George W. Lipe

Effects of a cold environment or age on methamphetamine-induced dopamine release in the caudate putamen of female rats

Extracellular levels of dopamine (DA) and metabolites as well as serotonin [5-hydroxytryptamine (5-HT)] and 5-hydroxyindoleacetic acid (5-HIAA) were determined in the caudate putamen (CPU) of either 6- or 12-month-old female rats using microdialysis and high-performance liquid chromatography with electrochemical detection (HPLC-ED) before, during, and after four consecutive injections (given at 2-h intervals) of methamphetamine (METH). In 6-month-old rats administered 4 x 5 mg/kg METH at an environmental temperature (ET) of 23 degrees C, peak extracellular DA levels (between 50 and 150 rho g/10 microliters) were attained 30-45 min after each dose of METH while dihydroxyphenylacetic acid (DOPAC) decreased steadily after the first doses of METH until it reached a plateau at 50% of control (550-700 pg/10 microliters) levels. Increases in 5-HT levels during METH administrations paralleled DA increases while 5-HIAA decreases paralleled DOPAC decreases. The total CPU DA and 5-HT content of these rats was about 65% of control at 3 days post-METH. Reducing the ET to 4 degrees C during dosing decreased the peak and average DA levels attained during the 4 x 5 mg/kg METH administration to about 50% of that observed at a 23 degrees C ET. Increasing the dose to 4 x 10 mg/kg METH (4 degrees C ET) increased peak and average CPU DA levels to 200% that observed during 4 x 5 mg/kg METH at a 23 degrees C ET. However, no significant decreases in total CPU DA content of any rats dosed with METH at a 4 degrees C ET were observed 3 days post-METH. In 12-month-old rats dosed with 4 x 5 mg/kg METH (23 degrees C ET), the peak and average extracellular DA levels were only 30-60% that of 6-month-old rats. However, the CPU DA content of older rats was significantly decreased both 3 (30% control) and 14 (60% control) days post-METH. In summary, METH toxicity may not be predicted solely by the extracellular levels of DA attained during METH administration; age and ET also greatly influence METH neurotoxicity.

Effect of manganese on the concentration of amino acids in different regions of the rat brain.

The present study was designed to determine if chronic exposure of weanlings and adult rats to Mn produces significant alterations in amino acid concentrations in different regions of the rat brain. Weanling (30 day old) and adult (90 day old) male rats were exposed to 10 and 20 mg Mn/kg body weight per day, by gavage, for 30 days. Forty-eight hours after the last dose, animals were sacrificed by decapitation and brains were dissected into different regions to determine the concentration of amino acids by HPLC/EC. A dose dependent decrease in body weight gain was found in the adult, but not in the weanling rats. Significant increases occurred in concentrations of aspartate, glutamate, glutamine, taurine and gamma-aminobutyric acid (GABA) in the cerebellum of the adult rats dosed with 20 mg/kg per day, Mn. A significant decrease in the concentration of glutamine was observed in caudate nucleus and hippocampus of weanling rats dosed with 10 mg/kg, Mn. These data suggest that chronic Mn exposure can produce a decrease in body weight gain in adult rats and alterations in amino acids in different regions of weanling and adult rat brains.

Determination of amino acids in different regions of the rat brain application to the acute effects of tetrahydrocannabinol (THC) and trimethyltin (TMT)

A modified HPLC method is described for the determination of amino acids [aspartic acid, glutamic acid, glutamine, glycine, taurine, and gamma-aminobutyric acid (GABA)] in brain tissue utilizing precolumn derivatization with o-phthalaldehyde (OPA)-tert-butyl-thiol and electrochemical detection. A simple extraction procedure was employed and DL-homoserine used as internal standard. A neurotoxin previously shown to affect brain amino acids (trimethyltin, TMT) and a psychoactive compound hypothesized to act on these neurochemicals (delta-9-tetrahydrocannabinol, THC) were administered to adult male rats and amino acids were measured. Results revealed a gradient of distribution of most amino acids, with lowest levels posteriorly in the brain stem and increasing to the highest values in anterior cortical regions. TMT increased glutamine significantly in all brain regions examined, but increased glycine and decreased taurine only in the frontal cortex and hippocampus. No significant changes in any amino acid were found in hippocampus after THC treatment. The results establish the validity and usefulness of this HPLC method for detecting neurotoxicity-related changes in brain amino acid metabolism.

The effects of chronic exposure of manganese on antioxidant enzymes in different regions of rat brain

The present study was designed to investigate if chronic exposure to manganese (Mn) produces an effect on antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) activities and reduced glutathione (GSH) content in different regions of rat brain. Adult male Sprague-Dawley (CD) rats were dosed with 0, 2.5 or 5.0 mg MnCl2/kg, for 3 months (5 days/week). The activity of total superoxide dismutase did not vary significantly in any region of the brain with either 2.5 or 5.0 mg MnCl2/kg. A significant increase of Mn-superoxide dismutase (Mn-SOD) activity was attained in hippocampus, cerebellum and brain stem. The Cu, Zn-superoxide dismutase activity was reduced in all regions of the brain, however, reduction was not statistically significant. No significant effect of Mn on glutathione peroxidase activity was observed in any region of the brain. Glutathione content was significantly reduced in cerebellum, whereas, no change was observed in other brain regions. The results show that chronic exposure to manganese significantly increased the Mn-superoxide dismutase activity in selected brain regions. Therefore, increased Mn-SOD may enhance the antioxidant ability of the brain to reduce oxidative stress.

Pharmacokinetics of doxylamine, a component of Bendectin, in the rhesus monkey.

The elimination of doxylamine and metabolites was determined after iv administration of [14C]doxylamine succinate at 0.7 and 13.3 mg/kg to the adult female rhesus monkey. Although the total recovery of radioactivity was the same for the low- and high-dose studies (90.2%), the rate of plasma elimination of doxylamine and its demethylated metabolite (desmethyldoxylamine) was slower for the high dose group. The 24 hr urinary excretion of doxylamine metabolites, desmethyl- and didesmethyldoxylamine, was significantly increased and the polar doxylamine metabolites were significantly decreased as the iv doxylamine succinate dose was increased. The plasma elimination of gas chromatograph (GC)-detected doxylamine was determined after oral administration of Bendectin (doxylamine succinate and pyridoxine hydrochloride) at 7, 13.3, and 27 mg/kg to adult female rhesus monkeys. As the dose increased, the clearance of doxylamine decreased. A statistically evaluated fit of the oral data to a single-compartment, parallel first-order elimination model and a single-compartment, parallel first- and second-order (Michaelis-Menten) elimination model indicated that the more complex model containing the second-order process was most consistent with the observed elimination data.

Comparison of Glutamine-Enhanced Glutamate Release from Slices and Primary Cultures of Rat Brain

Increased extracellular glutamate has been associated with a wide range of effects including production of neurotoxicity. Glutamine has previously been shown to cause increased release of glutamate from a variety of preparations. Extracellular central nervous system (CNS) glutamine levels are known to increase with neurotoxin exposures, hepatic failure, renal failure, head trauma or stroke. However, the action of glutamine to enhance the release of glutamate under nondepolarizing conditions has not been well studied. Since glutamine-mediated increases in extracellular glutamate are potentially of significance in cellular damage as a result of CNS insult, further examination of this phenomenon is important. Striatal and hippocampal slices or virtually neuron-free primary striatal glial cultures were employed in studies to further elucidate the mechanism(s) of glutamine-enhanced glutamate release. Elevated extracellular glutamine caused increased glutamate release in all three preparations. In hippocampal and striatal slices elevated glutamine caused an enhancement of N-methyl-D-aspartate (NMDA) receptor-mediated [3H]catecholamine release equivalent to that produced by high concentrations (up to 100 microM) of exogenous glutamate. In both striatal slices and primary cultures kynurenate increased glutamate release in the presence of 500 microM glutamine, while kainate either had no effect or decreased glutamate levels in the presence of glutamine. Since several presynaptic modulators of release did not affect the glutamate release produced by glutamine in slices, vesicular release of glutamate from nerve terminals was probably not involved in the effects of the exogenous glutamine. The similarities between striatal slices and primary striatal cultures indicate that enzymatic conversion of glutamine to glutamate within glia may be an important factor in the glutamine-mediated elevation of extracellular glutamate levels.

Age and Dietary Factors in Hippocampal Sensitivity to Trimethyltin

Prolonged restriction of the total daily calories or protein fed to rodents slows the kidney damage, myocardial degeneration, and the rate of appearance of neoplasms, but not the decline of sensorimotor function that normally occurs with age.’ Because these dietary regimes are now being practiced by people in an effort to extend their lifespan, it is important to understand the effects of such dxts on the brain. Trimethyltin (TMT) neurotoxicity is in some respects similar to age-related neurodegeneration. Both TMT (FIG. la,b) and age damage hippocampal neurons, produce Alzheimer‘s Type I1 ghosis (both nuclei and cytoplasm of astrocytes are enlarged) (FIG. lc,d), and impair passive avoidance perf~rmance.~-~ Both TMT and age may be hypothesized to act on the hippocampus through release of endogenous “excitotoxins.” Therefore we used TMT to “challenge? the aging brain to determine how dietary factors might alter its resistance to neurotoxic (and by inkrence, aging) processes. We administered single i.p. doses of TMT (0 to 4.5 mgkg) to 7or 17-month-old male Fischer 344 rats that had been either fed ud libitum with a 21% (“medium”) protein diet, or restricted (“CR”) to 60% of the ud &turn caloric intake of the medium protein diet from 14 weeks of age. Other groups had been maintained ud libitum fbr at least 2 months on isocaloric 13% (“low”) or 2996 (“high”) protein diets. Three weeks after TMT dosing, we evaluated passive avoidance performance (as an indicator of behavioral impairment) and we carried out histological studies using a FinkHeimer stain (to find degenerating axons) and immunohistochemical studies (for &al fibrillary acidic protein [GFAP]). We also measured several amino acids and analyzed hippocampal kainic acid (KA) receptors. TABLE 1 summarizes our investigtions, which are reported in more detail elsewhere.6 Glutamine (Gln), which increases glutamate release when superfused

Placental Steroid Dehydrogenases: Assessment with a Nonhuman Primate in Situ Placental Perfusion Model

The oxidoreductase activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) has been demonstrated in human placenta and catalyzes the interconversion of estradiol-17β (E2) and estrone (E1) (Langer and Engel, 1958; Jarabak and Sack, 1969; Strickler and Tobias, 1982). Preimplantation rat and mouse embryos have also been reported to possess 17β-HSD activity (Wu and Lin, 1982; Wu and Matsumoto, 1985). It has been postulated that 17β-HSD is responsible for the conversion of the potent estrogen, E2, to the less potent E1 in later-term non-human primate pregnancy (Slikker et al., 1982a) and regulates the ratio of E2 and E1 throughout the preimplantation period in the rodent (Wu and Matsumoto, 1985).