Noriyuki Morita

Nav1.1 Localizes to Axons of Parvalbumin-Positive Inhibitory Interneurons: A Circuit Basis for Epileptic Seizures in Mice Carrying an Scn1a Gene Mutation

Loss-of-function mutations in human SCN1A gene encoding Nav1.1 are associated with a severe epileptic disorder known as severe myoclonic epilepsy in infancy. Here, we generated and characterized a knock-in mouse line with a loss-of-function nonsense mutation in the Scn1a gene. Both homozygous and heterozygous knock-in mice developed epileptic seizures within the first postnatal month. Immunohistochemical analyses revealed that, in the developing neocortex, Nav1.1 was clustered predominantly at the axon initial segments of parvalbumin-positive (PV) interneurons. In heterozygous knock-in mice, trains of evoked action potentials in these fast-spiking, inhibitory cells exhibited pronounced spike amplitude decrement late in the burst. Our data indicate that Nav1.1 plays critical roles in the spike output from PV interneurons and, furthermore, that the specifically altered function of these inhibitory circuits may contribute to epileptic seizures in the mice.

Distribution of glucocorticoid receptor immunoreactivity and mRNA in the rat brain: an immunohistochemical and in situ hybridization study.

The localization of glucocorticoid receptor (GR) immunoreactivity and mRNA in the adult rat brain was examined by light microscopic and electron microscopic immunohistochemistries, and in situ hybridization. For the purpose of detailed investigation of the distribution and comparison of GR immunoreactivities and mRNAs, specific polyclonal antibodies against a part of the transcription modulation (TR) domain of rat GR were used in the immunohistochemistry, whereas fluorescein-labeled RNA probes, complementary to the TR domain in the GR cDNA were used in the in situ hybridization. In the rat brain, GR immunoreactivity was predominantly localized in the cell nucleus, and the expression of GR mRNA was detected in the cytoplasm. GR-immunoreactive and GR mRNA-containing cells were widely distributed from the olfactory bulb of the forebrain to the gracile-cuneate nuclei of the medulla oblongata. The highest densities of GR-immunoreactive and mRNA-containing cells were observed in the subfields of cerebral cortex, olfactory cortex, hippocampal formation, amygdala, septal region, dorsal thalamus, hypothalamus, trapezoid body, cerebellar cortex, locus coeruleus and dorsal nucleus raphe. The distributional pattern of GR immunoreactivity in many regions was well-correlated with that of GR mRNA, but in the CA3 and CA4 pyramidal layers of the hippocampus, different localization was noted. The present study provides the groundwork for elucidating the role of GRs in brain function.

REAL-TIME IMAGING OF GLUCOCORTICOID RECEPTOR DYNAMICS IN LIVING NEURONS AND GLIAL CELLS IN COMPARISON WITH NON-NEURAL CELLS

To investigate the intracellular trafficking of glucocorticoid receptor (GR) in response to various conditions in a single living cell, a green fluorescent protein (GFP) and rat GR chimera construct (GFP‐GR) was prepared. We transiently transfected GFP‐GR into primary cultured rat hippocampal neurons, cortical glial cells, and non‐neural cells, e.g. COS‐1 cells and CV‐1 cells, and compared the dynamic changes in subcellular localization of GFP‐GR in these cells. When GFP‐GR was expressed in the cells, GFP‐GR efficiently transactivated the mouse mammary tumour virus promoter in response to dexamethasone (DEX). The cytoplasm‐to‐nuclear translocation of GFP‐GR induced with 10–7 m DEX, a specific agonist of GR, at 37 °C was completed within 30 min in all cell types used, and the rate of nuclear translocation was dependent on the ligand dose. The translocation of GFP‐GR into the nucleus from the cytoplasm was induced in a ligand‐specific manner, similar to that of the native GR. The disruption of microtubules by colchicine or nocodazole showed no significant effect on the DEX‐induced GFP‐GR translocation from the cytoplasmic region to the nuclear region. The cells were not deteriorated during time‐lapse imaging analysis for 1 h at 37 °C. The present findings suggest that the subcellular localization of GFP‐GR is dynamically changed in response to extracellular and intracellular conditions, and that there are no conspicuous variations in the manner of trafficking of GR among different types of cells in vitro.

2008 Special Issue: Cerebellar development transcriptome database (CDT-DB): Profiling of spatio-temporal gene expression during the postnatal development of mouse cerebellum

A large amount of genetic information is devoted to brain development and functioning. The neural circuit of the mouse cerebellum develops through a series of cellular and morphological events (including neuronal proliferation and migration, axogenesis, dendritogenesis, synaptogenesis and myelination) all within three weeks of birth. All of these events are controlled by specific gene groups, whose temporal and spatial expression profiles must be encoded in the genome. To understand the genetic basis underlying cerebellar circuit development, we analyzed gene expression (transcriptome) during the developmental stages on a genome-wide basis. Spatio-temporal gene expression data were collected using in situ hybridization for spatial (cellular and regional) resolution and fluorescence differential display, GeneChip, microarray and RT-PCR for temporal (developmental time series) resolution, and were annotated using Gene Ontology (controlled terminology for genes and gene products) and anatomical context (cerebellar cell types and circuit structures). The annotated experimental data were integrated into a knowledge resource database, the Cerebellar Development Transcriptome Database (CDT-DB http://www.cdtdb.brain.riken.jp), with seamless links to the relevant information at various bioinformatics database websites. The CDT-DB not only provides a unique informatics tool for mining both spatial and temporal pattern information on gene expression in developing mouse brains, but also opens up opportunities to elucidate the transcriptome for cerebellar development.

Function of aberrant EGFR in malignant gliomas

The most common alteration of the epidermal growth factor receptor (EGFR) gene in human malignant gliomas is an in-frame deletion of exon 2–7 from the extracellular domain. To study the relationship between the expression of this aberrant EGFR and cell proliferation, as well as apoptosis in malignant gliomas, we have developed U-87MG cell transfectants that express the aberrant (mutant-type) or normal (wild-type) EGFR. We analyzed cell number, tumor volume, and MIB-1 positive rate as proliferation markers, and found that in tissue culture, tumors derived from U-87 MG cells (mutant-type) have the same proliferative activity as those derived from U-87 MG cells (wild-type). However, when cells expressing mutant EGFR were implanted into nude mice subcutaneously, the tumorigenic capacity was much enhanced. We also found that the apototic index of tumors derived from U-87 MG cells (mutant-type) was less than 0.1%, whereas that of wild-type tumor was 1%. These results suggest that aberrant EGFR affects the malignancy of glioma by stimulating proliferation and inhibiting apoptosis.

In vitro and in vivo immunocytochemistry for the distribution of mineralocorticoid receptor with the use of specific antibody

To examine the distribution of mineralocorticoid receptor (MR) and the interactions with glucocorticoid receptor (GR) in the brain, we raised a polyclonal antibody against the transcriptional modulation domain of rat MR using the GST-fusion system. Immunoblotting analysis revealed that this antibody recognized a band with the molecular mass of MR in MR-transfected COS-1 cells and in a homogenate of rat hippocampus, and showed no cross-reactivity with GR. In vitro immunocytochemistry of both primary cultured hippocampal neurons and MR-transfected cells revealed immunoreactivity detected by this antibody in both the cytoplasm and nucleus in the absence of aldosterone (ALD), a specific agonist of MR. After 1 h of treatment with 10(-7) M ALD, the MR-immunoreactivity was accumulated in the nuclear region. In the case of GR-transfected cells, our anti-MR antibody either detected no immunopositive cells in the presence or absence of GR agonist. In our in vivo study, MR-immunoreactivity was observed in the rat hippocampus, where cell nuclei showed immunopositive reactions. These results suggest that our antibody against rat MR shows high specificity for the receptor both in liganded and unliganded forms, with no cross-reactivity to GR, and will be useful for cell biological and neuroanatomical investigations of MR.

Opalin, a Transmembrane Sialylglycoprotein Located in the Central Nervous System Myelin Paranodal Loop Membrane

In contrast to compact myelin, the series of paranodal loops located in the outermost lateral region of myelin is non-compact; the intracellular space is filled by a continuous channel of cytoplasm, the extracellular surfaces between neighboring loops keep a definite distance, but the loop membranes have junctional specializations. Although the proteins that form compact myelin have been well studied, the protein components of paranodal loop membranes are not fully understood. This report describes the biochemical characterization and expression of Opalin as a novel membrane protein in paranodal loops. Mouse Opalin is composed of a short N-terminal extracellular domain (amino acid residues 1–30), a transmembrane domain (residues 31–53), and a long C-terminal intracellular domain (residues 54–143). Opalin is enriched in myelin of the central nervous system, but not that of the peripheral nervous system of mice. Enzymatic deglycosylation showed that myelin Opalin contained N- and O-glycans, and that the O-glycans, at least, had negatively charged sialic acids. We identified two N-glycan sites at Asn-6 and Asn-12 and an O-glycan site at Thr-14 in the extracellular domain. Site-directed mutations at the glycan sites impaired the cell surface localization of Opalin. In addition to the somata and processes of oligodendrocytes, Opalin immunoreactivity was observed in myelinated axons in a spiral fashion, and was concentrated in the paranodal loop region. Immunogold electron microscopy demonstrated that Opalin was localized at particular sites in the paranodal loop membrane. These results suggest a role for highly sialylglycosylated Opalin in an intermembranous function of the myelin paranodal loops in the central nervous system.

DSCAM Deficiency Causes Loss of Pre-Inspiratory Neuron Synchroneity and Perinatal Death

Down syndrome cell adhesion molecule (DSCAM) is a neural adhesion molecule that plays diverse roles in neural development. We disrupted the Dscam locus in mice and found that the null mutants (Dscam−/−) died within 24 h after birth. Whole-body plethysmography showed irregular respiration and lower ventilatory response to hypercapnia in the null mutants. Furthermore, a medulla–spinal cord preparation of Dscam−/− mice showed that the C4 ventral root activity, which drives diaphragm contraction for inspiration, had an irregular rhythm with frequent apneas. Optical imaging of the preparation using voltage-sensitive dye revealed that the pre-inspiratory neurons located in the rostral ventrolateral medulla and belonging to the rhythm generator for respiration, lost their synchroneity in Dscam−/− mice. Dscam+/− mice, which survived to adulthood without any overt abnormalities, also showed irregular respiration but milder than Dscam−/− mice. These results suggest that DSCAM plays a critical role in central respiratory regulation in a dosage-dependent manner.

Clustered Fine Compartmentalization of the Mouse Embryonic Cerebellar Cortex and Its Rearrangement into the Postnatal Striped Configuration

Compartmentalization is essential for a brain area to be involved in different functions through topographic afferent and efferent connections that reflect this organization. The adult cerebellar cortex is compartmentalized into longitudinal stripes, in which Purkinje cells (PCs) have compartment-specific molecular expression profiles. How these compartments form during development is generally not understood. To investigate this process, we focused on the late developmental stages of the cerebellar compartmentalization that occur from embryonic day 17.5 (E17.5), when embryonic compartmentalization is evidently observed, to postnatal day 6 (P6), when adult-type compartmentalization begins to be established. The transformation between these compartmentalization patterns was analyzed by mapping expression patterns of several key molecular markers in serial cerebellar sections in the mouse. A complete set of 54 clustered PC subsets, which had different expression profiles of FoxP2, PLCβ4, EphA4, Pcdh10, and a reporter molecule of the 1NM13 transgenic mouse strain, were distinguished in three-dimensional space in the E17.5 cerebellum. Following individual PC subsets during development indicated that these subsets were rearranged from a clustered and multilayered configuration to a flattened, single-layered and striped configuration by means of transverse slide, longitudinal split, or transverse twist spatial transformations during development. The Purkinje cell-free spaces that exist between clusters at E17.5 become granule cell raphes that separate striped compartments at P6. The results indicate that the ∼50 PC clusters of the embryonic cerebellum will ultimately become the longitudinal compartments of the adult cerebellum after undergoing various peri- and postnatal transformations that alter their relative spatial relationships.

Phospholipase D family member 4, a transmembrane glycoprotein with no phospholipase D activity, expression in spleen and early postnatal microglia.

Background Phospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)4-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4. Methodology/Principal Findings PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity. Conclusions/Significance Results showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells.