Development of Light Microscopy Based Approaches in Neurocartography

Abstract

The process of mapping neuroanatomy at multiple scales is defined as neurocartography and has the ultimate goal of revealing a complete wiring diagram of synaptic connections. Neurocartography is an important pursuit because brain function is intricately linked to neuroanatomy, similar to how the function of a protein depends on its structure. Here, we developed new light microscopy approaches for neurocartography at both microscale and nanoscale levels to map single neuron morphologies and synaptic connectivity respectively. Spatially sparse labeling of neurons has been necessary when studying neuron morphologies to minimize overlap and avoid ambiguities during reconstructions. We developed two strategies to overcome this limitation and achieve spatially dense labeling. First, we used a multicolor genetic labeling (Brainbow) approach to stochastically express fluorescent proteins in a spatially dense population of neurons, facilitating reconstruction of single neuron morphologies. We extend the Brainbow viral toolbox by 1) introducing 12 fluorescent proteins in the form of 6 new viruses, 2) using a membrane and cytoplasmic dual labeling strategy, and 3) adopting the AAV.PHP.eB capsid to systemically induce expression and better control color diversity. Second, we used expansion microscopy to increase confocal imaging resolution by physically magnifying brain samples. We developed a multi-round immunostaining Expansion Microscopy (miriEx) protocol that enables multiplexed protein detection at multiple imaging resolutions. We then combined Brainbow with miriEx to simultaneously map morphology, molecular markers, and connectivity in the same brain section. We define the derivation of these properties from hyperspectral fluorescent channels as spectral connectomics, a light microscopy based approach towards mapping neuroanatomy and connectivity with molecular specificity. We applied our multimodal profiling strategy to directly link inhibitory neuron cell types with their network morphologies. Furthermore, we showed that correlative Brainbow and endogenous synaptic machinery immunostaining can be used to define putative synaptic connections between spectrally unique neurons, as well as map putative inhibitory and excitatory inputs. We envision that spectral connectomics can be applied routinely in neurobiology labs to gain insights into normal and pathophysiological neuroanatomy across multiple animals and time points. We hope that the light microscopy approaches developed in this dissertation will facilitate the extraction of new biological insights from neurocartography.