Cell Specific Dissection of the Macromolecular Components of Synapses in the Mammalian Brain: Unraveling the Molecular Synaptic Code (with Nat Heintz, Rockefeller University)
The mammalian brain consists of interconnected neuronal networks. The formation of a functional brain requires several steps from the generation of numerous types of neurons through the establishment of a very precise connectivity between these neurons. The final steps leading to the highly specific connections of the adult brain involve a precise targeting of different inputs to different subcellular domains of specific neurons, as well as a period of refinement through selective stabilization/elimination of synapses. It has been postulated that molecular cues as well as synaptic activity participate in the formation of very specific neuronal connections, but relatively little is known about the molecules regulating this phenomenon. In particular, the following questions need to be answered. How does a given neuron select its synaptic partners? How do neurons segregate these synapses to different cellular subcompartments?
The hypothesis that the targeting of synapses involves a molecular synaptic code was formulated more than 40 years ago by Sperry. To date, it is not yet clear what this code is made of, nor how important it is for the establishment of functional neural networks. Whereas the involvement of several classes of molecules has been proposed, direct in vivo evidence of their role is still rather scarce. One class of molecules that is targeted to different synapses is the neurotransmitter receptors – for example, GABA receptors are at inhibitory synapses, whereas glutamate receptors, like GRID2 or GLUR2, are at excitatory synapses. Until now, studies of molecular components of synapses have concentrated either on the molecules that are present in synapses in general or on signaling pathways that are tethered to a given neurotransmitter receptor. Here we seek to identify the composition of individual synapses from specific neuronal populations and compare these compositions with one another. The goal of this project is to develop a method that will enable us to elucidate these different synaptic compositions and to throw light on the synaptic code in the brain.
Strategy for identifying the protein content of a specific synapse in a specific neuronal population
Our method involves the following steps (see Figure):
- Construct fusions between a tag (Venus, which is a bright GFP variant) and selected synaptic receptors, like the glutamate or GABA receptors, which are specifically localized to a given type of synapse, and express these tagged receptors in a specific, functionally coherent neuronal population in vivo, through the generation of transgenic mice using the Bacterial Artificial Chromosome (BAC) modification strategy (Heintz, 2001; Gong et al., 2002).
- Purify synapses from the brain of these transgenic mice and specifically immunoprecipitate only those synapses bearing the tagged receptor.
- Analyze the composition of these specific synapses from a specific neuronal population by mass spectrometry.
This method should enable us to find molecules involved in synapse formation and specificity without any preconceived idea of their nature. Moreover, the method is based on the use of transgenic mice, allowing us to directly gather information on the composition of synapses in vivo.
Our method will enable us to analyze synapses from very specific, functionally coherent neuronal populations throughout the central nervous system. This proves feasible through the use of drivers that target the expression of our tagged receptors in specific neuronal populations. Indeed, the GENSAT project at Rockefeller University (Heintz, 2004) has already provided extensive data on the use of BACs to target the expression of GFP in various functionally coherent neuronal populations throughout the brain (Gong et al., 2003).
- Gong S, Yang XW, Li C, Heintz N. 2002. Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin of replication. Genome Res 12:1992-1998.
- Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ, Joyner A, Leblanc G, Hatten ME, Heintz N. 2003. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425:917-925.
- Heintz N. 2001. BAC to the future: the use of bac transgenic mice for neuroscience research. Nat Rev Neurosci 2:861-870.
- Heintz N. 2004. Gene expression nervous system atlas (GENSAT). Nat Neurosci 7:483.