Improvement of learning and increase in dopamine level in the frontal cortex by methylphenidate in mice lacking dopamine transporter.

The symptoms of attention-deficit/hyperactivity disorder (ADHD) are characterized by inattention and hyperactivity-impulsivity. It is a common childhood neurodevelopmental disorder that often persists into adulthood. Improvements in ADHD symptoms using psychostimulants have been recognized as a paradoxical calming effect. The psychostimulant methylphenidate (MPH) is currently used as the first-line medication for the management of ADHD. Recent studies have drawn attention to altered dopamine-mediated neurotransmission in ADHD, particularly reuptake by the dopamine transporter (DAT). This hypothesis is supported by the observation that DAT knockout mice exhibit marked hyperactivity that is responsive to acute MPH treatment. However, other behaviors relevant to ADHD have not been fully clarified. In the present study, we observed learning impairment in shuttle-box avoidance behavior together with hyperactivity in a novel environment in DAT knockout mice. Methylphenidate normalized these behaviors and enhanced escape activity in the tail suspension test. Interestingly, the effective dose of MPH increased extracellular dopamine in the prefrontal cortex but not striatum, suggesting an important role for changes in prefrontal dopamine in ADHD. Research that uses rodent models such as DAT knockout mice may be useful for elucidating the pathophysiology of ADHD.

Specific Roles of NMDA Receptor Subunits in Mental Disorders.

N-methyl-D-aspartate (NMDA) receptor plays important roles in learning and memory. NMDA receptors are a tetramer that consists of two glycine-binding subunits GluN1, two glutamate-binding subunits (i.e., GluN2A, GluN2B, GluN2C, and GluN2D), a combination of a GluN2 subunit and glycine-binding GluN3 subunit (i.e., GluN3A or GluN3B), or two GluN3 subunits. Recent studies revealed that the specific expression and distribution of each subunit are deeply involved in neural excitability, plasticity, and synaptic deficits. The present article summarizes reports on the dysfunction of NMDA receptors and responsible subunits in various neurological and psychiatric disorders, including schizophrenia, autoimmune-induced glutamatergic receptor dysfunction, mood disorders, and autism. A key role for the GluN2D subunit in NMDA receptor antagonist-induced psychosis has been recently revealed.

The Selective Serotonin Reuptake Inhibitor Paroxetine, but not Fluvoxamine, Decreases Methamphetamine Conditioned Place Preference in Mice.

Monoamine transporters are the main targets of methamphetamine (METH). Recently, we showed that fluoxetine, a selective serotonin reuptake inhibitor (SSRI), decreased METH conditioned place preference (CPP), suggesting that serotonin transporter (SERT) inhibition reduces the rewarding effects of METH. To further test this hypothesis, in the present study we investigated the effects of additional SSRIs, paroxetine and fluvoxamine, on METH CPP in C57BL/6J mice. In the CPP test, pretreatment with 20 mg/kg paroxetine abolished the CPP for METH, whereas pretreatment with 100 mg/kg fluvoxamine prior to administration of METH failed to inhibit METH CPP. These results suggest that paroxetine, a medication widely used to treat depression, may be a useful tool for treating METH dependence. Further, these data suggest that molecules other than the SERT [such as G protein-activated inwardly rectifying K+ (GIRK) channels] whose activities are modulated by paroxetine and fluoxetine, but not by fluvoxamine, are involved in reducing METH CPP by paroxetine and fluoxetine.

MOP Reduction During Long-Term Methamphetamine Withdrawal was Restored by Chronic Post-Treatment with Fluoxetine.

Previously, we found fluoxetine reduces methamphetamine preference in mice. However, effects of fluoxetine on developed methamphetamine preference and on methamphetamine induced gene expression changes have been largely unknown. The present study investigates effects of post-treatment with fluoxetine on methamphetamine dependence and on gene expressions after long-term withdrawal in mice. First, we examined whether chronic post-treatment with fluoxetine attenuated methamphetamine-conditioned place preference. Next, we examined the changes in gene expression levels after long-term withdrawal (with saline or fluoxetine treatment) following chronic methamphetamine treatment. Using mRNA from the pooled frontal cortices of 10 mice per group, gene expression analyses were performed using a custom-developed cDNA array and a real-time quantitative reverse transcription-PCR. Chronic post-treatments with fluoxetine abolished the conditioned place preference developed by methamphetamine administrations. Even after long-term withdrawal from repeated methamphetamine administration, µ-opioid receptor (MOP) gene expression was significantly reduced in the frontal cortex. The reduced MOP gene expression in the frontal cortex was restored by chronic administration with fluoxetine. These changes were confirmed by Western blot analyses. These findings suggest that the chronic post-treatments with fluoxetine might be effective for restoring the reduction of MOP levels in the frontal cortex following long-term abstinence from methamphetamine.

Effects of MDMA on Extracellular Dopamine and Serotonin Levels in Mice Lacking Dopamine and/or Serotonin Transporters.

3,4-Methylendioxymethamphetamine (MDMA) has both stimulatory and hallucinogenic properties which make its psychoactive effects unique and different from those of typical psychostimulant and hallucinogenic agents. The present study investigated the effects of MDMA on extracellular dopamine (DA(ex)) and serotonin (5-HT(ex)) levels in the striatum and prefrontal cortex (PFC) using in vivo microdialysis techniques in mice lacking DA transporters (DAT) and/or 5-HT transporters (SERT). subcutaneous injection of MDMA (3, 10 mg/kg) significantly increased striatal DA(ex) in wild-type mice, SERT knockout mice, and DAT knockout mice, but not in DAT/SERT double-knockout mice. The MDMA-induced increase in striatal DA(ex) in SERT knockout mice was significantly less than in wildtype mice. In the PFC, MDMA dose-dependently increased DA(ex) levels in wildtype, DAT knockout, SERT knockout and DAT/SERT double-knockout mice to a similar extent. In contrast, MDMA markedly increased 5-HT(ex) in wildtype and DAT knockout mice and slightly increased 5-HT(ex) in SERT-KO and DAT/SERT double-knockout mice. The results confirm that MDMA acts at both DAT and SERT and increases DA(ex) and 5-HT(ex).

Usefulness of transanal endoscopic surgery for carcinoid tumor in the upper and middle rectum.

BACKGROUND: This study evaluated the indications and outcome for transanal endoscopic surgery (TES) used to manage rectal carcinoid tumor as compared with those of conventional transanal local resection (TAR). METHODS: The retrospective study subjects were 28 patients with rectal carcinoid tumor treated by TES (n = 17) or TAR (n = 11) between January 1995 and December 2001. Patient and tumor characteristics, operative results, and postoperative outcomes were compared between the two groups. RESULTS: The distance from the anal verge to the distal tumor margin in the TES group (range, 4-12 cm; median, 6.8 cm) was significantly greater than in the TAR group (range, 3-6 cm; median, 4.5 cm) (p = 0.001). The median tumor diameter was 5.5 mm (range, 3-11 mm) in the TES group and 5.0 mm (range, 3-8 mm) in the TAR group, showing no statistical difference. Microscopically, resected specimens in both groups were typical carcinoid tumors restricted to the submucosal layer. No recurrence was noted in either group. CONCLUSION: Whereas TES is useful for patients with small rectal carcinoid tumor of typical histology within the submucosal layer in the upper and middle rectum, TAR is effective for accessing the lower rectum.

Changes in expression of the mouse homologues of KIAA genes after subchronic methamphetamine treatment.

Amphetamine abuse may be associated with adaptive changes in gene expression in the brain. In the present study, a newly developed cDNA array system comprising mouse KIAA (mKIAA) cDNA clones was used to examine the gene expression affected by chronic methamphetamine treatment. Approximately 800 mKIAA clones were blotted onto a nylon membrane and hybridized with 33P-labeled cDNA derived from mRNAs isolated from the whole brains of mice that had been treated daily with saline or methamphetamine (2 mg/kg, i.p.) for 2 weeks. The arrays displayed robust hybridization for almost all transcripts. The results obtained from five experiments were averaged, each performed with triplicate samples. Several clones were chosen as positive candidates for methamphetamine-induced changes; however, only Per2 and mKIAA0099 genes showed a significantly increased expression (P < .05). Subsequently, with the focus on the period-related proteins, the expression of these proteins in various parts of the rat brain were assessed by immunoblot analysis. Chronic administration of methamphetamine (8 mg/kg, i.p., for 10 days) caused increased Per2 protein expression in the hippocampus. Interestingly, chronic methamphetamine treatment at a lower dose (4 mg/kg, i.p., for 10 days) induced an increase in SCN circadian oscillatory protein (SCOP) expression, also in the hippocampus. These data suggest that long-lasting alterations of the period-related gene expressions in the hippocampus might play an important role in methamphetamine addiction.

Catecholamines and serotonin are differently regulated by tetrahydrobiopterin. A study from 6-pyruvoyltetrahydropterin synthase knockout mice.

(6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for tyrosine hydroxylase (TH), tryptophan hydroxylase, phenylalanine hydroxylase, and nitric-oxide synthase. These enzymes synthesize neurotransmitters, e.g. catecholamines, serotonin, and nitric oxide (NO). We established mice unable to synthesize BH4 by disruption of the 6-pyruvoyltetrahydropterin synthase gene, the encoded protein of which catalyzes the second step of BH4 biosynthesis. Homozygous mice were born at the almost expected Mendelian ratio, but died within 48 h after birth. In the brain of homozygous mutant neonates, levels of biopterin, catecholamines, and serotonin were extremely low. The number of TH molecules was highly dependent on the intracellular concentration of BH4 at nerve terminals. Alteration of the TH protein level by modulation of the BH4 content is a novel regulatory mechanism. Our data showing that catecholaminergic, serotonergic, and NO systems were differently affected by BH4 starvation suggest the possible involvement of BH4 synthesis in the etiology of monoamine-based neurological and neuropsychiatric disorders.

[Effect of ATP-sensitive K+ channel blocker (quinine) on the dopamine decrease induced by selective D3 agonist (7-OH-DPAT) in the rat striatum].

In vivo brain microdialysis was used to investigate the role of D3 receptors relating to dopamine auto-regulated systems in the rat. A local infusion of the dopamine D3 selective agonist, 7-OH-DPAT (100 nM), into the striatum through the dialysis membrane produced a significant decrease in extracellular concentrations of dopamine. A local application of ATP-sensitive K+ (KATP) channel blocker, quinine (10 microM, 100 microM, 1 mM), produced dose-dependent increases in extracellular concentrations of dopamine. Quinine (100 microM, 1 mM) significantly blocked a 7-OH-DPAT-induced decrease in the striatal dopamine levels in the dose-dependent manner. Because of many previous reports, the autoregulated functions of dopamine release by D3 receptors in the striatum can be regarded mainly as nerve terminal autoreceptors and/or a short-loop negative feedback system. Therefore these results suggest that KATP channels may be present in nigrostriatal dopaminergic terminals and that in the striatum, presynaptic dopamine D3 autoreceptors and/or a post-synaptic D3 related short-loop negative feedback system inhibit dopamine release tonically by the activation of KATP channels.

Interaction between cytochrome P450 and other drug-metabolizing enzymes: evidence for an association of CYP1A1 with microsomal epoxide hydrolase and UDP-glucuronosyltransferase.

Protein-protein interactions between cytochrome P450 (P450) and other drug-metabolizing enzymes were studied by affinity chromatography using CYP1A1-, glycine-, and bovine serum albumin (BSA)-conjugated Sepharose 4B columns. Sodium cholate-solubilized microsomes from phenobarbital-treated rat liver were applied to the columns and the material eluted with buffer containing NaCl was analyzed by immunoblotting. Microsomal epoxide hydrolase (mEH) and UDP-glucuronosyltransferases (UGTs), as well as NADPH-P450 reductase, were efficiently trapped by the CYP1A1 column. Glycine and BSA columns exhibited no ability to retain these proteins. Protein disulfide isomerase and calnexin, non-drug-metabolizing enzymes expressed in the endoplasmic reticulum, were unable to associate with the CYP1A1 column. These results suggest that CYP1A1 interacts with mEH and UGT to facilitate a series of multistep drug metabolic conversions.

The distribution of serotonergic nerves in microencephalic rats treated prenatally with methylazoxymethanol.

Prenatal exposure of pregnant rats to methylazoxymethanol acetate (MAM) induces microencephaly in the offspring. In the present study of these microencephalic rats (MAM rats) we used quantitative autoradiography to investigate [3H] paroxetine binding sites, which are a selective marker of serotonin (5-HT) transporters (5-HTT). The binding in the accumbens, cortex, hippocampus, and dorsolateral thalamus was significantly increased in MAM rats, compared to the control rats, while there was a significant decrease in the dorsal raphe nucleus of the MAM rats. The levels of 5-HTT mRNA in the dorsal raphe nuclei were analyzed by in situ hybridization, which revealed a significant decrease in 5-HTT mRNA-positive neurons in the MAM rats compared to the control rats. The results imply serotonergic hyperinnervation in the cerebral hemispheres of MAM rats, while a target-dependent secondary degeneration of 5-HT neurons might be induced in the dorsal raphe nuclei of MAM rats.

Dopamine D3 receptor binding by D3 agonist 7-OH-DPAT (7-hydroxy-dipropylaminotetralin) and antipsychotic drugs measured ex vivo by quantitative autoradiography.

Because the dopamine D3 receptor is primarily expressed in regions of the limbic system of brain, it was proposed that it may represent a target for antipsychotic drugs that is free of extrapyramidal side effects. An ex vivo receptor binding technique employing [3H]7-OH-DPAT was used to evaluate in vivo occupancy of dopamine D3 receptors in the rat nucleus accumbens by selective D3 agonist 7-OH-DPAT (7-hydroxy-dipropylaminotetralin) and various antipsychotic drugs. With an ID50 value of 0.07 mg/kg, the selective D3 agonist (+)-7-OH-DPAT had the most potent inhibitory effect on ex vivo binding of [3H]7-OH-DPAT among all drugs tested. Clinical doses of phenothiazine drugs, such as chlorpromazine and levomepromazine, induce binding to D3 receptors in vivo, while atypical antipsychotic drugs, such as clozapine, pimozide, and sulpiride, are very weak in inhibiting ex vivo binding of [3H]7-OH-DPAT, indicating that the role of D3 receptors as targets of antipsychotic drugs free of extrapyramidal side effects may not be important.

GTP cyclohydrolase I from Tetrahymena pyriformis: cloning of cDNA and expression.

A full-length cDNA clone for GTP cyclohydrolase I (EC 3.5.4.16) was isolated from a Tetrahymena pyriformis cDNA library by plaque hybridization. The nucleotide sequence determination revealed that the length of the cDNA insert was 1516 bp. The coding region encoded a protein of 223 amino acid residues with a calculated molecular mass of 25 416 Da. The deduced amino acid sequence of Tetrahrymena GTP cyclohydrolase I showed sequence identity with that of Escherichia coli (55%). The identity of T. pyriformis GTP cyclohydrolase I with sequences of Dictyostelium discoideum, Saccharomyces cerevisiae, Drosophila melanogaster, mouse, rat, and human enzymes was less marked and was 30, 30, 25, 28, 28, and 27%, respectively. RNA blot analysis showed a single mRNA species of 2.1 kb in this protozoan. The mRNA level of GTP cyclohydrolase I increased during synchronous cell division induced by intermittent heat treatment. The results suggest that the mRNA expression is associated with the cell cycle of T. pyriformis.

Molecular cloning of the human Nurr1 gene: characterization of the human gene and cDNAs.

Nurr1 is a member of the nuclear receptor superfamily of transcription factors that is expressed predominantly in the central nervous system, including developing dopaminergic neurons. Recently, it was demonstrated that Nurr1 is critical for midbrain dopaminergic cell differentiation. In order to investigate a possible relation of Nurr1 with the pathogenesis of Parkinson's disease or other neuropsychiatric disorders, we have cloned and characterized the human Nurr1 gene. The gene exists as a single copy in the human genome and comprises eight exons spanning 8kb. We determined the complete nucleotide sequence and flanking regions of the gene. Potential regulatory regions included consensus binding sites for NF-kappaB, CREB, and Sp1. Isolation of human Nurr1 cDNAs from fetal brain suggested the presence of a new splicing variant of Nurr1 in the human brain.

The atypical antipsychotic sertindole enhances efflux of dopamine and its metabolites in the rat cortex and striatum.

Previous studies have shown that sertindole (1-[2-[4-[5-chloro-1-(4-fluorophenyl)-1H-indol-3-yl]-1-piperidinyl]ethyl ]-2 imidazolidinone), an atypical antipsychotic drug that is a potent 5-HT2A and dopamine D2 receptor antagonist, preferentially affects mesocorticolimbic rather than mesostriatal dopamine neurons. Using in vivo microdialysis in conscious rats, we investigated the effects of sertindole on dopamine release and metabolism in the striatum and the medial prefrontal cortex. Systemic administration of sertindole dose dependently enhanced dopamine release in the medial prefrontal cortex and the striatum to the same extent.

Enzymes related to catecholamine biosynthesis in Tetrahymena pyriformis. Presence of GTP cyclohydrolase I.

We first identified GTP cyclohydrolase I activity (EC 3.5.4.16) in the ciliated protozoa, Tetrahymena pyriformis. The Vmax value of the enzyme in the cellular extract of T. pyriformis was 255 pmol mg-1 protein h-1. Michaelis-Menten kinetics indicated a positive cooperative binding of GTP to the enzyme. The GTP concentration producing half-maximal velocity was 0.8 mM. By high-performance liquid chromatography (HPLC) with fluorescence detection, a major peak corresponding to D-monapterin (2-amino-4-hydroxy-6-[(1'R,2'R)-1',2',3'-trihydroxypropyl]pteridin e, D-threo-neopterin) and minor peaks of D-erythro-neopterin and L-erythro-biopterin were found to be present in the cellular extract of Tetrahymena. Thus, it is strongly suggested that Tetrahymena converts GTP into unconjugated pteridine derivatives. In this study, dopamine was detected as the major catecholamine, while neither epinephrine nor norepinephrine was identified. Indeed, this protozoa was shown to possess the activity of a dopamine synthesizing enzyme, aromatic L-amino acid decarboxylase. On the other hand, activities of tyrosine hydroxylase or tyrosinase which converts tyrosine into dopa, the substrate of aromatic L-amino acid decarboxylase, could not be detected in this protozoa. Furthermore, neither dopamine beta-hydroxylase activity nor phenylethanolamine N-methyltransferase activity could be identified by the HPLC methods.

Effect of chronic amitriptyline administration on serotonergic receptors in rats with methylazoxymethanol-induced microencephaly.

Methylazoxymethanol (MAM)-induced cortical hypoplasia resulted in a 20% decrease in the Bmax of 5-HT2A receptors in the frontal cortex with no change in the Bmax of 5-HT1A receptors. Chronic treatment with amitriptyline did not further decrease the Bmax of 5-HT2A receptors in the MAM-lesioned cortex, suggesting that the persistent down-regulation of cortical 5-HT2A receptors in MAM-lesioned rats was induced by serotonergic hyperinnervation.

Effects of prenatal methylazoxymethanol treatment on striatal dopaminergic systems in rat brain.

To further examine the effects of prenatal methylazoxymethanol (MAM) treatment on striatal dopaminergic systems, the status of presynaptic dopamine transporters was examined by quantitative autoradiography of [3H]GBR 12935 binding. Significantly higher [3H]GBR 12935 binding was seen in MAM-lesioned striatum in comparison to the controls, indicating relative dopaminergic hyperinnervation in MAM-induced hypoplastic striatum. The effect of prenatal MAM treatment on extracellular levels of dopamine and its metabolites in the striatum was also examined using in vivo microdialysis. As measured in conscious freely-moving rats, prenatal MAM treatment significantly increased basal dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) release in the striatum in comparison with control rats. These data suggest that in accordance with morphological dopaminergic hyperinnervation, dopaminergic functions are significantly augmented in MAM-lesioned brains. Thus, it is suggested that MAM-induced microencephalic rats should serve as a good animal model for the study of augmented dopaminergic functions in the striatum.

Nitric oxide donor NOR 3 inhibits ketogenesis from oleate in isolated rat hepatocytes by a cyclic GMP-independent mechanism.

Studies were conducted to clarify the effects of nitric oxide donors NOR 3 ((+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide, FK409), SIN-1 (3-morpholinosydnonimine) and SNAP (S-nitroso-N-acetylpenicillamine) on the accumulation of cGMP and cAMP and Ca2+ mobilization as well as ketogenesis from oleate in isolated rat hepatocytes. NOR 3 caused inhibition of ketogenesis from oleate along with stimulation of cGMP accumulation in rat hepatocytes, whereas SIN-1 and SNAP exerted no effect on ketogenesis despite their marked stimulation of cGMP accumulation. Although the nitric oxide trapping agent, carboxy-PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide), antagonized the stimulation by NOR 3 of cGMP accumulation, it failed to modulate the anti-ketogenic action of NOR 3. Furthermore, neither 8-bromoguanosine-3',5'-cyclic monophosphate nor N2,2'-O-dibutyrylguanosine-3',5'-cyclic monophosphate mimicked the anti-ketogenic action of NOR 3. It is concluded in the present study that NOR 3-induced inhibition of ketogenesis in rat hepatocytes is not mediated by cGMP. The present study revealed that the remaining structure of NOR 3 from which nitric oxide had been spontaneously released had no anti-ketogenic action. We first and clearly demonstrated that nitrite production was dramatically enhanced when NOR 3 was incubated in the presence of rat hepatocytes. The mechanism whereby NOR 3 inhibits ketogenesis in rat hepatocytes will be discussed.