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Facilitation of dopamine-induced Na+ current response by Rho GTPase and phospholipase D in Aplysia neurons
S138
Abstracts / Neuroscience Research 58S (2007) S1–S244
P2-b39 The genomic structure and the variants of nitric oxide synthase gene in the terrestrial slug Limax valentianus Ryota Matsuo, Etsuro Ito Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Sanuki, Japan In the terrestrial slug Limax, NO is necessary for the synchronous oscillation of the local field potential in the procerebrum, which is thought to be involved in the odor discrimination and/or odor aversion learning. But there is no description about the genomic structure of the molluscan NO synthase, nor has the evolutionary origin of neuronal NO synthase (NOS) of mammals been clarified. Here we identified two types of NOS mRNAs of the slug, which show differential expression patterns within the brain. One is expressed broadly in the brain but in the low level (limNOS1), whereas, interestingly, the other is almost exclusively in the procerebrum (limNOS2). We also determined the whole genomic structure for limNOS1. It is composed of as many as 32 exons comparable to human nNOS gene, and has very similar exon–intron structure to that of human nNOS. Our results indicated that Limax NOS and human nNOS share the prototypical gene structure of NOS, and that their organization is highly conserved during the evolutionary history.
Delayed reduction of the protein level in the striatum P2-b40 after genetic ablation of the tyrosine hydroxylase gene in the substantia nigra Sakane 1 ,
Sumi-Ichinose 2 ,
Kojima 1 ,
Hiroaki Chiho Masayo Yoshinori Aso 1 , Shin-ichi Muramatsu 3 , Imaharu Nakano 3 , Keiya Ozawa 4 , Takahide Nomura 2 , Ikuko Nagatsu 1 , Toshiharu Nagatsu 2 , Daniel Metzger 5 , Pierre Chambon 5 , Hiroshi Ichinose 1 1 Graduate School of Bioscience & Biotechnology, Tokyo Institute of Technology, Yokohama, Japan; 2 School of Medicine, Fujita Health University, Japan; 3 Division of Neurology, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan; 4 Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan; 5 IGBMC, Illkirch, France We generated floxed TH mice to disrupt the TH gene using adenoassociated viral vector expressing Cre recombinase (AAV-Cre). Injection of AAV-Cre into the substantia nigra pars compacta (SNc) of the floxed TH mice induced recombination of the TH gene, followed by the loss of TH protein in the SNc. A decrease of TH protein in the striatum was much slower than that in the SNc suggesting that the turnover rate of the TH protein would be different between the cell body and the termianal.
P2-b44 Calcium-dependent mechanisms underlying propaga-
tion of ␥PKC translocation along the dendrites of cerebellar Purkinje cell
Hiroshi Tsubokawa 1 , Kaori Kashiwagi 2 , Naoaki Saito 2 , Mitsuyuki Nakao 1 , Norihiro Katayama 1 1 Graduate School of Information Sciences, Tohoku University, Sendai, Japan; 2 Biosignal Research Center, Kobe University, Kobe, Japan Regardless of the importance of protein kinase C (PKC) targeting, little is known concerning PKC translocation in CNS neurons. Especially, it remains unclear how, when and where PKC is targeted in response to Gq/11 -coupled receptor activations. To address this issue, we have generated brain-region specific and inducible ␥PKC-GFP transgenic mice using Tet-regulated system, and found that in cerebellar Purkinje cells electrical stimulation of parallel fibers evoked not only local translocation but also propagation of the translocation along dendritic arbor. Calcium imaging studies have shown that the parallel fiber stimulation also induces slowly propagating calcium wave, which could lead propagation of PKC translocation. To understand mechanism underlying the PKC wave, we constructed a compartmental model of Purkinje cell dendrites. Computer simulations suggest interplay between ␥PKC, Ca2+ channels and K+ channels on the plasma membrane of dendrites.
1 Inhibitory effects of thiamylal on excitation propagation P2-c0 in brain slices Naoshi Fujiwara 1 , Mayumi Ogawa 2 , Kenji Seo 3 Medical Technology, Niigata University, Niigata, Japan; 2 Anesthesiology, Niigata University, Niigata, Japan; 3 Dental Anesthesiology, Niigata University, Niigata, Japan
1
Inhibitory effects of thiamylal (a barbital derivative) on excitation propagation in the cortical and hippocampal slices were characterized by optical imaging techniques. Brain slices of the C57BL6J mouse stained with a voltage sensitive dye RH414 and changes in membrane potential were recorded as video images. In cortical slices, excitation evoked by stimulation to layer V was propagated to layer II–III, and the response was widely extended and prolonged by application of strychnine (20 mol/l). By a successive application of thiamylal (200 mol/l), the initial excitation was evoked but following responses rapidly diminished and disappeared. In hippocampal slices, excitation propagation elicited by stimulation to the CA1 stratum radiatum was also enhanced and prolonged by strychnine. This enhanced and prolonged excitation remained unchanged even in the presence of thiamylal. The results suggest that thiamylal makes excited neurons calm down by enhancing GABAA receptor activity in the cerebral cortex.
Research funds: a grant (No. 17024017) from the program Grants-in-Aid for Scientific Research on Priority Areas, from the ministry if Education, Culture, Science, and Technology of Japan.
P2-b43 N-glycans govern the innate immune response in neu-
2 Facilitation of dopamine-induced Na+ current response P2-c0
rocysticercosis
by Rho GTPase and phospholipase D in Aplysia neurons
V. Prabhakaran, V. Rajshekhar, A. Oommen Neurological Sciences, Christian Medical College, Vellore, India
Satoshi Kawasaki 1 , Shuji Watanabe 1 , Shingo Kimura 1 , Reiko Fujita 2 , Kazuhiko Sasaki 1 1 Department of Physiology, Iwate Medical University, Morioka, Japan; 2 Department of Chemistry, Iwate Medical University, Morioka, Japan
Taenia solium neurocysticercosis is the most common parasitic infection of the CNS & accounts for one-quarter epilepsy cases in South India. Seizures result when cysts degenerate in the brain releasing inflammatory cytokines. It would be useful to identify cellular mediators that govern parasitic destruction and clear infection. Aim of the study was to evaluate protective innate immune responses in T. solium infection. Peripheral blood mononuclear cells from healthy volunteers (obtained with informed consent) were stimulated for 24 h with 6 infection specific T. solium glycoproteins, their peptides or N-glycan components. We identify nitric oxide and IL-12 in culture supernatants and HSP-70 in cells, as mediators of protective innate immunity. Neither T. solium glycoproteins nor their peptide components elicited a response from the cells. However T. solium N-glycans were strong inducers of nitric oxide in macrophages leading to IL-12 upregulation and induction of HSP-70 that was cytoprotective. The role of T. solium N-glycans as mediators of protective innate immunity in neurocysticercosis will be discussed. Research fund: ICMR, New Delhi
Stimulation of D1 type of dopamine (DA)-receptor induces a slow Na+ current response in identified Aplysia neurons under voltage clamp. We previously reported that the Na+ current response to DA is facilitated by the activation of Rho GTPase. In the present study, we investigated possible effector molecules located at the downstream of the Rho. The Na+ current response to DA was depressed after intracellular injection of ␣synuclein, an inhibitory protein for phospholipase D (PLD). Furthermore, application of 1-butanol, which is known to inhibit PLD activity, significantly depressed the DA-induced current response, whereas 2-butanol, an inactive isomer of 1-butanol, had no significant effect on the response appreciably. In addition, dancylcadaverine, which is known to block receptor internalization, increased the DA-induced current response. These results suggest that activation of Rho and subsequent PLD may regulate the expression of the DA receptors on the surface membrane of these cells.
Abstracts / Neuroscience Research 58S (2007) S1–S244
P2-b39 The genomic structure and the variants of nitric oxide synthase gene in the terrestrial slug Limax valentianus Ryota Matsuo, Etsuro Ito Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Sanuki, Japan In the terrestrial slug Limax, NO is necessary for the synchronous oscillation of the local field potential in the procerebrum, which is thought to be involved in the odor discrimination and/or odor aversion learning. But there is no description about the genomic structure of the molluscan NO synthase, nor has the evolutionary origin of neuronal NO synthase (NOS) of mammals been clarified. Here we identified two types of NOS mRNAs of the slug, which show differential expression patterns within the brain. One is expressed broadly in the brain but in the low level (limNOS1), whereas, interestingly, the other is almost exclusively in the procerebrum (limNOS2). We also determined the whole genomic structure for limNOS1. It is composed of as many as 32 exons comparable to human nNOS gene, and has very similar exon–intron structure to that of human nNOS. Our results indicated that Limax NOS and human nNOS share the prototypical gene structure of NOS, and that their organization is highly conserved during the evolutionary history.
Delayed reduction of the protein level in the striatum P2-b40 after genetic ablation of the tyrosine hydroxylase gene in the substantia nigra Sakane 1 ,
Sumi-Ichinose 2 ,
Kojima 1 ,
Hiroaki Chiho Masayo Yoshinori Aso 1 , Shin-ichi Muramatsu 3 , Imaharu Nakano 3 , Keiya Ozawa 4 , Takahide Nomura 2 , Ikuko Nagatsu 1 , Toshiharu Nagatsu 2 , Daniel Metzger 5 , Pierre Chambon 5 , Hiroshi Ichinose 1 1 Graduate School of Bioscience & Biotechnology, Tokyo Institute of Technology, Yokohama, Japan; 2 School of Medicine, Fujita Health University, Japan; 3 Division of Neurology, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan; 4 Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan; 5 IGBMC, Illkirch, France We generated floxed TH mice to disrupt the TH gene using adenoassociated viral vector expressing Cre recombinase (AAV-Cre). Injection of AAV-Cre into the substantia nigra pars compacta (SNc) of the floxed TH mice induced recombination of the TH gene, followed by the loss of TH protein in the SNc. A decrease of TH protein in the striatum was much slower than that in the SNc suggesting that the turnover rate of the TH protein would be different between the cell body and the termianal.
P2-b44 Calcium-dependent mechanisms underlying propaga-
tion of ␥PKC translocation along the dendrites of cerebellar Purkinje cell
Hiroshi Tsubokawa 1 , Kaori Kashiwagi 2 , Naoaki Saito 2 , Mitsuyuki Nakao 1 , Norihiro Katayama 1 1 Graduate School of Information Sciences, Tohoku University, Sendai, Japan; 2 Biosignal Research Center, Kobe University, Kobe, Japan Regardless of the importance of protein kinase C (PKC) targeting, little is known concerning PKC translocation in CNS neurons. Especially, it remains unclear how, when and where PKC is targeted in response to Gq/11 -coupled receptor activations. To address this issue, we have generated brain-region specific and inducible ␥PKC-GFP transgenic mice using Tet-regulated system, and found that in cerebellar Purkinje cells electrical stimulation of parallel fibers evoked not only local translocation but also propagation of the translocation along dendritic arbor. Calcium imaging studies have shown that the parallel fiber stimulation also induces slowly propagating calcium wave, which could lead propagation of PKC translocation. To understand mechanism underlying the PKC wave, we constructed a compartmental model of Purkinje cell dendrites. Computer simulations suggest interplay between ␥PKC, Ca2+ channels and K+ channels on the plasma membrane of dendrites.
1 Inhibitory effects of thiamylal on excitation propagation P2-c0 in brain slices Naoshi Fujiwara 1 , Mayumi Ogawa 2 , Kenji Seo 3 Medical Technology, Niigata University, Niigata, Japan; 2 Anesthesiology, Niigata University, Niigata, Japan; 3 Dental Anesthesiology, Niigata University, Niigata, Japan
1
Inhibitory effects of thiamylal (a barbital derivative) on excitation propagation in the cortical and hippocampal slices were characterized by optical imaging techniques. Brain slices of the C57BL6J mouse stained with a voltage sensitive dye RH414 and changes in membrane potential were recorded as video images. In cortical slices, excitation evoked by stimulation to layer V was propagated to layer II–III, and the response was widely extended and prolonged by application of strychnine (20 mol/l). By a successive application of thiamylal (200 mol/l), the initial excitation was evoked but following responses rapidly diminished and disappeared. In hippocampal slices, excitation propagation elicited by stimulation to the CA1 stratum radiatum was also enhanced and prolonged by strychnine. This enhanced and prolonged excitation remained unchanged even in the presence of thiamylal. The results suggest that thiamylal makes excited neurons calm down by enhancing GABAA receptor activity in the cerebral cortex.
Research funds: a grant (No. 17024017) from the program Grants-in-Aid for Scientific Research on Priority Areas, from the ministry if Education, Culture, Science, and Technology of Japan.
P2-b43 N-glycans govern the innate immune response in neu-
2 Facilitation of dopamine-induced Na+ current response P2-c0
rocysticercosis
by Rho GTPase and phospholipase D in Aplysia neurons
V. Prabhakaran, V. Rajshekhar, A. Oommen Neurological Sciences, Christian Medical College, Vellore, India
Satoshi Kawasaki 1 , Shuji Watanabe 1 , Shingo Kimura 1 , Reiko Fujita 2 , Kazuhiko Sasaki 1 1 Department of Physiology, Iwate Medical University, Morioka, Japan; 2 Department of Chemistry, Iwate Medical University, Morioka, Japan
Taenia solium neurocysticercosis is the most common parasitic infection of the CNS & accounts for one-quarter epilepsy cases in South India. Seizures result when cysts degenerate in the brain releasing inflammatory cytokines. It would be useful to identify cellular mediators that govern parasitic destruction and clear infection. Aim of the study was to evaluate protective innate immune responses in T. solium infection. Peripheral blood mononuclear cells from healthy volunteers (obtained with informed consent) were stimulated for 24 h with 6 infection specific T. solium glycoproteins, their peptides or N-glycan components. We identify nitric oxide and IL-12 in culture supernatants and HSP-70 in cells, as mediators of protective innate immunity. Neither T. solium glycoproteins nor their peptide components elicited a response from the cells. However T. solium N-glycans were strong inducers of nitric oxide in macrophages leading to IL-12 upregulation and induction of HSP-70 that was cytoprotective. The role of T. solium N-glycans as mediators of protective innate immunity in neurocysticercosis will be discussed. Research fund: ICMR, New Delhi
Stimulation of D1 type of dopamine (DA)-receptor induces a slow Na+ current response in identified Aplysia neurons under voltage clamp. We previously reported that the Na+ current response to DA is facilitated by the activation of Rho GTPase. In the present study, we investigated possible effector molecules located at the downstream of the Rho. The Na+ current response to DA was depressed after intracellular injection of ␣synuclein, an inhibitory protein for phospholipase D (PLD). Furthermore, application of 1-butanol, which is known to inhibit PLD activity, significantly depressed the DA-induced current response, whereas 2-butanol, an inactive isomer of 1-butanol, had no significant effect on the response appreciably. In addition, dancylcadaverine, which is known to block receptor internalization, increased the DA-induced current response. These results suggest that activation of Rho and subsequent PLD may regulate the expression of the DA receptors on the surface membrane of these cells.