大鼠下丘脑前部室周区内生长抑素样神经元与神经肽Y样传入纤维的关系

应用包埋前免疫电镜PAP双重反应技术对大鼠下丘脑前部室周区内的生长抑素和神经肽Y的分布进行了超微结构研究。先用DAB法显示神经肽Y反应，然后用钼酸-TMB法显示生长抑素免疫反应，再经DAB-氯化钴稳定后作免疫电镜包埋。光镜观察结果显示在下丘脑室周区内见到生长抑素样神经元和神经肽Y样神经纤维末梢。电镜观察结果显示，生长抑素的钻酸-TMB免疫反应产物呈针状或块状位于胞质内；神经肽Y样神经纤维末梢内见到颗粒状或絮状DAB免疫反应产物分布于小清亮囊泡周围和线粒体膜上。神经肽Y样神经纤维末梢与生长抑素样神经元胞体形成轴-体突触。此外神经肽Y样神经末梢内大颗粒囊泡与突触前膜融合而形成胞吐像。由此可见神经肽Y样神经纤维末梢经过突触或非实触方式作用于下丘脑前部室周区内生长抑素样神经元而调节其分泌活动。     

猫孤束核胶状质亚核的突触构筑学研究

用透射电镜对猫的孤束核胶状质亚核(SNG)的突触型式进行了观察,除看到已报导的轴—树突触、轴—体突触、树—树突触外,还发现该核内含有轴—轴突触及突触球等结构。SNG内轴—树突触最常见,而轴—体突触、轴—轴突触和树—树突触则较少。各类突触中的突触囊泡多为圆形清亮囊泡,而扁平清亮囊泡和大颗粒囊泡较少。扁平清亮囊泡多与圆形清亮囊泡共存于同一轴—轴突触终末内。轴—轴突触均为对称型突触,有时与树突或胞体相连形成轴—轴—树突触或轴—轴—体突触。突触球多为以树突和棘为中心的中心树突型突触球。此外在SNG内还观察到嵴突触,并联突触等连接形式。SNG内突触的复杂性表明传入冲动在该核中可能经过扩散、汇聚和突触前抑制等多种复杂的整合过程调节内脏活动。     

大鼠神经垂体催产素能神经元内神经分泌颗粒的免疫电镜观察

目的　观察大鼠神经垂体催产素（ＯＴ）能神经元轴突及终末内颗粒囊泡形态学及免疫细胞化学特点。方法　应用电镜包埋后免疫细胞化学方法,对大鼠神经垂体进行ＯＴ免疫染色。结果　在ＯＴ能神经元轴突终末内,可见ＯＴ免疫反应性的致密核芯大颗粒囊泡和无ＯＴ免疫反应性的清亮小泡存在。部分囊泡与轴膜靠近,并可见颗粒囊泡与轴膜融合形成的胞吐现象;在轴突非终末部分,也可见ＯＴ免疫反应性的颗粒囊泡,散在分布轴浆内,还可见无ＯＴ免疫反应性的颗粒囊泡,呈散在或聚集分布,两种囊泡均可位于微管之间。结论　位于轴突和轴突终末内的囊泡,在形态学和ＯＴ免疫染色方面有明显差异,推测它们在功能上也有不同。     

降钙素基因相关肽样免疫反应在人脊髓内的分布

本文应用ABC免疫组织化学方法,研究了6例正常人脊髓内降钙素基因相关肽(CGRP)的定位分布。结果表明,CGRP样免疫反应纤维和终末广泛分布在脊髓全长各个节段的后角。免疫反应的强度和免疫反应纤维的密度,可依次归纳为。骶>腰>颈>胸。但颈膨大的免疫反应强度和免疫反应纤维的密度,却明显高于其他颈段。在脊髓的横切面上,免疫反应产物位于后角的各个板层(第Ⅰ～Ⅵ层和Lissauer束)以及中央管周围灰质,其中以胶状质密度最高。在脊髓前角,未见明显的阳性反应细胞体和免疫反应纤维。本文并对CGRP在胶状质内分布可能具有的功能意义进行了讨论。     

大鼠背根切断后脊髓胶状质内鸟核苷酸调节蛋白的改变

用兔抗鸟核苷酸调节蛋白αｏ亚单位多克隆抗血清，采用免疫组织化学方法来观察大鼠脊髓内αｏ免疫反应性，对鸟核苷酸调节蛋白进行定位。及其在切断脊神经背根（简称背根）后的改变。结果发现：正常鼠和对照组鼠脊髓后角浅层均出现强的αｏ免疫反应性，第二层（胶状质）最为密集，外侧脊核区也出现α免疫反应性纤维网。切断背根后胶状质内αｏ免疫反应性及其光密度定量均明显降低，表明胶状质内与一级伤害性刺激传入有关的神经元末梢     

家兔脊髓下橄榄投射的突触特点——HRP顺行标记法电镜研究

将HRP注入家兔脊髓腰膨大灰质,在对侧下橄榄背侧副核内显示出大量HRP顺行标记的脊髓纤维终末。并用电子显微镜观察这些标记终末构成的突触。标记终末主要含圆形清亮囊泡,少数含扁形或多形清亮囊泡,清亮囊泡间常夹有散在的大致密芯囊泡。在下橄榄背侧副核内,大部分标记终末形成轴树突触,少数形成轴体突触。有一标记终末同时含圆形及扁形两种清亮囊泡,各与一树突及一胞体构成突触。特别是标记终末还参与构成轴轴突触及突触球。本文还对这些特殊结构的机能意义进行了初步探讨。     

大鼠后根第一级传入纤维在脊髓胶状质内的溃变和突触联系

用切断后根方法在透射电镜下观察了大鼠后根第一级传入纤维在脊髓胶状质内的溃变和突触联系。切断脊神经后根24小时后,胶状质内出现明显的溃变终末。量最多又持续时间最长的溃变是电子致密型溃变,此外,也观察到了少数的电子透明型溃变和神经微丝型溃变;溃变终末大部分位于胶状质突触小球的中央。据统计,电子致密型溃变作为突触前成分与胶状质内的树突或树突棘形成轴—树突触者占98.8％,作为突触后成分与周围轴突形成轴—轴突触者占1.2％。     

切断坐骨神经后相应节段脊神经节和脊髓胶状质抗氟化物酸性磷酸酶(FRAP)与乙酰胆碱酯酶(AChE)双重染色及实验研究

在正常组大鼠脊神经节中,除小神经元(B)外,发现少数大神经元(A_1)也呈浓染的FRAP阳性反应;A_1内的FRAP与脊髓胶状质内FRAP的关系,尚待研究。切断坐骨神经后,双重染色显示,脊髓胶状质FRAP活性缺失处,AChE活性仍然存在,可能说明胶状质的AChE活性与一级传入神经元外周突损伤及溃变无紧密关系。切断坐骨神经后继以电针刺,则FRAP活性缺失的范围较单纯切断坐骨神经者为小,提示:电针刺似能减轻切断坐骨神经所致的脊髓胶状质FRAP活性缺失。     

大鼠脊髓胶状质内含P物质(SP)及脑啡肽(ENK)神经终末的超微结构

水文应用超微免疫组织化学技术探讨正常及切断后根大鼠胶状质(SG)内P物质(SP)及脑啡肽(ENK)轴突终末的分布及其突触联系。电镜观察发现:SP或ENK免疫反应产物主要分布于轴突终末的大颗粒囊泡的内面,小清亮囊泡及线粒体的外表面,在未应用秋水仙素的情况下,ENK样免疫反应性也可出现于树突及核周质,但电子密度较低。大量的含SP或ENK的终末,分别和未标记树突形成突触。但也偶然见到肽能神经终末与未标记轴突或胞体接触或形成突触联系。常遇到三或四联体,亦偶可见到以含SP轴突终来为中心的突触球。切断后根,手术侧既可见到变性轴突(SP阳性)与未标记轴突接触,又可见到ENK阳性轴突终末与溃变轴突接触,但为数不多。因此认为:通过SP阳性一级传入细纤维传递的感觉信息既可汇集于神经元的一个树突又可多向传递于若干个神经元。ENK在胶状质的抑制作用主要为突触后,但ENK对含SP的一级传入纤维的突触前抑制也不能排除。     

大鼠三叉神经、面神经、迷走神经及颈丛皮支与胶状质的联系

分别切断分布于大鼠头面部的周围神经,观察在胶状质内酸性磷酸酶活性消失的部位,以追踪它们的一级传入纤维与三叉神经脊束核尾侧亚核及脊髓后角胶状质的节段联系。各神经切断后酶活性的消失区如下:眶上神经在颈髓第一节胶状质最外侧处;眶下神经主要在延髓胶状质中央及外侧;颏神经在延髓及第一颈节胶状质最内侧处;耳颞神经主要在第一、二颈髓胶状质的近内侧处;面神经主要在第二颈髓胶状质中央近内侧处;迷走神经在第一、二颈髓胶状质近内侧或近中央处;耳大神经及颈皮神经见于上四颈节胶状质,主要在第二颈髓的中央处。以上实验结果证明,分布于头面部穴位的周围神经一级躯体感觉纤维皆终止于三叉神经尾侧亚核及脊髓后角的胶状质,几乎各神经都与第一、二颈节胶状质有联系。     

Localization of Leu-enkephalin in dense core vesicles of axon terminals.

The localization of enkephalins by immunofluorescence has already shown that these pentapeptides are largely distributed in the central nervous system. In order to clearly identify the structures and organelles containing enkephalins, an immunohistochemical localization of Leu-enkephalin was performed at the ultrastructural level. Leu-enkephalin was detected in dense core vesicles (70--100 nm in diameter) of many nerve fibers in the substantia gelatinosa of the spinal cord, the globus pallidus and the nucleus papabrachialis. These findings support the hypothesis that Leu-enkephalin could act as a neuromodulator.     

A study of the cutaneous afferent input to substantia gelatinosa

The primary afferent input to substantia gelatinosa of the spinal cord was studied in the decerebrate cat. The responses of cells in the substantia gelatinosa to natural stimulation of the skin and to graded electrical stimulation of the sural nerve were compared. The results show that the responses of these neurones are not predictable from the known properties of the primary afferent input. Interactions between A-fibre and C-fibre input to substantia gelatinosa cells were studied using A-fibre polarization block and conditioning A-fibre stimulation during C-fibre activation of these cells. Whereas A-fibres have strong inhibitory effects on C-fibre input to large projection cells in the spinal cord, this is not true for substantia gelatinosa cells.The results do not support the idea of substantia geletinosa as a simple relay between primary afferent inputs and spinal cord projection systems.     

An ultrastructural morphometric study of developing rat substantia gelatinosa

A morphometric analysis has been done on developing rat substantia gelatinosa of the lower cervical and upper thoracic levels of the spinal cord starting on the 15th day of gestation. The following parameters were measured: cell body diameter, cytoplasmic/nuclear areas, synaptic density, synaptic type and vesicle morphology of the presynaptic terminal in axodendritic synapses. Cell body size and cytoplasmic/nuclear areas of gelatinosal cells increase until the 15th day postnatally and then decrease somewhat to the adult values. The first synapses are seen on gestation day 17. Synaptic density increases linearly until the third day postnatally. Axodendritic synapses are most common throughout development and in the adult, while the proportion of axoaxonic synapses increases and axosomatic synapses decreases during development. Most of the terminals in axodendritic synapses contain clear-spherical vesicles but the occurrence of clear-flat vesicles and dense-cored vesicles in the terminals increases during development. It appears that these morphological parameters provide a stable index of development in the substantia gelatinosa which can be correlated with functional development of the area. Hopefully, they will provide a means to assess subtle anomalies induced by nonteratogenic drugs or other environmental changes.     

Effects of in vivo estradiol and progesterone on tritiated flunitrazepam binding in rat spinal cord

Tritiated flunitrazepam binding was measured in autoradiograms of lumbar spinal cord from control ovariectomized female rats and from ovariectomized females treated with either estradiol-silastic implants or one subcutaneous injection of 500 micrograms progesterone 4-5 h before being killed or both estradiol and progesterone. Four areas of spinal cord were analyzed: the substantia gelatinosa, the region leading to and around the central canal, the remainder of the dorsal horn and the ventral horn. Specific flunitrazepam binding was greatest in the substantia gelatinosa and in the region around the central canal and was weakest in the ventral horn. Estradiol had no effect on flunitrazepam binding in the spinal cord despite estradiol concentrating cells in the substantia gelatinosa and around the central canal. In vivo progesterone enhanced flunitrazepam binding in the substantia gelatinosa while 10(-8) M progesterone in vitro was without effect. One day after spinal transection flunitrazepam binding in endocrine controls and estradiol-treated animals was unchanged but a progesterone injection 4-5 h before being killed had no effect on binding in the substantia gelatinosa.     

Identification of presumptive long axon neurons in the substantia gelatinosa of the rat lumbosacral spinal cord: a Golgi study

The present Golgi study identifies and describes 3 types of presumptive long axon neurons in the substantia gelatinosa of the rat lumbosacral spinal cord. These neurons were traced from prenatal stages to adulthood and were found to correspond to the 'limitrophe' and two variations of the 'centrale' cell originally described by Ramón y Cajal in fetal and newborn animals. These neurons can be distinguished from intrinsic, non-projection neurons of the substantia gelatinosa.     

Light and electron microscopic autoradiographic study of the dorsal root projections to the cat dorsal horn

The distribution of terminals arising from dorsal root primary afferents was examined in the lumbar spinal cord of cats using light- and electron-microscopic autoradiography. Tritiated proline or leucine was injected into either the L6 or L7 dorsal root ganglion. The light-microscopic spinal cord distribution of radioactivity in the ganglia was independent of the type of amino acid used. Likewise, the length of the survival time after injection had no effect. The projections to the substantia gelatinosa and the marginal zone were consistently the densest. However, the topography of the dorsal horn distribution, relative the the segment of entry, varied significantly especially in the gelatinosa, depending upon the ganglia injected. Those to the substantia gelatinosa were largely limited to the segment of entry; those to the marginal zone and nucleus proprius extended many segments beyond the level of entry. At all levels the projection was exclusively ipsilateral to the side of injection.At the electron-microscopic level the distribution of radioactivity was determined in each of the three easily recognizable areas of the dorsal horn: the marginal zone, the substantia gelatinosa and the nucleus proprius. In each dorsal horn area the total terminal population was divided into four basic categories. Each of these areas was found to contain a characteristic distribution of these four terminal categories. The difference between areas arose, primarily, as a consequence of the dorsal to ventral decreasing frequency gradient of two types of terminal: those containing large, dense-cored vesicles and the increasing gradient of those containing flattened vesicles. The terminals with small pleomorphic vesicles and those with large round vesicles were frequently encountered in all three areas without a detectable frequency gradient.Similarly, the primary afferent terminal population, that is the subset of the total terminal population labelled after dorsal root ganglion injection, was also characteristic of the area, and each area was dominated by a different terminal type. In the marginal zone the terminals containing large dense-cored vesicles dominated. In the substantia gelatinosa the terminals with pleomorphic vesicles (which included the so-called ‘C’ type terminals) dominated. And the terminals containing large round vesicles dominated the primary afferent population in the nucleus proprius. The terminals containing flattened vesicles were never found to be specifically labeled in any of the areas examined.     

Endomorphin-like immunoreactivity in the rat dorsal horn and inhibition of substantia gelatinosa neurons in vitro

Endomorphin 1 and 2 are two tetrapeptides recently isolated from bovine as well as human brains and proposed to be the endogenous ligand for the mu-opiate receptor. Opioid compounds expressing mu-receptor preference are generally potent analgesics. The spinal cord dorsal horn is considered to be an important site for the processing of sensory information including pain. The discovery that endomorphins produced greater analgesia in mice upon intrathecal as compared to intracerebroventricular injections raises the possibility that dorsal horn neurons may represent the anatomic site upon which endomorphins exert their analgesic effects. We report here the detection of endomorphin 2-immunuoreactive fiber-like elements in superficial layers of the rat dorsal horn by immunohistochemical techniques. Whole-cell patch recordings from substantia gelatinosa neurons of cervical spinal cord slices revealed two conspicuous effects of exogenously applied endomorphin 1 and 2: (i) depression of excitatory postsynaptic potentials evoked by stimulation of dorsal root entry zone, and (ii) hyperpolarization of substantia gelatinosa neurons. These effects were reversed by the selective mu-opiate receptor antagonist beta-funaltrexamine. Collectively, the detection of endomorphin-like immunoreactivity in nerve fibers of the superficial layers and the inhibitory action of endomorphins on substantia gelatinosa neurons provide further support for a potential role of these two peptides in spinal nociception.     

Binding sites for 125I-cholecystokinin in primate spinal cord are of the CCK-A subclass

Cholecystokinin (CCK) receptor binding was measured in sections of human, monkey and rat spinal cord using autoradiographical techniques. In each species, high levels of specific 125I-Bolton-Hunter CCK binding were detected in the superficial layers of the dorsal horn (the substantia gelatinosa). In monkey and human but not rat spinal cord, 125I-CCK binding was dose-dependently inhibited by low concentrations of the selective CCK-A antagonist L-364,718. Binding of [3H]L-364,718, which was saturable (Bmax = 29.0 +/- 0.95 pmol/g wet wt.) and of high affinity (pKd) = 9.92 +/- 0.16) was also detected in sections of monkey spinal cord and had a similar localization to that of specific 125I-CCK binding. These data indicate that in striking contrast to CCK receptors in rat spinal cord, those in the primate cord are of the CCK-A receptor subclass.     

Descending monoaminergic pathways in the primate spinal cord

The distribution of monoamine axons and terminals within the spinal cord of a primate (Macaca mulatta) was studied with the Falck-Hillarp histofluorescence technique for the demonstration of biogenic amines. Catecholamine and indoleamine varicosities appeared qualitatively similar to those previously reported for the rat although the indoleamine terminals were difficult to visualize and were not studied in great detail. Catecholamine fibers innervate the substantia gelatinosa, marginal layer, intermediolateral cell column, ventral horn and the region surrounding the central canal. The location of monoamine axons, as revealed by spinal cord ligation, corresponds to that in the rat and cat with the exception of the dorsolateral region of white matter where fluorescent axons are not visible in the primate.     

Adenosine 5'-triphosphate inhibits slow depolarization induced by repetitive dorsal root stimulation via P2Y purinoceptors in substantia gelatinosa neurons of the adult rat spinal cord slices with the dorsal root attached

We previously reported that slow depolarization of substantia gelatinosa neurons is evoked by repetitive stimulation of C-fibers of dorsal root in adult rat spinal cord transverse slices with the dorsal root attached, which was considered to be an event relevant to spinal nociception. In the present study, we investigated the effects of adenosine 5′-triphosphate (ATP) and its analogs on the slow depolarization. ATP (10–100 μM) significantly inhibited the amplitude of slow depolarization in a concentration-dependent manner. The inhibitory effect of ATP was not reversed by suramin, an antagonist for some P2-purinoceptors, and was mimicked by a P2Y selective agonist uridine 5′-triphosphate, but not a P2X selective agonist ,β-methylene ATP. These results suggest that ATP inhibits the slow depolarization of substantia gelatinosa neurons relevant to nociceptive transmission in the spinal dorsal horn, via suramin-insensitive P2Y purinoceptors.