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Optofluidic control of rodent learning using cloaked caged glutamate.

ABSTRACT: Glutamate is the major excitatory neurotransmitter in the brain, and photochemical release of glutamate (or uncaging) is a chemical technique widely used by biologists to interrogate its physiology. A basic prerequisite of these optical probes is bio-inertness before photolysis. However, all caged glutamates are known to have strong antagonism toward receptors of ?-aminobutyric acid, the major inhibitory transmitter. We have developed a caged glutamate probe that is inert toward these receptors at concentrations that are effective for photolysis with violet light. Pharmacological tests in vitro revealed that attachment of a fifth-generation (G5) dendrimer (i.e., cloaking) to the widely used 4-methoxy-7-nitro-indolinyl(MNI)-Glu probe prevented such off-target effects while not changing the photochemical properties of MNI-Glu significantly. G5-MNI-Glu was used with optofluidic delivery to stimulate dopamine neurons of the ventral tegmental area of freely moving mice in a conditioned place-preference protocol so as to mediate Pavlovian conditioning.

SUBMITTER: Durand-de Cuttoli R

PROVIDER: S-EPMC7104027 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Optofluidic control of rodent learning using cloaked caged glutamate.

Durand-de Cuttoli Romain R Chauhan Pradeep S PS Pétriz Reyes Adriana A Faure Philippe P Mourot Alexandre A Ellis-Davies Graham C R GCR

Proceedings of the National Academy of Sciences of the United States of America 20200309 12

Glutamate is the major excitatory neurotransmitter in the brain, and photochemical release of glutamate (or uncaging) is a chemical technique widely used by biologists to interrogate its physiology. A basic prerequisite of these optical probes is bio-inertness before photolysis. However, all caged glutamates are known to have strong antagonism toward receptors of γ-aminobutyric acid, the major inhibitory transmitter. We have developed a caged glutamate probe that is inert toward these receptors ...[more]

PMID: 32152102

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Project description:Fast uncaging of low affinity competitive receptor antagonists can in principle measure the timing and concentration dependence of transmitter action at receptors during synaptic transmission. Here, we describe the development, synthesis and characterization of MNI-caged ?-D-glutamyl-glycine (?-DGG), which combines the fast photolysis and hydrolytic stability of nitroindoline cages with the well-characterized fast-equilibrating competitive glutamate receptor antagonist ?-DGG. At climbing fiber-Purkinje cell (CF-PC) synapses MNI-caged-?-DGG was applied at concentrations up to 5 mM without affecting CF-PC transmission, permitting release of up to 1.5 mM ?-DGG in 1 ms in wide-field flashlamp photolysis. In steady-state conditions, photoreleased ?-DGG at 0.55-1.7 mM inhibited the CF first and second paired EPSCs by on average 30% and 60%, respectively, similar to reported values for bath applied ?-DGG. Photolysis of the L-isomer MNI-caged ?-L-glutamyl-glycine was ineffective. The time-course of receptor activation by synaptically released glutamate was investigated by timed photolysis of MNI-caged-?-DGG at defined intervals following CF stimulation in the second EPSCs. Photorelease of ?-DGG prior to the stimulus and up to 3 ms after showed strong inhibition similar to steady-state inhibition; in contrast ?-DGG applied by a flash at 3-4 ms post-stimulus produced weaker and variable block, suggesting transmitter-receptor interaction occurs mainly in this time window. The data also show a small and lasting component of inhibition when ?-DGG was released at 4-7 ms post stimulus, near the peak of the CF-PC EPSC, or at 10-11 ms. This indicates that competition for binding and activation of AMPA receptors occurs also during the late phase of the EPSC, due to either delayed transmitter release or persistence of glutamate in the synaptic region. The results presented here first show that MNI-caged-?-DGG has properties suitable for use as a synaptic probe at high concentration and that its photolysis can resolve timing and extent of transmitter activation of receptors in glutamatergic transmission.

Dataset Information

Optofluidic control of rodent learning using cloaked caged glutamate.

Publications

Optofluidic control of rodent learning using cloaked caged glutamate.

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