Spatial and temporal distribution patterns of enkephalinergic neurons in adult and developing retinas of the preproenkephalin-green fluorescent protein transgenic mouse

Enkephalin (ENK) peptides are present in the retina of several vertebrate species and play a crucial role in establishing specific circuits during retinal development. However, there is no information available concerning the development of ENKergic neurons in the mouse retina. To address this question, we used preproenkephalin-enhanced green fluorescent protein (GFP) transgenic mice, in which ENKergic neurons are revealed by GFP. Our results showed that most GFP-positive cells were located in the proximal part of the inner nuclear layer with a scattering of GFP-immunoreactive cells in the ganglion cell layer (GCL) in the adult retina. Double immunostaining with syntaxin indicates that GFP expression was restricted to a population of amacrine cells. The proportions of glycine transporter-1 and γ-aminobutyric acid-positive cells among ENKergic neurons were 57.3 ± 2.4% and 10.1 ± 1.8%, respectively. We then injected retrograde tracer into the superior colliculus and observed that none of the ENKergic neurons in the GCL were retrogradely labeled with the tracer. GFP-positive cells were first observed at embryonic day (E) 15 in the inner neuroblastic layer at only very low levels, which gradually increased until E18. After birth, there was a steep rise in GFP expression levels, reaching maximal activity by postnatal day (P) 7. The distribution and intensity of GFP-positive cells at P15 were similar to those of adult retina. It was found that immunoreactive processes in the inner plexiform layer formed strongly stained patches. The present results provide detailed morphological evidence of the cell type and spatial and temporal distribution of ENKergic neurons in the retina.     

小鼠脊髓神经元内脑啡肽与1型囊泡膜谷氨酸转运体的共存

目的:以PPE-GFP转基因小鼠为研究工具,观察绿色荧光蛋白(GFP)阳性的脑啡肽(ENK)能神经元与1型囊泡膜谷氨酸转运体(VGLUT1)在脊髓的分布及共存情况。方法:利用免疫组织化学和原位杂交双标染色的方法。结果:GFP标记的ENK能神经元主要位于脊髓背角,在I-ⅡI层最为密集,背角深层内侧部及中央管周围呈中等密度分布,散在分布于前角。VGLUT1 mRNA阳性细胞广泛分布在脊髓各层。GFP/VGLUT1双标细胞主要分布在脊髓背角,I-ⅡI层双标细胞占GFP阳性细胞的22.95±1.10%,占VGLUT1阳性细胞的27.91±2.42%;IV-VI层中21.49±4.99%GFP阳性细胞表达VGLUT1,10.35±2.81%VGLUT1阳性细胞表达GFP;前角双标细胞占VGLUT1阳性细胞的1.07±0.37%,占GFP阳性细胞的32.08±13.15%。结论:双标结果表明脊髓内部分ENK能神经元表达1型囊泡膜谷氨酸转运体,推测ENK能神经元可能通过调控谷氨酸的释放发挥感觉信息调控作用。     

利用GAD67-GFP基因敲入方法显示小鼠脊髓内表达GFP的GABA能神经元的分布(英文)

γ-氨基丁酸 (GABA)是脊髓背角、前角内主要的抑制性神经递质。为了更好地观察脊髓背角内 GABA能神经元的形态和功能 ,本研究使用了两种谷氨酸脱羧酶 67-绿色荧光蛋白 (GAD67-GFP)基因敲入小鼠 ,并观察了敲入小鼠脊髓内的 GFP表达状况。用免疫荧光组织化学双标记方法显示脊髓内所有的 GF P阳性神经元基本上都呈 GAD67和 GABA阳性 ;GFP阳性神经元在脊髓背角的 ～ 层最为密集 ,背角深层内侧部及中央管周围呈中等密度分布 ,而在脊髓背角其它部位及前角则呈散在分布。脊髓内 GFP阳性神经元的分布与 GABA能神经元的分布一致。本文作者等还进一步在 GAD67-GFP敲入小鼠中观察了 GFP和神经元标志物神经元核蛋白 (Neu N )的共存状况。脊髓背角内 GFP阳性神经元分别占 、 和 层的 Neu N阳性神经元的 3 1.5 %、3 3 .3 %和 44 .7% ,与以往的 GABA免疫组化研究结果基本一致。本研究表明 GAD67-GFP基因敲入小鼠脊髓内的 GFP在GAD67启动子的调节下正确地表达于 GABA能神经元 ,该基因敲入小鼠可用于脊髓 GABA能神经元的形态学特征和生理学特性及其发育规律等方面的研究     

一种鸡胚发育过程脊髓神经纤维投射研究方法的建立

目的建立一种鸡胚发育过程脊髓神经纤维投射的研究方法。方法采用鸡胚带壳开窗培养技术,在鸡胚胚龄3d(E3),通过活体电转基因技术将携带有报告基因绿色荧光蛋白(GFP)的质粒(pCAGGS-GFP)准确注射到脊髓腔进行定时定位活体电转,转染后3d在体视荧光显微镜下进行观察;取出GFP阳性表达的胚胎,剥离出脊髓,从顶板处破开之后将脊髓展开,用4%多聚甲醛固定1h后,对神经钙黏蛋白(N-cadherin)进行免疫荧光染色,用4’6-二脒基-2-苯基吲哚(DAPI)染细胞核;封片后在荧光显微镜下观察神经纤维投射情况。结果对比横向切片和脊髓展开标本,两者均观察到GFP阳性转染侧的神经元纤维穿过底板沿对侧脊髓白质区边缘投射到神经结节,在脊髓展开标本中还可观察到神经纤维穿过底板再纵向向脑部投射;而N-cadherin免疫荧光染色结果表明,GFP基因的转染对机体正常的发育无明显影响。结论本实验建立了一种鸡胚发育过程脊髓神经纤维投射的研究方法。     

神经示踪定位SD大鼠长下行脊髓固有神经元胞体及其轴突投射的解剖位置

目的 利用神经示踪技术探讨SD大鼠长下行脊髓固有神经元及其轴突投射的解剖位置.方法 将荧光金(FG)注射入第1腰髓(L1)节段逆行标记大鼠下行脊髓固有神经元(DPNs)胞体;将顺行神经示踪剂生物素葡聚糖胺(BDA)注射到脊髓第3和第4颈髓处标记此处的DPNs胞体及其长下行脊髓固有束(LDPT).固定取材与切片染色后,检...     

大鼠三叉神经脊束核尾侧亚核和脊髓向丘脑和臂旁核的强啡肽能和一氧化氮能投射

本文用荧光金逆行追踪与免疫荧光组化染色相结合的方法，对大鼠三叉神经脊束核尾侧亚核和颈髓背角浅层向丘脑腹基底复合体和臂旁核的强啡肽能和NO能投射进行了研究．强啡肽原前体样阳性胞体主要位于尾侧亚核和颈髓背角的Ⅰ层和Ⅱ层外侧部；NOS样阳性胞体主要位于尾侧亚核和颈够背角Ⅱ层，Ⅰ层较少。将荧光金注入丘脑腹基底复合体后，荧光金逆标神经元主要见于对侧尾侧亚核、颈髓背角的Ⅰ层和外侧网状核，Ⅱ层偶见；将荧光金注入臂旁核后，逆标神经元主要见于同侧尾侧亚核和颈髓背角的Ⅰ、Ⅱ层，少量位于外侧网状核。尾侧亚核向丘脑瓜基底复合体投射神经元的16．6％，向臂旁核投射神经元的24．8％呈强啡肽原前体样阳性；颈髓背角浅层向丘脑腹基底复合体投射神经元的19．2％，向臂旁核投射神经元的272％呈强啡肽原前体样阳性。向丘脑腹基底复合体和臂旁核投射的强啡肽原前体／荧光金双标神经元分别占尾侧亚核浅层内强啡肽原前体样阳性神经元总数的7％和18％，分别占颈髓背角浅层内强啡肽原前体样阳性神经元总数的8．1％和21．9％。这些双标神经元多呈大梭形及中等大圆形和梨形。由昆侧亚核向丘脑腹基底复合体投射神经元的5．1％呈NOS阳性，向臂旁核投射神经元的11．8％呈NOS阳性。由颈髓背角浅层向丘脑版?     

基于鸡胚电转技术研究脊髓神经干细胞相关基因功能方法的建立

目的建立一种基于鸡胚电转技术研究脊髓神经干细胞(NSCs)相关基因功能的方法。方法 RT-PCR检测鸡胚发育不同时期脊髓NSCs表面标志物;在鸡胚胚龄(E)E2.5~E3时,利用活体电转基因技术将p CAGGSGFP质粒转染到鸡胚脊髓,E6时体视荧光显微镜下筛选绿色荧光蛋白(GFP)阳性胚胎,每组至少取材5个;通过脊髓横切及open-book技术观察神经纤维投射情况;普通光学显微镜下剥离出3~5条脊髓,经胰蛋白酶消化、离心后,无血清NSCs培养基重悬获得细胞铺板,于37℃、5%CO_2细胞培养箱内培养,定时观察GFP阳性脊髓NSCs的形态变化。结果 RT-PCR结果表明,鸡胚脊髓中阳性表达NSCs表面标志物;随后的脊髓横切及open-book结果表明,GFP阳性转染侧的神经纤维能穿过底板,投射到脊髓对侧;而脊髓NSCs体外培养结果显示,GFP标记的脊髓细胞具有典型的NSCs形态,继续培养后有明显突起产生。结论本实验成功建立了一种基于鸡胚电转技术研究脊髓神经干细胞相关基因功能的方法。     

高压氧前处理脊髓损伤大鼠前角运动神经元的凋亡*☆

背景：脊髓损伤后难以修复,损伤后保护残存的神经元是促进神经再生的关键。 目的：验证高压氧预处理可以通过抑制早期的细胞凋亡来保护脊髓前角运动神经元。 方法：随机将26只雄性Wistar大鼠等分成模型组和实验组。实验组在给予高压氧5 d后与模型组同时制作脊髓T9~10全横断模型。 结果与结论：尼氏染色显示脊髓T9~T10全横断后8 h及1 d,脊髓前角的浓染的细胞多见,与模型组相比,实验组脊髓前角浓染的细胞较少。TUNEL染色也显示脊髓T9~T10全横断后脊髓损伤后8 h~1 d,2组大鼠脊髓前角内均可见大量的凋亡神经元,3 d时凋亡神经元数量减少。相比于模型组,高压氧预处理8 h,1 d后大鼠脊髓前角凋亡神经元较少(P < 0.05,        P < 0.01)。说明高压氧预处理能对脊髓损伤后前角运动神经元起保护作用。       

细胞周期标记物在不同胚胎发育阶段小鼠脊髓内的分布模式

本研究用免疫组织化学方法观察了细胞周期全程标记物Ki-67和有丝分裂期标记物磷酸化的组蛋白H3(P-H3)在不同胚胎发育阶段小鼠脊髓的分布状况。结果显示:在胚胎第12d(E12)和E13,Ki-67标记细胞密集分布在脑室带(VZ)。E14以后,随着胎龄的增长,VZ内的Ki-67标记细胞逐渐减少,而套层内Ki-67阳性细胞的数量逐渐增多并于E17达到高峰。在此阶段,套层内Ki-67阳性细胞的数量占其阳性细胞总量的87.3%。在各胚胎阶段的脊髓内,VZ和套层内Ki-67阳性细胞的分布无背腹方向上的差异。P-H3标记细胞在胚胎发育阶段脊髓内分布的时空模式与Ki-67相似,其在套层内的分布同样于E17时达到高峰。另外,我们在E16之后的脊髓白质内还观察到了Ki-67和P-H3的标记细胞,以E18时数量最多。本研究结果提示,在胚胎发育晚期的脊髓套层内存在着具有增殖活性的神经祖细胞,它们可能是脊髓神经细胞产生的另外的来源。     

大鼠脊髓内囊泡膜谷氨酸转运体1和囊泡膜谷氨酸转运体2阳性纤维和终末在生后发育中的分布和表达变化

目的 探讨囊泡膜谷氨酸转运体1（VGLUT1）和VGLUT2阳性纤维和终末在生后第0天（P0）至第22天（P22）大鼠脊髓内的分布情况和表达变化。 方法 对生后发育P0～P22大鼠的颈膨大和腰膨大部位,进行VGLUT1和VGLUT2免疫组织化学染色。 结果 P0～P22大鼠颈膨大和腰膨大脊髓内均可观察到VGLUT1和VGLUT2阳性纤维和终末,但未观察到胞体样结构。VGLUT1和VGLUT2阳性纤维和终末的分布呈现明显的互补分布,尤其是以脊髓后角更加明显。其中,VGLUT1阳性纤维和终末在P0主要见于颈膨大和腰膨大脊髓后角Ⅲ～Ⅴ层,中间部和前角很微弱。脊髓发育至P3,不仅Ⅲ~Ⅴ层VGLUT1的表达进一步增强,且向外侧部扩展,并在后角基底部Ⅵ层和前角的外侧部（Ⅸ层）也可观察到较强的VGLUT1阳性纤维,呈现一条明显由背内向腹外的带状分布趋势。P7时此带状分布更加明显,并随着发育逐渐向内、外扩展,至P22时已广泛分布于除Ⅱ层之外的整个脊髓。而VGLUT2阳性纤维和终末在P0时即密集出现于脊髓后角Ⅰ～Ⅱ层以及前角的外侧边缘区域;之后随着发育,VGLUT2阳性纤维和终末的分布模式并未发生明显改变,但其密度逐渐有所增加,特别是Ⅰ～Ⅱ层内VGLUT2阳性产物的表达尤为明显。另外,在脊髓白质后索内可见VGLUT1阳性皮质脊髓后束纤维由颈髓（P3）逐渐下降至腰髓（P7）的发育过程。 结论 VGLUT1和VGLUT2阳性纤维和终末在脊髓发育过程中呈现明显不同,且表现出互补分布的特点,这对于进一步理解VGLUT1和VGLUT2在脊髓生后发育过程中不同功能特点可能有意义。     

Development of GABAergic neurons from the ventricular zone in the superior colliculus of the mouse

The superior colliculus (SC) is a layered structure in the midbrain and is particularly rich in gamma-aminobutyric acid (GABA). The present investigation aimed to determine whether the development of GABAergic neurons in the SC is common to that of the neocortex in which they are produced in a distinct area called the ganglionic eminence and are transported by tangential migration. A green fluorescent protein (GFP) knock-in mouse was used in which a GFP gene was introduced into the gene for glutamic acid decarboxylase (GAD) 67 and all GABAergic neurons were fluorescent. At embryonic day (E) 11-14, GFP-positive cells increased strikingly. They were spindle-shaped with processes at both poles and oriented radially between the ventricular and pial surface, together with other GFP-negative cells. After the cutting of the embryonic SC, GFP-positive cells accumulated on one side of the injury as expected from their radial but not tangential migration. In the living slice preparations GFP-positive cells migrated radially during the observation. These results indicate that tangential migration of GABAergic neurons as observed in the neocortex is not applicable and that radial migration from the underlying ventricular zone is predominant in the SC. At E12-13, bundles of commissural GFP-positive fibers which appeared to originate outside the SC were distributed at the superficial layer. These superficial fibers were no longer observed at the later stages.     

大鼠脊髓后角神经元轴突终末中脑啡肽、P物质在移植坐骨神经中的观察

将成年大鼠一段自体坐骨神经植入脊髓胸节段右侧后角灰质内、存活半个月至一个月,时移植物及其相连脊髓行神经丝,脑啡肽、P物质免疫组化染色.结果发现大量阳性轴突通过移植物-脊髓吻合口再生进入移植物.脑啡肽、P物质阳性再生纤维可见两类,膨体样串珠状纤维和大串珠状纤维.结果表明:脊髓后角损伤后至少有脑啡肽、P物质两类肽能神经元保持着再生潜力;在无靶细胞情况下,再生肽能神经元有神经肽的合成且再生轴突出现膨体样结构.     

Spinal cord contains neurotrophic activity for spinal nerve sensory neurons. Late developmental appearance of a survival factor distinct from nerve growth factor

Several tissues of the developing chick embryo have been reported to contain neurotrophic activity which can sustain the survival of sensory neurons maintained in culture. In a previous study, however, we noted that such nerve growth promoting activity was exceptionally low, if not absent, from extracts of spinal cord from chick embryos of up to 16 days incubation. Since then the combined results from a number of tissue culture studies have suggested that the central nervous system may be the source of a neurotrophic growth factor essential during the late development of sensory neurons. We have therefore carried out an extended range study of the neurotrophic properties of avian spinal cord. Extracts of spinal cord tissue prepared from chicks at stages between the last wk of embryogenesis and 12 wks after hatching were tested for their ability to promote survival and neurite outgrowth from both explant and dissociated neuron-enriched cultures of dorsal root, trigeminal, nodose and paravertebral chain sympathetic ganglia from chick embryos between 8 and 16 days old. We conclude from our results that spinal cord is a potent source of neurotrophic activity for sensory neurons, although this activity appears relatively late in development of the spinal cord. The predominant ontogenic increase in spinal cord neurotrophic activity was seen to occur during the first week after hatching. Sensory neurons from both spinal and cranial nerve ganglia were sustained in culture by spinal cord extracts, whereas sympathetic neurons did not respond. Neurons from older sensory ganglia (12-16 day old embryos) were much more responsive than similar neurons from young embryos (8 day).(ABSTRACT TRUNCATED AT 250 WORDS)     

Responses and afferent pathways of C1-C2 spinal neurons to cervical and thoracic esophageal stimulation in rats

Because vagal and sympathetic inputs activate upper cervical spinal neurons, we hypothesized that stimulation of the esophagus would activate C(1)-C(2) neurons. This study examined responses of C(1)-C(2) spinal neurons to cervical and thoracic esophageal distension (CED, TED) and afferent pathways for CED and TED inputs to C(1)-C(2) spinal neurons. Extracellular potentials of single C(1)-C(2) spinal neurons were recorded in pentobarbital-anesthetized male rats. Graded CED or TED was produced by water inflation (0.1-0.5 ml) of a latex balloon. CED changed activity of 48/219 (22%) neurons; 34 were excited (E), 12 were inhibited (I), and 2 were E-I. CED elicited responses for 18/18 neurons tested after ipsilateral cervical vagotomy, for 12/14 neurons tested after bilateral vagotomy and for 9/11 neurons tested after bilateral vagotomy and C(6)-C(7) spinal cord transection. TED changed activity of 31/190 (16%) neurons (28E, 3 I). Ipsilateral cervical vagotomy abolished TED-evoked responses of 5/12 neurons. Bilateral vagotomy eliminated responses of 2/4 neurons tested, and C(6)-C(7) spinal transection plus bilateral vagotomy eliminated responses of 2/2 neurons. Thus inputs from CED to C(1)-C(2) neurons most likely entered upper cervical dorsal roots, whereas inputs from TED were dependent on vagal pathways and/or sympathetic afferent pathways that entered the thoracic dorsal roots. These results supported a concept that C(1)-C(2) spinal neurons play a role in integrating visceral information from cervical and thoracic esophagus.     

Characterisation of the consequences of maternal immune activation on distinct cell populations in the developing rat spinal cord

Maternal immune activation (MIA) during gestation has been implicated in the development of neurological disorders such as schizophrenia and autism. Epidemiological studies have suggested that the effect of MIA may depend on the gestational timing of the immune challenge and the region of the central nervous system (CNS) in question. This study investigated the effects of MIA with 100 μg/kg lipopolysaccharide at either Embryonic days (E)12 or E16 on the oligodendrocytes, microglia and astrocytes of the offspring spinal cord. At E16, MIA decreased the number of olig2⁺ and Iba‐1⁺ cells in multiple grey and white matter regions of the developing spinal cord 5 h after injection. These decreases were not observed at postnatal day 14. In contrast, MIA at E12 did not alter Olig2⁺ or Iba‐1⁺ cell number in the developing spinal cord 5 h after injection, however, Olig2⁺ cell number was decreased in the ventral grey matter of the P14 spinal cord. No changes were observed in glial fibrillary acidic protein (GFAP) expression at P14 following MIA at either E12 or E16. These data suggest that E16 may be a window of immediate vulnerability to MIA during spinal cord development, however, the findings also suggest that the developmental process may be capable of compensation over time. Potential changes in P14 animals following the challenge at E12 are indicative of the complexity of the effects of MIA during the developmental process.     

体感诱发电位评价慢性压迫性脊髓症神经功能的病理机制

目的 探讨慢性压迫性脊髓症不同体感诱发电位(somatosensory evoked potential,SEP)变化对应的病理学机制.方法 20只SD大鼠经后路手术、颈椎管内(C5～C6水平)植入吸性聚氨酯胶片,该植入体在硬膜外逐渐吸水膨胀,形成对脊髓的慢性持续压迫.术前和造模后6个月检测SEP,并对慢性压迫脊髓行Micro-CT、组织学(HE染色)和组织化学(FLB染色)检测.结果 20只造模大鼠脊髓均出现侧后方明显压迫性形态学改变,Micro-CT显示脊髓灰质和白质扭曲变形.依据SEP变化分为Ⅰ(n=6)、Ⅱa(n=5)、Ⅱb(n=4)、Ⅲ(n=5)、Ⅳ(n=0)5类.SEP异常者脊髓后索髓鞘FLB染色显著减少(SEP异常:106±27;SEP正常:121±8;P=0.036),Micro-CT显示脊髓后索对比剂密度明显增加(SEP异常:95±5;SEP正常:87±6;P=0.041),后角内神经元也明显较少[SEP异常:(21±8)/mm2;SEP正常:(29±6)/mm2;P＞0.05].病理学上,SEP-Ⅰ型表现为脊髓中央管扩大;Ⅱa型表现为灰质内出血、静脉扩张和中央管缩小;Ⅱb型表现为灰质、白质排列紊乱,血管增生;Ⅲ型表现为神经元明显减少、白质-灰质结构不清,基质海绵样变.结论 慢性压迫性脊髓症不同类型的SEP变化反映了脊髓后索和灰质神经元损伤的严重程度,SEP作为脊髓功能预后评估的判断指标具有相应的病理学特征.     

Enigmatic cerebrospinal fluid-contacting neurons arise even after the termination of neurogenesis in the rat spinal cord during embryonic development and retain their immature-like characteristics until adulthood

Despite the abundance of cerebrospinal fluid-contacting neurons (CSF-cNs) lining the central canal of the spinal cord of mammals, little information is known regarding the phenotype and fate of these cells during development and in adulthood. Using immunofluorescence of spinal cord tissue of rats from the first postnatal day (P1) until the end of the 5th postnatal week (P36), we observed that these neurons show both immature (doublecortin+, β-III-tubulin+, neurofilament 200 kDa−) and more mature (weak NeuN+, P2X2+, GAD65+) characteristics during the first postnatal weeks. Because of the gradually decreasing number of CSF-cNs in the central canal lining during development, we were also interested in the migration potential of these cells. However, the assessment of the number of CSF-cNs in the lining of the central canal during postnatal development revealed that this decline is most likely associated with the growth of the spinal cord. Lastly, to reveal the birth date of CSF-cNs, we performed 5-bromo-2-deoxyuridine administration and colocalization analyses. We found that production of these cells appears from day 12 of embryonic development (E12) until E22. The vast majority of CSF-contacting neurons arise on E14 and E15. In contrast with other types of spinal neurons, the production of CSF-cNs is not restricted to a particular neuroepithelial region and occurs even after what is thought to be the termination of neurogenesis.     

Loss of propriospinal neurons after spinal contusion injury as assessed by retrograde labeling

We studied the number, location and size of long descending propriospinal tract neurons (LDPT), located in the cervical enlargement (C3–C6 spinal levels), and short thoracic propriospinal neurons (TPS), located in mid-thoracic spinal cord (T5–T7 spinal levels), 2, 6 and 16 weeks following a moderate low thoracic (T9) spinal cord contusion injury (SCI; 25 mm weight drop) and subsequent injections of fluorogold into the upper lumbosacral enlargement (L2–L4 spinal levels). Retrograde labeling showed that ∼23% of LDPT and 10% of TPS neurons were labeled 2 weeks after SCI, relative to uninjured animals. No additional significant decrease in number of labeled LDPT and TPS cells was found at the later time points examined, indicating that the maximal loss of propriospinal neurons in these two subpopulations occurs within the first 2 weeks post-SCI. The distribution of labeled cells post-moderate SCI was similar to normal in terms of their location within the gray matter. However, there was a significant change in the size (cross sectional area) of labeled neurons following injury, relative to uninjured controls, indicating a loss in the number of the largest class of propriospinal neurons. Interestingly, the number of labeled LDPT and TPS neurons was not significantly different following different injury severities. Although the rostro–caudal extent of the lesion site expanded between 2 and 16 weeks following injury, there was no significant difference in the number of propriospinal neurons that could be retrogradely labeled at these time points. Possible reasons for these findings are discussed.