Shinsuke Kaku

Neuropsin is essential for early processes of memory acquisition and Schaffer collateral long-term potentiation in adult mouse hippocampus in vivo

Long‐term potentiation (LTP) is thought to be particularly important in the acquisition of hippocampus‐associated memory, in part because it develops quickly and persists for indefinite periods. Extracellular proteolysis has been hypothesized to contribute to LTP by modifying adhesive relations of synapses and thus the morphology of excitatory synapses. Here we report that neuropsin (NP), an extracellular serine protease, is critically involved in the formation of both the potentiation effect and hippocampus‐dependent forms of memory. NP‐knockout mice were significantly impaired in the Morris water maze and Y‐mazes and failed to exhibit early phase LTP induced by a single tetanus. Potentiation was also impaired or completely blocked by in vivo application of a specific inhibitor or a neutralizing monoclonal antibody for NP. Intriguingly, recombinant (r‐) NP alone, without tetanic stimulation, elicited either long‐lasting potentiation or depression, depending on the applied dose. The r‐NP‐elicited potentiation was occluded by prior induction of LTP, while theta‐burst‐elicited LTP was occluded by application of r‐NP alone, suggesting that the two forms of plasticity have a common signalling pathway. r‐NP‐elicited potentiation and depression increased phosphorylation at different sites on the GluR1 subunit of the AMPA receptor that had previously been associated with LTP or long‐term depression. Thus, we conclude that NP is necessary for establishment of LTP and has a significant role in memory acquisition.

Characteristics of binding of [3H]NE-100, a novel sigma-receptor ligand, to guinea-pig brain membranes

We examined the characteristics of binding of radiolabeled N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride ([3H] NE-100), a highly potent and selective sigma-receptor ligand, to guinea-pig brain membranes. [3H]NE-100 showed saturable and reversible binding to sigma binding sites. A dissociation constant (Kd) and maximal numbers of binding sites (Bmax) obtained from Scatchard plot analysis were 1.2 ± 0.1 nM and 1049.3 ± 115.1 fmol/mg protein (n = 3), respectively. NE-100 was the most potent inhibitor of [3H]NE-100 binding among several structurally dissimilar sigma-receptor ligands, including haloperidol and ( + )-pentazocine. ( + )-Benzomorphanes had more than a 10-fold potent inhibitory activity over (−)-benzomorphanes, with regard to [3H]NE-100 binding. The binding of [3H]NE-100 was not influenced by histaminergic, dopaminergic, adrenergic, serotonergic cholinergic or glutaminergic agents at 10−7 M. GTP-γ-S and phenytoin also did not affect the binding of [3H]NE-100. A higher [3H]NE-100 binding was observed in the cerbellum and medulla oblongata. Except for the nuclear fraction, the highest level of [3H]NE-100 binding to subcellular fractions was observed in microsomal fractions. These results suggest that NE-100 selectively binds, with a high affinity, to sigma-1 binding sites in guinea-pig brain membranes, as an “antagonist”.

Lithium inhibits stress-induced changes in tau phosphorylation in the mouse hippocampus.

Summary.It has been pointed out that hyperphosphorylation of microtubule-associated protein tau caused by stress might participate in the early stages of Alzheimer’s disease pathogenesis. In the present study, we investigated the effects of cold water stress (CWS) on tau phosphorylation in the mouse hippocampus and the effects of GSK-3β inhibitor, LiCl, on CWS-induced changes in tau phosphorylation levels by immunoblot analyses. CWS exposure caused an increase in tau phosphorylation at the Tau-1 (Ser199/202), AT8 (Ser202/Thr205) and Ser396 sites. CWS-induced changes in tau phosphorylation at the Ser199/202 and Ser396, but not at Ser202/Thr205, were significantly attenuated by LiCl pretreatment. Total tau levels also showed a decided tendency to increase after CWS, which tendency was also countered by LiCl. Finally, we showed that CWS increased the active form of GSK-3β that was phosphorylated at Tyr216. These results suggest that a CWS-induced increase in phosphorylated tau in the hippocampus is mediated, at least partly, by the activation of GSK-3β.

Effects of combined treatment with eldecalcitol and alendronate on bone mass, mechanical properties, and bone histomorphometry in ovariectomized rats: A comparison with alfacalcidol and alendronate

Eldecalcitol (ELD), a 2β-hydroxypropyloxy derivative of 1α,25 (OH) 2D3, inhibits bone resorption more potently than alfacalcidol (ALF) while maintaining osteoblastic function in an ovariectomized (OVX) osteoporosis rat model. Alendronate (ALN), which is the most common bisphosphonate used for the treatment of osteoporosis, increases the bone mineral density (BMD) by suppressing bone resorption. In this study, we investigated the effects of combination treatments with ELD and ALN or with ALF and ALN on bone mass and strength in OVX rats. Seventy female rats, 32 weeks old, were assigned to seven groups: (1) a sham-operated control group; (2) an OVX-control group; (3) an ELD group; (4) an ALF group; (5) an ALN group; (6) an ELD+ALN group; and (7) an ALF+ALN group. OVX rats were orally treated with ELD (0.015 μg/kg), ALF (0.0375 μg/kg), or ALN (0.2mg/kg) daily for 12 weeks. In both the lumbar spine and the femur, ELD and ALF monotherapy significantly increased the BMD, and ELD+ALN and ALF+ALN significantly increased the BMD, compared with ALN monotherapy, as an additive effect. In particular, ELD+ALN resulted in a significantly higher BMD than ALF+ALN in the femur. On mechanical testing of the lumbar spine, ELD and ALF monotherapy significantly increased the ultimate load, and ELD+ALN and ALF+ALN significantly increased the ultimate load compared with ALN monotherapy. In the femur, ELD, ELD+ALN, and ALF+ALN treatment significantly increased the ultimate load, compared with the OVX-control group, and ELD+ALN resulted in a significantly higher ultimate load than ALN monotherapy. A histomorphometric analysis showed that ELD monotherapy and ELD+ALN combination therapy had a potent inhibitory effect on bone resorption parameters (osteoclast surface and eroded surface), while maintaining bone formation parameters (osteoblast surface and osteoid surface). By contrast, ALF and ALF+ALN significantly lowered the histological parameters of both bone resorption and formation. These results suggested that ELD or ALF used in combination with ALN has therapeutic advantages over ALN monotherapy, with ELD+ALN combination treatment producing an especially beneficial anti-osteoporotic effect by inhibiting osteoclastic bone resorption and maintaining osteoblastic function, compared with ALF+ALN combination treatment.

Effects of quercetin on the sleep–wake cycle in rats: Involvement of gamma-aminobutyric acid receptor type A in regulation of rapid eye movement sleep

The bioflavonoid quercetin is widely found in plants and exerts a large number of biological activities such as anti-hypertensive and anti-inflammatory properties. However, the effect of quercetin on the sleep-wake cycle has not been investigated. In the present study, we investigated the effect of quercetin on sleep-wake regulation. Intraperitoneal administration of quercetin (200mg/kg) significantly increased non-rapid eye movement (non-REM) sleep during dark period in rats, while it significantly decreased REM sleep. The decrease in REM sleep induced by quercetin was blocked by intracerebroventricular (i.c.v.) injection of bicuculline, a GABA(A) receptor antagonist. In contrast, the increase in non-REM sleep induced by quercetin was not affected by i.c.v. injection of bicuculline. Therefore, the present results suggest that quercetin alters the sleep-wake cycle partly through activation of GABA(A) receptors.

Analgesic Effect of the Newly Developed S(+)-Flurbiprofen Plaster on Inflammatory Pain in a Rat Adjuvant-Induced Arthritis Model

Preclinical Research

Sigma receptor modulation of the muscle relaxant action of eperisone

We examined the mechanism of the muscle relaxant action of eperisone, using Straub tail and binding studies. In vivo, eperisone (50 and 100 mg/kg i.p.) produced a dose-dependent inhibition of the Straub tail in mice and this inhibitory effect was significantly reversed by haloperidol (0.5 mg/kg i.p.). This drug in itself had no effect on the Straub tail. Sulpiride (50 mg/kg i.p.) failed to reverse the inhibitory effect of eperisone. In vitro, (+)-3-(3-[3H]hydroxyphenyl)-N-(1-propylpiperidine) ((+)-[3H]3-PPP) specific binding, in rat brain membrane, was prevented by eperisone and the IC50 value was 0.43 nM. The muscle relaxant action of eperisone may be modulated by sigma receptors.

Topical Anti-Inflammatory and Analgesic Effects of Multiple Applications of S(+)-Flurbiprofen Plaster (SFPP) in a Rat Adjuvant-Induced Arthritis Model

Preclinical Research

Analgesic effect of S (+)‐flurbiprofen plaster in a rat model of knee arthritis: analysis of gait and synovial fluid prostaglandin E2 levels

We developed S (+)‐flurbiprofen plaster (SFPP), a novel NSAID patch containing S (+)‐flurbiprofen (SFP), a potent cyclooxygenase (COX) inhibitor. The purpose of this study was to assess efficacy of SFPP by analysing its effect on the gait disturbance and measuring the prostaglandin E2 (PGE2) production in synovial fluid in a rat model of knee arthritis.

Binding of [3H]FH-510 to σ ligand recognition sites in guinea-pig brain membranes

Abstract We examined the characteristics of the binding of radiolabeled 5,8-dimethyl-4-(2-di-n-propylaminoethyl)carbazol monohydrochloride ([ 3 H]FH-510), a highly potent and selective σ ligand, to guinea-pig brain membranes. [ 3 H]FH-510 showed saturable and reversible binding to σ binding sites. The association rate constant (k +1 ) and dissociation rate constant (k −1 ) and dissociation (k −1 ) of [ 3 H]FH-510 were 0.23 min −1 ·nM −1 and 0.081 min −1 , respectively. Scatchard plot analysis showed a dissociation constant (K d and maximal number of binding sites (B max ) of 6.0 ± 0.63 nM and 1763.3 ± 177.4 fmol/mg protein (n = 7), respectively. The rank order of potency (K i ) of several structurally dissimilar σ ligands obtained for the displacement of [ 3 H]FH-510 binding was highly correlated with that determined for [ 3 H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ([ 3 H](+)-3-PPP) binding. The binding of [ 3 H]FH-510 was not influenced by histaminergic, dopaminergic, adrenergic, serotonergic or cholinergic agents at 10 −7 M. Higher [ 3 H]FH-510 binding to brain regions was observed in the cerebellum and pons-plus-medulla. Except for the nuclear fraction, the highest level of [ 3 H]FH-510 and [ 3 H](+)-3-PPP binding to subcellular fractions was observed in the microsomal fraction. From these results, it is suggested that FH-510 selectivity binds with high affinity to σ binding sites in guinea-pig membranes.