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The amount, quality, and diurnal pattern of sleep change greatly during development. Developmental changes of sleep/wake architecture are in a close relationship to brain development. The fragmentation of wake episodes is one of the salient features in the neonatal period, which is also observed in mature animals and human individuals lacking neuropeptide orexin/hypocretin signaling. This raises the possibility that developmental changes of lateral hypothalamic orexin neurons are relevant to the development of sleep/wake architecture. However, little information is available on morphological and physiological features of developing orexin neurons. To address the cellular basis for maturation of the sleep/wake regulatory system, we investigated the functional development of orexin neurons in the lateral hypothalamus. The anatomical development as well as the changes in the electrophysiological characteristics of orexin neurons was examined from embryonic to postnatal stages in orexin-EGFP mice. Prepro-orexin promoter activity was detectable at embryonic day (E) 12.0, followed by expression of orexin A after E14.0. The number of orexin neurons and their membrane capacitance reached similar levels to adults by postnatal day (P) 7, while their membrane potentials, firing rates, and action potential waveforms were developed by P21. The hyperpolarizing effect of serotonin, which is a major inhibitory signal for adult orexin neurons, was detected after E18.0 and matured at P1. These results suggest that the expression of orexin peptides precedes the maturation of electrophysiological activity of orexin neurons. The function of orexin neurons gradually matures by 3 weeks after birth, coinciding with maturation of sleep/wake architecture.

At central glutamatergic synapses, neurotransmitter often saturates postsynaptic AMPA receptors (AMPARs), thereby restricting the dynamic range of synaptic efficacy. Here, using simultaneous pre- and postsynaptic whole-cell recordings, at the calyx of Held synapse of immature rats, we have investigated the mechanism by which transmitter glutamate saturates postsynaptic AMPARs. When we loaded l-glutamate (1–100 mm) into presynaptic terminals, the quantal EPSC (qEPSC) amplitude changed in a concentration-dependent manner. At physiological temperature (36–37°C), the qEPSC amplitude increased when intraterminal l-glutamate concentration was elevated from 1 mm to 10 mm, but it reached a plateau at 10 mm. This plateau persisted after bath-application of the low affinity AMPAR antagonist kynurenate, suggesting that it was caused by saturation of vesicular filling with glutamate rather than by saturation of postsynaptic AMPARs. In contrast to qEPSCs, action potential-evoked EPSCs remained unchanged by increasing intraterminal l-glutamate from 1 mm to 100 mm, even at room temperature, indicating that multi-quantal glutamate saturated postsynaptic AMPARs. This saturation could be relieved by blocking AMPAR desensitization using cyclothiazide (100 μm). The concentration of ambient glutamate in the slice, estimated from NMDA receptor current fluctuations, was 55 nm; this was far below the concentration required for AMPAR desensitization. We conclude that rapid AMPAR desensitization, caused by glutamate released from multiple vesicles during synaptic transmission, underlies postsynaptic AMPAR saturation at this immature calyceal synapse before the onset of hearing.

Paired-pulse facilitation (PPF) and depression (PPD) are forms of short-term plasticity that are generally thought to reflect changes in transmitter release probability. However, desensitization of postsynaptic AMPA receptors (AMPARs) significantly contributes to PPD at many glutamatergic synapses. To clarify the involvement of AMPAR desensitization in synaptic PPD, we compared PPD with AMPAR desensitization, induced by paired-pulse glutamate application in patches excised from postsynaptic cells at the calyx of Held synapse of developing rats. We found that AMPAR desensitization contributed significantly to PPD before the onset of hearing (P10–12), but that its contribution became negligible after hearing onset. During postnatal development (P7–21) the recovery of AMPARs from desensitization became faster. Concomitantly, glutamate sensitivity of AMPAR desensitization declined. Single-cell reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated a developmental decline of GluR1 expression that correlated with speeding of the recovery of AMPARs from desensitization. Transmitter release probability declined during the second postnatal week (P7–14). Manipulation of the extracellular Ca2+/Mg2+ ratio, to match release probability at P7–8 and P13–15 synapses, revealed that the release probability is also an important factor determining the involvement of AMPAR desensitization in PPD. We conclude that the extent of involvement of AMPAR desensitization in short-term synaptic depression is determined by both pre- and postsynaptic mechanisms.

Objective: Peroxisome proliferator–activated receptor (PPAR)-γ ligand, pioglitazone (PIO), is reported to induce edema especially in postmenopausal women. The aim of this study was to elucidate the mechanism for PIO-induced sodium retention and to discover the therapeutic strategy for the PIO-induced changes in renal sodium handling. Methods and Procedures: Zucker obese rats were ovariectomized and were given PIO for 8 weeks. Renal sodium excretion and renal expressions of several enzymes that generate natriuretic substances were examined. Results: Sodium excretion was decreased in ovariectomized (OVX) rats that were given PIO when compared with OVX rats that were not. Urinary nitrites/nitrates excretion was decreased in OVX rats, but was restored by PIO. The expressions of nitric oxide synthases (NOSs) and cyclooxygenases-1/2 (COX-1/2) were unaltered. Similarly, the expression of epithelial sodium channels (ENaC), identified as a PPAR-γ-regulated gene product, was unaffected. In contrast, the expression of cytochrome P450 4A (CYP4A) was increased in OVX rats, and was downregulated by PIO. Co-treatment of OVX rats with PIO and PPAR-α ligand, fenofibrate, a putative inducer of CYP4A, restored not only the impaired sodium excretion but also the downregulated CYP4A expression. Discussion: PIO-induced sodium retention is specific in female OVX rats. Ovariectomy decreases renal NO production, but upregulates renal CYP4A expression to compensate for renal sodium balance. In this setting, PIO downregulates CYP4A, leading to sodium retention. Furthermore, PPAR-α ligands can provide a novel strategy for preventing the PIO-induced sodium retention.

Background and aims We previously demonstrated that acetylcholine elicited nitric oxide-dependent sustained and endothelium-derived hyperpolarizing factor (EDHF)-dependent transient dilation of afferent arterioles. The present study examined whether free radicals interacted with nitric oxide-dependent and EDHF-dependent vasodilator mechanisms in renal microvessels of salt-sensitive hypertension, using the isolated perfused hydronephrotic kidney. Methods and results Following the pretreatment with indomethacin (100 micromol/L) with or without nitro- l-arginine methylester (100 micromol/L), the effect of acetylcholine on noradrenaline (0.3 micromol/L)-induced constriction was evaluated in kidneys from Dahl salt-sensitive and salt-resistant rats. Although acetylcholine (0.01-10 micromol/L) caused dose-dependent and sustained vasodilation of afferent arterioles, attenuated dilation was observed in Dahl salt-sensitive rats, compared with that in salt-resistant rats (58 +/- 4 vs 101 +/- 11% reversal at 10 micromol/L acetylcholine). In the presence of nitro- l-arginine methylester, acetylcholine elicited only transient dilation, with vasodilator response blunted in Dahl salt-sensitive rats (64 +/- 4 vs 100 +/- 9% reversal at 10 micromol/L acetylcholine). Furthermore, chronic (8-10 weeks) treatment with tempol caused partial restoration of acetylcholine (10 micromol/L)-induced sustained arteriolar dilation (71 +/- 3% reversal), but complete reversal of transient dilation (92 +/- 4% reversal). Finally, acute treatment with tempol not only improved the sustained component of the acetylcholine-induced dilation but also restored the impaired responsiveness of transient dilation in Dahl salt-sensitive rats. Conclusion Both sustained (nitric oxide-mediated) and transient (EDHF-mediated) components of acetylcholine-induced afferent arteriolar dilation were attenuated in Dahl salt-sensitive rats, which was attributed, in part, to enhanced free radical activity. A reversal of the sustained and transient vasodilation by the acute tempol treatment suggests possible interaction between free radicals and EDHF as well as increased bioavailability of nitric oxide.

Rho-kinase plays an important role in hypertension and is reported to interfere with insulin signaling through serine phosphorylation of insulin receptor substrate-1 (IRS-1) in cultured vascular smooth muscle cells. We therefore examined the role of Rho-kinase in the development of insulin resistance in Zucker obese rats. In skeletal muscles and aortic tissues of Zucker obese rats, activation of RhoA/Rho-kinase was observed. Long-term Rho-kinase inhibition by 4 wk treatment with fasudil (a Rho-kinase inhibitor) not only reduced blood pressure but corrected glucose and lipid metabolism, with improvement in serine phosphorylation of IRS-1 and insulin signaling in skeletal muscles. Direct visualization of skeletal muscle arterioles with an intravital CCD videomicroscope demonstrated that both acetylcholine- and sodium nitroprusside-induced vasodilations were blunted, which were restored by the fasudil treatment. Furthermore, both fasudil and Y-27632 prevented the serine phosphorylation of IRS-1 induced by insulin and/or tumor necrosis factor-alpha in skeletal muscle cells. Collectively, Rho-kinase is responsible for the impairment of insulin signaling and may constitute a critical mediator linking between metabolic and hemodynamic abnormalities in insulin resistance.

In order to make researches on the effect of exploitation upon fish populations, six populations of guppy were maintained during a period of 331 weeks under conditions of space, light, temperature, pH, and food controlled as much as possible. The six populations were divided into three groups two by two: a control group without exploitation, a 10% rate of exploitation group and a 20 and 33% rate of exploitation group.