Wavelength-Selective One- and Two-Photon Uncaging of GABA

Abstract

We have synthesized photolabile 7-diethylamino coumarin (DEAC) derivatives of γ-aminobutyric acid (GABA). These caged neurotransmitters efficiently release GABA using linear or nonlinear excitation. We used a new DEAC-based caging chromophore that has a vinyl acrylate substituent at the 3-position that shifts the absorption maximum of DEAC to about 450 nm and thus is named "DEAC450". DEAC450-caged GABA is photolyzed with a quantum yield of 0.39 and is highly soluble and stable in physiological buffer. We found that DEAC450-caged GABA is relatively inactive toward two-photon excitation at 720 nm, so when paired with a nitroaromatic caged glutamate that is efficiently excited at such wavelengths, we could photorelease glutamate and GABA around single spine heads on neurons in brain slices with excellent wavelength selectivity using two- and one-photon photolysis, respectively. Furthermore, we found that DEAC450-caged GABA could be effectively released using two-photon excitation at 900 nm with spatial resolution of about 3 μm. Taken together, our experiments show that the DEAC450 caging chromophore holds great promise for the development of new caged compounds that will enable wavelength-selective, two-color interrogation of neuronal signaling with excellent subcellular resolution.

Cloaked Caged Compounds: Chemical Probes for Two‐Photon Optoneurobiology

“…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).…”

“…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 GABA surrogate), towards GABA-A receptors ( Figure 1, for syntheses see Supplementary Information). Each compound ( Figure 1a) was bath applied to a brain slices isolated from adult male and female mice of 3-7 months of age.Pyramidal cells in layer 2/3 of the prefrontal cortex were patch-clamped and a stimulation pipette was used to evoke GABAergic responses, as previously described [18] (see Figure S1 for similar recordings of high affinity antagonists). We found that we could detect no interference from the G4 dendrimer at concentrations up to 1 mM (Figure 1b).…”

“…Since our antagonism data suggests the cloaked caged GABA is essentially inert up to about 0.6 mM ( Figure 2) we bath applied the probe to brain slices at this concentration. We did this because we knew CDNI-Glu is best used at about 1 mM [21,[23][24][25][26] , and has an IC 50 = 0.24 mM for GABA-A receptor blockade [18] . Thus, we patch-clamped a CA1 neuron and monitored voltage changes produced by two-color, 2P uncaging.…”

“…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.…”