Amniotic-fluid ingestion enhances morphine analgesia during morphine tolerance and withdrawal in rats.

Ingestion of placenta and amniotic fluid has been shown to enhance opioid-mediated analgesia in rats produced by morphine injection, footshock, vaginal/cervical stimulation, and during late pregnancy. The present study was designed to investigate the effects of amniotic fluid ingestion on the characteristics of morphine dependency and withdrawal. Tail-flick latencies in Long-Evans rats were determined before and after repeated daily injections of morphine sulfate. It was found that ingestion of amniotic fluid after establishment of the morphine dependency, coupled with an injection of an otherwise ineffective dose of morphine, enhanced analgesia in morphine-dependent rats, and reversed hyperalgesia seen during withdrawal from morphine dependency.     

Involvement of nitric oxide synthesis in sensitization to the rewarding effects of morphine

Knowledge about the specific brain changes and neural plasticity processes produced by repeated exposure to a drug is essential to progress in the field of neurobiology of addiction and the development of effective medication. In the present study, the influence of nitric oxide synthesis on sensitization to the rewarding effects of morphine has been evaluated. The effects of pre-treatment of mice with saline or 20 mg/kg of morphine plus the nitric oxide synthase inhibitor 7-nitroindazole (7NI) (12.5 or 25 mg/kg) on the place conditioning induced by a low dose of morphine (2 mg/kg) were assessed. The dose of 2 mg/kg of morphine was ineffective in animals pre-treated with saline but induced a clear conditioned place preference (CPP) in those pre-treated with morphine. Conversely, animals pre-treated with morphine plus 7NI did not acquire CPP. Our results demonstrate that the nitric oxide pathway is implicated in the development of sensitization to the conditioned rewarding effects of morphine.     

Stress during adolescence alters behavioral sensitization to amphetamine

In humans, chronic intermittent and uncontrollable stress during adolescence is viewed as a key factor for vulnerability to drug abuse and development of psychopathologies later in life. Less is known about the long-term effects of chronic stress in animals during the juvenile period. Although there is evidence of cross sensitization during prenatal period and adulthood between chronic stress and amphetamine-induced behavioral sensitization in the rat, no studies have been conducted on cross sensitization between chronic variable stress in adolescence and behavioral sensitization to amphetamine. To address this question, at the onset of adolescence (28 days) male rats were subjected to 28 days of intermittent non-habituating social stress (isolation, novel environment, crowding, litter-shifting, subordination), or physical stress (restraint, swim, cold, ether, noise), or were handled as controls. Twenty-four hours after the last stressor or handling, all groups were exposed to a novel environment for 1 h, after which they underwent a regimen of behavioral sensitization to amphetamine. Our results showed that socially stressed rats have low locomotor activity in the novel environment, when compared to the control and physical groups who were identical in the same test. Even though socially stressed rats had lower locomotor activity in response to amphetamine injections, there were no significant differences during the training phase between the three groups at this dose of amphetamine. However, when tested for behavioral sensitization to amphetamine control and physically stressed rats showed a robust sensitization, socially stressed rats were significantly inhibited. We conclude that our chronic variable social stress protocol during adolescence inhibits behavioral sensitization to amphetamine during adulthood.     

Chronic exposure to morphine does not alter the neural tissues subserving its acute rewarding properties: apparent tolerance is overshadowing.

Drug-naive, but not morphine-dependent, rats preferred places paired with morphine (2 mg/kg) over unfamiliar neutral places. Both drug-naive and morphine-dependent rats preferred places paired with higher doses of morphine (20 mg/kg) over unfamiliar places. Lesions of the tegmental pedunculopontine nucleus (TPP) blocked the conditioned place preferences produced by both 2 and 20 mg/kg morphine in drug-naive rats but not the preferences produced by 20 mg/kg morphine in dependent rats. When morphine-dependent animals received withdrawal-alleviating doses of morphine (20 mg/kg) 3.5 hr before pairing one environment with 2 mg/kg morphine, they showed morphine-conditioned place preferences that were abolished by TPP lesions. The apparent behavioral tolerance to the TPP-mediated rewarding effects may have resulted from overshadowing by separate withdrawal-related motivational mechanisms.     

Procedures that produce context-specific tolerance to morphine in rats also produce context-specific withdrawal

Rats previously injected with morphine in the presence of a distinct environment (paired animals) were more tolerant to the analgesic effects of morphine in that environment than were rats previously injected with morphine in another environment (unpaired animals). When injected with saline instead of morphine in the distinct environment, paired animals were more reactive to pain (hyperalgesic) than unpaired animals, but no more reactive to pain than animals never given morphine. More important, the paired animals also exhibited more withdrawal symptoms (wet dog shakes, genital licking, circling, rearing, and defecation) during abstinence and naltrexone-precipitated withdrawal in the distinct environment than did the unpaired and saline animals. Thus, procedures that are capable of producing context-specific opiate tolerance are also capable of producing context-specific opiate withdrawal.     

Anxiety-like symptoms induced by morphine withdrawal may be due to the sensitization of the dorsal periaqueductal grey

Withdrawal from morphine leads to the appearance of extreme anxiety accompanied of several physical disturbances, most of them linked to the activation of brainstem regions such as the locus coeruleus, ventral tegmental area, hypothalamic nuclei and periaqueductal grey (PAG). As anxiety remains one of the main components of morphine withdrawal the present study aimed to evaluating the influence of the dorsal aspects of the PAG on the production of this state, since this structure is well-known to be involved in defensive behaviour elicited by anxiety-evoking stimuli. Different groups of animals were submitted to 10 days of i.p. morphine injections, challenged 2 h after with an i.p. injection of naloxone (0.1 mg/kg), and submitted to the plus-maze, open-field and light-dark transition tests. The effects of morphine withdrawal on anxiety-induced Fos immunolabelling were evaluated in four animals that passed by the light-dark transition test randomly chosen for Fos-protein analysis. Besides the PAG, Fos neural expression was conducted in other brain regions involved in the expression of anxiety-related behaviours. Our results showed that morphine withdrawn rats presented enhanced anxiety accompanied of few somatic symptoms. Increased Fos immunolabelling was noted in brain regions well-known to modulate these states as the prelimbic cortex, nucleus accumbens, amygdala and paraventricular hypothalamus. Increased Fos labelling was also observed in the ventral and dorsal aspects of the PAG, a region involved in anxiety-related processes suggesting that this region could be a common neural substrate enlisted during anxiety evoked by dangerous stimuli as well as those elicited by opiate withdrawal.     

Extremely low-frequency electromagnetic field exposure during chronic morphine treatment strengthens downregulation of dopamine D2 receptors in rat dorsal hippocampus after morphine withdrawal

The aim of this study was to investigate the effect of extremely low-frequency electromagnetic field (ELF-EMF) exposure during morphine treatment on dopamine D2 receptor (D2R) density in the rat dorsal hippocampus following withdrawal. Rats were exposed to ELF-EMF (20 Hz, 14 mT) or sham exposed for 1h per day before injection of morphine (10mg/kg, i.p.) once daily for 12 days. The saline control group was sham exposed for the same period. Immunohistochemistry was used to detect the density of D2Rs on the 1st, 3rd and 5th morphine withdrawal days. The results showed that the density of D2Rs in sham-exposed morphine-treated rats on the 1st and 3rd days of morphine withdrawal was significantly lower than that of the saline control group. The ELF-EMF-exposed morphine group also exhibited a significantly lower density of D2Rs on the 1st and 3rd withdrawal days relative to the sham-exposed morphine group. However, the D2R density in both groups tended to recover as morphine withdrawal days increased. The results suggest that dorsal hippocampal D2Rs are sensitive to morphine withdrawal and that this is potentiated by ELF-EMF pre-exposure during morphine treatment.     

Lesions of ventral tegmental dopamine neurons delay the development of tolerance to morphine catalepsy

Selective lesions of the dopamine (DA) neurons of the ventral tegmental area (VTA) were found to substantially delay the development of tolerance to morphine-induced catalepsy, in comparison with sham-operated controls receiving morphine. Lesioned subjects receiving vehicle injections showed no catalepsy. The data suggest that tolerance to morphine catalepsy requires intact VTA DA neurons. Furthermore, since the acute cataleptic response was intact in lesioned animals, the data suggest that the mechanisms involved in the cataleptic response to morphine are dissociable from those which bring about tolerance to that response.     

Morphine-stimulated metabolism of striatal and limbic dopamine is dissimilarly sensitized in rats upon withdrawal from chronic morphine treatment

The effects of acute morphine on the release of dopamine (DA) in the striatum and limbic forebrain of rats upon 48 h withdrawal from 20-day morphine treatment were studied using 3-methoxytyramine (3-MT) in tissue as an index of DA release. Homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were also measured. The chronic morphine treatment did not alter the concentrations of DA metabolites. Acute morphine (10 mg/kg) elevated all three DA metabolites in both brain areas. Morphine withdrawal potentiated the elevation of striatal and limbic 3-MT as well as that of striatal but not limbic HVA. These findings show that both striatal and limbic DA mechanisms are sensitized to morphine upon withdrawal but that sensitization of DA metabolism in these two brain areas occurs differently.     

Perinatal undernutrition facilitates morphine sensitization and cross-sensitization to cocaine in adult rats: a behavioral and neurochemical study

The development of sensitization to the locomotor effects of morphine and cross-sensitization between morphine and cocaine were evaluated in adult rats submitted to a protein malnutrition schedule from the 14th day of gestation up to 30 days of age (D-rats), and compared with well-nourished animals (C-rats). Dose-response curves to morphine-induced locomotor activity (5, 7.5, 10 or 15 mg/kg, i.p., every other day for 5 days) revealed a shift to the left in D-rats compared to C-rats. This implies that D-rats showed behavioral sensitization to the lower dose of morphine used (5 mg/kg), which was ineffective in C-rats. Furthermore, when a cocaine challenge (10 mg/kg, i.p) was given 48 h after the last morphine administration, only D-rats exhibited cross-sensitization in morphine-pretreated animals (7.5 and 10 mg/kg). In order to correlate the differential response observed with the functioning of the mesocorticolimbic dopaminergic system, extracellular dopamine (DA) levels were measured in the nucleus accumbens (core and shell) and the dorsal caudate-putamen. A challenge with cocaine in morphine pre-exposed animals produced an increase in DA release, but only in the nucleus accumbens “core” of D-rats. Similar DA levels were found in the nucleus accumbens “shell” and in the dorsal caudate-putamen of both groups. Finally, these results demonstrate that D-rats had a lower threshold for developing both a progressive behavioral sensitization to morphine and a cross-sensitization to cocaine. In accordance with these behavioral findings, a higher responsiveness of the nucleus accumbens core, expressed by increased DA levels, both basal and after cocaine challenge, was observed in D-rats.     

Effect of acupuncture on behavioral hyperactivity and dopamine release in the nucleus accumbens in rats sensitized to morphine

Acupuncture as a therapeutic intervention has been used for the treatment of many functional disorders including substance abuse. However, there are still many unanswered question about the basic mechanism underlying acupuncture's effectiveness in the treatment of drug addiction. Repeated injection of psycostimulants or morphine can produce behavioral and neurochemical sensitization and have been used as a model for studying drug addiction. The present study was designed to investigate the effect of acupuncture on repeated morphine-induced changes in extracellular dopamine levels using in vivo microdialysis and repeated morphine-induced behavioral changes. Male Sprague-Dawley rats were treated with saline or increasing doses of morphine (10, 20 and 40 mg/kg, s.c., twice daily for 3 days). Following 15 days of withdrawal, acupuncture was applied at bilateral Shenmen (HT7) points for 1 min after the systemic challenge with morphine HCl (5 mg/kg, s.c.). Results showed that acupuncture at the specific acupoint HT7, but not at control points (TE8 and tail) significantly decreased both dopamine release in the nucleus accumbens and behavioral hyperactivity induced by a systemic morphine challenge. These results suggest that the therapeutic effect of acupuncture on morphine addiction occurs through inhibition of neurochemical and behavioral sensitization to morphine.     

Strain differences in the effects of adrenalectomy on the midbrain dopamine system: implication for behavioral sensitization to cocaine

Adrenalectomy (ADX) abolishes behavioral sensitization to cocaine in DBA/2, but not C57BL/6 inbred mice. The present study tests the hypothesis that this ADX effect on behavioral sensitization in the DBA/2 strain involves changes in midbrain dopamine systems that do not occur in the C57BL/6 strain.     

Heme oxygenase type 2 modulates behavioral and molecular changes during chronic exposure to morphine

The heme oxygenase (HO) enzyme system has been shown to participate in nociceptive signaling in a number of different models of pain. In these experiments we investigated the role of the HO type 2 (HO-2) isozyme in tolerance to the analgesic effects of morphine, and the hyperalgesia and allodynia which are measurable upon cessation of administration. Wild type C57Bl/6 wild type mice or HO-2 null mutants in that background strain were treated with morphine for 5 days. The morphine administration protocol consisted of either twice daily repeated s.c. boluses of 15 mg/kg or s.c. implantation of a morphine pellet. At the end of the treatment period wild type mice treated by either protocol exhibited tolerance, but the HO-2 null mutants did not. The HO-2 null mutants also exhibited less mechanical allodynia following cessation of morphine administration, though only modest differences in thermal hyperalgesia were noted. There was no correlation between the degree of tolerance obtained in the bolus and pellet protocols and the degree of hyperalgesia and allodynia observed after cessation of morphine administration in the wild type mice. Our final experiments analyzed increases in expression of mRNA for nitric oxide synthase type 1, N-methyl-D-aspartate (NMDA) receptor NMDAR1 subunit and prodynorphin in spinal cord tissue. In pellet-treated mice two- to three-fold increases were observed in the abundance of these species, but very little change was observed in the null-mutant mice. Taken together our results indicate that HO-2 participates in the acquisition of opioid tolerance, the expression of mechanical allodynia after cessation of opioid administration and in gene regulation occurring in the setting of treatment with morphine. Furthermore, these studies suggest that the mechanisms underlying analgesic tolerance and opioid-induced hypersensitivity are at least somewhat distinct.     

Adenosine A2a receptor/dopamine D2 receptor hetero-oligomerization: a hypothesis that may explain behavioral sensitization to psychostimulants and schizophrenia

The mechanisms underlying psychostimulant-induced behavioral sensitization and schizophrenia are yet to be fully elucidated. Evidence suggests that the dopamine D2 receptor (DRD2) as well as other neurotransmitter system receptors may be involved in the two conditions, and previous reports have hypothesized that oligomerization of dopamine receptors and analogs from other neurotransmitter systems may underlie the mechanism responsible. This paper describes findings which show co-localization of DRD2 and the adenosine A2a receptor (A2aAR) in the striatum, interaction between the two receptors and A2aAR/DRD2 hetero-oligomerization in the neuronal cell. The possibility that A2aAR/DRD2 hetero-oligomerization may be involved in the development of psychostimulant-induced behavioral sensitization or the pathogenesis of schizophrenia is explored. A2aAR/DRD2 hetero-oligomerization can enhance dopaminergic function, resulting in an increased behavioral response to psychostimulants or the development of psychotic symptoms. Receptor oligomerization can affect receptor degradation, which may affect the persistence of behavioral sensitization or psychotic symptoms. The proposed A2aAR/DRD2 hetero-oligomerization model may suggest new directions in the understanding and treatment of schizophrenia.     

Kinetics and properties of cortisolresistant lymphocyte population from guinea pig lymph nodes during protein sensitization

Lymphocytes from the cervical lymph nodes of guinea pigs were incubated in medium No. 199 for 24 h in the presence of cortisol in concentrations of 20 and 100 g/100 ml. The survival rate of the lymphocytes and their cortisol metabolism were determined and their nucleic acid concentration was estimated cytophotometrically. In intact and sensitized guinea pigs progesterone, in a concentration of 10^–5 M, inhibited both the lytic action of cortisol and its metabolism. A marked decrease in cortisol metabolism by the lymphocytes was found from the sixth day after sensitization, and it did not recover until the 90th day. On the 17th-30th day the cortisol-resistant lymphocyte population was increased. In a concentration of 100 g/100 ml cortisol reduced the nucleic acid concentration in lymphocytes both of the intact guinea pigs and also of the sensitized animals on the 17th-30th day, when the cortisol-resistant cell population was increased.Department of General Pathology, N. I. Pirogov Second Moscow Medical Institute. Allergologic Scientific-Research Laboratory, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 1, pp. 68–71, January, 1977.     

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.     

Retinoids during the in vitro transition from bovine morula to blastocyst

BACKGROUND: The conversion of retinol (ROH) to retinoic acid (RA) is crucial during development but has been not studied during blastocyst formation. METHODS AND RESULTS: In vitro-produced bovine morulae were treated for 24 h with citral (which inhibits the synthesis of RA from ROH), citral + all trans retinoic acid (ATRA), ATRA or no additives. Citral interfered with blastocyst development, whereas exogenous RA had no effect. RA, however, reversed the effect of citral on development and stimulated cell proliferation. Neither citral nor RA changed the apoptotic index, but RA triggered an increase in the apoptotic frequency of the inner cell mass. Citral and RA reduced the necrotic index. Na/K-ATPase alpha1-subunit mRNA concentrations (analysed by real-time PCR) increased after hatching and showed dependence on retinoid activity, but no evidence was found of any retinoid effect on p53 expression. Nevertheless, the p53 mRNA concentration increased in response to proliferation in hatched blastocysts. CONCLUSION: The preimplantation bovine embryo metabolizes endogenous ROH to RA, which participates in important cell processes. The true extent of the influence of RA is unknown, although the modulation of retinoid metabolism seems to be a means of increasing cell proliferation. This knowledge might be used to improve embryo quality and the efficiency of stem cell derivation.