Seiji Miyata

Characterization of a Novel Rat Brain Glycosylphosphatidylinositol-anchored Protein (Kilon), a Member of the IgLON Cell Adhesion Molecule Family

In the central nervous system, many cell adhesion molecules are known to participate in the establishment and remodeling of the neural circuit. Some of the cell adhesion molecules are known to be anchored to the membrane by the glycosylphosphatidylinositol (GPI) inserted to their C termini, and many GPI-anchored proteins are known to be localized in a Triton-insoluble membrane fraction of low density or so-called “raft.” In this study, we surveyed the GPI-anchored proteins in the Triton-insoluble low density fraction from 2-week-old rat brain by solubilization with phosphatidylinositol-specific phospholipase C. By Western blotting and partial peptide sequencing after the deglycosylation with peptide N-glycosidase F, the presence of Thy-1, F3/contactin, and T-cadherin was shown. In addition, one of the major proteins, having an apparent molecular mass of 36 kDa after the peptide N-glycosidase F digestion, was found to be a novel protein. The result of cDNA cloning showed that the protein is an immunoglobulin superfamily member with three C2 domains and has six putative glycosylation sites. Since this protein shows high sequence similarity to IgLON family members including LAMP, OBCAM, neurotrimin, CEPU-1, AvGP50, and GP55, we termed this protein Kilon (akindred of IgLON). Kilon-specific monoclonal antibodies were produced, and Western blotting analysis showed that expression of Kilon is restricted to brain, and Kilon has an apparent molecular mass of 46 kDa in SDS-polyacrylamide gel electrophoresis in its expressed form. In brain, the expression of Kilon is already detected in E16 stage, and its level gradually increases during development. Kilon immunostaining was observed in the cerebral cortex and hippocampus, in which the strongly stained puncta were observed on dendrites and soma of pyramidal neurons.

Differences in Fos expression in the rat brains between cold and warm ambient exposures

Fos expression in the rat diencephalon, brain stem, cerebellum, and spinal cord was examined after warm (33 degrees C) and cold (10 degrees C) ambient exposures. Fos expression was examined with use of immunohistochemical method and the number of Fos-positive neurons in each nucleus was quantitatively analyzed. When rats were exposed to cold ambient, significant number of Fos-positive neurons was found in the lateral septal nucleus (LS), preoptic hypothalamic area (POA), parvocellular paraventricular hypothalamic nucleus (pPVN), lateral preoptic area (LPO), zona incerta (ZI), paraventricular thalamic nucleus (PV), ventromedial hypothalamic nucleus (VMH), subparafascicular thalamic nucleus (SPF), posterior hypothalamic area (PH), supramammillary nucleus (SuM), microcellular tegmental nucleus (MiTg), lateral lemniscus nucleus (LL), lateral dorsal central grey (CGLD), lateral ventral central grey (CGLV), dorsal parabrachial nucleus (DPB), locus coeruleus (LC), dorsal tegmental nucleus (DTg), vestibular nucleus (Ves), nucleus of solitary tract (Sol), spinal cord, and cerebellum. When animals were exposed to warm ambient, the numbers of Fos-positive neurons in the LS, POA, PV, LPO, and SuM were significantly increased to be equal to those of cold ambient. However, after warm ambient exposure the numbers of Fos-positive neurons in the DPB and spinal cord were increased but less than those of cold ambient, and those in the pPVN, VMH, ZI, SPF, PH, CGLD, CGLV, MiTg, LL, LC, DTg, Ves, Sol, and cerebellum were not significantly increased as compared with those of control or cold ambient. Abdominal temperature was not changed during cold ambient exposure, but the temperature was significantly increased during warm ambient exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasticity of neurohypophysial terminals with increased hormonal release during dehydration: Ultrastructural and biochemical analyses

Arginine vasopressin‐ (AVP) and oxytocin‐ (OXT) secreting magnocellular neurons undergo gross structural changes with chronic physiological stimulation. Here, we investigated subcellular aspects of plasticity in rat neurohypophysial terminals during dehydration. Ultrastructural analyses demonstrated that chronic dehydration by 2% NaCl drinking for 7 days significantly decreased the numbers of neurosecretory granules and microvesicles but not the numbers of mitochondria. Moreover, in dehydrated rats, terminals making neurovascular contacts enlarged, whereas terminals in apposition to astrocytes, i.e., neuroglial contacts, became smaller. Western blot analyses demonstrated significant decreases in the levels of F3 and Thy‐1 together with those of AVP‐ and OXT‐neurophysin, but the levels of synaptophysin, SNAP‐25, and GAP‐43 were unchanged. Both F3 and Thy‐1 were recovered in the buffer‐insoluble pellet, and phosphatidyl inositol‐specific phospholipase C treatment released both molecules from the crude membrane fraction, indicating that they are attached to terminal membranes by glycosylphosphatidyl inositol anchors. Confocal microscopic observations demonstrated that F3 colocalized with Thy‐1 in the same terminals of magnocellular neurons. In contrast, the level of calretinin, a Ca2+ binding protein was significantly increased with chronic dehydration. Thus, the present results suggest that enhancement of neurovascular contacts results from rearrangement of terminal‐astrocyte and terminal‐vessel contacts rather than enlargement or sprouting of magnocellular terminals themselves. The down‐regulation of F3 and Thy‐1 may contribute to enhancement of neurovascular contacts that accompany increased peptide release during dehydration. J. Comp. Neurol. 434:413–427, 2001.

Molecular characterization of the detergent-insoluble cholesterol-rich membrane microdomain (raft) of the central nervous system

Many fundamental neurological issues such as neuronal polarity, the formation and remodeling of synapses, synaptic transmission, and the pathogenesis of the neuronal cell death are closely related to the membrane dynamics. The elucidation of functional roles of a detergent-insoluble cholesterol-rich domain (raft) could therefore provide good clues to the molecular understanding of these important phenomena, for the participation of the raft in the fundamental cell functions, such as signal transduction and selective transport of lipids and proteins, has been elucidated in nonneural cells. Interestingly, the brain is rich in raft and the brain-derived raft differs in its lipid and protein components from other tissue-derived rafts. Since many excellent reviews are written on the membrane lipid dynamics of this microdomain, signal transduction, and neuronal glycolipids, we review on the characterization of the raft proteins recovered in the detergent-insoluble low-density fraction from rat brain. Special focus is addressed on the biochemical characterization of a neuronal enriched protein, NAP-22, for the lipid organizing activity of this protein has become increasingly clear.

Temporal changes of c-fos expression in oxytocinergic magnocellular neuroendocrine cells of the rat hypothalamus with restraint stress

The present experiments were undertaken to examine c-fos expression in magnocellular neuroendocrine cells (MNCs) of the rat hypothalamus with restraint stress using dual immunohistochemistry for c-fos and oxytocin. Restraint stress induced c-fos expression in oxytocinergic MNCs in the supraoptic nucleus (SON) and paraventricular nucleus (PVN). Quantitative immunohistochemical analysis revealed that percentages of c-fos-positive cells to oxytocin-immunoreactive MNCs in the SON and PVN maximally increased at 2 h after restraint stress had started, and began to decline in spite of the fact that the restraint of animals were continued. Similar results were obtained from time course of c-fos expression in parvocellular neurons of the PVN. When animals were released to move freely in their home cages following the 3-h restraint, the plasma levels of oxytocin declined to reach basal levels within 30 min and c-fos immunoreactivity in the hypothalamic MNCs and parvocellular neurons disappeared faster than those of the continually restrained. These results demonstrate that restraint stress induces c-fos expression in oxytocinergic MNCs in the SON and PVN, and that time course of c-fos expression is transient even in the continuation of restraint stress.

Metabolic mapping of the brain in pregnant, parturient and lactating rats using fos immunohistochemistry.

The present study was designed to investigate Fos-positive neurons of the female rat brain at various reproductive states in order to analyze the metabolic map connected with pregnancy, parturition and lactation. The number of Fos-positive neurons in each brain nucleus was analyzed with a quantitative immunohistochemical method in virgin, pregnant, parturient, lactating and arrested lactating rats. In parturient rats, a significant number of Fos-positive neurons was observed as compared to virgin or pregnant females in the following brain regions; the bed nucleus of the stria terminalis (BST), lateral septal nucleus (LS), medial preoptic area (MPA), periventricular hypothalamic nucleus (Pe), parvocellular paraventricular hypothalamic nucleus (PaPVN), magnocellular paraventricular hypothalamic nucleus (MaPVN), supraoptic nucleus (SON), paraventricular thalamic nucleus (PV), anterior hypothalamic area (AHA), lateral hypothalamic area (LH), amygdaloid nucleus (AM), supramammillary nucleus (SuM), substantia nigra (SN), central grey (CG), microcellular tegmental nucleus (MiTg), subparafascicular thalamic nucleus (SPF), posterior hypothalamic area (PH), dorsal raphe nucleus (DR), locus coeruleus (LC), dorsal parabrachial nucleus (DPB), nucleus of solitary tract (Sol), and ventrolateral medulla (VLM). Significant differences were found in the number of Fos-positive neurons between parturient and lactating females, although localization of Fos-positive neurons in lactating females was quite similar to parturient ones. Between parturient and lactating rats: (1) In the MPA, PaPVN, AHA, arcuate hypothalamic nucleus (Arc), ventromedial hypothalamic nucleus (VMH), MLT, and Ge, the number of Fos-positive neurons of lactating females were significantly higher than those of parturient ones; (2) In the LS, Pe, PV, LH, AM, SuM, CG, MiTg, SPF, PH, DR, LC, and VLM, there was no significant differences in the number of Fos-positive neurons; (3) In the BST, MaPVN, SON, SN, DPB and Sol, the number of Fos-positive neurons of lactating rats were significantly lower than those of parturient ones. These different patterns of Fos expression among many brain regions may be owing to the functional differences in each region. Fos expression in lactating rats was apparently induced by suckling stimulation because the removal of their litters immediately after parturition completely eliminated expression of Fos protein in each nucleus. These results suggest that the localization of Fos-positive neurons in a number of neural populations throughout the brain may be revealing the neural circuits in response to parturition or lactation.

Structural dynamics of neural plasticity in the supraoptic nucleus of the rat hypothalamus during dehydration and rehydration

It has been known that magnocellular neuroendocrine cells (MNCs) of mammalian hypothalamus show structural plasticity in response to chronic osmotic stimulation. In this study, we investigated the relationships among plasma osmolarity and several structural changes such as alterations of soma size, juxtaposition, and synapses of the supraoptic nucleus (SON) in the rat hypothalamus during dehydration and rehydration. Male rats were osmotically stimulated by supplying with 2% NaCl solution instead of tap water for 10 days, and then they were rehydrated with tap water. Plasma osmolarity was gradually elevated with progress of salt loading and returned to control level on the seventh day of rehydration. Both the percentage of membrane contact (juxtaposition) and the soma size of MNCs were increased in response to the rise of plasma osmolarity, and decreased to control level on the seventh day of rehydration. The number of synapses including both single synapses and multiple synapses per 100 microns soma membrane was lower than control on the fifth day of dehydration, but it was not different from controls on the tenth day of dehydration, and on the seventh and fourteenth day of rehydration. The total number of synapses per 100 microns soma membrane, the synaptic density, was maintained relatively constant, although soma size was progressively changed during dehydration or rehydration. This synaptic reorganization seems to be mainly regulated by synaptic sprouting during dehydration and by degradation of synapses during rehydration.(ABSTRACT TRUNCATED AT 250 WORDS)

Taurine in rat posterior pituitary: Localization in astrocytes and selective release by hypoosmotic stimulation

Taurine, a γ‐aminobutyric acid (GABA)‐like acidic amino acid, has previously been shown to be prominently localized to astrocytes in the supraoptic nucleus, the neurons of which contain only small amounts, and to have inhibitory actions on supraoptic neuronal activity. In the present study, taurine distribution in the neurohypophysis was determined by using a well‐characterized monoclonal antibody against taurine itself. Preembedding immunohistochemistry was performed at light and electron microscopic levels by using diaminobenzidine and gold‐substituted silver‐intensified peroxidase (GSSP) methods. At the light microscopic level, the distribution pattern and cellular localization of taurine immunoreactivity corresponded to that of glial fibrillary acidic protein. Pituicyte cell bodies and processes displayed dense taurine immunoreactivity. Electron microscopic observations revealed strong taurine GSSP reactions in these neural lobe astrocytes, but weak taurine reactivity was seen within only some neurosecretory axons. High‐performance liquid chromatography analyses demonstrated that in vitro hypoosmotic stimulation (reduction of 40 mOsm/kg) of isolated posterior pituitaries resulted in preferential increases in taurine release into the bathing medium without increased release of other amino acids. Conversely, tissue concentrations of taurine significantly decreased with hypoosmotic perfusion, while glutamate, glutamine, and GABA concentrations were not reduced. These results indicate that taurine is mainly concentrated in neurohypophysial astrocytes, which are known to engulf the neurosecretory axonal processes and terminals. Taurine released from pituicytes under basal and hypoosmotic conditions may act to suppress axon terminal depolarization and thereby depress release of neurohypophysial peptides.J.Comp. Neurol. 381:513‐523, 1997.

LPS-induced Fos expression in oxytocin and vasopressin neurons of the rat hypothalamus.

The aim of this study was to examine the involvement of the hypothalamic oxytocin (OXT) and vasopressin (AVP) neurons in acute phase reaction using quantitative dual-labeled immunostaining with Fos and either OXT and AVP in several hypothalamic regions. Administration of low dose (5 microg/kg) and high dose (125 microg/kg) of LPS induced intense nuclear Fos immunoreactivity in many OXT and AVP neurons in all the observed hypothalamic regions. The percentage of Fos-positive nuclei in OXT magnocellular neurons was higher than that of AVP magnocellular neurons in the supraoptic nucleus (SON), the magnocellular neurons in the paraventricular nucleus (magPVN), rostral SON (rSON), and nucleus circularis (NC), whose axons terminate at the posterior pituitary for peripheral release. The percentage of Fos-positive nuclei in AVP parvocellular neurons in the paraventricular nucleus (parPVN) was higher than that of OXT parvocellular neurons, whose axons terminate within the brain for central release. Moreover, the percentage of Fos-positive nuclei in AVP magnocellular neurons of the SON and rSON was significantly higher than that of the magPVN and NC when animals were given LPS via intraperitoneal (i.p.)-injection. This regional heterogeneity was not observed in OXT magnocellular neurons of i.p.-injected rats or in either OXT or AVP magnocellular neurons of intravenous (i.v. )-injected rats. The present data suggest that LPS-induced peripheral release of AVP and OXT is due to the activation of the magnocellular neurons in the SON, magPVN, NC, and rSON, and the central release of those hormones is in part derived from the activation of parvocellular neurons in the PVN. It is also suggested that the activation of AVP magnocellular neurons is heterogeneous among the four hypothalamic regions, but that of OXT magnocellular neurons is homogenous among these brain regions in response to LPS administration.

Morphological plasticity and rearrangement of cytoskeletons in pituicytes cultured from adult rat neurohypophysis

The adult rat neurohypophysis reveals drastic morphological plasticity of neuron-glial organization during chronic physiological stimulation. Pituicytes are modified astrocytes in the neurohypophysis, and shape conversion of them largely contributes to the morphological plasticity. The present study aimed to investigate the receptor-mediated mechanism for shape conversion of the pituicyte morphology, particularly in relation with changes of cytoskeletal organization. The cultured pituicytes from adult rat neurohypophysis were mostly flat amorphous shape in normal salt solution. Histochemical experiments showed that thick bundle of microfilament (stress fibers) and fine fibers of microtubule distributed evenly within the pituicyte. When pituicytes were treated with adenosine (more than 1 microM), isoproterenol (IPR); beta-agonist, more than 10 nM), and dibutyryl cyclic AMP (dBcAMP, 1 mM), the pituicyte morphology changed from flat to stellate shape. Upon treatment with dBcAMP, stress fibers within pituicyte cytoplasm disappeared, and microtubule assembled in the cellular processes and cytoplasm surrounding the nucleus. Pretreatment with colchicine (microtubule-disrupting agent, 25 microM) and orthovanadate (tyrosine phosphatase inhibitor, 1 mM) prevented dBcAMP-induced stellation of the pituicyte morphology. Treatment with sphingosine (protein kinase C inhibitor, 10 microM), W-7 (calmodulin dependent protein kinase inhibitor, 40 microM), ML-9 (myosin light chain kinase inhibitor, 20 microM), and cytochalasinB (CytB; microfilament disrupting agent, 5 microM), induced stellation of the pituicyte morphology. Treatment of endothelin-1 (more than 0.1 nM) and endotheline-3 (more than 0.1 nM) reverted dBcAMP-induced stellation of the pituicyte morphology to original flat one and also reverted arrangement of cytoskeletons of stress fiber and microtubules as seen in control one. The present results reveal that pituicyte shape conversion is mediated via beta-adrenergic, adenosine and endotheline and depend on rearrangement of stress fibers and microtubules. In addition, the mechanism of shape conversion of pituicytes cultured from adult neurohypophysis is quite similar to that of astrocytes cultured from neonatal brains and possibly is useful for understanding morphological plasticity of adult brains.