The stress-induced cytokine interleukin-6 decreases the inhibition/excitation ratio in the rat temporal cortex via trans-signaling.
ABSTRACT Although it is known that stress elevates the levels of pro-inflammatory cytokines and promotes hyper-excitable central conditions, a causal relationship between these two factors has not yet been identified. Recent studies suggest that increases in interleukin 6 (IL-6) levels are specifically associated with stress. We hypothesized that IL-6 acutely and directly induces cortical hyper-excitability by altering the balance between synaptic excitation and inhibition.
We used patch-clamp to determine the effects of exogenous or endogenous IL-6 on electrically evoked postsynaptic currents on a cortical rat slice preparation. We used control subjects or animals systemically injected with lipopolysaccharide or subjected to electrical foot-shock as rat models of stress.
In control animals, IL-6 did not affect excitatory postsynaptic currents but selectively and reversibly reduced the amplitude of inhibitory postsynaptic currents with a postsynaptic effect. The IL-6-induced inhibitory postsynaptic currents decrease was inhibited by drugs interfering with receptor trafficking and/or internalization, including wortmannin, Brefeldin A, 2-Br-hexadecanoic acid, or dynamin peptide inhibitor. In both animal models, stress-induced decrease in synaptic inhibition/excitation ratio was prevented by prior intra-ventricular injection of an analog of the endogenous IL-6 trans-signaling blocker gp130.
Our results suggest that stress-induced IL-6 shifts the balance between synaptic inhibition and excitation in favor of the latter, possibly by decreasing the density of functional γ-aminobutyric acid A receptors, accelerating their removal and/or decreasing their insertion rate from/to the plasma membrane. We speculate that this mechanism could contribute to stress-induced detrimental long-term increases in central excitability present in a variety of neurological and psychiatric conditions.
- SourceAvailable from: Francisco Garcia-Oscos[Show abstract] [Hide abstract]
ABSTRACT: Abstract The ratio between synaptic inhibition and excitation (sI/E) is a critical factor in the pathophysiology of neuropsychiatric disease. We recently described a stress-induced interleukin-6 dependent mechanism leading to a decrease in sI/E in the rodent temporal cortex. The aim of the present study was to determine whether a similar mechanism takes place in the prefrontal cortex, and to elaborate strategies to prevent or attenuate it. We used aseptic inflammation (single acute injections of lipopolysaccharide, LPS, 10 mg/Kg) as stress model, and patch-clamp recording on a prefrontal cortical slice preparation from wild-type rat and mice, as well as from transgenic mice in which the inhibitor of IL-6 trans-signaling sgp130Fc was produced in a brain-specific fashion (sgp130Fc mice). The anti-inflammatory reflex was activated either by vagal nerve stimulation or peripheral administration of the nicotinic α7 receptor agonist PHA543613. We found that the IL-6-dependent reduction in prefrontal cortex synaptic inhibition was blocked in sgp130Fc mice, or -in wild-type animals- upon application sgp130Fc. Similar results were obtained by activating the “anti-inflammatory reflex” -a neural circuit regulating peripheral immune response- by stimulation of the vagal nerve or through peripheral administration of the α7 nicotinic receptor agonist PHA543613. Our results indicate that the prefrontal cortex is an important potential target of IL-6 mediated trans-signaling, and suggest a potential new avenue in the treatment of a large class of hyperexcitable neuropsychiatric conditions, including epilepsy, schizophrenic psychoses, anxiety disorders, autism spectrum disorders, and depression.Brain Behavior and Immunity 08/2014; · 5.61 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The ratio between synaptic inhibition and excitation (sI/E) is a critical factor in the pathophysiology of neuropsychiatric disease. We recently described a stress-induced interleukin-6 dependent mechanism leading to a decrease in sI/E in the rodent temporal cortex. The aim of the present study was to determine whether a similar mechanism takes place in the prefrontal cortex, and to elaborate strategies to prevent or attenuate it. We used aseptic inflammation (single acute injections of lipopolysaccharide, LPS, 10 mg/kg) as stress model, and patch-clamp recording on a prefrontal cortical slice preparation from wild-type rat and mice, as well as from transgenic mice in which the inhibitor of IL-6 trans-signaling sgp130Fc was produced in a brain-specific fashion (sgp130Fc mice). The anti-inflammatory reflex was activated either by vagal nerve stimulation or peripheral administration of the nicotinic α7 receptor agonist PHA543613. We found that the IL-6-dependent reduction in prefrontal cortex synaptic inhibition was blocked in sgp130Fc mice, or – in wild-type animals – upon application sgp130Fc. Similar results were obtained by activating the “anti-inflammatory reflex” – a neural circuit regulating peripheral immune response – by stimulation of the vagal nerve or through peripheral administration of the α7 nicotinic receptor agonist PHA543613. Our results indicate that the prefrontal cortex is an important potential target of IL-6 mediated trans-signaling, and suggest a potential new avenue in the treatment of a large class of hyperexcitable neuropsychiatric conditions, including epilepsy, schizophrenic psychoses, anxiety disorders, autism spectrum disorders, and depression.Brain Behavior and Immunity 08/2014; · 5.61 Impact Factor
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ABSTRACT: Patients with postoperative cognitive dysfunction have poor outcomes. Neuroinflammation may be the underlying pathophysiology for this dysfunction. We determined whether proinflammatory cytokines affect the trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors to the plasma membrane, a fundamental biochemical process for learning and memory.Journal of Neuroinflammation 05/2014; 11(1):93. · 4.35 Impact Factor
The Stress-Induced Cytokine Interleukin-6 Decreases
the Inhibition/Excitation Ratio in the Rat Temporal
Cortex via Trans-Signaling
Francisco Garcia-Oscos, Humberto Salgado, Shawn Hall, Feba Thomas, George E. Farmer,
Jorge Bermeo, Luis Charles Galindo, Ruben D. Ramirez, Santosh D’Mello, Stefan Rose-John, and
Background: Although it is known that stress elevates the levels of pro-inflammatory cytokines and promotes hyper-excitable central
6 (IL-6) levels are specifically associated with stress. We hypothesized that IL-6 acutely and directly induces cortical hyper-excitability by
altering the balance between synaptic excitation and inhibition.
a cortical rat slice preparation. We used control subjects or animals systemically injected with lipopolysaccharide or subjected to electrical
foot-shock as rat models of stress.
Results: In control animals, IL-6 did not affect excitatory postsynaptic currents but selectively and reversibly reduced the amplitude of
inhibitory postsynaptic currents with a postsynaptic effect. The IL-6-induced inhibitory postsynaptic currents decrease was inhibited by
drugs interfering with receptor trafficking and/or internalization, including wortmannin, Brefeldin A, 2-Br-hexadecanoic acid, or dynamin
peptide inhibitor. In both animal models, stress-induced decrease in synaptic inhibition/excitation ratio was prevented by prior intra-
ventricular injection of an analog of the endogenous IL-6 trans-signaling blocker gp130.
possibly by decreasing the density of functional ?-aminobutyric acid A receptors, accelerating their removal and/or decreasing their
increases in central excitability present in a variety of neurological and psychiatric conditions.
Key Words: 2-Br-hexadecanoic acid, postsynaptic, Brefeldin A, dy-
terleukin-6 (IL-6), lipopolysaccharide (LPS), PI3K/AKT, patch-clamp,
anxiety (4), depression (1), and autistic spectrum disorders (ASD)
(5–7), but also to neurological conditions, including epilepsy (8,9)
and tinnitus (10). Such increase in excitability can lead to impaired
mal perception (hyperacusis, hypersensitivity to touch, sensory-
induced seizures), and/or altered emotion (paranoia, delusions,
A plethora of factors (adenosine triphosphate depletion, infec-
tion, trauma, chronic fatigue, acute stress) potentially triggering
common to many psychiatric conditions, such as schizo-
phrenic psychoses (1,2), posttraumatic stress disorder (3),
hyper-excitable neurological or psychiatric conditions are also
known to elevate the synthesis and release of cytokines (11–14).
Pro-inflammatory cytokines—including interleukin (IL)-1, IL-6, and
tumor necrosis factor ? (TNF-?)—are known to affect the brain at
the behavioral, morphological, and functional level, inducing for
example, sickness behavior (15), neurogenesis (16), and synaptic
Recent studies have indicated a specific role of IL-6 in stress-
related pathophysiology. For example, messenger RNA levels of
hippocampus (19), are greatly increased by psychological stress
(20), whereas abnormal increases in systemic levels of IL-6 follow
atric patients with a history of childhood maltreatment but not in
control subjects (21). Furthermore, noninflammatory stressors se-
lectively activate IL-6-producing vasopressin-positive neurons of
the paraventricular and supraoptic nuclei of the hypothalamus,
which in turn release systemic IL-6 (22).
Although neuronal membranes do not seem to display IL-6
receptors (23), they possess gp130 receptors—which upon stimu-
by non-neuronal brain cells initiate the Janus kinase/signal trans-
ducer and activator of transcription/extracellular signal-regulated
kinase/phosphatidyl inositol 3 kinase (PI3K) signal transduction, a
process referred to as “trans-signaling pathway” (24,25). Impor-
tantly, an abnormally high level of IL-6 has the potential to induce
and ?2subunits of ?-aminobutyric acid-A receptors (GABAARs) in
several brain regions, including the temporal cortex (26). A tempo-
rary or long-term hyper-excitability of the temporal cortex is hy-
From the School of Behavioral and Brain Sciences (FG-O, FT, GEF, MA);
School of Natural Sciences (SH, RDR, SDM); Eric Jonsson School of Engi-
neering (JB), University of Texas at Dallas, Richardson Texas; Centro de
Investigaciones Regionales Hideyo Noguchi (HS), Merida, Yucatan; Uni-
the Institute of Biochemistry (SR-J), Christian Albrechts University, Kiel
Address correspondence to Marco Atzori, Ph.D., Laboratory of Cellular and
Synaptic Physiology, School for Behavioral and Brain Sciences, Univer-
sity of Texas at Dallas, GR41, 800 West Campbell Road, Richardson, TX
75080; E-mail: email@example.com.
Received Jul 11, 2011; revised Oct 26, 2011; accepted Nov 11, 2011.
BIOL PSYCHIATRY 2012;71:574–582
© 2012 Society of Biological Psychiatry
pothesized to underlie the onset of tinnitus and hyperacusia, posi-
tively correlated with increased levels of IL-6 (27). An exquisite
vulnerability of the temporal lobe to damage by diverse stressors
might contribute to its relevance to schizophrenic psychoses, au-
tism, and epilepsy (28).
The causal relationship linking the increase in IL-6 levels and
central hyper-excitability is yet poorly understood. We considered
the possibility that IL-6 increases neuronal excitability by a direct
action at the synaptic level. To test this hypothesis we determined
the effects of the exogenous application of IL-6 as well as the IL-6-
aptic transmission on two types of stress, on a rat temporal cortex
preparation. We found that acute administration of IL-6, lipopoly-
saccharide (LPS) systemic injection, or foot-shock (FS) shifts the
pharmacological sensitivity of the IL-6 modulation of GABAergic re-
sponses is consistent with an increased, possibly ligand-dependent,
Materials and Methods
We used a temporal cortex slice preparation similar to a previ-
ously described one (Supplement 1) (29). 6,7-Dinitroquinoxaline-
a series of experiments for blocking ?-amino-3-hydroxy-5-methyl-
4-isoxazolepropionic acid receptor- and N-methyl-D-aspartate re-
Drugs and Solutions
Recombinant rat IL-6 (R&D Systems, Minneapolis, Minnesota)
2 ng/mL) and systemic (up to 15 ng/mL) concentrations measured
after immune challenge (30,31). The blocker of the IL-6 trans-path-
way (32), soluble glycoprotein 130Fc (sgp130Fc), was produced in
the laboratory of S.R.-J. at the Department of Biochemistry at the
Christian Albrecht Universitat (Kiel, Germany). Lipopolysaccharide
(serotype 0127:B8) was purchased from Sigma (St. Louis, Missouri).
All other drugs were purchased from Sigma or Tocris (Ellisville,
Missouri). After recording an initial baseline for 10–15 min, drugs
were bath-applied for 10 min or longer, until reaching a stable
condition (as defined in Statistical Analysis). For slice incubation
with wortmannin (200 nmol/L), the drug was dissolved in ethanol
(final dilution 1/2000). Brefeldin A (400 nmol/L), similar to 2-Br-
hexadecanoic acid (20 ?mol/L) was also dissolved in ethanol (final
dilution 1/500). The dynamin inhibitory peptide (P4) (20 ?mol/L)
was dissolved as described previously (33).
Electrophysiological methods are discussed in detail in Supple-
ment 1. Electrically evoked postsynaptic currents were measured
by delivering one or two electric stimuli (90–180 ?sec, 10–50 ?A)
stimulation monopolar electrode filled with artificial cerebrospinal
fluid at approximately 100–200 ?m from the recorded neuron.
Synaptic responses were monitored at different stimulation inten-
sities before baseline recording. Detection threshold was set at
approximately 150% of the SD of the noise (typical noise approxi-
mately 4–5 pA, threshold approximately 7–8 pA). A ?2 mV 100-
msec-long voltage pulse was applied at the beginning of every
episode to monitor the quality of the recording. Access resistance
(10–20 M?) was monitored throughout the experiment. Record-
ings displaying ?20% change in input or access resistance were
discarded from the analysis. All signals were filtered at 2 kHz and
sampled at 10 kHz. Reversal potential for postsynaptic currents
were evaluated, determining current-voltage (I-V) relationships for
the evoked postsynaptic current (peak amplitude of 10 events
at each holding potential Vhin the range from Vh? ?90 mV up to
Vh? ? 60 mV). Evoked inhibitory postsynaptic currents (IPSCs)
reversed polarity close to the theoretical reversal potential of ?65
mV (?64 ? 2 mV, n ? 3), whereas evoked excitatory postsynaptic
currents (EPSCs) reversed at Vexc? 10.5 ? 3 mV (n ? 3). All experi-
ments were performed at room temperature (22°C).
Rats were anesthetized with isoflurane, and body temperature
and respiration were maintained at physiological levels. The surgi-
stereotaxic (David Kopf Instruments, Tujunga, California) instru-
to the surgical platform. Rats received stereotaxic implantation
with bilateral guide cannulae in the lateral ventricles under aseptic
conditions, as follows. Skull screw anchors were fixed to the skull,
and cannulae (23-gauge stainless steel) were implanted (coordi-
mm) and secured in place with dental acrylic. A dummy cannula was
received intraperitoneal (IP) injection with aspirin (150.0 mg/kg) after
surgery and then again 7–8 hours later to aid with postoperative dis-
the cannulae was confirmed by allowing 2 ?L of sterile saline to flow
via gravity into the lateral ventricles. If cannulae placement could not
were in accordance with the National Institutes of Health Guidelines
LPS Injections. On test day, rats were injected (CMA/100 mi-
with sterile saline containing .1% bovine serum albumin (BSA) (ve-
hicle) or 100 ng sgp130Fc dissolved in 2?L vehicle. One hour later,
rats received IP injection, with sterile saline (.3 mL) or a dose of LPS
(.33 mg/kg body weight) eliciting a pro-inflammatory cytokine re-
sponse in the brain (34) and were decapitated by a guillotine 8
hours after treatment for obtaining brain slices (29).
Footshock. Rats were placed in an FS chamber (30 cm ? 10
cm). A 1.6-mA FS lasting 5 sec was administered every 4 min for a
total session duration of 64 min. Immediately after treatment, rats
were sacrificed, and brains slices were obtained. One hour before
being subjected to the FS procedure, animals received injection
with the microinjector through the cannulae with either .1% BSA
(vehicle) or 100 ng sgp130Fc dissolved in 2 ?L vehicle.
Statistical methods are described in the online Analysis section
IL-6 Selectively Decreases GABAergic Postsynaptic Currents
IL-6 application (10 ng/mL or 45 nmol/L) did not change the
mean evoked excitatory postsynaptic currents (eEPSC) amplitude
(time course in Figure 1A) (Figure 1B: mean eEPSC amplitude in
F. Garcia-Oscos et al.
BIOL PSYCHIATRY 2012;71:574–582 575
the coefficient of variation (CV) (Figure 1D) changed after applica-
tion of IL-6, suggesting that on average excitatory synaptic trans-
IL-6 reliably resulted in the decrease of approximately 50% in the
amplitude of gabazine-sensitive evoked inhibitory postsynaptic
currents (eIPSC) (example of time course in Figure 1E, average in
changes in mean PPR (Figure 1G) or CV (Figure 1H), suggesting a
postsynaptic action of IL-6.
IL-6 Shifts the Balance Between Inhibition and Excitation
synaptic input we used an intracellular solution allowing separate
Materials and Methods). We determined the resting potential for
acid (Figure 2A; insert shows a sample of traces at increasing hold-
ing potentials), as the intersection of the I-V curve with the voltage
was close to the theoretical reversal potential for Cl?(ECl
mV). Synaptic currents recorded at Vh? ?10 mV were completely
blocked by application of gabazine, demonstrating their GABAer-
tic currents obtained at a holding potential (Vh) of ?65 mV were
confirming that they were mediated by GABAARs. Similarly, we
evaluated an I-V relationship in the presence of gabazine (Figure
with an inward rectification probably due to residual polyamine
internal block (35).
synaptic responses in control as well as after bath-application of
sity that produced a solid signal at Vh? ?65 mV as well as at Vh?
?10 mV, to activate both GABAergic and glutamatergic axons (ex-
experiment confirmed that IL-6 decreases the cellular synaptic in-
hibition/excitation ratio (SIER) (same sample, Figure 2G) without
changing eEPSC amplitude (n ? 9, Figure 2H) while decreasing
eIPSC amplitude (same sample, Figure 2I).
IL-6 Decreases Muscimol-Evoked Currents
tive of a postsynaptic effect of IL-6. To determine a postsynaptic
bath-application of IL-6 on the response to the pressure-applied
currents that greatly outlasted the duration of the muscimol appli-
cations themselves (duration of the responses was approximately
course of Figure 3A, application of IL-6 decreased the maximum
amplitude of muscimol-induced responses to an extent similar to
Figure 3B, n ? 9), confirming that IL-6 affected GABAAR-mediated
responses mainly or completely by a postsynaptic mechanism.
Pharmacologic Properties of the IL-6-Induced Decrease of
We considered the possibility that IL-6 decreased GABAAR-me-
diated signal by modifying the equilibrium between membrane
expression and internalization of GABAARs, similar to the action of
other modulators (36). To test this hypothesis we used a series of
pharmacological agents known to interfere with receptor traffick-
ing and/or internalization.
The process of receptor internalization requires the presence of
functional PI3K/protein kinase B (AKT) (37). Slice pre-incubation
nmol/L) completely prevented the IL-6-induced decrease in eIPSC
amplitude (representative time course and traces in Figure 4A, left
and center, respectively), suggesting that the process is PI3K-de-
nin is compared with vehicle (Figure 4A, right panel, n ? 8, p ?
nonsignificant [ns], Student t test).
A similar effect was obtained by adding to the pipette solution
ing the displacement of endosomes from the cytosol to the mem-
brane and vice versa. Representative time course and traces illus-
trating the absence of the IL-6 effect with Brefeldin A in the
tively. The normalized effect of Brefeldin A is compared with the
Figure 1. Interleukin (IL)-6 modulation of synaptic activ-
effect of IL-6 (10 ng/mL) on the amplitude of excitatory
postsynaptic currents (EPSCs) recorded from cortical
layer II/III neurons. The insert displays the average of 10
synaptic currents (eEPSCs) do not differ before and after
IL-6 application (p ? n.s., n ? 10). (C) The ratio between
second and first response amplitude (paired pulse ratio
[PPR] ? A2/A1) in a pair pulse protocol (interpulse inter-
n.s., same sample). (D) The EPSC coefficient of variation
(CV) (calculated across 50 responses) is independent of
the amplitude of inhibitory postsynaptic currents (IPSCs)
(13.7 ? 1.4 min from the start of IL-6 application to 50%
maximal effect, n ? 20). (F) The mean amplitudes of
evoked inhibitory postsynaptic currents (eIPSCs) are sig-
IL-6 (p ? n.s., same sample). (H) The IPSC CV is indepen-
dent of IL-6 (nonsignificantly different [n.s.]).
576 BIOL PSYCHIATRY 2012;71:574–582
F. Garcia-Oscos et al.
respective data with control intracellular solution in the rightmost
panel of Figure 4B (n ? 11, p ? ns, Student t test).
anchor and modify membrane protein distribution to the plasma
membrane, which is blocked by the competitive blocker of palmi-
toyl acyl transferase 2-Br-hexadecanoic acid by substituting palmi-
tate on the catalytic site of palmitoylating enzymes (39). The pres-
ence of 2-Br-hexadecanoic acid in the pipette solution prevented,
on average, the IL-6-induced decrease in eIPSC amplitude (repre-
sentative time course and traces in Figure 4C, left and center, re-
the rightmost panel of Figure 4C.
Dynamin inhibitory peptide specifically blocks the dynamin-
dependent internalization of GABAARs after activation of insulin-
like and other receptors (33). The presence of P4 in the pipette
solution completely blocked the IL-6 depression of the eIPSC am-
center, respectively). The normalized effect of P4 is compared with
the respective data with control intracellular solution in the right-
most panel of Figure 4D.
amplitude of GABAAR-mediated currents by altering the density of
GABAARs in the plasma membrane.
Effect of Stress on the Synaptic Inhibition/Excitation Ratio
Although the results of our in vitro experiments demonstrate
whether endogenous IL-6 released by actual stressors have the
same effect. We tested this hypothesis by using two types of stres-
sors known to increase endogenous levels of IL-6: single-shot sys-
temic administration of LPS (31), and electrical FS (30,40).
hours after animal sacrifice but not at a later time (n ? 13 at ?3.5
hours, p ? .05; n ? 7 at ?3.5 hours, ns) (Figure 5A, bars 1 and 2,
respectively), compared with control subjects. To establish the IL-6
ilar experiments but accessing the lateral ventricles with cannulae.
The IP injection of vehicle and ICV BSA did not change SIER (n ? 8)
(Figure 5A, p ? ns; bar 3). The IP injection of LPS and ICV BSA
produced the same SIER as LPS injections without any ICV treat-
after ICV injection of 100 ng of the IL-6 trans-signaling pathway
Vh = -65 mV
Vh = +10 mV
Vh = -65 mV
1030 5070 90
Vh = -65 mV
Vh = +10 mV
Figure 2. Interleukin-6 decreases synaptic balance be-
presence of the glutamate receptor blockers 6,7-Dinitro-
quinoxaline-2,3-dione (DNQX) (10 ?mol/L) and kynure-
nate (KYN) (2 mmol/L). The reversal potential for ?-ami-
nobutyric acid-A receptor (GABAAR)-mediated currents
(VGABA? ?64 ? 2 mV, n ? 3) is close to the Cl- Nernst
potential calculated theoretically (VCl?? ?65 mV). The
insert displays the inversion of inhibitory synaptic cur-
amplitude of the signal recorded at Vh? ?10 mV, con-
firming the GABAergic nature of the synaptic response.
N-methyl-D-aspartate receptor (NMDAR) blockers gaba-
zine and kynurenic acid. Synaptic currents display rectifi-
cation probably due to the presence of unchelated poly-
amines (n ? 3). The insert displays some of the traces. (D)
firming the glutamatergic nature of the synaptic re-
sponse. (E) Representative traces (above) and sketch of
in (F). Scale bars are calibrated as in the rightmost trace.
(F) After identification of the proper stimulation site and
(redtrianglesontheleft).Afterdepolarizationto Vh? ? 10
mV, a baseline of synaptic responses (eIPSCs, blue circles)
lasted for the whole remainder of the experiment. After
stabilization of the eIPSC, amplitude was shifted back to
Vh? ?65 mV to monitor possible changes in eEPSC (red
triangles on the right). Numbers 1–4 in (E) correspond to
consecutive events. (G) The IL-6 decreases the ratio be-
aptic inhibition/excitation ratio ? AIPSC/AEPSC) without
changing glutamatergic responses (??p ? .02) (H) but
only affecting inhibition (I) (n ? 7; ???p ? .002). AMPA,
other abbreviations as in Figure 1.
F. Garcia-Oscos et al.
BIOL PSYCHIATRY 2012;71:574–582 577
FS Decreases SIER in an IL-6-Dependent Manner.
IL-6 (30, 40). We stressed experimental animals with electrical FS
(1.5 mA, 5 sec every 4 min) during 64 min (see Material and Meth-
ods). The FS treatment decreased SIER (n ? 13, p ? .05) (Figure 5B,
not change the FS-induced SIER depression (n ? 7, p ? .05; Figure
5B, bar 2), whereas ICV administration of BSA with sgp130Fc pro-
in vitro experiment for comparison. These results suggest that the
SIER is subject to acute and reversible modulation by stressors
through an increase in IL-6.
tion of the pro-inflammatory cytokine IL-6 selectively decreases
the temporal cortex of the rat. As a result, the balance between
synaptic inhibitory and excitatory input to cortical neurons shifted
toward excitation. In general, cytokines might affect pre- and/or
postsynaptic function. In our experiments, the failure of IL-6 to
change eIPSC PPR or the CV suggests a postsynaptic nature of the
effect, which was further corroborated by the decrease in the re-
sponse to postsynaptic application of the GABAAR agonist musci-
mol after IL-6 application.
Several earlier studies suggest that pro-inflammatory cytokines
can alter synaptic function in different brain regions in a highly
variable and area-dependent manner. For example, somnogenic
and motor-depressant effects of IL-1? have been ascribed to an
augmentation of GABAAR-mediated muscimol-evoked signal in
synaptic responses in the visual system and in the hippocampus
(42,43), shifting the balance between excitation and inhibition in
ity is present in the spinal cord, where IL-6 increases postsynaptic
currents induced by applications of ?-amino-3-hydroxy-5-methyl-4-
taneous IPSC frequency as well as currents induced by GABA and
presynaptic downregulation of parvalbumin-positive GABAergic
neurons caused by nicotinamide adenosine dinucleotide phos-
Mechanisms of Action of IL-6
Two mechanisms could account for changes in postsynaptic
inhibition: direct biophysical modulation of GABAAR function, or
GABAAR altered trafficking or internalization. GABAARs are known
subunits (33,36,48). Our pharmacological data suggest that IL-6
impairs the translocation of GABAARs between plasma membrane
and cytosolic compartment in either direction. The Phosphatidyl
inositol 3 kinase/AKT is involved in the regulation of synaptic func-
tion (37,49) and is ubiquitously involved in the regulation of mem-
brane internalization in many different preparations (50). Phos-
phatidyl inositol 3 kinase/AKT is also required for GABAAR
experiments, the IL-6-induced decrease of IPSC amplitude was
blocked by the PI3K/AKT inhibitor wortmannin, indicating the in-
the expression of GABAARs while increasing the expression of glu-
tamate receptor-1 subunits (43).
as membrane protein translocation/internalization (53–55) by in-
proteins, responsible for or otherwise involved in endocytosis
(38,56,57). Specifically, Brefeldin A induces GABAAR channel redis-
tribution by inhibiting Brefeldin-A-GDP/GTP exchange factor 2
(BIG2, a postsynaptic mouse protein bound to ?2- and ?3-GABAAR
subunits, with 95% homology with its human homolog) and has a
critical role in vesicular trafficking of ?2,3-containing GABAAR sub-
muscimol-ind. resp. ampl. (pA)
muscimol-ind. resp. ampl. (pA)
poral course of points of the effect of IL-6 (10 ng/mL) on cIPSC amplitude.
agonist muscimol (100 ?mol/L), applied every 90 sec at approximately 100
?mol/L from the recording area produced. (B) A summary chart showing
that IL-6 significantly decreases cIPSC amplitude (???p ? .001, n ? 9). ind.
578 BIOL PSYCHIATRY 2012;71:574–582
F. Garcia-Oscos et al.
qualitatively and quantitatively similar to those obtained in HEK-
on protein palmitoylation (63). It is known, for instance, that the
internalization of the ?2subunit of the GABAAR is palmitoylation-
dependent (39,64,65). We found that intracellular postsynaptic
block of palmitoylation inhibits the IL-6-induced decrease of the
IPSCs amplitude, suggesting that the effect depends on a func-
tional anchoring processing of GABAARs to a lipid bilayer.
The trafficking of membrane proteins including ionotropic re-
accessibility of specific intracellular moieties to molecular motors
including the GTP-ase dynamin. The peptide P4 selectively targets
dynamin, blocking vesicular displacement from the membrane to
the cytosolic compartment (66). GABAAR trafficking is specifically
impaired in the presence of P4 in both heterologous systems
(33,67) as well as in acute slice preparations (68). In our study, P4 in
the recording electrode abolished the IL-6-induced depression of
by altering the cycle of insertion/internalization of GABAARs.
Trafficking and internalization of GABAARs is a highly regulated
process (61,69) whose short- and long-term dysregulation is a po-
tential cause of numerous pathologic conditions like epilepsy, par-
ticularly in the temporal lobe (26,70,71). Brain infiltration by leuko-
cytes that produce pro-inflammatory cytokines as well as
alterations in immune cell transcription were identified as contrib-
utors to the pathophysiology of temporal lobe epilepsy (72). Nu-
seizures as cause or precipitating factor (73,74). The temporal cor-
tex might be exquisitely sensitive to stress, possibly resulting in
etiology of schizophrenic psychosis (75,76).
The IL-6-dependent decrease in SIER after systemic LPS or FS
delivery reinforces the hypothesis of a critical role played by IL-6 in
the stress-induced control of the balance between excitation and
perinatal LPS injections increase the levels of pro-inflammatory
rons in the schizophrenia model of ventral hippocampus lesion
(77). Interestingly, a hypothesis on the immune origin of the dis-
activity and epilepsy, is gaining increasing support (78,79). An ex-
a hyperexcitable neocortex, as hypothesized in autism spectrum
We speculate that an enhanced excitability caused by a de-
rary stressors, whereas the prolonged presence of stressors might
result in unpredictable long-term plasticity and a potentially detri-
mental maladaptive response. An imbalance between synaptic in-
hibition and excitation caused by increased synthesis, release,
a large and heterogeneous group of neurological and psychiatric
conditions associated with behavioral and central hyperexcitabil-
ity. Our finding supplies an additional contribution to understand-
eIPSC amplitude (pA)
pre-incubation in wortmannin
eIPSC amplitude (pA)
2-Br-hexadecanoic in intr. sol.
eIPSC amplitude (pA)
brefeldin A in intr. sol.
eIPSC amplitude (pA)
dynamin inhib. pep. in intr. sol.
ctr. intr. sol.
w/intrac. 2-Br-hexad. acid
ctr. intr. sol.
w/intrac. bref. A
ctr. intr. sol.
w/intrac. inh. pep.
lation. (A) The panel on the left displays a representative
with wortmannin (400 nmol/L). Wortmannin prevented
Student t test between eIPSC amplitude before or after
IL-6 application), indicating the involvement of phos-
phatidyl inositol 3 kinase (PI3K)/protein kinase B (AKT).
Middle panel in (A) shows the corresponding traces in
control or after bath-application of IL-6. The rightmost
graph compares the normalized effect of IL-6 on eIPSC
wortmannin in the incubation solution. (B–D) As in the
preceding, leftmost, middle, and rightmost panels illus-
trate the action of drugs dissolved in the pipette solution
on a representative time course (left), corresponding
traces (middle), and comparison with the effect of IL-6 in
control (right). (B) Brefeldin A (400 nmol/L) completely
prevents the IL-6-induced depression of eIPSC amplitude
(n ? 11). (C) 2-Br-hexadecanoic acid (2-Br-hexad. acid; 20
?mol/L) also blocks the effect of IL-6 on eIPSC amplitude
(dynamin inhib. pep.; 20 ?mol/L) in the intrapipette solu-
eIPSC decrease (n ? 8). ***Significance level of the statis-
tics comparing eIPSC normalized amplitudes in the pres-
and D, respectively). ctr. intr. sol., control intracellular so-
lution; other abbreviations as in Figure 1.
F. Garcia-Oscos et al.
BIOL PSYCHIATRY 2012;71:574–582 579
ing why drugs that restore the balance between inhibition and
excitation, like GABAAR enhancers such as benzodiazepines and
barbiturates and/or antiglutamatergics such as lamotrigine (81),
are so effective in the early treatment of patients suffering from
stress-related conditions—who often are prescribed condition-
specific long-term treatments only at a later stage.
The study has been funded with the University of Texas at Dallas
School of Natural Sciences, and Mrs. A. Banerjee and Dr. L. Dinh from
the School of Behavioral and Brain Sciences at the University of Texas
at Dallas for insightful discussions and critical review of the manu-
script. Dr. Rose-John is funded by grants from the Deutsche For-
schungsgemeinschaft, Bonn, Germany (SFB654, project C5; SFB841,
Dr. Rose-John is an inventor on the patent describing the function
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