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Pregnant women at risk of preterm labor usually receive synthetic glucocorticoids (sGCs) to promote fetal lung development. Emerging evidence indicates that antenatal sGC increases the risk of affective disorders in offspring. Data from animal studies show that such disorders can be transmitted to the second generation. However, the molecular mechanisms underlying the intergenerational effects of prenatal sGC remain largely unknown. Here we show that prenatal dexamethasone (Dex) administration in late pregnancy induced depression-like behavior in first-generation (F1) offspring, which could be transmitted to second-generation (F2) offspring with maternal dependence. Moreover, corticotropin-releasing hormone (CRH) and CRH receptor type 1 (CRHR1) expression in the hippocampus was increased in F1 Dex offspring and F2 offspring from F1 Dex female rats. Administration of a CRHR1 antagonist to newborn F1 Dex offspring alleviated depression-like behavior in these rats at adult. Furthermore, we demonstrated that increased CRHR1 expression in F1 and F2 offspring was associated with hypomethylation of CpG islands in Crhr1 promoter. Our results revealed that prenatal sGC exposure could program Crh and Crhr1 gene expression in hippocampus across 2 generations, thereby leading to depression-like behavior. Our study indicates that prenatal sGC can cause epigenetic instability, which increases the risk of disease development in the offspring's later life.-Xu, Y.-J., Sheng, H., Wu, T.-W., Bao, Q.-Y., Zheng, Y., Zhang, Y.-M., Gong, Y.-X., Lu, J.-Q., You, Z.-D., Xia, Y., Ni, X. CRH/CRHR1 mediates prenatal synthetic glucocorticoid programming of depression-like behavior across 2 generations.

Physical stress is one of the most important factors affecting morphine-induced conditioned place preference (CPP). Convincing evidences demonstrate that physical stress can activate lateral habenular (LHb) neurons. However, the mechanism by which physical stress regulates morphine-induced CPP through LHb remains unclear. In this study, we examined the impact of forced swimming stress (FSS) on morphine-induced CPP in rats. We found that FSS significantly decreased the CPP scores of rats compared with the normal morphine administration rats. Meanwhile, we detected the expression of DARPP-32 phosphorylation (p-DARPP-32) in the nucleus accumbens (NAc), and CaMKII in LHb. The results show that FSS enhanced the expression of CaMΚII in LHb, while it reduced the level of p-DARPP-32 expression in the NAc. Furthermore, by microinjecting AAV-CaMKII or AAV-RNAi into LHb, we demonstrated that an overexpression of CaMKII could reduce morphine-induced CPP scores of rats, while knock-down CaMΚII could restore morphine-induced CPP scores, which were interfered by FSS. In addition, by microinjecting DiI into the ventral tegmental area (VTA) and tail of VTA (tVTA) unilaterally, and an anterograde tracing virus (AAV-CaMKII-mCherry) into LHb unilaterally, we verified the neural projections from LHb to tVTA. Taken together, our findings suggest that FSS could activate LHb neurons through CaMΚII, and inhibit morphine-induced CPP through the LHb-tVTA pathway.

Summary Aims The intravenous anesthetic propofol caused episodic memory impairments in human. We hypothesized propofol caused episodic-like spatial memory retention but not acquisition impairments in rats and rescuing cAMP response element-binding protein (CREB) signaling using selective type IV phosphodiesterase (PDEIV) inhibitor rolipram reversed these effects. Methods Male Sprague-Dawley rats were randomized into four groups: control; propofol (25 mg/kg, intraperitoneal); rolipram; and rolipram + propofol (pretreatment of rolipram 25 min before propofol, 0.3 mg/kg, intraperitoneal). Sedation and motor coordination were evaluated 5, 15, and 25 min after propofol injection. Invisible Morris water maze (MWM) acquisition and probe test (memory retention) were performed 5 min and 24 h after propofol injection. Visible MWM training was simultaneously performed to resist nonspatial effects. Hippocampal CREB signaling was detected 5 min, 50 min, and 24 h after propofol administration. Results Rolipram did not change propofol-induced anesthetic/sedative states or impair motor skills. No difference was found on the latency to the platform during the visible MWM. Propofol impaired spatial memory retention but not acquisition. Rolipram reversed propofol-induced spatial memory impairments and suppression on cAMP levels, CaMKIIα and CREB phosphorylation, brain-derived neurotropic factor (BDNF) and Arc protein expression. Conclusions Propofol caused spatial memory retention impairments but not acquisition inability possibly by inhibiting CREB signaling.

Purpose Effective therapy for visual loss caused by optic nerve injury or diseases has not been achieved even though the optic nerve has the regeneration potential after injury. This study was designed to modify amniotic epithelial cells (AECs) with basic fibroblast growth factor (bFGF) gene, preliminarily investigating its effect on transected optic nerve. Methods A human bFGF gene segment was delivered into rat AECs (AECs/hbFGF) by lentiviral vector, and the gene expression was examined by RT-PCR and ELISA. The AECs/hbFGF and untransfected rat AECs were transplanted into the transected site of the rat optic nerve. At 28 days post transplantation, the survival and migration of the transplanted cells was observed by tracking labeled cells; meanwhile retinal ganglion cells (RGCs) were observed and counted by employing biotin dextran amine (BDA) and Nissl staining. Furthermore, the expression of growth associated protein 43 (GAP-43) within the injury site was examined with immunohistochemical staining. Results The AECs/hbFGF was proven to express bFGF gene and secrete bFGF peptide. Both AECs/hbFGF and AECs could survive and migrate after transplantation. RGCs counting implicated that RGCs numbers of the cell transplantation groups were significantly higher than that of the control group, and the AECs/hbFGF group was significantly higher than that of the AECs group. Moreover GAP-43 integral optical density value in the control group was significantly lower than that of the cell transplantation groups, and the value in the AECs/hbFGF group was significantly higher than that of the AECs group. Conclusions AECs modified with bFGF could reduce RGCs loss and promote expression of GAP-43 in the rat optic nerve transected model, facilitating the process of neural restoration following injury.

The role of central oxytocin in inhibitory action of lithium on the development of morphine dependence was behavioral investigated in rats. Acute lithium could enhance the morphine-induced analgesia in rats with or without chronic morphine treatment; this effect could be inhibited by intraventricular injection of oxytocin antagonist d (CH2)5-Tyr (Me)-[Orn8]-Vasotocin (OVT). Lithium could attenuate naloxone-precipitated withdrawal signs in morphine dependent rats. The reduction of the expression of naloxone-precipitated withdrawal signs by lithium was reversed by ICV of OVT. The lithium significantly inhibited the conditioned place preference (CPP) induced by morphine, which inhibitory action of lithium could also reverse by ICV injection of OVT. These results suggested that lithium might inhibit the physical dependence on morphine as well as psychological dependence in rats, and that this inhibitory effect of lithium on the development of morphine dependence might be associated with oxytocin systems in the central nervous system.

Ultra-high pressure eclogites and granulites both occur in the Dabie Mountains, central China. A garnet porphyroblast from felsic granulite in the Dabie Mountains has been analysed for compositional zoning by electron microprobe. Two segments of the porphyroblast have opposite compositional variations. Segment I (from centre outward 9 mm to analytical point 18) has decreasing XSps and increasing XPyr, while Segment II (from analytical point 18, 1 mm outward to the rim) has increasing XSps and XAlm and decreasing XPyr and XGrs. The compositional zoning in segment I is considered as growth zoning and that in Segment II as diffusive retrograde zoning. Garnet growth zoning records a P–T path prior to the peak granulite metamorphism. The minimum P–T conditions are estimated to be 1.35 GPa and 850 °C for peak metamorphism, based on the highest Mg/(Fe+Mg) composition in the garnet (analytical point 18) and matrix hypersthene, biotite and plagioclase. A symplectitic corona surrounds the porphyroblast and appears to have formed at 0.6 GPa and 700 °C. The well-preserved growth zoning in garnet suggests a short residence time for the granulite at peak metamorphism and thus rapid tectonic uplift history. The P–T path is consistent with that of ultra-high-pressure eclogite in the area. Tectonic movements during a collisional event could have brought both the granulite and the eclogite to their present positions.

To analyze the level of the oxytocin (OT) and the expression of oxytocin receptor (OTR) in males with idiopathic infertility.Sixty-five infertile males aged 20 -45 years were divided according to their semen parameters into an idiopathic oligozoospermia group (OG, n = 20), an idiopathic asthenozoospermia group (AG, n = 25), and an idiopathic oligoasthenozoospermia group (OAG, n = 20). Another twenty 20-45 years old healthy male volunteers with a natural childbearing history were included in the control group (CG). All the subjects were detected for the contents of luteinizing hormone (LH), follicle stimulating hormone (FSH) , testosterone (T) and OT, and analyzed for the expression of OTR by sequencing the functional region of the OTR promoter (OTRP), OTR-mRNA, and OTR-COOH terminus. The gene sequences were compared using DNASTAR-MegAlign, Western blot values changed into enumeration data, and all the data analyzed by one-way ANOVA and Dunnette's multiple range t-test.A significantly lower content of OT was observed in CG ( [79.30 +/- 3.83] pg/ml) than in OG ([118.53 +/- 7.69] pg/ml, AG ([108.81 +/- 5.66] pg/ml) and OAG ([103.71 +/- 4.54] pg/ml) (F(0.05/2[2,82]) = 8.29, P0.01). The content of LH was significantly lower in AG ([4.26 +/- 0.31] IU/L) and OAG ([4.55 +/- 0.40] IU/L) than in OG ([6.77 +/- 0.57] IU/L) and CG ([7.19 +/- 0.50] IU/L) (F(0.05/2 [2,82]) = 11.64, P0.01), and so was the content of FSH in AG ( [5.02 + 0.39] IU/L) than in CG ([8.91 +/- 0.91] IU/L), OG ([11.86 +/- 1.76] IU/L) and OAG ([8.82 +/- 1.03] IU/L) (F(0.05/[2,82]) = 7.22, P0.01). There were no significant differences in the T content among the four groups (F(0.05/2[2,82] = 0.42, P = 0.739). No evident gene mutation was found in OTRP and OTR-mRNA gene sequencing. Human OTRs in the lymphocytes were monomers and oligomers, mostly tetramers and hexamers. There were obviously more monomers in AG (0.41 +/- 0.03) and OAG (0.13 +/- 0.01) than in OG (0.05 +/- 0.004) and CG (0.05 +/- 0.003) (F(0.05/2[2,82]) = 115.50, P0.01), while the number of oligomers was markedly decreased in 20% of the cases in AG.Significant differences in the content of OT and expression of OTR between fertile and infertile men suggested an association of OT with male infertility. The decreased expression of OTR oligomers and increased expression of monomers may be related to idiopathic asthenozoospermia, which has provided a new insight into the pathogenesis and treatment of male infertility.

To investigate the relationship between oxytocin (OT) and male infertility, serum OT baseline concentration and oxytocin receptor (OTR) gene expression in fertile and infertile men were investigated.Twenty obstructive azoospermia patients, twenty five idiopathic asthenozoospermia patients, twenty idiopathic oligozoospermia patients and twenty healthy subjects were taken into consideration. Serum OT baseline concentration was determined by radioimmunoassay. Moreover, serum concentration of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone (T) were determined by chemoluminescence to evaluate the correlation with OT. OTR gene promotor and OTR mRNA expressions were determined by polymerase chain reaction and reverse transcriptase-polymerase chain reaction, respectively. OTR protein expression was also performed by Western Blot.Serum OT baseline concentrations in infertile groups were significantly higher than in fertile group (F0.05/2(2,82) = 8.29, p 0.001). Serum baseline concentration of OT was not correlated with that of LH, FSH and T. There was no significant difference in gene sequences of OTR gene promotor and OTR mRNA when comparing infertile patients with fertile. Human OTR was in the form of oligomers and monomers, and the oligomers were in the majority containing tetramers and hexamers. Monomer expression was significantly higher in idiopathic asthenozoospermia and idiopathic oligozoospermia than that in obstructive azoospermia and control group (F0.05/2(2,82) = 115.50, p 0.001). There was no significant difference in oligomer expression between different groups, but 20% of idiopathic asthenozoospermia cases showed a decrease.Significantly different OT baseline concentrations and OTR expressions between fertile and infertile men strongly suggest that OT/OTR system is likely to be linked with male infertility, providing new insights into the pathogenesis and treatment of male infertility.

Recent evidence suggests that rapid eye movement (REM) sleep deprivation (REMSD) causes learning and memory deficits. However, the mechanism of REMSD-induced memory impairment remains unclear. Calcineurin (CaN) is involved in synaptic plasticity and is known as a negative constraint on learning and memory. Here we report that 72 h REMSD by the modified multiple platform method in rats resulted in spatial memory impairment in the Morris water maze and elevated hippocampal cytosolic CaN activity, both of which were reversed after 18 h sleep recovery. CaN expression in the whole-tissue homogenate of the hippocampus was not altered by REMSD. The results suggest that elevated hippocampal CaN activity is involved in REMSD-induced spatial memory impairment.

Aim: G protein-coupled inwardly rectifying potassium channels (GIRK) are important for neuronal signaling and membrane excitability. In the present study, we intend to find whether GIRK channels express functionally in adult rat dorsal root ganglion (DRG) neurons. Methods: We used RT-PCR to detect mRNA for 4 subunits of GIRK in the adult DRG. The whole-cell patch clamp recording was used to confirm GIRK channels functionally expressed. Results: The mRNA for the 4 subunits of GIRK were detected in the adult DRG. GTP γ S enhanced inwardly rectifying potassium (K + ) currents of the DRG neurons, while Ba 2+ inhibited such currents. Furthermore, the GIRK channels were shown to be coupled to the GABA B receptor, a member of the G protein-coupled receptor family, as baclofen increased the inwardly rectifying K + currents. Conclusion: GIRK channels are expressed and functionally coupled with GABA B receptors in adult rat DRG neurons.

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to beta(2) subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively. (c) 2006 Elsevier Inc. All rights reserved.

To observe the effects of Salvia miltiorrhiza (SM) on levels of neuropeptide Y1-36 and calcitonin gene-related peptide immune reactive substances (ir-NPY, ir-CGRP) in blood plasma and pons-oblongata after hypoxia-ischemic brain injury (HIBI) in neonatal rats.Seven-day old rats were randomized into HIBI group (A), HIBI + SM group (B) and sham operation group(C). And each group was subdivided into 4 subgroups according to the different time after operation. 0.5 ml SM was injected intraperitoneally immediately and every 12 hrs afterwards. Changes of ir-NPY and ir-CGRP levels in plasma and pons-oblongata were observed immediately and 12, 24 and 48 hrs after HIBI by radioimmunoassay.Plasma levels of ir-NPY and ir-CGRP in different times after HIBI were all significantly raised but those in pons-oblongata were either raised or lowered to a certain degree. Part of the elevated ir-NPY could be reversed by SM injection.Central and peripheral neuropeptide Y1-36 and calcitonin gene-related peptide take part in the pathophysiological process of HIBI, SM could partially reverse the abnormal post-HIBI elevation of ir-NPY, which may be one of the pathways of SM in promoting recovery of damaged brain function.

The biotinylated annexin V was generated by genetic engineering method. The encoding region of annexin V was fused with a gene coding for the carboxyl terminal 87 residues of Escherichia coli biotin carboxyl carrier protein. The fused gene was expressed in E.coli and the annexin V was biotinylated in vivo. The biotinylation efficiency was about 60% as detected by HPLC. The biotinylated annexin V was purified by avidin affinity chromatography and used to detect the apoptosis of the neurons induced by morphine.

The expression of G-protein-gated inwardly rectifying K + channels subunits GIRKI and GIRK2 mRNAs in the supraoptic nucleus (SON) was investigated in the rat by in situ hybridization with non-radioactive dig-labeled cRNA probes. Double-labeled methods were used to study the co-localization of GIRK and 2 and oxytocin (OT) and vasopressin (AVP) in the SON. The present study revealed wide and intense expression of GIRKI and GIRK2 mRNAs with high overlapping in the SON, indicating the heterologous channel of GIRK1/GIRK2 was a major functional channel in the SON. Given that 100% of OT-positive and 95% of (AVP)-positive neurons in the SON expressed GIRK1/GIRK2 mRNAs, it is possible that GIRKI/ GIRK2 channel, activated through G-protein coupled receptors, may be involved in the inhibitory regulation of the release of OT and AVP from the SON.

Effect of chronic morphine treatment on oxytocin (OT) release from the long term-cultured organotypic slice of the supraoptic nucleus (SON) was investigated using radioimmunoassay. The co-localization of oxytocin and mu-opioid receptor in neurons within the SON was observed with the double-labeled methods of in situ hybridization combined with immunohistochemistry. After exposure to morphine for 6days, the OT levels in culture media were significantly decreased. Naloxone caused much greater release of OT in chronic morphine treatment group than in controls. Naloxone has no effect after acute morphine treatment. 90% of OT-ir (immunoreactive) neurons expressed mu-opioid receptor mRNA in the SON and 45% of the neurons that expressed mu-opioid receptor mRNAs were OT-ir neurons. These results indicated that the neurons within SON could develop dependence on morphine in vitro, and these effects might be exerted via mu-opioid receptor in oxytocin neurons of the SON.

The changes of oxytocin content and mRNA expression in some nuclei were investigated in morphine-dependent rats using radioimmunoassay (RIA) and in situ hybridization (ISH). After chronic administration of morphine, the oxytocin content in supraoptic nucleus (SON) and nucleus accumbens (NAc) decreased, and increased in the ventral tegment area (VTA) and locus coeruleus (LC), but did not change in other nuclei including the paraventricular nucleus (PVN), lateral septum (SEPTUM), raphe magnus nucleus (NRM) and periaquaductal gray (PAG). In morphine-L dependent rats, naloxone increased the levels of oxytocin in SON and PVN, but decreased that in LC. ISH first showed that chronic morphine treatment inhibited the oxytocin synthesis in SON but not in PVN. The present study demonstrates that chronic morphine treatment alters the brain oxytocin system, suggesting that oxytocin might contribute to the behavioral and neuroendocrine responses to morphine.

Interleukin (IL)-2 is not only an immunoregulatory factor, but also an analgesic molecule. There are distinct domains of immune and analgesic functions in the IL-2 molecule. The analgesic domain is located around the 45th Tyr residue of human IL-2 in tertiary structure. Antiopioid (β-endorphin, Leu-enkephalin, Met-enkephalin and dynorphin A1–13) sera partially neutralized the analgesic activity of IL-2. Monoclonal antibody against the IL-2 receptor α subunit (Tac) could not block the analgesic activity of IL-2. There existed cross-reactivity between IL-2 and antiopioid sera by indirect ELISA. These studies show strong structural and biological similarities between IL-2 and opioid peptides. The tertiary structure around the 45th residue of IL-2 composes the analgesic domain that is similar to that of endogenous opioids. These results are consistent with the hypothesis that multiple domains of cytokines serve as the structural bases for the immunoregulatory and neuroregulatory effects of cytokines.