Remote control of neuronal signaling

Abstract

A significant challenge for neuroscientists is to determine how both electrical and chemical signals affect the activity of cells and circuits and how the nervous system subsequently translates that activity into behavior. Remote, bidirectional manipulation of those signals with high spatiotemporal precision is an ideal approach to addressing that challenge. Neuroscientists have recently developed a diverse set of tools that permit such experimental manipulation with varying degrees of spatial, temporal, and directional control. These tools use light, peptides, and small molecules to primarily activate ion channels and G protein-coupled receptors (GPCRs) that in turn activate or inhibit neuronal firing. By monitoring the electrophysiological, biochemical, and behavioral effects of such activation/inhibition, researchers can better understand the links between brain activity and behavior. Here, we review the tools that are available for this type of experimentation. We describe the development of the tools and highlight exciting in vivo data. We focus primarily on designer GPCRs (receptors activated solely by synthetic ligands, designer receptors exclusively activated by designer drugs) and microbial opsins (e.g., channelrhodopsin-2, halorhodopsin, Volvox carteri channelrhodopsin) but also describe other novel techniques that use orthogonal receptors, caged ligands, allosteric modulators, and other approaches. These tools differ in the direction of their effect (activation/inhibition, hyperpolarization/depolarization), their onset and offset kinetics (milliseconds/minutes/hours), the degree of spatial resolution they afford, and their invasiveness. Although none of these tools is perfect, each has advantages and disadvantages, which we describe, and they are all still works in progress. We conclude with suggestions for improving upon the existing tools.

Fig. 1.

Genetic approaches to achieving spatially regulated transgene expression. In the tet-regulated expression system (A), a tissue specific-promoter drives expression of the tTA (tet-off, left) or rtTA (tet-on, right) transgene to produce the tTA or rtTA transcription factor, respectively, in a driver mouse line. That line is crossed to a responsive mouse line in which the TRE promoter drives expression of the exogenous transgene of interest. The tTA or rtTA transcription factor binds to the promoter to initiate transcription of the endogenous transgene. In the tet-off system, dox inhibits tTA binding to the TRE promoter. In the tet-on system, doxycycline is required for rtTA binding to the promoter. In the Cre-flox system (B), a tissue-specific promoter controls expression of Cre recombinase in a driver line. In the responsive line, loxP recognition sequences flank a stop sequence 5′ of the exogenous transgene of interest. Cre recombinase excises the stop site to allow the ubiquitous Rosa26 promoter to drive transgene expression. For virally mediated recombination (C), virus is infused into the Cre driver line. The virus carries a plasmid with a doubly floxed, inverted open reading frame. Cre mediates recombination at one of the two sets of recognition sites to cause inversion of the open reading frame and to allow the synapsin-1 promoter to drive transgene expression in neurons. The remaining lox sites are incompatible, thereby preventing re-inversion.

Fig. 2.

Activation of second-messenger cascades by designer G protein-coupled receptors. The RASSLs and DREADDs activate G_q-, G_s-, and G_i-coupled GPCRs. Activation of G_q activates PLC-β to stimulate PIP₂ hydrolysis into inositol-trisphosphate (IP₃) and diacylglycerol (DAG), then DAG activates PKC, and IP₃ activates the IP₃ receptor (IP₃R) to cause calcium release from the ER, which causes ERK1/2 activation. Activation of G_s activates adenylyl cyclase (AC) to promote cAMP formation and subsequent PKA activation, whereas activation of G_i inhibits AC activity, cAMP formation, and PKA activation. The Gβγ subunit of G_i-coupled GPCRs opens GIRK to allow hyperpolarizing potassium flux. Finally, G protein-independent signaling occurs through β-arrestin, which activates ERK1/2.

Fig. 3.

DREADDs are mutant muscarinic receptors. A, DREADDs are formed by point mutations in the third and fifth transmembrane regions of muscarinic receptors (stars; Y149C and A239G in hM₃). In addition, the G_s-coupled DREADD contains the second and third intracellular loops of the β₁-AR in place of those of the M₃ muscarinic receptor (gray loops). B, in human pulmonary artery smooth muscle cells, the hM3Dq receptor (hM₃D) is selectively activated by CNO but not by ACh, resulting in PIP₂ hydrolysis. Conversely, the wild-type M₃ muscarinic receptor (hM₃) is potently activated by ACh but not by CNO. [Adapted from Armbruster BN, Li X, Pausch MH, Herlitze S, and Roth BL (2007) Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc Natl Acad Sci USA 104:5163–5168. Copyright © 2007 National Academy of Sciences, USA.]

Fig. 4.

Activation of optical tools by distinct wavelengths of light. Spectrum depicts the distinct wavelengths and corresponding colors of light that maximally activate each of the various microbial opsins. Opsins labeled in black text depolarize neuronal membranes; those labeled in green hyperpolarize membranes; and those labeled in red couple to G protein.