Recombinase-Dependent Mouse Lines for Chemogenetic Activation of Genetically Defined CellTypes.

Natale R Sciolino,Sabrina D Robertson,Yu-Wei Chen,Serena M Dudek,Zoe A Mcelligott,Patricia Jensen,Nicholas W Plummer,Georgia M Alexander

doi:10.1016/j.celrep.2016.05.034

Abstract

Chemogenetic technologies, including the mutatedhuman Gq-coupled M3 muscarinic receptor (hM3Dq), have greatly facilitated our ability to directly link changes in cellular activity to altered physiology and behavior. Here, we extend the hM3Dq toolkit with recombinase-responsive mouse lines that permit hM3Dq expression in virtually any cell type. These alleles encode a fusion protein designed to increase effective expression levels by concentrating hM3Dq to the cell body and dendrites. To illustrate their broad utility, we targeted three different genetically defined cell populations: noradrenergic neurons of the compact, bilateral locus coeruleus and two dispersed populations, Camk2a+ neurons and GFAP+ glia. In all three populations, we observed reproducible expression and confirmed that activation of hM3Dq is sufficient to dose-dependently evoke phenotypic changes, without extreme phenotypes associated with hM3Dq overexpression. These alleles offer the ability to non-invasively control activity of diverse cell types to uncover their function and dysfunction at any developmental stage.

Highlights

Since their initial description less than 10 years ago (Armbruster et al, 2007), the engineered G protein-coupled receptors known as designer receptors exclusively activated by designer drugs (DREADDs) have proven invaluable for linking cellular activity to changes in physiology and behavior
We demonstrate the broad utility of the alleles in three different cell populations: noradrenergic neurons of the small, bilateral locus coeruleus (LC) and two dispersed cell populations, Camk2a+ neurons and GFAP+ glia
In a proof-ofprinciple experiment using our Flp/Cre-responsive allele, we demonstrate that clozapine N-oxide (CNO) increases neuronal firing of hM3Dq-expressing LC neurons in vitro

Summary

Introduction

Since their initial description less than 10 years ago (Armbruster et al, 2007), the engineered G protein-coupled receptors known as designer receptors exclusively activated by designer drugs (DREADDs) have proven invaluable for linking cellular activity to changes in physiology and behavior. Among the most frequently used DREADDs is hM3Dq, a mutated Gq-coupled human M3 muscarinic receptor that is activated exclusively by the designer drug clozapine N-oxide (CNO), but not its native ligand acetylcholine. By coupling to endogenous signal transduction pathways, hM3Dq has provided insight into the unique functions of cells as diverse as neurons, glia, pancreatic b cells, and hepatocytes (as reviewed in Urban and Roth, 2015). Activation of hM3Dq by CNO leads to calcium influx and increased cell firing, thereby promoting neuronal excitability (Alexander et al, 2009). HM3Dq has been of particular value for identifying the functions encoded by specific neurons in the freely behaving animal

Methods

Results

Discussion

Conclusion