大鼠孤束核内脑啡肽样免疫反应阳性结构及其轴突终末的突触联系

本文应用免疫细胞化学方法在光镜与电镜下观察了大鼠孤束核内脑啡肽样免疫反应（ＥＮＫ－ＬＩ）阳性结构的分布特征和ＥＮＫ－ＬＩ轴突终末的突触联系以及非突触性关系。结果表明：（１）经秋水仙素处理的大鼠,其孤束核内有许多ＥＮＫ－ＬＩ胞体的分布;而未经秋水仙素处理的大鼠,其孤束核内可见密集的ＥＮＫ－ＬＩ纤维与终末;ＥＮＫ－ＬＩ胞体、纤维和终末主要分布于锥体交叉平面至闩平面的孤束核内侧亚核与胶状质亚核。（２）ＥＮＫ－ＬＩ阳性产物主要定位于小圆形清亮囊泡外表面、大颗粒囊泡内和线粒体外表面等处。（３）ＥＮＫ－ＬＩ轴突终末主要与阴性树突形成轴－树突触。（４）阴性轴突终末终止于ＥＮＫ－ＬＩ轴突终末上,形成轴－轴突触。（５）ＥＮＫ－ＬＩ轴突终末与阴性轴突终末形成非突触性的轴－轴并靠。以上结果提示孤束核内的ＥＮＫ－ＬＩ神经成分主要通过突触后机制、也不排除突触前作用,参与孤束核中内脏信息的整合过程,而且这一作用又受到非ＥＮＫ－ＬＩ神经成分的调控。     

锌及锌转运蛋白ZnT3在小鼠海马苔藓纤维的一致性分布

目的 研究游离锌离子和锌转运蛋白ZnT3在小鼠海马的定位以及二的分布是否具有一致性。方法 应用锌TSQ荧光技术、锌金属自显影技术检测含锌神经元内的游离锌离子;应用免疫电镜技术检测ZnT3在含锌神经元轴突终末的分布。结果 游离锌离子和ZnT3免疫反应产物的分布在海马苔藓纤维内的分布具有一致性。在齿状回和CA3区的苔藓纤维内,锌和ZnT3蛋白定位于轴突终末的突触小泡。富含锌离子的含锌神经元轴突终末与CA3区锥体细胞的胞体和树突形成突触。尚可见锌离子存在于突触间隙内。结论 ZnT3向突触小泡内转运锌离子使锌离子聚积在含锌神经元轴突终末的突触小泡内,发挥锌离子的神经生物学功能。     

大鼠脊髓胶状质含亮啡肽终末与辣椒素敏感的C纤维终末的超微联系

本文应用免疫电镜术观察到,在大鼠脊髓胶状质内,亮啡肽(LEK)阳性的轴突终末与某些未标记神经元的树突、或树突棘、或胞体形成突触。其中一些轴(LEK)树(未标记)突触为对称性的。因此。在胶状质内,LEK神经元可以突触后抑制方式影响其它神经元的活动。LEK终末之间的对称性轴轴突触提示LEK终末可能具有自身调节作用。我们也发现少量的LEK终末与由硬脊膜外注射辣椒泰所致的一级传入C纤维变性(DEG)终末之间也存在直接和间接联系。这些联系包括以下几种:(1)轴(DEG)→树(LEK)突触;(2)轴(LEK)→轴(DEG)突触样接触;(3)轴(DEG)→树(未标记)←轴(LEK)三联体。这些联系分别提示:(1)一级传入C纤维可直接兴奋胶状质内含LEK的中间神经元,这可能是LEK对痛信息传递进行负反馈调节的机制之一;(2)LEK通过轴轴突触对一级传入C纤维的活动进行突触前抑制的可能性不能排除;(3)LEK可通过对上述三联体中的未标记树突进行抑制来间接调节一级传入C纤维的传入信息。此外,由未标记轴突终末与变性终末所形成的轴轴突触的存在提示,胶状质内非亮啡肽神经元也可通过轴轴突触以突触前抑制影响一级传入C纤维的功能。     

人海马生长抑素样免疫反应神经元的胚胎发育

用免疫组织化学ＰＡＰ法,观察人胎海马生长抑素样免疫反应神经元（Ｓｏｍａｔｏｓｔａｔｉｎ,ｉｍｍｕｎｏｒｅａｃｔｉｖｅｎｅｒｏｎｓ,简称ＳＯＭ－ＩＲ细胞）的分布,数量和形态变化,结果如下;１．胎龄第４－１０个月,ＳＯＭ样免疫反应神经元的分布无明显变化。其细胞的大部分分布在多形层,分布在锥体层和分子层的较少。各区皆有,ＣＡ１和ＣＡ２区最多。２．在第４个月即有ＳＯＭ样免疫反应神经元,其数量随着胎龄增长而逐渐增加,特别是在第５个月和第８个月增加明显。３．第４个月ＳＯＭ样免疫反应神经元呈小圆形或卵圆形,细胞突起短而粗,至第１０个月,细胞呈校形或锥形,突起细而长,胞浆丰富,接近成熟形态。实验结果提示：４—５个月海马细胞较少分化,用此阶段的海马作供体进行海马移植研究可能较为适宜。     

神经元对于高频电刺激的动态响应

深部脑刺激(deep brain stimulation,DBS)在许多神经系统疾病的临床治疗上都展现出良好的应用前景,然而,其作用机制尚不明确.常规DBS采用高频刺激(high frequency stimulation,HFS)的脉冲序列,这种窄脉冲最容易激活神经元结构中的轴突部分,通过轴突的投射,将HFS的作用传播至下游神经元.因此,为了探讨DBS的作用机制,并鉴于海马脑区是治疗癫痫和痴呆症等疾病的重要靶点,我们研究了海马区轴突HFS对于下游神经元的作用.对麻醉大鼠的海马CA1区传入神经通路Schaffer侧支施加1 min的100 Hz高频刺激,记录并提取下游CA1区锥体神经元和中间神经元的单元锋电位.计算锋电位的发放率,以及它们与刺激脉冲之间的锁相值(phase-locking value,PLV)和潜伏期,以定量分析HFS期间神经元动作电位发放的变化趋势.结果显示,在传入轴突上施加HFS时,初期会诱发下游神经元群体同步产生动作电位(即群峰电位).在HFS后期(群峰电位消失之后),两类神经元的单元锋电位发放仍然持续,并且发放率较稳定.但是,锋电位与刺激脉冲之间的锁相性逐渐减弱、潜伏期逐渐延长.而且,与中间神经元相比较,锥体神经元锋电位的锁相性更弱、潜伏期更长.这些结果表明,持续的轴突HFS可以诱导下游神经元产生非同步的活动,高频脉冲刺激引起的不完全轴突传导阻滞可能是导致该现象产生的主要原因.本文的研究为揭示脑刺激的作用机制提供了重要信息.     

小鼠脊髓内存在抑制性含锌神经元

目的探讨小鼠脊髓中是否含有抑制性的含锌神经元。方法应用锌金属自显影技术、免疫电镜技术和共聚焦激光扫描显微术,研究游离锌离子、锌转运蛋白(zinc transporter 3,ZnT3)与(glutamic acid decarboxylate,GAD)在小鼠脊髓内的共存情况。结果小鼠脊髓内至少有三种含锌神经元轴突终末,其中大多数为GAD阳性即γ-氨基丁酸能含锌神经元轴突终末,另外两种分别为GAD阴性含扁圆形小泡的甘氨酸能含锌神经元轴突终末和含圆形清亮小泡的兴奋性谷氨酸能含锌神经元轴突终末。结论在哺乳动物脊髓内存在大量的抑制性含锌神经元。锌离子从抑制性含锌神经元轴突终末释放到突触间隙内,作为神经调质作用于突触后的GABA受体或甘氨酸受体,参与脊髓运动和感觉功能的调控。     

海马区锥体神经元轴突高频电刺激对胞体的影响

目的 深部脑刺激（deep brain stimulation,DBS）利用持续的电脉冲高频刺激（high-frequency stimulation,HFS）调控神经元的活动，可望用于治疗更多脑疾病。为了深入了解HFS的作用机制，促进DBS的发展，本文研究轴突HFS在引起轴突阻滞期间神经元胞体的改变。方法 在麻醉大鼠海马CA1区的锥体神经元轴突上施加脉冲频率为100 Hz的1 min逆向高频刺激（antidromic high-frequency stimulation,A-HFS）。为了研究胞体的响应，利用线性垂直排列的多通道微电极阵列，记录刺激位点上游CA1区锥体神经元胞体附近各结构分层上的诱发电位，包括A-HFS脉冲诱发的逆向群峰电位（antidromic population spike,APS）以及A-HFS期间施加的顺向测试脉冲诱发的顺向群峰电位（orthodromic population spike,OPS），并计算诱发电位的电流源密度（current-source density,CSD），用于分析A-HFS期间锥体神经元胞体附近动作电位的生成和传导。结果 锥体神经...     

皮层神经元动作电位不应期和阈电位与神经元动作电位编码的关系

目的：测量和比较感觉运动皮层Ⅱ/Ⅲ层锥体神经元和中间神经元的内在特性并研究其与动作电位编码频率和精确性的关系。方法：采用全细胞电流钳记录模式,获得的数据输入pClamp和Origin进行处理分析。结果：与锥体神经元相比,中间神经元群集动作电位具有较低的阈电位水平和较短的不应期,从而中间神经元具有较高的动作电位编码频率和精确性。结论：皮层神经元动作电位的阈电位水平和不应期调控动作电位的编码频率和精确性。     

中枢神经系统内神经元信号处理(英文)

一种新颖的轴突断端(axon bleb)膜片钳记录方法大力促进了中枢神经系统轴突功能的研究。我们的工作应用这一方法揭示了大脑皮层锥体神经元的数码信号(具全或无特性的动作电位)的爆发和传播机制。在轴突始段(axon initial segment,AIS)远端高密度聚集的低阈值Na+通道亚型Nav1.6决定动作电位的爆发;而在AIS近端高密度聚集的高阈值Na+通道亚型Nav1.2促进动作电位向胞体和树突的反向传播。应用胞体和轴突的同时记录,我们发现胞体阈下膜电位的变化可以在轴突上传播较长的距离并可到达那些离胞体较近的突触前终末。进一步的研究证明了胞体膜电位的变化调控动作电位触发的突触传递,该膜电位依赖的突触传递是一种模拟式的信号传递。轴突上一类特殊K+通道(Kv1)的活动调制动作电位的波形,特别是其波宽,从而调控各种突触前膜电位水平下突触强度的变化。突触前终末的背景Ca2+浓度也可能参与模拟信号的传递。这些发现深化了我们对中枢神经系统内神经信号处理基本原理的认识,进而帮助我们理解脑如何工作。     

具有极性的分泌运输是神经元树突发育的形态和走向的基础

神经元在生长发育过程中会定向地生长出一根轴突和多根形态、功能不一的树突。虽然已经有一些关于轴突和树突分化的学说,但是关于树突形成时膜成分加载的细胞学机制仍不清楚。2005年11月Duke大学的Michael D．Ehlers实验室报道,神经元具有极性的分泌性运输方式是产生各种形态和方向树突的基础。在一般细胞内,高尔基体在细胞核周围呈现扁平膜囊的堆叠结构;而在神经元中,高尔基体既包括胞体的膜囊堆叠,又包括了树突内离散的高尔基体“前哨”（outpost;Golgi outposts指一些存在于树突中的高尔基体膜囊,本文译为“高尔基体前哨”）。Ehlers等的实验证明,在培养的海马锥体神经元中,后高尔基体的膜运输是朝向较长的树突的;而在体内,皮层和海马区的锥体神经元都有一根特化的向皮层表面行走的顶树突,后高尔基体的膜运输就朝向这根顶树突。他们发现,小的高尔基体“前哨”选择性地分布在较长的树突中,但不进入轴突。在树突中,高尔基体“前哨”常常集中在树突的分支处进行后高尔基体的运输。     

Regional and cellular distribution of neural visinin-like protein immunoreactivities (VILIP-1 and VILIP-3) in human brain

Neural visinin-like proteins (VILIPs) are members of the neuronal subfamily of intracellular EF-hand calcium sensor proteins termed the NCS family, which are thought to play important roles in cellular signal transduction. While numerous studies suggest a wide but uneven distribution of these proteins in rat and chicken brain, their location in, and possible significance for, the human brain, remains to be established. We used specific polyclonal antisera to map the human brain for VILIP-1 and VILIP-3 immunoreactivities. VILIP-1 was detected in cortical pyramidal cells and interneurons, septal, subthalamic and hippocampal neurons (subfields CA1 and CA4 pyramidal cells and especially hilar interneurons) as well as in cerebellar Golgi, basket, granule, stellate and dentate nucleus neurons. Purkinje cells were free of immunoreaction. VILIP-3 was more restricted in its distribution. It was identified in cerebellar Purkinje cells and a subpopulation of granule neurons. Further, neurons belonging to different nuclei of the brain stem and multiple subcortical nerve cells stained for visinin-like protein 3. A weak immunoreaction appeared in cortical and hippocampal neurons. Intracellularly the immunoreactivity appeared in the perikarya, dendrites and some axons. Sometimes, immunostaining was found in the neuropil. Glia did not express visinin-like proteins. Our findings support, from a neuroanatomical viewpoint, the idea that these calcium sensor proteins may be of relevance for neuronal signalling in the human CNS.     

In Situ Analysis of Na,K-ATPase α1- and α3-Isoform mRNAs in Aging Rat Hippocampus

Abstract: Age-related changes in the expression of Na,K-ATPase α1- and α3-isoform mRNAs were analyzed by in situ hybridization in the Fischer-344 rat hippocampus. Quantification of signal density with cRNA probes in rat hippocampus at 3 months of age showed (a) α1 content is 1.5 times higher in granule than in pyramidal cell layers, whereas α3 content shows the opposite ratio and (b) α3 label is found in large clusters related to mossy cells and basket cells and in medium clusters corresponding to interneurons within the dendritic fields of CA1–3. In the 24-month-old rats as compared with the young animals, the α1 signal is increased more than sevenfold in the dendritic fields and is not significantly changed in perikaryal layers. The α3 signal is reduced about threefold ( p < 0.0001, ANOVA, n = 6) in perikaryal layers, is almost completely absent over the interneurons, basket cells, and mossy cells, and is not significantly changed in dendritic fields. These data indicate age-related, cell- and isoform-specific alterations in pretranslational regulation of Na,K-ATPase α isoforms. The striking changes in the dendritic fields, mossy cells, and GABAergic basket cells and interneurons may constitute early and sensitive markers for age-related alterations in hippocampal function, before cell loss.     

Electrophysiological Heterogeneity of Fast-Spiking Interneurons: Chandelier versus Basket Cells

In the prefrontal cortex, parvalbumin-positive inhibitory neurons play a prominent role in the neural circuitry that subserves working memory, and alterations in these neurons contribute to the pathophysiology of schizophrenia. Two morphologically distinct classes of parvalbumin neurons that target the perisomatic region of pyramidal neurons, chandelier cells (ChCs) and basket cells (BCs), are generally thought to have the same “fast-spiking” phenotype, which is characterized by a short action potential and high frequency firing without adaptation. However, findings from studies in different species suggest that certain electrophysiological membrane properties might differ between these two cell classes. In this study, we assessed the physiological heterogeneity of fast-spiking interneurons as a function of two factors: species (macaque monkey vs. rat) and morphology (chandelier vs. basket). We showed previously that electrophysiological membrane properties of BCs differ between these two species. Here, for the first time, we report differences in ChCs membrane properties between monkey and rat. We also found that a number of membrane properties differentiate ChCs from BCs. Some of these differences were species-independent (e.g., fast and medium afterhyperpolarization, firing frequency, and depolarizing sag), whereas the differences in the first spike latency between ChCs and BCs were species-specific. Our findings indicate that different combinations of electrophysiological membrane properties distinguish ChCs from BCs in rodents and primates. Such electrophysiological differences between ChCs and BCs likely contribute to their distinctive roles in cortical circuitry in each species.     

Of mice and men, and chandeliers

How does the human neocortex reliably propagate information through neural circuits? One mechanism appears to involve relying on strong connections from pyramidal neurons to interneurons and a depolarizing action of cortical chandelier cells.     

Temporal redistribution of inhibition over neuronal subcellular domains underlies state-dependent rhythmic change of excitability in the hippocampus

The behaviour-contingent rhythmic synchronization of neuronal activity is reported by local field potential oscillations in the theta, gamma and sharp wave-related ripple (SWR) frequency ranges. In the hippocampus, pyramidal cell assemblies representing temporal sequences are coordinated by GABAergic interneurons selectively innervating specific postsynaptic domains, and discharging phase locked to network oscillations. We compare the cellular network dynamics in the CA1 and CA3 areas recorded with or without anaesthesia. All parts of pyramidal cells, except the axon initial segment, receive GABA from multiple interneuron types, each with distinct firing dynamics. The axon initial segment is exclusively innervated by axo-axonic cells, preferentially firing after the peak of the pyramidal layer theta cycle, when pyramidal cells are least active. Axo-axonic cells are inhibited during SWRs, when many pyramidal cells fire synchronously. This dual inverse correlation demonstrates the key inhibitory role of axo-axonic cells. Parvalbumin-expressing basket cells fire phase locked to field gamma activity in both CA1 and CA3, and also strongly increase firing during SWRs, together with dendrite-innervating bistratified cells, phasing pyramidal cell discharge. Subcellular domain-specific GABAergic innervation probably developed for the coordination of multiple glutamatergic inputs on different parts of pyramidal cells through the temporally distinct activity of GABAergic interneurons, which differentially change their firing during different network states.     

Computational modeling of GABA<Subscript>A</Subscript> receptor-mediated paired-pulse inhibition in the dentate gyrus

Paired-pulse inhibition (PPI) of the population spike observed in extracellular field recordings is widely used as a read-out of hippocampal network inhibition. PPI reflects GABA_A receptor-mediated inhibition of principal neurons through local interneurons. However, because of its polysynaptic nature, it is difficult to assign PPI changes to precise synaptic mechanisms. Here we used a detailed network model of the dentate gyrus to simulate PPI of granule cell action potentials and analyze its network properties. Our computational analysis indicates that PPI results mainly from a combination of perisomatic feed-forward and feedback inhibition of granule cells by basket cells. Feed-forward inhibition mediated by basket cells appeared to be the most significant source of PPI. Our simulations suggest that PPI depends more on somatic than on dendritic inhibition of granule cells. Furthermore, PPI was modulated by changes in GABA_A reversal potential (E_GABA) and by alterations in intrinsic excitability of granule cells. In summary, computer modeling provides a useful tool for determining the role of synaptic and intrinsic cellular mechanisms in paired-pulse field potential responses.     

Temporal dynamics of distinct CA1 cell populations during unconscious state induced by ketamine

Ketamine is a widely used dissociative anesthetic which can induce some psychotic-like symptoms and memory deficits in some patients during the post-operative period. To understand its effects on neural population dynamics in the brain, we employed large-scale in vivo ensemble recording techniques to monitor the activity patterns of simultaneously recorded hippocampal CA1 pyramidal cells and various interneurons during several conscious and unconscious states such as awake rest, running, slow wave sleep, and ketamine-induced anesthesia. Our analyses reveal that ketamine induces distinct oscillatory dynamics not only in pyramidal cells but also in at least seven different types of CA1 interneurons including putative basket cells, chandelier cells, bistratified cells, and O-LM cells. These emergent unique oscillatory dynamics may very well reflect the intrinsic temporal relationships within the CA1 circuit. It is conceivable that systematic characterization of network dynamics may eventually lead to better understanding of how ketamine induces unconsciousness and consequently alters the conscious mind.     

Gene expression in developing rat hippocampal pyramidal neurons appears independent of mossy fiber innervation.

In the rat, neonatal gamma-irradiation of the hippocampus induces a selective destruction of dentate granule cells and prevents the development of the mossy fiber-CA3 pyramidal cell connection. In the absence of mossy fiber input, the CA3 pyramidal neurons exhibit morphological alterations and rats deprived of dentate granule cells fail to develop kainate-induced epileptic activity in the CA3 pyramidal neurons. Neonatal elimination of the granule cells also impairs learning and memory tasks in adult rats. In the present work, we assessed by in situ hybridization and semi-quantitative RT-PCR, whether in the pyramidal layers, the absence of mossy fiber input alters the expression of a number of genes involved in activity-dependent signal transduction, in GABAergic neurotransmitter signaling and in neurite development via microtubule organization. Surprisingly, we show that the expression and the developmentally regulated alternative splicing of the genes we examined in the developing hippocampus are not altered in the pyramidal neurons, whether the dentate granule afferents are present or absent. Our results suggest that in the CA3 pyramidal layer, the developmental expression patterns of the mRNAs we studied are independent of extrinsic cues provided by mossy fiber input.     

Recruitment of Perisomatic Inhibition during Spontaneous Hippocampal Activity In Vitro

It was recently shown that perisomatic GABAergic inhibitory postsynaptic potentials (IPSPs) originating from basket and chandelier cells can be recorded as population IPSPs from the hippocampal pyramidal layer using extracellular electrodes (eIPSPs). Taking advantage of this approach, we have investigated the recruitment of perisomatic inhibition during spontaneous hippocampal activity in vitro. Combining intracellular and extracellular recordings from pyramidal cells and interneurons, we confirm that inhibitory signals generated by basket cells can be recorded extracellularly, but our results suggest that, during spontaneous activity, eIPSPs are mostly confined to the CA3 rather than CA1 region. CA3 eIPSPs produced the powerful time-locked inhibition of multi-unit activity expected from perisomatic inhibition. Analysis of the temporal dynamics of spike discharges relative to eIPSPs suggests significant but moderate recruitment of excitatory and inhibitory neurons within the CA3 network on a 10 ms time scale, within which neurons recruit each other through recurrent collaterals and trigger powerful feedback inhibition. Such quantified parameters of neuronal interactions in the hippocampal network may serve as a basis for future characterisation of pathological conditions potentially affecting the interactions between excitation and inhibition in this circuit.