大鼠隔核中肽能神经元的分布与形态测量

用免疫组织化学方法研究了大鼠隔核各亚核中降钙素基因相关肽，血管活性肠肽，生长抑素，神经降压肽，促皮质释放因子，Ｐ物质，亮氨酸－脑腓肽，黄体生成素释放因子，甘丙肽，胆囊收缩素，促甲状腺素释放因子，甲硫氨酸－脑腓肽，等１２种肽能神经元的分布，并用图象分析仪对肽能神经元的面积，周长，最大径，最小径和灰度进行了测量。肽能神经元主要分布在外侧隔核中间部，内侧隔核；而隔伞核及三角隔核中较少。图象分析仪测量表明     

GABA能中间神经元在运输应激大鼠躯体感觉皮质的分布

目的:探讨运输应激对大鼠躯体感觉皮质γ-氨基丁酸(GABA)能中间神经元分布的影响。方法:采用摇床加束缚建立大鼠运输应激模型,将24只SD雄性大鼠随机分为对照组(Control)、运输应激1 d组(TS1d)、运输应激3 d组(TS3d)、运输应激7 d组(TS7d)。应用尼氏染色、免疫组织化学染色法观察运输应激状态下大鼠躯体感觉皮质GABA能不同亚型中间神经元的表达及分布。结果:在躯体感觉皮质,运输应激之后小清蛋白(PV)阳性神经元、生长抑素(SOM)阳性神经元、钙视网膜蛋白(CR)阳性神经元数量显著减少,在TS7d时下降最明显;PV阳性神经元数量在第Ⅱ～Ⅳ层显著减少;SOM阳性神经元数量在第Ⅱ、Ⅲ、Ⅴ层显著减少;CR阳性神经元数量在第Ⅰ～Ⅵ层显著减少。结论:运输应激后,大鼠躯体感觉皮质GABA能中间神经元显著减少,其中PV和CR这两种钙结合蛋白类中间神经元数量下降最明显,提示钙结合蛋白在调节应激方面起关键作用。     

用免疫组织化学方法显示电生理记录部位神经元的化学性质——离体脑片法研究

500μm厚的冠状脑片取自新鲜大鼠下丘脑前部。在人工孵育条件下进行电生理观察,看到前连合核和第三脑室前部室周区部分神经元的电活动可被人工脑脊液中加入血管紧张素Ⅱ所兴奋,被提高人工脑脊液中的Ca~(++)浓度所抑制。这些电生理反应与大细胞内分泌神经元的电生理特性一致。记录结束时,记录部位用记录电极中的染料标记,脑片浸泡固定4—8h,恒冷箱切片,用抗催产素和抗加压素抗体孵育,ABC反应。结果在记录电极尖端所在部位看到数量不等的催产素或加压素免疫阳性神经元。上述电生理和免疫组化结果表明,研记录到的部分神经元可能含催产素或加压素免疫反应物质。本文报告了用免疫组织化学方法显示离体电生理标本,特别是记录部位神经元的免疫化学特性的实验结果。     

人胚胎海马发育的形态学研究Ⅲ．肽能神经元的发生

利用Ｇｏｌｇｉ镀银法、免疫组织化学法，对不同胎龄人肛胎海马的肽能神经元进行了研究．发现ＳＳ阳性神经元于胚胎１９周出现，ＳＰ阳性神经元、ＮＰＹ阳性神经元于１７周出现，ＧＡＢＡ阳性神经元在１６周即已显示。随胎龄增加，肽能神经元不断增多．肽能神经元广泛分布于海马的各区，但以多形层最为多见．和锥体细胞及颗粒细胞相比，肽能神经元有下列形态特点：（１）细胞形态和大小差异较大；（２）细胞突起多少不一，出现部位也无一定规律；（３）轴突投射范围小，但分枝多．Ｇｏｌｇｉ可以同时显示多种不同肽能神经元．ＧＡＢＡ阳性细胞与ＳＳ阳性细胞在发生时间和形态特点方面具有高度一致性，说明ＳＳ可与ＧＡＢＡ共存。     

大鼠三叉神经脊束间质核内接受内脏伤害性信息的含CB神经元向孤束核的投射

目的 探讨大鼠三叉神经脊束间质核(INV)内接受内脏伤害性信息的含calbindin D-28K(CB)神经元与孤束核(NTS)的投射联系。方法 用福尔马林刺激上消化道，应用荧光金(FG)逆行束路追踪结合Fos和CB的免疫荧光组织化学三重标记法。结果 INV的背侧边缘旁核(PaMd)和三叉旁核(PaV)内可见到大量FG逆标细胞，以注射FG的同侧为主。大部分FG逆标细胞(约71.2％)为CB免疫反应阳性。部分FG和CB双标记神经元(约31．5％)同时呈Fos免疫反应阳性的三重标记。结论 INV内部分接受内脏伤害性刺激的CB神经元可直接投射至NTS，含CB的神经元在内脏伤害性信息经INV向NTS的传导通路中，可能发挥重要作用.     

GABA样神经元和GABA能三叉一丘脑投射神经元在三叉神经感觉核簇的分布及形态学特征

用直接抗GABA免疫组化方法和与HRP逆行追踪相结合的双标记法在光镜下观察了GABA样神经元和GABA能丘脑投射神经元在大鼠三又神经惑觉核簇的分布和形态特征。结果显示:GABA样神经元广泛存在于三叉神经感觉核簇的各亚核中,但在每一亚核和尾侧亚核的各层,其分布状况和细胞形态特征各异。在尾侧亚孩,GABA样神经元的分布密度为Ⅰ、Ⅱ层>Ⅳ、Ⅴ层>Ⅲ层;在极间亚核,GABA样细胞分为背、腹二群,以腹侧群为主;在吻侧亚核,分为背内侧群、内侧带群和腹外侧群三群;在感觉主核分为背内侧群和腹外侧群;在三叉上核分为吻内侧群和尾外侧群。同样,本文首次发现,GABA能丘脑投射神经元也广泛存在于三叉神经感觉核簇各核中,而且在各亚核及各层中其分布密度及细胞形态也不相同。在尾侧亚核,双标细胞占38.6%,主要分布在Ⅰ、Ⅱ层,具有较长附突;在极间亚核,双标细胞占40%,主要分布在腹侧群;在吻侧亚核中仅在腹侧群有少量小的双标细胞;在感觉主核,双标细胞占20-30%,主要集中在背内侧群,腹外侧群仅有少量;在三叉上核,双标细胞仅出现在尾外侧群。结果提示:GABA对三叉神经感觉核簇中的各种深浅感觉功能的传导和整合可产生广泛的抑制影响,但其影响形式、程度和机制可能各不相同。     

大鼠孤束核内儿茶酚胺能神经元接受面口部深层组织伤害性信息并向臂旁核投射的形态学研究

为了探讨孤束核(NTS)内儿茶酚胺能神经元是否与面口部深层组织的伤害性信息有关并向臂旁核投射,本研究运用荧光金(FG))逆行追踪,福尔马林刺激咬肌和免疫荧光技术相结合的三重标记方法,在荧光显微镜下观察了大鼠NTS内酪氨酸羟化酶(TH)阳性并表达FOS蛋白的神经元向臂旁核的投射。将2％FG注入一侧臂旁外侧核后,向同侧咬肌内注射2％福尔马林溶液,并行TH和FOS免疫荧光组织化学染色,荧光显微镜下在同侧NTS内的连合、内侧、中间内侧和腹侧亚核中可见重叠分布的FG、FOS、TH单标神经元以及FG／TH、FOS／TH、FOS／FG双标和FG／FOS／TH三标神经元。FG／TH和FOS／TH双标神经元分别占同侧NTS内TH阳性神经元总数的28.6％和34.8％;FOS／FG双标神经元占同侧FG逆标神经元总数的26.4％;FG／FOS／TH三标神经元分别占同侧TH阳性神经元和FG逆标神经元总数的23.7％和8.4％。本结果提示大鼠NTS中的部分儿茶酚胺能神经元接受面口部深层组织的伤害性信息并向臂旁外侧核传递。     

偏侧帕金森病大鼠模型丘脑底核谷氨酸能神经元的免疫组织化学研究

为探讨丘脑底核中谷氨酸能神经元在帕金森病时的变化 ,本研究应用免疫组织化学和形态计量学技术对用 6-OHDA脑内注射制备的偏侧帕金森病大鼠模型丘脑底核谷氨酸能神经元的变化进行了观察和计数。结果发现 ,6-OHDA损毁侧丘脑底核中谷氨酸免疫反应阳性神经元的数量显著高于非损毁侧 (P<0 .0 1)。本研究结果提示 ,帕金森病时黑质多巴胺能神经元的减少可能导致丘脑底核过度兴奋 ,这很可能是产生帕金森病症状及其病情渐进发展的原因之一。     

Molecular mechanisms and signaling by comenic acid in nociceptive neurons influence the pathophysiology of neuropathic pain

Comenic acid (CA), a specific agonist of opioid-like receptors, effectively and safely relieves neuropathic pain by decreasing the Na_V1.8 channel voltage sensitivity in the primary sensory neuron membrane. CA triggers downstream signaling cascades, in which the Na,K-ATPase/Src complex plays a key role. After leaving the complex, the signal diverges ‘tangentially’ and ‘radially’. It is directed ‘tangentially’ along the neuron membrane to Na_V1.8 channels, decreasing the effective charge of their activation gating system. In the radial direction moving towards the cell genome, the signal activates the downstream signaling pathway involving PKC and ERK1/2. A remarkable feature of CA is its ability to modulate Na_V1.8 channels, which relieves neuropathic pain while simultaneously stimulating neurite growth via the receptor-coupled activation of the ERK1/2-dependent signaling pathway.     

Morphological and electrophysiological characteristics of pyramidal tract neurons in the rat

Summary Responses evoked in neurons of rat sensorimotor cortex upon stimulation of the pyramidal tract and ipsilateral cerebral peduncle were analysed using intracellular recording. Neurons responding antidromically to pyramidal tract stimulation (PT cells) and neurons failing to respond anti-dromically but exhibiting orthodromic responses were both stained by intracellular injection of horse-radish peroxidase (HRP). Layer V pyramidal neurons, including those responding antidromically, exhibited prominent long lasting membrane hyperpolarizations and inhibitions of action potentials following pyramidal tract or cerebral peduncle stimulation. Upon passage of polarizing intracellular current two components were identified within the hyperpolarizing potential. A short duration initial component readily reversed with hyperpolarizing current. Frequently this earlier component over-lapped a period of early excitation consisting of action potentials arising from recurrent EPSPs or large slow depolarizing potentials (SDPs). The second, much longer duration hyperpolarizing component did not reverse with passage of hyperpolarizing current and was often followed by a rebound period of depolarization and action potential generation. Both the excitatory and the inhibitory portions of these responses could be demonstrated in animals with acute thalamic transections severing the ascending lemniscal pathway to cortex. Following intracellular staining with HRP, two types of PT cells were identified by their different intracortical axonal arborizations. Most of the injected neurons had local axonal fields extending widely in layers V and VI, but with few or no collaterals extending radially toward the more superficial layers. A second type of PT cell had axon collaterals limited to a narrow zone around the dendritic field but extending radially as far as layer I. Cells of both types were observed to send axon collaterals into neostriatum. Both types of neurons exhibited morphological and physiological characteristics of slow PT cells, and we could find no cells comparable to the fast conducting PT cells observed in other species.Supported by NIH grants NS 20743 (to CJW), NS 20702 (to STK) and a FRSQ fellowship (to PL)     

The role of the capsaicin receptor TRPV1 and acid-sensing ion channels (ASICS) in proton sensitivity of subpopulations of primary nociceptive neurons in rats and mice

A local elevation of H+-ion concentrations often occurs in inflammation and usually evokes pain by excitation of primary nociceptive neurons. Expression patterns and functional properties of the capsaicin receptor and acid-sensing ion channels suggest that they may be the main molecular substrates underlying this proton sensitivity. Here, we asked how the capsaicin receptor TRPV1 and acid-sensing ion channels (ASICS) contribute to the proton response in subpopulations of nociceptive neurons from adult rats and mice (wildtype C57/Bl6, Balb/C and TRPV1-null). In cultured dorsal root ganglion neurons, whole cell patch clamp recordings showed that the majority of capsaicin-sensitive rat dorsal root ganglion neurons displayed large proton-evoked inward currents with transient ASIC-like properties. In contrast, the prevalence of ASIC-like currents was smaller in both mouse wildtype strains and more frequent in capsaicin-insensitive neurons. Transient ASIC-like currents were more frequent in both species among isolectin B4-negative neurons. A significantly reduced proton response was observed for dissociated dorsal root ganglion neurons in TRPV1 deficient mice. Unmyelinated, but not thin myelinated nociceptors recorded extracellularly from TRPV1-null mutants showed a profound reduction of proton sensitivity. Together these findings indicate that there are significant differences between rat and mouse in the contribution of TRPV1 and ASIC subunits to proton sensitivity of sensory neurons. In both species ASIC subunits are more prevalent in the isolectin B4-negative neurons, some of which may represent thin myelinated nociceptors. However, the main acid-sensor in isolectin B4-positive and isolectin B4-negative unmyelinated nociceptors in mice is TRPV1.     

Arachidonic acid potentiates acid-sensing ion channels in rat sensory neurons by a direct action

Acid-sensing ion channels (ASICs) are activated by a decrease in extracellular pH. ASICs are expressed in nociceptive sensory neurons, and several lines of evidence suggest that they are responsible for signaling the pain caused by extracellular acidification, but little is understood of the modulation of ASICs by pro-inflammatory factors. Using whole-cell patch clamp we demonstrate that low pH evokes three distinct inward currents in rat dorsal root ganglion neurons: a slowly inactivating transient current, a rapidly inactivating transient current, and a sustained current. All three currents were potentiated by arachidonic acid (AA), to 123%, 171%, and 264% of peak current, respectively. Membrane stretch had no effect on proton-gated currents, implying that AA is unlikely to act via local membrane deformation. The current carried by heterologously expressed ASIC1a and ASIC3 was also potentiated by AA. AA potentiates ASIC activation by a direct mechanism, because inhibition of AA metabolism had no effect on potentiation, and potentiation of single ASIC2a channels could be observed in cell-free patches. Potentiation by lipids with the same chain length as AA increased as the number of double bonds was increased. AA is known to be released in inflammation and the results suggest that AA may be an important pro-inflammatory agent responsible for enhancing acid-mediated pain.     

Localization of P2X2 and P2X3 receptors in rat trigeminal ganglion neurons

Purine receptors have been implicated in central neurotransmission from nociceptive primary afferent neurons, and ATP-mediated currents in sensory neurons have been shown to be mediated by both P2X3 and P2X2/3 receptors. The aim of the present study was to quantitatively examine the distribution of P2X2 and P2X3 receptors in primary afferent cell bodies in the rat trigeminal ganglion, including those innervating the dura. In order to determine the classes of neurons that express these receptor subtypes, purine receptor immunoreactivity was examined for colocalization with markers of myelinated (neurofilament 200; NF200) or mostly unmyelinated, non-peptidergic fibers (Bandeiraea simplicifolia isolectin B4; IB4). Forty percent of P2X2 and 64% of P2X3 receptor-expressing cells were IB4 positive, and 33% of P2X2 and 31% of P2X3 receptor-expressing cells were NF200 positive. Approximately 40% of cells expressing P2X2 receptors also expressed P2X3 receptors and vice versa. Trigeminal ganglion neurons innervating the dura mater were retrogradely labeled and 52% of these neurons expressed either P2X2 or P2X3 or both receptors. These results are consistent with electrophysiological findings that P2X receptors exist on the central terminals of trigeminal afferent neurons, and provide evidence that afferents supplying the dura express both receptors. In addition, the data suggest specific differences exist in P2X receptor expression between the spinal and trigeminal nociceptive systems.     

犬和大鼠回肠壁丛内5-羟色胺能神经元免疫组织化学观察

目的　观察犬和大鼠回肠壁丛内 5 羟色胺能神经元。方法　应用 5 羟色胺 (5 HT)抗体的免疫组织化学改良法对正常小狗 (5只 )回肠切片标本、正常大鼠 (8只 )和 5 羟色胺酸前处理大鼠 (4只 )回肠外纵肌全层铺片标本内含 5 HT免疫反应性 (5 HT IR)神经元进行了观察研究。结果　正常大鼠回壁内神经节 (丛 )内可见少数 5 HT IR核周体 ,及肠肌丛周围和节间束中含有丰富的 5 HT IR纤维。 5 羟色胺酸 (5 HTP)处理后大鼠与正常鼠相比较 ,回肠壁丛内 5 HT IR胞体和带膨体纤维的可见数稍多 ,及免疫反应性增强。正常狗远端内 5 HT IR神经元胞体和纤维非常丰富 5 HT IR基础丛内有 1～ 4个 5 HT IR神经元胞体。结论　本研究对犬和大鼠肠内 5 HT能神经元的存在提供了直接的形态学证据 ,肠 5 HT能神经元与胃肠运动的调节功能及其可能的受体机制有关     

生长抑素阳性神经元在大鼠纹状体的形态分布

目的 探讨成年大鼠纹状体内生长抑素（SOM）阳性神经元的形态分布。方法 选取6只SD大鼠,应用免疫细胞化学染色ABC法观察脑纹状体SOM神经元的分布特征。 结果 在大鼠纹状体内SOM免疫反应阳性神经元呈棕黄色,为强阳性或中等阳性染色,免疫反应阳性物质主要存在于胞质和突起。SOM阳性神经元在纹状体呈散在分布,胞体中等大小［（111.81±21.42）μm2］,多为卵圆形、椭圆型或多边形。背侧［（113.52±22.36）μm2］SOM阳性神经元胞体面积大于腹侧［（110.14±20.59）μm2］,外侧［（126.23±23.45）μm2］大于内侧［（97.26±23.63）μm2］。腹内侧区SOM阳性细胞数量百分比最多［（28.34±3.20）％］,其次是背内侧区［（27.64±3.52）％］和腹外侧区［（2498±317）％］,背外侧区最少［（1904±312）％］。 结论 SOM神经元在纹状体结构各区分布的大小及数量不完全相同。     

Phosphorylation of extracellular signal-related protein kinase is required for rapid facilitation of heat-induced currents in rat dorsal root ganglion neurons

A subgroup of dorsal root ganglion (DRG) neurons responds to noxious heat with an influx of cations carried by specific ion channels such as the transient receptor potential channel of the vanilloid receptor type, subtype 1 (TRPV1). Application of capsaicin induces a reversible facilitation of these currents. This facilitation could be an interaction of two agonists at their common receptor or be caused by an influx of calcium ions into the cell. Calcium influx into the cell can activate protein kinases such as the extracellular signal-related protein kinase (ERK) pathway. This study explored the kinetics, calcium-dependency and intracellular signals following application of capsaicin and leading to facilitation of heat-induced currents (Iheat) in rat DRG neurons. Application of 0.5 microM capsaicin caused a 2.65-fold increase of Iheat within 2 s, which was significantly correlated to a small capsaicin-induced current. Intracellular application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a fast calcium chelator, did not change capsaicin-induced currents or Iheat itself, but inhibited facilitation of Iheat by capsaicin. ERK is activated by calcium influx and membrane depolarization via the mitogen-activated protein kinase/extracellular signal-related protein kinase kinase (MEK). Application of the MEK inhibitor U0126 also inhibited facilitation of Iheat by capsaicin. We conclude that the MEK/ERK cascade is an intracellular signaling pathway playing a vital role in the regulation of nociceptive neurons' sensitivity. The very fast kinetics (less than two seconds) are only explainable with a membrane-attached or at least membrane-near localization of these kinases.     

Noradrenergic mechanism involved in the nociceptive modulation of nociceptive-related neurons in the caudate putamen

Norepinephrine (NE) participates in pain modulation of the central nervous system. The caudate putamen (CPu) is one region of the basal ganglia that has been demonstrated to be involved in nociceptive perception. Our previous work has shown that microinjection of different doses of norepinephrine into the CPu produces opposing effects in the tail-flick latency (TFL) of rats. However, the mechanism of action of NE on the pain-related neurons in the CPu remains unclear. The present study examined the effects of NE and the alpha-adrenoceptor antagonist phentolamine on the pain-evoked response of pain-excitation neurons (PENs) and pain-inhibition neurons (PINs) in the CPu of rats. Trains of electric impulses were used for noxious stimulation, and were applied to the sciatic nerve. The electrical activities of pain-related neurons in the CPu were recorded by a glass microelectrode. The results revealed that intra-CPu microinjection of NE (8 μg/2 μl) increased evoked firing frequency of PEN and shortened the firing latency, but decreased the evoked firing frequency of PIN and prolonged the inhibitory duration (ID). Intra-CPu administration of phentolamine (4 μg/2 μl) showed the opposite effects. The above results suggest that NE in the CPu modulates nociception by affecting the baseline firing rates of PENs and PINs.     

B-vitamins enhance afferent inhibitory controls of nociceptive neurons in the rat spinal cord

Summary Afferent inhibition of spinal dorsal horn neuronal responses to noxious skin heating was induced by transcutaneous electrical nerve stimulation in pentobarbital-anesthetized rats. Pretreatment with B vitamins significantly enhanced this afferent inhibition, possibly due to an increase in the synthesis rate of inhibitory neurotransmitters in central neurones.