Retinogeniculate (RG) synapses connect retinal ganglion cells to the thalamic relay cells of the dorsal lateral geniculate nucleus (dLGN). They are critical for regulating the flow of visual information from retina to primary visual cortex (V1). RG synapses in dLGN are uniquely larger and stronger than their counterparts in other retinorecipient regions. Moreover, in dLGN, RG synapses can be classified into two groups: simple RG synapses, which contain glia-encapsulated single RTs synapsing onto relay cell dendrites, and complex RG synapses, which contain numerous RTs that converge onto the shared regions of relay cell dendrites. To identify target-derived molecules that direct the transformation of RTs into unique RG synapses in dLGN, I used RNAseq to obtain the whole transcriptome of dLGN and its adjacent retinorecipient nucleus, vLGN, at different time points during RG synapses development. Leucine-Rich Repeat Transmembrane Neuronal 1 (LRRTM1), a synaptogenic adhesion molecule, was the candidate I selected based on its expression pattern. Here, I discovered that LRRTM1 regulates the development of complex RG synapses. Mice lacking LRRTM1 (lrrtm1-/-) not only show a significant reduction in the number of complex RG synapses but they exhibit abnormal visual behaviors. This work reveals, for the first time, a high level of retinal convergence onto dLGN relay cells in thalamus and the functional significance of this convergence for vision. / Ph. D. / Our relationship with the environment is heavily reliant on vision, an intricately wired sensory system, much like a circuit. This circuit begins at the eyes, with the retina, and spreads to different visual centers in the brain. Retinal ganglion cells (RGCs) send their wires, called axons, carrying information about the visual world to over 40 different regions in the brain. A major target of these axons is the dorsal lateral geniculate nucleus (dLGN), a region critical to our ability to perceive the visual world. The sites where RGCs connect to the dLGN cells are called retinogeniculate (RG) synapses, and my studies focused on understanding how these RG synapses develop and how they function. I am the first to discover the fact that more than a dozen distinct RGC axons cluster within the same neighborhood of one shared target cell in the dLGN. Unique to the dLGN, these clusters, termed complex RG synapses, are not seen in any other RGC target regions in the brain. Moreover, I demonstrated a new molecular mechanism that forms these synapses by identifying a protein called LRRTM1, as a critical molecule required for the formation of these complex RG synapses in the dLGN. By studying the visual behavior of mutant mice lacking LRRTM1, I demonstrated that complex RG synapses are important for performing complex visual tasks. The discoveries detailed within this dissertation add to current efforts to restore vision in patients suffering from severe visual impairments, via regenerative therapies, by furthering our understanding of how neural wires connect in the visual circuit to reveal everything we will ever know about the visual world.

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.

Immune molecules, including complement proteins, C1q and C3, have emerged as critical mediators of synaptic refinement and plasticity. Complement proteins localize to synapses and refine the developing retinogeniculate system via C3-dependent microglial phagocytosis of synapses. Retinal ganglion cells (RGCs) express C1q, the initiating protein of the classical complement cascade, during retinogeniculate refinement; however, the signals controlling C1q expression and function remain elusive. RGCs grown in the presence of astrocytes significantly upregulated C1q compared to controls, implicating an astrocyte-derived factor in neuronal C1q expression. A major goal of my dissertation research was to identify the signals that regulate C1q expression and function in the developing visual system. In this study, I have identified transforming growth factor beta \((TGF-\beta)\), an astrocyte-secreted cytokine, as both necessary and sufficient for C1q expression in RGCs through an activity-dependent mechanism. Specific disruption of retinal \(TGF-\beta\) signaling resulted in a significant reduction in the deposition of C1q and downstream C3 at retinogeniculate synapses and significant synaptic refinement defects in the retinogeniculate system. Microglia engulfment of RGC inputs in the lateral geniculate nucleus (LGN) was also significantly reduced in retinal \(TGF\beta\)RII KOs, phenocopying the engulfment defects observed in C1q KOs, C3 KOs, and CR3 KOs. Interestingly, in C1q KOs and retinal \(TGF\beta\)RII KOs, microglia also failed to preferentially engulf less active inputs when retinal activity was manipulated, suggesting that retinal activity and \(TGF-\beta\) signaling cooperatively regulate complement mediated synaptic refinement. In support of this hypothesis, blocking spontaneous activity in RGC cultures significantly reduced C1q upregulation by \(TGF-\beta\). Moreover, manipulating spontaneous retinal activity in vivo modulated C1q expression levels in RGCs and C1q deposition in the LGN. Together these findings support a model in which retinal activity and \(TGF-\beta\) signaling control expression and local release of C1q in the LGN to regulate microglia-mediated, complement-dependent synaptic pruning. These results provide mechanistic insight into synaptic refinement and, potentially, pathological synapse loss which occurs in the early stages of neurodegenerative diseases concurrently with aberrant complement expression and reactive gliosis.

The dorsal lateral geniculate nucleus (dLGN) has become an important model for studying many aspects of visual system development. To date, studies have focused on the development of retinal projections and the role of activity in shaping the pattern of synaptic connections made with thalamocortical relay cells. By contrast, little is known about relay cells and the factors that regulate the growth and establishment of their dendritic architecture. In many systems, such growth seems consistent with the synaptotrophic hypothesis which states that synapse formation and dendritic growth work in a concerted fashion such that afferent input and the establishment of functional synapses are needed to shape the maturation of dendritic arbors. To address this, we characterized the development of relay cells in the dLGN of wild-type (WT) mouse. By adopting a loss of function approach, we assessed the manner in which growth and maturation of relay cells were affected by retinal innervation. For this, we made use of the math-null (math5-/-) mouse in which progenitors fail to differentiate into retinal ganglion cells (RGCs), and exhibit a >95% cell loss. Anterograde labeling of RGC axons with cholera toxin subunit B (CTB), immunolabeling of RGC-specific presynaptic machinery in dLGN (e.g. vesicular glutamate transporter 2), and ultrastructural analysis at the electron microscopy level demonstrated that the dLGN is devoid of retinal innervation. We examined the functional and morphological characteristics of relay cells in WT and math5-nulls during early postnatal life by conducting in vitro whole cell recordings in slices containing dLGN. Individual relay cells were labeled by intracellular injection of biocytin, and imaged by confocal microscopy to obtain the 3-D reconstructions of their dendritic trees. Morphometric analysis revealed that relay cells in WT undergo two growth spurts: an early one where cell class specification and dendritic complexity are established and a later one marked by an increase in dendritic field and length. Following the third week, relay cells growth was stabilized. In math5-nulls, relay cells maintained their morphological identity whereby cells could be classified in three groups (Y: spherical, X: bi-conical, W: hemi-spherical). However, the dLGN was highly reduced in size, and relay cells showed disrupted growth spurts. Relay cells had smaller somata and exhibited fluctuations in dendritic complexity and field extent compared to age-matched WTs. Exuberance in dendritic branching was noted in week 2, and by week 5, relay cells had significantly smaller surface area resulting from a loss of dendritic segments and a reduction in dendritic field extent. Control experiments using RT-PCR revealed that these changes were not due to the loss of math5 in the dLGN. Whole cell recordings and voltage responses to square wave current pulses showed that math5-nulls possess the full compliment of intrinsic membrane properties, such as relay cells displayed both burst and tonic firing modes. A cross of the math5-null with a transgenic mouse that expresses GFP in layer VI cortical neurons revealed a dense plexus of corticogeniculate terminals throughout the mature dLGN. However, the rate of corticogeniculate innervation was highly accelerated and was complete a week earlier than WT. Electric stimulation of cortical axons revealed that synapses are functional and responses were indistinguishable from WT. Taken altogether, these observations suggest that retinal innervation plays an important trophic role in the maturation of dLGN and is necessary for the continued maintenance of relay cells’ structural integrity. However, the general form and function of relay cells seem largely unaffected by the loss of retinal innervation.

We study the activity-dependent refinement of sensory systems by using the mouse retinogeniculate system as a model. Spontaneous retinal waves lead to robust excitatory post-synaptic activity in developing relay cells in the dorsal lateral geniculate nucleus (dLGN) of the thalamus and are reportedly needed to help guide the segregation of retinal inputs into eye-specific domains as well as for the pruning of extraneous retinal inputs onto single dLGN relay cells. The composition of retinally evoked post-synaptic activity activated by these retinal waves in dLGN is largely unknown, but based on our in vitro recordings, such activity seems well suited to activate large, long-lasting, high-amplitude depolarizations mediated by L-type Ca2+ channel activation, plateau potentials. Plateau activity prevails early in life, at the peak of retinogeniculate refinement, however, little is known about the factors that contribute to the activation of these events, or the potential role of plateau potentials in mediating activity-dependent remodeling. In this thesis, we examined the factors and stimulus conditions that lead to the activation of plateau activity. We found that many aspects of developing retinogeniculatecircuitry (e.g., the high degree of retinal convergence, the temporal summation of excitatory post-synaptic potentials, and the lack of inhibitory connections) seem to favor their activation at early postnatal ages. We then tested whether such activity is necessary for the refinement of retinal projections, as well as their functional connections onto dLGN cells. To address this, we took a loss-of-function approach and made use of a transgenic mouse that lacks the β3 subunit of the L-type Ca2+ channel. These mutants have far fewer membrane-bound L-type Ca2+ channels and greatly attenuated L-type activity. In β3 nulls, L-type plateau potentials are rarely observed in the dLGN, even at young ages or when repetitive pulses of electrical stimulation are applied to the optic tract. Although these mice have normal stage II and III spontaneous retinal waves, the retinogeniculate projections of β3 null mice fail to segregate properly. In addition, the degree of retinal pruning is impaired. These results suggest that post-synaptic L-type Ca2+ channel activity is necessary to implement the activity-dependent refinement of the retinogeniculate pathway.

The retinogeniculate synapse, the connection between retinal ganglion cells (RGCs) and thalamic relay neurons, undergoes extensive remodeling and refinement in the first few postnatal weeks. While many studies have focused on this process, little is known about the factors that influence excitatory transmission during this dynamic period. A major goal of my dissertation research was to identify mechanisms that regulate glutamate release and clearance at the developing synapse. First, we investigated the role of glutamate transporters and metabotropic glutamate receptors (mGluRs) in shaping excitatory transmission. Early in development, we found presynaptic group II/III mGluRs are present and are activated by glutamate released from RGCs following optic tract stimulation at natural frequencies. This response was found to diminish with age, but glutamate transporters continued to shape synaptic currents throughout development. The finding that glutamate is able to escape the synaptic cleft and bind extrasynaptic high-affinity mGluRs led us to speculate that glutamate might also diffuse to neighboring synapses and bind ionotropic glutamate receptors opposing quiescent release sites. Excitatory currents recorded from immature, but not mature, retinogeniculate synapses display a prolonged decay timecourse. We found evidence that both asynchronous release of glutamate as well as spillover of glutamate between neighboring synapses contributes to these slowly decaying synaptic currents. Furthermore, we uncovered and characterized a novel, purely spillover-mediated current from immature relay neurons, which strongly supports the presence of glutamate spillover between boutons of different RGCs. The results of my studies indicate that far more RGCs contribute to relay neuron firing than would be predicted by the anatomy alone. Finally, in an ongoing study, we investigated the functional role of the neuronal glutamate transporter GLT-1 at the immature retinogeniculate synapse. While GLT-1 has been found in both neurons and glia, excitatory currents at the retinogeniculate synapse were largely unaffected in mice lacking neuronal GLT-1, suggesting non-neuronal glutamate transporters are responsible for the majority of glutamate removal from the developing synapse. Taken together, these results provide insight into the synaptic environment of the developing retinogeniculate synapse and identify a number of mechanisms that shape excitatory transmission during this period of synaptic maturation and refinement.