Vesicular glutamate transporter 3 expression identifies glutamatergic amacrine cells in the rodent retina.
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.
Johnson
Juliette
J
Department of Ophthalmology, University of California School of Medicine, San Francisco, San Francisco, California 94143, USA. cjj@itsa.ucsf.edu
Sherry
David M
DM
Liu
Xiaorong
X
Fremeau
Robert T
RT
Seal
Rebecca P
RP
Edwards
Robert H
RH
Copenhagen
David R
DR
D025042:Amacrine Cells / Q000254:growth & development / Q000378:metabolism*; D026922:Amino Acid Transport Systems, Acidic / Q000096:biosynthesis*; D000818:Animals; D015153:Blotting, Western; D002940:Circadian Rhythm; D018698:Glutamic Acid / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D018377:Neurotransmitter Agents / Q000378:metabolism; D017729:Presynaptic Terminals / Q000378:metabolism; D020033:Protein Isoforms / Q000096:biosynthesis; D051381:Rats; D020318:Rats, Long-Evans; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050496:Vesicular Glutamate Transport Proteins
D026922:Amino Acid Transport Systems, Acidic; D018377:Neurotransmitter Agents; D020033:Protein Isoforms; C460879:SLC17A8 protein, human; D050496:Vesicular Glutamate Transport Proteins; D018698:Glutamic Acid