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

Mice Wild-type C57 mice were obtained from Charles River. Calb2-cre mice were obtained from Jackson Laboratory (stock #010774). All analyses conformed to NIH guidelines and protocols approved by the Virginia Polytechnic Institute and State University Institutional Animal Care and Use Committees. Intraocular Injections of Brainbow AAVs The following brainbow AAVs were obtained from the University of Pennsylvania Vector Core ( http://www.med.upenn.edu/gtp/vectorcore/ ): AAV9.hEF1a. lox.TagBFP.lox.eYFP.lox.WPRE.hGH-InvBYF (lot #V3809TI-R) and AAV9. hEF1a.lox.mCherry.lox.mTFP1.lox.WPRE.hGH-InvCheTF (lot #V3530TI-R). Each brainbow AAV is capable of driving the expression of two different fluorescent proteins (see Figure 1A ). Intraocular injection of brainbow AAVs was performed as described previously for the intraocular delivery of cholera toxin subunit B ( Jaubert-Miazza et al., 2005 ; Su et al., 2011 ). 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 and transcardially perfused with PBS and 4% paraformaldehyde, and retinas and brains were post-fixed in 4% paraformaldehyde for 12 hr. Fixed brains were coronally sectioned (80–100 μm) on a vibratome (Microm HM 650V, Thermo Scientific) and mounted in VectaShield (Vector Laboratories). Fixed retinas were either prepared as whole mounts or were sectioned on a Leica CM1850 cryostat (16-μm cross-sections) and in either case were mounted in VectaShield (Vector Laboratories) ( Su et al., 2011 ). RGCs and retinal projections were analyzed from six 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 0.1 M cacodylate buffer. Brains were immediately removed and vibratomed (300-μm coronal sections) and dLGN were dissected. Tissues were then stained, embedded, sectioned, and imaged by Renovo Neural. 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 40 μm × 40 μm × 12–20 μm. Four data sets were analyzed (from a total of three 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.

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