【作者】 黄静；

【导师】 武胜昔；

【作者基本信息】 第四军医大学 ， 人体解剖与组织胚胎学， 2009， 博士

【摘要】 γ-氨基丁酸(GABA)和脑啡肽(ENK)能神经元是脊髓背角重要的中间神经元,这些神经元与初级传入纤维、背角浅层的投射神经元、来自上位脑结构的下行投射纤维以及其他类型的中间神经元之间形成复杂的局部环路,在感觉信息传递和调节过程中发挥着重要的作用。由于成熟中间神经元的结构及功能形成于发育时期,它们的正常发育是上述局部环路正确形成的保证,因此很有必要从发育阶段对其开展研究。但目前有关脊髓背角GABA能和ENK能中间神经元的神经化学特点、产生模式、迁移特点及调控机制等尚存许多空白。本研究利用特异性显示GABA能神经元的谷氨酸脱羧酶(GAD)67-绿色荧光蛋白(GFP)基因敲入小鼠和特异性显示ENK能神经元的前原脑啡肽(PPE)-GFP转基因小鼠,综合运用神经形态学、分子神经生物学等手段,开展了以下三部分研究内容:1.脊髓背角GABA能和ENK能神经元的神经化学特点运用荧光原位杂交与免疫荧光双标、单细胞RT-PCR及实时定量PCR等技术,对GAD67-GFP基因敲入小鼠和PPE-GFP转基因小鼠的特异性进行了验证,并系统观察了脊髓背角GABA能和ENK能神经元的神经化学特点、相互的共存关系及5-HT3A受体的表达。结果如下:(1)免疫荧光双标结果显示GAD67-GFP基因敲入小鼠脊髓GFP阳性细胞均表达神经元标志物神经元核蛋白(NeuN),背角浅层GFP细胞分别占I、Ⅱ和Ⅲ层NeuN阳性胞核的31.51%、33.34%和44.70%。将近98%的GFP细胞表达GAD67和GABA。原位杂交组织化学与免疫组织化学双标结果显示所有GFP阳性细胞与GAD67 mRNA共存。PPE-GFP转基因小鼠不同脑区GFP与PPE mRNA的表达模式一致。脊髓GFP阳性细胞均与NeuN、PPE mRNA共存。脊髓背角GFP阳性细胞分别占I、Ⅱ和Ⅲ层NeuN阳性神经元的16.95%、40.68%和12.45%。上述结果提示这两种小鼠中GFP的表达可特异显示脊髓GABA能和ENK能神经元。(2)脊髓背角58.38%、19.63%和2.81%的GABA能神经元分别与钙视网膜蛋白(CR)、小白蛋白(PV)和维生素D依赖性钙结合蛋白(CB)共存;脊髓背角ENK能神经元与CR、PV和CB的共存率为61.15%、5.05%和24.74%;12.44%的ENK能神经元与一氧化氮合酶(NOS)共存;脊髓背角ENK能神经元与囊泡膜谷氨酸转运体1(VGLUT1)和VGLUT2的共存比例为20.61%和21.21%;脊髓背角GABA能神经元与PPE mRNA的共存细胞占GABA能神经元的44.41%,占PPE mRNA的53.93%;单细胞PCR结果表明脊髓背角28.07%的GABA能神经元和22.58%的ENK能神经元表达5-HT3A受体。上述结果为更好的理解GABA能和ENK能神经元在脊髓的功能提供了形态学依据。2.脊髓GABA能神经元的发育模式及调控机制运用GAD67-GFP基因敲入小鼠和Ebf2基因敲除小鼠,结合免疫荧光双标、BrdU标记、脊髓片培养、Time-lapse荧光显微镜动态观察及单细胞RT-PCR等技术,观察了脊髓GABA能神经元的时空分布、起源、迁移模式和调控机制。结果如下:(1) GABA能神经元最早出现在胚胎11.5天(E11.5)脊髓的腹侧部,至胚胎晚期和生后阶段,主要分布在脊髓背角,于E17达到高峰。至P14,GABA能神经元的分布达到与成年脊髓相似的模式。上述结果提示脊髓GABA能神经元的分布具有明显的时间和空间分布特点,呈由腹侧向背侧的分化趋势。(2)脊髓GABA能神经元的产生主要集中在E10.5-E14.5。E11.5和E13.5是两个产生高峰,E10.5产生的GABA能神经元较少,且主要位于脊髓前角。E11.5和E13.5产生的GABA神经元主要分布在脊髓背角浅层和中央管周围。脊髓背角深层的GABA能神经元多产生于E12.5。胚胎晚期仅产生较少的GABA能神经元。(3)在体和离体结果均表明胚胎晚期脊髓套层部分GABA能神经元表达细胞周期标记物Ki-67、5-溴-2′-脱氧尿苷(BrdU)和磷酸化的组蛋白H-3(P-H3),提示这些GABA能神经元具有增殖活性。推测在套层内这些具有增殖活性的GABA能神经元是胚胎晚期脊髓GABA能神经元的一个新的来源。(4)脊髓GABA能神经元产生后以放射状迁移的方式到达脊髓套层,然后向背腹方向移动到达其终止部位。E10.5产生的GABA能神经元主要迁移至脊髓前角,而E11.5产生的GABA能神经元则主要迁移至脊髓背角。不同阶段产生的神经元具有特定的迁移模式。(5) Ebf2基因敲除小鼠脊髓背角Pax2的表达下调, Lmx1b、Drg11和Tlx3的表达未发生改变。上述结果提示Ebf2参与了脊髓GABA能神经元的发育调控。3.脊髓ENK能神经元的发育模式运用PPE-GFP转基因小鼠,结合免疫荧光双标技术,观察了脊髓ENK能神经元的时空分布、发育阶段ENK能神经元的神经化学特点及其表达的调控分子。结果如下:(1) ENK能神经元最早出现在E11.5颈段脊髓腹内侧部,腰段脊髓ENK能神经元在E12.5出现。在E13.5,ENK能神经元主要分布在中间带。E14.5时脊髓背侧开始出现ENK能神经元,之后细胞数量逐渐增多,于胚胎晚期和生后阶段,ENK能神经元主要分布在脊髓背角,P21达到与成年类似的分布模式。上述结果提示脊髓ENK能神经元的分布具有明显的时间和空间分布特点,呈腹侧向背侧、吻侧向尾侧的发育趋势。(2)发育阶段脊髓背角ENK能神经元与GABA能神经元的共存比率较恒定,E16时共存细胞占ENK能神经元的43.35%,P3时双标细胞占ENK能神经元的45.02%。不同发育阶段ENK能神经元与CB、PV及CR的共存率不同,提示在ENK能神经元的发育成熟的不同时程中发挥不同的功能。(3) E15.5时ENK能神经元与Pax2的共存细胞占ENK能神经元的65.17%;至P3,GFP/Pax2双标神经元占ENK能神经元的57.74%,且主要分布在背角浅层。在脊髓背角Ⅰ层观察到ENK能神经元与Lmx1b的共存细胞,在E15和E18,共存细胞分别占脊髓背角ENK能神经元的3.48%和4.50%。上述结果为深入探讨脊髓ENK能神经元的发育调控分子机制提供了形态依据。通过上述研究得到了以下结论:(1)较系统地观察了脊髓背角GABA能和ENK能神经元所包含的神经活性物质和5-HT受体亚型,为阐明这两类神经元的神经化学特点提供了实验依据;(2)揭示了脊髓GABA能和ENK能神经元的时间和空间分布特点及GABA能神经元的迁移模式;(3)明确了脊髓GABA能神经元的产生时程规律;首次发现了胚胎发育晚期脊髓套层内存在GABA能神经元祖细胞,提出了脊髓GABA能神经元的新起源;(4)证实Ebf2基因参与脊髓GABA能神经元的发育调节。GABA能和ENK能神经元作为脊髓背角局部环路重要的组成部分,对感觉传递的调控发挥重要作用。因此探讨脊髓背角GABA能和ENK能中间神经元的神经化学特点、神经元的产生、分化特点、迁移方式及调控基因等,不仅有助于理解正常生理状态下脊髓背角GABA能和ENK能神经元及其参与的局部环路在感觉信息调节中的作用,更重要的是有助于理解病理状态下发生可塑性变化和脊髓背角局部环路重建的机制,从而为脊髓损伤后GABA能和ENK能神经元的修复以及相关的神经系统变性疾病的发病机理和治疗提供新的思路。更多还原

【Abstract】 γ-aminobutyric acid (GABA) ergic neurons and enkephalin (ENK) ergic neurons are important interneurons in the spinal dorsal horn (SDH). These interneurons together with primary afferent fibers, projection neurons, decending terminals and other types of interneurons in the superficial layers comprise the complex nociceptive circuitry, which play important roles in modulating transmission of nociceptive information. The assembly of the complex neuronal circuit depends on the generation of functionally distinct types of dorsal horn neurons during development. Any disturbance of the development of these elements will strongly affect the formation of nociceptive circuitry in the SDH. So it is necessary to examine them during the development course. However, the neurochemical features, temperal and spatial distribution, origin, migration and transcriptional regulation of GABAergic and ENKergic neurons in the spinal cord remain largely unknown.In the present study, we used glutamic acid decarboxylase (GAD)67 -green fluorescence protein (GFP) knock-in mouse to characterize GABAergic neurons and preproenkephalin (PPE)-GFP transegenic mouse to characterize ENKergic neurons. We performed the following three parts of the experiment by using morphological and molecular biological methods.1. The neurochemical features of GABAergic and ENKergic neurons in the SDH.By using double immunofluorescence labeling, fluorescent in situ hybridization combined with immunofluorescence labeling, single-cell reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR methods, we confirmed the validity of GAD67-GFP knock-in mouse and PPE-GFP transgenic mouse. And then we observed the neurochemical features, the expression of 5-hydroxytryptamine (5-HT)3A receptor and the co-existence of GABAergic and ENKergic neurons in the SDH. The results were as followings: (1) Double immunofluorescence labeling results showed that all the GFP-positive neurons were co-localized with neuronal nuclei protein (NeuN). More than 98% of the GFP-positive neurons were positive for GAD67 and GABA. The double labeled staining for GFP and in situ hybridization for GAD67 mRNA showed that GFP immunoreactive neurons expressed GAD67 mRNA in the spinal cord. GFP-positive neurons constituted 31.51%, 33.34%, and 44.70% of the NeuN-positive neurons in laminae I, II, and III, respectively. The expression pattern of GFP-positive neurons in the PPE-GFP transgenic mouse paralleled with that of PPE mRNA expression in different brain regions. All the GFP-positive neurons in the spinal cord of the PPE-GFP transgenic mouse co-localized with NeuN and PPE mRNA. ENKergic neurons constituted 16.95%, 40.68%, and 12.45% of the NeuN-positive neurons in laminae I, II, and III, respectively. Thus, the above double-lebeling study convinced us of the usefulness of the mice for the studies of GABAergic and ENKergic neurons in the spinal cord. (2) The proportions of calretinin (CR)-, parvalbumin (PV)- and calbinding DK28 (CB)-positive cells among GABAergic neurons in the SDH were 58.38%, 19.63%, and 2.81%, respectively. The proportions of CR-, PV- and CB-positive cells among ENKergic neurons in the SDH were 61.15%, 5.05% and 24.74%, respectively. About 12.44% of ENKergic neurons in the SDH were immunoreactive for nitric oxide synthase (NOS). We found that 20.61% of ENKergic neurons in the SDH expressed VGLUT1 and 21.21% of ENKergic neurons were positive for VGLUT2. Quantitative analysis indicated that more than 44.41% of GABAergic neurons showed signals for PPE mRNA in the SDH. While 53.93% of PPE mRNA-expressing neurons were immunoreactive for GABA. Single-cell RT-PCR results showed that 5-HT3A receptor subunit was detected in 28.07% of GABAergic neurons and 22.58% of ENKergic neurons. These detailed results have broad implications for understanding the functional roles of GABAergic and ENKergic neurotransmission in the SDH.2. The developmental pattern and transcriptional regulation of GABAergic neurons in the spinal cord.By using GAD67-GFP knock-in mouse and early B factor 2 (Ebf2) knock out mouse, we observed the temporal and spatial distribution, origin, migration and transcriptional regulation of GABAergic neurons in the spinal cord. Double immunofluorescent labeling, 5-bromo-2-deoxyuridine (BrdU) labeling, spinal cord slice culture, Time-lapse observation and single-cell RT-PCR were used in this part. The results were as followings:(1) GFP-positive GABAergic neurons appeared at embryonic day (E) 11.5 in the ventral region of the spinal cord and became abundant in the whole future gray matter at E12. Thereafter, GFP-positive neurons increased progressively in number and extended from ventral to dorsal regions. The intensity of GFP-positive neurons in the dorsal horn peaked at E17. At postnatal day 14, the distribution pattern of GFP immunoreactivity was similar to that of GABAergic neurons in adult spinal cord. Taken together, the present results suggest that the GFP immunoreactivity, and thus the expression of GABA, undergoes a ventral to dorsal shift in the spinal cord during development.(2) Birthdating studies revealed that GABAergic neurogenesis were present since E10.5. Then the generation of GABAergic neurons significantly increased, reaching a peak at E11.5. The two waves for the co-localization of GABA and BrdU in the spinal cord were seen at E11.5 and E13.5. The vast majority of GABAergic neurons were generated before E14.5. Then, GABA-positive neuron generation decreased drastically. The birthdates of GABAergic neurons in each lamina were different.(3) Both in vivo and in vitro results indicated that a small but significant fraction of GABAergic neurons in the spinal mantle layer were double-labeled with cell-cycle markers Ki-67, BrdU and phosphorylated histone H3 (P-H3). These double-labeled neurons characterized by cell-cycle markers were proliferative GABAergic nneurons which might contribute to the production of spinal GABAergic neurons at late embryonic stages.(4) Time-lapse observation results indicated that after production, GABAergic neurons migrate to the spinal mantle layer in a radial manner and then migrate to the final location. BrdU labeling results showed that GABAergic neurons born at E10.5 migrate ventrally, and do not contribute to the formation of the superficial layer of the SDH. GABAergic neurons born at E11.5 migrate dorsally and contribute to the formation of the superficial layer of the SDH.(5) The expression of Pax2 in the SDH of the Ebf2 knock out mouse decreased compared with that in the wild type mouse. While there was no change in the expression of Lmx1b, Drg11 and Tlx3. These results suggested that Ebf2 was required for the development of GABAergic neurons in the spinal cord.3. The developmental pattern of ENKergic neurons in the spinal cord.By using PPE-GFP transgenic mouse and double immunofluorescent labeling method, we observed the temperal and spatial distribution and neurochemical characteristics of ENKergic neurons during the spinal cord development.(1) GFP-positive ENKergic neurons appeared at E11.5 in the ventral region of the spinal cord. At E13.5, GFP-positive neurons were mainly present in the intermediate zone. No matter what level was considered, the first labeled GFP-positive cells were observed in the dorsal gray matter at E14. Thereafter,GFP positive neurons increased progressively in number and extended from ventra1 to dorsa1 regions. After birth, GFP-positive neurons were mainly restricted to the dorsal gray matter and also decreased in the staining intensity. At postnatal day 21, the distribution pattern of GFP immunoreactivity was similar to that of ENKergic neurons in the adult spinal cord. Taken together, the present results suggest that ENKergic neurons develop according to a rostro-caudal and ventro-dorsal gradient.(2) Double labeling results revealed a significant population of neurons expressing both GABA and ENK. Interestingly, this proportion remained stable during the course of development. The proportions of double-labeled neurons among ENKergic neurons were 43.35% at E16, 45.02% at P3. CB, CR and PV showed a dynamic pattern of co-localization with ENK in neurons of the spinal cord throughout development. The transient expression of calcium-binding proteins in ENKergic neurons might be related to the critical period of development. (3) The proportions of Pax2 among ENKergic neurons in the SDH at E15.5 and E17.5 were 65.17% and 57.74%, respectively. At P3, the GFP/Pax2 double-labeled neurons were primarily located in the superficial layers of the SDH. We also observed a portion of ENKergic neurons co-expressed Lmx1b at E15 (3.48%) or at E18 (4.50%). Double-labeled neurons were mainly observed in laminae I. The above results provide detailed morphological evidence for the regulation of ENKergic neuron development.In summary, from the above results we can draw the following conclusions. (1) We examined the expression of neurochemical substances, 5-HT receptor subtype in the GABAergic and ENKergic neurons in the spinal cord. Such detailed information will provide morphological evidence for the neurochemical characteristics of GABAergic and ENKergic neurons in the SDH. (2) Our results showed the dynamic expression pattern of GABAergic and ENKergic neurons and the migration of GABAergic neurons in the spinal cord. (3) The present study revealed the birthdating of GABAergic neurons in the spinal cord. We confirmed the presence of GABAergic neuron progenitor in the spinal mantle layer at late embryonic stages. These GABAergic neuron progenitors might be another source of GABAergic neurons of the spinal cord. (4) Our results provide evidence that Ebf2 was required for the GABAergic neuron development in the spinal cord.GABAergic and ENKergic neurons are important elements of the neuronal circuitry in the SDH and play crucial roles in the modulation of nociception. Therefore, exploring the neurochemical features, the ontogeny, origin, migration and transcriptional regulation of GABAergic and ENKergic neurons will far improve our understanding of the functional relevance of these interneurons. Most importantly, it will helpful to comprehend the mechanism on the neural plasticity under pathological conditions. The identification of neurochemical features and developmental patterns of these interneurons will aid in the design of new strategies for spinal cord injury or some developmental disorders.更多还原