Interactions of morphine with putative neurotransmitters in the mesencephalic reticular formation

Acetylcholine (ACh) and norepinephrine (NE) have been identified previously as putative nociceptive neurotransmitters in the mesencephalic reticular formation (MRF) of the rat because they frequently mimic the change in neuronal firing (usually an increase) evoked by a noxious stimulus (NS). The purpose of this study was to determine if 1.) morphine (M) acts to prevent the increase in firing evoked by a NS by blocking the effects of either of these two neurotransmitters and 2.) if this effect is a specific narcotic effect. Using the technique of microiontophoresis in conjunction with extracellular recording, we located single units in the MRF in which 1.) neuronal firing was accelerated by a NS: 2.) M blocked this response; and 3.) either ACh or NE mimicked the effect of the NS. Neurons meeting these three criteria were studied further to determined if morphine would also block the response to either of the neurotransmitters and if this was a specific narcotic effect. We found that morphine blocked the increase in neuronal firing evoked by the NS and ACh or the NS and NE in over 50% of the cells meeting the above criteria. Some neurons were found in which both ACh and NE mimicked the NS and M blocked all three responses. This blockade of these neurotransmitters was a specific narcotic effect because it could be reversed by the systematic administration of naloxone. These data lead to the tentative hypothesis that M, acting via an opiate receptor, blocks the increase in neuronal firing evoked by a NS by blocking the postsynaptic effects of either ACh or NE. This may be one of the mechanisms by which morphine acts to produce analgesia.     

Putative nociceptive neurotransmitters in the mesencephalic reticular formation

The neurotransmitter(s) involved in the transmission of nociceptive information in the mesencephalic reticular formation (MRF) of the rat have not been identified. Acetylcholine (ACh), substance P (SP), neurotensin (NT), norepinephrine (NE) and dopamine (DA) have all been implicated as putative neurotransmitters involved in nociception. All of these compounds were microiontophoretically administered in the MRF of rats to determine which, if any, mimicked the effects produced by a nociceptive stimulus (foot pinch). This is only one of several criteria that a substance should meet to be considered a nociceptive neurotransmitter in the MRF. ACh and NE mimicked the effects of the nociceptive stimulus in 61% and 67% respectively of the cells tested; NT, DA and SP mimicked the effects of the nociceptive stimulus less frequently (33%, 30%, 23% respectively). Therefore, the nociceptive neurotransmitters in the MRF appear to be ACh and NE; NT, DA and SP may be neurotransmitters with a less important role in nociception in the MRF.     

Calcitonin gene-related peptide: antinociceptive activity in rats, comparison with calcitonin

The effects of synthetic human calcitonin gene-related peptide (CGRP) on nociceptive response were evaluated in rats by two behavioral tests (tail-flick and hot-plate) and by electrophysiological recording of the firing of thalamic neurons evoked by peripheral noxious mechanical stimuli. CGRP was administered intracerebroventricularly (i.c.v.) and its effects were compared with that of salmon calcitonin (sCT). In the tail-flick test, CGRP (0.25, 2.5 and 5 micrograms/rat) dose-dependently increased response latencies, whereas sCT (0.125, 2.5, 5 and 10 micrograms/rat) did not. Conversely, in the hot-plate test CGRP was effective in enhancing response latencies only at the highest dose of 10 micrograms/rat, while sCT (0.125, 0.25 and 2.5 micrograms/rat) inhibited the hot-plate response dose-dependently. In electrophysiological studies, CGRP (2.5 micrograms/rat, i.c.v.) completely inhibited the evoked neuronal thalamic firing and the same dose of sCT induced only a partial reduction. Furthermore, the antinociceptive effects of CGRP in the tail-flick test and in the electrophysiological studies were not prevented by naloxone. These results demonstrate that central administration of CGRP is effective in inhibiting nociceptive responses and its action like that of sCT does not involve an opioid mechanism. The differences in the antinociceptive profiles of CGRP and sCT suggest that the inhibitory effects of these peptides may involve different neuronal pathways.     

Role of endogenous opiates and extracellular K+ accumulation in the inhibition of frog spinal reflexes by electrical skin stimulation

Electrical skin stimulation of the hind limb (10-100 Hz, 30 s-5 min) at the intensity which leads only to the excitation of low threshold afferents depressed (for 1-30 min) the flexor reflex evoked in spinal frogs by nociceptive stimuli. The inhibition, which lasted for longer than 5 min was blocked by naloxone. Short-term poststimulation effects were associated with an increase of extracellular K+ concentration (delta [K]e) and were not blocked by naloxone. Enkephalins or morphine applied to the spinal cord surface increased the threshold for flexor reflexes while naloxone decrease their threshold. The stimulation was followed by short-term hyperpolarization of primary afferents (PAH; 1-5 min) and by depression of dorsal root potentials (DPRs) which had a similar time course to the delta [K]e, and were not blocked by naloxone. This period was frequently followed by longlasting PAH and enhancement of DRPs (5-30 min), which were abolished by naloxone. Superfusion of the isolated spinal cord with opioids produced PAH and enhanced DRPs evoked by nociceptive stimuli, while naloxone or increase of [K] in Ringer solution depolarized primary afferents and depressed DRPs. It is suggested that the antinociceptive effects of electrical stimulation of low threshold cutaneous afferents in spinal frogs involves at least two mechanisms. The short-term effect may result from delta [K]e, especially at high stimulus strength and is equally effective against noxious and non-noxious stimuli. The longlasting effects selectively affecting nociceptive transmission appear to be produced by endogenous opioids.     

Modification of antiallodynic and antinociceptive effects of morphine by peripheral and central action of fluoxetine in a neuropathic mice model

We have previously reported that serotonin concentration was reduced in the brain of mice with neuropathic pain and that it may be related to reduction of morphine analgesic effects. To further prove this pharmacological action, we applied fluoxetine, a selective serotonin reuptake inhibitor, to determine whether it suppressed neuropathic pain and examined how its different administration routes would affect antinociceptive and antiallodynic effects of morphine in diabetic (DM) and sciatic nerve ligation (SL) mice, as models of neuropathic pain. Antiallodynia and antinociceptive effect of drugs were measured by using von Frey filament and tail pinch tests, respectively. Fluoxetine given alone, intracerebroventicularly (i.c.v., 15 microg/mouse) or intraperitoneally (i.p., 5 and 10 mg/kg) did not produce any effect in either model. However, fluoxetine given i.p. enhanced both antiallodynic and antinociceptive effects of morphine. Administration of fluoxetine i.c.v., slightly enhanced only the antiallodynic effect of morphine in SL mice. Ketanserine, a serotonin 2A receptor antagonist (i.p., 1 mg/kg) and naloxone, an opioid receptor antagonist (i.p., 3 mg/kg), blocked the combined antinociceptive effect of fluoxetine and morphine. Our data show that fluoxetine itself lacks antinociceptive properties in the two neuropathy models, but it enhances the analgesic effect of morphine in the periphery and suggests that co-administration of morphine with fluoxetine may have therapeutic potential in treatment of neuropathic pain.     

Magnetic fields inhibit opioid-mediated ‘analgesic’ behaviours of the terrestrial snail,Cepaea nemoralis

1. The terrestrial snail, Cepaea nemoralis, when placed on a warmed surface (40 degrees C) displays a thermal avoidance behaviour that entails an elevation of the anterior portion of the fully extended foot. The latency of this nociceptive response was increased by the prototypical mu and specific kappa opiate agonists, morphine and U-50, 488H, respectively, in a manner indicative of anti-nociception and the induction of 'analgesia'. Pretreatment with the prototypical opiate antagonist, naloxone, blocked the morphine- and reduced the U-50, 488H-induced analgesia. Naloxone had no effects on the thermal response latencies of saline treated animals. 2. Exposure to either cold (7 degrees C) or warm (38 degrees C) temperature stress increased the nociceptive thresholds of Cepaea in a manner indicative of the induction of 'stress-induced analgesia'. The warm stress-induced analgesia was opioid mediated, being blocked by naloxone, whereas, the cold stress-induced analgesia was insensitive to naloxone. 3. Exposure for 15-30 min to 0.5 Hz weak rotating magnetic fields (1.5-8.0 G) significantly reduced the analgesic effects of the mu and kappa opiate agonists in a manner similar to that observed with naloxone. The magnetic stimuli also inhibited the endogenous opioid mediated warm stress-induced analgesia and significantly reduced the cold stress-induced analgesia. The magnetic stimuli had no evident effects on the nociceptive responses of saline-treated animals. The dihydropyridine (DHP) and non-DHP calcium channel antagonists diltiazem, verapamil. and nifedipine differentially and significantly reduced, while the DHP calcium channel agonist, BAY K8644, significantly enhanced the inhibitory effects of the magnetic fields on morphine-induced analgesia.     

Accelerated communciation: Constitutive μ opioid receptor activation as a regulatory mechanism underlying narcotic tolerance and dependence

Chronic administration of narcotic μ opioid agonists results in tolerance and dependence. We propose that agonist stimulation causes a gradual conversion of μ receptors to a constitutively active state μ¹ as a key step in tolerance and physical dependence. We provide evidence in support of the existence of μ¹ in human neuroblastoma cells, SH-SY5Y, and μ¹ upregulation during morphine treatment. Naloxone blocked μ¹ activity, acting as an antagonist with negative intrinsic activity which accounts for its high potency in eliciting withdrawal. In contrast, the μ selective antagonist CTAP did not affect μ¹ activity but inhibited naloxone's effect. The protein kinase inhibitor H7 was found to suppress μ¹ formation, suggesting that μ¹ is phosphorylated. In a model of acute morphine tolerance/dependence in mice, H7 prevented naloxone induced withdrawal jumping and reversed morphine (antinociceptive) tolerance. CTAP cause only mild withdrawal and attenuated naloxone induced withdrawal, as predicted for an antagonist without negative activity. These results support a role for constitutive μ receptor activation in narcotic tolerance and dependence, affording potential separation of acute and chronic narcotic effects.     

Excitation and inhibition of neurons in the trigeminal nucleus caudalis following periaqueductal gray stimulation

Electrical stimulation (3-4 shocks, 300 Hz, 30-150 microamperemeter) of the periaqueductal gray matter (CG) or dorsal raphé nucleus (DR) of decerebrate cats reduced or abolished the jaw-opening reflex response evoked by stimulation of either the tooth pulp or infraorbital nerve. In addition, CG or DR stimulation inhibited the response of 12 out of 16 trigeminal nucleus caudalis neurons to activation of their sensory afferent inputs. Ten other neurons recorded in the same sites, and often at the same time, but which did not respond to the sensory inputs utilized, were excited by identical stimuli to CG or DR. This excitatory response was blocked by intravenously administered naloxone (0.1-0.2 mg/kg). It is suggested that those neurons which are excited by CG and DR stimulation may be interneurons involved in pre- and post-synaptic inhibition of sensory transmission during stimulus-produced or narcotic analgesia.     

Effects of narcotic opiates and serotonin on the electrical behavior of neurons in the guinea pig myenteric plexus

Action potentials were recorded extracellularly from spontaneously firing neurons in the myenteric plexus of the guinea pig ileum. Morphine, which inhibits acetylcholine release from the myenteric plexus, inhibited the spontaneous electrical activity of about half the cells studied, while serotonin elevated the firing rate of these cells. Units not stimulated by serotonin were not inhibited by morphine or levorphanol. Morphine also prevented the increase in firing rate caused by serotonin. These effects of morphine were stereospecific and blocked by naloxone, and are therefore considered to be specific opiate effects. This study demonstrates opposing effects of narcotic opiates and serotonin on the electrical activity of serotoninoceptive neurons in the myenteric plexus.     

Capsaicin-induced avoidance behavior in the terrestrial Gastropoda Megalobulimus abbreviatus: evidence for TRPV-1 signaling and opioid modulation in response to chemical noxious stimuli

The aim of this study was to measure the nociceptive response (avoidance latency) of the land snail Megalobulimus abbreviatus (N=8 in each group) after topical capsaicin exposure (0.1% and 0.5% in 20% ethanol) and to compare it to a well-studied stressful (50 degrees C) thermal stimulus model. We also tested if ruthenium red, and capsazepine, respectively nonselective and selective TRPV1 receptor antagonists, could modify both capsaicin- and thermal-evoked responses. Finally, animals were pretreated with morphine, naloxone or morphine plus naloxone prior to capsaicin stimuli. Latencies were measured when the animal lifted its head-foot complex 1 cm from the substrate. Data were compared using ANOVA and LSD post hoc, and the Student T Test (p<0.05). Capsaicin elicited dose-dependent withdrawal behavior. The capsaicin vehicle (20% ethanol) also evoked a less intense but significant avoidance reaction. Capsazepine and ruthenium red attenuated both capsaicin and heat withdrawal responses, when compared to vehicles. Morphine increased, and naloxone, either alone or in combination with morphine, reduced capsaicin-evoked latencies when compared to morphine or saline. These results indicate that the TRPV1 receptor plays a role in the nociceptive circuits of M. abbreviatus.     

Morphine excitation in the cerebral cortex.

Microiontophoretic administrations of morphine to cholino-excitable neurones in the cerebral cortex of decerebrate cats evoked a weak excitation which became more prominent upon repeated administrations of the alkaloid. This effect was not antagonized by naloxone. Iontophoresis of methylatropine prevented the excitation induced with acetylcholine and morphine, leaving that caused by glutamate relatively unaltered. Similar applications of morphine to neurones which were not excited by test applications of acetylcholine did not result in excitation but elicited mainly a depression of glutamate-evoked firing. It is suggested that the muscarinic effect of morphine in the cortex may be related to the excitation and convulsions, but not the analgesia, which occurs upon systemic administrations of the narcotic.     

IV. Discriminative stimulus effects of narcotics: Evidence for multiple receptor-mediated actions

Results of studies on the discriminative stimulus effects of narcotics are consistent with the hypothesis that multiple receptors mediate the effects of these compounds. In the rat, at least three subsets of discriminative effects exist, although some drugs appear to have effects that transcend more than one subset. The discriminative effects of morphine-like narcotics (μ agonists), for example, are often clearly distinguishable from the discriminative effects produced by κ agonists, such as ketazocine, and from those produced by phencyclidine-like agonists, such as SKF-10,047 and cyclazocine. Cyclazocine, however, has been reported to have discriminative effects in common with morphine (45) and fentanyl (17) and appears to have κ-like, in addition to phencyclidine-like, discriminative effects. The relative ability of pure narcotic antagonists to block the discriminative effects of these compounds also provides evidence for distinct pharmacologic actions of these drugs. In the rat, the discriminative effects of morphine are blocked by doses of naloxone that are considerably smaller than those that are needed to block the discriminative effects of cyclazocine (44). The discriminative effects of phencyclidine are not altered at all by naltrexone (63).     

The actions of opiates in the rat substantia nigra: An electrophysiological analysis

Single unit recording and micropressure ejection techniques were used to investigate the actions of opiates on dopaminergic and non-dopaminergic neurons in the rat substantia nigra. Systemic administration of morphine, 1 to 4 mg/kg, led to a naloxone-reversible increase in firing rate of all zona compacta dopaminergic (ZC) neurons examined (n=10). In a specifically defined subpopulation of non-dopaminergic nigral zona reticulata (ZR) neurons, systemically administered morphine led to a naloxone reversible decrease in activity (n=9). D-Ala²-d-leu⁵ (DADL)-enkephalin, when applied directly onto ZC neurons by micropressure ejection techniques, had no effect on their firing rate. In contrast, micropressure ejection of DADL enkephalin onto ZR neurons produced a decrease in firing rate which was blocked by systemically administered naloxone. Morphine sulfate applied by pressure ejection onto both ZC and ZR neurons produced mixed results which were not always blocked by naloxone. These results suggest that one of the mechanisms by which opiates increase dopaminergic neurotransmission is through disinhibition of dopaminergic neurons in the substantia nigra.     

Selective interaction of drugs with a discriminable stimulus associated with narcotic actions

Rats were trained to bar press on either one of two levers depending on whether they received an injection of morphine (10 mg/kg) or saline. The rats responded on the morphine-correct lever when injected with another narcotic, fentanyl, but responded on the saline-correct lever when injected with a narcotic antagonist or another CNS active, but non-narcotic, drug (e.g., amphetamine, apomorphine). The narcotic antagonist, naloxone, prevented the occurrence of the narcotic discriminable stimulus, but the rats responded on the morphine-correct lever when injected with morphine plus any of a number of potent CNS active, but non-narcotic compounds. These results are discussed with reference to the specificity of this procedure for detecting drugs with narcotic agonist or antagonist properties.     

A comparison of the analgesic and behavioral effects of [D-Ala2] Met-enkepha-linamide and morphine in the mesencephalic reticular formation of rats.

[D-Ala²]Met-enkephalinamide (DALA) injected intracerebrally (IC) at low doses into specific sites of the mesencephalic reticular formation (MRF), produced a profound, long-lasting analgesia that was blocked by naloxone, a specific opiate antagonist. Morphine was only half as potent as DALA because morphine, injected IC at similar sites in the MRF, yielded a comparable analgesia only when injected at twice the dose. The analgesic effects of morphine were also antagonized by naloxene. Both DALA and morphine produced specific behavioral effects. Naloxone blocked the behavioral effects of DALA, but not those produced by morphine.     

beta-Endorphin controls vasopressin release during foot shock-induced stress in the rat

This study tested the possibility that beta-endorphin is involved in the regulation of vasopressin release during stress induced by inescapable electric foot shock. To this end, a specific anti-beta-endorphin antiserum or a control serum lacking the specific anti-beta-endorphin antibodies was administered to male rats. Plasma vasopressin concentrations, measured by radioimmunoassay, were not affected by brief foot shock stress in control rats, but were raised significantly by the stress in animals which had received an intracerebroventricular (i.c.v.) injection of the anti-beta-endorphin antiserum. In contrast, when the same volume of the anti-beta-endorphin antiserum was injected into a tail vein, foot shock stress produced only a slight effect on vasopressin release. I.c.v. injection of the antiserum changed neither basal nociceptive threshold nor stress-induced analgesia as revealed by the tail-flick latency. Vasopressin release induced by an osmotic stimulus was not influenced by the anti-beta-endorphin antiserum given i.c.v. The opiate antagonist naloxone or the glucocorticoid dexamethasone raised plasma vasopressin concentration in stressed rats which had received the control serum (i.c.v.); however, after i.c.v. injection of the anti-beta-endorphin antiserum neither naloxone nor dexamethasone elevated the plasma vasopressin concentration beyond the level reached by the anti-beta-endorphin antiserum (i.c.v.) alone. These results suggest that beta-endorphin inhibits the release of vasopressin during foot shock-induced stress in the rat.     

Effect of naloxone on antinociceptive activity of phenoxybenzamine.

Phenoxybenzamine was found to have antinociceptive activity in the mouse writhing syndrome test. The percent protection against writhing was dose related from 2.0 to 164.6 μM/kg phenoxybenzamine (ED-50, 37.1 ± 2.0 μM/kg). The dose-response curve for phenoxybenzamine was shifted to the left when morphine was administered. Administration of morphine, 1.12 μM/kg, reduced the ED-50 for phenoxybenzamine to 4.5 ± 1.3 μM/kg. Administration of naloxone shifted the dose response curve for phenoxybenzamine to the right. Naloxone 0.03 μM/kg increased the ED-50 for phenoxybenzamine to 102.5 ± 22.7 μM/kg. Chronic treatment with phenoxybenzamine did not change the antinociceptive response to phenoxybenzamine. The α-adrenergic blockers, phenotolamine and tolazoline showed weak antinociceptive activity which was not blocked by naloxone.     

Antinociceptive effects of intrathecally administered human beta-endorphin in the rat and cat

Rats chronically implanted with intrathecal catheters displayed a dose-dependent increase in the hot-plate and tail-flick response latencies following the injection of human beta-endorphin into the lumbar spinal subarachnoid space through the indwelling catheter. beta-Endorphin was approximately 25 times more potent than morphine on a molar basis. Matching morphine and beta-endorphin doses such that approximately equal submaximal submaximal effects occurred, it was observed that the antinociception produced by beta-endorphin lasted approximately three times longer than that produced by morphine. Experiments with intrathecal injection of beta-endorphin into the spinal subarachnoid space of cats fitted with intrathecal catheters also revealed a potent antinociceptive effect which was completely antagonized by naloxone. In the rats, naloxone administered systemically in doses of 10--100 microgram/kg produced a parallel shift in the dose-response curves of both nociceptive measures suggesting a competitive antagonism. Using a dose ratio analysis, an in vivo pA2 of 7.1 for naloxone was obtained. These data and those derived from previous work based on the pA2 suggest that the interaction of morphine, certain pentapeptides, and beta-endorphin is the same with regard to the spinal opiate receptor population mediating behaviorally defined analgesia.     

In vivo catechol activity in the rat locus coeruleus following different nociceptive stimuli and naloxone.

The nucleus locus coeruleus (LC) has been implicated in the processing of spinal reflexes following noxious stimuli. It has been demonstrated that noxious stimuli activate LC neuronal firing, but little is known about the neurochemical changes that might occur following such activation. To determine the effects of different noxious stimuli on LC neuronal activity, anaesthetized rats were exposed to mechanical (tail pinch), thermal (55 degrees C water), and chemical (5% Formalin injected in the hind paw) stimuli; the catechol oxidation current (CA.OC), an index of noradrenergic neuronal activity, in the locus coeruleus was monitored using differential normal pulse voltammetry. In addition, the effect of the opioid antagonist naloxone on the CA.OC in the LC was examined. Exposure to both mechanical and chemical stimuli significantly increased CA.OC indicating an increase in LC noradrenergic neuronal activity, while the thermal stimulus had no effect. Treatment with naloxone (1 mg/kg i.v.) had no effect on CA.OC in the LC. The results show a differential responsiveness of LC noradrenergic neurons to different modes of noxious stimuli and fail to demonstrate a tonic opioid regulation of these neurons in the anaesthetized rat.     

The role of T-type calcium channels in morphine analgesia,development of antinociceptive tolerance and dependence to morphine,and morphine abstinence syndrome

Involvement of T-type voltage dependent Ca2+ channels (VDCCs) on morphine antinociception, in the development of tolerance and dependence to morphine, and naloxone-precipitated abstinence syndrome in morphine dependent mice was examined by using mibefradil, a T-type VDCCs blocker. Mice were rendered tolerant and dependent on morphine by subcutaneous (s.c.) implantation of a morphine pellet containing 75 mg of morphine base for 72 hr. The tail-flick test was used to assess the nociceptive threshold. Coadministration of acute mibefradil (10 mg/kg, i.p.) with morphine enhanced the antinociceptive effects of acute morphine. Repeated mibefradil administration (10 mg/kg, i.p., just before, 24 and 48 hr after morphine pellet implantation) completely blocked the development of tolerance to the antinociceptive effect of morphine and even by this effect reached supersensitivity to morphine. However, repeated mibefradil treatment did not alter the development of dependence to morphine assessed by the A(50) values of naloxone (s.c.) required to precipitate withdrawal jumping 72 hr after morphine pellet. But, acute mibefradil (10, 30, and 50 mg/kg, i.p.) dose dependently decreased the expression of morphine abstinence syndrome when given directly 30 min prior to naloxone (0,05 mg/kg, s.c.) 72 hr after morphine pellet. These results indicate a critical role of T-type VDCCs in morphine antinociception, the development of tolerance to the antinociceptive effects of morphine and in morphine abstinence syndrome.