Cloaked Caged Compounds: Chemical Probes for Two‐Photon Optoneurobiology

Abstract

Caged neurotransmitters, in combination with focused light beams, enable precise interrogation of neuronal function, even at the level of single synapses. However, most caged transmitters are, surprisingly, severe antagonists of ionotropic gamma-aminobutyric acid (GABA) receptors. By conjugation of a large, neutral dendrimer to a caged GABA probe we introduce a "cloaking" technology that effectively reduces such antagonism to very low levels. Such cloaked caged compounds will enable the study of the signaling of the inhibitory neurotransmitter GABA in its natural state using two-photon uncaging microscopy for the first time. Graphical abstractCaged neurotransmitters are known to be quite antagonistic toward the GABA-A receptor. The conjugation of a polyester dendrimer "cloak" to the caged compound DEAC450-GABA significantly decreased its GABA-A receptor antagonism. The chemical probe was inert at concentrations required for effective two-photon photolysis on living cells. Keywords caged compounds; GABA-A receptors; optical methods; two-photon; biologically inert Correspondence to: Graham C. R. Ellis-Davies. b These authors contributed equally to this work.Supporting information for this article is given via a link at the end of the document. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptPhotochemical probes have revolutionized modern physiological studies. In an era initiated by Galvani, electrodes were the best technology available for real-time stimulation and measurement. However, the photon is more precise and versatile than the electron, so the former is not merely starting to "replace" the latter, but light, in combination with chemistry, is being used to open completely new avenues of physiological discovery [1] . To deliver their potential, the new chemical technologies require novel light sources and so pulsed lasers have been vital for time-resolved chemical biology [2] . Enabled by ultra-fast, pseudocontinuous Ti:sapphire lasers, neurobiology now routinely uses two-photon (2P) microscopy for live cell imaging in complex tissue such as acutely isolated brain slices and living animals. But, as noted by Denk, et al. in their seminal 1990 paper [3] , 2P excitation "also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules." While initial reports of two-photon uncaging used probes [4,5] originally designed for linear actuation, more recently caged compounds designed for 2P excitation have been introduced for Glu, GABA, IP 3 , calcium, etc [6] . Some of these have even proved useful for independent 2P physiological studies [2] . It is striking that caged Glu probes, in particular, have proved very useful but caged GABA probes much less so. One reason for this must the inherent antagonism of all caged GABA compounds towards GABA-A receptors at concentrations required for effective 2P photolysis in brain tissue [7] . We have developed a new technology, which ...

Dendrimer Conjugation Enables Multiphoton Chemical Neurophysiology Studies with an Extended π‐Electron Caging Chromophore

“…It is well documented that most caged neurotransmitters block GABA-A receptors at concentrations required for multiphoton chemical physiology. [17] However,alack of antagonism was strikingly evident during individual electrophysiological sweeps in which uncaging-evoked and spontaneous currents were interspersed and indistinguishable when 1 was locally applied from ap atch pipette at 0.15 mm ( Figure 3a,b). Note that such currents could be blocked by the specific GABA-A receptor antagonist gabazine ( Figure S1 in the Supporting Information).…”

“…As econd, less obvious challenge,but perhaps one that is even more important for chemical physiology,i st hat photoactivatable probes that use the "Marder-Reinhardt" principles inevitably become insoluble in aqueous buffer,especially at concentrations required for neurophysiological studies. [15] In this report, we show how extended p-electron chromophoric systems such as dinitrobisstyrylthiophene (BIST [16] ) can be coupled to the major inhibitory neurotransmitter gaminobutyric acid (GABA) and rendered water-soluble at sufficiently high concentrations by attachment of ad endrimer [17] such that the neurotransmitter can be very effectively photoreleased using multiphoton excitation.…”

Light‐induced electrons suppressed by Eu3+ ions doped in Ca11.94−xSrxAl14O33 caged phosphors for LED and FEDs

“…34 However, the intensities of This result suggests that Sr doping can promote the reduction of Eu 3+ to Eu 2+ and suppress generation of lightinduced electrons. In Eu 3+ -doped Ca 12 Al 14 O 33 , the reduction processes of Eu 3+ to Eu 2+ in H 2 atmosphere might be described in the following equations (3)(4)(5)(6).…”

“…Inorganic and organic caged compounds have attracted more attentions and exhibited many potential applications due to their unique physical and chemical properties. [1][2][3] For example, mayenite (Ca 12 Al 14 O 33 ) is one kind of such multifunctional materials exhibiting its variety of possible applications, such as catalytic 4 , electronic devices, 5 sensors, 6 solid electrolytes 7 , and electrides. 8,9 These functions are related to its special nanocage structure, as shown in Figure 1A.…”

Intracellular Uncaging of cGMP with Blue Light

“…Whilst many laboratories are equipped with such light sources, the recent wide availability of blue lasers (or LEDs) for in vivo experiments 14 suggests that caged compounds that are highly active to such light would be advantageous to many biologically focused laboratories. Thus, we have recently developed a new caging chromophore that is easily photolyzed by blue light 15–19 (430–480 nm). Called “DEAC450”, this chromophore offers excellent photochemical efficiency to excitation below 500 nm.…”