Chitoshi Takayama

Impairment of suckling response, trigeminal neuronal pattern formation, and hippocampal LTD in NMDA receptor ε2 subunit mutant mice

Multiple epsilon subunits are major determinants of the NMDA receptor channel diversity. Based on their functional properties in vitro and distributions, we have proposed that the epsilon 1 and epsilon 2 subunits play a role in synaptic plasticity. To investigate the physiological significance of the NMDA receptor channel diversity, we generated mutant mice defective in the epsilon 2 subunit. These mice showed no suckling response and died shortly after birth but could survive by hand feeding. The mutation hindered the formation of the whisker-related neuronal barrelette structure and the clustering of primary sensory afferent terminals in the brainstem trigeminal nucleus. In the hippocampus of the mutant mice, synaptic NMDA responses and longterm depression were abolished. These results suggest that the epsilon 2 subunit plays an essential role in both neuronal pattern formation and synaptic plasticity.

Developmental changes in expression and distribution of the glutamate receptor channel δ2 subunit according to the Purkinje cell maturation

The glutamate receptor (GluR) channel delta 2 subunit is considered to be a functional molecule involved in motor coordination, Purkinje cell synapse formation and cerebellar long-term depression. We examined developmental changes in expression and distribution of the GluR delta 2 in the mouse cerebellum by in situ hybridization and immunohistochemistry. The GluR delta 2 mRNA was detected as early as embryonic day 15 (E15) in a cell mass consisting of Purkinje neuroblasts in the posterior cerebellum. During late embryonic and postnatal periods, the GluR delta 2 mRNA was expressed abundantly and specifically in Purkinje cells. By immunohistochemistry, immunoreactivity of the GluR delta 2 was found in both shafts and spines of Purkinje dendrites at early postnatal period. By P21, however, the intense immunoreactivity became restricted to the dendritic spines, especially along the postsynaptic membrane in contact with parallel fiber terminals. These findings suggested that the transcription of the GluR delta 2 subunit occurs in the Purkinje cells from fetal through adult stage, but the intracellular localization of the protein products undergoes an alteration from non-synaptic to synaptic site when active synaptogenesis takes place.

Reduced spontaneous activity of mice defective in the ε4 subunit of the NMDA receptor channel

In an attempt to examine the functional significance of the molecular diversity of theN-methyl-d-aspartate (NMDA) receptor channel, we generated mutant mice defective in the e4 subunit by gene targeting technique. The e4 subunit mutant mice grew and mated normally. No e4 subunit protein was detected in the homozygous mutant mice, and the amount of the e4 subunit protein of 155 kDa was reduced in the heterozygous mice. The expressions of the other NMDA receptor channel subunit mRNAs were not appreciably affected by the mutation. The mutant mice exhibited no obvious histological abnormalities in the various brain regions and in the formation of whisker-related neuronal patterns (barrels, barreloids and barrelettes). In an open field test, however, the e4 subunit mutant mice showed a reduced spontaneous activity. No significant difference was found between the heterozygous and mutant mice in motor activity and anxiety tests. These results suggest that the e4 subunit of the NMDA receptor channel plays a role in vivo in controlling the spontaneous behavioral activity.

Identification of cell types producing RANTES, MIP-1α and MIP-1β in rat experimental autoimmune encephalomyelitis by in situ hybridization

The chemokines RANTES, macrophage inflammatory protein (MIP)-1α and MIP-1β are members of the β-family of chemokines and potent chemoattractants for lymphocytes and monocytes. To investigate the factors which regulate lymphocyte traffic in experimental autoimmune encephalomyelitis (EAE), we studied, by in situ hybridization analysis, the kinetics of mRNA expression and the potent cellular sources of RANTES, MIP-1α and MIP-1β in the central nervous system (CNS) during the course of EAE. RANTES-positive cells appeared in the subarachnoid space and infiltrated the subpial region at around day 10, increased to a peak at days 12–13 and then decreased following the resolution of the acute phase of EAE, though elevated RANTES message expressions still remained on chronic subclinical stage. Most of RANTES positive cells were identified as T-lymphocytes located mainly around blood vessels, by combined studies of in situ hybridization and immunohistochemistry. The remainder of the RANTES-positive cells were astrocytes and macrophages/microglia. MIP-1α and MIP-1β mRNA-positive cells appeared around day 10, increased further on days 12–13 and then gradually decreased. Most of the MIP-1α- and MIP-1β-positive mononuclear cells were located around blood vessels. The kinetics of RANTES, MIP-1α and MIP-1β expression paralleled those of the recruitment of infiltrating inflammatory cells and disease severity. Our observations support the possibility that chemokine production by T-cells, macrophages and astrocytes lead to the infiltration of inflammatory cells into the CNS parenchyma during the acute phase of EAE.

The Postmitotic Growth Suppressor Necdin Interacts with a Calcium-binding Protein (NEFA) in Neuronal Cytoplasm

Necdin, a growth suppressor expressed predominantly in postmitotic neurons, interacts with viral oncoproteins and cellular transcription factors E2F1 and p53. In search of other cellular targets of necdin, we screened cDNA libraries from neurally differentiated murine embryonal carcinoma P19 cells and adult rat brain by the yeast two-hybrid assay. We isolated cDNAs encoding partial sequences of mouse NEFA and rat nucleobindin (CALNUC), which are Ca2+-binding proteins possessing similar domain structures. Necdin interacted with NEFA via a domain encompassing two EF hand motifs, which had Ca2+ binding activity as determined by 45Ca2+ overlay. NEFA was widely distributed in mouse organs, whereas necdin was expressed predominantly in the brain and skeletal muscle. In mouse brain in vivo, NEFA was localized in neuronal perikarya and dendrites. By immunoelectron microscopy, NEFA was localized to the cisternae of the endoplasmic reticulum and nuclear envelope in brain neurons. NEFA-green fluorescent protein (GFP) fusion protein expressed in neuroblastoma N1E-115 cells was retained in the cytoplasm and partly secreted into the culture medium. Necdin enhanced the cytoplasmic retention of NEFA-GFP and potentiated the effect of NEFA-GFP on caffeine-evoked elevation of cytosolic Ca2+ levels. Thus, necdin and NEFA might be involved in Ca2+ homeostasis in neuronal cytoplasm.

Light- and electron-microscopic localization of the glutamate receptor channel 62 subunit in the mouse Purkinje cell

The localization of the glutamate receptor channel delta 2 subunit was investigated by immunohistochemistry. The delta 2-immunoreactivity was observed exclusively in the molecular layer of the cerebellar cortex. The electron microscopic analysis showed that the delta 2 subunit was localized in the dendritic spines of the Purkinje cells. The immunopositive spines often formed synaptic contacts with parallel fiber varicosities. As for the Purkinje cells ectopically localized in the cerebellar nuclei and brain stem, the dendritic shafts and cell bodies were strongly labeled. These ectopic Purkinje cells also formed asymmetrical synapses at the delta 2-immunopositive dendritic spines. Considering the specific localization of the delta 2 subunit in the postsynaptic site of the Purkinje cells, the subunit is suggested to be involved in the excitatory synaptic transmission in the cells, as a component of the glutamate receptor channel.

Developmental localization of potassium chloride co-transporter 2 (KCC2), GABA and vesicular GABA transporter (VGAT) in the postnatal mouse somatosensory cortex

Gamma-amino butyric acid (GABA) mediates the hyperpolarization of membrane potential, negatively regulating glutamatergic activity in the adult brain, whereas, mediates depolarization in the immature brain. This developmental shift in GABA actions is induced by the expression of potassium chloride co-transporter 2 (KCC2). In this study, we focused on the developing mouse somatosensory cortex, where the barrel structure in layer 4 is altered by the whisker-lesion during the critical period, before postnatal day 4 (P4). First, to clarify the time-course of postnatal changes in GABA actions, we investigated the developmental localization of KCC2. Second, to reveal its spatial and temporal relationship with GABA synapse formation, we examined the developmental localization of GABA and vesicular GABA transporter. KCC2 was localized within the pyramidal cells in layer 5 after P3, granule cells in layer 4 after P5 and neurons in layers 2 and 3 after P7, indicating that KCC2 was expressed in the chronological order of neuronal settling at the destination. The onset of KCC2 localization was almost concomitant with the formation of GABA synapses, suggesting that GABA was inhibitory after GABA synapse formation. Furthermore, extrasynaptically released GABA might be involved in the maintenance of activity-dependent plasticity as an excitatory transmitter during the critical period.

Formation of GABAergic synapses in the cerebellum

In the adult central nervous system (CNS), γ-amino butyric acid (GABA) is a predominant inhibitory neurotransmitter, and is involved in the expression of various higher brain functions. In the cerebellum, formation of GABAergic synapses is crucial for cerebellar functions. However, it is not fully understood how GABAergic synapses and networks are formed. We are morphologically investigating the developmental changes in GABAergic signaling and the mechanisms underlying the assembly of GABAergic synapses using the cerebellum, which provides an ideal system for the investigation of brain development. The anatomy and development of GABAergic synapses and networks in the cerebellar cortex are reviewed, the key factors for the formation of GABAergic synapses are addressed, and the mechanisms underlying the formation of cerebellar GABAergic networks are discussed

Architecture of Purkinje cells of the reeler mutant mouse observed by immunohistochemistry for the inositol 1,4,5-trisphosphate receptor protein P400

P400 protein, which is identical to the inositol 1,4,5-trisphosphate receptor protein, is a glycoprotein closely associated with the membranes of Purkinje cells. Three types of monoclonal antibodies against P400 protein were employed for the immunohistochemical detection of Purkinje cells in the cerebellum and brainstem of the normal and reeler mouse. Purkinje cells in both types of mice were immunoreactive against anti-P400 antibodies, and the soma, dendrites, axon and even terminal boutons in the cerebellar and vestibular nuclei could be clearly visualized. In the cerebellum of the reeler mutant, the heterotopic Purkinje cells both within and below the granule cell layer were also immunopositive and could be clearly differentiated from the deep cerebellar nuclei, in which neurons were immunonegative. The molecular layer of the reeler cerebellum varied in thickness and certain parts were completely defective. The dendrites within the molecular layer extended from Purkinje cells whose cell bodies were located in the normal position, abnormally in the granule cell layer, or at the surface of the central mass. Outside the cortex of the cerebellum, ectopic Purkinje cells were demonstrated in 3 cerebellar nuclei, the cerebellar medulla and peduncle, and brainstem of the normal and reeler mouse.

Altered distribution of inhibitory synaptic terminals in reeler cerebellum with special reference to malposition of GABAergic neurons

In immunohistochemical reactions against glutamic acid decarboxylase (GAD), gamma-aminobutyric acid (GABA) and glycine (Gly), neurons in the mouse cerebellum showed the following reactivities: (1) the dendrites and cell bodies of the Purkinje cells were only GAD-positive, but their axonal terminals were GABA- and GAD-positive; (2) in both stellate and basket cells, the cell bodies and terminals were GABA- and GAD-positive but Gly-negative; (3) the Golgi cells were GABA-, GAD- and Gly-positive; (4) the granule cells were negative with all antibodies. Based on the populations of each type of neuron, identified by the properties mentioned above, the reeler cerebellum was divided into four regions, namely, (1) the molecular and Purkinje cell layers covering the surface of the cerebellum, where the stellate and basket cells were present as in normal mouse, (2) the granule cell layer, where the heterotopic Purkinje and stellate-type cells (including both stellate and basket cells) were present together with the granule and Golgi cells, (3) the region beneath the granule cell layer where Purkinje cells were present as clusters of several neurons, and in addition, the superficial zone of the central cell mass, where the stellate-type and Golgi cells were present among the Purkinje cells, and (4) the deep zone of the central cell mass, where the Golgi cells were exclusively present among the Purkinje cells. The heterologous synapses originating from inhibitory interneurons were formed on the Purkinje cells closely related to the distribution of these neurons.