Photolysis of a Caged, Fast-Equilibrating Glutamate Receptor Antagonist, MNI-Caged γ-D-Glutamyl-Glycine, to Investigate Transmitter Dynamics and Receptor Properties at Glutamatergic Synapses.

Francisco Palma-Cerda,George Papageorgiou,Boris Barbour,Céline Auger,David Ogden

doi:10.3389/fncel.2018.00465

Abstract

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 γ-D-glutamyl-glycine (γ-DGG), which combines the fast photolysis and hydrolytic stability of nitroindoline cages with the well-characterized fast-equilibrating competitive glutamate receptor antagonist γ-DGG. At climbing fiber-Purkinje cell (CF-PC) synapses MNI-caged-γ-DGG was applied at concentrations up to 5 mM without affecting CF-PC transmission, permitting release of up to 1.5 mM γ-DGG in 1 ms in wide-field flashlamp photolysis. In steady-state conditions, photoreleased γ-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 γ-DGG. Photolysis of the L-isomer MNI-caged γ-L-glutamyl-glycine was ineffective. The time-course of receptor activation by synaptically released glutamate was investigated by timed photolysis of MNI-caged-γ-DGG at defined intervals following CF stimulation in the second EPSCs. Photorelease of γ-DGG prior to the stimulus and up to 3 ms after showed strong inhibition similar to steady-state inhibition; in contrast γ-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 γ-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-γ-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.

Highlights

The time scale and spatial distribution of transmitter release are important in determining both the speed and independence of information transfer between pre- and postsynaptic elements in synaptic transmission
Flashlamp photolysis releases ligand over a large field and it is important for interpretation of results to know the time-course of release and the persistence of released γ-DGG in the photolysis region
The γ-DGG concentrations immediately after the flash were calculated from the photolysis efficiency at the flashlamp energy used and the bath concentration of MNI-caged γ-DGG measured by spectrometry at the end of the recording

Summary

INTRODUCTION

The time scale and spatial distribution of transmitter release are important in determining both the speed and independence of information transfer between pre- and postsynaptic elements in synaptic transmission. The use of low affinity antagonists to assess neurotransmitter concentration and time-course (Clements et al, 1992) has been evaluated by Beato (2008) at glycinergic synapses and reviewed by Scimemi and Beato (2009) In simulations they showed a correlation between transmitter concentration and exposure time that prevents separate determination and requires additional information, such as the effects of transporter inhibition, to obtain independent parameter estimate. Independent determination of the postsynaptic gating properties is required for analysis to estimate transmitter timing and concentration; these are usually obtained from patch experiments and do not take account of possible differences between excised patch and post-synaptic receptor properties, resulting for instance from different receptor subunit/auxiliary protein compositions In this context the precise timing and quantitation of photorelease proposed here would be useful since data are gathered at the synapse of interest. Photolysis of MNI-caged amino acids is readily adapted to localized laser photolysis (Trigo et al, 2009) to yield faster time and better spatial resolution

Ethical Approval

RESULTS

DISCUSSION