Chemical Targeting of Voltage Sensitive Dyes to Specific Cell Types in the Brain

Voltage sensitive fluorescent dyes (VSDs) are important tools for probing signal transduction in neurons and other excitable cells. These sensors, rendered highly lipophilic to anchor the conjugated p-wire molecular framework in the membrane, offer several favorable functional parameters including fast response kinetics and high sensitivity to membrane potential changes. The impact of VSDs has however been limited due to the lack of cell-specific targeting methods in brain tissue or living animals. We address this key challenge by introducing a non-genetic molecular platform for cell and molecule specific targeting of synthetic voltage sensitive dyes in the brain. We employ a dextran polymer particle to overcome the inherent lipophilicity of voltage sensitive dyes by dynamic encapsulation and target the construct to specific axonal extensions using the monoamine transporter ligand dichloropane. VoLDeMo (<u>Vo</u>ltage Sensor-<u>L</u>igand-<u>De</u>xtran Targeted to <u>Mo</u>noaminergic Neurons) probes label dense dopaminergic axons in the mouse striatum and sparse noradrenergic axons in the mouse cortex in acute brain slices. We also demonstrate in whole adult <i>Drosophila</i> brains that VoLDeMo targeting is ligand dependent. VoLDeMo variants bearing either a classical electrochromic ANEP dye or state-of-the-art VoltageFluor dye respond to membrane potential changes in a similar manner to the parent dyes, as demonstrated by whole-cell patch recording. The VoLDeMo platform enables targeting of diffusible VSD probes to specific neuronal cells using endogenous expression levels of native components of neurotransmission machinery. We envision that modularity of our platform will enable its application to a variety of molecular targets (other receptors and covalent labeling-based tags) and sensors (including those in other imaging modalities), as well as lipophilic drugs and signaling modulators. This work demonstrates the feasibility of a chemical targeting approach and expands the possibilities of cell-specific imaging and pharmacology.