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The interplay of neural discharge patterns involved in “respiration”, “circulation”, “opening movements in the mandible”, and “locomotion” was investigated electrophysiologically in a decerebrate and arterially perfused in situ rat preparation. Sympathetic tone increased with increases in perfusion flow rate. All nerve discharges became clearly organized into discharge episodes of increasing frequency and duration punctuated by quiescent periods as the perfusion flow rate increased at 26ºC. The modulated sympathetic tone at 10× total blood volume/ min activated the forelimb pattern generator and spontaneously generated fictive forelimb movement during discharge episodes. The coupling rhythm of respiration and locomotion during motion occurred at frequency ratios ranges of 1:2 and 1:3. Small increases in systemic pressure were always generated after the initiation of motion. Opening movements in the mandible, occurring during the inspiratory phase at all tested flow rates, were generated in both the inspiratory and expiratory phases during motion. Although the central mechanism for the entrainment of respiratory and locomotor rhythms has not been identified, a spinal-feedback mechanism generating fictive locomotion in the upper spinal cord contributed to generating the opening movement in the mandible in the expiratory phase during motion. The existence of this mechanism implies that there is a reciprocal functional interaction between the brainstem and the spinal cord, whereby the intake and output of air by the lungs is efficiently improved during movement by both nasal and mouth breathing. These results suggest that this reciprocal functional interaction plays an important role in increasing oxygenated blood flow during locomotion.

Abstract: Gas-phase ion/molecule reactions in tetrafluoroethylene (C2F4) were studied with a pulsed electron beam mass spectrometer. When a few Torr of major gas, N2, containing 1–10mTorr C2F4 was ionized by 2keV electrons, CF3 + and C2F4 + were formed as major ions. CF3 + reacted with C2F4 rapidly to form C3F7 +. The cluster ion C3F7 +(C2F4) started to appear below ∼280K. In the clustering reactions of C3F7 + with C2F4, the equilibria could not be observed but the cluster ions C3F7 +(C2F4) n were found to grow very slowly at the expense of the smaller cluster ions C3F7 +(C2F4) n−1. The rate constant of reaction C2F4 + +C2F4 =C3F5 + +CF3 was measured to be (1.5±0.5)×10−11 cm3/molecules and found to be temperature-independent in the range of 310–180K. The thermochemical stabilities of X−(C2F4) n (X− =F−, Cl− and Br−) were measured. A peculiar behavior in reaction of F− with C2F4 was observed. Two isomers for F−(C2F4) were predicted. [Copyright &y& Elsevier]

Background: Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a disease responsible for cognitive impairment in adult humans. It is caused by mutations in the colony stimulating factor 1 receptor gene (CSF1R) or alanyl-transfer (t) RNA synthetase 2 (AARS2) gene and affects brain white matter. Settlement of stages of the pathological brain lesions (Oyanagi et al. 2017) from the findings of brain imaging will be inevitably essential for prognostication., Methods: MRI images of eight patients with ALSP were analyzed semiquantitatively. White matter degeneration was assessed on a scale of 0 to 4 (none, patchy, large patchy, confluent, and diffuse) at six anatomical points, and brain atrophy on a scale 0 to 4 (none, slight, mild, moderate, and severe) in four anatomical areas. The scores of the two assessments were then summed to give total MRI scores of 0-40 points. Based on the scores, the MRI features were classified as Grades (0-4). Regression analysis was applied to mutual association between mRS, white matter degeneration score, brain atrophy score, the total MRI score and disease duration., Results: White matter degeneration score, brain atrophy score, and the total MRI score were significantly correlated with the disease duration. MRI Grades (2-4) based on the total MRI scores and the features of the images were well correlated with the pathological lesion stages (II - IV); i.e., 'large patchy' white matter degeneration in the frontal and parietal lobes (MRI Grade 2) corresponded to pathological Stage II, 'confluent' degeneration (Grade 3) to Stage III, and 'diffuse' degeneration (Grade 4) to Stage IV., Conclusion: MRI Grades (2-4) resulted from the total MRI scores were well correlated with the pathological lesion Stages (II - IV)., Competing Interests: Declaration of competing interest The authors declare the following financial interests which may be considered as potential competing interests: Profs. Mitsunori Yamada and Kiyomitsu Oyanagi belong to the Department (Division of Neuropathology, Department of Brain Disease Research, Shinshu University School of Medicine) for Co-operative Research financed by Alnylam Pharmaceuticals, Inc. However, they have no direct conflicts of interests on the research. The other authors have no conflicts of interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To date, research on the role of the brainstem and spinal cord in motor behavior has relied on in vitro preparations of the neonatal rodent spinal cord, with or without the brainstem; their spatial and temporal scope are subject to technical limitations imposed by low oxygen tension in deep tissues. Therefore, we created an arterially perfused in situ preparation that allowed us to investigate functional interactions in the CNS from the neonatal to adult period. Decerebrated rodents were kept alive via total artificial cardiopulmonary bypass for extracorporeal circulation; the plasma oxygen and ion components needed for survival were supplied through the blood vessels. Interferon regulatory factor 8 (IRF8) is a transcription factor that promotes myeloid cell development and stimulates innate immune responses. In the brain, IRF8 is expressed only in microglia and directs the expression of many genes that serve microglial functions. Recent evidence indicates that IRF8 affects behavior and modulates Alzheimer's disease progression in a mouse model. However, whether this immune deficiency arising from the absence of IRF8 influences the development of the neuronal network in the spinal cord is unknown. We applied the above methodology to mice of all ages and electrophysiologically explored whether the absence of IRF8 influences the development of lumbar central pattern generator (CPG) networks. In mice of all ages, bilateral neuronal discharges by the normal CPG networks activated by the modulated sympathetic tone via descending pathways at high flow rates became organized into discharge episodes punctuated by periods of quiescence. Similar discharge episodes were generated by the adult CPG networks (≥P14 days) activated by drug application. However, discharge episodes elicited by activating the neonatal-juvenile CPG networks (

Proteinase-activated receptor-1 (PAR1) is expressed in astrocytes of various brain regions, and its activation is involved in the modulation of neuronal activity. Here, we report effects of PAR1 selective agonist TFLLR on respiratory rhythm generation in brainstem-spinal cord preparations. Preparations were isolated from newborn rats (P0-P4) under deep isoflurane anesthesia and were transversely cut at the rostral medulla. Preparations were superfused with artificial cerebrospinal fluid (25-26 °C), and inspiratory C4 ventral root activity was monitored. The responses to TFLLR of cells close to the cut surface were detected by calcium imaging or membrane potential recordings. Application of 10 μM TFLLR (4 min) induced a rapid and transient increase of calcium signal in cells of the ventrolateral respiratory regions of the medulla. More than 88% of responding cells (223/254 cells from 13 preparations) were also activated by low (0.2 mM) K + solution, suggesting that they were astrocytes. Immunohistochemical examination demonstrated that PAR1 was expressed on many astrocytes. Respiratory-related neurons in the medulla were transiently hyperpolarized (-1.8 mV) during 10 μM TFLLR application, followed by weak membrane depolarization after washout. C4 burst rate decreased transiently in response to application of TFLLR, followed by a slight increase. The inhibitory effect was partially blocked by 50 μM theophylline. In conclusion, activation of astrocytes via PAR1 resulted in a decrease of inspiratory C4 burst rate in association with transient hyperpolarization of respiratory-related neurons. After washout, slow and weak excitatory responses appeared. Adenosine may be partially involved in the inhibitory effect of PAR1 activation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 IBRO. Published by Elsevier Ltd. All rights reserved.)

As blood oxygenation decreases (hypoxemia), mammals mount cardiorespiratory responses, increasing oxygen to vital organs. The carotid bodies are the primary oxygen chemoreceptors for breathing, but sympathetic-mediated cardiovascular responses to hypoxia persist in their absence, suggesting additional high-fidelity oxygen sensors. We show that spinal thoracic sympathetic preganglionic neurons are excited by hypoxia and silenced by hyperoxia, independent of surrounding astrocytes. These spinal oxygen sensors (SOS) enhance sympatho-respiratory activity induced by CNS asphyxia-like stimuli, suggesting they bestow a life-or-death advantage. Our data suggest the SOS use a mechanism involving neuronal nitric oxide synthase 1 (NOS1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). We propose NOS1 serves as an oxygen-dependent sink for NADPH in hyperoxia. In hypoxia, NADPH catabolism by NOS1 decreases, increasing availability of NADPH to NOX and launching reactive oxygen species-dependent processes, including transient receptor potential channel activation. Equipped with this mechanism, SOS are likely broadly important for physiological regulation in chronic disease, spinal cord injury, and cardiorespiratory crisis.

Astrocytes are thought to play a crucial role in providing structure to the spinal cord and maintaining efficient synaptic function and metabolism because their fine processes envelop the synapses of neurons and form many neuronal networks within the central nervous system (CNS). To investigate whether putative astrocytes and putative neurons distributed on the ventral horn play a role in the modulation of lumbar locomotor central pattern generator (CPG) networks, we used extracellular recording and optical imaging techniques and recorded the neural output from the left L5 ventral root and the calcium activity of putative astrocytes and neurons in the L5 ventral horn at the same time when activating an isolated L1-L5 spinal cord preparation from rats aged 0-2 days. Optical measurements detected cells that showed a fluorescence intensity change under all experimental conditions, namely, (1) 5-HT + NMDA, (2) TTX, and (3) TTX + Low K + . These cells were semiautomatically identified using an in-house MATLAB-based program, as putative astrocytes and neurons according to the cell classification, i.e., increased or decreased fluorescence intensity change (ΔF/F0), and subjective judgment based on their soma size. Coherence and its phase were calculated according to the calcium activity of the putative astrocytes and putative neurons, and neural output was calculated during fictive locomotion with in-house MATLAB-based programs. We found that the number of putative astrocytes activated by applying low K + tends not to differ from that activated by applying the protease-activated receptor 1 (PAR1) selective agonist TFLLR-NH 2 (TFLLR). Moreover, the calcium activity of several putative astrocytes and neurons synchronized with locomotor-like activity at a frequency range below 0.5 Hz and the time lag between peaks of cellular calcium activity and locomotor-like activity ranged from -1000 to + 1000 ms. These findings presumably indicates that these putative astrocytes and neurons in the left L5 ventral horn require -1000 to + 1000 ms to communicate with lumbar CPG networks and maintain efficient synaptic function and metabolism in activated lumbar CPG networks. This finding suggests the possibility that putative astrocytic and neuronal cells in the L5 ventral horn contribute to generating the rhythms and patterns of locomotor-like activity by activated CPG networks in the first to fifth lumbar spinal cord., (Copyright © 2021 Elsevier B.V. All rights reserved.)

It is supposed that the nucleus of the solitary tract (NTS) in the dorsal medulla includes gas sensor cells responsive to hypercapnia or hypoxia in the central nervous system. In the present study, we analyzed cellular responses to hypercapnia and hypoxia in the NTS region of newborn rat in vitro preparation. The brainstem and spinal cord were isolated from newborn rat (P0-P4) and were transversely cut at the level of the rostral area postrema. To detect cellular responses, calcium indicator Oregon Green was pressure-injected into the NTS just beneath the cut surface of either the caudal or rostral block of the medulla, and the preparation was superfused with artificial cerebrospinal fluid (25-26°C). We examined cellular responses initially to hypercapnic stimulation (to 8% CO 2 from 2% CO 2 ) and then to hypoxic stimulation (to 0% O 2 from 95% O 2 at 5% CO 2 ). We tested these responses in standard solution and in two different synapse blockade solutions: (1) cocktail blockers solution including bicuculline, strychnine, NBQX and MK-801 or (2) TTX solution. At the end of the experiments, the superfusate potassium concentration was lowered to 0.2 from 3 mM to classify recorded cells into neurons and astrocytes. Excitation of cells was detected as changes of fluorescence intensity with a confocal calcium imaging system. In the synaptic blockade solutions (cocktail or TTX solution), 7.6 and 8% of the NTS cells responded to hypercapnic and hypoxic stimulation, respectively, and approximately 2% of them responded to both stimulations. Some of these cells responded to low K + , and they were classified into astrocytes comprising 43% hypercapnia-sensitive cells, 56% hypoxia-sensitive cells and 54% of both stimulation-sensitive cells. Of note, 49% of the putative astrocytes identified by low K + stimulation were sensitive to hypercapnia, hypoxia or both. In the presence of a glia preferential blocker, 5 mM fluoroacetate (plus 0.5 μM TTX), the percentage of hypoxia-sensitive cells was significantly reduced compared to those of all other conditions. This is the first study to reveal that the NTS includes hypercapnia and hypoxia dual-sensitive cells. These results suggest that astrocytes in the NTS region could act as a central gas sensor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Onimaru, Yazawa, Takeda, Fukushi and Okada.)

AMP-activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP-170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated disks in the heart, a specific cell-cell junction present between cardiomyocytes. A contractile inhibitor, MYK-461, prevented the localization of AMPK at the intercalated disks, and the effect was reversed by the removal of MYK-461, suggesting that the localization of AMPK is regulated by mechanical stress. Time-lapse imaging analysis revealed that the inhibition of CLIP-170 Ser-311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated disks. Interestingly, MYK-461 increased the individual cell area of cardiomyocytes in CLIP-170 phosphorylation-dependent manner. Moreover, heart-specific CLIP-170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP-170 at the intercalated disks., (© 2020 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Psychological stress activates the hypothalamus, augments the sympathetic nervous output, and elevates blood pressure via excitation of the ventral medullary cardiovascular regions. However, anatomical and functional connectivity from the hypothalamus to the ventral medullary cardiovascular regions has not been fully elucidated. We investigated this issue by tract-tracing and functional imaging in rats. Retrograde tracing revealed the rostral ventrolateral medulla was innervated by neurons in the ipsilateral dorsomedial hypothalamus (DMH). Anterograde tracing showed DMH neurons projected to the ventral medullary cardiovascular regions with axon terminals in contiguity with tyrosine hydroxylase-immunoreactive neurons. By voltage-sensitive dye imaging, dynamics of ventral medullary activation evoked by electrical stimulation of the DMH were analyzed in the diencephalon-lower brainstem-spinal cord preparation of rats. Although the activation of the ventral medulla induced by single pulse stimulation of the DMH was brief, tetanic stimulation caused activation of the DMH sustained into the post-stimulus phase, resulting in delayed recovery. We suggest that prolonged excitation of the DMH, which is triggered by tetanic electrical stimulation and could also be triggered by psychological stress in a real life, induces further prolonged excitation of the medullary cardiovascular networks, and could contribute to the pathological elevation of blood pressure. The connectivity from the DMH to the medullary cardiovascular networks serves as a chronological amplifier of stress-induced sympathetic excitation. This notion will be the anatomical and pathophysiological basis to understand the mechanisms of stress-induced sustained augmentation of sympathetic activity.

Eugenol modulates neuronal activity through actions on voltage-gated ionic channels and/or transient receptor potential channels. We previously suggested that eugenol inhibited cellular (and/or network) mechanisms essential for the maintenance of the respiratory burst activity in a brainstem-spinal cord preparation from newborn rat (postnatal day 0-3). Study of the distinct effects of eugenol in neonatal and later developmental stage rats may offer new information about postnatal developmental changes of respiratory neuron networks. In the present study, therefore, we compared effects of eugenol in an in vitro newborn rat preparation with those in an arterially perfused in situ preparation from juvenile rat (postnatal day 12-15). In the former preparation, application of 1 mM eugenol decreased respiratory rate and inspiratory burst duration. In contrast, in the latter preparation, 1 mM eugenol induced a gradual decrease in the amplitude of integrated phrenic nerve activity. Phrenic nerve activity gradually recovered at 25-30 min after washout with a burst duration similar to control values. We hypothesized that the depressant effects of eugenol were caused by inhibition of cell excitability in the neonatal rat in vitro preparation but by a reduction of synaptic interactions in the juvenile rat in situ preparation., (Copyright © 2019 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.)

The respiratory chain (RC) transports electrons to form a proton motive force that is required for ATP synthesis in the mitochondria. RC disorders cause mitochondrial diseases that have few effective treatments; therefore, novel therapeutic strategies are critically needed. We previously identified Higd1a as a positive regulator of cytochrome c oxidase (CcO) in the RC. Here, we test that Higd1a has a beneficial effect by increasing CcO activity in the models of mitochondrial dysfunction. We first demonstrated the tissue-protective effects of Higd1a via in situ measurement of mitochondrial ATP concentrations ([ATP] mito ) in a zebrafish hypoxia model. Heart-specific Higd1a overexpression mitigated the decline in [ATP] mito under hypoxia and preserved cardiac function in zebrafish. Based on the in vivo results, we examined the effects of exogenous HIGD1A on three cellular models of mitochondrial disease; notably, HIGD1A improved respiratory function that was coupled with increased ATP synthesis and demonstrated cellular protection in all three models. Finally, enzyme kinetic analysis revealed that Higd1a significantly increased the maximal velocity of the reaction between CcO and cytochrome c without changing the affinity between them, indicating that Higd1a is a positive modulator of CcO. These results corroborate that Higd1a, or its mimic, provides therapeutic options for the treatment of mitochondrial diseases., (© 2019 Federation of American Societies for Experimental Biology.)

The brains of 10 Japanese patients with adult onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) encompassing hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) and pigmentary orthochromatic leukodystrophy (POLD) and eight Japanese patients with Nasu-Hakola disease (N-HD) and five age-matched Japanese controls were examined neuropathologically with special reference to lesion staging and dynamic changes of microglial subsets. In both diseases, the pathognomonic neuropathological features included spherically swollen axons (spheroids and globules), axon loss and changes of microglia in the white matter. In ALSP, four lesion stages based on the degree of axon loss were discernible: Stage I, patchy axon loss in the cerebral white matter without atrophy; Stage II, large patchy areas of axon loss with slight atrophy of the cerebral white matter and slight dilatation of the lateral ventricles; Stage III, extensive axon loss in the cerebral white matter and dilatation of the lateral and third ventricles without remarkable axon loss in the brainstem and cerebellum; Stage IV, devastated cerebral white matter with marked dilatation of the ventricles and axon loss in the brainstem and/or cerebellum. Internal capsule and pontine base were relatively well preserved in the N-HD, even at Stage IV, and the swollen axons were larger with a higher density in the ALSP. Microglial cells immunopositive for CD68, CD163 or CD204 were far more obvious in ALSP, than in N-HD, and the shape and density of the cells changed in each stage. With progression of the stage, clinical symptoms became worse to apathetic state, and epilepsy was frequently observed in patients at Stages III and IV in both diseases. From these findings, it is concluded that (i) shape, density and subsets of microglia change dynamically along the passage of stages and (ii) increase of IBA-1-, CD68-, CD163- and CD204-immunopositive cells precedes loss of axons in ALSP., (© 2016 International Society of Neuropathology.)

Relatively larger scale peptide libraries immobilized on a gel-type solid support consisting of 24 natural and non-natural amino acids by the "split and combine method" have been constructed to find interacting molecules. The diversity was ca. 200 millions of hexapeptides with cysteinyl residues forming cyclotide. Selected beads after screening can be sequenced by the conventional Edman degradation, although several restrictions and the problems are known. To resolve these, a novel combinatorial method involving partial acid hydrolysis followed by liquid chromatography with on-line mass spectrometric analyses has been established. Problems were uncovered in an early stage of the process. Uncertain assignment caused by byproducts derived from a cystine residue and other materials could be resolved by optimal hydrolysis conditions and derivatization before mass spectrometric analysis. Discrimination between Leu and Ile could be performed using high energy collision induced dissociation in the high resolution MALDI-TOF-MS/MS. The present optimized protocol is useful for discovery of sequences of interacting molecules and a second library construction.

The transient receptor potential (TRP) channels are widely distributed in the central nervous system (CNS) and peripheral nervous system. We examined the effects of TRP ankyrin 1 (TRPA1) agonists (cinnamaldehyde and allyl isothiocyanate) on respiratory rhythm generation in brainstem-spinal cord preparations from newborn rats [postnatal days 0-3 (P0-P3)] and in in situ-perfused preparations from juvenile rats (P11-P13). Preparations were superfused with modified Krebs solution at 25-26°C, and activity of inspiratory C4 ventral root (or phrenic nerve) was monitored. In the newborn rat, an in vitro preparation of cinnamaldehyde (0.5 mM) induced typically biphasic responses in C4 rate: an initial short increase and subsequent decrease, then a gradual recovery of rhythm during 15 min of bath application. After washout, the respiratory rhythm rate further increased, remaining 200% of control for >120 min, indicating long-lasting facilitation. Allyl isothiocyanate induced effects similar to those of cinnamaldehyde. The long-lasting facilitation of respiratory rhythm was partially antagonized by the TRPA1 antagonist HC-030031 (10 μM). We obtained similar long-lasting facilitation in an in situ-perfused reparation from P11-P13 rats. On the basis of results from transection experiments of the rostral medulla and whole-cell recordings from preinspiratory neurons in the parafacial respiratory group (pFRG), we suggest that the rostral medulla, including the pFRG, is important to the induction of long-lasting facilitation. A histochemical analysis demonstrated a wide distribution of TRPA1 channel-positive cells in the reticular formation of the medulla, including the pFRG. Our findings suggest that TRPA1 channel activation could induce long-lasting facilitation of respiratory rhythm and provide grounds for future study on the roles of TRPA1 channels in the CNS., (Copyright © 2015 the American Physiological Society.)

Hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) is a neurodegenerative disease clinically characterized by slowly progressive cognitive decline and motor dysfunction. Neuropathology shows diffuse degeneration in the white matter, with prominent presence of widespread axonal spheroids. To investigate the mechanism underlying HDLS neurodegeneration, we characterized spheroids and examined their development in the degenerated white matter. Analysis revealed that the spheroids are an early neuropathological manifestation in the white matter degeneration and involve axonal component proteins and α-synuclein. The development of spheroids facilitates in initiating neurodegeneration in HDLS., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Multiple system atrophy (MSA) is a neurodegenerative disease caused by α-synuclein aggregation in oligodendrocytes and neurons. Using a transgenic mouse model overexpressing human α-synuclein in oligodendrocytes, we previously demonstrated that oligodendrocytic α-synuclein inclusions induce neuronal α-synuclein accumulation and progressive neuronal degeneration. α-Synuclein binds to β-III tubulin, leading to the neuronal accumulation of insoluble α-synuclein in an MSA mouse model. The present study demonstrates that α-synuclein co-localizes with β-III tubulin in the brain tissue from patients with MSA and MSA model transgenic mice as well as neurons cultured from these mice. Accumulation of insoluble α-synuclein in MSA mouse neurons was blocked by the peptide fragment β-III tubulin (residues 235-282). We have determined the α-synuclein-binding domain of β-III tubulin and demonstrated that a short fragment containing this domain can suppress α-synuclein accumulation in the primary cultured cells. Administration of a short α-synuclein-binding fragment of β-III tubulin may be a novel therapeutic strategy for MSA., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The interplay of the neuronal discharge patterns regarding respiration and locomotion was investigated using electrophysiological techniques in a decerebrate and arterially perfused in situ mouse preparation. The phrenic, tibial, and/or peroneal nerve discharge became clearly organized into discharge episodes of increasing frequency and duration, punctuated by periods of quiescence as the perfusion flow rate increased at room temperature. The modulated sympathetic tone induced by the hyperoxic/normocapnic state was found to activate the locomotor pattern generator (LPG) via descending pathways and generate a left and right alternating discharge during discharge episodes in the motor nerves. The rhythm coupling of respiration and locomotion occurred at a 1:1 frequency ratio. Although the phrenic discharge synchronized with the tibial discharge at all flow rates tested, the time lag between peaks of the two discharges during locomotion was ≈400 ms rather than ≈200 ms, suggesting spinal feedback via ascending pathways. The incidence of the phrenic and tibial discharge episodes decreased by ≈50% after spinalization at the twelfth thoracic cord and the respiratory rhythm was more regular. These results indicate that: (i) locomotion can be generated in a hyperoxic/normocapnic state induced by specific respiratory conditions, (ii) the central mechanism regarding entrainment of respiratory and locomotor rhythms relies on spinal feedback via ascending pathways, initiated by the activated LPG generating locomotion, and (iii) the increase in respiratory rate seen during locomotion is caused not only by afferent mechanical and nociceptive inputs but also by impulses from the activated spinal cord producing a locomotor-like discharge via ascending pathways.

Multiple system atrophy is an intractable neurodegenerative disease caused by α-synuclein (α-syn) accumulation in oligodendrocytes and neurons. With the use of a transgenic mouse model overexpressing human α-syn in oligodendrocytes, we demonstrated that oligodendrocytic α-syn inclusions induce neuronal α-syn accumulation, resulting in progressive neuronal degeneration. The mechanism through which oligodendrocytic α-syn inclusions trigger neuronal α-syn accumulation leading to multiple system atrophy is unknown. In this study, we identified cystatin C, an oligodendrocyte-derived secretory protein that triggers α-syn up-regulation and insoluble α-syn accumulation, in neurons of the mouse central nervous system. Cystatin C was released by mouse oligodendrocytes overexpressing human α-syn, and extracellular cystatin C increased the expression of the endogenous α-syn gene in wild-type mouse neurons. These neurons then accumulate insoluble α-syn and may undergo apoptosis. Cystatin C is a potential pathogenic signal triggering neurodegeneration in multiple system atrophy., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Recent epidemiological studies have identified interferon regulatory factor 8 (IRF8) as a susceptibility factor for multiple sclerosis (MS). However, how IRF8 influences the neuroinflammatory disease has remained unknown. By studying the role of IRF8 in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we found that Irf8(-/-) mice are resistant to EAE. Furthermore, expression of IRF8 in antigen-presenting cells (APCs, such as macrophages, dendritic cells, and microglia), but not in T cells, facilitated disease onset and progression through multiple pathways. IRF8 enhanced αvβ8 integrin expression in APCs and activated TGF-β signaling leading to T helper 17 (Th17) cell differentiation. IRF8 induced a cytokine milieu that favored growth and maintenance of Th1 and Th17 cells, by stimulating interleukin-12 (IL-12) and IL-23 production, but inhibiting IL-27 during EAE. Finally, IRF8 activated microglia and exacerbated neuroinflammation. Together, this work provides mechanistic bases by which IRF8 contributes to the pathogenesis of MS., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: Hereditary diffuse leukoencephalopathy with neuroaxonal spheroids (HDLS) is an adult-onset white matter disease that presents clinically with cognitive, mental and motor dysfunction. Several autopsy reports have indicated that the corpus callosum (CC), the largest bundle of white matter, is severely affected in patients with HDLS. The aim of this study was to evaluate corpus callosum atrophy (CCA) quantitatively in HDLS patients., Methods: We assessed CCA in six genetically-proven HDLS patients (HDLS group), in comparison with that observed in 20 patients with vascular dementia (VaD group) and 24 age-matched patients without organic central nervous system (CNS) disease (non-CNS group). Using midsagittal MR images, five measurements of the CC were obtained: the width of the rostrum (aa'), body (bb') and splenium (cc'), the anterior to posterior length (ab) and the maximum height (cd). Next, the corpus callosum index (CCI) was calculated as (aa' + bb' + cc')/ab., Results: All HDLS patients had white matter lesions in the CC and frontoparietal lobes on the initial MRI scans. Compared with that observed in the VaD and age-matched non-CNS groups, the CCI was significantly decreased in the HDLS group (with VaD group, p<0.01; with non-CNS group, p<0.01)., Conclusion: This study showed significant atrophy of the CC in all HDLS patients on the initial MRI scans obtained 6-36 months after onset. We propose that the early appearance of CCA, frequently accompanied by high-intensity in the genu and/or splenium, on T2 images is an important diagnostic clue to HDLS.

To achieve more efficient separation of intact proteins for proteomics applications, three columns of differing diameters (4.0, 4.6 and 6.0 mm internal diameter) were chosen for comparison and investigated to identify optimal conditions. The column with the largest diameter gave the largest peak capacity, showing the efficient separation of intact proteins, such as two protein standards, glutathione S-transferase and β-lactoglobulin. On the other hand, a low-molecular-weight compound was separated effectively on the smaller diameter column, demonstrating that the separation mechanism seems to differ between high- and low-molecular-weight compounds. Finally, using the 6.0 mm i.d. column, 680 protein peaks were observed in mouse liver extracts, demonstrating that a wider diameter separation column is effective for intact protein separations., (Copyright © 2013 John Wiley & Sons, Ltd.)

Polyglutamine (polyQ) diseases result from expansion of CAG trinucleotide repeats in their responsible genes. Although gene products with polyQ expansions undergo conformational changes to aggregate in neurons, the relationship between inclusions and neurotoxicity remains unclear. Dentatorubral-pallidoluysian atrophy (DRPLA) is a polyQ disease, and DRPLA protein, also known as atrophin-1 (ATN1), carries an expanded polyQ tract. To investigate how an expanded polyQ tract influences ATN1 aggregation and localization, we compared the aggregation of ATN1 with a polyQ tract to that of ATN1 with a polyleucine (polyL) tract. In COS-7 cells, polyL-ATN1 triggered more aggregation than polyQ-ATN1 of similar repeat sizes. Immunocytochemical and biochemical studies revealed that replacement of the polyQ tract with polyL alters ATN1 localization, leading to retention of polyL-ATN1 in the cytoplasm. Despite this change in localization, polyL-ATN1 and polyQ-ATN1 demonstrate comparable repeat length dependent toxicity. These results suggest that expanded polyQ repeats in ATN1 may contribute to neurodegeneration via alterations in both protein aggregation and intracellular localization., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Multiple system atrophy is a neurodegenerative disease caused by abnormal α-synuclein (α-syn) accumulation in oligodendrocytes and neurons. We previously demonstrated that transgenic (Tg) mice that selectively overexpressed human α-syn in oligodendrocytes exhibited neuronal α-syn accumulation. Microtubule β-III tubulin binds to endogenous neuronal α-syn to form an insoluble complex, leading to progressive neuronal degeneration. α-Syn accumulation is increased in the presynaptic terminals of Tg mice neurons and may reduce neurotransmitter release. To clarify the mechanisms underlying its involvement in neuronal dysfunction, in the present study, we investigated the effects of neuronal α-syn accumulation on synaptic function in Tg mice. Using whole-cell patch-clamp recording, we found that the frequency of miniature inhibitory postsynaptic currents was reduced in Tg mice. Furthermore, a microtubule depolymerizing agent restored normal frequencies of miniature inhibitory postsynaptic currents in Tg mice. These findings suggest that α-syn and β-III tubulin protein complex plays roles for regulation of synaptic vesicle release in GABAergic interneurons, and it causes to reduce GABAergic inhibitory transmission., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Multiple system atrophy (MSA) is a neurodegenerative disease caused by α-synuclein (α-syn) accumulation in oligodendrocytes and neurons. We generated a transgenic (Tg) mouse model in which human α-syn was overexpressed in oligodendrocytes. Our previous studies have revealed that oligodendrocytic α-syn inclusions induced neuronal α-syn accumulation, thereby resulting in progressive neuronal degeneration in mice. We also demonstrated that an insoluble complex of α-syn and β-III tubulin in microtubules progressively accumulated in neurons, thereby leading to neuronal degeneration. In the present study, we demonstrated that neuronal accumulation of the insoluble complex was derived from binding of α-syn to β-III tubulin and not from α-syn self-aggregation. Thus, interaction between α-syn and β-III tubulin plays an important role in neuronal α-syn accumulation in an MSA mouse model., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A wide-pore (30 nm) reversed-phase column (Intrada WP-RP, particle size 3 μm) was recently utilized for protein separation in differential proteomics analysis with fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS), and exerted a tremendous effect on finding biomarkers (e.g., for breast cancer). Further high-performance separation is required for highly complex protein mixtures. A recently prepared non-porous small-particle reversed-phase column (Presto FF-C18, particle size: 2 μm) was expected to more effectively separate derivatized protein mixtures than the wide-pore column. A preliminary experiment demonstrated that the peak capacity of the former was threefold greater than that of the latter in gradient elution of a fluorogenic derivatized model peptide, calcitonin. The FD-LC-MS/MS method with a non-porous column was then optimized and applied to separate liver mitochondrial proteins that were not efficiently separated with the wide-pore column. As a result, high-performance separation of mitochondrial proteins was accomplished, and differential proteomics analysis of liver mitochondrial proteins in a hepatitis-infected mouse model was achieved using the FD-LC-MS/MS method with the non-porous column. This result suggests the non-porous small-particle column as a replacement for the wide-pore column for differential proteomics analysis in the FD-LC-MS/MS method., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Dentatorubral-pallidoluysian atrophy (DRPLA) is caused by the expansion of polyglutamine (polyQ) in atrophin-1 (ATN1), also known as DRPLA protein. ATN1 is ubiquitously expressed in the central nervous system (CNS), although selective regions of CNS are degenerated in DRPLA, and this selective neuronal damage gives rise to the specific clinical features of DRPLA. Accumulation of mutant ATN1 that carries an expanded polyQ tract seems to be the primary cause of DRPLA neurodegeneration, but it is still unclear how the accumulation of ATN1 leads to neu-rodegeneration. Recently, cleaved fragments of ATN1 were shown to accumulate in the disease models and the brain tissues of patients with DRPLA. Furthermore, proteolytic processing of ATN1 may regulate the intracellular localization of ATN1 and its fragments. Therefore, proteolytic processing of ATN1 may provide clues to disease pathogenesis and hopefully aid in the determination of molecular targets for effective therapeutic approaches for DRPLA.