A novel micro-ECoG recording method for recording multisensory neural activity from the parietal to temporal cortices in mice.

Characterization of inter-regional interactions in brain is essential for understanding the mechanism relevant to normal brain function and neurological disease. The recently developed flexible micro (µ)-electrocorticography (µECoG) device is one prominent method used to examine large-scale cortical activity across multiple regions. The sheet-shaped µECoG electrodes arrays can be placed on a relatively wide area of cortical surface beneath the skull by inserting the device into the space between skull and brain. Although rats and mice are useful tools for neuroscience, current µECoG recording methods in these animals are limited to the parietal region of cerebral cortex. Recording cortical activity from the temporal region of cortex in mice has proven difficult because of surgical barriers created by the skull and surrounding temporalis muscle anatomy. Here, we developed a sheet-shaped 64-channel µECoG device that allows access to the mouse temporal cortex, and we determined the factor determining the appropriate bending stiffness for the µECoG electrode array. We also established a surgical technique to implant the electrode arrays into the epidural space over a wide area of cerebral cortex covering from the barrel field to olfactory (piriform) cortex, which is the deepest region of the cerebral cortex. Using histology and computed tomography (CT) images, we confirmed that the tip of the µECoG device reached to the most ventral part of cerebral cortex without causing noticeable damage to the brain surface. Moreover, the device simultaneously recorded somatosensory and odor stimulus-evoked neural activity from dorsal and ventral parts of cerebral cortex in awake and anesthetized mice. These data indicate that our µECoG device and surgical techniques enable the recording of large-scale cortical activity from the parietal to temporal cortex in mice, including somatosensory and olfactory cortices. This system will provide more opportunities for the investigation of physiological functions from wider areas of the mouse cerebral cortex than those currently available with existing ECoG techniques.

Evaluation of Human-Induced Pluripotent Stem Cells Derived from a Patient with Schwartz-Jampel Syndrome Revealed Distinct Hyperexcitability in the Skeletal Muscles.

Schwartz-Jampel syndrome (SJS) is an autosomal recessive disorder caused by loss-of-function mutations in heparan sulfate proteoglycan 2 (HSPG2), which encodes the core basement membrane protein perlecan. Myotonia is a major criterion for the diagnosis of SJS; however, its evaluation is based solely on physical examination and can be challenging in neonates and young children. Furthermore, the pathomechanism underlying SJS-related myotonia is not fully understood, and effective treatments for SJS are limited. Here, we established a cellular model of SJS using patient-derived human-induced pluripotent stem cells. This model exhibited hyper-responsiveness to acetylcholine as a result of abnormalities in the perlecan molecule, which were confirmed via comparison of their calcium imaging with calcium imaging of satellite cells derived from Hspg2-/--Tg mice, which exhibit myotonic symptoms similar to SJS symptoms. Therefore, our results confirm the utility of creating cellular models for investigating SJS and their application in evaluating myotonia in clinical cases, while also providing a useful tool for the future screening of SJS therapies.

Myocyte Culture with Decellularized Skeletal Muscle Sheet with Observable Interaction with the Extracellular Matrix.

In skeletal muscles, muscle fibers are highly organized and bundled within the basement membrane. Several microfabricated substrate models have failed to mimic the macrostructure of native muscle, including various extracellular matrix (ECM) proteins. Therefore, we developed and evaluated a system using decellularized muscle tissue and mouse myoblasts C2C12 to analyze the interaction between native ECM and myocytes. Chicken skeletal muscle was sliced into sheets and decellularized to prepare decellularized skeletal muscle sheets (DSMS). C2C12 was then seeded and differentiated on DSMS. Immunostaining for ECM molecules was performed to examine the relationship between myoblast adhesion status, myotube orientation, and collagen IV orientation. Myotube survival in long-term culture was confirmed by calcein staining. C2C12 myoblasts adhered to scaffolds in DSMS and developed adhesion plaques and filopodia. Furthermore, C2C12 myotubes showed orientation along the ECM orientation within DSMS. Compared to plastic dishes, detachment was less likely to occur on DSMS, and long-term incubation was possible. This culture technique reproduces a cell culture environment reflecting the properties of living skeletal muscle, thereby allowing studies on the interaction between the ECM and myocytes.

Estimation of economic loss by carcass weight reduction of Japanese dairy cows due to infection with bovine leukemia virus.

Bovine leukemia virus (BLV) infection is endemic in Japanese dairy farms. To promote the participation of farmers in BLV infection control in Japan, it is important to provide estimates of the economic losses caused by this infection. We hypothesized that decreased immune function due to BLV infection would increase visceral abnormalities, in turn reducing carcass weight. We employed mediation analysis to estimate the annual economic loss due to carcass weight reduction caused by BLV infection. Culled Holstein cows from 12 commercial dairy farms in the Nemuro and Kushiro regions of Hokkaido, Japan, were traced. Information on age and the last delivery day were collected. A non-infected culled cow was defined as a cow from which BLV provirus was not detected. A high-proviral-load (H-PVL) cow was defined as a cow whose PVL titer was above 2465 copies/50 ng DNA or 56,765 copies/105 cells. A BLV-infected cow with PVL titer lower than the thresholds was categorized as low-proviral load (L-PVL). Post-mortem examination results for culled cows were collected from a meat inspection center. The hypothesis was tested by three models, using data from 222 culled dairy cows. Model 1, a generalized linear mixed-effects model, selected carcass weight as an outcome variable, BLV status and the potential confounders (lactation stage and age) as explanatory variables, and herd as a random effect. Model 2 additionally included the number of abnormal findings in the post-mortem examination (AFPE) as an explanatory variable. Model 3 applied a Bayesian generalized linear mixed model, which employed a mediator separately modeled for AFPE, to estimate the amount of direct, indirect, and total carcass weight loss with adjustment for known confounding factors. Compared to the mean carcass weight for the non-infected culled cows, the carcass weight for H-PVL culled cows was significantly decreased by 30.4 kg on average. For each increase of one in the number of AFPE, the mean carcass weight was decreased by 8.6 kg. Only the indirect effect of BLV H-PVL status on carcass weight loss through AFPE was significant, accounting for 21.6 % of the total effect on carcass weight reduction. In 2017, 73,650 culled dairy cows were slaughtered in Hokkaido, and the economic loss due to carcass weight loss caused by BLV infection that year was estimated to be US $1,391,649. In summary, unlike L-PVL cows, H-PVL status was associated with carcass weight reduction, which was partially mediated by an increase in the number of visceral abnormalities.

Biogeochemical impacts of flooding discharge with high suspended sediment on coastal seas: a modeling study for a microtidal open bay.

Freshwater, suspended sediment matter (SSM), and nutrients discharged from rivers into the ocean have large impacts on biological production. In particular, during floods, coastal areas are greatly stirred up and large amounts of nutrients are supplied to the sea surface. We investigate the biogeochemical impact of flooding river discharges containing a large amount of SSM by conducting numerical simulations for a specific flooding event of the Yura River, Japan. Parameters are varied over wide ranges of SSM properties and nutrient content in riverine water. Two qualitatively different regimes of the riverine plume, hypopycnal and hyperpycnal, appear within realistic parameter ranges. Compared with the reference case without SSM, the surface salinity (nutrients) within the riverine plume becomes lower (higher) in hypopycnal cases and higher (lower) in hyperpycnal cases within a few days after the flooding discharge. These results suggest the necessity of properly taking into account the effect of SSM in assessing the influence of high river discharges on coastal biogeochemistry. It is the case not only for the specific river and event we are dealing with but also for other flooding events and other rivers and connecting coastal seas.

Perlecan Facilitates Neuronal Nitric Oxide Synthase Delocalization in Denervation-Induced Muscle Atrophy.

Perlecan is an extracellular matrix molecule anchored to the sarcolemma by a dystrophin-glycoprotein complex. Perlecan-deficient mice are tolerant to muscle atrophy, suggesting that perlecan negatively regulates mechanical stress-dependent skeletal muscle mass. Delocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma to the cytosol triggers protein degradation, thereby initiating skeletal muscle atrophy. We hypothesized that perlecan regulates nNOS delocalization and activates protein degradation during this process. To determine the role of perlecan in nNOS-mediated mechanotransduction, we used sciatic nerve transection as a denervation model of gastrocnemius muscles. Gastrocnemius muscle atrophy was significantly lower in perinatal lethality-rescued perlecan-knockout (Hspg2-/--Tg) mice than controls (WT-Tg) on days 4 and 14 following surgery. Immunofluorescence microscopy showed that cell membrane nNOS expression was reduced by denervation in WT-Tg mice, with marginal effects in Hspg2-/--Tg mice. Moreover, levels of atrophy-related proteins-i.e., FoxO1a, FoxO3a, atrogin-1, and Lys48-polyubiquitinated proteins-increased in the denervated muscles of WT-Tg mice but not in Hspg2-/--Tg mice. These findings suggest that during denervation, perlecan promotes nNOS delocalization from the membrane and stimulates protein degradation and muscle atrophy by activating FoxO signaling and the ubiquitin-proteasome system.

Perlecan, a heparan sulfate proteoglycan, regulates systemic metabolism with dynamic changes in adipose tissue and skeletal muscle.

Perlecan (HSPG2), a heparan sulfate proteoglycan, is a component of basement membranes and participates in a variety of biological activities. Here, we show physiological roles of perlecan in both obesity and the onset of metabolic syndrome. The perinatal lethality-rescued perlecan knockout (Hspg2-/--Tg) mice showed a smaller mass and cell size of white adipose tissues than control (WT-Tg) mice. Abnormal lipid deposition, such as fatty liver, was not detected in the Hspg2-/--Tg mice, and those mice also consumed more fat as an energy source, likely due to their activated fatty acid oxidation. In addition, the Hspg2-/--Tg mice demonstrated increased insulin sensitivity. Molecular analysis revealed the significantly relatively increased amount of the muscle fiber type IIA (X) isoform and a larger quantity of mitochondria in the skeletal muscle of Hspg2-/--Tg mice. Furthermore, the perlecan-deficient skeletal muscle also had elevated levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) protein. PGC1α expression is activated by exercise, and induces mitochondrial biosynthesis. Thus, perlecan may act as a mechano-regulator of catabolism of both lipids and glucose by shifting the muscle fiber composition to oxidative fibers. Our data suggest that downregulation of perlecan is a promising strategy to control metabolic syndrome.

Electrically evoked local muscle contractions cause an increase in hippocampal BDNF.

High-intensity exercise has recently been shown to cause an increase in brain-derived neurotropic factor (BDNF) in the hippocampus. Some studies have suggested that myokines secreted from contracting skeletal muscle, such as irisin (one of the truncated form of fibronectin type III domain-containing protein 5 (FNDC5)), play important roles in this process. Thus, we hypothesized that locally evoked muscle contractions may cause an increase of BDNF in the hippocampus through some afferent mechanisms. Under anesthesia, Sprague-Dawley rats were fixed on a custom-made dynamometer and their triceps surae muscles were made to maximally contract via delivery of electric stimulations of the sciatic nerve (100 Hz with 1-ms pulse and 3-s duration). Following 50 repeated maximal isometric contractions, the protein expressions of BDNF and activation of its receptor in the hippocampus significantly increased compared with the sham-operated control rats. However, the expression of both BDNF and FNDC5 within stimulated muscles did not significantly increase, nor did their serum concentrations change. These results indicate that local muscular contractions under unconsciousness can induce BDNF expression in the hippocampus. This effect may be mediated by peripheral reception of muscle contraction, but not by systemic factors.

Postexercise blood flow restriction does not enhance muscle hypertrophy induced by multiple-set high-load resistance exercise.

To test the applicability of postexercise blood flow restriction (PEBFR) in practical training programmes, we investigated whether PEBFR enhances muscle hypertrophy induced by multiple-set high-load resistance exercise (RE). Seven men completed an eight-week RE programme for knee extensor muscles. Employing a within-subject design, one leg was subjected to RE + PEBFR, whereas contralateral leg to RE only. On each exercise session, participants performed three sets of unilateral knee extension exercise at approximately 70% of their one-repetition maximum for RE leg first, and then performed three sets for RE + PEBFR leg. Immediately after completion of the third set, the proximal portion of the RE + PEBFR leg was compressed with an air-pressure cuff for 5 min at a pressure ranging from 100 to 150 mmHg. If participants could perform 10 repetitions for three sets in two consecutive exercise sessions, the work load was increased by 5% at the next exercise session. Muscle thickness and strength of knee extensor muscles were measured before and after the eight-week training period and after the subsequent eight-week detraining period. There was a main effect of time but no condition × time interaction or main effect of condition for muscle thickness and strength. Both muscle thickness and strength increased after the training period independent of the condition. This result suggests that PEBFR would not be an effective training method at least in an early phase of adaptation to high-load resistance exercise.

Blood lactate concentration as diagnostic predictors of uterine necrosis and its outcome in dairy cows with uterine torsion.

In order to determine blood lactate concentrations (bLac) and their validity as a diagnostic marker in bovine uterine torsion, blood samples were taken from 54 Holstein cows with uterine torsion before the correction of torsion. bLac in a group of cows with and without uterine necrosis were 15.0 and 3.0 mmol/l, respectively (P<0.01). Moreover, bLac in a group of dead or culled dams and in that of survived dams were 10.2 and 3.1 mmol/l, respectively (P<0.01). Furthermore, the proposed diagnostic cutoffs for bLac based on ROC analysis for detection of uterine necrosis and poor prognosis in dams were set at >5.0 and >6.5 mmol/l, respectively. These findings suggest that in dairy cows with uterine torsion, an increase in bLac is a diagnostic predictor of uterine necrosis as well as poor prognosis in dams.

Correlation between Ribosome Biogenesis and the Magnitude of Hypertrophy in Overloaded Skeletal Muscle.

External loads applied to skeletal muscle cause increases in the protein translation rate, which leads to muscle hypertrophy. Although some studies have demonstrated that increases in the capacity and efficiency of translation are involved in this process, it remains unclear how these two factors are related to the magnitude of muscle hypertrophy. The present study aimed to clarify the roles played by the capacity and efficiency of translation in muscle hypertrophy. We used an improved synergist ablation in which the magnitude of compensatory hypertrophy could be controlled by partial removal of synergist muscles. Male rats were assigned to four groups in which the plantaris muscle was unilaterally subjected to weak (WK), moderate (MO), middle (MI), and strong (ST) overloading by four types of synergist ablation. Fourteen days after surgery, the weight of the plantaris muscle per body weight increased by 8%, 22%, 32% and 45%, in the WK, MO, MI and ST groups, respectively. Five days after surgery, 18+28S rRNA content (an indicator of translational capacity) increased with increasing overload, with increases of 1.8-fold (MO), 2.2-fold (MI), and 2.5-fold (ST), respectively, relative to non-overloaded muscle (NL) in the WK group. rRNA content showed a strong correlation with relative muscle weight measured 14 days after surgery (r = 0.98). The phosphorylated form of p70S6K (a positive regulator of translational efficiency) showed a marked increase in the MO group, but no further increase was observed with further increase in overload (increases of 22.6-fold (MO), 17.4-fold (MI), and 18.2-fold (ST), respectively, relative to NL in the WK group). These results indicate that increases in ribosome biogenesis at the early phase of overloading are strongly dependent on the amount of overloading, and may play an important role in increasing the translational capacity for further gain of muscular size.

Groundwater dynamics of Fongafale Islet, Funafuti Atoll, Tuvalu.

Geoelectric and hydrologic surveys during spring tides revealed the spatiotemporal distribution of groundwater quality produced by tidal forcing in Fongafale Islet, Funafuti Atoll, Tuvalu. The observed low resistivity showed that saline water largely immersed the surficial Holocene aquifer, indicating that there is no thick freshwater lens in Fongafale Islet, unlike in other atoll islands of comparable size. Half of the islet was constructed by reclaiming the original swamp with porous, highly permeable coral blocks; this reclaimed area should not be considered as part of the islet width for calculation of the expected thickness of the freshwater lens. The degree of aquifer salinization depends on the topographic characteristics and the hydrologic controls on the inland propagation of the tidal forcing. Large changes in bulk resistivity and the electrical conductivity of groundwater from wells indicate that periodic salinization in phase with the semidiurnal tides was occurring widely, especially in areas at lower elevation than the high-tide level and in reclaimed areas with high permeability. Thin sheets of nearly fresh and brackish water were observed in the surficial aquifer in areas above the high-tide level and in taro swamps, respectively. The thinness of the brackish and freshwater sheets suggests that the taro swamps and the fresh groundwater resources of the islet are highly vulnerable to salinization from anticipated sea-level rise. An understanding of the inherent geologic and topographic features of an atoll is necessary to evaluate the groundwater resources of the atoll and assess the vulnerability of its water resources to climate change.