Opiate withdrawal-induced hyperactivity of locus coeruleus neurons is substantially mediated by augmented excitatory amino acid input.

Single-cell activity was recorded in the locus coeruleus (LC) of morphine-dependent, halothane-anesthetized rats. Systemic administration of the opiate antagonist naloxone (0.1 mg/kg, i.v.) robustly increased the activity of LC neurons. Local microinjection of naloxone or of its hydrophilic derivative, naloxone methiodide, into LC (10 mM, 20-40 nl) did not activate LC neurons in dependent rats. Intracerebroventricular or intracoerulear injection of kynurenate, a broad-spectrum antagonist of excitatory amino acids (EAAs), substantially but incompletely attenuated the activation of LC cells induced by intravenous naloxone-precipitated withdrawal (more than 50% blockade). Intracoerulear microinjections of the non-NMDA-receptor antagonist 6-cyano-7-dinitroquinoxaline-2,3-dione (CNQX) or the selective NMDA-receptor antagonist AP5 significantly reduced the withdrawal-induced excitation. AP5 was the least effective among all antagonists tested. Similar microinjections of kynurenate or CNQX almost completely suppressed the excitation of LC neurons induced by electrical stimulation of a rear footpad. LC responses to footpad stimulation (mediated by endogenous EAAs) or iontophoretically applied glutamate were not modified by the chronic morphine treatment. These results indicate that a substantial part of LC hyperactivity during opiate withdrawal is mediated by an augmented EAA input to LC.     

Single neurone studies of opioid tolerance and dependence at the ventrobasal thalamic level in an experimental model of clinical pain, the arthritic rat

The aim of this electrophysiological study was to investigate the effects of an acute injection of morphine (1 mg/kg i.v.) or the opioid antagonist naloxone (0.6-2 mg/kg i.v.) on thalamic ventrobasal (VB) neuronal activities recorded in arthritic rats rendered tolerant/dependent by pretreatment with relatively low doses of morphine. Recordings were performed in animals immobilized by i.v. injections of gallamine triethiodide (Flaxedil) and artificially ventilated under a moderate gaseous anesthesia (mixture of one-third O2, two-thirds N2O, 0.5-0.6% halothane). This level of anesthesia, as checked by the electrocorticogram, was stable and appeared sufficiently deep, since no sign of suffering or stress could be detected. The efficacy of morphine on VB neuronal responses induced by mild stimulation of the joints was greatly reduced in morphine-pretreated arthritic rats, compared to naive animals (mean neuronal inhibition of 35 vs 85%, respectively). This indicates that the tolerance phenomena observed in behavioral studies are reflected at the VB level, on neurons involved in pain processes. In addition, naloxone (0.6, 1 and 2 mg/kg i.v.) induced a dramatic increase in the evoked (52, 88 and 93%) and spontaneous (64, 211 and 292%) VB neuronal activities recorded in morphine-pretreated arthritic rats, while these activities were not significantly altered in naive arthritic rats. The time-courses of the modifications induced by naloxone in morphine-pretreated arthritic animals were similar to those of the naloxone-precipitated morphine withdrawal observed in freely moving rats. These findings may represent the neuronal correlate at the VB level of the withdrawal response and/or the hyperalgesia induced in tolerant arthritic rats by high doses of naloxone.     

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.     

Cardiovascular and behavioural effects induced by naloxone-precipitated morphine withdrawal in rat: characterization with tachykinin antagonists

This study examined the intracerebroventricular (i.c.v.) effects of three selective tachykinin receptor antagonists on the cardiovascular and behavioural responses induced by naloxone-precipitated morphine withdrawal in rats. I.c.v. injection of naloxone (10 microg) to morphine pre-treated rats (i.c.v. for 5 days) induced an immediate increase in blood pressure ( approximately 10 mmHg) and behavioural activity (sniffing > rearing > face washing approximately grooming approximately wet dog shake) without causing significant heart rate changes. The prior i.c.v. injection of the NK(1) receptor antagonist (6.5 nmol LY306740) reduced face washing and grooming during morphine withdrawal. NK(2) and NK(3) receptor antagonists (6.5 nmol SR48968 and R820) did not affect behavioural effects, yet the co-injection of the three tachykinin antagonists reduced all behavioural activity. The pressor response was not affected by the selective inhibition of NK(1) and NK(3) receptors while both blood pressure and heart rate were markedly enhanced by SR48968 during morphine withdrawal. The potentiating effect of SR48968 was prevented following simultaneous blockade of the three tachykinin receptors. In addition to confirming the involvement of central tachykinins in behavioural manifestations to morphine withdrawal, data suggest a modulatory function for tachykinins, especially the NK(2) receptor, in brain autonomic control of blood pressure and heart rate in supraspinal noloxone-precipitated withdrawal.     

Excitatory action of N-methyl- -aspartate on the rat locus coeruleus is mediated by nitric oxide: an in vivo voltammetric study

Biochemical, electrophysiological and behavioural studies have provided evidence that activation of N-methyl- -aspartate (NMDA) receptors contributes to the hyperactivity of noradrenergic neurons of the locus coeruleus (LC) in precipitated opioid withdrawal. Recently, it was demonstrated that central administration of nitric oxide (NO) synthase inhibitors suppresses this hyperactivity suggesting that NO mediates the NMDA receptor activation of LC in opioid withdrawal. Using a combination of microdialysis and in vivo voltammetry, this study examined whether local application of NMDA to the LC in opioid naive animals mimics the NO-dependent LC response seen in opioid withdrawal. In the urethane anaesthetized rat, perfusion of the LC (2 μl min⁻¹) with a solution of NMDA (5 mmol) via a microdialysis probe for 9 min resulted in a rapid and robust increase (290.1±32.2% above baseline) in the catechol oxidation current (CA·OC) recorded from the LC using differential normal pulse voltammetry (DNPV). The NMDA microdialysis also produced a large increase in the blood pressure (150.4±6.9% above baseline). An injection of the non-competitive NMDA receptor antagonist (+)MK-801 (0.5 mg kg⁻¹ i.v.), given 45 min after the start of NMDA application, rapidly returned both the CA·OC signal and the blood pressure response to baseline levels. Pretreatment of animals with intraventricular nitric oxide synthase (NOS) inhibitor, N^ω-nitro- -arginine methyl ester ( -NAME) (100 μg) significantly inhibited NOS activity in the LC, PAG-PVG and cerebellum. This dose of -NAME, administered prior to application of NMDA by microdialysis abolished the NMDA-induced rise in the CAOC recorded in the LC and the increase in systolic blood pressure. The results show that in voltammetry experiments, NMDA produces hyperactivity of LC and hypertension, responses that are dependent upon the synthesis of NO. Thus, in opioid naive rats, regional NMDA application via microdialysis mimics characteristics of the LC response that occur during the antagonist-precipitated opioid withdrawal.     

Effects of opioid agonist and antagonist in dams exposed to morphine during the perinatal period

The aim of the present work was to further analyse the features of opioid dependence following chronic morphine treatment during pregnancy and lactation. Dams from the day of mating were treated either with saline or with morphine (10 mg/kg) subcutaneously once daily. Physical and behavioural signs of morphine withdrawal were investigated both in the early postpartum period (maternal behaviour) and after weaning (physical signals, locomotion, anxiety-like behaviour). Maternal behaviour was evaluated after acute challenge with naloxone (3 mg/kg s.c.) or morphine (10 mg/kg s.c.) and morphine plus naloxone (10 mg/kg s.c. and 3 mg/kg s.c., respectively). After weaning sensitivity to the rewarding effect of morphine was measured by conditioned place preference and to the aversive effect of naloxone by conditioned place aversion tests. The intensity of physical and behavioural indices of dependence was also investigated by precipitation of withdrawal with naloxone (10 mg/kg s.c) after weaning. Naloxone impaired the maternal behaviour in morphine-treated dams but not in saline-ones. Acute challenge with morphine impaired maternal responsiveness both in saline and in morphine-treated dams, this effect of morphine, however could be completely antagonised by naloxone only in the saline-treated but not in the morphine-treated ones. Significantly increased sensitivity to the rewarding stimulus of morphine and more pronounced aversion to naloxone were observed in morphine-treated dams. Naloxone precipitated only moderate physical withdrawal signals in morphine-treated dams, while anxiety and locomotor activity after administration of naloxone (behavioural withdrawal) were not changed in them. In summary chronic, moderate dose morphine treatment during pregnancy and lactation resulted in only mild dependence, but it affected opioid-receptor sensitivity and presumably disrupted the functioning of endogenous opioid system.     

Inhibitory effects of diltiazem, an L-type Ca channel blocker, on naloxone-increased glutamate levels in the locus coeruleus of opioid-dependent rats

To investigate the effects of diltiazem, an L-type Cat²⁺ channel blocker, on naloxone-precipitated withdrawal and elevations of glutamate levels in the locus coeruleus (LC) of opioid-dependent rats, animals were continuously infused with 26 nmol/μl/h of morphine or butorphanol intracerebroventricularly (i.c.v.) via osmotic minipumps for 3 days. l.c.v. injection of naloxone (an opioid-receptor antagonist, 48 nmol/5 μl) precipitated withdrawal signs and increased extracellular fluid levels of glutamate in the LC of morphineor butorphanol-dependent rats measured by in vivo microdialysis. Meanwhile, concomitant infusion of opioids with diltiazem (10 or 100 nmol/μl/h) inhibited the withdrawal signs and prevented the elevations of glutamate levels in the LC. These results suggest that an expeditious release of glutamate in the LC region regulated by L-type Ca²⁺ channels mediated system plays a role in the expression of withdrawal signs from opioids.     

Role of soluble guanylate cyclase in dilator responses of the cerebral microcirculation

Responses of cerebral blood vessels to nitric oxide (NO) are mediated by soluble guanylate cyclase (sGC)-dependent and potentially by sGC-independent mechanisms. One sGC-independent mechanism by which NO may produce vasodilatation is inhibition of formation of a vasoconstrictor metabolite produced through the cytochrome P450 pathway. In these experiments, we examined the hypothesis that dilatation of cerebral microvessels in response to NO is dependent on activation of sGC. Diameters of cerebral arterioles (baseline diameter=94+/-5 micrometers, mean+/-S.E.) were measured using a closed cranial window in anesthetized rabbits. Under control conditions, YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole], an NO-independent activator of sGC, produced vasodilation that was blocked by ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one)(10 microM), an inhibitor of sGC. These findings indicate that sGC is functionally important in cerebral arterioles. In addition, acetylcholine (which stimulates endogenous production of NO by endothelium) produced dilatation of cerebral arterioles that was inhibited by ODQ. For example, 1 microM acetylcholine dilated cerebral arterioles by 34+/-7 and 5+/-1% in the absence and presence of ODQ (10 microM), respectively. Increases in arteriolar diameter in response to sodium nitroprusside (1 microM, an NO donor) were inhibited by approximately 80% by ODQ, but were not affected by 17-ODYA (10 microM) or clotrimazole (10 microM), inhibitors of the cytochrome P450 pathway. Thus, dilatation of the cerebral microcirculation in response to exogenously applied and endogenously produced NO is dependent, in large part, on activation of sGC.     

Alpha-adrenergic mediation of the tail skin temperature response to naloxone in morphine-dependent rats

Studies were undertaken to evaluate the role of central noradrenergic neurons in the tail skin temperature (TST) surge that accompanies morphine withdrawal in the rat. A 5 degrees C increase in TST and a 1-2 degrees C decrease in rectal temperature (Tr) was observed following administration of a dose of naloxone HCl (NAL, 1 mg/kg, s.c.) which precipitated withdrawal in morphine-dependent rats. Intracerebroventricular (i.c.v.) injection of clonidine HCl, a partial alpha 2-adrenergic agonist did not alter TST in morphine-dependent animals. However, clonidine (10 or 50 micrograms/rat, i.c.v.) given 10 min prior to the administration of NAL completely blocked the TST response to the opiate antagonist in the morphine-dependent animals. Although NAL and clonidine reduced Tr to a similar extent in morphine-dependent rats, their effects were not additive when the drugs were administered sequentially. Treatment with the alpha-adrenergic antagonist phentolamine (11.9 or 60 micrograms/rat, i.c.v.) failed to alter TST when administered alone, but the highest dose significantly reduced the TST response to naloxone in the morphine-dependent rat. In addition, phentolamine, at high doses only, moderately reduced Tr, but the alpha-adrenergic antagonist failed to modify the decline in Tr associated with NAL-precipitated morphine withdrawal. Collectively, these data indicate that brain noradrenergic neurons play a role in the TST surge which accompanies NAL-precipitated morphine withdrawal, and that the TST and Tr responses can be dissociated in the morphine-dependent rat.     

Cross-tolerance between analgesic low doses of morphine and naloxone in arthritic rats

The effects of acute injections of naloxone (3-3000 micrograms/kg i.v.) and morphine (100-1000 micrograms/kg i.v.) on the vocalization threshold induced by pressure on the paw were analyzed in adjuvant-induced arthritic rats pretreated either with naloxone or with morphine administered at low doses (9 micrograms/kg s.c. and 3000 micrograms/kg s.c., respectively) over 4 consecutive days. In naloxone-pretreated arthritic rats, the paradoxical analgesic effect of low doses of naloxone was almost abolished, and the potent analgesic effects of low doses of morphine were also strongly and dose-dependently reduced. In morphine-pretreated arthritic animals, the analgesic effect of low doses of naloxone was significantly attenuated. These results attest that a cross-tolerance with low analgesic doses of morphine and naloxone can be demonstrated in these chronic suffering animals. By contrast, in rats pretreated either with naloxone or morphine, the hyperalgesic effect of naloxone produced by higher doses persisted and even was unmasked for doses which were analgesic before the pretreatment. These data emphasize the involvement of opiate receptors different in their sensitivity and/or their functions in the two opposite effects of naloxone. They also suggest that opiate receptors and endorphinergic systems differ in normal animals and animals which experience persistent pain.     

Supraspinal and spinal mediation of naloxone-induced morphine withdrawal in rats

Unanesthetized rats, made physically dependent over 5 days by chronic intra-arterial infusion of increasing concentrations of morphine (35-100 mg/kg/day) underwent withdrawal by naloxone (6 micrograms) injection into either the lateral ventricle (i.c.v.), fourth ventricle (V4), intrathecal subarachnoid space (i.t.), or intra-arterially (i.a.) and were evaluated for cardiovascular and behavioral signs of precipitated abstinence. Naloxone i.c.v. produced a significantly greater increase in the magnitude and duration of withdrawal hypertension than did V4 injection. Naloxone i.t. produced a distinctively different, persistent, pressor response as compared to i.c.v., V4 or i.a. routes of administration, although no quantitative differences in behavioral signs of withdrawal were observed. Morphine-dependent, spinal transected (C1) animals generated an augmented pressor response to i.c.v. or i.t. naloxone. This pressor response was accompanied by a significant reduction in core temperature (0.50-0.79 degrees C). Both the naloxone-induced pressor and hypothermic responses were abolished by ganglionic (hexamethonium, 100 mg/kg, i.a.) or peripheral alpha-adrenergic (phentolamine 4 mg/kg, i.a.) blockade. The hypertensive and hypothermic effects of naloxone also were prevented in transected dependent rats by prior spinal pithing. We conclude that in morphine-dependent rats: supraspinal sites rostral to the V4 mediate a more intense naloxone-induced pressor response than caudal regions; cardiovascular and behavioral signs of withdrawal can be precipitated via the spinal cord of intact animals; and the production of withdrawal hypertension and hypothermia in spinal transected morphine-dependent rats indicates that these abstinence signs can be mediated through neuronal pathways within the spinal cord.     

Dopamine microdialysis in the nucleus accumbens during acute and chronic morphine, naloxone-precipitated withdrawal and clonidine treatment.

This study shows the effect of opiate withdrawal on dopamine (DA) in the nucleus accumbens (NAC). Microdialysis was used to detect variations in extracellular DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the NAC of freely moving rats during acute and chronic morphine treatment followed by naloxone-precipitated withdrawal with and without clonidine. Basal levels of extracellular DA did not change between sessions, but morphine (20 mg/kg, i.p.) caused a significant and identical increase in extracellular DA and metabolites in both the acute phase (day 1) and the chronic phase (day 7). On day 8, naloxone (20 mg/kg i.p.) caused a significant decrease in DA levels accompanied by typical withdrawal symptoms such as wet dog shakes and teeth-chattering. Clonidine pretreatment (200 micrograms/kg, i.p.) eliminated both the withdrawal symptoms and the DA decrease. These results support the view that morphine increases extracellular DA at times when the drug is rewarding and also suggest that the converse may be true; morphine withdrawal decreases DA release in association with the aversive state.     

Dependence and withdrawal following intracerebroventricular and systemic morphine administration: functional anatomy and behavior

Regional cerebral glucose utilization (RCGU) and behavior during precipitated morphine withdrawal were studied in rats made dependent by either intracerebroventricular (i.c.v.) or subcutaneous (s.c.) administration of morphine. [14C]2-deoxy-D-glucose autoradiography revealed that RCGU increased in an anatomically related group of limbic and brainstem structures in rats that were in morphine withdrawal precipitated by naloxone administration compared to morphine-dependent controls that were not in precipitated withdrawal. Correlation of RCGU for 24 brain structures comparing i.c.v. vs s.c. morphine-treated rats was highly significant for groups in withdrawal and for controls (r values, 0.958 and 0.971, respectively). Withdrawal behaviors including autonomic signs of withdrawal, withdrawal jumping, and incidence of diarrhea were not different between the two groups in withdrawal (i.c.v. and s.c.). Weight loss during withdrawal increased (P less than 0.05) in rats made dependent by s.c. morphine administration compared to rats that received morphine by the i.c.v. route. Taken together, these results indicate that RCGU changes during morphine withdrawal result solely from effects of chronic morphine in the central nervous system, not in peripheral sites. The increased weight loss of s.c.-treated, morphine-dependent rats in withdrawal suggests an independent peripheral effect perhaps mediated by visceral opiate receptors.     

Baclofen prevents the elevated plus maze behavior and BDNF expression during naloxone precipitated morphine withdrawal in male and female mice

In previous studies we have shown that baclofen, a selective GABA_B receptor agonist, prevents the somatic expression and reestablishes the dopamine and μ‐opioid receptors levels, modified during naloxone‐precipitated morphine withdrawal syndrome in male and female mice. There are no previous reports regarding sex differences in the elevated plus maze (EPM) and the expression of BDNF in morphine‐withdrawn mice. The present study analyses the behavioral and biochemical variations during morphine withdrawal in mice of both sexes, and whether these variations are prevented with baclofen. Swiss‐Webster albino prepubertal mice received morphine (2 mg/kg, i.p.) twice daily, for 9 consecutive days. On the 10th day, one group of morphine‐treated mice received naloxone (opioid receptor antagonist; 6 mg/kg, i.p.) 1 h after the last dose of morphine to precipitate withdrawal. A second group received baclofen (2 mg/kg, i.p.) before naloxone administration. The EPM behavior was measured during 15 min after naloxone injection. The expression of BDNF‐positive cells was determined by immunohistochemistry. Withdrawn male mice showed a higher percentage of time spent and number of entries to the open arms compared to withdrawn female mice. Baclofen prevented this behavior in both sexes. BDNF expression decreased in the AcbC, BNST, CeC, and CA3 of the hippocampus while increased in the BLA of morphine withdrawn male. Baclofen pretreatment prevented the BDNF expression observed in morphine withdrawn male mice in all the brain areas studied except in the CeC. Baclofen prevention of the EPM behavior associated to morphine withdrawal could be partially related to changes in BDNF expression. Synapse, 2016 . © 2016 Wiley Periodicals, Inc. Synapse 70:187–197, 2016. © 2016 Wiley Periodicals, Inc.     

The effect of pituitary adenylate cyclase-activating polypeptide on elevated plus maze behavior and hypothermia induced by morphine withdrawal

The aim of the present investigation was to study the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on morphine withdrawal-induced behavioral changes and hypothermia in male CFLP mice. Elevated plus maze (EPM) and jump tests were used to assess naloxone-precipitated morphine withdrawal-induced behavior responses. Different doses of subcutaneous (s.c.) naloxone, (0.1 and 0.2 mg/kg, respectively) were used to precipitate the emotional and psychical aspects of withdrawal on EPM and 1 mg/kg (s.c.) was used to induce the somatic withdrawal signs such as jumping, and the changes in body temperature. In our EPM studies, naloxone proved to be anxiolytic in mice treated with morphine. Chronic intracerebroventricular (i.c.v.) administration of PACAP alone had no significant effect on withdrawal-induced anxiolysis and total activity at doses of 500 ng and 1 μg. At dose of 500 ng, however, PACAP significantly counteracted the reduced motor activity in the EPM test in mice treated with morphine and diminished the hypothermia and shortened jump latency induced by naloxone in mice treated with morphine. These findings indicate that anxiolytic-like behavior may be mediated via a PACAP-involved pathway and PACAP may play an important role in chronic morphine withdrawal-induced hypothermia as well.     

Differential effects of acute morphine, and chronic morphine-withdrawal on obsessive-compulsive behavior: Inhibitory influence of CRF receptor antagonists on chronic morphine-withdrawal

Recent studies have provided convincing evidences for co-morbidity between opioid addiction and obsessive-compulsive disorder (OCD), and the involvement of the corticotrophin-releasing factor (CRF) in the effects of morphine-withdrawal. Some scanty evidences also point towards the role of CRF in OCD and related disorders. But, no evidence indicated the role of CRF in morphine withdrawal associated obsessive-compulsive behavior (OCB). Therefore, the present study investigated the role of CRF in morphine-withdrawal induced OCB in mice. Marble-burying behavior in mice was used to assess OCB as this model has good predictive and face validity. The results revealed that acute morphine dose dependently attenuated the marble burying behavior, whereas withdrawal of chronic morphine was associated with significant rise in marble burying behavior. This indicates the differential effect of acute morphine and chronic morphine-withdrawal on OCB. Further, acute treatment with CRF receptor antagonists like antalarmin (2 and 4μg/mouse, i.c.v.) or astressin-2B (3 and 10nmol/mouse, i.c.v.) dose dependently attenuated the peak morphine-withdrawal induced increase in marble burying behavior. Moreover, concomitant treatment with antalarmin (4μg/mouse, i.c.v.) or astressin-2B (10nmol/mouse, i.c.v.) along with morphine blocked the morphine-withdrawal associated exacerbation of OCB. These results indicate that OCB associated with morphine withdrawal state is partly mediated by the activation of central CRF receptors.     

Microdialysis evidence that acetylcholine in the nucleus accumhens is involved in morphine withdrawal and its treatment with clonidine

This study used microdialysis to measure changes in extracellular acetylcholine (ACh) content in the nucleus accumbens (NAC) of freely moving rats during acute and chronic morphine treatment, and following naloxone-precipitated withdrawal. Morphine injection (20 mg/kg, i.p.) caused a significant decrease in extracellular ACh which was not apparent after repeated exposure to the opiate for 7 days. Basal recovery of ACh was not altered by chronic morphine treatment. On day 8, after morphine dependence had been established, naloxone caused a large increase in ACh levels accompanied by withdrawal symptoms such as wet dog shakes, diarrhea and teeth-chattering. Pretreatment with clonidine (200 μg/kg, i.p.) reduced these withdrawal symptoms and eliminated the ACh response. These results suggest that accumbens ACh is involved in some of the aversive aspects of opiate withdrawal.     

Microdialysis evidence that acetylcholine in the nucleus accumbens is involved in morphine withdrawal and its treatment with clonidine.

This study used microdialysis to measure changes in extracellular acetylcholine (ACh) content in the nucleus accumbens (NAC) of freely moving rats during acute and chronic morphine treatment, and following naloxone-precipitated withdrawal. Morphine injection (20 mg/kg, i.p.) caused a significant decrease in extracellular ACh which was not apparent after repeated exposure to the opiate for 7 days. Basal recovery of ACh was not altered by chronic morphine treatment. On day 8, after morphine dependence had been established, naloxone caused a large increase in ACh levels accompanied by withdrawal symptoms such as wet dog shakes, diarrhea and teeth-chattering. Pretreatment with clonidine (200 micrograms/kg, i.p.) reduced these withdrawal symptoms and eliminated the ACh response. These results suggest that accumbens ACh is involved in some of the aversive aspects of opiate withdrawal.     

Oxytocin and vasopressin release in discrete brain areas after naloxone in morphine-tolerant and -dependent anesthetized rats: push-pull perfusion study.

The effects of naloxone on the release of oxytocin and vasopressin in discrete brain areas were investigated in control and morphine-tolerant/dependent female rats anesthetized with urethane. Two or three consecutive push-pull perfusates were collected for 30-40 min each and the peptide contents measured by radioimmunoassay; naloxone (5 mg/kg, i.v.) was given after the first perfusion. In control rats, naloxone did not increase oxytocin release from any of the regions studied: mediolateral septum, dorsal hippocampus, nucleus of tractus solitarius, or supraoptic nucleus. After naloxone, vasopressin release was approximately doubled in the nucleus of tractus solitarius (p less than 0.05), indicating endogenous opioid inhibition of vasopressin release. Naloxone increased oxytocin concentration in the circulation 3.7-fold (p less than 0.001) but did not affect vasopressin secretion. In rats made morphine tolerant/dependent by intracerebroventricular infusion of morphine for 5 d, oxytocin and vasopressin release in the perfused brain was initially similar to that in control rats, indicating tolerance to any initial morphine effects. In these rats, naloxone increased oxytocin release in the septum threefold relative to control rats (p less than 0.02) but did not alter oxytocin release in hippocampus or nucleus of tractus solitarius. Thus, the oxytocin neurons projecting to septum can develop morphine dependence and may be inhibited acutely by opioids acting via mu-receptors. The results indicate morphine acts selectively on oxytocin neurons projecting to mediolateral septum compared with other central projection areas and compared with centrally projecting vasopressin neurons. In the supraoptic nucleus, naloxone increased oxytocin release 2.3-fold (from 9.2 +/- 3.1 pg/30 min) and increased oxytocin release from axons of these neurons fivefold (from 7.8 +/- 3.2 pg/30 min). Naloxone had no significant effect on vasopressin release from any of the central sites, or on vasopressin secretion into blood, although oxytocin secretion was increased 36-fold (from 17.2 +/- 2.6 pg/ml; p less than 0.001), confirming dependence of magnocellular oxytocin neurons. The central processes of magnocellular supraoptic neurons may be a major source of central oxytocin released during morphine withdrawal.     

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.