Miho Oyasu

Immunocytochemical localization of a neuron-specific thrombospondin-1-like protein, NELL2: light and electron microscopic studies in the rat brain

We have studied the cellular and intracellular localization of NELL2, a neural thrombospondin-1-like protein. NELL2 protein was detected as doublet bands of 140 and 90 kDa with the use of the specific antibodies raised against the C-terminal region of NELL2 and was recognized only in the brain but not in the peripheral tissues. Within the brain, NELL2 was abundantly present in the hippocampus and cerebral cortex, found moderately in the olfactory bulb and hypothalamus, and at a low level in the thalamus, cerebellum, and medulla. Immunocytochemically, NELL2 was seen only in neurons but not in glial cells or in the white matter. NELL2-immunoreactive cells were distributed throughout the brain with the highest density in the hippocampus and cerebral cortex. NELL2 was mainly found in the cell bodies of neurons and the immunoreactivity was often seen as dots in the perikarya. The distribution of NELL2 immunoreactivity did not completely correspond to that of any subtypes of protein kinase C (PKC). Under electron microscopy, NELL2 protein was associated with the endoplasmic reticulum (ER), especially with rough ER. NELL2 immunoreactivity was found in the restricted parts of the ER and found commonly inside the ER. These results suggest that NELL2 protein is synthesized by neurons and may be secreted from the neurons involved in certain neuronal functions.

IMMUNOHISTOCHEMICAL LOCALIZATION OF SEROTONIN TRANSPORTER IN NORMAL AND COLCHICINE TREATED RAT BRAIN

Distribution of serotonin transporter (SET) was examined immunohistochemically in the rat brain using two specific polyclonal antibodies raised against oligopeptides corresponding with 15 amino acids of carboxyl terminus and 14 amino acids of amino terminus of rat SET. The distribution and density of SET immunoreactive varicose fibers were quite similar to those of serotonin immunoreactive fibers, however no neuronal cell bodies in the brainstem raphe nuclei was stained in normal rat brain. Electron microscopic study showed that SET immunoreactivity was predominantly localized in the presynaptic terminals. After intraventricular infusion of colchicine, neuronal perikarya of dorsal, median, and pontine raphe nuclei became visible. These results suggest that SET is likely present at the synaptic terminals of serotonergic neurons and such localization may be in good agreement with its pharmacological action which includes reuptake of serotonin at presynaptic nerve terminals.

Immunogold Electron Microscopic Demonstration of Distinct Submembranous Localization of the Activated γPKC Depending on the Stimulation

We examined the precise intracellular translocation of γ subtype of protein kinase C (γPKC) after various extracellular stimuli using confocal laser-scanning fluorescent microscopy (CLSM) and immunogold electron microscopy. By CLSM, treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a slow and irreversible accumulation of green fluorescent protein (GFP)-tagged γPKC (γPKC–GFP) on the plasma membrane. In contrast, treatment with Ca2+ ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of γPKC–GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that γPKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of γPKC–GFP on the plasma membrane, Ca2+ ionophore and NMDA rapidly translocated γPKC–GFP to the plasma membrane and then restricted γPKC–GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca2+ influx alone induced the association of γPKC with the plasma membrane for only a moment and then located this enzyme at a proper distance in a touch-and-go manner, whereas diacylglycerol or TPA tightly anchored this enzyme on the plasma membrane. The distinct subcellular targeting of γPKC in response to various stimuli suggests a novel mechanism for PKC activation.

Cellular and intracellular localization of neuron-specific PKC binding protein, Nel

We investigated the effects of the infusion with antisense oligodeoxynucleotide (AsCN) against mitogen-activated protein kinase (p42/44 MAP kinase) with direct and HVJ-liposome method into adult rat hippocampus. The distribution of fluorescein-tagged AsCN observed by fluorescence microscopy was limited around the injection site when the AsCN was infused with HVJ-liposome method, The intensity of the fluorescence was very strong in the neural cell nuclei around the injection site. Infiltration of inflammatory cells were observed within 24 hours after infusion of AsCN containing HVJ-liposome. The nuclei with fluorescence were distributed more widely in the hippocampus after direct infusion of fluorescein-tagged AsCN, although the intensity of the fluorescence was not so strong as that with HVJ-Iiposome method We investigatedsimilar distributions of the effects of AsON by immunohistochemistry with anti-MAPK antibody. We also investigated MAP kinase activities in the hippocampus after intracerebroventricular injection of glutamate when AsON had been injected into hippocampus with the direct injection method.