- Email: [email protected]
Single-cell electroporation technique applied to cultured cerebellar neurons
Abstracts / Neuroscience Research 68S (2010) e109–e222
technique is used to detect protein-protein interaction with high S/N ratio. In this technique, firefly luciferase was cleaved into N-terminal and C-terminal segments. Each segment was fused with one of two interacting proteins. By the interaction between these two fusion proteins, split segments complement each other to be a functional luciferase that can emit light. In this report, we tried to analyze the relationship between actin polymerization and cell death. First, we observed that treatment with the F-actin disrupting agent latrunculin A had a protective or accelerative effect on UV-induced HEK293T cell death depending on the timing of the treatment. To analyze how F-actin organization is regulated after UV irradiation and latrunculin A treatment, we employed our split luciferase-based probe proteins that could measure the amount of F-actin in real time in live cells. We observed that F-actin was transiently upregulated and gradually decreased after UV irradiation. Interestingly, after the cells were treated with latrunculin A for 1 h and washed out, the amount of F-actin was also upregulated transiently, and the treatment induced a protective effect against UV-induced cell death. Our results with real-time imaging of F-actin suggest that upregulation of F-actin is a protective reaction of the cells against UV-induced cell death stimulus. This technique can be applied to analyze the dynamics of actin cytoskeleton in neurons. doi:10.1016/j.neures.2010.07.2547
P1-s10 In situ monitoring of differentiation of PC12 cells by drug response observation using enzyme-luminescene method Tanveer Ahamd Mir 1 , Hiroaki Shinohara 1,2 1
Graduate School of Innovative Life Science, University of Toyama 2 Graduate School of Science and Engineering, University of Toyama The purpose of this study is to develop new method which performs realtime monitoring of cell differentiaiton. We paied attention to cell response for drug stimulation. Dopamine release from PC12 cells upon muscarine stimulation before and after differentiation by NGF addition was measured in situ by enzyme-luminescence method with tyramine oxidase, peroxidase and luminol. The in situ monitoring of remarkable enhancement of dopamine release ability of the cells might be applicable for cell differentiation monitoring in living state except cell morphology observation. doi:10.1016/j.neures.2010.07.2548
P1-s11 Conditional suppression of rho GTPase signaling pathway in specific neuronal types with Cre-loxP recombination driven by double lentiviral vector system Kenta Kobayashi 1 Kobayashi 1
, Shigeki
Kato 1 , Kozo
Kaibuchi 2 , Kazuto
1
Dept Mol Genet, Fukushima Med Univ, Fukushima 2 Dept Cell Pharmacol, Nagoya Univ Grad School Med, Nagoya Rho GTPase regulates various neuronal functions, including axon patterning, synapse formation, apoptosis and survival, through some effector proteins. However, the role of Rho GTPase signaling pathway in the brain function remains to be elucidated. In the present study, we generated two kinds of recombinant lentiviral (rLV) vectors. One is pseudotyped with vesicular somatitis virus glycoprotein (VSV-G) and the other is pseudotyped with rabies virus glycoprotein (RV-G). RV-G-pseudotyped rLV vector was recently developed as an efficient gene transfer system through retrograde transport in the brain by us. The VSV-G-pseudotyped rLV vector contains Cre recombinase gene and the RV-G-pseudotyped rLV vector contains flox-EGFP/RhoA dominant-negative mutant (EGFP/RhoA DN). RV-G-pseudotyped rLV vector containing flox-EGFP/RhoA DN was injected into the dorsal striatum, and VSV-G-pseudotyped rLV vector containing Cre recombinase gene was injected into the cerebral cortex, thalamus, and ventral midbrain, each of which innervates the striatum. We observed the expression of EGFP/RhoA DN induced by Cre-loxP mediated recombination system in the cerebral cortex, thalamus, and ventral midbrain. The lentiviral vector system combined with Cre-loxP recombination system will provide a powerful tool to analyze the in vivo function of Rho GTPase signaling pathway. doi:10.1016/j.neures.2010.07.2549
e221
P1-s12 Single-cell electroporation technique applied to cultured cerebellar neurons Masahiko Tanaka 1 , Yuchio Yanagawa 2,3 , Naohide Hirashima 1 1
Dept Cell Biophys, Grad Sch Pharmaceut Sci, Nagoya City Univ, Nagoya Dept Genet Behav Neurosci, Grad Sch Med, Gunma Univ, Maebashi 3 CREST, JST, Tokyo
2
Single-cell electroporation using micropipettes (patch-clamp electrode) is a method to introduce polar and charged molecules into single and identified cells (Haas et al., 2001; Rae & Levis, 2002; Rathenberg et al., 2003). This feature is advantageous especially in investigations of the nervous system because the nervous system is composed of various types of cells. Using this method, we transferred several types of molecules into mouse cerebellar Purkinje and Golgi cells in dissociated cell cultures. Fluorescent dyes (Lucifer Yellow, Alexa Fluor) and calcium ion indicators (fluo-3, Oregon Green 488 BAPTA-1) were easily transferred, although the fluorescence of these dyes became very weak several hours to one day after electroporation. Next, we transferred small interfering RNA (siRNA) against green fluorescent protein (GFP) into GFP-expressing Purkinje and Golgi cells (J. Neurosci. Meth. 178:80, 2009). The temporal changes in the intensity of GFP fluorescence in the same electroporated cells were monitored in real time over 10 days after electroporation by confocal microscopic imaging. Under the optimal conditions, GFP siRNA markedly reduced GFP fluorescence in the electroporated cells by 4 days after electroporation at the latest, whereas negative control siRNA had no effects. Sufficient reduction in GFP fluorescence continued until at least 10 days after electroporation in many electroporated cells. siRNAs against several endogenous genes such as calcium/calmodulin-dependent protein kinase ␣ and  were also transferred. As the results, immunocytochemical signals for each protein were reduced, showing that this method is effective in RNA interference of endogenous as well as exogenous genes. So far, the expression of GFP by the electroporation of GFP expression plasmids has not been observed. Thus, single-cell electroporation of siRNAs is a useful method for examining roles of genes involved in neuronal differentiation, function and degeneration. doi:10.1016/j.neures.2010.07.2550
P1-s13 Japan-specific diagnostic criteria of disorders of consciousness can contribute to improvements of the clical practices Soichiro Toda 1 , Chiaki Kagawa 2 1
Dept Health Sciences, Univ of Tokyo, Tokyo Yamanashi, Chuoo, Japan
2
Dept Clin Ethics, Univ of
A vegetative state (VS) is a widely known but complex clinical condition resulted from severe brain injury. While the pathology of absence of consciousness and the nature of its recovery is still poorly understood, VS can be clearly distinguished from a minimally conscious state (MCS). Healthcare professionals in Western countries have striven to distinguish VS from MCS and to elucidate the “gray zone” between the two conditions in order to predict the prognosis, manage patients appropriately, and tackle the clinical issues regarding end-of-life care. On the other hand, the definition of the “vegetative state (termed as VSJ in this report)” in Japan encompasses both a VS and MCS, which reflects the country-specific ideology in clinical care. However, whether the ambiguity and the cultural influence of the diagnostic criteria could positively contribute to the care for patients as well as the studies of consciousness has not yet been discussed. This report discusses the possibility of utilizing VSJ criteria which could provide a different quality of care for patients from that of Western Countries. The clarification and refinement of the diagnostic categories and their criteria is necessary to unravel the neural substrate underlying consciousness. Nonetheless, the accumulation of the practical knowledge based on VSJ could contribute to the establishment of sound diagnostic procedures in Western countries by providing supplemental data. The analysis includes the rate of misdiagnosis, the quality of care, and the effects of the interventions. In doing so, the advantages and the future direction of the VSJ criteria and its relationship with the VS/MCS category will be illustrated. Balancing the scientific and ethical frameworks of VSJ and VS/MCS contributes to the refinement and the modification of the MCS criteria. Moreover, it helps the connected parties to make appropriate end-of-life decisions. doi:10.1016/j.neures.2010.07.2551
technique is used to detect protein-protein interaction with high S/N ratio. In this technique, firefly luciferase was cleaved into N-terminal and C-terminal segments. Each segment was fused with one of two interacting proteins. By the interaction between these two fusion proteins, split segments complement each other to be a functional luciferase that can emit light. In this report, we tried to analyze the relationship between actin polymerization and cell death. First, we observed that treatment with the F-actin disrupting agent latrunculin A had a protective or accelerative effect on UV-induced HEK293T cell death depending on the timing of the treatment. To analyze how F-actin organization is regulated after UV irradiation and latrunculin A treatment, we employed our split luciferase-based probe proteins that could measure the amount of F-actin in real time in live cells. We observed that F-actin was transiently upregulated and gradually decreased after UV irradiation. Interestingly, after the cells were treated with latrunculin A for 1 h and washed out, the amount of F-actin was also upregulated transiently, and the treatment induced a protective effect against UV-induced cell death. Our results with real-time imaging of F-actin suggest that upregulation of F-actin is a protective reaction of the cells against UV-induced cell death stimulus. This technique can be applied to analyze the dynamics of actin cytoskeleton in neurons. doi:10.1016/j.neures.2010.07.2547
P1-s10 In situ monitoring of differentiation of PC12 cells by drug response observation using enzyme-luminescene method Tanveer Ahamd Mir 1 , Hiroaki Shinohara 1,2 1
Graduate School of Innovative Life Science, University of Toyama 2 Graduate School of Science and Engineering, University of Toyama The purpose of this study is to develop new method which performs realtime monitoring of cell differentiaiton. We paied attention to cell response for drug stimulation. Dopamine release from PC12 cells upon muscarine stimulation before and after differentiation by NGF addition was measured in situ by enzyme-luminescence method with tyramine oxidase, peroxidase and luminol. The in situ monitoring of remarkable enhancement of dopamine release ability of the cells might be applicable for cell differentiation monitoring in living state except cell morphology observation. doi:10.1016/j.neures.2010.07.2548
P1-s11 Conditional suppression of rho GTPase signaling pathway in specific neuronal types with Cre-loxP recombination driven by double lentiviral vector system Kenta Kobayashi 1 Kobayashi 1
, Shigeki
Kato 1 , Kozo
Kaibuchi 2 , Kazuto
1
Dept Mol Genet, Fukushima Med Univ, Fukushima 2 Dept Cell Pharmacol, Nagoya Univ Grad School Med, Nagoya Rho GTPase regulates various neuronal functions, including axon patterning, synapse formation, apoptosis and survival, through some effector proteins. However, the role of Rho GTPase signaling pathway in the brain function remains to be elucidated. In the present study, we generated two kinds of recombinant lentiviral (rLV) vectors. One is pseudotyped with vesicular somatitis virus glycoprotein (VSV-G) and the other is pseudotyped with rabies virus glycoprotein (RV-G). RV-G-pseudotyped rLV vector was recently developed as an efficient gene transfer system through retrograde transport in the brain by us. The VSV-G-pseudotyped rLV vector contains Cre recombinase gene and the RV-G-pseudotyped rLV vector contains flox-EGFP/RhoA dominant-negative mutant (EGFP/RhoA DN). RV-G-pseudotyped rLV vector containing flox-EGFP/RhoA DN was injected into the dorsal striatum, and VSV-G-pseudotyped rLV vector containing Cre recombinase gene was injected into the cerebral cortex, thalamus, and ventral midbrain, each of which innervates the striatum. We observed the expression of EGFP/RhoA DN induced by Cre-loxP mediated recombination system in the cerebral cortex, thalamus, and ventral midbrain. The lentiviral vector system combined with Cre-loxP recombination system will provide a powerful tool to analyze the in vivo function of Rho GTPase signaling pathway. doi:10.1016/j.neures.2010.07.2549
e221
P1-s12 Single-cell electroporation technique applied to cultured cerebellar neurons Masahiko Tanaka 1 , Yuchio Yanagawa 2,3 , Naohide Hirashima 1 1
Dept Cell Biophys, Grad Sch Pharmaceut Sci, Nagoya City Univ, Nagoya Dept Genet Behav Neurosci, Grad Sch Med, Gunma Univ, Maebashi 3 CREST, JST, Tokyo
2
Single-cell electroporation using micropipettes (patch-clamp electrode) is a method to introduce polar and charged molecules into single and identified cells (Haas et al., 2001; Rae & Levis, 2002; Rathenberg et al., 2003). This feature is advantageous especially in investigations of the nervous system because the nervous system is composed of various types of cells. Using this method, we transferred several types of molecules into mouse cerebellar Purkinje and Golgi cells in dissociated cell cultures. Fluorescent dyes (Lucifer Yellow, Alexa Fluor) and calcium ion indicators (fluo-3, Oregon Green 488 BAPTA-1) were easily transferred, although the fluorescence of these dyes became very weak several hours to one day after electroporation. Next, we transferred small interfering RNA (siRNA) against green fluorescent protein (GFP) into GFP-expressing Purkinje and Golgi cells (J. Neurosci. Meth. 178:80, 2009). The temporal changes in the intensity of GFP fluorescence in the same electroporated cells were monitored in real time over 10 days after electroporation by confocal microscopic imaging. Under the optimal conditions, GFP siRNA markedly reduced GFP fluorescence in the electroporated cells by 4 days after electroporation at the latest, whereas negative control siRNA had no effects. Sufficient reduction in GFP fluorescence continued until at least 10 days after electroporation in many electroporated cells. siRNAs against several endogenous genes such as calcium/calmodulin-dependent protein kinase ␣ and  were also transferred. As the results, immunocytochemical signals for each protein were reduced, showing that this method is effective in RNA interference of endogenous as well as exogenous genes. So far, the expression of GFP by the electroporation of GFP expression plasmids has not been observed. Thus, single-cell electroporation of siRNAs is a useful method for examining roles of genes involved in neuronal differentiation, function and degeneration. doi:10.1016/j.neures.2010.07.2550
P1-s13 Japan-specific diagnostic criteria of disorders of consciousness can contribute to improvements of the clical practices Soichiro Toda 1 , Chiaki Kagawa 2 1
Dept Health Sciences, Univ of Tokyo, Tokyo Yamanashi, Chuoo, Japan
2
Dept Clin Ethics, Univ of
A vegetative state (VS) is a widely known but complex clinical condition resulted from severe brain injury. While the pathology of absence of consciousness and the nature of its recovery is still poorly understood, VS can be clearly distinguished from a minimally conscious state (MCS). Healthcare professionals in Western countries have striven to distinguish VS from MCS and to elucidate the “gray zone” between the two conditions in order to predict the prognosis, manage patients appropriately, and tackle the clinical issues regarding end-of-life care. On the other hand, the definition of the “vegetative state (termed as VSJ in this report)” in Japan encompasses both a VS and MCS, which reflects the country-specific ideology in clinical care. However, whether the ambiguity and the cultural influence of the diagnostic criteria could positively contribute to the care for patients as well as the studies of consciousness has not yet been discussed. This report discusses the possibility of utilizing VSJ criteria which could provide a different quality of care for patients from that of Western Countries. The clarification and refinement of the diagnostic categories and their criteria is necessary to unravel the neural substrate underlying consciousness. Nonetheless, the accumulation of the practical knowledge based on VSJ could contribute to the establishment of sound diagnostic procedures in Western countries by providing supplemental data. The analysis includes the rate of misdiagnosis, the quality of care, and the effects of the interventions. In doing so, the advantages and the future direction of the VSJ criteria and its relationship with the VS/MCS category will be illustrated. Balancing the scientific and ethical frameworks of VSJ and VS/MCS contributes to the refinement and the modification of the MCS criteria. Moreover, it helps the connected parties to make appropriate end-of-life decisions. doi:10.1016/j.neures.2010.07.2551