Projections from lateral habenular to tail of ventral tegmental area contribute to inhibitory effect of stress on morphine-induced conditioned place preference.

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.

CRH/CRHR1 mediates prenatal synthetic glucocorticoid programming of depression-like behavior across 2 generations.

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.

Positive effects of bFGF modified rat amniotic epithelial cells transplantation on transected rat optic nerve.

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.

Rescue of cAMP response element-binding protein signaling reversed spatial memory retention impairments induced by subanesthetic dose of propofol.

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.

[Neuropeptide oxytocin and male infertility].

OBJECTIVE: To analyze the level of the oxytocin (OT) and the expression of oxytocin receptor (OTR) in males with idiopathic infertility. METHODS: 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. RESULTS: 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, P < 0.01). 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, P < 0.01), 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, P < 0.01). 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, P < 0.01), while the number of oligomers was markedly decreased in 20% of the cases in AG. CONCLUSION: 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.

Association between neuropeptide oxytocin and male infertility.

PURPOSE: 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. METHODS AND PATIENTS: 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. RESULTS: 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. CONCLUSIONS: 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.

Calcineurin contributes to spatial memory impairment induced by rapid eye movement sleep deprivation.

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.

G protein-coupled inwardly rectifying potassium channels in dorsal root ganglion neurons.

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. GTPgammaS enhanced inwardly rectifying potassium (K+) currents of the DRG neurons, while Ba2+ 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.

Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells.

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to beta2 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.

[Effect of Salvia miltiorrhiza on neuropeptide Y1-36 and calcitonin gene-related peptide in neonatal rats with hypoxia-ischemic brain injury].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Construction of Biotinylated Annexin V for Detection of Apoptosis.

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.