Opiate antagonism of glycine-evoked membrane polarizations in cultured mouse spinal cord neurons

The effect of several opiate receptor agonists on the responses of spinal cord neurons to putative inhibitory and excitatory amino acids was studied using an in vitro model system, cultured fetal mouse spinal cord neurons, and bath application of opiates. Intracellular recordings were made from the cultured neurons with conventional voltage recording techniques or under voltage clamp conditions. The putative amino acid neurotransmitters were applied by iontophoresis or micropressure ejection.Our main finding is that the opiate agonists, morphine and levorphanol (5–100 μM), consistently depressed the responses evoked by the putative inhibitory amino acid neurotransmitters glycine and β-alanine but not GABA. Dextrorphan, the inactive isomer of levorphanol, also depressed the glycine and β-alanine responses, but higher concentrations were required. The excitatory glutamate response was unaltered by these opiates. Leucine enkephalin, an opioid peptide, had no effect on the amino acid responses in the neurons where it was also tested. The opiate antagonist naloxone (10–100 μM) did not reverse the morphine or levorphanol depression of the amino acid responses making it unlikely that opiate receptors mediate this effect.Strychnine was considerably more effective than morphine as a glycine antagonist, producing depressions at nM concentrations compared to the μM concentrations required for morphine. Preliminary studies indicate that both morphine and strychnine act in a non-competitive manner. However, additional studies will be required before the sites of action for these agents can be identified.The possible pharmacological or toxicological significance of the present work remains to be determined. Considering the high doses of opiates (μM concentrations) required to depress the glycine and β-alanine responses, it is unlikely that this action is relevant to normal therapeutic situations. However, such concentrations of opiates are often utilized in pharmacological studies and may be achieved when opiates are applied by iontophoresis. Our data indicate that consideration of the present opiate action should be made when μM concentrations or iontophoretic application of opiates are used for pharmacological studies of CNS tissue.     

Cold-restraint stress reduces [H]etorphine binding to rat brain membranes: Influence of acute and chronic morphine and naloxone

[³H]Etorphine binding was characterized in rat brain homogenates depleted of endogenous opioids from animals acutely and chronically treated with morphine or naloxone and either unstressed or subjected to a 3-h restraint period in the cold. There was significant reduction in the number of high-affinity opiate binding sites in brain tissue from stressed as compared to unstressed animals. Despite the fact that the opiate drug regimens used produce marked behavioral and physiological effects, stress-induced opiate receptor depletion was not influenced by the drugs or by withdrawal. The various drug treatments also failed to produce significant changes in opiate receptor site densities or affinities in either stressed or unstressed animals. We propose that persistent activation of opiate receptors by endogenous opioids released during restraint stress leads to receptor ‘down-regulation’.     

II. Examination of spinal monoamine receptors through which brainstem opiate-sensitive systems act in the rat

Microinjection of morphine (5 μg) through stereotaxically implanted microinjection cannulas into the periaqueductal gray (104 sites), medial (n. raphe magnus; 26 sites) and paramedial (n. reticulogigantocellularis; 49 sites) medulla resulted in an increase in the latency of supraspinally (hot-plate) and spinally (tail-flick)-mediated responses evoked by thermal stimuli. This effect of intracerebral morphine on both hot-plate and tail-flick was dose-dependent, and reversed by systemically administered naloxone as well as by naloxone administered by microinjection into the same site. On the basis of frequency of occurrence, time of onset and magnitude of effect of the minimum effective dose, we could demonstrate no difference between the efficacy of morphine acting at sites in the periaqueductal gray, n. raphe magnus or n. reticulogigantocellularis on the supraspinally mediated response. In all areas examined, morphine was able to produce the maximum elevation in response latency. The microinjection of morphine into the periaqueductal gray frequently produced a total block of the thermally evoked spinally mediated tail-flick reflex. Unlike the periaqueductal gray, the systems through which opiates act in the n. raphe magnus or the n. reticulogigantocellularis to suppress spinal reflex activity displayed a clear plateau in their physiological effects. Microinjections of morphine into the n. raphe magnus or n. reticulogigantocellularis never produced a complete block of the spinal reflex. Further increases in inhibition could not be achieved by either a 3-fold increase in dose or bilateral injections into the paramedial medulla. The failure to block spinal reflex activity often occurred at sites where morphine would completely block the hot-plate response. These observations indicate that opiate receptor-linked systems in the mesencephalon and medulla can significantly attenuate the coordinated escape behavior otherwise evoked by a high-intensity thermal stimuli. We find there is no difference in the physiological efficacy of morphine acting in those regions on supraspinally mediated measures of pain responding. The differential effect on spinally mediated reflex function suggests that these several opiate linked systems produce their effect by discriminable mechanisms.     

Local and distal effects induced by unilateral striatal application of opiates in the absence or in the presence of naloxone on the release of dopamine in both caudate nuclei and substantiae nigrae of the cat

Halothane-anesthetized cats implanted with push-pull cannulae in both caudate nuclei (CN) and substantiae nigrae (SN) were used to study the effects of naloxone and various opiates when applied into the left CN on the release of newly synthetized tritiated dopamine (DA) from nerve terminals and dendrites of the two nigro-striatal dopaminergic pathways. In all cases, the drugs (naloxone, opiates alone or in the presence of naloxone) were applied for 30 min into the left CN. When applied alone, naloxone (10⁻⁶ M) induced a delayed reduction in tritiated DA release both in the ipsilateral and contralateral CN. These effects were seen after removal of the drug from the superfusion fluid. Complementary experiments made with tritiated naloxone (10⁻⁶ M) revealed that the contralateral effect on DA release was not due to a diffusion of the opiate antagonist from its application site. Locally,d-Ala₂, Met-enkephalinamide (d-Ala₂, Met-Enk, 10⁻⁶ M) and the potent δ agonist Tyr-d-Ser-Gly-Phe-Leu-Thr (DSThr, 5 × 10⁻⁸ M) induced a biphasic increase in tritiated DA release. The local changes in tritiated DA release evoked by morphine (10⁻⁶ M) and μ agonists such as Tyr-d-Ala-Gly-NH-C₆H₁₃ (10⁻⁸ M) and fentanyl (10⁻⁸ M) differed from those of δ agonists and furthermore differed from each other. For instance, morphine induced a delayed increase in tritiated DA release whereas a biphasic increase followed by a delayed inhibition occurred with fentanyl. Among all the opiates testedd-Ala₂-Met-Enk was the only one which elicited a distal effect, that is a reduction of tritiated DA release in the ipsilateral SN. Marked differences in these opiates' effects on tritiated DA release occurred both locally and in distal structures when opiates were applied simultaneously with naloxone (10⁻⁶ M). Locally, the changes induced by μ agonists were particularly altered since during morphine's application with naloxone a reduction of tritiated DA release occurred. In addition, the opiate antagonist prevented the second increase and the delayed inhibition of tritiated DA release evoked by fentanyl (10⁻⁸ M). Interestingly, the combined application of naloxone with eitherd-Ala₂,Met-Enk (10⁻⁶ M) DSThr (5 × 10⁻⁸ M) or morphine (10⁻⁶ M) resulted in the appearance of changes in tritiated DA release in contralateral structures. The most striking effect was seen withd-Ala₂,Met-Enk which enhanced tritiated DA release in the contralateral CN and SN. These results are discussed in the light of the involvement of several types of opiate receptors and of the polysynaptic pathways responsible for the distal changes in dopaminergic transmission.     

Comparison of antinociceptive action of morphine in the periaqueductal gray, medial and paramedial medulla in rat

Microinjection of morphine (5 micrograms) through stereotaxically implanted microinjection cannulas into the periaqueductal gray (104 sites), medial (n. raphe magnus; 26 sites) and paramedial (n. reticulogigantocellularis; 49 sites) medulla resulted in an increase in the latency of supraspinally (hot-plate) and spinally (tail-flick)-mediated responses evoked by thermal stimuli. This effect of intracerebral morphine on both hot-plate and tail-flick was dose-dependent, and reversed by systemically administered naloxone as well as by naloxone administered by microinjection into the same site. On the basis of frequency of occurrence, time of onset and magnitude of effect of the minimum effective dose, we could demonstrate no difference between the efficacy of morphine acting at sites in the periaqueductal gray, n. raphe magnus or n. reticulogigantocellularis on the supraspinally mediated response. In all areas examined, morphine was able to produce the maximum elevation in response latency. The microinjection of morphine into the periaqueductal gray frequently produced a total block of the thermally evoked spinally mediated tail-flick reflex. Unlike the periaqueductal gray, the systems through which opiates act in the n. raphe magnus or the n. reticulogigantocellularis to suppress spinal reflex activity displayed a clear plateau in their physiological effects. Microinjections of morphine into the n. raphe magnus or n. reticulogigantocellularis never produced a complete block of the spinal reflex. Further increases in inhibition could not be achieved by either a 3-fold increase in dose or bilateral injections into the paramedial medulla. The failure to block spinal reflex activity often occurred at sites where morphine would completely block the hot-plate response. These observations indicate that opiate receptor-linked systems in the mesencephalon and medulla can significantly attenuate the coordinated escape behavior otherwise evoked by a high-intensity thermal stimuli. We find there is no difference in the physiological efficacy of morphine acting in those regions on supraspinally mediated measures of pain responding. The differential effect on spinally mediated reflex function suggests that these several opiate linked systems produce their effect by discriminable mechanisms.     

Occurrence of the opiate alkaloid-selective μ3 receptor in mammalian microglia, astrocytes and Kupffer cells

Evidence is presented for occurrence of opiate alkaloid-selective, opioid-peptide-insensitive receptor binding sites, labeled with [³H]morphine, in primary cultures of cat microglia and of cat astrocytes, as well as on highly purified preparations of rat Kupffer cells. These receptors have been designated μ₃ on the basis of their close similarity to receptors first found to be present on human peripheral blood monocytes. Exposure of the microglia to morphine and etorphine cause marked quantifiable changes in cellular morphology, including assumption of a more rounded shape and retraction of cytoplasmic processes; in contrast, several opioid peptides were without effect on morphology. The effects of morphine on microglial morphology were blocked by the opiate antagonist naloxone. These effects of drugs on morphology were as predicted for action via the μ₃ receptor. Opiate alkaloid binding sites previously detected on the rat C6 glioma cell line were also characterized here as of the μ₃ receptor subtype. It is proposed that μ₃ receptors have broad distribution in different macrophage cell types of bone marrow lineage, including microglia and Kupffer cells. Furthermore, these receptors are not restricted to cells of bone marrow lineage, since they are also present on astrocytes.     

Chronic naltrexone increases opiate binding in brain and produces supersensitivity to morphine in the locus coeruleus of the rat

Rats were implanted subcutaneously for 2-4 weeks with slow-release pellets of naltrexone (10 mg) or placebo and then the pellets were removed. One day after removal of the pellet, animals were either (1) sacrificed and various CNS regions examined for specific binding of [3H]naloxone, [3H]etorphine or [3H]rauwolscine or (2) they were anesthetized and prepared acutely for assessing morphine-induced changes in the spontaneous activity of neurons in the locus coeruleus (LC). Naltrexone treatment significantly increased the number of specific binding sites for opiates, but not for alpha 2-adrenergic antagonists, in spinal cord, hypothalamus, striatum and cortex. Specific binding of [3H]naloxone was also increased in the LC. The spontaneous activity of neurons in the LC was reduced by the chronic naltrexone treatment, suggesting that these neurons became supersensitive to the tonic inhibitory effect of endogenous opioid peptides. Moreover, neurons in the LC of chronic naltrexone-treated rats exhibited an enhanced response to the inhibitory effects of morphine administered systemically. These results demonstrate that chronic opiate receptor blockade increases the number of receptor sites for morphine and that this increase in receptors is accompanied by a neuronal supersensitivity in the LC to morphine which can be assessed electrophysiologically.     

Cold-restraint stress reduces [3H]etorphine binding to rat brain membranes: influence of acute and chronic morphine and naloxone

[3H]Etorphine binding was characterized in rat brain homogenates depleted of endogenous opioids from animals acutely and chronically treated with morphine or naloxone and either unstressed or subjected to a 3-h restraint period in the cold. There was significant reduction in the number of high-affinity opiate binding sites in brain tissue from stressed as compared to unstressed animals. Despite the fact that the opiate drug regimens used produce marked behavioral and physiological effects, stress-induced opiate receptor depletion was not influenced by the drugs or by withdrawal. The various drug treatments also failed to produce significant changes in opiate receptor site densities or affinities in either stressed or unstressed animals. We propose that persistent activation of opiate receptors by endogenous opioids released during restraint stress leads to receptor 'down-regulation'.     

Dose effects of morphine on the spontaneous unit activity recorded from the thalamus,hypothalamus, septum,hippocampus, reticular formation,central gray,and caudate nucleus

Spontaneous activity was recorded from 652 units in 8 subcortical structures of unanesthetized rats. Recordings were obtained in central gray, mesencephalic reticular formation, parafasciculus thalami, caudate nucleus, anterior and ventro- medial hypothalamus, lateral septum, and dorsal hippocampus. Eighty recordings were obtained from untreated animals and 80 from saline-injected controls, none of which showed any significant changes of unit activity during the 4- 5-hr observation period. The effect of morphine, given in 5 incremental doses from 0.5 to 30.0 mg/kg ip, was followed in 492 units. Morphine enhanced or depressed spontaneous discharge rates, or caused biphasic effects, ie enhancement alternating with depression and vice versa. Naloxone induced increase in firing after either effect of morphine, or reduced spontaneous activity after morphineinduced increases. However, when morphine reduced neuronal discharges, naloxone never caused further depression. In 86 units, morphine at any dosage failed to alter neuronal activity, but in 54 of these units naloxone nevertheless induced alterations in firing rates. The pattern of responses to morphine differed between all 8 brain regions examined and was characteristic for each individual structure. This is the first systematic study describing the dose-response characteristics of morphine in 8 brain sites recorded simultaneously. Furthermore, it utilized freely behaving animals without the interference of anesthetics, which are themselves known to interact with opiates. The variety of response patterns seen supports the neuro pharmacological evidence for multiple opiate receptors or multiple sites of opiate action.     

Characterization of mechanical withdrawal responses and effects of μ-, δ- and κ-opioid agonists in normal and μ-opioid receptor knockout mice

Clinical and experimental observations suggest that opiates can exert different influences on the perception of stimuli from distinct sensory modalities. Thermally-induced nociception is classically responsive to opiate agonists. μ-Opioid receptor-deficient transgenic mice are more sensitive to thermal nociceptive stimuli and morphine fails to attenuate the nociceptive responses to thermal stimuli in these animals. To enhance our understanding of opiate influences on mechanical sensitivity, we have examined withdrawal responses to a sequence of ascending forces of mechanical stimuli in mice with normal (wild type), half-normal (heterozygous) and absent (homozygous) μ-opioid receptor levels. We report data from mice examined without drug pretreatment or following pretreatment with morphine, the selective κ-opioid agonist, U50488H, and the selective δ-opioid agonist, DPDPE. Saline-pretreated mice of each genotype displayed similar, monotonically increasing frequency of withdrawal responses to the graded stimuli. Subcutaneously administered morphine produced a dose-dependent reduction in withdrawal responses in wild type and heterozygous mice, but had no significant effect in homozygous mice. Intraventricular administration of DPDPE also reduced the frequency of paw withdrawal (FPW) in wild type mice, but not in homozygous mice. In contrast, systemic U50488H produced a dose-dependent attenuation of paw withdrawal in both wild type and homozygous mice. These findings suggest that (1) interactions of endogenous peptides with μ-opioid receptors may not play a significant role in the response to mechanical stimuli in drug-free animals, and (2) deficiency of μ-opioid receptors has no functional consequence on the response to the prototypical κ-opioid receptor agonist, but decreases responses to the prototypical μ- and δ-opioid receptor agonists.     

Effects of morphine and naloxone on hippocampal CA3 field potentials following systemic administration in the freely-moving rat

The effect of IV morphine, 2, 6 and 15 mg/kg, on hilar-evoked CA3 field potentials was studied to determine if this area would be more sensitive to mu-type opiate agonists than the CA1 or dentate regions. In addition, the effect of IV naloxone, 2 and 25 mg/kg, on the same responses was studied to determine if endogenous opiates reported to be present in the mossy fibers are released by electrical stimulation of this pathway. Neither morphine nor naloxone had an effect on CA3 field potentials at any dose used. The CA1 region of the hippocampus is the area most sensitive to morphine, and this effect of morphine correlates best, anatomically, with the localization of mu-receptors identified by the binding of dihydromorphine. Physiological release of endogenous opiates from the hippocampus remains to be shown.     

Reinforcing effects of morphine in the nucleus accumbens

The possibility that morphine exerts its reinforcing effects via an action in the nucleus accumbens was investigated in the rat using the self-administration model and the method of intracerebral injection. Male, adult rats implanted with a chronic cannula aimed at this nucleus were tested for self-administration in a rectangular chamber equipped with two levers, one at each end. Each subject was given several sessions in blocks of two sessions for a given substance, the first session with the active lever at one end of the chamber, the next with the active lever at the other end. During a session, responses on the inactive lever were without consequences. Depression of the active lever was coupled with the presentation of a tone to facilitate discrimination between the two levers. All subjects received a sequence of 5 sessions, the first for habituation, the next two to test for effects of artificial cerebrospinal fluid, and the next two to test for effects of morphine. A few subjects in which the cannula had not been dislodged or plugged were further tested for effects of morphine and then for effects of morphine mixed with naloxone. Each response on the active lever resulted in an injection of 0.02 μl. During the morphine sessions, this volume contained 0.2 μg of the opiate; during the sessions of morphine and naloxone, this volume contained 0.2 μg morphine mixed with 0.2 μg naloxone.The results show that self-administration was induced and maintained when morphine was injected inside the nucleus accumbens, not in an area outside this region but at the same brain level. The rate of responding on the active lever for morphine was higher than for artificial cerebrospinal fluid and it showed a pattern of gradual increase over time. In contrast, the rate of responding on the inactive lever during the morphine sessions showed a decline. This pattern was not evident in these animals during control sessions and in the animals in which the injections were outside the n. accumbens. These results are interpreted to indicate that the n. accumbens may be part of a system of structures mediating the reinforcing effects of opiates.     

Opiate, enkephalin, and endorphin analgesia: relations to a single subpopulation of opiate receptors

Differences in the receptor mechanisms of opiate analgesia and respiratory depression have been studied with three novel irreversible opiates. A single injection of the irreversible agonist oxymorphazone produces analgesia in mice, lasting over 24 hours. Conversely, the irreversible antagonist naloxazone dramatically reduces the analgesic effectiveness of a variety of opiate alkaloids and enkephalin analogs for over a day. Despite this marked reduction in analgesia after naloxazone treatment, morphine lethality (LD50) is unchanged in similarly treated mice. Receptor binding studies show that naloxazone irreversibly and selectively blocks a subpopulation of opiate receptors (the mu1 sites) to which all classes of opiates and enkephalins bind with highest affinity, whereas the drug has little to no effect on their lower-affinity sites (mu, and delta). The return of high-affinity receptor (mu1) binding to normal levels corresponds closely to the return of analgesic sensitivity and possibly represents receptor turnover in the central nervous system. These studies suggest that both opiate and opioid peptide analgesia is mediated through a single receptor subpopulation distinct from those involved with respiratory depression, and raise the possibility of specific opiate analgesics without respiratory depression.     

The effects of local micro injections of opiates and enkephalins into the forebrain on the electrocorticogram of the rat.

The effects of various opiate compounds have been studied on the electrocorticogram (ECoG) of the rat following local injection into various brain areas. Injections of all compounds studied into both the caudate-putamen and the basal forebrain, in particular the olfactory tubercles, induced changes in the ECoG. Injections of saline vehicle into these areas were ineffective as were injections of morphine into the corpus callosum. Potency was etorphine greater than morphine = codeine greater than thebaine. Naloxone alone was inactive following injection but if combined with morphine markedly attenuated the normal morphine response and reversed the morphine response if injected following morphine. The endogenous opiate compound enkephalin and the synthetic analogue D-ala2-met5-encamide also induced electrocortical changes which were naloxone sensitive. The results are similar to those following systemic administration of opiates. It is possible that the areas studied represent the site of action of systemically applied opiates. It is suggested that the opiates and enkephalins produce their actions by acting at the same site. Since the areas studied are rich in dopaminergic terminals an interaction may exist between dopaminergic and opiate mechanism to bring about the observed changes.     

Influence of morphine and cylazocine on the cortical epileptic foci in rabbits

The effects of morphine, cyclazocine and naloxone on penicillin- and strychnine-induced epileptic foci were studied in rabbits. The intracortical injection of penicillin (75, 150 and 300 units) elicited isolated spikes followed by repeated ictal events. The application of strychnine (0.062 and 0.125%) over the cortical surface of one side induced appearance of ipsilateral spiking spreading to the contralateral cortex.Administration of morphine (0.25–0.75 mg/kg i.v.) or cyclazocine (0.05–3.0 mg/kg i.v.) inhibited the occurrence or the duration of the EEG and motor manifestations induced by penicillin (75 and 150 units) and strychnine (0.062 and 0.125%), while it did not influence the effect of 300 units of penicillin. High doses of morphine (up to 10 mg/kg i.v.) failed to affect the epileptic responses to penicillin and strychnine and at the same time significantly reduced the pO₂ in arterial blood.Naloxone per se potentiated the effects of the lower doses of penicillin and strychnine. Only at very high doses (20 mg/kg i.v.) displayed a weak antagonism towards the anticonvulsant effect of the two opiates. A full antagonism is only observed towards the effect of cyclazocine (2 mg/kg i.v.) administered after penicillin.Present data provide additional evidence of the heterogeneity of regulations by opioids of convulsive phenomena. One can hypothesize that the anticonvulsant effect of the two opiate agonists is mediated by naloxone-insensitive opiate receptors, while the proconvulsant-convulsant effect of naloxone might be related to an inhibition of GABA and glycine-mediated transmission.     

Actions of iontophoretically applied morphine on hypothalamic thermosensitive units

The effects of iontophoretically applied morphine and naloxone were examined on 62 neurons in the preoptic/anterior hypothalamus (POAH) of urethane-anesthetized Sprague-Dawley rats. Thirty-four temperature-insensitive cells responded variably to morphine application. Four cells were excited, 19 inhibited and 11 remained unaffected. Morphine increased the firing rate 12 of 20 warm-sensitive cells. The other 8 were unaffected. None were inhibited. The spontaneous activity of 7 cold-sensitive cells was decreased by morphine application. One cell was unaffected and none were excited. Naloxone (≤ 15 nA) antagonized morphine's excitatoory effects on warm-sensitive cells and its inhibitory effects on cold-sensitive cells. Naloxone failed to antagonize any of morphine's actions on temperature-insensitive cells.Our results demonstrate that morphine excited warm-sensitive cells, which are assumed to mediate heat-dissipation responses, and inhibited cold-sensitive cells, which are assumed to mediate heat-gain responses. These actions parallel morphine's hypothermic action in the intact animal and, therefore, suggest that morphine lowers body temperature by exerting a coherent action on POAH warm- and cold-sensitive neurons. Since these effects were antagonized by naloxone, the action of morphine on warm- and cold-sensitive cells seems to be mediated by an opiate receptor.     

Beneficial effect of i.c.v. naloxone in anaphylactic shock is mediated through peripheral β-adrenoceptive mechanisms

In mice, fatal shock induced by release of endogenous histamine by compound 48/80 was reversed by the intracerebroventricular (i.c.v.) administration of the opiate antagonist naloxone (10–25 μg) but not by the systemic administration of the selective peripherally acting antagonist, naltrexone methyl bromide (2–5 mg/kg). Moreover, systemac or i.c.v. administration of morphine (25 mg/kg and 25 μg, respectively) exacerbated shock induced by compound 40/80. This effect was blocked by i.c.v. naloxone (10 μg) or naltrexone methyl bromide (10 μg) but not by systemic naltrexone methyl bromide (5 mg/kg). The pathogenic effect of i.c.v. morphine was blocked by the systemic administration of the opiate antagonist Win 44,441-3 (5 mg/kg) but not by its inactive (+) isomer, Win 44,441-2. The results suggest possible involvement of central opiate (endorphin) mechanisms in the pathophysiology of fatal histamine shock in mice.     

Evidence for opiate-activated NMDA processes masking opiate analgesia in rats

The acute interaction between opioid receptors and N-methyl-D-aspartate (NMDA) receptors on nociception was examined in rats using tail-flick and paw-pressure vocalisation tests. When injected at various times (1 to 6 h) after morphine (5 to 20 mg/kg, i.v.) or fentanyl (4x40 microgram/kg, i.v.), the opioid receptor antagonist naloxone (1 mg/kg, s.c.) not only abolished the opiate-induced increase in nociceptive threshold, but also reduced it below the basal value (hyperalgesia). The noncompetitive NMDA receptor antagonist MK-801 (0.15 or 0.30 mg/kg, s.c.) prevented the naloxone-precipitated hyperalgesia and enhanced the antinociceptive effects of morphine (7.5 mg/kg, i.v.) and fentanyl (4x40 microgram/kg, i.v.). These results indicate that the antinociceptive effects of morphine and fentanyl, two opiate analgesics widely used in humans in the management of pain, are blunted by concomitant NMDA-dependent opposing effects which are only revealed when the predominant antinociceptive effect is sharply blocked by naloxone. This study provides new rationale for beneficial adjunction of NMDA receptor antagonists with opiates for relieving pain by preventing pain facilitatory processes triggered by opiate treatment per se.     

Effects of morphine and morphine withdrawal on adrenergic neurons of the rat rostral ventrolateral medulla

In urethane anesthetized rats, iontophoretic application of morphine or α-methylnoradrenaline (α-MNE) inhibited (80–100%) the discharges of all putative adrenergic (C1) cells of the rostral ventrolateral medulla (RVLM). The effect of morphine was blocked selectively by naloxone while that of α-MNE was blocked selectively by theα₂-adrenergic antagonist idazoxan. Putative C1 cells were inhibited (75–100%) by low i.v. doses of clonidine (10–15 μg/kg). Most cells (7/10) were also inhibited by morphine i.v. (81% at 7 mg/kg). Two cells were slightly excited at doses below 2 mg/kg and inhibited at higher doses. Three cells were excited only. All effects of morphine i.v. were reversed by naloxone (1 mg/kg, i.v.). Intravenous administration of naloxone to morphine-dependent rats increased significantly the firing rate of all putative C1 adrenergic cells (from 5.8 ± 0.9 spikes/s to 12.3 ± 1.5 spikes/s;n = 8). During withdrawal these cells could still be inhibited (80–100%) by i.v. injection of clonidine (15 μg/kg). C-Fos expression induced by naltrexone-precipitated withdrawal was examined in the brainstem of freely moving morphine-dependent rats pretreated with clonidine or saline before injection of the opioid antagonist. The locus coeruleus (LC) of the same rats was examined for comparison. Morphine withdrawal without clonidine treatment significantly increased the number of Fos-like-immunoreactive (Fos-LIR) cells in the RVLM and LC. Clonidine pretreatment (1 mg/kg, i.p.) reduced the number of withdrawal-activated Fos-LIR cells in LC by 81%. In the RVLM this reduction averaged 37% for all cell types and 48% for C1 adrenregic cells. Further, a very large proportion of RVLM neurons that expressed c-Fos during morphine withdrawal (83%) were immunoreactive forα₂A-adrenergic receptors. This study suggests that, like noradrenergic cells of the LC, C1 adrenergic neurons of the RVLM are: (i) inhibited by both opiate andα₂-adrenergic receptor agonists; and (ii) activated during naloxone-precipitated morphine withdrawal, Since C1 cells are considered essential to sympathetic tone generation, their inhibition by morphine may contribute to the hypotensive effects of this opioid agonist in non-dependent individuals. Their excitation during opiate withdrawal may also contribute to the autonomic activation that characterizes this syndrome. Finally, inhibition of C1 cells by clonidine may contribute to the clinically recognized efficacy of this drug to attenuate autonomic signs of opiate withdrawal.     

Mu-Selective Opioid Peptides Stimulate Prolactin Release in Lactating Rats

The fact that opiates elicit prolactin secretion is well known. However, we have recently discovered that morphine does not stimulate prolactin release in lactating rats. The physiological basis for this alteration in opiate sensitivity during lactation is not known. Since morphine-induced prolactin secretion in male rats is mediated via the mu opioid receptor subtype, one possible explanation is that mu receptors are down-regulated during lactation. To address this possibility, the effects of mu opioid peptides on prolactin secretion were examined in lactating rats. The presumed mu-selective peptides DAGO ([D-Ala², Me-Phe⁴, Gly-ol⁵]-enkephalin) and PLO-17 ([NMe-Phe³, D-Pro⁴]-morphiceptin) were administered to primiparous lactating rats and the resulting hormone responses measured. Both DAGO and PLO-17 caused a rapid and significant rise in plasma prolactin during lactation. The prolactin-releasing effects of both peptides were naloxone reversible, suggesting involvement of opioid receptors. Moreover, the DAGO-induced secretion of prolactin could be completely abolished by pretreatment with the irreversible mu antagonist β-funaltrexamine. In lactating rats, DAGO and PLO-17 were poor growth hormone-releasing agents, providing further evidence for the mu specificity of these peptides. These results imply that during lactation, as in other reproductive states, mu opioid receptor sites are positively coupled to the prolactin secretory mechanism. Thus, the previously observed inability of morphine to elicit prolactin release in lactating rats cannot be explained on the basis of down-regulation of mu opioid receptors.