Development And Synaptic Output Of Vesicular Glutamate Transporter 3 (VGLUT3) Expressing Cells In The Rodent Retina

Abstract

Abstract: : Purpose: Synaptic transmission from glutamatergic neurons requires vesicular glutamate transporters (VGLUTs) to concentrate cytosolic glutamate in synaptic vesicles. The recently identified VGLUT3 isoform was found in amacrine cells, which are generally considered to be inhibitory interneurons. To investigate the synaptic machinery and synaptic output of VGLUT3 cells, and to determine a potential functional role, we further investigated these putative glutamatergic amacrine cells in adult and developing rodent retina. Methods: We used Western blotting and electron and light immunohistochemistry to study the expression of VGLUT3 in adult and developing rodent retina. Results: VGLUT3 colocalized with the synaptic vesicle marker SV2, and electron microscopy showed that VGLUT3 immunostained synaptic vesicles. VGLUT3 cells immunostain for glycine in their cell body and primary dendrites. 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, consistent with glutamatergic synaptic exocytosis by these cells. In the maturing postnatal mouse retina, Western blots showed VGLUT3 expression at postnatal day 7/8 (P7/8), while VGLUT3 immunostaining in retinal sections was first observed at P8, and achieved an adult pattern at P12. Conclusions: VGLUT3 expression is localized to synapses, and VGLUT3 cells make synaptic output to ganglion cell dendrites. VGLUT3 function begins 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 these VGLUT3 neurons as part of the light–entrainable retina–based circadian system.

Keywords: retina: proximal (bipolar, amacrine, and ganglion cells) • retina: neurochemistry • retinal development