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Inducible expression of retrograde transynaptic genetic tracer in mice
Abstracts / Neuroscience Research 58S (2007) S1–S244
P3-j43 Lycopene uptake to Mongolian gerbil attenuates apop-
S241
P3-j47 Time dependent progesterone-nouroprotection against
tosis in hippocampus induced by ischemia
ischemic brain damages
Kimikazu Fujita 1 , Nobuko Yoshimoto 2 , Toshiaki Kato 3 , Masataka Yamane 4 , Takahiro Inakuma 4 , Yutaka Nagata 1 , Eiichi Miyachi 1 1 Department Physiol., Sch. Med. Fujita Health, UniversityToyoake, Aichi, Japan; 2 Department Nutrition and Food Sciences, Nagoya Bunri University, Col., Japan; 3 Department Cli. Lab. Sci., Fujita Health, University Col., Japan; 4 Research Institute KAGOME Co. Ltd., Japan
Weiyan Cai 1 , Ling Chen 1 , Masahiro Sokabe 2 1 Department of Physiology, Nanjing Medical University, Nanjing, China; 2 Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya, Japan
Lycopene from plant origin is known to have an antioxidant action in tissues. Application of transient ischemia/recirculation to gerbil produced apoptosis in hippocampus after several days. We examined antioxidant effect of lycopene administration to the animal, expecting to find effect of lycopene to attenuate the progress of apoptosis caused by ischemia. Weanings at 21st days after birth were fed on lycopene containing MF food (5 mg/100 g) freely, and then we burdened cerebral ischemia to the animal. Amount of bcl-2 was decreased in the animal without lycopene intake, whereas it was increased clearly in lycopene administered animal after surgery. Increased TUNEL positive cells after ischemia reduced in lycopene treated animal after a week. These results strongly suggest that lycopene can act as an inhibitory modulator against apoptosis in neurons caused by ischemia even at genetic level.
P3-j44 Roles of macrophage-like cells expressing NG2 proteoglycan in ischemic rat brain Junya Tanaka 1 , Hiroaki Matsumoto 2 , Hisaaki Takahashi 1 , Anna Smirkin 1 , Yoshinori Imai 1 , Yoshiaki Kumon 2 , Tkanori Ohnishi 2 1 Department of Molecular and Cellular Physiology, Ehime University, Ehime, Japan; 2 Department of Neurosurgery, Ehime University, Ehime, Japan Macrophage-like cells accumulate in ischemic core in the rat brain after transient middle cerebral artery occlusion (90 min) from 3 to 14 days postreperfusion (dpr). Because such macrophage-like cells expressed NG2 chondroitin sulfate proteoglycan (NG2) as well as a macrophage-marker Iba1+, they were termed BINCs (Brain Iba1+/NG2+ Cells). BINCs were highly proliferative as revealed by labeling with 5-bromodeoxyuridine (BrdU) at 2 dpr. In this study, to gain insights as to whether BINCs play favorable roles in the ischemic events, 5-fluorouracil (5 FU) was intraperitoneally injected to ischemic rats at 2 dpr to kill BINCs specifically. This treatment caused marked loss of BINCs in ischemic core. Nearly half of the 5 FU-injected ischemic rats died by 10 dpr. Ischemic region of the survived rat brains markedly enlarged. These results indicate that BINCs play protective roles in the ischemic events.
Ovarian hormone progesterone (P4) is a particularly interesting candidate agent in treating stroke because of its wide time-window for administration to protect neurons from ischemic neuronal damages. We previously reported in a rat model that a single administration of P4 (4 mg/kg) at either 1 h or 48 h before middle cerebral artery occlusion (MCAO) was able to prevent the ischemia-induced pyramidal cell loss and long-term potentiation (LTP) in the hippocampus as well as the deficits in spatial cognition. Herein, we report that the delayed (48 h) neuroprotection was mediated by P4 receptor, a nuclear transcription factor receptor, followed by Src/ERK signaling. On the other hand, the acute (1 h) neuroprotection seemed to be mediated by an antagonism of a sigma1 receptor by P4. These results indicate that the wide time-window of P4-neuroprotection against ischemic brain damages includes two independent processes, a sigma1 receptor-mediated non-genomic and a P4 receptor-mediated genomic signal pathways.
P3-j48 Inducible expression of retrograde transynaptic genetic tracer in mice Hiromi Sano 1,2 , Mineto Yokoi 1,2 Molecular Neurogenetics Unit, HMRO, Kyoto University Graduate School of Medicine, Kyoto, Japan; 2 CREST, Japan Science and Technology Agency, Kawaguchi, Japan
1
A key step toward understanding of neural function is characterizing the neuronal connectivity. Tetanus toxin C fragment (TTC) is transynaptically and retrogradely transported without the toxin’s pathogenic effect. We introduce a new fusion construct, APTTC, consisting of the truncated human placental alkaline phosphatase with TTC, and generated the transgenic mouse line, tetO-APTTC, for inducible expression of APTTC regulated by tetracycline operator (tetO). In order to examine the connectivity of striatal medium spiny neurons (MSNs), we generated double transgenic mice by mating the tetO-APTTC transgenic mice to the PDE10A2-tTA knock-in heterozygote mice (PDE10A2tTA/+ ), in which tTA is selectively expressed in striatal MSNs. In the double transgenic mice, we found alkaline phosphatase (AP) staining in the tTA expressing regions and other specific regions whose axons are reported to terminate in striatal MSNs with previous tracing studies. Our data indicate that APTTC works as a retrograde transynaptic genetic tracer in mice. Research fund: CREST.
P3-j46 Change and translocation of HSP60 in gerbil hippocampal CA1 region induced by transient ischemia and its protective effect against ischemic damage In Koo Hwang 1 , Ki-Yeon Yoo 2 , Jung Hoon Choi 1 , In Se Lee 1 , Yeo Sung Yoon 1 , Je Kyung Seong 1 , Moo Ho Won 2 1 Department of Anatomy and Cell Biology, Seoul Nat’l Univ, Seoul, South Korea; 2 Department of Anatomy and Neurobiology, Hallym Univ, Chunchon, South Korea We observed changes in heat shock protein 60 (HSP60) in the gerbil hippocampal CA1 region after transient forebrain ischemia and its protective effect against ischemic damage. HSP60 immunoreactivity was increased at 3 h after ischemia/reperfusion (I–R) and peaked 6 h after I–R. At these time points, HSP60 immunoreactivity was detected in the mitochondria. At 24 h after I–R, HSP60 immunoreactivity was detectable in the cytoplasm of hippocampal CA1 region. Thereafter, HSP60 immunoreactivity was nearly disappeared in the hippocampal CA1 region. The HSP60 transfection via the adenovirus significantly increased the neuronal survival after transient forebrain ischemia. This suggests that the translocation and decrease of HSP60 may be associated with neuronal cell death and the induction of HSP60 protects neurons from ischemic damage. Research fund: BK21 Program for Veterinary Science, Seoul National University.
P3-kØ1 Functional transplant of photoactivated adenylyl cyclase into Aplysia mechanosensory neurons Tatsumi Nagahama 1 , Takeshi Suzuki 2 , Shinya Yoshikawa 3 , Mineo Iseki 2 1 Department of Biophysics, Faculty of Pharmaceutical Science, Toho University, Funabashi, Japan; 2 PRESTO, JST/Nat. Inst. for Basic Biol., Okazaki, Japan; 3 Department of Mar. Biosci., Fukui Prefectural University, Yoshida, Japan In the present experiments we attempted functional transplant of a photoactivated adenylyl cyclase (PAC) isolated from Euglena into mechanosensory neurons in Aplysia pleural ganglia. When cAMP was injected into the neurons, spike amplitude temporarily decreased while spike width temporarily increased. This effect was sustained longer in a bath solution containing IBMX, the phosphodiesterase inhibitor. We, therefore, explored these changes as indicators of appearance of the PAC function. PAC or the PAC expression vector (pNEX-PAC) was injected into cell bodies of neurons. Spike amplitude decreased in both cases and spike width increased in the PAC injection when the neurons were stimulated with light, suggesting that the transplanted PAC works well in Aplysia neurons. These results indicate that we can control cAMP production in specific neurons with light by the functional transplant of PAC.
P3-j43 Lycopene uptake to Mongolian gerbil attenuates apop-
S241
P3-j47 Time dependent progesterone-nouroprotection against
tosis in hippocampus induced by ischemia
ischemic brain damages
Kimikazu Fujita 1 , Nobuko Yoshimoto 2 , Toshiaki Kato 3 , Masataka Yamane 4 , Takahiro Inakuma 4 , Yutaka Nagata 1 , Eiichi Miyachi 1 1 Department Physiol., Sch. Med. Fujita Health, UniversityToyoake, Aichi, Japan; 2 Department Nutrition and Food Sciences, Nagoya Bunri University, Col., Japan; 3 Department Cli. Lab. Sci., Fujita Health, University Col., Japan; 4 Research Institute KAGOME Co. Ltd., Japan
Weiyan Cai 1 , Ling Chen 1 , Masahiro Sokabe 2 1 Department of Physiology, Nanjing Medical University, Nanjing, China; 2 Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya, Japan
Lycopene from plant origin is known to have an antioxidant action in tissues. Application of transient ischemia/recirculation to gerbil produced apoptosis in hippocampus after several days. We examined antioxidant effect of lycopene administration to the animal, expecting to find effect of lycopene to attenuate the progress of apoptosis caused by ischemia. Weanings at 21st days after birth were fed on lycopene containing MF food (5 mg/100 g) freely, and then we burdened cerebral ischemia to the animal. Amount of bcl-2 was decreased in the animal without lycopene intake, whereas it was increased clearly in lycopene administered animal after surgery. Increased TUNEL positive cells after ischemia reduced in lycopene treated animal after a week. These results strongly suggest that lycopene can act as an inhibitory modulator against apoptosis in neurons caused by ischemia even at genetic level.
P3-j44 Roles of macrophage-like cells expressing NG2 proteoglycan in ischemic rat brain Junya Tanaka 1 , Hiroaki Matsumoto 2 , Hisaaki Takahashi 1 , Anna Smirkin 1 , Yoshinori Imai 1 , Yoshiaki Kumon 2 , Tkanori Ohnishi 2 1 Department of Molecular and Cellular Physiology, Ehime University, Ehime, Japan; 2 Department of Neurosurgery, Ehime University, Ehime, Japan Macrophage-like cells accumulate in ischemic core in the rat brain after transient middle cerebral artery occlusion (90 min) from 3 to 14 days postreperfusion (dpr). Because such macrophage-like cells expressed NG2 chondroitin sulfate proteoglycan (NG2) as well as a macrophage-marker Iba1+, they were termed BINCs (Brain Iba1+/NG2+ Cells). BINCs were highly proliferative as revealed by labeling with 5-bromodeoxyuridine (BrdU) at 2 dpr. In this study, to gain insights as to whether BINCs play favorable roles in the ischemic events, 5-fluorouracil (5 FU) was intraperitoneally injected to ischemic rats at 2 dpr to kill BINCs specifically. This treatment caused marked loss of BINCs in ischemic core. Nearly half of the 5 FU-injected ischemic rats died by 10 dpr. Ischemic region of the survived rat brains markedly enlarged. These results indicate that BINCs play protective roles in the ischemic events.
Ovarian hormone progesterone (P4) is a particularly interesting candidate agent in treating stroke because of its wide time-window for administration to protect neurons from ischemic neuronal damages. We previously reported in a rat model that a single administration of P4 (4 mg/kg) at either 1 h or 48 h before middle cerebral artery occlusion (MCAO) was able to prevent the ischemia-induced pyramidal cell loss and long-term potentiation (LTP) in the hippocampus as well as the deficits in spatial cognition. Herein, we report that the delayed (48 h) neuroprotection was mediated by P4 receptor, a nuclear transcription factor receptor, followed by Src/ERK signaling. On the other hand, the acute (1 h) neuroprotection seemed to be mediated by an antagonism of a sigma1 receptor by P4. These results indicate that the wide time-window of P4-neuroprotection against ischemic brain damages includes two independent processes, a sigma1 receptor-mediated non-genomic and a P4 receptor-mediated genomic signal pathways.
P3-j48 Inducible expression of retrograde transynaptic genetic tracer in mice Hiromi Sano 1,2 , Mineto Yokoi 1,2 Molecular Neurogenetics Unit, HMRO, Kyoto University Graduate School of Medicine, Kyoto, Japan; 2 CREST, Japan Science and Technology Agency, Kawaguchi, Japan
1
A key step toward understanding of neural function is characterizing the neuronal connectivity. Tetanus toxin C fragment (TTC) is transynaptically and retrogradely transported without the toxin’s pathogenic effect. We introduce a new fusion construct, APTTC, consisting of the truncated human placental alkaline phosphatase with TTC, and generated the transgenic mouse line, tetO-APTTC, for inducible expression of APTTC regulated by tetracycline operator (tetO). In order to examine the connectivity of striatal medium spiny neurons (MSNs), we generated double transgenic mice by mating the tetO-APTTC transgenic mice to the PDE10A2-tTA knock-in heterozygote mice (PDE10A2tTA/+ ), in which tTA is selectively expressed in striatal MSNs. In the double transgenic mice, we found alkaline phosphatase (AP) staining in the tTA expressing regions and other specific regions whose axons are reported to terminate in striatal MSNs with previous tracing studies. Our data indicate that APTTC works as a retrograde transynaptic genetic tracer in mice. Research fund: CREST.
P3-j46 Change and translocation of HSP60 in gerbil hippocampal CA1 region induced by transient ischemia and its protective effect against ischemic damage In Koo Hwang 1 , Ki-Yeon Yoo 2 , Jung Hoon Choi 1 , In Se Lee 1 , Yeo Sung Yoon 1 , Je Kyung Seong 1 , Moo Ho Won 2 1 Department of Anatomy and Cell Biology, Seoul Nat’l Univ, Seoul, South Korea; 2 Department of Anatomy and Neurobiology, Hallym Univ, Chunchon, South Korea We observed changes in heat shock protein 60 (HSP60) in the gerbil hippocampal CA1 region after transient forebrain ischemia and its protective effect against ischemic damage. HSP60 immunoreactivity was increased at 3 h after ischemia/reperfusion (I–R) and peaked 6 h after I–R. At these time points, HSP60 immunoreactivity was detected in the mitochondria. At 24 h after I–R, HSP60 immunoreactivity was detectable in the cytoplasm of hippocampal CA1 region. Thereafter, HSP60 immunoreactivity was nearly disappeared in the hippocampal CA1 region. The HSP60 transfection via the adenovirus significantly increased the neuronal survival after transient forebrain ischemia. This suggests that the translocation and decrease of HSP60 may be associated with neuronal cell death and the induction of HSP60 protects neurons from ischemic damage. Research fund: BK21 Program for Veterinary Science, Seoul National University.
P3-kØ1 Functional transplant of photoactivated adenylyl cyclase into Aplysia mechanosensory neurons Tatsumi Nagahama 1 , Takeshi Suzuki 2 , Shinya Yoshikawa 3 , Mineo Iseki 2 1 Department of Biophysics, Faculty of Pharmaceutical Science, Toho University, Funabashi, Japan; 2 PRESTO, JST/Nat. Inst. for Basic Biol., Okazaki, Japan; 3 Department of Mar. Biosci., Fukui Prefectural University, Yoshida, Japan In the present experiments we attempted functional transplant of a photoactivated adenylyl cyclase (PAC) isolated from Euglena into mechanosensory neurons in Aplysia pleural ganglia. When cAMP was injected into the neurons, spike amplitude temporarily decreased while spike width temporarily increased. This effect was sustained longer in a bath solution containing IBMX, the phosphodiesterase inhibitor. We, therefore, explored these changes as indicators of appearance of the PAC function. PAC or the PAC expression vector (pNEX-PAC) was injected into cell bodies of neurons. Spike amplitude decreased in both cases and spike width increased in the PAC injection when the neurons were stimulated with light, suggesting that the transplanted PAC works well in Aplysia neurons. These results indicate that we can control cAMP production in specific neurons with light by the functional transplant of PAC.