Morphine-withdrawn Mice Research Articles

Activity in projection neurons from prelimbic cortex to the PVT is necessary for retrieval of morphine withdrawal memory.

Previous work has shown that the paraventricular nucleus of the thalamus (PVT) is an important region that is involved in the conditioned context-induced retrieval of morphine withdrawal memory. However, the upstream neural circuits that activate the PVT to participate in the conditioned context-induced retrieval of morphine withdrawal memory remain unknown. In the present work, we find that the conditioned context activates projection neurons from the prelimbic cortex (PrL) to the PVT, and the inhibition of PrL-PVT projection neurons inhibits the conditioned context-induced retrieval of morphine withdrawal memory; the conditioned context induces an increase in Arc expression, intrinsic excitability, and glutamate output in PrL-PVT projection neurons in morphine-withdrawn mice. These results suggest that the activity of PrL-PVT projection neurons is necessary for the retrieval of morphine withdrawal memory, and the conditioned context causes a plastic change in the activity in these projection neurons during the withdrawal memory retrieval.

A Sensitization Of Projection Neurons from Prelimbic Cortex to Paraventricular Nucleus of Thalamus Induced By Retrieval of Morphine Withdrawal Memory

Exposure to context that is associated with drug withdrawal syndromes induces the retrieval of drug withdrawal memory and leads to drug relapse. Previous study showed that the paraventricular nucleus of the thalamus (PVT) was an important region that was involved in conditioned context-induced retrieval of drug withdrawal memory. However, the upstream neuronal circuits that activate the PVT to participate in conditioned context-induced retrieval of morphine withdrawal memory remain to be studied. In the present work, using retrograde labeling method combined with immunohistochemical, chemical genetic, optogenetic and whole-cell recording approaches, we found that that projection neurons from the prelimbic cortex to the PVT played an important role in conditioned context-induced retrieval of morphine withdrawal memory and the retrieval of morphine withdrawal memory induced an increase in intrinsic excitability and glutamate output in PrL-PVT projection neurons in morphine withdrawn mice, suggesting a sensitization of PrL-PVT projection neurons after exposure to conditioned context.

Hydrogen-rich saline attenuates anxiety-like behaviors in morphine-withdrawn mice

Hydrogen therapy is a new medical approach for a wide range of diseases. The effects of hydrogen on central nervous system-related diseases have recently become increasingly appreciated, but little is known about whether hydrogen affects the morphine withdrawal process. This study aims to investigate the potential effects of hydrogen-rich saline (HRS) administration on naloxone-precipitated withdrawal symptoms and morphine withdrawal-induced anxiety-like behaviors. Mice received gradually increasing doses (25–100 mg/kg, i.p.) of morphine over 3 days. In the naloxone-precipitated withdrawal procedure, the mice were treated with three HRS (20 μg/kg, i.p.) injections, and naloxone (1 mg/kg, i.p.) was given 30 min after HRS administration. Body weight, jumping behavior and wet-dog shakes were immediately assessed. In the spontaneous withdrawal procedure, the mice were treated with HRS (20 μg/kg, i.p.) every 8-h. Mice underwent naloxone-precipitated or spontaneous withdrawal were tested for anxiety-like behaviors in the elevated plus-maze (EPM) and light/dark box (L/D box) paradigm, respectively. In addition, the levels of plasma corticosterone were measured. We found that HRS administration significantly reduced body weight loss, jumping behavior and wet-dog shakes in mice underwent naloxone-precipitated withdrawal, and attenuated anxiety-like behaviors in the EPM and L/D box tests after naloxone-precipitated withdrawal or a 2-day spontaneous withdrawal period. Hypo-activity or motor impairment after HRS administration was not observed in the locomotion tests. Furthermore, HRS administration significantly decreased the levels of corticosterone in morphine-withdrawn mice. These are the first findings to indicate that hydrogen might ameliorate withdrawal symptoms and exert an anxiolytic-like effect in morphine-withdrawal mice.

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 GABAB 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.

Accumbal dopamine, noradrenaline and serotonin activity after naloxone-conditioned place aversion in morphine-dependent mice

Dopamine (DA) neurons not only show a pattern signaling the magnitude, delay and probability of rewards but also code negative motivation and aversive events. Beside DA, other systems such as noradrenaline (NA) and serotonin (5-HT) may also be implicated in naloxone-induced conditioned place aversion (CPA; an index of the aversive consequences of withdrawal). The purpose of the present study was to evaluate: (i) the turnover of DA, NA and 5-HT in the nucleus accumbens (NAc), one of the most important substrates for aversive states, (ii) the changes in tyrosine hydroxylase (TH) gene expression in the ventral tegmental area, and (iii) total TH protein levels and TH phosphorylation in the NAc after naloxone-induced morphine withdrawal. DA, NA and 5-HT turnover was evaluated by high-performance liquid chromatography (HPLC). TH gene expression was determined by real time quantitative PCR (RT-PCR) and total TH and TH phosphorylated at Ser31 and Ser40 were analyzed by Western blot. Present results show that the aversion for environmental cues paired with opioid withdrawal was higher than that observed in the saline group treated with naloxone, which indicates that morphine pretreatment potentiated the ability of naloxone to produce place aversion. In addition, present data show that naloxone-induced CPA positively correlated with an increase of DA and NA turnover in the NAc, which paralleled an increase in TH gene expression in the VTA and TH phosphorylation and enhanced TH protein levels in the NAc. Thus, the present study indicates that naloxone-induced aversion in morphine-dependent mice enhances DA and NA activity in the NAc and suggests that transcriptional and post-transcriptional regulation of TH could be involved in the hyperactivity of mesolimbic dopaminergic system observed in morphine-withdrawn mice.

Social influences on plasma testosterone levels in morphine withdrawn adolescent mice and their drug-naïve cage-mates

Opioid administration in males results in opioid-induced androgen deficiency which persists throughout the treatment. In adults, this quickly reverses once opioid administration is suspended. However, less is known about the duration of the effect following drug discontinuation in adolescents. Given the significant implications to sexual maturation in adolescent males, this study examined plasma testosterone levels in both morphine withdrawn mice and their drug-naïve (saline-injected) cage-mates as compared to drug-naïve mice housed physically and visually separate from the morphine-treated mice ('saline only'). Consistent with the literature, plasma testosterone levels in morphine withdrawn adults were reduced on withdrawal day 1 (WD1) and returned to baseline levels by WD9. No significant effects were observed in their saline cage-mates. In the adolescents, no significant differences were observed on WD1 between the morphine withdrawn mice, their saline cage-mates, and the saline only mice - all of which had significantly lower plasma testosterone levels than adults. By WD9, testosterone levels in the saline only adolescent mice had reached adult levels. Notably, plasma testosterone levels were reduced in both the morphine withdrawn adolescent mice and their saline cage-mates, as compared to saline only mice. The effect was not a drug effect per se, given that reduced plasma testosterone levels were not observed in individually housed morphine withdrawn mice. Moreover, our results also suggest that these social effects are not solely explained by stress. These results have numerous implications to the short term and long term health of both adolescents requiring pain management and of adolescent drug addicts.

Morphine Withdrawal Lowers Host Defense to Enteric Bacteria: Spontaneous Sepsis and Increased Sensitivity to Oral Salmonella enterica Serovar Typhimurium Infection

Understanding the consequences of drug withdrawal on immune function and host defense to infection is important. We, and others, previously demonstrated that morphine withdrawal results in immunosuppression and sensitizes to lipopolysaccharide-induced septic shock. In the present study, the effect of morphine withdrawal on spontaneous sepsis and on oral infection with Salmonella enterica serovar Typhimurium was examined. Mice were chronically exposed to morphine for 96 h by implantation of a slow-release morphine pellet. Abrupt withdrawal was induced by removal of the pellet. In the sepsis model, bacterial colonization was examined and bacterial species were identified by necropsy of various tissues. It was found that at 48 h postwithdrawal, morphine-treated mice had enteric bacteria that were detected in the Peyer's patches (4/5), mesenteric lymph nodes (4/5), spleens (4/10), livers (6/10), and peritoneal cavities (8/10). In placebo pellet-withdrawn mice, only 2/40 cultures were positive. The most frequently detected organisms in tissues of morphine-withdrawn mice were Enterococcus faecium followed by Klebsiella pneumoniae. Both organisms are part of the normal gastrointestinal flora. In the infection model, mice were orally inoculated with S. enterica 24 h post-initiation of abrupt withdrawal from morphine. Withdrawal significantly decreased the mean survival time and significantly increased the Salmonella burden in various tissues of infected mice compared to placebo-withdrawn animals. Elevated levels of the proinflammatory cytokines were observed in spleens of morphine-withdrawn mice, compared to placebo-withdrawn mice. These findings demonstrate that morphine withdrawal sensitizes to oral infection with a bacterial pathogen and predisposes mice to bacterial sepsis.

Increased Sensitivity to Salmonella enterica Serovar Typhimurium Infection in Mice Undergoing Withdrawal from Morphine Is Associated with Suppression of Interleukin-12

We have shown previously that withdrawal from morphine induces immunosuppression in mice. The present study reports the effects of morphine withdrawal on infection with Salmonella enterica serovar Typhimurium. Mice were made dependent on morphine by the implantation of a slow-release morphine pellet for 96 h. Controls received a placebo pellet. Withdrawal was induced by pellet removal. Mice were inoculated intraperitoneally with Salmonella 24 h postwithdrawal. Morphine withdrawal sensitized mice to Salmonella infection, as evidenced by increased mortality, shortened mean survival time, and increased bacterial load in the blood, spleen, and liver. Examination of the levels of a panel of proinflammatory cytokines in sera of infected, morphine-withdrawn mice showed that morphine withdrawal inhibited the elevation of interleukin-12p70 (IL-12p70). The production of IL-12p40 in morphine withdrawal mice was also suppressed. The administration of exogenous IL-12 significantly decreased the bacterial burden in morphine-withdrawn mice. These studies show a correlation between the suppression of IL-12 production and a heightened susceptibility to Salmonella infection in mice undergoing withdrawal from morphine.

Morphine withdrawal sensitizes mice to lipopolysaccharide: Elevated TNF-α and nitric oxide with decreased IL-12

Mice made dependent on morphine using slow-release morphine pellets for 96 h were withdrawn by removal of pellets, followed by a sublethal dose of LPS 24 h later. These animals exhibited 100% lethality. Animals withdrawn from placebo pellets receiving LPS all survived, as did morphine-withdrawn mice receiving saline. Morphine-withdrawn LPS-treated animals had elevated serum TNF-α and nitric oxide levels, and depressed IL-12 levels compared to controls. Anti-TNF-α antibody given prior to LPS challenge afforded significant protection to morphine-withdrawn animals. These studies show that morphine withdrawal sensitizes to LPS lethality via increased production of TNF-α.

Metabotropic glutamate receptor 2/3-dependent long-term depression in the nucleus accumbens is blocked in morphine withdrawn mice.

The nucleus accumbens (NAc) plays a crucial role in addiction. We have recently shown that activation of presynaptic metabotropic glutamate 2/3 receptors (mGlu2/3) induces long-term depression (LTD) at glutamatergic synapses in the mouse nucleus accumbens (NAc) through the long lasting inhibition of P/Q-type Ca2+ channels and the cAMP/protein kinase A (PKA) pathway. Because presynaptic mGlu2/3 functions are augmented in the ventral tegmental area of morphine-withdrawn rats, we have evaluated the consequences of opiate treatment on mGlu2/3 LTD at prelimbic NAc glutamatergic synapses. Here we report that mGlu2/3 LTD is abolished after 1 week of withdrawal from chronic morphine treatment; in the morphine-withdrawn group LTD measured 5.99 +/- 4.84% (P < 0.05) compared with 21.13 +/- 5.42% in the sham group. In contrast, chronic morphine treatment did not alter the mechanisms normally underlying mGlu2/3 LTD, such as the cAMP/PKA pathway or P/Q-type Ca2+ channels. This study shows that one long-term consequence of morphine treatment is an alteration of synaptic plasticity at glutamatergic synapses in the NAc. Considering that mGlu2/3 agonists (e.g. LY-354740 used in the present study to induce LTD) reduce behavioural symptoms of morphine withdrawal, these findings could be important in the understanding of the cellular events underlying the dependence-inducing properties of opiates.

Morphine withdrawal-facilitated aggression is attenuated by morphine-conditioned stimuli

There is a considerable body of evidence indicating that stimuli associated with drug administration may become conditioned and evoke drug-like effects. The purpose of this study was to evaluate the ability of morphine-paired stimuli to affect an expression of morphine withdrawal-facilitated aggression. Individually housed aggressive adult mice were subjected to the repeated subcutaneous administration of morphine (twice a day, 8 days, increasing doses 10–80 mg/kg). Morphine treatment cessation facilitated an aggressive behaviour of animals during the second day of withdrawal. Subcutaneous but not intraperitoneal injection of saline attenuated the aggressive behaviour in morphine-withdrawn mice. These results suggest that the site of drug injection may serve as a conditioned stimulus.

The involvement of noradrenergic transmission in the morphine‐induced locomotor hyperactivity in mice withdrawn from repeated morphine treatment

1. Our previous studies suggest that in addition to the cerebral dopaminergic systems the noradrenergic ones have a crucial role in the morphine-induced behavioural sensitization in mice. Therefore the effects of alpha2-adrenoceptor antagonist, idazoxan (1 and 3 mg kg(-1), i.p.) on morphine-induced locomotor hyperactivity as well as on morphine-induced changes in cerebral noradrenaline (NA) and striatal dopamine (DA) metabolism were studied in mice withdrawn for 3 days from 5 day repeated morphine treatment. The concentrations of NA, free 3-methoxy-4-hydroxyphenylethylene glycol (MOPEG), DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxytyramine (3-MT) were determined. 2. Acute morphine (10 mg kg(-1), s.c.) increased locomotor activity in control and in morphine-withdrawn mice; idazoxan alone did not alter the activity. Idazoxan pretreatment did not alter the locomotor hyperactivity induced by acute morphine in control mice but potentiated it in morphine-withdrawn mice. 3. Acute morphine elevated MOPEG less but increased DOPAC and HVA more clearly in morphine-withdrawn mice than in controls, and decreased 3-MT only in controls. Idazoxan alone did not alter the NA or DA metabolite concentrations in control mice, but elevated MOPEG as well as DOPAC in morphine-withdrawn mice. 4. In control mice idazoxan enhanced acute morphine's elevating effect on MOPEG. In withdrawn mice idazoxan counteracted the tolerance so that acute morphine elevated MOPEG in these mice to about similar level as in controls. 5. Idazoxan pretreatment abolished the HVA increasing effect of acute morphine both in control and withdrawn mice. In control mice idazoxan enhanced morphine's elevating effect on DOPAC and abolished morphine's decreasing effect on 3-MT. Idazoxan did not alter morphine's effects on DOPAC or 3-MT concentrations in withdrawn mice. 6. Our results show that in morphine-withdrawn mice idazoxan pretreatment reveals the morphine-induced locomotor sensitization. This most probably occurs by overcoming the tolerance towards the acute morphine-induced increase of cerebral NA turnover and release. It is suggested that in mice the cerebral noradrenergic in addition to the dopaminergic systems are major determinants of the behavioural sensitization to morphine.

Continuous treatment with morphine increases diazepam binding inhibitor mRNA in mouse brain.

Effects of acute and chronic morphine treatment on the expression of diazepam binding inhibitor (DBI) mRNA in the mouse brain were examined. Cerebral DBI mRNA expression significantly increased in morphine-dependent mice, and this increase is more remarkable in morphine-withdrawn mice, whereas a single administration of morphine (50 mg/kg) produced no changes in the expression. Simultaneous administration of naloxone (3 mg/kg) with morphine completely abolished the increase in cerebral DBI mRNA expression observed in morphine-dependent and -withdrawn mice. These results indicate that a chronic functional interaction between morphine and opioid receptors has a critical role in increases in DBI mRNA expression.

Regional differences in cerebral noradrenaline turnover in mice withdrawn from repeated morphine treatment and tolerance to the effects of acute morphine.

The effects of morphine withdrawal and challenge doses (10 or 30 mg/kg) on the alpha-methyl-p-tyrosine (alpha MT)-induced noradrenaline (NA) depletion as well as on the free 3-methoxy-4-hydroxyphenylethylene glycol (MOPEG) concentration were studied in various brain areas of NMRI mice. Morphine was given subcutaneously 3 times daily for 5 days followed by 1 or 3 days' withdrawal. In morphine withdrawn mice the alpha MT-induced NA depletion and the free MOPEG concentrations were differentially altered. At 1-day withdrawal the alpha MT-induced NA depletion was retarded and the NA concentration was elevated in the forebrain area indicating reduced release of NA. Simultaneously, however, the free MOPEG concentration was significantly elevated in the forebrain area and in the lower brain stem suggesting enhanced NA turnover. No withdrawal-induced changes were found in the hypothalamic NA turnover. Acute morphine elevated the free MOPEG concentration and accelerated the alpha MT-induced NA depletion in all brain areas of control mice but not in mice withdrawn for 1 day from repeated morphine treatment. At 3 days' withdrawal, however, the 30 mg/kg morphine dose slightly accelerated the NA depletion in the forebrain area. These results show that morphine withdrawal differentially alters the alpha MT-induced NA depletion and the free MOPEG concentration in various mouse brain areas. These effects are relatively modest suggesting that in mice the noradrenergic mechanisms play a minor role in morphine withdrawal syndrome. However, in all brain areas of the morphine-withdrawn mice tolerance was found towards the NA turnover and release accelerating effect of acute morphine.

Morphine withdrawal aggression: modification with D1 and D2 receptor agonists.

Morphine withdrawal increases aggressive behaviors, induces explosive motor behaviors, and disrupts homeostatic functions in mice and rats. While many of these effects appear to result from altered dopaminergic activity during morphine withdrawal, the relative contributions of the D1 and D2 receptor subtypes remain unclear. In the present experiments, the D1 agonist SKF 38393 and the D2 agonist quinpirole were administered to male "resident" Swiss-Webster mice 5 h after the removal of a subcutaneously-implanted morphine or placebo pellet. These mice were then observed alone to determine changes in various motor activities and in confrontation with a group-housed male "intruder" to assess changes in aggressive behaviors. SKF 38393 decreased the display of aggressive behaviors by placebo and morphine-withdrawn mice without consistently altering walking or rearing. Quinpirole greatly decreased the display of aggressive behaviors by placebo mice and decreased aggressive behaviors in morphine-withdrawn mice to a lesser degree. The inhibitory effects of quinpirole were not specific to aggressive behaviors; low quinpirole doses also decreased the display of walking and rearing. In mice which received a low dose of SKF 38393 preceding quinpirole injection, pretreatment with the D1 agonist did not alter the effects of the D2 agonist quinpirole on motor activities but maintained high levels of aggression in morphine-withdrawn mice. The differential modification of aggressive and motor behaviors by selective dopaminergic agonists during morphine withdrawal further supports the suggestion that aggressive and motor behaviors are controlled independently; furthermore, D1 receptor stimulation appears to have particular relevance for the display of aggressive behaviors during morphine withdrawal.

Heightened aggressive behavior during morphine withdrawal: effects of d-amphetamine.

The morphine withdrawal syndrome is composed of profound short- and long-term changes in autonomic, somato-motor and affective functions. In mice, morphine withdrawal produces heightened aggressive behavior and alterations in motor behavior; however, it is unclear whether these changes in behavior occur in unison and are dependent on a common mechanism or occur independently. In order to characterize the morphine withdrawal syndrome in mice, male Swiss-Webster mice were housed with female partners for 3-4 weeks before being implanted subcutaneously with morphine or placebo pellets. The pellets were removed 72 h after implantation and behavioral measurements were conducted 5, 48 and 96 h after pellet removal. During these tests, mice received d-amphetamine (0.3-10 mg/kg) or saline after which they were assessed for changes in motor behavior and for changes in aggressive behavior while confronting a group-housed male "intruder". In morphine-withdrawn mice, frequency of attack behavior was increased by approximately 30% and this effect persisted for at least 4 days. In contrast, explosive jumping was increased and walking and rearing were greatly decreased at the onset of the withdrawal period but declined within the first 24 h and returned to control levels within 48 h of pellet removal. d-Amphetamine maintained the elevated level of aggressive behavior and sharply increased locomotion in morphine-withdrawn mice; in placebo-pelleted mice, d-amphetamine dose-dependently decreased aggressive behaviors while increasing locomotion. The differential time course and the differential modification by d-amphetamine suggest that heightened aggressive behavior is a long-lasting consequence of morphine-withdrawal based on separate mechanisms than the short-lived alterations in motor activity.

Social, motor, and autonomic signs of morphine withdrawal: differential sensitivities to catecholaminergic drugs in mice.

Drugs that predominantly influence catecholamines were used in order to simultaneously determine their ability to alter salient signs of social, motor and autonomic activity during morphine withdrawal, and to compare the sensitivity of each of these signs to these drugs. Cocaine, d-amphetamine, apomorphine and L-dopa increased attack and threat, but did not induce defensive behavior in morphine-withdrawn resident mice who were more responsive to the aggression-enhancing effects of these drugs than placebo control mice. Concurrently measured withdrawal jumping was not affected by these drugs, and the sensitivity to the hypothermic effects of these drugs was reduced. In contrast, clonidine decreased attack and threat behaviors, and morphine-withdrawn mice were more sensitive to this inhibitory influence. But like the stimulant drugs, clonidine did not affect withdrawal jumping, and the hypothermic action of clonidine was attenuated in morphine-withdrawn mice. These findings show that in mice, opiate withdrawal leads to altered attack and threat that is further amplified by catecholaminergic drugs. The present pattern of results indicates differential drug effects on social, motor and autonomic functions when the behaviors are measured 48 h following withdrawal.

In vivo binding of naloxone to opioid receptors in morphine-dependent mice

In vivo binding assay of opioid receptors for naloxone was tested in morphine-dependent mice. The content of naloxone in the brain (whole brain minus cerebellum) and cerebellum was determined as the total binding and non-specific binding, respectively, 20 min after the intravenous injection of [ 3H]-naloxone. Acute treatment with 8 mg/kg of morphine sulfate markedly enhanced the non-specific binding of naloxone. In contrast, withdrawal for 6 hr after 72 hr of implantation of morphine decreased the non-specific binding of naloxone. The Scatchard analysis of the specific binding revealed two binding sites. The apparent low affinity K d values of both morphine-tolerant and morphine-withdrawn mice were significantly decreased when compared to their respective controls. The apparent high affinity K d and the high and low B max values were not altered by the morphine dependent-tolerant state.