Neurobiology of Disease 82 (2015) 311–320

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Neurobiology of Disease

journal homepage: www.elsevier.com/locate/ynbdi

GABAA currents are decreased by IL-1β in epileptogenic tissue of patients

with temporal lobe epilepsy: implications for ictogenesis
Cristina Roseti b,1, Erwin A. van Vliet c,1, Pierangelo Cifelli a,f, Gabriele Ruffolo a, Johannes C. Baayen h,
Maria Amalia Di Castro a, Cristina Bertollini a, Cristina Limatola a,g, Eleonora Aronica c,d,
Annamaria Vezzani e,⁎⁎, Eleonora Palma a,b,⁎
a
Department of Physiology and Pharmacology, Istituto Pasteur-Fondazione Cenci Bolognetti, University of Rome Sapienza, Rome, Italy
b
IRCCS San Raffaele Pisana, Rome, Italy
c
Department of (Neuro)Pathology, Academic Medical Center, University of Amsterdam, The Netherlands
d
Stichting Epilepsie Instellingen Nederland (SEIN-Heemstede), The Netherlands
e
Department of Neuroscience, IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Milano, Italy
f
Ri.MED Foundation, Palermo, Italy
g
IRCCS Neuromed, Pozzilli, Italy
h
Department of Neurosurgery, VU University Medical Center, Amsterdam, The Netherlands

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

Article history: Temporal lobe epilepsy (TLE) is the most prevalent form of adult focal onset epilepsy often associated with drug-
Received 17 April 2015 resistant seizures. Numerous studies suggest that neuroinflammatory processes are pathologic hallmarks of both
Revised 2 July 2015 experimental and human epilepsy. In particular, the interleukin (IL)-1β/IL-1 receptor type 1 (R1) axis is activated
Accepted 6 July 2015
in epileptogenic tissue, where it contributes significantly to the generation and recurrence of seizures in animal
Available online 11 July 2015
models. In this study, we investigated whether IL-1β affects the GABA-evoked currents (IGABA) in TLE tissue from
Keywords:
humans. Given the limited availability of fresh human brain specimens, we used the “microtransplantation”
Cytokine method of injecting Xenopus oocytes with membranes from surgically resected hippocampal and cortical tissue
GABAA receptor from 21 patients with TLE and hippocampal sclerosis (HS), hippocampal tissue from five patients with TLE with-
Neuroinflammation out HS, and autoptic and surgical brain specimens from 15 controls without epilepsy. We report the novel finding
Oocytes that pathophysiological concentrations of IL-1β decreased the IGABA amplitude by up to 30% in specimens from
Pharmacoresistant epilepsy patients with TLE with or without HS, but not in control tissues. This effect was reproduced by patch-clamp re-
cordings on neurons in entorhinal cortex slices from rats with chronic epilepsy, and was not observed in control
slices. In TLE specimens from humans, the IL-1β effect was mediated by IL-1R1 and PKC. We also showed that IL-
1R1 and IRAK1, the proximal kinase mediating the IL-1R1 signaling, are both up-regulated in the TLE compared
with control specimens, thus supporting the idea that the IL-1β/IL-R1 axis is activated in human epilepsy. Our
findings suggest a novel mechanism possibly underlying the ictogenic action of IL-1β, thus suggesting that this
cytokine contributes to seizure generation in human TLE by reducing GABA-mediated neurotransmission.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction

Temporal lobe epilepsy (TLE) is a form of adult focal onset epilepsy

Abbreviations: Gamma-aminobutyric acid, GABA; GABA type A receptor, GABAA-R;
Hippocampal sclerosis, HS; Interleukin-1β, IL-1β; IL-1 receptor type 1, IL-1R1; IL-1 recep-
tor antagonist, IL-1Ra; Interleukin-1 receptor-associated kinase1, IRAK1; N-methyl-D-as-
partate, NMDA; Protein kinase C, PKC; Temporal lobe epilepsy, TLE.
⁎ Correspondence to: E. Palma, Department of Physiology and Pharmacology, Università
di Roma Sapienza, P.le A. Moro 5, 00185 Roma, Italy.
⁎⁎ Correspondence to: A. Vezzani, Department of Neuroscience, IRCCS Istituto di Ricerche
Farmacologiche "Mario Negri", Via G. La Masa 19, 20156 Milano, Italy.
E-mail addresses: annamaria.vezzani@marionegri.it (A. Vezzani),
eleonora.palma@uniroma1.it (E. Palma).
1
Equal contribution.
Available online on ScienceDirect (www.sciencedirect.com).
often associated with pharmacoresistance (Kwan et al., 2010; Blümcke
et al., 2013). Pathological changes can be observed in patients with
TLE, the most prominent of which is a loss of neurons in the hippocam-
pus, known as hippocampal sclerosis (HS; Blümcke et al., 2013), and as
many as 75% of patients with mesial TLE are considered to have drug-
resistant epilepsy (Schmidt and Löscher, 2005). The search for mecha-
nisms implicated in the pathogenesis of seizures in TLE, with the inten-
tion of characterizing new targets for developing novel drugs, revealed a
key role played by neuroinflammation, in particular the activation of the
IL-1β/IL-1 receptor type 1 (IL-1R1) axis, in epileptogenic brain areas
(Vezzani et al., 2011a). In addition to IL-1β/IL-1R1 activation, IRAK1, a

http://dx.doi.org/10.1016/j.nbd.2015.07.003
0969-9961/© 2015 Elsevier Inc. All rights reserved.
312 C. Roseti et al. / Neurobiology of Disease 82 (2015) 311–320
serine/threonine kinase functionally associated with IL-1R1, has a pivot-
al role in the activation of this inflammatory pathway (Gottipati e al.,
2008; Iyer et al., 2012). Supportive evidence for a role of IL-1β signaling
in epilepsy includes (Henshall et al., 2000; Vezzani et al., 2011b):
(i) increased IL-1β biosynthesis and release by astrocytes and microglia
and induction of IL-1R1 in neurons, both in experimental models and
human TLE brain tissue; (ii) powerful anti-ictogenic effects of drugs
blocking the IL-1β/IL-1R1 signaling activation, resulting in delayed sei-
zure precipitation and decreased seizure recurrence in various experi-
mental models; (iii) genetic perturbations of the IL-1β system in mice
causing profound changes in their intrinsic seizure susceptibility. This
evidence strongly suggests that activation of IL-1R1 signaling by IL-1β
is not a by-stander phenomenon but significantly contributes to seizure
generation and recurrence. Studies of the mechanisms mediating the
ictogenic effects of IL-1β revealed direct neuromodulatory proper-
ties of this cytokine (Vezzani and Viviani, 2015). In particular, IL-1β
induces IL-1R1-mediated post-transcriptional signaling in neurons
that involves the Src kinase-dependent tyrosine phosphorylation of
the NR2B-NMDA receptor, thereby promoting hyperexcitability, sei-
zures, and excitotoxicity by enhancing neuronal Ca2 + influx (Viviani
et al., 2003; Balosso et al., 2008; Vezzani and Viviani, 2015).
It has been proposed since 1958 (Jung et al., 1958) that, seizures
occur as a result of altered excitatory/inhibitory balance; therefore, an
altered GABAA receptor (R) function may contribute to epileptogenesis
(Scharfman and Brooks-Kayal, 2014; González et al., 2015). In line with
this hypothesis, we have shown that GABAA-Rs in the epileptic brain,
when repetitively activated, display a marked current desensitization
known as rundown (Ragozzino et al., 2005; Palma et al., 2007), thereby
contributing to the GABAergic impairment in epilepsy. This phenome-
non is significantly prevented by brain derived neurotrophic factor,
phosphatase inhibitors and the chemokine fractalkine (Palma et al.,
2005; Roseti et al., 2013).
Experimental evidence in rodents suggests that the ictogenic effects
of IL-1β may involve the GABAA-R since IL-1β reduces the peak ampli-
tude of the GABA-evoked current in rat patch-clamped hippocampal
neurons (Wang et al., 2000) and decreases synaptic inhibition in CA3
pyramidal cells (Zeise et al., 1997).
We therefore tested the hypothesis that there is pathophysiological
modulation of GABAA-R by IL-1β in human brain specimens, and found
that this cytokine significantly reduces GABAA-R mediated currents in
the hippocampus and the temporal cortex of patients with TLE with or
without HS, but not in non-epileptic control tissue. The IL-1β effect
was concomitant with up-regulation of IL-1R1 and its proximal kinase
IRAK-1 in the same epileptogenic tissue and was mediated by activation
of IL-1R1 and PKC.
This novel mechanism may contribute to hyper-excitability in the
temporal lobe, thereby underlying seizure generation in TLE, and possi-
bly in other pharmacoresistant forms of epilepsy associated with neuro-
inflammation (Bernard and Shevell, 2008; Vezzani et al., 2011a).
2. Methods
2.1. Patients
The clinical cases and controls included in this study were selected
from the Departments of Neuropathology of the Academic Medical Cen-
ter (AMC, University of Amsterdam) and the VU University Medical
Center (VUMC). The clinical characteristics derived from the patients'
medical records are summarized in Table 1. In the text, the number
of patients used in each experiment is reported, and referred to
Table 1 using the symbol #. We used 26 surgical epilepsy specimens
(hippocampus and temporal cortex) from patients who underwent
surgery for medically intractable epilepsy (#1–26, Table 1). All patients
underwent presurgical evaluation with non-invasive tests. Patients
who underwent implantation of strip and/or grid electrodes for chronic
subdural invasive monitoring before resection were excluded from the
study. All surgeries were performed by the same neurosurgeon (J.C.B.)
at VUMC. In TLE patients, the surgery consisted of an extensive temporal
lobectomy (ETL) including microsurgical resection of the amygdala and
parahippocampal gyrus and en bloc excision of the hippocampal forma-
tion. The interventions differed in the extent of the neocortical resec-
tion. Nondominant ETL included excision of approximately 4–7 cm or
more cm of the superior, middle and inferior temporal gyrus, depending
on intraoperative EEG measurements, whereas dominant ETL included
excision of approximately 3–7 cm of the superior, middle and inferior
temporal gyrus, depending on language mapping and intraoperative
EEG measurements. The predominant seizure types were medically in-
tractable complex partial seizures, and all patients had seizures which
were resistant to maximal doses of different anti-epileptic drugs
(Table 1). Epilepsy duration was calculated as the interval in years
from age at seizure onset to age at surgery; no patients included in
our series had seizures in the 24 h before surgery. Twenty one patients
with TLE (#1–21, Table 1) had hippocampal sclerosis (HS) with neuro-
nal loss within all hippocampal subfields, including CA1 and CA4 (HS,
ILAE type 1). Five patients with TLE but not HS (#22–26, Table 1)
who underwent resection of the hippocampus for medically intrac-
table TLE were included to provide a comparison group. All cases
were reviewed independently by two neuropathologists, and the
diagnosis was confirmed according to the international consensus
classification (Blümcke et al., 2013).
As control tissue, we used specimens from autopsies of patients
without any neurological disease and no sign of brain inflammation
(#27–41, Table 1; the causes of death included heart failure, respiratory
failure, myocardial infarction) and specimens from patients (#42–45,
Table 1) who underwent surgery for meningioma (WHO grade III).
The absence of seizures was determined during scheduled neurology
visits, using 60 min of awake EEG recordings; seizures were classified
according to the Engel classification.
All autopsies were performed within 10 to 24 h of death. Hippocam-
pal tissue specimens were dissected at the Department of Neuropathol-
ogy in 5 mm slabs perpendicular to antero-posterior axis. A slice from
hippocampal mid-body level was fixed overnight in 10% formalin for
paraffin embedding for histopathology and adjacent tissue was snap-
frozen over liquid nitrogen and stored at −80 °C until use. Frozen tissue
was shipped by courier to University of Rome.
Examination of histologically normal tissues obtained at surgery
showed a pattern of immunoreactivity (IR) similar to that observed in
control tissues from autopsies, thus arguing in favor of antigen preser-
vation in autopsies (Conti et al., 2011; Roseti et al., 2013). Tissue was ob-
tained and used in accordance with the Declaration of Helsinki and the
AMC Research Code provided by the Medical Ethics Committee and ap-
proved by the science committee of the VUMC Biobank. The Ethics Com-
mittee of the University of Rome “Sapienza” approved the technical
procedures. Informed consent was obtained from all individuals in-
volved in this study.
2.2. Immunohistochemistry
Tissue was fixed in 10% buffered formalin (autopsy tissue for 2
weeks; surgical specimens for 24 h) and embedded in paraffin. In
all cases, a representative formalin-fixed, paraffin-embedded tissue
block was studied, selecting large resection specimens containing
normal cortex adjacent to abnormal cortex for comparison, as an in-
ternal control. Paraffin-embedded tissue was sectioned at 6 μm,
mounted on organosilane-coated slides (Star Frost, Waldemar
Knittel GmbH, Brunschweig, Germany) and two sections from each
paraffin block were used for immunohistochemical staining, as de-
scribed previously (Ravizza et al., 2006; Iyer et al., 2010). Sections
were deparaffinated in xylene, rinsed in ethanol (100%, 95%, 70%)
and incubated for 20 min in 0.3% hydrogen peroxide diluted in meth-
anol. Antigen retrieval was performed using a pressure cooker in
0.1 M citrate buffer pH 6.0 at 120 °C for 10 min. After cooling on
 C. Roseti et al. / Neurobiology of Disease 82 (2015) 311–320 313

Table 1
Clinical characteristics of patients.

P# Age (yrs)/sex Epilepsy onset Surgical zone Seizure type Number of seizures/month Pathology Medications

#1* 43/M 28 L-T CPS/GTCS 15 HS TPM, PHT, CLB

#2*° 54/F 15 R-T SPS/CPS 24 HS CBZ, VPA, CLB
#3* 39/F 12 R-T CPS/GTCS 10 HS LEV, CBZ, PB, CNP
#4*° 19/M 15 R-T SPS 18 HS CBZ
#5*° 47/M 16 L-T CPS 7 HS VPA, CBZ, CLB
#6* 34/M 4 L-T CPS 4 HS LMT, PB
#7* 29/F 13 R-T SPS/GTCS 32 HS LMT, TPM
#8* 27/M 10 R-T CPS 12 HS CBZ, LCB
#9*° 54/F 12 R-T CPS/GTCS 8 HS CBZ, LMT, PB
#10*° 29/M 18 L-T CPS 14 HS CBZ
#11*° 28/F 14 L-T SPS 120 HS CBZ, LMT
#12*° 37/F 15 R-T CPS 1 HS CBZ, LEV
#13*° 29/M 15 L-T CPS 36 HS CBZ, CLB
#14*° 26/M 6 L-T CPS 5 HS LEV, TPM
#15*° 57/F 47 R-T CPS 1 HS CNP
#16*° 39/M 33 R-T CPS 32 HS CBZ
#17° 42/F 4 R-T CPS/GTCS 1 HS PHT, OXC
#18° 37/F 4 L-T CPS 10 HS CBZ
#19° 66/F 10 R-T CPS 8 HS CBZ, PB, PHT
#20*° 24/F 15 L-T CPS 20 HS TPM, LEV, OXC
#21*° 52/M 10 L-T CPS 1 HS CBZ, PB, VGB
#22*° 21/F 10 L-T SPS 32 non-HS LEV, VPA
#23*° 61/M 36 L-T CPS 2 non-HS CBZ
#24*° 48/M 29 R-T SPS/CPS 32 non-HS LMT, VPA
#25*° 48/F 42 R-T CPS 4 non-HS LEV, LMT
#26*° 63/F 52 R-T CPS 8 non-HS CLB, LMT, TPM
#27* 52/F Absent R-T – – None
#28* 48/M Absent L-T − − None
#29° 81/F Absent R-T – – None
#30*° 68/F Absent L-T − − None
#31*° 86/F Absent R-T − − None
#32*° 30/M Absent L-T − − None
#33*° 65/F Absent L-T − − None
#34*° 71/F Absent R-T − − None
#35° 60/M Absent R-T – – None
#36° 44/F Absent L-T – – None
#37° 63/F Absent R-T – – None
#38*° 25/F Absent L-T – – None
#39*° 31/M Absent L-T – – None
#40*° 63/M Absent L-T – – None
#41*° 56/M Absent L-T – – None
#42* 47/F Absent R-T – – Meningioma
#43* 75/F Absent R-T – – Meningioma
#44* 38/M Absent R-T – – Meningioma
#45* 32/M Absent L-T – – Meningioma

Patient 1–21: Patients with TLE and hippocampal sclerosis (HS); Patient 22–26: Patients with TLE without hippocampal sclerosis; Patient 27–41: non-epileptic tissues from autopsies (hip-
pocampus and temporal cortex); Patient 42–45: surgical specimens from patients without epilepsy but with meningioma III WHO; T, temporal; L, left; R, right.
Seizures Type: SPS, simple partial seizures; CPS, complex partial seizures; GTCS, generalized tonic/clonic seizures.
Drugs: CLB, Clobazam; CBZ, Carbamazepine; LEV, Levetiracetam; OXC, Oxcabazepine; PB, Phenobarbital; PHT, Phenytoin; VPA, Valproate; LCS, Lacosamide; CNP, Clonazepam; LMT,
Lamotrigine; TPM, Topiramate; VGB Vigabatrin. * Tissues used for electrophysiology, ° tissues used for western blot.

ice, slides were washed with phosphate-buffered saline (PBS; 0.1 M,
pH 7.4) and incubated overnight in polyclonal goat anti-human IL-
1RI (1:50; R&D Systems, Abingdon UK) in PBS at 4 °C. Thereafter, sec-
tions were washed in PBS and stained with a polymer based peroxi-
dase immunocytochemistry detection kit (PowerVision Peroxidase
system, ImmunoVision, Brisbane, CA, USA). After washing, sections
were stained with 3,3′-diaminobenzidin tetrahydrochloride (50 mg
DAB, Sigma-Aldrich, Zwijndrecht, The Netherlands) and 5 μl 30% hy-
drogen peroxide in a 10 ml solution of Tris–HCl. Sections were coun-
terstained with hematoxylin, dehydrated in alcohol and xylene, and
coverslipped. Sections incubated without primary antibodies or with
pre-immune serum were essentially blank. These experiments were
performed at University of Amsterdam (E. V. and E. A.).
2.3. Western Blot
Patients with sufficient frozen material available were selected
for Western blot analysis. Freshly frozen tissue (patients shown in
Table 1) was homogenized in lysis buffer containing per 20 ml:
200 μl 1 M Tris pH 8.0; 1 ml 3 M NaCl; 2 ml 10% NP-40; 4 ml 50%
glycerol; 800 μl Na-orthovanadate (10 mg/ml); 200 μl 0.5 M EDTA
pH 8.0; 400 μl protease inhibitors; 200 μl 0.5 M NaF; 11.2 ml H2O. Fifty
μg total protein per lane, as determined using the bicinchoninic acid
method (Smith et al., 1985), was separated by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (10% acrylamide) and trans-
ferred to immobilon-P membranes (Millipore corporation, Billerica,
MA, USA) by electroblotting (BioRad, Transblot SD, Hercules, USA).
Blots were incubated overnight in Tris buffered saline with Tween
(TBST; 20 mM Tris, 150 mM NaCl, 0.1% Tween, pH 7.5)/5% non-fat dry
milk, containing the primary antibody: polyclonal rabbit anti-IRAK-1
(1:1000, SC-7883, Santa Cruz Biotechnology, Heidelberg, Germany) or
monoclonal mouse anti-ß-actin (1:50,000, clone AC-15, Sigma-Aldrich
Chemie, Zwijndrecht, The Netherlands). After washing three times for
10 min in TBST, blots were incubated for 1 h in the secondary antibody:
anti-rabbit (1:2500, Invitrogen, Breda, The Netherlands) or anti-mouse
(1:2500, Dako, Glostrup, Denmark) labeled with horseradish peroxi-
dase. After washing three times for 10 min in TBST, immunoreactivity
was visualized with WesternBright ECL HRP substrate (Advansta,
Menlo Park, CA, USA) and the blots were digitized using a Luminescent
Image Analyzer, LAS-4000 (Fuji Film, Japan). The optical density of each
314 C. Roseti et al. / Neurobiology of Disease 82 (2015) 311–320
sample was measured using Adobe Photoshop. For each sample the op-
tical density of IRAK-1 was calculated relative to the optical density of ß-
actin. These experiments were performed at University of Amsterdam
(E. V. and E. A.).
2.4. Membrane preparation and oocytes injection
The use of female Xenopus laevis frogs conformed to institutional
policies and guidelines of the Italian Ministry of Health. The preparation
of human membranes (frozen hippocampus and temporal cortex from
patients shown in Table 1), their injection in X. laevis oocytes, and
GABA current recordings were carried out as previously described
(Miledi et al., 2006; Eusebi et al., 2009; Roseti et al., 2013). In a set of ex-
periments, we performed intranuclear injection in Xenopus oocytes
(Miledi et al., 2006) of human complementary DNA (cDNAs) encoding
for α1, β2 and γ2 GABAA subunits (pcDNA3 vector) or co-injected
with cDNA encoding human IL-1R1 (pCMV6-XL4 vector). Human
α1β2γ2 cDNA was provided as a gift by Dr. Keith Wafford and human
IL-1R1 cDNA was purchased from Origene 9620 (USA).
2.5. Electrophysiology
From 12 to 48 h after injection, membrane currents were recorded
from voltage-clamped X. laevis oocytes using two microelectrodes
filled with 3 M KCl as previously described (Miledi et al., 2006).
The oocytes were placed in a recording chamber (0.1 ml volume)
and perfused continuously with oocyte Ringer solution (OR: NaCl
82.5 mM; KCl 2.5 mM; CaCl2 2.5 mM; MgCl2 1 mM; Hepes 5 mM, ad-
justed to pH 7.4 with NaOH) at room temperature (20–22 °C). To
apply GABA or OR we used a gravity driven multi-valve perfusion
system (9–10 ml/min) controlled by computer (Biologique RSC-
200; Claix, France) to ensure the exact duration of each application.
Using this system, 0.5 to 1 s are sufficient to reach the complete re-
placement of applied solution.
Unless otherwise indicated, GABA 500 μM (freshly dissolved in OR)
was applied for 4 s to oocytes to elicit inward currents (IGABA). To deter-
mine the effect of IL-1β on IGABA, oocytes were first exposed to GABA
alone (two repetitive applications at 4 min intervals). Only the oocytes
where IGABA was stable (less than 5% of peak amplitude after the second
application) were subsequently used either for co-application of GABA
and IL-1β (2.5, 25, 250 and 400 ng/ml), or for GABA application after
4 min, 10 min or 60–250 min pre-incubation with the various IL-1β con-
centrations. The outcome measure was the percentage change in IGABA
amplitude with respect to the current evoked before IL-1β application.
The IGABA decay was defined as the time required for the current to
decay from its peak to the half-peak value (T0.5). GABA current rundown
was defined as the percentage decrease of the current peak amplitude
after six 10s-applications of GABA at 40 s intervals (Eusebi et al., 2009;
Roseti et al., 2013). Unless otherwise indicated, numbers (n) refer either
to oocytes or neuronal cells used in each experiment.
In some experiments, the following drugs were incubated for vari-
ous times before and during IL-1β application: 10 μM IL-1 receptor an-
tagonist (IL-1Ra), 10 μM PP2, a Src inhibitor (Viviani et al., 2003),
1 μM BEZ 235, a PI3K inhibitor (Chi et al., 2014), 50 μM capsazepine, a
TRPV1 channel blocker (Musumeci et al., 2011), 5 μM staurosporine
and 1 μM bisindolylmaleimide, two PKC inhibitors (Palma et al.,
2005), and 50 nM phorbol 12-myristate 13-acetate (PMA), a PKC activa-
tor (Bright and Smart, 2013). All drugs were purchased from Sigma Ita-
lia with the exception of GABA (Tocris, Bristol, UK), PP2 (Calbiochem,
UK), bisindolylmaleimide (Santa Cruz, USA), human recombinant IL-
1β and IL-1Ra (R&D Systems, Minnesota, USA). Drugs were dissolved
in sterile buffered saline and used fresh. Staurosporine, PMA, and
bisindolylmaleimide were dissolved in DMSO (final concentration
≥1:3000) and stored at −20 °C as stock solution. All the electrophysio-
logical experiments were done at University of Rome Sapienza (P.C.,
G.R., E.P.) and at San Raffaele Pisana in Rome (C.R.).
2.6. Whole-cell recordings from cortical rat slices from epileptic rats
Adult male Sprague–Dawley rats (225–250 g; Charles-River, Calco,
Italy) were housed at constant temperature (23 °C) and relative humid-
ity (60 ± 5%) with free access to food and water and a fixed 12 h light/
dark cycle. Procedures involving animals and their care were conducted
in conformity with the institutional guidelines that are in compliance
with national (D.L.n.116, G.U., Suppl. 40, February 18, 1992) and inter-
national laws and policies (EEC Council Directive 86/609, OJ L 358, 1, De-
cember 12, 1987; Guide for the Care and Use of Laboratory Animals, U.S.
National Research Council, 1996).
Status epilepticus (SE) was induced in 11 rats by intraperitoneal in-
jection of 10–12 mg/kg kainic acid (KA) in phosphate buffered saline
(PBS, pH 7.4). Six control rats were injected with the corresponding vol-
ume of vehicle. As described previously (Röder et al., 1996), KA induced
a behavioral syndrome defined as SE which included early staring, “wet
dog shakes” and seizures which evolved from mild forehead nodding to
generalized motor convulsions with rearing and falling (stage 4/5 sei-
zures, Racine, 1972). Rats were observed for at least 3 h by two indepen-
dent investigators, and only animals showing stage 4/5 seizures (~80%
of injected animals) were selected for further analysis of spontaneous
recurrent seizures (SRS). Three rats died during or immediately after
SE. Three months after SE induction, remaining rats were video-
monitored for 4 h/day for one week to detect generalized motor sei-
zures. The manifestation of at least two generalized tonic–clonic sei-
zures during one week of observation was used as a criterion to select
4 epileptic rats for the subsequent electrophysiology experiments. The
animals were injected and selected for subsequent studies at Mario
Negri Institute (A.V.).
2.6.1. Acute slices preparation
Cortical slices were prepared from four epileptic rats and four
vehicle-injected age-matched controls. Rats were killed by decapitation
under deep halothane anesthesia, then the brain was rapidly removed
and placed in ice-cold oxygenated (95% O2, 5% CO2) sucrose-based arti-
ficial cerebrospinal fluid (ACSF) containing 87 mM NaCl, 75 mM su-
crose, 2 mM KCl, 7 mM MgCl2, 0.5 mM CaCl2, 25 mM NaHCO3, 1.2 mM
NaH2PO4 and 10 mM glucose, pH 7.4, 300–305 mOsm. Transverse slices
of the entorhinal cortex (350 μm, ThermoScientific HM 650 V) were cut
and placed in a slice incubation chamber at room temperature with ox-
ygenated ACSF containing 125 mM NaCl, 2 mM KCl, 1.2 mM MgCl2,
2 mM CaCl2, 25 mM Na HCO3, 1.2 mM NaH2PO4 and 10 mM glucose,
with pH and osmolarity as above, and transferred to a recording cham-
ber within 1 to 6 h of slice preparation.
2.6.2. Electrophysiology
Whole-cell patch clamp recording was performed on layer V pyra-
midal neurons at 24–25 °C. Cell capacitance was constantly monitored,
and experiments where access resistance changed more than 20% were
discarded. Glass electrodes (3–4 MΩ) were filled with 140 mM Cs-
Methanesulfonate, 10 mM HEPES, 0.5 mM EGTA, and 2 mM Mg-ATP,
0.3 mM Na3-GTP, 2 mM MgCl2. IGABA currents were recorded at 0 mV
to avoid spurious contributions of mechanosensitive Na+ currents
(Mazzuferi et al., 2010; Ragozzino et al., 2005), with glutamatergic cur-
rents at their reversal potential. Under these experimental conditions,
with inactivated voltage-gated channels, cells were stable and healthy
for at least 1 h. Spontaneous inhibitory postsynaptic currents were
present but did not affect the quantification of GABA-induced currents.
GABA (100 μM) was delivered to cells by pressure applications
(5–10 psi; 100 ms; Picospritzer II; General Valve) from glass micropi-
pettes positioned close to whole-cell voltage-clamped neurons. After
current amplitude stabilization had occurred (less than 5% of peak am-
plitude decrease after the second GABA application), IL-1β (25 ng/ml)
was applied to the bath using a gravity-driven perfusion system
(speed 1.5 ml/min); this was washed out after 15 min. In all recordings
IGABA was evoked every 5 min in the control period, and during IL-1β
 C. Roseti et al. / Neurobiology of Disease 82 (2015) 311–320 315

application and wash-out. Signals were acquired (sampling 10 kHz,

low-pass filtered 2 kHz) with DigiData-1440A using pCLAMP-v10 soft-
ware (Axon Instruments). These experiments have been performed at
University of Rome (M.DC, C.B. and C.L.).

2.7. Statistical analysis of data

Data are presented as mean ± s.e.m. The number of the patients' or

non-epileptic controls' specimens used in each experiment for oocyte
transplantation is reported in Table 1. The experiments and data analy-
sis were performed by different researchers (one of them operating
with blind procedures), and each tissue specimen was used several
times in different sets of experiments. The normality of data sets was
tested according to the Shapiro Wilk test. Statistical significance was
assessed by Student's test or by Kruskal–Wallis one-way ANOVA on
ranks when data set were not normally distributed. Statistical tests
were performed using Sigmaplot Systat (Systat Software GmbH, CA,
USA) or Origin 8 software (Origin Lab Corporation, MA, USA) using
raw data. Only differences considered significant (p b 0.05) are indicat-
ed in the text and figures legends.

3. Results

3.1. IL-1β signaling increases in TLE

To show whether IL-1β signaling was induced in the specimens from

patients with TLE, we measured IRAK-1, a recognized marker of IL-1β/
IL-1R1 signaling activation (Gottipati et al., 2008; Iyer et al., 2012)
using western blot. Fig. 1 shows a 4-fold increase on average in IRAK-1
levels in the hippocampus (Fig. 1A, B) and temporal cortex (Fig. 1C) of
16 patients with TLE and HS and five without HS compared with 13 con-
trols from autopsy without epilepsy (see Table 1).
We pooled specimens from patients with TLE with and without HS
in a single group since we found a similar increase in IRAK-1, thus sug-
gesting that the activation of this signaling is independent of HS, and is
most likely induced by recurrent seizures. In accordance with the IRAK-
1 up-regulation, immunohistochemical analysis of the temporal cortex
of patients with TLE showed an increased IL-1R1 immunoreactivity in
neurons and glia (Fig. 2C, D) compared with control cortex (Fig. 2A,
B), as previously reported in the hippocampus of patients with TLE
(Ravizza et al., 2006).

3.2. IL-1β reduces GABA currents in tissue from patients with TLE but not in
controls
To investigate whether IL-1R1 activation affects GABA-mediated
currents, we microtransplanted Xenopus oocytes with hippocampal
membranes from a randomly chosen cohort of 23 patients with TLE, in-
cluding the five patients without HS, and 15 control specimens (includ-
ing those used in Fig. 1; see Table 1), and exposed them to IL-1β. GABA
(500 μM) alone elicited inward currents (IGABA) with amplitude ranging
between − 15 and − 170 nA; these currents were blocked by the
GABAA-R antagonist bicuculline (100 μM; not shown). Co-application
of IL-1β (2.5–500 ng/ml) with GABA in specimens from patients with
TLE did not alter IGABA amplitude (n = 15, not shown; #10–13,
Table 1). When oocytes were incubated with 25 ng/ml IL-1β for 4 or
10 min before GABA (Fig. 3A), the IGABA amplitude was not modified.
However, when oocytes were exposed to the same concentration of
IL-1β from 60 to 250 min before GABA application, then IGABA amplitude
progressively decreased in 80% of oocytes, reaching a maximal ~30% re-
duction after 120 min incubation (GABA, 73.2 ± 8.3 nA; +IL-1β, 52.5 ±
7.1 nA; n = 273 oocytes from 32 frogs; p b 0.001; #1-16,20,21, Table 1
and Supplementary Table 2; Fig. 3A–C). This effect was slowly reversible
during a 3 to 5-h wash-out period (Fig. 3C). Similar changes in IGABA am-
plitude were obtained using higher IL-1β concentrations (250 and
400 ng/ml; not shown; #6–9, Table 1), while a lower IL-1β concentration
Fig. 1. IRAK-1 level is increased in tissue from patients with TLE. (A) Representative bands of
IRAK1 in western blot from tissue homogenates of controls (autopsy specimens without
epilepsy) and from patients with TLE with and without HS (B,C). Densitometric analysis
(mean ± s.e.m. of optical density units relative to corresponding β-actin) shows a 4-fold
increase on average of IRAK1 level in the hippocampus and cortex of patients with TLE
(patients # 2, 4–5, 9–26, Table 1) compared with control tissue (patients # 29 to 41,
Table 1). *p b 0.05, Student's t-test.
(2.5-ng/ml; 120 min pre-incubation) induced a 17% decrease of IGABA
amplitude (GABA, 58.0 ± 9.7 nA; + IL-1β, 48.2 ± 8.2 nA; n = 10;
#6,21, Table 1) that was not statistically significant. We found no mod-
ification of current amplitude in oocytes incubated for 10 to 120 min
with oocytes' Ringer only (n = 40, not shown; #1–16, 20–26, Table 1).
These findings suggest that the IL-1β effect on IGABA is time and dose-
dependent. Moreover, the IGABA decrease induced by 25 ng/ml IL-1β
was not associated with changes in current decay (T0.5, GABA, 3.4 ±
0.12 s; + IL-1β, 3.1 ± 0.15 s; n = 21; #6–10, Table 1) suggesting that
the decrease of IGABA amplitude is not related to a change in the rate of
current inactivation.
The lack of IL-1β effect in 20% oocytes was unrelated to which pa-
tients were used for membrane injection and it was not dependent on
the membrane potential or GABA reversal potential of the oocytes.
This phenomenon was previously observed in these cell preparations
316 C. Roseti et al. / Neurobiology of Disease 82 (2015) 311–320
Fig. 2. IL-1R1 expression increases in temporal cortex. Distribution of IL-1RI immunoreactivity (IR) in the temporal cortex of patients with TLE and controls. Panels A and B: control cortex (A,
white matter, WM; B, gray matter, GM) without detectable IL-1R1 IR. Panels C and D: temporal cortex of patients with TLE (C, white matter, WM; D, gray matter, GM) showing increased IL-
1R1 expression in both glial (arrows in C) and neuronal cells (arrows in D). Scale bar in D: A–D, 40 μm.
(Roseti et al., 2013), and it may be due to a low efficiency in the expres-
sion of IL-1R1 or related signaling molecules in some microinjected
oocytes.
Using oocytes transplanted with membranes from patients with TLE
without HS, we found that a 120 min pre-incubation with 25 ng/ml IL-
1β induced a significant decrease of IGABA (GABA, 22.4 ± 1.6 nA; +IL-
1β, 16.0 ± 1.6 nA; n = 48, p b 0.01; #22–26, Table 1), similar to that ob-
served in the hippocampi of patients with TLE and HS, in accordance
with the common increase in IRAK-1 levels.
Likewise, 120 min incubation with 25 ng/ml IL-1β reduced the
IGABA amplitude by about 30% in oocytes injected with temporal cor-
tex membranes of patients with TLE (GABA, 108.3 ± 6.9 nA; + IL-1β,
77 nA ± 5.6; n = 240, p = 0.014; #1–16,20,21, Table 1; Fig. 4). The
IGABA decrease, in both the hippocampus and cortex from patients
with TLE, was prevented by 30 min pre-treatment with 10 μM of
IL-1 receptor antagonist (GABA, 49.5 ± 3.4 nA; + IL-1Ra, 49.7 ±
4 nA; n = 29; #1, 9–11, 13, Table 1; Fig. 5A,B), thus demonstrating
that the effect of IL-1β was mediated by the activation of IL-1R1/
IRAK-1 signaling.
To confirm that the effect of IL-1β was mediated by the IL-1R1 sig-
naling machinery transplanted in oocytes by human epileptic tissue,
we intranuclearly injected a separate group of oocytes with the cDNAs
encoding α1β2γ2 GABAA-R, the predominant GABAA-R subtype
expressed in healthy brain (MacDonald et al., 2010). We found that
the currents evoked by the activation of α1β2γ2 GABAA-Rs were not af-
fected by IL-1β (GABA, 1550 ± 214 nA; +IL-1β, 1670 ± 172 nA; n = 10,
not shown). Likewise, we found no IL-1β effect on GABA evoked currents
in oocytes co-injected with the cDNAs encoding α1β2γ2 GABAA-R and
IL-1R1 (GABA, 1849 ± 230 nA; + IL-1β, 1877 ± 206 nA; n = 10, not
shown).
In contrast to the situation in tissue from patients with TLE, IL-1β did
not modify IGABA amplitude in oocytes injected with hippocampal
(GABA, 65.9 ± 12.5 nA; +IL-1β, 61.8 ± 13.3 nA; n = 81; Fig. 3B,C) or
cortical (GABA, 116.9 ± 15.4 nA; + IL-1β, 106.3 ± 13.4 nA; n = 20;
Fig. 4A) membranes from autopsies of controls without epilepsy (#27,
28, 30–34, 38–41, Table 1) . Notably, the lack of IL-1β effect on IGABA am-
plitude in control tissue was confirmed using surgical samples from the
temporal cortex of patients with meningioma but without epilepsy
(GABA, 115.1 ± 17.1 nA; GABA + IL-1β, 107.7 ± 16 nA; n = 36; #42–
45, Table 1; Fig. 4A, Inset). These findings confirm the similarity between
surgical and post-mortem control tissues, as previously reported (Conti
et al., 2011; Roseti et al., 2013).
In a different set of experiments, we evoked a repetitive activa-
tion of GABAA -Rs in microtransplanted oocytes from TLE patients
with HS in order to induce the IGABA rundown since we previously
showed that this phenomenon is a hallmark of GABAergic impair-
ment in TLE tissue (Ragozzino et al., 2005; Roseti et al., 2013). A
120 min pre-treatment with 25 ng/ml IL-1β did not affect IGABA run-
down (GABA, 52.5 ± 9.7%; + IL-1β, 48 ± 6.8%; n = 12, #3–6, Table 1)
while still decreasing IGABA amplitude (128 ± 12.5 nA vs 85.3 ±
14 nA).
Finally, in order to exclude that the IL-1β induced IGABA decrease was
a particular effect related to the oocyte expression system, we per-
formed whole-cell patch clamp recordings of IGABA in neurons of ento-
rhinal cortex from six control and four epileptic rats. Applications of
GABA (100 μM) by pressure ejection evoked whole-cell currents of
632 ± 139 pA (range 166–1165 pA; n = 10) in controls and of 974 ±
409 pA (range 692–1781 pA; n = 6) in epileptic rat slices. Bath applica-
tion of IL-1β (25 ng/ml) for 15 min, decreased the peak amplitude of
IGABA by ~ 40% (590 ± 360 pA; p b 0.01) in slices from epileptic rats
whereas no effect was observed in control slices (653 ± 140 pA)
(Fig. 1S, Supplementary data).
3.3. IL-1β effect on GABA currents in TLE is mediated by PKC
In another set of experiments we explored the intracellular pathway
mediating the IL-1β-induced IGABA decrease in oocytes injected with
hippocampal membranes from patients with TLE. We examined the
canonical signaling activated down-stream of IL-1R1/IRAK1, namely
Src and PI3K kinases, by pre-treating oocytes for 60 min with the
specific inhibitors PP2 (Viviani et al., 2003) (10 μM) and BEZ 235
(Chi et al., 2014) (1 μM), respectively. Both inhibitors failed to pre-
vent the IL-1β effect on IGABA (PP2, n = 14; BEZ, n = 16, not shown;
#9–11, Table 1). We also tested the involvement of TRPV1 channels
since they mediate the IL-1β decrease of GABAergic synaptic cur-
rents in mouse corticostriatal slices (Musumeci et al., 2011). Sixty
minutes pre-treatment with the TRPV1 channel blocker capsazepine
(50 μM) did not prevent the IL-1β effect (n = 14, not shown; #4,9,
Table 1). We showed, however, that 60 min pre-treatment with
5 μM staurosporine or 1 μM bisindolylmaleimide, two PKC inhibitors
(Palma et al., 2005) which per se did not affect I GABA amplitude,
prevented the IL-1β-induced decrease of IGABA (GABA, 66 ± 8.4 nA;
staurosporine, 64.1 ± 7.5 nA; n = 18; #5,12,13, Table 1; GABA,
40.9 ± 8.9 nA; bisindolylmaleimide, 39.8 ± 10.7 nA; n = 12; #6
and 8, Table 1; Fig. 5A,B). Accordingly, 120 min pre-treatment of oo-
cytes with 50 nM phorbol 12-myristate 13-acetate (PMA), a PKC ac-
tivator (Bright and Smart, 2013), decreased I GABA by about 50%
(GABA, 44.9 ± 8.8 nA; + PMA, 25.1 ± 5.7 nA, n = 15, p = 0.026;
#6,8,15,16, Table 1; Fig. 5A,B). PMA was ineffective in oocytes
injected with tissue from controls without epilepsy (n = 10, not
shown; #27,28, Table 1). These results show that PKC is involved in
the IL-1β-induced decrease of IGABA in tissue from patients with
epilepsy.
 C. Roseti et al. / Neurobiology of Disease 82 (2015) 311–320
4. Discussion
We report the novel finding that IL-1β decreases IGABA amplitude in
tissue from patients with TLE, specifically in hippocampal and temporal
cortex specimens, but not in control specimens. This effect was shown
by recording GABAA evoked currents in oocytes transplanted with
membranes from either surgically resected brain tissue from patients
with TLE, or from autoptic and surgical control tissues. In the same
TLE specimens, we found an increase in the level of IRAK-1, a key prox-
imal kinase associated with IL-1R1, thus supporting previous evidence
of the activation of IL-1β/IL-1R1 axis in human TLE (Henshall et al.,
317
2000; Ravizza et al., 2008; Vezzani et al., 2011b). Accordingly, IL-1R1
immunoreactivity was enhanced in temporal cortex from patients
with TLE, as previously shown in hippocampi from patients with TLE
(Ravizza et al., 2008).
To study the effect of IL-1β on IGABA, we took advantage of the
microtransplantation technique in Xenopus oocytes (Eusebi et al.,
2009). The exact glial or neuronal origin of the membrane patches
transplanted onto the oocytes surface is not known, but this approach
has a two-fold advantage by allowing: 1. investigation of “whole”, glial
and neuronal, GABA currents in oocytes expressing human GABAA-Rs
that retain their native properties, (Eusebi et al., 2009; Palma et al.,
2003); 2. use of human tissues from patients without epilepsy as suit-
able controls since these individuals did not have neurological diseases
or ongoing inflammatory processes. Due to the limited quantity of tissue
available, control specimens are usually unsuitable for obtaining viable
brain slices or isolated neurons for physiological investigations with al-
ternative techniques.
We found that pathophysiological concentrations (≥25 ng/ml) of IL-
1β decreased the IGABA amplitude by up to 30% in specimens from pa-
tients with TLE; this corresponds to a relatively high concentration of
IL-1β compared to the normal range reported in blood or brain (e.g.,
pg/ml or pg/mg), thereby arguing in favor of an IL-1β effect compatible
with a pathologic rather than physiological scenario. Similar high con-
centrations were previously shown to affect GABAergic neurotransmis-
sion in in vitro rodent preparations (Wang et al., 2000).
The effect on IGABA by IL-1β in oocytes transplanted with human tis-
sue was replicated in a limited number of neurons in cortical slices from
epileptic rats, and, as in human specimens, was absent in control rat
slices. This evidence suggests that the IL-1β induced decrease in IGABA
was not a particular effect related to the oocyte expression system.
These results suggest the need for more in-depth investigations in ex-
perimental models of epilepsy.
The decrease of IGABA provoked by IL-1β was receptor-mediated, as-
sociated with increased IL-1R1 and IRAK-1 in epileptogenic tissue from
humans, and concentration-dependent, all features with pathophysio-
logical relevance (Ravizza et al., 2008; Vezzani et al., 2011a). These find-
ings also imply that the effect of IL-1β on IGABA occurs in the presence of
an up-regulated signaling machinery, in line with the evidence that
neuroinflammation can lead to brain dysfunction when prolonged or
excessive (Vezzani et al., 2011a; Vezzani and Viviani, 2015). Persistent
up-regulation of the IL-1β/IL-1R1 signaling has been reported in
human TLE and in related experimental models (Henshall et al., 2000;
Ravizza et al., 2006; Ravizza et al., 2008; Maroso et al., 2011; Tan et al.,
2015), and is confirmed by increased IL-1R1 and IRAK-1 in our TLE spec-
imens. When IL-1R1 is activated by IL-1β, ΙRAK-1 associates to the cell
membrane-bound receptor, therefore the IL-1R1/IRAK-1 complex is
transplanted into the oocytes. Interestingly, the induction of IRAK-1
was similar in the hippocampi of patients with TLE with or without
HS, thus suggesting that the persistent activation of the IL-1R1 signaling
in TLE may be driven by recurrent seizures rather than the underlying
neuropathology, as also suggested by findings in experimental models
Fig. 3. IL-1β decreases IGABA amplitude in oocytes transplanted with TLE hippocampus.(A) Time-
course of IL-1β-induced decrease of IGABA amplitude in oocytes microtransplanted with hip-
pocampal membranes from specimens from patients with TLE and HS. GABA alone was ap-
plied at 500 μM at the beginning of the experiment (time 0), and at various time points after
treatment with 25 ng/ml IL-1β. Data (mean ± s.e.m. of 15 oocytes/time point; patients #1–7,
Table 1) represent the percentage decrease of the peak amplitude induced by IL-1β. (B). His-
tograms show the average % change in IGABA amplitude after the 120 min application of 25
ng/ml IL-1β. The holding potential was −60 mV. Tissue from patients with TLE (n = 273 oo-
cytes; #1–16,20,21, Table 1) and patients without epilepsy (n = 81 oocytes, * p b 0.001 vs
GABA, Kruskal–Wallis test; patients #27,28,30–34,38–41, Table 1).(C) top traces are record-
ings in one representative oocyte transplanted with TLE membranes: 120 min pre-incuba-
tion with IL-1β reduced IGABA amplitude (middle trace) compared with GABA alone (first
trace). The effect was reverted by a three hour wash-out (third trace). Bottom traces show lack
of IL-1β effect on IGABA amplitude in one representative oocyte transplanted with hippo-
campal membranes from an individual without epilepsy. Holding potential and concentra-
tions are as in (A). Black bars indicate GABA application.
318 C. Roseti et al. / Neurobiology of Disease 82 (2015) 311–320

Fig. 4. IL-1 β decreases IGABA amplitude in oocytes transplanted with the cortex of patients with
TLE. (A) Histograms show the average % change in IGABA amplitude after a 120 min appli-
cation of 25 ng/ml IL-1β in specimens from patients with TLE specimens (patients #1–
16,20,21, Table 1) and autoptic control tissue from patients without epilepsy (patients
#27,28,30–34,38–41, Table 1). The holding potential was −60 mV. Black bars indicate
GABA application, *p = 0.014 vs GABA alone, Kruskal–Wallis test. (Inset) Histograms
show the lack of IL-1β effect in oocytes injected with membranes from surgical control tis-
sue from patients without epilepsy undergoing surgery for meningioma (WHO grade III).
(n = 36; patients #42–45, Table 1). Sample traces in inset are from one representative oo-
cyte transplanted with cortical membranes from a patient with meningioma but without
epilepsy. (B) Sample traces in one representative oocyte transplanted with membranes
from a patient with TLE before (first trace) and after 120 min pre-treatment with IL-1β
(middle trace). The effect reverted by 3 h wash-out (third trace). Holding potential and
black bars as in (A).

(Dube' et al., 2005, 2010; Maroso et al., 2010; Akin et al., 2011). Thus, the
decrease of IGABA amplitude mediated by IL-1β was similar in patients
with TLE with or without HS.
We found that IL-1β does not affect IGABA rundown caused by repet-
itive GABAA receptor stimulation which is increased in TLE hippocam-
pus vs control tissue, and reduced by fractalkine (Roseti et al., 2013).
This evidence suggests that inflammatory cytokines and chemokines,
both of which contribute to the neuroinflammatory milieu (Fabene
et al., 2010; Vezzani et al., 2011b), have a different effect on the IGABA
in epileptogenic tissue.
The relevance of our in vitro findings to the clinical condition is
based on the compelling evidence that the activation of the IL-1β/IL-
1R1 axis in experimental models promotes hyperexcitability, seizures
and cell death (Vezzani et al., 2011a,b). Our data, therefore, suggest
that IL-1β, by decreasing GABA currents in brain tissue of patients
with TLE, mediates a reduction of the GABAergic inhibitory tone in
Fig. 5. IL-1β effect on IGABA amplitude in TLE is mediated by IL-1R1 and PKC. (A) Histograms
show IGABA amplitude (mean ± s.e.m., expressed as percentage of control peak amplitude)
after the indicated treatments in oocytes injected with hippocampal membranes from pa-
tients with TLE and HS. (10 μM IL-1Ra, n = 29; patients #1, 9–11, 13, Table 1; 5 μM
staurosporine, n = 18; patients #5, 12, 13, Table 1; 1 μM bisindolylmaleimide, n = 12; pa-
tients #6 and 8, Table 1; 50 nM PMA, n = 15,*p = 0.026 vs GABA alone, Kruskal–Wallis
test; patients #6,8,15,16, Table 1). The holding potential was −60 mV. GABA was applied
at 500 μM alone and after 120 min exposure to 25 ng/ml IL-1β. Inhibitors were given be-
fore IL-1β as indicated in the text. (B) Recordings of GABA currents in 3 representative oo-
cytes before and after drugs treatment as indicated. Holding potential and concentrations
as in (A). Black bars indicate GABA application.
seizure-prone areas, thereby reducing the threshold for seizure
generation.
We show that PKC is a key down-stream component mediating the IL-
1β/IL-1R1/IRAK1-dependent decrease in IGABA. Supporting evidence
shows that activation of PKC leads to down-regulation of GABAA-R medi-
ated inhibition through β3 subunit phosphorylation (Brandon et al., 2000;
 C. Roseti et al. / Neurobiology of Disease 82 (2015) 311–320
Bright and Smart, 2013). Although in our experiments we do not distin-
guish between synaptic and extrasynaptic inhibition, one possibility is
that GABAA β subunits, that are over-expressed in the hippocampi of pa-
tients with TLE (Sperk et al., 2009), may be targeted by the activation of
IL-1β/IL-R1/IRAK1 signaling. Indeed, this signaling activates neuronal
PKC in various in vitro and in vivo systems, thereby affecting neuronal
function and excitability (Vezzani and Viviani, 2015). Therefore, the pos-
sibility that IL-1R1 coupling to PKC is an oocyte-specific feature is unlikely.
Notably, IL-1β does not affect IGABA in control tissue. This lack of ef-
fect may be due either to the relatively low expression of IL-1R1 and
IRAK-1 in control compared with epileptic tissue, thus resulting in a
less efficient activation of down-stream molecular signaling affecting
GABAA-R, or to up-regulation in epileptic tissue of a specific GABAA-R
subunit composition (Brooks-Kayal et al., 1998; Pirker et al., 2003;
Sperk, 2007,2009) that is particularly sensitive to IL-1β; both scenarios
are conceivable and not mutually exclusive.
In summary, since the IL-1β/IL1R1/IRAK1 signaling is activated in
human and experimental TLE, it may cause a protracted reduction of
GABAA-R current, thus contributing to hyperexcitability and resulting
in seizure generation and recurrence. Since drugs specifically blocking
the IL-1β system are clinically available (Vezzani and Viviani, 2015),
we suggest that this mechanism may be therapeutically targeted in
drug-resistant epilepsies associated with neuroinflammation (Vezzani
et al., 2011a,b).
5. Conclusions and outlook
We report the novel finding that pathophysiological concentrations
of IL-1β significantly decrease GABAA-evoked currents in hippocampal
and cortical specimens of patients with TLE characterized by the induc-
tion of IL-1R1/IRAK1 signaling. This effect was not observed in control
specimens from patients without epilepsy and ongoing inflammatory
processes. Likewise, IL-1β reduced GABAA currents in cortical slices
from epileptic rats, but not in control rats. These findings should prompt
additional investigations into the GABAA-R subunits that are specifically
targeted by IL-1β, to see whether IL-1β affects synaptic or extrasynaptic
GABAergic transmission, or both. The IL-1β effect on GABA-mediated
neurotransmission may contribute to the well-established ictogenic
actions of this cytokine, thus supporting the use of anti-inflammatory
drugs as adjuvant treatment in pharmacoresistant forms of epilepsy.
Conflict of interest
None of the authors declares conflict of interest.
Acknowledgments
This work was supported by AICE-FIRE 2014 (E.P.), PRIN project
2010 (C.R.), Ri.MED Foundation (P. C.), EPITARGET (FP7/2007–2013),
grant agreement no. 602102 (A.V. and E.A.), and Fondazione Monzino
grant no. 5600 (A.V.). We thank Gail Bell (NIHR University College
London, UK) for language editing and Aldo Giovannelli (University of
Aquila, Italy) and Sergio Fucile (University of Rome Sapienza) for helping
with statistics.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.nbd.2015.07.003.
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