PROJECT SUMMARY
Synaptic dysfunction is a common feature of neuropsychiatric disease. For example, a hallmark of age-related
neurodegenerative diseases such as Alzheimer’s and Parkinson’s is synaptic fibrilization and aggregation of
key proteins that participate in synapse and cell loss. Maladaptive plastic changes in synapse structure and
function underlie key aspects of behavioral and mood disorders ranging from addiction to depression, as well
as neurodevelopmental diseases like schizophrenia and autism. It is for these reasons that many investigators
across a range of neuroscience disciplines study the synapse, and the reason that new tools to study synapse
structure and function within neural circuits of interest are sorely needed. Indeed, current tools to assess
synapse structure in defined cell types are not readily compatible with state-of-the-art 3D volume approaches
such as serial block face scanning electron microscopy, and are severely hampered by inadequate
computational tools for quantitative assessment of these massive datasets. However, advances in molecular
genetics, optics, engineering and computing provide new opportunities to develop information rich strategies to
peer into the synapse. Here, we combine such advances to achieve a new state-of-the-art in imaging and
analyzing microcircuit connectivity and synapse structure within neurotransmitter-defined neural networks.
Specifically, we leverage the fact that the bulk of signaling across the synapse is mediated by a relatively small
population of small molecule neurotransmitters that are synthesized and packaged into synaptic vesicles at the
site of release in axonal compartments. The bulk of neurotransmission is thus dependent on just seven well-
described vesicular transporters expressed in brain. Our overall goal is to build a rigorous, easily deployable,
cell-type-specific, expandable, multi-functional toolkit for imaging and quantifying neurotransmitter-defined
synaptic connections by both light and electron microscopy in mice. To accomplish this, we will use
CRISPR/Cas9 to insert electron microscopy-compatible tags into native vesicular transporters (Aim 1),
establish simplified procedures for their monochrome and ‘multicolor’ labeling in 3D ultrastructure (Aim 2), and
computational tools for automated segmentation and quantitative analysis of key pre- and post-synaptic
metrics (Aim 3). Though these Aims are independently meritorious, by synthesizing them we aim to generate a
complete toolkit that will allow investigators to render neurotransmitter-defined circuit connections into 3D
ultrastructure datasets with automated quantitative assessment of key features of pre- and post-synaptic
structure.
