Morphine withdrawal activates hypothalamic-pituitary-adrenal axis and heat shock protein 27 in the left ventricle: the role of extracellular signal-regulated kinase.

The negative affective states of withdrawal involve the recruitment of brain and peripheral stress circuitry [e.g., noradrenergic activity, induction of the hypothalamo-pituitary-adrenocortical (HPA) axis, and the expression and activation of heat shock proteins (Hsps)]. The present study investigated the role of extracellular signal-regulated protein kinase (ERK) and ß-adrenoceptor on the response of stress systems to morphine withdrawal by the administration of [amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile (SL327), a selective inhibitor of ERK activation, or propranolol (a ß-adrenoceptor antagonist). Dependence on morphine was induced by a 7-day subcutaneous implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by the injection of naloxone (2 mg/kg s.c.). Plasma concentrations of adrenocorticotropin and corticosterone were determined by radioimmunoassay; noradrenaline (NA) turnover in left ventricle was determined by high-performance liquid chromatography; and catechol-O-methyl transferase (COMT) and Hsp27 expression and phosphorylation at Ser82 were determined by quantitative blot immunolabeling. Morphine-withdrawn rats showed an increase of NA turnover and COMT expression in parallel with an enhancement of adrenocorticotropin and plasma corticosterone concentrations. In addition, we observed an enhancement of Hsp27 expression and phosphorylation. Pretreatment with SL327 or propranolol significantly reduced morphine withdrawal-induced increases of plasma adrenocorticotropin and Hsp27 phosphorylation at Ser82 without any changes in plasma corticosterone levels. The present findings demonstrate that morphine withdrawal is capable of inducing the activation of HPA axis in parallel with an enhancement of Hsp27 expression and Hsp27 phosphorylation at Ser82 and suggest a role for ß-adrenoceptors and ERK pathways in mediating morphine-withdrawal activation of the HPA axis and cellular stress response.

Regulation of mu-opioid receptors, G-protein-coupled receptor kinases and beta-arrestin 2 in the rat brain after chronic opioid receptor antagonism.

The aim of this study was to analyse the biochemical and behavioural consequences of chronic treatment with opioid receptor antagonists in rats. We have evaluated the respiratory depressant and antinociceptive effects of the mu-opioid agonist sufentanil, the density of brain mu-opioid receptors, and the expression of G-protein-coupled receptor kinases and beta-arrestin 2 in cerebral cortex and striatum, following sustained opioid receptor blockade. Our results demonstrate that 24 h after interruption of 7 days chronic infusion of naltrexone (120 microg/h), the respiratory depressant potency of the mu-opioid receptor agonist sufentanil was increased to a similar extent as the antinociceptive potency (about three-fold). This was accompanied by mu-opioid receptor up-regulation in several areas of the rat brain associated with opioid control of pain perception and breathing. Moreover, chronic treatment with either naltrexone (120 microg/h) or naloxone (120 microg/h) caused significant increases in the expression levels of G-protein-coupled receptor kinases types 2, 3, and 6, and of beta-arrestin 2 in brain cortex and striatum. Together our data suggest an increased constitutive receptor activity secondary to mu-opioid receptor up-regulation following chronic antagonist treatment.

Changes in the expression of G protein-coupled receptor kinases and beta-arrestin 2 in rat brain during opioid tolerance and supersensitivity.

We previously demonstrated that chronic treatment of rats with the mu-opioid receptor agonist sufentanil induced pharmacological tolerance associated with mu-opioid receptor desensitization and down-regulation. Administration of the calcium channel blocker nimodipine during chronic treatment with sufentanil prevented mu-opioid receptor down-regulation, induced down-stream supersensitization, and produced supersensitivity to the opioid effects. The focus of the present study was to determine a role for G protein-coupled receptor kinases (GRKs) and beta-arrestin 2 in agonist-induced mu-opioid receptor signalling modulation during chronic opioid tolerance and supersensitivity. Tolerance was induced by 7-day chronic infusion of sufentanil (2 microgram/h). Supersensitivity was induced by concurrent infusion of sufentanil (2 microgram/h) and nimodipine (1 microgram/h) for 7 days. Antinociception was evaluated by the tail-flick test. GRK2, GRK3, GRK6 and beta-arrestin 2 immunoreactivity levels were determined by western blot in brain cortices. Acute and chronic treatment with sufentanil induced analgesic tolerance, associated with up-regulation of GRK2, GRK6, and beta-arrestin 2. GRK3 expression only was increased in the acutely treated group. When nimodipine was associated to the chronic opioid treatment, tolerance expression was prevented, and immunoreactivity levels of GRK2, GRK6 and beta-arrestin 2 recovered the control values. These data indicate that GRK2, GRK3, GRK6 and beta-arrestin 2 are involved in the short- and long-term adaptive changes in mu-opioid receptor activity, contributing to tolerance development in living animals. These observations also suggest that GRKs and beta-arrestin 2 could constitute pharmacological targets to prevent opioid tolerance development, and to improve the analgesic efficacy of opioid drugs.

Autoradiographic mapping of mu-opioid receptors during opiate tolerance and supersensitivity in the rat central nervous system.

In this autoradiographic study we have analysed the regional changes in the density of mu-opioid receptors produced by the chronic administration of sufentanil alone and after concurrent administration with nimodipine. mu-Opioid receptors in the central nervous system (CNS) of rats were labelled using 5 nM [3H]DAMGO. Sufentanil, a high-efficacy agonist, was administered for 7 days by chronic infusion (2 microg/h). Another group of animals received a simultaneous infusion of sufentanil (2 microg/h) and nimodipine (1 microg/h) for 7 days. These two drug regimes have been previously shown to induce tolerance and supersensitivity to the analgesic effect of the opioid, respectively. Our results clearly demonstrate that opioid tolerance is associated with a generalised down-regulation of mu-opioid binding sites throughout the brain and the spinal cord. Compared with the findings in tolerant animals, the CNS of animals supersensitive to sufentanil showed less down-regulation of mu-opioid receptors, to the extent that, particularly in brain areas related to nociception, such as the somatosensory cortex, central grey, raphe magnus nucleus and dorsal horn of the spinal cord, no down-regulation occurred. These neurochemical findings may contribute to the functional interaction between nimodipine and sufentanil that we have previously observed in analgesic studies.

Involvement of the cyclic AMP system in the switch from tolerance into supersensitivity to the antinociceptive effect of the opioid sufentanil.

1. We have previously demonstrated that chronic and simultaneous treatment of rats with the mu-opioid receptor agonist sufentanil and the Ca(2+) channel blocker nimodipine, not only prevented tolerance development, but the animals became supersensitive to the antinociceptive effect of the opioid. The focus of the present work was to determine the possible involvement of cross interactions between the adenylyl cyclase pathway and L-type voltage-sensitive Ca(2+)-channels, in modulating the switch from opioid tolerance into supersensitivity. 2. The modulatory effect of sufentanil on adenylyl cyclase activity was determined by measuring cyclic AMP production in slices from the cortex of rats rendered tolerant or supersensitive to the antinociceptive effect of the opioid. Tolerance was induced by chronic infusion of sufentanil, at a rate of 2 microg h(-1), for 7 days. Supersensitivity was induced by concurrent infusion of sufentanil (2 microg h(-1)) and nimodipine (1 microg h(-1)) for 7 days. Antinociception was evaluated by the tail-flick test. 3. Tolerance to the analgesic effect of sufentanil was associated with a significant reduction in the response of adenylyl cyclase to forskolin. Furthermore, the effect of the opioid on forskolin-induced cyclic AMP accumulation was abolished. On the other hand, supersensitivity to the analgesic effect of the opioid was associated with an increase in both, the adenylyl cyclase response to forskolin, and the opioid inhibition of cyclic AMP production. 4. We suggest that sustained L-type Ca(2+) channel blockade may result in changes in the adenylyl cyclase effector system triggered by mu-opioid receptor activation, leading to the switch from opioid tolerance into supersensitivity.

Effects of kappa- and mu-opioid receptor agonists on Ca2+ channels in neuroblastoma cells: involvement of the orphan opioid receptor.

The effects of micro-, delta- and kappa-opioid receptor agonists, and orphanin FQ/nociceptin (Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln), on K+-induced [Ca2+]i increase were examined in SK-N-SH cells. Exposure to K+ (50 mM) resulted in a [Ca2+]i rise, which was blocked (-85%) by furaldipine (1 microM) and increased (63%) by BayK 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethyl-pyridine-5 -carboxylate) (0.5 microM), indicating the involvement of L-type Ca2+ channels. The kappa-opioid receptor agonists 3,4-dichloro-N-Methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U-50488H) (1-50 microM) and 5,7,8-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]benze neacetamide (U-69593) (25 microM), and the mu-opioid receptor agonist sufentanil (100 nM-3 microM) inhibited the amplitude of K+-induced [Ca2+]i increase. The agonist of the orphan opioid receptor, orphanin FQ/nociceptin (1 microM), induced dual excitatory and inhibitory effects on the depolarisation-induced Ca2+ influx. The effects of the opioid receptor agonists were not blocked by the kappa-opioid receptor antagonist nor-binaltorphimine (1 microM), only weakly prevented by naloxone (10-100 microM) and naltrexone (100 microM), and partially prevented by pertussis toxin (100 ng/ml, 24 h). The antagonist of the orphan opioid receptor, [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 (1 microM), prevented the inhibitory effect of U-50488H, sufentanil and orphanin FQ. The present study provides pharmacological evidence for the presence of L-type Ca2+ channels in SK-N-SH cells, that are modulated by opioids through orphan opioid receptor activation.

Nimodipine-enhanced opiate analgesia in cancer patients requiring morphine dose escalation: a double-blind, placebo-controlled study.

The ability of nimodipine, a dihydropyridine calcium antagonist, to reduce the daily dose of oral morphine in cancer patients who had developed dose escalation, was tested in 54 patients under randomized, double-blind, placebo-controlled conditions. We selected patients that required at least two successive increments of morphine to maintain pain relief. A possible pharmacokinetic interaction between nimodipine and morphine was also studied in 14 patients by assaying steady-state serum levels of morphine and its 3- and 6-glucuronides. A total of 30 patients completed the study, 14 and 16 in the nimodipine and placebo groups, respectively. Nimodipine controlled the escalation of the morphine dose in 9 patients (65%), and placebo in 4 (28%), the difference being statistically significant (P=0.03). The dose of morphine was reduced from 313+/-52 to 174+/-33 mg/day (P < 0.001) in the nimodipine group, and from 254+/-26 to 218+/-19 mg/day (not significant) in the placebo group. The percentages of reduction in the daily dose of morphine also showed significant differences between both groups (P=0.02). One week after introducing nimodipine or placebo, while the dose of morphine remained similar to that of the pre-test week, the serum levels of morphine and its glucuronides were not modified significantly. We conclude that the introduction of nimodipine in patients chronically treated with morphine may be a safe alternative to reduce the daily requirements of the opioid. It is suggested that interference with Ca2+-related events may attenuate the development and/or expression of tolerance to morphine in a clinically relevant way.

Mu-opioid receptor regulation during opioid tolerance and supersensitivity in rat central nervous system.

We have analyzed by radiometric procedures in rat central nervous system the changes in the properties of mu-opioid receptors associated with tolerance and supersensitivity to the opioid agonist sufentanil. This study has used [3H]-[D-Ala2,MePhe4,Gly- (ol)5(2)]-enkephalin, a highly selective ligand, to label mu-opioid receptors in both membranes and tissue sections. The induction of opioid tolerance by chronic infusion for 7 days of high doses of sufentanil, a high efficacy agonist, produced mu-opioid receptor down-regulation, with a significant decrease in their density in both cortical (-67%) and spinal cord membranes (-55%) and no changes in the affinity constant. Autoradiographic studies showed an overall decrease of[3H]-Ala2,MePhe4,Gly-(ol)5(2)]-enkephalin binding in the somatosensory cortex (around -30%). When the dihydropyridine-Ca++ channel antagonist nimodipine was administered alone for 7 days, no significant changes in the density or affinity constant of mu-opioid receptors were observed. However, the chronic and simultaneous administration of nimodipine and sufentanil (7 days), induced a pronounced modification on the density of mu-opioid receptors of the rat central nervous system and blocked the down-regulation observed in sufentanil-treated (tolerant) rats. These neurochemical findings may account for the functional interaction we have observed previously in the analgesic studies between nimodipine and sufentanil. Our data strongly suggest a functional role of L-type Ca++ channels in the mediation of opioid tolerance and super-sensitivity.

Regulation of dihydropyridine-sensitive Ca++ channels during opioid tolerance and supersensitivity in rats.

The changes in cerebral dihydropyridine (DHP)-sensitive Ca++ channels (L-type) associated with tolerance and supersensitivity to the antinociceptive effect of the mu-opioid receptor agonist sufentanil were analyzed in rats. The tail-flick test was used to assess the nociceptive threshold. DHP binding and autoradiographic assays were performed with [3H]nimodipine and [3H]PN 200-110 [isopropyl 4-(2,1,3-benzoxadizol-4-yl)- 1,4-dihydro-2,6-dimethyl-5-methoxycarbonylpyridine-3-carboxylate], respectively. Chronic s.c. infusion of sufentanil (2 micrograms/hr) for 7 days induced tolerance (tolerance index, 5.6) in association with up-regulation of DHP binding sites in cerebral cortex membranes (+36%), as well as in brain sections. Animals were rendered hypersensitive to the antinociceptive effect of sufentanil by chronic and simultaneous infusion of sufentanil (2 micrograms/hr) and nimodipine (1 microgram/hr) for 7 days (potentiation index, 40 vs. tolerant). Under these conditions, a greater increase in the number of DHP binding sites was observed in cortex membranes (+71%), and more evidently in brain sections. In these animals, withdrawal of nimodipine for 48 hr returned the dose-response curve of sufentanil to the tolerant values, whereas Ca++ channels remained increased. The role of an increased influx through L-type channels in opioid tolerance is reinforced. Our results also suggest that, although changes in neuronal Ca++ fluxes are not the only underlying mechanism, the increase and the sustained blockade of Ca++ channels with nimodipine is essential for the expression of opioid supersensitivity.

Kappa-opioid receptor mediated antinociception in rats is dependent on the functional state of dihydropyridine-sensitive calcium channels.

The modulatory effect of the dihydropyridine Ca2+ channel antagonist nimodipine on the analgesic action of the kappa-opioid receptor agonist U-69,593 was analyzed using the tail-flick test in rats. The antinociceptive effect of U-69,593 (0.25-4 mg/kg) was antagonized by L-type Ca2+ channel blockade with nimodipine (200 microgram/kg, i.p.), the ED50 being increased from 1.4 to 7.3 mg/kg. On the contrary, when an increase in the density of these channels was induced by means of chronic and simultaneous treatment with nimodipine (1 microgram/h, 7 days) and sufentanil (2 micrograms/h, 8 days), the analgesic effect of U-69,593 was potentiated by 5-fold. Our results suggest a functional coupling between kappa-opioid receptors and L-type Ca2+ channels in nociception.

Enhancement of opiate analgesia by nimodipine in cancer patients chronically treated with morphine: a preliminary report.

The ability of nimodipine, a calcium-channel blocker, to enhance morphine analgesia and/or modify the development of tolerance was studied in patients with cancer pain who had needed successive increments of morphine for periods ranging from 21 to 780 days. Assessment of daily morphine consumption was the primary effect parameter. Nimodipine succeeded in reducing the daily dose of morphine in 16 of 23 patients (oral, n = 13; intrathecal, n = 3), and failed to modify it in 2 patients. Total oral daily dose was reduced by nimodipine (120 mg/day) from 282.6 +/- 47.7 mg to 158.7 +/- 26.2 mg (n = 15, P < 0.001). Intrathecal morphine was also reduced by 1-5 mg/day. Nimodipine was withdrawn in 5 patients during the first week of treatment due to intolerance (n = 3) or aggravation of the disease (n = 2). These preliminary results support experimental findings showing that pharmacological interference with Ca(2+)-related events may modify chronic opioid effects, including the expression of tolerance.

Potentiation of acute opioid-induced respiratory depression and reversal of tolerance by the calcium antagonist nimodipine in awake rats.

The interaction between sufentanil, a mu-opioid agonist, and the Ca2+ antagonist nimodipine on respiration and on the development of opioid tolerance in awake rats has been analyzed. Our previous work demonstrated that chronic treatment with nimodipine together with sufentanil increases the analgesic potency of the opioid 50 fold. Therefore, we have investigated whether the opioid-induced respiratory depression is potentiated in parallel with the analgesia. Ventilation was measured by the whole body plethysmographic method. In naive rats, sufentanil (10-80 micrograms/kg) consistently induced a dose-dependent respiratory depression. Pretreatment with nimodipine (200 micrograms/kg) potentiated this effect but to a lesser extent than it potentiated analgesia. After chronic administration of the opioid (2 micrograms/h, 7 days) tolerance was manifested as a reduction in both the area under the time course curve and in the maximum effect. Nimodipine (1 microgram/h) administered concurrently with sufentanil for 7 days counteracted the tolerance to respiratory depression but no additional potentiation was observed. These results demonstrate that the interaction between nimodipine and sufentanil is not limited to antinociception but also extends to respiratory depression. However, compared with analgesia, the clinical relevance of a potential increase in opioid-induced respiratory depression by nimodipine may be negligible.

The Ca2+ channel agonist Bay K 8644 induces central respiratory depression in cats, an effect blocked by naloxone.

We analyzed the respiratory effects induced by the Ca2+ channel agonist Bay K 8644 and its enantiomers, Bay R 5417 and Bay R 4407, applied to the ventral medullary surface of cats. Bay K 8644 (10 to 100 micrograms) and Bay R 5417 (50 to 200 micrograms) elicited a dose-dependent respiratory depression. Naloxone (0.1 mg/kg), but not D-naloxone, reversed these effects, indicating that an endogenous opioid mechanism was involved. Bay R 4407 (100 micrograms) was ineffective. The respiratory depressant effects induced by Bay K 8644 and its (-) enantiomer were a consequence of their agonist properties on the L-type Ca2+ channel, since (1) the activity of Bay K 8644 was stereospecific, and (2) nimodipine prevented the effect. We suggest that potent activation of Ca2+ channels or other mechanisms by high doses of Ca2+ agonists elicits the release of endogenous opioid peptides in medullary respiration-related structures.

Ca2+ channel modulation by dihydropyridines modifies sufentanil-induced respiratory depression in cats.

We analyzed the interaction between sufentanil, a selective mu agonist, and two dihydropyridines, the Ca2+ antagonist, nimodipine, and the Ca2+ agonist, Bay K 8644, on the respiratory actions induced in the brainstem of cats. Drugs were applied topically to the ventral medullary surface. Sufentanil (0.26-26 nmol) consistently induced an immediate and dose-dependent reduction in tidal volume. Respiratory frequency was only depressed by the higher doses of the opiate. Pretreatment with nimodipine (0.19 and 0.38 mumol) potentiated the respiratory depression induced by sufentanil (0.26 nmol). The potentiation included both frequency and tidal volume. On the other hand, under the influence of Bay K 8644 (0.28 nmol), the respiratory effect of the opiate (7.8 nmol) was partially antagonized. Our results indicate that modulation of the L-type Ca2+ channels by dihydropyridines modifies sufentanil-induced respiratory depression at the controlling medullary mechanisms of breathing.

Calcium channel modulation by dihydropyridines modifies sufentanil-induced antinociception in acute and tolerant conditions.

The study was aimed at elucidating the possible participation of L-type Ca2+ channel in the acute analgesic effect of an opiate and the development of tolerance to this action. Sufentanil, a selective mu agonist, and two dihydropyridines, the Ca2+ antagonist nimodipine and the Ca2+ agonist Bay K 8644, were selected. The tailflick test was used to assess the nociceptive threshold. In naive rats, nimodipine (200 micrograms/kg) potentiated the analgesic effect of sufentanil reducing the ED50 from 0.26 to 0.08 microgram/kg. Similar results were observed with its (-)-enantiomer Bay N 5248, while the (+) enantiomer Bay N 5247 was ineffective. Tolerance to the opiate was induced by chronic subcutaneous administration of sufentanil with minipumps (2 micrograms/h, 7 days). In these conditions the dose-response curve to sufentanil was displaced to the right and the ED50 was increased to 1.49 micrograms/kg. In tolerant rats, nimodipine preserved its potentiating ability and prevented the displacement to the right of the sufentanil dose response-curve (ED50 = 0.48 microgram/kg). When nimodipine was pumped (1 microgram/h, 7 days) concurrently with sufentanil, the development of tolerance to the opioid was not disturbed. However, the expression of tolerance was abolished and even the effect of acutely administered sufentanil was markedly potentiated (ED50 = 0.03 microgram/kg). Similar experiments were performed with Bay K 8644. In naive rats, Bay K 8644 at a low dose (20 micrograms/kg) that behaves as a calcium agonist, antagonized the analgesic effect of sufentanil (ED50 = 0.58 microgram/kg), whereas at a high dose (200 micrograms/kg) it potentiated this action (ED50 = 0.15 microgram/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of interaction between cholecystokinin and opioid systems in the central control of breathing.

The present study investigates the interaction between CCK and opioid systems on the central control of breathing by analyzing the respiratory effects of CCK-8S (0.09 nmol) and met-enkephalin (0.7 and 1.4 mumol) applied to the ventral medullary surface of cats under the influence of the opiate antagonist naloxone and the CCK antagonist proglumide. Neither naloxone nor proglumide were able to modify the respiratory effects of CCK-8S and met-enkephalin, respectively. Therefore, the hypothesis that CCK and the opioid systems might operate as parallel and antagonic forces throughout the central nervous system, is no longer tenable.

Respiratory actions induced by cholecystokinin at the brainstem level.

A functional differentiation of the action of cholecystokinin octapeptide (CCK-8) on the respiratory centers was accomplished by the topical application to the ventral surface of the medulla and to the dorso-rostral pontine surface in cats. In the medulla, CCK-8S at doses ranging from 0.09 nmol to 0.88 nmol, stimulated tidal volume in a dose-dependent fashion, with minimal or no changes in frequency. The antagonist proglumide (30 nmol) inhibited specifically the action on the respiratory amplitude. In the pons, CCK-8S did not modify the respiratory activity even at the dose of 8.8 nmol. The results suggest a specific involvement of CCK-8S in the mechanisms controlling respiratory amplitude, which appear mostly restricted to the medullary level. The lack of effect of the peptide in the pons is in agreement with the absence of CCK receptors in the respiration related nuclei located at that level, as evidenced by autoradiographic studies.

Mechanism of the respiratory action of pentobarbital at the medullary and pontine levels.

A functional differentiation of the respiratory action of pentobarbital was made by applying the drug in cats to the ventral surface of the medulla and to the dorso-rostral surface of the pons. The involvement of a GABAergic mechanism was assessed by studying the interaction of bicuculline with pentobarbital at both levels of the brain-stem. In the medulla, pentobarbital (4 to 16 mg) caused an immediate and dose-dependent depression of tidal volume down to apnea with a minimal or no change in frequency. In the pons, the depression affected selectively the frequency (-45%) without modification in amplitude. Medullary structures were much more sensitive to the action of pentobarbital. Doses that induced apnea in the medulla did not attain 50% depression of the minute volume in the pons. Bicuculline (300 micrograms) completely antagonized the effect of pentobarbital in both regions, suggesting that a GABAergic mechanism may be involved in the respiratory action of pentobarbital.

Differential respiratory patterns induced by opioids applied to the ventral medullary and dorsal pontine surfaces of cats.

The purpose of the present study was to make a functional dissection of the respiratory action of opioids, by their restricted application to the ventral surface of the medulla oblongata and to the rostro-dorsal surface of the pons in cats. The effects were compared to those induced after intracerebroventricular (i.c.v.) injection. Two mu-agonists, morphine and D-Ala2-Me-Phe4-Met(O)ol5-enkephalin (FK-33824), and the delta-agonist D-Ala2-D-Leu5-enkephalin (DADLE) were used. When applied to the ventral medullary surface, the opioids selectively depressed the generating mechanisms for tidal volume and the response to CO2, whereas the frequency was increased. The application to the rostral dorsal surface of the pons was followed by a selective depression of the respiratory frequency. By intracerebroventricular administration, the opioids depressed both the tidal volume and frequency generating mechanisms. The effects were always reversed by naloxone. The pontine structures were more sensitive to the action of the opioids than were the medullary centres. These findings suggest that the opioids can interact with different populations of respiratory neurones and that the respiratory output differs depending on the group of neurones selectively affected and the function they subserve in regulating respiratory activity.