Tadatoshi Hashimoto

Contribution of serotonin neurons to the functional recovery after spinal cord injury in rats

The contribution of serotonin neurons to the functional recovery after spinal cord injury was studied pharmacologically in rats with moderately severe neurologic impairment (complete paraplegia but responsive to tail pinching) 24 h after thoracic spinal cord (T11) compression-induced injury. Fourteen days after cord injury the levels of endogenous norepinephrine (NE, -33%), dopamine (DA, -50%) and serotonin (5-HT, -55%) in the lumbar cord in the injury control rats were decreased and there were significant correlations between the neurologic score and the NE level (rs = 0.562, P less than 0.01) and the 5-HT level (rs = 0.745, P less than 0.001) but not the DA level. Bilateral i.c.v. injection of 5,7-dihydroxytryptamine (200 micrograms/rat) 24 h after cord injury significantly retarded the neurologic recovery during the 14 days after injury, accompanied by a further reduction in the 5-HT level (-86%) but not in the NE or DA level. On the other hand, neither p-chlorophenylalanine (PCPA) (300 mg/kg, i.p., once daily starting 24 h after injury for 13 consecutive days) nor reserpine (1 mg/kg, i.p., 4 times, once 24 h after injury and then every fourth day) had any influence on the time course of the neurologic recovery during the 14 days after injury, although PCPA treatment further reduced the levels of NE (-50%) and 5-HT (-91%), and reserpine treatment further reduced the levels of NE (-95%), DA (-73%) and 5-HT (-85%).(ABSTRACT TRUNCATED AT 250 WORDS)

Apelin-transgenic mice exhibit a resistance against diet-induced obesity by increasing vascular mass and mitochondrial biogenesis in skeletal muscle

BACKGROUND Apelin is an endogenous ligand for the G-protein-coupled 7-transmembrane receptor, APJ. The administration of apelin-13, a truncated 13-amino acid apelin peptide, in diet-induced obese mice is reported to result in a decrease in adiposity due to the increase of energy expenditure with an increase in the expression of uncoupling proteins. METHODS We systematically compared the phenotype of human apelin-transgenic (apelin-Tg) mice fed standard or high-fat diets (HFD) with that of non-Tg control mice to clarify the effect of apelin on obesity. The beneficial effects of apelin were evaluated by multiple assay methods including indirect calorimetrical measurements, gene expression analysis, and immunohistochemical staining. RESULTS Apelin-Tg mice inhibited HFD-induced obesity without altering food intake and exhibited increased oxygen consumption and body temperature compared to non-Tg controls. Interestingly, the mRNA expressions of angiopoietin-1 (Ang1), a key molecule for vascular maturation, and its receptor, endothelium-specific receptor tyrosine kinase 2 (Tie2), were significantly upregulated in the skeletal muscle of HFD-fed apelin-Tg mice, and the areas of anti-CD31 antibody-positive endothelial cells also increased. Furthermore, both the aerobic type-I muscle fibre ratio and the DNA copy number of mitochondrial NADH dehydrogenase subunit 1 increased 2.0- and 1.4-fold in skeletal muscle, respectively. CONCLUSIONS These findings suggest that apelin stimulates energy expenditure via increase vascular mass and mitochondrial biogenesis in skeletal muscle. GENERAL SIGNIFICANCE Apelin is a prerequisite factor for anti-obesity by stimulating energy expenditure via regulating homeostatic energy balance.

Derivation of rat embryonic stem cells and generation of protease-activated receptor-2 knockout rats

One of the remarkable achievements in knockout (KO) rat production reported during the period 2008–2010 is the derivation of authentic embryonic stem (ES) cells from rat blastocysts using a novel culture medium containing glycogen synthase kinase 3 and mitogen-activated protein kinase kinase inhibitors (2i medium). Here, we report gene-targeting technology via homologous recombination in rat ES cells, demonstrating its use through production of a protease-activated receptor-2 gene (Par-2) KO rat. We began by generating germline-competent ES cells from Dark Agouti rats using 2i medium. These ES cells, which differentiate into cardiomyocytes in vitro, can produce chimeras with high ES cell contribution when injected into blastocysts. We then introduced a targeting vector with a neomycin-resistant gene driven by the CAG promoter to disrupt Par-2. After a 7-day drug selection, 489 neomycin-resistant colonies were obtained. Following screening by polymerase chain reaction (PCR) genotyping and quantitative PCR analysis, we confirmed three homologous recombinant clones, resulting in chimeras that transmitted the Par-2 targeted allele to offspring. Par-2 KO rats showed a loss of Par-2 messenger RNA expression in their stomach cells and a lack of PAR-2 mediated smooth muscle relaxation in the aorta as indicated by pharmacological testing. Compared with mice, rats offer many advantages in biomedical research, including a larger body size; consequently, they are widely used in scientific investigation. Thus, the establishment of a gene-targeting technology using rat ES cells will be a valuable tool in human disease model production and drug discovery.

Anatomical Transcriptome of G Protein-Coupled Receptors Leads to the Identification of a Novel Therapeutic Candidate GPR52 for Psychiatric Disorders

Many drugs of abuse and most neuropharmacological agents regulate G protein-coupled receptors (GPCRs) in the central nervous system (CNS)_ENREF_1. The striatum, in which dopamine D1 and D2 receptors are enriched, is strongly innervated by the ventral tegmental area (VTA), which is the origin of dopaminergic cell bodies of the mesocorticolimbic dopamine system_ENREF_3 and plays a central role in the development of psychiatric disorders_ENREF_4. Here we report the comprehensive and anatomical transcript profiling of 322 non-odorant GPCRs in mouse tissue by quantitative real-time PCR (qPCR), leading to the identification of neurotherapeutic receptors exclusively expressed in the CNS, especially in the striatum. Among them, GPR6, GPR52, and GPR88, known as orphan GPCRs, were shown to co-localize either with a D2 receptor alone or with both D1 and D2 receptors in neurons of the basal ganglia. Intriguingly, we found that GPR52 was well conserved among vertebrates, is Gs-coupled and responsive to the antipsychotic drug, reserpine. We used three types of transgenic (Tg) mice employing a Cre-lox system under the control of the GPR52 promoter, namely, GPR52-LacZ Tg, human GPR52 (hGPR52) Tg, and hGPR52-GFP Tg mice. Detailed histological investigation suggests that GPR52 may modulate dopaminergic and glutamatergic transmission in neuronal circuits responsible for cognitive function and emotion. In support of our prediction, GPR52 knockout and transgenic mice exhibited psychosis-related and antipsychotic-like behaviors, respectively. Therefore, we propose that GPR52 has the potential of being a therapeutic psychiatric receptor. This approach may help identify potential therapeutic targets for CNS diseases.

Effect of thyrotropin-releasing hormone on the neurologic impairment in rats with spinal cord injury: treatment starting 24 h and 7 days after injury

The effect of treatment with thyrotropin-releasing hormone (TRH) or naloxone on the neurologic impairment after spinal cord injury was studied in rats with the severest neurologic impairment (complete paraplegia, no withdrawal response upon tail pinching, and urinary incontinence) 24 h and 7 days after injury. Subcutaneous treatment with TRH (2.5, 10 and 40 mg/kg per day) once daily for 7 consecutive days starting 24 h or 7 days after injury improved the neurologic function in the rats with cord injury in a dose-related manner, with a minimum effective dose of less than 2.5 mg/kg per day in both cases. However, subcutaneous treatment with naloxone (40 mg/kg per day) once daily for 7 consecutive days starting 24 h after injury did not exert any beneficial effects on neurologic function. These results indicate that TRH but not naloxone treatment starting 24 h and as late as 7 days after injury is effective in rats with the severest neurologic impairment following spinal cord injury. Thus, it is suggested that the duration of the effectiveness of late treatment with TRH on the neurologic impairment in rats with spinal cord injury is more than 1 week, while the duration with naloxone is less than 24 h.

Comparative Study on the Behavioral and EEG Changes Induced by Diazepam, Buspirone and a Novel Anxioselective Anxiolytic, DIM-2327, in the Cat

Behavioral and EEG effects of 2-(7-chloro-1,8-naphthyridin-2-yl)-3-[(1,4)-dioxa-8-(azas piro-[4.5]dec-8- yl)carbonylmethyl]isoindolin-1-one (DN-2327; 1, 5 and 20 mg/kg p.o.) were compared to those of diazepam (0.2 and 1 mg/kg p.o.) and buspirone (1 and 5 mg/kg p.o.) in freely moving cats. DN-2327 did not affect motor coordination or the relative percentages of the three sleep-wakefulness stages. Diazepam (1 mg/kg) increased wakefulness and non-REM sleep, and buspirone (5 mg/kg) also increased wakefulness and decreased REM sleep. In addition, diazepam (1 mg/kg) caused severe motor disturbance, but buspirone did not. The cortical EEG power density spectra during wakefulness were changed almost dose-dependently by DN-2327 (decreased: 2-7.75 Hz; increased: 20-49.75 Hz), and dose-dependently by diazepam (decreased: 2-7.75 Hz; increased 13-49.75 Hz) and buspirone (decreased: 4-9.75 and 13-19.75 Hz). The effect of DN-2327 on the cortical EEG varied with the sleep-wakefulness stage. The power of the 4- to 7.75-Hz frequency (theta) band of the hippocampal EEG during wakefulness was decreased by diazepam and buspirone but not by DN-2327, while the peak frequency of its spectra was decreased only by diazepam. On the other hand, during non-REM sleep, DN-2327 decreased the power of the theta band as did diazepam. These results indicate that the behavioral and EEG effects of DN-2327 differ completely from those of buspirone and considerably from those of diazepam and that the EEG effect of DN-2327 varies with the sleep-wakefulness stage.

Effect of Thyrotropin-releasing Hormone on Pentobarbitone-induced Sleep in Rats: Continuous Treatment with a Sustained Release Injectable Formulation

Abstract— The mode of action and the time course of the effects of continuous thyrotropin‐releasing hormone (TRH) treatment using a two‐week sustained release injectable formulation of TRH‐containing copoly(±)‐lactic/glycolic acid) microspheres (TRH‐SR) on pentobarbitone‐induced sleeping time were studied in rats. Subcutaneous treatment with TRH‐SR at doses corresponding to 0·05 and 0·2 mg of TRH kg−1 day−1 caused a dose‐related shortening of pentobarbitone‐induced sleeping time with a minimum effective dose (MED) of 0·05 mg kg−1 day−1, without affecting the body weight gain. On the other hand, the MED of TRH when given as a bolus subcutaneous injection was 40 mg kg−1. The effect of TRH‐SR treatment was blocked by intraperitoneal scopolamine (0·1 mg kg−1) and mecamylamine (2 mg kg−1) but not by scopolamine methyl bromide (0·1 mg kg−1). The results indicate that continuous TRH treatment using TRH‐SR causes shortening of pentobarbitone‐induced sleeping time at doses lower than those required using bolus injection and probably by a mechanism involving the central cholinergic system.

Hypercholesterolemia and atherosclerosis in low density lipoprotein receptor mutant rats

To establish low density lipoprotein receptor (LDLR) mutant rats as a hypercholesterolemia and atherosclerosis model, we screened the rat LDLR gene for mutations using an N-ethyl-N-nitrosourea mutagenesis archive of rat gene data, and identified five mutations in its introns and one missense mutation (478T>A) in exon 4. The C160S mutation was located in the ligand binding domain of LDLR and was revealed to be equivalent to mutations (C160Y/G) identified in human familial hypercholesterolemia (FH) patients. The wild type, heterozygous, and homozygous mutant rats were fed a normal chow diet or a high fat high cholesterol (HFHC) diet from the age of 10 weeks for 16 weeks. The LDLR homozygous mutants fed the normal chow diet showed higher levels of plasma total cholesterol and LDL cholesterol than the wild type rats. When fed the HFHC diet, the homozygous mutant rats exhibited severe hyperlipidemia and significant lipid deposition from the aortic arch to the abdominal aorta as well as in the aortic valves. Furthermore, the female homozygous mutants also developed xanthomatosis in their paws. In conclusion, we suggest that LDLR mutant rats are a useful novel animal model of hypercholesterolemia and atherosclerosis.

Characteristics of TRK-130 (Naltalimide), a Novel Opioid Ligand, as a New Therapeutic Agent for Overactive Bladder

We characterized TRK-130 (N-[(5R,6R,14S)-17-(cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxymorphinan-6-yl]phthalimide; naltalimide), an opioid ligand, to clarify the therapeutic potential for overactive bladder (OAB). In radioligand-binding assays with cells expressing human µ-opioid receptors (MORs), δ-opioid receptors (DORs), or κ-opioid receptors (KORs), TRK-130 showed high selectivity for MORs (Ki for MORs, DORs, and KORs = 0.268, 121, and 8.97 nM, respectively). In a functional assay (cAMP accumulation) with cells expressing each human opioid receptor subtype, TRK-130 showed potent but partial agonistic activity for MORs [EC50 (Emax) for MORs, DORs, and KORs = 2.39 nM (66.1%), 26.1 nM (71.0%), and 9.51 nM (62.6%), respectively]. In isovolumetric rhythmic bladder contractions (RBCs) in anesthetized guinea pigs, TRK-130 dose-dependently prolonged the shutdown time (the duration of complete cessation of the bladder contractions) (ED30 = 0.0034 mg/kg i.v.) without affecting amplitude of RBCs. Furthermore, TRK-130 ameliorated formalin-induced frequent urination at doses of higher than 0.01 mg/kg p.o. in guinea pigs under the freely moving condition. Meanwhile, TRK-130 showed only a negligible effect on the gastrointestinal transit at doses of up to 10 mg/kg s.c. in mice. These results indicate that TRK-130 is a potent and selective human MOR partial agonist without undesirable opioid adverse effects such as constipation and enhances the storage function by suppressing the afferent limb of the micturition reflex pathway, suggesting that TRK-130 would be a new therapeutic agent for OAB.

Effects of TAK-802, a novel acetylcholinesterase inhibitor, and tamsulosin, an α1-adrenoceptor antagonist, and their synergistic effects on the urodynamic characteristics in a guinea-pig model of functional bladder outlet obstruction

To investigate the effects of TAK‐802, a potent acetylcholinesterase inhibitor, and tamsulosin, an α1‐adrenoceptor antagonist, and their concomitant administration on the urodynamic characteristics in a guinea‐pig model of functional bladder outlet obstruction.