Optical control of pain in vivo with a photoactive mGlu5 receptor negative allosteric modulator

Article abstract of DOI:10.7554/eLife.23545

Light-operated drugs constitute a major target in drug discovery, since they may provide spatiotemporal resolution for the treatment of complex diseases (i.e. chronic pain). JF-NP-26 is an inactive photocaged derivative of the metabotropic glutamate type

Full text of DOI:10.7554/eLife.23545

RESEARCH ARTICLE
Optical control of pain in vivo with a
photoactive mGlu receptor negative
5
allosteric modulator
1
,2,3†
4,5†
6
6
Joan Font
, Marc L o´ pez-Cano , Serena Notartomaso , Pamela Scarselli ,
6
7
6
8
Paola Di Pietro , Roger Bresol ´ı -Obach , Giuseppe Battaglia , Fanny Malhaire ,
8
1
2,3,9
8
Xavier Rovira , Juanlo Catena , Jes u´ s Giraldo
, Jean-Philippe Pin ,
4
,5
8
7
V ´ı ctor Fern a´ ndez-Due n˜ as , Cyril Goudet , Santi Nonell ,
6
,10
1
4,5
Ferdinando Nicoletti , Amadeu Llebaria *, Francisco Ciruela
*
1
MCS, Laboratory of Medicinal Chemistry, Institute for Advanced Chemistry of
2
Catalonia (IQAC-CSIC), Barcelona, Spain; Institut de Neuroci e` ncies, Universitat
Aut o` noma de Barcelona, Bellaterra, Spain; Unitat de Bioestad ı´ stica, Universitat
Aut o` noma de Barcelona, Bellaterra, Spain; Departament de Patologia i Terap e` utica
3
4
Experimental, Facultat de Medicina i Ci e` ncies de la Salut, IDIBELL, Universitat de
5
Barcelona, Barcelona, Spain; Institut de Neuroci e` ncies, Universitat de Barcelona,
6
7
Barcelona, Spain; I.R.C.C.S. Neuromed, Pozzilli, Italy; Institut Qu ı´ mic de Sarri a` ,
8
Universitat Ramon Llull, Barcelona, Spain; IGF, CNRS, INSERM, Univ. Montpellier,
Montpellier, France; Network Biomedical Research Center on Mental Health
CIBERSAM), Instituto de Salud Carlos III, Madrid, Spain; Department of
9
1
0
(
Physiology and Pharmacology, University Sapienza, Rome, Italy
*
For correspondence: amadeu.
Abstract Light-operated drugs constitute a major target in drug discovery, since they may
provide spatiotemporal resolution for the treatment of complex diseases (i.e. chronic pain). JF-NP-
llebaria@iqac.csic.es (AL);
fciruela@ub.edu (FC)
5
26 is an inactive photocaged derivative of the metabotropic glutamate type 5 (mGlu ) receptor
†
negative allosteric modulator raseglurant. Violet light illumination of JF-NP-26 induces a
photochemical reaction prompting the active-drug’s release, which effectively controls mGlu
These authors contributed
equally to this work
5
receptor activity both in ectopic expressing systems and in striatal primary neurons. Systemic
administration in mice followed by local light-emitting diode (LED)-based illumination, either of the
thalamus or the peripheral tissues, induced JF-NP-26-mediated light-dependent analgesia both in
neuropathic and in acute/tonic inflammatory pain models. These data offer the first example of
Competing interests: The
authors declare that no
competing interests exist.
Funding: See page 16
5
optical control of analgesia in vivo using a photocaged mGlu receptor negative allosteric
modulator. This approach shows potential for precisely targeting, in time and space, endogenous
receptors, which may allow a better management of difficult-to-treat disorders.
DOI: 10.7554/eLife.23545.001
Received: 22 November 2016
Accepted: 19 March 2017
Published: 11 April 2017
Reviewing editor: Gary L
Westbrook, Vollum Institute,
United States
Introduction
Copyright Font et al. This
article is distributed under the
terms of the Creative Commons
Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited.
Metabotropic glutamate (mGlu) receptors are widely distributed along the pain neuraxis and modu-
late pain transmission (Kolber, 2015) at different anatomical levels. Hence, subtype-selective mGlu
receptor ligands are considered as promising candidate drugs for the treatment of chronic pain.
Accordingly, selective negative allosteric modulators (NAMs) of mGlu
1 5
or mGlu receptors, and ago-
nists or positive allosteric modulators (PAMs) of mGlu or mGlu receptors have consistently been
2
4
shown to display analgesic activity in experimental animal models of chronic pain (Montana and
Gereau, 2011). In particular, mGlu receptor NAMs (i.e. raseglurant) are under development for the
5
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treatment of neuropathic pain and migraine; however, their systemic use may be limited by mecha-
nism-related adverse effects, such as hepatotoxicity, cognitive impairment and psychotomimetic
effects (Friedmann et al., 1980; Gravius et al., 2005; Homayoun et al., 2004; Kinney et al., 2003).
The use of light in optogenetics (Tye and Deisseroth, 2012) and photopharmacology
(Kramer et al., 2013; Lerch et al., 2016) to control activity of living neurons is a ground-breaking
approach that is reaching a high impact in experimental neuroscience (Boyden, 2015) and drug dis-
covery (Song and Kn o¨ pfel, 2016). Photopharmacology is based on the use of photoactive drugs
that can trigger biological responses upon light irradiation at appropriate wavelengths. Accordingly,
temporal and spatial remote control of drug activity is possible with a highly precise regulation of its
in vivo effects. Differently from optogenetics, the application of photoregulated ligands and optical
techniques allows the modulation of native proteins without genetic manipulation, thus facilitating
translation into therapeutics with protocols similar to those employed in conventional drug discovery
and clinical practice. The potential application of this strategy to pain treatment is highlighted by
the evidence that a photoisomerizable molecule entering nociceptive neurons through the TrpV1 ion
channel causes analgesia by inhibiting voltage-gate ion channels in response to illumnation
(
Mourot et al., 2012). We developed alloswitch-1, a selective photoswitchable mGlu5 receptor
NAM, which allowed the optical control of endogenous receptors (Pittolo et al., 2014), in an
attempt to achieve a light-dependent control of mGlu receptors within the pain neuraxis. However,
alloswitch-1 and related photoswitchable compounds show mGlu NAM activity in its natural trans
configuration in the dark, thereby causing light-independent analgesic effects in animals (G o´ mez-
Santacana et al., 2017). Therefore, we aimed to synthesize an inactive mGlu receptor NAM, which
5
5
5
is converted into an effective analgesic drug upon illumination. An effective strategy to achieve this
would consist of developing inactive photosensitive mGlu receptor-based drugs, which may there-
5
after be activated by light administration (photo-uncaging) exclusively in brain regions critically
involved in the control of pain. This approach may allow the release of the active drug in an anatomi-
cally restricted fashion and with regulated dosing. The photodelivery of biologically active molecules
has been usually addressed using either direct compound photolysis (Ellis-Davies, 2007) or light-
triggered release from nanosystems (Fomina et al., 2012), although uncaging in vivo has been sel-
dom used in rodents (Crowe et al., 2010; Takano et al., 2006). Here, we aimed to develop a photo-
activatable mGlu5 receptor NAM, which upon local illumination in the hind paw or the thalamus
would exert analgesic effects.
Results
5
Design and synthesis of a caged mGlu receptor NAM. We applied a caging strategy to generate a
photocaged compound based on the chemical binding of the mGlu5 receptor NAM, raseglurant
ADX-10059), to a coumarinyl phototrigger (Figure 1). Thus, we synthesized JF-NP-26 (Figure 1) by
(
modifying the aromatic amine group in raseglurant to generate a carbamate derivative of the violet-
light absorbing coumarin DEACM in a one pot procedure (Figure 1).
Transformation of the amino group of raseglurant into the coumarinyl carbamate shifted its main
UV-vis absorption band from 338 nm to around 313 nm, and a second absorption peak appeared at
386 nm due to the coumarinyl phototrigger (Figure 2A). Subsequently, we assessed the photochem-
ical behaviour of JF-NP-26 upon exposure to 405 nm light (Figure 2B), which is safer than UV radia-
tion for our in vivo biological experiments. The UV-visible absorption spectrum of JF-NP-26 showed
dramatic changes upon 405 nm irradiation (Figure 2C), consistent with the photo-induced release of
raseglurant and leaving the coumarinyl alcohol DEACM (Figure 2A). The photouncaging quantum
yield was determined by potassium ferrioxalate actinometry (Kuhn et al., 1989), as ’chem = 0.18
(Figure 2D). This process is likely involving the photolysis of the benzylic bond in JF-NP-26, thus
generating a carbamic acid derivative that spontaneously decarboxylates to give raseglurant.
Optical modulation of mGlu receptor activity with JF-NP-26 in cultured
5
cells and in primary neurons
Subsequently, we assessed the JF-NP-26-mediated negative allosteric modulation of mGlu recep-
5
tor-induced responses to the orthosteric agonist quisqualate, by using an inositol phosphate (IP)
accumulation assay (Figure 3A). Interestingly, while JF-NP-26 didn’t show activity in dark conditions,
its NAM activity was rescued upon 405 nm visible light illumination (pIC50 = 7.1; Figure 3A). Thus, a
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Figure 1. Design and synthesis of JF-NP-26. The synthesis of JF-NP-26 from raseglurant involves a one-pot procedure using raseglurant and 4-
hydroxymethyl-7-diethylaminocoumarin (DEACM). In brief, a first reaction with triphosgene, NEt and toluene for 3 hr was followed by an incubation
3
with DEACM, NaH and THF at 100 C for 12 hr (see Materials and methods). Upon irradiation with 405 nm visible light the irreversible photolytic reaction
˚
produced raseglurant. The following figure supplements are available for Figure 1.
DOI: 10.7554/eLife.23545.002
The following figure supplements are available for figure 1:
Figure supplement 1. Supporting spectra for the synthesis of raseglurant.
DOI: 10.7554/eLife.23545.003
Figure supplement 2. Supporting spectra for the synthesis of JF-NP-26.
DOI: 10.7554/eLife.23545.004
5
light-dependent gain of function was demonstrated for JF-NP-26 in the mGlu -mediated IP accumu-
lation assay. In addition, we evaluated the ability of JF-NP-26 to photomodulate the mGlu recep-
5
tor-mediated intracellular calcium accumulation in cultured cells ectopically expressing the receptor
5
(Figure 3B). Agonist challenge induced a robust mGlu receptor-mediated intracellular calcium rise
both in dark and under 405 nm illumination, which was blocked by raseglurant (Figure 3B). Interest-
ingly, again while JF-NP-26 was unable to restrain agonist-mediated signalling in dark conditions, it
abolished mGlu5 receptor-mediated intracellular calcium accumulation upon 405 nm irradiation
(Figure 3C), thus demonstrating a light-dependent NAM activity. Next, we performed similar
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Figure 2. Photochemical properties of JF-NP-26. (A) UV-visible absorption spectra of raseglurant (Ras), the coumarine DEACM, and JF-NP-26 (JF) in
PBS. The sum of the free Ras and DEACM spectra shows important differences relative to that of their conjugate JF (B) Neuronal viability upon 405 nm
irradiation. The neuronal viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)À2,5-diphenyltetrazolium bromide (MTT) assay (see Supplementary
Information). Cultured neurons were irradiated with 405 nm light during 5, 15 and 160 min before the MTT incubation 3% hydrogen peroxide (HP) was
used a positive control of cell death. All values were normalized using intact neurons as a 100% of viability threshold and expressed as mean ± S.E.M. of
three independent experiments performed in triplicate. (C) Changes in the absorption spectrum of JF-NP-26 upon illumination in PBS with 405 nm
light. The spectrum of the irradiated sample shows an excellent match with the sum of the free Ras and DEACM spectra in panel A. (D) Determination
of the quantum yield of raseglurant photouncaging by comparison of the rate of this process with the rate of a standard photochemical reaction,
r
namely ferrioxalate photoreduction (’ = 1.14 at 405 nm, see Experimental Section).
DOI: 10.7554/eLife.23545.005
calcium experiments in cultured neurons from mouse striatum (Figure 3D) to assess the light-depen-
dency of NAM activity in a native system (Figure 3E). Results (Figure 3F) were comparable to those
obtained in heterologous expression systems (Figure 3C), thus indicating that optical modulation of
JF-NP-26 could also be performed in a native receptor environment.
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Figure 3. Optical modulation of mGlu
Determination of mGlu receptor-mediated inositol phosphate accumulation in transiently transfected HEK-293T
cells. Normalized dose-response of raseglurant (Ras)- and JF-NP-26 (JF)-mediated inhibition of quisqualate (1 mM)-
induced IP accumulation in dark or upon irradiation at 405 nm (violet trace) in mGlu receptor expressing cells.
Each data point corresponds to mean± SEM of three independent experiments performed in triplicate. The pEC50
value determined for quisqualate was 7.52. (B) Transiently transfected HEK-293T cells with the mGlu receptor
were pretreated with vehicle (veh), raseglurant (Ras) and JF-NP-26 (JF) at 1 mM in the dark (left panel) or upon 405
nm irradiation (right), and then challenged with quisqualate (Quis, 100 mM). (C) Quantification of mGlu receptor
5
receptor activity in cultured cells and in primary striatal neurons. (A)
5
5
5
5
inhibition. The percentage of receptor inhibition is calculated as described in Materials and methods and
expressed as mean ± S.E.M. (n = 10). ***p<0.001, one-way ANOVA with Dunnett’s multiple comparison test using
the Veh+dark condition as a control. (D) Expression of mGlu
neurons were fixed, permeabilized and immunostained using a rabbit anti-mGlu
primary antibody was detected using Cy3-conjugated donkey anti-rabbit antibody (1/200). Neurons were analyzed
by single immunofluorescence with a confocal microscopy. Scale bar: 10 mm. (E) Determination of mGlu receptor-
5
receptor in primary striatal neurons. Primary striatal
5
receptor (1 mg/ml) antibody. The
5
mediated intracellular calcium accumulation in primary striatal neurons. Neurons were incubated with vehicle (veh),
raseglurant (Ras) or JF-NP-26 (JF), then kept in the dark (left panel) or irradiated at 405 nm (right panel) before
5
being challenged with quisqualate (Quis) (see Materials and methods). (F) Quantification of mGlu receptor
inhibition. The percentage of receptor inhibition is calculated as described in the Material and methods section
and expressed as mean ± S.E.M. (n = 5–10). **p<0.005 and ***p<0.001, one-way ANOVA with Dunnett’s multiple
comparison test using the Veh+dark condition as a control.
DOI: 10.7554/eLife.23545.006
Peripheral and central antinociceptive action of JF-NP-26 upon in vivo
photoactivation
We assessed the in vivo analgesic activity of JF-NP-26 activated by light in two established models
of pain in mice: the chronic constriction injury (CCI) model of neuropathic pain and the formalin test
(Figure 4A) (Mogil, 2009).
First, we proved the validity of our approach in mice subjected to unilateral CCI of the sciatic
5
nerve, in which we explored the contribution of central mGlu receptors by photomodulating these
receptors in the ventrobasal thalamus, a pivotal relay and processing point for somatosensory infor-
mation ascending from the spinal cord to the cerebral cortex (Kolber, 2015; Palazzo et al., 2014).
Accordingly, we implanted optical fibers in the brain to allow precise light delivery into the ventro-
basal thalamus (Figure 4A). Importantly, we used a LED lamp illumination system, not requiring the
use of laser sources to achieve the photorelease of the active compound, and assessed mechanical
pain thresholds by means of von Frey filaments (Figure 4A). Interestingly, a single injection of rase-
glurant (10 mg/kg, i.p.) significantly increased pain thresholds in CCI mice regardless of light
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Figure 4. In vivo assessment of JF-NP-26 light-dependent analgesic efficacy in neuropathic pain. (A) Scheme
showing the different LED-mediated irradiation points within the pain neuraxis (i.e. hind paw and thalamus) and
pain triggering (i.e. hind paw formalin administration and chronic constriction injury -CCI- of the sciatic nerve).
VPM, ventral posteromedial nucleus. (B) Chronic constriction injury (CCI) of the sciatic nerve causing mechanical
allodynia. Mechanical thresholds were measured in 21 days post-surgery CCI mice. Thus, animals were
intraperitoneally injected with vehicle (Veh, saline), raseglurant (Ras, 10 mg/kg) or JF-NP-026 (JF, 10 mg/kg) 20 min
before the thalamus was irradiated at 405 nm (or dark) for 5 min. Subsequently, the mechanical thresholds were
assessed before and after light irradiation. Values are means ± S.E.M. of 7–9 mice per group. **p<0.05, Student t
test, JF-NP-26 dark vs. JF-NP-26 irradiated and raseglurant dark vs. Veh dark.
DOI: 10.7554/eLife.23545.007
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irradiation (Figure 4B; data with raseglurant after irradiation are not shown). In contrast, systemic
injection of JF-NP-26 (10 mg/kg, i.p.) significantly (p<0.01) increased pain thresholds in CCI mice
only after thalamic irradiation (Figure 4B).
On the basis of this proof of principle success, we took advantage of a different mice model of
pain to further investigate the analgesic activity of light-delivered JF-NP-26 either in the periphery or
in ventrobasal thalamus. The formalin test, which allows an objective analysis of pain based on noci-
fensive behavior, displays a first phase of pain (5 min after formalin injection in the hind paw), which
models acute inflammatory pain, and a second phase (20–30 min after formalin injection), which
models chronic inflammatory pain and reflects the development of central sensitization
(Mogil, 2009). Formalin injection into the mouse hind paw induced an innate licking behavior that
was significantly reduced by systemic administration of raseglurant (10 mg/kg, i.p., Figure 5, middle
panel), thus demonstrating an antinociceptive efficacy both in phase I (70 ± 3%, p<0.001) and phase
II (97 ± 1%, p<0.001) of the formalin test. We then directly irradiated the ipsilateral hind paw with
external 405 nm light (Figure 4A) following the experimental regimen shown in Figure 5 (upper
panel). Noteworthy, while JF-NP-26 (10 mg/kg, i.p.) was unable to promote antinociception in dark
conditions (Video 1), it elicited antinociception following direct hind paw irradiation both at phase I
(54 ± 5%, p<0.001) and phase II (34 ± 5%, p<0.001) (Figure 5, middle panel; Video 2), demonstrat-
ing that JF-NP-26 was able to be peripherally photoactivated. Interestingly, under the same experi-
mental conditions when the contralateral hind paw was irradiated no antinociceptive effect was
observed (Figure 5, middle panel, column C), thus demonstrating that a non-site specific peripheral
photoconversion of JF-NP-26 does not yield systemic raseglurant-mediated analgesia. On the other
hand, we also evaluated the effects of JF-NP-26 by delivering light into the ventrobasal thalamus. As
shown in Figure 5 (lower panel), implantation of the illumination system by itself did not affect noci-
fensive responses in the formalin test and did not alter the analgesic activity of raseglurant, as
expected. While JF-NP-26 (10 mg/kg, i.p.) was inactive under basal conditions, it was able to cause
substantial analgesia both in phase I (45 ± 9%, p<0.01) and phase II (90 ± 4%, p<0.001) of the forma-
lin test in response to thalamic irradiation (Figure 5, lower panel). Importantly, under the same
experimental conditions when the striatum was irradiated no antinociceptive effect was observed
(Figure 5, lower panel, column S), indicating that a CNS region of the pain neuraxis must be specifi-
cally irradiated to obtain pain control by JF-NP-26. Significantlly, uncaging of JF-NP-26 in the thala-
mus produced a stronger analgesic effect in phase II of the formalin test, which reflects mechanisms
of central sensitization. Finally, the thalamic irradiation effects of JF-NP-26 were obtained after intra-
peritoneal administration, demonstrating its effective brain penetration in mice. Overall, upon local
and thalamic photoactivation, JF-NP-26 demonstrated analgesic effects in two different models of
pain, thus raising the interesting possibility that localized light targeting of peripheral and thalamic
mGlu receptors represent a valuable strategy for the treatment of pain diseases.
5
Systemic administration and in vivo photoactivation of JF-NP-26 does
not impair memory in mouse
Raseglurant has been shown to possess effectiveness on migraine treatment (Keywood and Wake-
field, 2008). However, despite good clinical efficacy, tolerability and safety in a 2-week double-
blind, placebo-controlled, multicentre trial (Zerbib et al., 2011), the long-term administration of
raseglurant in migraine patients resulted in a disappointing high incidence of hepatic transaminase
abnormalities, thus halting any further clinical development (Marin and Goadsby, 2010). Hence, we
aimed to assess if raseglurant and JF-NP-26 induced liver toxicity in mice. To this end, we deter-
mined serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) after a
3-day high dose (i.e. 50 mg/kg) raseglurant and JF-NP-26 treatment, thus harsher conditions to that
used in our antinociceptive experiments (i.e. single dose of 10 mg/kg, ip). Interestingly, while a 3-
day acetaminophen treatment (300 mg/kg, ip) induced a significant increase in serum ALT and AST
(Figure 6A), as described (Zhang et al., 2015), the 3-day raseglurant and JF-NP-26 treatment (50
mg/kg, ip) did not alter ALT and AST serum levels (Figure 6A). Thus, these results demonstrated
that raseglurant and JF-NP-26 treatments used in our pain animal models were safe because no liver
toxicity was observed.
On the other hand, it has been reported that mGlu5 receptor NAMs may produce behavioural
undesirable side effects. For instance, systemic administration of MPEP, a mGlu5 receptor NAM,
reduced reference memory in rats (Christoffersen et al., 2008). Thus, we aimed to explore whether
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Figure 5. Peripheral and central light-dependent JF-NP-26-mediated antinociception in mice. In the upper panel a
scheme of the irradiation regime at 405 nm light (violet rectangles) and liking recordings (green rectangles - Phase
I and Phase II) in the formalin animal model of pain is shown. Thus, animals were intraperitoneally injected with
vehicle (Veh, 20%DMSO + 20%tween80 in saline), raseglurant (Ras, 10 mg/kg) or JF-NP-026 (JF, 10 mg/kg) 20 min
before the hind paw or the thalamus was irradiated at 405 nm (or dark) for 5 min. As a control, the contralateral
hind paw (C) or the striatum (S) were also irradiated. Subsequently, the total hind paw licking was measured for 0–
5
min (Phase I) and 15–30 min (Phase II) after intraplantar injection of 20 ml of formalin solution (2.5%
paraformaldehide). The antinociceptive effect was calculated as the percentage of the maximum possible effect
mean ± S.E.M., n = 5–6 mice per group). **p<0.01 and ***p<0.001, one-way ANOVA with Dunnett’s multiple
(
comparison test using Veh+dark as a control.
DOI: 10.7554/eLife.23545.008
systemic administration and local photoactivation of JF-NP-26 circumvented raseglurant-associated
adverse effects. To this end, we assessed the impact of raseglurant and JF-NP-26 in mouse working/
reference memory by using the novel object recognition test. As expected, naive mice injected with
vehicle spent significantly more time in exploring the novel object than the familiar object
(
Figure 6B). Interestingly, raseglurant impaired novel object recognition at the dose of 20 mg/kg
Figure 6B), although it was inactive at 10 mg/kg (not shown). Optic fiber implanted mice treated
(
with vehicle or JF-NP-26 (10 or 20 mg/kg) and irradiated bilaterally at the ventrobasal thalamus (see
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above) showed a normal performance in the
novel object recognition test (Figure 6A). Over-
all, these results demonstrated that neither the
systemic administration nor the thalamic photo-
activation of JF-NP-26 cause memory impairment
in mice.
Discussion
We report a new pharmacological approach
based on analgesic ligands endowed with light-
dependent activity. This strategy allows the pho-
tocontrol of mGlu
5
receptors at different stations
of the pain neuraxis.
Video 1. Video showing formalin-induced hind paw
To our knowledge, JF-NP-26 is the first caged
mGlu5 receptor NAM that has been shown to
induce light-dependent analgesia in models of
inflammatory and neuropathic pain in freely
behaving animals. The ability to resolve analgesia
licking in a JF-NP-26 -treated mouse upon dark
conditions from Figure 5.
15 cm ø x 20 cm height), was administered with JF-NP-
6 (10 mg/kg, i.p.). After 20 min, the LED-based
irradiation system was attached to the thalamic
Mouse, in a glass beaker
(
2
implanted optic fiber and mock irradiated (dark) during with a fine resolution in time and space confers
min. Then, formalin solution (20 mL) was intraplantarly to our experimental approach a high significance
5
(
i.pl.) injected and total licking or biting time of the
for further development of new caged drugs act-
ing at different levels of the pain neuraxis.
hind paw was recorded. A 30 s’ video fragment
exemplifying the formalin test is shown.
DOI: 10.7554/eLife.23545.010
Raseglurant is a mGlu receptor NAM that has
5
been shown to possess efficacy on the treatment
of migraine (Keywood and Wakefield, 2008).
However, its clinical development was discontin-
ued because of liver toxicity (Marin and
Goadsby, 2010). Accordingly, we synthesized a caged compound (JF-NP-26) that upon illumination
would release raseglurant only in the areas of
interest, thus avoiding potential systemic unde-
sired effects associated to mGlu receptor func-
5
tioning (i.e. memory impairment). Of note,
uncaging of JF-NP-26 was designed to be eli-
cited with light pulses in the visible spectrum
(
405 nm). This property differentiates JF-NP-26
from the majority of caged compounds that
require UV light for activation. This is highly
advantageous for translational research because
light within the visible spectrum has a good tis-
sue penetration and causes less tissue damage
with respect to UV light (Regan and Parrish,
1993). Remarkably our LED-based illumination
allowed the effective transdermal uncaging of
JF-NP-26 at the mouse hind paw, just where
many noxious stimuli are detected and trans-
Video 2. Video showing formalin-induced hind paw
licking in a JF-NP-26-treated mouse upon 405 nm
duced by peripheral nociceptors. In fact, mGlu
5
irradiation from Figure 5.
15 cm ø x 20 cm height), was administered with JF-NP-
6 (10 mg/kg, i.p.). After 20 min, the LED-based
Mouse, in a glass beaker
receptors are expressed in peripheral nocicep-
tors, where they contribute to mechanisms of
nociceptive sensitization by enhancing the activ-
ity of TRPV1 channels through a complex mecha-
nism mediated by inositol phospholipid
hydrolysis, prostaglandin formation, and protein
kinase A-dependent phosphorylation of TRPV1
(Bhave et al., 2001; Karim et al., 2001; Neuge-
bauer, 2001; Hu et al., 2002). Thus, raseglurant
(
2
irradiation system was attached to the thalamic
implanted optic fiber and irradiated with 405 nm visible
light during 5 min. Then, formalin solution (20 mL) was
intraplantarly (i.pl.) injected and total licking or biting
time of the hind paw was recorded. A 30 s’ video
fragment exemplifying the formalin test is shown.
DOI: 10.7554/eLife.23545.011
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Figure 6. Effect of systemic administration and in vivo photoactivation of JF-NP-26 in liver toxicity and memory in
mouse. (A) Effect of Raseglurant and JF-NP-26 on serum transaminases. Mice were treated with vehicle (veh),
acetaminophen treatment (APAP; 300 mg/kg, ip), raseglurant (Ras; 50 mg/kg, ip) or JF-NP-26 (JF; 50 mg/kg, ip) for
three consecutive days. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were
determined as described in Materials and methods. The results are expressed as means ± S.E.M. of three mice per
group. **p<0.01, ***p<0.001 (One-Way ANOVA followed by Dunnett’s post hoc test using the vehicle condition as
a control. (B) Novel object recognition test. The time spent in exploring novel and familiar objects were measured
35 min after drug administration. Naive mice were intraperitoneally injected with vehicle (6% DMSO +6% tween80
in saline) or raseglurant (10 or 20 mg/kg). Mice bilaterally implanted with optic fibers in the thalamus were injected
with vehicle or JF-NP-26 3 (10 or 20 mg/kg) 15 min before light irradiatiation (405 nm for 5 min). Values are means
±
S.E.M. of 4–7 mice per group. *p<0.05 (One-Way ANOVA + Fisher’s LSD test) vs. the respective values of the
time spent with the familiar object (F(7,38) = 7.28). Doses of 10 mg/kg of either raseglurant in naive mice or JF-NP-
6 three in light-irradiated mice did not affect recognition memory. These data are not included in the graph.
2
DOI: 10.7554/eLife.23545.009
released from JF-NP-26 in response to light irradiation in the hind paw may interact with mGlu
5
receptors present in peripheral nociceptors, thereby causing analgesia in the formalin test. In line
with this, intraplantar JF-NP-26 uncaging showed higher antinociceptive efficacy in the first phase of
the formalin test, which reflects the activation of peripheral nociceptors (Hunskaar and Hole, 1987).
One of the major findings of the present work consists of the local photoactivation of JF-NP-26
to give the active compound after its systemic (i.p.) administration. Accordingly, mice were systemi-
cally injected with JF-NP-26, and fast antinociceptive effects were produced by light irradiation in
the ventrobasal thalamus, the main relay station of the ascending pain pathway modulating the
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sensory, cognitive, and emotional components of pain. Light-mediated delivery of raseglurant in the
thalamus showed a greater antinociceptive efficacy during the second phase of the formalin test,
which reflects the development of central sensitization to inflammatory pain. Hence, our data sug-
5
gested that mGlu receptors located at two different stations of the pain neuraxis (i.e., peripheral
nociceptors and ventrobasal thalamic nuclei) might be contributing differentially to acute inflamma-
tory pain and mechanisms of central sensitization Importantly, JF-NP-26 also showed fast analgesic
activity in the CCI model of neuropathic pain in response to thalamic irradiation. Neuropathic pain is
characterized by a robust and long-lasting nociceptive sensitization that reflects a maladaptive pro-
cess of activity-dependent synaptic plasticity (Basbaum et al., 2009). Interestingly, mGlu receptors
5
participate on the expression of this pathological form of synaptic plasticity at different levels of the
pain neuraxis, from the spinal cord to the regions of the pain matrix including the amygdala and
cerebral cortex (Neugebauer et al., 2003; Li and Neugebauer, 2004; Hu et al., 2007; Hu and Ger-
eau, 2011; Lin et al., 2015). Our data indicate that thalamic mGlu5 receptors are critical for the
expression of nociceptive sensitization and can be specifically targeted by analgesic drugs in the
treatment of neuropathic pain. Overall, our research provided valuable information regarding the
5
contribution of mGlu receptors to the processing of nociceptive transmission at specific anatomic
loci within the pain neuraxis (e.g., central vs. peripheral) as well as its participation in the modulation
of both acute inflammatory and chronic neuropathic pain.
In conclusion, systemic administration of inactive drugs that are activated by light on demand
may provide a powerful strategy to reach a rapid control of pain limiting the development of dose-
and mechanism-related adverse effects that are typically encountered with conventional analgesic
5
drugs. Our positive results constitute a proof of principle that mGlu receptor NAM uncaging may
be a suitable therapeutic approach for the local control of pain, which could be applied in general to
conditions of severe pain that are difficult to treat, and, in particular, to conditions of fast-onset
severe pain that requires a rapid and highly localized treatment. However, the transition from pre-
to clinical settings still has to solve a number of practical problems, including assessment of photoac-
tive compound stability and toxicity, as well as the development of implantable and remotely con-
trolled LEDs covering the photochemical needs to release the active compounds with light.
Materials and methods
Synthesis of drugs
All the chemicals and solvents were provided from commercial suppliers and used without purifica-
tion, except the anhydrous solvents, which were treated previously through a system of solvent puri-
fication (PureSolv), degasified with inert gases and dried over alumina or molecular sieve (dimethyl
formamide). Dry triethylamine was distilled over calcium hydride. Building blocks 2-bromo-4,6-dime-
thylpyridin-3-amine (OR7028) and 1-ethynyl-3-fluorobenzene (PC5956, 98%), were purchased from
Apollo Scientific, bis(triphenylphosphine)palladium (II) dichloride (412740, ꢀ 99% trace metal basis)
and copper iodide (03140, purum, ꢀ99,5%) from Sigma-Aldrich (St. Louis, MO, USA). DCEAM cou-
1
marin was obtained following the protocol reported in the literature , starting from 7-diethylamino-
4-methylcoumarin (M063, ꢀ98%), purchased from TCI. Reactions were monitored by thin layer chro-
matography (60 F, 0.2 mm, Macherey-Nagel) by visualisation under 254 and/or 365 nm lamp.
Reverse phase column chromatography KP-C18-HS 12g (Biotage), automated with Isolera One with
UV-Vis detection (Biotage) using CV (Column volumes) as units for elution gradient, corresponding 1
CV to 1,28 min at 14 mL/min of flux. Flash column chromatography SNAP KP Sil 50 microns (Biot-
age), automated with IsoleraOnewith UV-Vis detection (Biotage). mp: melting point B-545 (B u¨ chi),
ramp 2 C/min. NMR: Variant-Mercury 400 MHz (Agilent Technologies). Chemical shifts are reported
˚
in parts per million (ppm) against the reference compound tetramethysilane using the signal of the
1
13
residual non-deuterated solvent (Chloroform d = 7.26 ppm ( H), d = 77.16 ppm ( C), DMSO
1
1
13
d = 2.50 ppm ( H) Methanol d = 3.34 ppm ( H), d = 49 ppm ( C)). High-Performance Liquid Chroma-
tography (HPLC) Alliance 2695 separation module (Waters) coupled to Waters 2996 photodiode
detector (DAD) (Waters), 5 mL of sample 2.5 mM in DMSO was injected, using a KinetexC18 2.6 mm
4
.6 Â 50 mm (Phenomenex) column. The mobile phase used was a mixture of A = NaH
Na HPO aqueous buffer 10 mM pH = 7 and B = methanol, with the method described as follows:
flow 1 mL/min 5%B-70%B 1 min, 70%B-80%B 1 min, 80%B-100%B 6 min, 100%B 1 min, runtime 15
2 4
PO /
2
4
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min. Ultrahigh-Performance Liquid Chromatography (UPLC) Aquity(Waters) coupled to an Acquity
TUV UV-Vis detector (Waters) and a LCT Premier Orthogonal Accelerated Time of Flight Mass Spec-
trometer (TOF) (Waters). HRMS: UPLC/MS chromatograms were obtained by injection of 2 ‘L of sam-
ple 25 ‘M in AcN/DMSO 99:1, using aAcquity UPLC BEH C18 1.7 ’m 2.1 Â 100 mm column (Waters).
The mobile phase used was a mixture of A = aqueous formic acid 20 mM and B = formic acid 20 nM
in acetonitrile, with the method described as follows: flow 0.3 mL/min 5–95%B 2.69 min, 95%B 6
min, runtime 14 min. Data from mass spectra were analyzed by electrospray ionization in positive
and negative mode. Spectra were scanned between 50 and 1500 Da with values every 0.2 s and
peaks are given m/z (% of basis peak). ESI were analyzed by FIA (flux injected analysis) with Acquity
(
Waters) coupled to LCT Premier Orthogonal Accelerated Time of Flight Mass Spectrometer (TOF)
Waters). Data were obtained in the same conditions as for samples of UPLC/MS.
(
The synthesis of 2-((3-fluorophenyl)ethynyl)À4,6-dimethylpyridin-3-amine, raseglurant (ADX-
10059) (Figure 1) was previously described in the literature (Bolea et al., 2004) using a general con-
ditions of Sonogashira reaction. A solution of 2-bromo-4,6-dimethylpyridin-3-amine (200 mg, 0.995
mmol), bis(triphenylphosphine)palladium (II) dichloride (35 mg,0,05 mmol) and copper iodide (10
mg, 0,05 mmol), previously purged with argon, in 1 mL of anhydrous DMF, 1-ethynyl-3-fluoroben-
zene (0,12 mL,1.094 mmol) and dry triethylamine (0,42 mL, 2.98 mmol) were added, and the reaction
mixture was stirred at 40 C for 8 hr. After this time, 40 mL of ethyl acetate was added, and the mix-
˚
ture was washed with 40 mL of saturated solution of NaHCO
3
and 40 mL of brine, the organic layer
was dried over Na
column chromatography with ethyl acetate-hexane 1:4. A palid brown solid was isolated (197 mg,
3%). A portion of this compound was dissolved in ether and HCl 1N was added, the precipitate
2 4
SO filtered and evaporated under vacuum. The residue was purified trough flash
8
1
was collected by filtration, to give the hydrochloride salt as a yellow solid. H NMR (400 MHz,
DMSO-d6) d 7.75–7.71 (m, 1H), 7.63 (dd, J = 7.6, 1.2 Hz, 1H), 7.56 (td, J = 8.0, 5.9 Hz, 1H), 7.50 (s,
1H), 7.39 (td, J = 8.6, 2.5 Hz, 1H), 4.00 (s, 4H), 2.54 (s, 3H), 2.34 (s, 3H). 13C NMR (101 MHz, DMSO-
d6) d 162.92, 160.49, 145.61, 141.44, 140.80, 131.08, 131.00, 128.21, 128.18, 127.78, 122.66,
+
1
22.56, 118.59, 118.36, 117.57, 117.36, 113.15, 101.11, 79.92, 18.37, 17.88. HRMS (m/z): [M+H] -
calcd. for C15
RT = 3.34 min.
2
H13FN , 241.1141; found, 241.1112 HPLC/DAD: purity (abs = 254 nm)=100%;
The synthesis of synthesis of (7-(diethylamino)-2-oxo-2H-chromen-4-yl)methyl (2-((3-fluorophenyl)
ethynyl)-4,6-dimethylpyridin-3-yl)carbamate, JF-NP-26 (Figure 1), was performed using conventional
chemical methods. A solution of triphosgene (100 mg, 0.336 mmol) in 2 mL of toluene, a mixture of
2-((3-fluorophenyl)ethynyl)-4,6-dimethylpyridin-3-amine (218 mg, 0.90 mmol) and dry triethylamine
(
(
0,22 mL, 1.68 mmol) in 2 mL of toluene was added, and the reaction mixture was stirred over 3 hr
the reaction was monitored by TLC).
Afterwards, the reaction mixture was purged with a gentle flow of nitrogen over 20 min to
remove the phosgene produced during the reaction, the crude residue was dissolved in 2 mL of tolu-
ene and a solution of 7-(diethylamino)-4-(hydroxymethyl)-2H-chromen-2-one (83 mg, 0.337 mmol)
and sodium hydride (14 mg, 0.337 mmol) in 1 mL of THF previously stirred at room temperature for
1
0 min, was added and heated at 100 C overnight. The mixture was evaporated under vacuum and
˚
4
2 4
0 mL of ethyl acetate were added, washed with 40 mL of brine, dried over Na SO , filtered and
evaporated under vacuum. The residue was purified by flash column chromatography in ethyl ace-
tate-hexane 3:2. A yellow-orange solid was obtained and it was purified again at isolerabiotage in
reverse phase conditions: 4VC (H O:Acetonitrile 95:5), 3VC (H O:Acetonitrile 40:60), 10 VC (H O:
2
2
2
Acetonitrile 0:100), the crude was lyophilized overnight. A yellow solid was obtained (95 mg, 55%).
Mp: 77–79 C,1H NMR (400 MHz, Methanol-d4) d 7.50 (s, 1H), 7.41–7.29 (m, 3H), 7.28–7.21 (m, 2H),
˚
7
6
1
9
5
.19–7.12 (m, 1H), 6.67 (d, J = 9.1 Hz, 1H), 6.53 (s, 1H), 6.19 (s, 1H), 5.42 (s, 2H), 3.46 (q, J = 7.1 Hz,
H), 1.20 (t,J = 7.1 Hz,9H). 13C NMR (101 MHz, cd3od) d 163.55, 156.93, 156.10, 151.01, 147.16,
30.23, 130.14, 127.60, 127.57, 125.33, 124.78, 118.02, 117.78, 116.28, 116.07, 108.94, 105.74,
+
6.83, 44.15, 21.87, 16.38, 11.28. HRMS (m/z): [M+H] calcd. for C30
14.2125 HPLC/DAD: purity (abs = 254 nm)=100%; RT = 5.88 min.
3 4
H28FN O , 514.2142; found,
Photochemical characterization
The absorption spectra of raseglurant and JF-NP-026 were recorded in DMSO solutions (100 mM)
with a Varian Cary 300 UV-Vis spectrophotometer (Agilent Technologies). Full absorption spectra
were obtained by scanning between 300 nm to 600 nm with an average time of 33 ms at 5 nm
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intervals. To determine the optimal illumination wavelength for uncaging, we recorded UV-Vis spec-
tra plotting the values giving a maximum absorption at 385 nm for JF-NP-026. The photouncaging
quantum yield (jchem) was measured by comparison of the rate of photouncaging (kchem) with the
rate of potassium ferrioxalate photoreduction (k
r
) upon excitation at 405 nm. The solutions of JF-NP-
26 and potassium ferrioxalate were optically-matched at 405 nm and the quantum yield of potassium
r
ferrioxalate reduction was taken as ’ = 1.14 (see [Kuhn et al., 1989]). Potassium ferrioxalate was
synthesized as described elsewhere (Kl a´ n et al., 2013).
Cell culture
Human embryonic kindney (HEK) 293 T cells obtained from ATCC (CRL-321, RRID:CVCL_0063) were
transiently transfected with the mGlu receptor either by polyethylenimine (PEI) method or by elec-
5
troporation technique. Then, 24 hr after transfection cells were seeded in a 96-well plate (10.000
cells/well) using Dulbecco’s modified eagles medium (Sigma-Aldrich) supplemented with 100 U/mL
penicillin (Biowest), 100 mg/mL streptomycin (Biowest), 10% v/v fetal bovine serum (Invitrogen), 1
mM pyruvic acid (Biowest), non-essential aminoacids (Biowest) and 2 mM L-Glutamine (Biowest).
After 24 hr, intracellular calcium determinations experiments were performed. Cells were kept at 5%
2
CO , 37 C, and 95% humidity conditions. Cells were tested for mycoplasma content, thus only myco-
˚
plasma-free cells were used.
Striatal primary neurons and immunocytochemistry
Primary neurons from striatum were cultured from E18 CD-1 mice embryos as previously
described (Gratacos et al., 2001). In brief, the dissected striatum was treated with 1.25% trypsin
(Sigma-Aldrich) for 10 min before being mechanically dissociated with a flame polished Pasteur
pipette. Neurons were plated onto poly-D-lysine (0.1 mg/mL)/laminin (0.01 mg/ml)-coated 96-well
2
black plate at a density of 80,000 cells/cm in minimum essential medium (Invitrogen) supplemented
with 10% horse serum(Invitrogen), 10% bovine serum(Invitrogen), 1 mM pyruvic acid (Biowest), and
0.59% glucose (Biowest). After 4–14 hr, the medium was substituted with Neurobasal medium (Invi-
trogen) supplemented with penicillin (100 U/mL) (Biowest), streptomycin (100 mg/mL) (Biowest),
0
.59% glucose (Biowest), and B27 supplement (Invitrogen). Neurons were kept at 5% CO , 37 C and
2
˚
9
5% humidity for 21 days before the calcium determination experiments.
For immunocytochemistry, primary neurons from striatum growing on coverslips were fixed in 4%
paraformaldehyde for 15 min and exposed to a rabbit anti-mGlu receptor antibody (1 mg/ml; Milli-
5
pore, Billerica, MA, USA; RRID:AB_2295173). Primary antibodies were detected using a Cy3-conju-
gated donkey anti-rabbit antibody (1/200; Jackson ImmunoResearch Laboratories Inc., West Grove,
PA, USA). Coverslips were rinsed for 30 min, mounted with Vectashield immunofluorescence
medium (Vector Laboratories, Peterborough, UK) and examined using a Leica TCS 4D confocal scan-
ning laser microscope (Leica Lasertechnik GmbH, Heidelberg, Germany) (Luj a´ n et al., 2001).
Neuronal viability
The neuronal viability upon irradiation with 405 nm was calculated using the 3-(4,5-dimethylthiazol-
2
-yl)À2,5-diphenyltetrazolium bromide (MTT) assay (adapted from [Lopachev et al., 2016]). In brief,
cultured neurons in 96 well plates were irradiated with 405 nm light for different periods of time (5,
5 or 160 min) before the incubation with 0.5 mg/mL of MTT. Subsequently, the medium was
1
removed and 100 mL of dimethylsulfoxide (DMSO) was added to each well. The absorbance at 540
nm wavelength of each sample was measured in a POLARstar Omega multi-mode microplate reader
(BMG Labtech). All values were normalized using intact neurons as a 100% of viability threshold. 3%
hydrogen peroxide (HP) was used a positive control of cell death.
Inositol phosphate determinations
The mGlu
assay IP-One HTRF kit (Cisbio Bioassays) according to the manufacturer’s instructions
Trinquet et al., 2006). Glutamate levels were maintained at minimal concentrations by co-express-
ing the excitatory amino acid transporter one (EAAC1). Importantly, all the mGlu receptor con-
5
-mediated inositol phosphate (IP) accumulation was determined using the FRET-based
(
5
tained a haemagglutinin (HA) epitope tag in their N terminus to allow cell surface expression by
ELISA for subsequent normalization. After transfection cells were seeded in black clear-bottom 96-
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5
well plates at a concentration of 1.5 Â 10 cells/well. The medium was replaced by glutamate-free
DMEM GlutaMAX-I (Invitrogen) after 6 hr. Next day cells were challenged with increasing concentra-
tions of the test compounds in the presence of quisqualate EC80 (1 mM) for 30 min, at 37 C and 5%
˚
CO
2
before we determined IP accumulation (Trinquet et al., 2006). When necessary the compounds
were irradiated at 405 nm for 5 min before being added to the cells. Finally, the cells were washed
once with stimulation buffer (Cisbio Bioassays) to remove any interfering chromophore and then the
cells were lysed for IP determination. The IP associated fluorescence was determined in a RUBYstar
multimode microplate reader (BMG Labtech).
Intracellular calcium determinations
Fluo-4 NW Calcium Assay Kit (ThermoFisher Scientific) was used to measure intracellular calcium
accumulation. In brief, HEK-293T cells or striatal primary neurons grown in 96-well black plates were
incubated with Fluo4 NW as indicated by the manufacturer. Subsequently, cells were pretreated for
5
min with vehicle (HBSS), raseglurant or JF-NP-26 (1 mM) and then maintained in the dark or irradi-
ated at 405 nm for 5 min before being challenged with a saturating concentration (100 mM) of quis-
qualate. Real-time Fluo4 NW emission (i.e. 535 nm) was measured after 485 nm excitation in a
POLARstar Omega multi-mode microplate reader.
The results were expressed as the percentage of mGlu receptor inhibition induced by the treat-
5
ments following the equation:
veh
drug
veh
Inhibitionð%Þ ¼ ½ðAUC À AUC Þ=AUC Š  100
Where AUCveh and AUCdrug represent the area under curve value in the vehicle- and drug-treated
conditions, respectively.
Animals
Animals were housed and tested in compliance with the guidelines described in the Guide for the
Care and Use of Laboratory Animals (Clark et al., 1996) and following the European Union direc-
tives (2010/63/EU). All efforts were made to minimize animal suffering and the number of animals
used. For the formalin animal model of pain adult male CD-1 mice (Charles River Laboratories,
L’Arbresle, France; RRID:MGI:3785721) weighing 20–25 g were used and for the neuropathic pain
animal model adult male C57BL/6J mice (Charles River, Calco, Italy; RRID:IMSR_JAX:000664) weigh-
ing 20–25 g were employed. All animals were housed in groups of five in standard cages with ad-libi-
tum access to food and water and maintained under 12 hr dark/light cycle (starting at 7:30 AM),
2
2 C temperature, and 66% humidity (standard conditions). All animal experimentation was carried
˚
out by a researcher blind to drug treatments.
Brain optic fiber implantation
Mice were anesthetized with a combination of ketamine/xylacine (dose of 100 mg/kg body weight
for ketamine and 10 mg/kg for xylacine) and implanted with optic fibers (TFC_400/475–0.53_3.5
mm_TSM4.0_B45, Doric Lenses Inc., Quebec, Canada) using dental cement and chirurgic screws
(Agnthos, Liding o¨ , Sweden) in a Kopf or Stoelting stereotaxic frame (Stoelting Co., Wood Dale, IL,
USA). The site of implantation was either the left and right thalamus (coordinates: À1.8 mm poste-
rior to the bregma, ±1.5 mm lateral to the midline, 3.5 mm ventral from the surface of skull) or the
left and right striatum (coordinates: +1.2 X mm posterior to the bregma, ±1.5 mm lateral to the mid-
line, 3.5 mm ventral from the surface of skull), following to the atlas of Paxinos and
Franklin (Franklin and Paxinos, 2008). After optic fiber implantation mice were treated with Meta-
cam (BoehringerIngelheim, Ingelheim am Rhein, Germany) during three days and they were checked
routinely for one week.
Measurement of liver function
Serum ALT and AST activities were determined by using an Olympus AU400 Automatic Biochemical
Analyzer (Olympus, Tokyo, Japan) in the Clinical Veterinary Service at the Autonomous University of
Barcelona.
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Formalin animal model of pain
The formalin animal model of pain was performed as previously described (Lapa et al., 2009) and
explained in more detail at Bio-protocol (L o´ pez-Cano et al., 2017). In brief, mice (n = 5–6) were
administered intraperitoneally (i.p.) with vehicle, raseglurant or JF-NP-26 20 min before a diluted for-
malin solution (20 mL of 2.5% formalin/0.92% formaldehyde, Sigma-Aldrich) was intraplantarly (i.pl.)
injected in the mid-plantar surface of the right hind paw of the mouse. The formalin-induced noci-
ceptive behaviour in dark and light conditions was quantified as the time spent licking or biting the
injected paw during the 30 min after the injection of formalin. The initial acute phase (0–5 min; phase
I), reflecting the acute peripheral pain, was followed by a relatively short quiescent period, which
was then followed by a prolonged response (15–30 min; phase II) which is related to the develop-
ment of central nociceptive sensitization. For the peripheral JF-NP-026 uncaging the hind paw was
directly irradiated with a LED-based fiberoptic system (Doric Lenses Inc.) at 405 nm light (or dark) for
5
min before the recording of each phase. For the central nervous system uncaging both thalamic
nuclei were irradiated through the brain implanted optic fibers (Doric Lenses Inc.) with 405 nm light
or dark) for 5 min before the recording of each phase. The 405 nm light was administered at 2000
(
mA intensity and 500 Hz frequency.
The antinociception induced by the treatments in the formalin test was calculated with the
equation:
Antinociceptive effectð%Þ ¼ ½ðLTV À LTDÞ=LTVŠ  100
where LTV and LTD represent the licking/biting time in the vehicle- and drug-treated animals,
respectively.
Mechanical allodynia assessment in a neuropathic pain animal model
The assessment of the mechanical allodynia in the chronic constriction injury (CCI) of the sciatic nerve
model was explained in more detail at Bio-protocol (Notartomaso et al., 2018). In brief, the sciatic
nerve CCI induction was carried out under isoflurane anesthesia (5% for induction and 2% for main-
tenance), using a modified version of a previously described method (Bennett and Xie, 1988). The
biceps femoris and the gluteus superficialis were separated by blunt dissection, and the right sciatic
nerve was exposed. CCI was produced by tying one ligature (6–0 silk, Ethicon, LLC, San Lorenzo,
PR, USA) around the sciatic nerve. The ligature was tied loosely around the nerve, until it elicited a
brief twitch in the respective hind limb, which prevented over-tightening of the ligation, taking care
to preserve epineural circulation. The incision was cleaned and the skin was closed with 2–3 ligatures
of 5–0 dermalon. After performing the ligature of the sciatic nerve, during the same anesthesia ses-
sion, mice were implanted with optic fibers (see above). After surgery, mice returned to their home
cages and checked routinely for 72 hr.
Mechanical allodynia was assessed 7, 14 and 21 days after surgery by measuring the hind paw
withdrawal response to von Frey filament stimulation. Mice were placed in a dark box (20 x 20 Â 40
cm) with a wire grid bottom through which the von Frey filaments (North Coast Medical, Inc., San
Jose, CA, USA), bending force range from 0.008 to 3.5 g, were applied by using a modified version
of the up-down paradigm previously described (Chaplan et al., 1994). Briefly, lack of response to a
filament indicated the next higher filament in the following stimulation, whereas a positive response
indicated the next lower filament. Each filament was applied and pressed perpendicularly to the
plantar surface of the hind paw until it bent for five times with a 3 min interval. The filament that
evoked at least three paw withdrawals was assigned as the pain threshold in grams. Mice were
treated either with raseglurant (10 mg/kg, i.p. in 6% DMSO, 6% Tween 80 in saline) orJF-NP-26, (10
mg/kg, i.p. in 6% DMSO, 6% Tween 80 in saline). Both thalamic nucleus were irradiated (or dark)
through the brain implanted optic fibers (Doric Lenses Inc.) with 405 nm light (or dark) for 5 min, 20
min after drug administration. The light was administered at 2000 mA intensity and 500 Hz fre-
quency and the mechanical thresholds were quantified before and after light irradiation.
Novel object recognition test
To evaluate reference memory, we performed the novel object recognition test as previously
described (Christoffersen et al., 2008) with minor modifications. In brief, mice were divided into six
subgroups: vehicle-injected mice, raseglurant (10 or 20 mg/kg, i.p.)-injected mice, vehicle-injected
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mice implanted with optic fibers and JF-NP-26 (10 or 20 mg/kg, i.p.)-injected mice implanted with
optic fibers. All mice were injected 20 min before the training session. On the first three days, all
mice were handled for 3 min once daily and 24 hr later they were habituated for 20 min to the empty
arena. On day 5, mice underwent a 5 min training period. Afterwards, mice return to their home
cages for 5 min, and were subsequently subjected to a 5 min retention test. The 405 nm light was
administered at 2000 mA intensity and 500 Hz frequency in vehicle and JF-NP-26-injected mice 5
min immediately before the training session. Two identical objects were used as familiar objects dur-
ing the training trial and the third object was used as novel object during the 5 min retention ses-
sion. As an index of recognition memory, the time spent exploring the familiar and novel object was
measured during the retention session.
Statistics
The number of samples (n) in each experimental condition is indicated in figure legends. When two
experimental conditions were compared, statistical analysis was performed using an unpaired t test.
Otherwise, statistical analysis was performed by one-way analysis of variance (ANOVA) followed by
Bonferroni post-hoc test. In cell experiments and formalin test results Dunnett’s multiple comparison
post-hoc test was performed using Veh+dark as a control. Statistical significance was set as p<0.05.
Acknowledgements
This work was supported by MINECO/ISCIII (SAF2014-55700-P, PCIN-2013–019 C03-03 and PIE14/
00034), the Catalan Government (2014 SGR 1054), ICREA (ICREA Academia-2010), Fundaci o´ la Mar-
at o´ de TV3 (Grant 20152031) and IWT (SBO-140028) to FC. MINECO (PCIN-2013–018 C03-02 and
SAF2014-58396-R) to JG. MINECO (PCIN-2013–017 C03-01 and CTQ2014-57020-R), the Catalan
Government (2014SGR109 and 2014CTP0002) to AL. ERANET Neuron project ‘LIGHTPAIN’ (to AL,
JG, PC and FN). MINECO (CTQ2013-48767-C3-1-R and CTQ2015-71896-REDT), the Catalan Gov-
ernment (2014SGR304) to SN RB-O thanks the European Social Funds and the SUR del DEC de la
Generalitat de Catalunya for a predoctoral fellowship (2016 FI_B1 00021).
Additional information
Funding
Funder
Grant reference number
Author
Ministerio de Econom ´ı a y
Competitividad
PCIN-2013-018-C03-02
Jes u´ s Giraldo
Ministerio de Econom ´ı a y
Competitividad
SAF2014-58396-R
Jes u´ s Giraldo
Jean-Philippe Pin
Cyril Goudet
Cyril Goudet
Santi Nonell
Fondation pour la Recherche DEQ20130326522
M e´ dicale
European Commission
Neuron eranet, ANR-12-
NEUR-0003-05
Agence Nationale de la Re-
cherche
ANR-12-NEUR-0003
Ministerio de Econom ´ı a y
Competitividad
CTQ2013-48767-C3-1-R
CTQ2015-71896-REDT
Ministerio de Econom ´ı a y
Competitividad
Santi Nonell
Generalitat de Catalunya
Ministero della Salute
2014SGR304
Santi Nonell
ERA-NET NEURON
Ferdinando Nicoletti
"
LIGHTGLUPAIN"
Ministerio de Econom ´ı a y
Competitividad
PCIN-2013-017-C03-01
Amadeu Llebaria
Amadeu Llebaria
Ministerio de Econom ´ı a y
Competitividad
CTQ2014-57020-R
Font et al. eLife 2017;6:e23545. DOI: 10.7554/eLife.23545
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Research article
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Generalitat de Catalunya
Generalitat de Catalunya
2014SGR109
Amadeu Llebaria
Amadeu Llebaria
Francisco Ciruela
2014CTP0002
SAF2014-55700-P
Ministerio de Econom ´ı a y
Competitividad
Instituto de Salud Carlos III
Fundaci o´ la Marat o´ de TV3
PIE14/00034
Francisco Ciruela
Francisco Ciruela
Francisco Ciruela
Grant 20152031
Instituci o´ Catalana de Recerca i ICREA Academia-2010
Estudis Avan c¸ ats
Agentschap voor Innovatie
door Wetenschap en Techno-
logie
SBO-140028
Francisco Ciruela
Ministerio de Econom ´ı a y
Competitividad
PCIN-2013-019-C03-03
2014 SGR 1054
Francisco Ciruela
Francisco Ciruela
Generalitat de Catalunya
The funders had no role in study design, data collection and interpretation, or the decision to
submit the work for publication.
Author contributions
JF, FM, Data curation, Investigation, Methodology; ML-C, Formal analysis, Investigation, Methodol-
ogy; SNot, PS, PDP, RB-O, Data curation, Formal analysis, Investigation, Methodology; GB, Concep-
tualization, Data curation, Supervision, Validation, Methodology, Project administration; XR, Formal
analysis, Supervision, Validation, Investigation; JC, Formal analysis, Supervision, Investigation, Visuali-
zation; JG, Software, Supervision, Validation, Investigation, Visualization; J-PP, Conceptualization,
Resources, Supervision, Validation, Visualization, Methodology; VF-D, Formal analysis, Supervision,
Validation, Investigation, Visualization, Writing—original draft; CG, Supervision, Validation, Investiga-
tion, Visualization, Methodology; SNon, Resources, Formal analysis, Supervision, Methodology, Writ-
ing—original draft; FN, Conceptualization, Data curation, Formal analysis, Supervision, Validation,
Investigation, Visualization, Writing—original draft; AL, Conceptualization, Resources, Supervision,
Validation, Visualization, Methodology, Writing—original draft; FC, Conceptualization, Resources,
Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodol-
ogy, Writing—original draft, Writing—review and editing
Author ORCIDs
Serena Notartomaso, http://orcid.org/0000-0003-4374-9233
Pamela Scarselli, http://orcid.org/0000-0002-4245-0849
Paola Di Pietro, http://orcid.org/0000-0003-1327-1961
Roger Bresol ı´ -Obach, http://orcid.org/0000-0002-7819-7750
Giuseppe Battaglia, http://orcid.org/0000-0001-7571-3417
Xavier Rovira, http://orcid.org/0000-0002-9764-9927
Jes u´ s Giraldo, http://orcid.org/0000-0001-7082-4695
Jean-Philippe Pin, http://orcid.org/0000-0002-1423-345X
V ´ı ctor Fern a´ ndez-Due n˜ as, http://orcid.org/0000-0001-7834-2965
Cyril Goudet, http://orcid.org/0000-0002-8255-3535
Ferdinando Nicoletti, http://orcid.org/0000-0003-0917-443X
Amadeu Llebaria, http://orcid.org/0000-0002-8200-4827
Francisco Ciruela, http://orcid.org/0000-0003-0832-3739
Ethics
Animal experimentation: Animals were housed and tested in compliance with the guidelines
described in the Guide for the Care and Use of Laboratory Animals and following the European
Union directives (2010/63/EU). All efforts were made to minimize animal suffering and the number
of animals used. For the formalin animal model of pain adult male CD-1 mice (Charles River Labora-
tories, L’Arbresle, France) weighing 20-25 g were used and for the neuropathic pain animal model
adult male C57BL/6J mice (Charles River, Calco, Italy) weighing 20-25 g were employed. All animals
Font et al. eLife 2017;6:e23545. DOI: 10.7554/eLife.23545
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were housed in groups of five in standard cages with ad-libitum access to food and water and main-
tained under 12 h dark/light cycle (starting at 7:30 AM), 22 C temperature, and 66% humidity (stan-
˚
dard conditions).
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