Light microscopy based approach for mapping connectivity with molecular specificity

Abstract

Mapping neuroanatomy is a foundational goal towards understanding brain function. Electron microscopy (EM) has been the gold standard for connectivity analysis because nanoscale resolution is necessary to unambiguously resolve chemical and electrical synapses. However, molecular information that specifies cell types is often lost in EM reconstructions. To address this, we devised a light microscopy approach for connectivity analysis of defined cell types called spectral connectomics. We combined multicolor genetic labeling (Brainbow) of neurons with a m ult i-r ound i mmunostaining Ex pansion Microscopy (miriEx) strategy to simultaneously interrogate morphology, molecular markers, and connectivity in the same brain section. 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.

Decrowding Expansion Pathology: Unmasking Previously Invisible Nanostructures and Cells in Intact Human Brain Pathology Specimens

“…Some expansion protocols have been shown to be capable of preserving protein antigens throughout the expansion process (Supp. Table 1) [17][18][19][20][21][22][23][24][25] , and are thus compatible with postexpansion immunostaining. However, most of these existing post-expansion staining protocols either required specialized fixative compositions 17,18,21,22,24 , and thus are incompatible with archival clinical samples, or they showed incomplete softening with tissue cracks and anisotropy 19 , or had uncharacterized nanoscale isotropy 20 .…”

“…Fig. 1), a strategy previously demonstrated by other post-expansion staining protocols but not on human tissues 18,20,31 . High grade glioma tissues are known to undergo abnormal endothelial proliferation, leading to some areas of tissue with abnormally large amounts of vascularity and extracellular matrix (ECM).…”

“…We used higher levels of sodium dodecyl sulfate (SDS) (20% weight/volume (w/v)) than in earlier protein-preserving protocols (proExM, 1%; mExM, 4%; MAP/U-ExM/miriEx/pan-ExM/ExR, 5.8%; for other protein-preserving protocols, see Supp. Table 1) [17][18][19][20][21][22][23][24] , reasoning that this could help with isotropic expansion by better converting proteins to a denatured state, and minimizing non-covalent intra-and inter-protein interactions that could potentially hinder molecular separation and tissue expansion 26,27 . We also included a new ingredient, β-mercaptoethanol (100 mM), a reducing agent, which we reasoned could help with isotropic expansion by cleaving inter-molecular disulfide bridges between structural components of the tissue [25][26][27][28][29][30] .…”

“…1E; see Supp. Table 2 for antibodies used in this work); staining is performed at the partially expanded [~2.3x] state, instead of the fully expanded [~4x] state, because full expansion requires sample immersion in deionized water, a low-ionicstrength environment that hinders antibody binding, consistent with earlier post-expansion immunostaining protocols [17][18][19][20][21][22] . Multiplexing is possible because these antibodies can be stripped using the same buffer, and then new antibodies applied (Supp.…”

Improved blind demixing methods for recovering dense neuronal morphology from barcode imaging data

“…The number of neurons that can be uniquely labeled and identified by the original method was limited to several hundreds, because of limitations on the number of distinct colors that can be generated and distinguished. Recent studies have focused on reducing these bottlenecks [8,37,38].…”