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Brain barriers disruption and acute seizures and kindling by pentylenetetrazole in mice
Abstracts / Neuroscience Research 68S (2010) e335–e446
P3-b39 Brain barriers disruption and acute seizures and kindling by pentylenetetrazole in mice Kouichi Itoh 1 Nakamura 2
, Shuntaro
Kohnomi 1 , Sonoko
Danjo 2 , Yu
1 Lab. for Brain Sci., Kagawa Sch. of Pharmaceuti Sci., Tokushima Bunri University, Kagawa, Japan 2 Department of Neuropyschia, Kagawa University, Sch. of Med., Kagawa, Japan
Gadolinium (Gd)-Enhanced Magnetic Resonance Imaging (GdEMRI) is receiving increased interest as a valuable tool for monitoring the brain barriers (BB) functions in the animal brain based the impermeability of Gd contrast agent into brain parenchyma. Here the possibility that in vivo GdEMRI can detect the entry of Gd in the brain of mouse behaving with intended stimulation is tested. In pentylenetetrazole (PTZ)-induced clonic-tonic convulsive seizures in mice, intensities of Gd in parenchyma dramatically increased in midbrain and cerebral cortex. These Gd siganl intensities were significantly increased under awaking in comparison to under anesthesia. To determine relationship between Gd signal enhancement and glutamatergic activities, the effects of noncompetitive and competitive N-Methyl-d-Aspartate receptor (NMDAR) antagonist, MK801 and CGP39551, were examined on GdEMRI in PTZ-injected mice. The PTZ-induced Gd signal enhancement was completely antagonized by MK-801 and CGP39551. These data indicated that the Gd-dependent signal enhancement took place as a result of BB disruption by the overactivation of glutamatergic neurons through NMDAR in the epileptic brain. doi:10.1016/j.neures.2010.07.1553
P3-b40 LPS enhance the expression of nitric oxide synthases in the choroid plexus epithelial cell line ECPC-4 cells Mami Ohkusa , Mieko Otani, Keiji Sano, Masaoki Takano Laboratory of Molecular Cell Biology, Kobe Gakuin University The choroid plexus plays a key role in the production of cerebrospinal fluid (CSF) proteins as well as in CSF dynamics. Murine choroid plexus cell lines, ECPC-4 retains the characteristics of choroid plexus epithelial cells in that they express such choroid plexus cell markers or related proteins as transthyretin and alpha2-macroglobulin. These cells may be useful material for analyses of choroid plexus functions. Here we showed that lipopolysaccharide (LPS) enhanced the expression of nitric oxide synthases (NOSs) in ECPC-4 cells. Toll like receptor, LTR-2 and -4 were expressed in ECPC-4 detected by RT-PCR. The three types NOS, eNOS, iNOS and nNOS were expressed, and iNOS and eNOS were induced within 24–72 h by LPS treatment. Furthermore, expression of pro-inflammatory mediators interleukin-1 (IL-1), tumor necrosis factor-␣ (TNF-␣), and cyclooxygenase-2 mRNA increased within 4–8 h after addition of LPS to ECPC4 cells. The addition of IL-1 and TNF-␣ to investigate the major mediator for iNOS expression in ECPC4 cells remarkably induced expression of iNOS mRNAs. In addition, NFkB, a transcription factor in inflammatory process, were expressed in ECPC-4, and NFkB gene silencing inhibits the expression of iNOS by LPS.These results suggest that LPS activates iNOS in the choroid plexus via autocrine induction of IL-1 and TNF-␣, NFkB is a key role factor in iNOS transcriptional process. It may hypothesize that choroid plexus epithelial cells has an important role as the inflammatory sensor in the CNS. doi:10.1016/j.neures.2010.07.1554
e351
pictures. Similar results were obtained after injection of ruthenium red in the perfusate, which indicates that La3++ ions and ruthenium red molecules could diffuse into perivasucular spaces without obvious hindrance, presumably passing through the incomplete blood–brain barrier. In electrophysiological experiments on pinealocytes, which were whole-cell clamped at −50 mV, definite inward currents were induced in pinealocytes when external saline containing 10 M nicotine was applied. Other nicotinic agonists, including ACh, choline and cytisine also induced similar inward currents. These findings suggest that pinealocytes may monitor the concentration of various substances in blood vessel and cause specific responses in their membrane. doi:10.1016/j.neures.2010.07.1555
P3-c02 Proteomic identification of co-factors for the methyl-CpG binding protein, MeCP2 Keita Tsujimura 1 , Akiya Suzuki 1 , Yoichiro Fukao 2 , Masayuki Fujiwara 2 , Kinichi Nakashima 1 1 Laboratory of molecular neuroscience, Nara Institute of Science and Tecnology (NAIST) 2 The Plant Science Education Unit, Nara Institute of Science and Tecnology (NAIST)
Rett syndrome (RTT) is recognized as a disease correlated to mutations of X-linked gene, methyl-CpG binding protein 2 (MeCP2). Although MeCP2 has been identified as a methylation-dependent transcriptional repressor, expression profiling of MeCP2 deficient mice did not reveal significant changes in gene expression. Moreover, ChIP-on chip analysis demonstrated that MeCP2 associates preferentially with the promoters of transcriptionally active genes rather than of silent genes. These findings suggest that MeCP2 may not function simply as a global repressor for gene transcription. It is generally known that MeCP2 is highly expressed in neurons, yet it is becoming apparent that it is also expressed in other CNS cell types including neural stem cells (NSCs), astrocytes and oligodendrocytes, implying that MeCP2 plays some roles in these cells as well. However, it is far from clear to date how MECP2 mutations cause Rett-like phenotypes. Thus, it is important to ask whether additional functions of MeCP2 play a role in the pathogenesis. To reveal novel functions of MeCP2, we sought to identify MeCP2-interacting proteins in the four major cell types in the CNS (NSCs, neurons, astrocytes and oligodendrocytes) using comprehensive proteomic approaches. We expressed FLAG-tagged MeCP2 in these cells and performed immunoprecipitation with anti-FLAG antibody. The precipitants were then subjected to LC–MS/MS analysis. Among co-precipitated protein with MeCP2, we were able to detect HDAC2, ATRX, YB-1 and other chromatin related molecules that were previously reported as MeCP2-associating proteins, indicating that our experimental system, at least in part, can detect functional complexes of MeCP2. We also identified several common and cell-type specific MeCP2-associating proteins. Interestingly, many RNA-binding proteins were commonly identified in all cell types. These results raise the intriguing possibility that MeCP2 has other as yet unknown functions. doi:10.1016/j.neures.2010.07.1556
P3-c03 Regional characteristics of gene expression in the brain of the rhesus monkey Takao Oishi 1 , Akira Sato 2 , Sayuri Higaki 1 , Noriyuki Higo 3 , Toshio Kojima 2,4 1
P3-c01 Monitoring-mode responses to nicotine in the pinealocytes of the rodents: A patch-clamp study combined with electron-microscopy Kazuyoshi Kawa Neurophysiology, Tohoku University Graduate School of Medicine, Japan Using isolated pineal glands from ddy mice or Wistar rats, responsive properties to nicotine and their specific morphological characters of pinealocytes (principal cells in the pineal gland) were studied. Observation with transmission electron microscopy (EM; Hitachi H7600, 80 kV) shows that pineal gland consists of principal cells, interstitial cells, and perivasucular spaces containing nerve fibers and blood capillaries. Each component of cells was clearly distinguished by their characteristic electron density and nuclear form. Close observation of EM pictures at 6,000-fold showed presence of open gaps and/or fenestrations on endothelium of the blood capillaries. Injection of La3++ ions in perfusate during glutaraldehyde fixation revealed appearance of electron-dense deposits in perivasucular spaces of the pineal gland in EM
Sect. Systems Neurosci, Primate Res. Inst., Kyoto Univ, Inuyama Computational Systems Biol. Res. Group, RIKEN, Yokohama 3 Human Tech. Res. Inst., AIST, Tsukuba 4 Res. Equipment Center, Hamamatsu Univ School of Med., Hamamatsu
2
We investigated gene expression patterns in several brain regions of the rhesus monkey, including various neocortical areas, the hippocampus, the putamen, and the cerebellar cortex by using DNA microarray system (Agilent, 4x44K). To compare gene expression patterns among neocortical areas from several points of view, such as neuron number, cytoarchitecture, and degree of myelination, we selected following seven areas: the dorsolateral prefrontal area (FD of von Bonin and Bailey), the premotor area (FB), the primary motor area (FA), the primary somatosensory area (PB), the parietal association area (PE), the temporal association area (TE), and the primary visual area (OC). Cluster analysis revealed distinct gene expression pattern in the cerebellar cortex from those in the neocortical areas. Differential gene expression profiles were also observed among the neocortical areas. The degree of similarity in the gene expression between the given cortical
P3-b39 Brain barriers disruption and acute seizures and kindling by pentylenetetrazole in mice Kouichi Itoh 1 Nakamura 2
, Shuntaro
Kohnomi 1 , Sonoko
Danjo 2 , Yu
1 Lab. for Brain Sci., Kagawa Sch. of Pharmaceuti Sci., Tokushima Bunri University, Kagawa, Japan 2 Department of Neuropyschia, Kagawa University, Sch. of Med., Kagawa, Japan
Gadolinium (Gd)-Enhanced Magnetic Resonance Imaging (GdEMRI) is receiving increased interest as a valuable tool for monitoring the brain barriers (BB) functions in the animal brain based the impermeability of Gd contrast agent into brain parenchyma. Here the possibility that in vivo GdEMRI can detect the entry of Gd in the brain of mouse behaving with intended stimulation is tested. In pentylenetetrazole (PTZ)-induced clonic-tonic convulsive seizures in mice, intensities of Gd in parenchyma dramatically increased in midbrain and cerebral cortex. These Gd siganl intensities were significantly increased under awaking in comparison to under anesthesia. To determine relationship between Gd signal enhancement and glutamatergic activities, the effects of noncompetitive and competitive N-Methyl-d-Aspartate receptor (NMDAR) antagonist, MK801 and CGP39551, were examined on GdEMRI in PTZ-injected mice. The PTZ-induced Gd signal enhancement was completely antagonized by MK-801 and CGP39551. These data indicated that the Gd-dependent signal enhancement took place as a result of BB disruption by the overactivation of glutamatergic neurons through NMDAR in the epileptic brain. doi:10.1016/j.neures.2010.07.1553
P3-b40 LPS enhance the expression of nitric oxide synthases in the choroid plexus epithelial cell line ECPC-4 cells Mami Ohkusa , Mieko Otani, Keiji Sano, Masaoki Takano Laboratory of Molecular Cell Biology, Kobe Gakuin University The choroid plexus plays a key role in the production of cerebrospinal fluid (CSF) proteins as well as in CSF dynamics. Murine choroid plexus cell lines, ECPC-4 retains the characteristics of choroid plexus epithelial cells in that they express such choroid plexus cell markers or related proteins as transthyretin and alpha2-macroglobulin. These cells may be useful material for analyses of choroid plexus functions. Here we showed that lipopolysaccharide (LPS) enhanced the expression of nitric oxide synthases (NOSs) in ECPC-4 cells. Toll like receptor, LTR-2 and -4 were expressed in ECPC-4 detected by RT-PCR. The three types NOS, eNOS, iNOS and nNOS were expressed, and iNOS and eNOS were induced within 24–72 h by LPS treatment. Furthermore, expression of pro-inflammatory mediators interleukin-1 (IL-1), tumor necrosis factor-␣ (TNF-␣), and cyclooxygenase-2 mRNA increased within 4–8 h after addition of LPS to ECPC4 cells. The addition of IL-1 and TNF-␣ to investigate the major mediator for iNOS expression in ECPC4 cells remarkably induced expression of iNOS mRNAs. In addition, NFkB, a transcription factor in inflammatory process, were expressed in ECPC-4, and NFkB gene silencing inhibits the expression of iNOS by LPS.These results suggest that LPS activates iNOS in the choroid plexus via autocrine induction of IL-1 and TNF-␣, NFkB is a key role factor in iNOS transcriptional process. It may hypothesize that choroid plexus epithelial cells has an important role as the inflammatory sensor in the CNS. doi:10.1016/j.neures.2010.07.1554
e351
pictures. Similar results were obtained after injection of ruthenium red in the perfusate, which indicates that La3++ ions and ruthenium red molecules could diffuse into perivasucular spaces without obvious hindrance, presumably passing through the incomplete blood–brain barrier. In electrophysiological experiments on pinealocytes, which were whole-cell clamped at −50 mV, definite inward currents were induced in pinealocytes when external saline containing 10 M nicotine was applied. Other nicotinic agonists, including ACh, choline and cytisine also induced similar inward currents. These findings suggest that pinealocytes may monitor the concentration of various substances in blood vessel and cause specific responses in their membrane. doi:10.1016/j.neures.2010.07.1555
P3-c02 Proteomic identification of co-factors for the methyl-CpG binding protein, MeCP2 Keita Tsujimura 1 , Akiya Suzuki 1 , Yoichiro Fukao 2 , Masayuki Fujiwara 2 , Kinichi Nakashima 1 1 Laboratory of molecular neuroscience, Nara Institute of Science and Tecnology (NAIST) 2 The Plant Science Education Unit, Nara Institute of Science and Tecnology (NAIST)
Rett syndrome (RTT) is recognized as a disease correlated to mutations of X-linked gene, methyl-CpG binding protein 2 (MeCP2). Although MeCP2 has been identified as a methylation-dependent transcriptional repressor, expression profiling of MeCP2 deficient mice did not reveal significant changes in gene expression. Moreover, ChIP-on chip analysis demonstrated that MeCP2 associates preferentially with the promoters of transcriptionally active genes rather than of silent genes. These findings suggest that MeCP2 may not function simply as a global repressor for gene transcription. It is generally known that MeCP2 is highly expressed in neurons, yet it is becoming apparent that it is also expressed in other CNS cell types including neural stem cells (NSCs), astrocytes and oligodendrocytes, implying that MeCP2 plays some roles in these cells as well. However, it is far from clear to date how MECP2 mutations cause Rett-like phenotypes. Thus, it is important to ask whether additional functions of MeCP2 play a role in the pathogenesis. To reveal novel functions of MeCP2, we sought to identify MeCP2-interacting proteins in the four major cell types in the CNS (NSCs, neurons, astrocytes and oligodendrocytes) using comprehensive proteomic approaches. We expressed FLAG-tagged MeCP2 in these cells and performed immunoprecipitation with anti-FLAG antibody. The precipitants were then subjected to LC–MS/MS analysis. Among co-precipitated protein with MeCP2, we were able to detect HDAC2, ATRX, YB-1 and other chromatin related molecules that were previously reported as MeCP2-associating proteins, indicating that our experimental system, at least in part, can detect functional complexes of MeCP2. We also identified several common and cell-type specific MeCP2-associating proteins. Interestingly, many RNA-binding proteins were commonly identified in all cell types. These results raise the intriguing possibility that MeCP2 has other as yet unknown functions. doi:10.1016/j.neures.2010.07.1556
P3-c03 Regional characteristics of gene expression in the brain of the rhesus monkey Takao Oishi 1 , Akira Sato 2 , Sayuri Higaki 1 , Noriyuki Higo 3 , Toshio Kojima 2,4 1
P3-c01 Monitoring-mode responses to nicotine in the pinealocytes of the rodents: A patch-clamp study combined with electron-microscopy Kazuyoshi Kawa Neurophysiology, Tohoku University Graduate School of Medicine, Japan Using isolated pineal glands from ddy mice or Wistar rats, responsive properties to nicotine and their specific morphological characters of pinealocytes (principal cells in the pineal gland) were studied. Observation with transmission electron microscopy (EM; Hitachi H7600, 80 kV) shows that pineal gland consists of principal cells, interstitial cells, and perivasucular spaces containing nerve fibers and blood capillaries. Each component of cells was clearly distinguished by their characteristic electron density and nuclear form. Close observation of EM pictures at 6,000-fold showed presence of open gaps and/or fenestrations on endothelium of the blood capillaries. Injection of La3++ ions in perfusate during glutaraldehyde fixation revealed appearance of electron-dense deposits in perivasucular spaces of the pineal gland in EM
Sect. Systems Neurosci, Primate Res. Inst., Kyoto Univ, Inuyama Computational Systems Biol. Res. Group, RIKEN, Yokohama 3 Human Tech. Res. Inst., AIST, Tsukuba 4 Res. Equipment Center, Hamamatsu Univ School of Med., Hamamatsu
2
We investigated gene expression patterns in several brain regions of the rhesus monkey, including various neocortical areas, the hippocampus, the putamen, and the cerebellar cortex by using DNA microarray system (Agilent, 4x44K). To compare gene expression patterns among neocortical areas from several points of view, such as neuron number, cytoarchitecture, and degree of myelination, we selected following seven areas: the dorsolateral prefrontal area (FD of von Bonin and Bailey), the premotor area (FB), the primary motor area (FA), the primary somatosensory area (PB), the parietal association area (PE), the temporal association area (TE), and the primary visual area (OC). Cluster analysis revealed distinct gene expression pattern in the cerebellar cortex from those in the neocortical areas. Differential gene expression profiles were also observed among the neocortical areas. The degree of similarity in the gene expression between the given cortical