Neurons are highly polarized and use compartmentalized information processing at synapses for communication with other neurons. Synapses differ in shape, size, and function and exhibit ongoing plasticity during development and modification of neural circuits. This plasticity is essential for correct wiring of the nervous system and its ability to encode, maintain and remember facts, procedures and experiences. While the structural diversity of synapses is largely known, their potential molecular diversity is much less well understood. Molecules residing at synapses have been identified by diverse approaches but the complement of proteins at individual synapse types and their stoichiometric relationships with other molecules is unknown. Current classification mostly relies on neurotransmitter/receptor phenotypes, leading to broad descriptors like “excitatory” or “inhibitory” synapses. This relatively coarse understanding of molecular diversity of synapses is mostly due to technical limitations associated with the small size of synapses and difficulties in purifying them, particularly those associated with different cell types. As the function of the synapse and its ability to change is largely determined by the molecules (mRNAs, proteins, lipids) inhabiting it, it is essential to understand the molecular diversity of synapses. Indeed, the quality, quantity and interactions of proteins at synapses largely determine the physiological properties of synaptic transmission and on a larger scale, the organization and function of neuronal circuits. The aims of this project are to determine the transcriptomes and proteomes of genetically-identifiable synaptic populations in different brain areas, to determine the molecular diversity in these same synapse populations using transcriptomic analysis of individual synapses, and to assess how these synaptic proteomes, transcriptomes and the transcriptomic diversity respond to plasticity. We use fluorescence-activated synaptosome sorting (FASS) to purify different synaptic populations from different brain areas, and next generation RNA sequencing (RNA-NGS) and liquid chromatography tandem mass spectrometry (LC-MS/MS), optimized for quantitative transcriptomic and proteomic profiling of very small amounts of sorted synapses. We further develop a method, SynDrops, for the transcriptomic analysis of individual synapses. These studies will reveal the diversity within synapse types and across brain areas and states, and allow the field to probe “diseased” synapses in the future.
