Abstract
Purpose :
In photoreceptors, light-dependent changes in the membrane potential drive glutamate release from the synaptic terminal onto the bipolar cells (BCs) and horizontal cells (HCs). In rods, the single ribbon synapse facilitates the high rate of glutamate release through exocytosis (vesicle fusion), which is complemented by endocytosis (vesicle retrieval). Endocytosis commonly requires the GTPase dynamin, whose two isoforms, Dnm1 and Dnm3, are expressed specifically in neurons. Here, we investigated the impact of deleting Dnm1 and Dnm3 on rod synaptic transmission and morphology.
Methods :
Dnm1- and Dnm3-floxed mice were bred with transgenic iCre75 mice to generate rod-specific single (rodDnm1-/- and rodDnm3-/-) and double knockouts (rodDnm1-/-/3-/-). Scotopic ERG recordings were used to evaluate rod synaptic transmission to rod BCs. Confocal and electron microscopy were used to assess the structure of the rod photoreceptors and rod BCs.
Results :
Immunostaining confirmed the conditional deletion of Dnm1 and Dnm3 from rods at P28. In rodDnm1-/-/3-/- mice, scotopic ERG a-waves were normal, consistent with normal outer segment ultrastructure and trafficking of rhodopsin, whereas scotopic b-waves were significantly impaired, consistent with reduced staining of ribbon synapse-related proteins (RIBEYE and bassoon) and synaptic vesicle-related proteins (VGLUT1 and Syt1). Rod synaptic terminals progressively degenerate at P90 and P180, as impaired vesicle endocytosis causes accumulation of enlarged endosome-like vesicles and disassembly of the ribbon at ultrastructural level, respectively. In rodDnm1-/-/3-/- retinas, HC axon terminals (anti-calbindin, anti-ZNP, sparse labeling by AAV-GFP) and rod BCs dendrites (anti-PKCα, anti-mGluR6) progressively degenerate without the loss of cell number. In contrast, cone pedicles and the cone-driven ERG in rodDnm1-/-/3-/- mice remained intact. No changes in rod synaptic function or structure were observed in single knockouts, suggesting that Dnm1/3 function is redundant.
Conclusions :
Dual deletion of Dnm1 and Dnm3 from rods causes functional impairment and degeneration of the synaptic terminal. This degeneration is accompanied by structural defects in postsynaptic neurons and loss of light-driven signals in rod BCs, showing that synaptic transmission in rods requires dynamin-dependent endocytosis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.