A. Western blot quantification of total mTOR protein levels in rat brain demonstrates a significant downregulation at P3–P11 in the hippocampus (32.0±6.1% of adult, n = 5, p<0.01 at P3, 44.7±2.8 of adult, n = 4, p<0.05 at P6, 69.0±6.1% of adult, n = 5, p<0.05 at P10/11) and P3–P16 in the neocortex (28.0±5.4% of adult, n = 4, p<0.001 at P3, 36.4±2.1 of adult, n = 4, p<0.001, 54.0±5.2% of adult, n = 5, p<0.001 at P10/11, 70.0±7.8% of adult, n = 4, p<0.05 at P16), compared to adulthood (100%). B. Phospho-mTOR (Ser2448)/mTOR ratios demonstrate a modest peak at P3 in the hippocampus (1.76±0.14 fold, n = 5, p>0.05), and a significant upregulation at P3–P10/11 in the neocortex (28.8±4.5 fold increase, n = 4, p<0.001 at P3, 21.0±2.5 fold increase, n = 4, p<0.001 at P6, 17.7±1.7 fold increase, n = 5, p<0.001 at P10/11), as compared to adult standard (1). C. Phospho-p70S6K (Thr389)/p70S6K ratios demonstrate a significant upregulation at P6–P11 in the hippocampus (54.2±15.3 fold increase, n = 4, p<0.05 at P6, 43.8±12.3 fold increase, n = 5, p<0.05 at P10/11) and at P3–P11 in the neocortex (98.5±12.6 fold, n = 5, p<0.001 at P3, 130.3±12.6 fold, n = 4, p<0.001 at P6, 70.7±8.4 fold, n = 7, p<0.001 at P10/11) as compared to adult standard (1). D. Phospho-S6 (Ser235/236)/S6 ratios are significantly increased at P6–P16 in the hippocampus (2.9±0.2 fold increase, n = 5, p<0.001 at P6, 4.9±0.9 fold increase, n = 5, p<0.001 at P10/11, 3.0±0.3 fold increase, n = 4, p<0.01 at P16), and at P10–P21 in the neocortex (3.1±0.4 fold, n = 5, p<0.05 at P10/11, 8.5±1.7 fold, n = 5, p<0.01 at P16, 4.9±0.3 fold, n = 4, p<0.001 at P21), relative to adult standard (1). Mean values for each age group are expressed relative to the mean adult levels, represented by the dotted line. *: p<0.05. Error bars indicate S.E.M. Insets are the representative western blots for individual proteins.

A. Phospho-4E-BP1 levels significantly increase in both hippocampus (1.47±0.13 fold, n = 12, p<0.001) and neocortex (1.55±0.19 fold, n = 11, p<0.05) at 12 h after HS induction in P10 rat pups. B. Phospho-p70S6K is simultaneously significantly upregulated in both hippocampus (1.34±0.14 fold, n = 7, p<0.05) and neocortex (1.81±0.34 fold, n = 7, p<0.05) 12 h following HS onset. C. Subsequent increases in phospho-S6 are observed in both hippocampus (1.88±0.31 fold, n = 12, p<0.01) and neocortex (1.35±0.08 fold increase, n = 9, p<0.01) at 24 h post-HS. Histograms represent averaged optical density normalized to actin, and expressed relative to the mean control values (represented by the dotted line). No significant difference is observed in the total levels of any of these proteins (p>0.05). Insets are the representative western blots for individual proteins. C: control; HS: hypoxic seizures. *: p<0.05, **: p<0.01. Error bars indicate S.E.M.

A-B. Confocal images of CA1 hippocampal region double labeled with NeuN (red) and phospho-S6 (green) show a moderate increase in phospho-S6 expression in pyramidal neuron cell bodies and a marked phospho-S6 upregulation in the dendrites (arrows) in the post-HS group (B, B1), relative to controls (A, A1). C-D. Confocal microscopy of neocortical layer V demonstrates a similar predominant increase in phospho-S6 (green) expression in the apical (arrows) and basal (arrowheads) dendrites of NeuN (red) positive neurons in the HS group (D, D1), relative to controls (C, C1). Confocal images represent z-stacks composed of multiple plane images collected at 1 µm intervals. Scale bars are 100 µm for A-D. Insets (A1–D1) show corresponding higher magnification of individual cells.

A. Representative EEG traces from HS+R and HS+V rats, with HS+V showing an example of an observed seizure. Scale bar  = 1 sec, 50 µV for both traces. Inset shows an expanded view of the EEG trace in the HS+V group. B. Subdermal wire electrode EEG recordings show that rats that had received vehicle with HS at P10 (HS+V) have significantly more spontaneous seizures at P36–38 (0.89±0.25 seizures/h, n = 15) than control normoxic litter mates (C+V), (0.12±0.07 seizures/h, n = 16, p<0.01). Rapamycin treatment at HS (HS+R) attenuates the HS induced later life epilepsy (0.25±0.10 seizures/h, n = 15, p<0.05). Rapamycin treatment of sham P10–11 littermates (C+R) has no significant effect on the EEG pattern (0.30±0.08 seizures/h, n = 28, p = 0.70), as compared to the C+V. C. Rapamycin also reverses the effects of HS on cumulative ictal EEG activity/h. Rats with prior HS treated with vehicle (HS+V) exhibit significantly greater ictal activity per hour (6.30±3.31sec/h, n = 16 rats) compared to naïve control rats treated with either vehicle (C+V), (0.60±0.37 sec/h, n = 16), or rapamycin (C+R), (1.45±0.58 sec/hr, n = 15). In contrast, rapamycin treatment in HS rats (HS+R) shows significant protection (1.48±0.42sec/h, n = 28, p<0.01). *: p<0.05, **: p<0.01, ***: p<0.001. Error bars indicate S.E.M.

Neonatal seizures induce intense synaptic activity followed by rapid increases in extracellular glutamate and BDNF levels (within 1 h post-HS; Figure 4A). Activation of glutamate receptors, including NMDA receptors (NMDARs) and Ca²⁺-permeable AMPA receptors (CP-AMPARs) [44], followed by opening of L-type voltage-gated Ca²⁺ channels (VGCCs), results in increased Ca²⁺ influx. Enhanced expression and function of BDNF activates the membrane-bound tyrosine kinase B receptors (TRKBRs) and, together with Ca²⁺ influx, leads to the recruitment of the PI3K/PDK1/Akt and the Ras/MEK/ERK signaling pathways (within 1–3 h post-HS; Figure 4B–C). Akt- and ERK-dependent inactivation of Tsc1/Tsc2 complex results in activation of Rheb GTP-ase, and subsequent induction of mTORC1 activity (within 12–24 h post-HS; Figure 3). In turn, the mTORC1 may inhibit 4EBP1 and enhance the activity of p70S6K at 12 h post-HS, leading to enhanced eIF4E and S6 signaling, and increased protein translation at 24 h post-HS. The negative feedback inhibition between p70S6K and PI3K may account for the time lag between p70S6K and S6 phosphorylation changes. Altered TORC1-dependent translation of synaptic plasticity proteins (such as PSD-95, CamKII, Arc, GluR1) [40], [91], among with other important cellular processes regulated by the mTORC1 kinase (autophagy, ribosomal biogenesis and cellular metabolism) [92], may lead to both short-term and long-term neuronal hyperexcitability (Figure 6B), enhancement of synaptic function (Figure 6C-F), epilepsy (Figure 7), and autism-like behaviors (Figure 8). Treatment with the mTORC1 inhibitor rapamycin immediately before and after neonatal seizures reverses both early and late neurological and behavioral consequences (Figures 6–8).

Coronal sections double labeled with NeuN (red) and phospho-S6 (Ser235/236) (green) demonstrate high phospho-S6 expression in hippocampal CA3 region (A1–C1) and neocortical layer V (A2–C2) at P11. Double labeling with GAD-67 (red) in combination with phospho-S6 (green) demonstrates no co-expression in hippocampal (D1–F1) or neocortical (D2–F2) interneurons. GFAP and phospho-S6 double labeling shows no phospho-S6 expression in the hippocampal (G1–I1) and neocortical (G2–I2) astrocytes. Scale bars 50 µm.

A. Quantification of BDNF protein levels at 1, 3, 12, 24, 48 h post-HS shows a bi-phasic upregulation in the hippocampus at 1 h (1.31±0.12 fold, n = 7, p<0.05) and 12 h (1.25±0.08 fold, n = 7, p<0.01), and a single increase at 1 h (1.27±0.06 fold, n = 10, p<0.01) in the neocortex. Of note, only the mature 14 kD BDNF band is quantified because of the relevance of this isoform for synaptic plasticity and epileptogenesis. B. Phospho-Akt (Thr308) is significantly increased at 1 h post-HS in the hippocampus (1.24±0.09 fold, n = 8, p<0.05), while in the cortex levels are upregulated at 3 h (1.3±0.11 fold, n = 7, p<0.05) and downregulated at 24 h after HS (0.7±0.04 fold, n = 7, p<0.01). C. Phospho-ERK1/2 expression is significantly higher at 3 h post-HS in the hippocampus (1.46±0.18 fold, n = 7), but not in the neocortex (1.37±0.22 fold, n = 11, p = 0.11). Averaged optical density normalized to actin are expressed relative to the mean control values (dotted line). No significant difference is observed in the total levels of phospho-Akt (Thr308) or phospho-ERK1/2 (Thr202/Tyr204) (p>0.05). Insets are the representative western blots for individual proteins. C: control; HS: hypoxic seizures. *: p<0.05, **: p<0.01. Error bars indicate S.E.M.

A. Representative western blots of hippocampal and neocortical tissue, collected at 12–24 h post-HS, show that rapamycin treatment almost completely blocks phospho-4EBP1, phospho-p70S6K and phospho-S6 in HS and normoxic control animals, with no effect on total protein levels. The rapamycin-treated HS rats (HS+R) demonstrate a significant reduction of phospho-4EBP1/4EBP1 (0.48±0.07, n = 8, p<0.05 in hippocampus, and 0.52±0.04, n = 8, p<0.01 in neocortex), phospho-p70S6K/p70S6K (0.43±0.04, n = 4, p<0.05 in hippocampus, and 0.19±0.06, n = 8, p<0.001 in neocortex), and phospho-S6/S6 (0.03±0.02, n = 4, p<0.001 in hippocampus, and 0.08±0.02, n = 4, p<0.001 in neocortex), relative to vehicle-treated controls (C+V) (1). Sham control rats treated with rapamycin (C+R) also show a significant decrease in phospho-4EBP1/4EBP1 (0.56±0.06, n = 8, p<0.05 in hippocampus, and 0.53±0.04, n = 8, p<0.01 in neocortex), phospho-p70S6K/p70S6K (0.45±0.05, n = 4, p<0.05 in hippocampus, and 0.16±0.01, n = 8, p<0.001 in neocortex), and phospho-S6/S6 (0.1±0.02, n = 4, p<0.001 in hippocampus, and 0.08±0.03, n = 4, p<0.001 in neocortex), compared to vehicle-treated sham controls (C+V). B. Rapamycin treatment reduces subacute increases in seizure susceptibility induced by kainic acid (KA) administration. When exposed to KA at P13, vehicle-treated HS rats (HS+V) demonstrate a significant increase in the average seizure severity (2.8±0.12, n = 13; modified Racine scale, see methods), compared to vehicle-treated normoxic control littermates (C+V), (2.2±0.11, n = 27, p<0.05). Rapamycin-treated HS rats (HS+R) show a significant decrease in KA seizure severity, relative to the HS+V group (2.13±0.09, n = 15; p<0.01). C. Representative traces of AMPA receptor-mediated spontaneous excitatory post-synaptic currents (sEPSCs), pharmacologically isolated by blocking NMDA and GABA_A receptors, in hippocampal ex vivo slices from control and HS rats treated in vivo with either vehicle (C+V, HS+V) or rapamycin (C+R, HS+R) and removed at 48 h post-HS (P12). D. Normalized cumulative distribution of AMPA receptor sEPSCs, recorded at 48 h post-HS demonstrates a significant increase in amplitude in the vehicle-treated HS (HS+V) animals compared to vehicle (C+V) or rapamycin-treated controls (C+R), and this is significantly attenuated by in vivo rapamycin treatment (HS+R), (p<0.001, Kolmogorov–Smirnov test). E. AMPA receptor sEPSCs amplitude is significantly higher in the vehicle-treated HS group (HS+V), relative to vehicle-treated controls (C+V), (137.62±3.39% of vehicle-treated controls, n = 6; p<0.001), and reduced significantly in the rapamycin-treated HS rat pups (HS+R), (107.93±4.64% of vehicle-treated controls, n = 6; p<0.01). In vivo rapamycin treatment has no effect on AMPA receptor sEPSCs amplitude in the normoxic control rats (C+R), (102.28±5.4% of vehicle-treated controls; n = 6, p>0.05). F. AMPA receptor sEPSCs show a trend of increased frequency in the HS+V group, compared to C+V and C+R groups (164.98±37.98% of vehicle-treated controls, n = 6, p = 0.16), which is attenuated by in vivo rapamycin treatment (122.35±26.5% of vehicle-treated controls, n = 6). In vivo rapamycin treatment has no effect on AMPA receptor sEPSCs frequency in the normoxic control rats (C+R) (108.39±18.6% of vehicle-treated controls, n = 6, p>0.05). *: p<0.05; **: p<0.01. Error bars indicate S.E.M.

A. Schematic of behavioral testing protocol B. Distance traveled and time spent exploring the center zone during a 30-minute open field test does not vary significantly between the treatment groups (n = 16–29, p>0.05) (C+V: vehicle-treated sham control litter mates; C+R: rapamycin-treated sham control litter mates; HS+V: vehicle-treated HS rats; HS+R: rapamycin-treated HS rats). C. The olfactory habituation/dishabituation assay confirms no significant differences in the time spent sniffing non-social odors or social odors, with similar habituation and dishabituation, as the time spent sniffing the first swab of each new odor increases (two-way ANOVA for treatment [F(3,420) = 1.18, p = 0.32], odor [F(14,420) = 22.59, p<0.0001] and interaction [F(42,420) = 0.33, p = 1.00]). D. All groups spent significantly more time interacting with the novel rat than with a novel object in the three-chamber sociability test (two-way ANOVA for treatment [F(3,146) = 0.58, p = 0.63], stimulus [F(1,146) = 255.9, p<0.0001] and interaction [F(3,146) = 1.32, p = 0.27]). E. In contrast, there were significant differences in the preference for social novelty among treatment groups (Bonferroni post-tests). C+V rats had a significant preference for social novelty, spending greater time interacting with the novel rat (90.88±15.93 vs. 32.13±6.09 sec, n = 16, p = 0.0013), while the HS+V rats lacked a preference for social novelty (76.97±14.11 vs. 50.73±9.76 sec, n = 16, p = 0.14). Importantly, rapamycin treatment reversed the deficits in social novelty in HS rats (HS+R), (113.24±10.67 vs. 70.74±8.41 sec, n = 29, p = 0.0017). Rapamycin-treated control rats (C+R) did not significantly shift their preference for the social novelty (82.46±12.99 vs. 69.96±13.52 sec, n = 16, p = 0.49) (two-way ANOVA for treatment [F(3,146) = 3.41, p = 0.02], stimulus [F(1,146) = 17.21, p<0.0001] and interaction [F(3,146) = 1.29, p = 0.28]).