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Understanding Synaptic Partner Recognition

The Question - How do neurons find their correct synaptic partners?
The nervous system is composed of an immensely complex network of neural circuits that govern perception, thought and behavior.  For instance, for an organism to sense its environment, sensory neurons must make synaptic connections onto interneurons that convey input to the correct region of the brain for processing.  At each step of this sensory circuit,neurons must identify appropriate synaptic targets among the many neurites they contact before forming synaptic connections – a process known as synaptic partner recognition.  Despite its central role in circuit formation, the mechanisms neurons employ to choose the correct synaptic partner are poorly understood.
The Model Organism - C. elegans
We chose to study this question in the microscopic nematode (roundworm) C. elegansbecause it is an ideal model organism for genetic studies due to the extensive genetic and molecular tools available.  Cell specific promoters have been characterized which allow the study of interactions between individual neurons.  In addition, previous work indicates that molecules regulating synapse formation and synaptic transmission in C. elegans are conserved in humans.  Most importantly, it is the only organism for which a complete map of synaptic connections has been generated through decades of study making it ideal for the study of synaptic partner recognition.
Our Project - Utilizing NLG-1 GRASP to discover molecules that mediate synaptic partner recognition
Our goal is to discover new genes and molecular mechanisms governing synaptic partner recognition in complex nerve bundles, where parallel neural processes must distinguish among multiple potential targets to form appropriate en passant connections.  The majority of the nervous system is composed of similarly complex environments with multiple potential partners, yet little is known about how synaptic partner recognition is mediated in these environments.  However, a forward genetic screen to isolate new genes was not possible, due to the lack of a method to rapidly identify inappropriate connections.  Visualizing alignment of existing pre- and postsynaptic markers was not possible in such a complex environment due to the resolution limit of conventional light microscopy.  Reconstruction of synaptic connections using electron microscopy would take months to years for each animal, making it impractical for this purpose.  

Figure 1. NLG-1 GRASP is a novel transgenic marker that labels correct synaptic partner recognition with green fluorescence. Circles represent presynaptic vesicles, crosshatch marks represent the postsynaptic density.
To address these questions, we developed a novel trans-synaptic marker called NLG-1 GRASP that allows visualization of changes in synaptic connectivity in live animals with conventional fluorescence microscopy.  We fused complementary fragments of a split GFP (Green Fluorescent Protein) to Neuroligin 1, which we found to localize both pre- and postsynaptically (click for sequence information). This system offers a simple and rapid means to query synaptic specificity: the presence (or absence) of GFP fluorescence indicates the formation (or lack) of the appropriate synapses.  
Using NLG-1 GRASP, we have successfully visualized changes in connectivity in three characterized circuits using known synaptic specificity mutants in live animals.  We are now taking genetic approaches to discover novel molecular mechanisms guiding synaptic partner recognition in complex nerve bundles, utilizing NLG-1 GRASP to detect defects in connectivity.  This marker will allow us to rapidly identify synaptic partner recognition mutants that would not be isolated with conventional methods, allowing the identification of new pathways mediating this fundamental process.