Supplementary Material for: 3-Dimensional ultrastructural analysis of the rat pinealocyte; presence of secretory bulbous projections delineated from the cell body by junctional complexes.

Introduction: The superficial pineal gland of the Sprague Dawley rat is a neuroendocrine structure secreting the hormone melatonin. By use of block face scanning electron microscopy, our aim here was to identify the 3-dimensional ultrastructure of the gland.
Methods: A series of 2,731 block face images of the rat pineal tissue, 30 nm in thickness, was obtained in a Teneo Video Volume scanning electron microscope and used for 3-dimensional reconstruction by use of the TrakEM2-plugin in the ImageJ software. Thin sections of the tissue were cut for transmission electron microscopy.
Results: Our analyses revealed cellular bulbous processes, containing 50 – 100 nm clear vesicles, that emerged from a neck-like area at the cell body of the pinealocyte. These bulbous processes extend into small canaliculi located in the center of parenchymal folliculi of the gland as well as into the perivascular spaces. Junctional complexes, comprising both gap and tight junctions, connected the lateral cellular membranes of the pinealocytes, where the bulbous processes emerged from the cell bodies. The canaliculi were via the extracellular space connected to the perivascular spaces.
Discussion: The junctional complexes reported here would prevent a substance, released from the vesicles in the bulbous processes, from targeting the cell body from which they emerge. In line with previous combined morphological and biochemical demonstrations of glutamate located in clear vesicles of bulbous processes in the rat pineal gland, our ultrastructural data support the concept that bulbous processes could participate in a paracrine glutamatergic inhibition of the melatonin secretion in the pineal gland.
Conclusion: Bulbous secretory projections separated from the cell body by a junctional complex is a new feature of neuroendocrine cells.

引言：斯普拉格-道利大鼠（Sprague Dawley rat）的浅表松果体（pineal gland）是一种分泌褪黑素（melatonin）的神经内分泌结构。本研究采用块面扫描电子显微镜（block face scanning electron microscopy）技术，旨在解析该腺体的三维超微结构。
方法：本研究在Teneo Video Volume扫描电子显微镜下获取了2731张大鼠松果体组织的块面图像，单张图像对应组织厚度为30 nm；随后借助ImageJ软件中的TrakEM2插件（TrakEM2-plugin）完成三维重建。此外，我们还切取了组织超薄切片用于透射电子显微镜（transmission electron microscopy）观察。
结果：分析结果显示，松果体细胞（pinealocyte）胞体的颈样区域会伸出球状突起，该突起内含直径50~100 nm的透明囊泡。此类球状突起可延伸至腺体实质滤泡（parenchymal folliculi）中央的细小小管（canaliculi），以及血管周间隙（perivascular spaces）中。连接复合体（junctional complexes）同时包含缝隙连接（gap junctions）与紧密连接（tight junctions），负责连接松果体细胞的侧细胞膜，而球状突起正是从这些细胞的胞体处伸出。上述细小小管通过细胞外间隙（extracellular space）与血管周间隙相连通。
讨论：本研究报道的连接复合体可阻止球状突起囊泡释放的物质靶向其起源的胞体。此前已有研究结合形态学与生化手段证实，大鼠松果体球状突起的透明囊泡中含有谷氨酸（glutamate），本研究的超微结构数据与此一致，支持球状突起可通过旁分泌谷氨酸能信号，抑制松果体褪黑素分泌的假说。
结论：由连接复合体与胞体分隔开的球状分泌突起，是神经内分泌细胞的一种全新特征。