Histone methylation-mediated microRNA-32-5p down-regulation in sensory neurons regulates pain behaviors via targeting Cav3.2 channels.

microRNA (miRNA)­mediated gene regulation has been studied as a therapeutic approach, but its functional regulatory mechanism in neuropathic pain is not well understood. Here, we identify that miRNA-32-5p (miR-32-5p) is a functional RNA in regulating trigeminal-mediated neuropathic pain. High-throughput sequencing and qPCR analysis showed that miR-32-5p was the most down-regulated miRNA in the injured trigeminal ganglion (TG) of rats. Intra-TG injection of miR-32-5p agomir or overexpression of miR-32-5p by lentiviral delivery in neurons of the injured TG attenuated established trigeminal neuropathic pain. miR-32-5p overexpression did not affect acute physiological pain, while miR-32-5p down-regulation in intact rats was sufficient to cause pain-related behaviors. Nerve injury increased the methylated histone occupancy of binding sites for the transcription factor glucocorticoid receptor in the miR-32-5p promoter region. Inhibition of the enzymes that catalyze H3K9me2 and H3K27me3 restored the expression of miR-32-5p and markedly attenuated pain behaviors. Further, miR-32-5p­targeted Cav3.2 T-type Ca2+ channels and decreased miR-32-5p associated with neuropathic pain caused an increase in Cav3.2 protein expression and T-type channel currents. Conversely, miR-32-5p overexpression in injured TG suppressed the increased expression of Cav3.2 and reversed mechanical allodynia. Together, we conclude that histone methylation-mediated miR-32-5p down-regulation in TG neurons regulates trigeminal neuropathic pain by targeting Cav3.2 channels.

Repeated exposure to propofol in the neonatal period impairs hippocampal synaptic plasticity and the recognition function of rats in adulthood.

Anesthesia of neonates with propofol induces persistent behavioral abnormalities in adulthood. Although propofol-triggered apoptosis of neurons in the developing brain may contribute to the development of cognitive deficits, the mechanism of neurotoxicity induced by neonatal exposure to propofol remains unclear. In this study, the effects of neonatal propofol anesthesia on synaptic plasticity and neurocognitive function were investigated. Postnatal day 7 (PND-7) Sprague-Dawley rats were intraperitoneally injected with fat emulsion or 20, 40 or 60 mg/kg propofol for three consecutive days. The expression of brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrkB) and postsynaptic density protein 95 (PSD-95) in the rat hippocampus at PND-10 and PND-12 was measured by Western blotting. The number of dendritic branches, total dendritic length and dendritic spine density were observed by Golgi-Cox staining 24 h and 72 h after the last propofol administration. Long-term potentiation (LTP) was measured electrophysiologically in hippocampus of PND-60 rats to evaluate the synaptic function. The learning and memory abilities of rats were evaluated by Morris water maze (MWM) experiments, Novel object recognition test (NORT) and Object location test (OLT) at PND-60. Our results showed that neonatal exposure to propofol significantly inhibited the expression of BDNF, TrkB and PSD-95 in the rat hippocampus. The number of dendritic branches, total dendritic length and dendritic spine density of neurons in the rat hippocampus were markedly reduced after neonatal propofol anesthesia. LTP was significantly diminished in hippocampus of PND-60 rats after repeated exposure to propofol in the neonatal period. Morris water maze experiments showed that repeated neonatal exposure to propofol significantly prolonged the escape latency and decreased the time spent in the target quadrant and the number of platform crossings. NORT and OLT showed that repeated neonatal exposure to propofol markedly reduced the Investigation Time for novel object or location. All of the results above indicate that repeated exposure to propofol in the neonatal period can impair hippocampal synaptic plasticity and the recognition function of rats in adulthood.

Electrical stimulation of the superior sagittal sinus suppresses A-type K+ currents and increases P/Q- and T-type Ca2+ currents in rat trigeminal ganglion neurons.

BACKGROUND: Migraine is a debilitating neurological disorder involving abnormal trigeminovascular activation and sensitization. However, the underlying cellular and molecular mechanisms remain unclear. METHODS: A rat model of conscious migraine was established through the electrical stimulation (ES) of the dural mater surrounding the superior sagittal sinus. Using patch clamp recording, immunofluorescent labelling, enzyme-linked immunosorbent assays and western blot analysis, we studied the effects of ES on sensory neuronal excitability and elucidated the underlying mechanisms mediated by voltage-gated ion channels. RESULTS: The calcitonin gene-related peptide (CGRP) level in the jugular vein blood and the number of CGRP-positive neurons in the trigeminal ganglia (TGs) were significantly increased in rats with ES-induced migraine. The application of ES increased actional potential firing in both small-sized IB4-negative (IB4-) and IB4+ TG neurons. No significant changes in voltage-gated Na+ currents were observed in the ES-treated groups. ES robustly suppressed the transient outward K+ current (IA) in both types of TG neurons, while the delayed rectifier K+ current remained unchanged. Immunoblot analysis revealed that the protein expression of Kv4.3 was significantly decreased in the ES-treated groups, while Kv1.4 remained unaffected. Interestingly, ES increased the P/Q-type and T-type Ca2+ currents in small-sized IB4- TG neurons, while there were no significant changes in the IB4+ subpopulation of neurons. CONCLUSION: These results suggest that ES decreases the IA in small-sized TG neurons and increases P/Q- and T-type Ca2+ currents in the IB4- subpopulation of TG neurons, which might contribute to neuronal hyperexcitability in a rat model of ES-induced migraine.

Serotonin type-1D receptor stimulation of A-type K(+) channel decreases membrane excitability through the protein kinase A- and B-Raf-dependent p38 MAPK pathways in mouse trigeminal ganglion neurons.

Although recent studies have implicated serotonin 5-HT1B/D receptors in the nociceptive sensitivity of primary afferent neurons, the underlying molecular and cellular mechanisms remain unclear. In this study, we identified a novel functional role of the 5-HT1D receptor subtype in regulating A-type potassium (K(+)) currents (IA) as well as membrane excitability in small trigeminal ganglion (TG) neurons. We found that the selective activation of 5-HT1D, rather than 5-HT1B, receptors reversibly increased IA, while the sustained delayed rectifier K(+) current was unaffected. The 5-HT1D-mediated IA increase was associated with a depolarizing shift in the voltage dependence of inactivation. Blocking G-protein signaling with pertussis toxin or by intracellular application of a selective antibody raised against Gαo or Gß abolished the 5-HT1D effect on IA. Inhibition of protein kinase A (PKA), but not of phosphatidylinositol 3-kinase or protein kinase C, abolished the 5-HT1D-mediated IA increase. Analysis of phospho-p38 (p-p38) revealed that activation of 5-HT1D, but not 5-HT1B, receptors significantly activated p38, while p-ERK and p-JNK were unaffected. The p38 MAPK inhibitor SB203580, but not its inactive analogue SB202474, and inhibition of B-Raf blocked the 5-HT1D-mediated IA response. Functionally, we observed a significantly decreased action potential firing rate induced by the 5-HT1D receptors; pretreatment with 4-aminopyridine abolished this effect. Taken together, these results suggest that the activation of 5-HT1D receptors selectively enhanced IA via the Gßγ of the Go-protein, PKA, and the sequential B-Raf-dependent p38 MAPK signaling cascade. This 5-HT1D receptor effect may contribute to neuronal hypoexcitability in small TG neurons.

Necrostatin-1 protection of dopaminergic neurons.

Necroptosis is characterized by programmed necrotic cell death and autophagic activation and might be involved in the death process of dopaminergic neurons in Parkinson's disease. We hypothesized that necrostatin-1 could block necroptosis and give protection to dopaminergic neurons. There is likely to be crosstalk between necroptosis and other cell death pathways, such as apoptosis and autophagy. PC12 cells were pretreated with necroststin-1 1 hour before exposure to 6-hydroxydopamine. We examined cell viability, mitochondrial membrane potential and expression patterns of apoptotic and necroptotic death signaling proteins. The results showed that the autophagy/lysosomal pathway is involved in the 6-hydroxydopamine-induced death process of PC12 cells. Mitochondrial disability induced overactive autophagy, increased cathepsin B expression, and diminished Bcl-2 expression. Necrostatin-1 within a certain concentration range (5-30 µM) elevated the viability of PC12 cells, stabilized mitochondrial membrane potential, inhibited excessive autophagy, reduced the expression of LC3-II and cathepsin B, and increased Bcl-2 expression. These findings suggest that necrostatin-1 exerted a protective effect against injury on dopaminergic neurons. Necrostatin-1 interacts with the apoptosis signaling pathway during this process. This pathway could be a new neuroprotective and therapeutic target in Parkinson's disease.

Glial cell-derived neurotrophic factor attenuates neuropathic pain in a mouse model of chronic constriction injury: possible involvement of E-cadherin/p120ctn signaling.

Treating neuropathic pain is a major clinical challenge, and several key molecules associated with nociception have been suggested as potential targets for novel analgesics. Many studies have reported the anti-nociceptive effects of glial cell-derived neurotrophic factor (GDNF), but the underlying mechanism remains largely unknown. The present study was performed to assess the effects of GDNF in a mouse model of chronic constriction injury (CCI)-induced neuropathic pain. We also determined the potential role of E-cadherin/p120 catenin (p120ctn) signaling in these effects. Mice received an intrathecal acute injection of PBS, GDNF, and DECMA-1 (an E-cadherin functional blocking antibody) or a combination of DECMA-1 with GDNF on the testing days. Our results demonstrated that CCI caused a rapid decrease in E-cadherin and membrane-associated p120ctn in the spinal dorsal horn. Together, these data demonstrated that E-cadherin-associated p120ctn was upregulated by GDNF and that this upregulation was inhibited by pre-treatment with DECMA-1. Moreover, DECMA-1 significantly inhibited the effect of GDNF on thermal hyperalgesia. These data suggest that GDNF might have a therapeutic potential for the treatment of CCI-induced neuropathic pain and that the E-cadherin/p120ctn might play a role in GDNF-induced attenuation of thermal hyperalgesia.

NCAM signaling mediates the effects of GDNF on chronic morphine-induced neuroadaptations.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for midbrain dopamine (DA) neurons, while the DA neurons in the ventral tegmental area (VTA) is a crucial part of the neural circuits associated with drug addiction. Recently, more and more evidence suggests that GDNF plays an important role in negatively regulating the neuroadaptations induced by chronic exposure to drugs, which was thought to be the neurobiological basis of drug addiction, but the underlying mechanism is still unknown. More recently, the neural cell adhesion molecule (NCAM), which plays an important role in the process of neural plasticity, has been identified as an alternative signaling receptor for GDNF. The purpose of this study was to investigate whether NCAM was involved in the effects of GDNF on the neuroadaptations induced by chronic morphine exposure. Immunostaining results showed that NCAM was widely expressed in the VTA of rats, including all the DA neurons. The results also showed that the phosphorylation of NCAM-associated FAK, but not the total NCAM, was upregulated by GDNF, and this upregulation was inhibited by pre-treatment with the NCAM function-blocking antibody. Moreover, pre-treatment with the antibody could antagonize the effect of GDNF on inhibiting the neuroadaptations induced by chronic morphine exposure, including the decreases of the number and length of neurites and the size of cell bodies of VTA dopamine neurons, as well as the increase of tyrosine hydroxylase in the VTA dopamine neurons. These results suggest that NCAM signaling is involved in the negative regulatory effects of GDNF on chronic morphine-induced neuroadaptations.

[Short-term curative efficacy of autologous cytokine induced killer cells combined with low-dose IL-2 regimen containing immune enhancement by thymic peptide in elderly patients with B-cell chronic lymphocytic leukemia].

This study was purposed to evaluate the safety and curative effect of autologous cytokine induced killer cells (CIK) combined with low-dose IL-2 regimen containing immune enhancement of thymic peptide on elderly patients with B-cell chronic lymphocytic leukemia (B-CLL). Thymic peptide α1 was subcutaneously given as the immunoenhancement agent at a dose of 1.6 mg/d, 14 days as one cycle. Peripheral blood mononuclear cells (PBMNC) from 5 patients with B-CLL were isolated once a week to induce ex vivo CIK cells through culture in the context of interferon (IFN)-γ, interleukin (IL)-2 and anti-CD3 monoclonal antibody. The PBMNC were separated from patients before and after 14 days as one cycle of thymic peptide α1 administration. Parameters of amplification ability, effector cells quantity, lymphocyte subgroups percentage and antitumor cytotoxicity were compared before and after thymic peptide administration. The 5 patients were treated with CIK cells combined with low-dose IL-2 regimen immediately after injection of thymic peptide α1. The CIK cells plus low-dose IL-2 regimen containing thymic peptide enhancement was defined as: thymic peptide α1 1.6 mg/d was subcutaneously administered once every other day; (4 - 6) ×10(9) of CIK cells were transfused followed by IL-2 subcutaneous administration of 1 mU/d on days 1-10, 28 days as one cycle. Clinical evaluation parameters including cellular immunity function, CLL related biomarkers, disease state and infectious frequency and degree were investigated before and after CIK cells infusion puls IL-2. The results showed that the amount of amplified CIK cells, the percentage and amplification times of effector cells and antitumor cytotoxicity more significantly increased after thymic peptide α1 treatment than before its use (P < 0.05). The total 46 cycles of CIK cells infusion plus IL-2 were completed in the 5 CLL patients. No adverse reaction was observed. After treatment of CIK cells plus IL-2, the general conditions of 5 CLL patients were to different extent improved. Simultaneously, percentages of CD3(+), CD3(+)CD8(+), and CD3(+)CD56(+) cells in peripheral blood remarked by raised (P < 0.05), the serum level of ß2 microglobulin was significantly declined (P < 0.05), and the frequency and degree of infection was also decreased (P < 0.05). Following CIK cells plus IL-2 therapy, the transformation of disease state from partial remission (PR) to complete remission was seen in 3 patients, from stable disease (SD) to PR in 1 patient, and from progress of disease to SD in 1 patient. It is concluded that the regimen of autologous CIK cells combined with low-dose IL-2 containing immune enhancement of thymic peptide is safety and effective for the treatment of elderly patients with B-CLL.

A new alternative NF-κB pathway mediated the neuroprotection of GDNF on 6-OHDA-induced DA neurons neurotoxicity.

Glial cell line-derived neurotrophic factor (GDNF) is a potent protective factor for dopaminergic (DA) neurons, but the signaling mechanisms underlying the effect of GDNF on these neurons remain obscure. Here, both our in vivo and in vitro studies demonstrate that the majority of DA neurons express the NF-κB-inducing kinase (NIK), which is the essential kinase for mediating activation of the new alternative NF-κB signaling pathway. Additionally, we also show that GDNF induced the time/dose-dependent phosphorylation of IκB kinase α (IKKα) and p100, facilitated the processing of p100 to p52 and accelerated the translocation of NF-κB dimmers into the nuclei of DA neurons. We furtherly found that the dimmer which translocate into the nucleus was RelA/p52 not RelB/p52. Meanwhile, the attenuation of 6-OHDA-induced DA neuronal apoptosis due to GDNF was reversed subsequent to the inhibition of p100 expression by RNAi while the neuroprotective effect of GDNF on injured DA neurons was strengthened by the overexpression of p100. Our data, therefore, indicate that a new alternative NF-κB signaling pathway, which was not the classic pathway but different from the non-canonical pathway, exists in DA neurons and mediates the neuroprotective effect of GDNF on these neurons.

Genetic diversity of Newcastle disease viruses isolated from domestic poultry species in Eastern China during 2005-2008.

Seventy-nine Newcastle disease viruses (NDV) isolated from clinical specimens of different poultry species including chickens, pigeons (Columba livia), geese and ostriches in Eastern China during 2005-2008 were characterized biologically and phylogenetically. The results showed genetic diversity of these viruses: three class I viruses and one genotype I and 12 genotype II viruses of class II circulating in chickens were avirulent; four genotype VIb viruses isolated from pigeons were moderately virulent; and two genotype III viruses and 57 genotype VIId viruses were highly virulent. The three class I viruses were further classified as genotypes 2 and 3. The very high F protein sequence identity of one genotype I virus with strain Queensland V4 and 12 genotype II viruses with strain La Sota indicated that these viruses originated from the two vaccine strains. Two genotype III viruses shared greater than 99% sequence identity with the moderately virulent vaccine strain Mukteswar but exhibited significantly higher virulence, suggesting that they evolved from the vaccine virus and that the Mukteswar vaccine should be banned in China. Fifty-seven of the 63 virulent NDVs in this study belonged to genotype VIId, indicating its predominance in Eastern China. Genotype VIId viruses could be further classified into two subgroups. Four of the five NDVs isolated from pigeons belonged to genotype VIb, indicating its host-specific preference. Both the genotype VIb and VIId NDVs showed low amino acid similarity to the vaccine strains currently used in China, implying the urgent need to develop better vaccines against the most prevalent NDVs in China.

[Genome sequencing and genetic analysis of a natural reassortant H5N1 subtype avian influenza virus possessing H9N2 internal genes].

Abstract:One H5N1 subtype avian influenza virus, A/duck/Shandong/009/2008 (Dk/SD/009/08), was isolated from apparently healthy domestic ducks in some live bird market in East China during our epidemiological surveillance. To investigate the genetic composition, Dk/SD/009/08 was subjected to genome sequencing. The amino acid motif of cleavage site was "PLRERRRK-R/GL", which was consistent with the characterization of the HPAIV. According to the newest unified nomenclature system of H5N1, Dk/SD/ 009/08 was classified into Clade 2.3.4. The BLAST results showed that four gene segments (HA, NA, NP and NS) had the highest nucleotide identities with H5N1 subtype AIVs whereas the remaining four (PB2, PB1, PA and M) displayed the closest relationship with H9N2 subtype. Therefore, Dk/SD/009/08 might be a natural reassortant virus. The phylogenetic analysis further indicated that G1-like H9N2 subtype AIVs which was prevalent mainly in quails of Southern China might provide the internal genes for Dk/ SD/009/08.

Calbindin-D28K expression induced by glial cell line-derived neurotrophic factor in substantia nigra neurons dependent on PI3K/Akt/NF-kappaB signaling pathway.

Calbindin-D28K is a calcium-binding protein in neuronal cytoplasm, which has the capability to protect neurons from degeneration. It was reported that glial cell line-derived neurotrophic factor (GDNF) increased calbindin-D28K expression in dopaminergic neurons in vitro. It was observed in our research that GDNF also enhanced the expression of calbindin-D28K in adult rat substantia nigra neurons in vivo. To investigate the intracellular signaling pathways underlying the calbindin-D28K expression induced by GDNF, immunoblot and immunoprecipitation analyses were performed in our present study. Our results showed that injection of GDNF alone into substantia nigra of an adult rat brain increased the calbindin-D28K expression; meanwhile, the phosphorylation level of protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) increased. However, the calbindin-D28K expression induced by GDNF was specifically blocked by the inhibitor of phosphatidylinositol 3-kinase (PI3K), but the inhibitor of ERK1/2 did not block the calbindin-D28K expression. Furthermore, GDNF administration also caused the nuclear factor kappaB (NF-kappaB/p65), to translocate from cytoplasm into the nucleus, and the inhibitor of PI3K effectively blocked the translocation. Immunoprecipitation assay results further demonstrated that it was the p65/p52 complex of NF-kappaB, rather than the p65/p50 complex that translocated into the neuronal nucleus. The calbindin-D28K expression induced by GDNF was also inhibited when the NF-kappaB signaling pathway was blocked by Helenalin. These results described a novel mechanism by which the activation of PI3K/Akt-->NF-kappaB (p65/p52) signaling pathway could play a role in the calbindin-D28K expression induced by GDNF.

Involvement of NCAM in the effects of GDNF on the neurite outgrowth in the dopamine neurons.

Glial cell line-derived neurotrophic factor (GDNF) exerts its biological effects via a multi-component receptor system including the ligand binding receptor--GDNF family receptor-alpha1 (GFRalpha1) and the signaling receptor--RET tyrosine kinase. Recently, the neural cell adhesion molecule (NCAM) has been identified as an alternative signaling receptor for GDNF. The purpose of this study was to investigate whether NCAM could mediate the protective effect of GDNF on injured dopamine (DA) neurons and to determine which cytoplasmic signal molecule associated with NCAM was activated while GDNF performing this effect. The results showed that the phosphorylation of NCAM-associated Fyn was upregulated with GDNF treatment, and this upregulation was inhibited by pre-treatment with the NCAM function-blocking antibody. Moreover, pre-treatment with the antibody could abolish the effect of GDNF on promoting the neurite outgrowth of these DA neurons, except for the effect of GDNF on promoting the expression of tyrosine hydroxylase (TH) in these DA neurons. These results suggest that NCAM is involved in the promotive effect of GDNF on the neurite outgrowth in lesioned DA neurons, but not involved in the promotive effect of GDNF on TH expression in these neurons.

Integrin beta1 is involved in the signaling of glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for the substantia nigra (SN) dopamine (DA) neurons. The transmembrane signaling of GDNF is mediated by a unique receptor system, including the ligand binding receptor GDNF family receptor alpha (GFRalpha) and the transmembrane signaling receptor Ret or neural cell adhesion molecule-140 (NCAM-140). Here, we found that another transmembrane cell adhesion molecule, integrin, a heterodimer consisting of alpha and beta subunits, also mediates the transmembrane signaling of GDNF. The results showed that the level of phosphorylated Src homology 2 domain containing (Shc), which was associated with the cytoplasmic domain of integrin beta1, increased after GDNF administration. Coimmunoprecipitation analysis demonstrated that integrin beta1 could form a complex with GFRalphal. The simulation of molecular modeling showed that four H-bonds were formed between integrin beta1 and GFRalpha. These data indicate that integrin beta1 is involved in the transmembrane signaling of GDNF and suggest that integrin beta1 may be an alternative signaling receptor for GDNF.

Role of PI3-K/Akt pathway and its effect on glial cell line-derived neurotrophic factor in midbrain dopamine cells.

AIM: To explore the intracellular mechanisms underlying the survival/differentiation effect of the glial cell line-derived neurotrophic factor (GDNF) on dopamine (DA) cells. METHODS: Midbrain slice culture and primary cell culture were established, and the cultures were divided into 3 groups: control group, GDNF group, and the phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) pathway-inhibited group. Then the expression of tyrosine hydroxylase (TH) was detected by immunostaining as well as Western blotting. RESULTS: GDNF treatment induced an increase in the number of TH-immunoreactive (ir) cells and the neurite number of TH-ir cells, as well as in the level of TH expression in cultures (Number of TH-ir cells in the slice culture: control group, 8.76+/-0.75; GDNF group, 18.63+/-0.95. Number of TH-ir cells and neurite number of TH-ir cells in cell culture: control group, 3.65+/-0.88 and 2.49+/-0.42; GDNF group, 6.01+/-0.43 and 4.89+/-0.46). Meanwhile, the stimulation of cultured cells with GDNF increased the phosphorylation of Akt, which is a downstream effector of PI3-K/Akt. The effects of GDNF were specifically blocked by the inhibitor of the PI3-K/Akt pathway, wortmannin (Number of TH-ir cells in slice culture: PI3-K/Akt pathway-inhibited group, 6.98+/-0.58. Number of TH-ir cells and neurite number of TH-ir cells in cell culture: PI3-K/Akt pathway-inhibited group, 3.79+/-0.62 and 2.50+/-0.25, respectively). CONCLUSION: The PI3-K/Akt pathway mediates the survival/differentiation effect of GDNF on DA cells.

The distributions and signaling directions of the cerebrospinal fluid contacting neurons in the parenchyma of a rat brain.

Many studies have been made on the distributions of CSF contacting neurons (CSF-CNs) in the parenchyma of the brain with horseradish peroxidase (HRP) or autoradiographics. A significant amount of data has shown that both HRP and autoradiographical substances could pass freely through the spaces of ependyma into the parenchyma of the brain. It is therefore possible that the results were not exact. We found that CB-HRP was a dependable tracer to CSF-CNs and studied the distributions and the signaling directions of cerebrospinal fluid contacting neurons (CSF-CNs) in the parenchyma of the brain with the cholera toxin subunit B with horseradish peroxidase (CB-HRP) tracing combined with transmission electron microscopy. The results were as follows: (1) CSF contacting tanycytes existed not only in the wall of the third ventricle (3V), but also in the walls of the lateral ventricle (LV), the fourth ventricle (4V) and the central canal (CC) of the spinal cord. (2) Some CSF contacting glia cells were observed in the lateral septal nucleus (LS). (3)The distal CSF-CNs in the parenchyma were found in LS, the anterodorsal thalamic nucleus (AD), the supramammillary nucleus (SuM), the dorsal raphe nucleus (DR), the floor of 4V and the lateral superior olive (LSO), but they were mainly found in DR and divided into groups A and B. (4) Axon terminals labeled by CB-HRP were found in the cavity of the brain ventricle. (5) The synaptic relationships between the neurons were labeled by CB-HRP in DR and no-labeled by CB-HRP in the parenchyma. Both synapses Gray I and II were found. It was significant that the presynaptic elements were formed by the neurons no-labeled CB-HRP and the postsynaptic elements labeled CB-HRP. Our results suggested firstly that the signaling directions of CSF-CNs in DR were only from the parenchyma to CSF.

Visual gene diagnosis of HBV and HCV based on nanoparticle probe amplification and silver staining enhancement.

A visual gene-detecting technique using nanoparticle-supported gene probes is described. With the aid of gold nanoparticle-supported 3'-end-mercapto-derivatized oligonucleotide serving as detection probe, and 5'-end -amino-derivatized oligonucleotide immobilized on glass surface acting as capturing probe, target DNA was detected visually by sandwich hybridization based on highly sensitive "nano-amplification" and silver staining. Different genotypes of Hepatitis B and C viruses in the serum samples from infected patients were detected using home-made HBV, HCV, and HBV/HCV gene chips by the gold/silver nanoparticle staining amplification method. The present visual gene-detecting technique may avoid limitations with the reported methods, for its high sensitivity, good specificity, simplicity, speed, and cheapness. This technique has potential applications in many fields, especially in multi-gene detection gene chips coupled with the detection will find applications in clinic. Additionally, resonance Rayleigh light scattering (RLS) spectroscopy is used, for the first time, to judge and monitor the immobilization of gene probes on gold nanoparticle surfaces.