Optogenetics has become an emerging technique for neuroscience investigations owing to the great spatiotemporal precision and the target selectivity it provides. Here we extend the optogenetic strategy to GABAA receptors (GABAARs), the major mediators of inhibitory neurotransmission in the brain. We generated a light-regulated GABAA receptor (LiGABAR) by conjugating a photoswitchable tethered ligand (PTL) onto a mutant receptor containing the cysteine-substituted α1-subunit. The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500 and 380 nm light, respectively, resulting in photoswitchable receptor antagonism. In hippocampal neurons, this LiGABAR enabled a robust photoregulation of inhibitory postsynaptic currents. Moreover, it allowed reversible photocontrol over neuron excitation in response to presynaptic stimulation. LiGABAR thus provides a powerful means for functional and mechanistic investigations of GABAAR-mediated neural inhibition.

A photo-activatable aziridinium precursor has been developed to investigate the possibility of a photo-initiated traditional nucleophilic reaction. The photolysis of a quaternary amine yields a tertiary amine and has allowed us to temporally control aziridinium formation and subsequent alkylation of a colorimetric nucleophilic reporter molecule. We have also used this photo-initiated reaction to alkylate a sulfhydryl group. This new photo-initiated alkylation strategy is water-soluble and expands the toolkit of photo-activated crosslinkers for protein labeling research.

Photochromic ligands, molecules that can be induced to change their physical properties through applied light, are currently the topic of much chemical biology research. This specialized class of small organic structures are, surprisingly to many, fairly common in nature. At the core of a number of natural biological processes lies a small molecule that changes shape or some other measurable property in response to light absorption. For instance, conformational changes invoked by reversible photoisomerization of a retinoid small molecule found in the photoreceptors of the human eye leads to vision. In plants, photoisomerization of a cinnamate moiety leads to altered gene expression. The photosensitive molecule can be viewed simply as a nanosensor of light, much like a photosensitive electrical component might be added to a circuit to sense day versus night to turn an electrical circuit on or off. Synthetic organic chemists and chemical biologists have been, for at least the last 15 years, trying to either mimic or exploit the native photochromism found in nature. Here, we describe the design process to develop a photochromic molecule to be used in neurobiology.

•Medicinal chemistry can be used to direct pharmacophore modifications.•Vectoring out of binding site allows for linked cargo (i.e. fluorophores).•Multiple pharmacophores are discussed along with both failed and successful designs.•Labeling of endogenously expressed receptors and transporters are discussed.Fluorescently labeled, small molecule ligands designed for the labeling and tracking of neuronal receptors have become an increasingly popular tool in neurobiology. The small size of these probes allows for subcellular imaging of proteins in their native state with minimal perturbation of the system. Several factors such as the selectivity of the pharmacophore, the size and composition of linkers used, and the fluorescence stability of the fluorophore can all influence the effectiveness of the small molecule probe. Here we discuss a few key molecular targets of this technology including the NMDA receptor, serotonin transporter, dopamine transporter, and adenosine receptor due to their involvement in numerous neurodegenerative diseases. Future iterations of these probes will allow for a better understanding of many important neurological proteins as well as the development of new and potent therapeutic drugs. This review will cover probe design considerations and discuss examples of specific small molecule fluorescent ligands that have been used to study a multitude of neuronal receptors through fluorescent imaging.This article is part of the Special Issue entitled ‘Fluorescent Tools in Neuropharmacology’.

Hyperexcitation in the central nervous system is the root cause of a number of disorders of the brain ranging from acute injury to chronic and progressive diseases. The major limitation to treatment of these ailments is the miniscule, yet formidable blood–brain barrier. To deliver therapeutic agents to the site of desired action, a number of biomedical engineering strategies have been developed including prodrug formulations that allow for either passive diffusion or active transport across this barrier. In the case of prodrugs, once in the brain compartment, the active therapeutic agent is released. In this review, we discuss in some detail a number of factors related to treatment of central nervous system hyperexcitation including molecular targets, disorders, prodrug strategies, and focused case studies of a number of therapeutics that are at a variety of stages of clinical development.Download high-res image (188KB)Download full-size image