Though our understanding of the protein composition of the synapse and our recognition of the involvement of synaptic mechanisms in psychiatric disease both continue to grow, our ability to study the synaptic function of genes is still hampered by low throughput, high overhead assays. We propose to address this need by developing pooled screens of the effect of genetic perturbations on synaptic function in cultured neurons. Compared to arrayed screens, pooled screens are more robust to condition-to-condition variability and are thus better suited for studies of highly culture-dependent phenotypes such as synaptic transmission. Despite this advantage, it has been unclear how to translate pooled screening technology from studying cell survival or growth coupled phenotypes to dynamical functional phenotypes like those involved in neuronal function. We propose an innovative way around this problem by harnessing new optical methods. Our approach will be to perform barcoded genetic perturbations on a pool of cultured neurons, to perform time-resolved imaging assays of their postsynaptic or presynaptic function, and to subsequently use multiplexed RNA or protein readout methods to read out barcodes and match synaptic phenotypes to perturbations. Postsynaptic function will be read out using simultaneous voltage imaging and optogenetics, while presynaptic function will be read out by monitoring vesicle recycling with a pH sensitive fluorescent protein. The postsynaptic and presynaptic protocols are independent of each other and constitute separate specific aims. We will extensively characterize the performance of both assays and perform a pilot screen on the synaptic functions of 50 genes identified in Schizophrenia whole exome sequencing studies. Accomplishing these aims is possible by innovations in expression strategies, measurement protocols, and barcoding approaches. The chief significance of this work is to enable studies of synaptic function to occur earlier in the discovery process when studying new families of genes. In addition, the pilot screen we perform will generate valuable information about the basic neurobiological function of Schizophrenia associated genes. We will validate these results with traditional patch clamp approaches, and they will serve as the launching point for further investigations into the mechanisms of action of these genes.
Neurons communicate with each other via synapses, but studying the genes which affect synaptic function has been technically difficult due to the low throughput of synaptic assays. We propose to develop two new pooled functional assays for presynaptic and postsynaptic function to remove this barrier. We will then leverage these assays to screen poorly understood Schizophrenia associated genes for direct effects on synaptic transmission.
