The interactive effects of cocaine/gender on immune function in mice. An observation of in vivo acute cocaine exposure.

The present experiments used a mouse model including both sexes to study the impact of acute cocaine exposure on the function of four major immunocompetent cell types (T, B cells, NK, and macrophages). Cocaine hydrochloride, 5 mg or 40 mg/kg, was administrated by i.p. injection to C57BL/6 mice. Thymocytes and splenocytes were obtained 24 h after injection. Acute in vivo cocaine exposure inhibited the proliferation of T lymphocytes in response to Con-A in both thymocytes and splenocytes. However, the attenuated IL-2 production was only seen in thymocytes. These effects on T cells were greater in male mice than in female mice. The function of macrophages was also impaired by acute cocaine exposure; however, the impact was greater in female than in male mice. In conclusion, the effects of acute cocaine exposure altered the functions of immunocompetent cells and the effects varied with gender.

A murine model of prenatal cocaine exposure: effects on the mother and the fetus.

To develop and characterize a murine model for investigating the long-term effects of prenatal cocaine exposure, the present study established the route of drug administration and the doses to be used for pregnant C57BL/6 mice. Comparison of the effects of a high dose of cocaine (60 mg/kg) when gavaged or injected subcutaneously (SC) established patterns of pathology characteristic of administration route but no dominating logic for selecting one over the other route for prenatal studies; however, because of the fourfold greater brain levels, with no evidence of greater pathology, the SC route was selected. When injected daily during gestation days 12-18, the period of prenatal development of dopamine systems, cocaine at doses producing plasma concentrations consistent with its stimulatory effects reduced food ingestion and weight gains during pregnancy and fetal body and brain weights at term. The extent of these reductions was comparable to reports on babies exposed to cocaine prenatally. Furthermore, the present study suggests that maternal undernutrition is not a likely mediator of these perinatal effects and that differences in the amount of cocaine exposure may cause the contrasting effects of maternal cocaine noted in the human literature.

Cocaine concentrations in fetal C57BL/6 mouse brain relative to maternal brain and plasma.

Cocaine concentrations in maternal plasma and brain and fetal brain of mice were evaluated as a model for fetal brain exposure during maternal cocaine use. On days 12-18 of gestation, mice (C57BL/6; N = 5-7/group) received SC cocaine-HCI: 20 or 40 mg/kg. Maternal plasma and brain (accumbens and caudate nuclei removed), and fetal brain were collected at 0.5, 1, and 2 h following the last injection. Analysis was by GC-MS. Brain cocaine levels in the dams declined from 9.6 to 3.4 and 20.9 to 12.5 micrograms/g [corrected] during the 0.5-1-h period after the low and high doses, respectively, and were 7.5-14.3 times greater than plasma levels. The corresponding fetal brain concentrations changed from 1.6 to 1.3 and 2.9 to 3.4 micrograms/g [corrected]. By 2 h, brain cocaine concentrations in dams declined to approximately 10% of their 0.5-h values, with a slower drug decay occurring in fetal brain. Maternal plasma cocaine concentrations correlated with those of maternal brain (r = 0.94, p < 0.01) and fetal brain (r = 0.69, p < 0.01). The present results indicate that cocaine accumulates to a lesser extent in fetal brain than in maternal brain of C57BL/6 mice; however, the duration of exposure appears to be more sustained in the fetus, a phenomenon that may have toxicological implications for human in utero cocaine exposure.

Cocaine/sex type effects on T lymphocytes: a preliminary report.

The influence of gender on the effects of cocaine (5 or 40 mg/kg) on immune function of sexually immature (35-day-old) C57BL/6J mice was assessed. The proliferation of T cells in thymus and spleen stimulated with phytohemagglutinin (PHA) and concanavalin-A(Con-A) was determined by 3H thymidine incorporation 24 hours after cocaine injections. The drug produced a dose-dependent reduction (25-51%) of Con-A and PHA-stimulated T cell proliferation in thymus from both male and female mice. Additionally, the impairment occurred at a lower cocaine dose for female than for male mice when the T cells were challenged with PHA. Under the experimental conditions, cocaine did not alter weights or cell numbers of thymus and spleen or T cell proliferation in spleen.

Effects of prenatal ethanol exposure on dopamine systems in C57BL/6J mice.

Young rats prenatally exposed to ethanol exhibit heightened responses to dopaminergie (DA) drugs, altered brain concentrations of dopamine, and its metabolite dihydroxyphenylacetic acid (DOPAC), and transient reductions in DA receptor binding. Adult mice exposed to ethanol prenatally also exhibit increased responses to DA drugs; however, brain concentrations of DA and DOPAC are unaltered. The effects of prenatal ethanol exposure on DA or DOPAC concentrations in young mice or on DA receptor binding in mice of any age are unknown. Therefore, to determine if the different effects of prenatal ethanol exposure on rats and mice are due to age at time of testing or species, we determined its effects on DA concentrations and turnover in young mice under conditions previously reported for adult offspring and on DA D1 and D2 receptor binding in both young and adult offspring. Consistent with our previous report for adult offspring, prenatal ethanol exposure did not alter DA concentrations or turnover. The treatment did, however, diminish periadolescent growth as previously reported and produced a transient increase in DA D1, but not DA D2 receptor binding. DA receptor binding was not altered in adult offspring. Although unrelated to prenatal ethanol exposure, the sexes differed on all of the DA measures. Combined with previous reports, the present study suggests that species rather than age is more likely to account for the different effects of prenatal ethanol exposure on DA systems, and that sex differences in DA systems should be further examined.

Perinatal maternal ethanol effects on pregnant mice and on offspring viability and growth: influences of exposure time and weaning diet.

C57BL/6 mice were maintained on liquid diets containing 27.5% ethanol-derived calories beginning on gestational day 12. For one group, the liquid diet was replaced with laboratory chow on the morning of gestational day 18, approximately 24 hr before delivery. For a second group, the diet was continued for 7 days postnatally. Two additional groups were pair-fed isocaloric sucrose diets, and a final group was maintained on normal laboratory chow. The mice consumed sufficient amounts of diet to provide near 30 g ethanol/kg/day during pregnancy and slightly greater amounts during the first postpartum week. The ethanol diet produced no maternal deaths and did not alter weight gain during gestational days 12-17 of pregnancy. The results confirmed previous reports that dietary ethanol exposure under conditions that have rather minor effects on the pregnant mouse can substantially affect the fetus. As previously reported, when exposure was terminated 24 hr before delivery, neonatal mortality was elevated by approximately 30%, but birth weight was not altered. When the diet was extended into the lactation period, mortality was delayed but not reduced, possibly by reducing the severity of ethanol withdrawal. Birthweight was reduced by 10% when exposure continued until birth, suggesting that low birthweights for fetal ethanol-exposed (FEE) mice can be eliminated by terminating exposure approximately 24 hr before delivery. As in previous reports, FEE mice with normal birthweights had an attenuated preadolescent growth spurt, suggesting that low birthweights and attenuated postnatal growth are likely mediated by different mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of plasma cocaine and ethylcocaine (cocaethylene) in mice using gas chromatography-mass spectrometry and deuterated internal standards.

A gas chromatographic-mass spectrometric method is described for the determination of cocaine and ethylcocaine (cocaethylene) from mouse plasma microsamples (50 microliters). [2H3]Cocaine and [2H5]ethylcocaine served as internal standards, analytical separations were performed on a (5% phenyl)methylpolysiloxane capillary column, and detection was by selected-ion monitoring of electron-impact generated fragment ions [M - CO2Ph]. Pilot study plasma concentrations of ethylcocaine following coadministration of cocaine and ethanol were less than 5% of the parent drug.

Prenatal ethanol effects and dopamine systems of adult C57 male mice.

Behavioral, pharmacological, and neurochemical studies indicate that prenatal ethanol exposure can alter dopamine (DA) systems of developing rats. In addition, some of the behavioral changes described for prenatal-ethanol-exposed rats and mice (e.g., reduced responding for food and other rewards) as well as their response to various psychoactive drugs (e.g., amphetamines, methylphenidate, haloperidol) suggest that the DA system changes might extend into adulthood. Neurochemical studies on the effects of prenatal ethanol on DA systems of adults have not been reported for either species. The present study provides a neurochemical assessment of prenatal ethanol effects on DA systems of fully mature mice. Compared to chow and sucrose controls, adult offspring of mice fed a diet containing 25% ethanol derived calories had preadolescent growth deficits as observed in previous studies which also showed long-term behavioral deficits. Prenatal ethanol exposure in the present study, however, did not alter the concentration of DA or dihydroxyphenylacetic acid (DOPAC) nor the progressive decline in the concentration of these compounds in either striatum or nucleus accumbens of mature mice at intervals after synthesis inhibition by alpha-methyl-DL-p-tyrosine. Thus, the present study provides no neurochemical confirmation of altered DA systems resulting from prenatal ethanol exposure under conditions previously observed to alter adult behavior.

Effects of prenatal ethanol exposure on later sensitivity to the low-dose stimulant actions of ethanol in mouse offspring: possible role of catecholamines.

The purpose of this study was to examine whether prenatal ethanol (EtOH) exposure alters later sensitivity to the low-dose stimulant effects of EtOH. Because the locomotor stimulant effects of EtOH are thought to be mediated, at least in part, by activation of brain monoamine systems, and because prenatal EtOH exposure has been shown to alter brain monoamine activity, it was hypothesized that prenatal EtOH exposure may alter sensitivity to the stimulant actions of EtOH. To test this hypothesis, sensitivity to the locomotor stimulant effects of various challenge doses of EtOH was examined in male and female offspring from prenatal alcohol (A), pair-fed (PF), and lab chow (LC) groups at different ages. In addition, to address the hypothesis further, sensitivity to the catecholamine synthesis inhibitor alpha-methyl-p-tyrosine (AMPT) was examined in these offspring, as well. Results indicated that male offspring prenatally exposed to EtOH exhibited reduced baseline activity and a blunted stimulant response to all challenge doses of EtOH (0.75-1.5 g/kg) in comparison with control offspring at 30 days of age, but these effects appeared to "normalize" at 70 days of age. Female EtOH-exposed offspring also exhibited a reduced baseline level of activity relative to control offspring, as well as a blunted stimulant response to the lowest challenge dose of EtOH (0.75 g/kg) at 30 days of age, and these effects persisted into adulthood. The stimulant response to higher doses of EtOH did not significantly differ among prenatal treatment groups in young or adult female offspring. However, because baseline activity was significantly lower in female EtOH-exposed offspring than control offspring, the stimulant response to these doses of EtOH (1.125 and 1.5 g/kg) was relatively greater than that for PF and LC offspring. Importantly, none of the differences in performance among the prenatal treatment groups could be attributed to an alteration in EtOH pharmacokinetics, because blood EtOH levels measured immediately following the 10-min test session were similar for all prenatal treatment groups across all of the EtOH test doses. Further, a similar response profile as that observed following EtOH challenge at 70 days of age was obtained following phenobarbital challenge (10-40 mg/kg). Finally, whereas AMPT (50-400 mg/kg) dose-dependently antagonized the stimulant effects of EtOH in all prenatal treatment groups, this effect of AMPT was significantly greater in mice prenatally exposed to EtOH in comparison with control offspring.(ABSTRACT TRUNCATED AT 400 WORDS)

Gas chromatographic-mass spectrometric determination of plasma and brain cocaine in mice.

A gas chromatographic-mass spectrometric method is described for the determination of cocaine in mouse plasma microsamples and brain. Cocaine and [2H3]cocaine were extracted with pentane-isopropyl alcohol (97:3, v/v), chromatographed on a (5% phenyl) methylpolysiloxane capillary column, and detected by selected-ion monitoring of electron impact generated m/z 182 and 185 fragment ions. The small sample size (50 microliters), simplicity of workup, and high response linearity (mean r = 0.9993) distinguish the method. Cocaine was found in mouse brain at approximately 5 times greater concentration than in plasma after 20 or 40 mg/kg subcutaneous doses.

Gender differences in the effects of ethanol on C57BL/6 mice.

The influence of gender on the stimulatory and depressant effects of ethanol was examined in C57BL/6 (C57) mice. In Experiment 1, locomotor activity was assessed in young (2-month-old) male and female mice injected intraperitoneally (IP) with stimulatory (1.5 g/kg) or depressant (2.5 g/kg) doses of ethanol. Both the stimulatory and the depressive effects of ethanol were greater in young male than female C57 mice, and the gender difference was unrelated to blood ethanol concentration (BEC). In Experiment 2, older (9-month-old) male and female mice were given ethanol (2.5 g/kg) either by IP injection or gavage to determine if the gender differences in BEC and ethanol effects observed in the first experiment depended upon the route of ethanol administration. In this experiment, ethanol reduced locomotor activity more in males than females whether given by gavage or IP injection, and the males had higher BECs than females at the time of testing. Thus, the differences in the behavioral effects of ethanol appeared to be related to BEC. The greater depressive effect of ethanol on older male mice in this experiment is consistent with an earlier report of prolonged ethanol-hypnosis in older male C57 mice. Therefore, differences in BEC could account for the gender differences in the behavioral effects of ethanol on older but not young mice. The gender difference in BEC of mice obtained in the present and earlier reports is opposite to that reported for humans.(ABSTRACT TRUNCATED AT 250 WORDS)

Postnatal growth deficits in prenatal ethanol-exposed mice: characteristics and critical periods.

We previously reported that offspring of C57BL/6 mice maintained on liquid diets containing 20% or 25% ethanol-derived calories throughout pregnancy had birth weights comparable with controls but had weight reductions that became manifest around 23 to 28 days postnatally. Since this pattern of weight reduction may represent an unrecognized condition for human ethanol exposure, we completed a number of experiments to more thoroughly characterize the altered growth of prenatal ethanol exposed C57BL/6 mice. The results of this study indicate that consumption of liquid diets containing either 17% or 25% ethanol-derived calories during pregnancy can reduce growth of male and female offspring. Although attenuated postnatal growth of prenatal ethanol-exposed rodents is not commonly reported, prospective studies in humans suggest that in addition to having lower birth weights, children prenatally exposed to ethanol are postnatally growth retarded. Mice exposed to the low ethanol doses used in the present study had normal birth weights; however, their growth was attenuated between 19 and 28 days of age (preadolescent growth spurt) resulting in a weight reduction for at least 35 days, and which according to our previous report could extend into adulthood. The latter stages of gestation appear to be more sensitive to the postnatal growth retarding effect of prenatal ethanol exposure than early gestation. Caloric deficiency and postnatal maternal factors were eliminated as possible mechanisms for the growth deficit.

Ethanol stimulation after chronic exposure in C57 mice.

The effects of chronic exposure to ethanol via an ethanol containing diet on locomotor activity and on the response to depressive and stimulatory doses of ethanol were examined in C57BL/6cr (C57) mice. Mice maintained for approximately 3 weeks on a liquid diet in which 25% of the calories derived from ethanol had blood levels ranging from undetectable to 60 mg% when sampled at 2200 hr. They were less active than nutritionally equivalent controls, were less depressed by a high dose of ethanol (3 g/kg), and were more stimulated by a low dose of ethanol (2 g/kg). The results establish that chronic EtOH exposure via liquid diets attenuate its depressive effect and exacerbate its stimulatory effect on locomotor activity of C57 mice as previously shown for other strains or for rats. The increased activity elevation in chronically exposed mice may reflect an unmasking of excitation upon developing tolerance to EtOH depression.

Effect of dopaminergic drugs on enhancement of the binding of quinuclidinyl benzilate by phencyclidine in vivo.

Phencyclidine has been shown to enhance the specific binding of [3H]quinuclidinyl benzilate (QNB) in the brain of the mouse when both compounds are given in vivo. Since a variety of studies indicate that dopaminergic (DA) systems are involved in the action of phencyclidine, it seemed that DA systems might mediate the enhancement of the binding of QNB by phencyclidine. The results of the present studies demonstrate that the neurotoxin 6-hydroxydopamine, the DA D1 antagonist (R)-8-[chloro]-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1-H-3-benzazepine-7- ol) (SCH23390), the DA D1 agonist p-dibenzylamino benzonitrile (SKF38393) and the DA D2 antagonist spiperone, all failed to modify the ability of phencyclidine to enhance the accumulation of QNB in brain. Quinpirole, a DA D2 agonist diminished accumulation of QNB after phencyclidine, however, this effect was confounded by quinpirole decreasing the non-specific binding of QNB, as well as increasing concentrations of QNB in plasma, when given with phencyclidine. In contrast, haloperidol, a DA antagonist, decreased the enhanced specific binding of QNB after phencyclidine without any apparent confounding influences. Since haloperidol is also known to strongly inhibit the binding of ligands to the sigma receptors, it was hypothesized that the actions of phencyclidine and haloperidol on the binding of QNB in vivo may be through sigma receptors rather than DA systems.

Effect of prenatal ethanol administration on the urogenital system of mice.

The purpose of the present series of studies was to determine whether an obstruction in the urogenital system or vesicoureteral reflux (reflux, the retrograde passage of urine from the bladder into the kidney) existed in mice prenatally exposed to ethanol which might account for the high incidence of hydronephrosis and hydroureter observed. In order to examine these possibilities, indigo carmine was injected into the bladder of 19-day fetuses previously exposed to ethanol on Day 10 of gestation and the presence of hydronephrosis and/or reflux determined. As expected, we found a greatly increased incidence of hydronephrosis and hydroureter. In addition, there was a significant increase in reflux in the ethanol-treated mice. The incidence of reflux appeared to be related to the severity of the hydronephrosis observed, though cases of hydronephrosis without reflux and reflux without hydronephrosis were found. These data suggest both hypotheses may be salient and that a multiplicity of urogenital abnormalities are found following prenatal ethanol exposure.

Effect of central administration of phencyclidine on plasma concentrations of luteinizing hormone.

Previous research has demonstrated that excitatory amino acids acting at N-methyl-D-aspartate (NMDA) receptors stimulate the release of luteinizing hormone (LH). Castration also elevates LH, an effect that may also involve NMDA receptors since the specific NMDA antagonist, DL-2-amino-5-phosphonopentanoic acid (AP5), antagonizes this action. Since PCP antagonizes a variety of actions of NMDA agonists, we hypothesized that it would diminish the ability of glutamate, homocysteic acid and castration to elevate LH. Our data support this hypothesis.

Parametric studies on phencyclidine enhancement of 3H quinuclidinyl benzilate accumulation in vivo.

The purpose of these experiments was to define the temporal parameters involved in the phencyclidine (PCP) enhancement of 3H quinuclidinyl benzilate (QNB) accumulation in mouse brain. PCP enhanced QNB accumulation in brain if given intraperitoneally (IP) 1 and 4, but not 16 hours before intravenous (IV) administration of QNB. This effect was found in hypothalamus, striatum, cortex and hippocampus, but not cerebellum. PCP given after QNB did not alter QNB accumulation. The PCP enhancement persisted for at least 72 hours after QNB administration. These results confirm previous studies demonstrating that PCP must be present prior to QNB administration to enhance the accumulation and show that the effect persists for an extended period of time.

Discriminative properties of phencyclidine in mice: generalization to ketamine and monohydroxy metabolites.

The discriminative properties of phencyclidine (PCP) and their generalization to the effects of ketamine and monohydroxylated PCP metabolites were examined in C57BL/6cr mice utilizing two-lever operant procedures. As previously reported for pigeons and rats, PCP was discriminable in this species at a training dose of 3.0 mg/kg. PCP discriminability generalized to test doses of the drug that did not influence response rates (as low as 1.75 mg/kg) and also to ketamine (10 mg/kg). Both PCP monohydroxylated metabolites were active in mice. PCP partially generalized to the monohydroxylated metabolite, 1-(1-phenylcyclohexyl)4-hydroxy piperidine (PCHP) but not to 1-(1-phenyl-4-hydroxycyclohexyl) piperidine (PPC), which is consistent with previous reports on rats. The generalization of the PCP stimulus to PCHP was not as extensive in mice as previously reported for rats, suggesting that it may be less potent in this species. Although PCP discriminability generalized to PCHP, this generalization required PCHP doses that would produce tissue concentrations much higher than could result from discriminable doses of PCP. Therefore, the PCHP metabolite does not appear to mediate PCP discriminability in C57BL/6cr mice.

Stimulatory effects of ethanol in C57BL/6 mice.

Although ethanol stimulation is well documented in several species including humans, there is some controversy about whether the stimulation occurs in the highly inbred mouse strain, C57BL/6. Since inbred mouse strains are frequently used to elucidate mechanisms for individual differences in reaction to alcohol, the present study was undertaken to more completely characterize the behavioral effects of ethanol and to help resolve some of the controversy regarding the drug's stimulatory effect on C57 mice. Activity of female C57BL/6cr mice was assessed in either a lighted or dark environment for 20 min after injections of water or ethanol at doses of 0.5, 1.0, 2.0, 4.0 g/kg. Elevated activity (stimulation) was observed in mice injected with relatively low ethanol doses and tested in the light. The 2.0 g/kg dose produced a transient elevation in activity which declined rapidly across time. Animals tested under the dark condition were not stimulated by the drug but had activity reductions to high doses of ethanol. The detection of ethanol-induced stimulation appears to be related to the performance of control mice rather than a light-related difference in ethanol sensitivity.

Doubtful role for phencyclidine metabolites in PCP enhancement of QNB binding.

In vivo administration of phencyclidine (PCP) has been shown to enhance the accumulation of intravenously administered 3H quinuclidinyl benzilate (QNB) in certain regions of mouse brain. Since this effect can be blocked by prior administration of atropine, it has been interpreted as an enhancement by PCP of the specific binding of QNB. The present studies replicated this earlier work and determined that two major hydroxy metabolites of PCP found in rodents 1-(1-phenylcyclohexyl)4-hydroxy piperidine (4-OH-pip PCP) and 1-(1-phenyl-4-hydroxycyclohexyl)piperidine (4-OH-cyclo PCP) probably do not contribute substantially to the PCP effect on QNB binding. Though QNB accumulation in brain was increased by injection of 4-OH-cyclo PCP, the doses necessary for the effect were substantially higher than those needed for PCP. Furthermore, pretreatment of the animals with beta-diethylaminoethyl diphenylpropylacetate (SKF525A), a compound known to block the metabolism of PCP in liver microsomes and thus the formation (at least in part) of these metabolites, did not attentuate the PCP effect on QNB binding.