Unparalleled control of neural activity using orthogonal pharmacogenetics

Abstract

Studying the functional architecture of the brain requires technologies to precisely measure and perturb the activity of specific neural cells and circuits in live animals. Substantial progress has been made in recent years to develop and apply such tools. In particular, technologies that provide precise control of activity in genetically defined populations of neurons have enabled the study of causal relationships between and among neural circuit elements and behavioral outputs. Here, we review an important subset of such technologies, in which neurons are genetically engineered to respond to specific chemical ligands that have no interfering pharmacological effect in the central nervous system. A rapidly expanding set of these "orthogonal pharmacogenetic" tools provides a unique combination of genetic specificity, functional diversity, spatiotemporal precision, and potential for multiplexing. We review the main classes of orthogonal pharmacogenetic technologies, including neuroreceptors to control neuronal excitability, systems to control gene transcription and translation, and general constructs to control protein-protein interactions, enzymatic function, and protein stability. We describe the key performance characteristics informing the use of these technologies in the brain, and potential directions for improvement and expansion of the orthogonal pharmacogenetics toolkit to enable more sophisticated systems neuroscience.

Figure 1

Illustrated example of multiplexed orthogonal pharmacogenetics. (A) Two cell types (blue and orange) involved in a particular neural circuit (top) are genetically modified to express orthogonal actuators responding to several distinct ligands that can be administered orally to the model organism (bottom). (B) One neuron (orange) expresses four distinct OP constructs, enabling temporally specific, multiplexed control of excitation (ion channel controlled by ligand A), inhibition (ion channel controlled by ligand B), gene transcription (transcriptional transactivator controlled by ligand C), and decreased presynaptic transmitter release (vesicle protein multimerization controlled by ligand D). A second neuron (blue) has an orthogonal GPCR coupled to an endogenous potassium channel, enabling orthogonal inhibition under control of ligand E. (C) Using the five ligands corresponding to different orthogonal actuators, it is possible to test 32 binary (ligand on or off) experimental conditions in this system.

Figure 2

Mechanisms of orthogonal neuroreceptors. GPCRs form the basis for both excitatory and inhibitory OP systems (A, D) based on interactions with different endogenous G proteins. GPCR signaling cascades leading to excitation and inhibition are described in the text. Cys-loop LGICs (B, E) are also used to effect inhibition and excitation based on pore domain ion selectivity. TRPV1 (C) excites cells through a nonselective cation conductance.