Johnson Juliette, Sherry David M, Liu Xiaorong, Fremeau Robert T, Seal Rebecca P, Edwards Robert H, Copenhagen David R
Department of Ophthalmology, University of California School of Medicine, San Francisco, San Francisco, California 94143, USA.
J Comp Neurol. 2004 Sep 27;477(4):386-98. doi: 10.1002/cne.20250.
Synaptic transmission from glutamatergic neurons requires vesicular glutamate transporters (VGLUTs) to concentrate cytosolic glutamate in synaptic vesicles. In retina, glutamatergic photoreceptors and bipolar cells exclusively express the VGLUT1 isoform, whereas ganglion cells express VGLUT2. Surprisingly, the recently identified VGLUT3 isoform was found in presumed amacrine cells, generally considered to be inhibitory interneurons. To investigate the synaptic machinery and conceivable secondary neurotransmitter composition of VGLUT3 cells, and to determine a potential functional role, we further investigated these putative glutamatergic amacrine cells in adult and developing rodent retina. Reverse transcriptase-PCR substantiated VGLUT3 expression in mouse retina. VGLUT3 cells did not immunostain for ganglion or bipolar cell markers, providing evidence that they are amacrine cells. VGLUT3 colocalized with synaptic vesicle markers, and electron microscopy showed that VGLUT3 immunostained synaptic vesicles. VGLUT3 cells were not immunoreactive for amacrine cell markers gamma-aminobutyric acid, choline acetyltransferase, calretinin, or tyrosine hydroxylase, although they immunostain for glycine. VGLUT3 processes made synaptic contact with ganglion cell dendrites, suggesting input onto these cells. VGLUT3 immunostaining was closely associated with the metabotropic glutamate receptor 4, which is consistent with glutamatergic synaptic exocytosis by these cells. In the maturing mouse retina, Western blots showed VGLUT3 expression at postnatal day 7/8 (P7/8). VGLUT3 immunostaining in retinal sections was first observed at P8, achieving an adult pattern at P12. Thus, VGLUT3 function commences around the same time as VGLUT1-mediated glutamatergic transmission from bipolar cells. Furthermore, a subset of VGLUT3 cells expressed the circadian clock gene period 1, implicating VGLUT3 cells as part of the light-entrainable retina-based circadian system.
谷氨酸能神经元的突触传递需要囊泡谷氨酸转运体（VGLUTs）将胞质中的谷氨酸浓缩到突触小泡中。在视网膜中，谷氨酸能光感受器和双极细胞只表达VGLUT1亚型，而神经节细胞表达VGLUT2。令人惊讶的是，最近发现的VGLUT3亚型存在于假定的无长突细胞中，这些细胞通常被认为是抑制性中间神经元。为了研究VGLUT3细胞的突触机制和可能的继发性神经递质组成，并确定其潜在的功能作用，我们进一步研究了成年和发育中的啮齿动物视网膜中的这些假定的谷氨酸能无长突细胞。逆转录酶 - PCR证实了VGLUT3在小鼠视网膜中的表达。VGLUT3细胞对神经节细胞或双极细胞标志物不进行免疫染色，这证明它们是无长突细胞。VGLUT3与突触小泡标志物共定位，电子显微镜显示VGLUT3免疫染色的突触小泡。VGLUT3细胞对无长突细胞标志物γ - 氨基丁酸、胆碱乙酰转移酶、钙视网膜蛋白或酪氨酸羟化酶无免疫反应，尽管它们对甘氨酸进行免疫染色。VGLUT3突起与神经节细胞树突形成突触接触，表明这些细胞对神经节细胞有输入。VGLUT3免疫染色与代谢型谷氨酸受体4密切相关，这与这些细胞的谷氨酸能突触胞吐作用一致。在成熟的小鼠视网膜中，蛋白质免疫印迹显示出生后第7/8天（P7/8）有VGLUT3表达。视网膜切片中的VGLUT3免疫染色在P8首次观察到，在P12达到成年模式。因此，VGLUT3的功能与VGLUT1介导的双极细胞谷氨酸能传递大约在同一时间开始。此外，一部分VGLUT3细胞表达生物钟基因period 1，这表明VGLUT3细胞是基于视网膜的可被光调节的生物钟系统的一部分。
