Brain Region Specific Single-Molecule Fluorescence Imaging

Abstract

We developed an approach utilizing nanoscale vesicles extracted from brain regions combined with single molecule imaging to monitor how an animal's physiological condition regulates the dynamics of protein distributions in different brain regions. This method was used to determine the effect of nicotine on the distribution of receptor stoichiometry in different mouse brain regions. Nicotine induced upregulation of α4β2 nicotinic acetylcholine receptors (nAChRs) is associated with changes in their expression, trafficking, and stoichiometry. The structural assembly of nAChRs has been quantified in cell culture based systems using single molecule techniques. However, these methods are not capable of quantifying biomolecule assembly that takes place in a live animal. Both nicotine induced upregulation and changes in nAChR stoichiometry differ across brain regions. Our single molecule approach revealed that nicotine acts differentially across brain regions to alter assembly in response to exposure and withdrawal.

Upregulation of nAChRs and Changes in Excitability on VTA Dopamine and GABA Neurons Correlates to Changes in Nicotine-Reward-Related Behavior

“…Additionally, we can detect changes in stoichiometry but not define specific stoichiometries. The former problem (plasma membrane changes) is partially reconciled by our electrophysiology assays (discussed below) but the latter can now be addressed using newly developed single-molecule microscopy assays (Fu et al, 2019).…”

Green Apple e-Cigarette Flavorant Farnesene Triggers Reward-Related Behavior by Promoting High-Sensitivity nAChRs in the Ventral Tegmental Area

“…Nicotine or flavorant induced nAChR upregulation and changes in stoichiometry occur over the course of 10-12 d in vivo Nashmi et al, 2007), whereas the in vitro system findings occur in 24 h (Srinivasan et al, 2011). This difference in time dependency for in vitro preparations is accompanied by the lacking complexity of a cell's environment in vivo (Fu et al, 2019). Stoichiometry changes within in vivo systems are not only brain region specific (Fu et al, 2019), but they also rely on unique cellular machinery that is absent in our in vitro model.…”

“…This difference in time dependency for in vitro preparations is accompanied by the lacking complexity of a cell's environment in vivo (Fu et al, 2019). Stoichiometry changes within in vivo systems are not only brain region specific (Fu et al, 2019), but they also rely on unique cellular machinery that is absent in our in vitro model. Despite this, our transfection methods follow validated protocols that have matched several in vivo mouse and human studies (Srinivasan et al, 2011;Henderson et al, 2017), and we are able to detect a change in nAChR stoichiometry that is consistent with our findings using brain slice electrophysiology as both suggest the presence of more high-affinity nAChRs.…”

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

“…This application has resulted in new insights into synaptic contacts and communication among neurons and glial cells in the brain 71 . From brain cells to vesicles, fluorescent staining protocols support inspection and capture of cell functions via confocal microscopy for advancements in therapeutic techniques 72 , 73 .…”