Malarkey Erik B, Parpura Vladimir
Departments of Neurobiology and Cell Biology, Center for Glial Biology inMedicine, Atomic Force Microscopy & Nanotechnology Laboratories, Civitan International Research Center, Evelyn F. McKnight Brain Institute, University of Alabama, Birmingham, AL, USA.
J Physiol. 2011 Sep 1;589(17):4271-300. doi: 10.1113/jphysiol.2011.210435. Epub 2011 Jul 11.
Astrocytes can release various gliotransmitters in response to stimuli that cause increases in intracellular Ca(2+) levels; this secretion occurs via a regulated exocytosis pathway. Indeed, astrocytes express protein components of the vesicular secretory apparatus. However, the detailed temporal characteristics of vesicular fusions in astrocytes are not well understood. In order to start addressing this issue, we used total internal reflection fluorescence microscopy (TIRFM) to visualize vesicular fusion events in astrocytes expressing the fluorescent synaptobrevin 2 derivative, synapto-pHluorin. Although our cultured astrocytes from visual cortex express synaptosome-associated protein of 23 kDa (SNAP23), but not of 25 kDa (SNAP25), these glial cells exhibited a slow burst of exocytosis under mechanical stimulation; the expression of SNAP25B did not affect bursting behaviour. The relative amount of two distinct types of events observed, transient and full fusions, depended on the applied stimulus. Expression of exogenous synaptotagmin 1 (Syt1) in astrocytes endogenously expressing Syt4, led to a greater proportion of transient fusions when astrocytes were stimulated with bradykinin, a stimulus otherwise resulting in more full fusions. Additionally, we studied the stability of the transient fusion pore by measuring its dwell time, relation to vesicular size, flickering and decay slope; all of these characteristics were secretagogue dependent. The expression of SNAP25B or Syt1 had complex effects on transient fusion pore stability in a stimulus-specific manner. SNAP25B obliterated the appearance of flickers and reduced the dwell time when astrocytes were mechanically stimulated, while astrocytes expressing SNAP25B and stimulated with bradykinin had a reduction in decay slope. Syt1 reduced the dwell time when astrocytes were stimulated either mechanically or with bradykinin. Our detailed study of temporal characteristics of astrocytic exocytosis will not only aid the general understanding of this process, but also the interpretation of the events at the tripartite synapse, both in health and disease.
星形胶质细胞可响应导致细胞内钙离子水平升高的刺激而释放多种神经胶质递质；这种分泌通过一种受调控的胞吐途径发生。事实上，星形胶质细胞表达囊泡分泌装置的蛋白质成分。然而，星形胶质细胞中囊泡融合的详细时间特征尚未得到很好的理解。为了开始解决这个问题，我们使用全内反射荧光显微镜（TIRFM）来观察表达荧光突触结合蛋白2衍生物（突触pHluorin）的星形胶质细胞中的囊泡融合事件。尽管我们从视觉皮层培养的星形胶质细胞表达23 kDa的突触体相关蛋白（SNAP23），但不表达25 kDa的（SNAP25），但这些神经胶质细胞在机械刺激下表现出缓慢的胞吐爆发；SNAP25B的表达并不影响爆发行为。观察到的两种不同类型事件（瞬时融合和完全融合）的相对数量取决于所施加的刺激。在内源性表达Syt4的星形胶质细胞中外源表达突触结合蛋白1（Syt1），当用缓激肽刺激星形胶质细胞时，会导致更高比例的瞬时融合，而缓激肽刺激否则会导致更多的完全融合。此外，我们通过测量其停留时间、与囊泡大小的关系、闪烁和衰减斜率来研究瞬时融合孔的稳定性；所有这些特征都依赖于促分泌素。SNAP25B或Syt1的表达以刺激特异性方式对瞬时融合孔稳定性产生复杂影响。当星形胶质细胞受到机械刺激时，SNAP25B消除了闪烁的出现并缩短了停留时间，而表达SNAP25B并用缓激肽刺激的星形胶质细胞衰减斜率降低。当星形胶质细胞受到机械刺激或用缓激肽刺激时，Syt1都会缩短停留时间。我们对星形胶质细胞胞吐时间特征的详细研究不仅有助于对这一过程的总体理解，也有助于对健康和疾病状态下三方突触处事件的解释。
