Jun Tanaka

Gq protein alpha subunits Galphaq and Galpha11 are localized at postsynaptic extra-junctional membrane of cerebellar Purkinje cells and hippocampal pyramidal cells.

Following cell surface receptor activation, the α subunit of the Gq subclass of GTP‐binding proteins activates the phosphoinositide signalling pathway. Here we examined the expression and localization of Gq protein α subunits in the adult mouse brain by in situ hybridization and immunohistochemistry. Of the four members of the Gq protein α subunits, Gαq and Gα11 were transcribed predominantly in the brain. The highest transcriptional level of Gαq was observed in cerebellar Purkinje cells (PCs) and hippocampal pyramidal cells, while that of Gα11 was noted in hippocampal pyramidal cells. Antibody against the C‐terminal peptide common to Gαq and Gα11 strongly labelled the cerebellar molecular layer and hippocampal neuropil layers. In these regions, immunogold preferentially labelled the cytoplasmic face of postsynaptic cell membrane of PCs and pyramidal cells. Immunoparticles were distributed along the extra‐junctional cell membrane of spines, dendrites and somata, but were almost excluded from the junctional membrane. By double immunofluorescence, Gαq/Gα11 was extensively colocalized with metabotropic glutamate receptor mGluR1α in dendritic spines of PCs and with mGluR5 in those of hippocampal pyramidal cells. Together with concentrated localization of mGluR1α and mGluR5 in a peri‐junctional annulus on PC and pyramidal cell synapses ( Baude et al. 1993 , Neuron, 11, 771–787; Luján et al. 1996 , Eur. J. Neurosci., 8, 1488–1500), the present molecular‐anatomical findings suggest that peri‐junctional stimulation of the group I metabotropic glutamate receptors is mediated by Gαq and/or Gα11, leading to the activation of the intracellular effector, phospholipase Cβ.

Extra-junctional localization of glutamate transporter EAAT4 at excitatory Purkinje cell synapses

WE used silver-enhanced immunogold electron microscopy to reveal synaptic localization of the glutamate transporter EAAT4 in mouse cerebellar Purkinje cells (PCs). Gold—silver particles representing the EAAT4 were densely localized on extra-junctional membrane, but not on junctional membrane of PC spines in contact with parallel fiber or climbing fiber terminals. No particle accumulations were observed at inhibitory synapses formed on cell body and dendritic shafts of PCs. Therefore, the EAAT4 is selectively targeted to the extra-junctional site of excitatory PC synapses. The finding suggests that the EAAT4 transports glutamate or its related amino acids from outside the synaptic cleft, which would facilitate glutamate diffusion from the synaptic cleft to the extrasynaptic space and restrict glutamate spillover to adjacent synapses.

Pregnancy during hibernation in Japanese black bears: effects on body temperature and blood biochemical profiles

Abstract Bears from the family Ursidae are the only terrestrial mammals that go through gestation, parturition, and lactation during hibernation. This is the 1st study to examine the influence of reproductive status (i.e., nonpregnant, pseudopregnant, or pregnant) on body temperature and blood biochemical profiles in hibernating black bears. Pregnant bears appeared to have higher and more stable body temperatures (37–38°C) than nonpregnant ones (34–36°C) during pregnancy, which was followed by a rapid drop to levels comparable to those of nonpregnant individuals after parturition. In midpregnancy (i.e., January), pregnant bears had higher blood glucose and lower triglyceride concentrations than did nonpregnant ones, whereas blood concentrations of free fatty acids, glycerol, and ketone bodies did not differ significantly. Plasma urea, creatinine, and urea/creatinine levels were significantly lower in pregnant bears than in nonpregnant ones. Pseudopregnant bears showed similar changes in body temperature and blood profiles to pregnant ones, but blood glucose levels in January were significantly lower than those in pregnant bears. These results suggest that pregnant bears maintain homeothermic conditions and supply nutrients to the fetus by stimulating thermogenesis, gluconeogenesis, and urea recycling during hibernation. In addition, these physiological changes appear to be mainly caused by corpus luteum–derived factors (e.g., progesterone) but also were facilitated by placental factors.

Seasonal changes in the expression of energy metabolism-related genes in white adipose tissue and skeletal muscle in female Japanese black bears.

Bears undergo annual cycles in body mass: rapid fattening in autumn (i.e., hyperphagia), and mass loss in winter (i.e., hibernation). To investigate how Japanese black bears (Ursus thibetanus japonicus) adapt to such extreme physiological conditions, we analyzed changes in the mRNA expression of energy metabolism-related genes in white adipose tissues and skeletal muscle throughout three physiological stages: normal activity (June), hyperphagia (November), and hibernation (March). During hyperphagia, quantitative real-time polymerase chain reaction analysis revealed the upregulation of de novo lipogenesis-related genes (e.g., fatty acid synthase and diacylglycerol O-acyltransferase 2) in white adipose tissue, although the bears had been maintained with a constant amount of food. In contrast, during the hibernation period, we observed a downregulation of genes involved in glycolysis (e.g., glucose transporter 4) and lipogenesis (e.g., acetyl-CoA carboxylase 1) and an upregulation of genes in fatty acid catabolism (e.g., carnitine palmitoyltransferase 1A) in both tissue types. In white adipose tissues, we observed upregulation of genes involved in glyceroneogenesis, including pyruvate carboxylase and phosphoenolpyruvate carboxykinase 1, suggesting that white adipose tissue plays a role in the recycling of circulating free fatty acids via re-esterification. In addition, the downregulation of genes involved in amino acid catabolism (e.g., alanine aminotransferase) and the TCA cycle (e.g., pyruvate carboxylase) indicated a role of skeletal muscle in muscle protein sparing and pyruvate recycling via the Cori cycle. These examples of coordinated transcriptional regulation would contribute to rapid mass gain during the pre-hibernation period and to energy preservation and efficient energy production during the hibernation period.

Biological Activity and Chemistry of the Compound Ascidian Eusynstyela tincta

The extract of the compound ascidian Eusynstyela tincta, collected from Palk Bay and Gulf of Mannar, India was subjected through a battery of biological screens. The crude methanolic extract and subsequent fractions exhibited antibacterial, antifungal, cytotoxic and feeding deterrent activities. The extracts showed broad-spectrum antibiotic activity, and inhibited both bacterial and fungal pathogens. It also showed 100% cytotoxicity (25(μg/ml) against Dalton’s lymphoma and Ehrlich ascites tumour cells. A consistent feeding deterrent activity was established in both laboratory and field trials. It deterred the gold fish Carassius auratus. Among the extract/fractions assayed, methanol/ chloroform fraction had remarkable activity. The crude extract was separated through bioassay-guided fractionation and the active metabolite was identified as a pyridoacridine alkaloid, Kuanoniamine A. This study demonstrates that the ascidian metabolite, which exhibits antibiotic, cytotoxic and feeding deterrent activities, may play a role in the chemical defense mechanism.

910 Extrasynaptic localization of the glutamate transport EAAT4 at Purkinje cell synapses

YOSHIHIDE TANIWAKI, MOTOHIRO KATO We reported previously that microglial cells were activated transsynaptically through the propagation pathway of the hippocampal seizure induced by kainic acid in the rat. In the present study, we report an expression of heat shock protein (HSP) with the similar intracerebral distribution in the same seizure model. The time lags between seizure induction and the phenomena were about 4 hours for microglial activation and about 72 hours for HSP expression. These results suggests that. although the processes involved in the two phenomena are different, the HSP expression also occur transsynaptically in association with activation of the neurons by seizure activity.