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Myosin II ATPase activity is involved in drebrin translocation in dendritic spines
Abstracts / Neuroscience Research 71S (2011) e108–e415
found that the relationship was enhanced in SDRs, indicating that the efficacy of basal synaptic transmission was increased. Western blot analysis showed that there was no significant difference in the expression of GluA1, GluA2, PSD95 and presynaptic proteins between SDRs and control rats. However, the expression of NMDAR subunits, GluN1, GluN2A and GluN2B, was increased in the CA1 region of SDRs. Correspondingly, SDRs showed an increase in the ratio of NMDAR- to AMPAR-mediated synaptic currents in whole-cell patchclamp recordings from CA1 pyramidal cells. Furthermore, SDRs exhibited enhanced LTP induced by tetanic stimulation, as expected from the increased NMDAR expression. Taken together, our results suggest that GH regulates the expression of NMDA receptors in the hippocampus, which may result in modification of abilities of learning and memory in the whole animal. doi:10.1016/j.neures.2011.07.951
P3-b19 Myosin II ATPase activity is involved in drebrin translocation in dendritic spines Nobuhiko Kojima , Toshiyuki Mizui, Yuko Sekino, Tomoaki Shirao Gunma Univ., Grad. Sch. of Med., Dep. of Neurobiol. Behav., Maebashi, Japan Recent evidence indicates that myosin II is involved in dynamic changes in actin cytoskeletal organization in dendritic spines during synaptic plasticity. Since drebrin binds tightly with F-actin in dendritic spines of mature neurons, the immunopositive signal of drebrin can be a good indicator for drebrin-bound actin filaments. We have previously found that glutamate stimulation rapidly decreases drebrin immunopositive signal in dendritic spines of cultured neurons (Sekino et al., 2006). In the present study we measured the signal intensities of drebrin separately in dendritic spines and their parent dendritic shafts, and calculated the spine-dendrite ratio (SDR) under the various conditions. We found that the drebrin SDR was increased and decreased by inhibition and activation of the NMDA receptor, respectively. EGTA application showed that influx of extracellular Ca2+ through the NMDA receptors regulates the drebrin SDR. Furthermore, blebbistatin, a myosin II ATPase blocker, inhibited the glutamate-induced decrease in the drebrin SDR. These data indicate that glutamate-induced translocation of drebrin is regulated by myosin II ATPase activity through a Ca2+ -dependent mechanism. However, ML-7, a myosin light chain (MLC) kinase blocker, did not inhibit the glutamate-induced decrease in the drebrin SDR, indicating that myosin II ATPase activity is not regulated by Ca2+ -dependent MLC phosphorylation, rather can be regulated by an actin-linked mechanism. Finally, we found that change in the drebrin SDR transiently occurred during chemical LTP stimulation (bath application of 200 M glycine + 0 M Mg2+ ). Thus, Ca2+ influx through the NMDA receptors triggers myosin-II-driven transient translocation of drebrin-bound actin filaments from dendritic spines during LTP induction. Research fund: KAKENHI (16300117, 19200029, 20021002). doi:10.1016/j.neures.2011.07.952
P3-b20 Dendritic spine formation depends on the density of existing spines Yuki Oe , Keiko Tominaga-Yoshino, Akihiko Ogura Osaka University Graduate School of Frontier Biosciences, Suita, Japan It is assumed that long-term memory is substantiated by structural plasticity including the formation of new synapses. We previously reported that the repeated inductions of LTP led to a long-lasting enhancement of synaptic strength accompanied by synaptogenesis in the stable cultures of hippocampal slice and named this phenomenon RISE (Repetitive-LTP-Induced Synaptic Enhancement). It is of great interest how and when new synapses are formed. Here we pursue the dynamics of dendritic spines (postsynaptic structure) of apical dendrite of CA1 pyramidal neuron after the RISE-producing stimulation, using a line of transgenic mouse (Thy1-YFP-H) that expresses yellow fluorescent protein sparsely in the CA1 region. The density of spines showed no increase during days 0–3, but it did at day 6, confirming the previously reported results. However, the detailed examination of individual spines revealed that new spines began to be formed already during days 0–3, while the loss of spines also increased in this period to cancel out the formation. The balance of gain/loss of spines shifted to gain to result in the increase of spines in total at day 6. In other words, the spines increased in a biasedrandom walk manner of yuragi (wobble) process. The density of spines is not uniform among the dendrites. When we classified the dendrites to highspine-density and low-spine density populations, setting the boundary at 1.3 spines/m dendrite length, we found that the above imbalanced spine turnover occurred limitedly in the low-spine-density populations, resulting in the increase of spine density in the low-spine-density populations only.
e219
Research fund: This work is supported by KAKENHI (19300108) to A.O. doi:10.1016/j.neures.2011.07.953
P3-b21 Turning on of CICR in LTD induction after critical period of NR2 subunits switch Hiroki Yasuda ERSC, Grad. Sch. Med., Gunma Univ., Maebashi, Japan Developmental plasticity is hypothesized to mediate activity dependent refinement of neural circuits during the maturation of the central nervous system. Long-term depression (LTD) of synaptic transmission in the CA1 region of the hippocampus is especially dominant in the developing hippocampus and triggered by activation of NMDA receptors at postsynaptic sites. Calcium influx through NMDA receptors induce calcium-induced calcium release (CICR) from internal calcium stores, however whether the CICR and ryanodine receptors, one of the internal store channels, are involved in LTD induction throughout development remains to be elucidated. Here I report that internal calcium stores and ryanodine receptors start entirely mediating induction of LTD in the hippocampus during the prepubertal period. Intracellular application of both thapsigargin and ryanodine, inhibitors of Ca2+ -ATP pumps on internal stores and a ryanodine receptors, respectively, did not at all affect LTD in the CA1 of the hippocampus at <15 postnatal days (P < 15), but completely block LTD in the P > 19 hippocampus. Furthermore I found that ifenprodil, an antagonist of NMDA receptors with NR2B subunits, had no effects on LTD in P > 20 hippocampus, but did reduce it at P < 15, when CICR does not contribute to LTD induction. I conclude that CICR through ryanodine receptors at postsynaptic sites rapidly starts to compensate calcium rise for LTD induction instead of reducing NR2B subunits after postnatal 2 weeks in the hippocampus. Research fund: KAKENHI (21200016), KAKENHI (20500354). doi:10.1016/j.neures.2011.07.954
P3-c01 ␣- and CaMKII regulate inhibitory synaptic plasticity oppositely in the cerebellum Nobuhiro Nagasaki , Tomoo Hirano, Shin-ya Kawaguchi Dept. Biophys., Grad. Sch. Sci., Kyoto Univ. At inhibitory synapses on a cerebellar Purkinje neuron (PN), long lasting potentiation of GABAA receptor responsiveness (called rebound potentiation: RP) is induced by an increase in intracellular Ca2+ concentration and resultant activation of Ca2+ /CaM-dependent protein kinase II (CaMKII). CaMKII sustains its activity through autophosphorylation at Thr286, which is implicated in the RP establishment. CaMKII works as a holoenzyme consisting of ␣ and  subunits, and the latter specifically binds to F-actin and has a higher affinity for Ca2+ /CaM. However, it remains unknown whether two subunits equally contribute to RP or not. To address this issue, we first knocked-down each subunit in a cultured PN using RNAi. Knock-down of CaMKII suppressed RP, whereas that of ␣CaMKII did not. These results indicate that CaMKII is necessary for the RP induction. Next, we overexpressed each CaMKII in a PN. ␣CaMKII overexpression impaired RP, while CaMKII did not. The RP suppression by ␣CaMKII overexpression was rescued by additional overexpression of CaMKII. These results suggested that ␣CaMKII negatively controls RP and the expression balance of two subunits is critical for the RP induction. Finally, we investigated the molecular basis of the opposite roles of ␣- and CaMKII in RP by evaluating the effects of deletion of two unique regions in CaMKII. The results suggested that CaMKII contributes to the RP induction not through the binding to F-actin but through the higher affinity for Ca2+ /CaM. Taken together, the expression ratio of ␣and CaMKII plays a pivotal role in the RP induction due to their different affinity for Ca2+ /CaM. Research fund: KAKENHI21700349, gCOE program MEXT. doi:10.1016/j.neures.2011.07.955
P3-c02 Substrate-free FRET-based enzymological analysis of CaMKK Masatoshi Inoue 1 , Hajime Fujii 1 , Sayaka Takemoto-Kimura 1,2 , Hiroyuki Okuno 1,3 , Haruhiko Bito 1,3 1 3
Dept. of Neurochem., Grad. Sch. of Med., Univ. of Tokyo, Japan 2 PRESTO-JST CREST-JST
The CaMKK-CaMKIV cascade plays a major role in transforming neural activity into CREB-dependent gene expression. While previous studies showed that CaMKK␣ and CaMKK are essential for late phase-neuronal plasticity
found that the relationship was enhanced in SDRs, indicating that the efficacy of basal synaptic transmission was increased. Western blot analysis showed that there was no significant difference in the expression of GluA1, GluA2, PSD95 and presynaptic proteins between SDRs and control rats. However, the expression of NMDAR subunits, GluN1, GluN2A and GluN2B, was increased in the CA1 region of SDRs. Correspondingly, SDRs showed an increase in the ratio of NMDAR- to AMPAR-mediated synaptic currents in whole-cell patchclamp recordings from CA1 pyramidal cells. Furthermore, SDRs exhibited enhanced LTP induced by tetanic stimulation, as expected from the increased NMDAR expression. Taken together, our results suggest that GH regulates the expression of NMDA receptors in the hippocampus, which may result in modification of abilities of learning and memory in the whole animal. doi:10.1016/j.neures.2011.07.951
P3-b19 Myosin II ATPase activity is involved in drebrin translocation in dendritic spines Nobuhiko Kojima , Toshiyuki Mizui, Yuko Sekino, Tomoaki Shirao Gunma Univ., Grad. Sch. of Med., Dep. of Neurobiol. Behav., Maebashi, Japan Recent evidence indicates that myosin II is involved in dynamic changes in actin cytoskeletal organization in dendritic spines during synaptic plasticity. Since drebrin binds tightly with F-actin in dendritic spines of mature neurons, the immunopositive signal of drebrin can be a good indicator for drebrin-bound actin filaments. We have previously found that glutamate stimulation rapidly decreases drebrin immunopositive signal in dendritic spines of cultured neurons (Sekino et al., 2006). In the present study we measured the signal intensities of drebrin separately in dendritic spines and their parent dendritic shafts, and calculated the spine-dendrite ratio (SDR) under the various conditions. We found that the drebrin SDR was increased and decreased by inhibition and activation of the NMDA receptor, respectively. EGTA application showed that influx of extracellular Ca2+ through the NMDA receptors regulates the drebrin SDR. Furthermore, blebbistatin, a myosin II ATPase blocker, inhibited the glutamate-induced decrease in the drebrin SDR. These data indicate that glutamate-induced translocation of drebrin is regulated by myosin II ATPase activity through a Ca2+ -dependent mechanism. However, ML-7, a myosin light chain (MLC) kinase blocker, did not inhibit the glutamate-induced decrease in the drebrin SDR, indicating that myosin II ATPase activity is not regulated by Ca2+ -dependent MLC phosphorylation, rather can be regulated by an actin-linked mechanism. Finally, we found that change in the drebrin SDR transiently occurred during chemical LTP stimulation (bath application of 200 M glycine + 0 M Mg2+ ). Thus, Ca2+ influx through the NMDA receptors triggers myosin-II-driven transient translocation of drebrin-bound actin filaments from dendritic spines during LTP induction. Research fund: KAKENHI (16300117, 19200029, 20021002). doi:10.1016/j.neures.2011.07.952
P3-b20 Dendritic spine formation depends on the density of existing spines Yuki Oe , Keiko Tominaga-Yoshino, Akihiko Ogura Osaka University Graduate School of Frontier Biosciences, Suita, Japan It is assumed that long-term memory is substantiated by structural plasticity including the formation of new synapses. We previously reported that the repeated inductions of LTP led to a long-lasting enhancement of synaptic strength accompanied by synaptogenesis in the stable cultures of hippocampal slice and named this phenomenon RISE (Repetitive-LTP-Induced Synaptic Enhancement). It is of great interest how and when new synapses are formed. Here we pursue the dynamics of dendritic spines (postsynaptic structure) of apical dendrite of CA1 pyramidal neuron after the RISE-producing stimulation, using a line of transgenic mouse (Thy1-YFP-H) that expresses yellow fluorescent protein sparsely in the CA1 region. The density of spines showed no increase during days 0–3, but it did at day 6, confirming the previously reported results. However, the detailed examination of individual spines revealed that new spines began to be formed already during days 0–3, while the loss of spines also increased in this period to cancel out the formation. The balance of gain/loss of spines shifted to gain to result in the increase of spines in total at day 6. In other words, the spines increased in a biasedrandom walk manner of yuragi (wobble) process. The density of spines is not uniform among the dendrites. When we classified the dendrites to highspine-density and low-spine density populations, setting the boundary at 1.3 spines/m dendrite length, we found that the above imbalanced spine turnover occurred limitedly in the low-spine-density populations, resulting in the increase of spine density in the low-spine-density populations only.
e219
Research fund: This work is supported by KAKENHI (19300108) to A.O. doi:10.1016/j.neures.2011.07.953
P3-b21 Turning on of CICR in LTD induction after critical period of NR2 subunits switch Hiroki Yasuda ERSC, Grad. Sch. Med., Gunma Univ., Maebashi, Japan Developmental plasticity is hypothesized to mediate activity dependent refinement of neural circuits during the maturation of the central nervous system. Long-term depression (LTD) of synaptic transmission in the CA1 region of the hippocampus is especially dominant in the developing hippocampus and triggered by activation of NMDA receptors at postsynaptic sites. Calcium influx through NMDA receptors induce calcium-induced calcium release (CICR) from internal calcium stores, however whether the CICR and ryanodine receptors, one of the internal store channels, are involved in LTD induction throughout development remains to be elucidated. Here I report that internal calcium stores and ryanodine receptors start entirely mediating induction of LTD in the hippocampus during the prepubertal period. Intracellular application of both thapsigargin and ryanodine, inhibitors of Ca2+ -ATP pumps on internal stores and a ryanodine receptors, respectively, did not at all affect LTD in the CA1 of the hippocampus at <15 postnatal days (P < 15), but completely block LTD in the P > 19 hippocampus. Furthermore I found that ifenprodil, an antagonist of NMDA receptors with NR2B subunits, had no effects on LTD in P > 20 hippocampus, but did reduce it at P < 15, when CICR does not contribute to LTD induction. I conclude that CICR through ryanodine receptors at postsynaptic sites rapidly starts to compensate calcium rise for LTD induction instead of reducing NR2B subunits after postnatal 2 weeks in the hippocampus. Research fund: KAKENHI (21200016), KAKENHI (20500354). doi:10.1016/j.neures.2011.07.954
P3-c01 ␣- and CaMKII regulate inhibitory synaptic plasticity oppositely in the cerebellum Nobuhiro Nagasaki , Tomoo Hirano, Shin-ya Kawaguchi Dept. Biophys., Grad. Sch. Sci., Kyoto Univ. At inhibitory synapses on a cerebellar Purkinje neuron (PN), long lasting potentiation of GABAA receptor responsiveness (called rebound potentiation: RP) is induced by an increase in intracellular Ca2+ concentration and resultant activation of Ca2+ /CaM-dependent protein kinase II (CaMKII). CaMKII sustains its activity through autophosphorylation at Thr286, which is implicated in the RP establishment. CaMKII works as a holoenzyme consisting of ␣ and  subunits, and the latter specifically binds to F-actin and has a higher affinity for Ca2+ /CaM. However, it remains unknown whether two subunits equally contribute to RP or not. To address this issue, we first knocked-down each subunit in a cultured PN using RNAi. Knock-down of CaMKII suppressed RP, whereas that of ␣CaMKII did not. These results indicate that CaMKII is necessary for the RP induction. Next, we overexpressed each CaMKII in a PN. ␣CaMKII overexpression impaired RP, while CaMKII did not. The RP suppression by ␣CaMKII overexpression was rescued by additional overexpression of CaMKII. These results suggested that ␣CaMKII negatively controls RP and the expression balance of two subunits is critical for the RP induction. Finally, we investigated the molecular basis of the opposite roles of ␣- and CaMKII in RP by evaluating the effects of deletion of two unique regions in CaMKII. The results suggested that CaMKII contributes to the RP induction not through the binding to F-actin but through the higher affinity for Ca2+ /CaM. Taken together, the expression ratio of ␣and CaMKII plays a pivotal role in the RP induction due to their different affinity for Ca2+ /CaM. Research fund: KAKENHI21700349, gCOE program MEXT. doi:10.1016/j.neures.2011.07.955
P3-c02 Substrate-free FRET-based enzymological analysis of CaMKK Masatoshi Inoue 1 , Hajime Fujii 1 , Sayaka Takemoto-Kimura 1,2 , Hiroyuki Okuno 1,3 , Haruhiko Bito 1,3 1 3
Dept. of Neurochem., Grad. Sch. of Med., Univ. of Tokyo, Japan 2 PRESTO-JST CREST-JST
The CaMKK-CaMKIV cascade plays a major role in transforming neural activity into CREB-dependent gene expression. While previous studies showed that CaMKK␣ and CaMKK are essential for late phase-neuronal plasticity