Synaptic development in the nematode brain

Glial cells are the most abundant cell type in the vertebrate nervous system, yet their role during the development of the nervous system is not well understood. Our studies demonstrate the importance of glia in specifying precise neural connectivity in vivo. These findings, however, were limited to the synapses formed between AIY and RIA. What other roles do glial cells play in orchestrating the development of neural circuits in the nematode brain?

We have observed that there are at least nine other circuits that form synapses in the glia-specified coordinate where AIY innervates RIA. We are currently exploring the glial requirement for the development of these circuits by ablating the glia (using genetic and laser ablations methods). We are also identifying the molecular cues that could be used by glia in directing the innervation of the circuits in this region. Our preliminary data suggests that glia employ different signaling cues to orchestrate the innervation of multiple circuits. Therefore, we are currently employing forward and reverse genetic approaches to identify the molecular pathways activated by glia in directing the innervation of these glia-dependent circuits. Given the evolutionary conservation of glial cells, the developmental and molecular events uncovered in these studies could be conserved in higher organisms.

Serotonergic pathways, ranging from neurotransmitter biogenesis to signaling, are well conserved across evolution. Nematodes, like vertebrates, have only a handful of neurons that produce serotonin, and these neurons exhibit morphological features similar to those seen in vertebrate serotonergic neurons. For instance, the main serotonergic neuron in C. elegans, called NSM, elaborates axonal arbors within a precise neuroanatomical coordinate overlying the nerve ring (Axang et al., 2008). We recently developed a system that allows us to assay NSM morphogenesis and synaptogenesis in vivo, in real time and with single cell resolution. This gives us an unprecedented opportunity to dissect the cellular and molecular mechanisms that regulate serotonergic synapse development in vivo. To identify how precise targeting is achieved in serotonergic neurons in vivo, we are working toward the following 3 goals:

1. How do ENT-containing arbors form with SPATIAL specificity in the nematode?
2. How is the TEMPORAL specificity of arborization and ENT formation regulated in the nematode?
3. What are the molecular mechanisms that contribute to the physical assembly and plasticity of ENTs and associated arbors?

The Colon-Ramos lab is interested in studying how synapses form. We use the nematode C. elegans as our model system, focusing specifically on the development of synapses between two neurons, the AIY and RIA interneurons, in the head of the animal. The AIY interneurons act as the presynaptic partners to the RIA interneurons, forming synapses at specific points within a portion of the AIY neurite we refer to as zone 2. Traditionally, synapse formation is thought to take place after neurite outgrowth has occurred, but we recently observed that Zone 2 is the first part of the AIY neurite to form. This observation raises the possibility that synapse formation between AIY and RIA takes place concurrently with neurite outgrowth. We are currently testing this hypothesis by visualizing synapse formation in AIY during embryogenesis.

In addition to observing how synapses form in the developing embryo, we are also interested in understanding the pathways and molecules involved in directing where synapses form between AIY and RIA. Previous research has demonstrated that the UNC-6/Netrin pathway is important for directing synapse formation in AIY and axon guidance in RIA. UNC-6 is produced by a glial cell which ensheathes one side of Zone 2, and UNC-6 signals through its receptor UNC-40 to promote synapse localization in AIY. Interestingly, RIA (the AIY postsynaptic partner) is located on the opposite side of the AIY neurite as the glial cell, suggesting that there might be a secondary mechanism directing synapse formation between AIY and RIA besides glia-derived UNC-6/Netrin. We are currently examining whether other molecules known to signal through the UNC-40 receptor may play a role in directing AIY synapse formation.

Five different neuron pairs have been identified as essential for the proper functioning of the thermotaxis circuit. These neurons contact many other neurons in the nerve ring, but form specific synaptic connections to assemble the thermotaxis behavioral circuit. Our previous studies focused on examining one such set of synapses: those between interneuron AIY and interneuron RIA. We are now continuing this work by determining the developmental events and molecular signals that direct the assembly of the rest of the thermotaxis behavioral circuit.

We are focusing on the different types of synapses formed between the presynaptic AIY and its postsynaptic partners. For instance, AIY innervates another major interneuron, AIZ, at a specific subcellular region of its axon that is distinct from the region where AIY innervates RIA. What are the molecular and cellular requirements for innervation of AIY to AIZ? How do they compare to the signals that direct innervation of AIY to RIA? How is the architectural specificity achieved? We are using the same basic approach to identify the molecular signals which direct innervation between AIY and AIZ.

UNC-40/DCC receptor was originally identified as an axon guidance molecule, and has been shown to regulate cell migration and neuronal polarization. We identified an unexpected role for UNC-40/DCC as a regulator of presynaptic assembly. How can the same receptor and ligand pair elicit axon guidance or presynaptic assembly in a cell-specific manner? We are using genetic and molecular approaches to answer this question.

In particular, we are focusing on the newly identified capacity of the UNC-40/DCC receptor to direct presynaptic assembly. We are conducting structure/function studies aimed at identifying domains important for UNC-40 synaptogenic function in the AIY interneuron. Determining the roles of different UNC-40 domains will be critical for understanding how UNC-40/DCC specifies correct synaptic location in the AIY interneuron and how this function relates to its conventional role as a guidance molecule. We are also using genetic approaches to identify the signal transduction pathways that lead to the assembly of presynaptic terminals. Our preliminary data suggest that the UNC-40-mediated axon guidance and presynaptic assembly pathways are genetically separable. Given how conserved the Netrin pathway is across metazoans, and the importance of UNC-40/DCC in coordinating the innervation of circuits in the nematode brain, we expect our studies will be relevant to our future understanding on how these signaling pathways could be utilized in the innervation of more complex circuits, such as those found in vertebrate brains.