Si‐LENT [Brain] Slice Recordings: a Novel Method for the Ex Vivo Genetic Modification of Neurons
Authors: Rodriguez Larry , You Chang , Brodie Mark , Davies Daryl 
Published: June 2021

Understanding the function of a specific receptor in a cell is key for developing new therapies for diseases. Current early‐stage drug development strategies often utilize non‐specific or “dirty” drugs/molecules to determine receptor function, as more specific agonists or antagonists may not be available. A second strategy incorporates genetic modifications to determine the role of a receptor in an animal model of disease. This method is time‐consuming, expensive, and can produce artifacts that confound or skew results. Furthermore, certain modifications may not produce viable offspring. These issues make developing drugs for neurological conditions more challenging, as the function of a receptor can depend on the brain region where a neuron is located or can require an interaction partner (receptor cross‐talk). One way to study neurons is to perform extracellular recordings on mouse or rat brain slices. In these experiments, an electrode is placed near, but not onto or into, a neuron. This electrode, usually filled with a saline solution, measures the number of action potentials over time, a.k.a. the firing rate. Agonists and/or drugs are then perfused on to the neuron, and the effects are reported as a change in the firing rates. Here, we describe a new method: “SiRNA‐Loaded Electrodes kNocksdown Target (Si‐LENT)” slice electrophysiology. Si‐LENT slice is a new method for performing extracellular recordings in brain slice that can knockdown target genes over the course of a recording session. Si‐LENT slice incorporates SiRNA into the recording electrode, which is delivered to the cell during the experiment. For the current study, we targeted P2X4 receptors, which we have previously linked to regulation of alcohol consumption. We recorded from dopaminergic (DAergic) neurons located in the ventral tegmental area (VTA) of the mouse brain. DAergic neurons in the VTA of the brain have been shown to be associated with learning, reward, and addiction, but the function of P2X4 receptors in this region is not well understood. We found a significant reduction in receptor activity after two hours of recording, and a sustained reduction for up to two hours thereafter. Five hours after the start of the experiment, P2X4 receptor response returned to baseline, suggesting adaptation to the inclusion of the siRNA over a longer time period (or recovery over time as the electrode and the cell may drift apart.) We also report an interaction between P2X4 receptors and ethanol, which was ablated in Si‐LENT slice recordings. These data demonstrate that this method that can be used in the cascade of experiments linked to target validation in drug discovery, which is also faster, cheaper, and more efficient than generating animal knockouts, and subverts the issue of developmental compensatory changes.

Support or Funding Information

NIH R01AA022448; AFPE Pre‐doctoral fellowship; USC School of Pharmacy

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.