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

Article abstract of DOI:10.3389/fncel.2018.00465

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.

Full text of DOI:10.3389/fncel.2018.00465

ORIGINAL RESEARCH
published: 05 December 2018
doi: 10.3389/fncel.2018.00465
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-Cerda1, George Papageorgiou2, Boris Barbour3, Céline Auger1
and David Ogden1,2
*
1Brain Physiology Lab, UMR8118 Université Paris Descartes, Paris, France, 2The Francis Crick Institute, London, United
Kingdom, 3Institut de Biologie de l’Ecole Normale Supérieure (IBENS), Ecole Normale Supérieure, CNRS, INSERM, PSL
University, Paris, France
Edited by:
Marco Canepari,
UMR5588 Laboratoire
Interdisciplinaire de Physique (LIPhy),
France
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
Reviewed by:
Annalisa Scimemi,
University at Albany, United States
Julie Perroy,
Centre National de la Recherche
Scientifique (CNRS), France
*Correspondence:
David Ogden
david.ogden@parisdescartes.fr
Received: 01 August 2018
Accepted: 15 November 2018
Published: 05 December 2018
Citation:
Palma-Cerda F, Papageorgiou G,
Barbour B, Auger C and Ogden D
(2018) 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.
Front. Cell. Neurosci. 12:465.
doi: 10.3389/fncel.2018.00465
Frontiers in Cellular Neuroscience | www.frontiersin.org
1
December 2018 | Volume 12 | Article 465

Palma-Cerda et al.
Caged Fast Antagonist at Glutamate Synapses
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.
Keywords: photolysis, synaptic transmission, caged fast antagonist, glutamate receptor, climbing fiber, Purkinje
cell, cerebellum, nitroindoline
the analysis of L-glutamate concentration and time-course at
synapses. In one study, Liu et al. (1999) applied a concentration-
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. Synapses with a single or few release
sites and short-lived transmitter transients show greater
independence and postsynaptic specificity than multisite
synapses that activate a larger postsynaptic volume. This
distinction has been formalized as synaptic vs. parasynaptic
(‘‘spillover’’ or volume transmission; Szapiro and Barbour, 2007,
2009; Coddington et al., 2014) and relates directly to the time
for which transmitter interacts with postsynaptic receptors
following release. Fast, timed application of an antagonist
to the post-synaptic receptors would enable dissection of
the time-course of receptor activation to distinguish modes
of transmission. However, there are few examples of caged
photolabile derivatives of receptor antagonists applied in
neuroscience, although they are potentially useful tools in
probing synaptic function on the time scale of transmission.
They offer the possibility of timed release and competition with
transmitter binding at postsynaptic receptors, thereby giving
time-resolved data of transmitter concentration at the target
receptors. With further development of selective ligands there
is the possibility of targeting specific signaling pathways as has
been done with selective agonists (Palma-Cerda et al., 2012).
To be useful caged antagonists require fast photochemistry
relative to the synaptic timescale, with uncaging
rates >10,000 s−1 following a light pulse. Additionally, to
facilitate repeated applications in wide-field photolysis, it is
also desirable that the photoreleased antagonist dissociates
rapidly from receptors, requiring a low affinity, contrary
to the high affinity generally useful for pharmacological
studies at synapses. Because of the dependence of block
on transmitter concentration, the low affinity competitive
receptor antagonists also permit a measure of the relative
concentration of the competing neurotransmitter during
transmission. Here, we report the development and application
of a fast-equilibrating caged glutamate receptor antagonist to
study excitatory synaptic mechanisms. Photolysis is with the
well-established nitroindoline photochemistry, which is both
fast (half-time for release 0.15 µs; Morrison et al., 2002) and
stable to hydrolysis (Papageorgiou et al., 1999). Nitroindoline
photochemistry has been applied to synaptic physiology since
1999 and is well established as an effective experimental tool in
neuroscience.
ratio analysis to show low affinity competitive antagonism by
γ-DGG with L-glutamate at hippocampal AMPA-R synapses
(estimated equilibrium dissociation constant K = 0.55 mM)
and used the dependence of block on glutamate concentration
to show fast equilibration of γ-DGG with receptors. At
climbing fiber-Purkinje cell (CF-PC) synapses Wadiche and
Jahr (2001) used the glutamate concentration dependence
of γ-DGG block to test multivesicular release during paired
responses. The analysis was based on fast application patch
data and kinetic simulations, estimating a fast dissociation
rate constant of 104 −1. The use of low affinity antagonists
s
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. Furthermore, 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.
In this report, we describe the chemical synthesis and
characterization of MNI-caged γ-DGG and tests for interference
with synaptic transmission at high (mM) concentration at
cerebellar climbing fiber to Purkinje cell synapses. Both
enantiomers γ-DGG and γ-LGG were prepared in caged form.
The latter endogenous dipeptide (Kanazawa et al., 1965) served
as a readily available model reagent in the synthesis and has
been reported to have a lower affinity for glutamate receptors
(Francis et al., 1980; Davies and Watkins, 1981), making it useful
as a control probe. Structures of the two caged compounds
are shown in the Supplementary Information. The results
show that MNI-caged γ-DGG can be used at concentrations
up to 5 mM to release mM concentrations by flashlamp or
laser photolysis and that timing of competition with synaptic
L-glutamate can be resolved with 1 ms precision with flashlamp
photolysis. This article describes the properties of MNI-caged
γ-DGG in relation to synaptic investigations and presents results
of transmitter timing and duration at the CF-PC synapse
Previous use of the low-affinity antagonist γ-D-glutamyl-
glycine (γ-DGG, Davies and Watkins, 1981) has been in
Frontiers in Cellular Neuroscience | www.frontiersin.org
2
December 2018 | Volume 12 | Article 465

Palma-Cerda et al.
Caged Fast Antagonist at Glutamate Synapses
with wide-field flashlamp photolysis. 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.
Strathclyde) and analyzed in Igor Pro 7 (Wavemetrics, Lake
Oswego, OR, USA).
For climbing fiber (CF) stimulation, cathodal pulses of
50 µs duration and supramaximal amplitude were delivered
from a constant voltage stimulator (DS2, Digitimer, Welwyn
UK) through glass pipettes with 2 MΩ resistance filled with
bath solution, positioned at the surface of the slice in the
granule cell layer. Voltage thresholds were 30–70 V (15–35 µA)
determined regularly during recordings. The stimulus comprised
two pulses separated by 40 ms or 150 ms to test paired
pulse depression. To inhibit Na-current in the Purkinje neuron
with CF stimulation the intracellular solution contained 5 mM
QX314.HCl salt. The extracellular solution was supplemented
with 3 µM SR 95531 and 50 µM D-AP5 to block GABAA
and NMDA receptors, respectively, or as specified in Figure
legends.
MATERIALS AND METHODS
Ethical Approval
Sprague–Dawley rats were provided by Janvier (St Berthevin,
France) and subsequently housed in agreement with the
European Directive 2010/63/UE regarding the protection of
animals used for experimental and other scientific purposes.
Experimental procedures were approved by the French
Ministry of Research and the ethical committee for animal
experimentation of Paris Descartes.
Slice Preparation
Flash Photolysis
Experiments were made with parasagittal slices 200 µm thick
cut from the cerebellum of Sprague-Dawley rats, age 12–21 days.
Slices were prepared and stored at 34◦C throughout in a solution
containing (mM): 115 NaCl, 2.5 KCl, 1.3 NaH2PO4, 26 NaHCO3,
0.1 L-ascorbic acid, 25 glucose, 1 MgSO4, 2 CaCl2 equilibrated
with 95% O2–5% CO2.
Flash photolysis was through the condenser of the transmitted
light path with a Xenon Arc Flashlamp (Rapp Optoelectronic,
Hamburg, Germany) producing a 0.5–1.5 ms light pulse,
bandpass filtered 275–395 nm, energy measured as 120 mJ on
discharge at 300V with three capacitors. Because of the high cage
concentration it is not feasible to use epi-illumination for near
UV wide spectrum photolysis, the light is absorbed too strongly
by mM cage over 3 mm pathlength. The optical arrangement
and calibration procedures are summarized in Supplementary
Figure S2. The flashlamp was coupled with a 3 mm liquid
light guide into the transmitted-illumination path by a silica
lens and 425 nm LP dichroic reflector and focused by a silica
condenser through the slice to illuminate uniformly a 200 µm
diameter region at the top surface. Calibration of photolysis
was in separate experiments by comparison with photolysis of
caged fluorophore NPE-HPTS (the nitrophenylethyl ether of
HPTS) which becomes fluorescent on uncaging to release free
HPTS (exc 470/40 nm, em 525/50 nm; see Supplementary
Figure S2 caption in Supplementary Information; also Canepari
et al., 2001). Briefly, NPE-HPTS at 100 µM in borate buffer
(10 mM pH 9) is suspended by stirring into Sylgard 184 resin
to form vesicles of 5–20 µm diameter that cure to form a thin
layer on coverslips. The progressive increase in fluorescence
in each vesicle with consecutive flashlamp pulses depends
on the efficiency of photolysis in the microscope at the
flashlamp parameters used, the conversion/flash obtained by
fitting an exponential under conditions where bleaching of
released HPTS is minimal. The conversion of MNI-glutamate
relative to NPE-HPTS with wide-field flashlamp illumination
and band-pass near UV filter was determined as 2.1, and will
be the same for MNI-caged γ-DGG or MNI-caged γ-LGG, since
no additional chromophores are present. A further correction
was applied for transmission of photolysis light through the
slice, estimated from transmission of the flashlamp pulse as
32% in the molecular layer of cerebellar slices 200 µm thick
from 18 day rats, 40% at 15 days and 44% at 13 days. At the
end of the experiment the bath concentration of MNI-γ-DGG
was measured in a microliter spectrophotometer (Nanodrop,
ThermoFisher Scientific) from the absorbance at 322 nm with
Electrophysiological Recording
A Hepes-buffered saline was used containing a low concentration
of bicarbonate to maintain intracellular pH, composition (mM):
130 NaCl, 4 KCl, 2.5 NaHCO3, 10 Hepes, 25 glucose, 0.1 ascorbic
acid, 1 mM MgSO4 and 2 mM CaCl2, pH 7.3 with NaOH,
perfused at 5 mL/min. Once a recording was established
perfusion was stopped and the cage, dissolved at 20 mM
in Hepes-buffered isotonic saline, added to the bath volume.
Air was blown over the surface of the static bath solution
to produce mixing of the caged compound; ambient pCO2
is equilibrated at 2.5 mM bicarbonate and pH is controlled
by Hepes. The presence of bicarbonate facilitates intracellular
pH regulation by CO2 diffusion across the membrane. The
intracellular solution for Purkinje neurons was (in mM): 150 K
gluconate, 10 Hepes, 0.01 EGTA, 2.5 MgCl2, 2 ATPNa2 and
0.4 GTPNa, 5 mM QX314, pH adjusted to 7.3 with KOH
and osmolality to 300 mosmol/kg. Alexa 488 20 µM was
included to record cell morphology (Andor Ixon EMCCD;
Oxford, UK). Recordings were at 21–24◦C. The preparation
was viewed with a Zeiss Axioskop1FS (Oberkochen, Germany)
with Olympus 40× 0.8w objective. To avoid photolysis,
transmitted illumination was long pass filtered at 425 nm.
The optical arrangement is described in Supplementary
Figure S2.
Recording pipettes had resistances of 2.5–3.5 MΩ. Whole
cell voltage clamp recordings from Purkinje neurons were with
an Axopatch 200B amplifier (Molecular Devices, San Jose,
CA, USA). Series resistance was 7–10 MΩ and compensated
60%–80%. The pipette potential was held at −60 to −30 mV,
data were not corrected for the junction potential of 12 mV.
Data acquisition was with a CED P1401 interface (Cambridge
Electronic Design, Cambridge, UK) LP filtered at 3 kHz, sampled
at 50 kHz with WINWCP (Dr. John Dempster, University of
Frontiers in Cellular Neuroscience | www.frontiersin.org
3
December 2018 | Volume 12 | Article 465

Palma-Cerda et al.
Caged Fast Antagonist at Glutamate Synapses
molar absorption coefficient 4800 M−1cm−1. To obtain the
concentration released, the cage concentration was multiplied
by the conversion/flash of NPE-HPTS at the flash intensity
used, multiplied by the factor of 2.1 for relative efficiencies of
MNI-glutamate to NPE-HPTS with this excitation spectrum,
and by the transmission factor through the slice dependent
on the age of the animal. Measurements of several vesicles
in the microscope field showed that photolysis was uniform
over the 200 µm field viewed by the camera (Andor Ixon,
Oxford Instruments, Oxford, UK). Conversion efficiencies for
MNI-caged-γ-DGG at the flashlamp settings used here, as in
Figures 1A,B, were estimated as 14%/flash for 300 V one
capacitor and 30%/flash for 300 V three capacitors in 13-day
200-µm thick slices.
Chemical Synthesis and Photochemistry of
MNI-Caged γ-D-Glutamylglycine
Synthesis and purification of both D and L optical isomers
of MNI-caged γ-glutamylglycine were based on published
protocols for MNI-glycine (Papageorgiou et al., 2011) and
for substituted nitroindolines (Papageorgiou and Corrie,
2000). Full experimental details of the synthesis of the two
isomers and their structural verification are given in the
Supplementary Information (Data sheet 1), together with
circular dichroism spectra (Supplementary Figure S1) to
confirm the configurations. The photocleavage reaction is
outlined in Scheme 1 (below).
RESULTS
Drugs
Time-Course of γ-DGG Concentration
Following Photorelease From MNI-Caged
γ-DGG
Drugs used were D-AP5, SR 95531, NBQX, Cyclothiazide,
Amiloride and QX314.HCl from Tocris Bioscience UK or Sigma-
Aldrich.
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 release of carboxylate ligands from nitroindoline-
caged precursors was shown by nanoseconds laser flash
photolysis to have a halftime of 150 ns (Morrison et al., 2002).
Thus, on the 0.1 ms timescale relevant for synaptic function,
photolysis closely follows the light intensity, the quantity of
γ-DGG released and its concentration in the irradiated volume
are given by the time integral of the flash. A photodiode was used
to monitor light intensity applied by photolysis. The photodiode
output (dotted gray traces) and their integral (dashed traces)
are shown in Figure 1 for low (300 V, one capacitor, panel A)
and high energy (three capacitors, panel B) flashes. Simultaneous
voltage clamp recording in the Purkinje neuron shows transient
current (black solid traces) evoked by photolysis; these are
discussed below. 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.
The photolysis efficiencies were calculated for low or high flash
energy from independent experiments in the same microscope
as described above in the ‘‘Materials and Methods’’ section
and in Supplementary Information.
FIGURE 1 | Time-course of γ-D-glutamyl-glycine (γ-DGG) concentration
during and following photorelease. Panels (A,B) show photodiode records of
the flash (dotted gray traces, scale bar 0.5 V) and their integrals over the pulse
representing the time-course of the γ-DGG concentration (dashed traces), and
the whole cell current generated by photolysis of MNI-caged γ-DGG in a
Purkinje neuron at −60 mV (black traces, scale bar 100 pA). Panel (A) shows
the response to a low intensity flash as used in synaptic experiments,
producing 14% conversion to release γ-DGG and proton concentrations of
0.65 mM. Panel (B) shows the response to a strong flash releasing 1.3 mM
γ-DGG and protons. The bath concentration of MNI-caged γ-DGG was
4.6 mM in (A,B). (C) Following wide-field photolysis of NPE-HPTS the
time-course of diffusional loss of HPTS fluorescence was monitored from a
region defined by an aperture in a conjugate image plane corresponding to
10 µm in the slice. HPTS and amino acids have similar diffusion coefficients in
extracellular space. A single flash released HPTS from 100 µM NPE-HPTS
equilibrated in the slice. Fluorescence of photoreleased HPTS (em 525/50 nm)
from the 10 µm spot was measured every 5 s thereafter, corrected for
background counts and normalized to the maximum fluorescence immediately
after the flash. The data are from three trials, the dotted lines show the mean
half-time for the fluorescence decline, estimated as 60 s; the time-course is
expected to be similar for photoreleased γ-DGG.
Time-Course of Diffusional Loss of
Photoreleased γ-DGG From the Irradiated
Region
After fast release the concentration of γ-DGG in the region
of the Purkinje neuron will decline by diffusional exchange
of photoreleased γ-DGG between the irradiated volume,
approximately 10 µl, and the bulk bath volume of 1.5 ml.
This will determine the concentration of γ-DGG adjacent to
synaptic sites with time following photorelease. The highly polar
fluorophore HPTS (pyranine) photoreleased in extracellular
space has previously been used to measure diffusion within
tissues (Xia et al., 1998); it has a diffusion coefficient
Frontiers in Cellular Neuroscience | www.frontiersin.org
4
December 2018 | Volume 12 | Article 465

Palma-Cerda et al.
Caged Fast Antagonist at Glutamate Synapses
330 µm2s−1, similar to that estimated for synaptic L-glutamate
(Nielsen et al., 2004). HPTS was photoreleased from NPE-caged-
HPTS releasing fluorophore quickly and uniformly over the
field of view (rate of photolysis of 550 s−1 at pH 7 following
the flash; Jasuja et al., 1999). The fluorescence was monitored
after the flash at 5 s intervals with 100 ms camera frames over
an aperture placed in a conjugate image plane corresponding
to 10 µm (referred to the specimen plane) and corresponding
in size to a PC soma. The results are shown for three slices
in Figure 1C, giving an average half-time for diffusional loss
from the region of the cell of 60 s. In relation to the paired
pulse protocol used here the concentration of HPTS over an
interval of 50 or 150 ms declined to 90%–95%, and in the
30 s interval between paired stimuli declined to 60%–70% of
the concentration released by photolysis associated with the
preceding paired pulse stimulation. Rapid mixing of the bath
between experimental protocols was by a perfusion tube and
remote syringe.
FIGURE 2 | Current evoked by photolysis of MNI-caged γ-DGG or MNI-caged
γ-LGG. (A) Currents evoked by photorelease of L-glutamate at 3 µM and
7 µM in a Purkinje cell. (B) Currents of similar amplitude evoked by
photorelease of 0.5 or 1.0 mM γ-DGG, in the absence (solid black traces) or
presence (dotted gray traces) of the AMPA/Kainate receptor antagonist NBQX
(30 µM). Currents evoked by γ-DGG are faster than the glutamate evoked
currents and are not affected by the high affinity AMPA receptor competitive
antagonist NBQX, indicating that γ-DGG is not an agonist of AMPA/Kainate
receptors. (C) To test for an effect of the protons co-released with γ-DGG and
γ-LGG (see Scheme 1), photorelease of 1.0 mM γ-LGG was compared in
saline buffered with 70 mM HEPES (black trace) or 10 mM HEPES (gray
trace). High proton buffering reduced the amplitude of the currents. Time of
release indicated by the arrow.
Transient Inward Current During Photolysis
Due to Proton Release
The photolysis of MNI-caged γ-DGG shown in Figures 1A,B
evokes a transient inward current. Release of 0.5 or 1 mM of
either γ-DGG (shown in Figures 1A,B, 2B) or γ-LGG (not shown)
generated transient currents of approximately 0.25 nA and 0.6 nA
amplitude that followed closely the light intensity in rise-time,
and subsequently declined with a fast milliseconds time-course.
As shown in Scheme 1, the photolysis of MNI-caged γ-DGG
generates γ-DGG, a proton and a nitrosoindole by-product
in stoichiometric proportion. Each of these products could
activate the transient inward current observed on photolysis.
γ-DGG released at high concentration by fast photolysis might
bind and activate AMPA, Kainate or NMDA receptors on a
milliseconds timescale, functioning as a weak or partial agonist.
By comparison with the γ-DGG evoked currents however,
photorelease of L-glutamate (3 µM or 7 µM) from MNI-caged
L-glutamate to generate similar peak amplitudes evoked currents
with much slower onset and decline (Figure 2A), suggesting
that activation of AMPA-receptors by γ-DGG or γ-LGG is
not responsible for the fast current. Moreover, photorelease of
γ-DGG or γ-LGG at 0.5 mM or 1 mM in presence of NBQX
(30 µM, Figure 2B) and D-AP5 (50 µM, data not shown)
evoked transient currents with the same amplitudes as control.
As a further test for AMPA receptor activation and rapid
desensitization, the effect of cyclothiazide (100 µM), a blocker
of AMPA receptor desensitization, affected neither amplitude
nor time-course of the currents. The data show that γ-DGG
is not acting as a weak agonist at AMPA-Kainate receptors
nor promoting desensitization, supporting the conclusion from
steady-state experiments that it acts as a fast-competitive
antagonist (Liu et al., 1999; Wadiche and Jahr, 2001).
A second possibility to account for the transient current
may be the activation of a proton-gated channel or proton
exchanger by the H+ released stoichiometrically with γ-DGG
during photolysis, shown in Scheme 1. A first test was to increase
the pH buffering from 10 mM HEPES (buffer capacity 5 mM/pH
at pH 7.3) to 70 mM, a 7-fold greater equilibrium buffer capacity.
The result is illustrated in Figure 2C. The peak amplitude in
70 mM HEPES (black trace) was reduced to 50% of that in
SCHEME 1 | Photolysis of MNI-caged γ-DGG releases γ-DGG, a nitrosoindole by-product and a proton for each caged molecule photolysed.
Frontiers in Cellular Neuroscience | www.frontiersin.org
5
December 2018 | Volume 12 | Article 465

Palma-Cerda et al.
Caged Fast Antagonist at Glutamate Synapses
10 mM HEPES (gray trace), and the decay of the current was
shortened from 5 ms to 2 ms, indicating a role of protons in
the activation of the current. However, the partial inhibition and
faster time-course of the currents by high HEPES concentration
suggest that the kinetics of buffering by HEPES is too slow to bind
protons on the timescale of photorelease.
ASIC channels are known to be present in Purkinje neurons
and to respond rapidly to protons (Lingueglia et al., 1997).
As a further test of the activation of a proton gated channel,
an inhibitor of ASIC channels, IEPA, an analog of amiloride,
was tested at the concentration of 30 µM. In two PC the
current was inhibited by 30%, however tests of recovery were
not possible because the concentration that could be applied was
limited by toxicity of the vehicle DMSO. Amiloride itself also
produced incomplete inhibition at high concentration, tested at
concentrations up to 500 µM. ASIC channels are the most likely
candidates for the observed current however known blockers
amiloride and derivatives tested here were ineffective and the
origin of this conductance will require further pharmacological
testing.
Finally, the nitrosoindole by-product generated by the
photolysis reaction could in principle be responsible for the
transient current. This possibility cannot easily be tested
independently since there are no fast-acting reagents available
to quench potential oxidative actions. The reducing thiols DTT
and glutathione have been shown to react too slowly to be
useful in this context (half-time 120 s; Papageorgiou et al., 2008).
Evidence from previous experiments however, has suggested that
any interference by the nitroso by-products are slow and not
on the time-course of the photolysis pulse (Papageorgiou et al.,
2011). Thus, it is unlikely that the fast currents are generated by
this product of the photolysis reaction.
FIGURE 3 | Effect of MNI-caged γ-DGG on CF-PC synaptic transmission.
Paired pulse stimulation at 150 ms interval before (black trace) and 5 min after
addition of 4.6 mM MNI-caged γ-DGG to the bath (gray trace). Pipette
potential −40 mV, 21◦C.
the second. No change of stimulation threshold was observed
immediately following addition of the cage, although threshold
often increased progressively during the experiment.
Block of Climbing Fiber Transmission at
Constant γ-DGG Concentration Generated
by Photolysis
γ-DGG is a low affinity competitive antagonist that binds to
receptors with fast kinetics similar to L-glutamate. Consequently,
there is competition between γ-DGG and L-glutamate that
reaches equilibrium on the time-scale of the synaptic glutamate
concentration transient; this property has been used to estimate
changes in the amount of transmitter released from the reduced
inhibition by γ-DGG at high glutamate concentration (Liu et al.,
1999; Wadiche and Jahr, 2001). The CF-PC synapse shows
short-term depression attributed to reduced transmitter release
in the second pulse of a pair, resulting in reduced competition
and greater fractional block by equal concentrations of γ-DGG
in the second compared to the first response (Wadiche and Jahr,
2001). The slow diffusion of γ-DGG from the region of photolysis
of MNI-caged-γ-DGG shown above permits estimates of the
block at near steady concentration following flash photolysis
and may be compared with published steady state data. In
experiments to test this, CF were stimulated on a 30 s cycle
with pairs of stimuli separated by 40 ms. After acquiring control
data over two or more cycles, γ-DGG was released by photolysis
coinciding with the first stimulus of a pair. Since γ-DGG diffuses
slowly from the region of photolysis the concentration remained
high during the second stimulus. The steady state inhibition
was calculated by subtraction of the EPSC in γ-DGG from
the control followed by normalization to the control EPSC
amplitude.
The evidence suggests that the transient conductance
activated upon photolysis of MNI-caged γ-DGG is due
to activation of ASIC channels and other conductances,
such as electrogenic exchangers, by the protons released
stoichiometrically with γ-DGG. Current evoked by proton
release during photolysis of MNI-caged ligands has not been
demonstrated previously because L-glutamate or other agonists
have not been applied with wide-field photolysis at the high
concentrations used here for release of γ-DGG.
Tests for Interference of MNI-Caged γ-DGG
With Climbing Fiber-Purkinje Cell (CF-PC)
Synaptic Transmission
Although MNI-caged ligands have been widely used and
evaluated since 1999 the concentrations of cage used here are
high and were tested for interference with synaptic transmission
at CF-PC synapses. The example presented in Figure 3 shows
paired CF EPSCs separated by 150 ms before (black trace)
and 5 min after addition of MNI-caged γ-DGG to give
a concentration of 4.6 mM in the bath (gray trace). The
threshold for CF stimulation and the synaptic charge of the
first and second EPSC of the pair were compared before and
after addition of MNI-caged γ-DGG; data in seven cells with
2.7–5.2 mM caged γ-DGG showed charge in the first EPSC was
0.97 ± 0.023 (SD) of pre-cage control and 0.99 ± 0.018 in
The results are illustrated in Figure 4 which shows the
EPSCs elicited by a pair of CF stimuli separated by 40 ms
in the presence of MNI-caged γ-DGG before (gray trace,
control) and after photolysis timed with the first stimulus
(black trace). The fraction of current blocked by γ-DGG was
Frontiers in Cellular Neuroscience | www.frontiersin.org
6
December 2018 | Volume 12 | Article 465

Palma-Cerda et al.
Caged Fast Antagonist at Glutamate Synapses
FIGURE 4 | Steady state inhibition of climbing fiber paired pulse EPSCs. (A) Control paired EPSCs in gray and test EPSCs in black following photorelease of 1.1 mM
γ-DGG at the time of stimulation of the first EPSC. Paired stimulations 40 ms interval. γ-DGG inhibits the second EPSC more than the first. (B) The current blocked by
γ-DGG (dashed line) was obtained by subtracting test (black trace) from control (gray trace) and is compared to control. (C) Fractional block of the peak current of
1st and 2nd EPSC following a flash to release γ-DGG at the first stimulus; data from 10 cells, Mean ± SEM. Range of γ-DGG concentrations released 0.55–1.7 mM.
Note that diffusional loss of photoreleased γ-DGG is negligible on this time scale (see Figure 1C). Panels (B,C) show that the γ-DGG blocked component is a greater
fraction of the control in the second EPSC than the first, indicating a lower competing transmitter concentration in the second EPSC.
obtained by subtracting the current recorded after photolysis
from the control, giving the γ-DGG-sensitive current (dotted
traces in Figure 4B). Data from 10 cells are summarized in
the bar graph Figure 4C. The average fractional block of the
first pulse was 0.29 ± 0.06 (SEM) and of the second by
0.60 ± 0.06 (SEM) respectively, measured in Figure 4C at
the peak current. The greater block of the second EPSC is
consistent with the interpretation that transmitter release and
concentration are less in the second pulse than the first during
paired pulse depression at CF-PC synapses (Wadiche and Jahr,
2001).
to transmitter binding to postsynaptic receptors is predicted to
produce block similar to the steady state block shown for the
second EPSC in Figure 4. Release of γ-DGG after transmitter
release will produce inhibition if transmitter is present and
postsynaptic receptors are available.
Each panel of Figure 5 shows the EPSC recorded with
photolysis triggered at the time indicated following CF
stimulation (black traces), superimposed with the control EPSC
recorded 30 s earlier (gray traces). The results show that γ-DGG
release with 0–1 ms or 2–3 ms delay from the stimulus resulted
in a reduced peak amplitude, similar to the steady state inhibition
of 60% described in Figure 4C. At longer delays γ-DGG released
over 4–5 ms, 5–6 ms, 6–7 ms from the stimulus showed the
same peak EPSC as control but a faster decay. γ-DGG released
in the interval 3–4 ms reduced the peak amplitude of the EPSC
much less than at earlier times and speeds the decline, indicating
competition with transmitter during this interval. The records
are superimposed in Figure 6A to permit comparison of changes
in the EPSC waveform.
The block by γ-DGG was quantified by integrating the EPSC
to obtain the total synaptic charge in the absence and presence
of γ-DGG release. The charge blocked by γ-DGG was obtained
by subtracting the integrals and then normalizing to control so
as to permit comparisons among experiments. The data were
integrated over the whole EPSC (from the CF stimulus for 60 ms;
data in Figure 6B) or from the time of the flash (to 60 ms post-
stimulus; Figure 6C).
Timed Inhibition of the CF EPSC by
Photoreleased γ-DGG: Delayed Release
and Sustained Transmitter Presence
The fast photorelease of γ-DGG from MNI-caged-γ-DGG was
used here to probe transmitter action at postsynaptic receptors
during the CF EPSC in order to test the timing and duration
of release, and the possible contribution of receptor activation
late in the EPSC. The analysis was made on the second of
paired EPSCs with 150 ms interval in a 30 s stimulus cycle. The
second EPSC was used because the smaller second EPSCs are less
affected by poor voltage clamp spatial uniformity. The flashlamp
pulse was 1 ms duration (see low intensity flash of Figure 1A)
and delayed by 0–10 ms with respect to the CF stimulation.
The protocol and results are illustrated by the records shown
in Figure 5, traces in gray are controls and in black after
photorelease of 0.74 mM γ-DGG at the times indicated. Timing
was set with respect to the stimulus, the γ-DGG concentration
rising with the integral of light intensity over 1 ms from the time
indicated, as shown in Figure 1A and Figure 6B. Release prior
The data from eight cells are summarized in Figure 6B as
the fraction of synaptic charge blocked by γ-DGG photorelease at
different times delayed with respect to the stimulus. The fraction
of EPSC charge blocked by γ-DGG is given for each delay as
Mean ± SD of 4–8 determinations plotted at the end of the
Frontiers in Cellular Neuroscience | www.frontiersin.org
7
December 2018 | Volume 12 | Article 465

Palma-Cerda et al.
Caged Fast Antagonist at Glutamate Synapses
FIGURE 5 | Timed inhibition of CF-PC EPSC by photorelease of γ-DGG. Each panel shows the second EPSCs of a pair separated by 150 ms, the control (gray
traces) and 30 s later with photorelease of 0.74 mM γ-DGG (black traces), released in a 1 ms flash from the indicated times relative to the stimulus. The bath
contained 5.9 mM MNI-caged γ-DGG. At early times (0 and 2 ms delay from the stimulus) inhibition is comparable to steady-state inhibition. At later times of γ-DGG
release (triggered at 3–10 ms) the EPSC approaches the control EPSC peak and only the decay is affected. Age 14 days, −50 mV, 24◦C.
flash. The γ-DGG concentration range was 0.34–0.74 mM. The
results show that release of γ-DGG in the intervals 0–1 ms and
2–3 ms after the stimulus produced block at similar levels, both
with mean 60%, and similar to the steady-state block of the
second EPSC shown in Figure 4C. This indicates that receptor
activation by transmitter release had not started after 3 ms from
the stimulus. γ-DGG released over the interval 3–4 ms after the
stimulus produced a reduced mean block of 40%, indicating
competition with transmitter for a shorter time in this interval.
Also, the SD is larger, approximately double that seen at other
time points, indicating jitter in the timing of transmitter release
between measurements. Later, with γ-DGG release at 4–5 ms and
5–6 ms, the block is smaller and less variable. The data therefore
suggest that glutamate binding of postsynaptic receptors in
competition with photoreleased γ-DGG occurs mainly at 3–4 ms
following CF stimulation, a time corresponding to the initial rise
of the EPSC. A second observation from Figure 6B (seen also
in Figures 5, 6A) is the continued inhibition by γ-DGG when
released at 5–6 ms, close to the peak EPSC, or at longer delays.
This suggests that transmitter is available to bind free receptors
late in the EPSC. As a better measure of γ-DGG block at late times
the data were expressed as the fraction of EPSC charge blocked
relative to control by integrating and comparing the EPSC charge
only over the period following the flash. The results plotted in
Figure 6C show a substantial fraction, average 23%, of the EPSC
charge is blocked by γ-DGG released at 6 ms and later times
post-stimulus. Although the change in EPSC is small at 5–6 ms
and later, the synaptic charge is significant and the observations
nonetheless indicate the presence of transmitter at the later time
points. The results imply receptor availability and reactivation by
transmitter, due either to delayed release or persistence in the
synapse. It may be noted that late in the EPSC the fractional
block at low transmitter concentration is expected to be constant
in absence of competition, simply related to the concentration
of γ-DGG and its equilibrium constant for receptor binding (Liu
et al., 1999).
DISCUSSION
MNI-caged-γ-DGG was developed as a tool to probe the
activation of postsynaptic receptors during transmission at
glutamatergic synapses, to investigate the roles of spillover
or volume transmission and the functional independence of
synaptic sites. A main purpose of the experiments reported
was the verification of MNI-caged-γ-DGG as a probe suitable
for testing receptor activation in synaptic and parasynaptic
transmission. The CF-PC synapse was studied as a single
connection permitting an assessment of the new cage. No
detrimental effects of the cage itself were seen on CF-PC
transmission, as judged by threshold for stimulation, synaptic
properties or short-term plasticity tested by paired pulse
depression, at concentrations up to 5 mM of MNI-caged γ-DGG
or MNI-caged γ-LGG. The cages synthesized and purified
with the methods described above and in Supplementary
Information were soluble in physiological saline at 20 mM
concentration, enabling use from stock solutions. It is also
important that there was no loss of chiral specificity in the
synthesis, as shown by the results of CD spectrometry (see SI,
Supplementary Figure S1). The MNI cages have well tested
properties of hydrolytic stability (Papageorgiou et al., 1999),
Frontiers in Cellular Neuroscience | www.frontiersin.org
8
December 2018 | Volume 12 | Article 465

Palma-Cerda et al.
Caged Fast Antagonist at Glutamate Synapses
FIGURE 6 | Inhibition by γ-DGG photoreleased during the EPSC: estimating the timing of the synaptic transmitter transient. Panel (A) shows the EPSCs presented in
Figure 5 superimposed for comparison of the time-course; the record obtained by release of γ-DGG between 0 and 1 ms, coincident with the stimulus, is marked 0;
records corresponding to release over 2–3 ms and 3–4 ms post-stimulus are labeled 2 and 3 respectively. Vertical arrows indicate the trigger timing of photolysis
pulses releasing γ-DGG as delay times from the stimulus. Panel (B) summarizes data from 4 to 8 cells of the fraction of total synaptic charge blocked by γ-DGG. Data
are Mean ± SD plotted at the end of the 1 ms photolysis pulse. The integration of the light pulse is indicated by the gray shading for each data point and represents
the time-course of γ-DGG concentration increase during the flash, uniform over the microscope field. To obtain the fractional charge blocked by γ-DGG test EPSCs
were integrated over 60 ms following the stimulus, subtracted from and normalized to the integral of control EPSCs recorded 30 s earlier. MNI-caged-γ-DGG
concentration range 2.7–5.9 mM, γ-DGG released range 0.34–0.74 mM. Panel (C) to quantify γ-DGG fractional block late in the EPSC, integrations were made from
the time of the flash to 60 ms, subtracted from and normalized to the same period in control. To control for the proton current artifact a record was taken with the
flash but without stimulation and subtracted from the test records obtained with stimulation. As a further check the records in (C) were integrated from 1 ms after the
flash, requiring no subtraction of initial current. Results with or without subtraction were similar. Animal ages 13 days in (A), 13–14 days in (B,C). Temperature
21–24◦C.
sub-microsecond release (Morrison et al., 2002) and good
pharmacological properties at glutamate synapses (Canepari
et al., 2001; Trigo et al., 2009; Palma-Cerda et al., 2012). The
ability to use high concentrations without detrimental effects
on excitability or transmitter release, the known efficiency with
405 nm laser photolysis of the MNI-cages (Trigo et al., 2009), and
the time-resolution of photolysis suggest MNI-caged- γ-DGG is a
useful synaptic probe.
To our knowledge, this is the first study that tests the fast
release of a fast equilibrating antagonist at AMPA-Kainate and
NMDA receptors and we were interested to know whether
transient activation of receptors might be apparent under these
release conditions. There was no partial agonist activity of γ-DGG
seen when applied at mM concentration by photorelease on
a sub-millisecond timescale. This indicates that the receptor
binding of γ-DGG does not induce conformation changes that
result in transient channel gating and desensitization. It is
consistent with the equilibrium data of Liu et al. (1999) that
γ-DGG has properties of a competitive receptor antagonist,
apparently showing no efficacy for channel gating. We also tested
the ability of MNI-caged-LGG to block CF-PC synapses upon
photolysis, the results confirming that γ-LGG is much less potent
than γ-DGG at similar high concentrations.
The immediate physiological results presented here, an
analysis of the EPSC at the CF-PC synapse, show two interesting
aspects. The first is a long delay of 3–4 ms from the stimulus
to the first evidence of receptor activation. Since the stimulus
site is close, within 50 µm of the Purkinje soma, the conduction
delay is predicted to be short, less than 100 µs. At synapses
where the delay from presynaptic spike to postsynaptic activation
has been measured it is 0.5–1 ms at room temperature (Katz
and Miledi, 1965; Forsythe, 1994). The delay from stimulus to
release here is therefore approximately 1 ms longer than can
be accounted for by synaptic delay and conduction velocity in
small myelinated fibers. It may point to other processes such as
additional conduction delays in the CF terminals that may delay
release.
The second finding is a late component of receptor activation
occurring after the major period of activation at 3–4 ms
post-stimulus. Vesicular release is known to continue at a
Frontiers in Cellular Neuroscience | www.frontiersin.org
9
December 2018 | Volume 12 | Article 465

Palma-Cerda et al.
Caged Fast Antagonist at Glutamate Synapses
reduced rate for several milliseconds after the peak release at
about 1 ms (Katz and Miledi, 1965; Trigo et al., 2012) and may
contribute a late component. An additional factor contributing
to the activation of postsynaptic current late in the EPSC may
be persistence of glutamate in the synapse. The CF terminals
are protected by Bergmann glial processes and glutamate
transporters at high density on both the PC and the glial
membranes which will quickly buffer and take up the transmitter.
The experiments here were done at 21–24◦C and both release and
uptake will be slower compared with physiological temperature
(Auger and Attwell, 2000). The relative contributions of the two
mechanisms are not addressed here and will be better examined
at physiological temperature.
The possibility that γ-DGG acts at presynaptic glutamate
receptors to modify transmitter release as well as postsynaptically
has not been excluded. Presynaptic effects were tested by
Wadiche and Jahr (2001) by monitoring transporter currents,
with the finding that these were unaffected by addition of 2 mM
γ-DGG. However, the transporter currents in their study were
isolated by a high concentration of NBQX which would mask an
effect of γ-DGG on presynaptic AMPA-K receptors. While ruling
out direct effects of γ-DGG on release, the possibility of block
by γ-DGG of receptor mediated modulation of transmission
by presynaptic AMPA-K or NMDAR is difficult to test. There
are, however, no reports of presynaptic AMPAR or NMDAR at
climbing fiber terminals.
We have noted previously a selectivity of γ-DGG for
Bergmann glial AMPAR with about 10-fold higher affinity
than Purkinje AMPAR (Bellamy and Ogden, 2005). This
may relate to the different receptor composition, GluA1 and
GluA4 in Bergmann glia and GluA1, GluA2 and GluA3 in
Purkinje neurons, and the presence of auxiliary proteins,
particularly TARP γ2. Thus, there is the additional possibility
of time resolved experiments with pharmacological specificity
for AMPA receptors of different subunit and auxiliary protein
composition by the photorelease of γ-DGG at low or high
concentrations to distinguish receptor types pharmacologically.
Further, evidence has been presented that kinetic parameters
and functional affinity of AMPAR for transmitter L-glutamate
differs among TARP vs. non-TARP associated receptors (Zhang
et al., 2014; Carbone and Plested, 2016; Devi et al., 2016).
If the affinity for L-glutamate differs among AMPA-R of
different compositions and gating mode, the estimates of
transmitter concentration with steady-state application of γ-DGG
will be modified due to competition and to differences in
affinity.
An important result obtained here in the context of
uncaging methodology was the activation in Purkinje cells of
a small fast inward current gated by mM release of protons.
Protons are released during nitrobenzyl and nitroindoline
photolysis stoichiometrically with the ligand, however usually the
concentrations are low and, in the case of local laser photolysis,
rapidly dissipated by diffusion. The high concentrations released
over a large volume here are unusual. The data shown in
Figure 2 indicate that even raising the buffer capacity from
10 mM to 70 mM HEPES was not effective in completely
suppressing the current; even if the protonation rate is high
in aqueous solution, estimated as 1010 L.mole−1.s−1, the pK
of 7.3 indicates a deprotonation rate in the milliseconds range
for HEPES. Further, the normal bicarbonate-CO2 buffer is
known to have kinetics on a second timescale and will be
ineffective on a millisecond timescale. These considerations
apply also to the strong alkalinization produced by photolysis
of DMnitophen and NPE-EGTA to release Ca intracellularly
(Trigo et al., 2012). The origin of the inward current
activated by protons here is not clear. ASIC channels are
known to be present in Purkinje neurons and to respond
rapidly to protons (Lingueglia et al., 1997), however known
blockers amiloride and derivatives tested here were ineffective.
Other possibilities are electrogenic transporters with coupled
proton flux or proton permeabilities which may be tested
pharmacologically.
The time resolution in the experiments reported here is
determined by the 1 ms duration of the flashlamp discharge,
measured for each record to determine the precise timing of
γ-DGG release but longer than the 0.1 ms limit imposed by
equilibration in receptor binding by γ-DGG (Liu et al., 1999;
Wadiche and Jahr, 2001). The flashlamp provides widefield
photolysis and was necessary because of the large field of CF
dendritic innervation. At small synaptic contacts laser photolysis
over a smaller area will give time resolution of 0.1 ms with
MNI-caged γ-DGG at individual synapses with the methods
described by Trigo et al. (2009). As well as improved time
resolution, the extent of photolysis is greater with laser activation,
permitting greater economy with the cage and better control of
the concentration released.
Recently the time resolution of tethered genetically encoded
fluorescent glutamate reporters has been improved to the
milliseconds level (Helassa et al., 2018) providing another way to
investigate transmitter concentration profiles at single synapses.
The two approaches are complementary, the present method
more readily applicable because there is no requirement for
transgene or viral labeling. Combining the two approaches is
unlikely because of the strong bleaching action of near-UV on
the GFP reporter fluorescence.
Although MNI cages have properties of speed, stability
and pharmacological inertness it is generally known that they
are very inefficient in two-photon photolysis, requiring high
concentrations and high intensities, releasing a few percent of
the cage concentration at non-toxic intensities. The two-photon
cross-section measured by us and in other studies is 0.05 GM,
whereas the cross-section required for 50% photolysis in the
laser spot is two orders larger at the threshold for non-toxic
exposure, an average power of 5 mW. Phototoxicity of ultra-
short pulsed irradiation is seen presynaptically, characterized by
aberrant release at average powers greater than 5 mW (Hopt and
Neher, 2001; Kiskin et al., 2002). Two-photon excitation of MNI-
caged-γ-DGG to release useful concentrations of γ-DGG would
be phototoxic and too localized to be useful in testing distributed
transmitter release.
In conclusion, we present the synthesis and evaluation of
a fast-caged antagonist MNI-caged-γ-DGG and the methods to
apply it in the analysis of synaptic transmission in this case with
wide-field photolysis at the CF-PC synapse. The results define
Frontiers in Cellular Neuroscience | www.frontiersin.org
10
December 2018 | Volume 12 | Article 465

Palma-Cerda et al.
Caged Fast Antagonist at Glutamate Synapses
the time-window of receptor activation by transmitter, present
evidence for a delay in transmission or release, and persistence of
receptor activation by transmitter late in the EPSC.
University (ANR-11-IDEX-0001-02), Labex (ANR-10-LABX-
54 MEMOLIFE) and Fondation pour la Recherche Médicale
(DEQ20160334927).
AUTHOR CONTRIBUTIONS
ACKNOWLEDGMENTS
FP-C, GP, BB, CA and DO contributed to the conception and
design of the study. GP synthesized and characterized reagents.
FP-C and DO performed experiments and analyzed data. DO
wrote the first draft of the manuscript. All authors contributed to
manuscript revision, read and approved the submitted version.
We thank the animal facility at Universite Paris Descartes
St. Peres for animal care. We thank Dr. Steve Martin
(Francis Crick Institute) for expert help with CD spectra.
We gratefully acknowledge the contribution of Dr. John E.T.
Corrie to the chemical synthesis, and for advice, discussion and
encouragement.
FUNDING
SUPPLEMENTARY MATERIAL
The research was supported by the ANR (FP-C, CA, DO;
ANR-16-CE16-0016), the CNRS and Université Paris Descartes
(DO, CA), the UK Medical Research Council and Francis
Crick Institute (GP). BB was supported by Idex PSL Research
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fncel.
2018.00465/full#supplementary-material
REFERENCES
Jasuja, R., Keyoung, J., Reid, G. P., Trentham, D. R., and Khan, S. (1999).
Chemotactic responses of Escherichia coli to small jumps of photoreleased
L-aspartate. Biophys. J. 76, 1706–1719. doi: 10.1016/s0006-3495(99)
77329-7
Kanazawa, A., Kakimoto, Y., Nakajima, T., Shimizu, H., Takesada, M., and Sano, I.
(1965). Isolation and identification of γ-L-glutamylglycine from bovine brain.
Biochim. Biophys. Acta 97, 460–464. doi: 10.1016/0304-4165(65)90157-1
Katz, B., and Miledi, R. (1965). The effect of temperature on the synaptic delay
at the neuromuscular junction. J. Physiol. 181, 656–670. doi: 10.1113/jphysiol.
1965.sp007790
Kiskin, N. I., Chillingworth, R., McCray, J. A., Piston, D., and Ogden, D.
(2002). The efficiency of two-photon photolysis of a ‘‘caged’’ fluorophore,
o-1–(2-nitrophenyl)ethylpyranine, in relation to photodamage of synaptic
terminals. Eur. Biophys. J. 30, 588–604. doi: 10.1007/s00249-001-0187-x
Lingueglia, E., de Weille, J. R., Bassilana, F., Heurteaux, C., Sakai, H.,
Waldmann, R., et al. (1997). A modulatory subunit of acid sensing ion channels
in brain and dorsal root ganglion cells. J. Biol. Chem. 272, 29778–29783.
doi: 10.1074/jbc.272.47.29778
Liu, G., Choi, S., and Tsien, R. W. (1999). Variability of neurotransmitter
concentration and nonsaturation of postsynaptic AMPA receptors at synapses
in hippocampal cultures and slices. Neuron 22, 395–409. doi: 10.1016/s0896-
6273(00)81099-5
Morrison, J., Wan, P., Corrie, J. E. T., and Papageorgiou, G. (2002).
Mechanisms of photorelease of carboxylic acids from 1-acyl-7-nitroindolines
in solutions of varying water content. Photochem. Photobiol. Sci. 1, 960–969.
doi: 10.1039/b206155d
Nielsen, T. A., DiGregorio, D. A., and Silver, R. A. (2004). Modulation of glutamate
mobility reveals the mechanism underlying slow-rising AMPAR EPSCs and the
diffusion coefficient in the synaptic cleft. Neuron 42, 757–771. doi: 10.1016/j.
neuron.2004.04.003
Palma-Cerda, F., Auger, C., Crawford, D. J., Hodgson, A. C. C., Reynolds, S. J.,
Cowell, J. K., et al. (2012). New caged neurotransmitter analogs selective
for glutamate receptor sub-types based on methoxynitroindoline and
nitrophenylethoxycarbonyl caging groups. Neuropharmacology 63, 624–634.
doi: 10.1016/j.neuropharm.2012.05.010
Papageorgiou, G., Beato, M., and Ogden, D. (2011). Synthesis and photolytic
evaluation of a nitroindoline-caged glycine with a side chain of high negative
charge for use in neuroscience. Tetrahedron 67, 5228–5234. doi: 10.1016/j.tet.
2011.05.045
Auger, C., and Attwell, D. (2000). Fast removal of synaptic glutamate
by postsynaptic transporters. Neuron 28, 547–558. doi: 10.1016/s0896-
6273(00)00132-x
Beato, M. (2008). The time course of transmitter at glycinergic synapses onto
motoneurons. J. Neurosci. 28, 7412–7425. doi: 10.1523/JNEUROSCI.0581-
08.2008
Bellamy, T. C., and Ogden, D. (2005). Short-term plasticity of Bergmann glial cell
extrasynaptic currents during parallel fiber stimulation in rat cerebellum. Glia
52, 325–335. doi: 10.1002/glia.20248
Canepari, M., Nelson, L., Papageorgiou, G., Corrie, J. E. T., and Ogden, D. (2001).
Photochemical and pharmacological evaluation of 7-nitroindolinyl-and
4-methoxy-7-nitroindolinyl-amino
neurotransmitters. J. Neurosci. Methods 112, 29–42. doi: 10.1016/s0165-
0270(01)00451-4
Carbone, A. L., and Plested, A. J. (2016). Superactivation of AMPA receptors by
auxiliary proteins. Nat. Commun. 8:10178. doi: 10.1038/ncomms10178
Clements, J. D., Lester, R. A., Tong, G., Jahr, C. E., and Westbrook, G. L. (1992).
The time course of glutamate in the synaptic cleft. Science 258, 1498–1501.
doi: 10.1126/science.1359647
Coddington, L. T., Nietz, L. K., and Wadiche, J. I. (2014). The contribution of
extrasynaptic signalling to cerebellar information processing. Cerebellum 13,
513–520. doi: 10.1007/s12311-014-0554-7
Davies, J., and Watkins, J. C. (1981). Differentiation of kainate and quisqualate
receptors in the cat spinal cord by selective antagonism with γ-D (and
L)-glutamylglycine. Brain Res. 206, 172–177. doi: 10.1016/0006-8993(81)
90111-6
Devi, S. P., Howe, J. R., and Auger, C. (2016). Train stimulation of parallel fiber to
Purkinje cell inputs reveals two populations of synaptic responses with different
receptor signatures. J. Physiol. 594, 3705–3727. doi: 10.1113/jp272415
Forsythe, I. D. (1994). Direct patch recording from identified presynaptic
terminals mediating glutamatergic EPSCs in the rat CNS, in vitro. J. Physiol.
479, 381–387. doi: 10.1113/jphysiol.1994.sp020303
Francis, A. A., Jones, A. W., and Watkins, J. C. (1980). Dipeptide antagonists
of amino acid-induced and synaptic excitation in the frog spinal cord.
J. Neurochem. 35, 1458–1460. doi: 10.1111/j.1471-4159.1980.tb09025.x
Helassa, N., Dürst, C.-D., Coates, C., Kerruth, S., Arif, U., Schulze, C., et al. (2018).
Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral
synapses. Proc. Natl. Acad. Sci. U S A 115, 5594–5599. doi: 10.1073/pnas.
1720648115
acids
as
novel,
fast
caged
Papageorgiou, G., and Corrie, J. E. T. (2000). Effects of aromatic substituents
on the photocleavage of 1-acyl-7-nitroindolines. Tetrahedron 56, 8197–8205.
doi: 10.1016/s0040-4020(00)00745-6
Papageorgiou, G., Ogden, D., Barth, A., and Corrie, J. E. T. (1999). Photorelease
of carboxylic acids from 1-acyl-7-nitroindolines in aqueous solution: rapid
Hopt, A., and Neher, E. (2001). Highly nonlinear photodamage in two-photon
fluorescence microscopy. Biophys. J. 80, 2029–2036. doi: 10.1016/s0006-
3495(01)76173-5
Frontiers in Cellular Neuroscience | www.frontiersin.org
11
December 2018 | Volume 12 | Article 465

Palma-Cerda et al.
Caged Fast Antagonist at Glutamate Synapses
and efficient photorelease of L-glutamate. J. Am. Chem. Soc. 121, 6503–6504.
doi: 10.1021/ja990931e
Wadiche, J. I., and Jahr, C. E. (2001). Multivesicular release at climbing
fiber-Purkinje cell synapses. Neuron 32, 301–313. doi: 10.1016/s0896-
6273(01)00488-3
Xia, P., Bungay, P. M., Gibson, C. C., Kovbasnjuk, O. N., and Spring, K. R.
(1998). Diffusion coefficients in the lateral intercellular spaces of Madin-Darby
canine kidney cell epithelium determined with caged compounds. Biophys. J.
74, 3302–3312. doi: 10.1016/s0006-3495(98)78037-3
Zhang, W., Devi, S. P., Tomita, S., and Howe, J. R. (2014). Auxiliary proteins
promote modal gating of AMPA- and kainate-type glutamate receptors. Eur.
J. Neurosci. 39, 1138–1147. doi: 10.1111/ejn.12519
Papageorgiou, G., Ogden, D., and Corrie, J. E. T. (2008). An antenna-sensitised
1-acyl-7-nitroindoline that has good solubility properties in the presence of
calcium ions and is suitable for use as a caged L-glutamate in neuroscience.
Photochem. Photobiol. Sci. 7, 423–432. doi: 10.1039/b800683k
Scimemi, A., and Beato, M. (2009). Determining the neurotransmitter
concentration profile at active synapses. Mol. Neurobiol. 40, 289–306.
doi: 10.1007/s12035-009-8087-7
Szapiro, G., and Barbour, B. (2007). Multiple climbing fibers signal to molecular
layer interneurons exclusively via glutamate spillover. Nat. Neurosci. 10,
735–742. doi: 10.1038/nn1907
Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Szapiro, G., and Barbour, B. (2009). Parasynaptic signalling by fast
neurotransmitters: the cerebellar cortex. Neuroscience 162, 644–655.
doi: 10.1016/j.neuroscience.2009.03.077
Trigo, F. F., Corrie, J. E. T., and Ogden, D. (2009). Laser photolysis of caged
compounds at 405 nm: photochemical advantages, localisation, phototoxicity
and methods for calibration. J. Neurosci. Methods 180, 9–21. doi: 10.1016/j.
jneumeth.2009.01.032
Trigo, F. F., Sakaba, T., Ogden, D., and Marty, A. (2012). Readily releasable pool
of synaptic vesicles measured at single synaptic contacts. Proc. Natl. Acad. Sci.
U S A 109, 18138–18143. doi: 10.1073/pnas.1209798109
Copyright © 2018 Palma-Cerda, Papageorgiou, Barbour, Auger and Ogden. This
is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums
is permitted, provided the original author(s) and the copyright owner(s) are credited
and that the original publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution or reproduction is permitted which does not
comply with these terms.
Frontiers in Cellular Neuroscience | www.frontiersin.org
12
December 2018 | Volume 12 | Article 465