Ⅰ型囊泡膜谷氨酸转运体在生后发育大鼠三叉神经运动核中的表达

目的观察Ⅰ型囊泡膜谷氨酸转运体在大鼠三叉神经运动核生后发育过程中的表达变化。方法取生后不同发育阶段大鼠脑干,行冰冻切片和Ⅰ型囊泡膜谷氨酸转运体免疫染色,光镜观察。结果刚出生大鼠三叉神经运动核背外侧部即可以观察到Ⅰ型囊泡膜谷氨酸转运体免疫染色阳性结构,随着发育进展,免疫染色阳性逐渐增加,出生7天后Ⅰ型囊泡膜谷氨酸转运体免疫染色模式接近成年水平。结论大鼠三叉神经运动核内Ⅰ型囊泡膜谷氨酸转运体阳性终末在出生后1周内较快地成熟。     

激光共聚焦扫描显微镜和透射电镜观察大鼠纹状体内谷氨酸能突触连接的对比观察

目的 探讨使用激光共聚焦扫描显微镜 (Laser scanning confocal microscope,LSCM)观察大鼠纹状体内谷氨酸能突触连接的方法的可行性.方法 12只正常大鼠分为两组,6只大鼠进行纹状体中等棘刺神经元的CM-DiI 单细胞标记,然后Ⅰ型囊泡膜谷氨酸转运体(vesicular glutamate transporter 1,VGluT1 )免疫荧光标记,LSCM层扫后三维重建,观察VGluT1阳性位点在中等棘刺神经元树突上的分布.另外6只大鼠用TEM观察不对称性突触在纹状体神经元树突上的分布.对两种方法的结果进行比较.结果 用LSCM 和TEM方法观察到的纹状体神经元上谷氨酸能突触连接分布情况一致,没有统计学差异.但LSCM更具优越性的是,可以对图像进行三维重构,从而有利于对神经元之间突触连接的空间分布观察和定量分析.结论 神经细胞荧光标记技术结合LSCM观察是考察纹状体神经元上谷氨酸能突触连接的有效方法.     

Ⅱ型囊泡膜谷氨酸转运体阳性终末与γ-氨基丁酸阳性神经元在小鼠腰髓背角的分布及联系

目的 研究Ⅱ型囊泡膜谷氨酸转运体(vesicular glutamate transporter 2,VgluT2)阳性终末与γ-氨基丁酸(γ-aminobutyric acid,GABA)阳性神经元在小鼠腰髓背角的分布和联系。方法采用免疫组织化学方法研究VgluT2阳性终末与GABA阳性神经元在小鼠腰髓背角的分布;采用免疫荧光组织化学双重标记方法研究VgluT2阳性终末与GABA阳性神经元在小鼠腰髓背角的联系。结果VgluT2阳性终末与GABA阳性神经元在小鼠腰髓背角各层均有分布,特别是在Ⅱ层内侧部二分布都较为密集,免疫荧光双重标记后在激光共聚焦显微镜下可见GABA阳性神经元周围有许多VGluT2阳性终末与其胞体或突起密切接触。结论小鼠腰髓背角Ⅱ层内侧部GABA阳性神经元直接接受兴奋性传入。     

迷走神经运动背核内心迷走神经元的GABA能突触前支配受谷氨酸能紧张性调控

谷氨酸能和GABA能支配是心迷走节前神经元(cardiac vagal neuron,CVN)的主要兴奋性和抑制性突触传入.在CVN的活动调节中,这两种支配是否有相互作用、以及如何相互作用目前尚不清楚.本研究用神经元逆行荧光染料标记法和电压膜片钳方法证明,谷氨酸NMDA型和非NMDA型受体拮抗剂AP5和CNQX在全脑片应用条件下,对疑核(nucleus ambiguus,NA)内CVN的GABA能突触前活动无明显影响,而对迷走神经运动背核(dorsal motor nucleus ofthe vagus,DMNX)内CVN的GABA能突触前活动有显著的抑制作用.这些观察结果提示支配迷走神经运动背核内CVN的GABA能神经元可能接受紧张性谷氨酸能支配,而支配疑核内CVN的GABA能神经元则没有这种紧张性谷氨酸能支配.疑核内和迷走神经运动背核内CVN的这种调节差异,是两个核团的CVN在心率和心功能调节中功能分工的可能机制之一.     

与咬肌运动神经元直接联系的运动前神经元在脑干的分布

目的为了定位向咬肌运动神经元投射的最后一级运动前神经元在脑干内的分布。方法注射麦芽凝集素结合的辣根过氧化物酶(WGA-HRP)至咬肌神经逆行跨突触追踪,然后通过免疫组织化学方法显示了该类神经元。结果这类神经元分布在双侧三叉上核(Vsup)、三叉神经感觉主核背侧部(Vpdm)、小细胞网状结构(PCR)和三叉神经脊束核吻侧亚核背侧部(Vodm),以及对侧三叉神经运动核(Vmo)。数量上,Vsup,特别是注射侧Vsup中,标记的神经元数量最多;其他核团内,双侧标记的神经元的数量无明显差别。结论一侧咬肌运动神经元直接接受脑干双侧多个区域调控。     

中枢神经系统谷氨酸转运体的研究进展

在中枢神经系统,谷氨酸转运体在谷氨酸一谷氨酰胺循环中发挥着重要作用。谷氨酸转运体有高亲和力转运体,即兴奋性氨基酸转运体（excitatory amino acid transporters,EAATs）和低亲和力转运体,即囊泡谷氨酸转运体（vesicular glutamate transporters,VGLUTs）两种类型。其中,VGLUTs的功能是特异地将突触囊泡外的谷氨酸转运进入突触囊泡内,它包括三个成员,分别是VGLUT1、VGLUT2和VGLUT3。一方面,VGLUT1和VGLUT2标记了所有的谷氨酸能神经元,是谷氦酸能神经元和它们轴突末端高度特异的标志;另一方面,VGLUT1标志着皮质一皮质投射,而VGLUT2则标志着丘脑一皮层投射,VGLUT3则位于抑制性突触末端。     

大鼠脑内代谢型谷氨酸受体5亚型(mGluR5)定位分布的免疫细胞化学研究

本研究用免疫细胞化学技术观察了大鼠脑内参与兴奋性突触传递的代谢型谷氨酸受体5亚型(mGluR5)的精确定位分布.mGluR5阳性浓染的神经元胞体和纤维密集地分布于大脑皮质浅层、嗅球、伏核、尾壳核、前脑基底部、隔区、苍白球、腹侧苍白球、海马CA1和CA2区、下丘中央核、被盖背侧核和三叉神经脊束核尾侧亚核浅层;淡染而稀疏的mGluR5阳性神经元胞体和纤维见于屏状核、终纹床核、杏仁中央核、丘脑部分核团、上丘浅灰质层、外侧丘系背侧核和延髓中央灰质.     

大鼠第三脑室及中脑水管多巴胺能触液神经元的分布

目的观察大鼠第三脑室、中脑水管及中缝背核内多巴胺能触液神经元的分布情况.方法应用CB逆行追踪、TH免疫组织化学和CB/TH免疫荧光双重标记技术,观察多巴胺能触液神经元在间脑及中脑内的分布情况.结果 TH免疫阳性触液神经元分布在第三脑室尾侧部和中脑水管全程的腹侧室管膜上及室管膜内,其胞体呈倒置梨形、圆形或椭圆形、多角形和梭形;在中缝背核内可见少量CB/TH免疫荧光双重标记的远位触液神经元;另在正中隆起部位TH免疫阳性神经末梢含量丰富.结论大鼠第三脑室、中脑水管及中缝背核内存在多巴胺能触液神经元,其在脑-脑脊液之间的信息传递中有着重要的作用.     

友鼠外侧膝状体中继神经元HRP示踪结合谷氨酸免疫组织化学研究

采用 HRP逆行追踪结合谷氨酸免疫组织化学方法观察大鼠外侧膝状体背侧核 (d L GN)中继神经元的化学递质。光镜下 HRP标记细胞与谷氨酸免疫阳性细胞清晰可辩。HRP单标记细胞位于外侧膝状体背侧核内 ,胞浆及树突基部充满棕色颗粒。免疫金银法 (IGSS)单标记的谷氨酸免疫阳性神经元分布于外侧膝状体背侧核与腹侧核 ,胞体内充满黑色银颗粒。在外侧膝状体背侧核内可见 HRP和谷氨酸双标记细胞 ,其数目占 HRP标记细胞总数的 70 .9± 6 .4%。本文提示 ,谷氨酸可能是外侧膝状体背侧核投射至视皮质的中继神经元的神经递质之一。     

大鼠中缝核群中生长抑素样神经元对咽肌前运动神经元的支配-PRV和SOM免疫荧光双标记法研究

用PRV和SOM免疫荧光双标记法研究了大鼠中缝核群中SOM样神经元对咽肌前运动神经元的调控。PRV注射大鼠咽肌后,在中缝苍白核、中缝隐核、中缝大核和中缝背核中可见少量PRV和SOM双标记细胞。首次证明了大鼠中缝核群中SOM样神经元对咽肌前运动神经元的支配。推测中缝核群中的SOM可能和咽肌运动的精确调控有关。     

Vesicular glutamate transporter 1 immunoreactivity in extrinsic and intrinsic innervation of the rat esophagus

Encouraged by the recent finding of vesicular glutamate transporter 2 (VGLUT2) immunoreactivity (-ir) in intraganglionic laminar endings (IGLEs) of the rat esophagus, we investigated also the distribution and co-localization patterns of VGLUT1. Confocal imaging revealed substantial co-localization of VGLUT1-ir with selective markers of IGLEs, i.e., calretinin and VGLUT2, indicating that IGLEs contain both VGLUT1 and VGLUT2 within their synaptic vesicles. Besides IGLEs, we found VGLUT1-ir in both cholinergic and nitrergic myenteric neuronal cell bodies, in fibers of the muscularis mucosae, and in esophageal motor endplates. Skeletal neuromuscular junctions, in contrast, showed no VGLUT1-ir. We also tested for probable co-localization of VGLUT1-ir with markers of extrinsic and intrinsic esophageal innervation and glia. Within the myenteric neuropil we found, besides co-localization of VGLUT1 and substance P, no further co-localization of VGLUT1-ir with any of these markers. In the muscularis mucosae some VGLUT1-ir fibers were shown to contain neuronal nitric oxide synthase (nNOS)-ir. VGLUT1-ir in esophageal motor endplates was partly co-localized with vesicular acetylcholine transporter (VAChT)/choline acetyltransferase (ChAT)-ir, but VGLUT1-ir was also demonstrated in separately terminating fibers at motor endplates co-localized neither with ChAT/VAChT-ir nor with nNOS-ir, suggesting a hitherto unknown glutamatergic enteric co-innervation. Thus, VGLUT1-ir was found in extrinsic as well as intrinsic innervation of the rat esophagus.     

Identification of alpha and gamma trigeminal motoneurons by the vibratome paraplast technique for HRP histochemistry

A morphometric analysis of the masseteric motoneuron pool of the trigeminal motor nucleus was performed in the rat using horseradish peroxidase as a marker. Thick (40 microns) cryosections and thin (7 microns) Paraplast sections were compared. Two types of motoneurons related to the masseter muscle were observed. Small motoneurons, which had a high nuclear index, were found interspersed between large motoneurons, which had more cytoplasm. Evidence is provided that the small trigeminal motoneurons are gamma neurons that innervate the intrafusal muscle fibers of the masseteric muscle spindles.     

Distribution of vesicular glutamate transporter 1 (VGLUT1) in the mouse esophagus

In rat and mouse esophagus, vesicular glutamate transporter 2 (VGLUT2) has been demonstrated to identify vagal intraganglionic laminar endings (IGLEs); this has recently also been shown for VGLUT1 in rat esophagus. In this study, we have investigated the distribution of VGLUT1 in the mouse esophagus and compared these results with the recently published data from the rat esophagus. Unexpectedly, we have discovered that VGLUT1 mostly fails to identify IGLEs in the mouse esophagus. This is surprising, since the distribution of VGLUT2 shows comparable results in both species. Confocal imaging has revealed substantial colocalization of VGLUT1 immunoreactivity (-ir) with cholinergic and nitrergic/peptidergic markers within the myenteric neuropil and in both cholinergic and nitrergic myenteric neuronal cell bodies. VGLUT1 and cholinergic markers have also been colocalized in fibers of the muscularis mucosae, whereas VGLUT1 and nitrergic markers have never been colocalized in fibers of the muscularis mucosae, although this does occur in fibers of the muscularis running to motor endplates. Thus, VGLUT1 is contained in the nitrergic innervation of mouse esophageal motor endplates, another difference from the rat esophagus. VGLUT1-ir is therefore present in extrinsic and intrinsic innervation of the mouse esophagus, but the significant differences from the rat indicate species variations concerning the distribution of VGLUTs in the peripheral nervous system. This study was supported by the Johannes und Frieda Marohn-Stiftung, Erlangen.     

Doublecortin is expressed in trigeminal motoneurons that innervate the velar musculature of lampreys: considerations on the evolution and development of the trigeminal system

Studies in lampreys have revealed interesting aspects of the evolution of the trigeminal system and the jaw. In the present study, we found a marker that distinguishes subpopulations of trigeminal motoneurons innervating two different kinds of oropharyngeal muscles. Immunofluorescence with an antibody against doublecortin (DCX; a neuron-specific phosphoprotein) enabled identification of the trigeminal motoneurons that innervate the velar musculature of larval and recently transformed sea lampreys. DCX-immunoreactive (-ir) motoneurons were observed in the rostro-lateral part of the trigeminal motor nucleus of these animals, but not in lampreys 1 month or more after metamorphosis. Combined double DCX/tubulin and serotonin/tubulin immunofluorescence and tract-tracing experiments with neurobiotin (NB) were also performed in larvae for further characterization of this system. Rich innervation by DCX-ir fibers was observed on the muscle fibers of the velum but not on the upper lip or lower lip muscles, which were innervated by tubulin-ir/DCX-negative fibers. No double-labelled DCX-ir motoneurons were observed in experiments in which the tracer NB was applied to the upper lip. Innervation of velar muscles by serotonergic fibers is also reported. The present results indicate that development of the trigeminal motoneurons innervating the velum differs from that of the trigeminal motoneurons innervating the lips, which is probably related to the dramatic regression of the velum during metamorphosis. The absence of data on a similar subsystem in the trigeminal motor nucleus of gnathostomes suggests that they may be lamprey-specific motoneurons. These results provide support for the "heterotopic theory" of jaw evolution and are inconsistent with the theories of a velar origin for the gnathostome jaw.     

Influence of peripheral targets on the expression of calcitonin gene-related peptide immunoreactivity in rat cranial motoneurones

Calcitonin gene-related peptide-like immunoreactivity (CGRP-ir) is displayed by motoneurons that innervate striated muscle but is absent from preganglionic parasympathetic motoneurons. One hypothesis to explain this is that CGRP gene expression in motoneurons is, in part, dependent on influences from the innervated organ. To test this hypothesis, we cross-anastomosed the right hypoglossal and cervical vagal nerves of rats so that the vagal motoneurons grew to innervate the musculature of the tongue. Following a recovery period of 17 to 52 weeks, the distribution of CGRP-ir in the dorsal motor vagal nucleus was determined in both cross-anastomosed animals and self-anastomosed control animals. Successful reinnervation of the tongue musculature by vagal motoneurons was demonstrated by showing that electrical stimulation of the central vagus/peripheral hypoglossal nerve produced a twitch of the tongue muscles. Motoneurones of the dorsal motor vagal nucleus, which now innervated the tongue were found to express CGRP-ir, which was evident from the double labeling of neurons with both horseradish peroxidase and CGRP-ir. Motoneurones of the dorsal motor vagal nucleus contralateral to the cross-anastomosis remained CGRP negative. Similarly, motoneurons of the dorsal motor vagal nucleus in control animals where the vagus nerve was self-anastomosed remained CGRP negative, showing that an induction of CGRP expression is not a result of nerve section itself. We suggest that a signal from the striated muscle transported retrogradely via the motor axon regulates expression of CGRP-ir in motoneurons. © 1995 John Wiley & Sons, Inc.     

雌激素Beta受体在大鼠脑内表达的免疫组化定位研究

为了探讨雌激素作用于神经系统的机理,采用硫酸镍铵增强显色的免疫组化SP法研究了新的雌激素受体（ER－β）在成年雌雄大鼠脑内的分布。研究证实ER－β免疫阳性物质主要位于神经元的细胞核内,但在个别脑区也可在胞浆甚至突起内检测到。最强的ER－β免疫阳性信号见于前嗅核、大脑皮质、小脑浦肯野细胞、斜角带垂直部、蓝斑和三叉神经运动核等部位;中等强度的染色见于隔内侧核、杏仁外侧核、黑质、中央灰质等部位;较弱的阳性反应见于下丘脑与杏仁复合体的部分核团。在一些部位还存在表达水平甚至细胞内定位模式的性别差异,如前庭上核内的表达只见于雌性;雄性大鼠三叉神经运动核内ER－β蛋白主要表达于胞浆内,细胞核为阴性;而在雌性大鼠该部位ER－β蛋白主要位于细胞核等。以上结果表明ER－β蛋白在大鼠脑内分布广泛并具有一定的性别差异,在与学习记忆有关的脑区如大脑皮质和基底前脑内有很高的表达,提示在脑组织内雌激素可能通过ER－β这一新的信号途径发挥多种重要的调控作用,如学习记忆等。     

Endogenous presynaptic nitric oxide supports an anterograde signaling in the central nervous system

The source size and density determine the extent of nitric oxide (NO) diffusion which critically influences NO signaling. In the brain, NO released from postsynaptic somas following NMDA-mediated activation of neuronal nitric oxide synthase (nNOS) retrogradely affects smaller presynaptic targets. By contrast, in guinea pig trigeminal motor nucleus (TMN), NO is produced presynaptically by tiny and disperse nNOS-containing terminals that innervate large nNOS-negative motoneurons expressing the soluble guanylyl-cyclase (sGC); consequently, it is uncertain whether endogenous NO supports an anterograde signaling between pre-motor terminals and postsynaptic trigeminal motoneurons. In retrogradely labeled motoneurons, we indirectly monitored NO using triazolofluorescein (DAF-2T) fluorescence, and evaluated sGC activity by confocal cGMP immunofluorescence. Multiple fibers stimulation enhanced NO content and cGMP immunofluorescence into numerous nNOS-negative motoneurons; NOS inhibitors prevented depolarization-induced effects, whereas NO donors mimicked them. Enhance of cGMP immunofluorescence required extracellular Ca(2+), a nNOS-physiological activator, and was prevented by inhibiting sGC, silencing neuronal activity or impeding NO diffusion. In conclusion, NO released presynaptically from multiple cooperative tiny fibers attains concentrations sufficient to activate sGC in many motoneurons despite of the low source/target size ratio and source dispersion; thus, endogenous NO is an effective anterograde neuromodulator. By adjusting nNOS activation, presynaptic Ca(2+) might modulate the NO diffusion field in the TMN.     

Neuromechanisms of reciprocal interrelation between jaw-opening and jaw-closing muscles in the cat (author's transl)]

Neural controlling mechanisms between the digastric (jaw-opening) and masseter (jaw-closing) muscles were studied in the cat. High threshold afferent impulses from the anterior belly of the digastric muscle to masseteric montoneurons in the trigeminal motor nucleus induced an EPSP-IPSP sequence of potentials with long latency, and high threshold afferent impulses from the masseter muscle also exerted a similar effect on digastric motoneurons in the same nucleus innervating the anterior belly of the digastric muscle. These results suggest that reciprocal inhibition via Ia interneurons as observed between the flexor and extensor muscles in the spinal cord does not exist between the digastric and masseter muscles in the cat. However, the respective motoneurons innervating the masseter and digastric muscles receive inputs of early excitation-late inhibition via high threshold afferent nerve fibers from each antagonistic muscle. As such, since EPSPs preceding IPSPs are recognized, these high threshold afferent impulses may exert not only a reciprocal inhibitory effect, but also a synchronous excitatory or inhibitory effect on the antagonistic motoneurons.     

Immunohistochemical localization of insulin-like growth factor binding protein 2 in the central nervous system of SOD1<Superscript>G93A</Superscript> transgenic mice

In the present study, we performed immunohistochemical studies to investigate the changes of insulin-like growth factor binding protein 2 (IGFBP2) in the central nervous system of SOD1^G93A mutant transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS). Decreased immunoreactivity for IGFBP2 was observed in the cerebral cortex, hippocampus and brainstem of SOD1^G93A transgenic mice. In the cerebral cortex, the number of IGFBP2-positive cells was decreased in the somatomotor area, somatosensory area, auditory area, visual area, entorhinal area, piriform area and prefrontal area. In the hippocampal formation, IGFBP2 immunoreactivity was significantly decreased in the CA1-3 areas and the dentate gyrus. In the brainstem, few IGFBP2-immunoreactive cells were observed in the medullary and pontine reticular formation, vestibular nucleus, trigeminal motor nucleus, facial nucleus, hypoglossal nucleus and raphe nucleus. In the spinal cord, IGFBP2 immunoreactivity was not significantly decreased in SOD1^G93A transgenic mice. This study showing decreased IGFBP2 in different brain regions of SOD1^G93A transgenic mice may provide clues for understanding differential susceptibility of neural structures in ALS. S. E. Sim and Y. H. Chung have contributed equally to this work.