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Involvement of zinc transport system in zinc-induced microglial activation
Abstracts / Neuroscience Research 68S (2010) e109–e222
e127
[3 H]thymidine incorporation into cultured astrocytes in the presence and absence of H2 O2 were suppressed by the removal of extracellular Na+ . In the condition of the removal of extracellular Na+ [3 H]thymidine incorporation into cultured astrocytes in the presence and absence of H2 O2 were suppressed by 1 mM dipyridamole, which inhibit ENT2, but not 100 nM nitrobenzylthioinosine, which inhibit ENT1. And it is known that thymidine is not selectively incorporated via CNT2. H2 O2 -induced delayed cell death was promoted by ENT inhibitors and by the removal of extracellular Na+ . These finding indicate that the decrease of H2 O2 -induced thymidine incorporation via ENT2 and CNT3 might be relative to delayed cell death in cultured astrocytes.
of mGluR5s in processes was 1.5 times higher than that in the soma. To elucidate the mechanism underlying this polarized distribution of mGluR5, we examined the diffusion dynamics of mGluR5 molecules on the cell surface. By FRAP experiment with pHluorin-tagged mGluR5, we demonstrated that the migration of mGluR5s from the process into the soma was significantly slowed down. Furthermore, single molecule imaging of endogenous mGluR5s labeled with quantum dots revealed that the lateral diffusion of mGluR5s on the process was confined in the same process. These results strongly suggest the existence of diffusion barrier between the soma and the process of astrocytes, and that this diffusion barrier could contribute to maintain higher mGluR5 density and the higher Ca2+ responsiveness in astrocytic processes.
doi:10.1016/j.neures.2010.07.2131
doi:10.1016/j.neures.2010.07.2133
P1-c19 Developmental change of VGAT-Venus expression in organotypic cultured cerebellar cortex and cultured glial cells
P1-c21 Strain difference in regulatory mechanism of P2X7 receptor expressed by cultured mouse astrocytes
1 Department of Environmental and Life Sciences, Toyohashi University of Technology 2 Honda Electronics Co., Ltd., Toyohashi 3 Aichi Institute of Technology, Toyota 4 Dept. of Anat. 2, Univ. of the Ryukyu Fac. of Med. Okinawa 5 Hamamatsu Univ. Sch. of Med. Hamamatsu
Dept. of Environ. Biochem., Kyoto Pharm. Univ
Yui Kurimoto 1 , Tadashi Ogo 1 , Kazuto Kobayashi 2 , Naohiro Hozumi 3 , Chitoshi Takayama 4 , Atsuo Fukuda 5 , Sachiko Yoshida 1
␥-Amino butyric acid (GABA) is known to play important roles as a modulator in the survival and development of cerebellar cortex. To assist an observation of GABAergic neurons, we produced the vesicular GABA transporter (VGAT)Venus (VGAT-Venus) transgenic rats. In cerebellar cortex of the VGAT-Venus rats, most GABAergic neurons involving Golgi cells, basket cells and stellate cells were positive for Venus, whereas Purkinje cells were almost negative in vivo. In developing cerebellum, on the other hand, many Venus positive cells were observed underneath the Purkinje cell layer and in the external granular layer (EGL), where ambient GABA was observed. Although no GABAergic neurons located there, fibrous glial cells expressed VGAT during the first postnatal week. Thereafter, VGAT-Venus in the glial cells was eliminated. In this study, we observed distribution and elimination of VGAT in culture of dissociated glial cells and organotypic culture of neonatal cerebellar cortex. Venus fluorescence and ambient GABA in the EGL of organotypic culture was observed after 24 h in vitro, then, both Venus and GABA release in the EGL decreased gradually. This ectopic VGAT expression was also observed even in the undifferentiated dissociated glial culture, which was remained for several weeks. Differentiated fibrous glial cells in vitro, however, exhibited weak Venus fluorescence. We would report an interaction between VGAT expression and intracellular fibrous structure in glial cells using the highresolution impedance microscopy, and discuss physiological roles as well as an elimination process of glial VGAT. doi:10.1016/j.neures.2010.07.2132
Misa Arizono 1,2 , Takeshi Nakamura 3 , Kyoko Nakamura 3 , Masahiro Enomoto 1 , Akitoshi Miyamoto 1,2 , Hiroko Bannai 1 , Katsuhiko Mikoshiba 1 Lab. Dev. Neurobiol., BSI RIKEN Wako ICORP
Objective: Activation of P2X7 receptor (P2X7R) by high levels of ATP under pathological conditions such as ischemia, forms cationic channel, and its prolonged activation triggers non-selective pore opening, resulting cell death. Previously, we demonstrated that P2X7R expressed by cultured astrocytes obtained SJL-strain mice was constitutively activated without any exogenous stimulation, suggesting its possible role in physiological conditions such as cellular communication. SJL-strain mice are characterized to have strong fighting tendency, and thus it is interesting to clarify the molecular mechanism underlying constitutive activation of P2X7R expressed by this strain astrocytes. In this study, we characterized the functional expression of P2X7R in cultured astrocytes obtained from ddY mice characterized to be mild, and compared the obtained findings with those of SJL-strain mouse astrocytes. Methods: Primary cultured astrocytes were prepared as previously reported. P2X7R channel opening was determined by YO-PRO-1 (YP) uptake. siRNA was transfected to cells by a lipofection method. Expression of P2X7R was evaluated by western blot. Result and Discussion: Under basal conditions, YP uptake by ddY mouse astrocytes was less than that by SJL mouse astrocytes. Differing from the case of SJL mouse astrocytes, YP uptake by the ddY mouse astrocytes was only partially inhibited by a selective antagonist for P2X7R, oxATP and was not affected by P2X7R-knockdown with siRNA transfection. Expression level of P2X7R in ddY mouse astrocytes was almost comparable with that in SJL mouse astrocytes, and their nucleotide sequences were the same. These results suggested that there might be differences in regulatory mechanism of P2X7R activity between astrocytes obtained from these mouse strains. The more detailed investigations are in progress in our laboratory. doi:10.1016/j.neures.2010.07.2134
P1-c20 Astrocyte processes exhibit confined mGluR5 diffusion to maintain high mGluR5 density and high sensitivity to mGluR agonist
1
Yuka Kido , Chiyo Kawahara, Yasuko Terai, Manami Fukagawa, Jyunki Kato, Yukari Dohi, Hiroto Okuda, Nishida Kentaro, Kazuki Nagasawa
2
Inst. Med. Sci., Univ. Tokyo
3
JST,
Astrocytes in the central nervous system actively regulate two critical factors in brain function, namely neuronal transmission and local blood flow, and Ca2+ elevation in astrocytic processes plays crucial role in the regulation of its interacting partners such as synapses and blood vessels. One of the main pathways responsible for the Ca2+ elevation in astrocytes is the Ca2+ release from the endplasmic reticulum through the IP3 receptor evoked by the activation of the metabotropic gluatamate receptor type 5 (mGluR5). In the present study, we aimed to characterize this mGluR-mediated Ca2+ signal in the processes of astrocytes. A genetically-encoded Ca2+ indicator GCaMP2 transfected in cultured hippocampal astrocytes enabled us to monitor detailed Ca2+ dynamics in fine processes. We report here that the astrocytic processes have higher Ca2+ responsiveness to the mGluR agonist than the soma, while the machinery for IP3 -induced Ca2+ release was equally functional both at the soma and processes. The immunocytochemistry revealed that the distribution of mGluR5s was polarized: the density
P1-c22 Involvement of zinc transport system in zincinduced microglial activation Takeshi Nishiura 1 , Shohei Segawa 1 , Shogo Nakamura 1 , Yuki Ohsato 1 , Misaki Tani 1 , Kentaro Nishida 1 , Youichirou Higashi 2 , Kazuki Nagasawa 1 1 2
Department of environmental biochemistry, Kyoto Pharm. Deparment of Neurosurgery, Medical School, Kochi University
Univ
Objective: Zinc has an important role as a modulator of synaptic transmission. Under ischemic conditions, excess amount of zinc is released from presynaptic vesicles of neurons, and triggers microglial activation and eventually neuronal death. We previously showed that ATP release from microglia via hemichannels and activation of P2 receptors in autocrine/paracrine manners were involved in zinc-induced microglial activation. However, it is not clear whether zinc should be taken up into microglia to activate them. Recently, Zrt- and Irt-like proteins (ZIP) family has been identified as zinc influx system, and some of the family are revealed to be expressed ubiquitously in mammals. In the present study, we examined whether zinc influx into microglia via ZIP isoforms mediated zinc-induced microglial activation. Methods: Primary cultured microglial cells were maintained in FBSsupplemented EMEM. 65Zn uptake was analyzed by an inhibitor-stop method. Expression of mRNA for ZIP isoforms was determined by real-time PCR. Microglial activation was evaluated by morphological changes, superoxide generation mediated by NADPH oxidase and PAR accumulation as a
e128
Abstracts / Neuroscience Research 68S (2010) e109–e222
marker for PARP activation. ATP release from microglia was measured with a two-enzyme assay system. Results & Discussion: 65Zn uptake by microglia exhibited time- and temperature-dependence and saturation kinetics. Among ZIP isoforms, ZIP1 and ZIP7 were highly expressed by microglia. NiCl2, relatively ZIP1selective substrate/inhibitor, showed cis- and trans-inhibitory effect on 65Zn uptake. Furthermore, an intracellular zinc chelator N,N,N ,N -tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) almost completely blocked morphological changes, superoxide generation, PAR accumulation and also ATP release in microglia exposed to zinc. Collectively, these results suggest that zinc influx through ZIP1 might be a key step in zinc-induced microglial activation. doi:10.1016/j.neures.2010.07.2135
P1-c23 Astrocytes modulate action potential waveforms in the middle of axonal conduction Takuya Sasaki 1 , Norio Matsuki 1 , Yuji Ikegaya 1,2 1
Lab Chem Pharmacol, Grad Sch Pharm Sci, Univ of Tokyo, Tokyo 2 Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Japan Once initiated, an action potential is believed to propagate uniformly to the axon terminal. Here we report, however, that astrocytes surrounding the axon modulate the waveform of an action potential in the middle of axonal conduction and exert a long-range modulation of downstream synaptic transmission. In hippocampal slices ex vivo, synaptically coupled neuron pairs were patch-clamped, and the axon pathway was visualized with fluorescent dye. Astrocytes near the presynaptic axon were loaded with calcium fluorescent indicator and caged-calcium compound and activated by uncaging with UV flash . The uncaging led to an increase in the amplitude of unitary synaptic currents that occurred in the downstream postsynaptic neurons. This effect was abolished by application of glutamate receptor antagonists to the uncaged region. We simultaneously carried out axonal and somatic patch-clamp recordings from a single pyramidal cell. Photoactivation of astrocytes near the axon increased the duration of an action potential in the axon. These results suggest that calcium activity of axonal astrocytes facilitates synaptic transmission onto postsynaptic neurons far apart from these astrocytes by modulating presynaptic axon conduction. This new form of the neuro-glial interactions indicates that astrocytes’ influence reaches more widely than previously believed. doi:10.1016/j.neures.2010.07.2136
P1-c24 Real time imaging analysis of microglial condition in acute brain slice Junya Masuda , Hidetoshi Tozaki-Saitoh, Makoto Tsuda, Kazuhide Inoue Dept. Mol. Syst. Pharmacol., Grad. Sch. Pharm. Sci., Kyushu Univ Microglia play main roles in immune defense of central nervous system (CNS). Recent evidence showing microglial highly dynamic function has also supported this concept by real time imaging of their unique response to local CNS injury. This response is observed as rapid process extension toward the lesion site, which is the well-characterized reaction of resident microglia in order to maintain CNS homeostasis. These rapid microglial behaviors are directly observable by using two-photon microscopy in vivo or ex vivo. However, the influence of experimental procedure on sensitive microglial activity remains to be carefully assessed especially in ex vivo tissue slice preparation. Therefore we analyzed microglial state in acute brain slices by observing their morphological and motile changes with two-photon imaging. In the early time after slice preparation, microglia in slice behaved similarly as those in vivo. Then, they gradually retracted processes, reduced process branching, and finally became amoeboid form. By tracking process ends of microglia in short time movie and measuring process motility rate at each time point after slicing, it was found that microglial dynamic process motility, an indication of sensing activity, started to increase before morphological transformation occurred. These changes were also affected by the depth of microglial location from sliced surface and the temperature condition in imaging chamber. Our present findings suggest that we should consider the dynamic aspect of microglia to refer to their activation state and provide new information that there is time-, depth-, and temperature-restriction in optimal ex vivo experiments for the study of native microglia. doi:10.1016/j.neures.2010.07.2137
P1-c25 Observation of glial cell damage using enzymelinked optical assay and a high-resolution acoustic impedance microscope Keiichi Maruko 1 Kobayashi 3 , Seiji Yoshida 1
, Keigo Aoki 1 , Michio Yamamoto 4 , Naohiro
Horiuchi 2 , Kazuto Hozumi 5 , Sachiko
1 Department of Environmental and Life Sciences, Toyohashi University of Technology 2 System Instruments Co.,Ltd., Tokyo, Japan 3 Honda Electronics Co., Ltd., Toyohashi 4 Hamamatsu Univ. School of Medicine, Hamamatsu 5 Aichi Inst. of Technology, Toyota
Cell damage and death occur due to inflammatory lesions or canceration, and progress surrounding organs. We have reported that spatial distribution of Lactate dehydrogenase (LDH) leakage could be detected using optical assay device and CCD camera. LDH leakage was increased with heat shock or the application of cytotoxin. In addition, we have reported intracellular cytoskeletal change could be visualized with the high-resolution acoustic impedance microscopy with 320 MHz transducer. The intracellular impedance would reflect on the intracellular localization of F-actin and/or microtubules. In this report, to investigate the progress of cell damage to several cytotoxins, actinomycin D or anisomycin, we observed the change of intracellular impedance to cytotoxin and compared their LDH leakage. Rat cerebral glial cells were cultured on OptiCellTM , and observed with the acoustic microscopy through the OptiCellTM film, or with the optical assay device on cut OptiCellTM film. Intracellar impedance was affected mainly by F-actin, not by microtubules, and treatment of actinomycin-D decreased intracellular impedance remarkably. Furthermore, both actinomycin-D and anisomycin showed linear increment of LDH leakage, whereas ultraviolet light showed transient increment of LDH. We suggested that the high-resolution acoustic microscopy and LDH photoassay device would be fine tools to detect cell damages and deformation of cytoskeletons. doi:10.1016/j.neures.2010.07.2138
P1-c26 Novel assays for neurotoxicity, neural development and neuronal/glia interactionsvia multiplexed HCA of neurons and astrocytes Hitomi Fukada 1 , Janet L. Anderl 2 , Andrew J. Ball 2 1
Life Sciences Division, Nihon Millipore K.K 2 Millipore Corporation
In the brain, astrocytes are integral to maintenance of homeostasis. Glial Fibrillary Acidic Protein (GFAP) is an intermediate filament protein expressed in astrocytes. GFAP is also upregulated in gliosis, commonly accompanied by astrocyte proliferation and hypertrophy. Gliosis is a hallmark of nervous system injury, thus GFAP can serve as a biomarker of neurotoxicity. HCA represents a novel tool to simultaneously measure multiple features associated with gliosis within a single assay. Neuronal tolerance of some toxins is substantially increased by the proximity of astrocytes. We hypothesized that HCA could be utilized to develop novel assays for neurotoxicity using astrocytes and neuronal-astrocytic co-cultures. After screening a panel of antibodies for antigen specificity, cell type specificity, signal-to background ratio and ability to quantitate cellular differentiation and cytotoxicity, we obtained a combination of detection reagents including GFAP and III-tubulin antibodies suitable for multiplexed analysis of neuronal and astrocytic parameters. We show that HCA can be used successfully to measure changes in astrocyte number, morphology, and GFAP expression, and that changes in each of these parameters in response to a panel of neurotoxins can readily be quantified. In neuronalastrocytic co-culture models analyzing expression of GFAP and III-tubulin, we were able to simultaneously quantitate neuronal and astrocytic neurotoxic endpoints, and to demonstrate a neuroprotective effect of astrocytes upon neurons in response to some toxins. We also showed that additional parameters e.g. synaptophysin and vimentin expression may be incorporated into these assays, enabling four-color screens generating additional phenotypic/mechanistic data. In summary, we have developed novel HCA assays that offer a means of large-scale, non-subjective, quantitative neurotoxicity assessment via multiple parameters in astrocytes, neurons, and neuronal-astrocytic cocultures. doi:10.1016/j.neures.2010.07.2139
e127
[3 H]thymidine incorporation into cultured astrocytes in the presence and absence of H2 O2 were suppressed by the removal of extracellular Na+ . In the condition of the removal of extracellular Na+ [3 H]thymidine incorporation into cultured astrocytes in the presence and absence of H2 O2 were suppressed by 1 mM dipyridamole, which inhibit ENT2, but not 100 nM nitrobenzylthioinosine, which inhibit ENT1. And it is known that thymidine is not selectively incorporated via CNT2. H2 O2 -induced delayed cell death was promoted by ENT inhibitors and by the removal of extracellular Na+ . These finding indicate that the decrease of H2 O2 -induced thymidine incorporation via ENT2 and CNT3 might be relative to delayed cell death in cultured astrocytes.
of mGluR5s in processes was 1.5 times higher than that in the soma. To elucidate the mechanism underlying this polarized distribution of mGluR5, we examined the diffusion dynamics of mGluR5 molecules on the cell surface. By FRAP experiment with pHluorin-tagged mGluR5, we demonstrated that the migration of mGluR5s from the process into the soma was significantly slowed down. Furthermore, single molecule imaging of endogenous mGluR5s labeled with quantum dots revealed that the lateral diffusion of mGluR5s on the process was confined in the same process. These results strongly suggest the existence of diffusion barrier between the soma and the process of astrocytes, and that this diffusion barrier could contribute to maintain higher mGluR5 density and the higher Ca2+ responsiveness in astrocytic processes.
doi:10.1016/j.neures.2010.07.2131
doi:10.1016/j.neures.2010.07.2133
P1-c19 Developmental change of VGAT-Venus expression in organotypic cultured cerebellar cortex and cultured glial cells
P1-c21 Strain difference in regulatory mechanism of P2X7 receptor expressed by cultured mouse astrocytes
1 Department of Environmental and Life Sciences, Toyohashi University of Technology 2 Honda Electronics Co., Ltd., Toyohashi 3 Aichi Institute of Technology, Toyota 4 Dept. of Anat. 2, Univ. of the Ryukyu Fac. of Med. Okinawa 5 Hamamatsu Univ. Sch. of Med. Hamamatsu
Dept. of Environ. Biochem., Kyoto Pharm. Univ
Yui Kurimoto 1 , Tadashi Ogo 1 , Kazuto Kobayashi 2 , Naohiro Hozumi 3 , Chitoshi Takayama 4 , Atsuo Fukuda 5 , Sachiko Yoshida 1
␥-Amino butyric acid (GABA) is known to play important roles as a modulator in the survival and development of cerebellar cortex. To assist an observation of GABAergic neurons, we produced the vesicular GABA transporter (VGAT)Venus (VGAT-Venus) transgenic rats. In cerebellar cortex of the VGAT-Venus rats, most GABAergic neurons involving Golgi cells, basket cells and stellate cells were positive for Venus, whereas Purkinje cells were almost negative in vivo. In developing cerebellum, on the other hand, many Venus positive cells were observed underneath the Purkinje cell layer and in the external granular layer (EGL), where ambient GABA was observed. Although no GABAergic neurons located there, fibrous glial cells expressed VGAT during the first postnatal week. Thereafter, VGAT-Venus in the glial cells was eliminated. In this study, we observed distribution and elimination of VGAT in culture of dissociated glial cells and organotypic culture of neonatal cerebellar cortex. Venus fluorescence and ambient GABA in the EGL of organotypic culture was observed after 24 h in vitro, then, both Venus and GABA release in the EGL decreased gradually. This ectopic VGAT expression was also observed even in the undifferentiated dissociated glial culture, which was remained for several weeks. Differentiated fibrous glial cells in vitro, however, exhibited weak Venus fluorescence. We would report an interaction between VGAT expression and intracellular fibrous structure in glial cells using the highresolution impedance microscopy, and discuss physiological roles as well as an elimination process of glial VGAT. doi:10.1016/j.neures.2010.07.2132
Misa Arizono 1,2 , Takeshi Nakamura 3 , Kyoko Nakamura 3 , Masahiro Enomoto 1 , Akitoshi Miyamoto 1,2 , Hiroko Bannai 1 , Katsuhiko Mikoshiba 1 Lab. Dev. Neurobiol., BSI RIKEN Wako ICORP
Objective: Activation of P2X7 receptor (P2X7R) by high levels of ATP under pathological conditions such as ischemia, forms cationic channel, and its prolonged activation triggers non-selective pore opening, resulting cell death. Previously, we demonstrated that P2X7R expressed by cultured astrocytes obtained SJL-strain mice was constitutively activated without any exogenous stimulation, suggesting its possible role in physiological conditions such as cellular communication. SJL-strain mice are characterized to have strong fighting tendency, and thus it is interesting to clarify the molecular mechanism underlying constitutive activation of P2X7R expressed by this strain astrocytes. In this study, we characterized the functional expression of P2X7R in cultured astrocytes obtained from ddY mice characterized to be mild, and compared the obtained findings with those of SJL-strain mouse astrocytes. Methods: Primary cultured astrocytes were prepared as previously reported. P2X7R channel opening was determined by YO-PRO-1 (YP) uptake. siRNA was transfected to cells by a lipofection method. Expression of P2X7R was evaluated by western blot. Result and Discussion: Under basal conditions, YP uptake by ddY mouse astrocytes was less than that by SJL mouse astrocytes. Differing from the case of SJL mouse astrocytes, YP uptake by the ddY mouse astrocytes was only partially inhibited by a selective antagonist for P2X7R, oxATP and was not affected by P2X7R-knockdown with siRNA transfection. Expression level of P2X7R in ddY mouse astrocytes was almost comparable with that in SJL mouse astrocytes, and their nucleotide sequences were the same. These results suggested that there might be differences in regulatory mechanism of P2X7R activity between astrocytes obtained from these mouse strains. The more detailed investigations are in progress in our laboratory. doi:10.1016/j.neures.2010.07.2134
P1-c20 Astrocyte processes exhibit confined mGluR5 diffusion to maintain high mGluR5 density and high sensitivity to mGluR agonist
1
Yuka Kido , Chiyo Kawahara, Yasuko Terai, Manami Fukagawa, Jyunki Kato, Yukari Dohi, Hiroto Okuda, Nishida Kentaro, Kazuki Nagasawa
2
Inst. Med. Sci., Univ. Tokyo
3
JST,
Astrocytes in the central nervous system actively regulate two critical factors in brain function, namely neuronal transmission and local blood flow, and Ca2+ elevation in astrocytic processes plays crucial role in the regulation of its interacting partners such as synapses and blood vessels. One of the main pathways responsible for the Ca2+ elevation in astrocytes is the Ca2+ release from the endplasmic reticulum through the IP3 receptor evoked by the activation of the metabotropic gluatamate receptor type 5 (mGluR5). In the present study, we aimed to characterize this mGluR-mediated Ca2+ signal in the processes of astrocytes. A genetically-encoded Ca2+ indicator GCaMP2 transfected in cultured hippocampal astrocytes enabled us to monitor detailed Ca2+ dynamics in fine processes. We report here that the astrocytic processes have higher Ca2+ responsiveness to the mGluR agonist than the soma, while the machinery for IP3 -induced Ca2+ release was equally functional both at the soma and processes. The immunocytochemistry revealed that the distribution of mGluR5s was polarized: the density
P1-c22 Involvement of zinc transport system in zincinduced microglial activation Takeshi Nishiura 1 , Shohei Segawa 1 , Shogo Nakamura 1 , Yuki Ohsato 1 , Misaki Tani 1 , Kentaro Nishida 1 , Youichirou Higashi 2 , Kazuki Nagasawa 1 1 2
Department of environmental biochemistry, Kyoto Pharm. Deparment of Neurosurgery, Medical School, Kochi University
Univ
Objective: Zinc has an important role as a modulator of synaptic transmission. Under ischemic conditions, excess amount of zinc is released from presynaptic vesicles of neurons, and triggers microglial activation and eventually neuronal death. We previously showed that ATP release from microglia via hemichannels and activation of P2 receptors in autocrine/paracrine manners were involved in zinc-induced microglial activation. However, it is not clear whether zinc should be taken up into microglia to activate them. Recently, Zrt- and Irt-like proteins (ZIP) family has been identified as zinc influx system, and some of the family are revealed to be expressed ubiquitously in mammals. In the present study, we examined whether zinc influx into microglia via ZIP isoforms mediated zinc-induced microglial activation. Methods: Primary cultured microglial cells were maintained in FBSsupplemented EMEM. 65Zn uptake was analyzed by an inhibitor-stop method. Expression of mRNA for ZIP isoforms was determined by real-time PCR. Microglial activation was evaluated by morphological changes, superoxide generation mediated by NADPH oxidase and PAR accumulation as a
e128
Abstracts / Neuroscience Research 68S (2010) e109–e222
marker for PARP activation. ATP release from microglia was measured with a two-enzyme assay system. Results & Discussion: 65Zn uptake by microglia exhibited time- and temperature-dependence and saturation kinetics. Among ZIP isoforms, ZIP1 and ZIP7 were highly expressed by microglia. NiCl2, relatively ZIP1selective substrate/inhibitor, showed cis- and trans-inhibitory effect on 65Zn uptake. Furthermore, an intracellular zinc chelator N,N,N ,N -tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) almost completely blocked morphological changes, superoxide generation, PAR accumulation and also ATP release in microglia exposed to zinc. Collectively, these results suggest that zinc influx through ZIP1 might be a key step in zinc-induced microglial activation. doi:10.1016/j.neures.2010.07.2135
P1-c23 Astrocytes modulate action potential waveforms in the middle of axonal conduction Takuya Sasaki 1 , Norio Matsuki 1 , Yuji Ikegaya 1,2 1
Lab Chem Pharmacol, Grad Sch Pharm Sci, Univ of Tokyo, Tokyo 2 Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Japan Once initiated, an action potential is believed to propagate uniformly to the axon terminal. Here we report, however, that astrocytes surrounding the axon modulate the waveform of an action potential in the middle of axonal conduction and exert a long-range modulation of downstream synaptic transmission. In hippocampal slices ex vivo, synaptically coupled neuron pairs were patch-clamped, and the axon pathway was visualized with fluorescent dye. Astrocytes near the presynaptic axon were loaded with calcium fluorescent indicator and caged-calcium compound and activated by uncaging with UV flash . The uncaging led to an increase in the amplitude of unitary synaptic currents that occurred in the downstream postsynaptic neurons. This effect was abolished by application of glutamate receptor antagonists to the uncaged region. We simultaneously carried out axonal and somatic patch-clamp recordings from a single pyramidal cell. Photoactivation of astrocytes near the axon increased the duration of an action potential in the axon. These results suggest that calcium activity of axonal astrocytes facilitates synaptic transmission onto postsynaptic neurons far apart from these astrocytes by modulating presynaptic axon conduction. This new form of the neuro-glial interactions indicates that astrocytes’ influence reaches more widely than previously believed. doi:10.1016/j.neures.2010.07.2136
P1-c24 Real time imaging analysis of microglial condition in acute brain slice Junya Masuda , Hidetoshi Tozaki-Saitoh, Makoto Tsuda, Kazuhide Inoue Dept. Mol. Syst. Pharmacol., Grad. Sch. Pharm. Sci., Kyushu Univ Microglia play main roles in immune defense of central nervous system (CNS). Recent evidence showing microglial highly dynamic function has also supported this concept by real time imaging of their unique response to local CNS injury. This response is observed as rapid process extension toward the lesion site, which is the well-characterized reaction of resident microglia in order to maintain CNS homeostasis. These rapid microglial behaviors are directly observable by using two-photon microscopy in vivo or ex vivo. However, the influence of experimental procedure on sensitive microglial activity remains to be carefully assessed especially in ex vivo tissue slice preparation. Therefore we analyzed microglial state in acute brain slices by observing their morphological and motile changes with two-photon imaging. In the early time after slice preparation, microglia in slice behaved similarly as those in vivo. Then, they gradually retracted processes, reduced process branching, and finally became amoeboid form. By tracking process ends of microglia in short time movie and measuring process motility rate at each time point after slicing, it was found that microglial dynamic process motility, an indication of sensing activity, started to increase before morphological transformation occurred. These changes were also affected by the depth of microglial location from sliced surface and the temperature condition in imaging chamber. Our present findings suggest that we should consider the dynamic aspect of microglia to refer to their activation state and provide new information that there is time-, depth-, and temperature-restriction in optimal ex vivo experiments for the study of native microglia. doi:10.1016/j.neures.2010.07.2137
P1-c25 Observation of glial cell damage using enzymelinked optical assay and a high-resolution acoustic impedance microscope Keiichi Maruko 1 Kobayashi 3 , Seiji Yoshida 1
, Keigo Aoki 1 , Michio Yamamoto 4 , Naohiro
Horiuchi 2 , Kazuto Hozumi 5 , Sachiko
1 Department of Environmental and Life Sciences, Toyohashi University of Technology 2 System Instruments Co.,Ltd., Tokyo, Japan 3 Honda Electronics Co., Ltd., Toyohashi 4 Hamamatsu Univ. School of Medicine, Hamamatsu 5 Aichi Inst. of Technology, Toyota
Cell damage and death occur due to inflammatory lesions or canceration, and progress surrounding organs. We have reported that spatial distribution of Lactate dehydrogenase (LDH) leakage could be detected using optical assay device and CCD camera. LDH leakage was increased with heat shock or the application of cytotoxin. In addition, we have reported intracellular cytoskeletal change could be visualized with the high-resolution acoustic impedance microscopy with 320 MHz transducer. The intracellular impedance would reflect on the intracellular localization of F-actin and/or microtubules. In this report, to investigate the progress of cell damage to several cytotoxins, actinomycin D or anisomycin, we observed the change of intracellular impedance to cytotoxin and compared their LDH leakage. Rat cerebral glial cells were cultured on OptiCellTM , and observed with the acoustic microscopy through the OptiCellTM film, or with the optical assay device on cut OptiCellTM film. Intracellar impedance was affected mainly by F-actin, not by microtubules, and treatment of actinomycin-D decreased intracellular impedance remarkably. Furthermore, both actinomycin-D and anisomycin showed linear increment of LDH leakage, whereas ultraviolet light showed transient increment of LDH. We suggested that the high-resolution acoustic microscopy and LDH photoassay device would be fine tools to detect cell damages and deformation of cytoskeletons. doi:10.1016/j.neures.2010.07.2138
P1-c26 Novel assays for neurotoxicity, neural development and neuronal/glia interactionsvia multiplexed HCA of neurons and astrocytes Hitomi Fukada 1 , Janet L. Anderl 2 , Andrew J. Ball 2 1
Life Sciences Division, Nihon Millipore K.K 2 Millipore Corporation
In the brain, astrocytes are integral to maintenance of homeostasis. Glial Fibrillary Acidic Protein (GFAP) is an intermediate filament protein expressed in astrocytes. GFAP is also upregulated in gliosis, commonly accompanied by astrocyte proliferation and hypertrophy. Gliosis is a hallmark of nervous system injury, thus GFAP can serve as a biomarker of neurotoxicity. HCA represents a novel tool to simultaneously measure multiple features associated with gliosis within a single assay. Neuronal tolerance of some toxins is substantially increased by the proximity of astrocytes. We hypothesized that HCA could be utilized to develop novel assays for neurotoxicity using astrocytes and neuronal-astrocytic co-cultures. After screening a panel of antibodies for antigen specificity, cell type specificity, signal-to background ratio and ability to quantitate cellular differentiation and cytotoxicity, we obtained a combination of detection reagents including GFAP and III-tubulin antibodies suitable for multiplexed analysis of neuronal and astrocytic parameters. We show that HCA can be used successfully to measure changes in astrocyte number, morphology, and GFAP expression, and that changes in each of these parameters in response to a panel of neurotoxins can readily be quantified. In neuronalastrocytic co-culture models analyzing expression of GFAP and III-tubulin, we were able to simultaneously quantitate neuronal and astrocytic neurotoxic endpoints, and to demonstrate a neuroprotective effect of astrocytes upon neurons in response to some toxins. We also showed that additional parameters e.g. synaptophysin and vimentin expression may be incorporated into these assays, enabling four-color screens generating additional phenotypic/mechanistic data. In summary, we have developed novel HCA assays that offer a means of large-scale, non-subjective, quantitative neurotoxicity assessment via multiple parameters in astrocytes, neurons, and neuronal-astrocytic cocultures. doi:10.1016/j.neures.2010.07.2139