Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity. Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

In vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus.

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

Imaging neural circuit pathology of autism spectrum disorders: autism-associated genes, animal models and the application of in vivo two-photon imaging.

Recent advances in human genetics identified genetic variants involved in causing autism spectrum disorders (ASDs). Mouse models that mimic mutations found in patients with ASD exhibit behavioral phenotypes consistent with ASD symptoms. These mouse models suggest critical biological factors of ASD etiology. Another important implication of ASD genetics is the enrichment of ASD risk genes in molecules involved in developing synapses and regulating neural circuit function. Sophisticated in vivo imaging technologies applied to ASD mouse models identify common synaptic impairments in the neocortex, with genetic-mutation-specific defects in local neural circuits. In this article, we review synapse- and circuit-level phenotypes identified by in vivo two-photon imaging in multiple mouse models of ASD and discuss the contributions of altered synapse properties and neural circuit activity to ASD pathogenesis.

Advanced Technologies for Local Neural Circuits in the Cerebral Cortex.

The neural network in the brain can be viewed as an integrated system assembled from a large number of local neural circuits specialized for particular brain functions. Activities of neurons in local neural circuits are thought to be organized both spatially and temporally under the rules optimized for their roles in information processing. It is well perceived that different areas of the mammalian neocortex have specific cognitive functions and distinct computational properties. However, the organizational principles of the local neural circuits in different cortical regions have not yet been clarified. Therefore, new research principles and related neuro-technologies that enable efficient and precise recording of large-scale neuronal activities and synaptic connections are necessary. Innovative technologies for structural analysis, including tissue clearing and expansion microscopy, have enabled super resolution imaging of the neural circuits containing thousands of neurons at a single synapse resolution. The imaging resolution and volume achieved by new technologies are beyond the limits of conventional light or electron microscopic methods. Progress in genome editing and related technologies has made it possible to label and manipulate specific cell types and discriminate activities of multiple cell types. These technologies will provide a breakthrough for multiscale analysis of the structure and function of local neural circuits. This review summarizes the basic concepts and practical applications of the emerging technologies and new insight into local neural circuits obtained by these technologies.

[The Basic Modules of the Neocortex].

The mammalian neocortex contains diverse cell types but whether they organize into repeated modular circuits remains unknown. We discovered that major cell types in neocortical layer 5 form a lattice structure in many areas of the brain. Large-scale three-dimensional imaging revealed that distinct types of excitatory and inhibitory neurons form cell type-specific radial clusters termed microcolumns. Microcolumns form a hexagonal lattice tessellating a wide region of the neocortex. Neurons within individual microcolumns exhibit synchronized in vivo activity and visual responses with similar orientation preference and ocular dominance. During early postnatal development, microcolumns are coupled by cell type-specific gap junctions and later received convergent synaptic inputs. Thus, layer 5 neurons organize into a brain-wide modular system providing a template for cortical processing.

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex.

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Lattice system of functionally distinct cell types in the neocortex.

The mammalian neocortex contains many cell types, but whether they organize into repeated structures has been unclear. We discovered that major cell types in neocortical layer 5 form a lattice structure in many brain areas. Large-scale three-dimensional imaging revealed that distinct types of excitatory and inhibitory neurons form cell type-specific radial clusters termed microcolumns. Thousands of microcolumns, in turn, are patterned into a hexagonal mosaic tessellating diverse regions of the neocortex. Microcolumn neurons demonstrate synchronized in vivo activity and visual responses with similar orientation preference and ocular dominance. In early postnatal development, microcolumns are coupled by cell type-specific gap junctions and later serve as hubs for convergent synaptic inputs. Thus, layer 5 neurons organize into a brainwide modular system, providing a template for cortical processing.

Periodic organization of a major subtype of pyramidal neurons in neocortical layer V.

A major question in neocortical research is the extent to which neuronal organization is stereotyped. Previous studies have revealed functional clustering and neuronal interactions among cortical neurons located within tens of micrometers in the tangential orientation (orientation parallel to the pial surface). In the tangential orientation at this scale, however, it is unknown whether the distribution of neuronal subtypes is random or has any stereotypy. We found that the tangential arrangement of subcerebral projection neurons, which are a major pyramidal neuron subtype in mouse layer V, was not random but significantly periodic. This periodicity, which was observed in multiple cortical areas, had a typical wavelength of 30 µm. Under specific visual stimulation, neurons in single repeating units exhibited strongly correlated c-Fos expression. Therefore, subcerebral projection neurons have a periodic arrangement, and neuronal activity leading to c-Fos expression is similar among neurons in the same repeating units. These results suggest that the neocortex has a periodic functional micro-organization composed of a major neuronal subtype in layer V.

NMDA receptor-dependent synaptic translocation of insulin receptor substrate p53 via protein kinase C signaling.

The activity-dependent remodeling of postsynaptic structure is a fundamental process underlying learning and memory. Insulin receptor substrate p53 (IRSp53), a key player in cytoskeletal dynamics, is enriched in the postsynaptic density (PSD) fraction, but its significance in synaptic functions remains unclear. We report here that IRSp53 is accumulated rapidly at the postsynaptic sites of cultured hippocampal neurons after glutamate or NMDA stimulation in an actin cytoskeleton-dependent manner. Pharmacological profiles showed that a PKC inhibitor, but not other kinase inhibitors, specifically suppressed the synaptic translocation of IRSp53 in response to NMDA, and the selective activation of PKC with phorbol ester markedly induced the synaptic translocation. Reverse transcriptase-PCR and Western blotting showed that IRSp53-S is the major isoform expressed in cultured hippocampal neurons. The synaptic targeting of IRSp53-S was found to be mediated through N-terminal coiled-coil domain and the PDZ (PSD-95/Discs large/zona occludens-1)-binding sequence at its C-terminal end and regulated by the PKC phosphorylation of its N terminus. In electrophysiological experiments, overexpression of IRSp53-S wild type and IRSp53-S mutant that is spontaneously accumulated at the postsynaptic sites enhanced the postsynaptic function as detected by an increased miniature EPSC amplitude. These data suggest that IRSp53 is involved in NMDA receptor-linked synaptic plasticity via PKC signaling.

Collaboration of PSD-Zip70 with its binding partner, SPAR, in dendritic spine maturity.

Recent studies have reported on the molecular mechanisms underlying dendritic spine (spine) dynamics. Because most of these studies investigated spine dynamics by overexpressing constitutively active or dominant-negative PSD (postsynaptic density) proteins in cultured mature neurons, the results represent the enlargement of mature spines or their return to an immature state. Here, we developed the technique of in utero electroporation to investigate spine dynamics. Using this technique, we demonstrated the suppression of spine maturation by the C-terminal variants of PSD-Zip70 in vitro and in vivo. Transient overexpression of the C terminus of PSD-Zip70 and knock-down of PSD-Zip70 also displayed the destabilization of mature spines. We further found the PSD-Zip70 and SPAR (spine-associated RapGAP) interaction via the short C-terminal region of PSD-Zip70 and the GK-binding domain of SPAR. In association with immature spines induced by overexpression of the PSD-Zip70 C terminus or knock-down of PSD-Zip70, SPAR lost its spine localization. Overexpression of the GK-binding domain of SPAR also induced to form immature spines without affecting the localization of PSD-Zip70 in the small heads of filopodial spines. Our results suggest that PSD-Zip70 in collaboration with SPAR is critically involved in spine maturity, especially in the mature spine formation and the maintenance of spine maturity.

Involvement of the phosphatidylinositol 3-kinase/rac1 and cdc42 pathways in radial migration of cortical neurons.

During cortical development, newly generated neurons migrate radially toward their final positions. Although several candidate genes essential for this radial migration have been reported, the signaling pathways regulating it are largely unclear. Here we studied the role of phosphatidylinositol (PI) 3-kinase and its downstream signaling molecules in the radial migration of cortical neurons in vivo and in vitro. The expression of constitutively active and dominant-negative PI 3-kinases markedly inhibited radial migration. In the neocortical slice culture, a PI 3-kinase inhibitor suppressed the formation of GTP-bound Rac1 and Cdc42 and radial migration. Constitutively active and dominant-negative forms of Rac1 and Cdc42 but not Akt also significantly inhibited radial migration. In migrating neurons, wild-type Rac1 and Cdc42 showed different localizations; Rac1 localized to the plasma membrane and Cdc42 to the perinuclear region on the side of the leading processes. These results suggest that both the PI 3-kinase/Rac1 and Cdc42 pathways are involved in the radial migration of cortical neurons and that they have different roles.

MALS is a binding partner of IRSp53 at cell-cell contacts.

Insulin receptor substrate p53 (IRSp53) is a key player in cytoskeletal dynamics, interacting with the actin modulators WAVE2 and Mena. Here, we identified a PDZ protein, MALS, as an IRSp53-interacting protein using a yeast two-hybrid screen. A pull-down assay showed that IRSp53 and MALS interact through the PDZ domain of MALS and the C-terminal PDZ-binding sequence of IRSp53. Their interaction in MDCK cells was also demonstrated by co-immunoprecipitation. Immunocytochemistry showed the colocalization of IRSp53 and MALS at cell-cell contacts. Cytochalasin D induced the redistribution of both proteins to the cytosol. Thus, MALS is a partner of IRSp53 anchoring the actin-based membrane cytoskeleton at cell-cell contacts.

Synaptic targeting of PSD-Zip45 (Homer 1c) and its involvement in the synaptic accumulation of F-actin.

PSD-Zip45/Homer1c, which contains an enabled/VASP homology 1 (EVH1) domain and leucine zipper motifs, is a postsynaptic density (PSD) scaffold protein that interacts with metabotropic glutamate receptors and the shank family. We studied the molecular mechanism underlying the synaptic targeting of PSD-Zip45 in cultured hippocampal neurons. The EVH1 domain and the extreme C-terminal leucine zipper motif were molecular determinants for its synaptic targeting. The overexpression of the mutant of the EVH1 domain or deletion of the extreme C-terminal leucine zipper motif markedly suppressed the synaptic localization of endogenous shank but not PSD-95 or GKAP. In contrast, an overexpressed GKAP mutant lacking shank binding activity had no effect on the synaptic localization of shank. Actin depolymerization by latrunculin A reduced the synaptic localization of PSD-Zip45, shank, and F-actin but not of PSD-95 or GKAP. Overexpression of PSD-Zip45 enhanced the accumulation of synaptic F-actin. Additionally, overexpression of PSD-Zip45 and an isoform of shank induced synaptic enlargement in association with the further accumulation of synaptic F-actin. The EVH1 domain and extreme C-terminal leucine zipper motif of PSD-Zip45 were also critical for these events. Thus, these data suggest that the PSD-Zip45-shank and PSD-95-GKAP complexes form different synaptic compartments, and PSD-Zip45 alone or PSD-Zip45-shank is involved in the synaptic accumulation of F-actin.

The postsynaptic density and dendritic raft localization of PSD-Zip70, which contains an N-myristoylation sequence and leucine-zipper motifs.

The postsynaptic site of the excitatory synapse, which is composed of the postsynaptic density (PSD) attached to the postsynaptic membrane, is a center for synaptic plasticity. To reveal the molecular organization and functional regulation of the postsynaptic site, we cloned a 70 kDa protein that is concentrated in PSDs using a monoclonal antibody against the PSD. This protein, named PSD-Zip70, is highly homologous to the human FEZ1/LZTS1 gene product. PSD-Zip70 contains an N-myristoylation consensus sequence, a polybasic cluster in the N-terminal region and four leucine-zipper motifs in the C-terminal region. Light and electron microscopy showed that this protein was localized to the dendritic spines, especially in the PSD and the postsynaptic membrane. Fractionation of the synaptic plasma membrane demonstrated that PSD-Zip70 was localized to the PSD and the dendritic raft. In Madin-Darby canine kidney (MDCK) cells, exogenous PSD-Zip70 was targeted to the apical plasma membrane of microvilli, and its N-myristoylation was necessary for this targeting. In hippocampal neurons, N-myristoylation was also required for the membrane localization and the C-terminal region was critically involved in the synaptic targeting. These results suggest that PSD-Zip70 may be involved in the dynamic properties of the structure and function of the postsynaptic site.

Study on the inhibition of protein production by L-cysteine derivatives of nucleic acid bases.

Isopoly (S-carboxymethyl-L-cysteine) derivatives of nucleic acids bases were prepared as antisense compounds. In previous studies, we investigated the properties of these compounds in vitro, and revealed that these compounds in vivo regulated the cell death presumably due to the inhibition of protein production. In this study, western and northern blots were carried out in order to reveal the mechanism of this inhibition for N-methyl-D-aspartate receptor in neuroblastoma x glioma hybrid NG108-15 cell line. In addition, we investigated the resistance of these compounds against cell extract and the metabolism. In conclusion, we proved that these compounds inhibited the protein production by antisense mechanism.