Kazuaki Shimosato

Simultaneous Monitoring of Conditioned Place Preference and Locomotor Sensitization Following Repeated Administration of Cocaine and Methamphetamine

The paradigm of conditioned place preference has been widely used to demonstrate the rewarding properties of psychomotor stimulants. Such drugs also stimulate locomotor activity. Repeated administration of low doses of psychomotor stimulants causes progressive increases in the locomotor stimulating effect, a phenomenon termed behavioral sensitization. Using a new activity monitor (SCANET MV-10LD) that simultaneously measures the amount of time spent and the distance traveled in each side of a two-compartment chamber, the present study assessed place preference conditioning and locomotor sensitization following repeated administration of cocaine or methamphetamine (MAP) in mice. We examined the effect of environmental factors on these activities using two different types of chamber: one having a single cue, and the other having dual cues for the discrimination of compartments. In both types of chamber, cocaine (5-20 mg/kg) and MAP (1-2 mg/kg) similarly produced conditioned place preference. However, repeated cocaine administration caused the development of locomotor sensitization only in the single-cue chamber. On the other hand, repeated administration of MAP resulted in the development of sensitization in both types of chamber. The findings indicate that environmental factors differentially affect the development of locomotor sensitization, but not place preference conditioning, following repeated administration of cocaine or methamphetamine. The advantages of this new system will be discussed.

Concurrent evaluation of locomotor response to novelty and propensity toward cocaine conditioned place preference in mice

Neural and physiological factors underlying individual differences in the vulnerability to drug abuse are the major questions remained to be determined. The present study described new methods that concurrently assess the relationship between locomotor response to novelty and conditioned place preference (CPP) for cocaine in mice using a novel activity monitor, SCANET. The activity monitor simultaneously measured the distance traveled and the amount of time spent in each side of a two-compartment CPP chamber, and the latency to the first entry into the other side of the CPP chamber. Mice were divided into two groups according to their locomotor response to the first exposure to the CPP chamber during a preconditioning session; high-responder (HR) and low-responder (LR) mice. Following conditioning sessions, the CPP score was shown to be higher in LR mice than in HR mice, especially at low doses of cocaine. In a separate experiment with SCANET, we measured the latency to the first entry into the white-floored compartment from the black-floored compartment of the CPP chamber as an index of anxiety-like behavior. The latency was significantly prolonged in the LR group as compared with the HR group. Thus, the new methods described here are considered to be useful for examining animal models of drug addiction with respect to anxiety-like behavior.

Urinary excretion of p-hydroxylated methamphetamine metabolites in man. I: A method for determination by high-performance liquid chromatography-electrochemistry

Abstractp-Hydroxymethamphetamine and p-hydroxyamphetamine in urine samples from methamphetamine addicts were analyzed by high-performance liquid chromatography-electrochemistry (HPLC-EC). The urine samples were hydrolyzed with equal volumes of 12 N HCl at 60 °C for 4 h and were then diluted with water and neutralized with NaOH solution. The neutralized urine was passed through a solid phase extraction column, Bond-Elut® C18, and after washing, the substances were eluted with acidified acetonitrile. The eluate was evaporated under a stream of nitrogen. The residue was dissolved in 0.1 N PCA and a small volume of the aliquots was injected into the HPLC. This procedure for determination quantitated both free and conjugated forms of the metabolites together. Thereby we could determine concentrations of the metabolites in minute urine samples; i.e., from 2.5 μl urine. The free form of the metabolites alone was analyzed by the same procedure except for hydrolysis of the conjugates.Concentrations of methamphetamine, p-hydroxymethamphetamine and p-hydroxyamphetamine in the urine samples of addicts collected at arbitrary times were determined by this procedure or by gas chromatography. It was found that there was no correlation between the concentration of methamphetamine and that of the metabolites. This investigation also revealed that various ratios between the concentrations generally were scattered over a wide range of percentages.

Differential effects of NMDA receptor and dopamine receptor antagonists on cocaine toxicities

Cocaine produces not only euphoric effects but also a wide range of detrimental effects, including seizures and lethality. The present study examined the involvement of the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptors and the dopamine D1 and D2 receptors in seizure activity and lethality observed following single and repeated injections of cocaine in ddY mice. Repeated injections of 60 mg/kg cocaine resulted in the development of sensitization to the convulsant effects of cocaine during an initial 3 or 4 days, followed by the development of tolerance at day 5 and day 6. Repeated injections of 90 mg/kg cocaine augmented the lethal effect of cocaine progressively over the course of treatment. Treatment with 0.1-0.4 mg/kg of the noncompetitive NMDA receptor antagonist, MK-801, prevented the development of sensitization to cocaine-induced seizures in a dose-dependent manner, and attenuated partially the cocaine-induced lethality. In contrast, treatment with 10-50 mg/kg of the dopmaine D2 receptor antagonist, sulpiride, had no effects on the development of sensitization and tolerance to cocaine-induced seizures. On the other hand, treatment with 0.1-0.5 mg/kg of the dopamine D1 receptor antagonist, SCH 23390, not only prolonged the latency to 90 mg/kg cocaine-induced seizures but also delayed the development of sensitization to the convulsant effects of cocaine. The increased lethality observed following repeated injection of cocaine was unaffected by treatment with SCH 23390, but was severely aggravated by treatment with sulpiride.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary excretion of p-hydroxylated methamphetamine metabolites in man. II. Effect of alcohol intake on methamphetamine metabolism

The effect of drinking alcoholic beverages on methamphetamine metabolism was investigated in man. The subjects, 97 males and 9 females, were divided into three groups by evaluation of their urinary pH; i.e., acidic, subacidic and neutral groups. The subjects in each group were further divided into ethanol-positive subjects and ethanol-negative subjects, depending on the presence or absence of ethanol in their urine. Gas chromatographic analysis showed the urinary concentrations of methamphetamine in the ethanol-positive subjects to be higher than those in the ethanol-negative subjects in both the acidic and subacidic urinary pH groups. Liquid chromatography, on the other hand, showed the urinary concentrations of p-hydroxymethamphetamine and p-hydroxyamphetamine for the ethanol-positive subjects to be lower than those for the ethanol-negative subjects in all three groups. The relative proportions of p-hydroxylated metabolites to unchanged methamphetamine in urine, therefore, were severely reduced in the ethanol-positive subjects. These results suggest that drinking alcoholic beverages probably results in a suppression of methamphetamine metabolism in man.

An enzyme-linked immunosorbent assay for plasma-paraquat levels of poisoned patients

To investigate the efficacy of medical treatments, plasma-paraquat levels were measured in patients who had ingested the liquid weedkiller, Gramoxon, containing paraquat. We determined the levels by an enzyme-linked immunosorbent assay (ELISA) using a murine paraquat-specific monoclonal antibody developed by Niewola et al. (Clin. Chim. Acta, 148 (1985) 149-156). Using this ELISA, concentrations of paraquat in the range 1.56-100 ng/ml could be measured. This assay method had good precision and was suitable for measurement of low levels of paraquat. The therapeutic treatments were most effective on the day of admission. The plasma-paraquat levels of poisoned patients decreased significantly. However, the levels tended to increase again during the pause period of the haemoperfusion and this increase was serious for fatal cases. It is clear that elimination of poison not only from blood but also from tissues is necessary to lower the mortality rate.

Increased polyamine levels and changes in the sensitivity to convulsions during chronic treatment with cocaine in mice

Polyamines have been demonstrated to modulate seizure activity in animals. Repeated administration of a subthreshold dose of cocaine resulted in the development of sensitization to cocaine-induced seizures during an initial 3 or 4 days, followed by the development of tolerance to seizures on days 5 and 6. In the present study, polyamines, such as putrescine, spermidine and spermine, were measured in regions of the brain obtained from mice that showed differential sensitivity in seizure activity during repeated cocaine injections. Animals were sacrificed for polyamine measurements 24 h after the second and the fifth injections of either cocaine or saline (on day 3 and day 6, respectively), and 3 days after the last injection. On day 3, there were significant increases in putrescine in the striatum, hippocampus and cerebellum, and in spermine in the cerebellum of cocaine-treated mice, as compared to saline-treated mice. On day 6, treatment with cocaine significantly increased putrescine in all regions, and spermidine in striatum and hippocampus, as compared to saline treatment. Cocaine treatment had no effect on any polyamine levels measured 3 days after the last injection, except for spermidine in the cortex. Because putrescine has been shown to be an antagonist of the polyamine-binding site on the N-methyl-D-aspartate receptor and to retard the development of amygdala-kindling, the present results suggest that the increases in putrescine content may be associated with the development of tolerance to convulsant effects observed during the later period of repeated administration of cocaine.

Effects of methotrexate and cyclophosphamide on polyamine levels in various tissues of rats

The effects of methotrexate (MTX) and cyclophosphamide (CYP) on body weight, organ weight, and the concentration of putrescine, spermidine, and spermine in 14 different tissues were measured in rats that had been given these compounds for 5 consecutive days. These three polyamines in both the thymus and spleen of rats treated with MTX and CYP showed a statistically significant decrease. Further, putrescine in the seminal vesicles, kidney, liver, and small intestine of MTX-treated rats, and in the prostate, seminal vesicles, kidney, heart, liver, small intestine, and lung of CYP-treated rats, spermidine in the prostate, seminal vesicles, testis, thymus, spleen, kidney, heart, small intestine, and skeletal muscle of CYP-treated rats, and spermine in the prostate, seminal vesicles, kidney, heart, small intestine, and stomach of CYP-treated rats showed statistically significant decreases. Recognition of the significance of polyamine levels and attention to their response in anti-cancer drug therapy may have clinical implications.

Long-term sensitization to the behavioral effects of naltrexone is associated with regionally specific changes in the number of μ and δ opioid receptors in rat brain

Enhanced sensitivity to some of the behavioral effects of the opioid antagonist naltrexone (NTX) develops following once-weekly injections of cumulative doses of the drug. Rats treated with this regimen of NTX injections show enhanced sensitivity to the operant response rate decreasing effects of NTX and NTX-induced salivation. The enhanced sensitivity is long-lasting and appears to be produced through conditioning processes. We have conducted saturation binding assays to assess possible changes in the number and affinity of mu and delta opioid receptors in cortical, midbrain and hindbrain membrane preparations from Long-Evans rats treated once weekly for 8 weeks with cumulative doses of the drug (1, 3, 10, 30 and 100 mg/kg). 3H-DAMGO (0.5-21 nM) and 3H-pCl-DPDPE (0.04-4 nM) were used to characterize mu and delta receptors, respectively. NTX treatment had no effect on 3H-DAMGO binding in cortex, but decreased binding in midbrain and increased binding in hindbrain relative to saline-treated controls. Saturation analyses revealed that these differences reflected changes in the number, but not the affinity of mu receptors. NTX treatment also increased the amount of 3H-pCl-DPDPE bound to delta receptors in midbrain and hindbrain, but not in cortex. Again, these changes were due to changes in the number of receptors. Thus, chronic NTX differentially affects the number of mu and delta opioid receptors in various brain regions.

Rapid determination of p-hydroxylated methamphetamine metabolites by column liquid chromatography—electrochemistry

A method for electrochemical determination of p-hydroxymethamphetamine and p-hydroxyamphetamine in human urine was investigated by column liquid chromatography. A rapid and simple extraction of these substances from urine was performed using a solid-phase extraction column. The detection limit for quantitation was approximately 50 pg for the metabolites. The simplicity and sensitivity of this method allows for analysis of these metabolites in the fields of toxicology and forensic medicine.