New caged neurotransmitter analogs selective for glutamate receptor sub-types based on methoxynitroindoline and nitrophenylethoxycarbonyl caging groups
Résumé
Photolysis is widely used in experimental neuroscience to isolate post-synaptic receptor activation from
presynaptic processes, to determine receptor mechanisms in situ, for pharmacological dissection of
signaling pathways, or for photostimulation/inhibition in neural networks.We have evaluated new caged
neuroactive amino acids that use 4-methoxy-7-nitroindolinyl- (MNI) or 1-(2-nitrophenyl)ethoxycarbonyl
(NPEC) photoprotecting groups to make caged ligands specific for glutamate receptor subtypes.
Each was tested for interference with synaptic transmission and excitability and for receptorspecific
actions in slice preparations. No adverse effects were found at glutamate receptors. At high
concentration, MNI-caged, but not NPEC-caged ligands, interfered with GABA-ergic transmission.
MNI-caged amino acids have sub-microsecond release times suitable for investigating mechanisms at fast
synaptic receptors in situ. MNI-NMDA and MNI-kainate were synthesized and tested. MNI-NMDA showed
stoichiometric release of chirally pure NMDA. Wide-field photolysis in cerebellar interneurons produced
a fast-rising sustained activation of NMDA receptors, and localized laser photolysis gave a fast, transient
response. Photolysis of MNI-kainate to release up to 4 mMkainate generated large inward currents at resting
membrane potential in Purkinje neurons. Application of GYKI 53655 indicated that 40% of the current was
due to AMPA receptor activation by kainate. Signaling via metabotropic glutamate receptors (mGluR) does
not require fast release rates. NPEC cages are simpler to prepare but have slower photorelease. Photolysis of
NPEC-ACPDorNPEC-DHPGinPurkinje neurons generated slowinwardcurrents blockedbythemGluRtype1
antagonist CPCCOEt similar to the slowsEPSC seenwith parallel fiber burst stimulation.NPEC-AMPAwas also
tested in Purkinje neurons and showed large sustained inward currents selective for AMPA receptors with
little activation of kainate receptors. MNI-caged L-glutamate, NMDA and kainate inhibit GABA-A receptors
with IC50 concentrations close to the maximum concentrations useful in receptor signaling experiments.
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