Assessing effects of tamoxifen on tolerance, dependence, and glutamate and glutamine levels in frontal cortex and hippocampus in chronic morphine treatment.

Tamoxifen has been shown to reduce glutamate release from presynaptic glutamatergic nerves and reverse tolerance to morphine-induced respiratory depression. Changes in glutamatergic neurotransmission in the central nervous system contribute to morphine tolerance, dependence, and withdrawal. This study, therefore, evaluated effects of tamoxifen on development of analgesic tolerance and dependence, and brain glutamate and glutamine levels in chronic morphine administration. Mice implanted with placebo or morphine pellets were injected with tamoxifen (0.6-2 mg/kg) or vehicle twice daily for 3 days. Nociceptive response was evaluated in the hot plate and tail immersion tests, 4, 48 and 72 h post-implant, and following a challenge dose of morphine (10 mg/kg). Withdrawal signs were determined after naloxone (1 mg/kg) administration. Morphine increased nociceptive threshold which declined over time. At 72 h, acute morphine elicited tolerance to the analgesic effect in the hot plate test in vehicle or tamoxifen administered animals. In the tail immersion test, however, tolerance to morphine analgesia was observed in tamoxifen, but not vehicle, co-administration. Tamoxifen did not reduce withdrawal signs. In contrast to previous reports, glutamate and glutamine levels in the hippocampus and frontal cortex did not change in the morphine-vehicle group. Confirming previous findings, tamoxifen (2 mg/kg) decreased glutamate and glutamine concentrations in the hippocampus in animals with placebo pellets. Both doses of tamoxifen significantly changed glutamate and/or glutamine concentrations in both regions in morphine pellet implanted animals. These results suggest that tamoxifen has no effect on dependence but may facilitate tolerance development to the antinociception, possibly mediated at the spinal level, in chronic morphine administration.

Effect of oxytocin pretreatment on the development of morphine tolerance and dependence in rats.

Increased opioid synthesis and release, and enhanced alpha-2 adrenoceptor signaling have been suggested to mediate repeated oxytocin-induced long-lasting effects including elevated pain threshold in rats. This study evaluated whether oxytocin pretreatment would influence development of dependence and tolerance to the nociceptive and body temperature responses to morphine and enhance effects of alpha-2 adrenergic agonist clonidine on nociceptive threshold, body temperature and morphine withdrawal signs. Rats injected subcutaneously with saline or 1 mg/kg oxytocin for 5 days were implanted with placebo or morphine pellets 24 h after the treatment period. Body temperature and nociception were assessed, with nociception determined via by hot plate and tail immersion tests, before and 4, 24 and 48 h after pellet implantation, and following a challenge dose of morphine. Withdrawal signs were determined after naloxone administration. Oxytocin produced analgesia, as evidenced by increased paw withdrawal latency in the hot plate test. Morphine increased body temperature and nociceptive threshold which declined over time. Morphine challenge could not demonstrate tolerance to the body temperature response. Analgesic tolerance was observed in the hot plate test in saline and in both tests in oxytocin pretreated rats. Naloxone-precipitated withdrawal appeared to be less severe in oxytocin pretreatment. Clonidine was ineffective on the withdrawal signs but decreased body temperature and increased tail flick latency in the tail immersion test in oxytocin pretreated animals. These results, while producing evidence for a hyperresponsiveness in alpha-2 adrenoceptors, provide contrasting effects on morphine tolerance and dependence, and their partial mediation by opioidergic and adrenergic activation in repeated oxytocin treatment.

Effects of tamoxifen and glutamate and glutamine levels in brain regions in repeated sleep deprivation-induced mania model in mice.

Protein kinase C inhibitor tamoxifen reduces symptoms of acute mania in bipolar patients and mania-like behaviors in animals. Memory impairment and altered levels of glutamate and glutamate/glutamine ratio have been reported in mania. Tamoxifen suppresses glutamate release which plays an important role in memory. The present study evaluated whether tamoxifen's activity participates in its antimanic efficacy in repeated sleep deprivation mania model. Mice were divided into control and 24-h sleep-deprived groups and were treated with vehicle or 1 mg/kg tamoxifen twice daily for 8 days. Sleep deprivation was repeated three times at intervals of 2 days. Square crossing and rearing were recorded as measures of locomotor activity. Memory and risk taking behavior were evaluated using novel object recognition and staircase tests, respectively. Glutamate and glutamine levels were measured in the frontal cortex and hippocampus. Behavioral tests were conducted 24 h after the second or immediately after the third sleep deprivations. Sleep deprivation increased locomotor activity and risk taking. Glutamate and glutamine levels and glutamate/glutamine ratio in the frontal cortex and hippocampus were unaffected. Locomotor hyperactivity was prevented by tamoxifen treatment. No change in the recognition index suggested lack of memory impairment in the model. These findings confirm the relevance of repeated sleep deprivation as a mania model and tamoxifen as an antimanic agent. However, future research is needed to further address lack of memory impairment in the model and lack of glutamatergic influence on the model and antimanic effect of tamoxifen.

Ketamine and its combinations with valproate and carbamazepine are ineffective against convulsions induced by atropine treatment and food intake in fasted mice.

OBJECTIVES: Fasted rodents treated with antimuscarinics develop convulsions after refeeding. Food deprivation for 48 hr produces changes in [3H]glutamate binding suggesting glutamatergic contribution to the underlying mechanism of the seizures that are somewhat unresponsive to antiepileptics. Studies in animals and epileptic patients yielded considerable information regarding the anticonvulsant effect of the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist ketamine. Thus, this study evaluated the efficacy of ketamine and its combinations with valproate and carbamazepine on convulsions in fasted animals. MATERIALS AND METHODS: Following 24 hr of fasting, mice were given saline, 5 or 10 mg/kg ketamine, 250 mg/kg sodium valproate, 24 mg/kg carbamazepine, 5 mg/kg ketamine+sodium valproate, or 5 mg/kg ketamine+carbamazepine and then were treated with saline or 2.4 mg/kg atropine (5-9 mice per group). The animals were observed for the occurrence of convulsions after being allowed to eat ad libitum. RESULTS: Ketamine, valproate and carbamazepine pretreatments were ineffective in preventing the convulsions developed after atropine treatment and food intake in fasted animals. The incidence of convulsions was significantly higher in 5 and 10 mg/kg ketamine, carbamazepine, and carbamazepine+ketamine groups, but not in the valproate and valproate+ketamine treated animals. CONCLUSION: In contrast to previous findings obtained with the NMDA antagonist dizocilpine (MK-801), ketamine lacks activity against convulsions developed after fasting. The drug does not enhance the efficacy of valproate and carbamazepine either. Using different doses of ketamine or other NMDA antagonists, further studies may better clarify the anticonvulsant effect of ketamine and/or role of glutamate in these seizures.

Contribution of M1 and M2 muscarinic receptor subtypes to convulsions in fasted mice treated with scopolamine and given food.

Treatment of fasted mice and rats with the nonselective muscarinic antagonist, scopolamine or atropine, causes convulsions after food intake. This study evaluated the effect of fasting on the expression of M1 and M2 muscarinic receptors in the brain regions, the relationship between receptor expression and seizure stages, and the muscarinic receptor subtype which plays a role in the occurrence of convulsions. Mice were grouped as allowed to eat ad lib (fed) and deprived of food for 24h (fasted). Fasted animals developed convulsions after being treated with scopolamine (60%) or the selective M1 receptor antagonist pirenzepine (10mg/kg; 20% and 60mg/kg; 70%) and given food. Fasting increased expression of M1 receptors in the frontal cortex and M2 receptors in the hippocampus, but produced no change in the expression of both receptors in the amygdaloid complex. Food intake after fasting decreased M1 receptor expression in the frontal cortex and M1 and M2 receptor expression in the hippocampus. Seizure severity was uncorrelated with muscarinic receptor expression in the brain regions. Taken together, these findings provide evidence for the role of M1 muscarinic receptor antagonism and fasting-induced increases in M1 and M2 expression possible underlying mechanism in the occurrence of convulsions in fasted animals.

Antimuscarinic-induced convulsions in fasted mice after food intake: No evidence of spontaneous seizures, behavioral changes or neuronal damage.

Prolonged or repeated seizures have been shown to cause spontaneous recurrent seizures, increased anxiety­related behavior, locomotor hyperactivity, impaired functions of learning and memory, and neuronal damage in the hippocampus and other brain regions in animals. Mice and rats treated with antimuscarinic drugs after fasting for two days or less develop convulsions after being allowed to eat ad libitum. To address whether such behavioral and neuroanatomic changes occur following these convulsions, mice treated i.p. with saline (control) or 2.4 mg/kg atropine and given food after 24 h of fasting were grouped according to seizure scores for behavioral and histological analysis. Following convulsions, the occurrence of spontaneous recurrent seizures was observed for 30 days. Motor activity and grooming behavior were assessed in the open field, and memory was assessed using the novel object recognition test 4 and 7 days after onset of convulsions, respectively. Animals allocated for the histological analysis were decapitated 7 days after onset of convulsions and hippocampal slices were evaluated for the percentage of degenerating neurons stained with Fluoro­Jade C. Spontaneous recurrent seizures, locomotor alterations, anxiety­related behavior, memory impairment, and neuronal loss in the granular layer of the dentate gyrus were not detected in the animals with seizure score 1-2 or 3-5. These results are in accordance with those related to the absence of behavioral changes, cognitive deficits, and hippocampal neuronal damage after single brief seizures in animals and patients with epilepsy.

Learning and memory in the forced swimming test: effects of antidepressants having varying degrees of anticholinergic activity.

The antidepressant-induced reduction in immobility time in the forced swimming test may depend on memory impairment due to the drug's anticholinergic efficacy. Therefore, the present study evaluated learning and memory of the immobility response in rats after the pretest and test administrations of antidepressants having potent, comparatively lower, and no anticholinergic activities. Immobility was measured in the test session performed 24 h after the pretest session. Scopolamine and MK-801, which are agents that have memory impairing effects, were used as reference drugs for a better evaluation of the memory processes in the test. The pretest administrations of imipramine (15 and 30 mg/kg), amitriptyline (7.5 and 15 mg/kg), trazodone (10 mg/kg), fluoxetine (10 and 20 mg/kg), and moclobemide (10 and 20 mg/kg) were ineffective, whereas the pretest administrations of scopolamine (0.5 mg/kg) and MK-801 (0.1 mg/kg) decreased immobility time suggesting impaired "learning to be immobile" in the animals. The test administrations of imipramine (30 mg/kg), amitriptyline (15 mg/kg), moclobemide (10 mg/kg), scopolamine (0.5 and 1 mg/kg), and MK-801 (0.1 mg/kg) decreased immobility time, which suggested that the drugs exerted antidepressant activity or the animals did not recall that attempting to escape was futile. The test administrations of trazodone (10 mg/kg) and fluoxetine (10 and 20 mg/kg) produced no effect on immobility time. Even though the false-negative and positive responses made it somewhat difficult to interpret the findings, this study demonstrated that when given before the pretest antidepressants with or without anticholinergic activity seemed to be devoid of impairing the learning process in the test.

Antimuscarinic-induced convulsions in fasted animals after food intake: evaluation of the effects of levetiracetam, topiramate and different doses of atropine.

This study evaluated the effects of different doses of atropine and new antiepileptics, levetiracetam and topiramate, on the development of convulsions triggered by food intake in antimuscarinic-treated fasted animals. Mice deprived of food for 24 h and treated i.p. with atropine at a dose of 2.4 or 24 mg/kg developed convulsions after being allowed to eat ad libitum. No convulsions were observed in fasted animals treated with 0.24 mg/kg atropine. There was no difference in the incidence of convulsions between the two atropine treatments, but latency to convulsions was longer in 24 mg/kg atropine treated animals. The lowest dose of atropine, 0.24 mg/kg, caused stage 1 and stage 2 activity, but did not provide the convulsive endpoint (stage 3, 4, 5 activity). Administration of levetiracetam (50 or 200 mg/kg) or topiramate (50 or 100 mg/kg) to another group of 24-h fasted mice was ineffective in reducing the incidence of convulsions developed in the animals after 2.4 mg/kg atropine treatment and food intake. However, the higher dose of levetiracetam prolonged the onset of convulsions. Present results demonstrated the efficacy of low and high doses of atropine on the development of convulsions in fasted animals and provided additional evidence for the ineffectiveness of antiepileptic treatment in these seizures.

Scopolamine-induced convulsions in fasted animals after food intake: sensitivity of C57BL/6J mice and Sprague-Dawley rats.

Food intake triggers convulsions in fasted BALB/c mice and Wistar albino rats treated with antimuscarinic drugs, scopolamine or atropine. Inbred strain studies have yielded considerable information regarding genetic influences on seizure susceptibility and factors contribute to epileptogenesis in rodents. This study, therefore, investigated sensitivity to antimuscarinic-induced seizures in C57BL/6J mice and Sprague-Dawley rats. Food deprivation for 48h in mice and 52h in rats did not produce strain differences in body weight loss. Fasted animals treated i.p. with 3mg/kg scopolamine developed convulsions after food intake. The incidence of convulsions was indifferent in comparison to BALB/c mice and Wistar albino rats. Number of animals developing stage 5 was more and onset of convulsions was longer in C57BL/6J mice than in BALB/c mice. Strain-related differences in sensitivity to seizures in C57BL/6J mice may need further evaluation for investigating genetic influences on scopolamine-induced seizures.

Spontaneous withdrawal in intermittent morphine administration in rats and mice: effect of clonidine coadministration and sex-related differences.

BACKGROUND/AIM: Treating animals repeatedly with intermittent and increasing morphine doses has been suggested to allow some withdrawal during each dosing interval, which causes repeated stress. The present study aimed to test this hypothesis and assess sex-related differences in withdrawal signs and their suppression by clonidine. MATERIALS AND METHODS: Male and female rats and mice were administered with increasing doses of morphine twice daily at different dosing intervals. Rats were given clonidine in drinking water (5 µg/mL). Spontaneous and naloxone-precipitated withdrawal signs and novelty-induced grooming were evaluated. RESULTS: Male rats and male and female rats displayed manifestations of morphine withdrawal at the end of 14-h and 24-h dosing intervals, respectively. Clonidine attenuated the severity of the withdrawal signs. Male but not female mice displayed withdrawal signs at the end of 12-h and 17-h dosing intervals. Female mice exhibited less pronounced naloxone-precipitated withdrawal syndrome. Grooming did not reflect a "stress-like state" in morphine-treated animals. CONCLUSION: These findings indicate intermittent morphine treatment-induced spontaneous withdrawal in rats and mice and sex-related differences in spontaneous and naloxone-precipitated withdrawal signs in mice. Since the treatment protocol closely parallels the drug use pattern in opioid addicts, further experiments are needed to clarify the stress associated with the treatment and the efficacy of sedatives.

Evaluation of anxiolytic effect and withdrawal anxiety in chronic intermittent diazepam treatment in rats.

This study evaluated the effect of intermittent administration in the development of dependence to diazepam in chronic use of the drug. Gabapentin was used to provide an anxiolytic effect on drug-free days. During a 28-day treatment schedule, rats were given diazepam (15 mg/kg) once daily continuously, or intermittently with saline or gabapentin (50 mg/kg) on days 5, 10, 15, 20, and 25. Anxiety-like behavior was assessed on days 10 and 30 using the elevated plus-maze test and novelty-induced grooming test. Contrary to continuous administration, intermittent diazepam did not provide anxiolytic-like activity on day 10; instead, it prevented withdrawal anxiety on day 30. Gabapentin produced anxiolytic-like effects during the withdrawal period, but not on day 10. These results suggest that intermittent administration of diazepam (given either alone or alternatively with a drug possessing anxiolytic activity) may be of value in preventing the development of physical dependence during the chronic use of the drug. However, further studies are needed to demonstrate that this protocol could effectively produce anxiolytic activity on diazepam-free days.

Assessment of rewarding and reinforcing properties of biperiden in conditioned place preference in rats.

Biperiden is one of the most commonly abused anticholinergic drugs. This study assessed its motivational effects in the acquisition of conditioned place preference in rats. Biperiden neither produced place conditioning itself nor enhanced the rewarding effect of morphine. Furthermore, biperiden in combination with haloperidol also did not affect place preference. These findings suggest that biperiden seems devoid of abuse potential properties at least at the doses used.

Seizures triggered by food intake in antimuscarinic-treated fasted animals: evaluation of the experimental findings in terms of similarities to eating-triggered epilepsy.

Food intake triggers convulsions in fasted mice and rats treated with antimuscarinic drugs, scopolamine or atropine. Bearing some similarities in triggering factor and manifestations of the seizures in patients with eating-evoked epilepsy, seizures in fasted animals may provide insight into the mechanism(s) of this rare and partially controlled form of reflex epilepsy.

Scopolamine-induced convulsions in fasted mice after food intake: evaluation of the sedative effect in the suppression of convulsions.

Food intake triggers convulsions in fasted mice and rats treated with antimuscarinic drugs, scopolamine or atropine. Most of the drugs produced anticonvulsant efficacy in these convulsions have sedative effects. Thus, the present study was performed to evaluate the contribution of sedation in the suppression of convulsions by using sedative drugs chlorpromazine, morphine, amitriptyline and diphenhydramine. Mice fasted for 24h and treated with 3mg/kg scopolamine developed convulsions soon after refeeding. Treatment of chlorpromazine and morphine during food deprivation did not provide a preventive effect in the development of convulsions observed after food intake in fasted animals. Pretreatment of amitriptyline, but not diphenhydramine, before scopolamine treatment suppressed the incidence of convulsions. Present results could not clearly demonstrate the role played by sedative effect in suppression of convulsions in fasted animals.

Scopolamine-induced convulsions in fasted mice after food intake: the effect of duration of food deprivation.

It has been shown that mice and rats treated with antimuscarinic drugs, scopolamine or atropine, after fasting for 48 h develop convulsions soon after refeeding. The present study was performed to evaluate whether mice also develop convulsions after being deprived of food for 1-24 h. The effect of day-night fasting on the development of convulsions was also determined in 12-h deprived animals. Mice were deprived of food for periods of 1, 2, 3, 6, 9, 12, 18, 24, and 48 h. Animals fasted for 12 h during the day or night were deprived of food at 08:00 or 20:00 h, respectively. At the time of testing, animals were treated with intraperitoneal (i.p.) saline or 3 mg/kg scopolamine. Twenty minutes later, they were given food and allowed to eat ad lib. All animals were observed for 30 min for the incidence and onset of convulsions. Fasted animals treated with scopolamine developed clonic convulsions after food intake. Incidence of convulsions was significant in 2-, 3-, 12-, 18-, 24-, and 48-h deprived animals. Convulsions observed after deprivation of food for 12 h during the day or at night were almost similar in both regimens. Our results indicate that food deprivation itself, rather than its duration, seems to be the principal factor in the development of these convulsions.

Evaluation of the acute effects of amitriptyline and fluoxetine on anxiety using grooming analysis algorithm in rats.

The tricyclic amitriptyline and the selective serotonin reuptake inhibitor fluoxetine have distinct actions in animal models of anxiety, though both antidepressants are used against anxiety disorders. Grooming behavioural sequencing, rather than its general "activity" measures, has been suggested to measure effectively the pharmacologically induced anxiolytic and anxiogenic-like effects in rats and mice. In the present study, the acute effects of amitriptyline and fluoxetine on anxiety were re-evaluated by using an analysis algorithm in novelty-induced grooming activity in rats. Additionally, the effects on anxiety-like behaviour in the hole board were examined. Amitriptyline (5 and 10 mg/kg) and fluoxetine (5 and 10 mg/kg) not only affected the traditional gross measures, but also produced changes in incorrect transitions and regional distribution of grooming behaviour. High dose of fluoxetine showed an anxiogenic-like profile by reducing head dipping and rearing in the hole board. Depending on the effects on the behavioural microstructure of grooming activity, present findings imply that amitriptyline may possess anxiogenic and fluoxetine may possess anxiolytic activities. However, measures of hole board do not fully support this suggestion.

The evaluation of antimuscarinic-induced convulsions in fasted rats after food intake.

The present study was performed to evaluate convulsions after food intake in fasted rats pretreated with scopolamine or atropine and to determine whether these convulsions respond to drugs found effective in fasted mice. Scopolamine (2.4 mg/kg) and atropine (2.4 mg/kg) were given intraperitoneally (i.p.) to rats fasted for 52h. Both drugs induced convulsions after animals were allowed to eat ad lib. Another group of fasted rats pretreated with saline, MK-801 (0.1mg/kg), clonidine (0.1mg/kg), chlorpromazine (2 and 4 mg/kg), valproate (200mg/kg), diazepam (1.5 and 2mg/kg) or gabapentin (50mg/kg) were treated i.p. with saline or scopolamine (2.4 mg/kg) and were allowed to eat ad lib. Clonidine, MK-801, chlorpromazine (4 mg/kg) and diazepam (2 mg/kg) reduced the incidence of scopolamine-induced convulsions in fasted rats. Gabapentin could only prolong the onset of convulsions. Neither treatment was effective against myoclonus of hindlimbs. Present results showed that fasted rats also develop antimuscarinic-induced convulsions which do not completely respond to treatments found effective in convulsions of fasted mice.

Scopolamine-induced convulsions in fasted mice after food intake: effects of glucose intake, antimuscarinic activity and anticonvulsant drugs.

The present study was performed to further evaluate the contribution of antimuscarinic activity and hypoglycaemia to the development of scopolamine-induced convulsions in fasted mice after food intake. The effects of anticonvulsant drugs on convulsions were also evaluated. Antimuscarinic drugs atropine (3 mg/kg) and biperiden (10 mg/kg) were given intraperitoneally (i.p) to animals fasted for 48 h. Like scopolamine, both drugs induced convulsions after animals were allowed to eat ad libitum. Another group of animals was given glucose (5%) in drinking water during fasting. These animals, although they had normoglycaemic blood levels after fasting, also developed convulsions after treated with scopolamine i.p. (3 mg/kg), atropine (3 mg/kg) or biperiden (10 mg/kg) and allowed to eat ad libitum. Among the drugs studied, only valproate (340 mg/kg), gabapentin (50 mg/kg) and diazepam (2.5 and 5 mg/kg) markedly reduced the incidence of scopolamine-induced convulsions. The present results indicate that antimuscarinic activity, but not hypoglycaemia, underlies these convulsions which do not respond to most of the conventional anticonvulsant drugs.