温阳解郁颗粒调节BDNF/TrkB/ERK信号通路保护皮质酮诱导损伤型海马神经细胞的作用机制研究＊

目的 观察温阳解郁颗粒（Wenyang Jieyu granule，WYJY）对皮质酮（Corticosterone，CORT）诱导损伤型小鼠海马神经细胞（TH22 cell）的保护作用，基于脑源性神经营养因子（Brain derived neurotrophic factor， BDNF）/酪氨酸激酶B（Tyrosine kinase B， TrkB）/细胞外信号调节蛋白激酶（Extra cellular regulated protein kinases， ERK）信号通路探讨WYJY保护海马神经细胞的作用机制。方法 体外构建小鼠海马神经细胞皮质酮诱导损伤模型，以不同浓度的WYJY和氟西汀（Fluoxetine，FXT）含药血清作用于模型细胞，细胞增殖-毒性检测（Cell Counting Kit-8， CCK-8）法分析细胞活性，倒置显微镜下观察给药前后细胞形态结构的改变，采用蛋白免疫印迹法（Western Blot）、实时荧光定量PCR（Quantitative real-time PCR， qPCR）法检测神经细胞内凋亡因子（BCL2-Associated X， Bax）、抗凋亡因子（B-cell lymphoma-2， Bcl-2）、BDNF、Trkb、ERK以及丝氨酸/苏氨酸激酶（Phospho-p90RSK， RSK）、环磷腺苷效应元件结合蛋白（cAMP-response element binding protein， CREB）蛋白表达水平以及相关基因的表达水平。结果 在浓度为459.5 μmol·L^-1的CORT作用24 h后，HT22细胞的活性抑制率达到50%，在此条件作用下细胞形态结构损伤明显，凋亡程度严重，细胞上清中BDNF的含量显著减少（P<0.05），细胞内凋亡相关因子Bax/Bcl-2的比值明显升高（P<0.01），BDNF、Trkb、ERK、RSK、CREB磷酸化蛋白表达水平和mRNA表达水平明显降低（P<0.01）；以5%的浓度为2.85 g·kg^-1的WYJY和10%的FXT含药血清作用于受损的HT22细胞后，HT22细胞存活率明显提升（P<0.01），细胞结构的损伤明显改善，细胞凋亡程度减轻，细胞外BDNF的含量显著升高（P<0.05），细胞内Bax/Bcl-2比值显著下调（P<0.01），BDNF、Trkb、ERK、RSK、CREB磷酸化蛋白表达水平和mRNA表达水平显著提升（P<0.05，P<0.01）。结论 温阳解郁颗粒可有效保护高浓度CORT造成的小鼠海马神经细胞损伤。调控BDNF/Trkb/ERK通路，放大CREB信号传导，影响Bcl-2、BDNF水平，可能是其保护海马神经元，发挥抗抑郁疗效的重要机制。     

左旋四氢巴马汀对大鼠海马脑片缺氧损伤电生理的影响

 目的在海马脑片水平观察左旋四氢巴马汀(L-tetrahydropalmatine,L-THP)对脑组织缺氧后及恢复给氧早期突触功能的影响。方法大鼠离体海马脑片以人工脑脊液孵育,观察缺氧后海马CA1区诱发群峰电位(population spike,PS)开始减小的时间和消失时间,缺氧损伤电位(hypoxic injury potential,HIP)出现时间、出现率和保持时间,复氧后PS的恢复率和恢复程度等指标并观察L-THP对这些指标的影响。结果L-THP(0.028,0.14,0.28 mmol·L^-1)组脑片PS减小时间、消失时间与缺氧组相比后延,并有显著差异(P<0.05),但其作用强度随剂量递减。缺氧组脑片平均在658.8 s出现HIP,L-THP 0.028 mmol·L^-1组,0.14 mmol·L^-1组和0.28 mmol·L^-1组脑片出现时间分别推迟为1 152.4和851.1,723.5 s,与缺氧组相比均有显著差异(P<0.05)。各组的HIP出现率分别为100%,33.3%,67.0%,83.3%。复氧30 min后,氧-葡萄糖剥夺(oxygen-glucose dep-rivation,OGD)组的恢复率为8.33%,恢复程度为19.8%。在L-THP组(0.028,0.14,0.28 mmol·L^-1),PS的恢复率分别为92%、75%和33.3%,恢复程度分别为82.5%,70.4%和58.6%,与对照组比有非常显著的差异(P<0.01)。结论L-THP能减缓缺氧时PS幅度的降低速度,减轻和防止脑片突触功能的不可逆损伤,提高脑片的耐缺氧能力,但随着剂量增加而作用减弱。     

苏郁胶囊对抑郁模型小鼠行为及海马神经元损伤的影响

目的：探究苏郁胶囊对慢性应激抑郁模型小鼠行为、海马神经元损伤及海马突触体内游离Ca²⁺浓度的影响。方法:将60只昆明种雄性小鼠分为正常组、模型组、苏郁胶囊高、中、低剂量组（22.8,11.4,5.7 g·kg^-1）。采用长期不可预见性中等强度应激造成小鼠抑郁模型。测定各组小鼠体重变化,Open-field法和糖水消耗实验测定各组小鼠的行为变化；Nissl染色法观察海马CA1,CA3区神经元形态及锥体细胞数目；以Fura-2负载及荧光分光光度计检测海马突触体内游离Ca²⁺浓度。结果:与正常组比较,模型组小鼠体重增长缓慢(P<0.01),水平活动得分和垂直活动得分显著减少(P<0.01),糖水消耗量明显降低(P<0.01)；海马CA3区锥体细胞数量明显减少(P<0.01)；海马突触体内游离Ca²⁺浓度明显升高(P<0.01)；苏郁胶囊高剂量组于第14,21天时小鼠体重增长数明显高于模型组（P<0.05）；苏郁胶囊高剂量能增加抑郁小鼠的水平活动得分(P<0.05)；苏郁胶囊高、中剂量组能增加抑郁小鼠的垂直活动得分(P<0.01,P<0.05)；苏郁胶囊高、中、低剂量能明显增加抑郁小鼠的糖水消耗量(P<0.01,P<0.05,P<0.05)；苏郁胶囊高、中、低剂量组能明显增加海马CA3区锥体细胞数量 (P<0.01,P<0.05,P<0.05)；苏郁胶囊高、中、低剂量组能明显降低抑郁小鼠海马突触体内游离Ca²⁺浓度(P<0.01,P<0.01,P<0.05)。结论:苏郁胶囊能改善小鼠的抑郁样行为；保护抑郁模型小鼠海马神经元损伤,其可能与苏郁胶囊抑制海马神经细胞外Ca²⁺内流,阻止Ca²⁺超载相关。     

Proteomic analysis of human hippocampal subfields provides new insights into the pathogenesis of Alzheimer’s disease and the role of glial cells

The hippocampus and entorhinal cortex (EC), the earliest affected areas, are considered relative to early memory loss in Alzheimer''s disease (AD). The hippocampus is composed of heterogeneous subfields that are affected in a different order and varying degrees during AD pathogenesis. In this study, we conducted a comprehensive proteomic analysis of the hippocampal subfields and EC region in human postmortem specimens obtained from the Chinese human brain bank. Bioinformatics analysis identified region‐consistent differentially expressed proteins (DEPs) which associated with astrocytes, and region‐specific DEPs which associated with oligodendrocytes and the myelin sheath. Further analysis illuminated that the region‐consistent DEPs functioned as connection of region‐specific DEPs. Moreover, in region‐consistent DEPs, the expression level of S100A10, a marker of protective astrocytes, was increased in both aging and AD patients. Immunohistochemical analysis confirmed an increase in the number of S100A10‐positive astrocytes in all hippocampal subfields and the EC region of AD patients. Dual immunofluorescence results further showed that S100A10‐positive astrocytes contained apoptotic neuron debris in AD patients, suggesting that S100A10‐positive astrocytes may protect brain through phagocytosis of apoptotic neurons. In region‐specific DEPs, the proteome showed a specific reduction of oligodendrocytes and myelin markers in CA1, CA3, and EC regions of AD patients. Immunohistochemical analysis confirmed the loss of myelin in EC region. Above all, these results highlight the role of the glial cells in AD and provide new insights into the pathogenesis of AD and potential therapeutic strategies.     

Differential localization of carbachol- and bicuculline-sensitive pontine sites for eliciting REM sleep-like effects in anesthetized rats

Carbachol, a cholinergic agonist, and GABA_A receptor antagonists injected into the pontine dorsomedial reticular formation can trigger rapid eye movement (REM) sleep-like state. Data suggest that GABAergic and cholinergic effects interact to produce this effect but the sites where this occurs have not been delineated. In urethane-anesthetized rats, in which carbachol effectively elicits REM sleep-like episodes (REMSLE), we tested the ability of 10 nL microinjections of carbachol (10 m m ) and bicuculline (0.5 or 2 m m ) to elicit REMSLE at 47 sites located within the dorsal pontine reticular formation at the levels -8.00 to -10.80 from bregma (B) (Paxinos and Watson, The Rat Brain in Stereotaxic Coordinates , Academic Press, San Diego, 1997). At rostral levels, most carbachol and some bicuculline injections elicited REMSLE with latencies that gradually decreased from 242 to 12 s for carbachol and from 908 to 38 s for bicuculline for more caudal injection sites. As the latencies decreased, the durations of bicuculline-elicited REMSLE increased from 104 s to over 38 min, and the effect was dose dependent, whereas the duration of carbachol-elicited REMSLE changed little (104–354 s). Plots of REMSLE latency versus the antero-posterior coordinates revealed that both drugs were maximally effective near B-8.80. At levels caudal to B-8.80, carbachol was effective at few sites, whereas bicuculline-elicited REMSLE to at least B-9.30 level. Thus, the bicuculline-sensitive sites extended further caudally than those for carbachol and antagonism of GABA_A receptors both triggered REMSLE and controlled their duration, whereas carbachol effects on REMSLE duration were small or limited by its concurrent REMSLE-opposing actions.     

Differential effects of melatonin on amyloid‐β peptide 25–35‐induced mitochondrial dysfunction in hippocampal neurons at different stages of culture

Abstract: β‐Amyloid (Aβ) is strongly involved in the pathogenesis of Alzheimer’s disease (AD), and mitochondria play an important role in neurodegenerative disorders. To determine whether any different effect of melatonin on cultured neurons treated with Aβ in vitro and which may be produced through its different action on mitochondria at different stages of culture, we investigated the damage of cultured rat hippocampal neurons mitochondrial function induced by Aβ in young neurons [days in vitro 10 (DIV 10)] and senescent neurons (DIV 25) and the protective effect of melatonin. Rat hippocampal neurons were incubated with amyloid‐β peptide 25–35 (Aβ25‐35) alone or pretreatment with melatonin. Cell viability, mitochondrial membrane potential (Δψm), ATP and the activity of the respiratory chain complexes were measured. Data showed that Aβ25‐35 caused a reduction in Δψm, inhibited the activity of the respiratory chain complexes and led to ATP depletion, melatonin attenuated Aβ25‐35‐induced mitochondrial impairment in young neurons, whereas melatonin had no effect on Aβ25‐35‐induced mitochondrial damage in senescent neurons. These results demonstrate that melatonin has differential effect on Aβ25‐35‐induced mitochondrial dysfunction at different stages of culture and suggest that melatonin is useful for the prevention of AD, rather than treatment.     

丹皮酚对大鼠离体海马神经元NMDA受体的影响

目的:研究丹皮酚对大鼠海马神经元缺糖缺氧的保护作用。方法:以原代培养的新生大鼠海马神经元作为研究对象,建立缺糖缺氧损伤模型,倒置相差显微镜下进行一般形态学的动态观察,用MTT法测定神经元存活率。通过放射配基结合实验测定海马NMDA受体结合力变化。结果:丹皮酚可显著降低神经元缺糖缺氧时细胞死亡率,减弱NMDA受体结合力。结论:丹皮酚对离体培养的大鼠海马神经元缺糖缺氧具有保护作用,其作用机制与减弱NMDA受体结合力有关。     

Effects of the ephedra alkaloid methylephedrine on the basal evoked potential transportation and the long-term potentiation (LTP) in the rat hippocampal dentate granule cells in vivo

Objective The effect of the ephedra/ephedrine alkaloid methylephedrine(dl-methylephedrine hydrochloride for testing in this paper)on cognitive related synaptic plasticity was investigated by recording extracellular field evoked potentials and its LTP in hippocampal dentate granule cells in urethane-anaesthetized rats in vivo.Methods Single pathway recording of evoked field potentials was made from the dentate granule cells of hippocampal hemisphere in response to stimulation of the ipsilateral medial perforant path(MPP).Two parameters,the amplitude of population spike(PS amplitude)and the latency of the PS,were employed to evaluate the effects of drug on the overall changes in cellular responses.Results The present study show that methylephedrine 90 mg·kg-1 intraperitoneally,about 1/3 LD50,could increase the latency of the PS in hippocampal dentate granule cells by constant single stimulation of the MPP as the basal ransportation.However,the 30 mg·kg-1 and 10 mg·kg-1 dosage had no effect on the latency,and there are no influences of PS amplitude for all examinational groups.The methylephedrine 90 mg·kg-1 group significant enhanced the development of amplitude LTP in hippocampal dentate granule cells that induced by 60 Hz,60 pulses conditional tetanus in medial perforant path area.Also,the 30 mg·kg-1 group can promoted the maintenance of LTP induced by this tetanus,but no promotion on PS amplitude LTP appeared in this dosage and no any changes been found in 10 mg·kg-1 dosage group.Conclusions The ephedra/ephedrine alkaloid methylephedrine can modulate the synaptic plasticity in the lateral perforant path.A possible mechanism of methylephedrine on hippocampal LTP is been discussed.     

Topographical and laminar organization of subicular projections to the parahippocampal region of the rat

In this study, we analyzed in detail the topographic organization of the subiculoparahippocampal projection in the rat. The anterograde tracers Phaseolus vulgaris leucoagglutinin-L and biotinylated dextran amine were injected into the subiculum at different septotemporal and transverse levels. Deep layers of the ento-, peri-, and postrhinal cortices are the main recipients of subicular projections, but in all cases we noted that a small fraction of the projections also terminates in the superficial layers II and III. Analysis of the fiber patterns in the parahippocampal region revealed a topographic organization, depending on the location of the cells of origin along both the transverse and the septotemporal axes of the subiculum. Projections originating from subicular cells close to CA1, i.e., proximal part of subiculum, terminate exclusively in the lateral entorhinal cortex and in the perirhinal cortex. In contrast, projections from cells closer to the subiculum-presubiculum border, i.e., distal part of subiculum, terminate in the medial entorhinal cortex and in the postrhinal cortex. In addition, cells in septal portions of the subiculum project to a lateral band of entorhinal cortex parallel to the rhinal sulcus and to peri- or postrhinal cortices, whereas cells in more temporal portions project to more medial parts of the entorhinal cortex. These results indicate that subicular projections to the parahippocampal region precisely reciprocate the known inputs from this region to the hippocampal formation. We thus suggest that the reciprocal connectivity between the subiculum and the parahippocampal region is organized as parallel pathways that serve to segregate information flow and thus maintain the identity of processed information. Although this parallel organization is comparable to that of the CA1-parahippocampal projections, differences exist with respect to the degree of collateralization.     

Dentate granule cells in reeler mutants and VLDLR and ApoER2 knockout mice

We have studied the organization and cellular differentiation of dentate granule cells and their axons, the mossy fibers, in reeler mutant mice lacking reelin and in mutants lacking the reelin receptors very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). We show that granule cells in reeler mice do not form a densely packed granular layer, but are loosely distributed throughout the hilar region. Immunolabeling for calbindin and calretinin revealed that the sharp border between dentate granule cells and hilar mossy cells is completely lost in reeler mice. ApoER2/VLDLR double-knockout mice copy the reeler phenotype. Mice deficient only in VLDLR showed minor alterations of dentate organization; migration defects were more prominent in ApoER2 knockout mice. Tracing of the mossy fibers with Phaseolus vulgaris leukoagglutinin and calbindin immunolabeling revealed an irregular broad projection in reeler mice and ApoER2/VLDLR double knockouts, likely caused by the irregular wide distribution of granule cell somata. Mutants lacking only one of the lipoprotein receptors showed only minor changes in the mossy fiber projection. In all mutants, mossy fibers respected the CA3-CA1 border. Retrograde labeling with DiI showed that malpositioned granule cells also projected as normal to the CA3 region. These results indicate that ( 1 ) reelin signaling via ApoER2 and VLDLR is required for the normal positioning of dentate granule cells and (2) the reelin signaling pathway is not involved in pathfinding and target recognition of granule cell axons.