Introduction
The previous installment of the Journal Club focused on genes involved in the assembly of synaptic connections between brain cells. In this issue, we will explore how synaptic assembly genes have been paired with novel fluorescence technology to label synapses in specific neural pathways in vivo.

Mapping the functional connectivity of the brain is both a very old and currently rather hot topic in neuroscience. Remarkably, using the simple technique of the Golgi stain, Ramon y Cajal was able to describe the morphology of numerous cell classes in the brain and also infer the functional anatomy of circuits such as the hippocampus. He was helped by the highly stereotyped circuit architecture of some brain areas. Despite a century of follow-up work, our knowledge of the overall wiring diagram of the brain remains at a rudimentary stage. Modern neuroscientists need a reliable and simple method to identify the connections between genetically selected cell types in complex local circuits and from long distance connections.

A recent paper by Feinberg et al, from Cori Bargmann’s lab, should help us make sense of these tangled webs of connections. The authors propose a clever new technique, Genetic Reconstitution Across Synaptic Partners, to track the locations of selected synaptic connections backed by an impressive set of in vivo proof-of-principal experiments in c. elegans. The gist of the strategy is to split a fluorescent marker into two non-functional components and then distribute each half on different sides of circuit’s connection. Only at synaptic connections would the two components be close enough to undergo trans-complementation and reconstitute a functional marker.

A number of labs had previously shown that the green fluorescent protein (GFP) could be split into two non-fluorescent halves, which can trans-complement and become fluorescent. However, these probes generally had poor kinetics and required tethering by fusion to tightly dimerizing accessory components to prevent probe degradation. These requirements presented too much of a challenge for trans-synaptic complementation. Thankfully, in 2005 the Waldo group published their ‘superfolder’ GFP, which also turned out to be more much stable when split to two components. Results
Feinberg et al. took this split superfolder GFP and genetically expressed each half on the surface of neurons. They used a variety of tethering strategies, including fusion to the broadly distributed transmembrane protein CD4, the presynaptically targeted PTP-3A and the pre and postsynaptically targeted (in the worm) neuroligin homolog (NLG-1). When expressed in worm lines, each technique was able to generate stable fluorescent labels at the sites of synaptic contact between genetically selected sells. With fluorescence microscopy, they used the probe to visualize sites of known synaptic contact and to discover novel synaptic mistargeting in several mutant lines.

GRASP shows great promise to define the connectivity map of the brains of drosophila, zebrafish and mammals. This technique could find significant use in studying the patterns of experience dependent rewiring of synaptic connections in small transparent in vivo systems. Similar work in mammalian systems would likely be restricted by light scattering, but single timepoint measurements could be taken across the entire brain with careful tissue fixation. The relatively small genetic size of the probes should facilitate methods of viral introduction, making transgenic line generation unnecessary for many questions. Speaking of questions, this paper makes a few discussion questions come to mind…

Questions:

1. In some of the images there are high levels of autofluorescence that are brighter than the labeled synapses. What is the source of this background? Would it be as much of a problem in mammalian brains? 2. How would you confer greater synapse specificity in mammalian systems? What effects would you have to be watchful for if you overexpressed synaptic adhesion molecules in a mouse? How could you alleviate these problems? 3. Interestingly, the spilt GFP site is very asymmetric, with a 16 residue peptide (215-230) complementing a 214 residue GFP barrel. What impact would this have on extending the technique to include multiple fluorescent colors, such as CFP and Citrine What about for the mFruits? 4. This technique labels the close juxtaposition of two neurons. In what ways could you prove the label was targeting functional synaptic connections? How could you determine the polarity (excitatory vs. inhibitory) of the connection? 5. Would it be possible to reconstitute functional activity sensors at synapses? For example could this strategy be used to reconstitute GluSnFR and image glutamate at a specific neuronal connections? What additional challenges might this entail? 6. What advantages does GRASP have over Steve Smith’s Array Tomography, or Jeff Lichtman’s Brainbow technique for mapping synaptic connections? What are the disadvantages?