NMDA receptor activity regulates synaptic connections between retinal ganglion and bipolar cells

Abstract

Purpose : To determine the significance of NMDA receptors on the regulation of synaptic connections between retinal ganglion cells (RGCs) and bipolar cells (BCs).

Methods : 1) We used a genetically identified subtype of RGC (J-RGC) as our working model to reveal the RGC subtypes specific synaptic connections to BCs.
2) We used a transgenic and viral approach to illustrate the synaptic connections between J-RGCs and their presynaptic BCs. The approach uses a Cre-Dependent AAV2-mCherry-WGA-Flpo vector on CreER-J-RGC:FRT-EGFP (J-RGC-FRT) double transgenic mice to express mCherry and EGFP in J-RGCs, and express EGFP in BCs that are synaptically connected to J-RGCs.
3) We generated CreER-J-RGC:FRT-EGFP:Grin1^flox/flox (J-RGC-Grin1-FRT) triple transgenic mice to conditionally deleted NMDA receptors in J-RGCs in order to investigate the significance of NMDA receptor deletion on the synaptic connections between J-RGCs and BCs.
4) We used confocal/2P imaging and 3D reconstruction to determine the subtypes of BCs that are labeled through J-RGC specific transcellular gene expression in both J-RGC-FRT and J-RGC-Grin1-FRT mice.
5) We used BC specific antibodies to confirm the subtypes of BCs that are transcellularly labeled through J-RGCs.

Results : 1) We identified eight different subtypes of BCs that are transcellularly labeled through J-RGCs.
2) The predominate BC subtype was rod BCs, and a new subtype of BC which we call Aii-BC.
3) In Grin1 KO mice, there is a reduction in the proportion of subtype 2 and 4 cone BCs (CBCs), but an increase in rod BCs (Χ² 2x2 analysis).
4) The proportion of Aii-BCs remains unchanged in the Grin1 KO mice.

Conclusions : There are a wide variety of BCs that provide input to J-RGCs, with the majority of them being OFF CBCs, rod BCs, and Aii-BCs. Grin1 KO significantly modifies the proportional distribution of some BC inputs. This indicates that glutamate signaling may be important for normal circuit formation.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

A) 3D modeling of cell types in the retina. B-E) EGFP image (left) with a 3D rendering of the BC in red (right). B) 3 CBC, C) 5 CBC, D) Rod BC and Aii-amacrine cell (purple) E) Aii-BC. F) Comparison of BC proportions found in JAMB WT and Grin1 KO.

 

A1) J-RGC-FRT section shows a BC close to J-RGC. A2) J-RGC and CBC separated for clarity. B-F) Antibody staining for various BC subtypes. B) ZPN-Syt2 (2, 6 CBC), C) HCN4 (3a CBC), D) PkARIIβ (3b CBC), E) CaB5 (3, 5 CBC and Rod BC) F) PKC (Rod BC).