Multiple Retinal Axons Converge onto Relay Cells in the Adult Mouse Thalamus

SUMMARY Activity-dependent refinement of neural circuits is a fundamental principle of neural development. This process has been well studied at retinogeniculate synapses—synapses that form between retinal ganglion cells (RGCs) and relay cells within the dorsal lateral geniculate nucleus. Physiological studies suggest that shortly after birth, inputs from ~20 RGCs converge onto relay cells. Subsequently, all but just one to two of these inputs are eliminated. Despite widespread acceptance, this notion is at odds with ultrastructural studies showing numerous retinal terminals clustering onto relay cell dendrites in the adult. Here, we explored this discrepancy using brainbow AAVs and serial block face scanning electron microscopy (SBFSEM). Results with both approaches demonstrate that terminals from numerous RGCs cluster onto relay cell dendrites, challenging the notion that only one to two RGCs innervate each relay cell. These findings force us to re-evaluate our understanding of subcortical visual circuitry.


dLGN. A.
Retinal terminals in the adult dLGN were immuno-labeled with antibodies directed against vesicular glutamate transporter 2 (VGluT2). High magnification images of terminals in the "shell" and "core" of dLGN are shown in A' and A'', respectively ( participating in simple retinogeniculate synapses had significantly larger diameters, more active zones, more dendritic protrusion and a higher active zone to bouton diameter ratio (which suggests that the increase in active zone number is not merely caused by larger bouton size).

Mice
Wild-type C57 mice were obtained from Charles River. Calb2-cre mice were obtained from Jackson Laboratory (Stock #010774). All analyses conformed to National Institutes of Health guidelines and protocols approved by the Virginia Polytechnic Institute and State University Institutional Animal Care and Use Committees.
Briefly, mice were anesthetized with isoflurane vapors at P12-14. The sclera was pierced with a sharp-tipped glass pipette and excess vitreous was drained. Another pipette, filled with a 1:1 mixture of both brainbow AAVs, was inserted into the hole made by the first pipette. The pipette containing the AAVs was attached to a Picospritzer and a prescribed volume (3-5 μl) of solution was injected into the eye. After 21 days, mice were euthanized, transcardially perfused with PBS and 4% paraformaldehyde, and retinas and brains were post-fixed in 4% paraformaldehyde for 12 hours. Fixed brains were coronally sectioned (80-100 µm) on a vibratome (Microm HM 650V, Thermo Scientific) and mounted in ProLong Gold (Invitrogen).
Fixed retinas were either prepared as whole-mounts or were sectioned on a Leica CM1850 cryostat (16µm cross-sections) and in either case were mounted in ProLong Gold (Invitrogen) . RGCs and retinal projections were analyzed from 6 animals.
Images were acquired on a Zeiss LSM 700 confocal microscope and color analysis of maximum projections images was performed in Photoshop.

Serial Block Face Scanning Electron Microscopy
Mice were transcardially perfused sequentially with PBS and 4% paraformaldehyde / 2% glutaradehyde in 0.1M cacodylate buffer. Brains were immediately removed, vibratomed (300 µm coronal sections) and dLGN were dissected. Tissues were then stained, embedded, sectioned and imaged by Renovo Neural Inc. (Cleveland, OH). Images were acquired at a resolution of 5 nm/pixel and image sets included > 200 serial sections (with each section representing 75 nm in the z axis). SBFSEM data sets were 40µm x 40 µm x 12-20 µm. 4 data sets were analyzed for each region (from a total of 3 P42 wild-type mice). Data sets were traced and analyzed in TrakEM2 (Cardona et al. 2012). Retinal terminals were identified (and distinguished from non-retinal terminals) by the presence of synaptic vesicles and pale mitochondria as previously described (Lund and Cunningham 1972;Bickford et al. 2010;Hammer et al. 2014). Synaptic sites were identified by the presence of active zones and postsynaptic densities. Analysis of data sets was performed independently by three researchers to ensure unbiased results.