The purpose of this study is to determine the locations throughout the rat subcortex that receive monosynaptic inputs from retinal ganglion cells. In this study cholera toxin B subunit (CTb) was used to obtain efficient staining of both axons and axon terminals. With high resolution scanning this allowed the exact boundaries of retinal termination zones to be visualized at the whole-brain scale and to single-cell resolution. We made intra-occular injections of CTb unilaterally in 6 adult male Long-Evans rats, and sectioned two brains in each of the three planes at 30 um to allow us to form an inclusive picture of the retinal projection fields in three dimensions. We immunohistochemically processed the tissue with an avidin/biotinylated complex and 3-3-diaminobenzidine (DAB), and counterstained with Geimsa to more clearly visualize retinorecipient cell bodies.

We present a macroscopic view of the location and structure of all the retinorecipient zones. For the dorsal-lateral geniculate nucleus (dLGN) we have analyzed the projections in greater detail at single-cell resolution. Based on monocular injections it was unclear whether the ipsilateral and contralateral projection fields were mutually exclusive (non-overlapping). To understand the detailed sub-structure of these terminations we intra-occularly injected two different fluorophor-conjugated CTb subunits, one in each eye. This allows the ipsilateral and contralateral projections to be visualized simultaneously, revealing how the two sets of retinal inputs interweave in the dLGN.

View of retinal projections in the contralateral thalamus viewed from a coronal section using DAB staining. Dorsal LGN (above) and ventral LGN (below) are shown. Holes lacking contralateral input are reproducible across animals; at least some of these have corresponding blobs on the ipsilateral side (not shown).	Retinal terminations in the tectum (superior colliculous) visualized in coronal section, dark field. In addition to dense contralateral staining (right), individual ganglion cell termini can be visualized in the sparse ipsilateral projection (left).

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