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Proteome analyses of the postsynaptic density (PSD), a proteinaceous specialization beneath the postsynaptic membrane of excitatory synapses, have identified several thousands of proteins. While proteins with predictable functions have been well studied, functionally uncharacterized proteins are mostly overlooked. In this study, we conducted a comprehensive meta-analysis of 35 PSD proteome datasets, encompassing a total of 5, 869 proteins. Employing a ranking methodology, we identified 97 proteins that remain inadequately characterized. From this selection, we focused our detailed analysis on the highest-ranked protein, FAM81A. FAM81A interacts with PSD proteins, including PSD-95, SynGAP, and NMDA receptors, and promotes liquid–liquid phase separation of those proteins in cultured cells or in vitro. Down-regulation of FAM81A in cultured neurons causes a decrease in the size of PSD-95 puncta and the frequency of neuronal firing. Our findings suggest that FAM81A plays a crucial role in facilitating the interaction and assembly of proteins within the PSD, and its presence is important for maintaining normal synaptic function. Additionally, our methodology underscores the necessity for further characterization of numerous synaptic proteins that still lack comprehensive understanding.

Dendritic spines constitute the major compartments of excitatory post-synapses. They undergo activity-dependent enlargement, which is thought to increase the synaptic efficacy underlying learning and memory. The activity-dependent spine enlargement requires activation of signaling pathways leading to promotion of actin polymerization within the spines. However, the molecular machinery that suffices for that structural plasticity remains unclear. Here, we demonstrate that shootin1a links polymerizing actin filaments in spines with the cell-adhesion molecules N-cadherin and L1-CAM, thereby mechanically coupling the filaments to the extracellular environment. Synaptic activation enhances shootin1a-mediated actin-adhesion coupling in spines. Promotion of actin polymerization is insufficient for the plasticity; the enhanced actin-adhesion coupling is required for polymerizing actin filaments to push against the membrane for spine enlargement. By integrating cell signaling, cell adhesion, and force generation into the current model of actin-based machinery, we propose molecular machinery that is sufficient to trigger the activity-dependent spine structural plasticity.

Synaptic plasticity plays a crucial role in memory formation by regulating the communication between neurons. Although actin polymerization has been linked to synaptic plasticity and dendritic spine stability, the causal link between actin polymerization and memory encoding has not been identified yet. It is not clear whether actin polymerization and structural changes in dendritic spines are a driver or a consequence of learning and memory. Using an extrinsically disordered form of the protein kinase LIMK1, which rapidly and precisely acts on ADF/cofilin, a direct modifier of actin, we induced long-term enlargement of dendritic spines and enhancement of synaptic transmission in the hippocampus on command. The activation of extrinsically disordered LIMK1 in vivo improved memory encoding and slowed cognitive decline in aged mice exhibiting reduced cofilin phosphorylation. The engineered memory by an extrinsically disordered LIMK1 supports a direct causal link between actin-mediated synaptic transmission and memory.

In this reply we thank both authors for their thoughtful insights on our original opinion piece "Guiding principles for hydrologists conducting interdisciplinary research and fieldwork with participants." We believe these discussions will help to inspire and guide current and future researchers and illustrate how to continue to bring together physical and social data, experiences, and perspectives, and bridge the gap between the two disciplines with respect to socio-hydrological topics. Furthermore, we are confident that these insights and experiences will help foster a deeper understanding for hydrologists and natural scientists engaging with these discussions and research. Here we focus on two important themes that cut across both Quandt and Haeffner's replies: (1) further discussions on the importance of perceptions and lived experiences; and (2) further discussions on collaborative working and some of the major external barriers.

In this reply to Thaler, we take the opportunity to explore two main aspects of his piece to continue the discussion: (1) the integration of data from the social and natural sciences, and (2) the importance of transdisciplinary research. We agree, and highlight that necessary learning, reflections and participation processes are time-intensive for researchers, practitioners and participants. We believe that these discussion pieces are important for informing, engaging, and debating challenges and practices, providing continued opportunities to learn from one another, enabling new forms of research and collaborations to flourish.

In this reply we thank both authors for their thoughtful insights on our original opinion piece “Guiding principles for hydrologists conducting interdisciplinary research and fieldwork with participants.” We believe these discussions will help to inspire and guide current and future researchers and illustrate how to continue to bring together physical and social data, experiences, and perspectives, and bridge the gap between the two disciplines with respect to socio-hydrological topics. Furthermore, we are confident that these insights and experiences will help foster a deeper understanding for hydrologists and natural scientists engaging with these discussions and research. Here we focus on two important themes that cut across both Quandt and Haeffner’s replies: (1) further discussions on the importance of perceptions and lived experiences; and (2) further discussions on collaborative working and some of the major external barriers.

In the hippocampus, spatial and nonspatial information are jointly represented as a neural map in which locations associated with salient features are over-represented by increased densities of relevant place cells. Although we recently demonstrated that experience-dependent establishment of these disproportionate maps is governed by selective stabilization of salient place cells following their conversion from non-place cells, the underlying mechanism for pre-established map reorganization remained to be understood. To this end, we investigated the changes in CA1 functional cellular maps imaged using two-photon calcium imaging in mice performing a reward-rearrangement task in virtual reality. Mice were pre-trained on a virtual linear track with a visual landmark and a reward in two distinct locations. Then, they were re-trained on the same track with the exception that the location of reward was shifted to match the landmark location. We found that, in contrast to de novo map formation, robust map reorganization occurred through parallel coordination of new place field formation, lateral shifting of existing place fields, and selective stabilization of place fields encoding salient locations. Our findings demonstrate that intricate interplay between multiple forms of cellular dynamics enables rapid updating of information stored in hippocampal maps.

Transient information input to the brain leads to persistent changes in synaptic circuits, contributing to the formation of memory engrams. Pre- and postsynaptic structures undergo coordinated functional and structural changes during this process, but how such changes are achieved by their component molecules remains largely unknown. We found that activated CaMKII, a central player of synaptic plasticity, undergoes liquid–liquid phase separation with the NMDA-type glutamate receptor subunit GluN2B. Due to CaMKII autophosphorylation, the condensate stably persists even after Ca2+ is removed. The selective binding of activated CaMKII with GluN2B cosegregates AMPA receptors and the synaptic adhesion molecule neuroligin into a phase-in-phase assembly. In this way, Ca2+-induced liquid–liquid phase separation of CaMKII has the potential to act as an activity-dependent mechanism to crosslink postsynaptic proteins, which may serve as a platform for synaptic reorganization associated with synaptic plasticity. © 2021, The Author(s), under exclusive licence to Springer Nature America, Inc.

Transient information input to the brain leads to persistent changes in synaptic circuits, contributing to the formation of memory engrams. Pre- and postsynaptic structures undergo coordinated functional and structural changes during this process, but how such changes are achieved by their component molecules remains largely unknown. We found that activated CaMKII, a central player of synaptic plasticity, undergoes liquid–liquid phase separation with the NMDA-type glutamate receptor subunit GluN2B. Due to CaMKII autophosphorylation, the condensate stably persists even after Ca2+ is removed. The selective binding of activated CaMKII with GluN2B cosegregates AMPA receptors and the synaptic adhesion molecule neuroligin into a phase-in-phase assembly. In this way, Ca2+-induced liquid–liquid phase separation of CaMKII has the potential to act as an activity-dependent mechanism to crosslink postsynaptic proteins, which may serve as a platform for synaptic reorganization associated with synaptic plasticity. © 2021, The Author(s), under exclusive licence to Springer Nature America, Inc.

To explore and address complex water-related issues, true collaborative, interdisciplinary research at the interface of hydrology and social science is necessary. Accordingly, hydrologists are increasingly working with social sciences and becoming involved in fieldwork with participants. With the overarching aim of facilitating collaboration and interdisciplinary water research, here we discuss important considerations and guiding principles for hydrologists, both those new to and those already familiar with interdisciplinary research, who are: (i) involved in fieldwork with participants; and (ii) working more collaboratively with social scientists. Drawing on first-hand experiences, this paper combines theory and experience from hydrologists and social scientists from their various interdisciplinary research projects to better understand key ethical, theoretical and practical considerations when working with participants. Complementary to this, we discuss the barriers and opportunities in collaborative interdisciplinary research. Facilitating these practices and understandings for hydrologists is essential to strengthen collaboration and to develop more holistic, successful research.

To explore and address complex water-related issues, true collaborative, interdisciplinary research at the interface of hydrology and social science is necessary. Accordingly, hydrologists are increasingly working with social sciences and becoming involved in fieldwork with participants. With the overarching aim of facilitating collaboration and interdisciplinary water research, here we discuss important considerations and guiding principles for hydrologists, both those new to and those already familiar with interdisciplinary research, who are: (i) involved in fieldwork with participants; and (ii) working more collaboratively with social scientists. Drawing on first-hand experiences, this paper combines theory and experience from hydrologists and social scientists from their various interdisciplinary research projects to better understand key ethical, theoretical and practical considerations when working with participants. Complementary to this, we discuss the barriers and opportunities in collaborative interdisciplinary research. Facilitating these practices and understandings for hydrologists is essential to strengthen collaboration and to develop more holistic, successful research.

To explore and address complex water-related issues, true collaborative, interdisciplinary research at the interface of hydrology and social science is necessary. Accordingly, hydrologists are increasingly working with social sciences and becoming involved in fieldwork with participants. With the overarching aim of facilitating collaboration and interdisciplinary water research, here we discuss important considerations and guiding principles for hydrologists, both those new to and those already familiar with interdisciplinary research, who are: (i) involved in fieldwork with participants; and (ii) working more collaboratively with social scientists. Drawing on first-hand experiences, this paper combines theory and experience from hydrologists and social scientists from their various interdisciplinary research projects to better understand key ethical, theoretical and practical considerations when working with participants. Complementary to this, we discuss the barriers and opportunities in collaborative interdisciplinary research. Facilitating these practices and understandings for hydrologists is essential to strengthen collaboration and to develop more holistic, successful research.

Dendritic spines constitute the major compartments of excitatory post-synapses. They undergo activity-dependent enlargement, which is thought to increase the synaptic efficacy underlying learning and memory. The activity-dependent spine enlargement requires activation of signaling pathways leading to promotion of actin polymerization within the spines. However, the molecular machinery that suffices for that structural plasticity remains unclear. Here, we demonstrate that shootin1a links polymerizing actin filaments in spines with the cell-adhesion molecules N-cadherin and L1-CAM, thereby mechanically coupling the filaments to the extracellular environment. Synaptic activation enhances shootin1a-mediated actin-adhesion coupling in spines. Promotion of actin polymerization is insufficient for the plasticity; the enhanced actin-adhesion coupling is required for polymerizing actin filaments to push against the membrane for spine enlargement. By integrating cell signaling, cell adhesion, and force generation into the current model of actin-based machinery, we propose molecular machinery that is sufficient to trigger the activity-dependent spine structural plasticity., identifier:2211-1247

To explore and address complex water-related issues, true collaborative, interdisciplinary research at the interface of hydrology and social science is necessary. Accordingly, hydrologists are increasingly working with social sciences and becoming involved in fieldwork with participants. With the overarching aim of facilitating collaboration and interdisciplinary water research, here we discuss important considerations and guiding principles for hydrologists, both those new to and those already familiar with interdisciplinary research, who are: (i) involved in fieldwork with participants; and (ii) working more collaboratively with social scientists. Drawing on first-hand experiences, this paper combines theory and experience from hydrologists and social scientists from their various interdisciplinary research projects to better understand key ethical, theoretical and practical considerations when working with participants. Complementary to this, we discuss the barriers and opportunities in collaborative interdisciplinary research. Facilitating these practices and understandings for hydrologists is essential to strengthen collaboration and to develop more holistic, successful research.

In the hippocampus, spatial and nonspatial information are jointly represented as a neural map in which locations associated with salient features are over-represented by increased densities of relevant place cells. Although we recently demonstrated that experience-dependent establishment of these disproportionate maps is governed by selective stabilization of salient place cells following their conversion from non-place cells, the underlying mechanism for pre-established map reorganization remained to be understood. To this end, we investigated the changes in CA1 functional cellular maps imaged using two-photon calcium imaging in mice performing a reward-rearrangement task in virtual reality. Mice were pre-trained on a virtual linear track with a visual landmark and a reward in two distinct locations. Then, they were re-trained on the same track with the exception that the location of reward was shifted to match the landmark location. We found that, in contrast to de novo map formation, robust map reorganization occurred through parallel coordination of new place field formation, lateral shifting of existing place fields, and selective stabilization of place fields encoding salient locations. Our findings demonstrate that intricate interplay between multiple forms of cellular dynamics enables rapid updating of information stored in hippocampal maps.

Dendritic spines constitute the major compartments of excitatory post-synapses. They undergo activity-dependent enlargement, which is thought to increase the synaptic efficacy underlying learning and memory. The activity-dependent spine enlargement requires activation of signaling pathways leading to promotion of actin polymerization within the spines. However, the molecular machinery that suffices for that structural plasticity remains unclear. Here, we demonstrate that shootin1a links polymerizing actin filaments in spines with the cell-adhesion molecules N-cadherin and L1-CAM, thereby mechanically coupling the filaments to the extracellular environment. Synaptic activation enhances shootin1a-mediated actin-adhesion coupling in spines. Promotion of actin polymerization is insufficient for the plasticity; the enhanced actin-adhesion coupling is required for polymerizing actin filaments to push against the membrane for spine enlargement. By integrating cell signaling, cell adhesion, and force generation into the current model of actin-based machinery, we propose molecular machinery that is sufficient to trigger the activity-dependent spine structural plasticity.

Dendritic spines constitute the major compartments of excitatory post-synapses. They undergo activity-dependent enlargement, which is thought to increase the synaptic efficacy underlying learning and memory. The activity-dependent spine enlargement requires activation of signaling pathways leading to promotion of actin polymerization within the spines. However, the molecular machinery that suffices for that structural plasticity remains unclear. Here, we demonstrate that shootin1a links polymerizing actin filaments in spines with the cell-adhesion molecules N-cadherin and L1-CAM, thereby mechanically coupling the filaments to the extracellular environment. Synaptic activation enhances shootin1a-mediated actin-adhesion coupling in spines. Promotion of actin polymerization is insufficient for the plasticity; the enhanced actin-adhesion coupling is required for polymerizing actin filaments to push against the membrane for spine enlargement. By integrating cell signaling, cell adhesion, and force generation into the current model of actin-based machinery, we propose molecular machinery that is sufficient to trigger the activity-dependent spine structural plasticity., journal article

In the hippocampus, spatial and nonspatial information are jointly represented as a neural map in which locations associated with salient features are over-represented by increased densities of relevant place cells. Although we recently demonstrated that experience-dependent establishment of these disproportionate maps is governed by selective stabilization of salient place cells following their conversion from non-place cells, the underlying mechanism for pre-established map reorganization remained to be understood. To this end, we investigated the changes in CA1 functional cellular maps imaged using two-photon calcium imaging in mice performing a reward-rearrangement task in virtual reality. Mice were pre-trained on a virtual linear track with a visual landmark and a reward in two distinct locations. Then, they were re-trained on the same track with the exception that the location of reward was shifted to match the landmark location. We found that, in contrast to de novo map formation, robust map reorganization occurred through parallel coordination of new place field formation, lateral shifting of existing place fields, and selective stabilization of place fields encoding salient locations. Our findings demonstrate that intricate interplay between multiple forms of cellular dynamics enables rapid updating of information stored in hippocampal maps.

Transient information input to the brain leads to persistent changes in synaptic circuits, contributing to the formation of memory engrams. Pre- and postsynaptic structures undergo coordinated functional and structural changes during this process, but how such changes are achieved by their component molecules remains largely unknown. We found that activated CaMKII, a central player of synaptic plasticity, undergoes liquid–liquid phase separation with the NMDA-type glutamate receptor subunit GluN2B. Due to CaMKII autophosphorylation, the condensate stably persists even after Ca2+ is removed. The selective binding of activated CaMKII with GluN2B cosegregates AMPA receptors and the synaptic adhesion molecule neuroligin into a phase-in-phase assembly. In this way, Ca2+-induced liquid–liquid phase separation of CaMKII has the potential to act as an activity-dependent mechanism to crosslink postsynaptic proteins, which may serve as a platform for synaptic reorganization associated with synaptic plasticity. © 2021, The Author(s), under exclusive licence to Springer Nature America, Inc.

To explore and address complex water-related issues, true collaborative, interdisciplinary research at the interface of hydrology and social science is necessary. Accordingly, hydrologists are increasingly working with social sciences and becoming involved in fieldwork with participants. With the overarching aim of facilitating collaboration and interdisciplinary water research, here we discuss important considerations and guiding principles for hydrologists, both those new to and those already familiar with interdisciplinary research, who are: (i) involved in fieldwork with participants; and (ii) working more collaboratively with social scientists. Drawing on first-hand experiences, this paper combines theory and experience from hydrologists and social scientists from their various interdisciplinary research projects to better understand key ethical, theoretical and practical considerations when working with participants. Complementary to this, we discuss the barriers and opportunities in collaborative interdisciplinary research. Facilitating these practices and understandings for hydrologists is essential to strengthen collaboration and to develop more holistic, successful research.

Hippocampal sclerosis, the major neuropathological hallmark of temporal lobe epilepsy, is characterized by different patterns of neuronal loss. The mechanisms of cell-type-specific vulnerability and their progression and histopathological classification remain controversial. Using single-cell electrophysiology in vivo and immediate-early gene expression, we reveal that superficial CA1 pyramidal neurons are overactive in epileptic rodents. Bulk tissue and single-nucleus expression profiling disclose sublayer-specific transcriptomic signatures and robust microglial pro-inflammatory responses. Transcripts regulating neuronal processes such as voltage channels, synaptic signaling, and cell adhesion are deregulated differently by epilepsy across sublayers, whereas neurodegenerative signatures primarily involve superficial cells. Pseudotime analysis of gene expression in single nuclei and in situ validation reveal separated trajectories from health to epilepsy across cell types and identify a subset of superficial cells undergoing a later stage in neurodegeneration. Our findings indicate that sublayer- and cell-type-specific changes associated with selective CA1 neuronal damage contribute to progression of hippocampal sclerosis.

Hippocampus receives dense serotonergic input specifically from raphe nuclei. However, what information is carried by this input and its impact on behavior has not been fully elucidated. Here we used in vivo two-photon imaging of activity of hippocampal median raphe projection fibers in behaving male and female mice and identified two distinct populations: one linked to reward delivery and the other to locomotion. Local optogenetic manipulation of these fibers confirmed a functional role for these projections in the modulation of reward-induced behavior. The diverse function of serotonergic inputs suggests a key role in integrating locomotion and reward information into the hippocampal CA1.

In the hippocampus, locations associated with salient features are represented by a disproportionately large number of neurons, but the cellular and molecular mechanisms underlying this over-representation remain elusive. Using longitudinal calcium imaging in mice learning to navigate in virtual reality, we find that the over-representation of reward and landmark locations are mediated by persistent and separable subsets of neurons, with distinct time courses of emergence and differing underlying molecular mechanisms. Strikingly, we find that in mice lacking Shank2, an autism spectrum disorder (ASD)-linked gene encoding an excitatory postsynaptic scaffold protein, the learning-induced over-representation of landmarks was absent whereas the over-representation of rewards was substantially increased, as was goal-directed behavior. These findings demonstrate that multiple hippocampal coding processes for unique types of salient features are distinguished by a Shank2-dependent mechanism and suggest that abnormally distorted hippocampal salience mapping may underlie cognitive and behavioral abnormalities in a subset of ASDs.

There are active oxygen species that contribute to oxidative coupling or the partial oxidation during the oxidative dehydrogenation of methane when using solid oxide catalysts, and those species have not been definitively identified. In the present study, we clarify which of the active oxygen species affect the oxidative dehydrogenation of methane by employing photo-catalysts such as TiO2 or WO3, which generate active oxygen from UV-LED irradiation conditions under an oxygen flow. These photo-catalysts were studied in combination with Sm2O3, which is a methane oxidation coupling catalyst. For this purpose, we constructed a reaction system that could directly irradiate UV-LED to a solid catalyst via a normal fixed-bed continuous-flow reactor operated at atmospheric pressure. Binary catalysts prepared from TiO2 or WO3 were either supported on or kneaded with Sm2O3 in the present study. UV-LED irradiation clearly improved the partial oxidation from methane to CO and/or slightly improved the oxidative coupling route from methane to ethylene when binary catalysts consisting of Sm2O3 and TiO2 are used, while negligible UV-LED effects were detected when using Sm2O3 and WO3. These results indicate that with UV-LED irradiation the active oxygen of O2− from TiO2 certainly contributes to the activation of methane during the oxidative dehydrogenation of methane when using Sm2O3, while the active oxygen of H2O2 from WO3 under the same conditions afforded only negligible effects on the activation of methane.

In the hippocampus, locations associated with salient features are represented by a disproportionately large number of neurons, but the cellular and molecular mechanisms underlying this over-representation remain elusive. Using longitudinal calcium imaging in mice learning to navigate in virtual reality, we find that the over-representation of reward and landmark locations are mediated by persistent and separable subsets of neurons, with distinct time courses of emergence and differing underlying molecular mechanisms. Strikingly, we find that in mice lacking Shank2, an autism spectrum disorder (ASD)-linked gene encoding an excitatory postsynaptic scaffold protein, the learning-induced over-representation of landmarks was absent whereas the over-representation of rewards was substantially increased, as was goal-directed behavior. These findings demonstrate that multiple hippocampal coding processes for unique types of salient features are distinguished by a Shank2-dependent mechanism and suggest that abnormally distorted hippocampal salience mapping may underlie cognitive and behavioral abnormalities in a subset of ASDs.Using longitudinal two-photon calcium imaging in mice during virtual navigation, Sato et al. demonstrate that persistent and separable neuronal subsets mediate the hippocampal over-representation of reward and landmark locations. Learning-induced over-representation of landmarks is absent while rapid over-representation of rewards is enhanced, in a mouse model of autism lacking Shank2.

Extracellular signal-regulated kinase 5 (ERK5) regulates diverse physiological responses such as proliferation, differentiation, and gene expression. Previously, we demonstrated that ERK5 is essential for neurite outgrowth and catecholamine biosynthesis in PC12 cells and sympathetic neurons. However, it remains unclear how ERK5 regulates the activity of ion channels, which are important for membrane excitability. Thus, we examined the effect of ERK5 on the ion channel activity in the PC12 cells that overexpress both ERK5 and the constitutively active MEK5 mutant. The gene and protein expression levels of voltage-dependent Ca²⁺ and K⁺ channels were determined by RT-qPCR or Western blotting. The A-type K⁺ current was recorded using the whole-cell patch clamp method. In these ERK5-activated cells, the gene expression levels of voltage-dependent L- and P/Q-type Ca²⁺ channels did not alter, but the N-type Ca²⁺ channel was slightly reduced. In contrast, those of Kv4.2 and Kv4.3, which are components of the A-type current, were significantly enhanced. Unexpectedly, the protein levels of Kv4.2 were not elevated by ERK5 activation, but the phosphorylation levels were increased by ERK5 activation. By electrophysiological analysis, the inactivation time constant of the A-type current was prolonged by ERK5 activation, without changes in the peak current. Taken together, ERK5 inhibits an inactivation of the A-type current by phosphorylation of Kv4.2, which may contribute to the neuronal differentiation process.

Synapses store information by long-lasting modifications of their structure and molecular composition, but the precise chronology of these changes has not been studied at single-synapse resolution in real time. Here we describe the spatiotemporal reorganization of postsynaptic substructures during long-term potentiation (LTP) at individual dendritic spines. Proteins translocated to the spine in four distinct patterns through three sequential phases. In the initial phase, the actin cytoskeleton was rapidly remodeled while active cofilin was massively transported to the spine. In the stabilization phase, cofilin formed a stable complex with F-actin, was persistently retained at the spine, and consolidated spine expansion. In contrast, the postsynaptic density (PSD) was independently remodeled, as PSD scaffolding proteins did not change their amount and localization until a late protein synthesis-dependent third phase. Our findings show how and when spine substructures are remodeled during LTP and explain why synaptic plasticity rules change over time., RIKEN Brain Science Institute, National Institutes of Health (U.S.) (Grant R01DA17310), Human Frontier Science Program (Strasbourg, France), Paul and Anne Punzak Marcus Fund

The postsynaptic density (PSD) is crucial for synaptic functions, but the molecular architecture retaining its structure and components remains elusive. Homer and Shank are among the most abundant scaffolding proteins in the PSD, working synergistically for maturation of dendritic spines. Here, we demonstrate that Homer and Shank, together, form a mesh-like matrix structure. Crystallographic analysis of this region revealed a pair of parallel dimeric coiled coils intercalated in a tail-to-tail fashion to form a tetramer, giving rise to the unique configuration of a pair of N-terminal EVH1 domains at each end of the coiled coil. In neurons, the tetramerization is required for structural integrity of the dendritic spines and recruitment of proteins to synapses. We propose that the Homer-Shank complex serves as a structural framework and as an assembly platform for other PSD proteins., Massachusetts Institute of Technology. Biophysical Instrumentation Facility, RIKEN Brain Science Institute, National Institutes of Health (U.S.) (Grant R01DA17310), Japan. Ministry of Education, Culture, Sports, Science and Technology (Grant-in-Aid for Scientific Research)

Dendritic spines are small, highly motile structures on dendritic shafts that provide flexibility to neuronal networks. Spinules are small protrusions that project from spines. The number and the length of spinules increase in response to activity including theta burst stimulation and glutamate application. However, what function spinules exert and how their formation is regulated still remains unclear. Phosphatidylinositol-3,4,5-trisphosphate (PIP[subscript 3]) plays important roles in cell motility such as filopodia and lamellipodia by recruiting downstream proteins such as Akt and WAVE to the membrane, respectively. Here we reveal that PIP[subscript 3] regulates spinule formation during structural long-term potentiation (sLTP) of single spines in CA1 pyramidal neurons of hippocampal slices from rats. Since the local distribution of PIP[subscript 3] is important to exert its functions, the subcellular distribution of PIP[subscript 3] was investigated using a fluorescence lifetime-based PIP[subscript 3] probe. PIP[subscript 3] accumulates to a greater extent in spines than in dendritic shafts, which is regulated by the subcellular activity pattern of proteins that produce and degrade PIP[subscript 3]. Subspine imaging revealed that when sLTP was induced in a single spine, PIP[subscript 3] accumulates in the spinule whereas PIP[subscript 3] concentration in the spine decreased., National Institutes of Health (U.S.) (Grant R01DA17310)

Breakthroughs in imaging techniques and optical probes in recent years have revolutionized the field of life sciences in ways that traditional methods could never match. The spatial and temporal regulation of molecular events can now be studied with great precision. There have been several key discoveries that have made this possible. Since green fluorescent protein (GFP) was cloned in 1992, it has become the dominant tracer of proteins in living cells. Then the evolution of color variants of GFP opened the door to the application of Förster resonance energy transfer (FRET), which is now widely recognized as a powerful tool to study complicated signal transduction events and interactions between molecules. Employment of fluorescent lifetime imaging microscopy (FLIM) allows the precise detection of FRET in small subcellular structures such as dendritic spines. In this review, we provide an overview of the basic and practical aspects of FRET imaging and discuss how different FRET probes have revealed insights into the molecular mechanisms of synaptic plasticity and enabled visualization of neuronal network activity both in vitro and in vivo., National Science Foundation (U.S.) (Grant R01DA17310)

Unbalanced visual input during development induces persistent alterations in the function and structure of visual cortical neurons. The molecular mechanisms that drive activity-dependent changes await direct visualization of underlying signals at individual synapses in vivo. By using a genetically engineered Förster resonance energy transfer (FRET) probe for the detection of CaMKII activity, and two-photon imaging of single synapses within identified functional domains, we have revealed unexpected and differential mechanisms in specific subsets of synapses in vivo. Brief monocular deprivation leads to activation of CaMKII in most synapses of layer 2/3 pyramidal cells within deprived eye domains, despite reduced visual drive, but not in nondeprived eye domains. Synapses that are eliminated in deprived eye domains have low basal CaMKII activity, implying a protective role for activated CaMKII against synapse elimination., National Basic Research Program of China (973 Program) (2010CB327901), Japan. Ministry of Education, Culture, Sports, Science and Technology. Foundation of Synapse and Neurocircuit Pathology. (Grant-in-Aid for Scientific Research on Innovative Area), RIKEN Brain Science Institute, National Institutes of Health (U.S.) (Grant R01DA17310), National Institutes of Health (U.S.) (Postdoctoral Fellowship), Japan Society for the Promotion of Science (Grant-In-Aid for Scientific Research)

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the central nervous system and is critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, due to lack of a specific method. Here, based on our previous work, we attempted to generate an optical probe for fluorescence lifetime imaging (FLIM) of CaMKII activity by fusing the protein with donor and acceptor fluorescent proteins at its amino- and carboxyl-termini. We first optimized the combinations of fluorescent proteins by taking advantage of expansion of fluorescent proteins towards longer wavelength in fluorospectrometric assay. Then using digital frequency domain FLIM (DFD-FLIM), we demonstrated that the resultant protein can indeed detect CaMKII activation in living cells. These FLIM versions of Camui could be useful for elucidating the function of CaMKII both in vitro and in vivo.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the central nervous system and is critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, due to lack of a specific method. Here, based on our previous work, we attempted to generate an optical probe for fluorescence lifetime imaging (FLIM) of CaMKII activity by fusing the protein with donor and acceptor fluorescent proteins at its amino- and carboxyl-termini. We first optimized the combinations of fluorescent proteins by taking advantage of expansion of fluorescent proteins towards longer wavelength in fluorospectrometric assay. Then using digital frequency domain FLIM (DFD-FLIM), we demonstrated that the resultant protein can indeed detect CaMKII activation in living cells. These FLIM versions of Camui could be useful for elucidating the function of CaMKII both in vitro and in vivo.

The Co/Pd and Co/Pd artificial lattice films have attracted much interest by their special magnetization properties. We discussed the effect of the Pt, Pd layer thickness on the magnetic anisotropy, and we showed the effect of the hydrogen ion implantation on the magnetic properties of multi-layered films. The Co/Pt and Co/Pd multi-layered films were formed on Si(111) substrates with molecular beam epitaxy. We did structure analysis, magnetic domain analysis and magnetic properties evaluation with XRD, MFM and VSM, respectively. Among the series of films of 0.4 nm Co layer, XRD showed that the film of 1.0 nm Pt layer had a highest periodicity and that they had (111) plane orientation completely. The magnetic domain size reduced with the increase of the thickness of Pt layer. We found out that the coercivity decreased linearly as a function of the length of magnetic domain wall in the unit area. The result of VSM showed that the multi-layered films of Pt thickness of less than 2.8 nm had perpendicular magnetic anisotropy. The perpendicular anisotropy energy changed by the nonmagnetic layer thickness and had a maximum value for 0.4 nm Co 0.4 nm/nonmagnetic metal 1.0 nm multi-layered film. After hydrogen implantation into the films, XRD showed that the lattice spacing was swelled with hydrogen dose. Also, MFM observed that the magnetic domain size reduced with the increase of the hydrogen dose. The easy axis of magnetization changed from perpendicular to parallel in the plane with the increase of the hydrogen dose. After evacuation of hydrogen at 473 K, perpendicular anisotropy was partially recovered. This phenomenon suggested that the origin of magnetic anisotropy was mainly the lattice mismatch and distortion in the layer interface. But Co/Pd film was not recovered by this thermal treatment. This means that Pd made stable hydride and did not evacuate hydrogen at this temperature., Pub. in Materials Research Society symposium proceedings; v. 674 pT1.3.1-T1.3.6, 2001. This article is from ADA402512 Applications of Ferromagnetic and Optical Materials, Storage and Magnetoelectronics: Symposia Held in San Francisco, California, U.S.A. on April 16-20, 2001

AMPA-type glutamate receptors (AMPA-Rs) mediate a majority of excitatory synaptic transmission in the brain. In hippocampus, most AMPA-Rs are hetero-oligomers composed of GluR1/GluR2 or GluR2/GluR3 subunits. Here we show that these AMPA-R forms display different synaptic delivery mechanisms. GluR1/GluR2 receptors are added to synapses during plasticity; this requires interactions between GluR1 and group I PDZ domain proteins. In contrast, GluR2/GluR3 receptors replace existing synaptic receptors continuously; this occurs only at synapses that already have AMPA-Rs and requires interactions by GluR2 with NSF and group II PDZ domain proteins. The combination of regulated addition and continuous replacement of synaptic receptors can stabilize long-term changes in synaptic efficacy and may serve as a general model for how surface receptor number is established and maintained.

To examine how functional circuits are established in the brain, we studied excitatory transmission in early postnatal hippocampus. Spontaneous neural activity was sufficient to selectively deliver GluR4-containing AMPA receptors (AMPA-Rs) into synapses. This delivery allowed non-functional connections to transmit at resting potentials and required NMDA receptors (NMDA-Rs) but not CaMKII activation. Subsequently, these delivered receptors were exchanged with non-synaptic GluR2-containing AMPA-Rs in a manner requiring little neuronal activity. The enhanced transmission resulting from this delivery and subsequent exchange was maintained for at least several days and required an interaction between GluR2 and NSF. Thus, this sequence of subunit-specific trafficking events triggered by spontaneous activity in early postnatal development may be crucial for initial establishment of long-lasting functional circuitry.

To elucidate mechanisms that control and execute activity-dependent synaptic plasticity, a-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA-Rs) with an electrophysiological tag were expressed in rat hippocampal neurons. Long-term potentiation (LTP) or increased activity of the calcium/ calmodulin-dependent protein kinase II (CaMKII) induced delivery of tagged AMPA-Rs into synapses. This effect was not diminished by mutating the CaMKII phosphorylation site on the GluR1 AMPA-R subunit, but was blocked by mutating a predicted PDZ domain interaction site. These results show that LTP and CaMKII activity drive AMPA-Rs to synapses by a mechanism that requires the association between GluR1 and a PDZ domain protein.