大鼠GAD样、甘氨酸样和SP样阳性终末与三叉神经中脑核神经元接触--Confocal显微镜观察

应用免疫荧光组织化学双重染色和三重染色技术,在激光共聚焦显微镜下观察了大鼠三叉神经中脑核(Vme)内谷氨酸脱羧酶(GAD)样、甘氨酸(Gly)样和P物质(SP)样阳性终末与磷酸激活的谷氨酰胺酶(PAG)样阳性神经元之间的联系.结果显示:①几乎所有的Vme神经元均呈PAG样免疫阳性,这些神经元绝大多数为大的假单极神经元.②密集分布的GAD样或Gly样免疫阳性的神经终扣分别聚集于PAG样阳性的Vme神经元胞体周围,并与之形成密切接触.③SP样免疫阳性终末与PAG样阳性的Vme神经元形成接触,且这些终末中有部分与5-羟色胺(5-HT)免疫活性物质共存.以上结果表明:GABA能、Gly能和SP能投射至Vme的神经终末,可能在初级神经元即Vme水平对谷氨酸介导的口面部本体觉信息的传递具有抑制性调控作用.     

大鼠GAD样,甘氨酸样和SP样阳性终末与三叉神经中脑核神经 …

应用免疫荧乐组织化学双重染色和三重染色技术,在激光共聚焦显微镜下观察了大鼠三叉神经中脑核（Ｖｍｅ）内谷氨酸脱羧酶（ＧＡＤ）样、甘氨酸（Ｇｌｙ）样和Ｐ物质（ＳＰ）样阳性终末与磷酸激活的谷氨酰胺酶（ＰＡＧ）样阳性神经元之间的联系。结果显示：①几乎所有的Ｖｍｅ神经元均呈ＰＡＧ样免疫阳性,这些神经元绝大多数为大的假单极神经元。②密集分布的ＧＡＤ样或Ｇｌｙ样免疫阳性的神经终扣分别聚集于ＰＡＧ样阳性的Ｖｍｅ神     

大鼠三叉神经中脑核内囊泡膜谷氨酸转运体样和GAD样阳性末梢与GABAA受体α1亚单位阳性神经元的联系

最近已在哺乳类动物脑内克隆出两种新的脑内特殊的Na^ 依赖的无机磷酸转运体，它们均属于囊泡膜谷氨酸转运体，被分别命名为VGluT1(Vesicular glutamate transporter of typel)和DNPI（differentiation-associated Na^ -dependent inorganic phosphate cotransporter).本研究应用免疫荧光组织化学三重染色技术，在激光共聚焦显微镜下观察了大鼠三叉神经中脑核（Vme）内VGluT1样和DNPI样以及谷氨酸脱羧酶（GAD）样阳性末梢与GABAA受体α1亚单位（GABAA Rα1）样阳性神经元之间的联系。结果显示：①几乎所有的Vme神经元均呈GABAA受体α1亚单位样免疫阳性，吻尾方向在其全长出现，这些神经元绝大多数为大的假单极神经元（直径为25－50μm），但也有小部分为直径小于25μm的神经元。②大量的VGluT1样长DNPI样免疫阳性的神经纤维末梢广泛分布于Vme内，其中DNPI样阳性纤维和末梢的分布密度高于VGluT1样和DNPI样免疫阳性的神经纤维和末梢广泛分布于Vme内，其中DNPI样阳性纤维和末梢的分布密度高于VGluT1；同时还观察到GAD样阳性纤维和末梢也密集分布于Vme内。③VGluT1样、DNPI样和GAD样免疫阳性终扣分别包绕在GABAA受体α1亚单位样阳性Vme神经元胞体周围，并与之形成密切接触。以上结果提示Vme神经元在介导顺口部本体感觉信息的传递中，可能同时接受中枢其他来源的谷氨酸能和GABA能末梢的调控，其中GABA能的投身末梢可能通过位于Vme神经元内的GABAA受体对面部口部本体感觉信息的传递发挥抑制性调控作用。     

大鼠三联单叉神经中脑核内囊泡膜谷氨酸转运体样和GAD样阳性末梢与GABAA受体α1亚单位样阳性神经元的联系

最近已在哺乳类动物脑内克隆出两种新的脑内特殊的Na+依赖的无机磷酸转运体,它们均属于囊泡膜谷氨酸转运体,被分别命名为VGluTl(Vesicular glutamate transporter of type1)和DNPI(differentiation-associatedNa+-dependent inorganicphosphate eotransporter).本研究应用免疫荧光组织化学三重染色技术,在激光共聚焦显微镜下观察了大鼠三叉神经中脑核(Vine)内VGIuT1样和DNPI样以及谷氨酸脱羧酶(GAD)样阳性末梢与GABAA受体α1亚单位(GABAARa1)样阳性神经元之间的联系.结果显示:①几乎所有的Vme神经元均呈GABAA受体α1亚单位样免疫阳性,吻尾方向在其全长出现,这些神经元绝大多数为大的假单极神经元(直径为25～50um),但也有小部分为直径小于25um的神经元.②大量的VGluT1样和DNPI样免疫阳性的神经纤维和末梢广泛分布于Vine内,其中DNPI样阳性纤维和末梢的分布密度高于VGIuT1;同时还观察到GAD样阳性纤维和末梢也密集分布于Vme内.③VGluT1样、DNPI样和GAD样免疫阳性终扣分别包绕在GABAA受体α1亚单位样阳性Vine神经元胞体周围,并与之形成密切接触.以上结果提示:Vme神经元在介导面口部本体感觉信息的传递中,可能同时接受中枢其他来源的谷氨酸能和GABA能末梢的调控,其中GABA能的投射末梢可能通过位于Vine神经元内的GABAA受体对面口部本体感觉信息的传递发挥抑制性调控作用.     

大鼠三叉-小脑投射神经元表达磷酸激活的谷氨酰胺酶——双标法研究

磷酸激活的谷氨酰胺酶(phosphate activated glutaminase,PAG)可作为谷氨酸能神经元的标志物.采用逆行束路追踪技术和PAG免疫荧光组织化学法,研究脑干三叉核团复合体中向小脑袢状小叶Crusl区投射神经元是否呈PAG样免疫反应阳性.结果表明:将荧光金(Fluoro-Gold,FG)注入小脑袢状小叶Crusl区后,在三叉神经感觉主核(Pr5)、三叉神经脊束核极间亚核(Sp5I)和吻侧亚核背内侧部(Sp5DM)、三叉间核(15)以及三叉神经运动核与上橄榄核之间的一个小区域(nucleus J)中均可见到FG逆行标记细胞.标记细胞出现在上述核团的双侧,但以同侧为主.PAG免疫荧光组织化学反应结果显示:许多FG标记神经元呈强PAG样免疫阳性,其中FG/PAG双标神经元占FG逆行标记神经元总数的73%～94%.结果提示:脑干三叉神经核团中的谷氨酸能神经元可能参与触觉信息从面口部向小脑的传递.     

大鼠三叉—小脑投射神经元表达磷酸激活的谷氨酰胺酶——双标法研究

磷酸激活的谷氨酰胺酶 (phosphateactivatedglutaminase,PAG)可作为谷氨酸能神经元的标志物。采用逆行束路追踪技术和PAG免疫荧光组织化学法 ,研究脑干三叉核团复合体中向小脑袢状小叶Crus1区投射神经元是否呈PAG样免疫反应阳性。结果表明 :将荧光金 (Fluoro Gold ,FG)注入小脑袢状小叶Crus1区后 ,在三叉神经感觉主核 (Pr5 )、三叉神经脊束核极间亚核 (Sp5I)和吻侧亚核背内侧部 (Sp5DM)、三叉间核 (I5 )以及三叉神经运动核与上橄榄核之间的一个小区域 (nucleusJ)中均可见到FG逆行标记细胞。标记细胞出现在上述核团的双侧 ,但以同侧为主。PAG免疫荧光组织化学反应结果显示 :许多FG标记神经元呈强PAG样免疫阳性 ,其中FG/PAG双标神经元占FG逆行标记神经元总数的 73%～ 94 %。结果提示 :脑干三叉神经核团中的谷氨酸能神经元可能参与触觉信息从面口部向小脑的传递。     

大鼠三叉神经尾侧亚核内GABAA受体和GABA,GAD的分布

免疫细胞化学方法观察了GABAA受体（GABAAR）亚单位、GABA和GAD在大鼠三叉神经尾测亚核内的分布及其匹配关系。结果表明：GABAARα亚单位免疫阳性纤维主要分布于二层内侧都（Ⅱi），而GABAARβ亚单位免疫阳性纤维在鳖个Ⅱ层和Ⅲ层均有密集分布．GABAA的α亚单位免疫阳性胞体只在Vc的Ⅰ、Ⅲ层有散在分布．GABAAR的β亚单位免疫阳性胞体在Vc的Ⅲ层有密集分布．GABAA及其合成酶GAD免疫阳性终末在Vc浅层有密集分布。以上结果表明GABAR其合成酶GAD与GABAAR的分布总体上是相互匹配的，但GABAR各亚单位在Vc内的分布在在差异，提示Vc内的GABAA可能通过作用于CABAAR的不同亚单位而产生多种生理功能。     

痛刺激后大鼠三叉神经尾侧亚核星形细胞和神经元相互关系的电镜观察

目的：观察大鼠上唇皮下注射福尔马林后，三叉神经尾侧亚核（Sp5C)内反应性星形细胞和神经元之间相互关系的超微结构。方法：用DAB染色的抗胶质原纤维酸性蛋白（GFAP）、抗connexin43(Cx43）和金颗粒标记抗Cx32双标记免疫电镜方法。结果：电镜下观察到Sp5C内反应性星形细胞和神经元之间存在4种联系结构：第一种是突触样结构；第二种是三种成分的突触复合体，第三种是缝隙连接；第四种是由Cx32和Cx43构成的异源性缝隙连接（HGJ）。HGJ表现为两侧膜增厚，Cx43阳性物质和Cx32阳性金颗粒分别位于星形细胞和神经元一侧，痛刺激后HGJ数明明显增加。结论：神经元和星形细胞之间有多种信息通道，HGJ可能是一种快速，适应性信息通道。Sp5C星形细胞可能通过HGJ调节神经元的活动，共同参与中枢神经系统对刺激反应的调节。     

大鼠三叉神经脊束核尾侧亚核浅层内GABA样、L-ENK样、GlyT2样阳性末梢与Sindibis病毒转染表达GFP神经元之间的联系

应用GFP基因重组病毒标记技术和免疫荧光组织化学技术,在荧光显微镜和激光共聚焦扫描显微镜下观察了大鼠三叉神经脊束核尾侧亚核(Vc)浅层内GABA样、L-ENK样、GlyT2样阳性纤维和末梢与Sindibis病毒转染表达GFP神经元之间的联系.结果显示:①将GFP基因重组Sindibis病毒注入一侧Vc浅层后,仅在同侧vc注射部位附近的Ⅰ～Ⅲ层内观察到4～8个逆标的GFP神经元,这些神经元的胞体为小型(直径≤15 μm)圆形、梭形或不规则形;②Vc浅层内均可见大量的GABA样、L-ENK样、GlyT2样阳性纤维和末梢,以Ⅰ和Ⅱ层分布最为密集;③GABA样、L-ENK样、GlyT2样阳性纤维和末梢分别聚集于GFP标记神经元的胞体或树突周围,并与之形成密切接触.以上结果表明:Vc浅层内GFP神经元可能与GABA能、L-ENK能、Gly能阳性纤维和末梢形成突触联系并接受这些抑制性神经末梢的调控.     

Response characteristics of primary periodontal mechanoreceptive neurons in the trigeminal mesencephalic nucleus to trapezoidal mechanical stimulation of a single tooth in the rat

The response characteristics of primary periodontal mechanoreceptive neurons in the trigeminal mesencephalic nucleus (MeNV) were studied by changing the rate and magnitude of trapezoidal pressure applied to the upper incisor in very lightly anesthetized rats.Using a metal microelectrode in the MeNV either for recording or for stimulation, the projection site of primary afferents in the anterior superior alveolar nerve innervating the upper incisor was determined. Its stereotaxic coordinates were 1.0–2.0 mm posterior to the interaural plane and 1.2–1.3 mm lateral to the mid-sagittal plane, corresponding to the caudal part of the MeNV.From this site of 19 animals, single unitary activity from 41 primary periodontal mechanoreceptive neurons was recorded, which were identified by: (1) the constant, brief latency of 1.0–4.0 ms (2.0 ± 0.6ms, mean±S.D.) from the onset of single tooth tapping; (2) the wave forms of their unitary spike responses; and (3) the ability to follow faithfully trains of repetitive stimuli applied to the anterior superior alveolar nerve at rates of more than 100 Hz for 2 s. The responses of the identified primary mechanoreceptive neurons in the MeNV were tested for repetitive tapping and trapezoidal pressure to the ipsilateral upper incisor. The highest frequency of one-to-one following was observed in repetitive tapping at 100 Hz. All of the 27 tested neurons exhibited such very rapid adaptation as to show only on-off responses to trapezoidal pressures, and did not respond at all unless the pressures were applied more rapidly than 0.6 Newton/s (N/s). In most of these neurons, the number of evoked spikes was greater in on-responses than in off-responses, and the number and frequency of spikes were increased with an increase in the rate of pressure application. But in several neurons only one spike was triggered even when the pressures were applied at the rate of more than 83.1 N/s.     

Enkephalin-, substance P- and serotonin-like immunoreactive axonal varicosities in close apposition to perikarya of mesencephalic trigeminal nucleus neurons in the cat

A light microscopic study in adult cats provided evidence suggesting that neuronal cell bodies of mesencephalic trigeminal nucleus neurons were often in direct contact with axonal varicosities showing enkephalin-, substance P- or serotonin-like immunoreactivity.     

Glycine-immunoreactive terminals in the rat trigeminal motor nucleus: light- and electron-microscopic analysis of their relationships with motoneurones and with GABA-immunoreactive terminals

Post-embedding immunolabelling methods were applied to semi-thin and ultrathin resin sections to examine the relationships between glycine- and γ-aminobutyric acid (GABA)-immunoreactive terminals on trigeminal motoneurones, which were identified by the retrograde transport of horseradish peroxidase injected into the jaw-closer muscles. Serial sections were cut through boutons and alternate sections were incubated with antibodies to glycine and GABA. Light-microscopic analysis of semi-thin sections revealed a similar pattern of glycine and GABA-immunoreactive boutons along the motoneurone soma and proximal dendrites, and of immunoreactive cell bodies in the parvocellular reticular and peritrigeminal areas surrounding the motor nucleus. Immunoreactive synaptic terminals on motoneurones were identified on serial ultrathin sections at electron-microscopic level using a quantitative immunogold method. Three populations of immunolabelled boutons were recognized: boutons immunoreactive for glycine alone (32%), boutons immunoreactive for GABA alone (22%), and boutons showing co-existence of glycine and GABA immunoreactivities (46%). Terminals which were immunoreactive for glycine only contained a higher proportion of flattened synaptic vesicles than those which were immunoreactive for GABA only, which contained predominantly spherical vesicles. Terminals which exhibited both immunoreactivities contained a mixture of vesicle types. All three classes of terminal formed axo-dendritic and axo-somatic contacts onto retrogradely labelled motoneurones. A relatively high proportion (25%) of boutons that were immunoreactive for both transmitters formed synapses on somatic spines. However, only GABA-immunoreactive boutons formed the presynaptic elements at axo-axonic contacts: none of these were found to contain glycine immunoreactivity. These data provide ultrastructural evidence for the role of glycine and GABA as inhibitory neurotransmitters at synapses onto jaw-closer motoneurones, but suggest that presynaptic control of transmission at excitatory (glutamatergic) synapses on motoneurones involves GABAergic, but not glycinergic inhibition.     

大鼠三叉神经脊束核尾侧亚核浅层内GABA样、L—ENK样，GlyT2样阳性末梢与Sindibis病毒转染表达GFP神经元之间的联系

应用GFP基因重组病毒标记技术和免疫荧光组织化学技术 ,在荧光显微镜和激光共聚焦扫描显微镜下观察了大鼠三叉神经脊束核尾侧亚核 (Vc)浅层内GABA样、L ENK样、GlyT2样阳性纤维和末梢与Sindibis病毒转染表达GFP神经元之间的联系。结果显示 :①将GFP基因重组Sindibis病毒注入一侧Vc浅层后 ,仅在同侧Vc注射部位附近的Ⅰ～Ⅲ层内观察到 4～ 8个逆标的GFP神经元 ,这些神经元的胞体为小型 (直径≤ 15 μm)圆形、梭形或不规则形 ;②Vc浅层内均可见大量的GABA样、L ENK样、GlyT2样阳性纤维和末梢 ,以Ⅰ和Ⅱ层分布最为密集 ;③GABA样、L ENK样、GlyT2样阳性纤维和末梢分别聚集于GFP标记神经元的胞体或树突周围 ,并与之形成密切接触。以上结果表明 :Vc浅层内GFP神经元可能与GABA能、L ENK能、Gly能阳性纤维和末梢形成突触联系并接受这些抑制性神经末梢的调控。     

Dopamine-immunoreactivity in the rat mesencephalic trigeminal nucleus: an ultrastructural analysis

The ultrastructure and distribution of dopaminergic boutons within the rat mesencephalic trigeminal (Me5) nucleus was examined with the use of electronmicroscopic immunocytochemistry. A total of 5102 boutons, comprising axosomatic and axodendritic synaptic terminals as well as non-synaptic boutons (or varicosities), located in the ventrocaudal portion of Me5 was analysed. Approximately 20% of these boutons were dopamine-immunoreactive. Morphological analysis showed that the dopaminergic synaptic terminals, axodendritic as well as axosomatic, were exclusively of the S- and G-bouton type; they contained, respectively, small spherical vesicles or small pleomorphic vesicles in combination with large granular dense-cored vesicles. All dopaminergic varicosities in the Me5 were of the G-bouton type. Quantitative analysis revealed that most of the dopaminergic synaptic terminals in the Me5 nucleus contacted dendrites, while only a minority (12%) contacted Me5 somata. This dopaminergic somatic input comprised about half (52%) of the total axosomatic input on Me5 neurons. The present results and previous findings with respect to the prominent serotonergic component of the axosomatic input to Me5 neurons indicate that dopamine and serotonin account for most of the axosomatic input in the ventrocaudal part of the Me5 nucleus. In fact, the present results seem to support previous observations regarding the existence of a population of afferent neurons in which dopamine and serotonin are colocalized.     

Morphological characteristics and terminating patterns of masseteric neurons of the mesencephalic trigeminal nucleus in the rat: an intracellular horseradish peroxidase labeling study

In order to study the morphological characteristics and terminating patterns of the neurons of the trigeminal mesencephalic nucleus (Vme), 55 masseteric neurons in Vme in the rat were stained by intracellular injection of horseradish peroxidase (HRP). Labeled cells were distributed throughout the nucleus. These neurons were divided into three types: uni- or pseudounipolar (type A, n = 43), bipolar (type B, n = 5), and multipolar cells (type C, n = 7). Each type was further divided into two subtypes according to the largest diameter of the perikarya (type a greater than or equal to 30 microns, type b less than 30 microns). The central processes of type Aa neurons projected to the following three groups of target nuclei: 1) nuclei functioning as interneurons, including supratrigeminal nucleus (Vsup), intertrigeminal nucleus (Vint), juxta-trigeminal region (Vjux), and parvicellular nucleus of the pontomedullary reticular formation (PcRF); 2) motor nuclei, including the trigeminal motor nucleus (Vmo), accessory facial nucleus (NVIIacs), accessory abducens nucleus (NVIacs), and a small number of labeled axons in the oculomotor nucleus and trochlear nucleus; 3) sensory nuclei, including the dorsomedial part of the principal trigeminal sensory nucleus (Vpdm) and the dorsomedial part of subnucleus oralis of the trigeminal spinal nucleus (Vodm). Labeled processes were dense in the Vsup, Vmo, and Vpdm. The proprioceptive pathway of the fifth nerve is discussed. Direct projections from type Aa neurons of Vme to the Vpdm and dorsolateral part of the Vsup contribute to conduction of the proprioceptive information from spindles of masticatory muscle to the contralateral thalamus in the rat. Different axon morphology, distribution, terminal branch density, and terminating patterns of type Aa neurons were noted in different functional groups of the projecting nuclei, especially in the Vsup, Vmo, and Vpdm. The highest terminal branching density, the most extensive distribution, and two different types of branching patterns (claw-like and comb-like) were observed in Vsup. Selective distribution and single-beaded or "Y"-shaped terminal branches were observed in Vmo. In the Vppdm the axonal branches were sparser than in the Vsup or Vmo, and had an arrangement like the branches of a weeping willow tree. These characteristics of anatomical organization might be related to the function of each projecting nucleus.     

Differential distribution of cell bodies and central axons of mesencephalic trigeminal nucleus neurons supplying the jaw-closing muscles and periodontal tissue: A transganglionic tracer study in the cat

Distribution of cell bodies and central axons of mesencephalic trigeminal nucleus (MTN) neurons were examined in the cat by the method of transganglionic transport of horseradish peroxidase (HRP). Jaw-closing muscle afferent MTN neurons were distributed throughout the whole rostrocaudal extent of the MTN, and sent their axons ipsilaterally to the supratrigeminal and intertrigeminal regions, dorsolateral division of the motor trigeminal nucleus, lateral part of the medullary reticular formation, lamina VI of C1-C3 cord segments, and cerebellum. On the other hand, periodontal receptor afferent MTN neurons were located mainly in the caudal part of the MTN, and sent their axons ipsilaterally to the supratrigeminal region and cerebellum. The existence of multipolar MTN neurons with 1-9 smooth dendrites was also confirmed; most of them were jaw-closing muscle afferent neurons.