Circadian rhythm of metabolic oscillation in suprachiasmatic nucleus depends on the mitochondrial oxidation state, reflected by cytochrome C oxidase and lactate dehydrogenase.

Metabolic activity in the suprachiasmatic nucleus (SCN), a center of biological rhythm, is higher during the daytime than at 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., Arg-vasopressin). Similar transcriptional regulation by Npas2/Bmal1 heterodimer formation operates in the brain, which is dependent on the redox state (i.e., NAD/NADH). To clarify the metabolic function of SCN in relation to the redox state and glycolysis levels, we measured glucose, lactate dehydrogenase (LDH), LDH mRNA, and cytochrome C oxidase, energy-producing biochemical materials from mitochondria/cytosol, in rats kept under a light-dark cycle. Mitochondrial cytochrome C oxidase activity, measured by the changes in absorption at 550 nm, was higher during the light period than during the dark period. Glucose concentration was higher during the light period. In contrast, LDH and its coding mRNA were higher during the dark period. Mitochondrial aggregation, which is reflected by mitochondrial membrane potential, indexed by JC-1 fluorescence, was higher during the light period. The results indicate that the glycolysis energy pathway in the SCN, which exhits higher metabolic activity during the day than at night, might be involved in the generation of circadian rhythm.

Circadian rhythm of enolase in suprachiasmatic nucleus depends on mitochondrial function.

Metabolic activity in the suprachiasmatic nucleus (SCN), a center of biological rhythm, is higher during the daytime than at 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., Arg- vasopressin). Similar transcriptional regulation by NPAS2/BMAL1 heterodimer formation operates in the brain, which depends on the redox state (i.e., NAD/NADH). To clarify the metabolic function of SCN in relation to the redox state, two-dimensional electrophoresis was carried out on the mitochondrial fraction of SCN, obtained from rats kept under a light:dark cycle and constant under dim light. The electrophoretic pattern with TOF-mass spectrometry analysis revealed that enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. The enolase activity, coupled with lactate dehydrogenase, was higher during the light period than that in the dark. However, enolase mRNA, analyzed by RT-PCR, showed higher levels during the dark period than in the light. The clock gene products Per2, Bmal1, Rev-erbα, and AVP mRNA in the mitochondrial fraction of SCN developed a circadian rhythm showing almost the same peak time as that in whole SCN. These mRNA rhythms ran free except for that of Rev-erbα mRNA. The results indicate that, in the glycolysis-related energy pathway, enolase might be involved in higher metabolic activity during the day than at night, at least in part.

Enhanced neurogenesis from neural progenitor cells with G1/S-phase cell cycle arrest is mediated by transforming growth factor beta1.

We have previously demonstrated that a G1/S-phase cell cycle blocker, deferoxamine (DFO), increased the number of new neurons from rat neurosphere cultures, which correlated with prolonged expression of cyclin-dependent kinase (cdk) inhibitor p27(kip1) [H. J. Kim et al. (2006)Brain Research, 1092, 1-15]. The present study focuses on neuronal differentiation mechanisms following treatment of neural stem/progenitor cells (NPCs) with a G1/S-phase cell cycle blocker. The addition of DFO (0.5 mm) or aphidicolin (Aph) (1.5 microm) to neurospheres for 8 h, followed by 3 days of differentiation, resulted in an increased number of neurons and neurite outgrowth. DFO induced enhanced expression of transforming growth factor (TGF)-beta1 and cdk5 at 24 h after differentiation, whereas Aph only increased TGF-beta1 expression. DFO-induced neurogenesis and neurite outgrowth were attenuated by administration of a cdk5 inhibitor, roscovitine, suggesting that the neurogenic mechanisms differ between DFO and Aph. TGF-beta1 (10 ng/mL) did not increase neurite outgrowth but rather the number of beta-tubulin III-positive cells, which was accompanied by enhanced p27(kip1) mRNA expression. In addition, TGF-beta receptor type II expression was observed in nestin-positive NPCs. Results indicated that DFO-induced TGF-beta1 signaling activated smad3 translocation from the cytoplasm to the nucleus. In contrast, TGF-beta1 signaling inhibition, via a TGF-beta receptor type I inhibitor (SB-505124), resulted in decreased DFO-induced neurogenesis, in conjunction with decreased p27(kip1) protein expression and smad3 translocation to the nucleus. These results suggest that cell cycle arrest during G1/S-phase induces TGF-beta1 expression. This, in turn, prompts enhanced neuronal differentiation via smad3 translocation to the nucleus and subsequent p27(kip1) activation in NPCs.

Pretreatment with low doses of erythropoietin ameliorates brain damage in periventricular leukomalacia by targeting late oligodendrocyte progenitors: a rat model.

BACKGROUND: One of the pathological hallmarks of periventricular leukomalacia (PVL) is the selective vulnerability of late oligodendrocyte progenitors (preoligodendrocytes; preOLs) to hypoxia-ischemia (H-I). It is unknown whether recombinant human erythropoietin (rhEPO) protects preOLs in vivo. OBJECTIVES: To develop a rat PVL model in which preOLs are selectively damaged and exhibit similar pathological changes to diffuse-type human PVL, various conditions of H-I were compared in P2-P7 rats (P2 = postnatal day 2). To evaluate the effect of rhEPO on oligoprotection (preOLs), rhEPO was administered to P3 PVL rats. METHODS: After counts of NG2-positive and O4-positive cells were performed in P2-P7 rats, right common carotid artery occlusion followed by 6% O(2) for 0-120 min was performed in P2-P4 rats. The mortality and histological alterations after hematoxylin/eosin staining and ED1 immunostaining were assessed 2 days after H-I. Various doses of rhEPO (1-30,000 U/kg i.p.) were administered to PVL rats 15 min before administration of 6% O(2). RESULTS: Double-positive cells for NG2 and O4 were detected from P2, and their number gradually increased until P7. Although right common carotid artery occlusion with 6% O(2) for 60 min resulted in a relatively high proportion of deaths in P2-P4 rats, typical histological changes in the PVL diffuse component were found in most surviving P3 animals. With 50-100 U/kg rhEPO, the histological damage was attenuated. CONCLUSIONS: Histological changes similar to those seen in the PVL diffuse component were induced by H-I in P3 rats, in which preOLs were gradually developing, and a low dose of rhEPO was effective in the treatment of brain damage induced by H-I.

Altered striatal vulnerability to 3-nitropropionic acid in rats due to sex hormone levels during late phase of brain development.

Systemic administration of 3-nitropropionic acid (3-NPA) leads to a shortage of cellular ATP and induces striatum-specific lesions that resemble Huntington's disease. Gender differences, in terms of vulnerability of striatum to 3-NPA, have been shown in male rats. The goal of the present study was to determine whether changes in sex hormone levels during the critical period of sexual differentiation (E17-P4) influence striatal vulnerability to 3-NPA. An androgen receptor antagonist, flutamide, or an aromatase-inhibitor, fadrozole hydrochloride, which block conversion of testosterone to estradiol, were administered to embryonic rats during E17-E20 or E18-E20, respectively, with subsequent 3-NPA (20mg/(kg day) for 2 days) treatment during adulthood (8-9 weeks old). Motor behavior and histological changes (IgG exudation due to blood-brain barrier dysfunction and glial fibrillary acidic protein immunoreactivity) were assessed. Treatment with flutamide significantly decreased the 3-NPA-induced motor behavior in male rats, while administration of fadrozole hydrochloride increased atypical motor behavior in female rats. IgG exudation, as well as decreased glial fibrillary acidic protein reactivity, was observed in animals with motor defects. Flutamide decreased testosterone levels in male rats, while fadrozole hydrochloride increased testosterone levels in female rats. These results suggest that prenatal modulation of sexual hormonal levels greatly influences vulnerability to 3-NPA during adulthood and directly correlates to serum testosterone levels.

Increase in dopaminergic neurons from mouse embryonic stem cell-derived neural progenitor/stem cells is mediated by hypoxia inducible factor-1alpha.

A reliable method to induce neural progenitor/stem cells (NPCs) into dopaminergic (DAergic) neurons has not yet been established. As well, the mechanism involved remains to be elucidated. To induce DAergic differentiation from NPCs, a cytokine mixture (C-Mix) of interleukin (IL)-1beta, IL-11, leukemia-inhibitory factor (LIF), and glial-derived neurotrophic factor or low oxygen (3.5% O(2): L-Oxy) was used to treat embryonic stem (ES) cell-derived NPCs. Treatment with C-Mix increased the number of tyrosine hydroxylase (TH)-positive cells compared with controls (2.20-fold of control). The C-Mix effect was induced by mainly LIF or IL-1beta treatment. Although L-Oxy caused an increase in TH-positive cells (1.34-fold), the combination of L-Oxy with C-Mix did not show an additive effect. Increases in DA in the medium were shown in the presence of C-Mix, LIF, and L-Oxy by high-performance liquid chromatography. Gene expression patterns of neural markers [tryptophan hydroxylase (TPH), GAD67, GluT1, beta-tubulin III, glial fibrillary acidc protein, and TH] were different in C-Mix and L-Oxy treatments. Because increases in hypoxia-inducible factor (HIF)-1alpha protein were found in both treatments, we investigated the effect of HIF-1alpha on differentiation of NPCs to DAergic neurons. Inhibition of HIF-1alpha by the application of antisense oligodeoxynucleotides (ODNs) to NPCs caused a decrease in TH-positive cells induced by LIF treatment. Gene expressions of TH, GAD67, and GluT1 were decreased, and those of TPH, beta-tubulin III, and S-100beta were increased by treatment with just ODNs, indicating the importance of the endogenous effect of HIF-1alpha on neuronal differentiation. These data suggest that enhanced differentiation into DAergic neurons from ES cell-derived NPCs was induced by C-Mix or L-Oxy mediated by HIF-1alpha.

Diethylnitrosamine-induced hepatic lesions are greater in rats maintained under a light-dark cycle than under constant light, related to the locomotor activity rhythm.

Environmental lighting conditions affect circadian rhythm and carcinogenesis. The effect of diethylnitrosamine (DEN, i.p., 200 mg/kg) on carcinogenesis and circadian rhythmicity under a light-dark (LD) cycle, constant dark (DD) and constant light (LL) was analyzed in rats. After the recognition of entrainment in locomotor activity rhythm to LD cycle, animals remained under the LD cycle or were released into DD or LL. Liver carcinogenicity, measured by GST-P immunostaining, was higher under the LD cycle than under DD and LL. Two weeks after DEN injection, locomotor activity in 24 hr had increased under the LD. Circadian rhythmicity might be coupled with the carcinogenicity of DEN.

Increase in neurogenesis and neuroblast migration after a small intracerebral hemorrhage in rats.

Neural stem/progenitor cells (NPCs) reside in the subventricular zone (SVZ) and dentate gyrus in the adult mammalian brain. It has been reported that endogenous NPCs are activated after brain insults such as ischemic stroke. We investigated whether proliferation and migration of endogenous NPCs are increased after a collagenase-induced small intracerebral hemorrhage (ICH) near the internal capsule in rats. Bromodeoxyuridin (BrdU) administration for 14 days after ICH (post-labeling) resulted in an increase in the number of BrdU-positive cells as shown in both ipsilateral and contralateral SVZs. BrdU treatment given for 2 days before ICH to label endogenous NPCs (pre-labeling), caused more BrdU-positive cells to be detected in the ipsilateral dorsal striatum (dSTR) compared to those in the contralateral dSTR 14 days after ICH. BrdU- and doublecortin (Dcx)-positive cells were found in the ipsilateral STR. An increase in the number of Dcx-positive migrating immature neurons was found in the dSTR and peri-hemorrhage area 14 days after ICH, and a cluster of Dcx-positive cells was found in the STR around the lesion 28 days after ICH. Matrix metalloproteinase-2 (MMP-2) was strongly expressed in wide area of the injured brain, particularly around the lesion 14 and 28 days after ICH. Dcx- and MMP-2-positive cells were detected in the ipsilateral STR near the lesion. These data suggest that collagenase-induced ICH enhances the proliferation of endogenous NPCs and the migration of newly born neuroblasts toward the hemorrhage area.

Pleiotrophin promotes functional recovery after neural transplantation in rats.

Pleiotrophin promotes survival of dopaminergic neurons in vitro. To investigate whether pleiotrophin promotes survival of grafted dopaminergic neurons in vivo, donor cells from ventral mesencephalon were treated with pleiotrophin (100 ng/ml) during cell preparation and grafted into striatum of hemi-Parkinson model rats. Functional recovery in methamphetamine-induced rotations was improved, and more tyrosine hydroxylase-positive cells survived in the striatum in the pleiotrophin-treated group. Pleiotrophin addition to cells just before transplantation also resulted in better functional recovery; however, no caspase-3 activation was seen during cell preparation. Interestingly, the effect of pleiotrophin on the survival was additive to that of glial-cell line-derived neutropic factor. These results revealed that pleiotrophin had effects on donor cells in neural transplantation in vivo.

Oral administration of metal chelator ameliorates motor dysfunction after a small hemorrhage near the internal capsule in rat.

Cerebral hemorrhage leads to local production of free iron, radicals, cytokines, etc. To investigate whether a decrease of iron-mediated radical production influences functional recovery after intracerebral hemorrhage (ICH), a modified ICH rat model with a small hemorrhage near the internal capsule (IC) accompanied with relatively severe motor dysfunction was first developed. Then clioquinol (CQ), an iron chelator that reduces hydroxyl radical production, was orally administrated. Injection of different doses of Type IV collagenase (1.4 mul 1-200 U/ml) into the left striatum near the IC in Wistar rats showed that injection of 7.5 U/ml collagenase resulted in a small hemorrhoidal lesion near the IC with relatively severe motor dysfunction (IC model). Retrograde labeling of neurons in the sensory-motor cortex and axons in the corticospinal tract using Fluoro-gold (FG) injection into the spinal cord (C3-C4) showed that few labeled neurons in the sensory-motor cortex were detected in the IC model, FG-labeled axons disappeared, and FG-including ED-1-positive cells appeared within 24 hr in the IC. Assessments of behavior and histologic analysis after oral administration of CQ in the IC model indicated that oral administration of CQ prevented a decrease of FG-labeled neurons, and resulted in better motor-function recovery. CQ inhibited hydrogen peroxide-induced cell toxicity in oligodendrocytes in vitro, but not in neurons. Our data suggests that CQ ameliorated motor dysfunction after a small hemorrhage near the IC by a mechanism that is related to reduction of chain-reactive hydroxyl radical production in oligodendrocytes.

Involvement of nitric oxide in 3-nitropropionic acid-induced striatal toxicity in rats.

The roles of nitric oxide (NO) in 3-nitropropionic acid (3-NPA)-induced toxicity were investigated using in vivo and in vitro models. Chronic 3-NPA administration (10 mg/kg) to rats produced selective striatal lesions that were associated with abnormal motor and EMG activities. In these animals, there was loss of glial fibrillary acidic protein (GFAP)-positive cells with extravasation of IgG in the lesion center, although microtubule-associated protein (MAP)-2-positive cells remained, indicating that astrocytes were involved. 3-NPA increased the NO(2)(-)/NO(3)(-) levels in microdialysates obtained from the striatum, thalamus and cerebellum. The basal NO(3)(-) level was much higher in the striatum than in the other areas. The NO(2)(-)/NO(3)(-) levels in the striatum were much higher in animals exhibiting abnormal muscular activity. Expression of endothelial NO synthase (eNOS), but not neuronal NOS (nNOS), was greatly increased in the striatum at 5 h after a second 3-NPA exposure, but not in other areas. In astrocyte cultures, the toxic effects of 3-NPA were associated with corresponding increases in the NO(2)(-) level, and this toxicity was attenuated by hemoglobin (Hb; 20 microM), which quenches NO. The NO(2)(-) generated by 3-NPA, even without cells, was also antagonized by Hb. 3-NPA, S-nitroso-n-acetyl-dl-penicillamine (SNAP) and sodium nitroprusside (SNP) all increased the NO current (detected by NO-sensitive electrodes) in concentration-dependent manners, and Hb significantly attenuated the NO generation induced by 3-NPA, SNAP or SNP. Taken together, these results suggest that 3-NPA generates NO both directly as a donor and indirectly by enhancing NOS expression to produce toxic effects on astrocytes and neuronal toxicity.

Treatment with deferoxamine increases neurons from neural stem/progenitor cells.

Neural transplantation is a promising approach for treating neurodegenerative disease. Neural stem/progenitor cells (NPCs) are self-renewing and multipotent and thus are good candidates for donor cells when they have been clearly defined to differentiate into neurons. As neuronal differentiation follows cell cycle exit, we investigated whether neuron production from NPCs is increased by treatment with cell cycle blockers. NPCs from E12.5 rat ventral mesencephalon were cultured as neurospheres in DMEM/F12 medium containing N2 supplements and bFGF. Treatment of NPCs with deferoxamine, a G1/S phase blocker, increased the number of beta-tubulin III-positive cells after differentiation, concomitant with increases of MAP2 mRNA and protein, and a decrease of GFAP protein. Further, an increase in beta-tubulin III/BrdU double-positive cells and a decrease in GFAP/BrdU double-positive cells were confirmed. In real-time PCR, the expressions of p21(cip1), p27(kip1) and p57(kip2) mRNAs remained unaltered for 8 h after treatment with deferoxamine but were significantly elevated after 1 day. Deferoxamine specifically enhanced the elevation of p27(kip1) mRNA at 1-2 days and the accumulation of p27(kip1) protein at 3 days, along with the activation of neuroD promoter and the elevation of neuroD mRNA. Transfection of p27(kip1) into NPCs induced activation of neuroD promoter and increase of number of beta-tubulin III-positive cells. These data suggest that pretreatment with deferoxamine increases the number of neurons from NPCs related to prolonged p27(kip1) elevation and activation of the neuroD signaling pathway. In this way, regulation of the cell cycle should be a useful first step in engineering NPCs for neural transplantation.

Homeotic factor ATBF1 induces the cell cycle arrest associated with neuronal differentiation.

The present study aimed to elucidate the function of AT motif-binding factor 1 (ATBF1) during neurogenesis in the developing brain and in primary cultures of neuroepithelial cells and cell lines (Neuro 2A and P19 cells). Here, we show that ATBF1 is expressed in the differentiating field in association with the neuronal differentiation markers beta-tubulin and MAP2 in the day E14.5 embryo rat brain, suggesting that it promotes neuronal differentiation. In support of this, we show that ATBF1 suppresses nestin expression, a neural stem cell marker, and activates the promoter of Neurod1 gene, a marker for neuronal differentiation. Furthermore, we show that in Neuro 2A cells, overexpressed ATBF1 localizes predominantly in the nucleus and causes cell cycle arrest. In P19 cells, which formed embryonic bodies in the floating condition, ATBF1 is mainly cytoplasmic and has no effect on the cell cycle. However, the cell cycle was arrested when ATBF1 became nuclear after transfer of P19 cells onto adhesive surfaces or in isolated single cells. The nuclear localization of ATBF1 was suppressed by treatment with caffeine, an inhibitor of PI(3)K-related kinase activity of ataxa-telangiectasia mutated (ATM) gene product. The cytoplasmic localization of ATBF1 in floating/nonadherent cells is due to CRM1-dependent nuclear export of ATBF1. Moreover, in the embryonic brain ATBF1 was expressed in the cytoplasm of proliferating stem cells on the ventricular zone, where cells are present at high density and interact through cell-to-cell contact. Conversely, in the differentiating field, where cell density is low and extracellular matrix is dense, the cell-to-matrix interaction triggered nuclear localization of ATBF1, resulting in the cell cycle arrest. We propose that ATBF1 plays an important role in the nucleus by organizing the neuronal differentiation associated with the cell cycle arrest.

6-Hydroxydopamine-induced lesions in a rat model of hemi-Parkinson's disease monitored by magnetic resonance imaging.

Injection with 6-hydroxydopamine (6-OHDA) into the nigrostriatal pathway results in loss of nigrostriatal dopaminergic neurons, which has been used widely as an animal model of Parkinson's disease. In the present study, location and extent of lesions 1 day after 6-OHDA injections (2, 4, 8, or 16 microg as a free base) in the substantia nigra (SN) were evaluated in rats by T(2)-weighted magnetic resonance imaging (MRI). The changes in MRI were also compared to immunohistochemical and behavioral changes. Hyperintense area in MRI was found at the region corresponding to 6-OHDA injection in a dose-dependent manner and was accompanied by a loss of tyrosine hydroxylase (TH)-positive cells. The shape of hyperintense area in the SN appeared to be composed of two components (i.e., circular and longitudinal regions). Administration of a larger dose of 6-OHDA (8-16 microg) was accompanied by an increase in hyperintense area and loss of TH-positive cells beyond the SN. The hyperintense area was observed on the first and third days after 6-OHDA injection, but the size and intensity declined to near normal levels on the ninth day. Rotational behavior induced by methamphetamine reached maximal levels at 4 microg 6-OHDA, and the behavior was maintained with doses up to 16 microg of 6-OHDA. Intrastriatal injection with 6-OHDA was less effective. These results suggest that MRI provides highly valuable information for verifying the size and location of intended lesions as well as for determining the optimal dose of neurotoxins in individual animals.

Dexamethasone induces different wheel running activity than corticosterone through vasopressin release from the suprachiasmatic nucleus.

During the analysis of wheel running activity, we found that corticosterone (1 mg/100 g BW) injection decreased wheel activity, while dexamethasone (0.1 mg/100 g) increased the activity. To clarify the functional differences between corticosterone and dexamethasone, we measured Arg-vasopressin (AVP) release from the suprachiasmatic nucleus (SCN) slice culture in vitro and AVP coding mRNA in the SCN in vivo. The corticosterone (0.2 and 2 microg/ml, final concentration in medium) decreased the AVP release, while it increased by dexamethasone (0.2 and 2 microg/ml). An AVP mRNA in the SCN was decreased by both corticosterone (1 mg/100 g) and dexamethasone (0.1 mg/100 g). The differences in wheel activity by corticosterone and dexamethasone are discussed from the changes of AVP in the SCN.

High titer retroviral gene transduction to neural progenitor cells for establishment of donor cells for neural transplantation to parkinsonian model rats.

Neural progenitor cells (NPCs) are expected to be useful donor sources for cell transplantation therapy in Parkinson's disease. However, control of the differentiational lineage, especially into dopaminergic neurons, is still difficult. Thus, genetic modification of NPCs to produce l-dopa is potentially useful. The present study prepared high titer retrovirus carrying human tyrosine hydroxylase-1 (HTH-1) gene. HTH-1 gene could be efficiently transduced into NPCs obtained from the E12.5 rat mesencephalon. This retroviral gene transduction caused no apparent changes in survival, proliferation, or differentiation. In vitro, HTH-1 gene-transduced NPCs released little l-dopa and addition of tetrahydrobiopterin, the cofactor of tyrosine hydroxylase, was required for production of l-dopa. In vivo, three of seven hemi-parkinsonian model rats that received HTH-1 gene-transduced donor NPCs achieved functional recovery. High titer retroviral vector for gene transduction could be used to prepare NPCs for transplantation to hemi-parkinsonian model rats. However, functional recovery after transplantation of HTH-1 gene-transduced NPCs was incomplete.

Pleiotrophin mRNA is highly expressed in neural stem (progenitor) cells of mouse ventral mesencephalon and the product promotes production of dopaminergic neurons from embryonic stem cell-derived nestin-positive cells.

Neural stem cells are promising candidates for donor cells in neural transplantation. However, the mechanism by which neural stem cells differentiate into neurons is not well understood. In the present study, a serial analysis of gene expression (SAGE) was carried out to generate a gene file of neural stem (progenitor) cells from the mouse ventral mesencephalon. Among the 15,815 tags investigated, the mRNA of the housekeeping genes (elongation factor 1-alpha, ATPase subunit 6, GAPDH, actin), laminin receptor 1, HSP 70, pleiotrophin, and nestin were highly expressed. Because pleiotrophin (PTN) exhibits mitogenic and trophic effects on neural development and exhibits trophic effects on survival of dopaminergic (DAergic) neurons, we investigated the role of PTN in neurogenesis, especially to DAergic neurons. Here, we show that PTN increased the production of tyrosine hydroxylase (TH)-positive neurons from embryonic stem (ES) cell-derived nestin-positive cells. The expression of Nurr1 mRNA was enhanced by PTN. L-dopa in the culture medium was increased by PTN. This effect was as strong as with sonic hedgehog. Data suggest that PTN mRNA is highly expressed in neural stem (progenitor) cells of mouse ventral mesencephalon, and PTN promotes the production of DAergic neurons from ES cell-derived nestin-positive cells.

Signal transmission from the suprachiasmatic nucleus to the pineal gland via the paraventricular nucleus: analysed from arg-vasopressin peptide, rPer2 mRNA and AVP mRNA changes and pineal AA-NAT mRNA after the melatonin injection during light and dark periods.

Arg-vasopressin (AVP) containing neurons are one of the output paths from the suprachiasmatic nucleus (SCN), the center of the biological clock. AVP mRNA transcription is controlled by a negative feedback loop of clock genes. Circadian rhythm of melatonin release from the pineal gland is regulated by the SCN via the paraventricular nucleus (PVN). To clarify the transduction system of circadian signals from the SCN to the pineal gland, we determined the effects of melatonin injection (1 mg/kg, i.p.) during light and dark periods on Per2 and AVP mRNAs in the SCN and PVN, in addition to arylalkylamine N-acetyltransferase (AA-NAT) and inducible cAMP early repressor (ICER) mRNAs in the pineal gland of rats using RT-PCR. AVP peptide contents were also measured in the SCN and PVN. AVP content in the SCN decreased during the light period, while no changes were observed in the PVN. In the SCN, Per2 mRNA increased during both light and dark periods. In the PVN, Per2 decreased during the light period and increased during the dark period at 180 min after melatonin injection. In the pineal gland, Per2 mRNA increased between 60 and 180 min after the melatonin injection during the light period, while it did not significantly change during the dark period. The AA-NAT mRNA varied similar to the Per2 mRNA changes. These results might suggest that the different responses to melatonin in the pineal gland during the light and dark periods was originated in the changes of Per2 in the PVN via SCN.

Melatonin suppresses cerebral edema caused by middle cerebral artery occlusion/reperfusion in rats assessed by magnetic resonance imaging.

Melatonin, a pineal secretory product synthesized from tryptophan, has been found to be effective against neurotoxicity. The present study was aimed at demonstrating the effectiveness of melatonin in vivo in reducing ischemia-induced cerebral edema using magnetic resonance imaging (MRI). Rats were subjected to middle cerebral artery (MCA) occlusion/reperfusion surgery. Melatonin was administered twice (6.0 mg/kg, p.o.) just prior to 1 hr of MCA occlusion and 1 day after the surgery. T2-weighted multislice spin-echo images were acquired 1 day after the surgery. In the saline-treated control rats, increases in T2-weighted signals (water content) were clearly observed in the striatum and in the cerebral cortex. In the melatonin-treated group, total volume of edema was reduced by 51.6% compared with control group (P < 0.01). The protective effect of melatonin against edema was more clearly observed in the cerebral cortex (reduced by 59.8%, P < 0.01) than in the striatum (reduced by 34.2%, P < 0.05). Edema volume in a coronal slice was the greatest at the level of the bregma. Suppression of cerebral edema by melatonin was more effective posterior than anterior to the bregma. Melatonin appeared to reduce the volume of the edematous sites rather than to shift the signal intensity distribution. The present MRI study clearly demonstrates the effectiveness of melatonin against cerebral edema formation in ischemic animals in vivo, especially in the cerebral cortex. Melatonin may be highly useful in preventing cortical dysfunctions such as motor, sensory, memory, and psychological impairments associated with ischemic stroke.

Regulation mode of evaporative cooling underlying a strategy of the heat-tolerant FOK rat for enduring ambient heat.

Compared with other rat strains, the inbred FOK rat is extremely heat tolerant. This increased heat tolerance is due largely to the animal's enhanced saliva spreading abilities. The aims of the present study were to 1) quantify the heat tolerance capacity of FOK rats and 2) determine the regulatory mode of the enhanced salivary cooling in these animals. Various strains of rats were acutely exposed to heat. In the heat-intolerant strains, saliva spreading was insufficient and the core temperature (Tc) rose rapidly. In contrast, FOK rats maintained an elevated Tc plateau (39.5 +/- 0.7 degrees C) for 5-6 h over a wide range of ambient temperatures (Ta) (37.5-42.5 degrees C). In hot environments the FOK rats secreted copious amounts of saliva and spread it over more than the entire ventral body surface. FOK rats had a low Tc threshold for salivation, and the salivation rate increased linearly in proportion to the Tc deviation from the threshold. No strain difference or temperature effect was observed in the saliva secretion rate from in vitro submandibular glands perfused by sufficient doses of ACh. These results suggest that 1) the ability of FOK rats to maintain a moderate steady-state hyperthermia (39.5 +/- 0.7 degrees C) over a wide Ta range is enabled by a lowered threshold Tc for salivation and functional negative-feedback control of saliva secretion and 2) strain differences in ability to endure heat stress are mainly attributable to changes in the thermoregulatory control system rather than altered secretory abilities of the salivary glands.