Withdrawal-like symptoms in young and adult rats maternally exposed to methadone

Rats of 30, 45, 60 and 120 days of age, maternally exposed to methadone (5 mg/kg daily) during gestation and /or lactation, were evaluated on a variety of behavioral and physiological parameters related to drug withdrawal. Animals were tested before and after an acute injection of naloxone (10 mg/kg). Prior to maloxone injection, methadone-exposed rats were subnormal in body temperature at 30 days of age, hypoalgesic at 45 days, and weighed less than controls at 60 days. Additionally, and in contrast to control rats, methadone-exposed animals at most ages displayed head shake and wet-dog shake behaviors. After naloxone administration, methadone-exposed rats exhibited an increase in the mean number of head and wet-dog shakes over pre-injection levels. Although control rats injected with naloxone also demonstrated head shakes (at all ages) and wet-dog shakes (at 45 days), these behaviors were usually not of the magnitude as noted for methadone-exposed offspring receiving naloxone. Perturbations in body weight and hypothermia during development, along with head shake and wet-dog shake behaviors which were exacerbated following naloxone administration, suggest a protracted state of physical dependence/withdrawal and/or permanent damage as a result of perinatal exposure to methadone.     

Opiate self-administration in adult offspring of methadone-treated female rats

Chronic treatment of female Sprague-Dawley rats with methadone 5 mg/kg IP throughout gestation and lactation resulted in an increased oral self-administration (S.A.) of morphine by their 85-90-day-old offspring. By day 16 of the S.A. treatment schedule methadone offspring were taking 75 to 80% of their total fluid intake as morphine solution when given a choice between morphine solution and water, while control offspring under the same conditions took 33% of their total fluids as morphine solution. When the subjects were again given a choice between water and morphine solution following a 12-day drug free period, methadone offspring drank a significantly greater percentage of morphine solution than controls. Methadone S.A. in methadone offspring was not different from controls. The reasons for this marked difference between morphine and methadone S.A. are not clear. However, it does appear that chronic maternal exposure to methadone may facilitate development of a morphine-S.A. behavior in their offspring.     

Adolescent and adult female rats differ in sensitivity to nicotine's activity effects

More than 90% of cigarette smokers begin smoking during adolescence. This between-subjects repeated-measures experiment examined: (1) nicotine's acute effects on activity in adolescent and adult female Sprague–Dawley rats (Drug Phase I); (2) the effects of age of initial nicotine exposure on activity when nicotine was not administered (Interim Phase); and (3) the effects of age of initial nicotine exposure on later responses to nicotine (Drug Phase II). The experiment consisted of three separate phases. In Drug Phase I, animals were administered either 0 (saline), 0.01, 0.10, 0.50, or 1.0 mg/kg nicotine via subcutaneous injections for 12 days and horizontal activity was measured daily. During the Interim Phase (no drug phase), activity was measured but nicotine was not administered. During Drug Phase II, the same animals were administered the same nicotine dosages as in Drug Phase I for 12 days and activity was measured daily. Drug Phase I revealed dose–response differences between adolescent and adult female rats. In addition, animals initially exposed to nicotine in adolescence exhibited greater sensitivity to nicotine's activity-increasing effects than did females initially exposed to nicotine in adulthood (i.e., Drug Phase II).     

Disorders in bone metabolism of female rats chronically exposed to cadmium

The effect of cadmium (Cd) on bone metabolism during skeletal development and maturity was investigated on a rat model of human exposure. Young female Wistar rats were exposed to 1, 5, or 50 mg Cd/l in drinking water for 3, 6, 9, and 12 months. Total bone mineral density (T-BMD), bone mineral content (BMC), density (BMD), and bone area at the femur and lumbar spine (L1-L5) were measured densitometrically. Alkaline phosphatase (ALP) and osteocalcin (OC) as bone formation markers, and carboxy-terminal cross-linking telopeptides of type I collagen (CTX) in bone (trabecular and cortical) or serum as bone resorption markers were measured. Renal calcium (Ca) handling and Cd body burden were evaluated as well. At the stage of intensive skeletal development (the first 6 months of the experiment), at all exposure levels, Cd inhibited the processes of bone formation and as a result disturbed the accumulation of bone mass leading to osteopenia (- 1 > Z score/T score BMD > -2.5) and at 5 and 50 mg Cd/l even to more advanced disorders in the BMD. Continuation of the exposure up to skeletal maturity led to high bone turnover with increased resorption enhancing the prevalence of osteopenia or the BMD values having the Z score/T score < -2.5. The results allow for the conclusion that chronic, even low-level exposure to Cd disturbs bone metabolism during skeletal development and maturity by affecting bone turnover most probably through a direct influence on bone formation and resorption, and indirectly via disorders in Ca metabolism. Our findings confirm the hypothesis that environmental exposure to Cd may be a risk factor for low BMD.     

Lack of response inhibition in rats prenatally exposed to alcohol

Offspring of mothers who consumed either 32, 19, 8, or 0% of their daily caloric intake in the form of ethanol during pregnancy were tested for passive avoidance. At 18 days of age, the number of trials to criterion and the within-group variability were direct functions of the amount of ethanol consumed by the mother during pregnancy. At 41–53 days of age, alcohol-treated pups still required more trials to criterion than controls and had faster speeds into the shock compartment on the first trial. When the progeny of mothers consuming either 35, 17, or 0% ethanolderived calories during pregnancy were compared for conditioned taste aversion to a lithium chloride solution, a linear dose-response function was again evident. Animals in the alcohol-treated groups showed less suppression of drinking than controls. These investigations indicated that the effects of alcohol exposure in utero were manifested in behavioral out-comes involving response inhibition that were not correlated with the more familiar physical symptoms.     

Postnatal orotate treatment: Effects on learning and memory in adult rats

During the postnatal period, male Wistar rats were treated with orotate, either from the 6th to 15th, 16th to 25th, or 26th to 35th day of life. Learning and memory were tested in adulthood. Rats that received orotate from the 6th to 15th day showed a better retention of a learned brightness discrimination (Y-maze) than controls. An active avoidance (pole jumping) was learned more quickly by the rats orotatetreated from the 6th to 15th day than by controls. The spontaneous locomotor activity of previously orotatetreated rats was the same as in controls. Body weight measurements revealed no differences between orotate rats and control rats. The results suggest that memory retention in adulthood can be improved by postnatal orotate treatment.     

Route of administration influences substitution patterns in rats trained to discriminate methadone vs. vehicle

Replacement therapy with the synthetic μ-opioid agonist methadone is an efficacious treatment for opioid abuse. While much is known about methadone's pharmacology, its discriminative stimulus properties remain largely unexplored. The present study sought to establish methadone discrimination in rats. Moreover, some research suggests that route of administration alters the discriminative stimulus of methadone. Thus, the present study also compared intraperitoneal (i.p.) and subcutaneous (s.c.) routes of administration. Male Sprague–Dawley rats were trained to discriminate 3.0 mg/kg methadone (i.p.) from vehicle in a two-lever discrimination procedure. Generalization tests were conducted with a variety of compounds administered i.p. and s.c. Methadone fully substituted for itself, yielding ED₅₀s of 1.5 mg/kg (i.p.) and 0.2 mg/kg (s.c.). Naltrexone (i.p.), an opioid antagonist produced a dose-dependent reduction in methadone-appropriate responding. The methadone stereoisomers fully substituted for methadone when given s.c.; however, when administered i.p., (+) and (−) methadone produced partial and no substitution, respectively. Heroin fully generalized to methadone regardless of administration route, while morphine fully substituted when given s.c., but not i.p. The kappa-agonist U50-488 failed to generalize to methadone with either route of administration. These results demonstrated that methadone's discriminative stimulus is mediated through μ-opioid receptor activity and is similar to that of commonly abused opioids (heroin, morphine). Additionally, route of administration produced differential results for many of the drugs tested, suggesting decreased drug bioavailability following i.p. administration due to hepatic first pass metabolism. Taken together, these results suggest that methadone's shared subjective effects with abused opioids, as well as its unique metabolic properties contribute to its efficacy in opioid maintenance therapy.     

Luteinizing-hormone-releasing hormone modifies retention of passive and active avoidance responses in rats

The influence of posttraining subcutaneous administration of luteinizing-hormone-releasing hormone (LHRH) was tested on the retention of either active or passive avoidance conditioning in male rats. Injection of LHRH (200/kg) immediately after the acquisition of an active avoidance response (two-way shuttle behavior) enhanced retention of the response, assessed 7 days later. When the neuropeptide was injected immediately after a passive avoidance conditioning training, the effects varied with the intensity of the footshock applied. LHRH enhanced retention of avoidance training with weak footshock (0.20 and 0.35 mA) but impaired retention of training with strong footshock (0.70 and 1.0 mA). The effects of LHRH seem to be unspecific since they are similar to those observed after treatment with several hormones. The results are discussed based on the interactions between peripherally injected hormones and endogenous substances released following footshock. A modulatory effect on the monoaminergic pathway involved in memory storage processes is postulated.     

Sensitivity to acute methadone dose changes in maintenance patients

This study assessed whether methadone patients can identify acute dose changes in their maintenance dose, and explored the relationships between self-reported drug effects and real or perceived dose changes. Four times each week patients (N = 10) unpredictably received either 80%, 90%, 100%, 110% or 120% of their usual daily dose (50–100 mg). Approximately 24 hr later they indicated which dose they had received on the previous day, and rated the previous day's dose in terms of good effects, bad effects, and change in medication taste. Correct estimation of the doses received was always at the levels expected by chance alone. Furthermore, this sample of patients could not detect dose-related changes in medication taste. However, self-reports of good effects were significantly higher when patients believed that they had received a dose increment, and ratings of bad effects were higher when patients believed that they had received a dose decrement.     

Impairments of learning and memory following intracerebroventricular administration of AF64A in rats

Three types of learning and memory tests (Morris water maze, active and passive avoidance) were performed in rats following intracerebroventricular infusion of ethylcholine aziridium (AF64A). In Morris water maze, AF64A-treated rats showed the delayed latencies to find the platform from 6th day after the infusion. In pretrained rats, AF64A caused the significant delay of latency at 7th day, but not 8th day. In the active avoidance for the pre-trained rats, the escape latency was significantly delayed in AF64A-treatment. The percentages of avoidance in AF64A-treated rats were less increased than those in the control. Especially, the percentage of no response in the AF64A-treated rats was markedly increased in the first half trials. In the passive avoidance, AF64A-treated rats shortened the latency 1.5 h after the electronic shock, but not 24 h. AF64A also caused the pretrained rats to shorten the latency 7th day after the infusion, but not 8th day. These results indicate that AF64A might impair the learning and memory. However, these results indicate that the disturbed memory by AF64A might rapidly recover after the first retrain. Furthermore, these results suggest that AF64A may be a useful agent for the animal model of learning for spatial cognition.     

Behavioral effects of nicotine withdrawal in adult male and female rats

Nicotine withdrawal may differ between men and women but clinical reports are inconsistent. Two experiments were conducted to examine behavioral effects of nicotine withdrawal in male and female adult rats in dimly-lit and brightly-lit environments. Ninety-six Sprague-Dawley male and female rats received 7 days continuous subcutaneous infusion via ALZET osmotic minipumps filled with saline or 3.16 mg/kg/day nicotine hydrogen tartrate expressed as base. Behavioral observations were made before, during, and after drug administration. During observations, occurrences of empty-mouth-chewing, whole-body-shakes, abnormal grooming, abnormal posture/movement, diarrhea, ptosis, eyeblinks, and any other abnormal behaviors were counted. Cessation of nicotine administration upon pump removal caused a significant increase in withdrawal behaviors in males and females in both environments. In the dimly-lit environment, females showed more withdrawal behavior than males; there was no sex difference in the brightly-lit environment. Males that had received nicotine displayed more withdrawal behavior in the brightly-lit environment than in the dimly-lit environment, while females that had received nicotine displayed similar amounts of withdrawal behavior in both environments. Behavioral symptoms of withdrawal may be more affected by the environment in male rats than in female rats. These experiments are the first to compare nicotine withdrawal in adult male and female rats.     

Subsequent anxiety-related behavior in rats exposed to low-dose methadone during gestation, lactation or both periods consecutively

In order to assess the long-term behavioral consequences of exposing rats to methadone during gestation, lactation or both periods consecutively, pregnant Wistar dams were provided with drinking water containing approximately 2.39 mg/kg/day methadone. Soon after birth, litters of offspring were assigned to methadone-naïve foster mothers. Half of these foster mothers were then provided with drinking water containing methadone (approximately 2.86 mg/kg/day), while the other half received unadulterated water. Maternal weight gain, pregnancy duration, litter sizes, sex ratios and average pup weights were recorded. Following weaning on postnatal day (PND) 28, individual rats were weighed and inspected for physical abnormalities and stress reactions at PND20, 60 and 120. At these same ages, observations were also made of the rats' behavior in an emergence apparatus, and an open field. Apart from a smaller number of full-term pregnancies, there were no effects of any type of methadone treatment on physical measurements recorded at any age. Nor were there any behavioral effects of gestational methadone experienced on its own. However, methadone experienced during lactation (without gestational exposure) decreased emergence speed at PND30, and for all testing ages combined, increased open-field ambulation (males only), walking, rearing and occupancy of the center of the apparatus. Exposure to methadone during both gestation and lactation decreased emergence latencies at PND30 and, for all ages combined, decreased ambulation (males only), center occupancy and defecation. The subsequent behavioral effects of methadone were largely confined to lactational exposure and, when combined with gestational exposure, suggested increased anxiety.     

Neuroendocrine changes in adult female rats prenatally exposed to phenytoin

Neuroendocrine changes in fetal hydantoin syndrome have not been described yet. This study was aimed to verify the hypothesis that prenatal exposure to phenytoin influences the stress response of adult female offspring in an animal model. To study possible development of depression like state, hedonic behavior and long-term changes in neuropeptide gene expression in the hypothalamus were investigated. Treatment consisted of per os administration of 150 mg/kg of phenytoin or water daily, from day 7-18 of gestation. Adult female offspring (6 animals per group) were acutely stressed by 1 min handling. Blood was collected in conscious rats via tail artery cannulas before, 1, 15 and 30 min after the handling. Exposure to phenytoin in uterus resulted in increased catecholamine and corticosterone concentrations in response to a mild stressor of 1 min handling in adult offspring. The gestational treatment used in this study did not induce a depression like state nor long-term changes in neuropeptide gene expression in the adult offspring. In conclusion, prenatal exposure to phenytoin treatment enhanced the stress response of adult female offspring. Possible new component of fetal hydantoin syndrome is the increase in catecholamine release in response to a mild stressor in adulthood.     

Genital tract abnormalities in female rats exposed todiethylstilbestrol in utero

Genital tract morphology in 14-month old female rats exposed prenatally to diethylstilbestrol (DES) was analyzed as part of an examination of the effects of transplacental exposure to DES on estrogen sensitive tissues. Pregnant Sprague-Dawley rats were injected with sesame oil alone or with DES in sesame oil on days 10 and 13 of gestation (total dose 1.2 μg DES) or on days 15 and 18 (total dose 1.2μg or 120 μg DES). Female offspring (9–15 per group) were sacrificed at 14 months of age. Effects of DES exposure varied with the dose given and with the stage of differentiation of the fetal tissues. In the ovaries of rats exposed to 120 μg of DES on days 15 and 18 of gestation, follicular elements were reduced and replaced by dense sheets of stromal cells; oophoritis was noted in five of nine rats. Hypercellularity of oviductal stroma was another common feature, as was suppurative salpingitis. Ovaries of rats exposed to 1.2 μg DES on days 10 and 13 of gestation were more likely to contain numerous corpora lutea than the other DES-exposed groups or controls. An increased incidence of benign uterine abnormalities was observed in DES-exposed offspring, including squamous metaplasia and suppurative endometritis. In the cervices of all nine rats exposed to 120 μg DES on days 15 and 18 of gestation, the epithelial surface showed a convoluted pattern, lined by stratified squamous and stratified cuboidal cells. Thus, prenatal exposure to DES, especially at the higher dose used, has long-term consequences on reproductive tract morphology in Sprague-Dawley rats.     

Behavioural observations in gunn rats

The Gunn rat is a hooded mutant of albino rat with various biochemical defects, including a low UDP-glucuronosyl-transferase activity. As a consequence, about half of their offspring are jaundiced from birth, due to high free bilirubin levels, and develop widespread brain damage. The behaviour of both jaundiced and nonjaundiced Gunn rats was studied in four different tests in a shuttle-box and in a stepthrough passive avoidance situation, and compared with that of normal hooded rats. No differences among groups were found in performance of shuttle responses to a tone in a pseudoconditioning paradigm in which tones and shocks were given at random. However, rats from the two Gunn groups made less shuttlings to the tone in two tests that involved an avoidance contingency (each response cancelled one shock). In addition, nonicteric Gunn rats also performed poorly in a classical conditioning test in the shuttle-box (tones and shocks paired on every trial regardless of responses). This last deficiency of non-icteric Gunn rats may be explained by their higher tendency to freeze in situations involving stimulus-stimulus interactions. They also showed a higher latency than that of the two other groups to enter the dark side of the step-through apparatus on their first exposure to it. All animals seemed to learn the passive-avoidance task to the same extent, however, as shown in a retest carried out 48 h later. Both Gunn groups were hypersensitive to the stereotyped-behaviour-inducing action of apomorphine (0.125–1.0 mg/kg, i.p.), but all groups were about equally sensitive to that of d-amphetamine sulfate (0.5–4.0 mg/kg). Since apomorphine is disposed of by glucuronidation, this might be explained by the low UDP-glucuronosyl-transferase activity known to exist in the Gunn animals. The present results show that additional genetic defects have developed by in-breeding in the Gunn population, which are unrelated to brain damage caused by bilirubin, and which can be well characterized from a behavioural standpoint.     

Cannabinoid receptor binding and mRNA levels in several brain regions of adult male and female rats perinatally exposed to delta9-tetrahydrocannabinol.

The present study was designed to elucidate whether perinatal delta9-tetrahydrocannabinol (delta9-THC) exposure results in changes in cannabinoid receptor binding and mRNA levels in adulthood. Most of the brain areas studied, including the basal ganglia, the cerebellum, the limbic structures, and most of the hippocampal regions exhibited no changes in cannabinoid receptor binding and mRNA levels in adulthood as a consequence of the perinatal delta9-THC exposure. However, some subtle changes could be appreciated in specific regions, although their physiological relevance seems uncertain. For example, delta9-THC-exposed males exhibited a small decrease in binding in the superficial layer of the cerebral cortex, an effect that was not seen in delta9-THC-exposed females and in mRNA levels for both males and females. In the CA2 layer of the Ammon's horn, there was an increase in mRNA levels of delta9-THC-exposed animals, although this was statistically significant only in males. However, the more marked and probably relevant changes were seen in the arcuate nucleus, where delta9-THC-exposed males exhibited an increase in binding, whereas this tended to decrease in delta9-THC-exposed females. In an additional experiment, we analyzed the motor response of these animals to a challenge with SR141716, a specific antagonist for cannabinoid receptors. The delta9-THC-exposed animals tended to show a higher response to SR141716 challenge, with changes apparently more marked in delta9-THC-exposed females, although they did not reach statistical significance. In summary, perinatal cannabinoid exposure does not appear to significantly alter cannabinoid receptor binding and mRNA expression in the brain of adult rats, as well as the motor response caused by the blockade of these receptors with a specific antagonist. There were some changes in the status of cannabinoid receptors but they were very small and, hence, of debatable physiological relevance. The most significant of these effects was the increase in binding observed in the arcuate nucleus of delta9-THC-exposed males.     

Hospital morphine preparation for abstinence syndrome in newborns exposed to buprenorphine or methadone

Objective This study was undertaken to evaluate the adequacy of a hospital formulated oral morphine preparation for management of neonatal abstinence syndrome (NAS) and to compare clinical features in infants exposed to methadone or buprenorphine in utero. Method Between October 1998 and October 2004 all infants born to mothers treated with buprenorphine or methadone during pregnancy were enrolled into this prospective study. Morphine hydrochloride solution (0.2 mg/ml) was prepared without preservatives under a flow laminar air box (class 100). Mean outcome measure Morphine solution: quantitative and qualitative HPLC analysis and microbiological study at regular intervals during storage at 4°C for 6 months. Maternal characteristics: age, opiate dose during pregnancy. Neonatal characteristics: gestational age at delivery, birth weight, Lipsitz scores. Morphine dose: daily morphine dose, maximum morphine dose, duration of NAS, and duration of treatment required to achieve stable Lipsitz scores below 4. Statistics: Kruskal–Wallis test for comparison of median values. Results Microbiological and HPLC analysis showed that the morphine preparation remained stable for 6 months at 4°C. Nine methadone-exposed infants and 13 buprenorphine-exposed infants were included in the study. All infants presented NAS requiring treatment with the morphine solution. Lipsitz scores at birth were significantly different in the methadone and buprenorphine groups (P < 0.05). The methadone group required significantly higher doses of morphine preparation than the buprenorphine group during the first 38 days of treatment (P < 0.05): 0.435 ± 0.150 mg/kg/day vs. 0.257 ± 0.083 mg/kg/day. Conclusion This hospital morphine solution is adequate for management of NAS. Preparations showed good stability and doses could be adjusted with a margin of 0.02 mg. The onset of NAS occurred within 24 h after birth in methadone-exposed infants (range 6–24 h) and within 48 h after birth in buprenorphine-exposed infants (range 24–168 h). Due to the possibility of delayed onset of NAS up to 7 days, infants born to mothers treated with buprenorphine should be kept in the hospital for an appropriate surveillance period. Treatment time was significantly longer (45 vs. 28 days) and the mean morphine doses were higher (1.7 fold) in methadone-exposed than buprenorphine-exposed infants.     

Effect of low-level lifetime exposure to cadmium on calciotropic hormones in aged female rats

The effect of low-level lifetime exposure to cadmium (Cd) on calciotropic hormones and the possible association between the Cd-induced disorders in bone metabolism and these hormones were investigated on a female rat model of human environmental exposure in areas unpolluted by this metal. For this purpose, the concentrations of 25-hydroxyvitamin D (25OHD), 1,25-dihydroxyvitamin D (1,25(OH)₂D), calcitonin (CT) and parathormone (PTH) were measured in the serum of control and Cd-exposed (1 mg Cd/l in drinking water for 24 months) female rats. Calcium (Ca) and inorganic phosphorus (P_i) serum concentrations, renal tubular reabsorption of Ca (TRCa) and phosphate (TRP) and the glomerular filtration rate (GFR) were estimated as well. Moreover, 1,25(OH)₂D, metallothionein (MT) and Cd were determined in the kidney. The exposure to Cd led to a decrease in the serum concentrations of 25OHD and 1,25(OH)₂D (by 50 and 31%, respectively) and the concentration of 1,25(OH)₂D in the kidney mitochondrial fraction (by 55%). The serum concentrations of CT and PTH increased (5.2-fold and by 29%, respectively) and those of Ca and P_i were unchanged, whereas the TRCa, TRP and GFR decreased due to the exposure to Cd. The results give evidence that the low lifetime exposure to Cd disturbs the metabolism of calciotropic hormones and damages the reabsorptive and filtrative function of the kidney in aged female rats. Numerous correlations noted between calciotropic hormones and the indices of kidney function, and indices of bone turnover and bone mineral status (bone mineral content and density) of these females indicate a relationship between these hormones and the kidney functional status and bone metabolism. The results of the present study together with our previous findings on the bone status in the experimental model allow for the conclusion that the low lifetime exposure to Cd by affecting the metabolism and proper function of calciotropic hormones may contribute to the advancement of bone damage at the elderly.     

PA-learning in young rats with dorsal hippocampal- and hippocampo-entorhinal atropine.

Twenty-one-day-old rats injected with atropine into dorsal hippocampus and trained on a white-black step-through passive avoidance task, did not perform differently from their controls in acquisition or extinction. In contrast, when atropine was administered into the ventral hippocampo-entorhinal area, the animals displayed a passive avoidance deficit. The results support the finding that the posteroventral but not the anterodorsal part of the hippocampal complex is implicated in passive avoidance learning and suggest a cholinergic mediation of this effect.     

The induction of mounting behavior in female rats by p-chlorophenylalanine.

The effect of p-chlorophenylalanine (pCPA) and testosterone propionate (TP) on mounting behavior of ovariectomized female rats towards receptive and non-receptive stimulus females was studied. Both pCPA and TP facilitated mounting and the effect was intensified when these compounds were administered together. pCPA increased mounting behavior in the non-receptive stimulus condition. The results are interpreted as further evidence that pCPA affects sexual behavior in two ways: at first by a direct influence on the neutral substrate underlying masculine sexual behavior, and secondly by changing information processing of the sensory input from the partner.