Staff member

Glycine receptors (GlyRs) are indispensable for maintaining excitatory/inhibitory balance in neuronal circuits that control reflexes and rhythmic motor behaviors. Here we have developed Glyght, a GlyR ligand controlled with light. It is selective over other Cys-loop receptors, is active in vivo, and displays an allosteric mechanism of action. The photomanipulation of glycinergic neurotransmission opens new avenues to understanding inhibitory circuits in intact animals and to developing drug-based phototherapies.

JTD Keywords: Glycine receptors, Photopharmacology, Optopharmacology, Inhibitory neurotransmission, CNS, Photoswitch

Photoswitches are molecules that change their conformation with light of specific wavelength. These light-regulated molecules can be designed to target ion channels, thus providing a unique tool for precise spatial and temporal control of ion channel functioning. Recently, we have applied a multidisciplinary approach to design, synthesize and functionally characterize two of such photoswitches, azo-NZ1 [Maleeva et al. Br. J. Pharmacol. 2019] and Glyght [Gomila-Juaneda et al. BioRxiv 2019], targeting GABA and glycine receptors, respectively. Using homology modeling and molecular docking, we have provided a molecular explanation of the light-dependent effect of these two photoswitchable ligands, as observed in in vitro electrophysiology experiments and in vivo tadpole behavioral assays. Azo-NZ1 is composed of a nitrazepam moiety merged to an azobenzene photoisomerizable group, yet it has an inhibitory effect on GABA A receptors under visible light and also inhibits benzodiazepine-insensitive GABA C (rho2) receptors. Molecular modeling, combined with electrophysiology and mutagenesis experiments, shows that addition of the sulfonyl azobenzene unexpectedly converts the ligand into a pore blocker. Glyght is also an azobenzene-containing benzodiazepine, yet it acts selectively on glycine receptors as a negative modulator and its inhibitory action increases under UV light. Molecular modeling suggests that Glyght binds to a novel allosteric site located at the interface between the extracellular and transmembrane domains. The two aforementioned photoswitches pave the way towards photomanipulation of inhibitory (gabaergic and glycinergic) neurotransmission, with potential applications in understanding inhibitory circuits in intact animals and in development of drug-based phototherapies

JTD

Manipulation of neuronal activity using two-photon excitation of azobenzene photoswitches with near-infrared light has been recently demonstrated, but their practical use in neuronal tissue to photostimulate individual neurons with three-dimensional precision has been hampered by firstly, the low efficacy and reliability of NIR-induced azobenzene photoisomerization compared to one-photon excitation, and secondly, the short cis state lifetime of the two-photon responsive azo switches. Here we report the rational design based on theoretical calculations and the synthesis of azobenzene photoswitches endowed with both high two-photon absorption cross section and slow thermal back-isomerization. These compounds provide optimized and sustained two-photon neuronal stimulation both in light-scattering brain tissue and in Caenorhabditis elegans nematodes, displaying photoresponse intensities that are comparable to those achieved under one-photon excitation. This finding opens the way to use both genetically targeted and pharmacologically selective azobenzene photoswitches to dissect intact neuronal circuits in three dimensions.

JTD

The physiological activity of proteins is often studied with loss-of-function genetic approaches, but the corresponding phenotypes develop slowly and can be confounding. Photopharmacology allows direct, fast, and reversible control of endogenous protein activity, with spatiotemporal resolution set by the illumination method. Here, we combine a photoswitchable allosteric modulator (alloswitch) and 2-photon excitation using pulsed near-infrared lasers to reversibly silence metabotropic glutamate 5 (mGlu5) receptor activity in intact brain tissue. Endogenous receptors can be photoactivated in neurons and astrocytes with pharmacological selectivity and with an axial resolution between 5 and 10 µm. Thus, 2-photon pharmacology using alloswitch allows investigating mGlu5-dependent processes in wild-type animals, including synaptic formation and plasticity, and signaling pathways from intracellular organelles.

JTD Keywords: Photopharmacology, Photoactivation, Pharmacological selectivity, Functional silencing, 2-photon pharmacology

Synapses learn and remember by persistent modifications of their internal structures and composition but, due to their small size, it is difficult to observe these changes at the ultrastructural level in real time. Two-photon fluorescence microscopy (2PM) allows time-course live imaging of individual synapses but lacks ultrastructural resolution. Electron microscopy (EM) allows the ultrastructural imaging of subcellular components but cannot detect fluorescence and lacks temporal resolution. Here, we describe a combination of procedures designed to achieve the correlated imaging of the same individual synapse under both 2PM and EM. This technique permits the selective stimulation and live imaging of a single dendritic spine and the subsequent localization of the same spine in EM ultrathin serial sections. Landmarks created through a photomarking method based on the 2-photon-induced precipitation of an electrodense compound are used to unequivocally localize the stimulated synapse. This technique was developed to image, for the first time, the ultrastructure of the postsynaptic density in which long-term potentiation was selectively induced just seconds or minutes before, but it can be applied for the study of any biological process that requires the precise relocalization of micron-wide structures for their correlated imaging with 2PM and EM.

JTD Keywords: Correlated imaging, DAB, Dendritic spine, Photobranding, Photoetching, Photomarking, Postsynaptic density, Serial-section transmission electron microscopy, Synapse, Time-lapse live two-photon fluorescence microscopy