Suppression of PKR Promotes Network
Excitability and Enhanced Cognition
by Interferon-g-Mediated Disinhibition
Ping Jun Zhu,1,2Wei Huang,1,2Djanenkhodja Kalikulov,1,7Jong W. Yoo,3Andon N. Placzek,1,6Loredana Stoica,1,2
Hongyi Zhou,1,2John C. Bell,4Michael J. Friedlander,1,7Kre? simir Krnjevi? c,5Jeffrey L. Noebels,3
and Mauro Costa-Mattioli1,2,*
1Department of Neuroscience
2Molecular and Cellular Biology, Center for Addiction, Learning, and Memory
3Department of Neurology
Baylor College of Medicine, Houston, TX 77030, USA
4Ottawa Health Research Institute, Ottawa K1H 8L6, Canada
5Physiology Department, McGill University, Montreal H3G 1Y6, Canada
6Present address: Division of Basic Medical Sciences, Mercer University School of Medicine, 1550 College Street, Macon, GA 31207, USA
7Present address: Virginia Tech Carilion Research Institute, Virginia Tech, Roanoke, VA 24016, USA
The double-stranded RNA-activated protein kinase
(PKR) was originally identified as a sensor of virus
infection, but its function in the brain remains
unknown. Here, we report that the lack of PKR
enhances learning and memory in several behavioral
tasks while increasing network excitability. In addi-
tion, loss of PKR increases the late phase of long-
lasting synaptic potentiation (L-LTP) in hippocampal
slices. These effects are caused by an interferon-g
(IFN-g)-mediated selective reduction in GABAergic
synaptic action. Together, our results reveal that
PKR finely tunes the network activity that must be
maintained while storing a given episode during
learning. Because PKR activity is altered in several
neurological disorders, this kinase presents a prom-
ising new target for the treatment of cognitive
dysfunction. As a first step in this direction, we
show that a selective PKR inhibitor replicates the
Pkr?/?phenotype in WT mice, enhancing long-term
memory storage and L-LTP.
The double-stranded (ds) RNA-activated protein kinase (PKR) is
widely present in vertebrates, and its activation leads to the
phosphorylation of several substrates, the major known cyto-
plasmic target being the translation initiation factor eIF2a (Dever
etal.,2007).Although PKRisactivated inresponseto avariety of
cellular stresses, such as viral infection (Garcı ´a et al., 2007) and
status epilepticus (Carnevalli et al., 2006), and in several neuro-
pathologies, including Alzheimer’s (Couturier et al., 2010; Peel
and Bredesen, 2003), Parkinson’s (Bando et al., 2005), Hunting-
ton’s (Bando et al., 2005; Peel et al., 2001), and Creutzfeldt-
Jakob’s diseases (Paquet et al., 2009), little is known about its
role in normal brain function.
Cognitive functions are believed to arise from the finely
coordinated interactions of a large number of neurons widely
distributed throughout the brain. A fundamental yet unresolved
question of modern neuroscience is how optimal synchroniza-
tion is achieved without degrading information flow. Though
transient synchronizations of neuronal discharges have been
proposed to promote memory consolidation (Beenhakker
and Huguenard, 2009; Buzsaki, 2006; Girardeau et al., 2009;
Paulsen and Moser, 1998), seizure activity can develop in hyper-
excitable oscillatory networks (Huguenard and McCormick,
2007; Steriade, 2005). GABAergic synaptic transmission plays
a pivotal role in maintaining this balance. GABAergic inhibitory
neurons not only suppress the activity of principal cells, but
also serve as a generator of oscillations in hippocampal
networks (Freund, 2003; Klausberger and Somogyi, 2008;
Mann and Mody, 2010; Sohal et al., 2009), which appear to be
crucial for memory consolidation (Beenhakker and Huguenard,
2009; Buzsaki, 2006; Girardeau et al., 2009; Paulsen and Moser,
1998). Furthermore, GABAergic inhibition also helps to terminate
these rhythmic events, thus preventing runaway epileptiform
network activity. However, little is known about the molecular
mechanisms critical for dynamically scaling GABAergic control
of neuronal synchrony during memory formation.
A reduction of GABAergic synaptic transmission is classically
associated with epileptiform activity (Beenhakker and Hugue-
nard, 2009; Cossart et al., 2005; Noebels, 2003). Though many
kinds of epilepsy lead to cognitive impairment (Holmes and
Lenck-Santini, 2006), some seizure disorders spare memory,
and a few rare forms have been associated with extraordinary
mental abilities (Heaton and Wallace, 2004; Hughes, 2010).
However, little is known about the causes or possible genetic
1384 Cell 147, 1384–1396, December 9, 2011 ª2011 Elsevier Inc.
mutations associated with this type of epilepsy. Here, we report
that loss of PKR or pharmacological inhibition of PKR activity
promotes network hyperexcitability and enhances L-LTP and
long-term memory (LTM). Importantly, we show that PKR regu-
lates these processes via a selective control of GABAergic
synaptic transmission mediated by interferon-g (IFN-g), uncov-
ering a new molecular signaling pathway that regulates network
rhythmicity, synaptic plasticity, and memory storage in the adult
Deficient PKR Kinase Activity Leads to Synchronous
Network Discharges In Vivo and In Vitro
PKR knockout (Pkr?/?) mice are viable, fertile, and of normal size
and are phenotypically indistinguishable from their wild-type
(WT) littermates (Abraham et al., 1999). Nissl staining and
synaptic markers for the vesicular glutamate transporter 1
(VGLUT1, a marker of presynaptic glutamatergic terminals),
postsynaptic density protein 95 (PSD95, a marker of postsyn-
aptic terminals), and glutamic acid decarboxylase 67 (GAD67,
a marker of GABAergic terminals) show no gross abnormalities
in Pkr?/?mouse brain (Figures S1A–S1D available online). As
expected, PKR protein is undetectable in Pkr?/?brain, as deter-
mined by immunohistochemistry and western blotting (Figures
S1E and S1F).
To determine whether PKR regulates network rhythmicity, we
first monitored spontaneous cortical brain rhythms in freely
moving WT and Pkr?/?mice by simultaneous video and electro-
encephalogram (videoEEG) recording. In recordings from Pkr?/?
mice, we found intermittent abnormal spike discharges (Fig-
ure 1A; at a mean frequency of 40 ± 9 interictal spike/hr), as
at a mean frequency of 1.1 ± 0.021 seizures/hr), that were not
accompanied by convulsive behavioral manifestations. Neither
abnormality ever appeared in the EEG from WT mice (Figure 1B).
Simultaneous recordings from the neocortex and hippocampus
from Pkr?/?mice showed comparable epileptiform activity (see
Movie S1). Thus, loss of PKR leads to aberrant hyperactivity of
neuronal networks. Because the excitability imbalance in Pkr?/?
mice might arise during development, we suppressed PKR
activity acutely in adult WT mice by systemic administration of
the injections of PKRi rapidly induced both interictal spikes (Fig-
ure 1D; at a mean frequency of 25 ± 7.8 interictal spike/hr) and
abnormal EEG rhythmic bursting activity (Figure 1E), like those
occurring spontaneously in Pkr?/?mice (compare Figure 1E to
Figure 1A). As in the case of Pkr?/?mice, the spontaneous
discharges in PKRi-treated mice were not accompanied by
changes in ongoing behavior. Together, these observations
reveal a pivotal role for this kinase as a regulator of network
To determine whether the abnormal synchronous network
activity in Pkr?/?mice or WT mice treated with PKRi could be
recapitulated in vitro, we recorded field EPSPs (fEPSPs) and
population spikes (in CA1) in hippocampal slices from WT and
Pkr?/?mice or in WT slices treated with PKRi. The population
spike is a useful index of the responsiveness of many pyramidal
neurons to excitatory inputs: its size reflects the neuron’s
intrinsic excitability as well as the balance between excitatory
and inhibitory synaptic inputs. An appropriate single electrical
stimulus applied to the stratum radiatum elicited a similar field
EPSP and population spike in WT and Pkr?/?slices (Figures
2A and 2B, insets). However, in the presence of a very low
priming concentration of bicuculline (2 mM), the same stimulus
evoked a prominent after-discharge in Pkr?/?slices, but not in
WT slices (compare Figure 2A to 2B; see also Figures 2D and
2E), revealing a latent hyperexcitability of hippocampal networks
in Pkr?/?slices. A similar after-discharge was observed when
PKRi was applied to WT slices (Figure 2C; see also Figures 2D
and 2E), demonstrating a comparable increase in excitability
when PKR was inhibited pharmacologically.
An increased tendency to fire spikes in Pkr?/?slices was also
revealed when field responses were elicited in CA1 by stimu-
lating the afferent input over a wide range of intensities. Plots
of afferent fiber volley (AV) versus stimulus intensity, initial slope
iRKP + TWiRKP + TW
Figure 1. Synchronized EEG Activity in Pkr?/?Mice
or WT Mice Treated with PKRi In Vivo
(A–C) Traces from bilateral cortical electrodes (left hemi-
sphere reference, L-r; right hemisphere reference, R-r)
show abnormal synchronous activity, including solitary
interictal spikes followed by brief wave discharges (A) and
electrographic seizures (C) in freely moving Pkr?/?mice,
but not in WT mice (B).
(D and E) Injection of PKR inhibitor (PKRi; 0.1 mg/kg i.p.)
induced acute spiking (D) and rhythmic bursts (E) in adult
WT mice. Calibration: 1 s and 200 mV. Abnormal EEG
activity was absent from all WT control recordings (n = 7)
PKRi-injected mice (recorded 1 hr after of PKRi injection).
By Fisher’s exact test, p values were < 0.001 and < 0.01,
lack of change in brain morphology; Figure S6B for EEG
interictal spikes in mice with constitutively reduced eIF2a
phosphorylation (eIF2a+/S51Amice); and Figure S7D for the
effect of PKRi in vivo.
Cell 147, 1384–1396, December 9, 2011 ª2011 Elsevier Inc. 1385
of field EPSPs versus AV size, and paired-pulse facilitation (PPF)
did not significantly differ between Pkr?/?and WT slices (Figures
2F–2H), indicating no change in the excitability of afferent fibers
or the efficacy of excitatory synaptic transmission in Pkr?/?
slices. Relatively small fEPSPs (<half-maximal) evoked similar
population spikes in Pkr?/?and WT slices (Figure 2I), indicating
no difference in intrinsic excitability of the postsynaptic cells.
Hence, the intrinsic membrane properties and responsiveness
of CA1 pyramidal neurons did not differ between WT and Pkr?/?
slices (Figures 2J–2K). However, fEPSPs elicited by stronger
stimulation evoked larger population spikes in Pkr?/?slices,
thus reaching a higher ceiling in the sigmoidal relationship
Bicuculline 2 μM
Bicuculline 2 μM
Bicuculline 2 μM
Burst duration (ms)
Number of spikes/burst
Afferent volley (mV)
EPSP slope (v/s)
Stimulation intensity (v)
Afferent volley (mV)
Interpulse interval (ms)
0100 200 300 400 500
PP facilitation ratio
EPSP slope (v/s)
Population spike (mV)
0 1 2 3 4
Number of spikes
Current injection (pA)
100 ms 20 mV
RP=-59 mVRP=-60 mV
Figure 2. Synchronized Activity and Reduced Inhibition in Pkr?/?Hippocampal Slices or WT Slices Treated with PKRi
(A–C) Population spikes were elicited by half-maximal electrical stimulation at 0.03 Hz (indicated by arrow).Insets in (A), (B), and (C) are averaged traces recorded
before applying a very low concentration of bicuculline (2 mM), which generated pronounced after-discharges in Pkr?/?slices (B) or WT slices treated with PKRi
(1 mM) (C), but not in WT slices (A). For all plots, n R 5; calibrations: 2 ms and 3 mV for insets and 10 ms and 5 mV for slow traces.
(D and E) Compared to WT slices, the number of evoked spikes (D, F(2, 24)= 66.3; **p < 0.01) and the duration of burst (E, F(2.24)= 100.2; **p < 0.01) were increased
in Pkr?/?slices or WT slices treated with PKRi.
(F–K) Similar intrinsic neuronal properties and basal synaptic transmission but maximal fEPSPs elicited larger population spikes in Pkr?/?slices.
(F) Input-output data show similar amplitudes of presynaptic fiber volleys over a wide range of stimulus intensities in WT and Pkr?/?slices. Mean afferent volley
versus stimulus strength were fitted by linear regression (R2= 0.989 for WT and 0.993 for Pkr?/?slices).
(G) fEPSPs as a function of presynaptic fiber volley did not differ between WT and Pkr?/?slices (linear regression; R2= 0.643 for WT and 0.638 for Pkr?/?slices).
(H) Paired-pulse facilitation of fEPSPs did not differ between WT and Pkr?/?slices, as shown by the plots of the PP ratio (fEPSP2/ fEPSP1) for various intervals of
(I) Sigmoidal relationship of population spikes versus fEPSP, though initially similar, reached a higher ceiling in Pkr?/?slices.
(J and K)Inwhole-cellrecordings inthepresence ofglutamateand GABAantagonists,resting membrane potential was ?59± 1.23 mVforWT and ?60± 1.22mV
for Pkr?/?slices (p > 0.05), and input resistance was 397 ± 24 MU for WT and 381 ± 39 MU for Pkr?/?slices (p > 0.05). Inward and outward current pulses
generated similar voltages changes (J) and numbers of spikes (K) in WT and Pkr?/?slices.
For information about the effect of PKRi in vitro, see Figure S7C.
1386 Cell 147, 1384–1396, December 9, 2011 ª2011 Elsevier Inc.
between population spikes and fEPSPs (Figure 2I). Given that
excitatory transmission was unchanged (Figure 2G), these data
suggest that population spikes reached a higher ceiling in Pkr?/?
slices because inhibition was impaired. A comparable increase
in the population spike amplitude ceiling is generated by
a GABA antagonist (Chavez-Noriega et al., 1989). Accordingly,
pharmacological inhibition of PKR (with PKRi) also enhanced
population spikes (see Figures S3F and S3G).
Deficient PKR Kinase Activity Reduces Inhibitory
Because impaired inhibition is a common feature of genetic
this hypothesis, we studied inhibitory synaptic transmission in
a series of experiments on hippocampal slices from WT and
Pkr?/?mice and WT slices treated with PKRi. In whole-cell
patch-clamp recordings from CA1 neurons, the frequency (but
not the amplitude) of both spontaneous and miniature inhibitory
postsynaptic currents (sIPSCs and mIPSCs) was significantly
reduced in Pkr?/?slices (Figures S2A and 3A) or in WT slices
treated with PKRi (Figures S2B and 3B). The absence of change
in mIPSC amplitude or duration is a strong indication that PKR
does not affect the sensitivity of pyramidal cells to synaptically
released GABA. Next, we found that, in CA1 neurons from
PKR-deficient slices (Pkr?/?slices and PKRi-treated WT slices),
stimulation intensities (Figures S2D and 3C). In contrast to its
effect in WT slices, PKRi did not alter the amplitude of evoked
IPSCs in Pkr?/?slices (compare Figure 3D to 3E), confirming
that its effect was not due to an off-target action. Providing
evidence of reduced GABA release, paired-pulse depression,
a sensitive index of changes in evoked GABA release (Thomson,
2000), was significantly decreased in PKR-deficient slices (Fig-
ure 3F). Moreover, PKR seems to regulate inhibitory transmis-
sion presynaptically, as there was no difference in the time
course of mIPSCs and sIPSCs between WT slices and PKR-
deficient slices (Figures 3A, 3B, S2A, S2B, and Table S1), which
is consistent with a lack of change in postsynaptic receptor-
tion in PKR-deficient slices is the much better preservation
of CA1 population spikes during high-frequency stimulation
(compare Figure S3A to S3C and S3D; see also Figure S3E),
cuculline (Figures S3B and S3E). Finally, in keeping with input/
output data from Pkr?/?slices (Figures 2F and 2G), PKRi had
no effect on the afferent volley and the initial slope of field EPSPs
in WT slices (Figure S3F), but it enhanced population spikes (Fig-
ure S3G), as expected if pyramidal neurons were more excitable
owing to reduced ongoing inhibition. As expected, PKRi had no
effect on population spikes in Pkr?/?slices, where PKRi’s target
(PKR) is absent (Figure S3H) or when inhibition was already
blocked (Figure S3I).
PKR acts selectively on GABAergic inhibition, as neither the
amplitude nor the frequency of spontaneous excitatory postsyn-
aptic currents (sEPSCs), miniature EPSCs (mEPSCs), or evoked
EPSCs (eEPSCs) was significantly changed in PKR deficient
slices (Figure 4). On the basis of our genetic and pharmacolog-
ical evidence, we conclude that PKR’s normal function is to
maintain a relatively low level of excitability by enhancing
GABAergic synaptic transmission.
Deficient PKR Kinase Activity Facilitates L-LTP
Because the induction of long-term potentiation (LTP) is facili-
tated by a decrease in GABA tone (Abraham et al., 1986; Davies
et al., 1991; Wigstro ¨m and Gustafsson, 1983), we wondered
whether reduced synaptic inhibition in Pkr?/?slices (or WT slices
treated with PKRi) could facilitate the induction of LTP. Early LTP
(E-LTP), typically induced by a single train of high-frequency
(tetanic) stimulation, lasts only 1–2 hr and depends on modifica-
tion of pre-existing proteins, whereas late LTP (L-LTP), generally
induced by several (typically four) tetanic trains separated by
5–10 min, persists for many hours and requires new protein
synthesis (Kandel, 2001). In WT slices, a single high-frequency
stimulus train (100 Hz for 1 s) elicited only a short-lasting
E-LTP (Figure 5A). By contrast, in Pkr?/?slices, the same stimu-
lation generated a long-lasting late LTP (L-LTP) (Figure 5A),
which was blocked by the protein synthesis inhibitor anisomycin
(Figure 5B). Four tetanic trains (at 100 Hz) elicited a similar L-LTP
in WT and Pkr?/?slices (Figure 5C). In agreement with the find-
ings in Pkr?/?slices, incubation with PKRi converted a transient
E-LTP into a sustained L-LTP in WT slices (Figure 5D) but did
not induce any further potentiation in Pkr?/?slices (Figure 5E),
confirming the specificity of PKRi. These data demonstrate
thatgeneticdeletion orpharmacological inhibitionofPKRlowers
the threshold for the induction of L-LTP.
If L-LTP is facilitated in Pkr?/?slices due to reduced inhibition,
then reinforcing GABAergic tone should convert the effect of
a single train from long-lasting to short-lasting. Indeed, incuba-
tion with a low concentration of diazepam (1 mM), which potenti-
ates GABAAaction (Haefely, 1990), markedly reduced L-LTP in
Pkr?/?slices (Figure 5F) but had no effect on L-LTP induced by
four tetanic trains in WT slices (Figure 5G). Only a very high
concentration of diazepam (50 mM) reduced L-LTP in WT slices
(Figure 5H). These data support our hypothesis that the facilita-
tion of L-LTP in Pkr?/?slices is a consequence of decreased
Deficient PKR Kinase Activity Enhances Learning
GABAergic function is crucial for memory consolidation
(Izquierdo and Medina, 1991; McGaugh and Roozendaal,
2009). To address whether learning and memory are enhanced
in Pkr?/?mice, which exhibit impaired hippocampal GABA-
mediated inhibition, mice were tested for hippocampus-depen-
dent spatial memory in the Morris water maze (Morris et al.,
1982). Because weak tetanic stimulation (one train at 100 Hz)
induced L-LTP in slices from Pkr?/?mice, we trained mice
using a weak protocol (only one training session per day) for
8 days. Pkr?/?mice found the platform significantly faster than
did WT control littermates (Figure 6A); and in the probe test,
performed on day 9, when the platform was removed, only
Pkr?/?mice remembered the platform location (target quadrant;
Figure 6B). Thus, genetic deletion of PKR strengthened
Cell 147, 1384–1396, December 9, 2011 ª2011 Elsevier Inc. 1387
Mice were also studied in two forms of Pavlovian fear condi-
tioning. Contextual fear conditioning was induced by pairing
a context (conditioned stimulus [CS]) with a foot shock (the
unconditioned stimulus [US]), whereas in auditory fear condi-
tual fear conditioning involves both the hippocampus and
amygdala, whereas auditory fear conditioning requires only the
amygdala (LeDoux, 2000). When mice were subsequently
exposed to the same CS, fear responses (freezing) were taken
as an index of the strength of the CS-US association. Naive WT
and Pkr?/?mice showed a similar amount of freezing prior to a
weak training protocol (a single pairing of a tone with a 0.35 mA
foot shock), whereas Pkr?/?mice exhibited more freezing than
did WT control littermates when tested 24 hr later (Figure 6C).
Similarly, Pkr?/?mice showed enhanced long-term auditory
mice was unlikely because both baseline freezing (Figures 6C
and 6D) and anxiety-reflecting behavior (in the elevated plus
Stimulus intensity (mA)
eIPSC amplitude (pA)
Intersimulus interval (ms)
0.1 0.50.7 0.91.10
mIPSC amplitude (pA)
mIPSC amplitude (pA)
PKRi 1 μM (n=6)
05 10 15 20
eIPSC amplitude (%)
PKRi 1 μM (n=7)
Figure 3. Reduced Inhibitory Synaptic Responses in CA1 of Pkr?/?Slices or WT Slices Treated with PKRi
(A) Sample traces (top) and summary data (bottom) show reduced frequency (t = 4.7; **p < 0.01) but no change in the amplitude (Mann-Whitney U test, U = 66.0;
p = 0.84) of mIPSCs (recorded at ?60 mV with a KCl-containing patch pipette and in the presence of the wide-spectrum glutamate antagonist kynurenic acid
[2 mM] and tetrodotoxin [TTX, 1 mM]) in CA1 neurons from Pkr?/?mice. Traces at right (each is an average of at least 100 mIPSCs) do not differ between WT
(uppermost) and Pkr?/?slices (middle), as confirmed by superimposed traces (lowest).
(B) Similarly, in WT slices, PKRi decreased the frequency of mIPSCs (t = 3.42; *p < 0.05), but not their amplitude (t = 0.46; p = 0.65). Summary data and individual
events are as in (A).
(A and B) Calibrations: 1 s and 50 pA for slow traces; 20 ms and 20 pA for fast traces.
(C) eIPSC amplitude as a function of stimulation intensity is shown superimposed and plotted (below) as input/output curves (recorded at 0 mV in the presence of
APV [50 mM], CNQX [10 mM], and CGP55845 [10 mM]) (*p < 0.05; **p < 0.01). Calibrations: 50 ms and 200 pA.
(D and E) PKRi reduced the amplitude of evoked IPSCs in WT slices (D) (t = 3.2; p < 0.01), but not in Pkr?/?slices (E) (Mann-Whitney U test, U = 20.2; p = 0.62).
Horizontal bar indicates PKRi application. Inset traces were obtained at times ‘‘a’’ and ‘‘b.’’
(F) Paired IPSCs at 50, 100, 200, and 400 ms interstimulus intervals (ISIs) are superimposed (at left) after subtracting the first IPSC from paired responses.
Corresponding ratios of IPSC2/IPSC1are plotted at right. Note the reduced paired-pulse depression at 50 ms in Pkr?/?slices (t = 7.85; **p < 0.01) and WT slices
treated with PKRi (t = 3.47; **p < 0.01).
See Figure S2 for further information about sIPSCs and eIPSCs in PKR-deficient slices; Figure S3 for the role of PKR in cumulative GABAergic inhibition; Fig-
ure S6A for the reduced mIPSCs in slices from eIF2a+/S51Amice; and Figure S7C for the effect of PKRi in vitro.
1388 Cell 147, 1384–1396, December 9, 2011 ª2011 Elsevier Inc.
eEPSC amplitude (% )
PKRi 1 μM (n=7)
0 5 10 15 20
sEPSC amplitude (pA)
mEPSC amplitude (pA)
Figure 4. Excitatory Synaptic Transmission Is
Unaltered in Pkr?/?Slices or WT Slices Treated
(A) Whole-cell recordings of EPSCs were performed with
gluconate-containing pipettes at ?70 mV in the presence
of 100 mM picrotoxin. Sample traces (top) and summary
data (bottom) show similar frequency (Mann-Whitney
U test, U = 20.5; p = 0.95) and amplitude (t = 0.48; p = 0.64)
of spontaneous EPSCs (sEPSCs) in WT and Pkr?/?slices.
(B) Sample traces (top) and summary data (bottom) show
and amplitude (t = 0.34; p = 0.74) of miniature EPSCs
and TTX [1 mM]) in WT and Pkr?/?slices.
(C) PKRi (1 mM) had no effect on EPSCs evoked (for ‘‘a’’
versus ‘‘b’’; illustrated by inset traces, t = 0.40; p = 0.69) in
the presence of 100 mM picrotoxin. Horizontal bar indi-
cates the period of incubation with PKRi.
(A and B) Calibrations: 1 s and 20 pA. (C) Calibrations:
10msand 100pA. Forinformation about theeffect ofPKRi
in vitro, see Figure S7C.
Cell 147, 1384–1396, December 9, 2011 ª2011 Elsevier Inc. 1389
fEPSP slope (% change)
WT untetanized (n=4)
Pkr-/- untetanized (n=6)
Diazepam 1 μM
050 100 150 200250
Anisomycin 40 μM
050 100150200 250
Vehicle untetanized (n=5)
PKRi untetanized (n=4)
Diazepam 50 μM
200250 0 50 100 150
PKRi 1 μM
Vehicle (n=7) PKRi (n=6)
050 100150 200250
Pkr-/- (n=7)WT (n=7)
Figure 5. Facilitated L-LTP in Pkr?/?Slices or WT Slices Treated with PKRi
(A) A single high-frequency train (100 Hz for 1 s) elicited a short-lasting early LTP (E-LTP) in WT slices but generated a sustained late LTP (L-LTP) in Pkr?/?slices
(F(1,14)= 76.8; p < 0.001).
(B) The facilitated L-LTP in Pkr?/?slices was suppressed by anisomycin (F(1,11)= 22.2; p < 0.001).
(C) L-LTP induced by four tetanic trains at 100 Hz is similar in WT and Pkr?/?slices (F(1,12)= 0.48; p = 0.49).
(D and E) PKRi converted E-LTP into L-LTP in WT slices (D) (F(1,10)= 61.2; p < 0.001) but in Pkr?/?slices (E) did not further potentiate the sustained LTP elicited by
a single high-frequency train (F(1,11)= 1.1; p = 0.18).
(F and G) A low concentration of diazepam (1 mM) impaired L-LTP induced by a single tetanus in Pkr?/?slices (F) (F(1,10)= 11.3; p < 0.001), but not the L-LTP
induced by four tetani in WT slices (G) (F(1,10)= 0.23; p = 0.64).
(H) In WT slices, a high concentration of diazepam (50 mM) impaired L-LTP induction by four trains at 100 Hz (F(1,9)= 15.4; p < 0.01). Horizontal bars indicate the
periods of drug application. (Inset) Superimposed traces obtained before and 220 min after tetanic stimulation. All comparisons were done at 220 min.
Calibrations: 5 ms and 3 mV.
For information about the role of PKRi in L-LTP maintenance, see Figure S7A. See Figure S7C for the effect of PKRi in vitro.
1390 Cell 147, 1384–1396, December 9, 2011 ª2011 Elsevier Inc.
maze and open field; Figure S4) were normal in Pkr?/?mice.
Hence, the lack of PKR improved both auditory and contextual
long-term fear memories. Enhanced cognition is also associated
with rapid memory extinction (Lee and Silva, 2009) whenanimals
shock is applied. As expected, Pkr?/?mice showed faster
contextual fear extinction than did WT controls (Figure 6E).
ical inhibition of PKR should also potentiate long-term fear
memories. To test this prediction, WT mice were injected with
either vehicle or PKRi immediately after Pavlovian fear condi-
tioning. Both contextual and auditory long-term fear memories
were enhanced in PKRi-treated mice when measured 24 hr after
training (Figures 6F and 6G).
CS + US
SCSC- erPrh 42ev i aN
MWM acquisition curve
MWM probe test
Quadrant occupancy (s)
Freezing (% at 24 hr)
24 48 72 96
CSCS + US
# Egr-1-positive nuclei
Figure 6. Enhanced Spatial and Fear Memory in
Pkr?/?Mice or WT Mice Treated with PKRi
(A) Mean escape latencies as a function of training days in
the Morris water maze (one trial/day). Compared to WT
controls, escape latencies were significantly shorter for
Pkr?/?mice after day 6 (day 7, F(1,24)= 7.4; day 8, F(1, 24)=
6.4; *p < 0.05).
(B) In the probe test performed on day 9, only Pkr?/?mice
preferred the target quadrant (F(1, 24)= 11.109; **p < 0.01).
(C) For contextual fear conditioning, freezing times were
recorded before conditioning (naive, during 2 min period)
and then 24 hr after training (during 5 min period).
(D) For auditory fear memory, freezing times were
measured 24 hr posttraining either before the onset of the
tone (pre-CS, for 2 min) or during the tone presentation
(for 3 min). Enhanced freezing 24 hr after training indicates
stronger fear memory in Pkr?/?mice (C, F(1, 21)= 10.2;
**p < 0.01; D, F(1,21)= 5.2; *p < 0.05).
(E) Pkr?/?mice exhibited significantly faster extinction of
freezing in response to the context, as compared to WT
littermates (at 48 hr, WT versus Pkr?/?mice; F(1, 15)= 4.7;
*p < 0.05; Pkr?/?mice within group, 24–72 hr; F(2, 8)= 12.8;
**p < 0.01).
(F and G) PKRi injection (0.1 mg/kg i.p.) immediately after
training enhanced both contextual (F, F(1,
*p < 0.05) and auditory (G, F(1, 14)= 19.1; ***p < 0.001) long-
term fear memories.
(H) The expression of the immediate-early gene Egr-1 was
similar in CA1 neurons from WT and Pkr?/?mice exposed
to context (CS). In response to contextual fear training
(CS+US), there was a significantly greater number of
Egr-1-positive neurons in CA1 from Pkr?/?mice (F(1,9)=
11.94; **p < 0.01; Pkr?/?CS versus CS+US, F(1,8)= 30.7;
**p < 0.01).
For information about the role of PKRi in LTM mainte-
nance, see Figure S7B. See Figure S7D for the effect of
PKRi in vivo and Figure S4 for the lack of anxiety-reflecting
behavior in Pkr?/?mice.
14) = 6.6;
Because PKR deficiency enhanced long-term
memory storage, we asked whether memory
allocation, the process by which neurons or
synapses are specifically activated or incorpo-
rated in a neural circuit during learning (Silva
et al., 2009), is also enhanced in Pkr?/?mice.
To identify neurons selectively activated during
learning, we analyzed the expression of the
immediate-early gene Egr-1 (also called Zif/268). Egr-1 has
been extensively used for this purpose (Frankland et al., 2004;
Hall et al., 2000), and its deletion blocks L-LTP and memory
consolidation (Jones etal.,2001).WT andPkr?/?micewere sub-
jected to a weak fear conditioning protocol (a single pairing of
a tone with a 0.35 mA foot shock), and the expression of Egr-1
in the CA1 region was quantified by immunohistochemistry, as
previously described (Frankland et al., 2004). Egr-1 expression
was not significantly different when animals of both genotypes
were exposed to the context alone. In contrast, a weak training
paradigm, which triggered a more robust long-lasting memory
only from Pkr?/?mice (Figure 6H). Thus, the lack of PKR favors
the recruitment of CA1 neurons into the encoding process.
Cell 147, 1384–1396, December 9, 2011 ª2011 Elsevier Inc. 1391
Inhibition of Interferon-g in PKR-Deficient Mice
Restores Normal Excitability, LTP, and Memory
Given that IFN-g increases neuronal excitability in hippocampal
slices by reducing GABA release (Mu ¨ller et al., 1993) and that
PKR suppresses the translation of IFN-g mRNA (Ben-Asouli
et al., 2002; Cohen-Chalamish et al., 2009), we hypothesized
that the hyperexcitability of neural networks in PKR-deficient
mice might be caused byIFN-g-mediated disinhibition. In accor-
dance with this prediction, IFN-g levels were increased in the
hippocampus from Pkr?/?mice (Figure 7A). Similar to treatment
Bicuculline 2 μM
Bicuculline 2 μM
Bicuculline 2 μM
Bicuculline 2 μM
Burst duration (ms)
Number of spikes/burst
Burst duration (ms)
Number of spikes/burst
bicuculline 10 μM
IFN-γ (200 U/ml)
PS amplitude (% change)
IFN-γ (200 U/ml)
fEPSP slope (%)
0 10 20 30 40 50 60 70
0 10 20 30 40
PS amplitude (% change)
5 ms 2 mV
Figure 7. Inhibition of IFN-g Rescues Hyperexcitability, Facilitated L-LTP, and LTM Caused by PKR Deficiency
(A) IFN-g was increased in pooled hippocampal extracts from Pkr?/?mice (Mann-Whitney U test, U = 0.00; **p < 0.01).
(B) IFN-g (200 U/ml) enhanced the amplitude of population spikes in WT slices (t = 2.65; p < 0.05), but not in the presence of bicuculline (C, t = 1.03; p = 0.33).
of bicuculline (2 mM) in Pkr?/?slices.
(F and G) NAb-IFN-g greatly reduced the number of evoked spikes (F) (t = 6.52; ***p < 0.001) and burst duration (G) (Mann-Whitney U test, U = 0.00, **p < 0.01).
(H and I) Combined application of PKRi (1 mM) with a low concentration of bicuculline (2 mM) induced prominent after-discharges in WT slices (H), but not in
(J and K) Note the great reduction in the number of evoked spikes (J, t = 6.21; ***p < 0.001) and burst duration (K, t = 4.21; ***p < 0.001) in Ifn-g?/?slices.
(L) PKRi induced a sustained L-LTP in WT slices, but not in Ifn-g?/?slices (F(1,14)14.9; p < 0.01).
effect in Ifn-g?/?mice (N) (F(1,14)= 0.62; p = 0.44).
For further information about IFN-g and PKRi in hyperexcitability and auditory long-term fear memory, see Figure S5. For information about the effect of PKRi
in vitro and in vivo, see Figures S7C and S7D.
1392 Cell 147, 1384–1396, December 9, 2011 ª2011 Elsevier Inc.
with PKRi (see Figure S3G), addition of exogenous IFN-g
enhanced the amplitude of CA1 population spikes in WT slices
(Figure 7B) but had no effect in slices preincubated with bicucul-
line (10 mM), in which GABAergic synaptic transmission was
already blocked (Figure 7C), or in those treated with a mono-
clonal neutralizing antibody against IFN-g (NAb-IFN-g; Fig-
ure S5A), which specifically binds to IFN-g and blocks its
biological activity (Buchmeier and Schreiber, 1985; Schreiber
et al., 1985).
To test whether inhibition of IFN-g could restore normal excit-
ability in PKR-deficient slices, we blocked IFN-g’s function with
NAb-IFN-g. After-discharges induced by a low concentration of
bicuculline in Pkr?/?slices were drastically reduced by bath
application of NAb-IFN-g, but not by a heat-inactivated form of
the same antibody (I-NAb-IFN-g, compare Figure 7D to 7E;
see also Figures 7F and 7G). Accordingly, in WT slices, PKRi
failed to increase the amplitude of population spikes in the
presence of a Nab-IFN-g (compare Figure S3G to S5B) or
the inhibitor of translation anisomycin (compare Figure S3G
to S5C). Taken together, these data indicate that the increased
excitability in PKR-deficient slices is mediated by endogenous
IFN-g. In contrast to WT slices (Figure 7H; see also Figures
7J and 7K), the combination of PKRi and 2 mM bicuculline
failed to generate epileptiform after-discharges in Ifn-g?/?slices
(Figure 7I; see also Figures 7J and 7K), thus demonstrating that
the increased excitability of WT slices induced by PKRi requires
IFN-g. Therefore, two quite independent approaches—genetics
(Ifn-g?/?mice) and immunology (NAb?IFN-g)—showed that the
hyperexcitability of PKR-deficient slices was primarily due to
To determine whether PKR deficiency facilitates L-LTP and
enhances LTM via IFN-g, Ifn-g?/?mice and WT mice were
treated with PKRi. In contrast to its effect in WT slices, in Ifn-g?/?
slices, PKRi failed to promote L-LTP induced by a single high-
frequency train (Figure 7L). Similarly, PKRi enhanced long-term
fear memories in WT mice, but not in Ifn-g?/?mice (Figures
7M, 7N, S5D, and S5E). These data provide evidence that the
facilitation of both L-LTP and LTM in PKRi-treated mice is due,
at least in part, to IFN-g-mediated disinhibition.
Our data reveal that the lack of a double-stranded RNA-
activated protein kinase PKR, originally identified as a sensor
of virus infection, unexpectedly leads to hyperexcitability in
cortical and hippocampal networks. In addition, L-LTP and
behavioral learning are enhanced in Pkr?/?mice or when PKR
activity is pharmacologically blocked. The increased excitability
Thus, PKR regulates network rhythmicity, synaptic plasticity,
and memory storage by potentiating GABAergic synaptic
GABAergic inhibition not only controls the efficacy and
plasticity ofexcitatory synapses,but alsopromotes the synchro-
nized firing of large assemblies of principal cells at certain
preferred frequencies (Mann and Paulsen, 2007). Slow theta
and faster gamma oscillations and ripples appear to be crucially
involved in mnemonic processes (Buzsaki, 2006; Maurer and
McNaughton, 2007). Multiple lines of evidence support the
idea that GABAergic control of synaptic plasticity is a key mech-
Moser, 1998). First, reduced GABAergic-mediated inhibition
facilitates the induction of LTP (Abraham et al., 1986; Davies
et al., 1991; Wigstro ¨m and Gustafsson, 1983). Second, long-
term disinhibition of a subset of CA1 pyramidal neurons corre-
lates with the acquisition of spatial memory (Gusev and Alkon,
2001). Third, modest pharmacological reduction of GABAergic
transmission enhances memory consolidation (Izquierdo and
Medina, 1991; McGaugh and Roozendaal, 2009). Finally,
GABAergic neurons of the medial septum drive theta rhythmicity
in the hippocampal network (Hangya et al., 2009).
Our results provide new insight into the function of PKR in
the adult brain and how the suppression of PKR promotes
network hypersynchrony and enhances L-LTP and cognitive
performance. Wepropose a model in which PKRregulates these
processes by IFN-g-mediated disinhibition. This model is
consistent with thefollowingobservations.First, IFN-g increases
excitability (Figure 7B) and generates paroxysmal discharges in
hippocampal slices by reducing GABA release at inhibitory
synapses (Mu ¨ller et al., 1993). Second, through a pseudoknot
in its 50UTR, IFN-g mRNA locally activates PKR and eIF2a to
inhibit its own translation, but not translation of other mRNAs
(Ben-Asouli et al., 2002; Cohen-Chalamish et al., 2009). Hence,
Ifn-g mRNA translation is enhanced when PKR is genetically or
pharmacologically inhibited or when eIF2a phosphorylation is
reduced (Ben-Asouli et al., 2002). Accordingly, the levels of
IFN-g were enhanced in the hippocampus (Figure 7A; and
possibly also in the amygdala) from Pkr?/?mice. Third, in WT
slices, PKRi increases neuronal excitability in a translation-
dependent manner (Figures S3G and S5C). Fourth, like Pkr?/?
mice, animals with constitutively reduced eIF2a phosphorylation
(eIF2a+/S51Amice) showed facilitated L-LTP and LTM (Costa-
Mattioli et al., 2007), reduced GABAergic synaptic transmission
(Figures S6A and S6B), and EEG synchronous spike discharges
(Figures S6C and S6D), supporting the notion that PKR, via the
phosphorylation of eIF2a, regulates translation of Ifn-g mRNA.
Fifth, chemically mediated increase in eIF2a phosphorylation
blocks L-LTP and LTM in the murine forebrain (Jiang et al.,
2010). Sixth, Ifn-g?/?mice are resistant to both virus- and kainic
acid-induced limbic seizures (Getts et al., 2007). Finally, spatial
memory is enhanced in transgenic mice overexpressing small
amounts of IFN-g (Baron et al., 2008). Thus, our observations
show that PKR and IFN-g—both crucial components of the anti-
viral and inflammatory response (Farrar and Schreiber, 1993;
Garcı ´a et al., 2007) —play an important role in activity-depen-
dent changes in synaptic strength and network rhythmicity in
the adult brain.
In contrast to PKR, which selectively regulates inhibitory
presynaptic terminals and controls the induction of L-LTP and
LTM (but not their maintenance; Figures S7A and S7B), protein
kinase Mzeta (PKMzeta), which targets excitatory synapses by
factor that is both necessary and sufficient for the maintenance
of L-LTP and LTM (Sacktor, 2011). Another well-characterized
kinase complex controlling protein synthesis-dependent L-LTP
and LTM is the mammalian target of rapamycin complex 1
Cell 147, 1384–1396, December 9, 2011 ª2011 Elsevier Inc. 1393
(mTORC1) (Richter and Klann, 2009; Stoica et al., 2011).
However, the nature and the precise synaptic mechanism by
which mTORC1-dependent proteins promote L-LTP and LTM
In conclusion, we report here a mouse model in which the lack
of the eIF2a kinase PKR, through IFN-g-mediated reduction of
GABAergic inhibition, results in network hyperexcitability,
enhanced long-lasting synaptic plasticity, and improved cogni-
In view of the robust and consistent enhancement in learning
and memory produced by loss of PKR or by treatment with
PKRi, it is reasonable to suggest that PKRi (or agents which
riencing age-related memory loss or patients suffering from the
most devastating memory loss associated with Alzheimer’s
disease, in which PKR activity is indeed known to be abnormally
Pkr knockout (Pkr?/?) mice (Abraham et al., 1999) were backcrossed for at
least eight generations to 129SvEv mice. eIF2a+/S51Amice have been previ-
ously described (Costa-Mattioli et al., 2007). Ifn-g?/?mice and WT CF57/Bl6
control mice were obtained from Jackson Laboratories. All experiments
were performed on 8- to 16-week-old males. Mice were kept on a 12 hr
light/dark cycle, and the behavioral experiments were conducted during the
light phase of the cycle. Mice had access to food and water ad libitum, except
during tests. Animal care and experimental procedures were performed with
approval from the animal care committees of Baylor College of Medicine.
Chronic Videoelectroencephalographic Monitoring
Videoelectroencephalographic (VideoEEG) recordings were performed as
previously described (Price et al., 2009). Pkr?/?mice and WT littermates
were anesthetized with Avertin (1.25% tribromoethanol/amyl alcohol solution,
i.p.) at a dose of 0.02 ml/g. Teflon-coated silver wire electrodes (120 mm diam-
subdural space over frontal, central, parietal, and occipital cortices and
2.0 mm deep into the dorsal hippocampus bilaterally. All recordings were
done at least 24 hr after surgery on mice freely moving in the test cage. Digi-
tized videoEEG data were obtained daily for up to 2 weeks during prolonged
and random 2 hr sample recordings (Stellate Systems, Harmonie software
version 5.0b). The EEG data were interpreted by two experienced mouse elec-
trocorticographers blinded to genotype, and interictal spikes (100–300 mV,
20–100 ms duration) and seizure frequency were included for analysis.
All electrophysiology experiments were done by investigators blind to the
genotype. Field recordings were from CA1 in horizontal hippocampal slices
(350 mm), maintained at 28?C–29?C in interface chamber, as previously
described (Costa-Mattioli et al., 2007; Stoica et al., 2011; for additional
information, see Extended Experimental Procedures). Whole-cell recordings
were performed at 28?C–29?C using conventional patch-clamp techniques
and an Axopatch 200B amplifier (Molecular Devices, Union City, CA). CA1
neurons were visually identified by infrared differential interference contrast
video microscopy on the stage of an upright microscope (Axioskope FS2,
Carl Zeiss, Oberkochen, Germany). Patch pipettes (resistances 4–6 MU)
creatine, 2 Mg3-ATP, and 0.2 Na3-GTP; pH was adjusted to 7.2 and osmolarity
to 290 mOsm using a Wescor 5500 vapor pressure osmometer (Wescor,
Logan, UT). Synaptic responses were evoked with a bipolar stimulating
electrode positioned in stratum radiatum. For recording of inhibitory synaptic
currents, gluconate was replaced with KCl. sIPSCs were recorded in the pres-
ence of 2 mM kynurenic acid, and miniature IPSCs (mIPSCs) were recorded in
the presence of kynurenic acid (2 mM) and tetrodotoxin (TTX; 1 mM). Evoked
IPSCs were recorded in the presence or absence of D-AP5 (50 mM), CNQX
(10 mM), and CGP55845 (10 mM). Excitatory postsynaptic currents (EPSCs)
were recorded in the presence of 10 mM bicuculline or 100 mM picrotoxin. All
synaptic events were sampled 10–15 min after washing in PKRi. The electrical
signals were filtered online at 5 kHz and digitized at 10 kHz. Series and input
resistance were measured continually during recording by injecting a ?5 mV
3 25 ms test pulse prior to stimulus. If they varied more than ± 20%, recording
was abandoned and the data were discarded. All drugs were obtained from
Tocris (Ellisville, MO) (unless otherwise indicated).
Contextual and Auditory Fear Conditioning
The investigators were blind to the genotype for all behavioral tests. Fear
conditioning was performed as previously described (Costa-Mattioli et al.,
2007; Stoica et al., 2011). Mice were first handled for 3–5 min for 3 days and
then habituated to the conditioning chamber for 20 min for another 3 days.
On the training day, after 2 min in the conditioning chamber, mice received
a pairing of a tone (2800 Hz, 85 db, 30 s) with a coterminating foot shock
(0.35 mA, 1 s), after which they remained in the chamber for 2 additional min
and were then returned to their home cages. Mice were tested 24 hr after
training for ‘‘freezing’’(immobilitywith the exception of respiration) in response
to the tone (in a chamber to which they had not been conditioned) and to the
training context (training chamber).
During testing for auditory fear conditioning, mice were placed in the
chamber and freezing responses were recorded during the initial 2 min (pre-
CS period) and during the last 3 min when the tone was played (CS period).
Mice were returned to their cages 30 s after the end of the tone. For tests of
contextual fear conditioning, mice were returned to the conditioning chamber
for 5 min. Tests of responses to the training context (chamber A) and to the
tone (chamber B) were done in a counterbalanced manner. Extinction was
studied in a different set of animals, and freezing in response to the condi-
tioned context was assessed for 5 min at 24 hr, 48 hr, 72 hr, and 96 hr after
training and normalized to the amount of freezing obtained at 24 hr. For all
tests, freezing behavior was hand scored at 5 s intervals during the 5 min
period by a rater who was blind to the genotype. The percent of time spent
by the mouse freezing was taken as an index of learning and memory. PKRi
was freshly dissolved in saline and then i.p. injected immediately after fear
conditioning at a dose of 0.1 mg/kg, which is known to block PKR activity in
the hippocampus in vivo (Ingrand et al., 2007; for additional information, see
Extended Experimental Procedures).
Morris Water Maze
In a circular pool (140 cm diameter) of opaque water (kept at 22?C–23?C),
WT and Pkr?/?littermates were trained daily for 8 days using a relatively
weak training protocol, one trial per day (Costa-Mattioli et al., 2007). The laten-
cies of escape from the water onto the hidden (submerged) platform (10 cm of
diameter) were monitored by an automated video tracking system (HVS
Image, Buckingham, UK). For the probe trial, which was performed on day
9, the platform was removed from the pool and the animals were allowed to
search for 60 s. The percentage of time spent in each quadrant of the pool
(quadrant occupancy) was recorded. There was no significant difference in
swimming speed between Pkr?/?mice and WT littermates (16.9 ± 0.87 cm/s
were trained at the same time of day during their light phase.
Immunohistochemistry, Western Blotting, and ELISA
For all immunohistochemical experiments, the investigators were blind to the
genotype. Hippocampal celllysates,western blotting, andimmunohistochem-
istry were performed as previously described (Costa-Mattioli et al., 2007;
Stoica et al., 2011).
All data are presented as means ± SEMs. The statistics were based on
Student’s t test or one-way ANOVA and between group comparisons by
Tukey’s Test, unless otherwise indicated. p < 0.05 was considered significant.
*p < 0.05, **p < 0.01, and ***p < 0.001.
1394 Cell 147, 1384–1396, December 9, 2011 ª2011 Elsevier Inc.
figures, one table, and one movie and can be found with this article online at
We thank G. Buzsa ´ki, H. Zoghbi, and M. Rasband for comments on an early
version of the manuscript. This work was supported by funds to M.C.-M.
(Searle award 09-SSP-211; George and Cynthia Mitchell Foundation, Baylor
IDDRC), J.L.N. (NINDS NS 29709, NICHD HD24064, Baylor IDDRC), and
M.J.F. (NEI EY12782). The authors declare no competing financial interests.
Received: June 7, 2011
Revised: September 6, 2011
Accepted: November 2, 2011
Published: December 8, 2011
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1396 Cell 147, 1384–1396, December 9, 2011 ª2011 Elsevier Inc.
EXTENDED EXPERIMENTAL PROCEDURES
Micewereweanedatthethirdpostnatalweek andgenotypedbyPCR.Briefly,themutantandcorresponding WTalleles aredetected
by a four-primer PCR assay in which Oligo-1 (50-GGAACTTTGGAGCAATGGA-30) and Oligo-2 (50-TGCCAATCAGAAAATCTAAAAC-
30) give a WT band of 225 base-pair fragment and Oligo-3 (50-TGTTCTGTGGCTATCAGGG-30) and Oligo-4 (50-TGAGGAGTTCTTCT
GAGGG-30) give a 432 base-pair fragment from the deleted allele.
As indicated, the following drugs were added to ACSF superfusing slices: anisomycin (Calbiochem, CA), PKRi (Calbiochem, CA),
diazepam (Sigma-Aldrich, St. Louis, MO), mouse IFN-g (R & D systems, Minneapolis, MN), monoclonal Nab-IFN-g (H22; R & D
Systems, Minneapolis, MN) and bicuculline (Tocris, Ellisville, MO). The latter was bicuculline free base which blocks only GABAA
picrotoxin and CGP55845 were all purchased from Tocris Bioscience (Ellisville, MO). PKRi (Calbiochem, San Diego), a potent ATP-
binding-site-directed inhibitor ofPKRactivity(Jammi etal.,2003;Shimazawa andHara,2006)was appliedto slices ata finalconcen-
tration of 1 mM (0.01% DMSO), which is known to block PKR activity ex-vivo (Page et al., 2006; Wang et al., 2007). For the in vivo
studies PKRi was dissolved in saline and then injected intraperitoneally (i.p.) at a dose of 0.1 mg/kg, which is known to block PKR
activity in the hippocampus in vivo (Ingrand et al., 2007). EEG was recorded 1 hr after PKRi injection.
Briefly, horizontal hippocampal slices (350 mm) were cut from brains of WT or age-matched Pkr?/?littermates in 4?C artificial cere-
brospinal fluid (ACSF) and kept in ACSF at room temperature for at least one hr before recording. Slices were maintained in an inter-
face-type chamber perfused with oxygenated ACSF (95% O2and 5% CO2) containing in mM: 124 NaCl, 2.0 KCl, 1.3 MgSO4, 2.5
to stimulate Schaffer collateral and commissural fibers. Field potentials were recorded at 28-29?C using ACSF-filled micropipettes.
The recording electrodes were placed in the stratum radiatum for field excitatory postsynaptic potentials (fEPSPs), and stratum pyr-
fEPSPs, and 50% of maximal response for population spikes. A stable baseline of responses was established for at least 30 min at
0.033 Hz. Tetanic LTP was induced by high-frequency stimulation in brief trains (100 Hz for 1 s), applied either as a single train or four
trains separated by 5 min intervals as previously described (Costa-Mattioli et al., 2007; Stoica et al., 2011). To reduce day-to-day
variations, on a given day we recorded from WT and Pkr?/?slices, or from slices of both groups treated with drugs. Furthermore,
we recorded from WT and Pkr?/?slices in parallel using only one slice per genotype (in the same chamber to ensure uniformity in
experimental conditions across groups). Thus, the n’s refer to both the number of slices and the number of mice. Statistical analyses
were performed with the t test and one-way ANOVA. All data are presented as means ± SEM.
Elevated Plus Maze Test
The elevated plus-maze apparatus consisted of two open arms (35 3 5 cm) and two enclosed arms of the same size (with 15 cm high
opaque walls). The arms and central square were made of plastic plates and were elevated 40 cm above the floor. Mice were placed
in the central square of the maze (5 3 5 cm). Behavior was recorded during a 5 min period. Data acquisition and analysis were per-
formed automatically with ANYMAZE software.
Mice were placed in the center of a 40 3 40 3 40-cm arena, and their activity was quantified over a 10 min period by a computer-
operated optical animal activity system (ANYMAZE).
Immunohistochemistry, Western Blotting, and ELISA
For immunohistochemical experiments mice were deeply anesthetized and perfused intracardially with cold PBS and subsequently
with4%paraformaldehyde (PFA)inicecold0.1Mphosphatase buffer(PBS). Brainswereremovedfromtheskull,storedina4%PFA
solution overnight (at 4?C), and 40 mm horizontal sections were cut on a microtome (Leica VT1000S, Germany). Free-floating method
was used while rinsing between steps. Sections were first placed in a blocking solution (5% BSA, 0.3% Triton and 4% Normal Goat
Biotechnology, CA), GAD67 (Millipore, Billerica, MA), V-Glut 1 (Synaptic Systems, Goettingen, Germany) and PSD95 (NeuroMab,
CA)] and then rinsed four times (for 20 min) with PBS before incubation with the secondary antibody (for 4 hr). After four washes
(each for 20 min) with PBS, the sections were mounted on Superfrost Plus slides (VWR, West Chester, PA). Finally, the sections
were coverslipped with VECTASHIELD? Hard Set mounting medium (Vector Lab, Burlingame, CA). Digital photos were taken
with a Zeiss LSM 510 laser confocal microscope.
Cell 147, 1384–1396, December 9, 2011 ª2011 Elsevier Inc. S1
Egr-1 Staining Download full-text
Prior to contextual fear conditioning, WT and Pkr?/?mice were handled for three consecutive days. They were then trained as
described above. Control groups were exposed to the context, except that they received no shock during training. Ninety minutes
after training, brain sections were cut as described above and pre-treated in 0.3%H2O2in PBS. The sections were incubated with an
triton and 4% normal goat serum in PBS) for 48 hr; and then incubated for 60 min at room temperature with a biotinylated goat-anti
rabbit antibody (1:500; Vector Laboratories, Burlingame, CA) followed by an avidin–biotin–horseradish peroxidase (HRP; ABC kit;
Vector Laboratories, Burlingame, CA). The bound peroxidase was located by incubating sections in 0.1% 3,30-diaminobenzidine
(DAB) and 0.025% H2O2at room temperature for 5–10 min, which generated the visible substrate. Immunoreactive CA1 neurons
were counted within a given area (0.07 mm2), as described earlier (Frankland et al., 2004; Hall et al., 2001).
The hippocampi were dissected out of brain from WT and Pkr?/?mice and homogenized in a buffer containing 320 mM sucrose,
5 mM HEPES, 1 mM NaHCO3, 1 mM EDTA, 1 mM, 1% CHAPS, 0.5% BSA, 20 mg/ml PMSF, 20 mg/ml aprotinin. Homogenates
were centrifuged at 12,000 rpm/min for 15 min at 4?C and the supernatant was recovered. The levels of murine IFN-g production
were determined by ELISA according to the manufacturer’s procedure (R & D systems Minneapolis, MN).
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S2 Cell 147, 1384–1396, December 9, 2011 ª2011 Elsevier Inc.