Shinji Shibanoki

Hippocampal degeneration inducing impairment of learning in rats: model of dementia?

In the pharmacological field, the development of drugs effective for dementia is now widely anticipated because of the increase in the elderly population. Dementia has some histological degeneration in the brain, including the hippocampus. Preclinical evaluations of such drugs use animal models with memory impairment, since memory impairment is a major criterion of dementia. We therefore investigated two animal models with hippocampal degeneration. Neonatal administration of monosodium glutamate (MSG) induced specific degeneration of hippocampal pyramidal cells in the CA1 region of Wistar rats in adulthood. In these animals, the correct response rate during the acquisition period of light-dark discrimination learning was significantly lower than that in the control group. No significant changes were noted in the hippocampal concentrations of neurotransmitter substances, including acetylcholine and glutamate. In the second model, similar histological changes were observed at 3 weeks after oral administration of trimethyltin (TMT). These histological changes were accompanied by a reduction in the intrahippocampal concentrations of acetylcholine and glutamate. In the case of light-dark discrimination learning, neither pre- nor post-training administration of TMT affected the correct response rate during both the acquisition and retention test periods. In the case of 8-arm radial maze learning, the increase in correct response rate was significantly suppressed in comparison with that of the control group when TMT was administered at 4 weeks before starting the acquisition trial. This suppression was followed by a lower response rate in the retention test. On the other hand, the correct response rates in retention tests were not affected when TMT was administered after completion of the acquisition trial. These findings indicate that sole degeneration of the hippocampus was able to induce different types of memory impairment, and single evaluation of a drug with one learning paradigm was difficult to justify that a drug is effective for dementia.

Effect of systemic administration of N-methyl-D-aspartic acid on extracellular taurine level measured by microdialysis in the hippocampal CA1 field and striatum of rats.

Abstract: The extracellular concentrations of amino acids in the hippocampal CA1 field and striatum of conscious freely moving rats were monitored simultaneously by in vivo brain microdialysis using HPLC with electrochemical detection. Under basal conditions, aspartate, glutamate, glutamine, glycine, taurine, and alanine were detected, but γ‐aminobutyric acid was undetectable in both regions. In‐traperitoneal injection of N‐methyl‐d‐aspartic acid (NMDA; 10 mg/kg) caused a significant increase (three‐to fivefold) in the taurine concentration in the dialysate obtained from both the hippocampal CA1 and striatum, whereas other amino acids (aspartate, glutamate, and alanine) did not show significant changes. Local application of NMDA (300 γ) to both regions via the dialysis probes also caused a similar increase (three‐to fivefold) in both regions. Under infusion of hypertonic Ringers solution containing 150 mM sucrose, the effect of NMDA on the level of taurine in both the regional dialysates was not affected. The effect of NMDA was totally reduced by intraperitoneal administration of MK‐801 (0.3–1.0 mg/kg), a noncompetitive antagonist of NMDA receptors. Continuous infusion of dl‐2‐amino‐5‐phosphonovaleric acid (1.0 mM), a competitive antagonist of NMDA receptors, via the dialysis probes completely inhibited the effect of NMDA. These findings suggest that systemic administration of NMDA is effective as well as local administration into the brain and that NMDA receptors might be involved in the regulation of the extracellular taurine level in the brain without dependence on cell swelling.

Measurement and pharmacokinetic analysis of imipramine and its metabolite by brain microdialysis

1 The feasibility of the brain microdialysis method for direct measurement and pharmacokinetic study of imipramine (Imip) and its metabolite desipramine (DMI) was investigated in the rat brain. 2 A dialysis tube was inserted into the right striatum of male Wistar rats, which were administered i.p. with 12.5 mg kg−1 Imip. Thirty μl dialysate was collected every 15 min, and the levels of Imip and DMI were measured by high‐performance liquid chromatography with electrochemical detection (h.p.l.c.–e.c.d.). SKF‐525A and aminopyrine were concomitantly administered in order to assess their respective effects on the pharmacokinetics of Imip and DMI in the brain. 3 The intracerebral half life (t1/2) of Imip was 2.4 ± 0.3 h with Imip alone. Premedication with SKF‐525A, an inhibitor of drug‐metabolizing enzymes, significantly prolonged the t1/2 of Imip, while at the same time production of DMI from Imip was accordingly inhibited. Concomitant administration of aminopyrine did not induce any significant change in the concentrations of Imip, but significantly inhibited the concentrations of DMI through its competitive antagonism in the demethylation pathway. 4 The present results suggest that the brain microdialysis method reflects the intracerebral pharmacokinetics of Imip and DMI well and may be applicable to further pharmacokinetic investigations of psychotropic agents.

Further characterization of the in vitro hydrolysis of [Leu]- and [Met]enkephalin in rat plasma : HPLC-ECD measurement of substrate and metabolite concentrations

Hydrolysis of [Leu]- and [Met]enkephalin was determined in whole rat plasma in vitro by using HPLC-ECD to measure Tyr, Tyr-Gly and Tyr-Gly-Gly formation. Although [Leu]- and [Met]enkephalin did not differ in Tyr or Tyr-Gly accumulation, the amount of Tyr-Gly-Gly resulting from [Met]enkephalin hydrolysis was greater than that resulting from [Leu]enkephalin hydrolysis, and [Met]enkephalins half-life in plasma was slightly shorter than that of [Leu]enkephalin. By comparing metabolite formation in the presence and absence of peptidase inhibitors with high selectivity for their respective enzymes, these studies demonstrated that aminopeptidase M and angiotensin converting enzyme are the major peptidases that hydrolyze enkephalins in rat plasma.

Developmental changes in serotonin levels in the chick spinal cord and brain.

Developmental changes in 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the developing chick spinal cord and brain were examined using high-performance liquid chromatography with electrochemical detection and immunohistochemistry. On embryonic day (E)6 only small amounts of 5-HT (0.086 ng) and 5-HIAA (0.0144 ng) were found in the spinal cord. By contrast, large amounts of 5-HT (x30) and 5-HIAA (x60) were detected in non-neuronal tissue outside the spinal cord; a similar distribution of 5-HT was also detected by immunohistochemistry. Up to E10, the highest concentrations of 5-HT in the spinal cord were found in the cervical region, followed by the thoracic and lumbar regions. In embryos older than E16, as well as in posthatched chicks, however, the highest and lowest concentrations of 5-HT were found in the lumbar and thoracic spinal cord, respectively. The concentration of spinal cord 5-HT reached maximal values on posthatching day (P)7, after which there was a marked decrease. By P120, 5-HT levels in the spinal cord decreased to the same level as on E10-E16. Concentrations in the brain, however, gradually increased with development. The basic pattern of development of 5-HIAA was similar to that of 5-HT.

Liquid chromatographic determination of free and conjugated 3-methoxy-4-hydroxyphenylethylene glycol with wide-ranging substances related to monoamine metabolism

A simple and sensitive procedure was developed for the simultaneous determination of substances metabolically related to monoamine transmitters including 3-methoxy-4-hydroxy-phenylethylene glycol (MOPEG) in dissected brain regions of rats using high-performance liquid chromatography combined with electrochemical detection. The tissue sample was homogenized in HCl solution. The homogenate was divided into two portions, of which one was used for the assay of MOPEG after enzymatic hydrolysis with sulfatase. A butanol extraction process was performed on the remaining portion to obtain the sample of monoamine transmitters, precursor amino acids, and acidic metabolites. The monoamines and precursor amino acids were finally recovered in HCl solution, while the acidic metabolites shifted into the alkaline buffer from the organic layer. The basic and neutral substances were separated with a 0.1 M sodium citrate/citric acid buffer system (pH 4.0) containing 1% tetrahydrofuran, and the acidic ones with 0.075 M sodium citrate/citric acid buffer (pH 3.5) containing 1% tetrahydrofuran, 10% methanol, and 12% acetic acid. The steady-state concentrations of three monoamine transmitters (noradrenaline, dopamine, and 5-hydroxytryptamine) were determined together with their precursors and metabolites. Changes in the concentrations of these substances were examined for various drugs, of which the effects had been previously confirmed. The changes reflected putative drug effects and demonstrated that the procedure was applicable to the regional determination of monoamines and their metabolically related substances.

Enkephalin hydrolysis by mouse plasma in vitro

Hydrolysis of [Leu]- and [Met]enkephalin was determined in samples of pooled whole mouse plasma in vitro by using HPLC-ECD to measure accumulation of Tyr-containing metabolites. More Tyr-Gly-Gly accumulated from [Met]enkephalin than from [Leu]enkephalin hydrolysis, and [Met]enkephalins half-life in mouse plasma was approximately half that of [Leu]enkephalin. Comparisons of metabolite formation in the presence versus the absence of inhibitors with high selectivity for various peptidases demonstrated that a bestatin-sensitive aminopeptidase, presumably aminopeptidase M, as well as enkephalinase and angiotensin converting enzyme, participate in the hydrolysis of enkephalin in mouse plasma.

Supranormal levels of serotonin and its metabolite after raphe cell transplantation in serotonin-denervated rat hippocampus ☆

Transplantation of fetal raphe cells (14 days of gestation) into the adult rat hippocampus, 2 weeks following serotonin (5-HT)-denervation with intracisternal injection of 5,7-dihydroxytryptamine, can restore 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels in the hippocampus to far beyond normal values. Transplantation into the unilateral hippocampus produces asymmetrical turning behavior after administration of the 5-HT releasor, p-chloroamphetamine (IP), comparable to the behavior reported for rats with 5-HT denervation of the unilateral hippocampus. The effect is blocked by prior depletion of 5-HT with p-chlorophenylalanine (IP). The asymmetry in 5-HT levels are correlated with the behavioral change. These data indicate that a large amount of 5-HT is released from nerve terminals of transplanted raphe cells, and suggest that the supranormal levels of 5-HT and 5-HIAA after raphe cell transplantation are neurochemical correlates of 5-HT hyper-innervation of the hippocampus which has been reported previously.

Sensitive method for measuring hydrolysis of enkephalins in plasma, using high-performance liquid chromatography with electrochemical detection

This paper describes a simple and sensitive method for detection of [Leu]- and [Met]enkephalin and their N-terminal tyrosine-containing metabolic fragments (Tyr, Tyr-Gly, Tyr-Gly-Gly, and Tyr-Gly-Gly-Phe), using high-performance liquid chromatography with electrochemical detection. The method employs a carbon graphite working electrode with increased working electrode surface area (40 mm2). The procedures were applied to assay of the activities of enkephalin-degrading enzymes in whole plasma collected from rats, mice, and chicks.

Direct correlation between level of morphine and its biochemical effect on monoamine systems in mouse brain. Evidence for involvement of dopaminergic neurons in the pharmacological action of acute morphine.

The pharmacokinetics (uptake and elimination) and pharmacodynamics (biochemical effects on monoamine systems) of morphine in the CNS were investigated concurrently. ICR mice, weighing about 25 g, were injected intravenously with several doses (2.5-80 mg/kg) of morphine. The animals were killed by microwave irradiation (5 kW, 0.6 sec) at 10 and 30 min, and 1, 2, 4, 8 and 24 hr after the injection. The intracerebral levels of morphine and metabolically related substances consisting of monoamines [noradrenaline, dopamine (DA), 5-hydroxytryptamine (5-HT), 3, 4-dihydroxyphenylacetic acid (DOPAC), 3-methoxy-4-hydroxyphenylacetic acid [homovanillic acid (HVA)], 5-hydroxyindoleacetic acid (5-HIAA), tyrosine and tryptophan] were determined in identical samples by a combination of organic extraction and high-performance liquid chromatography with electrochemical detection. The intracerebral level of morphine was found to depend on the dose injected, and the biological half-life of the drug was estimated to be about 1 hr. The morphine injection (2.5-80 mg/kg) caused significant increases in monoamine metabolites although only slight changes occurred in the concns of parent transmitters. The intracerebral level of morphine was significantly correlated with the ratios DOPAC/DA and HVA/DA (r = 0.7033, P less than 0.0001; and r = 0.6455, P less than 0.0001, respectively). On the other hand, the correlation between the morphine level and 5-HIAA/5-HT was lower than those for DOPAC/DA and HVA/DA. These results suggest that monoamine systems, especially DA, are closely involved in the biochemical effects of morphine. Furthermore, the proposed procedure is demonstrated to be useful as a new approach in biochemical pharmacology, where the direct correlation between the distribution of a drug (pharmacokinetics) and the biochemical effects of the drug (pharmacodynamics) can be measured.