Long-distance polarizing microscope system combined with solenoid-type magnet for microscopy and simultaneous measurement of physical parameters.

We have developed a long-distance polarizing microscope system combined with a solenoid-type superconducting magnet. By inserting an infinity-corrected objective lens into the magnet, direct or polarizing microscope images are observed in magnetic fields of up to 12 T at various temperatures down to 2 K. Through magneto-optical measurements in the transmission geometry, the local magnetization process of a transparent magnet is evaluated in areas of 10 × 10 µm2. This system enables simultaneous measurements of other physical properties over a wide range of temperatures and magnetic fields. The basic principle of the proposed long-distance microscopy can be applied to imaging experiments in various research fields, particularly biology and chemistry.

Mice with cleavage-resistant N-cadherin exhibit synapse anomaly in the hippocampus and outperformance in spatial learning tasks.

N-cadherin is a homophilic cell adhesion molecule that stabilizes excitatory synapses, by connecting pre- and post-synaptic termini. Upon NMDA receptor (NMDAR) activation by glutamate, membrane-proximal domains of N-cadherin are cleaved serially by a-disintegrin-and-metalloprotease 10 (ADAM10) and then presenilin 1(PS1, catalytic subunit of the γ-secretase complex). To assess the physiological significance of the initial N-cadherin cleavage, we engineer the mouse genome to create a knock-in allele with tandem missense mutations in the mouse N-cadherin/Cadherin-2 gene (Cdh2 R714G, I715D, or GD) that confers resistance on proteolysis by ADAM10 (GD mice). GD mice showed a better performance in the radial maze test, with significantly less revisiting errors after intervals of 30 and 300 s than WT, and a tendency for enhanced freezing in fear conditioning. Interestingly, GD mice reveal higher complexity in the tufts of thorny excrescence in the CA3 region of the hippocampus. Fine morphometry with serial section transmission electron microscopy (ssTEM) and three-dimensional (3D) reconstruction reveals significantly higher synaptic density, significantly smaller PSD area, and normal dendritic spine volume in GD mice. This knock-in mouse has provided in vivo evidence that ADAM10-mediated cleavage is a critical step in N-cadherin shedding and degradation and involved in the structure and function of glutamatergic synapses, which affect the memory function.

Attenuation of pain behaviour by local administration of alpha-2 adrenoceptor antagonists to dorsal root ganglia in a rat radiculopathy model.

BACKGROUND: There were several reports suggesting α-adrenoceptor antagonists are effective to treat neuropathic pain. The aims of this study were as follows: (1) to introduce drug delivery system for dorsal root ganglion (DRG) neurons; (2) to elucidate the effects of α-adrenoceptor antagonists in acute, subacute or chronic phase and (3) to determine which subtype of adrenoceptor was mainly involved. METHOD: We used 130 male Sprague-Dawley rats. After root constriction, rats received three local injections of α-adrenoceptor antagonists around DRG. We administered the non-selective α-adrenoceptor antagonist phentolamine for 3 consecutive days from day 0, 4 or 11 after the surgery, and the α1-adrenoceptor antagonist prazosin, the α1-adrenoceptor antagonist silodosin, the more preferred α1-adrenoceptor than prazosin and the α2-adrenoceptor antagonist yohimbine for 3 consecutive days from day 0 after the surgery. RESULTS: Phentolamine and yohimbine continually attenuated pain behaviour. Prazosin at high dose attenuated pain behaviour, however, prazosin at low dose did not attenuate pain behaviour every experimental day. Silodosin had no analgesic effect. Phentolamine injections from day 4 after surgery attenuated pain behaviour that had been established on the 3rd experimental day until the 28th post-operative day, although effect of phentolamine wore off. Phentolamine injections from day 11 after surgery temporarily attenuated pain behaviour that had been established on the 3rd, 7th and 10th experimental days. CONCLUSIONS: This study showed α-adrenoceptor antagonists could suppress pain behaviour via α2-adrenoceptor in acute phase and temporary attenuate pain behaviour in chronic phase. These findings presented potentials sympathetic nerve blockade contributed to treat neuropathic pain.

Detection of an immature dentate gyrus feature in human schizophrenia/bipolar patients.

Hippocampus-associated cognitive impairments are a common, highly conserved symptom of both schizophrenia (SCZ) and bipolar disorder (BPD). Although the hippocampus is likely an impacted region in SCZ/BPD patients, the molecular and cellular underpinnings of these impairments are difficult to identify. An emerging class of mouse models for these psychiatric diseases display similar cognitive impairments to those observed in human patients. The hippocampi of these mice possess a conserved pathophysiological alteration; we term the 'immature dentate gyrus' (iDG), characterized by increased numbers of calretinin-positive immature neuronal progenitors, a dearth of calbindin-positive mature neurons and (often) constitutively increased neurogenesis. Although these models provide a link between cellular dysfunction and behavioral alteration, limited translational validity exists linking the iDG to human pathophysiology. In this study, we report the initial identification of an iDG-like phenotype in the hippocampi of human SCZ/BPD patients. These findings suggest a new motif for the etiology of these diseases and link an emerging class of mouse models to the human disease condition.

Sympathectomy attenuates excitability of dorsal root ganglion neurons and pain behaviour in a lumbar radiculopathy model.

OBJECTIVES: In order to elucidate the influence of sympathetic nerves on lumbar radiculopathy, we investigated whether sympathectomy attenuated pain behaviour and altered the electrical properties of the dorsal root ganglion (DRG) neurons in a rat model of lumbar root constriction. METHODS: Sprague-Dawley rats were divided into three experimental groups. In the root constriction group, the left L5 spinal nerve root was ligated proximal to the DRG as a lumbar radiculopathy model. In the root constriction + sympathectomy group, sympathectomy was performed after the root constriction procedure. In the control group, no procedures were performed. In order to evaluate the pain relief effect of sympathectomy, behavioural analysis using mechanical and thermal stimulation was performed. In order to evaluate the excitability of the DRG neurons, we recorded action potentials of the isolated single DRG neuron by the whole-cell patch-clamp method. RESULTS: In behavioural analysis, sympathectomy attenuated the mechanical allodynia and thermal hyperalgesia caused by lumbar root constriction. In electrophysiological analysis, single isolated DRG neurons with root constriction exhibited lower threshold current, more depolarised resting membrane potential, prolonged action potential duration, and more depolarisation frequency. These hyperexcitable alterations caused by root constriction were significantly attenuated in rats treated with surgical sympathectomy. CONCLUSION: The present results suggest that sympathectomy attenuates lumbar radicular pain resulting from root constriction by altering the electrical property of the DRG neuron itself. Thus, the sympathetic nervous system was closely associated with lumbar radicular pain, and suppressing the activity of the sympathetic nervous system may therefore lead to pain relief.

An increased dose of insulin detemir improves glycaemic control and reduces body weight of Japanese patients with diabetes.

AIMS: The aim of study was to evaluate the safety and efficacy of insulin detemir as a basal insulin switching from neutral protamine Hagedorn insulin (NPH) and insulin glargine in patients with diabetes on an intensive insulin therapy regimen. METHODS: This 6-month multicentre, prospective, treat-to-target [glycosylated haemoglobin (HbA(1c) ) less than 6.5%] trial included 92 people with diabetes (61 type 1, 29 type 2 and two unknown diabetes types). Detemir was administered first with fixed dose and injection times and then adapted to optimal dose after 3 months. RESULTS: Mean HbA(1c) (%) of all the subjects at months 4 to 6 of the study was improved compared with month 0 (7.34 ± 0.87, 7.28 ± 0.88, 7.25 ± 0.93 vs. 7.55 ± 1.18; p < 0.05 paired t-test). However, significant improvement was seen only among the patients who had previously used NPH as a basal insulin. Twice-daily injection of basal insulin increased among people in the type 1 previously injected insulin glargine. Total insulin dose increased in the type 1 glargine group. The mean body weight change in the highest quartile body mass index (BMI) group was from 70.7 to 69.3 kg over the 6 months. Quality of life (QoL) relating to the patients' glycaemic control tended to improve without a change in frequency of hypoglycaemia. CONCLUSIONS: The results suggest that insulin detemir has a greater effect on glycaemic control in subjects with poor glycaemic control using NPH; can reduce or maintain body weight in obese patients; and obtains perceptive stability for patients with unstable glycaemic control.

Comprehensive behavioural study of GluR4 knockout mice: implication in cognitive function.

Fast excitatory transmission in the mammalian central nervous system is mediated by AMPA-type glutamate receptors. The tetrameric AMPA receptor complexes are composed of four subunits, GluR1-4. The GluR4 subunit is highly expressed in the cerebellum and the early postnatal hippocampus and is thought to be involved in synaptic plasticity and the development of functional neural circuitry through the recruitment of other AMPA receptor subunits. Previously, we reported an association of the human GluR4 gene (GRIA4) with schizophrenia. To examine the role of the GluR4 subunit in the higher brain function, we generated GluR4 knockout mice and conducted electrophysiological and behavioural analyses. The mutant mice showed normal long-term potentiation (LTP) in the CA1 region of the hippocampus. The GluR4 knockout mice showed mildly improved spatial working memory in the T-maze test. Although the retention of spatial reference memory was intact in the mutant mice, the acquisition of spatial reference memory was impaired in the Barnes circular maze test. The GluR4 knockout mice showed impaired prepulse inhibition. These results suggest the involvement of the GluR4 subunit in cognitive function.

Autism spectrum disorder is related to endoplasmic reticulum stress induced by mutations in the synaptic cell adhesion molecule, CADM1.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with an unknown molecular pathogenesis. A recent molecular focus has been the mutated neuroligin 3, neuroligin 3(R451C), in gain-of-function studies and for its role in induced impairment of synaptic function, but endoplasmic reticulum (ER) stress induced by mutated molecules also deserves investigation. We previously found two missense mutations, H246N and Y251S, in the gene-encoding synaptic cell adhesion molecule-1 (CADM1) in ASD patients, including cleavage of the mutated CADM1 and its intracellular accumulation. In this study, we found that the mutated CADM1 showed slightly reduced homophilic interactions in vitro but that most of its interactions persist. The mutated CADM1 also showed morphological abnormalities, including shorter dendrites, and impaired synaptogenesis in neurons. Wild-type CADM1 was partly localized to the ER of C2C5 cells, whereas mutated CADM1 mainly accumulated in the ER despite different sensitivities toward 4-phenyl butyric acid with chemical chaperone activity and rapamycin with promotion activity for degradation of the aggregated protein. Modeling analysis suggested a direct relationship between the mutations and the conformation alteration. Both mutated CADM1 and neuroligin 3(R451C) induced upregulation of C/EBP-homologous protein (CHOP), an ER stress marker, suggesting that in addition to the trafficking impairment, this CHOP upregulation may also be involved in ASD pathogenesis.

Reproducibility of a method for analysis of morphological changes in perioral soft tissue in children using video cameras.

The purpose of the present study was to determine the appropriate reference facial expression and to examine the reproducibility of our system using two commercially available video cameras in consideration of functional diagnosis of perioral muscles. The following items were investigated: 1) influence of forward and backward movements and the experiment sequence from measurement to analysis, 2) usefulness of head fixation by a restraining device during video recording for evaluation of morphological changes in facial expression, 3) conditions for establishment of reference points for determination of movements of perioral soft tissue, and 4) reproducibility of perioral soft tissue movements as assessed by changes in landmarks. The results of this study showed that accurate static and dynamic evaluation of perioral soft tissue is possible by using our method of video recording with the subject's head restrained and our method of analysis. The results also showed that centric occlusion of the most appropriate reference facial expression as for measurement of perioral soft tissue movements and that protrusion of the lips and lateral extension of the angel of the mouth are facial expressions with high degrees of reproducibility.

Simulation of exercise-dependent difference in metabolism with a mathematical model for analyses of measurements using near-infrared spectroscopy.

Near-infrared spectroscopy (NIRS) is a useful technique for noninvasive measurement of muscle oxygenation. However, analyses of the dynamic changes in muscle metabolism based only on experimental observations of NIRS are difficult. Therefore, we constructed a mathematical model of muscle metabolism, comprising of the ATP synthesizing systems and O2 diffusion system, to identify the mechanisms responsible for those observations. A customized NIRS instrument was used to measure the changes in muscle oxygenation of the forearm flexor muscles during intermittent and continuous isometric flexion exercises when healthy male subjects participated in exercises tests. The exercise-dependent difference in changes could be distinguished and the simulated results agreed well with that measured experimentally. Although the contraction intensity for both exercises was identical, the magnitude of energy needed to perform the respective exercises was different. This difference was reflected by the changes in the ATP synthesizing systems, in which the energy needed during the latter-half of continuous exercise was mostly supplied by anaerobic system, whereas that during intermittent exercise was supplied by the aerobic and anaerobic systems that operated synergistically. From the results, we conclude that the model could be a useful tool for the elucidation of the relationship between experimental observations of NIRS and muscle metabolism.

Imaging of Regional Differences of Muscle Oxygenation during Exercise Using Spatially Resolved NIRS.

The development of imaging systems using near-infrared spectroscopy (NIRS) has enabled noninvasive measurement of regional changes in muscle oxygenation. A spatially resolved NIRS (SR-NIRS) imaging instrument was utilized for real-time measurement of spatial-temporal changes in muscle oxygenation during exercises. Changes in muscle oxygenation and localized O2consumption in the quadriceps muscle were measured during sustained isometric knee extension without and with leg-press to the ground exercises. In the former exercise, the level of tissue oxygen saturation (TOS) of the rectus femoris (RF) muscle was found to be lower than that of vastus lateralis (VL) and vastus medialis (VM) muscles. The highest localized O2consumption rate, VO2, reflecting the localization in distribution of muscle metabolism, was detected in the RF muscle at the initial stage of exercise. As exercise progressed, VO2of the RF muscle decreased whereas that of the VL and VM muscles increased. In contrast, TOS decreased to about the same level for the VL, RF and VM muscles in the latter exercise. Also, VO2of all three muscles decreased as exercise progressed. Initial results demonstrated that the SR-NIRS instrument enables measurement of regional differences in muscle oxygenation in the quadriceps muscle during different exercises.

Squamous metaplasia of Paget's disease.

A patient had triple extramammary Paget's disease of both axillary and genital regions. Right inguinal lymphadenopathy was found 1 year after excision of all the skin lesions. Excisional biopsy of the lymph node demonstrated a mixture of Paget cells and atypical squamoid cells with horn pearls suggestive of keratinization. The squamoid cells were positive for cytokeratin 10, a marker of suprabasal epidermis, and also positive for laminin gamma2 which is often expressed in invasive squamous cell carcinoma. The coexistence of these different cells within the same tumour island suggested that the squamoid cells derived from metaplasia of Paget cells.

M1-M5 muscarinic receptor knockout mice as novel tools to study the physiological roles of the muscarinic cholinergic system.

A large body of evidence indicates that muscarinic acetylcholine receptors (mAChRs) play critical roles in regulating the activity of many important functions of the central and peripheral nervous systems. However, identification of the physiological and pathophysiological roles of the individual mAChR subtypes (M(1)-M(5)) has proven a difficult task, primarily due to the lack of ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues and organs express multiple mAChRs. To circumvent these difficulties, we used gene targeting technology to generate mutant mouse lines containing inactivating mutations of the M(1)-M(5) mAChR genes. The different mAChR mutant mice and the corresponding wild-type control animals were subjected to a battery of physiological, pharmacological, behavioral, biochemical, and neurochemical tests. The M(1)-M(5) mAChR mutant mice were viable and reproduced normally. However, each mutant line displayed specific functional deficits, suggesting that each mAChR subtype mediates distinct physiological functions. These results should offer new perspectives for the rational development of novel muscarinic drugs.

Forebrain-specific calcineurin knockout selectively impairs bidirectional synaptic plasticity and working/episodic-like memory.

Calcineurin is a calcium-dependent protein phosphatase that has been implicated in various aspects of synaptic plasticity. By using conditional gene-targeting techniques, we created mice in which calcineurin activity is disrupted specifically in the adult forebrain. At hippocampal Schaffer collateral-CA1 synapses, LTD was significantly diminished, and there was a significant shift in the LTD/LTP modification threshold in mutant mice. Strikingly, although performance was normal in hippocampus-dependent reference memory tasks, including contextual fear conditioning and the Morris water maze, the mutant mice were impaired in hippocampus-dependent working and episodic-like memory tasks, including the delayed matching-to-place task and the radial maze task. Our results define a critical role for calcineurin in bidirectional synaptic plasticity and suggest a novel mechanistic distinction between working/episodic-like memory and reference memory.

PKC1, a protein kinase C homologue of Saccharomyces cerevisiae, participates in microtubule function through the yeast EB1 homologue, BIM1.

BACKGROUND: RSC is a chromatin-remodelling complex of Saccharomyces cerevisiae and essential for growth. Its catalytic subunit is encoded by the NPS1/STH1 gene. At the present time, little is known regarding the cellular function of RSC. RESULTS: To identify genes with functions related to NPS1, we screened high-copy suppressor genes for the temperature- and thiabendazole (TBZ)-sensitive mutant allele of NPS1, nps1-105. Amongst the suppressors we cloned PKC1/STT1 and BIM1 that encoded a homologue of mammalian protein kinase C and a conserved microtubule binding protein homologous to human EB1, respectively. Both the temperature sensitive mutation of PKC1, stt1, and the bim1 null mutation caused synthetic growth defects with nps1-105. A genetic analysis of the functional relationships between these genes revealed that PKC1 suppressed the defect of nps1-105 through the BIM1 function but not by the activation of the MPK1/MAPK pathway. The stt1 mutation alone showed TBZ sensitivity and delayed the G2-phase progression at semi-permissive temperatures. Both of these stt1 phenotypes were suppressed by the over-expression of BIM1. In addition, stt1 as well as nps1-105, mis-segregated a mini-chromosome at frequencies higher than the wild-type at a permissive temperature. The mis-segregation was enhanced in the nps1-105 stt1 double mutant. CONCLUSION: These results suggest that Pkc1p plays a role which is relevant to microtubule functions and that this role is mediated by a hitherto unknown PKC signalling pathway and by Bim1p

Differences between the tolerance characteristics of two anticonvulsant benzodiazepines in the amygdaloid-kindled rat.

The characteristics of the development of tolerance to the anticonvulsant effects of chronic treatment by dipotassium clorazepate and diazepam using amygdaloid-kindled rats were investigated. Dipotassium clorazepate (5 mg/kg) or diazepam (5 mg/kg) were intraperitoneally administered for 10 consecutive days. Tolerance to the anticonvulsant effect of dipotassium clorazepate developed in seizure stage on day 6, after-discharge duration on day 7 and seizure latency on day 4. In contrast, tolerance to the effects of diazepam developed more rapidly in seizure stage on day 4, after-discharge duration on day 4 and seizure latency on day 3. Thus tolerance to the anticonvulsive effect of dipotassium clorazepate developed relatively slower than that to diazepam. All rats had stage 5 convulsions 24 hr after cessation of the administration of dipotassium clorazepate and diazepam. Concomitant determinations of plasma concentrations of the main metabolite of dipotassium clorazepate and diazepam, desmethyldiazepam, showed no statistical difference during treatment, suggesting that the developed tolerance was not metabolic but functional.

Repression of rRNA synthesis due to a secretory defect requires the C-terminal silencing domain of Rap1p in Saccharomyces cerevisiae.

A secretory defect causes specific transcriptional repression of both ribosomal protein and ribosomal RNA genes, suggesting the coupling of plasma membrane and ribosome syntheses. We previously reported that the rap1-17 allele, which produced C-terminally truncated Rap1p, derepressed transcription of ribosomal protein genes when the secretory pathway was blocked. In this paper, we demonstrate that the rap1-17 mutation also leads to significant attenuation of transcriptional repression of rRNA genes due to a secretory defect. In contrast, the rap1-2 temperature-sensitive allele containing a unique missense mutation in the middle of the coding sequence has only a weak effect on repression. These results suggest that the C-terminal silencing domain of Rap1p is required for transcriptional repression of rDNA in response to a secretory defect. We also demonstrated that transcriptional regulation of ribosomal protein genes in response to nitrogen limitation was not affected by the rap1-17 allele, suggesting that the mechanism of nitrogen response is distinct from that of the secretory response.