Optogenetic pharmacology for control of native neuronal signaling proteins

Abstract

The optical neuroscience revolution is transforming how we study neural circuits. By providing a precise way to manipulate endogenous neuronal signaling proteins, it also has the potential to transform our understanding of molecular neuroscience. Recent advances in chemical biology have produced light-sensitive compounds that photoregulate a wide variety of proteins underlying signaling between and within neurons. Chemical tools for optopharmacology include caged agonists and antagonists and reversibly photoswitchable ligands. These reagents act on voltage-gated ion channels and neurotransmitter receptors, enabling control of neuronal signaling with a high degree of spatial and temporal precision. By covalently attaching photoswitch molecules to genetically tagged proteins, the newly emerging methodology of optogenetic pharmacology allows biochemically precise control in targeted subsets of neurons. Now that the tools for manipulating endogenous neuronal signaling proteins are available, they can be implemented in vivo to enhance our understanding of the molecular bases of brain function and dysfunctions.

Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics

“…Molecular 2P absorption cross-sections (σ 2 ) in GoeppertMayer units ( (Table 1) are remarkably consistent with those of the parent compounds and related compounds from both classes of azobenzenes (36,38). Notably, the σ 2 of both MAGs are orders of magnitude larger than that of the most widely used caged neurotransmitter, MNI-glutamate (σ 2 Quantum Yield of MAG Photoisomerization. Brightness depends not only on the strength of absorption, but also on the quantum efficiency of the process of interest.…”

“…optogenetics | pharmacology | multiphoton | photoswitch | azobenzene M odern neurobiology relies heavily on optical microscopy to observe, and, increasingly, to manipulate (1,2), biological processes in live tissue. Among these methods, 2-photon-excited fluorescence microscopy (2PM) with near-infrared (NIR) light has emerged as an important technique for extending optical microscopy to highly scattering tissue (3,4).…”

“…In particular, the inherent spatial confinement of 2P absorption is critical for optical manipulation of individual cells (10) in the intact mammalian brain, where it is difficult to control the spatial extent of gene expression or confine soluble reagents. However, 2P-excited optogenetics is not yet as widespread in adoption as 2PM, being widely perceived to require sophisticated optical techniques (11)(12)(13) or reagent concentrations that compromise pharmacological specificity (2,14).…”

“…PTLs comprise a modular class of genetically-targeted photochemical tools first conceived in the 1970s by Eranger and Lester for acetylcholine receptors (19,20). PTLs have since been developed for a variety of other receptors and channels (2). Maleimideazobenzene-glutamate (MAG) compounds are designed to be conjugated to the extracellular ligand binding domains of genetically engineered mammalian glutamate receptors (GluRs), enabling pharmacological manipulation of signaling pathways dependent on the excitatory neurotransmitter glutamate (21)(22)(23)(24)(25).…”

A solution to dependency: using multilevel analysis to accommodate nested data

“…A common feature of all these techniques is that they shift the n from the animal or tissue level to the cellular or even subcellular level, and invariably yield data with a nested structure. For instance, super resolution light microscopy allows imaging and advanced understanding of neuronal compartments 18 , immunogold cytochemistry allows determination of subcellular localization of pro teins 19 , and recent advances in optogenetics and optopharmacology facilitate selective control of electrical and protein activity, respec tively, in circuits, individual cells or subcellular compartments 20,21 . All these techniques concern the collection of multiple observations from one cell, thereby yielding nested data.…”