Tac1-Expressing Neurons in the Periaqueductal Gray Facilitate the Itch-Scratching Cycle via Descending Regulation

1. Download high-res image (244KB)
2. Download full-size image

     

Characterization of Basal and Morphine-Induced Histamine Release in the Rat Periaqueductal Gray

Abstract: Previous studies have shown that antinociceptive doses of systemic morphine increase extracellular histamine (HA) levels in the rat periaqueductal gray (PAG), although the cellular origin of basal and morphine-induced HA release in the PAG is unknown. Treatment with α-fluoromethylhistidine (FMH; 100 mg/kg, i.p.), the irreversible inhibitor of histidine decarboxylase, decreased basal HA release by a maximum of 80% and prevented morphine-induced HA release in the PAG. In addition, perfusion of this area with the sodium channel blocker tetrodotoxin (10⁻⁶ M ) decreased basal HA release by a maximum of 57% from baseline levels. When the perfusion medium was modified by substitution of magnesium for calcium, extracellular HA levels in the PAG decreased by a maximum of 72%, and morphine-induced HA release was prevented. Thioperamide (5 mg/kg, i.p.), an H₃ antagonist, increased HA release in the PAG to a maximum of 249% within the first 30–60-min period. Taken together, these results suggest that basal and morphine-induced HA release in the rat PAG have a neuronal origin.     

Opposite Effects of Neuropeptide FF on Central Antinociception Induced by Endomorphin-1 and Endomorphin-2 in Mice

Neuropeptide FF (NPFF) is known to be an endogenous opioid-modulating peptide. Nevertheless, very few researches focused on the interaction between NPFF and endogenous opioid peptides. In the present study, we have investigated the effects of NPFF system on the supraspinal antinociceptive effects induced by the endogenous µ-opioid receptor agonists, endomorphin-1 (EM-1) and endomorphin-2 (EM-2). In the mouse tail-flick assay, intracerebroventricular injection of EM-1 induced antinociception via µ-opioid receptor while the antinociception of intracerebroventricular injected EM-2 was mediated by both µ- and κ-opioid receptors. In addition, central administration of NPFF significantly reduced EM-1-induced central antinociception, but enhanced EM-2-induced central antinociception. The results using the selective NPFF₁ and NPFF₂ receptor agonists indicated that the EM-1-modulating action of NPFF was mainly mediated by NPFF₂ receptor, while NPFF potentiated EM-2-induecd antinociception via both NPFF₁ and NPFF₂ receptors. To further investigate the roles of µ- and κ-opioid systems in the opposite effects of NPFF on central antinociception of endomprphins, the µ- and κ-opioid receptors selective agonists DAMGO and {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593, respectively, were used. Our results showed that NPFF could reduce the central antinociception of DAMGO via NPFF₂ receptor and enhance the central antinociception of {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 via both NPFF₁ and NPFF₂ receptors. Taken together, our data demonstrate that NPFF exerts opposite effects on central antinociception of endomorphins and provide the first evidence that NPFF potentiate antinociception of EM-2, which might result from the interaction between NPFF and κ-opioid systems.     

Functionally Selective Signaling for Morphine and Fentanyl Antinociception and Tolerance Mediated by the Rat Periaqueductal Gray

Functionally selective signaling appears to contribute to the variability in mechanisms that underlie tolerance to the antinociceptive effects of opioids. The present study tested this hypothesis by examining the contribution of G protein-coupled receptor kinase (GRK)/Protein kinase C (PKC) and C-Jun N-terminal kinase (JNK) activation on both the expression and development of tolerance to morphine and fentanyl microinjected into the ventrolateral periaqueductal gray of the rat. Microinjection of morphine or fentanyl into the periaqueductal gray produced a dose-dependent increase in hot plate latency. Microinjection of the non-specific GRK/PKC inhibitor Ro 32-0432 into the periaqueductal gray to block mu-opioid receptor phosphorylation enhanced the antinociceptive effect of morphine but had no effect on fentanyl antinociception. Microinjection of the JNK inhibitor SP600125 had no effect on morphine or fentanyl antinociception, but blocked the expression of tolerance to repeated morphine microinjections. In contrast, a microinjection of Ro 32-0432 blocked the expression of fentanyl, but not morphine tolerance. Repeated microinjections of Ro 32-0432 blocked the development of morphine tolerance and inhibited fentanyl antinociception whether rats were tolerant or not. Repeated microinjections of SP600125 into the periaqueductal gray blocked the development of tolerance to both morphine and fentanyl microinjections. These data demonstrate that the signaling molecules that contribute to tolerance vary depending on the opioid and methodology used to assess tolerance (expression vs. development of tolerance). This signaling difference is especially clear for the expression of tolerance in which JNK contributes to morphine tolerance and GRK/PKC contributes to fentanyl tolerance.     

大鼠延髓腹外侧表面呼吸调节化学感受区的感受机制分析

用免疫组化（ＨＲＰ）、Ｈ＾＋表面透入、核团微量注射、微电泳及损毁等方法探讨了延髓腹侧表面中枢化学感受机制。结果表明它与其浅层核团：斜方体核、外周橄榄腹外侧核（ＬＶＰＯ）、斜方体后核、巨细胞旁外侧核和外侧网状核等有神经结构联系。表面Ｈ＾＋可能被上述核团的突起或胞体感受。非呼吸相关神经元（ＬＶＰＯ）与呼吸相关神经元,同样可能参与中枢化学感受而调节呼吸活动。     

Capturing the Mechanism Underlying TOP mRNA Binding to LARP1

1. Download : Download high-res image (163KB)
2. Download : Download full-size image

     

rBTI及其突变体对C. elegans寿命的影响

重组荞麦胰蛋白酶抑制剂（recombinant buckwheat trypsin inhibitor,rBTI）是一种来源于荞麦Potato Ⅰ抑制剂家族的丝氨酸蛋白酶抑制剂,具有很好的生物活性及功能。先前的研究表明,rBTI在秀丽隐杆线虫（Caenorhabditis elegans）中具有很好的延长寿命的性质,但其具体的作用机制还不太清楚。本文的研究证明,rBTI能够调节转录因子DAF-16的转录活性,进而影响线虫的寿命,且该性质与其胰蛋白酶抑制活性密切相关。通过定点突变技术,分别对rBTI的45位、53位和44位氨基酸活性位点进行突变,获得了4种不同胰蛋白酶抑制活性的rBTI突变体,分别命名为rBTI-R45A,rBTI-R45F,rBTI-W53R和rBTI-P44T。经典模式生物秀丽隐杆线虫寿命检测实验显示,野生型rBTI可以明显延长C.elegans的寿命,且在0～10 μmol/L 范围内具有浓度依赖性。和未处理对照组相比,10 μmol/L 野生型rBTI延长寿命幅度可达到14.5%,但是突变体rBTI-R45A,rBTI-R45F和rBTI W53R均不同程度失去了延长寿命的功能。利用荧光显微观察及qRT-PCR等方法进一步研究发现,野生型rBTI 可增强寿命调控转录因子DAF-16的转录活性。与寿命检测实验结果一致,4种 rBTI突变体均不能使DAF-16转录活性增强。上述结果表明,在C.elegans中,rBTI可增强长寿因子DAF 16的转录活性,进而延长虫体寿命,且该功能的发挥依赖于其适当的胰蛋白酶抑制活性。本文的结果为进一步研究开发rBTI的功能提供了实验支持和理论基础。     

Effect of Dorsal and Ventral Hippocampal Lesions on Contextual Fear Conditioning and Unconditioned Defensive Behavior Induced by Electrical Stimulation of the Dorsal Periaqueductal Gray

The dorsal (DH) and ventral (VH) subregions of the hippocampus are involved in contextual fear conditioning. However, it is still unknown whether these two brain areas also play a role in defensive behavior induced by electrical stimulation of the dorsal periaqueductal gray (dPAG). In the present study, rats were implanted with electrodes into the dPAG to determine freezing and escape response thresholds after sham or bilateral electrolytic lesions of the DH or VH. The duration of freezing behavior that outlasted electrical stimulation of the dPAG was also measured. The next day, these animals were subjected to contextual fear conditioning using footshock as an unconditioned stimulus. Electrolytic lesions of the DH and VH impaired contextual fear conditioning. Only VH lesions disrupted conditioned freezing immediately after footshock and increased the thresholds of aversive freezing and escape responses to dPAG electrical stimulation. Neither DH nor VH lesions disrupted post-dPAG stimulation freezing. These results indicate that the VH but not DH plays an important role in aversively defensive behavior induced by dPAG electrical stimulation. Interpretations of these findings should be made with caution because of the fact that a non-fiber-sparing lesion method was employed.     

Persistent Activity of Metabotropic Glutamate Receptor 5 in the Periaqueductal Gray Constrains Emergence of Chronic Neuropathic Pain

1. Download : Download high-res image (166KB)
2. Download : Download full-size image

     

The Molecular Mechanism Underlying Mechanical Anisotropy of the Protein GB1

Mechanical responses of elastic proteins are crucial for their biological function and nanotechnological use. Loading direction has been identified as one key determinant for the mechanical responses of proteins. However, it is not clear how a change in pulling direction changes the mechanical unfolding mechanism of the protein. Here, we combine protein engineering, single-molecule force spectroscopy, and steered molecular dynamics simulations to systematically investigate the mechanical response of a small globular protein GB1. Force versus extension profiles from both experiments and simulations reveal marked mechanical anisotropy of GB1. Using native contact analysis, we relate the mechanically robust shearing geometry with concurrent rupture of native contacts. This clearly contrasts the sequential rupture observed in simulations for the mechanically labile peeling geometry. Moreover, we identify multiple distinct mechanical unfolding pathways in two loading directions. Implications of such diverse unfolding mechanisms are discussed. Our results may also provide some insights for designing elastomeric proteins with tailored mechanical properties.     

Rostrocaudal Somatotopy in the Neural Connections Between the Lateral Hypothalamus and the Dorsal Periaqueductal Gray of the Rat Brain

Summary 1. The lateral hypothalamus (LH) and the dorsal periaqueductal gray area (dPAG) are two important brain structures involved in central cardiovascular control.2. In the present study, we searched for possible rostrocaudal somatotopy in the neural connections from the three subdivisions of the LH (anterior—LHa; tuberal—LHt and posterior—LHp) to the different rostrocaudal portions of the dPAG.3. The bidirectional neuronal tracer biotinylated-dextran-amine (BDA) was microinjected into different rostrocaudal coordinates of the dPAG (AP 3.4–2.7 mm) of male Wistar rats. One week later, animals were sacrificed and brain slices were processed and analyzed to detect neuronal efferent projections from the LH to the dPAG.4. Neuronal cell body staining was observed along all the rostrocaudal axis of the LH when BDA was microinjected in more rostral dPAG coordinates. When the BDA was microinjected into more caudal dPAG regions, labeled neurons were observed only in the caudal portion of the LH.5. Efferent projections from the LHa were directed only to the rostral portion of the dPAG. Projections from the rostral and medial portions of the LHt were also directed to the rostral dPAG, whereas both rostral and caudal dPAG received projections from the caudal portion of the LHt. Efferent projections from the anterior portion of the LHp were directed to both rostral and caudal dPAG, whereas projections from the caudal LHp were only directed to the rostral portion of the dPAG.6. The results suggest a somatotopic correlation in LH projections to the dPAG with main connections to the rostral dPAG, which are efferent from the three divisions of the LH. More caudal regions of the dPAG received afferents only from posterior sites in the LH.7. Moreover, the results point out to extensive and complex neural somatotopic projections from all LH subdivisions to different rostrocaudal portions of the dPAG, reinforcing the idea of significant functional interactions between the brain structures.     

NAAG Peptidase Inhibition in the Periaqueductal Gray and Rostral Ventromedial Medulla Reduces Flinching in the Formalin Model of Inflammation

ABSTRACT: BACKGROUND: Metabotropic glutamate receptors (mGluRs) have been identified as significant analgesic targets. Systemic treatments with inhibitors of the enzymes that inactivate the peptide transmitter N-acetylaspartylglutamate (NAAG), an mGluR3 agonist, have an analgesia-like effect in rat models of inflammatory and neuropathic pain. The goal of this study was to begin defining locations within the central pain pathway at which NAAG activation of its receptor mediates this effect. RESULTS: NAAG immunoreactivity was found in neurons in two brain regions that mediate nociceptive processing, the periaqueductal gray (PAG) and the rostral ventromedial medulla (RVM). Microinjection of the NAAG peptidase inhibitor ZJ43 into the PAG contralateral, but not ipsilateral, to the formalin injected footpad reduced the rapid and slow phases of the nociceptive response in a dose-dependent manner. ZJ43 injected into the RVM also reduced the rapid and slow phase of the response. The group II mGluR antagonist LY341495 blocked these effects of ZJ43 on the PAG and RVM. NAAG peptidase inhibition in the PAG and RVM did not affect the thermal withdrawal response in the hot plate test. Footpad inflammation also induced a significant increase in glutamate release in the PAG. Systemic injection of ZJ43 increased NAAG levels in the PAG and RVM and blocked the inflammation-induced increase in glutamate release in the PAG. CONCLUSION: These data demonstrate a behavioral and neurochemical role for NAAG in the PAG and RVM in regulating the spinal motor response to inflammation and that NAAG peptidase inhibition has potential as an approach to treating inflammatory pain via either the ascending (PAG) and/or the descending pain pathways (PAG and RVM) that warrants further study.     

Hypocretin-2 Saporin Lesions of the Ventrolateral Periaquaductal Gray (vlPAG) Increase REM Sleep in Hypocretin Knockout Mice

Ten years ago the sleep disorder narcolepsy was linked to the neuropeptide hypocretin (HCRT), also known as orexin. This disorder is characterized by excessive day time sleepiness, inappropriate triggering of rapid-eye movement (REM) sleep and cataplexy, which is a sudden loss of muscle tone during waking. It is still not known how HCRT regulates REM sleep or muscle tone since HCRT neurons are localized only in the lateral hypothalamus while REM sleep and muscle atonia are generated from the brainstem. To identify a potential neuronal circuit, the neurotoxin hypocretin-2-saporin (HCRT2-SAP) was used to lesion neurons in the ventral lateral periaquaductal gray (vlPAG). The first experiment utilized hypocretin knock-out (HCRT-ko) mice with the expectation that deletion of both HCRT and its target neurons would exacerbate narcoleptic symptoms. Indeed, HCRT-ko mice (n = 8) given the neurotoxin HCRT2-SAP (16.5 ng/23nl/sec each side) in the vlPAG had levels of REM sleep and sleep fragmentation that were considerably higher compared to HCRT-ko given saline (+39%; n = 7) or wildtype mice (+177%; n = 9). However, cataplexy attacks did not increase, nor were levels of wake or non-REM sleep changed. Experiment 2 determined the effects in mice where HCRT was present but the downstream target neurons in the vlPAG were deleted by the neurotoxin. This experiment utilized an FVB-transgenic strain of mice where eGFP identifies GABA neurons. We verified this and also determined that eGFP neurons were immunopositive for the HCRT-2 receptor. vlPAG lesions in these mice increased REM sleep (+79% versus saline controls) and it was significantly correlated (r = 0.89) with loss of eGFP neurons. These results identify the vlPAG as one site that loses its inhibitory control over REM sleep, but does not cause cataplexy, as a result of hypocretin deficiency.     

The Analgesic Effect and Mechanism of the Combination of Sodium ferulate and Oxymatrine

Sodium ferulate (SF) and Oxymatrine (OMT) were compounds extracted from Chinese herbs, and have been used in clinical treatment of heart and hepatic diseases, respectively, in China for many years. The objective of this study was to examine the analgesic effect and the mechanism of the combined treatment of SF and OMT. Using the animal pain models by applying Acetic Acid Writhing Test and Formalin Test, the combination of SF and OMT showed significant analgesic effect in dose-dependent manner. In vitro, the combined treatment inhibited the increase in intracellular calcium concentration evoked by capsaicin in the dorsal root ganglion neurons. Importantly, a synergistic inhibitory effect of SF and OMT on the capsaicin-induced currents was demonstrated by whole-cell patch-clamp. Our results suggest that SF and OMT cause significant analgesic effect which maybe related to the synergistic inhibition of transient receptor potential vanilloid-1.     

BAK1调控植物免疫信号识别和转导的分子机制

植物通过类受体激酶感知环境变化,产生相应的信号来调控机体生长发育。BAK1 (BRI1-associated kinase 1)是其中研究最深入的类受体激酶之一。它调控多种生理过程的信号转导,如植物生长发育、细胞死亡和植物免疫等。本文综述了BAK1作为模式识别受体的共受体以及信号转导的调控子,调控免疫信号识别和转导的机理。以期为深入研究BAK1基因家族在植物抗病反应中的作用,阐明植物免疫信号转导途径提供信息。     

Mechanism Underlying the Protective Effect of Selenium on NO-Induced Oxidative Damage in Bovine Mammary Epithelial Cells

This experiment was conducted to investigate the effects and mechanism of selenium (Se) on antioxidant and immune function of bovine mammary epithelial cells (BMEC) damaged by nitric oxide (NO). The third-generation BMEC was randomly divided into eight treatments with six replicates. The BMEC in the control group was cultured in the medium without Se and diethylenetriamine/NO (DETA/NO) for 30 h. For the DETA/NO group and Se protection group BMEC were exposed to different concentrations of Se (0, 10, 20, 50, 100, 150, and 200 nmol/L) for 24 h, followed by treatment with DETA/NO (1000 μmol/L) for 6 h. Compared with the control group, DETA/NO decreased proliferation rate and activity of thioredoxin reductase (TrxR; P < 0.05). Additionally, DETA/NO decreased the gene expression of both nuclear factor-E2-related factor 2 (Nrf2) and TrxR, as well as the protein expression level of TrxR. However, the activity, and expression levels of inducible nitric oxide synthase (iNOS), as well as the concentration and gene expression level of interleukin-1β (IL-1β) and the concentration of NO significantly increased (P < 0.05). The gene expression levels of indexes related to the mitogen-activated protein kinase (MAPK) signaling pathway showed similar changes. Treatment of BMEC with Se significantly reversed DETA/NO-induced changes in a linear or quadratic dose-dependent manner (P < 0.05), with greatest benefit at 50 nmol/L. These data suggests that Se improves the antioxidant function of BMEC, and protects cells from DETA/NO-induced oxidative damage, primarily by enhancing the activity of TrxR and decreasing the concentration of NO through modulation of Nrf2 and MAPK signaling pathways.

     

MFG-E8 and HMGB1 Are Involved in the Mechanism Underlying Alcohol-Induced Impairment of Macrophage Efferocytosis

Efferocytosis is a unique phagocytic process for macrophages to remove apoptotic cells in inflammatory loci. This event is maintained by milk fat globule-EGF factor 8 (MFG-E8), but attenuated by high mobility group box 1 (HMGB1). Alcohol abuse causes injury and inflammation in multiple tissues. It alters efferocytosis, but precise molecular mechanisms for this effect remain largely unknown. Here, we showed that acute exposure of macrophages to alcohol (25 mmol/L) inhibited MFG-E8 gene expression and impaired efferocytosis. The effect was mimicked by hydrogen peroxide. Moreover, N-acetylcysteine (NAC), a potent antioxidant, blocked acute alcohol effect on inhibition of macrophage MFG-E8 gene expression and efferocytosis. In addition, recombinant MFG-E8 rescued the activity of alcohol-treated macrophages in efferocytosis. Together, the data suggest that acute alcohol exposure impairs macrophage efferocytosis via inhibition of MFG-E8 gene expression through a reactive oxygen species dependent mechanism. Alcohol has been found to suppress or exacerbate immune cell activities depending on the length of alcohol exposure. Thus, we further examined the role of chronic alcohol exposure on macrophage efferocytosis. Interestingly, treatment of macrophages with alcohol for seven days in vitro enhanced MFG-E8 gene expression and efferocytosis. However, chronic feeding of mice with alcohol caused increase in HMGB1 levels in serum. Furthermore, HMGB1 diminished efferocytosis by macrophages that were treated chronically with alcohol, suggesting that HMGB1 might attenuate the direct effect of chronic alcohol on macrophage efferocytosis in vivo. Therefore, we speculated that the balance between MFG-E8 and HMGB1 levels determines pathophysiological effects of chronic alcohol exposure on macrophage efferocytosis in vivo.     

植物响应联合胁迫机制的研究进展

自然界中, 植物通常面对多重联合胁迫。在全球气候变化日益加剧的背景下, 多重联合胁迫对植物生长发育及作物产量形成的不利影响日益显著。阐明植物响应和适应联合胁迫的生理与分子机制, 对人们理解植物对自然环境的适应机理, 及培育耐受联合胁迫的新品种有重要意义。研究表明, 植物响应联合胁迫的机制是特异的, 不能简单地从单一胁迫响应叠加来推断。植物遭受联合胁迫时, 各种生理、代谢和信号途径相互作用, 使得植物响应联合胁迫非常复杂。该文综述了植物响应联合胁迫的生理与分子机理的最新进展, 并阐述了植物响应联合胁迫的研究方法。     

丹参多酚酸盐对斑马鱼胚胎血管新生的影响及分子机制初步研究

目的:研究丹参多酚酸盐对斑马鱼发育过程中血管新生的影响,并初步探讨其机制。方法:取血管内皮具有绿色荧光蛋白标记的转基因斑马鱼卵后,胚胎分别给0.5%二甲基亚砜(DMSO,对照组)、丹参多酚酸盐(1mg/ml,实验组)处理24h,观察斑马鱼血管发育的变化并记录节间血管长度。定量RT-PCR检测前述药物处理后4、12和24h的斑马鱼胚胎中血管内皮生长因子(VEGF)的表达变化。结果:与对照组相比,使用丹参多酚酸盐药物处理后明显促进斑马鱼节间血管发育,其长度差别具有统计学意义([79.67±2.96)umvs(61.11±2.56)um,n=10,P<0.01)]。实验组较对照组VEGFmRNA表达量升高,在药物作用12h和24h时差异有统计学意义(P<0.05)。结论:丹参多酚酸盐可促进斑马鱼血管新生,可能与通过上调VEGF的表达有关。