大鼠中脑导水管周围灰质腹外侧区5-羟色胺样、P物质样和亮氨酸-脑啡肽样免疫反应阳性亚微结构的电镜观察

本文用免疫电镜技术研究了大鼠中脑导水管周围灰质腹外侧区内5-HT样、SP样和L-ENK样的免疫反应阳性亚微结构。5-HT样免疫反应阳性的胞体较多,常见5-HT样阳性树突与阴性轴突终末形成多为非对称性的轴-树突触;偶见阳性轴突终末与阴性树突以及阴性轴突终末与阳性胞体分别构成轴-树和轴-体突触.SP样阳性胞体数目较少,可见少量含多形性小泡的阴性轴突终末与之形成轴-体突触;由阴性轴突终末与阳性树突所形成的轴-树突触最常见;阳性轴突终末与阴性胞体和阳性树突分别构成轴-体突触和轴-树突触。L-ENK样阳性胞体数目也较少,L-ENK样阳性树突与阴性轴突终末所形成的轴-树突触最多见,可见L-ENK样阳性胞体与阴性轴突终末构成轴-体突触;偶见阳性轴突终末与阴性树突形成轴-树突触。上述各种突触均主要含圆形小泡,有时有少量扁平小泡、椭圆形小泡和颗粒囊泡。     

Ultrastructural characterization of relationship between serotonergic and GABAergic structures in the ventral part of the oral pontine reticular nucleus

The ventral part of the oral pontine reticular nucleus (vRPO) is involved in the generation and maintenance of rapid eye movement (REM) sleep. Both GABAergic and serotonergic neurotransmission have been implicated in the control of the sleep–wakefulness cycle. Nevertheless, the synaptic organization of serotonergic terminals in the vRPO has not yet been characterized. We performed an electron microscope study of serotonin-immunoreactive (5-HT-IR) terminals using immunoperoxidase or immunogold–silver methods. In a second set of experiments, combining GABA immunoperoxidase and 5-HT immunogold–silver techniques, we examined inputs from GABA-immunoreactive (GABA-IR) terminals to serotonergic neurons. 5-HT-IR terminals were located primarily on dendrites and occasionally on somata of unlabeled and 5-HT-IR neurons. The majority of the synapses formed by 5-HT-IR terminals were of the symmetrical type, making contacts primarily with unlabeled dendritic profiles. Moreover, 5-HT-IR terminals contacted unlabeled axon terminals that formed asymmetric synapses on dendrites. Double immunolabeling experiments showed 5-HT-IR and GABA-IR afferents, in apposition to each other, making synapses with the same dendrites. Finally, GABA-IR terminals innervated 5-HT-IR and GABA-IR dendrites. Our findings indicate that serotonin would modulate the neuronal activity through inhibitory or excitatory influences, although the action of serotonin on the vRPO would predominantly be inhibitory. Moreover, the present results suggest that the serotonin modulation of vRPO neurons might involve indirect connections. In addition, GABA might contribute to the induction and maintenance of REM sleep by inhibiting serotonergic and GABAergic neurons in the vRPO.     

大鼠杏仁体基底外侧核中DA能与GABA能神经元之间突触关系的免疫电镜研究

目的 了解杏仁体中多巴胺(DA)能神经末梢和7-氨基丁酸(GABA)能神经元之间的相互关系、探索两者在精神分裂症中作用的神经解剖学机制。方法 用免疫电镜双标技术，分别以抗DA和抗谷氨酸脱羧酶(GAD、GABA的合成酶)标记大鼠杏仁体基底外侧核中DA能神经末梢和GABA能神经元，在电镜下观察DA能神经末梢和GABA能神经元之间的突触联系。结果 由DA免疫阳性神经末梢形成的突触中，有43％直接或间接形成于DA免疫阳性神经末梢与GAD免疫反应阳性树突结构之问，其中单一性突触为38％、汇聚性突触为30％、连续性突触为20％、末梢间突触样联系为12％。另外的57％由DA免疫阳性神经末梢与未标记的神经结构形成，其中在未标记的神经元胞体形成10％、树突上82％、轴突末梢上8％。由DA免疫反应阳性末梢形成的所有突触均为对称性即抑制性突触。结论 在大鼠杏仁体基底外侧核中，DA能神经末梢系统以对称性突触支配着GABA能中间神经元，同时，又与核内的谷氨酸能投射神经元形成突触联系．并影响其活动。     

大鼠杏仁体基底外侧核中小白蛋白反应阳性神经元受抑制性神经网络支配(英文)

小白蛋白 (PV)神经元作为杏仁核簇基底外侧核 (BL)中局部神经环路成分 ,对杏仁核的情绪、学习和记忆过程等机能发挥重要作用。为探讨 BL中 PV中间神经元的突触形成状态 ,本研究用抗 PV抗体标示 PV神经元 ,以抗多巴胺 (DA)抗体标示多巴胺能轴突及末梢作为传入纤维的标志 ,对大鼠杏仁核做了免疫电镜双标记研究。结果表明 ,突触主要见于 PV免疫阳性神经元的树突结构上 ,包括从树突干到中间及小型树突的各级分支。其中 68%的突触由未标记的轴突终末形成 ,3 2 %分别由 DA(2 1% )和 PV(11% )免疫阳性轴突末梢形成。 PV免疫阳性神经元与未标记末梢所形成的突触大多数是对称性的 ,仅少数为非对称性。这些非对称性突触见于 PV神经元的树突小棘和连续性突触 ,即一个未标记轴突末梢与另一个未标记轴突末梢形成对称性突触 ,后者又与 PV免疫阳性神经元树突形成非对称性突触。 DA和 PV免疫阳性神经元轴突终末与 PV免疫阳性神经元树突之间的突触全部是对称性的。以上结果表明 ,大鼠杏仁核 BL 的 PV中间神经元受非对称性突触所构成的包括多巴胺系统在内的抑制性神经网络支配     

GABAB and group I metabotropic glutamate receptors in the striatopallidal complex in primates

Glutamate and GABA neurotransmission is mediated through various types of ionotropic and metabotropic receptors. In this review, we summarise some of our recent findings on the subcellular and subsynaptic localisation of GABA_B and group I metabotropic glutamate receptors in the striatopallidal complex of monkeys. Polyclonal antibodies that specifically recognise GABA_BR1, mGluR1a and mGluR5 receptor subtypes were used for immunoperoxidase and pre‐embedding immunogold techniques at the light and electron microscope levels. Both subtypes of group I mGluRs were expressed postsynaptically in striatal projection neurons and interneurons where they aggregate perisynaptically at asymmetric glutamatergic synapses and symmetric dopaminergic synaptic junctions. Moreover, they are also strongly expressed in the main body of symmetric synapses established by putative intrastriatal GABAergic terminals. In the globus pallidus, both receptor subtypes are found postsynaptically in the core of striatopallidal GABAergic synapses and perisynaptically at subthalamopallidal glutamatergic synapses. Finally, extrasynaptic labelling was commonly seen in the globus pallidus and the striatum. Moderate to intense GABA_BR1 immunoreactivity was observed in the striatopallidal complex. At the electron microscope level, GABA_BR1 immunostaining was commonly found in neuronal cell bodies and dendrites. Many striatal dendritic spines also displayed GABA_BR1 immunoreactivity. Moreover, GABA_BR1‐immunoreactive axons and axon terminals were frequently encountered. In the striatum, GABA_BR1‐immunoreactive boutons resembled terminals of cortical origin, while in the globus pallidus, subthalamic‐like terminals were labelled. Pre‐embedding immunogold data showed that postsynaptic GABA_BR1 receptors are concentrated at extrasynaptic sites on dendrites, spines and somata in the striatopallidal complex, perisynaptically at asymmetric synapses and in the main body of symmetric striatopallidal synapses in the GPe and GPi. Consistent with the immunoperoxidase data, immunoparticles were found in the presynaptic grid of asymmetric synapses established by cortical‐ and subthalamic‐like glutamatergic terminals. These findings indicate that both GABA and glutamate metabotropic receptors are located to subserve various modulatory functions of the synaptic transmission in the primate striatopallidal complex. Furthermore, their pattern of localisation raises issues about their roles and mechanisms of activation in normal and pathological conditions. Because of their ‘modulatory’ functions, these receptors are ideal targets for chronic drug therapies in neurodegenerative diseases such as Parkinson's disease.     

大鼠臂旁核内接受孤束核内脏传入之神经元同时接受中央杏仁核的反馈调节──溃变、HRP法结合包埋后免疫电镜研究

为研究来自孤束核的内脏传导信息在臂旁核水平是否接受中央杏仁核的反馈调节及其递质性质，以及孤束核—臂旁核—中央杏仁核传导通路中，在臂旁核水平是否接受ＧＡＢＡ的调节，本文将ＨＲＰ注入中央杏仁核进行顺、逆行标记，同时将兴奋性氨基酸毒素海人酸注入孤束核进行损毁，观实其顺行溃变终末，取外侧臂旁核超薄切片后结合抗ＧＡＢＡ的免疫电镜染色，观察发现有下列几种标记；（１）顺行溃变终末，所有的都与臂旁核神经元形成非对称性突触；（２）ＨＲＰ标记终末有两类：第一类和臂旁核神经元形成对称性突触，占ＨＲＰ标记终末总数的８０％以上，第二类与臂旁核神经元形成非对称性突触，另外有大量的ＨＲＰ标记的胞体和树突；（３）胶体金标记的ＧＡＢＡ阳性终末，皆与突触后结构形成对称性突触；（４）ＧＡＢＡ／ＨＲＰ双标记终末，具有ＧＡＢＡ免疫阳性终末和第一类ＨＲＰ标记终末的共同特征。上述几种标记在臂旁核内有以下几种关系：（１）溃变终末和ＧＡＢＡ阳性终末与同一个ＨＲＰ标记或非标记的突形成轴－树突触；（２）溃变终末和第一类ＨＲＰ标记终末共同终止于同一非标记讨突；（３）溃变终末与ＨＲＰ标记树突或胞位形成非对称性突触；（４）ＧＡＢＡ／ＨＲＰ双标记终末与非标记树突或胞体?     

Neuroanatomical and neurochemical organization of projections from the central amygdaloid nucleus to the nucleus retroambiguus via the periaqueductal gray in the rat

The periaqueductal gray (PAG)-nucleus retroambiguus (NRA) pathway has been known to be involved in the control of vocalization and sexual behavior. To know how the amygdaloid complex influences the PAG-NRA pathway, here we first examined the synaptic organization between the central amygdaloid nucleus (CeA) fibers and the PAG neurons that project to the NRA by using anterograde and retrograde tract-tracing techniques in the rat. After ipsilateral injections of biotinylated dextran amine (BDA) into the CeA and cholera toxin B subunit (CTb) into the NRA, the prominent overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found ipsilaterally in the lateral/ventrolateral PAG, where some of the BDA-labeled terminals made symmetrical synaptic contacts with somata and dendrites of the CTb-labeled neurons. After CTb injection into the lateral/ventrolateral PAG, CTb-labeled neurons were distributed mainly in the medial division of the CeA. After BDA injection into the lateral/ventrolateral PAG, BDA-labeled fibers were distributed mainly in and around the NRA within the medulla oblongata. Using a combined retrograde tracing and in situ hybridization technique, we further demonstrated that more than half of the CeA neurons labeled with Fluoro-Gold (FG) injected into the lateral/ventrolateral PAG were positive for glutamic acid decarboxylase 67 mRNA and that the vast majority of PAG neurons labeled with FG injected into the NRA expressed vesicular glutamate transporter 2 mRNA. The present results suggest that the glutamatergic PAG-NRA pathway is under the inhibitory influence of the GABAergic CeA neurons.     

Ultrastructural evidence that substance P neurons form synapses with noradrenergic neurons in the A7 catecholamine cell group that modulate nociception.

Potent antinociception can be produced by electrical stimulation of spinally projecting noradrenergic neurons in the A7 catecholamine cell group and this effect is blocked by intrathecal injection of alpha2-adrenoceptor antagonists. Microinjection of substance P near A7 neurons also produces antinociception that is blocked by intrathecal injection of alpha2-adrenoceptor antagonists. These observations suggest that substance P produces antinociception by activating noradrenergic A7 neurons. However, it is not known whether this effect of substance P is produced by a direct or an indirect action on A7 neurons. Although light microscopic studies have demonstrated the existence of both substance P-containing axon terminals and neurokinin-1 receptors in the region of the A7 cell group, it is not known whether substance P terminals form synapses with noradrenergic A7 neurons. These experiments used double-labeling immunocytochemical methods and electron microscopic analysis to determine whether substance P-containing axons form synapses with noradrenergic neurons in the A7 cell group. Pre-embedding immunocytochemistry, combined with light and electron microscopic analysis, was used to provide ultrastructural evidence for synaptic connections between substance P-immunoreactive terminals labeled with immunoperoxidase and tyrosine hydroxylase-immunoreactive A7 neurons labeled with silver-enhanced immunogold. Tyrosine hydroxylase labeling was found in perikarya and dendrites in the A7 region, and substance P labeling was found in axons and synaptic terminals. Substance P-labeled terminals formed asymmetric synapses with tyrosine hydroxylase-labeled dendrites, but only a few of these were present on tyrosine hydroxylase-labeled somata. Substance P-labeled terminals also formed asymmetric synapses with unlabeled dendrites, and many unlabeled terminals formed both symmetric and asymmetric synapses with tyrosine hydroxylase-labeled dendrites. These results demonstrate that substance P neurons form a significant number of synapses with the dendrites of noradrenergic A7 neurons and support the conclusion that microinjection of substance P in the A7 cell group produces antinociception by direct activation of spinally projecting noradrenergic neurons.     

Pre- and postsynaptic localization of GABAB receptors in the basal ganglia in monkeys

GABAergic neurotransmission involves ionotropic GABA_A and metabotropic GABA_B receptor subtypes. Although fast inhibitory transmission through GABA_A receptors activation is commonly found in the basal ganglia, the functions as well as the cellular and subcellular localization of GABA_B receptors are still poorly known. Polyclonal antibodies that specifically recognize the GABA_BR1 receptor subunit were produced and used for immunocytochemical localization of these receptors at the light and electron microscope levels in the monkey basal ganglia. Western blot analysis of monkey brain homogenates revealed that these antibodies reacted specifically with two native proteins corresponding to the size of the two splice variants GABA_BR1a and GABA_BR1b. Preadsorption of the purified antiserum with synthetic peptides demonstrated that these antibodies recognize specifically GABA_BR1 receptors with no cross-reactivity with GABA_BR2 receptors. Overall, the distribution of GABA_BR1 immunoreactivity throughout the monkey brain correlates with previous GABA_B ligand binding studies and in situ hybridization data as well as with recent immunocytochemical studies in rodents. GABA_BR1-immunoreactive cell bodies were found in all basal ganglia nuclei but the intensity of immunostaining varied among neuronal populations in each nucleus. In the striatum, interneurons were more strongly stained than medium-sized projection neurons while in the substantia nigra, dopaminergic neurons of the pars compacta were much more intensely labeled than GABAergic neurons of the pars reticulata. In the subthalamic nucleus, clear immunonegative neuronal perikarya were intermingled with numerous GABA_BR1-immunoreactive cells. Moderate GABA_BR1 immunoreactivity was observed in neuronal perikarya and dendritic processes throughout the external and internal pallidal segments. At the electron microscope level, GABA_BR1 immunoreactivity was commonly found in neuronal cell bodies and dendrites in every basal ganglia nuclei. Many dendritic spines also displayed GABA_BR1 immunoreactivity in the striatum. In addition to strong postsynaptic labeling, GABA_BR1-immunoreactive preterminal axonal segments and axon terminals were frequently encountered throughout the basal ganglia components. The majority of labeled terminals displayed the ultrastructural features of glutamatergic boutons and formed asymmetric synapses. In the striatum, GABA_BR1-containing boutons resembled terminals of cortical origin, while in the globus pallidus and substantia nigra, subthalamic-like terminals were labeled.Overall, these findings demonstrate that GABA_B receptors are widely distributed and located to subserve both pre- and postsynaptic roles in controlling synaptic transmission in the primate basal ganglia.     

Neuroanatomical and neurochemical organization of projections from the central amygdaloid nucleus to the nucleus retroambiguus via the periaqueductal gray in the rat

The periaqueductal gray (PAG)-nucleus retroambiguus (NRA) pathway has been known to be involved in the control of vocalization and sexual behavior. To know how the amygdaloid complex influences the PAG-NRA pathway, here we first examined the synaptic organization between the central amygdaloid nucleus (CeA) fibers and the PAG neurons that project to the NRA by using anterograde and retrograde tract-tracing techniques in the rat. After ipsilateral injections of biotinylated dextran amine (BDA) into the CeA and cholera toxin B subunit (CTb) into the NRA, the prominent overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found ipsilaterally in the lateral/ventrolateral PAG, where some of the BDA-labeled terminals made symmetrical synaptic contacts with somata and dendrites of the CTb-labeled neurons. After CTb injection into the lateral/ventrolateral PAG, CTb-labeled neurons were distributed mainly in the medial division of the CeA. After BDA injection into the lateral/ventrolateral PAG, BDA-labeled fibers were distributed mainly in and around the NRA within the medulla oblongata. Using a combined retrograde tracing and in situ hybridization technique, we further demonstrated that more than half of the CeA neurons labeled with Fluoro-Gold (FG) injected into the lateral/ventrolateral PAG were positive for glutamic acid decarboxylase 67 mRNA and that the vast majority of PAG neurons labeled with FG injected into the NRA expressed vesicular glutamate transporter 2 mRNA. The present results suggest that the glutamatergic PAG-NRA pathway is under the inhibitory influence of the GABAergic CeA neurons.     

大鼠三叉神经脊束核尾侧亚核Ⅱ层内CB样、PV样阳性终末与向臂旁核投射神经元的突触联系

为了观察三叉神经脊束核尾侧亚核 层神经元与向臂旁核投射神经元的突触关系 ,选用 Calbindin D-2 8k和 Parvalbu-min作为标志物 ,采用 HRP逆行追踪与免疫组织化学技术相结合的双重反应技术对 层内的 Calbindin D-2 8k样和 Parvalbumin样阳性神经元与三叉神经脊束核尾侧亚核向臂旁核投射神经元之间的突触联系进行了观察。HRP注入臂旁核后 ,逆标神经元主要位于同侧三叉尾侧亚核 层至 层。Calbindin D-2 8k和 Parvalbumin样阳性胞体、纤维和终末主要集中在 层内侧带。电镜下观察 ,此二者的阳性反应产物呈弥散分布 ;两者的阳性终末与 HRP逆标神经元的胞体和树突之间主要形成轴 -树突触 ,偶见轴 -体突触 ;Calbindin D-2 8k样阳性终末与 HRP逆标神经元的树突形成的非对称性突触占 83% ,Parvalbumin样阳性终末与 HRP逆标神经元的树突形成的对称性突触占 85 %。此外 ,还观察到参与突触小球组成的初级传入纤维终末与 HRP逆标神经元的树突形成突触联系。以上结果提示 :(1)三叉神经脊束核尾侧亚核 层的 Calbindin D-2 8k样或 Parvalbumin样阳性神经元可通过突触传递机制对三叉神经脊束核尾侧亚核向臂旁核投射的神经元进行调控 ,影响外周伤害性信息向上位脑结构的传递 ;(2 )三叉神经脊束核尾侧亚核向臂旁核投?     

大鼠三叉神经脊束核Ⅱ尾侧亚核层内CB样、PV样阳性结构的分布及其突触联系

本研究用免疫组织化学方法观察了 Calbindin D-2 8k( CB)样和 Parvalbumin ( PV)样胞体、纤维和终末在三叉神经脊束核尾侧亚核 ( Vc) 层内的分布及它们的突触联系。在光镜下观察到 CB样和 PV样阳性胞体、纤维和终末在 II层内侧带 ( IIi)最为密集 ,PV样阳性神经元的胞体稍大 ,但数量少于 CB样阳性神经元。在电镜下观察到 CB样或 PV样阳性结构主要形成下列 4种突触联系 :( 1)阳性轴突终末与阳性或阴性轴突终末形成对称性轴 -轴突触和少量非对称性轴 -轴突触 ;( 2 )阳性轴突终末与阳性树突形成非对称性和对称性轴 -树突触 ;( 3 ) CB样阳性轴突终末与阴性树突主要形成非对称性轴 -树突触 ,PV样阳性轴突终末与阴性树突主要形成对称性轴 -树突触 ;( 4 )阴性轴突终末与阳性树突形成非对称性和对称性轴 -树突触。另外还可见到 CB样或PV样阳性或阴性树突、轴突及终末与 CB样、PV样阳性或阴性的初级传入纤维终末形成 型和 II型突触小球。 型突触小球数量较多 ,有典型的扇贝样初级传入纤维终末和不均一的小泡 ,线粒体少 ;II型突触小球的初级传入纤维终末粗大而清亮 ,外观不规则 ,有均匀一致的小泡和丰富的线粒体。根据上述结果可以推知在面口部伤害性信息的传递和调控过程中 ,Vc II层神经元发挥着重要的作用     

大鼠杏仁体基底外侧核中含D2受体的γ氨基丁酸神经元受多巴胺能末梢支配(英文)

杏仁体中的多巴胺(DA)和γ -氨基丁酸(GABA)递质系统均参与精神分裂症的病理过程,临床上一般用多巴胺II型受体(D2)阻断剂予以治疗。然而,目前尚不清楚GABA与D2受体是否共存,也不清楚DA能神经末梢与GABA能神经元之间的联系方式。本实验用共聚焦激光扫描显微镜(CLSM)和免疫电镜(IEM)研究了杏仁体关键性核团基底外侧核中GABA与D2受体的共存关系以及DA神经能末梢与GABA能神经元之间的突触关系。CLSM显示由谷氨酸脱羧酶(GAD)标记的GABA能神经元全部对D2受体呈免疫阳性反应,表明GABA能神经元含有D2受体。IEM显示,在 980个DA能神经末梢形成的突触中,45%的突触是由DA免疫反应阳性神经末梢直接(36% )或间接(9% )与GAD免疫反应阳性神经元的树突形成,另 55%是由DA免疫反应阳性神经末梢与未标记的神经元成分形成。DA GABA的直接性突触进而可区分为单突触 (16% )、汇聚突触 (14% )及轴 轴突触(6% )。而DA- GABA的间接性突触是个突触复合体。在该复合体中,DA免疫反应阳性末梢在一个未标记的末梢上形成对称性突触,而该未标记末梢又与GAD免疫反应阳性树突形成非对称性突触。在DA与未标记神经元成分之间的突触中,AD免疫反应阳性末梢分别与未标记胞体(4% )、树突(42% )及轴突末梢(9% )形成突触。所有DA突触无一例外均为?     

Ultrastructural morphometric analysis of enkephalin-immunoreactive terminals in the ventrocaudal periaqueductal gray: analysis of their relationship to periaqueductal gray-raphe magnus projection neurons

The periaqueductal gray plays an important role in the descending modulation of nociception. While the importance of endogenous opioids to periaqueductal gray circuits that modulate nociception is supported by many studies, the ultrastructural relationships between enkephalin-immunoreactive axon terminals and the surrounding periaqueductal gray neuropil have not been quantitated in the rat. Further, the possible interaction between enkephalin-immunoreactive axon terminals and periaqueductal gray neurons that project to the rostroventral medulla has not been described. The present study utilized electron microscopic immunocytochemistry to quantitate the normal neuronal associations of enkephalin-immunoreactive terminals in the caudal periaqueductal gray of the rat. A primary focus of this analysis was to ascertain whether any interaction exists between enkephalin-immunoreactive axon terminals and periaqueductal gray neurons that were retrogradely-labeled from the nucleus raphe magnus and adjacent medullary reticular nuclei. We examined the ventrolateral periaqueductal gray and the ventral periaqueductal gray immediately subjacent to the aqueduct and found that both the average terminal diameters and the volume fractions of enkephalin-immunoreactive terminals were very similar. In these two regions, most terminals were observed to be in close apposition to either two or three dendrites that were neither retrogradely-labeled nor enkephalin-immunoreactive, although axonal and perikaryal associations were also observed. In the ventrolateral periaqueductal gray, 22% of all enkephalin-immunoreactive terminals were adjacent to periaqueductal gray-nucleus raphe magnus and periaqueductal gray-reticular nucleus projection neurons. In the periaqueductal gray subjacent to the aqueduct, 32% of all enkephalin-immunoreactive terminals were adjacent to periaqueductal gray-nucleus raphe magnus and periaqueductal gray-reticular nucleus projection neurons. Symmetrical synapses with these retrogradely-labeled neurons were formed by 5.5% of enkephalin-immunoreactive terminals in the ventrolateral periaqueductal gray, and by 4.3% of enkephalin-immunoreactive terminals located subjacent to the aqueduct. We also noted that enkephalin-immunoreactive terminals formed symmetrical synapses with non-retrogradely-labeled, enkephalin-immunoreactive dendrites in the periaqueductal gray. Direct opioid input onto putative excitatory periaqueductal gray output neurons that are hypothesized to modulate nociception was an unexpected finding.(ABSTRACT TRUNCATED AT 400 WORDS)     

小鼠中脑导水管周围灰质下行投射的SP能终末与中缝大核内SP受体阳性神经元的突触联系

P物质(SP)在中枢神经系统内具有镇痛作用,中脑导水管周围灰质(PAG)和中缝大核(NRM)是中枢内源性镇痛系统的关键结构。本研究采用顺行追踪与免疫组化双重染色相结合的三标方法,观察了起源于PAG的SP能阳性终末与NRM内SP受体(SPR)阳性神经元之间的突触联系。结果显示:将生物素化葡聚糖胺(BDA)注入小鼠PAG的腹外侧区后,BDA顺行标记终末可见于脑干的许多区域,但主要位于NRM。其中的部分BDA顺行标记终末呈SP阳性。NRM内可见到散在分布的SPR阳性神经元。在电镜下可见来自PAG的SP/BDA双标终末与NRM内的SPR阳性神经元的胞体和树突形成以非对称性为主的突触联系。本研究的结果提示起源于PAG的下行投射终末所释放的SP是激活中枢内源性镇痛系统并使之发挥镇痛作用的重要神经活性物质。     

Ultrastructural localization and afferent sources of substance P in the rat parabrachial region

The ultrastructural morphology and afferent sources of terminals containing substance P-like immunoreactivity were examined in the rat parabrachial region. In the first portion of the study, a polyclonal antiserum to substance P was localized in the ventrolateral parabrachial region using the peroxidase-antiperoxidase labeling technique combined with electron microscopy. The antiserum was tested for cross-reaction with substance P, physalaemin, substance K and neuromedins B, C and K. Cross-reactivity was most intense with substance P. However, substance K, neuromedin K and physalaemin also exhibited limited cross-reactions with the antiserum. In the ventrolateral parabrachial region of untreated adult animals, substance P-like immunoreactivity was localized in axon terminals containing numerous small (40-60 nm) clear vesicle and 1-3 large (90-120 nm) dense-core vesicles. At least 54% of the labeled terminals formed asymmetric synapses with unlabeled dendrites; and at least 30% of the recipient dendrites received more than one labeled axon terminal. In addition, the labeled terminals were associated less frequently with other unlabeled soma, axon terminals and blood vessels. In the second part of the study, we examined whether or not perikarya in various extrinsic regions contributed to the substance P-like immunoreactivity in axon terminals in the parabrachial region. Wheat-germ agglutinin conjugated horseradish peroxidase was injected unilaterally into the parabrachial region of adult rats two days prior to being killed and one day prior to intraventricular injection of colchicine (100 micrograms in 7.5 microliter saline) which enhanced the detection of immunoreactivity in perikarya. Sections were first processed by a tetramethylbenzidine reaction stabilized with cobalt-diaminobenzidine for demonstration of the transported peroxidase then were immunocytochemically labeled for substance P. Perikarya containing both the black granular retrograde labeling and brown peroxidase-immunoreactivity were found in the nuclei of the solitary tracts, the caudal ventrolateral reticular formation, the lateral dorsal tegmental nucleus and the paraventricular, dorsomedial and lateral hypothalamic nuclei. The projections were largely, but not exclusively, from perikarya located on the same side as the parabrachial injection. We conclude that substance P, or a closely related tachykinin, is a putative transmitter or modulator within a number of pathways to the parabrachial region and that these afferents act primarily through axodendritic synapses with intrinsic neurons.     

Ultrastructural analysis of ventrolateral periaqueductal gray projections to the A7 catecholamine cell group

Stimulation of neurons in the ventrolateral periaqueductal gray produces antinociception that is mediated in part by pontine noradrenergic neurons. Previous light microscopic analysis provided suggestive evidence for a direct projection from neurons in the ventrolateral periaqueductal gray to noradrenergic neurons in the A7 cell group that innervate the spinal cord dorsal horn. Therefore, the present ultrastructural study used anterograde tracing combined with tyrosine hydroxylase immunoreactivity to provide definitive evidence that neurons in the ventrolateral periaqueductal gray form synapses with the somata and dendrites of noradrenergic neurons of the A7 cell group. Injections of the anterograde tracers biotinylated dextran amine or Phaseolus vulgaris leucoagglutinin into the ventrolateral periaqueductal gray of Sasco Sprague-Dawley rats yielded a dense innervation in the region of the lateral pons containing the A7 cell group. Electron microscopic analysis of anterogradely labeled terminals (n=401) in the region of the A7 cell group indicated that approximately 10% of these formed plasmalemmal appositions to tyrosine hydroxylase-immunoreactive dendrites with no intervening astrocytic processes. About 23% of these were asymmetric synapses, 10% were symmetric synapses, and 67% did not exhibit clearly differentiated synaptic specializations. The majority of anterogradely labeled terminals (60%) formed plasmalemmal appositions with dendrites and somata that lacked detectable tyrosine hydroxylase immunoreactivity. About 35% of these were symmetric synapses, 9% were asymmetric synapses and 56% did not form synaptic specializations. Approximately 30% of all anterogradely labeled terminals displayed features characteristic of axo-axonic synapses.The present results provide direct ultrastructural evidence to support the hypothesis that the analgesia produced by stimulation of neurons in the ventrolateral periaqueductal gray is mediated, in part, by activation of spinally projecting noradrenergic neurons in the A7 catecholamine cell group.     

大鼠三叉神经本体感觉中枢通路二、三级核团内Parvalbumin样阳性神经元突触联系的电镜研究

本教研室以往的研究证实 Parvalbum in样免疫阳性细胞广泛分布于三叉神经本体感觉中枢四级通路的各级中继核团 ,其中有 30 %～ 5 0 %为投射神经元。本研究应用电镜免疫组织化学技术进一步对此通路第二、三级神经元所在地的 Parvalbumin样阳性神经元及其纤维和终末的突触联系进行了观察。结果显示 Parvalbum in样阳性结构主要形成以下几种突触联系 :( 1) Par-valbumin样阳性轴突与 Parvalbumin样阳性胞体或树突形成轴 -体或轴 -树突触 ,其中以非对称性突触为主 ,对称性突触较少 ;( 2 )Parvalbumin样阳性轴突分别与 Parvalbumin样阴性神经元的胞体或树突形成轴 -体或轴 -树突触 ,这些突触联系以对称性为主 ,非对称性大约占 30 %左右 ;( 3) Parvalbumin样阴性终末与 Parvalbumin样阳性树突形成以对称性为主的轴 -树突触 ,这种突触大约占所有突触联系的 5 0 %。以上结果表明 :面口部本体感觉信息由三叉神经中脑核神经元向丘脑腹后内侧核传递的过程中 ,Par-valbumin样阳性轴突终末可通过突触传导机制而兴奋或抑制二、三级核团内的投射或中间神经元而发挥其重要作用     

大鼠中脑导水管周围灰质-大中缝核-脊髓通路——顺行溃变和HRP逆行追踪结合法的电镜观察

本文用海人酸注入中脑导水管周围灰质(PAG)和HRP注入脊髓(SpC)相结合的方法在电镜水平对PAG-NRM-SpC通路进行了研究。在大中缝核(NRM)内可见四种突触:(1)溃变轴突终末与HRP逆标胞体形成轴-体突触;(2)溃变轴突终末与非HRP标记的远端树突形成轴-树突触;(3)HRP顺标轴突末梢与非HRP标记的树突和HRP逆标胞体形成轴-树和轴-体突触。我们认为:PAG-NRM-SpC间接通路在NRM中可能有单突触和通过中间神经元两种联系方式。NRM和SpC之间有往返联系。