, but neither the α₂-adrenoceptor antagonist yohimbine nor the α₁-adrenoceptor antagonist WB4101 altered latencies. However, two sequential doses of yohimbine blocked LH-induced antinociception on both tests. In contrast, two sequential doses of WB4101 increased nociceptive responses on both the tail-flick and foot-withdrawal tests. These findings, and those of published reports, suggest that neurons in the LH activate spinally projecting methionine enkephalin neurons, as well as two populations of A7 noradrenergic neurons that exert a bidirectional effect on nociception. One of these populations increases nociception through the action of α₁-adrenoceptors and the other inhibits nociception through the action of α₂-adrenoceptors in the spinal cord dorsal horn.     

Differential influence of two serotonin 5-HT3 receptor antagonists on spinal serotonin-induced analgesia in rats

We tested the antinociceptive effect of intrathecal (i.t.) administration of 5-HT and the 5-HT₃ receptor agonist, 1-(m-chlorophenyl)-biguanide (mCPBG), in rats submitted to a mechanical noxious stimulus and the influence of the 5-HT₃ receptor selective antagonists, tropisetron and granisetron. Both 5-HT and mCPBG (0.01, 0.1, 1, 10, 20 μg/rat) produced a significant dose-dependent antinociception. The lowest active doses were 0.1 and 1 μg for 5-HT and mCPBG, respectively. The effect, observed with 20 μg, was significantly lower with mCPBG (+33±6%) than with 5-HT (+63±7%). For 5-HT-induced antinociception, the minimal inhibitory doses were 0.001 μg/rat for tropisetron and 10 μg/rat for granisetron. In contrast, the same doses of the two antagonists (from 0.1 μg/rat) similarly inhibited the effect of mCPBG. This study provides evidence that contrary to tropisetron, doses of granisetron able to inhibit the effect of a 5-HT₃ receptor agonist failed to reduce that of 5-HT. This demonstrates a heterogeneity between 5-HT₃ receptor antagonists and questions the true involvement of these receptors in spinal 5-HT-induced antinociception.     

Evidence for central α2-adrenergic modulation of rat pineal melatonin synthesis

This study was performed to distinguish central and peripheral α₂-adrenoceptors in the inhibition of rat pineal melatonin synthesis. The rats received lipo- or hydrophilic α₂-adrenoceptor ligand injections at middark; after 1 or 2 h the pineal melatonin contents were measured. The lipophilic agonist medetomidine (100 μg/kg s.c.) suppressed the melatonin contents significantly, while the hydrophilic agonists ST-91 and p-aminoclonidine (10 or 100 μg/kg i.v.) did not. The suppression by medetomidine was counteracted by the lipophilic antagonist yohimbine (0.3–3.0 mg/kg i.p.) but not by the hydrophilic antagonist L-659,066 (1–10 mg/kg i.v.). In conclusion, the suppression of nocturnal melatonin synthesis by α₂-adrenoceptor agonists is mainly of central origin.     

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.     

Antinociceptive effect of ketanserin in mice: involvement of supraspinal 5-HT2 receptors in nociceptive transmission

The involvement of 5-HT₂ receptors in pain transmission was investigated in mice. Subcutaneous administration of the selective 5-HT₂ receptor antagonist ketanserin produced dose-dependent antinociception in the hot-plate and acetic acid-induced writhing tests withED₅₀ values (95% confidence limit) of 1.51 (1.13–1.89) and 0.62 (0.10–1.40) mg/kg, respectively, but was without any significant effect on the tail-flick test. Pretreatment with the catecholamine depletors 6-hydroxydopamine (2.5 μg, i.c.v.) orα-methyl-p-tyrosine (200 mg/kg, s.c.), or the serotonin synthesis inhibitorp-chlorophenylalanine methylester (200 mg/kg, s.c.), resulted in a significant decrease in the antinociceptive effect of ketanserin. Likewise, intrathecal (i.t.) administration of 1 μg/mouse of idazoxan (anα₂-antagonist), methysergide (mixed 5-HT₁, and 5-HT₂ antagonist) or ketanserin also reversed the antinociceptive effect of s.c. administered ketanserin. The results of this work indicate that 5-HT₂ receptors located supraspinally may inhibit descending nociceptive neurotransmission. In addition, these studies suggest that 5-HT₂ receptors located at the spinal level modulate nociception.     

Intrathecal GABAB antagonists attenuate the antinociception produced by microinjection ofl-glutamate into the ventromedial medulla of the rat

This study examined whether the antinociception produced by glutaminergic stimulation of neurons in the nucleus raphe magnus (NRM) and nucleus reticularis gigantocellularis pars α (NGCpα) is mediated by an action of GABA at GABA_B receptors in the spinal cord. Rats were pretreated with intrathecal (i.t.) administration of the selective GABA_B receptor antagonists phaclofen (100 μg) or CGP 35348 (30 μg), the serotonin receptor antagonist methysergide (30 μg), or vehicle. Fifteen min later, 30 nmoll-glutamate was microinjected into the NRM, NGCpα, or sites in the medulla outside these two regions. Microinjection ofl-glutamate into the NRM or NGCpα in vehicle-pretreated rats significantly increased tail flick latency. This increase was antagonized, but not abolished, by i.t. pretreatment with 30 μg CGP 35348 or 100 μg phaclofen. Pretreatment with 30 μg methysergide completely antagonized the antinociception produced byl-glutamate. Microinjection ofl-glutamate at medullary sites outside the NRM or NGCpα did not produce antinociception. In an ancillary experiment, the possibility that the ability of methysergide, phaclofen or CGP 35348 to antagonize glutamate-induced antinociception was related to non-specific increases in tail skin temperature was explored. Although phaclofen or methysergide increased tail skin temperature, the magnitude and time course of this increase were not consistent with the antagonism of glutamate-induced antinociception. Moreover, administration of CGP 35348 resulted in a modest decrease in tail skin temperature. Thus, antagonism of glutamate-induced antinociception does not appear to result from non-specific alterations in tail skin temperature. Taken together, these results indicate that the antinociception produced by activation of neurons in the NRM and NGCpα is at least partially mediated by GABA_B receptors 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.     

Spinal monoamine mediation of stimulation-produced antinociception from the lateral hypothalamus

Stimulation-produced antinociception can be evoked from a wide variety of sites in the brain, including the lateral hypothalamus (LH). The present study, in rats lightly anesthetized with pentobarbital, examined descending inhibition of the nociceptive tail flick (TF) reflex produced by focal electrical stimulation in the LH and the neurotransmitter(s), at the level of the lumbar enlargement, mediating the inhibition. Systematic tracking studies demonstrated that stimulation in the diencephalon dorsal to the hypothalamus did not reliably inhibit the TF reflex. Inhibition of the TF reflex was produced, however, throughout the hypothalamus at intensities of stimulation typically between 50 and 200 μA. The area requiring low intensities of stimulation (50–100 μA) to inhibit the TF reflex was a diffuse region of the LH, inferior to the mammillothalamic tract and internal capsule, medial to the supraoptic decussation and including the medial forebrain bundle. Microinjections of S-glutamate (100 mM, 0.5μl) in the LH did not inhibit the TF reflex, suggesting that activation of fibers of passage by stimulation was responsible for inhibition of the TF reflex produced from the LH. The intrathecal administration of pharmacologic antagonists (15–30 μg; naloxone, methysergide, phentolamine, prazosin or yohimbine) revealed that the α-adrenoceptor antagonists phentolamine and yohimbine produced the greatest increases in stimulation thresholds in the LH for inhibition of the TF reflex (83.7% and 89.8%, respectively). The intrathecal administration of methysergide produced a lesser, but statistically significant 11% increase in the stimulation threshold for inhibition of the TF reflex. These results indicate that spinal α₂-adrenoceptors primarily mediate the descending inhibition of the TF reflex produced by electrical stimulation in the LH.     

A role for presynaptic α2-adrenoceptors in angiotensin II-induced drinking in rats

Studies from this laboratory have shown that either central or peripheral administration of clonidine, the α₂-adrenoceptor agonist, can attenuate a variety of dipsogenic stimuli in rats. Further, yohimbine and tolazoline, α₂-adrenoceptor antagonists, augment the drinking response to both peripherally administered isoproterenol and angiotensin II. Studies reported here establish a dose-inhibition relationship between the dose of clonidine administered (2 to 32 μg/kg) intracerebroventricularly (IVT) and inhibition of the drinking response to peripherally administered angiotensin II (200 μg/kg, SC). DI₅₀ was approximately 4 μg/kg. Yohimbine (300 μg/kg, SC) reversed the antidipsogenic effect of centrally administered clonidine (32 μg/kg, IVT) on angiotensin II-induced (200 μg/kg, SC) water intake. Phenylephrine, an α₂-adrenoceptor agonist, administered IVT (40 and 80 μg/kg) also inhibited angiotensin II-induced drinking in a dose-related fashion. The antidipsogenic effect of phenylephrine (80 μg/kg) was blocked by administration of yohimbine (300 μg/kg, SC). Thus, this effect of phenylephrine most likely occurs by way of α₂-adrenoceptors. These results support a role for the pre-synaptic α₂-adrenoceptor in the mediation of drinking in rats. Activation of α₂-adrenoceptors is accompanied by reduced water intake while inhibition of these receptors enhances water intake.     

Effects of intracerebroventricularly administered chimeric peptide of metenkephalin and FMRFa—[D-Ala]YFa—on antinociception and its modulation in mice

An enzymatically stable analog of YGGFMKKKFMRFamide (YFa), a chimeric peptide of metenkephalin and FMRFa, was synthesised. The antinociceptive effects of intracerebroventricular injections of this analog—[D-Ala²]YAGFMKKKFMRFamide ([D-Ala²]YFa)—was then investigated using the mouse radiant-heat tail-flick test. [D-Ala²]YFa produced modest to good antinociception at 1, 2, and 5 μg/mouse (0.64, 1.28, and 3.22 nmol, respectively). This antinociceptive effect was completely reversed by the opioid receptor antagonist naloxone (1.5 μg/mouse: 4.12 nmol, intracerebroventricular [i.c.v.]), administered 5 min prior. Pretreatment (5 min) with either neuropeptides FF (1 μg/mouse: 0.92 nmol, i.c.v.) or FMRFa (1 μg/mouse: 1.69 nmol, i.c.v.) significantly attenuated the antinociceptive effects induced by [D-Ala²]YFa (1 μg/mouse, i.c.v.). Intracerebroventricular administration of [D-Ala²]YFa at 1 μg/mouse dose with morphine (2 μg/mouse: 5.86 nmol, i.c.v.) produced an additive antinociceptive effect, suggesting that [D-Ala²]YFa may have a modulatory effect on opioid (morphine) analgesia. These results provide further support for a role of such amphiactive sequences in antinociception and its modulation.     

Serotonergic neurotransmission in the dorsal raphe nucleus recruits in situ 5-HT(2A/2C) receptors to modulate the post-ictal antinociception

The post-ictal immobility syndrome is followed by a significant increase in the nociceptive thresholds in animals and humans. The aim of this study was to assess the involvement of the dorsal raphe nucleus (DRN) in the post-ictal antinociception. The second aim was to study the role of serotonergic intrinsic mechanisms of the DRN in this hypo-algesic phenomenon. Pentylenetetrazole (PTZ), an ionophore GABA-mediated Cl⁻ influx antagonist, was peripherally used to induce tonic–clonic seizures in Wistar rats. The nociceptive threshold was measured by the tail-flick test. Neurochemical lesions of the DRN, performed with microinjection of ibotenic acid (1.0 μg/0.2 μL), caused a significant decrease of tonic–clonic seizure-induced antinociception, suggesting the involvement of this nucleus in this antinociceptive process. Microinjections of methysergide (1.0 and 5.0 μg/0.2 μL), a non-selective serotonergic receptor antagonist, into DRN caused a significant decrease in the post-ictal antinociception in seizing animals, compared to controls, in all post-ictal periods presently studied. These findings were corroborated by microinjections of ketanserin (at 1.0 and 5.0 μg/0.2 μL) into DRN. Ketanserin is an antagonist with large affinity for 5-HT_2A/2C serotonergic receptors, which, in this case, caused a significant decrease in the tail-flick latencies in seizing animals, compared to controls after the first 20 min following tonic–clonic convulsive reactions. These results indicate that serotonergic neurotransmission of the DRN neuronal clusters is involved in the organization of the post-ictal hypo-algesia. The 5-HT_2A/2C receptors of DRN neurons seem to be critically involved in the increase of nociceptive thresholds following tonic–clonic seizures.     

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.     

Opioid antagonists in the periaqueductal gray inhibit morphine and β-endorphin analgesia elicited from the amygdala of rats

In addition to brainstem sites of action, analgesia can be elicited following amygdala microinjections of morphine and μ-selective opioid agonists. The present study examined whether opioid analgesia elicited by either morphine or β-endorphin in the amygdala could be altered by either the general opioid antagonist, naltrexone, the μ-selective antagonist, β-funaltrexamine (BFNA) or theδ₂ antagonist, naltrindole isothiocyanate (Ntii) in the periaqueductal gray (PAG). Both morphine (2.5–5 μg) and β-endorphin (2.5–5 jig) microinjected into either the baso-lateral or central nuclei of the amygdala significantly increased tail-flick latencies and jump thresholds in rats. The increases were far more pronounced on the jump test than on the tail-flick test. Placements dorsal and medial to the amygdala were ineffective. Naltrexone (1–5 μg) in the PAG significantly reduced both morphine (tail-flick: 70–75%; jump: 60–81%) and β-endorphin (tail-flick: 100%; jump: 93%) analgesia elicited from the amygdala, indicating that an opioid synapse in the PAG was integral for the full expression of analgesia elicited from the amygdala by both agonists. Both BFNA (68%) and Ntii (100%) in the PAG significantly reduced morphine, but not β-endorphin analgesia in the amygdala on the tail-flick test. Ntii in the PAG was more effective in reducing morphine (60%) and β-endorphin (79%) analgesia in the amygdala on the jump test than BFNA (15–24%). Opioid agonist-induced analgesia in the amygdala was unaffected by opioid antagonists administered into control misplacements in the lateral mesencephalon, and the small hyperalgesia elicited by opioid antagonists in the PAG could not account for the reductions in opioid agonist effects in the amygdala. These data indicate that PAGδ₂ and to a lesser degree, μ opioid receptors are necessary for the full expression of morphine and β-endorphin analgesia elicited from the amygdala.     

Depression of C fiber-evoked activity by intrathecally administered reactive red 2 in rat thalamic neurons

To investigate the possible role of spinal purinoceptors in nociception, the potent P₂-purinoceptor antagonist reactive red 2 was studied in rats under urethane anesthesia in which nociceptive activity was elicited by electrical stimulation of afferent C fibers in the sural nerve and recorded from single neurons in the ventrobasal complex of the thalamus. Intrathecal (i.t.) application of reactive red 2 (6–200 μg) caused a dose-dependent reduction of the evoked activity in thalamic neurons. The estimated ED₅₀ was 30 μg, and the maximum depression of nociceptive activity amounted to about 70% of the control activity at a dose of 100 μg. Morphine, administered i.t. at a maximally effective dose (80 μg), inhibited the evoked nociceptive activity by only up to 55% of the control activity. An i.t. co-injection of reactive red 2 (100 μg) and morphine (80 μg) caused a maximum reduction of the evoked thalamic activity by up to 85% of the control activity, thus, exceeding significantly the effect elicited by either drug alone. Similarly, i.t. co-injection of almost equipotent dosages of reactive red 2 (30 μg) and morphine (30 μg) caused a maximum reduction of the evoked activity by up to 72% of the control activity, which again exceeded significantly the effect of either drug alone. The results suggest that in rats reactive red 2 exerts antinociception by blockade of P₂-purinoceptors in the spinal cord and, hence, support the idea that ATP may play an important role in spinal transmission of nociceptive signals. An activation of the spinal opioid system does not seem to contribute to the effect of reactive red 2 but might act additive or even synergistically with its antinociceptive action.     

Antinociceptive effects of isoleucine derivatives of deltorphin I and deltorphin II in rat spinal cord: a search for selectivity of delta receptor subtypes

Deltorphins show a high affinity and selectivity for δ opioid receptors. Analogs of deltorphins with substitution of Val^5,6 residues with more hydrophobic lle^5,6 appear to have a higher in vitro activity and selectivity than parent deltorphins. In our study, changes in the nociceptive threshold after intrathecally injected deltorphin I (DELT I), deltorphin II (DELT II) and their lle^5,6-derivatives (ILE-DELT I and ILE-DELT II, respectively) were investigated in a tail-flick (TF) and a paw pressure (PP) tests. Male Wistars rats (260–350 g) with a chronically implanted catheter in the lumbar enlargement of the spinal cord were used. DELT I and DELT II, injected i.th. in doses of 0.15, 1.5 and 15 μg, increased the TF latency in a dose-dependent manner. The effect of their derivatives was similar, but the action of ILEDELT II was shorter than that of the parent peptide. In the PP test, the antinociceptive effects of DELT I and their derivative ILE-DELT I were similar, but the effect of a higher dose of ILE-DELT I lasted longer in comparison with the parent peptide. Both DELT II and ILE-DELT II exhibited a low and short-lasting antinociceptive potency in the PP test. The effect of DELT 1 (1.5 μg) was antagonized by pretreatment with NTI (30 μg), a non-selective δ opioid receptor antagonist, as well as by the δ2 receptor antagonist NTB (3 μg) and the δ1 antagonist BNTX (1 μg) in both those tests used. The antinociceptive effect of DELT II(1.5 μg) was antagonized by pretreatment with NTI (30 μg) and NTB (3 μg) in the TF test, but not in the PP test. In the latter test, the antinociceptive effect of DELT II was potentiated by pretreatment with BNTX (1 μg). The effects of both the derivatives ILE-DELT I and ILE-DELT II were antagonized by NTl (30 μg) in the TF test, and by NTI (30 μg) and NTB (3 μg) in the PP test. Like in the case of the parent peptide, the effect of ILE-DELT II was potentiated by pretreatment with the δ1 antagonist BNTX (1 μg). Summing up, modification of the DELT I and II by substituting lle^5,6 for Val^5,6 residues appears to influence the δ1 selectivity rather then the potency of the peptides at spinal δ receptors.     

μ-, but not δ-, opioid receptor-mediated antinociception in mice is attenuated by γ-irradiation

Mice were exposed to whole-body irradiation (500 rads) from a ¹³⁷Cs γ-source and tested 2 h later for antinociception (tail-flick test) produced by intracerebroventricular administration of morphine or the more δ-selective opioid peptide, [ -Pen², -Pen⁵]enkephalin (DPLPE). Irradiation significantly attenuated the antinociception produced by morphine, but not by DPLPE. These results demonstrate a differential sensitivity of μ- and δ-opioid receptors to γ-irradiation and, in addition, may be of clinical relevance for cancer patients receiving concurrent radiation therapy and opioid analgesics.