Interaction between interleukin-1β and type-1 cannabinoid receptor is involved in anxiety-like behavior in experimental autoimmune encephalomyelitis
Gentile et al. Journal of Neuroinflammation
Interaction between interleukin-1β and type-1 cannabinoid receptor is involved in anxiety-like behavior in experimental autoimmune encephalomyelitis
Antonietta Gentile 0 2
Diego Fresegna 0 2
Alessandra Musella 0 2
Helena Sepman 0 2
Silvia Bullitta 0 2
Francesca De Vito 0 2
Roberta Fantozzi 1
Alessandro Usiello 5 6
Mauro Maccarrone 0 4
Nicola B. Mercuri 0 2
Beat Lutz 3
Georgia Mandolesi 0
Diego Centonze 1 2
0 Laboratory of Neuroimmunology and Synaptic Transmission, IRCCS Fondazione Santa Lucia, Centro Europeo di Ricerca sul Cervello (CERC) , 00143 Rome , Italy
1 Unit of Neurology and of Neurorehabilitation, IRCCS Istituto Neurologico Mediterraneo Neuromed , 86077 Pozzilli, IS , Italy
2 Department of Systems Medicine, Tor Vergata University , 00133 Rome , Italy
3 Institute of Physiological Chemistry
4 Centro di Ricerca Integrata, Facoltà di Medicina e Chirurgia, Università Campus Bio-Medico , 00128 Rome , Italy
5 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples (SUN) , Caserta , Italy
6 Behavioural Neuroscience Laboratory, CEINGE Biotecnologie Avanzate , 80145 Naples , Italy
Background: Mood disorders, including anxiety and depression, are frequently diagnosed in multiple sclerosis (MS) patients, even independently of the disabling symptoms associated with the disease. Anatomical, biochemical, and pharmacological evidence indicates that type-1 cannabinoid receptor (CB1R) is implicated in the control of emotional behavior and is modulated during inflammatory neurodegenerative diseases such as MS and experimental autoimmune encephalomyelitis (EAE). Methods: We investigated whether CB1R could exert a role in anxiety-like behavior in mice with EAE. We performed behavioral, pharmacological, and electrophysiological experiments to explore the link between central inflammation, mood, and CB1R function in EAE. Results: We observed that EAE-induced anxiety was associated with the downregulation of CB1R-mediated control of striatal GABA synaptic transmission and was exacerbated in mice lacking CB1R (CB1R-KO mice). Central blockade of interleukin-1β (IL-1β) reversed the anxiety-like phenotype of EAE mice, an effect associated with the concomitant rescue of dopamine (DA)-regulated spontaneous behavior, and DA-CB1R neurotransmission, leading to the rescue of striatal CB1R sensitivity. Conclusions: Overall, results of the present investigation indicate that synaptic dysfunction linked to CB1R is involved in EAE-related anxiety and motivation-based behavior and contribute to clarify the complex neurobiological mechanisms underlying mood disorders associated to MS.
Type-1 cannabinoid receptor; Striatum; Interleukin-1β; Anxiety; Experimental autoimmune encephalomyelitis
(Continued from previous page)
glycoprotein 35-55; MS, Multiple sclerosis; MSNs, Medium spiny projection neurons; NB, Nest building; PBS,
Phosphatebuffered saline; sEPSCs, Spontaneous excitatory postsynaptic currents; sIPSCs, Spontaneous inhibitory postsynaptic
currents; TNF, Tumor necrosis factor; TRPV1, Transient receptor potential vanilloid 1 channels; WB, Western blot;
WT, Wild type
Multiple sclerosis (MS) is the most common cause of
neurological disability in young adults, affecting 1 in
800–1000 people in Western countries. Mood
disturbances are frequent in MS, even in its early phases and
in the absence of physical disability [1, 2].
Rather than merely representing a subjective reaction to
a chronic and potentially disabling disease, anxiety and
depression in MS are increasingly recognized to follow the
effect of the inflammatory milieu on neuronal function
and connectivity [3–6], thus sharing with the
inflammatory neurodegenerative process of the disease of some
neurobiological underpinnings. For example,
proinflammatory cytokines such as interleukin-1β (IL-1β) and
tumor necrosis factor (TNF), released during MS attacks,
have been implicated both in delayed neurodegeneration
in MS brains and in mood alterations , therefore
suggesting common determinants for both phenomena.
Studies in the experimental autoimmune encephalomyelitis
(EAE), the most characterized murine model of MS, have
clearly highlighted the independence of behavioral
alterations from motor disability and, most notably, the
involvement of cytokines [7–10].
Type-1 cannabinoid receptor (CB1R) are crucial
regulators of both excitatory and inhibitory synaptic
transmission in different brain areas [11, 12], including the
striatum [13, 14] and the brain area involved in MS .
CB1R plays a substantial role in MS disease course [16–18]
and in mood control . Reduced CB1R signaling in mice
lacking the CB1R gene (CNR1), in fact, results in more
severe motor deficits and synaptic pathology linked to
neurodegenerative damage after EAE [20, 21], and human
subjects carrying a genetic variant of CNR1 associated with
reduced CB1R protein expression have higher risk of
progressive MS course , and more severe relapsing MS
disease course , and neurodegenerative damage . On
the other hand, endocannabinoid signaling enhancement
has antidepressant and anxiolytic actions in humans 
and in rodents [26, 27], and genetic or pharmacological
blockade of CB1R promotes depression- and anxiety-like
behavior in humans [28, 29] and in rodents [30–32].
Whether CB1R plays a role in MS-associated mood
alterations is however entirely speculative. To answer
this question, we investigated the link between CB1R
function and the emotional consequences of brain
inflammation in EAE mice. In particular, we addressed
the link between CB1R function and IL1-β effects at
GABA synapses in the striatum of EAE mice. Indeed,
IL1-β has been previously demonstrated to control the
sensitivity of CB1R on GABAergic transmission and to
induce anxiety-like behavior in naïve mice .
Moreover, we have recently shown that IL1-β is implicated
in depressive-like behavior in EAE mice .
Our results point to CB1R as involved in EAE-associated
anxiety and establish a previously unrecognized link
between mood alterations and IL-1β-dependent
inflammatory synaptic dysfunction in EAE and, possibly, MS.
The subjects in this study were 7–8-week-old female
mice, C57BL/6N, obtained from Charles-River (Italy)
and CNR-EMMA Mouse Clinic facility
(MonterotondoRome, Italy). Animals were randomly assigned to
standard cages, with four to five animals per cage, and kept at
standard housing conditions with a light/dark cycle of
12 h and free access to food and water. Only
minipumpimplanted mice were housed in isolated cages endowed
with special bedding (TEK-FRESCH, Harlan) in order to
avoid skin infections around the surgical scar. Since
1 week before immunization, all animals were kindly
handled once a day to reduce the stress induced by
operator manipulation during behavioral experiments.
All experiments were carried out in accordance with
the Guide for the Care and Use of Laboratory Animals
and the European Communities Council Directive of 24
November, 1986 (86/609/EEC).
EAE induction and clinical evaluation
Chronic EAE was induced in 7–8-week animals as
previously described [10, 34]. Furthermore, EAE was induced
in female mice totally lacking CB1Rs (CB1R-KO) 
and respective wild-type (WT) littermate controls. Mice
were injected subcutaneously at the flanks with 200 μg of
myelin oligodendrocyte glycoprotein 35-55 (MOG35-55)
emulsion to induce EAE by active immunization. The
emulsion was prepared under sterile conditions using
MOG35-55 (>85 % purity, Espikem, Florence, Italy) in
300 μl of complete Freund’s adjuvant (CFA, Difco,
Lawrence, KS, USA) containing Mycobacterium
tuberculosis (8 mg/ml; strain H37Ra, Difco) and emulsified with
phosphate-buffered saline (PBS). All animals were injected
with 500 ng pertussis toxin (Sigma, St. Louis, MO, USA)
intravenously on the day of immunization and 2 days later.
Control animals received the same treatment as EAE
mice without the immunogen, MOG peptide, including
complete CFA and Pertussis toxin (referred to as
“CFA”). Animals were daily scored for clinical
symptoms of EAE, according to the following scale: 0 =
healthy; 1 = flaccid tail; 2 = ataxia and/or paresis of
hindlimbs; 3 = paralysis of hindlimbs and/or paresis of
forelimbs; 4 = tetraparalysis; and 5 = moribund or death
due to EAE. Intermediate clinical signs were scored by
adding 0.5 value [10, 35]. The presymptomatic phase
was kept in the range 8–11 days post immunization
(dpi), before the onset day, when immunized animals
showed the first clinical manifestation (12 dpi), as
previously shown .
Minipump implantation and continuous intracranial
One week before immunization, mice were implanted
with a minipump in order to allow continuous
intracerebroventricular (icv) infusion of either vehicle or
interleukin 1 receptor antagonist (IL1-ra) (150 ng/day; R&D
Systems) for 4 weeks. Alzet osmotic minipumps (model
1004; Durect Corporation, Cupertino, CA) connected via
catheter tube to intracranial cannula (Alzet Brain Infusion
Kits 3) delivered vehicle or IL1-ra into the right lateral
ventricle at a continuous rate of 0.11 μl/h. The
coordinates used for icv minipump implantation were
anteroposterior = −0.4 mm from the bregma; lateral = −1 mm;
and depth: 2.5 mm from the skull [10, 36].
In vivo amphetamine treatment
EAE mice were given intraperitoneal injection of
amphetamine sulfate (5 mg/kg) in a volume of 10 ml/kg or vehicle
 and after 24 h were sacrificed for both
electrophysiological and western blot experiments. Control CFA and
EAE mice received intraperitoneal injection of saline
solution (NaCl 0.9 %). Each experimental group consisted of
four to five animals (three sets of experiments).
Behavioral experiments were performed during the
presymptomatic phase of the disease (8–9 dpi). The animals
were tested during light period (9:00–12:00 am) in a
dedicated room with a constant temperature (26 ± 1 °C).
All tests were performed in different days with distinct
groups of animals. Each session was preceded by at least
1 h habituation in the behavioral room.
The light/dark test (LDT) is based on the innate
aversion of rodents to brightly lit areas . The test
apparatus consisted of an open white compartment (30 × 20 ×
20 cm, 300 lux) joined by a 3 × 3-cm opening to a dark
compartment (15 × 20 × 20 cm, 0 lux) which was painted
black and covered with a lid. The anxiogenic nature of
the white compartment was increased by additional
illumination from a 60-W angle poise lamp placed 45 cm
above the center of the apparatus. Mice were allowed to
move freely between the two chambers with door open
for 10 min. The score for the transition was assigned
from the analysis of the video recordings, when the
animal came out of the dark chamber with all four paws.
The apparatus was cleaned with 10 % ethanol after each
trial to effectively remove the scent of the previously
tested animal. The time spent in each chamber (referring
to the last 5 min of the test) was recorded by ViewPoint
video tracking software.
Nest building is a natural and instinctual behavior that
involves species-typical sensorimotor actions important to
the survival of the animal. These behaviors are dependent
upon motivation . To evaluate the quality of nest
construction, mice were individually housed 1 h before the
onset of dark phase in a clean cage overnight with no
enrichment aside a pre-weighted roll of cotton in the
cagetop food hopper. The morning after (9 am), the quality of
the nest were evaluated using the following scoring
system: (1) no nest, (2) platform-type nest consisting of a
pallet on the floor of the cage, (3) bowl- or cupshaped nest
with sides, or (4) bowl- or cup-shaped nest with sides and
a cover . An investigator blind to treatment and
experimental group scored the quality of the nests.
Mice were killed by cervical dislocation, and
corticostriatal coronal slices (200 μm) were prepared from fresh
tissue blocks of the brain with the use of a vibratome .
A single slice was transferred to a recording chamber
and submerged in a continuously flowing artificial
cerebrospinal fluid (ACSF) (34 °C, 2–3 ml/min) gassed with
95 % O2–5 % CO2. The composition of the control ACSF
was (in mM) 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4,
2.4 CaCl2, 11 Glucose, and 25 NaHCO3. The striatum
could be readily identified under low power magnification,
whereas individual neurons were visualized in situ using a
differential interference contrast (Nomarski) optical
system. This employed an Olympus BX50WI (Japan)
noninverted microscope with 40× water immersion objective
combined with an infra-red filter, a monochrome CCD
camera (COHU 4912), and a PC compatible system for
analysis of images and contrast enhancement (WinVision
2000, Delta Sistem, Italy). Recording pipettes were
advanced towards individual striatal cells in the slice under
positive pressure and visual control (WinVision 2000,
Delta Sistemi, Italy) and, on contact, tight GΩ seals were
made by applying negative pressure. The membrane patch
was then ruptured by suction and membrane current and
potential monitored using an Axopatch 1D patch clamp
amplifier (Molecular Devices, Foster City, CA, USA).
Whole-cell access resistances measured in voltage clamp
were in the range of 5–20 MΩ. Whole-cell patch clamp
recordings were made with borosilicate glass pipettes
(1.8 mm o.d.; 2–3 MΩ), in voltage-clamp mode, at the
holding potential of −80 mV.
To study GABA-mediated spontaneous inhibitory
postsynaptic currents (sIPSCs), the recording pipettes were
filled with internal solution of the following composition
(mM): 110 CsCl, 30 K+-gluconate, 1.1 EGTA, 10 HEPES,
0.1 CaCl2, 4 Mg-ATP, 0.3 Na-GTP. MK-801, and CNQX
were added to the external solution to block, respectively,
NMDA and non-NMDA glutamate receptors. Drugs were
first dissolved in water or in DMSO (HU-210) and then in
the ACSF to the desired final concentration. The
concentrations of the various drugs were chosen according to
previous in vitro studies on corticostriatal brain slices [33, 41]
and were as follows (in μM): 10 CNQX, 25 MK-801, and 1
HU-210 (Tocris Bioscience).
Synaptic events were stored by using P-CLAMP 9
(Axon Instruments) and analyzed offline on a personal
computer with Mini Analysis 5.1 (Synaptosoft, Leonia,
NJ, USA) software. The detection threshold of sIPSCs
was set at twice the baseline noise. The fact that no false
events would be identified was confirmed by visual
inspection for each experiment. Offline analysis was
performed on spontaneous synaptic events recorded during
fixed time epochs (1–2 min), sampled every 2–3 min
(5–12 samplings) .
Only data from putative GABAergic medium spiny
projection neurons (MSNs) were included in the present study
and identified immediately after rupture of the GΩ seal, by
evaluating their firing response to the injecting of
depolarizing current (typically tonic, with little or no adaptation).
One to five cells per animal were recorded. For each
type of experiment and time-point, at least four mice per
group were employed. Electrophysiological results from
neurons recorded from the same animal were treated as a
separate sample and averaged before calculating statistics.
One animal per day was used for the electrophysiological
experiment. Throughout the text “n” refers to the number
of cells, unless otherwise specified.
Striatal total protein extracts preparation and WB
At least 4 animals per group were included in all western
blot (WB) experiments. Mice were sacrificed through
cervical dislocation, and both left and right striata were quickly
removed and frozen until use. Tissues were homogenized
in RIPA buffer plus protease and phosphatase inhibitors
cocktail (SIGMA) as previously described [10, 36].
Primary antibodies were used as following: mouse
antiβ-actin (1:20,000, 1 h RT; Sigma-Aldrich), rabbit
antidopamine (DA)- and cAMP-regulated phosphoprotein
32 kDa (DARPP32; 1:50,000, 1 h RT; Abcam), rabbit
antip-Th34-DARPP32 (1:1000, overnight 4 °C; Merck), goat
anti-p-Ser316-CB1R (1:200, overnight 4 °C; SantaCruz),
and goat anti-CB1R (1:200, overnight 4 °C; SantaCruz).
Membranes were incubated with the following secondary
antibodies: anti-mouse IgG HRP (1:10,000; GE Healthcare,
formerly Amersham Biosciences), anti-rabbit IgG HRP
(1:2000; GE Healthcare, formerly Amersham Biosciences),
anti-goat IgG HRP (1:2000; GE Healthcare, formerly
Amersham Biosciences), and diluted in 1 % milk for 1 h at
RT. Blots were stripped with Restore Western Blot
Stripping Buffer (Thermo Scientific), after detection of
phosphosites. Complete stripping was assessed by incubation with
proper secondary antibody immunodetection was
performed by ECL reagent (Amersham) and membrane was
exposed to film (Amersham). Densitometric analysis of
protein levels was performed by NIH ImageJ software
(http://rsb.info.nih.gov/ij/). Phospho-CB1R band
densitometry was normalized respect to β-actin, since in a different
blot of the same samples, we did not detect changes in
CB1R unphosphorylated protein, while DARPP32
phosphoprotein levels were normalized to DARPP32
unphosphorylated protein to account for changes in the amount of
DARPP32 unphosphorylated protein. WB results are
presented as data normalized to control CFA values.
RNA extraction and qPCR
Total RNA was extracted according to the standard
miRNeasy Micro kit protocol (QIAGEN). The RNA quantity
and purity were analyzed with NanoDrop 2000c
spectrophotometer (Thermo Scientific). The quality of RNA was
assessed by visual inspection of the agarose gel
electrophoresis images. Next, 250 ng of total RNA was
reversetranscribed using high-capacity cDNA reverse
transcription kit (Applied Biosystem) according to the
manufacturer’s instructions and 20 ng of cDNA was amplified with
SensiMix SYBR Hi-Rox Kit (Bioline; Meridian Life Science)
in triplicate using the Applied Biosystem 7900HT Fast Real
Time PCR system. Relative quantification was performed
using the ΔΔCT method. β-actin was used as internal
controls. The following primer sequences were used.
Brain-derived neurotrophic factor (BDNF) (NM_007540):
5′-ACCATAAGGACGCGGACTTGT-3′ (sense); 5′-AAG
AGTAGAGGAGGCTCCAAAGG-3′ (antisense); β-actin
A-3′ (sense); and
Data were presented as mean ± SEM. Throughout the
text, “n” refers to the number of animals, with the
exception of electrophysiological experiments, where “n”
refers to the number of the cells. Two-sample
comparisons were carried out using the Student’s T test for
parametric measures or Mann-Whitney for
nonparametric variables, while multiple comparisons were
made using one-way ANOVA followed by Tukey’s HSD
or non-parametric Kruskal–Wallis test followed by
Dunn’s comparisons. The main effects of the two
conditions (genotype and EAE) on the dependent behavioral
variables and the interactions genotype × EAE were
analyzed by performing two-way ANOVAs. The significance
level was established at p < 0.05.
EAE-induced anxiety is associated with CB1R
desensitization in the striatum
We investigated the anxiety-like phenotype associated
with MOG-induced central inflammation in
presymptomatic EAE mice. In these mice, we assessed
anxiousdepressive-like responses by using the LDT and the nest
building (NB) paradigm, a motivation-based task .
Significant differences between EAE and controls
emerged at the LDT (Fig. 1a–a”), since the time spent in
the light zone (EAE 16.42 ± 4.6 %; CFA: 42.21 ± 4.72 %;
unpaired T test; p < 0.01; Fig. 1a) and the number of
rearing episodes during LDT (CFA: 23 ± 3.32, n = 8;
EAE: 11.38 ± 2.33, n = 8, unpaired T test: p < 0.05;
Fig. 1a’) were reduced in EAE, indicating both
anxietylike behavior and reduced motivation-based activity,
respectively, in accordance with our previous findings .
Next, we measured goal-directed behavior of EAE and
control mice by using the NB test. Nesting ability is a
natural, instinctive motivation-driven behavior in
rodents . Murine NB skills have been found altered
during inflammation challenge [42, 43] and, of note, in
EAE mice during the acute phase of the disease
independently of motor defects . Preclinical EAE mice
performed significantly worse than controls in terms of
nesting score (CFA 3.5 ± 0.18; EAE 2.07 ± 0.31;
MannWhitney non-parametric test p < 0.01) (Fig. 1b, b’), again
indicating that EAE is associated with reduction of social
behavior, which is a characteristic trait of sickness
behavior, affecting EAE mice .
The endocannabinoid system (ECS) is deeply involved
in mood control [25, 26]. We asked whether CB1Rs could
be involved in EAE-behavioral syndrome. One week
before immunization, we evaluated the spontaneous
responses of WT and CB1R-KO mice at LDT, in order to
monitor anxiety-like behavior under basal condition. After
2 weeks, during EAE (7–9 dpi), the mice were tested again
for anxiety by means of LDT.
CB1R-KO mice spent less time in the lit compartment
than WT mice (n = 8 for each groups; F = 13.54, p < 0.01),
showing increased anxiety-related behavior as previously
reported . Such behavior was exacerbated by EAE
induction. EAE induction had a substantial effect on
behavioral performance, accounting for 47.01 % of total
variance (F = 40.76, p < 0.0001), with a significant
interaction with genotype (F = 4.403, p < 0.05) (Fig. 1c),
suggesting the role of CB1Rs in anxiety-like behavior
associated to EAE.
We have previously reported that the downregulation
of CB1R-mediated control of GABA synapses in the
striatum represents a reliable synaptic correlate of anxiety
induced by chronic psychoemotional stress  or by a
single intracerebroventricular injection of IL-1β . Of
note, in a previous study, we demonstrated that CB1R
sensitivity on GABA synapses is lost in the striatum of
EAE mice during the symptomatic phase of the disease
. We therefore assessed whether EAE-induced anxiety
was also associated with alterations of CB1R function.
Thus, control and EAE mice were sacrificed to study
synaptic transmission in the striatum (9–11 dpi). Here, we
found that application of HU210 (1 μM, 10 min)
significantly reduced the frequency of sIPSCs in CFA mice (79.8
± 3.5 %; n = 10; p < 0.01), while this CB1R agonist failed to
alter GABA transmission in the presymptomatic EAE
mice (96.3 ± 3.4 %; n = 9; p > 0.05), suggesting that this
synaptic alteration is precocious and, more interestingly, is
linked to behavioral alterations in EAE (Fig. 1d, d’).
Overall, these data indicate that CB1Rs are involved in
EAE-mediated anxiety-like behavior occurring in the
presymptomatic phase of the disease.
Blockade of IL-1β signaling reduces anxiety, rearing
defects, and CB1R dysfunction in EAE
To investigate the role of IL-1β in EAE-induced anxiety,
we chronically inhibited IL-1β signaling in these mice by
icv delivery of IL-1ra. We have previously shown that this
preventive treatment reduced motor disability and
inflammatory reaction in the cerebellum, the hippocampus, and
the striatum of EAE mice during the symptomatic phase
of the disease [10, 36, 47]. Moreover, increased IL1-beta
expression in the striatum of EAE mice with preserved
motor functions was associated to depressive-like and
motivation-based behaviors observed in these mice .
Here, we investigated the anxious-like behavior of EAE
mice treated with IL-1ra during the preclinical phase of
EAE, when motor defects are not detectable.
In the LDT, IL-1ra partially corrected the behavioral
alterations of pre-symptomatic EAE mice. The time spent
in the lit zone by EAE-IL-1ra mice was not different with
respect to both CFA-vehicle and EAE-vehicle
(CFA-vehicle 49.90 ± 4.98, EAE-vehicle 27.18 ± 6.34, EAE-IL-1ra
41.34 ± 3.76; one-way ANOVA post hoc comparisons:
CFA-vehicle vs EAE-vehicle p < 0.05; CFA-vehicle vs
EAEIL-1ra p > 0.05; EAE-vehicle vs EAE-IL-1ra p > 0.05),
although very similar to CFA-vehicle (Fig. 2a, a”). However,
rearing activity during the LDT was fully corrected by icv
IL-1ra delivery in EAE mice (EAE-IL-1ra: 21.13 ± 2.88,
n = 8, vs EAE-VH: 11.38 ± 2.33, n = 8; p < 0.05; Fig. 2a’).
Fig. 1 Anxiety-like behavior in EAE is associated to CB1R dysfunction in the striatum. a Exploratory behavior of EAE mice was investigated firstly
by LDT confirmed increased anxiety-like behavior in EAE group as showed by time spent in the light zone of the task. a’ Rear episodes in EAE mice were
severely reduced in comparison to CFA mice. a” Representative video-recording tracking of CFA and EAE mice performance in the light zone of the LDT
apparatus. b, b’ EAE and CFA control mice were evaluated for their ability to construct nests (rated on a 4-point scale) to investigate motivation-based
behavior. Bar graph shows a reduction in the quality of nest in EAE mice (b). b’. Representative photographs of nests built by CFA and EAE mice. c. The
performance of CB1R-KO mice at the LD test (% of time spent in the bright compartment) revealed an anxiety-related behavior, which is heavily affected
by EAE induction. d, d’ Bath application of the CB1R agonist HU210 on striatal slices induced sIPSC frequency reduction in CFA mice (p < 0.01). In EAE
striatal slices, the effect of HU210 was abolished (p > 0.05) (d). Representative electrophysiological traces are depicted in d’. Values are means ± SEM.
Statistical differences were analyzed by unpaired T-test or Mann-Whitney test (behavior) and by paired T-test (electrophysiology). *p < 0.05, **p < 0.01.
Two-way ANOVA analysis for genotype factor: ##p < 0.01, ### p < 0.0001
We next addressed the effects of the IL-1ra icv delivery
in EAE mice at the NB test. Consistently with the results
in non-minipump-implanted animals, the nesting score of
EAE mice receiving icv vehicle (9 dpi, n = 14; 1.78 ± 0.15)
was worse than CFA-vehicle animals (n = 14; 3.25 ± 0.17),
and IL-1ra icv treatment corrected these behavioral
alterations (2.67 ± 0.16; n = 14; non-parametric Kruskal–Wallis
test followed by Dunn’s comparisons: CFA-vehicle vs
EAEvehicle p < 0.001; CFA-vehicle vs EAE-IL-1ra p > 0.05;
EAE-vehicle vs EAE-IL-1ra p < 0.05) (Fig. 2b, b’).
We performed electrophysiological measurements of
striatal CB1R sensitivity in these mice 1 week after the
behavioral tests in order to recover the effect of the stress
induced by behavioral manipulation on endocannabinoid
Fig. 2 IL-1ra preventive treatment improves EAE behavioral syndrome and restores striatal CB1R function. a–a” At the LDT, the IL-1ra treatment
improved the behavioral alterations of EAE mice; the time spent in the light zone, EAE-IL-1ra mice showed values similar to CFA-vehicle,
although being not significantly different to both CFA and EAE-vehicle (a). a’ Vertical activity increased in EAE-IL-ra mice, indicating an ameliorated
explorative response induced by IL1-ra treatment. Examples of LDT video-tracking are depicted in a”. b, b’ Consistently with the results obtained in
non-minipump-implanted animals (shown in Fig. 1b, b’), the nesting score of EAE-vehicle mice was worse than CFA-vehicle animals and IL-1ra icv
treatment corrected these behavioral alterations (b). Representative photographs in b’. c, c’. The lack of the depressant effect mediated by HU210 in EAE
slices was rescued in EAE mice receiving in vivo treatment of IL-1ra by minipump implantation (c). In c’, there are examples of electrophysiological traces
showing the lack of the effect of HU210 only in EAE-vehicle mice. Values are means ± SEM. Statistical differences were analyzed by one-way ANOVA for
multiple comparisons (followed by Tukey HSD or Dunn's comparisons) or by unpaired T-test for rear numbers in LDT. *p < 0.05, **p < 0.01, ***p < 0.001
system . Of note, the loss of CB1R sensitivity on
GABA synapses is a synaptic dysfunction that occurs
throughout the disease progression (; present paper])
and IL-1ra treatment was previously shown to ameliorate
both the depressive-like behavior of EAE mice in the acute
phase of the disease and the dopaminergic dysfunction
affecting the striatum .
We observed that the preventive and central treatment
with IL-1ra rescued the physiological response of CB1R
to HU210 (% of sIPSC frequency pre-HU210:
CFAvehicle 81.60 ± 5.6, n = 8; EAE-vehicle 108.4 ± 5.8, n = 9;
EAE-IL-1ra 82.4 ± 3.9, n = 10; one-way ANOVA post hoc
comparisons: CFA-vehicle vs EAE-vehicle p < 0.01;
EAEvehicle vs EAE-IL-1ra p < 0.05) (Fig. 2c, c’).
Altogether, these data are consistent with the idea that
EAE anxiety-like behavior is linked to IL-1β-mediated
Dopamine system is involved in EAE-induced alteration of
striatal CB1R function
Striatal CB1R function is regulated by the DA system.
Accordingly, pharmacological enhancement of DA signaling
in vivo sensitizes CB1R  and DA receptor inhibition
blocks them . Recent biochemical and
electrophysiological experiments demonstrated impaired DA
transmission in the striatum of EAE mice , raising the possibility
that also CB1R dysfunction is mediated by defective DA
signaling in this neuroinflammatory condition. Of note,
genetic and pharmacological blockade of DA pathway is
known to reduce rearing activity in mice [50–52]. The
reduced rearing activity observed in EAE mice confirmed
defective DA signaling in EAE mice  and suggested that
CB1R dysfunction could indeed be secondary to this
alteration in EAE.
We tested therefore the hypothesis that defective DA
transmission could be responsible of CB1R downregulation
in EAE by evaluating striatal CB1R function in EAE mice
treated in vivo with amphetamine, to favor DA release
from DA nerve terminals. In striatal brain slices prepared
from these animals, HU210 caused the expected inhibition
of sIPSC frequency (EAE-amphetamine: 80.06 ± 3.2 %, n =
11, paired T test p < 0.05; EAE-vehicle: 97.8 ± 3.6, n = 10;
paired T test p > 0.05), confirming the rescue of CB1R
function by DA system stimulation in EAE mice (Fig. 3a).
We have recently found that phosphorylation at
threonine 34 (P-Th34) of the DA- and cAMP-regulated
phosphoprotein 32 kDa (DARPP32), marker of striatal
MSNs, is increased in EAE, possibly due to unbalanced
signaling through D1- and D2-like receptors . By WB
experiments, we checked the phosphorylation of DARPP32
at Th34 site after in vivo treatment with amphetamine to
assess whether amphetamine could interfere with DA
signaling in MSNs. Amphetamine treatment in CFA mice did
not change neither the extent of DARPP32 phosphorylation
at Th34 (p-Th34-DARPP32/DARPP32: CFA-vehicle 1 ±
0.06, n = 6; CFA-amphetamine 1.13 ± 0.04, n = 7; unpaired
T test p > 0.05; Fig. 3b, b’), nor the total amount of
DARPP32 (DARPP32/β-actin ratio: CFA-vehicle 1 ± 0.07
n = 6; CFA-amphetamine 1.17 ± 0.06, n = 7; unpaired T
test p > 0.05; Fig. 3b–b”). Amphetamine reduced, although
not completely, the hyper-phosphorylation of
Th34DARPP32 induced by EAE (p-Th34-DARPP32/DARPP32
ratio: CFA-vehicle 1 ± 0.25, EAE-vehicle 2.7 ± 0.23,
EAEamphetamine 1.91 ± 0.13; CFA-vehicle n = 5 vs EAE-vehicle
n = 5, one-way ANOVA post hoc comparison: p < 0.001;
CFA-vehicle vs EAE-amphetamine n = 4; one-way ANOVA
post hoc comparison: p < 0.05; Fig. 3c, c’). However, the
amphetamine treatment induced a slight increase in the
expression of total DARPP32 in the EAE striatum
(DARPP32/β-actin ratio: CFA-vehicle 1 ± 0.07,
EAEvehicle 1.03 ± 0.07, EAE-amphetamine 1.34 ± 0.03;
oneway ANOVA: CFA-vehicle vs EAE-amphetamine p < 0.01;
EAE-vehicle vs EAE-amphetamine p < 0.05) (Fig. 3c–c”),
which accounted for the quantification of p-Th34-DARPP32,
suggesting the triggering of compensatory mechanism
induced by amphetamine to cope with the EAE-induced
overstimulation of DA system.
We investigated the possible mechanisms underpinning
the link between the CB1R- and DA-mediated
neurotransmission. We first assessed the expression of the BDNF,
which was previously shown to be a downstream effector
of D2Rs in the modulation of CB1R in the mouse striatum
. By qPCR, we found that BDNF transcript was
significantly increased in the striatum of EAE-vehicle mice (n = 6,
fold change mRNA: 5.95 ± 1.90) compared to CFA-vehicle
mice (n = 8, fold change mRNA: 1.15 ± 0.22), and that
amphetamine treatment was not able to re-establish normal
levels of BDNF expression (n = 6, fold change mRNA:
5.23 ± 1.69; one-way ANOVA Tukey post hoc
comparisons: CFA-vehicle vs EAE-vehicle p < 0.05, CFA-vehicle
vs EAE-amphetamine p > 0.05, EAE-vehicle vs
EAEamphetamine p > 0.05; Fig. 4a). This result indicated that
BDNF modulation was not involved in the observed
recovery of neurotransmission by amphetamine.
We next asked whether amphetamine treatment could
modify the phosphorylation status of CB1R. CB1R
undergoes phosphorylation at serine 316 (p-Ser316), and such
phospohorylation has been linked to the internalization of
the receptor-ligand complex . WB experiments showed
that CB1R levels did not differ among the three
experimental groups EAE-vehicle (n = 5, CB1R/β-actin ratio: 1 ± 0.02),
CFA-vehicle (n = 5, CB1R/β-actin ratio: 1.13 ± 0.28), and
EAE-amphetamine (n = 4; CB1R/β-actin ratio: 0.84 ± 0.18;
one-way ANOVA Tukey post hoc comparison: p > 0.05 for
all comparisons; Fig. 4b, b’). Conversely, compared to
control (CFA-vehicle n = 5, p-Ser316-CB1R/β-actin ratio: 1 ±
0.19) EAE striatal lysates (n = 5; p-Ser316-CB1R/β-actin
ratio: 0.40 ± 0.12) showed a strong downregulation of the
Fig. 3 CB1R function is linked to dopamine system in the EAE striatum. a The inhibitory effect of HU210 on sIPSC frequency of striatal neurons
was completely restored in EAE mice receiving i.p. injection of amphetamine (p < 0.05). b–b” Amphetamine treatment did not affect DARPP32
phosphorylation in the striatum of control CFA mice, as shown by the WB image in b and the densitometric analysis in b’. The treatment did not
change the amount of unphosphorylated protein, as shown in b and b”. c–c” Representative WB of striatal protein extracts from EAE-vehicle and
EAE-amphetamine mice: the densitometric analysis of the bands displays an upregulation of Th34-DARPP32 in EAE-vehicle samples and a partial
recovery of such phosphorylation in EAE-amphetamine group, as shown by graph in c’. Amphetamine treatment induced a significant upregulation of
DARPP32 expression in EAE striatum (c”). WB data are normalized to CFA values. Values are means ± SEM. Statistical differences were analyzed by paired
Student’s T test (electrophysiology) and one-way ANOVA followed by Tukey HSD (WB). *p < 0.05 EAE-amphetamine vs EAE-vehicle
p-Ser316-CB1R, which was not recovered by amphetamine
treatment (n = 4, p-Ser316-CB1R/β-actin ratio: 0.43 ± 0.10;
one-way ANOVA Tukey post hoc comparisons:
CFAvehicle vs EAE-vehicle p < 0.05, CFA-vehicle vs
EAEamphetamine p > 0.05, EAE-vehicle vs EAE-amphetamine
p > 0.05, Fig. 4c, c’), excluding this mechanism for
amphetamine-mediated recovery of CB1R sensitivity on
The results of the present investigation suggest the
involvement of CB1Rs in the anxious phenotype of EAE
mice. EAE-induced anxiety was in fact associated with a
dramatic downregulation of CB1R-mediated presynaptic
inhibition of GABA transmission in the striatum, a brain
area increasingly recognized to play a substantial role in
anxiety control in humans and rodents [54, 55] and
Fig. 4 BDNF and CB1R signaling in EAE striatum. a BDNF mRNA in
EAE-vehicle striatum is upregulated with respect to control CFA-vehicle
(p < 0.05). Such alteration is not corrected by amphetamine treatment.
b shows WB of striatal lysates of CFA-vehicle, EAE-vehicle, and
EAEamphetamine. CB1R levels in the striatum are not affected by EAE, as
indicated by WB analysis of protein lysates (b’). Amphetamine given 24 h
before the sacrifice of the animals did not affect the total amount of
CB1R in the EAE striatal lysates. CB1R signal was normalized to β-actin
bands. WB data are normalized to CFA values. c, Representative WB of
lysates from CFA-vehicle, EAE-vehicle, and amphetamine-vehicle probed
with antibody specific for the carboxy-terminal phosphorylation of CB1R
(p-Ser316): the histogram in c’ indicates a remarkable reduction of CB1R
phosphorylation, normalized to β-actin, which is not modulated by
amphetamine treatment. Values are means ± SEM. Statistical
differences were analyzed by one-way ANOVA followed by
Tukey HSD (WB and qPCR). *p < 0.05 EAE-vehicle vs CFA-vehicle
affected in both EAE  and MS . Anxiety was
exacerbated in EAE mice lacking CB1Rs, and both the
anxiety-like phenotype and striatal CB1R sensitivity were
rescued in EAE mice by central inhibition of IL-1β
signaling with IL-1ra. Moreover, CB1R function was
restored by amphetamine treatment, providing further
evidence for the cannabinoid-dopamine interaction in
the striatum of EAE mice.
Despite the high prevalence and severity of mood
disturbances in the MS population, depression and anxiety
are generally underestimated and undertreated  and
poor knowledge of the pathophysiological mechanisms
of mood alteration in MS explains, at least in part, the
scarce attention paid to this serious comorbid condition.
Several studies in the EAE model have highlighted the
independence of behavioral alterations in EAE mice
from motor disability and linked these to the effects of
pro-inflammatory cytokines, like TNF and IL1-β, in
different brain circuits, like the amygdala, the
hippocampus, and the striatum [7–10]. TNF and IL-1β, like
other pro-inflammatory cytokines, have been
convincingly associated with mood disorders both in humans
[57, 58] and rodents , and their levels increased in
serum and CSF of MS patients [3, 6, 60], and in EAE
brains [8–10, 36].
We previously associated EAE anxious-like behavior
to TNF-induced altered glutamatergic transmission in
the striatum . The present investigation links the
anxiety-like behavior of presymptomatic EAE mice to
IL-1β-induced CB1R dysfunction on GABA synapses.
Overall, the studies coming from our and other labs
indicate that the behavioral syndrome associated to
EAE reflects the complex and parallel action of
proinflammatory molecules on specific synaptic targets.
In this respect, here, we showed that the loss of CB1R
sensitivity on GABA synapses, previously described in
the symptomatic phase of the disease , occurs early,
before the appearance of motor symptoms, raising the
possibility that this synaptic defect could be involved in
EAE-anxiety-like behavior. In mice lacking CB1R, EAE
caused a significant increase of anxiety after EAE in both
WT and CB1R-KO mice at the LDT. CB1R-KO mice,
however, developed a much more marked anxiety-like
behavior in the preclinical phase of EAE compared to
their WT counterpart, indicating high vulnerability to
the effects of neuroinflammation in both EAE-induced
motor deficits  and anxiety (present study). CB1-KO
mice lack the inhibitory function of CB1Rs in controlling
both the glutamatergic and the GABAergic transmission
and the effect of pro-inflammatory cytokines on neurons
[21, 33, 60], predisposing them to be more sensitive to
EAE induction. In fact, pharmacological activation of
CB1R dampens the TNF-mediated potentiation of
striatal spontaneous glutamate-mediated excitatory
postsynaptic currents (sEPSCs), which is believed to cogently
contribute to the inflammation-induced
neurodegenerative damage observed in EAE mice. Furthermore, mice
lacking CB1R showed a more severe clinical course and,
in parallel, exacerbated alterations of sEPSC duration
after induction of EAE, indicating that endogenous
cannabinoids activate CB1R and mitigate the synaptotoxic
action of TNF in EAE .
The interaction between IL1β and the ECS is also
emerging, based on the evidence that IL-1β effects on
striatal spontaneous excitatory and inhibitory currents
are regulated by transient receptor potential vanilloid 1
(TRPV1) channels, members of the ECS .
Furthermore, IL-1β has also been shown to modulate the
sensitivity of CB1Rs controlling synaptic transmission in the
striatum (, present study]). Of note, IL-1β is involved
in mood alterations associated with inflammatory
illnesses and with stress. In line with this, a single icv
injection of IL-1β caused anxiety in mice and abrogated
the sensitivity of CB1Rs controlling GABA synapses in
the striatum. Identical behavioral and synaptic results
were obtained following social defeat stress, and icv
injection of IL-1ra reverted both effects . The present
findings reported in EAE are consistent with our
previous observations and reinforce them. In fact, both
behavioral and synaptic dysfunctions were abrogated by
in vivo inhibition of IL1-β.
IL-1β-dependent inhibition of striatal CB1R function
was mediated by the interference of this pro-inflammatory
cytokine with the DA system, since DAergic stimulation
with amphetamine abrogated the effects of EAE on
CB1Rs, and IL-1ra reversed the biochemical and
molecular , as well as the behavioral defects and [10, present
study] of DA transmission.
The role of DA in the control of striatal CB1R function
has already been reported in previous studies, showing that
in vivo facilitation of DA release with cocaine enhances the
sensitivity of these receptors , while DA receptor
blockade with haloperidol causes the opposite effect .
Interestingly, the role of dopamine in immune function and
fatigue perception in MS is also emerging and other studies
are needed to explore the mechanisms of dopamine
imbalance in MS . Here, we found that amphetamine
treatment was able to restore normal CB1R functioning at
GABA terminals in the striatum of EAE mice, confirming
the link between the two systems in this brain area. We
therefore asked whether amphetamine treatment could
also recover mood disturbance in EAE mice, but we could
not assess behavior in the treated mice. Indeed, in our
experimental condition, about 70 % of the mice showed
hypo-locomotion (number of the line crossing between the
LD compartments: EAE amphetamine: 1.58 ± 0.47, n = 17;
EAE vehicle: 3.78 ± 0.42, n = 14; unpaired T test **p =
0.0021, data not shown), in accordance with the literature
reporting that amphetamine affects locomotion in a
doseand time-dependent manner [64, 65].
Since we have previously hypothesized a D1R-D2R
altered signaling in the EAE striatum due to increased
phosphorylation of DARPP32 at th34 , we analyzed
the effect of amphetamine on this biochemical feature of
the DA system in the EAE striatum. Amphetamine as
well as D1R agonists is reported to induce a rapid
increase in the phosphorylation of DARPP32 at Th34
site (15 or 30 min after injection) [37, 66], by activating
D1R and inducing a potentiation of the PKA pathway. In
our experimental settings, we found that in control mice,
amphetamine did not change the phosphorylation status
of DARPP32, possibly due to the later time-point of
evaluation (24 h) and the dose administered. However,
amphetamine induced an upregulation of the total DARPP32,
which accounted for the reduction of the DARPP32
phosphorylation compared to EAE. In the light of the reduced
D2R signaling in the striatum of EAE mice , we
suppose that the increased DA levels due to amphetamine
treatment restore D2Rs functioning counteracting the
aberrant condition of D1 oversensitization in EAE. This
ultimately induces compensatory mechanisms aimed at
enhancing DARPP32 availability for phosphorylation.
Our electrophysiological recordings suggest that
increasing DA levels induces presynaptic rearrangements
that recover CB1R function on GABA terminals. Thus, we
explored possible mechanisms to explain this result. One
possibility was given by the interaction between D2R and
CB1R mediated by BDNF as described in a previous study:
D2R stimulation induces BDNF downregulation, which in
turn regulates CB1R function in striatum . The
proposed mechanism was linked to the location of both kinds
of receptors at lipid raft. In line with the hypothesis of
D2R-CB1R desensitization in the EAE striatum, we found
for the first time that BDNF mRNA is increased in the
striatum of EAE mice. However, amphetamine treatment
recovers CB1R function in a BDNF-independent manner.
Another possibility was given by the phosphorylation of
CB1R at ser316. Although the role of CB1R
phosphorylation in the endocannabinoid-mediated
neurotransmission is poorly studied, it has been proposed that it
regulates the internalization of the receptor-ligand
complex , a process necessary for the normal functioning
of the receptor signaling. Upregulation of CB1R
phosphorylation has been interpreted as a self-regulating
mechanism to reduce CB1R signaling  and linked to
increased social play .
Here, we found that CB1R phosphorylation is
reduced in the striatum of EAE mice, indicating an
altered CB1R signaling and functioning and corroborating
the electrophysiological results. Amphetamine treatment
was not able to restore normal phosphorylation status of
CB1R, leading to the conclusion that also this mechanism
was not involved in amphetamine-induced recovery of
CB1R sensitivity on GABA synapses.
It is worth mentioning that the D2R-CB1R signaling is
the subject of extensive investigation. Several other
mechanisms, here not explored, may take place, such as the
formation of D2R-CB1R heteromers or the involvement of
CB1R accessory proteins, like the cannabinoid receptor
interacting protein type 1 (CRIP1) . Although the
results of the present investigation do not allow exhaustive
conclusions about the mechanisms regulating the
D2RCB1R signaling in the EAE striatum, they clearly indicate
that in the EAE striatum, the DA system is compromised
and that this affect the function of CB1R.
Collectively, our data contribute to clarify the synaptic
and, at least in part, molecular basis of mood
disturbances in EAE and, possibly, MS. Understanding the
neurobiological underpinning of anxiety-like behavior in
EAE mice is of crucial importance to optimize the
treatment of mood disturbance in MS and, possibly, other
neuroinflammatory diseases. In this direction, targeting
the endocannabinoid system may be a valid therapeutic
tool for the treatment of both psychiatric and motor
symptoms in MS patients.
This investigation was supported by the Italian National Ministero dell’Università
(Grant No. 2010BN3MXM_007) to DC, by the German Research Foundation DFG
(FOR926) to BL, and by the Italian National Ministero della Salute (Progetto
Giovani Ricercatori, Code GR-2011-02351422 and GR-2011-02347036) to AM and
GM. DF is recipient of a Fondazione Italiana Sclerosi Multipla (FISM) fellowship.
Availability of data and materials
Data supporting the conclusions of this article are presented in the manuscript.
AG, DF, AM, HS, SB, and FDV performed the research. RF performed the analysis
and interpretation of data. AU supervised the behavioral experiments. BL provided
the CB1KO mice. NBM, MM, and BL contributed to the study conception and
design and critically revised the manuscript. AG, DF, GM, and DC designed the
research and drafted the manuscript. All authors read and approved the final
Ethics approval and consent to participate
All animal experiments described in this study were conducted at IRCCS
Fondazione Santa Lucia, according to the guidelines set by the Internal
Institutional Review Committee, the European Directive 2010/63/EU and the
European Recommendations 526/2007, and the Italian D.Lgs 26/2014. All efforts
were made to minimize the number of animals used and their suffering.
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