Abstract
Purpose :
While EM studies have defined the shape and connectivity of the mammalian cone photoreceptor synapse, the nanoscale organization of the cleft proteins that mediate neurotransmission are less well understood. Here, we use quadruple fluorescent labeling and 2D and 3D STED super-resolution microscopy to define how receptors, transporters, and synaptic connections are organized at the cone synapse.
Methods :
Wholemount retinas from ground squirrel and mouse of either sex were removed and fixed in 2% glyoxal solution for 2 days at 4oC. Vertical slices were cut at either 100 or 300 µm thickness for mounting in the cross-sectional or en face orientation. Tissue was blocked in donkey serum and incubated in primary for 4-6 and secondary for 2-4 days. Donkey species specific secondaries were labeled with the NHS-esters of fluorophores from ATTO, Abberior, and Biotium. Tissue was embedded in melamine (index ref = 1.52) and imaged with a Leica sp8 STED or Abberior STEDYCON or INFINITY microscopes in both 2D and 3D modes using a 775 nm depleting laser. Images were deconvolved in Huygens Professional (SVI) and reconstructed and analyzed with Blender and Imaris software. In some cases, bipolar cells were labeled prior to fixation via whole cell pipettes that contained neurobiotin or by a bipolar cell selective AAV2 that expressed GFP. Particle density and nearest neighbor spacing were measured by standard methods.
Results :
Antibodies against the cone glutamate transporter EAAT5 labeled ~75 puncta per terminal. Puncta density was uniform across the terminal with a minimal inter-particle distance of ~230 nm. EAAT5 puncta were randomly organized with respect to GluA4 puncta, which served as markers for invaginations and which themselves formed an array with a minimal spacing. EAAT5 labeling was uncorrelated with the contacts of labeled On and Off bipolar cell types. GluR4 had a uniform density across the terminal with the exception of a distinct dip in in the center, an organization shared with basal GluK1 labeling and opposite to that of the dendritic contacts of Off cb1a and On cb7b cells. Labeling for mGluR6 and GluK1 was anticorrelated, but in both cases extended along dendrites to a depth of >1 µm beneath the terminal, features shared with the mouse cone terminal.
Conclusions :
The cone terminal has two types of nanostructural organization: spatially uniform/semi-regular and zonal with circular symmetry (donut with hole).
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.