The visual system is one of the most widely used and best understood sensory systems and the dorsal lateral geniculate nucleus (dLGN) of the mouse has emerged as a model for investigating the cellular and molecular mechanisms underlying the development and activity-dependent refinement of sensory connections. Thalamic organization is highly conserved throughout species and the dLGN of the mouse possesses many features common to higher mammals, such as carnivores and primates. Two general classes of neuron are present within the dLGN, thalamocortical relay cells and interneurons, both of which receive direct retinal input. Axons of relay cells exit dLGN and convey visual information to layer IV of cortex, whereas interneurons are involved in local circuitry. In addition, dLGN receives rich nonretinal input from numerous areas of the brain. Studies thus far have focused on the retinogeniculate pathway and the development of connections between retinal ganglion cells (RGCs) and relay cells has been well characterized. However, there are still a number of unanswered questions about circuit development in dLGN. Here we examined two aspects that are not well understood, the pattern of retinal convergence onto interneurons and the structural and functional innervation of nonretinal projections. To address the first issue we conducted in vitro whole-cell recordings from acute thalamic slices of GAD67-GFP mice, a transgenic strain in which dLGN interneurons express GFP. We also did 3-D reconstructions of biocytin-labeled interneurons using multi-photon laser scanning microscopy in conjunction with anterograde labeling of retinogeniculate projections to examine the distribution of retinal contacts. To begin to examine the development of nonretinal connections in dLGN we made use of a transgenic mouse (golli-τ-GFP) to visualize corticogeniculate projections, one of the largest sources of nonretinal input to dLGN. Using this mouse we studied the timing and patterning of corticogeniculate innervation in relation to the development of the retinogeniculate pathway. We also used binocular enucleation and genetic deafferentation to test whether the retina plays a role in regulating nonretinal innervation. We found that there is a coordination of retinal and nonretinal innervation in dLGN. Projections from the retina were the first to innervate and they entered dLGN at perinatal ages. They also made functional connections with both relay cells and interneurons at early postnatal ages. Interestingly, relay cells underwent a period of retinogeniculate refinement, whereas the degree of retinal convergence onto interneurons was maintained. This possibly reflects the different roles that these two cell types have in dLGN. Both structural and functional corticogeniculate innervation was delayed in comparison and occurred postnatally, however in the absence of retinal input the timing of corticogeniculate innervation was accelerated. RGCs transmit the visual information encoded in the retina to dLGN so it may be necessary for these connections to be formed before those from nonretinal projections, which serve to modulate that signal on its way to cortex. Thus precise timing of retinal and nonretinal innervation may be important for the appropriate formation of connections in the visual system and the retina seems to be playing an important role in regulating this timing.

Visual system development requires the formation of precise circuitry in the dorsal lateral geniculate nucleus (dLGN) of the thalamus. Although much work has examined the molecular mechanisms by which retinal axons target and form synapses in dLGN, much less is known about the mechanisms that coordinate the formation of non-retinal inputs in dLGN. These non-retinal inputs represent ~90% of the terminals that form in dLGN. Interestingly, recently reports show that the targeting and formation of retinal and non-retinal inputs are temporally orchestrated. dLGN relay neurons are first innervated by retinal axons, and it is only after retinogeniculate synapses form that axons from cortical layer VI neurons are permitted to enter and arborize in dLGN. The molecular mechanisms governing the spatiotemporal regulation of corticogeniculate innervation are unknown. Here we screened for potential cues in the perinatal dLGN that might repel the premature invasion of corticogeniculate axons prior to the establishment of retinogeniculate circuitry. We discovered aggrecan, an inhibitory chondroitin sulfate proteoglycan (CSPG), was highly enriched in the perinatal dLGN, and aggrecan protein levels dropped dramatically at ages corresponding to the entry of corticogeniculate axons into the dLGN. In vitro assays demonstrated that aggrecan is sufficient to repel axons from layer VI cortical neurons, and early degradation of aggrecan, with chondroitinase ABC (chABC), promoted advanced corticogeniculate innervation in vivo. These results support the notion that aggrecan is necessary for preventing premature innervation of the dLGN by corticogeniculate axons. To understand the mechanisms that control aggrecan distribution, we identified a family of extracellular enzymes (the a disintegrin and metalloproteinase with thromobospondin motifs [ADAMTS] family) expressed in postnatal dLGN that are known to contain aggrecan-degrading activity. Importantly, ADAMTS family members are upregulated in dLGN after retinogeniculate synapses form, and intrathalamic injection of ADAMTS4 (also known as aggrecanase-1) resulted in premature invasion of dLGN by corticogeniculate axons. Taken together these results implicate aggrecan and ADAMTSs in the spatial and temporal regulation of non-retinal inputs to the dLGN.