Photochemical control of endogenous ion channels and cellular excitability

Fortin, Doris L.; Banghart, Matthew R.; Dunn, Timothy W; Borges, Katharine; Wagenaar, Daniel A.; Gaudry, Quentin; Karakossian, Movses H.; Otis, Thomas S.; Kristan, William B.; Trauner, Dirk; Krämer, Richard

doi:10.1038/nmeth.1187

Cited by 224 publications

(235 citation statements)

References 34 publications

(50 reference statements)

Supporting

Mentioning

224

Contrasting

Unclassified

Order By: Relevance

“…The generation of smaller photocurrents by 2P-DH was consistent with the smaller fraction of the cell that was illuminated (compare blue region to red region in Fig. 4 A and B, respectively) considering that studies showed that LiGluR overexpressed in neurons is distributed throughout the plasma membrane (23,46). Additional analysis of photoswitching efficiency is presented in SI Appendix (SI Appendix, Fig.…”

Section: Resultssupporting

confidence: 75%

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

Carroll

Berlin

Levitz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

116

View full text Add to dashboard Cite

Mammalian neurotransmitter-gated receptors can be conjugated to photoswitchable tethered ligands (PTLs) to enable photoactivation, or photoantagonism, while preserving normal function at neuronal synapses. "MAG" PTLs for ionotropic and metabotropic glutamate receptors (GluRs) are based on an azobenzene photoswitch that is optimally switched into the liganding state by blue or near-UV light, wavelengths that penetrate poorly into the brain. To facilitate deep-tissue photoactivation with near-infrared light, we measured the efficacy of two-photon (2P) excitation for two MAG molecules using nonlinear spectroscopy. Based on quantitative characterization, we find a recently designed second generation PTL, , to have a favorable 2P absorbance peak at 850 nm, enabling efficient 2P activation of the GluK2 kainate receptor, LiGluR. We also achieve 2P photoactivation of a metabotropic receptor, LimGluR3, with a new mGluR-specific PTL, D-MAG0 460 . 2P photoswitching is efficiently achieved using digital holography to shape illumination over single somata of cultured neurons. Simultaneous Ca 2+ -imaging reports on 2P photoswitching in multiple cells with high temporal resolution. The combination of electrophysiology or Ca 2+ imaging with 2P activation by optical wavefront shaping should make second generation PTL-controlled receptors suitable for studies of intact neural circuits.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). Remarkably, barely 25 years after the first 2P-excited fluorescence image was published (5), 2PM is now performed in awake, behaving animals (4, 6-8). Naturally, optical manipulations in the brain can benefit from the many advantages of 2PM (9). 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 (11-13) or reagent concentrations that compromise pharmacological specificity (2, 14).The rapid time to adoption of 2PM owes at least some credit to the availability of spectroscopic data on the 2P-excited efficacy, or brightness, of synthetic and genetically encoded fluorophores (15-17). Brightness, defined for fluorophores as the product of absorption cross-section and fluorescence quantum yield, gives the experimenter an objective metric to assess fluorescent reporters and identify appropriate optical parameters, such as the optimal excitation wavelength and range of light intensities (18). By ...

show abstract

Section: Resultssupporting

confidence: 75%

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

Carroll

Berlin

Levitz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

116

View full text Add to dashboard Cite

show abstract

“…4,5 Due to their promising applications in both the material and life sciences, the interest in such materials has notably increased over the past few decades. [6][7][8] A more fundamental interest is triggered by the fact that photochromic reactions can be studied with extremely high time-resolution down to the femtosecond regime. This fact allows real-time observation of fundamental chemical processes, e.g., bond rotation, bond-formation or cleavage, and also allows a detailed comparison with quantum-chemical calculations.…”

Section: Introductionmentioning

confidence: 99%

The photochemical ring opening reaction of chromene as seen by transient absorption and fluorescence spectroscopy

Herzog

Ryseck

Ploetz

et al. 2013

Photochem Photobiol Sci

View full text Add to dashboard Cite

In this paper we investigate the photochromic ring-opening reaction of 2,2-diphenyl-5,6-benzo(2H)-chromene. In particular, we study the uncertainties and contradictions in various published reaction models using a combination of transient absorption and fluorescence spectroscopy with femtosecond time resolution. We propose a simplified reaction scheme which is in good agreement with theoretical studies. Here, photoexcitation populates a Franck-Condon state, whose fast vibrational wave packet motion, vibrational relaxation, bond-alternation and/or solvent rearrangement processes occur on the sub-picosecond timescale. Our data suggest that the resulting excited state minimum with picosecond lifetime still features structural characteristics of the closed form. Subsequently, the ring-opened photoproducts are formed in a concerted step from the excited state. The velocity of the photoreaction hence only depends on the time that the molecule needs to reach the transition region between the ground and excited states where the crucial bond breakage occurs.

show abstract

“…The light-gated chloride pump halorhodopsin (NpHR) from the archaebacterium Natronomonas pharaonis has recently been introduced into neuroscience along with enhanced derivatives (9)(10)(11)(12)(13)(14) and enables superior temporal and spatial control. Other light-controlled silencing methods are being developed (15)(16)(17), but require covalent attachment of a photo-switchable affinity label. NpHR silencing has been demonstrated electrophysiologically (10,12) and has been used to reversibly paralyze Caenorhabditis elegans expressing NpHR in motor peripheries (12).…”

mentioning

confidence: 99%

Optical control of zebrafish behavior with halorhodopsin

Arrenberg

Bene

Baier

2009

Proc. Natl. Acad. Sci. U.S.A.

231

218

View full text Add to dashboard Cite

Expression of halorhodopsin (NpHR), a light-driven microbial chloride pump, enables optical control of membrane potential and reversible silencing of targeted neurons. We generated transgenic zebrafish expressing enhanced NpHR under control of the Gal4/ UAS system. Electrophysiological recordings showed that eNpHR stimulation effectively suppressed spiking of single neurons in vivo. Applying light through thin optic fibers positioned above the head of a semi-restrained zebrafish larva enabled us to target groups of neurons and to simultaneously test the effect of their silencing on behavior. The photostimulated volume of the zebrafish brain could be marked by subsequent photoconversion of co-expressed Kaede or Dendra. These techniques were used to localize swim command circuitry to a small hindbrain region, just rostral to the commissura infima Halleri. The kinetics of the hindbrain-generated swim command was investigated by combined and separate photo-activation of NpHR and Channelrhodopsin-2 (ChR2), a light-gated cation channel, in the same neurons. Together this ''optogenetic toolkit'' allows loss-of-function and gain-of-function analyses of neural circuitry at high spatial and temporal resolution in a behaving vertebrate.echnology for the inactivation of specific neurons within an otherwise intact circuit promises to accelerate research into the neural basis of behavior (1-8). The light-gated chloride pump halorhodopsin (NpHR) from the archaebacterium Natronomonas pharaonis has recently been introduced into neuroscience along with enhanced derivatives (9-14) and enables superior temporal and spatial control. Other light-controlled silencing methods are being developed (15-17), but require covalent attachment of a photo-switchable affinity label. NpHR silencing has been demonstrated electrophysiologically (10, 12) and has been used to reversibly paralyze Caenorhabditis elegans expressing NpHR in motor peripheries (12). Despite its promise, however, NpHR has so far found only limited applications for circuit analysis in vivo. In this study, we have devised a versatile and cost-effective optical stimulation strategy for manipulation of animal behavior with this tool. These advances were made possible by our choice of zebrafish as the experimental system.Zebrafish are ideal models for testing and applying lightcontrolled channels and pumps in vertebrates, since they are translucent and display a number of quantifiable behaviors during the first 2 weeks of larval development (18-21). Accordingly, Wyart et al. (22) used a re-engineered, light-gated glutamate receptor (LiGluR), to induce swimming by photostimulation of a rare type of spinal neuron. Douglass et al. (23) succeeded in triggering escape responses by activating ChR2 in single zebrafish mechanosensory cells. The adaptation of the Gal4/UAS method from Drosophila melanogaster (24) to zebrafish enables targeting transgene expression to specific brain areas and cell types (25-29) and will further contribute to the refinement of an optogenetic toolkit in thi...

show abstract

Photochemical control of endogenous ion channels and cellular excitability

Cited by 224 publications

References 34 publications

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

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

The photochemical ring opening reaction of chromene as seen by transient absorption and fluorescence spectroscopy

Optical control of zebrafish behavior with halorhodopsin

Contact Info

Product

Resources

About