摘要目的：观察大鼠海马CA1区突触发育的超微结构，提出其突触发育可塑性的形态学特征和参考突触数密度。方法：采用连续超薄切片,观察突触发育的形态特征，结合体视学disector法测量突触总体及穿孔突触数密度。结果：突触生后发育过程中, 形态具有复杂的三维结构，穿孔突触的突触接触区曲面凸向突触前区域，20日龄曲度大于30日龄。可见突触小泡偏侧分布现象。20日龄突触后致密物质比30日龄组厚，均可见T-terminals和C-terminals两种类型突触下致密小体分布。海马CA1区生后20至30日龄突触数密度增加。结论：穿孔突触接触面曲度较大，并凸向突触前区域，突触后致密物质较厚,可见突触下致密小体，均可作突触发育和可塑性突触的形态特征。

中国图书资料分类法分类号R332.81-332

Study of the synaptogenesis and plasticity in rat hippocampus

SHEN Li, MAI Hong-Yan, NAN Yan, ZHANG Lin, WANG Shu-Ling

(Department of Anatomy, Beijing Medical University, Beijing100083)

MeSHSynapses/growthNeuronal plasticityHippocampus/growthSynapses/ultrastruct

ABSTRACTObjective: To observe the ultrastructure of the synaptogenesis in rats hippocampus. CA1 during postnatal 20 d and 30 d and provide the morphological characters and the reference numerical densities of synapses in development processes.Methods:The data of the numerical densities of perforated synapses and total synapses were determined using successive electron microscopy sections and a new stereological technique, the disector method.Results:It was appeared the feature of synapse was in complicated three-dimensional structure. The contact surfaces of perforated synapses showed a definite curvature, and curved surfaces projected to pre-synaptic area. The curvature of postnatal 20 d group was larger than that of postnatal 30 d group. The phenomena of vesicular lateralization was found. The postsynaptic densities of postnatal 20 d group were thicker than that of postnatal 30 d group. Subsynaptic dense bodies were associated with subsynaptic dense “Taxi” bodies (T-terminals) and with subsynaptic cisternae (C-terminals). The items of numerical densities of total synapses increased with increasing from postnatal 20 d to 30 d.Conclusion: The larger curved contact surfaces of perforated synapses and projectin to pre-synaptic area, as well as the thicker postsynaptic densities with subsynaptic dense bodies were consider as the morphological feature of synaptogenesis and its plasticity.

（J Beijing Med Univ, 1999,31:209-211)

中枢神经系统(central nervous system, CNS)发育及成熟后的结构与功能联系复杂。CNS的复杂性与精确性表现在相关神经元以突触联系组成高度特异、十分精确的功能回路^［1］。突触在发育过程中的产生、增减、形态特征等是精确的、有一定规律的。生后早期神经系统仍处于发育期，表现出较强的可塑性。有研究认为，突触发育及可塑性与学习记忆、儿童早期智力开发、衰老和CNS损伤、修复和再生等一些神经系统疾病有关^［2］。因此，神经系统的可塑性除取决于神经元的形态、突起、出芽等，另外突触的数量、形态与功能状态，也是反映神经系统功能状态的重要参数。已往研究证明，大鼠中枢突触发育过程主要在生后3周完成^［3］，我们采用连续电镜切片和体视学Disector法^［4］，观察并测量大鼠生后20d和30d海马CA1区突触发育的形态特征和数密度，提供突触发育和可塑性的依据及参考数密度。

1材料与方法

出生后20和30日龄Wistar大鼠各3只（北京医科大学实验动物部提供）。腹腔注射麻醉，经左心室灌注固定液250ml［2%(体积分数)多聚甲醛、2．5％（体积分数)戊二醛的0.1mol·L^－1PBS,pH7.4］。剥取海马，再固定4h后，0.1mol·L^－1PBS4℃过夜，振动切片机额状切片（50～75μm）。1%(体积分数)锇酸后固定1h（4℃），脱水及618环氧树脂平板包埋。随机抽取包埋厚片，在解剖镜下定位取CA1区进行半薄切片，1%(体积分数)甲苯胺蓝染色，显微镜下半薄切片定位海马CA1区锥体细胞层和分子层。超薄切片机连续切片，制备6～10片连续超薄切片构成的切片带裱于独眼铜网。醋酸铀-枸椽酸铅双重染色。电镜下观察切片带的第一张切片，辨认海马分子层，以神经毡周围的毛细血管和多个线粒体为参考定位标志，随机选取神经毡摄片（14000倍）。相继选取相邻切片，拍摄与第一张切片在同一纵轴上的视野，即相邻视野，每条切片带连续摄片6～7张。

洗印放大3.5倍电镜照片，选定测试面积为1.39×10^－5mm²,以微皱褶法测量切片厚度为(61±5)nm，计数突触截面数依据Jones等^［5］标准，代入N_V=Q^－/tA公式,计算突触数密度。其中N_V为突触数密度；Q^－为计数切片上出现，而核查切片上不出现的突触截面数；t为超薄切片的厚度；A为测试面积。所得两组计量资料进行两组间比较，采用t检验。

2结果

2.1突触接触区二维重建和形态观察

通过对相邻连续穿孔突触的突触后致密物质截面位置和长度实现突触连续平面的二维重建（图1a，b）观察20日龄和30日龄突触形态。尽管由于连续切片只有6～7张的限制，未能找到从头至尾的完整突触，但仍能证明，突触发育过程中有多种突触类型，突触形态具有复杂的三维结构。

a, planar reconstruction of the perforated synapse from successive section of postnatal 30d group in Figure 2; b, planar reconstruction of the perforated synapse from successive section of postnatal 20d group in Figure 3.

图1二维重建海马CA1区穿孔突触

Figure 1Planar reconstruction of the perforated synapse

of hippocampus CA1

穿孔突触:20日龄和30日龄组均可见穿孔突触（图2，3），属典型的GrayⅠ型非对称型突触。在突触侧切面上，由于孔洞的存在，出现不连续的突触后致密物质，多以圆盘状或马蹄状的穿孔突触为主。穿孔突触的突触接触区多呈现出一定的曲度，且曲面凸向突触前区域，20日龄的曲度大于30日龄。突触前小泡20日龄比30日龄小而密集，均有突触小泡偏侧分布现象，即大量突触小泡集中分布于其中某一段突触后致密物质（postsynaptic densities, PSD）相对的突触前区域中，而余部PSD所对应的突触前区域则没有或极少数目的突触小泡。观察发现20日龄突触后致密物质比30日龄组厚，均可见突触下致密小体（subsynaptic dense bodies）,有“Taxi”小体（subsynaptic dense “Taxi”bodies, T-terminals）和网状小体（subsynaptic cisternae, C-terminals）两种类型（图2～4）。

图230日龄鼠海马CA1区穿孔突触的连续切片，可见突触小泡偏侧分布。

箭头示突触下致密小体-Taxi小体×49 000

Figure 2The successive transverse section of postnatal 30d group one perforated synapse of hippocampus CA1，showed the phenomena of vesicular lateralization. Arrow showed the subsynaptic dense “Taxi” bodies (T-terminals)×49 000

图320日龄鼠海马CA1区穿孔突触的连续切片，可见突触后致密物较厚，

箭头示突触下致密小体-网状小体。穿孔突触的突触接触面呈曲面，凸向突触前区域×49 000

Figure 3The successive transverse section of postnatal 20 d group. One perforated synapse of hippocampus CA1 had the more thick postsynaptic density, and arrow showed the subsynaptic cisternae (C-terminals). The contact surfaces of perforated synapses showed a definite curvature, and curved surfaces projected

to presynaptic area×49 000

图420日龄鼠海马CA1区非穿孔突触的连续切片，可见突触后致密物较厚，箭头示突触下致密小体——Taxi小体×49 000

Figure 4The successive transverse section of postnatal<