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Localization of voltage-dependent calcium channel subunit alpha 1A (Cav2.1) in the rat cerebellum
e110
Abstracts / Neuroscience Research 68S (2010) e109–e222
P1-a05 The regulatory site of pore dilation and current facilitation in P2X7 receptor
P1-a07 Localization of voltage-dependent calcium channel subunit alpha 1A (Cav2.1) in the rat cerebellum
Keisuke Migita 1 , Terrance M. Egan 2 , Junko Yamada 1 , Masahiko Tomiyama 1 , Shinya Ueno 1
Dwi Wahyu Indriati 1 , Naomi Kamasawa 1 , Masahiko Watanabe 2 , Ryuichi Shigemoto 1
1
Dept Neurophysiol, Hirosaki Univ, Hirosaki 2 Dept Pharmacol Physiol Sci, St. Louis Univ, USA
1
The P2X receptors are a family of ligand-gated ion channels composed of seven subunits (P2X1 -P2X7 ). P2X receptors contain two putative poreforming transmembrane segments, a large cysteine-rich ligand-binding extracellular domain, and intracellularly located N and C termini. P2X7 receptors are known to be localized and functional in microglial cells. Unlike other P2X receptors which are activated by low doses of their natural agonist ATP, P2X7 receptors are activated by high concentrations of ATP. The receptor operates as a nonselective cationic channel during initial agonist application, but with prolonged application, the receptor provides a permeation pathway to molecules with a molecular weight of up to 800 Da, including a fluorescent dye YO-PRO-1. P2X7 receptor activity has been demonstrated to be involved in chronic pain associated with inflammatory diseases. Therefore, these receptors are the target of new anti-inflammatory antagonists. However, it is still poorly known P2X7 receptor structure-function relationships. We thus investigated the character of current facilitaion and YO-PRO-1 uptake in P2X7 receptor and its N-terminal T15E mutant. The biphasic current was observed in P2X7 receptor during the prolonged ATP application. The late phase of current was facilitated. The current facilitation was increased when Ca2+ and Mg2+ were removed from the bath solution. However, the current facilitation was not observed in T15E mutant. Moreover, P2X7 receptor showed YO-PRO-1 uptake, whereas T15E mutant do not have permeability to YO-PRO-1. Therefore, P2X7 receptor dilates in physiological solution, leading to current facilitation, and this phenomenon is regulated by 15 residue of N-terminal.
AbstractCalcium channels play a pivotal role in mediating the synaptic transmission. Among the six different types of calcium channels, the alpha 1A subunit of calcium channel is most abundantly expressed in the cerebellum. The purpose of this study was to investigate the precise subcellular localization of alpha 1A subunit of calcium channel by means of SDS digested freeze fracture replica labeling (SDS-FRL). Based on our previous finding with preembeding immunogold method, at the light microscopic level, immunoreactivity for the alpha 1A protein was prevalent in the molecular layer whereas immunostaining was moderate in the soma of Purkinje cells and weak in the granule cell layer. At the electron microscopic level immunogold particles for alpha 1A were found on protoplasmic face (Pface) of Purkinje cell dendrites and soma. The particles were often seen to be associated with a distinct type of intramembrane particle (IMP) which was bigger in size in compared to the surrounding IMPs. Whereas the majority of the immunogold particles in the Purkinje cell dendrite and soma were seen to form clusters, some others were rather sparsely distributed. For identification of synaptic contact, replicas were double labeled for alpha 1A and AMPA-type glutamate receptors (AMPAR). We found labeling for AMPAR in exoplasmic face (E-face) of postsynaptic site and immunogold particles for alpha 1A were found to be localized to the presynaptic membrane specialization in the adjacent protoplasmic face (P-face) of presynaptic terminals. In alpha 1A knock-out mice, the clusters of IMPs were found without alpha 1A labeling on Purkinje cell soma and dendrite and the labeling in the presynaptic active zone was also gone. These results confirm our previous findings with the preembedding immunogold labeling and further suggest hot spot formation of P/Q type calcium channels on Purkinje cell dendrites.
doi:10.1016/j.neures.2010.07.2054
P1-a06 Regional and subcellular distribution of the ␣1E subunit of voltage-gated calcium channel in the adult mouse brain Laxmi Kumar Parajuli 1,2 Ryuichi Shigemoto 1,2
, Yugo Fukazawa 1,2 , Akos Kulik 3 ,
1
Division of Cerebral Structure, National Institute for Physiological Sciences 2 Department of Physiological Sciences, The Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8787, Japan 3 Department of Anatomy and Cell Biology, University of Freiburg, Albertstrasse 17, Freiburg, Germany Rise in intracellular Ca2+ arising from the openings of the voltage-gated calcium channels (VGCC) trigger multiple cellular and subcellular responses. Among ten different subunits of VGCC, the ␣1E subunit, the pore-forming subunit of the high voltage-activated R-type calcium channel, has been reported to be important for inducing synaptic plasticity, triggering neuronal exocytosis, generation of action potential bursts and afterdepolarizations. Though electrophysiological and in situ hybridization studies have previously described the regional distribution of the ␣1E subunit, the morphological evidence for its precise localization is missing due to the lack of a selective antibody against this subunit. Here, we generated a specific antibody against the ␣1E subunit to study its distribution both at the light microscopic (LM) and electron microscopic (EM) levels. At the LM level, the staining was more prominent in the forebrain compared to the midbrain and hindbrain structures. Neuropils of the superficial layer of neocortex, hippocampus, striatum, amygdala and inferior olive displayed high level of immunoreactivity. In the hippocampus, no immunoreactivity was detected in the pyramidal cell layer and the granule cell layer. In addition, a remarkable difference in immunoreactivity was seen between the CA1 and CA3 stratum radiata. The immunoreactivity in the thalamus and midbrain regions was below the detection limit. The EM immunogold analysis in hippocampus and the neocortex revealed that this channel is postsynaptically localized in the dendritic shafts and spines and considerably less abundant in the somata of principal cells. Presynaptically, the immunogolds were localized in the extrasynaptic region remote from the active zone. Our data provide the comprehensive picture of the cellular and subcellular localizations of the ␣1E subunit of voltage-gated calcium channel in various brain regions. doi:10.1016/j.neures.2010.07.2055
2
Department of Physiological Sciences, National Institute of Natural Sciences Dept. of Anatomy, Hokkaido University, Sapporo, Japan
doi:10.1016/j.neures.2010.07.2056
P1-a08 Information processing on the dendrite in hippocampal granule cells Hirofumi Hayakawa 1 , Tadanobu Kamijou 1 , Makoto Yoneyama 2 , Yasuhiro Fukushima 2 , Takeshi Aihara 1 1
Department of Technology, Tamagawa University, Tokyo Institute, Tamagawa University, Tokyo
2
Brain Science
Dentate granule cells have inputs though the lateral perforant path (LPP) with non-spatial information, smell etc., and inputs though the medial perforant path (MPP) with spatial information, place. Inputs though the LPP and those though the MPP are projected to the lateral dendrite and the medial dendrite of dentate granule cells from layer II in the entorhinal cortex (EC), respectively. In addition, it has been experimentally confirmed that memory is improved by the presence of smell information. Therefore, there is an possibility that the processing of spatial information is influenced by non-specific information in dentate granule. To investigate how input information with different modalities are integrated on the dendrite of dentate granule cells, two input stimulus were applied to lateral and medial dendrite, respectively. As the result, the responses to the LPP stimulation show different characteristics from that of the MPP stimulation depending on the stimulus frequency; sustained for LPP stimulus and transient for MPP stimulus. Furthermore, nonlinear components of superposed amplitude mediated by LPP stimulus and MPP stimulus were significantly observed only if MPP stimulus was applied before MPP stimulus. The result suggests that there is nonlinear integration on the dendrite of hippocampal dentate granule cells, in which the processing of spatial information is influenced by non-specific information with facilitatory effect. doi:10.1016/j.neures.2010.07.2057
Abstracts / Neuroscience Research 68S (2010) e109–e222
P1-a05 The regulatory site of pore dilation and current facilitation in P2X7 receptor
P1-a07 Localization of voltage-dependent calcium channel subunit alpha 1A (Cav2.1) in the rat cerebellum
Keisuke Migita 1 , Terrance M. Egan 2 , Junko Yamada 1 , Masahiko Tomiyama 1 , Shinya Ueno 1
Dwi Wahyu Indriati 1 , Naomi Kamasawa 1 , Masahiko Watanabe 2 , Ryuichi Shigemoto 1
1
Dept Neurophysiol, Hirosaki Univ, Hirosaki 2 Dept Pharmacol Physiol Sci, St. Louis Univ, USA
1
The P2X receptors are a family of ligand-gated ion channels composed of seven subunits (P2X1 -P2X7 ). P2X receptors contain two putative poreforming transmembrane segments, a large cysteine-rich ligand-binding extracellular domain, and intracellularly located N and C termini. P2X7 receptors are known to be localized and functional in microglial cells. Unlike other P2X receptors which are activated by low doses of their natural agonist ATP, P2X7 receptors are activated by high concentrations of ATP. The receptor operates as a nonselective cationic channel during initial agonist application, but with prolonged application, the receptor provides a permeation pathway to molecules with a molecular weight of up to 800 Da, including a fluorescent dye YO-PRO-1. P2X7 receptor activity has been demonstrated to be involved in chronic pain associated with inflammatory diseases. Therefore, these receptors are the target of new anti-inflammatory antagonists. However, it is still poorly known P2X7 receptor structure-function relationships. We thus investigated the character of current facilitaion and YO-PRO-1 uptake in P2X7 receptor and its N-terminal T15E mutant. The biphasic current was observed in P2X7 receptor during the prolonged ATP application. The late phase of current was facilitated. The current facilitation was increased when Ca2+ and Mg2+ were removed from the bath solution. However, the current facilitation was not observed in T15E mutant. Moreover, P2X7 receptor showed YO-PRO-1 uptake, whereas T15E mutant do not have permeability to YO-PRO-1. Therefore, P2X7 receptor dilates in physiological solution, leading to current facilitation, and this phenomenon is regulated by 15 residue of N-terminal.
AbstractCalcium channels play a pivotal role in mediating the synaptic transmission. Among the six different types of calcium channels, the alpha 1A subunit of calcium channel is most abundantly expressed in the cerebellum. The purpose of this study was to investigate the precise subcellular localization of alpha 1A subunit of calcium channel by means of SDS digested freeze fracture replica labeling (SDS-FRL). Based on our previous finding with preembeding immunogold method, at the light microscopic level, immunoreactivity for the alpha 1A protein was prevalent in the molecular layer whereas immunostaining was moderate in the soma of Purkinje cells and weak in the granule cell layer. At the electron microscopic level immunogold particles for alpha 1A were found on protoplasmic face (Pface) of Purkinje cell dendrites and soma. The particles were often seen to be associated with a distinct type of intramembrane particle (IMP) which was bigger in size in compared to the surrounding IMPs. Whereas the majority of the immunogold particles in the Purkinje cell dendrite and soma were seen to form clusters, some others were rather sparsely distributed. For identification of synaptic contact, replicas were double labeled for alpha 1A and AMPA-type glutamate receptors (AMPAR). We found labeling for AMPAR in exoplasmic face (E-face) of postsynaptic site and immunogold particles for alpha 1A were found to be localized to the presynaptic membrane specialization in the adjacent protoplasmic face (P-face) of presynaptic terminals. In alpha 1A knock-out mice, the clusters of IMPs were found without alpha 1A labeling on Purkinje cell soma and dendrite and the labeling in the presynaptic active zone was also gone. These results confirm our previous findings with the preembedding immunogold labeling and further suggest hot spot formation of P/Q type calcium channels on Purkinje cell dendrites.
doi:10.1016/j.neures.2010.07.2054
P1-a06 Regional and subcellular distribution of the ␣1E subunit of voltage-gated calcium channel in the adult mouse brain Laxmi Kumar Parajuli 1,2 Ryuichi Shigemoto 1,2
, Yugo Fukazawa 1,2 , Akos Kulik 3 ,
1
Division of Cerebral Structure, National Institute for Physiological Sciences 2 Department of Physiological Sciences, The Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8787, Japan 3 Department of Anatomy and Cell Biology, University of Freiburg, Albertstrasse 17, Freiburg, Germany Rise in intracellular Ca2+ arising from the openings of the voltage-gated calcium channels (VGCC) trigger multiple cellular and subcellular responses. Among ten different subunits of VGCC, the ␣1E subunit, the pore-forming subunit of the high voltage-activated R-type calcium channel, has been reported to be important for inducing synaptic plasticity, triggering neuronal exocytosis, generation of action potential bursts and afterdepolarizations. Though electrophysiological and in situ hybridization studies have previously described the regional distribution of the ␣1E subunit, the morphological evidence for its precise localization is missing due to the lack of a selective antibody against this subunit. Here, we generated a specific antibody against the ␣1E subunit to study its distribution both at the light microscopic (LM) and electron microscopic (EM) levels. At the LM level, the staining was more prominent in the forebrain compared to the midbrain and hindbrain structures. Neuropils of the superficial layer of neocortex, hippocampus, striatum, amygdala and inferior olive displayed high level of immunoreactivity. In the hippocampus, no immunoreactivity was detected in the pyramidal cell layer and the granule cell layer. In addition, a remarkable difference in immunoreactivity was seen between the CA1 and CA3 stratum radiata. The immunoreactivity in the thalamus and midbrain regions was below the detection limit. The EM immunogold analysis in hippocampus and the neocortex revealed that this channel is postsynaptically localized in the dendritic shafts and spines and considerably less abundant in the somata of principal cells. Presynaptically, the immunogolds were localized in the extrasynaptic region remote from the active zone. Our data provide the comprehensive picture of the cellular and subcellular localizations of the ␣1E subunit of voltage-gated calcium channel in various brain regions. doi:10.1016/j.neures.2010.07.2055
2
Department of Physiological Sciences, National Institute of Natural Sciences Dept. of Anatomy, Hokkaido University, Sapporo, Japan
doi:10.1016/j.neures.2010.07.2056
P1-a08 Information processing on the dendrite in hippocampal granule cells Hirofumi Hayakawa 1 , Tadanobu Kamijou 1 , Makoto Yoneyama 2 , Yasuhiro Fukushima 2 , Takeshi Aihara 1 1
Department of Technology, Tamagawa University, Tokyo Institute, Tamagawa University, Tokyo
2
Brain Science
Dentate granule cells have inputs though the lateral perforant path (LPP) with non-spatial information, smell etc., and inputs though the medial perforant path (MPP) with spatial information, place. Inputs though the LPP and those though the MPP are projected to the lateral dendrite and the medial dendrite of dentate granule cells from layer II in the entorhinal cortex (EC), respectively. In addition, it has been experimentally confirmed that memory is improved by the presence of smell information. Therefore, there is an possibility that the processing of spatial information is influenced by non-specific information in dentate granule. To investigate how input information with different modalities are integrated on the dendrite of dentate granule cells, two input stimulus were applied to lateral and medial dendrite, respectively. As the result, the responses to the LPP stimulation show different characteristics from that of the MPP stimulation depending on the stimulus frequency; sustained for LPP stimulus and transient for MPP stimulus. Furthermore, nonlinear components of superposed amplitude mediated by LPP stimulus and MPP stimulus were significantly observed only if MPP stimulus was applied before MPP stimulus. The result suggests that there is nonlinear integration on the dendrite of hippocampal dentate granule cells, in which the processing of spatial information is influenced by non-specific information with facilitatory effect. doi:10.1016/j.neures.2010.07.2057