MNI-caged-L-glutamate

New caged neurotransmitter analogs selective for glutamate receptor sub-types based on methoxynitroindoline and nitrophenylethoxycarbonyl caging groups.[Pubmed:22609535]

Neuropharmacology. 2012 Sep;63(4):624-34.

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 sub-types. Each was tested for interference with synaptic transmission and excitability and for receptor-specific 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 muM kainate 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-ACPD or NPEC-DHPG in Purkinje neurons generated slow inward currents blocked by the mGluR type 1 antagonist CPCCOEt similar to the slow sEPSC seen with parallel fiber burst stimulation. NPEC-AMPA was 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 IC(5)(0) concentrations close to the maximum concentrations useful in receptor signaling experiments.

Comparative analysis of inhibitory effects of caged ligands for the NMDA receptor.[Pubmed:15652611]

J Neurosci Methods. 2005 Mar 15;142(1):1-9.

Photolytic release of neurotransmitters from caged precursors is a useful method to study synaptic processes with high temporal and spatial resolution. At present, the two most widely used classes of caged precursors for studies on glutamate receptors are based on derivatives of the 2-nitrobenzyl caging group (alpha-carboxy-2-nitrobenzyl, CNB) and the nitroindoline caging group (7-nitroindoline, NI, and 4-methoxy-7-nitroindoline, MNI). Besides NI- and MNI-caged amino acids being thermally more stable than the CNB-caged amino acids, there have been no other major advantages reported of using compounds from either of these two classes. Here, we show inhibitory effects of CNB-glutamate and a number of other CNB-caged agonists on N-methyl-D-aspartate (NMDA) receptors at non-saturating concentrations of the co-agonist glycine. In contrast, NI- and MNI-glutamate and most other NI-/MNI-caged agonists that we tested were inert under these conditions. Furthermore, we demonstrate that carboxynitroindoline-caged glycine (CNI-glycine), which was previously found to inhibit glycine receptors, has no such effect on NMDA receptors. Together, these findings underline the usefulness of NI- and MNI-caged ligands and show that CNB-caged compounds should be avoided in studies involving NMDA receptors.

Photochemical and pharmacological evaluation of 7-nitroindolinyl-and 4-methoxy-7-nitroindolinyl-amino acids as novel, fast caged neurotransmitters.[Pubmed:11640955]

J Neurosci Methods. 2001 Nov 15;112(1):29-42.

Reagents capable of rapid and efficient release of neuroactive amino acids (L-glutamate, GABA and glycine) upon flash photolysis of thermally stable, inert precursors have been elusive. 7-Nitroindolinyl (NI)-caged and 4-methoxy-7-nitroindolinyl (MNI)-caged compounds that fulfil these criteria are evaluated here. These caged precursors are highly resistant to hydrolysis. Photolysis is fast (half time< or =0.26 ms) and the conversion achieved with a xenon flashlamp is about 15% for the NI-caged L-glutamate and about 35% for the MNI-caged L-glutamate. A procedure is described for calibration of photolysis in a microscope-based experimental apparatus. NI-caged L-glutamate itself showed no agonist or antagonist effects on AMPA and NMDA receptors in cultured neurones, and had no effect on climbing fibre activation of Purkinje neurones. A control compound with identical photochemistry that generated an inert phosphate upon photolysis was used to confirm that the intermediates and by-products of photolysis have no deleterious effects. MNI-caged L-glutamate is as stable and fast as NI-caged L-glutamate and similarly inert at glutamate receptors, but about 2.5 times more efficient. However, NI-caged GABA is an antagonist at GABA(A) receptors and NI-glycine an antagonist at glycine receptors. The results show the utility and limitations of these fast and stable caged neurotransmitters in the investigation of synaptic processes.

Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons.[Pubmed:11687814]

Nat Neurosci. 2001 Nov;4(11):1086-92.

Dendritic spines serve as preferential sites of excitatory synaptic connections and are pleomorphic. To address the structure-function relationship of the dendritic spines, we used two-photon uncaging of glutamate to allow mapping of functional glutamate receptors at the level of the single synapse. Our analyses of the spines of CA1 pyramidal neurons reveal that AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors are abundant (up to 150/spine) in mushroom spines but sparsely distributed in thin spines and filopodia. The latter may be serving as the structural substrates of the silent synapses that have been proposed to play roles in development and plasticity of synaptic transmission. Our data indicate that distribution of functional AMPA receptors is tightly correlated with spine geometry and that receptor activity is independently regulated at the level of single spines.