Neuropharmacology 96 (2015) 105e112

Contents lists available at ScienceDirect

Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

Invited review

The interplay between inflammatory cytokines and the

endocannabinoid system in the regulation of synaptic transmission
Silvia Rossi a, b, Caterina Motta a, b, Alessandra Musella a, b, Diego Centonze a, b, *
a
Tor Vergata, 00133 Rome, Italy
Clinica Neurologica, Dipartimento di Medicina dei Sistemi, Universita
b
Fondazione Santa Lucia/Centro Europeo per la Ricerca sul Cervello (CERC), 00143 Rome, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Excessive glutamate-mediated synaptic transmission and secondary excitotoxicity have been proposed
Available online 28 September 2014 as key determinants of neurodegeneration in many neurological diseases.
Soluble mediators of inflammation have recently gained attention owing to their ability to enhance
Keywords: glutamate transmission and affect synaptic sensitivity to neurotransmitters. In the complex crosstalk
TNF between soluble immunoactive molecules and synapses, the endocannabinoid system (ECS) plays a
IL-1b
central role, exerting an indirect neuroprotective action by inhibiting cytokine-dependent synaptic al-
Glutamate
terations, and a direct neuroprotective effect by limiting glutamate transmission and excitotoxic damage.
GABA
TRPV1
On the other hand, the endocannabinoid (eCB)-mediated control of synaptic transmission is altered by
Multiple sclerosis proinflammatory cytokines with consequent effects in central nervous system (CNS) disorders.
CB1 receptor In this review, we summarize the interactions, at the pre- and postsynaptic level, between major
inflammatory cytokines and the ECS. In addition, the behavioral and clinical consequences of the
modulation of synaptic transmission during neuroinflammation are discussed.
This article is part of a Special Issue entitled ‘Neuroimmunology and Synaptic Function’.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction
In recent years, the classical dichotomy between neuro-
inflammation and neurodegeneration has been challenged by a
series of discoveries showing that the two processes co-exist in
central nervous system (CNS) diseases. Common molecular path-
ways bringing together the two pathological processes have, in fact,
been described. Synaptic alterations and late excitotoxicity have
been proposed to link neuroinflammation to neurodegeneration
(Centonze et al., 2009; Rossi et al., 2012a). The crucial role of
endocannabinoid (eCB) system (ECS), able to modulate both
inflammation and synaptic transmission, has been indeed indi-
cated. Its activation determines a protective mechanism aimed at
reducing both neurodegenerative and inflammatory damage
through various and partially converging mechanisms. Best known
are the effects of the soluble mediators of inflammation and of eCBs
in the regulation of the inflammatory response that leads to cell
damage (Parolaro, 1999; Piomelli, 2003). Recently, direct and
* Corresponding author. Clinica Neurologica, Dipartimento di Medicina dei Sis-
temi, Universit

a Tor Vergata, Via Montpellier 1, 00133 Rome, Italy. Tel.: þ39 06 7259
6010; fax: þ39 06 7259 6006.
E-mail address: centonze@uniroma2.it (D. Centonze).
distinct effects on synapses were described for both. New and less
studied are the synaptic effects of their interaction and this is the
specific objective of this review.
Inflammation involves complex biological processes that act as a
protective mechanism to remove the injurious stimuli, whereas it
can lead to tissue damage if self-perpetuating. Many soluble me-
diators are released by activated T lymphocytes and microglia
during neuroinflammatory diseases. Among these factors, inter-
leukin-1b (IL-1b) and tumor necrosis factor (TNF) initiate and
propagate inflammation, mainly by inducing a local cytokine
network, but also have the potential to directly and extensively
influence synaptic transmission (Stellwagen et al., 2005;
Stellwagen and Malenka, 2006; Lai et al., 2006; Rossi et al.,
2012a, 2013). Both neurotransmitters and cytokines affect their
target cells through surface receptors and other molecular mech-
anisms. Cytokine receptors are present also in neurons in many
brain areas, such as the hippocampus, hypothalamus, striatum, and
cerebellum (Alboni et al., 2009; Gardoni et al., 2011).
ECBs are lipid messengers derivatives of integral components of
cellular membranes, anandamide (AEA; Devane et al., 1992) and 2-
arachidonoylglycerol (2-AG; Sugiura et al., 1995) being the best
characterized. A wide range of receptors, biosynthetic and hydro-
lyzing enzymes, and putative membrane transporters completes
the description of the ECS, a master system that is deeply involved

http://dx.doi.org/10.1016/j.neuropharm.2014.09.022
0028-3908/© 2014 Elsevier Ltd. All rights reserved.
106 S. Rossi et al. / Neuropharmacology 96 (2015) 105e112
in a variety of regulatory functions throughout the body
(Basavarajappa, 2007; Kano et al., 2009; De Petrocellis and Di
Marzo, 2009). It has been suggested as a pro-homeostatic and
pleiotropic signaling system activated in a time- and tissue-specific
way during physiological and pathological conditions. ECS disrup-
tion is strongly involved in neuropsychiatric disorders, particularly
in affective disturbances, such as anxiety and depression. To date,
two subtypes of cannabinoid Gi-protein-coupled receptors, type-1
(CB1Rs) and type-2 cannabinoid receptors (CB2Rs), have been fully
characterized and cloned. CB1Rs are the predominant cannabinoid
receptors in the CNS (Herkenham et al., 1991; Moldrich and
Wenger, 2000; Piomelli, 2003), acting as modulators of excitatory
and inhibitory neurotransmission (Vaughan and Christie, 2005),
but lower expression levels have been found also in the periphery
such as blood vessels, immune cells, and reproductive tissues
(Parolaro, 1999; Hillard, 2000; Habayeb et al., 2008; Bo €rner et al.,
2009). CB2Rs are expressed predominantly by microglia (Cabral
and Marciano-Cabral, 2005) and peripheral immune cells (Lee
et al., 2001; Bo€rner et al., 2009), but relatively low CB2R expres-
sion has been identified also in neurons (Van Sickle et al., 2005;
Brusco et al., 2008).
In this review, the synaptic effects of both proinflammatory
cytokines and the ECS are discussed, with focus on their mutual
interaction in the modulation of both excitatory and inhibitory
transmission. Limiting excitotoxic neurodegeneration sustained by
inflammatory mechanisms might explain the neuroprotective role
of the ECS. On the other hand, the disruption of ECS control of
synaptic transmission mediated by inflammation might be
involved in mood alterations and neurodegeneration processes in
neuroinflammatory disorders such as multiple sclerosis (MS). Data
supporting the behavioral and clinical counterpart of the synaptic
interplay between inflammatory cytokines and the ECS during
neuroinflammation will be presented.
2. Proinflammatory cytokines modulate synaptic
transmission
A contemporary view of the role of cytokines as neuro-
modulators is emerging from studies in humans and experimental
models. Cytokines act on neurons in normal adult brain, modu-
lating neural functions. They in fact are likely involved in synaptic
plasticity, neural transmission, and calcium signaling. Different
molecular mechanisms at pre- and postsynaptic levels can be
differentially activated by the same cytokines in different brain
structures.
In the cerebellum, IL-1b increases the duration of AMPA
receptor-mediated spontaneous excitatory postsynaptic currents
(sEPSCs) through a rapid downregulation of glutamateeaspartate
glial transporter (Mandolesi et al., 2013), but in hippocampal neu-
rons it enhances NMDA receptor function through activation of
tyrosine kinases and subsequent NR2A/B subunit phosphorylation
(Viviani et al., 2003). AMPA receptor expression and phosphoryla-
tion are also regulated in the hippocampus by IL-1b (Lai et al.,
2006). Of note, these effects on synaptic transmission involve
exclusively the postsynaptic sensitivity of the neurons to gluta-
mate, whereas the alterations that we have recently described to be
mediated by IL-1b in the striatum are compatible with a presyn-
aptic action of this cytokine. In fact, only the frequency of sEPSCs
increased in corticostriatal slices incubated with IL-1b, as for
increased glutamate release from presynaptic terminals, whereas
classical postsynaptic parameters of glutamate transmission
(amplitude and kinetic properties of sEPSCs) were unaltered (Rossi
et al., 2012a). We also ruled out the involvement of NMDA receptors
in this effect, since NMDA antagonists failed to prevent the effect of
IL-1b on sEPSC frequency (Rossi et al., 2012a).
Inhibitory GABA synapses provide a robust counterbalance to
glutamate signaling in neurons, and their possible alterations
might influence neuronal excitability and survival. A reduced
GABAA receptor-mediated neuronal inhibition has been demon-
strated to follow IL-1b action at GABA synapses. IL-1b, in fact,
reduced the amplitude of spontaneous inhibitory postsynaptic
currents (sIPSCs) recorded from striatal neurons, as for a post-
synaptic modulation of GABA transmission (Rossi et al., 2012b). In
conclusion, IL-1b has the property of increasing glutamate-
mediated synaptic transmission and limiting GABA-mediated
inhibitory transmission, resulting in unbalanced hyperexcitation
and possibly excitotoxic neurodegeneration.
Another proinflammatory cytokine extensively involved in the
modulation of synaptic transmission is TNF. TNF has been shown to
induce significant changes of glutamate transmission in the hip-
pocampus and striatum, by altering the expression and the
composition of glutamate AMPA receptor subunit (Stellwagen et al.,
2005; Stellwagen and Malenka, 2006; Leonoudakis et al., 2008;
Centonze et al., 2009). TNF is released in response to microglial
activation (Block and Hong, 2005). We have demonstrated that
Incubation of brain slices with activated microglia mimicked the
effects of TNF on the kinetic properties of glutamatergic sEPSCs, by
increasing the duration and decay time of the synaptic currents.
Furthermore, blockade of TNF signaling fully prevented the alter-
ations of sEPSCs induced by microglia, suggesting that, at least
in vitro, TNF is the main effector of the synaptic action of microglia
(Centonze et al., 2009).
On the other hand, growing evidence suggests that inflamma-
tory cytokines are involved in molecular mechanisms of brain
plasticity and in complex affective, motivational and cognitive
processes (Beattie et al., 2002; Ross et al., 2003; Yirmiya and
Goshen, 2011; Rossi et al., 2012c; Marin and Kipnis, 2013; Mori
et al., 2014). IL-1b is necessary for long-term potentiation (LTP)
maintenance (Ross et al., 2003) and is able to lower the threshold of
LTP induction (Mori et al., 2014). LTP is a long-lasting increase in
synaptic efficacy, which is thought to be an important underlying
mechanism of learning and memory formation (Bliss and
Collingridge, 1993) and, recently, described at the basis of brain
reserve, which is the ability to limit the clinical expression of brain
damage (Weiss et al., 2014). However there is also evidence of
detrimental effects of proinflammatory cytokines on CNS plasticity.
IL-1b was found to impair LTP induction and maintenance in a dose
dependent manner (Murray and Lynch, 1998). Similarly although
physiologically low levels of TNF may be important in the regula-
tion of synaptic plasticity (Stellwagen and Malenka, 2006), patho-
physiological levels of this cytokine have deleterious effects on
synaptic plasticity (Cunningham et al., 1996; Khairova et al., 2009).
The increase sensitivity of AMPA receptors to synaptically
released glutamate, described for both IL-1b and TNF, has been
involved in both synaptic plasticity (Beattie et al., 2002) and exci-
totoxic neurodegeneration (Centonze et al., 2009; Rossi et al.,
2012a, 2014a). Considerable overlap, in fact, exists in the receptor
and post-receptor mechanisms required for both LTP and excito-
toxicity, and a plasticity-pathology continuum has been proposed
(McEachern and Shaw, 1999; Calabresi et al., 2003). In line with
this, a dual role of inflammation in the modulation of synaptic
transmission could be considered, depending on the cytokine levels
and site of action.
3. The ECS modulates synaptic transmission
ECBs serve as retrograde messengers released from post-
synaptic nerve terminals, whose classical function is to bind spe-
cific receptors located at the pre-synaptic site and involved in the
modulation of neurotransmitter release. However, their impact on
 S. Rossi et al. / Neuropharmacology 96 (2015) 105e112
synaptic transmission is widespread and more complex than
initially thought. CB1Rs are massively expressed at the synaptic
terminals of excitatory and inhibitory neurons (Marsicano and Lutz,
2006), interacting with dopaminergic, glutamatergic and GABAer-
gic signaling (Szabo et al., 1998; Huang et al., 2001; Gubellini et al.,
2002; Vaughan and Christie, 2005; Di Filippo et al., 2008).
Neuronal activity is a potent stimulus for eCB synthesis and
release from postsynaptic neurons (Kano et al., 2009). Recently,
several ionotropic and G-protein-coupled receptors were also
shown to be capable of triggering eCB release, such as dopamine
D2 receptors (Centonze et al., 2004; Yin and Lovinger, 2006),
group I metabotropic glutamate receptors (Varma et al., 2001;
Centonze et al., 2007a), muscarinic receptors (Kim et al., 2002;
Musella et al., 2010), AMPA and NMDA receptors (Brown et al.,
2003; Ohno-Shosaku et al., 2007). Upon postsynaptic activa-
tion, these lipid mediators traverse the synapse to bind pre-
synaptic CB1Rs, thereby suppressing neurotransmitter release at
both excitatory and inhibitory synapses in a short- and long-
term manner. Although many signaling cascades have been
described, the CB1R is best-known for binding to members of
the Gi/o group of G proteins, resulting in decreased cyclic AMP
production and protein kinase A activation. Ultimately, these
changes trigger a decrease in calcium influx and an increase in
potassium conductance at presynaptic sites, leading to a
reduction of neurotransmitter release. Additional downstream
targets of the CB1Rs include receptor tyrosine kinases and
mitogen-activated protein kinases, among others (Dalton et al.,
2009).
However, a picture is emerging where the two most studied
eCBs, AEA and 2-AG, are functionally interconnected and different
regulators of glutamatergic and GABAergic transmission. Evidence
exists that in the striatum the two sets of CB1Rs controlling
glutamate or GABA transmission differ for the preferential eCB
activating them, and also have different regulation mechanisms.
CB1Rs controlling striatal glutamate synapses [CB1R(glu)], but not
those regulating GABA transmission [CB1R(GABA)] seem to be
targeted preferentially by AEA. In fact, the genetic or pharmaco-
logical inhibition of the AEA degrading enzyme fatty acid amide
hydrolase (FAAH), that selectively increases AEA levels, has been
shown to inhibit glutamate but not GABA transmission in a CB1R-
dependent manner (Maccarrone et al., 2008; Rossi et al., 2010a).
On the other hand, the stimulation of endogenous 2-AG synthesis
only affects GABA synapses by activating CB1R(GABA) (Maccarrone
et al., 2008). Of note, the elevation of AEA concentrations, induced
by pharmacological or genetic inhibition of AEA degradation, re-
duces levels, metabolism and physiological effects of 2-AG through
the stimulation of transient receptor potential vanilloid 1 (TRPV1)
channels (Maccarrone et al., 2008), indicating a functional inter-
action between these molecules.
Although the principal mechanism by which eCBs regulate
synaptic function is through retrograde signaling, a role for post-
synaptic CB1R signaling has also been described. ECBs, in fact, can
modulate neural function and synaptic transmission by engaging
TRPV1 channels and also CB1Rs located on or within the post-
synaptic cell (Bacci et al., 2004; Toth et al., 2005; Musella et al.,
2009; Grueter et al., 2010; Rossi et al., 2011a). In the cerebral cor-
tex, autocrine activation of postsynaptic CB1Rs evokes long-lasting
self-inhibition of a specific subset of interneurons (Bacci et al.,
2004). We have also demonstrated that pharmacological stimula-
tion of TRPV1 channels selectively and transiently enhances the
frequency of sEPSCs recorded from striatal neurons (Musella et al.,
2009). AEA acts as an endovanilloid substance in the striatum, since
genetic inhibition of anandamide degradation resulted in a tonic
activation of TRPV1 channels modulating glutamate but not GABA
release (Musella et al., 2009).
107
In the nervous system, glial cells were long considered passive
elements, their major role involving serving as structural support to
neurons. Today, glial cells are considered key components of and
players in synaptic communication. This applies to the role of glial
cells in eCB signaling as well. CB1Rs are expressed in hippocampal
astrocytes, and its activation results in an increase in intracellular
calcium (Navarrete and Araque, 2008). Furthermore, it has been
shown that astrocytic CB1-dependent increase in calcium concen-
tration determines the release of glutamate from astrocytes and
subsequent activation of neuronal presynaptic NMDA receptors,
resulting in time-dependent long-term depression of synaptic
transmission (Min and Nevian, 2012).
In the complex scenario from brain plasticity to excitotoxicity,
modulators of synaptic transmission, as discussed for inflammatory
cytokines, play dual, even opposite, roles. ECB signaling is respon-
sible for both depolarization-induced suppression of inhibition
(Wilson and Nicoll, 2001; Ohno-Shosaku et al., 2001) and
depolarization-induced suppression of excitation (Kreitzer and
Regehr, 2001), identifying it as a powerful and widespread modu-
lator of synaptic strength. Besides short-term forms of plasticity
thought to be involved in regulating over-excitability and pro-
moting synaptic homeostasis, eCBs are described as modulators of
processes underlying long-term plasticity and plasticity of elec-
trical synapses (Gerdeman et al., 2002; Chevaleyre and Castillo,
2003; Connors and Long, 2004; Heifets and Castillo, 2009). On
the other hand, the stimulation of CB1Rs located on presynaptic
glutamatergic nerve terminals limits glutamate release and exci-
totoxic damage, showing a direct neuroprotective effect of eCBs
(Eshhar et al., 1993; Marsicano et al., 2003; Centonze et al., 2007b,c;
Loría et al., 2010).
Collectively, these observations propose that the ECS is very
malleable and can establish specializations that lead to a much
wider array of final effects than the traditional short- and long-term
depression of glutamatergic and GABA-mediated transmission.
Recent evidence points to the membrane environment as a critical
regulator of signal transduction triggered by CB1Rs (Maccarrone
et al., 2009), and calls for further studies aimed at better clari-
fying the contribution of membrane lipids to eCB signaling.
4. ECS modulate the effect of proinflammatory cytokines on
synaptic transmission
The ECS has been recently proposed as a key modulator of the
synaptic effects of inflammation. Increasing evidence suggests the
extensive involvement of ECS in cytokine modulation of synaptic
transmission.
CB1Rs interfere with the neuronal effects of TNF, a proin-
flammatory cytokine that enhances glutamatergic synaptic cur-
rents and excitotoxic damage (Centonze et al., 2009; Rossi et al.,
2011a, 2014a). We have, in fact, demonstrated that TNF effect on
sEPSC duration is inhibited by concomitant pharmacological stim-
ulation of CB1R in slices, and additionally it is exacerbated in whole
CB1R-KO mice, while being attenuated in FAAH-KO mice (Rossi
et al., 2011a). Increased postsynaptic expression and phosphory-
lation of glutamate AMPA receptors have been recognized to
mediate TNF activity on glutamate sEPSCs (Centonze et al., 2009),
and consistently the stimulation of CB1R blocks TNF-induced in-
crease of surface AMPA receptors, exerting an antiexcitotoxic effect
in neurons (Zhao et al., 2010). Intracellular signaling between CB1
and TNF receptors competes to regulate AMPAR trafficking, as
suggested in studies of cortical pyramidal neurons (Hill et al., 2007;
Kim et al., 2006) and spinal cord neurons (Salio et al., 2002).
The synaptic effects of TNF can be also modulated by TRPV1
channels (Musumeci et al., 2011). TRPV1 channels are involved in
several inflammatory diseases, but their action is still controversial,
108 S. Rossi et al. / Neuropharmacology 96 (2015) 105e112
since both pro-inflammatory and anti-inflammatory roles have
been described (Rashid et al., 2003; Wang and Wang, 2005). When
applied to striatal slices from TRPV1-KO mice, TNF induced a higher
increase of sEPSC decay time and duration than in slices from wild-
type (WT) animals. This effect involved changes in glutamate AMPA
receptor activity, since the selective glutamate NMDA receptor
antagonist MK-801 did not alter TNF effect on sEPSCs in both
groups of animals (Musumeci et al., 2011). Of note, TRPV1 channels
exert opposite effects on the synaptic alterations induced by IL-1b,
showing a permissive role on these effects. IL-1b significantly re-
duces, at the postsynaptic level, the amplitude of GABA-mediated
sIPSCs (Musumeci et al., 2011; Rossi et al., 2012b), while in-
creases, at the presynaptic terminal, the frequency of glutamate-
mediated sEPSCs (Rossi et al., 2012a). Genetic or pharmacological
blockade of TRPV1 channels prevented the action of IL-1b on both
GABAergic and glutamatergic currents (Musumeci et al., 2011; Rossi
et al., 2012a). These data point at TRPV1 channels as essential
mediators of the synaptic effects of IL-1b.
The opposing interplay with the synaptic actions of the two
major pro-inflammatory cytokines suggests that the inflamma-
tory milieu determines whether TRPV1 channels exert prefer-
entially aversive or protective effects on neurons during
neuroinflammation.
5. Proinflammatory cytokines modulate synaptic
transmission through the ECS
The interplay between inflammation and the ECS bidirectionally
acts at the synapses. If eCBs are crucial modulators of cytokine-
dependent synaptic alterations, inflammation can directly alter
the eCB effects on synaptic transmission. An even closer interaction
between pro-inflammatory cytokines and the ECS has been, in fact,
recently identified. We have found that IL-1b abrogates the sensi-
tivity of CB1R controlling both glutamate and GABA transmission,
through different mechanisms. The depressant effect of CB1R ag-
onists on sEPSC and sIPSC frequency was completely abolished in
the presence of IL-1b (Rossi et al., 2012c; De Chiara et al., 2013). The
regulatory mechanisms of striatal CB1R(GABA) and CB1R(glu) are
remarkably different. While AEA seems to be the eCB molecule
targeting striatal CB1Rs(glu) (Rossi et al., 2010a), striatal
CB1Rs(GABA) are activated by 2-AG (Maccarrone et al., 2008;
Musella et al., 2010). Furthermore, CB1Rs(GABA) but not
CB1Rs(glu) are under the control of the dopaminergic system and
of brain-derived neurotrophic factor (BDNF) (Centonze et al.,
2007d; De Chiara et al., 2010a), and are regulated by environ-
mental factors such as stress (Rossi et al., 2008), and rewarding
experiences (De Chiara et al., 2010b). IL-1b blocks the sensitivity of
cannabinoid CB1R(GABA) in the striatum, through a mechanism
again dependent on TRPV1 channels, and also on lipid raft integrity,
where these receptors normally reside (Rossi et al., 2012c). The
inhibitory effects of IL-1b over striatal CB1Rs(GABA) are indeed
caused by increased cholesterol content in lipid rafts, and the
BDNF/trkB pathway plays a permissive role in such an effect (Rossi
et al., 2012c). Partial genetic deletion of BDNF or pharmacological
inhibition of trkB, in fact, abolished the effects of IL-1b on
CB1Rs(GABA), a result in line with evidence showing that BDNF
increases cholesterol content in lipid rafts (Suzuki et al., 2007), and
inhibits per se CB1R(GABA) function (De Chiara et al., 2010a). Of
note, the depletion of cholesterol in striatal slices blocks IL-
1beCB1R(GABA) interaction (Rossi et al., 2012c), pointing at
cholesterol-lowering agents as potential modulator of CB1R effects,
especially in an inflammatory environment.
IL-1beCB1R(GABA) and IL-1beCB1R(glu) interactions are
differentially regulated by the BDNF- and trkB-dependent compo-
sition of membrane lipid rafts, but are both dependent upon the
protein kinase C (PKC)/TRPV1 pathway. BDNF gene partial deletion,
pharmacological blockade of trkB and membrane cholesterol
removal, all left unaltered the sensitivity of CB1Rs(glu) to IL-1b (De
Chiara et al., 2013). On the other hand, genetic deletion of TRPV1
and pharmacological blockade of PKC, a major activator of TRPV1
channels (Sikand and Premkumar, 2007; Musella et al., 2009),
blocked IL-1b-mediated inhibition of both subtypes of CB1Rs (Rossi
et al., 2012c; De Chiara et al., 2013). Along with CB1Rs, striatal
glutamate and GABA synapses are controlled by other presynaptic
receptor subtypes, such as GABAB receptors. Application of the
GABAB receptor agonist significantly reduced striatal sEPSC and
sIPSC frequency even in the presence of IL-1b (Rossi et al., 2012c; De
Chiara et al., 2013), indicating the specificity of IL-1b action on
cannabinoid control of neurotransmitter release.
6. Synaptic interplay between cytokines and the ECS in
neuroinflammatory diseases
Neuroinflammation is a hallmark of MS, a chronic disease
affecting the CNS and presenting both demyelinating and neuro-
degenerative features. The described effects of inflammatory mol-
ecules on synaptic transmission provide a plausible link between
inflammatory reaction and neurodegenerative damage in this dis-
orders. On the other hand, the interplay between eCBs and cyto-
kines at the synapses suggests a potential way to modulate
neuronal dysfunctions.
6.1. Clinical implications
An exacerbated glutamate-mediated transmission has been re-
ported in experimental autoimmune encephalomyelitis (EAE), a
mouse model of MS, even in the absence of overt demyelinating
foci, but in association with microglial activation and release of
proinflammatory cytokines (Centonze et al., 2009). In mice with
EAE (Centonze et al., 2009; Haji et al., 2012) and in patients with
progressive MS (Rossi et al., 2014a), we have demonstrated that
glutamate transmission is altered by TNF, and that CB1R modulates
the synaptic effects of this proinflammatory cytokine (Rossi et al.,
2011a). Pharmacological activation of CB1R, in fact, dampened the
TNF-mediated potentiation of glutamatergic EPSCs (Rossi et al.,
2011a), which is believed to contribute to the inflammation-
induced excitotoxic damage observed in EAE mice (Centonze
et al., 2009). Furthermore, we also found that mice lacking CB1R
showed a more severe clinical course and, in parallel, exacerbated
alterations of sEPSCs. Consistently, we found that mice lacking
FAAH, and thus expressing elevated brain levels of AEA, developed
a less severe disease, and were resistant to the effects of TNF on
sEPSCs (Rossi et al., 2011a).
Also TRPV1 channels contrast the TNF-mediated synaptic al-
terations. The genetic or pharmacological blockade of TRPV1
channels, in fact, enhances the synaptic effects of TNF. Besides the
in vitro modulation, we have demonstrated that TRPV1-KO mice
exhibit an exacerbation of EAE clinical score and of synaptic ab-
normalities at the peak phase of the disease (Musumeci et al., 2011).
Excitotoxic insults observed in EAE are mediated not only by
TNF altering the postsynaptic properties of sEPSCs, but also by other
mechanisms altering glutamate release and the frequency of
sEPSCs. Among these mechanisms, the reverse mode of axonal
sodium/calcium exchanger (Rossi et al., 2010b), overactive NMDA
receptors (Grasselli et al., 2013) and IL-1b (Rossi et al., 2012a) have
been identified. Mice lacking CB1R on glutamate terminals were
characterized by worse presynaptic alterations during EAE, since
sEPSC frequency, a reliable measure of presynaptic transmitter
release, was increased in CB1R-KO(glu) mice as compared to CB1R-
KO(GABA) and to WT EAE mice (Musella et al., 2014).
 S. Rossi et al. / Neuropharmacology 96 (2015) 105e112 109

Fig. 1. Overall scheme of the synaptic interactions between the two proinflammatory cytokines, IL-1b and TNF, and the ECS in the striatum. IL-1b increases glutamate release at
presynaptic terminals and reduces postsynaptic GABAA receptor function, resulting in unbalanced neuronal hyperexcitation. IL-1b abrogates the sensitivity of CB1Rs controlling
glutamate and GABA release. TRPV1 channels are permissive for IL-1b synaptic effects on both glutamate and GABA transmission, while the BDNF/trkB regulates CB1ReIL-1b
interaction only on GABAergic terminals. In the last panel, TNF-mediated potentiation of postsynaptic AMPA receptors, limited by activation of CB1 receptors and of TRPV1 channels,
is shown.

The synaptic deficits of glutamatergic transmission are promi-
nent during the peak phase of EAE (Centonze et al., 2009; Rossi
et al., 2010b), whereas a downregulation of inhibitory GABAergic
transmission is observed during the chronic phase of the disease
(Rossi et al., 2011b). Evidence exists that IL-1b contributes to alter
GABAergic transmission in MS patients (Rossi et al., 2012b) and in
EAE model (Musumeci et al., 2011), and that TRPV1 channels, at
least in the model, are necessary for IL-1b synaptic effects. We have
indeed demonstrated that the genetic deletion of TRPV1 channels
attenuates the alterations of GABA synapses and favors a better
recovery in the chronic phase of EAE (Musumeci et al., 2011). On the
other hand, the crucial role of IL-1b on synaptic hyperexcitability,
neuronal damage and disability progression has been established in
MS (Rossi et al., 2012a, 2014b), but the potential modulation of
TRPV1 channels in such effects remains to be clarified.
In line with the idea that CB1R is neuroprotective in MS, we also
found that MS patients bearing a variant of the CB1R gene associ-
ated with reduced expression of the receptor developed a more
severe disease course, associated with increased neurodegenera-
tive damage (Rossi et al., 2011c, 2013). We found, in fact, that MS
patients with long AAT repeats within the CNR1 gene (12 in both
alleles) had more pronounced neuronal degeneration in response
to inflammatory white matter damage both in the optic nerve and
in the cortex, and in parallel, worse visual abilities and cognitive
performances (Rossi et al., 2013).
The implications of the specific cross-talk between inflamma-
tory cytokines and eCBs at the synapses extend beyond MS, and
might bear relevance for other neurodegenerative disorders.
Several neurodegenerative disorders have been linked to excitatory
neuronal damage, including Parkinson's disease and Huntington
diseases, Alzheimer disease and amyotrophic lateral sclerosis
(Mattson, 2003; Koutsilieri and Riederer, 2007). Hyperactive
microglia, a common feature of these neurodegenerative diseases,
secretes proinflammatory cytokines, which significantly contribute
to neuronal damage. Also the ECS is extensively altered in these
disorders (Centonze et al., 2007c; Rossi et al., 2010c; Di Filippo et al.,
2008) but the evidence of the synaptic interplay between cytokines
and eCBs is still lacking in this diseases.
6.2. Behavioral effects
MS causes a variety of motor and sensory deficits and it is also
associated with mood disturbances. It is increasingly recognized
that anxiety and depression in MS depend on specific effects of
neuroinflammation in neuronal circuits, rather than reflecting
subjective reactions to a chronic disease. In this respect, both in-
flammatory cytokines and eCBs play a fundamental role in
emotional disorders. High levels of proinflammatory cytokines
have been found in peripheral blood and in cerebrospinal fluid of
depressed patients (Zorrilla et al., 2001), and blockade of cytokine
110 S. Rossi et al. / Neuropharmacology 96 (2015) 105e112
signaling is associated with relevant mood-enhancing effects in
humans (Tyring et al., 2006). We have also demonstrated that the
anxious phenotype of mice exposed to stress is associated with loss
of sensitivity of CB1Rs in the striatum (Rossi et al., 2008, 2010a), and
that potentially rewarding experiences contrast the behavioral
consequences of stress by enhancing CB1R function (De Chiara
et al., 2010a,b).
A plausible synaptic mechanism at the basis of anxiety associated
with the inflammatory response has been identified in the interac-
tion between IL-1b and CB1Rs. We found that a single intra-
cerebroventricular (icv) injection of IL-1b causes anxiety in mice,
and abrogates the sensitivity of CB1R(GABA) in the striatum (Rossi
et al., 2012c). Identical behavioral and synaptic results were ob-
tained following social defeat stress, and reverted by icv injection of
IL-1 receptor antagonist (Rossi et al., 2008). IL-1b-mediated inhibi-
tion of CB1R function was secondary to altered cholesterol compo-
sition within membrane lipid rafts, and required intact function of
the TRPV1 channel (Rossi et al., 2012c). CB1Rs reduce transmitter
release by inhibiting cAMP levels in presynaptic nerve terminals
(Howlett, 2004), implying that IL-1b-mediated inhibition of CB1R
sensitivity results in enhanced cAMP signaling in neurons. In line
with this, the enhancement of cAMP signaling in the striatum has
been associated with increased anxious-depressive behavior in mice
(Favilla et al., 2008). The involvement of striatal neuron activity in
the control of anxiety-related behavior has been, in fact, recently
emphasized (Mathew and Ho, 2006; Favilla et al., 2008). To this re-
gard, it has been shown that EAE mice present high anxiety indexes
and TNF-mediated potentiation of excitatory synaptic transmission
in the striatum (Haji et al., 2012). Interestingly, the inhibition of TNF
signaling corrected the synaptic defects of pre-symptomatic EAE
mice, while icv injection of TNF in control mice altered sEPSCs,
mimicking the synaptic effects of EAE. CB1R activation is involved in
the control of post-synaptic alterations induced by TNF (Rossi et al.,
2011a), suggesting another potential interaction between eCB sys-
tem and inflammation in mood control.
7. Conclusions
Synaptic alterations are proposed as crucial determinants of the
link between neuroinflammation and neurodegeneration. Soluble
cytokines, capable of altering synaptic transmission and eCBs, able
to modulate cytokine-dependent synaptic deficits, are involved in
the control of neuronal function and survival. The two major
proinflammatory cytokines, IL-1b and TNF, acting preferentially at
pre- or postsynaptic sites, enhance glutamatergic transmission and
induce dendritic pathology. IL-1b is also involved in the inhibition
of GABAergic transmission. The specific interplay of the different
components of ECS with the synaptic actions of the two cytokines,
might explain the complex clinical effects of TRPV1 channel and
CB1R stimulation in the experimental model of MS, and suggests
that the inflammatory milieu determines whether ECS exerts
preferentially aversive or protective effects on neuronal survival
during neuroinflammatory disorders. In fact, TRPV1 channel is
permissive for the synaptic action of IL-1b, whereas CB1R limits the
TNF-induced alteration of excitatory transmission, as exemplified
in Fig. 1.
Targeting the cytokineeeCBs interaction at the synapses might
provide neuroprotective effects, and represent a novel route for the
treatment of mood disorders and neurodegenerative damage
associated or not with neuroinflammatory diseases.
Acknowledgments
This investigation was supported by the Italian National Minis-
tero della Salute (GR-2008-1144980), the Italian National Ministero

(MIUR-PRIN 2010-2011), and by Fondazione Baroni
dell'Universita
to DC; and by the Italian National Ministero della Salute (GR-2011-
02351422) to AM.
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