(A–C) Traces from bilateral cortical electrodes (left hemisphere 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 µV. Abnormal EEG activity was absent from all WT control recordings (n = 7) but present in all Pkr^−/− mice (n = 10) and in six out of seven PKRi-injected mice (recorded 1 hr after of PKRi injection). By Fisher’s exact test, p values were < 0.001 and < 0.01, respectively.
For additional information, see Movie S1. See Figure S1 for lack of change in brain morphology; Figure S6B for EEG interictal spikes in mice with constitutively reduced eIF2α phosphorylation (eIF2α^+/S51A mice); and Figure S7D for the effect of PKRi in vivo.

(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 µM), which generated pronounced after-discharges in Pkr^−/− slices (B) or WT slices treated with PKRi (1 µM) (C), but not in WT slices (A). For all plots, n ≥ 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 paired stimulation.
(I) Sigmoidal relationship of population spikes versus fEPSP, though initially similar, reached a higher ceiling in Pkr^−/− slices.
(J and K) In whole-cell recordings in the presence of glutamate and GABA antagonists, resting membrane potential was −59 ± 1.23 mV for WT and −60 ± 1.22 mV for Pkr^−/− slices (p > 0.05), and input resistance was 397 ± 24 MΩ for WT and 381 ± 39 MΩ 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.

(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 µM]) 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 µM], CNQX [10 µM], and CGP55845 [10 µM]) (*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/IPSC₁ are 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; Figure S6A for the reduced mIPSCs in slices from eIF2α^+/S51A mice; and Figure S7C for the effect of PKRi in vitro.

(A) Whole-cell recordings of EPSCs were performed with gluconate-containing pipettes at −70 mV in the presence of 100 µM 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 similar frequency (Mann-Whitney U test, U = 16.2; p = 0.34) and amplitude (t = 0.34; p = 0.74) of miniature EPSCs (mEPSCs) (recorded in the presence of picrotoxin [100 µM] and TTX [1 µM]) in WT and Pkr^−/− slices.
(C) PKRi (1 µM) 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 µM picrotoxin. Horizontal bar indicates the period of incubation with PKRi.
(A and B) Calibrations: 1 s and 20 pA. (C) Calibrations: 10 ms and 100 pA. For information about the effect of PKRi in vitro, see Figure S7C.

(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 µM) 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 µM) 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.

(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, 14)} = 6.6; *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 maintenance, 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.

(A) IFN-γ was increased in pooled hippocampal extracts from Pkr^−/− mice (Mann-Whitney U test, U = 0.00; **p < 0.01).
(B) IFN-γ (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).
(D and E) A neutralizing antibody against IFN-γ (NAb-IFN-γ; D), but not its heat-inactivated form (E), prevented epileptiform activity enabled by a low concentration of bicuculline (2 µM) in Pkr^−/− slices.
(F and G) NAb-IFN-γ 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 µM) with a low concentration of bicuculline (2 µM) induced prominent after-discharges in WT slices (H), but not in Ifn-γ^−/− slices (I).
(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-γ^−/− slices.
(L) PKRi induced a sustained L-LTP in WT slices, but not in Ifn-γ^−/− slices (F_(1,14) 14.9; p < 0.01).
(M and N) Injection of PKRi (0.2 mg/kg i.p.) immediately after training enhanced long-term contextual fear memory in WT mice (M) (F_(1,16) = 6.9, p < 0.05) but had no effect in Ifn-γ^−/− mice (N) (F_(1,14) = 0.62; p = 0.44).
For further information about IFN-γ 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.