Two-photon uncaging of γ-aminobutyric acid in intact brain tissue

Abstract

We have synthesized a photosensitive (or caged) 4-carboxymethoxy-5,7-dinitroindolinyl (CDNI) derivative of γ-aminobutyric acid (GABA). Two-photon excitation of CDNI-GABA produced rapid activation of GABAergic currents in neurons in brain slices with an axial resolution of approximately 2 micrometers, and enabled high-resolution functional mapping of GABA-A receptors. Two-photon uncaging of GABA, the major inhibitory neurotransmitter, should allow detailed studies of receptor function and synaptic integration with subcellular precision.

Cloaked Caged Compounds: Chemical Probes for Two‐Photon Optoneurobiology

“…The slopes of the two data groups (25 vs 0.8) provides an estimate of the relative effectiveness of CDNI versus G5-DEAC450 for GABA uncaging. Even though the QY was 0.23, we found much more consistent results with G5-DEAC450 than CDNI [15] , and we believe this is because of the reduced antagonism of the former towards GABA-A receptors in the concentration ranges used.Next, we searched for the minimum concentration of CDNI-GABA or G5-DEAC450-GABA that could be used for reliable 2P-evoked GABAergic currents ( Figure 4). As we reported previously, CDNI-GABA at 400 μM could be used for 2P uncaging at 720 nm [15] ( Figure 4a), but much lower concentrations (< 100 μM) were only useful for 1P uncaging ( Figure 4b).…”

“…Even though the QY was 0.23, we found much more consistent results with G5-DEAC450 than CDNI [15] , and we believe this is because of the reduced antagonism of the former towards GABA-A receptors in the concentration ranges used.…”

“…This off-target effect was substantiated by other reports and applies to other caged glutamate probes such CDNI-Glu [16] and RuBi-Glu [16,17] . Much less surprising was that caged GABA probes such as CDNI-GABA [15] , RuBi-GABA [18] and PEG-DEAC450-GABA [18] were also found to be quite antagonistic towards GABA-A receptors.A clue to the potential solution to this "on-target" problem for caged GABA was provided by testing BOC-protected PEG-DEAC450-GABA (i.e. the precursor to the caged compound [18] ) when it was found to have substantially reduced GABA-A antagonism (Mike Higley, personal communication).…”

Multi-photon excitation in uncaging the small molecule bioregulator nitric oxide

“…(Online version in colour.) [48]. The species shown in figure 4 use π -unsaturated aromatic compounds as the TPE chromophores, but it should be noted that metal complexes such as ruthenium amines have similarly been used as chromophores for TPE uncaging of neurotransmitters [46,51].…”

“…The three-dimensional resolution opportunities provided by TPE have extensively been exploited in neuroscience both in tissue samples and in vivo [43][44][45][46][47][48][49][50]. The NIR TPE induced uncaging of neurotransmitters such as glutamate (figure 4) and has allowed the functional mapping of the respective receptors in neurons with high-resolution (μm), sub-cellular precision Figure 4.…”