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The present study was designed to characterize the antinociception produced by the administration of a muscarinic agonist, (+)-cis-methyldioxolane, into the rostral ventral medulla (RVM) of male Sprague-Dawley rats. Seven days after the implantation of 25-gauge stainless-steel guide cannulae, animals were injected with graded doses of (+)-cis-methyldioxolane, and antinociception was assessed by using the 52 degrees C hot-plate and tail-flick tests in a single-blind design. (+)-cis-Methyldioxolane produced dose-related hot-plate and tail-flick antinociception for 30 to 45 min peaking 5 to 10 min after RVM injection. The ED50 values of (+)-cis-methyldioxolane in the hot-plate and tail-flick tests were 2.4 and 1.7 nmol, respectively. Five-minute preinjections with 0.35 nmol of the muscarinic M1 receptor blocker pirenzepine competitively antagonized the antinociception produced by (+)-cis-methyldioxolane. The antinociception produced by RVM injections of the muscarinic M2 receptor blocker methoctramine was additive to the antinociceptive effects of (+)-cis-methyldioxolane when the two antinociceptive effects of (+)-cis-methyldioxolane when the two agents were combined. Twenty four-hour pretreatment of the RVM with the irreversible muscarinic receptor antagonist 4-diphenylacetoxy-N-[2-chloroethyl]-piperidine mustard blocked the antinociceptive effects of (+)-cis-methyldioxolane completely. Administration of 6 nmol of the nitric oxide synthase inhibitor L-NG-nitroarginine into the RVM competitively antagonized the antinociception produced by (+)-cis-methyldioxolane and (+)-muscarine in the hot-plate and tail-flick tests. Pretreatment with 100 nmol of L-arginine, but not D-arginine, significantly reversed the inhibitory effects of L-NG-nitroarginine on (+)-cis-methyldioxolane-produced antinociception. Pretreatment with 100 nmol of the guanylyl cyclase inhibitor methylene blue into the RVM profoundly antagonized the antinociception produced by (+)-cis-methyldioxolane. Neither buffer L-NG-nitroarginine, L-arginine-D-arginine or methylene blue altered hot-plate or tail-flick nociception when injected alone into the RVM. In contrast, either dibutyryl cyclic GMP or 8-bromo cyclic GMP, membrane-permeable cyclic GMP analogs, produced hot-plate and tail-flick antinociception when injected alone into the RVM. These data are consistent with the hypothesis that the antinociception produced by muscarinic stimulation of the RVM is mediated by an L-arginine/nitric oxide/cyclic GMP cascade.

This study was designed to determine if the antinociception produced by intrathecally (i.t.) administered muscarinic agonists in male Sprague-Dawley rats is mediated by an L-arginine/nitric oxide/cyclic GMP cascade. Seven days after implantation of intrathecal catheters, antinociception was produced with graded doses of (+)-cis-methyldioxolane (CD). The ED50 values for CD in the hot-plate and tail-flick tests were 2.6 and 2.0 nmol, respectively. The corneal and righting reflexes, as well as stepping and negative geotaxic responses, were not affected by CD. Six-minute pretreatment with 50 nmol of the nitric oxide synthase inhibitor L-NG-nitroarginine (NNR) significantly inhibited CD-produced hot-plate and tail-flick antinociception as evidenced by 6-fold shifts to the right of the CD dose-response curves. Coadministration of 150 nmol of L-arginine with NNR reversed the NNR-induced inhibition of the antinociception produced by CD. D-Arginine was without effect. Pretreatment with 500 nmol of the guanylyl cyclase inhibitor methylene blue also antagonized the antinociception produced by CD in both the hot-plate and tail-flick tests. In parallel with CD, coadministration of L-arginine with NMR reversed the NMR-induced inhibition of (+)-muscarine-produced antinociception in both nociceptive tests. Intrathecal administration of buffer, 50 nmol of NNR, 150 nmol of L- or D-arginine or 500 nmol of methylene blue, did not alter nociceptive responses when injected alone. In contrast, i.t. administration of the membrane-permeable cyclic GMP analogs, dibutyryl cyclic GMP and 8-bromo cyclic GMP, produced antinociception in both nociceptive tests when injected alone.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of spinal mechanisms in subcutaneous (s.c.) nicotine-induced antinociception was examined in male Sprague-Dawley rats using the hot-plate and tail-flick tests. Nicotine (0.125, 0.25, 0.375 or 0.5 mg/kg s.c.) produced a dose-related inhibition of nociception in both tests. Although increasing negative geotaxis response times slightly, no significant alteration of other motor reflexes was observed with 0.375 mg/kg of s.c. nicotine. Microinjection of 7 nmol of the high-affinity choline uptake inhibitor hemicholinium-3 into the rostral ventral medulla completely inhibited the antinociception produced by 0.375 mg/kg of s.c. nicotine. Intrathecal (i.t.) injection of 61 nmol of nicotine (in 10 microliter buffer) produced no changes in hot-plate or tail-flick test response latencies. Nicotine-induced antinociception was blocked by a variety of i.t. antagonists injected 12 min before s.c. injection of 0.375 mg/kg of nicotine. In both tests, i.t. pretreatment with 0.1 mumol (in 10 microliter buffer) of scopolamine, methysergide, yohimbine, idazoxan, mecamylamine or 0.2 mumol of atropine attenuated nicotine-induced increases in test response latencies. Pretreatment with 0.1 mumol of atropine attenuated nicotine-induced increases in tail-flick test, but not in the hot-plate test. Pretreatment with 0.1 mumol of i.t. prazosin or naloxone produced no changes in nicotine-induced increases in test response latencies in either test. These data suggest that the antinociception produced by s.c. nicotine is mediated via a number of sites in the spinal cord, including alpha-2 adrenergic, serotonergic and muscarinic cholinergic.(ABSTRACT TRUNCATED AT 250 WORDS)

The antinociceptive effect of subcortically administered nicotine was investigated in the rat using the hot-plate and tail-flick tests. Adult male Sprague-Dawley rats were implanted with guide cannulas aimed at 185 sites in the forebrain, midbrain and hindbrain. After 1 week, nicotine was injected in 0.5 microliter of 50 mM phosphate buffer, pH 7.4. The pedunculopontine tegmental nucleus (PPTg) of the mesopontine tegmentum and the nucleus raphe magnus (NRM) of the ventral medulla were the most sensitive sites of nicotine-induced antinociception. The median effective doses of nicotine to inhibit hot-plate or tail-flick nociception after PPTg or NRM administration ranged between 1.4 and 3 nmol. The lack of effect of s.c. injections of naloxone on the antinociception induced by nicotine in the PPTg or NRM ruled out endogenous opioid mechanisms. Coadministration of mecamylamine or pirenzepine with nicotine into the NRM competitively antagonized nicotine-induced antinociception. The administration of the muscurinic cholinergic type 2 receptor antagonist methoctramine into the NRM produced a strong antinociceptive response which was blocked by prior treatment of the NRM with hemicholinium-3. Hemicholinium-3 pretreatment of either the PPTg or the NRM antagonized the antinociception induced by nicotine at these sites. Hemicholinium-3 pretreatment of the NRM also antagonized the antinociception produced by s.c. administered nicotine. The antinociceptive effects of nicotine injected in the PPTg were blocked by procainamide injections in the NRM; however, the antinociceptive effects of nicotine injected in the NRM were not blocked by bilateral injections of procainamide in the PPTg. Both lesioning the PPTg with ibotenic acid and pretreating the NRM with hemicholinium-3 abolished completely the antinociception induced by nicotine or (+)-cis-dioxolane microinjections into the PPTg. However, neither ibotenic acid-induced nor electrolytic lesions of the PPTg alone altered CRL. The data support the existence of a tonically active cholinergic pathway which is under autoinhibitory control that originates in the PPTg, terminates in the NRM and modulates nociception by activating descending pain inhibitory systems relaying within the NRM.

The ability of nicotine to induce antinociception after subcortical administration was investigated in the rat. Adult male Sprague-Dawley rats were implanted unilaterally with guide cannulas aimed at the pedunculopontine tegmental nucleus of the mesopontine tegmentum. After 1 week, nicotine was injected in 0.5 microliter of 0.2 M pH 7.4 phosphate buffer. Antinociception was assessed using the 52 degrees C hot-plate test and the tail-flick method; for the most part, the results in the hot-plate test parallelled those in the tail-flick test. Nicotine inhibited nociceptive responses at a median effective antinociceptive dose (A5O) of 1.6 nmol in the hot-plate test and 3.4 nmol in the tail-flick test. Mecamylamine, 0.8 nmol coadministered with nicotine, antagonized nicotine antinociception as evidenced by 5- to 8-fold increases in the nicotine A5O. Nicotine antinociception was also antagonized by coadministrations of either 0.8 nmol of (-)-scopolamine or 0.4 nmol of the M1 antagonist pirenzepine by over 12-fold in the hot-plate test and 5-fold in the tail-flick test. The M2 antagonist methoctramine had antinociceptive effects of its own when injected into the mesopontine tegmentum at a dose of 0.1 nmol; when coinjected with nicotine, the effects of the methoctramine-nicotine combination appeared to be additive. One hour preinjection into the mesopontine tegmentum with 13.5 nmol of (-)-vesamicol, an agent which interferes with acetylcholine storage and/or release, markedly inhibited nicotine antinociception; only a 24% antinociceptive response could be elicited by nicotine in the hot-plate test whereas the nicotine A5O was increased 3-fold in the tail-flick test. Pretreatment with the inactive isomer (+)-vesamicol had no effect. In other experiments, mesopontine tegmental injection of (+)-cis-dioxolane, a high affinity muscarinic cholinergic agonist, elicited strong antinociceptive responses which were potently antagonized by coadministration with 0.5 nmol of pirenzepine but not by 0.8 nmol of mecamylamine. The data indicate that nicotine-induced antinociception may depend upon intact neurotransmission at M1 sites in addition to nicotinic sites within the mesopontine tegmentum.

In mice implanted with a morphine pellet for 3 days and subsequently withdrawn for various periods of time, a single injection of morphine results in a rapid and marked sensitization of abstinent mice to naloxone-induced jumping behavior when compared to abstinent mice pretreated with saline or to placebo-withdrawn mice given a single morphine injection. Maximum sensitization of abstinent mice occurs at morphine doses of about 10 to 30 mg/kg and peaks at 0.5 to 2 hours after morphine administration. The ability of morphine to sensitize abstinent mice to naloxone declines with time after pellet removal and appears to consist of two components, one with a short T1/2 and one with a much longer T1/2. Abstinent mice are also sensitized to naloxone by levorphanol and methadone, but not by dextrorphan. It is proposed that the administration of a narcotic drug to abstinent mice uncovers a latent, pre-existing state of physical dependence and restores the responsiveness of this state to naloxone.

Unilateral lesions of the substantia nigra zona compacta (SNC) in rats were produced by electrolytic coagulation or by an injection of 6-hydroxydopamine. Two to 5 weeks later, after being preselected for amphetamine-induced ipsilateral circling behavior, the animals were administered narcotic agonists or antagonists and their circling behavior was observed. Morphine, methadone, levorphanol, nalorphine or pentazocine induced ipsilateral circling movements; both naloxone and dextrorphan were without effect. Ipsilateral circling was also observed in rats with unilateral electrolytic lesions after administration of agents that are thought to enhance central dopaminergic activities: d-amphetamine, l-dopa and apomorphine. In rats with unilateral electrolytic or 6-hydroxydopamine SNC lesions that were rendered highly morphine-dependent by multiple-morphine pellet implantation, contralateral (C) circling behavior was observed within 1 to 2 minutes after a naloxone challenge; the onset and duration of C circling behavior coincided with the initial appearance and duration of precipitated-morphine withdrawal signs. C circling was also observed after administration of putative dopamine receptor blockers, haloperidol and pimozide in rats with either unilateral electrolytic or 6-hydroxydopamine SNC lesions. Morphine pretreatment diminished both the C circling intensity and the appearance of withdrawal signs observed after a naloxone challenge in morphine-dependent, SNC-lesioned rats. The naloxone-precipitated withdrawal in unilaterally lesioned morphine-dependent rats was accompanied by a 20% elevation of neostriatal dopamine in the intact side. In contrast to the effects of a chronic SNC lesion in decreasing neostriatal dopamine, a 77% increase was observed in the lesioned side 30 minutes after electrolytic coagulation. Thus, narcotic agonists and partial agonists may enhance central dopaminergic activities and naloxone-precipitated withdrawal may involve a diminution in central dopaminergic activities of the nigroneostriatal pathway.

In mice rendered morphine-dependent by pellet implantation for 3 days, the administration of pargyline 6 hours after pellet removal intensified narcotic abstinence behavior, particularly the narcotic withdrawal jumping response. Pargyline, 75 mg/kg i.p., caused a 6- to 9-fold increase in the incidence of jumping in mice withdrawing from morphine 6 hours after removal of the pellet, whereas this effect was not observed: 1) 1 hour after the injection of pargyline or 2) in animals still implanted with the morphine pellet. The median effective dose (ED50) of pargyline required to elicit withdrawal jumping in mice implanted with morphine decreased with increasing physical dependence. The ED50 for 72 hours was about one-sixth that after 24 hours of implantation. Additionally, pargyline potentiated naloxone-precipitated withdrawal jumping as evidenced by a reduction of the naloxone ED50 by approximately one-half. Administration of other monoamine oxidase inhibitors such as pheniprazine, iproiazid or tranylcypromine failed to alter the indicence of jumping in dependent mice undergoind abrupt morphine with drawal. Further, dopamine receptor stimulation by amphetamine, pheniprazine or amantadine antagonized the pargyline-induced jumping response. These data suggest that the increased incidence of withdrawal jumping observed after pargyline in morphine-dependent mice is not related to monoamine oxidase inhibition but rather to a possible pargyline-induced decrease in dopaminergic activity.

The present study was designed to characterize the antinociception produced by the administration of a potent muscarinic agonist into the intrathecal space of the lumbar spinal cord of male Sprague-Dawley rats. Seven days after surgical implantation of intrathecal catheters, animals were injected with graded doses of (+)-cis-methyldioxolane. (+)-cis-Methyldioxolane produced hot-plate and tail-flick antinociception for up to 90 min, peaking 5 to 30 min after injection. The dose of (+)-cis-methyldioxolane that inhibited nociception by 50% was 12 nmol in both the hot-plate and tail-flick tests. This antinociception was not accompanied by a general depression of other spontaneous motor responses. The tissue concentration of (+)-cis-methyl-dioxolane in the lumbar spinal cord present at the time of maximal hot-plate and tail-flick antinociception was approximately 12 microM. In similarity to (+)-cis-methyldioxolane, intrathecally administered (+)-muscarine also produced strong hot-plate and tail-flick antinociceptive responses. In contrast, intrathecally administered N-methylcarbachol, a nicotinic agonist, had no effect on nociception. Five-minute pretreatment with graded doses of pirenzepine, methoctramine, idazoxan, LY53857, or S-(-)-zacopride each significantly antagonized hot-plate and tail-flick antinociception produced by 37.5 nmol of (+)-cis-methyldioxolane in a dose-related manner with median effective antagonist doses in the range of 0.4 to 2.2 nmol. Intrathecal pretreatment with graded doses of prazosin or naloxone enhanced the antinociception produced by (+)-cis-methyldioxolane in the tail-flick but not the hot-plate tests. Intrathecal vehicle, S(-)-propranolol or mecamylamine did not alter (+)-cis-methyldioxolane-induced antinociception. The data suggest that the antinociceptive responses produced by intrathecally administered (+)-cis-methyldioxolane involve the stimulation of muscarinic M1 and/or M2 cholinergic receptors, and may also involve activation of alpha-2 adrenergic, 5-hydroxytryptamine1c/2 and 5-hydroxytryptamine3 serotonergic receptor systems at the level of the lumbar cord.

The present study was designed to determine the degree of participation of opioid, noradrenergic, serotonergic and cholinergic systems of the lumbar spinal cord in the antinociception produced by nicotinic stimulation of the pedunculopontine tegmental nucleus (PPTg) or nucleus raphe magnus (RMg). Adult, male Sprague-Dawley rats were implanted subcortically with 25-gauge cannulas into the PPTg or RMg. The animals were also implanted with intrathecal catheters terminating in the subarachnoid space just rostral to the lumbar enlargement. Seven days after surgery, animals were injected with 15 to 100 nmol of various receptor antagonists intrathecally 10 min before microinjections of 40 nmol of N-methylcarbachol (NMC) into the PPTg or RMg. Doses of antagonists were chosen which did not induce motor disturbances and did not alter hot-plate or tail-flick nociception when administered alone. NMC produced hot-plate and tail-flick antinociception for 20 to 25 min, peaking 5 to 10 min after either PPTg or RMg microinjection. Intrathecal administration of 50 nmol of idazoxan, 100 nmol of S-(-)-propranolol, 20 nmol of LY53857, 25 nmol of S-(-)-zacopride, 100 nmol of pirenzepine or 50 nmol of methoctramine each antagonized in part the antinociception produced by PPTg or RMg microinjections of 40 nmol of NMC. Intrathecal administration of 100 nmol of naloxone or 100 nmol of prazosin enhanced the antinociceptive effects of NMC. Intrathecal vehicle or 100 nmol of mecamylamine did not alter NMC-induced antinociception. Complete antagonism of PPTg-administered NMC antinociception was achieved only when animals were pretreated intrathecally with combinations of at least three of the following four antagonists, 20 nmol of S-(-)-zacopride, 15 nmol of LY53857, 25 nmol of idazoxan and 50 nmol of methoctramine. The data suggest that the antinociceptive responses produced by nicotinic stimulation of the PPTg or RMg have similar pharmacologic profiles, and are redundantly mediated via alpha-2 adrenergic, 5-HT1c/2 and 5-HT3 serotonergic, and M2 cholinergic receptor interactions in the lumbar spinal cord.