During development, tightly regulated mechanisms establish the proper balance between excitatory and inhibitory synaptic inputs made onto each neuronal cell type. However, the mechanisms coordinating the development of these two types of synapses are still poorly understood. We recently discovered that SRGAP2 is a postsynaptic protein playing key roles in vivo in promoting the rate of excitatory and inhibitory synaptic maturation and limiting the density of both types of synapses made onto pyramidal neurons in the developing cortex. Additionally, we and others discovered that SRGAP2 has undergone several partial gene duplications specifically in the human lineage. Only one of these gene duplications, called SRGAP2C (the ancestral copy of the human gene was renamed SRGAP2A) has been fixed in the human population and is expressed in the developing human brain. We discovered that SRGAP2C binds to and inhibits the functions of SRGAP2A during synaptic development. When human-specific SRGAP2C is expressed in mouse cortical pyramidal neurons in vivo, it induces significant delay (neoteny) of synaptic maturation and significant increase in both excitatory and inhibitory synaptic density. However, at this point one of the most significant limitations in studying the roles of SRGAP2A or SRGAP2C in synaptic development in vivo is that it is extremely challenging to obtain quantitative information on excitatory and inhibitory synapses across the entire dendritic arbor of well-defined groups of pyramidal neurons. The objective of this application is to create a platform for creating whole-neuron synaptic input maps and to use this platform to investigate how SRGAP2A and SRGAP2C influence synaptic development throughout the dendritic arbor. This application's central hypothesis is that by regulating the scale and timing of synaptic development, SRGAP2A and SRGAP2C control the spatial organization of excitatory and inhibitory synapses across dendritic domains.
Aim 1 will enable labeling of inhibitory synapses throughout whole pyramidal neurons using in vivo genome editing with CRISPR-Cas9. I will use in utero electroporation to insert a FLAG tag at the 5? end of Gephyrin, which encodes an inhibitory synaptic scaffolding protein.
In Aim 2, I will use neuron reconstruction software developed in collaboration with the Peng lab to create whole-cell synaptic input maps of cortical pyramidal neurons from transgenic mice expressing SRGAP2C or lacking SRGAP2A. These maps will provide unprecedented insight into the structured organization of synapses throughout the dendritic tree and allow us to determine how SRGAP2A and SRGAP2C regulate global synaptic development.
We identified a gene called SRGAP2A that plays a critical role in coordinating the rate of maturation of both excitatory and inhibitory synapses in the developing cortex. Interestingly, this gene has been duplicated specifically in the human lineage and expression of this human-specific version called SRGAP2C inhibits SRGAP2A function and results in slower synapse maturation and increased number of synapses in cortical neurons. This project will test the roles of SRGAP2A and its human-specific gene copy in excitatory and inhibitory synaptic organization across the dendritic arbor of whole neurons throughout development.
