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）浅表松果体是一种分泌褪黑素的神经内分泌结构。本研究采用块面扫描电子显微镜技术，旨在解析该腺体的三维超微结构。
方法：本研究使用Teneo Video Volume扫描电子显微镜，获取了2731张厚度为30纳米的大鼠松果体组织块面图像，并通过ImageJ软件中的TrakEM2插件完成三维重构。此外，我们还切取组织超薄切片用于透射电子显微镜观察。
结果：本研究通过分析发现，松果体细胞（pinealocyte）可从胞体的颈状区域伸出球状突起，该突起内含有直径50~100纳米的清亮囊泡。这些球状突起可延伸至腺体实质滤泡中央的细小小管内，以及血管周间隙中。连接复合体（包含缝隙连接与紧密连接）连接了松果体细胞的侧细胞膜，而球状突起正是从该侧细胞膜处由胞体伸出。上述小管通过细胞外间隙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来源胞体。结合此前针对大鼠松果体球状突起清亮囊泡中谷氨酸的形态学与生物化学联合验证结果，本研究的超微结构数据支持下述假说：球状突起可通过旁分泌谷氨酸能信号途径，对松果体的褪黑素分泌产生抑制作用。
结论：通过连接复合体与胞体分离的球状分泌突起，是神经内分泌细胞的一种全新特征。