William D. Wessinger

Approaches to the study of drug interactions in behavioral pharmacology

This paper discusses some of the approaches which have been used for analyzing drug interactions with an emphasis on applications for behavioral pharmacology. Two broad categories of drug interactions are defined: heterergic, when only one of the drugs is active in the behavioral measure employed, and homergic, when both of the interacting drugs have similar action. Two distinct models are presented for homergic drug interactions. The effect-addition model predicts that the combined action of two drugs is equal to the arithmetic sum of the individual effects. This is referred to as effect-additive and deviations from the predicted effects are described accordingly. The dose-addition model takes both dose and effect into account, and thus has a sounder theoretical basis. Leftward shifts in the dose-effect curves are described as equal to (dose-additive), or greater than (supra-additive) or less than (infra-additive) predicted on the basis of the relative potencies of the interacting drugs. Isobolographic methods facilitate data reduction and allow a graphic depiction of dose-addition analysis. A survey of the literature utilizing isobolographic techniques is presented.

(+)-Methamphetamine-induced spontaneous behavior in rats depends on route of (+)METH administration

These studies examined the role of (+)-methamphetamine ((+)METH) administration route on spontaneous behavioral activity vs. time relationships, and pharmacokinetic mechanisms for differences in effects. Male Sprague-Dawley rats (n=6 per administration route) received saline and three doses (0.3, 1.0 and 3.0 mg/kg) of (+)METH in a mixed-sequence design by intravenous (iv), subcutaneous (sc) or intraperitoneal (ip) administration. Locomotion and stereotypy were quantified by video-tracking analysis. The effects of (+)METH on spontaneous behavior were dose- and route-dependent. In particular, total locomotor activity was greatest following 3.0 mg/kg intraperitoneally (P<0.05) and stereotypy ratings were greatest following 3.0 mg/kg subcutaneously (P<0.05). In addition, the duration of locomotor effects was greatest after 3.0 mg/kg subcutaneously (P<0.05). Serum pharmacokinetic parameters were determined in separate rats given 3.0 mg/kg by subcutaneous and intraperitoneal administration (n=4 per administration route). The (+)METH elimination half-life was not different between the routes, but the (+)METH AUC was greater (P<0.05), and the (+)METH and (+)-amphetamine (AMP) maximum concentrations occurred later following subcutaneous than after intraperitoneal dosing (P<0.05), increasing and prolonging drug exposure. In conclusion, the overall pattern of (+)METH effects on locomotor activity depend on dose and the route of administration, which affects serum concentration and the time course of behavioral effects.

Determination of dextromethorphan and its metabolites in rat serum by liquid-liquid extraction and liquid chromatography with fluorescence detection.

Dextromethorphan is an effective and safe antitussive, but has liabilities with respect to its abuse potential at doses above the therapeutic dose. At these higher doses, people report phencyclidine-like effects from the drug. A number of animal models have suggested that dextrorphan, an active metabolite of dextromethorphan, is responsible for the abuse liability of the parent compound when dextromethorphan is taken at high doses. Full pharmacokinetic profiles in single animals have not been demonstrated in these studies due to a lack of analytical sensitivity and/or selectivity for dextromethorphan and its metabolites. We have developed a low-cost liquid chromatographic method capable of characterizing the concentration-time profile for dextromethorphan and dextrorphan for 8 h in rats following an 18 mg/kg i.p. dose of dextromethorphan. Limits of quantitation (S/N=10) in 100 microL of serum were 0.25, 0.19, 0.27, and 0.22 nmol/mL for 3-hydroxymorphinan, dextrorphan, 3-methoxymorphinan, and dextromethorphan, respectively. Inter-day precision was better than 11% across the dynamic range of the method.

Metabolic changes and DNA hypomethylation in cerebellum are associated with behavioral alterations in mice exposed to trichloroethylene postnatally

Previous studies demonstrated that low-level postnatal and early life exposure to the environmental contaminant, trichloroethylene (TCE), in the drinking water of MRL+/+ mice altered glutathione redox homeostasis and increased biomarkers of oxidative stress indicating a more oxidized state. Plasma metabolites along the interrelated transmethylation pathway were also altered indicating impaired methylation capacity. Here we extend these findings to further characterize the impact of TCE exposure in mice exposed to water only or two doses of TCE in the drinking water (0, 2, and 28mg/kg/day) postnatally from birth until 6weeks of age on redox homeostasis and biomarkers of oxidative stress in the cerebellum. In addition, pathway intermediates involved in methyl metabolism and global DNA methylation patterns were examined in cerebellar tissue. Because the cerebellum is functionally important for coordinating motor activity, including exploratory and social approach behaviors, these parameters were evaluated in the present study. Mice exposed to 28mg/kg/day TCE exhibited increased locomotor activity over time as compared with control mice. In the novel object exploration test, these mice were more likely to enter the zone with the novel object as compared to control mice. Similar results were obtained in a second test when an unfamiliar mouse was introduced into the testing arena. The results show for the first time that postnatal exposure to TCE causes key metabolic changes in the cerebellum that may contribute to global DNA methylation deficits and behavioral alterations in TCE-exposed mice.

Sexual dimorphic effects of chronic phencyclidine in rats

The behavioral effects of phencyclidine (PCP) were studied in male and female Sprague-Dawley rats to determine if chronic infusions would result in sexually dimorphic effects. Rats were trained to make operant responses for food during 30-min response periods that occurred 4 times each day. After attaining stable baseline behaviors, 10 mg of PCP/kg/day was infused s.c. for 10 days. Females were more profoundly affected than males. In the females, response rates were suppressed to 30-71% of control rates during the first 7 days of infusion. In contrast, response rate in male rats never fell below 77% of control during the infusion period. By the eighth infusion day both sexes had become tolerant to these behavioral effects. After stopping infusions there was clear evidence that behavioral dependence had developed; however, the abstinence effects in males and females were similar. Saturation studies of [3H]dizocilpine (MK-801; (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine) binding to brain membranes were conducted to determine if there were sex-dependent receptor differences. There were no significant differences in Kd +/- S.D. (7.6 +/- 1.5 and 7.1 +/- 0.9 nM for males and females, respectively) or Bmax +/- S.D. (4.1 +/- 0.2 and 4.0 +/- 0.5 pmol/mg protein for males and females, respectively).

Centrally mediated opioid induced depression of hepatic glutathione: Effects of intracerebroventricular administration of mu kappa, sigma and delta agonists

It has recently been demonstrated that morphine produces a loss of hepatocellular glutathione in mice by virtue of its action within the central nervous system. The ability of opioid receptor antagonists to abolish morphines effect on hepatic glutathione suggests that this action is opioid-receptor mediated. The involvement of opioid receptors in this phenomenon is confirmed in the present study in mice by the ability of naltrexone, 100 micrograms administered intracerebroventricularly (i.c.v.), to completely block the decrease in hepatic glutathione induced by an i.c.v. injection of 100 micrograms of morphine. Intracerebroventricular administration of the selective mu (mu) opioid receptor agonist, (D-Ala2,N-MePhe4,Gly-ol5)enkephalin (DAGO; 25-50 micrograms), or the selective delta (delta) opioid agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE; 3-50 micrograms), like morphine, produced significant decreases in hepatic glutathione 3 h after administration. The selective kappa (kappa) opioid receptor agonists, ethylketocyclazocine (1-30 micrograms) and trans-(+/-)3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide-methane sulfonate (U50 488; 10-300 micrograms), as well as the selective sigma (sigma) opioid agonists, phencyclidine (PCP; 50-300 micrograms) and N-allylnormetazocine (SKF 10,047; 1-30 micrograms), had no effect on the concentrations of glutathione in the liver. It appears from these data that stimulation of mu- or delta-, but not kappa- or sigma-opioid receptors within the central nervous system results in a loss of hepatocellular glutathione.

Tolerance to and dependence on MK-801 (dizocilpine) in rats

Rats were trained to respond under a fixed-ratio 30 schedule for food presentation during four daily 0.5-h sessions occurring every 6 h. After stable baseline response was established, osmotic minipumps were implanted that infused vehicle or (+)-5 methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine hydrogen maleate (dizocilpine; MK-801), SC. Behavioral sessions continued to be conducted daily. After 10 days the infusion pumps were removed. Vehicle and 0.10 mg/kg per day MK-801 did not affect behavior during infusions or after cessation of dosing. Dosing with 0.32 and 0.56 mg/kg per day initially suppressed responding, but tolerance developed to these effects. After the infusions were stopped, a dose-dependent disruption of operant behavior occurred. Response rates for the 0.32 and 0.56 mg/kg per day infusion groups were suppressed to 41 and 27% of preinfusion control response rates, respectively, the day after dosing stopped; however, no physical signs of abstinence were observed. Response rates recovered toward control over the next 2-4 days. In a separate experiment, the suppression of response produced by abstinence from 0.32 mg/kg per day of MK-801 (SC) for 10.5 days was reversed by readministration of MK-801 (IP). These results demonstrate that MK-801 produces dependence, as evidenced by the emergence of a behavioral abstinence syndrome after cessation of dosing.

Behavioral dependence of phencyclidine in rats

The abuse of PCP continues to be an important medical problem in many urban areas. The probability that dependence on PCP may contribute to its compulsive use and relapse is supported by animal studies demonstrating its dependence liability. In the present study, five rats were housed in operant chambers and trained to respond on a lever under a fixed-ratio 30 schedule of food presentation. They obtained all their daily food during four 30-min response periods occurring every 6 hr. After stable baselines of behavior were established the rats were injected with PCP (3.0-7.5 mg/kg/injection), i.p., 1 hr before each response session for 7-10 days. Following chronic dosing, the drug injections were replaced with saline injections for 10 days. Disruptions in behavior were observed upon cessation of relatively brief chronic exposure to PCP (as little as 7 days) and at relatively low doses (5.6 mg/kg/6 hr = 22.4 mg/kg/day). The behavioral disruption was not accompanied by overt signs of abstinence and persisted for up to 48 hr.

Discriminative-stimulus control by morphine in the pigeon under a fixed-interval schedule of reinforcement.

The stimulus control by morphine under a fixed-interval (FI) 180-s schedule of reinforcement was examined in five pigeons trained to discriminate 5.0 mg/kg of i.m. morphine from saline. After training, dose-response relationships were determined for morphine, d-amphetamine, pentobarbital, fentanyl, and MK-801. At low doses of morphine, responding of individual subjects was predominantly on the saline-appropriate key. At intermediate doses responding occurred on both keys throughout the interval. Following doses of 5.6 mg/kg and higher, responding on both keys was observed in the early portions of the interval with responding shifting to the morphine-appropriate key as the interval progressed. Thus, a graded dose-effect curve for morphine was obtained under the FI schedule. The fentanyl dose-effect curve was similar to that obtained with morphine, but the other test drugs were not generalized. Five other pigeons were trained to discriminate 5.0 mg/kg of morphine from saline under a fixed-ratio (FR)100 schedule of reinforcement. During testing with morphine, responses were confined to the saline-appropriate key at 0.3 and 1.0 mg/kg of morphine, but at higher doses responses were confined to the drug key. In contrast to the results obtained using the FI schedule, a quantal relationship for responding on the two keys was observed when a FR schedule was employed.

Inflammatory and oxidative stress-related effects associated with neurotoxicity are maintained after exclusively prenatal trichloroethylene exposure

&NA; Trichloroethylene (TCE) is a widespread environmental toxicant with immunotoxic and neurotoxic potential. Previous studies have shown that continuous developmental exposure to TCE encompassing gestation and early life as well as postnatal only exposure in the drinking water of MRL+/+ mice promoted CD4+ T cell immunotoxicity, glutathione depletion and oxidative stress in the cerebellum, as well increased locomotor activity in male offspring. The purpose of this study was to characterize the effects of exclusively prenatal exposure on these parameters. Another goal was to investigate potential plasma oxidative stress/inflammatory biomarkers to possibly be used as predictors of TCE‐mediated neurotoxicity. In the current study, 6 week old male offspring of dams exposed gestationally to 0, 0.01, and 0.1 mg/ml TCE in the drinking water were evaluated. Our results confirmed that the oxidized phenotype in plasma and cerebellum was maintained after exclusively prenatal exposure. A Phenotypic analysis by flow cytometry revealed that TCE exposure expanded the effector/memory subset of peripheral CD4+ T cells in association with increased production of pro‐inflammatory cytokines IFN‐&ggr; and IL‐17. Serum biomarkers of oxidative stress and inflammation were also elevated in plasma suggesting that systemic effects are important and may be used to predict neurotoxicity in our model. These results suggested that the prenatal period is a critical stage of life by which the developing CNS and immune system are susceptible to long‐lasting changes mediated by TCE. HighlightsPrenatal TCE exposure enhanced oxidative stress biomarkers in the cerebellum.Prenatal TCE exposure promoted increased locomotor activity.Prenatal TCE exposure increased peripheral CD4+ T cell activated/memory cells.Prenatal TCE exposure increased variance ratio of cytokines IL‐17 and IFN‐&ggr;.Prenatal TCE exposure increased plasma biomarkers of oxidative stress/inflammation.