大鼠海马内胆碱能纤维损伤后的再生和抽芽

<正> 观察穹窿海马伞损伤鼠海马胆碱能纤维损伤后的再生状况.方法:切断SD成年大鼠穹窿海马伞,用染AChE纤维的组织化学方法结合网格测试,分析术后1、2、3、4周海马CA1区和齿状回的分子层胆碱能纤维的再生和侧支抽芽.结果:损伤1周时CA1区和齿状回的分子层胆碱能纤维明显减少,分别减少到67.50%和66.91%;从第2周开始纤维数量逐渐增多,至第4周CA1区分子层纤维恢复到正常的82.42%,而齿状回分子层纤维恢复到97.82%.结论:成年哺乳类海马内     

海马结构基因表达差异及其功能分析

海马结构(hippocampal formation)的主要组成是海马和齿状回(dentate gyrus,DG),它们与大脑皮质和皮质下中枢有广泛的纤维联系,参与形成学习记忆.海马皮质从海马沟至脑室回依次为分子层、锥体层和多形层;齿状回皮质也分3层:分子层、颗粒细胞层和多形层,其神经细胞发出的纤维不超出海马结构范围.海马内锥体细胞规则排列,故结构比较一致.尽管如此,依据细胞形态、不同皮质区发育差异以及纤维排列不同,海马皮质亦能分为4个沿其长轴分布的不同亚区,即CA1、CA2、CA3、CA4区,且研究表明各亚区在基因表达方面也存在显著差别.     

神经干细胞移植对192-IgG-saporin老年性痴呆模型鼠的学习记忆及海马胆碱能纤维的影响

目的探讨神经干细胞(NSCs)移植对192-IgG-saporin致老年性痴呆模型鼠学习记忆和海马胆碱能纤维再生的影响。方法采用192-IgG-saporin(2.5μg/5μL)侧脑室注射SD大鼠建立痴呆模型后,行基底前脑神经干细胞移植,4周后行Y迷宫检测,并观察大鼠海马胆碱能纤维数的变化。结果Y迷宫检测显示大鼠的学习、记忆能力,模型组(107.38±9.34、3.75±0.71)与正常组比较明显下降(P<0.01),而移植组(75.26±5.33、5.45±0.51)有所改善(P<0.05);免疫组化显示模型组大鼠海马CA1区辐射层和齿状回分子层胆碱能阳性纤维,模型组与正常组比较,CA1辐射层和齿状回分子层纤维密度分别减少到11.07%和12.96%(P<0.01),与正常组比较均而干细胞移植组则分别恢复到正常组的81.39%和75.30%(P>0.05)。结论神经干细胞能促进192-IgG-saporinAD动物模型鼠学习记忆能力的恢复及海马胆碱能纤维的再生。     

氯化锂匹罗卡品诱导癫痫大鼠海马神经元GPER1表达的变化

目的:观察G蛋白偶联雌激素受体1(GPER1)在癫痫大鼠海马神经元中表达的变化。方法:成年雄性SD大鼠分成对照组(control)和癫痫组(epilepsy),利用腹腔注射氯化锂-匹罗卡品方法制备癫痫模型,分别在1、2、3、7、14 d和28 d,利用Morris水迷宫检测大鼠学习记忆能力,利用尼氏染色观察大鼠海马神经元形态变化;利用免疫组化和Western Blot技术观察GPER1在海马的表达。结果:水迷宫结果显示,与Control组相比,造模14 d的大鼠逃逸潜伏时间明显延长(P 0. 05),穿越目标象限区域的次数较Control组显著降低(P 0. 05)。尼氏染色结果显示:与Control组相比,造模1 d和2 d的大鼠CA1及CA3区锥体细胞层细胞及DG区颗粒细胞层细胞体积缩小,细胞间距增加,尼氏染色减弱;造模3和7 d的大鼠细胞体积明显缩小,细胞间隙明显增大,尼氏染色加深,CA1及CA3细胞数量明显减少;造模14 d和28 d的大鼠神经元体积逐渐向正常恢复,但仍较Control组小。免疫组化结果显示:GPER1免疫阳性细胞以海马锥体细胞和齿状回颗粒细胞为主,主要分布在细胞膜。与Control组相比,造模2 d和3 d的大鼠海马CA1及CA3区GPER1表达增加(P 0. 05),7 d后增加最明显(P 0. 01),14、28 d后表达下降;在DG区,造模3 d及7 d的大鼠GPER1表达增加(P 0. 05),14 d后表达下降(P 0. 05),28 d大鼠无显著差异。Western Blot结果显示:与Control组比较,造模2 d和3 d的大鼠GPER1相对表达量开始增高,7 d后明显增高(P 0. 05),14 d及28 d的大鼠表达降低。结论:GPER1在海马神经元的表达随着神经元损伤的加重而增高,随着神经元损伤的恢复逐渐降低,提示其表达变化与神经元的损伤与修复有关。     

蒙古种沙土鼠背海马胆碱能神经局部分布

目的 :对蒙古种沙土鼠背海马乙酰胆碱酯酶阳性纤维和末梢的局部分布作分区分层的研究。方法 :应用乙酰胆碱酯酶纤维染色法及计算机图像扫描和统计学分析等方法。结果 :背海马局部乙酰胆碱酯酶纤维和末梢的分布按密度从大到小排列为 :CA2、CA3区辐射层 >CA4区 (齿状回门区 ) >齿状回多形层 >齿状回下支分子层 >齿状回上支分子层、背海马分子层 >CA1、CA2、CA3区多形层。结论 :实验结果为蒙古种沙土鼠背海马胆碱能投射的定位分布的进一步研究提供了一定的形态学基础     

急性酒精中毒损伤新生鼠海马细胞分子结构

目的 研究胎儿酒精中毒脑损伤的细胞分子机制.方法 腹腔注射中毒剂量酒精至生后3～10dSD大鼠,制作急性酒精中毒模型;6小时后提取海马细胞DNA,并用琼脂糖凝胶电泳分离DNA;同时制作海马结构的石蜡切片,尼氏染色观察海马及齿状回细胞的结构.结果 酒精注射组海马细胞DNA断裂,断裂片段集中在200bp、400bp、900bp等;海马CAI区,齿状回颗粒层、多形层的细胞数均明显减少;CAl区锥体细胞胞核染色体明显浓缩呈团块状并聚集在核膜周围;颗粒细胞层胶质细胞增生;多形层细胞肿胀,细胞核染色体消失.结论 酒精破坏发育期大鼠海马结构,引起海马细胞凋亡或坏死.确切机制有待进一步阐明.     

NMDA受体亚单位NR1、NR2A和NR2B在大鼠海马的免疫组织化学表达

目的 :观察N 甲基 D 门冬氨酸受体亚单位 1 (N methyl D aspartatereceptorsubunit 1 ,NR1 )、亚单位 2A(NR2A)和亚单位 2B(NR2B)在成年大鼠海马结构各区的表达特点 ,为研究三者在海马生理和病理过程中的作用提供形态学资料。方法 :大鼠脑 2 0 μm厚冰冻切片 ,免疫组织化学ABC法显色 ,图像分析。结果 :NR1、NR2A和NR2B在海马CA1～CA3区锥体细胞以及齿状回颗粒细胞普遍表达 ,三者中以NR1免疫组织化学反应最强 ,NR2A最弱 ,NR2B居中。NR1与NR2A在海马各区间的表达水平都无显著差异 ;NR2B在海马CA1区的表达明显强于其在CA3区及齿状回的表达 ,尤其是CA1区锥体细胞的顶树突在贯穿辐射层及腔隙分子层的全长中都呈高表达。结论 :NR1、NR2A和NR2B在正常海马结构各区的表达强度和形式存在差异 ,提示各区间天然N 甲基 D 门冬氨酸(N methyl D aspartate ,NMDA)受体的亚单位构成比例可能有所不同     

大鼠下丘脑腹内侧核与海马神经纤维联系研究

目的:探讨大鼠下丘脑腹内侧核与海马的传入、传出神经纤维联系,为摄食和能量代谢等内脏活动的神经调节机制的深入研究提供形态学依据。方法:参考大鼠脑立体定位图谱,借助脑立体定位仪将逆行示踪剂伊文思蓝(EB)分别注入SD大鼠左侧下丘脑腹内侧核(A组,18只)或海马CA2区(B组,18只),大鼠存活3 d后,4%多聚甲醛心脏灌注,冰冻切片,荧光显微镜下观察。结果:A组双侧海马的CA2,CA3处均显示有荧光标记细胞,且对侧荧光强于同侧;B组下丘脑腹内侧核处未见荧光标记细胞。结论:下丘脑腹内侧核与海马有广泛的纤维联系,可接受来自双侧海马CA2,CA3区的神经纤维投射,两者可能在内脏活动调节中发挥重要作用。     

Expression of calcium/calmodulin-dependent protein kinase Ⅱ in the hippocampal subregions of the rat during postnatal development

目的 探讨Wistar大鼠生后海马发育过程中钙/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)的表达.方法 应用免疫荧光方法检测CaMKⅡ在生后不同时期大鼠海马CA1、CA3区和齿状回(DG)中的表达情况(n=48). 结果 CaMKⅡ于生后各期海马CA1区和DG的表达逐渐增强,生后第10天(P10)达高峰期,此后逐渐减弱;于CA3区的表达在P4和P10时均较高.其中,CaMKⅡ在CA3区的表达高于在CA1区和DG的表达,在多形层和分子层的表达高于在锥体细胞层或颗粒细胞层的表达. 结论 CaMKⅡ在CA1、CA3区和DG中的表达具有特异性的时空分布模式,这可能与其在生后发育过程中的突触发生,树突、轴突形成,海马的成熟以及学习记忆功能相关.     

钙/钙调蛋白依赖性蛋白激酶Ⅱ在大鼠生后海马发育中的表达

目的 探讨Wistar大鼠生后海马发育过程中钙/钙调蛋白依赖性蛋白激酶Ⅱ（CaMKⅡ）的表达。 方法 应用免疫荧光方法检测CaMKⅡ在生后不同时期大鼠海马CA1、CA3区和齿状回（DG）中的表达情况（n =48）。结果 CaMKⅡ于生后各期海马CA1区和DG的表达逐渐增强，生后第10天（P10）达高峰期，此后逐渐减弱；于CA3区的表达在P4和P10时均较高。其中，CaMKⅡ在CA3区的表达高于在CA1区和DG的表达，在多形层和分子层的表达高于在锥体细胞层或颗粒细胞层的表达。结论 CaMKⅡ在CA1、CA3区和DG中的表达具有特异性的时空分布模式，这可能与其在生后发育过程中的突触发生，树突、轴突形成，海马的成熟以及学习记忆功能相关。     

Contributions of mossy fiber and CA1 pyramidal cell sprouting to dentate granule cell hyperexcitability in kainic acid-treated hippocampal slice cultures

Axonal sprouting like that of the mossy fibers is commonly associated with temporal lobe epilepsy, but its significance remains uncertain. To investigate the functional consequences of sprouting of mossy fibers and alternative pathways, kainic acid (KA) was used to induce robust mossy fiber sprouting in hippocampal slice cultures. Physiological comparisons documented many similarities in granule cell responses between KA- and vehicle-treated cultures, including: seizures, epileptiform bursts, and spontaneous excitatory postsynaptic currents (sEPSCs) >600 pA. GABAergic control and contribution of glutamatergic synaptic transmission were similar. Analyses of neurobiotin-filled CA1 pyramidal cells revealed robust axonal sprouting in both vehicle- and KA-treated cultures, which was significantly greater in KA-treated cultures. Hilar stimulation evoked an antidromic population spike followed by variable numbers of postsynaptic potentials (PSPs) and population spikes in both vehicle- and KA-treated cultures. Despite robust mossy fiber sprouting, knife cuts separating CA1 from dentate gyrus virtually abolished EPSPs evoked by hilar stimulation in KA-treated but not vehicle-treated cultures, suggesting a pivotal role of functional afferents from CA1 to dentate gyrus in KA-treated cultures. Together, these findings demonstrate striking hyperexcitability of dentate granule cells in long-term hippocampal slice cultures after treatment with either vehicle or KA. The contribution to hilar-evoked hyperexcitability of granule cells by the unexpected axonal projection from CA1 to dentate in KA-treated cultures reinforces the idea that axonal sprouting may contribute to pathologic hyperexcitability of granule cells.     

Lesion-induced CA1 mossy fibers in the rat represent a neoinnervation

Summary The developmental pattern of hippocampal mossy fiber (dentate granule cell axon) innervation to the pyramidal cell layer was examined with anterograde transport methods. Injection of ³H-leucine into the dentate gyrus on PN 1 resulted in labeling of the incipient stratum lucidum extending to, but not beyond, the CA3 region on PN 3 and 5. Since destruction of CA3 pyramidal cells on PN 5 results in aberrant mossy fiber innervation to CA1 pyramidal cells (Cook and Crutcher 1985), these results suggest that the presence of mossy fibers in CA1 of the rat represents a neoinnervation (perhaps representing a more primitive pattern of connectivity) and not the persistence of a transient developmental projection.     

Postnatal development of the mouse dentate gyrus in organotypic cultures of the hippocampal formation

The hippocampal formation of newborn mice was explanted and maintained in Maximow culture assemblies for up to 35 days. At the time of explantation, only the suprapyramidal limb of the dentate gyrus was cytoarchitectonically distinct, and electron microscopy of newborn hippocampus revealed no definitive synapses. Histogenesis, as indicated by the development of the infrapyramidal limb of the dentate gyrus, and synaptogenesis, as indicated by the in vitro formation of mossy fiber synapses on the dendrites of hippocampal pyramidal neurons were studied by light and electron microscopy. At 12 days and thereafter in culture, mossy fiber terminals were found in synapsis with dendritic spines probably belonging to pyramidal cells of the hippocampal zone CA4. Near dentate granule cell somata a few axosomatic and many axospinous and axodendritic synapses were found. The data indicate that granule cells of the developing dentate gyrus are capable of differentiation in vitro into a structure essentially equivalent to that developed in vivo. The granule cells may become arranged into a recognizable granule cell layer, and develop dendritic processes which receive synapses virtually identical to those found in the intact organ. The differentiation of these features proceeds in the absence of the extrinsic afferents from the septum or from the contralateral hippocampal formation.     

The multifarious hippocampal mossy fiber pathway: a review

The hippocampal mossy fiber pathway between the granule cells of the dentate gyrus and the pyramidal cells of area CA3 has been the target of numerous scientific studies. Initially, attention was focused on the mossy fiber to CA3 pyramidal cell synapse because it was suggested to be a model synapse for studying the basic properties of synaptic transmission in the CNS. However, the accumulated body of research suggests that the mossy fiber synapse is rather unique in that it has many distinct features not usually observed in cortical synapses. In this review, we have attempted to summarize the many unique features of this hippocampal pathway. We also have attempted to reconcile some discrepancies that exist in the literature concerning the pharmacology, physiology and plasticity of this pathway. In addition we also point out some of the experimental challenges that make electrophysiological study of this pathway so difficult.Finally, we suggest that understanding the functional role of the hippocampal mossy fiber pathway may lie in an appreciation of its variety of unique properties that make it a strong yet broadly modulated synaptic input to postsynaptic targets in the hilus of the dentate gyrus and area CA3 of the hippocampal formation.     

Presynaptic inhibition of excitatory afferents to hilar mossy cells

The hippocampus contains one very strong recurrent excitatory network formed by associational connections between CA3 pyramidal cells and another that depends largely on a disynaptic excitatory pathway between dentate granule cells. The recurrent excitatory network in CA3 has long been considered a possible location of autoassociative memory storage, whereas changes in the level and arrangement of recurrent excitation between granule cells are strongly implicated in epileptogenesis. Hilar mossy cells are likely to receive collateral input from CA3 pyramidal cells and they are key intermediaries (by mossy fiber inputs) in the recurrent excitatory network between granule cells. The current study uses minimal stimulation techniques in an in vitro preparation of the rat dentate gyrus to examine presynaptic modulation of both mossy fiber and non-mossy fiber inputs to hilar mossy cells. We report that both mossy fiber and non-mossy fiber inputs to hilar mossy cells express presynaptic gamma-aminobutyric acid type B (GABA(B)) receptors that are subject to tonic inhibition by ambient GABA. We further find that only non-mossy fiber inputs express presynaptic muscarinic acetylcholine receptors, but that bath application of cholinergic agonists produces action potential-dependent increases in ambient GABA that can indirectly inhibit mossy fiber inputs. Finally, we demonstrate that mossy cells express high-affinity postsynaptic GABA(A) receptors that are also capable of detecting changes in ambient GABA produced by cholinergic agonists. Our results are among the first to directly characterize these important collateral inputs to hilar mossy cells and may help facilitate informed comparison between primary and collateral projections in two major excitatory pathways.     

Loss of dynorphin-mediated inhibition of voltage-dependent Ca2+ currents in hippocampal granule cells isolated from epilepsy patients is associated with mossy fiber sprouting

The endogenous kappa receptor selective opioid peptide dynorphin has been shown to inhibit glutamate receptor-mediated neurotransmission and voltage-dependent Ca2+ channels. It is thought that dynorphin can be released from hippocampal dentate granule cells in an activity-dependent manner. Since actions of dynorphin may be important in limiting excitability in human epilepsy, we have investigated its effects on voltage-dependent Ca2+ channels in dentate granule cells isolated from hippocampi removed during epilepsy surgery. One group of patients showed classical Ammon's horn sclerosis characterized by segmental neuronal cell loss and astrogliosis. Prominent dynorphin-immunoreactive axon terminals were present in the inner molecular layer of the dentate gyrus, indicating pronounced recurrent mossy fiber sprouting. A second group displayed lesions in the temporal lobe that did not involve the hippocampus proper. All except one of these specimens showed a normal pattern of dynorphin immunoreactivity confined to dentate granule cell somata and their mossy fiber terminals in the hilus and CA3 region. In patients without mossy fiber sprouting the application of the kappa receptor selective opioid agonist dynorphin A ([D-Arg6]1-13, 1 microM) caused a reversible and dose-dependent depression of voltage-dependent Ca2+ channels in most granule cells. These effects could be antagonized by the non-selective opioid antagonist naloxone (1 microM). In contrast, significantly less dentate granule cells displayed inhibition of Ca2+ channels by dynorphin A in patients with mossy fiber sprouting (Chi-square test, P < 0.0005). The lack of dynorphin A effects in patients showing mossy fiber sprouting compares well to the loss of kappa receptors on granule cells in Ammon's horn sclerosis but not lesion-associated epilepsy. Our data suggest that a protective mechanism exerted by dynorphin release and activation of kappa receptors may be lost in hippocampi with recurrent mossy fiber sprouting.     

Lesion-induced transneuronal plasticity in the adult rat hippocampus

The process of reactive synaptogenesis has been demonstrated in several areas of the central nervous system, including the hippocampal dentate gyrus. After a complete unilateral entorhinal lesion, approximately 85% of the input to the outer two-thirds of the ipsilateral dentate molecular layer is lost. Bilateral fluctuations in synaptic density within non-denervated zones of the dentate molecular layer predict further alterations in neural circuitry at sites located transneuronally to the denervated dentate granule cells. Using quantitative electron microscopy, our study demonstrates a complete cycle of synapse loss and reacquisition within the ipsilateral but not contralateral CA4/hilus region of the hippocampal formation. This area is one of the terminal fields for the dentate granule cell mossy fiber axons. In addition the granule cell mossy fiber axons sprout during the postlesion time course and form a significantly increased number of new mossy fiber terminals within the ipsilateral and contralateral CA4/hilus area. Our results indicate that responses to brain injury may no longer be confined to a local denervated site, but probably include polyneuronal circuitry loops, which may encompass one or more areas of the central nervous system. Previous difficulties in providing a close behavioral or functional correlation to localized structural events may be explained by a more global brain response to an injury.     

Timing and efficacy of transmitter release at mossy fiber synapses in the hippocampal network

It is widely accepted that the hippocampus plays a major role in learning and memory. The mossy fiber synapse between granule cells in the dentate gyrus and pyramidal neurons in the CA3 region is a key component of the hippocampal trisynaptic circuit. Recent work, partially based on direct presynaptic patch-clamp recordings from hippocampal mossy fiber boutons, sheds light on the mechanisms of synaptic transmission and plasticity at mossy fiber synapses. A high Na⁺ channel density in mossy fiber boutons leads to a large amplitude of the presynaptic action potential. Together with the fast gating of presynaptic Ca²⁺ channels, this generates a large and brief presynaptic Ca²⁺ influx, which can trigger transmitter release with high efficiency and temporal precision. The large number of release sites, the large size of the releasable pool of vesicles, and the huge extent of presynaptic plasticity confer unique strength to this synapse, suggesting a large impact onto the CA3 pyramidal cell network under specific behavioral conditions. The characteristic properties of the hippocampal mossy fiber synapse may be important for pattern separation and information storage in the dentate gyrus-CA3 cell network.     

The C5a anaphylatoxin receptor CD88 is expressed in presynaptic terminals of hippocampal mossy fibres

Background  

In the periphery, C5a acts through the G-protein coupled receptor CD88 to enhance/maintain inflammatory responses. In the brain, CD88 can be expressed on astrocytes, microglia and neurons. Previous studies have shown that the hippocampal CA3 region displays CD88-immunolabelling, and CD88 mRNA is present within dentate gyrus granule cells. As granule cells send dense axonal projections (mossy fibres) to CA3 pyramidal neurons, CD88 expression could be expressed on mossy fibres. However, the cellular location of CD88 within the hippocampal CA3 region is unknown.