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Glutamate receptor δ2 is essential for input pathway-dependent regulation of synaptic AMPAR contents in cerebellar Purkinje cells
Abstracts / Neuroscience Research 71S (2011) e46–e107
e93
gia (HSP), suggesting the role of protrudin in maintaining neuronal integrity. HSPs are characterized by progressive spasticity in the lower limbs and a part of HSPs are accompanied by other symptom such as seizures, mental retardation, or dementia. We generated mice deficient in protrudin, and have found that the mutant mice exhibited reduction of LTP. Rab11 is known to promote the trafficking of AMPA receptor in response to PKA signaling at the dendritic spines during LTP. We thus examined whether protrudin is required for the Rab11-dependent trafficking of AMPA receptor. Mice deficient in either protrudin exhibited the defects in AMPA receptor transport at the dendritic spine. Furthermore, these mutant mice showed impairment in spine maturation and synaptic formation. In addition, we found that FYVE domain of protrudin is specifically phosphorylated by PKA, which promotes the interaction of protrudin with Rab11. Our results indicate that protrudin promotes the trafficking of Rab11-positive recycling endosomes to the post-synaptic plasma membrane via the PKA-signaling. Protrudin thus facilitates the AMPA receptor transport, suggesting that protrudin possibly plays a critical role in cognitive functions of the brain.
or GluD2), which is known to be efficiently trafficked to and selectively expressed at PF-PC synapses. We applied postembedding immunogold electron microscopy to GluR␦2-knockout (KO) and control mice, and measured labeling density for GluA1–4 at three excitatory synapses in the cerebellar molecular layer. In both control and GluR␦2-KO mice, GluA1–3 were localized at PF and CF synapses in PCs, while GluA2–4 were at PF synapses in interneurons. In control mice, labeling density for each of GluA1–3 was 4to-6 times lower at PF-PC synapses than at CF-PC synapses. In GluR␦2-KO mice, however, their labeling density displayed a 3-to-5-fold increase at PF synapses, but not at CF synapses, thus effectively eliminating input pathwaydependent disparity between the two PC synapses. Furthermore, we found an unexpected 2-fold increase in labeling density for GluA2 and GluA3, but not GluA4, at PF-interneuron synapses, where we identified low but significant expression of GluR␦2. These results suggest that GluR␦2 is involved in a common mechanism that restricts the number of synaptic AMPARs at PF synapses in PCs and molecular layer interneurons. Research fund: Grant-in-Aid for Scientific Research (S), CREST.
doi:10.1016/j.neures.2011.07.397
doi:10.1016/j.neures.2011.07.399
O4-E-2-2 Roles of Purkinje cell activity in climbing fiber synapse elimination in an organotypic coculture preparation of the cerebellum and medulla oblongata
O4-E-2-4 Cornichon-2 dictates physical and pharmacological properties of AMPA receptors by controlling AMPA receptor-TARP assembly
Takayasu Mikuni , Naofumi Uesaka, Masanobu Kano
Akihiko Kato 1 , Martin Tomita 2 , David Bredt 1
Dept. of Neurophysiol., Grad. Sch. of Med., Univ. of Tokyo, Tokyo, Japan
Gill 1 , Matthew
Roberts 2 , Susumu
1
Understanding of how functional neural circuits are built during postnatal development is one of the most important issues in neuroscience. It has been reported that neural activity is crucial for circuit refinement including synapse formation, maturation, elimination and maintenance. However, how neural activity regulates these processes has yet to be elucidated. The climbing fiber (CF) to Purkinje cell (PC) synapse in the cerebellum has provided a good model to study the cellular and molecular mechanisms of activitydependent synapse elimination in the developing central nervous system. We explored the mechanisms of CF to PC synapse elimination by modifying postsynaptic PC activity in an in vitro coculture preparation of cerebellar slices with medulla explants containing the inferior olive, the origin of CFs. In this preparation, multiple CF to PC synapses are formed during the initial one week of coculture, and then surplus CFs are eliminated thereafter with similar molecular mechanisms to those found in vivo. We used a lentiviral gene transfer technique to express in PCs channelrhodopsin-2 (ChR2) or microRNA targeting P/Q-type voltage-dependent Ca2+ channel (VDCC), a major VDCC in PCs, to up- or down-regulate PC activity, respectively. Using these methods, we found that CF to PC synapse elimination was impaired by PC-specific RNAi knockdown of P/Q-type VDCCs. On the other hand, increasing PC activity by blue light stimulation of ChR2-expressing PCs for two days accelerated CF to PC synapse elimination. Furthermore, acceleration of CF to PC synapse elimination by ChR2 stimulation was not observed when P/Q-type VDCCs were knocked down. These observations suggest that CF to PC synapse elimination requires postsynaptic PC activity, followed by Ca2+ influx through P/Q-type VDCCs. Research fund: Grants-in-Aid for Scientific Research (S) No. 21220006, from MEXT, Japan. The Strategic Research Program for Brain Sciences, project D, from MEXT, Japan.
Neuroscience Discovery Research, Lilly Research Laboratory 2 CNNR, Dept of Cellular and Molecular Physiology, Yale Univ, New Heaven, CT AMPA-type glutamate receptors are crucial for excitatory synaptic transmission in brain. Neuronal AMPA receptor complexes comprise a tetramer of GluA pore-forming subunits as well as accessory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and cornichon2/3 (CNIH-2/3). Here we describe that CNIH-2 regulates the channel properties of TARP containing AMPA receptors by decreasing the number of TARPs per receptor. In recombinant systems, AMPA receptors derived from GluA subunits with TARP, ␥-8, show slow onset “resensitization” during continuous glutamate application. However, ␥-8-containing hippocampal AMPA receptors do not display resensitization. We found that CNIH-2 abrogates ␥8-mediated resensitization and modifies AMPA receptor pharmacology and gating to match those of hippocampal neurons. We hypothesized that CNIH2 controls the stoichiometry of TARPs to AMPA receptors. Using tandem GluA/TARP constructs to constrain stoichiometry, we found that resensitization occurs in channels with four TARP subunits per complex. Reducing the number of TARPs per complex produces AMPA receptors with neuronlike kinetics and pharmacology, suggesting a neuronal mechanism controls GluA/TARP assembly. Importantly, we find that co-expression of CNIH-2 with GluA/TARP complexes reduces TARP stoichiometry within AMPA receptors. In both rat and mouse hippocampal neurons, CNIH-2 also associates with AMPA receptors on the neuronal surface and synapses. Taken together, these studies show that CNIH-2 plays critical roles in AMPA-TARP assembly. doi:10.1016/j.neures.2011.07.400
doi:10.1016/j.neures.2011.07.398
O4-E-3-1 Invaginating inhibitory synapse with particularly rich endocannabinoid signaling machinery in the basal nucleus of the amygdala
O4-E-2-3 Glutamate receptor ␦2 is essential for input pathway-dependent regulation of synaptic AMPAR contents in cerebellar Purkinje cells
Takayuki Yoshida 1,2,3 , Motokazu Uchigashima 2 , Miwako 2 4 Yamasaki , Istvan Katona , Maya Yamazaki 5 , Kenji Sakimura 5 , Masanobu Kano 3,6 , Mitsuhiro Yoshioka 1 , Masahiko Watanabe 2
Miwako Yamasaki 1 , Taisuke Miyazaki 1 , Hirotsugu Azechi 2 , Manabu Abe 2 , Rie Natsume 2 , Teruki Hagiwara 2 , Atsu Aiba 4 , Masayoshi Mishina 3 , Kenji Sakimura 2 , Masahiko Watanabe 1 1
Dept. of Anat., Grad. Sch. of Med., Hokkaido Univ., Sapporo, Japan 2 Dept. Cellular Neurobiol., BRI, Niigata Univ., Niigata, Japan 3 Dept. Mol. Neurobiol. & Pharmacol., Grad. Sch. Med., Univ. of Tokyo, Tokyo, Japan 4 Lab. Animal Resources, Center for Disease Biol. and Integrative Medicine, Faculty of Medicine, Univ. of Tokyo, Tokyo, Japan The number of synaptic AMPA receptors (AMPARs) is the major determinant of synaptic strength, and is differently regulated in input pathway-dependent and target cell type-dependent manners. In cerebellar Purkinje cells (PCs), the density of synaptic AMPARs is approximately 5 times lower at parallel fiber (PF) synapses than at climbing fiber (CF) synapses. However, molecular mechanisms underlying this biased synaptic distribution remain unclear. As a candidate molecule, we focused on glutamate receptor ␦2 (GluR␦2
1
Dept Neuropharmacol, Hokkaido Univ Grad Sch Med, Sapporo 2 Dept Anat, Hokkaido Univ Grad Sch Med, Sapporo 3 Dept Cellular Neurosci, Osaka Univ, Suita 4 Inst Exp Med, Hungarian Acad Sci, Budapest, Hungary 5 Dept Cell Neurobiol, Brain Res Inst, Niigata Univ, Niigata 6 Dept Neurophysiol, Grad Sch Med, Univ Tokyo, Tokyo
2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of synaptic transmission in the brain. 2-AG is synthesized in activated postsynaptic neurons by sn-1-specific diacylglycerol lipase (DGL), binds to presynaptic cannabinoid CB1 receptors, suppresses neurotransmitter release, and is degraded mainly by monoacylglycerol lipase (MGL). In the basolateral amygdala complex, it has been demonstrated that CB1 is particularly enriched in axon terminals of cholecystokinin (CCK)positive GABAergic interneurons, induces short- and long-term depression at inhibitory synapses, and is involved in extinction of fear memory. Here we clarified a unique molecular convergence of DGL␣, CB1 , and MGL at spe-
e93
gia (HSP), suggesting the role of protrudin in maintaining neuronal integrity. HSPs are characterized by progressive spasticity in the lower limbs and a part of HSPs are accompanied by other symptom such as seizures, mental retardation, or dementia. We generated mice deficient in protrudin, and have found that the mutant mice exhibited reduction of LTP. Rab11 is known to promote the trafficking of AMPA receptor in response to PKA signaling at the dendritic spines during LTP. We thus examined whether protrudin is required for the Rab11-dependent trafficking of AMPA receptor. Mice deficient in either protrudin exhibited the defects in AMPA receptor transport at the dendritic spine. Furthermore, these mutant mice showed impairment in spine maturation and synaptic formation. In addition, we found that FYVE domain of protrudin is specifically phosphorylated by PKA, which promotes the interaction of protrudin with Rab11. Our results indicate that protrudin promotes the trafficking of Rab11-positive recycling endosomes to the post-synaptic plasma membrane via the PKA-signaling. Protrudin thus facilitates the AMPA receptor transport, suggesting that protrudin possibly plays a critical role in cognitive functions of the brain.
or GluD2), which is known to be efficiently trafficked to and selectively expressed at PF-PC synapses. We applied postembedding immunogold electron microscopy to GluR␦2-knockout (KO) and control mice, and measured labeling density for GluA1–4 at three excitatory synapses in the cerebellar molecular layer. In both control and GluR␦2-KO mice, GluA1–3 were localized at PF and CF synapses in PCs, while GluA2–4 were at PF synapses in interneurons. In control mice, labeling density for each of GluA1–3 was 4to-6 times lower at PF-PC synapses than at CF-PC synapses. In GluR␦2-KO mice, however, their labeling density displayed a 3-to-5-fold increase at PF synapses, but not at CF synapses, thus effectively eliminating input pathwaydependent disparity between the two PC synapses. Furthermore, we found an unexpected 2-fold increase in labeling density for GluA2 and GluA3, but not GluA4, at PF-interneuron synapses, where we identified low but significant expression of GluR␦2. These results suggest that GluR␦2 is involved in a common mechanism that restricts the number of synaptic AMPARs at PF synapses in PCs and molecular layer interneurons. Research fund: Grant-in-Aid for Scientific Research (S), CREST.
doi:10.1016/j.neures.2011.07.397
doi:10.1016/j.neures.2011.07.399
O4-E-2-2 Roles of Purkinje cell activity in climbing fiber synapse elimination in an organotypic coculture preparation of the cerebellum and medulla oblongata
O4-E-2-4 Cornichon-2 dictates physical and pharmacological properties of AMPA receptors by controlling AMPA receptor-TARP assembly
Takayasu Mikuni , Naofumi Uesaka, Masanobu Kano
Akihiko Kato 1 , Martin Tomita 2 , David Bredt 1
Dept. of Neurophysiol., Grad. Sch. of Med., Univ. of Tokyo, Tokyo, Japan
Gill 1 , Matthew
Roberts 2 , Susumu
1
Understanding of how functional neural circuits are built during postnatal development is one of the most important issues in neuroscience. It has been reported that neural activity is crucial for circuit refinement including synapse formation, maturation, elimination and maintenance. However, how neural activity regulates these processes has yet to be elucidated. The climbing fiber (CF) to Purkinje cell (PC) synapse in the cerebellum has provided a good model to study the cellular and molecular mechanisms of activitydependent synapse elimination in the developing central nervous system. We explored the mechanisms of CF to PC synapse elimination by modifying postsynaptic PC activity in an in vitro coculture preparation of cerebellar slices with medulla explants containing the inferior olive, the origin of CFs. In this preparation, multiple CF to PC synapses are formed during the initial one week of coculture, and then surplus CFs are eliminated thereafter with similar molecular mechanisms to those found in vivo. We used a lentiviral gene transfer technique to express in PCs channelrhodopsin-2 (ChR2) or microRNA targeting P/Q-type voltage-dependent Ca2+ channel (VDCC), a major VDCC in PCs, to up- or down-regulate PC activity, respectively. Using these methods, we found that CF to PC synapse elimination was impaired by PC-specific RNAi knockdown of P/Q-type VDCCs. On the other hand, increasing PC activity by blue light stimulation of ChR2-expressing PCs for two days accelerated CF to PC synapse elimination. Furthermore, acceleration of CF to PC synapse elimination by ChR2 stimulation was not observed when P/Q-type VDCCs were knocked down. These observations suggest that CF to PC synapse elimination requires postsynaptic PC activity, followed by Ca2+ influx through P/Q-type VDCCs. Research fund: Grants-in-Aid for Scientific Research (S) No. 21220006, from MEXT, Japan. The Strategic Research Program for Brain Sciences, project D, from MEXT, Japan.
Neuroscience Discovery Research, Lilly Research Laboratory 2 CNNR, Dept of Cellular and Molecular Physiology, Yale Univ, New Heaven, CT AMPA-type glutamate receptors are crucial for excitatory synaptic transmission in brain. Neuronal AMPA receptor complexes comprise a tetramer of GluA pore-forming subunits as well as accessory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and cornichon2/3 (CNIH-2/3). Here we describe that CNIH-2 regulates the channel properties of TARP containing AMPA receptors by decreasing the number of TARPs per receptor. In recombinant systems, AMPA receptors derived from GluA subunits with TARP, ␥-8, show slow onset “resensitization” during continuous glutamate application. However, ␥-8-containing hippocampal AMPA receptors do not display resensitization. We found that CNIH-2 abrogates ␥8-mediated resensitization and modifies AMPA receptor pharmacology and gating to match those of hippocampal neurons. We hypothesized that CNIH2 controls the stoichiometry of TARPs to AMPA receptors. Using tandem GluA/TARP constructs to constrain stoichiometry, we found that resensitization occurs in channels with four TARP subunits per complex. Reducing the number of TARPs per complex produces AMPA receptors with neuronlike kinetics and pharmacology, suggesting a neuronal mechanism controls GluA/TARP assembly. Importantly, we find that co-expression of CNIH-2 with GluA/TARP complexes reduces TARP stoichiometry within AMPA receptors. In both rat and mouse hippocampal neurons, CNIH-2 also associates with AMPA receptors on the neuronal surface and synapses. Taken together, these studies show that CNIH-2 plays critical roles in AMPA-TARP assembly. doi:10.1016/j.neures.2011.07.400
doi:10.1016/j.neures.2011.07.398
O4-E-3-1 Invaginating inhibitory synapse with particularly rich endocannabinoid signaling machinery in the basal nucleus of the amygdala
O4-E-2-3 Glutamate receptor ␦2 is essential for input pathway-dependent regulation of synaptic AMPAR contents in cerebellar Purkinje cells
Takayuki Yoshida 1,2,3 , Motokazu Uchigashima 2 , Miwako 2 4 Yamasaki , Istvan Katona , Maya Yamazaki 5 , Kenji Sakimura 5 , Masanobu Kano 3,6 , Mitsuhiro Yoshioka 1 , Masahiko Watanabe 2
Miwako Yamasaki 1 , Taisuke Miyazaki 1 , Hirotsugu Azechi 2 , Manabu Abe 2 , Rie Natsume 2 , Teruki Hagiwara 2 , Atsu Aiba 4 , Masayoshi Mishina 3 , Kenji Sakimura 2 , Masahiko Watanabe 1 1
Dept. of Anat., Grad. Sch. of Med., Hokkaido Univ., Sapporo, Japan 2 Dept. Cellular Neurobiol., BRI, Niigata Univ., Niigata, Japan 3 Dept. Mol. Neurobiol. & Pharmacol., Grad. Sch. Med., Univ. of Tokyo, Tokyo, Japan 4 Lab. Animal Resources, Center for Disease Biol. and Integrative Medicine, Faculty of Medicine, Univ. of Tokyo, Tokyo, Japan The number of synaptic AMPA receptors (AMPARs) is the major determinant of synaptic strength, and is differently regulated in input pathway-dependent and target cell type-dependent manners. In cerebellar Purkinje cells (PCs), the density of synaptic AMPARs is approximately 5 times lower at parallel fiber (PF) synapses than at climbing fiber (CF) synapses. However, molecular mechanisms underlying this biased synaptic distribution remain unclear. As a candidate molecule, we focused on glutamate receptor ␦2 (GluR␦2
1
Dept Neuropharmacol, Hokkaido Univ Grad Sch Med, Sapporo 2 Dept Anat, Hokkaido Univ Grad Sch Med, Sapporo 3 Dept Cellular Neurosci, Osaka Univ, Suita 4 Inst Exp Med, Hungarian Acad Sci, Budapest, Hungary 5 Dept Cell Neurobiol, Brain Res Inst, Niigata Univ, Niigata 6 Dept Neurophysiol, Grad Sch Med, Univ Tokyo, Tokyo
2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of synaptic transmission in the brain. 2-AG is synthesized in activated postsynaptic neurons by sn-1-specific diacylglycerol lipase (DGL), binds to presynaptic cannabinoid CB1 receptors, suppresses neurotransmitter release, and is degraded mainly by monoacylglycerol lipase (MGL). In the basolateral amygdala complex, it has been demonstrated that CB1 is particularly enriched in axon terminals of cholecystokinin (CCK)positive GABAergic interneurons, induces short- and long-term depression at inhibitory synapses, and is involved in extinction of fear memory. Here we clarified a unique molecular convergence of DGL␣, CB1 , and MGL at spe-