Development of Anionically Decorated Caged Neurotransmitters: In Vitro Comparison of 7‐Nitroindolinyl‐ and 2‐(p‐Phenyl‐o‐nitrophenyl)propyl‐Based Photochemical Probes

Abstract

Neurotransmitter uncaging, especially that of glutamate, has been used to study synaptic function for over 30 years. One limitation of caged glutamate probes is the blockade of GABA-A receptor function. This problem comes to the fore when the probes are applied at the high concentrations required for effective 2-photon photolysis. To mitigate such problems one could improve the photochemical properties of caging chromophores and/or remove receptor blockade. We show that addition of a dicarboxylate unit to the widely used MNI-Glu reduced the off-target effects by about 50-70%. When the same strategy was applied to an electron rich 2-(p-phenyl-onitrophenyl)-propyl (PNPP) cage, the pharmacological improvements were not as significant as for MNI. Finally, we used very extensive biological testing of the PNPP-caged Glu (more than 250 uncaging currents at single dendritic spines) to show that nitro-biphenyl caging chromophores have a 2-photon uncaging efficacy similar to that of MNI-Glu.Photochemically protected neurotransmitters offer physiologists a powerful means of controlling important aspects of chemical and electrical signaling in cells from the central nervous system [1][2][3] . Such optical probes, called caged compounds, initially used the orthonitrobenzyl [4] and ortho-nitroveratryl [5] chromophores developed, respectively, by Baltrop and Patchornik. Whilst such chromophores have proved the most widely used of all photochemical protecting groups in both chemistry and biology [6][7][8] , benzyl ester derivatives of biomolecules such as Glu and GABA have some predictable (e.g. hydrolysis at physiological pH), and some unpredictable weaknesses (e.g. GABA-A antagonism) in the context of synaptic physiology [3] .Several studies have sought to address these issues. For example, in 1999 Corrie and coworkers showed that a 7-nitroindolinyl-caged Glu [9] is very stable at physiological pH (i.e. 7.4). Subsequently several studies published by the Corrie/Ogden and Ellis-Davies/Kasai

Two-color, one-photon uncaging of glutamate and GABA

“…However, the concentration range that is used for 1P uncaging is substantially lower, suggesting that DEAC454-GABA could be effective [ 7 ]. With the recent development of the caged glutamate probe dcPNPP-Glu [ 20 ] with a λ max close to the λ min of DEAC454-GABA ( Fig 1b ), these compounds could form a complimentary optical pair for wavelength-selective 1P uncaging. Thus, orthogonal two-color, 1P uncaging of GABA and glutamate could be possible.…”

“…Near-UV light (365 nm) was used for photolysis of caged glutamate to evoke AMPA receptor currents in pyramidal neurons of the hippocampal CA1 region. A solution of dcPNPP-Glu (330 μM) was re-circulated [ 20 ]. Flashes of near-UV light (365 nm, 5 and 10 mW) evoked inward currents that were dependent on the duration and power of the flash ( Fig 2a ).…”

“…Analytical HPLC was performed on a Beckman System Gold device with diode array detector using a Grace Altima C18 column (250 mm x 4.6 mm). dcPNPP-Glu was synthesized as previously described [ 20 ].…”

Thermodynamically Stable, Photoreversible Pharmacology in Neurons with One‐ and Two‐Photon Excitation

“…Thus, 4FAB‐QA photochemistry essentially preserves all the important photophysical properties reported for the core chromophore . However, favorable chemical properties of optical probes are often not maintained when tested on living cells . Therefore, we evaluated the photopharmacological efficacy of 1 in neurons natively expressing voltage‐gated ion channels targeted by QA molecules.…”

Photoactivation Strategies for Therapeutic Release in Nanodelivery Systems

“…Ortho-Nitrobenzene Heterolysis: ONB linkers are most actively used and broadly defined to include ortho-nitrophenylethyl (NPE), [132][133][134] ortho-nitrodibenzofuran (NDBF), [124] and orthonitromandelic acid (NM). [135][136][137] Their photolysis occurs very effectively by UV absorption, [23,24,[138][139][140][141][142] or less effectively by visible light absorption via two-photon absorption (710 nm, [124] 750 nm [123,141] ).…”