Metabolic activity in a suprachiasmatic nucleus (SCN), a center of biological rhythm, is higher during the daytime than at the night. The rhythmic oscillation in the SCN is feedback controlled by the CLOCK/BMAL1 heterodimer binding to the E-box in target genes (e.g., Per2, Arg-vasopressin). Similar transcriptional regulation by NPAS2/BMAL1 heterodimer formation is operating in the forebrain, which is depending on the redox state (i.e., NAD/NADH). To clarify the metabolic function of SCN in relation to the redox state, we measured biochemicals in mitochondria and cytosol in the SCN homogenate. The integrity of JC-1 fluorescent dye, which is the index of mitochondrial electric potential gradients was higher at the light-dark transition time. Mitochondrial cyctochrome C oxidase activity, measured by the changes in absorption at 550nm, was higher during the light period than during the dark period. The proteins, extracts from mitochondrial fraction in SCN, obtained from the rats kept under the light dark cycle, were analyzed by two-dimension electrophoresis. The electrophoretic pattern revealed that an enolase, catalyzing the interconversion of 2-phosphoglycorate and 2-phosphoenol-pyruvate content in mitochondria was higher during the subjective daytime than that in the night. However, the enolase mRNA content in cytosol fraction of SCN, measured using RT-PCR, showed higher levels during the dark period than during the light. The results suggest the glycolysis with energy producing pathway might concern to preserve the higher metabolic activity during the day than at night. [Jpn J Physiol 54 Suppl:S208 (2004)]

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In mammals, circadian rhythms are driven by a pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. The pacemaker composed of an ensemble of multiple, single-cell oscillators in the SCN. We have reported circadian oscillation of arginine vasopressin (AVP) release in dispersed SCN cell culture. The organotypic SCN slice culture also showed circadian oscillation of AVP release. The period length of the slice containing whole SCN was almost same as that of behavioral rhythm observed in vivo (23.78 +/- 0.04h, mean +/- SEM). When the ventral part was removed by surgical cut across the slice in the horizontal plane, however, it became shorter (23.19 +/- 0.10h). On the other hand, the removal of the dorsal part did not affect the period length. These results suggest that the oscillators in ventral and dorsal cells have difference in period length and that the dorsal oscillators are regulated by ventral one to form a single integrated oscillator. [Jpn J Physiol 54 Suppl:S209 (2004)]

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Inhibition periods (less than 10 times/min) for dozens of minutes were often observed in a long-term (24 hours) intracranial self-stimulation (ICSS; 50-60 times/min) in rats; electrical stimulations were applied to the hemi-lateral medial forebrain bundle of rats when the rats pressed a lever. The inhibition was not induced by thermal effect on neural function or by muscular fatigue, since the animals in the inhibition period started ICSS again when more intensive stimulations to the ipsi-lateral medial forebrain bundle or stimulations to the contra-lateral medial forebrain bundle were applied to the animals. In such a long-term ICSS, the inhibition period was significantly decreased in the animals pharmacologically treated with 5,7-dihydroxy-tryptamine just after their birth for inducing degeneration of serotonergic neurons in the dorsal raphe nucleus, although the treatment did not affect ICSS in short duration. These observations indicate that the serotonergic nervous system facilitates or modulates the inhibition of the long-term ICSS and would be involved in weariness or fatigue sensation. [Jpn J Physiol 54 Suppl:S209 (2004)]

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Song of passerine (like zebra finch) is a learned motor behavior which used by males to attract females and protect territories. Generally, bird song is consists of several different song notes (elements), and order of these notes is stereotyped. Among the passerine birds, however, male Bengalese finches (Lonchura striata var. domestica; BF) sing complex song which follows finite state syntax. The song control system in the forebrain of songbird consists of a set of discrete nuclei include HVC, RA, Area X, NIf and LMAN. In this study, we injected neural tracers (dexstran amines) in 3 song nuclei (HVC, RA and Area X) to elucidate the input-output connections and local circuitry (cyto-architecture) of HVC. Result shows that HVC receives input from Field L and NIf, and it sends axons to RA (analog of mammalian primary motor cortex that innervates nXII and DM) and Area X (analog of basal ganglia). We observed that 3-5 middle to large sized neurons formed clusters in HVC, and these clusters were distributed dispersedly throughout the nucleus. Most of the clusters innervated both RA and Area X. We also recorded neural responses to bird's own song (BOS) in HVC of BF by 1-3 tetrodes. Clustered multi-neuron activities were synchronized to temporal structure of BOS. This result suggests that the finite state syntax of complex bird song represent dispersedly in HVC. [Jpn J Physiol 54 Suppl:S209 (2004)]

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In previous recording experiments it has been shown that the laterodorsal tegmental nucleus (LDT) neurons increase their activity when penile erection occurs during paradoxical sleep. LDT neurons are thus involved in penile erection physiology. However their role during erection still remains to be determined.In head-restrained unanesthetized rats, brainstem areas from the caudal midbrain to the rostral pons were electrically stimulated using carbon fiber electrodes. Electrical stimulation consisted of rectangular pulses of less than 200 μA, 3 s, 50 Hz. In addition, cortical EEG, neck muscle EMG, Bulbospongious muscle (BS) EMG and pressure of the corpus spongiosum of the penis (CSP) were simultaneously recorded. Penile erection pressure was measured using a telemetric transducer. Histological brain sections were then made to confirm the position of the electrical stimulus. Penile erection could be evoked in and around the LDT and the midline area at the level of LDT. This study suggests that the LDT is involved in the regulation of penile erection. [Jpn J Physiol 54 Suppl:S209 (2004)]

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There are two entrainment stimuli of circadian rhythm, restricted feeding and light. Light-induced expression of mPer1 and mPer2 in the suprachiasmatic nucleus (SCN) closely correlated with the light-induced entrainment of locomotor activity rhythm. Previously, we investigated the effects of restricted feeding (RF) on the expression of mPer1 and mPer2 mRNA in the paraventricular nucleus (PVN), pyriformcortex, hippocampus and cerebral cortex of mice. To understand the relation between clock genes and RGS16, we investigated mRNA of RGS16 in restricted feeding.
In situ hybridization revealed the circadian expression of RGS16 mRNA. The expression of the RGS16 mRNA showed a clear peak in SCN at daytime, as well as mPer1 and mPer2. Contrary, a peak in PVN (paraventricular nucleus), pyriformcortex, cerebral cortex and hippocampus has seen at nighttime. After 6 days of scheduled-RF (4 hours in the daytime) RGS16 mRNA level showed a peak in the cerebral cortex, hippocampus, PVN and pyriform cortex in the daytime. Scheduled-RF attenuated these rhythmicity outside SCN. However, the expression pattern of RGS16 mRNA in SCN didn't show the phase shift on scheduled-RF.
These results show the relation between clock genes and G-protein signaling related gene for food stimuli outside SCN regions. [Jpn J Physiol 54 Suppl:S210 (2004)]

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Macaque monkeys are able to discriminate between food and non-food, or male and female monkeys, by using visual information. It is known that visual preference is modified by endogenous or exogenous factors. In this study we have examined the effects of several environmental factors on visually-guided category discrimination tasks in two female rhesus monkeys, (macaca mulatta). Each trial of the task was started with a visual presentation of one of the following pictures. In food/non-food discrimination task, a picture of either food or non-food object was presented. In male/female task, a picture from one of the sexes of Japanese monkeys was presented. Pre-determined target, for example category of male, was assigned on a lever, and the monkey has to press the lever when the "target" category, male in this case, was presented on a screen to get water reword. Tasks were composed of blocked-fashion, and in each block, a monkey had to discriminate the category of a presented picture. We examined the effects of the plant-derived odor on this task. We used green odors, which were cis-3-hexenol and trans-2-hexenal extracted from green leaves. Aditionally oak lactone and jasmine lactone, which are major ingredient of wiskey and oolong, were used. As a result, green odors and oak lactone negatively affected the performance of food/non-food task, in particular trans-2-hexenal reduced the score during exposure and extended latency of the onset of bar press . By contrast, The latency for male/female task was reduced by exposure of plant-derived odor. [Jpn J Physiol 54 Suppl:S210 (2004)]

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To examine whether the estrous cycle affects the spontaneous locomotor activity and body temperature rhythms, locomotor activity and body temperature were measured in cycling female rats using a radiotelemetry system. All rats were housed in constantly controlled conditions (12L12D), and data were collected under freely moving conditions. Although all rats showed distinct daily rhythms in motor activity and body temperature, the estrous cycle affected these daily changes. The activity counts and body temperature from the night of proestrus through the early morning of estrus were significantly greater than those in the corresponding dark phases on diestrus 1 or diestrus 2. In addition, the activity counts and body temperature in the early morning of estrus were significantly greater than those in the corresponding phase on diestrus 1, diestrus 2, or proestrus. Furthermore, motor activity and body temperature significantly decreased in ovariectomized rats, but were maintained at control levels in ovariectomized estrogen-primed rats. These results suggest an essential role of circulating estrogen to sustain the normal level of locomotor activity and body temperature in cycling female rats. [Jpn J Physiol 54 Suppl:S210 (2004)]

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Migration and apoptosis of selected neurons is essential for developing a properly arranged neural network. Although many migrate to the cortical regions, only those neurons that differentiate into a functional neural net are maintained while the others are eliminated through an apoptotic mechanism. As a model of this neuronal development, P19 embryonic carcinoma cells differentiate into neuronal cells when grown as aggregates in the presence of retinoic acid (RA). During RA-induced neuronal differentiation, the aggregated cells lose their malignant phenotype and many undergo apoptosis to provide a robust model of the apoptosis seen in the developing brain. On the other hand, reelin plays an important role in the migration of embryonic neurons. Even after neuronal migration is complete, reelin is expressed in all layers of the mature cerebral cortex and weak immunoreactivity of reelin is routinely detected in selected populations of cortical neurons throughout life. The persistence of reelin throughout life suggested to us that reelin may play a novel role in the maturing brain after neuronal migration is completed. In our study, reelin protected P19 embryonal cells from apoptosis during retinoic-acid induced neuronal differentiation. This protective effect of reelin included activating the Src-family kinases/PI3 kinase/Akt pathway. [Jpn J Physiol 54 Suppl:S211 (2004)]

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We have reported that pleiotrophin (PTN) expression is enhanced in the dopamine (DA)-depleted striatum, and PTN specifically promoted survival of DAergic neurons in vitro. We investigated whether PTN treatment to donor cells during cell preparation in neural transplantation promoted the survival of grafted DAergic neurons. Donor cells were obtained from E15 ventral mesencephalon, suspended to single cells with 0.25% trypsin in PBS (20 min, 37°C) and kept in DMEM on ice. PTN (100 ng/ml) was treated during all procedure in cell preparation. Cells were grafted into the striatum of Parkinson model rats that were received 6-hydroxydopamine into the left substantia nigra 2-3 weeks before transplantation. To test the optimal number of donor cells for our purpose at first, we transplanted cells (1.0-7.5x10⁴ per animal) with no PTN treatment (control) followed by methamphetamine-induced rotational test. Cells of 3.0 x 10⁴ were optimal number of grafted cells for our purpose: some animals showed functional recovery 5 weeks after transplantation, however most animals did not. In PTN-treated group, recovery of motor function was obtained earlier, and the improvement was significantly greater in PTN-treated group. There were more DAergic cells in the striatum in PTN-treated group (192.2 ± 24.3, n=6) compared to that in control (137.8 ± 22.6, n=5). Data suggest that PTN treatment during cell preparation in neural transplantation promoted survival of grafted DAergic neurons. [Jpn J Physiol 54 Suppl:S211 (2004)]

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It was reported that brain-derived neurotrophic factor (BDNF) plays an important role in developmental plasticity of the visual cortex and its expression is regulated in activity-dependent manner. Althogh it is known that protein kinases such as , extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase A (PKA) or calcium-calmodulin kinase type IV (CaMK IV) regulate BDNF level in vitro, it is not clear how these kinases regulate BDNF level in vivo. So we measured level of BDNF protein in the visual cortex of young rats treated with various kinase inhibitors or activators to examine the contribution of each kinase to BDNF level. Surprisingly, inhibition of PKA increased BDNF protein level. Inhibition of ERK1/2 or CaMK for 2 days induced no detectable change in the BDNF level. These results suggest that BDNF level in the visual cortex might be regulated by combination of various kinases by other untested pathway. Increase of BDNF by PKA inhibition may reflect accumulation of unreleased BDNF, suggesting a role of PKA in BDNF release. [Jpn J Physiol 54 Suppl:S211 (2004)]

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We have previously demonstrated that the peripheral vasopressin system participates in the control of renal sympathetic nerve activity (RSNA) and Fos-like immunoreactivity (FLI), as a neuronal marker, in the paraventricular nucleus of the hypothalamus, via the vasopressin V₁ receptor during central salt loading in conscious rats. However, it is important to examine the [Na⁺] near the osmosensitive region during central salt loading and determine whether or not the stimulation has physiological relevance. The [Na⁺]_CSF electrode was prepared using a modification of a previous procedure [Nose H, et al., J. Appl. Physiol., 73 (1992)]. The tip of the double-barreled Na⁺-electrode was located at AP = -2.8, L = 0.9, V = 7.5 mm, lateral angle = 7.5; using a 22-mm 18-gauge stainless steel guide cannula. Rats were administered infusions of 0.15, 0.3, 0.67, and 1.0 M HS (hypertonic saline) at 1 μl/min for 20 min into the left LV under anesthesia, and changes in [Na⁺]_CSF in the V3 were measured. For the 0.3, 0.67, and 1.0 M HS groups, the increase in [Na⁺]_CSF reached the maxima (13.7 ± 1.2, 59.9 ± 3.8, and 89.7 ± 7.0 mEq/kg H₂O) at 16, 12, and 20 min, respectively. The changes in cerebrospinal fluid [Na⁺] during i.c.v. administration of 0.3 M hypertonic saline were compatible with those expected for thermal dehydration. This smaller than predicted increase (38.8, 134.7, and 220.1 mEq/kg H₂O, respectively) in [Na⁺]_CSF might be due to rapid buffering by free water movement from extraventricular spaces in response to the osmotic gradient. [Jpn J Physiol 54 Suppl:S212 (2004)]

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Experiments were carried out to examine whether γ-aminobutyric acid (GABA) receptor mechanisms participate in the release of serotonin (5-hydroxytryptamine, 5-HT) in the subfornical organ (SFO) using microdialysis methods. Perfusion of the GABA receptor antagonists as well as agonists was performed in the region of the SFO through a microdialysis probe and extracellular concentrations of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured in freely moving rats. Perfusion of the GABA_A receptor antagonist bicuculline (10 and 50 μM), but not the GABA_B receptor antagonist phaclofen (10 and 50 μM), enhanced dialysate 5-HT and 5-HIAA concentrations in the SFO area, suggesting that the GABAergic system may tonically inhibit the 5-HT release in the SFO area through GABA_A receptors. Higher perfusion of the GABA_A receptor agonist muscimol (50 μM) or the GABA_B receptor agonist baclofen (250 μM) decreased the 5-HT and 5-HIAA concentrations. Hypovolemia caused by subcutaneous injection of polyethylene glycol (PEG, 30%, 5 ml) increased the 5-HT and 5-HIAA concentrations in the SFO area, and the increase in the 5-HT and 5-HIAA levels was reduced by perfusion of muscimol (10 μM), but not by baclofen (50 μM). These results show the involvement of both GABA_A and GABA_B receptors in the control of 5-HT release in the SFO area, and imply that the GABA_A receptor mechanism may be importance for the serotonergic regulatory system of fluid balance. [Jpn J Physiol 54 Suppl:S212 (2004)]

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Intracisternal injection of L-proline in concious rat increased blood pressure through peripheral vasopressin release (Jpn J Physiol 1995), and microinjection of L-proline into the caudal ventral medulla of anesthetized rat decreased blood pressure through vascular sympathetic suppression (Annual Meeting 2002 in Hiroshima). These neural effects of L-proline suggest existence of proline sensitive receptors in the rat brain. However, there are no data for precise localization of L-proline within the brain. In the present study, we raised antibody against L-proline in the rabbit and purified by an affinity chromatography the antibody which specifically recognized L-proline. Immunohistochemical staining of the rat brain slices using the antibody detected positively stained neurons in the hypothalamus and brainstem indicating that endogenous L-proline is localized in a group of neurons in the rat brain. Our observation supports roles of proline as a neurotransmitter candidate. [Jpn J Physiol 54 Suppl:S212 (2004)]

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Several treatments which regulate tyrosine hydroxylase (TH) transcription, such as stress in vivo, or 12-O-tetradecanoylphorbol-13-acetate (TPA) in cell culture, induce both Egr1 and AP1 factors. Previously, we identified that a functional Egr1 motif overlapped with Sp1 site in the rat TH promoter and determined that induction of TH promoter activity by Egr1 also required an AP1/Ebox motif in addition to the Egr1 motif. Here, we examined the mechanism of the cross-talk between these motifs. Increasing the distance between the AP1/Ebox and the Sp1/Egr1 motifs, as well as mutation of either or both motifs, reduced the ability of Egr1 to up-regulate the reporter activity controlled by proximal 272 nucleotides of the rat TH promoter. Electrophoretic mobility shift assays with nuclear extract from TPA treated PC12 cells were used to identify the composition of the factors which bound the AP1/Ebox motif. The complexes formed with labeled AP1/Ebox oligonucleotide were reduced or supershifted with antisera to Fos family, Fra-2, c-Fos and Jun D. Excess Sp1/Egr1 oligonucleotide or anti Egr1 antisera did not compete. Transfection of PC12 cells with Fra-2 induced reporter activity requiring the AP1, but not the Egr1 motif. However, when cotransfected with Fra-2, Egr1 expression vectors elicited lower induction of luciferase activity than observed with Egr1 alone. These results suggest that although Egr1 does not compete for binding of AP1 factors to the promoter, it can modulate the regulation of TH transcription by AP1 factors. [Jpn J Physiol 54 Suppl:S212 (2004)]

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Recently the presence of the crosstalks among neuro- and immune systems involved in maintaining the homeostasis has been established. Cytokines and catecholamines may constitute a significant portion of the regulatory networks. It has been reported that IκB expression is regulated by norepinephrine (NE) at both transcriprional and post-transcriptional levels, which could contribute to the observed anti-inflammatory properties of NE. We examined the alteration of inflammatory cytokine expression levels and NE content at locus coeruleus (LC) after peripheral lipopolysaccharide (LPS) injection.The mRNA expression levels of genes encoding IκBα, IL-1β, TNF-α within the LC of C3H/HeN mice (male, 8W) were measured after 5 μg of LPS injection. The IκBα mRNA expression level increased within 1 hr and reached a maximum level at 2 hr. The mRNA expression levels of IL-1β, TNF-α increased within 2 hr, reached a maximum level at 4 hr, and increased at 15-24 hr again. By contrast, NE content at LC decreased at 24 hr. Pretreatment of animals with CP-154, a corticotropin releasing factor type 1 receptor antagonist, did not affect the increase in mRNA expression caused by LPS injection. Collectively, the quick up-regulation of inflammatory cytokine mRNA expression at LC after peripheral LPS injection may be caused by the activation of neural circuit, not by humoral factors transmitted into brain. The research is currently in progress in order to elucidate the relationship between the change of NE content and inflammatory cytokine upregulation at the LC. [Jpn J Physiol 54 Suppl:S213 (2004)]

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The olfactory system has been known to be modulated by a systemic inflammatory stress such as sepsis. The inflammatory stress is intervened by many cytokines such as tumor necrosis factor (TNF)-α. Therefore, the olfactory bulb (OB) which is a major constituent of olfactory system was investigated of its cytokine production as a rapid response to bacterial endotoxin lipopolysaccharide (LPS) and the occurrence of apoptosis as a final event in the OB was also evaluated. Male C3H/HeN mice (eight-week-old; 25-28 g) were injected intraperitoneally with LPS (50 μg/mice) or saline (control). At different time points from 0.5 h to 24 h after the injection the mice were sacrificed and their OB was used for the quantitation of mRNA expression levels by using real-time PCR amplification and the immunohistochemical detection. Peripheral LPS challenge rapidly enhanced the mRNA expression levels in the OB of the genes encoding IκBα, NF-κB, TNF-α and iNOS. The induction of TNF-α mRNA was followed by an increased expression of TNF-α protein. All TNF-α-positive cells, which were mainly present in the granule cell layer or the olfactory nerve layer, were double-labeled with anti-GFAP antibody. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive cells were observed in the granule cell layer. These findings provide evidence for rapid OB response to endotoxemia, and suggest that olfactory system may play a role in the modulation of endotoxemia-induced cognitive and behavioral changes. [Jpn J Physiol 54 Suppl:S213 (2004)]

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Semaphorins are a family of secreted and membrane-bound proteins, known to control axonal pathfinding. Semaphorin4A(Sema4A) has recently been shown to be involved in immune cell activation. However, the role of Sema4A in the nervous system is totally unknown. To examine if Sema4A functions as a chemo-repulsive cue to growth cones of developing hippocampal neurons, growth cone collapse assay with recombinant Sema4A was performed in primary hippocampal neurons cultured from E17 rats or mice. As a result, Sema4A induced a significant growth cone collapse in hippocampal neurons as compared with the culture without Sema4A. The Sema4A-induced growth cone collapse could be blocked by Y-27632, the Rho-kinase inhibitor. The neurite outgrowth assay suggested that Sema4A could work as a retraction inducer by inhibiting neurite outgrowth of hippocampal neurons. Furthermore, an immunocytochemical analysis using the antibodies to Sema4A directly demonstrated the binding of recombinant Sema4A to the growth cones of hippocampal neurons. Thus, our data indicated that Sema4A could function as a chemo-repulsive cue by activating an unknown receptor whose signal is transmitted to Rho-kinase to induce growth cone collapse of hippocampal neurons. Consistent with the data above, our preliminary analyses of Sema4A knockout mice suggested that the infrapyramidal mossy fiber projection in the hippocampus was abnormally longer than that of wild-type mice. The overshooting phenotype implies that Sema4A can be a guidance cue controlling the proper formation of neuronal network in vivo. [Jpn J Physiol 54 Suppl:S213 (2004)]

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Unlike the mammals, fish optic nerve can regenerate their axons after axotomy. There is much knowledge about axon guidance molecules revealed by classical biochemical techniques. The previous studies demonstrated that nitric oxide synthase (NOS) recognized as NADPH-diaphorase and growth associated protein-43 (GAP-43) were upregulated after fish optic nerve injury by histochemical and two-dimmensional electrophoresis. To further investigate molecular mechanism of neural type of NOS and GAP-43 in the goldfish retina during optic nerve regeneration. We isolated cDNA fragments for nNOS and GAP-43 with probes of PCR products amplified by primers constructed from zebrafish and goldfish sequences, respectively. We got 490 bp fragment of nNOS cDNA and 639 bp fragment of GAP-43 cDNA. By using these cDNA clones, Northern blot hybridization was done in the goldfish retinal samples following optic nerve injury. The level of GAP-43 mRNA significantly increased about 2-fold 1 week after treatment and then gradually declined by 30 days after optic nerve injury. We are now in progress to investigate the localization of nNOS and GAP-43 mRNA in the fish retina with in situ hybridization and to further investigate the functional role of these molecules on neurite outgrowth with in vitro culture system. [Jpn J Physiol 54 Suppl:S213 (2004)]

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Mature neurons in the mammalian CNS fail to regenerate their axons after injury. Unlike mammals, retinal ganglion cells (RGCs) in the goldfish can successfully regrow their axons, whereas RGCs in the mammals become apoptotic following injury. Focusing on this difference between rat and fish after optic nerve injury, we compared both goldfish and rat RGCs after optic nerve crush with respect to cell death and survival signals. Fish RGCs could survive, whereas rat RGCs died gradually after 6 day treatment. The crude enzyme activity of caspase-3 in the rat retina was specifically activated, accompanying the cell death. By contrast, the activity of caspase-3 in the fish retina was reduced during 10-20 day treatment. We further measured levels of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins in both goldfish and rat retinas after injury by Western blotting analysis. In goldfish, the retinal level of Bcl-2 and p-Akt, an activated form of survival signal significantly increased 10-20 days after injury. Successively, the p-Akt inactivated Bad, an another pro-apoptotic protein. By contrast, in rat the retina level of Bax significantly increased 6 days after injury. The different behaviors of Bcl-2 family proteins and p-Akt in the goldfish and rat retina were morphologically confirmed to be limited to localize the RGCs by immunohistochemical analysis. The opposing properties of fish and rat retinas in cell death and survival signals offer us some clues for solving the mystery of CNS regeneration. [Jpn J Physiol 54 Suppl:S214 (2004)]

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Unlike the mammalian CNS, the fish optic nerve can regenerate their axons after optic nerve transection. TG_R, one type of transglutaminase that derived from regenerating goldfish retina, was upregulated after optic nerve injury. We have isolated a cDNA clone for TG_R that comprised of 678 amino acids. In situ hybridization and immunohistochemical study showed that the increase of TG_R expression was limited to be only in the ganglion cells. The TG_R enzyme activity and mRNA content were peaked at 20 days after optic nerve transection. In contrast to goldfish, the expression of TG of rat disappeared for 1 week from ganglion cells after optic nerve crush. To investigate the molecular mechanism of TG_R in the optic nerve regeneration, we made recombinant TG_R protein with the expression vector pFLAG-CMV-1 transfected in HEK 293 cellsUsing an in vitro culture system, addition of the recombinant TG_R protein lead to promotion of neurite outgrowth from early stage of culture. Furthermore, an in vivo assay system of retinotectal connection traced with WGA-HRP was clearly blocked by daily treatment of TG inhibitors into goldfish orbit for 30 days. These results indicate that upregulation of TG_R is an indispensable event for regrowing the optic nerve fibres in vitro and in vivo. We are now in progress to investigate the effect of blocking TG_R expression with RNAi on neurite outgrowth. [Jpn J Physiol 54 Suppl:S214 (2004)]

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Generalized absence epilepsy is a neurological childhood disorder, which is characterized by behavioral arrest with string and by 3Hz spike and wave discharges (SWDs) in electroencephalography (EEG). γ-Butyrolactone (GBL) is the most used drug for animal model of absence seizure. We previously showed a dissociation of absence seizure and CRE-binding activity induced by GBL in the developing rat brain. The GBL is metabolized to γ-hydroxybutyric acid (GHB) and GHB further affects both on GABA_B and GHB receptors. Thus, GABA_B and GHB receptors are thought to be involved in generation of absence seizure. However the molecular mechanism of absence epilepsy mediated via GABA_B and GHB receptors is not so clear until today. In the present study, we compared effect of GABA_B and GHB on the EEG, behavior and CRE-binding activity in the adult mouse brain by using receptor-specific agonists and antagonists. A low dose (40 mg/kg) of baclofen, a specific GABA_B receptor agonist induced a typical absence seizure of arrest in behavior, SWDs in EEG and CRE-binding activity in the mouse brain. The change in behavior, EEG and transcriptional activity continued for a long time (>2 hr). In contrast, a high dose (200mg/kg) of GHB induced only CRE-binding activity but not typical change of EEG. The action of GHB did not continue so long (<45 min). These results also show a dissociation of absence seizure and CRE-binding activity in the adult mouse brain. [Jpn J Physiol 54 Suppl:S214 (2004)]

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In a previous study, we reported fast and slow recovery phases of goldfish behavior after optic nerve transection. To understand the molecular mechanism of regrowing optic axons and reforming the retinotectal topography, we identified genes from tectum whose expression is upregulated in the late stage of optic nerve regeneration. A cDNA library constructed from goldfish tectum was screened by differential hybridization with cDNA probes derived from normal or axotomized tecta. One clone was identified as ferritin heavy subunit by high sequence homology. The level of the mRNA in the tectum increased at 30 days and peaked at 60 days after optic nerve transection. The increased expression of the mRNA was detected in the SFGS layer of the tectum by in situ hybridization. On the other hand, Eph/ephrine and neuropirin/semaphorin family are well characterized as an axon guidance molecule for retinotectal topography during development of the visual system. In the present study, therefore the full length of ephrine A2 and semaphorin IIIA cDNAs were cloned from goldfish tectal cDNA library. Their expression pattern and localization in the tectum were determined by in situ hybridization. Our data suggest that ephrine A2 and semaphorin IIIA also work as axon guidance molecules for retinotectal topography after optic nerve lesion, as seen as visual development. [Jpn J Physiol 54 Suppl:S214 (2004)]

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occ1 is a gene that is transcribed preferentially in the primary visual cortex (V1) of macaque monkeys in an activity-dependent and a developmental regulated manner (Tochitani et al. 2001, 2003). Since the visual pathway is most evolved in the primate, the occ1 transcription pattern might be different between mammals. In the present study, transcription patterns of occ1 (and its mouse orthologue frp/tsc-36) were examined in mice, ferrets, marmosets and macaques. Although occ1/frp was transcribed strongly in various nuclei in the subcortical regions of each animal in similar patterns, expression patterns in cerebral cortices were quite different. occ1/frp mRNA expressions in the neocortex of mice and ferrets were very weak and had no regional specificity. The marmoset pattern was similar to the macaque pattern, although the region specificity in marmosets was less conspicuous than that in macaques. Since occ1/frp was transcribed in some sensory relay nuclei in each animal, not only macaque V1, the regulation of occ1/frp transcription by sensory input was examined with the deprivation of eye, olfactory epithelium and cochlear of mice. The occ1/frp transcription in each relay nucleus was not significantly affected by these sensory deprivations, showing occ1 in macaque V1 is regulated in a specific manner. These results imply that occ1/frp is involved in the evolution of the visual pathway. [Jpn J Physiol 54 Suppl:S215 (2004)]

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β amyloid peptide (Aβ) accumulation with aging is one of the main pathological features of Alzheimer Disease (AD). Aβ is produced from amyloid precursor protein (APP) by the action of β and γ secretase, it is degraded by some proteases such as neprilysin. Recently, β amyloid cleaving enzyme (BACE)was identified as β secretase and presenilin 1 is essential for the action of γ secretase. The alteration of activities of these substances with aging may induce to changes in turnover of Aβ and elevate the risk of AD. We therefore examined age-related changes of mRNA expressions in these substances. The hippocampus and cerebral cortex were dissected from young (4 weeks old)and aged (95 weeks old) rats and APP, BACE, presenilin 1 and neprilysin mRNA expressions in these tissues were measured by semi-quantitative RT-PCR method. GAPDH mRNA expressions were used for standardization of the template. In the hippocampus, BACE, presenilin 1 and neprilysin mRNA expressions were significantly reduced by aging, but there was no age difference in APP mRNA expression. In aged rats, the expression levels of BACE, presenilin 1 and neprilysin mRNA in the hippocampus were significantly lower than those in the cerebral cortex. Howevere, the mRNA expressions were examined didn't differ between the two brain regions in young rats. These results suggest that BACE, presenilin 1 and neprilysin mRNA expressions reduce only in the hippocampus with advancing age in rats. [Jpn J Physiol 54 Suppl:S215 (2004)]

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A growing evidence has accumulated that glycoproteins are known to be involved in various biological events such as cell recognition and differentiation. Protein and lipid glycosylation changes are observed during development, differentiation and importantly, numerous disease state. In order to clarify the mechanism by which sugar chains of glycoproteins undergo changes during myelination and certain neurological diseases of peripheral nerves, we have performed glycome analysis of glycan chains released from glycoproteins in peripheral nerves. "Glycome" is a term meaning the whole set of glycans produced by individual organisms, as the third bioinformative macromolecules to be elucidated next to the genome and proteome. Glycans were released by glycopeptidase-A digestion of glycopeptides derived from the glycoproteins in bovine or rat peripheral nerves, pyridylaminated and then mapped by two-dimensional (2D) HPLC; ion-exchange HPLC and followed by ODS-HPLC. Characterization of each glycan was performed by MALDI-TOF mass spectrometry before and after appropriate glycosidase digestions or solvolysis to remove sulfate. More than one hundred glycan species were assigned based on these methods. Most of the glycans were of hybrid-type oligosaccharide. Several novel sugars containing non sulfated or sulfated glucuronic acid, which is known as HNK-1 carbohydrate epitope, will be reported. [Jpn J Physiol 54 Suppl:S215 (2004)]

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Hippocalcin is a member of the neuronal calcium sensors (NCS) family predominantly expressed in the hippocampal pyramidal cells. Here we show the interaction of hippocalcin with mixed- lineage kinase (MLK) 3, which has been identified as a hippocalcin-binding protein by using yeast two-hybrid screening of mouse brain cDNA library, and indicate the possible involvement of hippocalcin in MLK3 activation process. Immunoprecipitation study using antigen-purified anti-hippocalcin antibody displayed the co-precipitation of MLK3 in 1% TritonX-100 soluble hippocampal lysate. In vitro co-precipitation analysis using domain specific constructs of MLK3 revealed that hippocalcin bound with C-terminal Pro/Ser/Thr-rich region of MLK3. Recombinant fragments for each EF-hand domain of hippocalcin failed to bind with MLK3, indicating that whole hippocalcin molecule involved in the interaction with MLK3. Immunocomplex kinase assay displayed that resting kinase activity of MLK3 in hippocalcin deficient mice was higher than that in wild type. Then we detected the phosphorylation state of MLK3 because phosphorylation events in MLK3 play crucial roles in its activation process. Immunoblot analysis using anti-phospho-Ser/Thr antibodies revealed that immunoprecipitated MLK3 in hippocalcin deficient mice was higher phosphorylated than that in wild type. These observations suggest that hippocalcin inhibits the MLK3 activation via regulating MLK3 phosphorylation state. [Jpn J Physiol 54 Suppl:S215 (2004)]

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The purpose of this study is to evaluate whether the level of serotonergic activity in brain can be estimated by measurement of serotonin (5-HT) concentration in blood. Although it has been long suggested that the monoamines including 5-HT does not transport across the blood-brain barrier (BBB), a recent study demonstrated an existence of 5-HT transport system at BBB by in vitro study. The present study was conducted to evaluate this notion by in vivo study. We examined a change in 5-HT level in whole blood, when brain 5-HT level was elevated by intravenous administration of 5-HTP in an anesthetized, spontaneously breathing rat with and without gastrointestinal resection, namely non-GI rat and control rat. Brain 5-HT level was measured by microdialysis method and HPLC system: samples were corrected every 10 minutes in medial prefrontal cortex in non-GI rat and control rat. We also measured 5-HT concentration in whole blood before and after 5-HTP loads. We found that blood 5-HT level showed a clear augmentation whenever brain 5-HT level was elevated by 5-HTP either non-GI rat or control rat. These results indicated that an increase in blood 5-HT level was induced an increase in brain 5-HT level. [Jpn J Physiol 54 Suppl:S216 (2004)]

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Extracelluar signal regulated kinase (ERK) or N-terminal c-Jun kinase (JNK) has been implicated as survival enhancer or death inducer in the central nervous system (CNS), respectively. Another MAP kinase, p38 MAP kinase, has been also found in the CNS cells. By the experiments using several cell types such as pheochromocytoma cell line PC12, neuroblastoma cell line N1E-115, cerebellar granule cells, endothelial cells, smooth muscle cells, it has been supposed that p38 MAP kinase might play multiple roles such as process extension, differentiation, survival, synaptic plasticity and migration, the precise function of p38 MAP kinase in the CNS cells remains unknown. To investigate the function of p38 MAP kinase in the CNS cells, first, we prepared cultured oligodendrocytes, because the predominant p38 MAP kinase expression has been reported in oligodendrocyte under physiological condition. SB202190, specific p38 MAP kinase inhibitor, dramatically reduced the number of 2', 3'-cyclic nucleotide 3'-phosphodiesterase-positive cultured oligodendrocytes by inducing the apoptosis indicated by TUNEL reaction after 24 hours, although negative pyridinyl-imidazole compound did not. Apoptosis induced by p38 MAP kinase inhibitor could be blocked by the pretreatment of JNK-inhibitor, but not caspase inhibitor, DEVD-CHO. These results suggest that p38 MAP kinase could play significant role of oligodendrocytes survival. [Jpn J Physiol 54 Suppl:S216 (2004)]

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Ghrelin is a gut-brain peptide, which was identified from the rat stomach as the first endogenous ligand for the growth-hormone secretagogue receptors. Ghrelin has been known to increase food intake and body weight by intravenous (IV) injection and intracerebroventricular (ICV) administration. In addition, ghrelin also elicits a decrease in mean arterial pressure (MAP) and heart rate (HR) by IV and ICV administration. However, little is known about central effects of ghrelin on cardiovascular regulation. In order to investigate whether ghrelin has effect on cardiovascular actions in the restricted region of the CNS, we examined cardiovascular response to microinjection of ghrelin into the nucleus tractus solitarius (NTS) of rat. After administration of vehicle solution into the NTS, no significant changes in MAP (-3 ± 0 mmHg) and HR (-4 ± 3 beats/min) were observed. In contrast, intra-NTS injection of 40 pmol/100 nl of ghrelin reduced 12 ± 3 mmHg of MAP and 22 ± 4 beats/min of HR, respectively (n=5). These reductions were significant (p<0.05) against the changes in vehicle control. The intra-NTS injection of ghrelin evoked hypotension and bradycardic responses. These results suggest that ghrelin may contribute to regulation of the cardiovascular control. [Jpn J Physiol 54 Suppl:S216 (2004)]

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Once the functional MRI has been developed in 1990's, it has promptly prevailed as a vital tool for investigation of the human brain function as well as for clinical diagnosis. Although it is 1.5 T in currently prevailing MRI system, the strength of magnetic filed is planning to be elevated up to 7T in order to achieve a higher spatiotemporal resolution and a higher signal to noise ratio of MR images. However, biological effects of the intense magnetic filed are still unclear, and the guide line currently applied for MRI as a safety criteria in the world is based on the experimental results accumulated before dozen years. The following factors should be reconsidered as the biological effects of magnetic field: 1) strength of magnetic field, 2) area and duration exposed to the magnetic field, and 3) dynamic property of the magnetic field. Here, we show changes in the distribution of flowing erythrocytes in a model branched vessels under an inhomogeneous magnetic field as follows: 1) attraction of paramagnetic erythrocytes induced by a gradient magnetic field was demonstrated by an increase in the concentration of erythrocytes, 2) the degree of attraction was proportional to the "magnetic susceptibility" of the erythrocytes and also proportional to "magnetic flux density" x "magnetic field gradient" as long as it was up to 10 T^2/m. Further, we discuss effects of gradient magnetic fields on the in vivo observation from the viewpoint of imaging artifacts and safety criteria on human brain mapping. [Jpn J Physiol 54 Suppl:S216 (2004)]

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Hyperphosphorylated tau is a chemical deposit in brain of Alzheimer's disease(AD) patient and characterized by double phosphorylation of closely located residues such as Ser²⁰² and Thr²⁰⁵, Thr²¹² and Ser²¹⁴. Such double phosphorylation seems to require complex regulations. For example involvement of more than two tau kinases or activators is considered.
We have focused on sequential phosphorylation of tau by CDK5, glycogen synthase kinase 3β(GSK3β) and cyclic-AMP dependent kinase (PKA), which regulates AT100 reactivity on Ser²⁰², Thr²⁰⁵, Thr²¹², and Ser²¹⁴.
First we had conduced in vitro tau phosphorylation by single kinase. Western blot analysis clearly showed phosphorylation on Ser²⁰², Thr²⁰⁵, and Thr²¹² was fully dependent on CDK5. Truncation of CDK5 activator p35 enhanced most of phosphorylation sites except Ser²⁰². Phosphorylation on Ser²¹⁴ is highly functioned by PKA. GSK3β mostly shared phosphorylation sites with CDK5 and had less activity than CDK5.
Second we had examined sequential phosphorylation by CDK5, GSK3β, then PKA. The change in phosphorylation preference of CDK5, resulted from cleavage of p35 to p25, had little effect on sequential phosphorylation. Phosphorylation of Ser²⁰² or Thr²⁰⁵ by sequential regulation was dependent on CDK5 and Ser²¹⁴ was phosphorylated by PKA. Surprisingly GSK3β evoked striking enhancement of phosphorylation on Thr²¹² after CDK5 preincubation even faint phosphorylation on Thr²¹² was detected by 48hr incubation of GSK3βalone.
These results indicated that sequential phosphorylation was highly responsible for hyperphosphorylation of tau in consequence of stimulation of AD specific phosphorylation. [Jpn J Physiol 54 Suppl:S217 (2004)]

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To investigate whether diabetes mellitus (DM) induced during early postnatal period affects myelinogenesis of the optic nerve or not, we first made neonatal-DM model rats and then carried out electron-microscopic (EM) observation and immunostaining of myelin basic protein (MBP). Neonatal-DM model rats were made by Streptozotocin injection (i.p., 100 μg/g body weight) to P2 Wistar rats. Triphasic change in the blood glucose level was found: initial sever hyperglycemia during the first 3 postnatal weeks (P4~P21), normoglycemia lasted for about two weeks (P22~P34), and mild progressive hyperglycemia after P34. Electron-microscopically, it was found in DM-rats that number of ensheathed axons was decreased in P7 and number of myelinated axons was slightly decreased in P12 and P33. Most nodes of Ranveir were abnormally formed (broad, swollen nodes) with partial axoglial dysjunction in P19. In P33, myelin lamellae were disrupted in most of myelinated axons. Immunohistochemical staining with MBP in serial sections along the entire length of the optic nerve was carried out. Migration of the oligodendrocyte precursors (OPCs) along the optic nerve from chiasmal end to eye end and sequential expression of OPCs antigens were detected in both DM group and controls. We found that number of MBP-positive cells was reduced at all levels along the entire length of the nerve in DM rats at P10. Data suggest that DM, induced during the early critical postnatal period, caused delay in the process of myelination with subsequent abnormal formation of nodal and paranodal domains. [Jpn J Physiol 54 Suppl:S217 (2004)]

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Peripheral administration of cholecystokinin (CCK)-8 causes an activation of oxytocin (OXT) -secreting neurons in the paraventricular (PVN) and supraoptic nuclei (SON) with elevation of plasma OXT level in rats. In the present study, we investigated the effects of intravenous (iv) administration of CCK-8 on OXT-secreting neurons in the PVN and SON of Otsuka Long-Evans Tokushima Fatty (OLETF) rats that have a congenital defect in the expression of the CCK-A receptor gene, using in situ hybridization histochemistry (ISH) for c-fos mRNA and immunohistochemistry for Fos. Although c-fos mRNA was detected in the PVN and the dorsal parts of the SON of LETO (control) rats 30 minutes after iv administration of CCK-8, c-fos mRNA was not detected in the PVN and SON of OLETF rats. The distributions of Fos-like immunoreactivity (LI) were identical to the results obtained from ISH. After iv administration of saline, c-fos mRNA and Fos-LI were not detected in the hypothalamus of both OLETF and LETO rats. Dual staining for OXT and Fos revealed that Fos-LI was mainly observed in OXT-secreting neurons in the PVN and SON of LETO rats after iv administration of CCK-8. These results suggest that CCK-8 may activate OXT-secreting neurons in the rat PVN and SON through CCK-A receptor. [Jpn J Physiol 54 Suppl:S217 (2004)]

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Monitoring the immediate early genes (IEGs) is a useful method for following stress-induced cellular response in the neuroendocrine system. We have examined the transcriptional activities of IEGs (c-fos, jun-B, NGFI-A and NGFI-B) and arginine vasopressin (AVP) gene in the hypothalamic paraventicular (PVN) and supraopic nuclei (SON) of rats after acute osmotic stimulus, using in situ hybridization histochemistry. After intraperitoneal (i.p.) administration of hypertonic saline (2% body weight, 900 mOsm/kg), the expressions of all IEGs in the PVN and SON increased significantly as early as 10 min, peaked at 30 min and maintained until 60 min. The level of AVP heteronuclear (hn) RNA also peaked at 30 min, although maintained until 180 min. The expressions of all IEGs also increased dose-dependently in response to the concentration of i.p. administration of saline (more than 600 mOsm/kg). These results showed that induction of IEGs as well as the level of AVP hnRNA in the PVN and SON is a useful marker for following the neuroendocrine responses to acute osmotic change in rats. [Jpn J Physiol 54 Suppl:S218 (2004)]

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The nitric oxide (NO) is assumed to be a modulator of the secretion of pancreatic hormones. Recently, it has been reported that a constitutive nitric oxide synthase (cNOS) located in all kinds of endocrine cells in the rat islets of Langerhans (Alm, et al., 1999). In the present study, we attempted to demonstrate the actual production of NO in the A- and B-cells of the islets of rat pancreas. Exocrine cells, islets, and other cells of the pancreas were dispersed by the collagenase method. The islets were picked up under a stereoscope and superfused with Krebs solution under the fluorescence microscope. After loading with 4,5-diaminofluorescein-2 diacetate (DAF-2 DA, 2.5 μM), a bright green fluorescence indicating production of NO was developed within a large number of islet cells. A- and B-cells of the islets were fixed and stained by immunohistochemistry for glucagon and insulin respectively. The stained islets were observed with confocal microscope (FV500, Olympus). The green fluorescence was observed in many insulin-immunoreactive B-cells. NO was also produced in glucagon-positive cells scattered in peripheral region of the islet. The results suggest that NO is continuously produced in A- and B-cells of the islet and modulates the secretions of glucagon and insulin. [Jpn J Physiol 54 Suppl:S218 (2004)]

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In a classical conditioning paradigm, postnatal day (PND)-12 rats show aversion to the odor that has been paired with foot shock in a 30-min training session on PND-11. Impaired olfactory learning, however, is observed in young rats with intrauterine growth restriction (IUGR) induced by bilateral occlusion of the utero-ovarian artery for 20 min on day 16 of pregnancy. Transient feto-placental ischemia evokes ischemia/reperfusion injury. It is well known that melatonin is a powerful scavenger of oxygen free radicals. Biochemical analyses demonstrated that the administration of melatonin to pregnant rats protects against ischemia/reperfusion-induced lipid and DNA damage in fetal brain as well as oxidative damage in the mitochondria of placentas. Therefore we examined whether melatonin administration to pregnant rats is capable of restoring aversive olfactory learning in young rats with IUGR. IUGR rats born of the mother rat that had received melatonin administration during the whole pregnancy period showed aversion to the conditioned odor. Aversive olfactory learning was normally observed in non-IUGR rats born of the sham-operated mother rat. Melatonin, when infused directly into the olfactory bulb during odor-shock stimulation, failed to restore olfactory learning in IUGR rats. These results suggest that melatonin administration to pregnant rats is not able to restore olfactory learning in young rats with IUGR, but can preserve learning ability from IUGR by protection against ischemia/reperfusion-induced oxidative damage. [Jpn J Physiol 54 Suppl:S218 (2004)]

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The use of saliva as an alternative specimen for the determination of various stress hormones has gained increasing attention, because saliva sampling has the advantage that it is making multiple sampling and stress free. We examined the effects of psychological stressor and soother on the levels of salivary cortisol and β-endorphin as the indexes of hypothalamic-pituitary-adrenocortex activity, and amylase as the index of sympatho-adrenomedullary activity. The subjects were 83 healthy volunteers (23.3 yrs). State-Trait Anxiety Inventory (STAI) was used to assess the responsibility to anxiety. The video of corneal transplant surgery (stressful video) and scenic beauty (soothing video) were served for 15 min. The whole saliva was collected at every 3 min. The amylase level was significantly increased just after the beginning of stressful video viewing (p<0.01) and immediately returned to the pre-stress level just after the video end. The cortisol and β-endorphin levels were increased later than the amylase, and returned to the pre-stress level later 3-12 min than that of amylase. Soothing video viewing significantly decreased in amylase level, and increased in β-endorphin level (p<0.05), but did not affected on the cortisol level. Correlation between the amylase level and the STAI score was highly significant (r = 0. 535, p<0, 01). However, the cortisol and β-endorphin levels did not correlate to STAI score. It is suggested that the salivary amylase is a useful tool of psychological stress of young adult, and is more sensitive and specific than cortisol and β-endorphin measurements. [Jpn J Physiol 54 Suppl:S218 (2004)]

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We have previously proposed a role of NMDA receptors in the anteroventral third ventricular region (AV3V) in a hypovolemic VP secretion, on the basis of the potency of AV3V infusion of its antagonist (MK) to inhibit the plasma VP response to hemorrhage. However, as it causes two other stimuli for VP release, the rises in plasma (p) osmolality (Osm) and angiotensin (ANG) II, it is possible that the MK treatment might have blunted the response by interfering these events. Moreover, it could be suspected that MK applied to the AV3V, a juxta-ventricular area, might have diffused into the ventricular space to produce the effect via actions on remote regions. The aim of this study was to further explore validity of the view by examining those problems. In conscious and unrestrained rats, normotensive (H1) and hypotensive hemorrhages (H2) were induced successively by removing arterial blood twice at a 10-min interval. The H2, but not H1, augmented VP, Osm, glucose and ANG II in plasma, and reduced its K+. PNa+ and heart rate were not changed. MK administered into the AV3V 15 min preceding H1 was verified to block the VP response to H2, whereas it did not affect the responses of pANG II, pOsm, glucose, blood pressure or other variables. When infused into the cerebral ventricle, MK did not influence the VP response. Thus, the present results provide further support for our proposal that NMDA receptors in the AV3V may operate to mediate VP secretion in the state with hypotensive hypovolemia. [Jpn J Physiol 54 Suppl:S219 (2004)]

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Expressional regulation of estrogen-responsive genes in adult female rats. Wada-Kiyama Y.1), Rho J.-y.2), Kiyama R.3), Sakuma Y.1) 1) Nippon Med. Sch., Tokyo, Japan. 2) Korea Insti. Biosci. Biotech., Daejeon, Korea. 3) RCG, AIST, Ibaraki, Japan. In rats, estrogen plays critical roles in sexual differentiation of the brain and regulation of the sexual behavior. To examine the regulatory mechanism of estrogen in rats, we prepared 8-weeks ovariectomized rats and treated with or without estrogen for two weeks. mRNA was obtained from the hypothalamus, uterus, liver, kidney and skeletal muscle and analyzed by Northern blotting and/or RT-PCR. Neuronal system-related genes (Amphiregulin, Neuropeptide Y-Y1 receptor, Bassoon, N-Cadherin) and estrogen-susceptible cancer-related gene (C-terminal binding protein interacting protein) were selected based on the results of a cDNA microarray analysis which profiled estrogen-responsive genes in MCF-7 cell line. In the hypothalamus, all except the Amphiregulin gene were down-regulated. In other tissues, the expression showed marked differences. We then analyzed the time course of the estrogen-responsiveness of these genes, finding changes in expression patterns especially in the skeletal muscle. Our results show that the estrogen-responsive genes, which were demonstrated simply as either up- or down-regulation by estrogen in MCF-7, exhibit tissue and temporal-specific expression patterns in adult female rats. [Jpn J Physiol 54 Suppl:S219 (2004)]

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Cellular functions of the GnRH neuron have been widely studied by using its clonal cell line GT1-7. To further characterize GT1-7 cells, we investigated the diversity of voltage-gated Ca²⁺ channels in GT1-7 cells with perforated-patch clamp and RT-PCR. An R-type Ca²⁺ channel blocker SNX-482 inhibited the Ca²⁺ currents by 76% in all cells examined (n=9). A T-type Ca²⁺ channel blocker Ni²⁺ inhibited the Ca²⁺ currents by 13% in all cells examined (n=9). An L-type Ca²⁺ channel blocker nimodipine inhibited the Ca²⁺ currents by 18% in 5 out of 11 cells examined. An N-type Ca²⁺ channel blocker ω-conotoxin-GVIA inhibited the Ca²⁺ currents by 14% in 3 out of 20 cells examined. A P/Q-type Ca²⁺ channel blocker ω-agatoxin-IVA had no effect on the currents (n=9). The contribution of the L-type was much greater in the current elicited by ramp depolarization than in that evoked by a square pulse. On the contrary, the contribution of the R-type was less in the current by ramp depolarization. RT-PCR revealed that GT1-7 cells expressed the α_1B, _1D, _1E and _1H subunit mRNA. Furthermore, SNX-482 and nifedipine inhibited the high K⁺ induced increase in the intracellular Ca²⁺ concentration and GnRH release. These results suggest that GT1-7 cells express R-, L-, N- and T-type voltage-gated Ca²⁺ channels and the R-type is a major current component, and the L- N- and T-type are minor ones. The R- and L-type Ca²⁺ channels play a critical role in the regulation of Ca²⁺-dependent GnRH release. [Jpn J Physiol 54 Suppl:S219 (2004)]

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Interleukin-18 (IL-18), known as interferon-γ inducing-factor, exerts pleiotropic roles as a potent pro-inflammatory cytokine. In addition to immune cells, it has been demonstrated in rat adrenal cortex, posterior pituitary gland, and medial habenular nucleus and bovine somatotrophs. Although IL-18 has been demonstrated to increase in obesity, diabetus mellitus, ischemic heart diseases and autoimmune diseases, the mechanism by which the induction of IL-18 occurs in vivo remains to be elucidated. To clarify the secretary mechanism of IL-18 into circulation, the present study investigated IL-18 levels in response to immobilization, cold, sleep deprivation, and lipopolysaccharide (LPS). In addition, to speculate the source of IL-18, adrenectomy was conducted. The mean IL-18 level under control condition was 189 ± 1.0 pg/ml. In immobilization stress for 1 h and 2 h, its levels significantly increased to 222.3 ± 2.2 pg/ml (p<0.05) and 220.7 ± 0.8 pg/ml (p<0.001), respectively. Of important notice, its levels further went up in 2 h after the 2-h immobilization stress (320.5 ± 7.7 pg/ml, p<0.05). A significant increase was also observed in cold stress (226 ± 2.9 pg/ml, p<0.05), while no significant change occurred in sleep deprivation. In adrenectomized mice IL-18 levels significantly increased in response to LPS, but not to stressors. LPS significantly up-regulated IL-18 mRNA expression in the spleen, but not in the adrenal gland. The present results show the tissue-specific induction of IL-18 gene expression, and suggest that adrenal cortex may be the main source of IL-18 which increases in blood during stress. [Jpn J Physiol 54 Suppl:S219 (2004)]

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Cyclin-denpendent kinase 5 (Cdk5) is a serine/threonine kinase with close structural homology to the Cdks. Cdk5 forms a complex with its activators, p35 or p39. Previous studies have shown that both p35 and p39 are only expressed in neuron, and Cdk5/p35 has important roles in the regulation of neuronal functions such as coricogenesis and fasciculation of the axon fibers. Moreover, recent studies have revealed that Cdk5/p35 is involved in neurotransmitter release. Cdk5/p35 phosphorylates many synaptic proteins such as P/Q- type voltage-dependent calcium channel (P/Q-type VDCC), Dynamin 1 and Amphiphysin 1. Phosphorylation of P/Q-type VDCC by Cdk5 suppresses neurotransmitter release by downregulation of its channel activity. These previous observations strongly suggest that Cdk5/p35 has an important role in the regulation of neurotransmission. It is known that the secretion of insulin in pancreatic islets has a similar mechanism with neurotransmitter releasing. However, the expression level of Cdk5/p35 and its physiological functions during the secretion of insulin are unknown. In order to investigate the role of Cdk5/p35 in islets, we examined the expression of Cdk5/p35 in isolated islets and β-cell line MIN6. [Jpn J Physiol 54 Suppl:S220 (2004)]

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Neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus (ARC) are the site of convergence of central and peripheral feeding regulating signals and play an important role for feeding stimulation. In this study, we investigated how leptin, ghrelin and orexin signals are integrated in NPY neurons. We isolated single neurons from the ARC of rats and measured the cytosolic Ca²⁺ concentration ([Ca²⁺]i) with fura-2 fluorescence imaging. To identify NPY neurons, we did immunocytochemical staining after [Ca²⁺]i measurements. Leptin infusion suppressed ghrelin- and orexin-induced [Ca²⁺]i increases in single NPY neurons, and the suppression was temporal for ghrelin-induced [Ca²⁺]i increases. Because our previous research showed that [Ca²⁺]i increases induced by ghrelin were suppressed by a PKA inhibitor and those by orexin were suppressed by a PKC inhibitor in NPY neurons, we investigated whether leptin could suppress the [Ca²⁺]i increases induced by adenylyl cyclase activator and PKC activator. Leptin suppressed both forskolin- and TPA-induced [Ca²⁺]i increases, while those induced by a high concentration of KCl were not affected. These results demonstrate that leptin suppresses the ghrelin-and orexin-induced [Ca²⁺]i increases at least partly by counteracting the cAMP-PKA and PKC signaling, respectively, but not by direct inhibition of Ca²⁺ channels. These interactions in NPY neurons may play an important role in integrating metabolic, nutritional and central signals of the body and thereby producing an appropriate feeding-controlling output. [Jpn J Physiol 54 Suppl:S220 (2004)]

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Obese patients are at risk for development of cardiovascular disease, which can in part be explained by disturbances in the haemostatic and fibrinolytic systems. Recently, it has been demonstrated that the adipocyte itself is able to produce a primary fibrinolytic inhibitor, PAI-1, possibly explaining the high levels found in obesity. To gain insight into molecular mechanism of up-regulation of PAI-1 gene expression in fat tissues, we investigated the role of an adipocyte-enriched transcription factor, PPARγ. To identify cis-acting genetic elements required for induction of PAI-1 gene transcription, we constructed the plasmids containing a 5' deletion series of the PAI-1 promoter. When sequences between -762 and -723 were deleted, the level of induction significantly fell. Gel mobility shift assay indicated that atypical PPRE overlapping C/EBP binding site (-752 to -733) is important for PPARγ binding. Chromatin immunoprecipitation assay demonstrated that PPARγ is physically associated with PAI-1 promoters in vivo. Furthermore, mutation of the atypical PPRE abrogated the increase in transcriptional activities of PAI-1 promoter. Functional assay revealed that besides PPARγ/RXRα expression vectors, transfection of CBP expression vectors increased the luciferase activity to 7.5 fold and 11 fold of control value in COS-1 cells and 3T3-L1 preadipocyte, respectively. These data indicate that up-regulation of PAI-1 gene expression during adipogenesis is due to the functional cooperation between CBP and PPARγ/RXRα through atypical PPRE. [Jpn J Physiol 54 Suppl:S220 (2004)]

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To examine the role of gonadal steroid hormones in the stress-induced acetylcholine (ACh) release in the hippocampus, an in vivo microdialysis study was performed in gonadectomized (Gdx), gonadectomized and estrogen-primed (Gdx+E), and gonadectomized and testosterone-primed (Gdx+T) rats. Using both sexes of rats, the Gdx and steroid priming were performed at least 2 weeks before the experiment. ACh concentration in dialysates was assayed by the HPLC system, and the restraint stress was applied from 1200 h to 1300 h. In male rats, the ACh release in the hippocampus promptly increased within 15 min after the onset of the restraint stress in all groups of rats (P < 0.01 vs pre). Although the magnitude of ACh release response was low in Gdx or Gdx+E male rats, it was successfully ameliorated in Gdx+T male rats (P < 0.01). Also in female rats, the ACh release in the hippocampus promptly increased within 15 min after the onset of the restraint stress in all groups of rats (P < 0.01 vs pre). The magnitude of ACh release response was low in Gdx female rats, but was successfully ameliorated in Gdx+E and Gdx+T female rats (P < 0.05). In addition, Gdx+T male rats showed greater stress response than Gdx+E female rats (P < 0.05), that is indentical to the sex difference in gonadally intact rats. These results suggest that circulating testosterone in male rats as well as circulating estrogen in female rats contribute to sustain the stress response of ACh release in the hippocampus. [Jpn J Physiol 54 Suppl:S220 (2004)]

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We previously reported that the BST, as the medial preoptic area (mPOA), in female rats has significantly more CRH neurons than in male rats. In the present study, we examined whether low dose testosterone propionate (TP) affected sex difference in CRH neurons in association with changes in the estrous cyclicity. Neonatal female rats were injected sc with 1 μg or 5 μg TP at the age of 5 days. Control male and female rats were injected with sesame oil alone. Daily virginal smears were obtained from the age of 7 weeks and rats were seved to immunocytochemical processing at the age of 9-11 weeks. In control male and female rats, there was a significant sex difference in the number of CRH neurons in the BST and the mPOA, as we reported previously. One μg TP had no effect on the estrous cyclicity and the number of CRH neurons both in the BST and the mPOA. Sixty% of female rats injected with 5 μg TP were persistently estrous. The number of CRH neurons in the BST of these persitent estrous rats was significantly small, but that in the mPOA was not changed, compared to control female rats. We suggest that a low dose testosterone causes loss of sex difference in the number of CRH neurons in the BST and the estrous cyclicity in female rats and that CRH neurons in the BST is more sensitive than the mPOA to steroid milieu during the neonatal period. [Jpn J Physiol 54 Suppl:S221 (2004)]

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PCBs are known as environmental contaminants that cause various effects in many organs including the central nerve system. Some PCBs pass through blood brain barrier (BBB) to accumulate in brain and cause disruptions of brain development. Previously we reported that very low dose, as low as 10-10M of hydroxylated PCBs suppressed TR-mediated transcription. However, the mechanism of suppression by PCB is not clarified. To understand the mechanism, we first examined the effects of PCB on coactivator and coreppressor interaction with TR using mammalian two-hybrid assay in CV-1 cell and GST pull-down study. TR-mediated transcription stimulated by steroid receptor coactivator-1 (SRC-1) was suppressed by PCBs. However, the binding of SRC-1 to TR was not dissociated by PCBs. PCBs did not recruit nuclear receptor corepressor (N-CoR) to TR in the presence of T3. Next, we examined PCB effects on TR binding to TRE by electorophoretic mobility shift assay. TR/retinoid X receptor (RXR) heterodimer complex was partially dissociated from TRE in the presence of PCBs. These results indicate that PCB suppressed TR-mediated transcriptional activation by partial dissociation of TR/RXR heterodimer complex from TRE. [Jpn J Physiol 54 Suppl:S221 (2004)]

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Recently, we have cloned a general coactivator named coactivator activator (CoAA). It contains RNA-recognition motifs (RRMs). Further, CoAA interacts with DNA-dependent protein kinase (DNA-PK) that may associate with basal transcriptional machinery. Thus, we hypothesized that CoAA may locate the RNA polymerase II (Pol II)-containing basal transcriptional machinery. We investigated the interaction between CoAA and RNA using monoribopolymer interacting assay, which showed that RRM of CoAA bound with RNA directly. Steroid receptor RNA activator (SRA) has been cloned and characterized as a RNA-form coactivator. Using transient transfection-based reporter assay, CoAA showed a synergistic activation with SRA on glucocorticoid receptor (GR)-, estrogen receptor (ERα)-, and thyroid hormone receptor (TRβ1)-mediated transcription. RRM was not required on SRA action. Thus, we tested if SRA affects the ligand-binding domain (LBD) of TR in the presence of CoAA using mammalian one-hybrid assay. Transcription was activated when Gal4-fused TRβ1-LBD was cotranscfected with CoAA and SRA into CV-1 cells. SRA synergistically activated transcription with CoAA. Suggesting that SRA may contribute to the LBD-mediated transcriptional activation in the presence of CoAA. We concluded that intrinsic CoAA may located the Pol II-containing basal transcriptional machinery, and may associate with newly produced RNA. Also CoAA interacts with SRA-containing HAT complex. Taken together, CoAA could interact both newly produced RNA and SRA. [Jpn J Physiol 54 Suppl:S221 (2004)]

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A variety of stimuli deriving from the brain and peripheral endocrine organs modify the secretion and synthesis of prolactin (PRL) in lactotrophs. Although there are numerous studies of in vivo and in vitro regulation of PRL secretion and mRNA expression by these stimuli, the regulation of prolactin transcription has been elucidated only in lactotroph cell lines. This is due to low efficiencies for transfection of transcriptional reporter genes in primary cultures and in vivo. Here we report the establishment of lactotroph-specific luciferase expression mediated by infection of recombinant adenovirus vector to rat anterior pituitary primary cultures. The luciferase gene located downstream of the rat PRL promoter gene (3.3 kb) was inserted as a reporter gene into Adeno-X vector. Double fluorescence immunocytochemistry revealed that the luciferase protein was expressed specifically in lactotrophs and not in other pituitary cell types. The luciferase assay was sensitive enough to detect the activity even in pituitary primary cultures containing 10⁵ cells. The luciferase activity was elevated after the treatment with several stimuli including insulin-like growth factor I. These results suggest that adenovirus-mediated luciferase gene expression under the control of PRL promoter gene is a useful method for determination of PRL promoter activity in primary lactotroph cultures. [Jpn J Physiol 54 Suppl:S221 (2004)]

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