Infantile amnesia reflects a developmental critical period for hippocampal learning

Journal name:
Nature Neuroscience
Volume:
19,
Pages:
1225–1233
Year published:
DOI:
doi:10.1038/nn.4348
Received
Accepted
Published online
Corrected online

Abstract

Episodic memories formed during the first postnatal period are rapidly forgotten, a phenomenon known as 'infantile amnesia'. In spite of this memory loss, early experiences influence adult behavior, raising the question of which mechanisms underlie infantile memories and amnesia. Here we show that in rats an experience learned during the infantile amnesia period is stored as a latent memory trace for a long time; indeed, a later reminder reinstates a robust, context-specific and long-lasting memory. The formation and storage of this latent memory requires the hippocampus, follows a sharp temporal boundary and occurs through mechanisms typical of developmental critical periods, including the expression switch of the NMDA receptor subunits from 2B to 2A, which is dependent on brain-derived neurotrophic factor (BDNF) and metabotropic glutamate receptor 5 (mGluR5). Activating BDNF or mGluR5 after training rescues the infantile amnesia. Thus, early episodic memories are not lost but remain stored long term. These data suggest that the hippocampus undergoes a developmental critical period to become functionally competent.

At a glance

Figures

  1. Latent infantile memories are rapidly forgotten but reinstate later in life with reminders.
    Figure 1: Latent infantile memories are rapidly forgotten but reinstate later in life with reminders.

    Experimental schedule is shown above each panel. Acquisition (Acq) and memory retention are expressed as mean latency ± s.e.m. (ac) Mean latency ± s.e.m. of naive, shock-only rats and rats trained (Tr, training) at PN17 and tested (T): (a) immediately (immediate test, I.T.; n = 5, 8; two-way ANOVA followed by Bonferroni post hoc test, condition F1,22 = 11.53, P = 0.0026; testing F1,22 = 10.71, P = 0.0035; interaction F1,22 = 9.209, P = 0.0061; 3 independent experiments); (b) 30 min; n = 9, 11, 11; two-way ANOVA followed by Bonferroni post hoc test, condition F2,56 = 14.60, P < 0.001; testing F1,56 = 5.48, P = 0.023; interaction F2,56 = 2.73, P = 0.074; 3 independent experiments); (ac) 1 d; and (c) 7 d after training (n = 8, 8, 8; two-way ANOVA followed by Bonferroni post hoc test, condition F2,42 = 2.437, P = 0.0997; testing F1,42 = 0.4311, P = 0.515; interaction F2,42 = 0.9929, P = 0.379; 3 independent experiments). (df) Mean latency ± s.e.m. of naive, shock-only rats and rats trained at PN24 and tested: (d) immediately (n = 7, 10; two-way ANOVA followed by Bonferroni post hoc test, condition F1,30 = 153.6, P < 0.0001; testing F1,30 = 0.7410, P = 0.3962; interaction F1,30 = 0.6629, P = 0.4220; 3 independent experiments); (e) 30 min (n = 9, 5, 11; two-way ANOVA followed by Bonferroni post hoc; condition F2,44 = 55.51, P < 0.001; testing F1,44 = 0.97, P = 0.33; interaction F2,44 = 1.72, P = 0.19; 3 independent experiments); (df) 1 d; and (f) 7 d after training (n = 8, 8, 8; two-way ANOVA followed by Bonferroni post hoc test, condition F2,42 = 183.8, P < 0.0001; testing F1,42 = 0.48, P = 0.489; interaction F2,44 = 0.5949, P = 0.5562; 3 independent experiments). (g) Mean latency ± s.e.m. of naive, shock-only rats and rats trained at PN17 and tested 1 d, 7 d, 10 d and 16 d after training (n = 10, 9, 8; two-way ANOVA followed by Bonferroni post hoc test, condition F2,96 = 5.542, P = 0.0053; testing F3,96 = 0.9441, P = 0.4226; interaction F6,96 = 1.056, P = 0.3945; 3 independent experiments). (h) Mean latency ± s.e.m. of naive, shock-only rats and rats trained at PN17 and tested 1 d and 7 d after training, and after a RS (red arrow) given 2 d thereafter in a different context (n = 11, 10, 12; two-way ANOVA followed by Bonferroni post hoc, condition F2,138 = 43.48, P < 0.0001; testing F4,138 = 21.81, P < 0.0001; interaction F8,138 = 12.27, P < 0.0001; 3 independent experiments). Four days after T4 the rats were tested in a novel context (NC). (i) Mean latency ± s.e.m. of naive, shock-only rats and rats trained at PN17 and given a RS 9 d after training and tested 1 d (T1) and again 6 d later (T2) (n = 8, 5, 6; two-way ANOVA followed by Bonferroni post hoc test, condition F2,32 = 0.8669, P = 0.4299; testing F1,32 = 0.0259, P = 0.8731; interaction F1,32 = 0.1791, P = 0.8368; 3 independent experiments). (j,k) Mean latency ± s.e.m. of naive, shock-only rats and rats trained at PN17 and tested: (j) 7 d (n = 12, 8, 12; two-way ANOVA followed by Bonferroni post hoc test, condition F2,116 = 24.0, P < 0.0001; testing, F3,116 = 6.733, P = 0.0003; interaction F6,116 = 4.137, P = 0.0008; 3 independent experiments) or (k) 4 weeks after training (T1) (n = 6, 6, 9; two-way ANOVA followed by Bonferroni post hoc test, condition F2,71 = 13.18, P < 0.0001; testing F3,71 = 3.98, P < 0.011; interaction F6,71 = 3.54, P = 0.004; 3 independent experiments). A RS was given 2 d later, and the rats were tested 1 d (T2) and again 6 d later (T3). Four days after T3 the rats were tested in a NC. *P < 0.05, **P < 0.01, ***P < 0.001.

  2. The latent infantile memory trace is hippocampus dependent.
    Figure 2: The latent infantile memory trace is hippocampus dependent.

    Experimental schedule is shown above each panel. Memory retention is expressed as mean latency ± s.e.m. (a,b) Mean latency ± s.e.m. of rats injected (↑) in the dorsal hippocampus with vehicle or muscimol 30 min before training (Tr) at (a) PN17 (n = 8, 10; two-way ANOVA followed by Bonferroni post hoc test, treatment F1,48 = 17.74, P = 0.0001; testing F2,48 = 32.02, P < 0.0001; interaction F2,48 = 17.43, P < 0.0001; 3 independent experiments) or (b) PN24 (n = 8, 7; two-way ANOVA followed by Bonferroni post hoc test, treatment F1,39 = 22.57, P < 0.0001; testing F2,39 = 16.65, P < 0.0001; interaction F2,39 = 5.108, P = 0.0107; 3 independent experiments) and tested (T) at the indicated times. At T2, upon entering the shock compartment, rats were trained again (Tr) and tested 1 d later. (c,d) Mean latency ± s.e.m. of rats trained at PN17 and injected (↑) in the dorsal hippocampus with vehicle or muscimol 30 min before (c) T1, given 7 d after training (n = 11, 10; two-way ANOVA followed by Bonferroni post hoc test, treatment F1,57 = 0.0013, P = 0.9719; testing F2,57 = 27.68, P < 0.0001; interaction F2,57 = 0.03027, P = 0.9702; 3 independent experiments), or (d) a reminder shock (RS), given 2 d after T1 (n = 8, 7; two-way ANOVA followed by Bonferroni post hoc test, treatment F1,39 = 0.4162, P < 0.5226; testing F2,39 = 60.59, P < 0.0001; interaction F2,39 = 0.1302, P = 0.8783; 3 independent experiments). Rats were tested again 1 d after RS (T2). *P < 0.05, **P < 0.01, ***P < 0.001.

  3. Training at PN17 increases pTrkB and switches the ratio of GluN2B/GluN2A levels in the dorsal hippocampus.
    Figure 3: Training at PN17 increases pTrkB and switches the ratio of GluN2B/GluN2A levels in the dorsal hippocampus.

    (a) Examples and densitometric western blot analyses of dHC total extracts from naive rats euthanized at PN17, PN24 or PN80 (adult; n = 8, 8, 8). Data are expressed as mean percentage ± s.e.m. of adult naive rats (one-way ANOVA followed by Newman-Keuls multiple comparison test, pTrkB F2,21 = 3.342, P = 0.0549; BDNF F2,21 = 7.125, P = 0.0043; GluN2A F2,21 = 1.524, P = 0.2410; GluN2B F2,21 = 12.41, P = 0.0003; GluN2A/GluN2B ratio F2,21 = 17.68, P < 0.0001; 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001. (b,c) Examples and densitometric western blot analyses of dHC total extracts from rats trained in IA at PN17 or PN24, and euthanized 30 min, 9 h, 24 h, 48 h after training (n = 6–10 rats per group). To account for developmental differences, two groups of naive rats were used: PN17 (n = 8) and PN19 (n = 6) or PN24 (n = 8) and PN26 (n = 8). Data are expressed as mean percentage ± s.e.m. (b) PN17 naive rats (n = 8, 6, 10, 7, 6, 6, one-way ANOVA followed by Dunnett's multiple comparison test, pTrkB F3,27 = 10.29, P = 0.0001; BDNF F3,27 = 1.998, P = 0.1381; GluN2A F3,27 = 8.580, P = 0.0004; GluN2B F3,27 = 2.923, P = 0.0527; GluN2A/GluN2B F3,27 = 3.243, P = 0.0389; 3 independent experiments). (c) PN24 naive rats (n = 8, 6, 6, 7, 7, 8; one-way ANOVA followed by Dunnett's multiple comparison test, pTrkB F3,23 = 4.489, P = 0.0128; BDNF F3,23 = 5.256, P = 0.0066; GluN2A F3,23 = 0.7538, P = 0.5314; GluN2B F3,23 = 0.08686, P = 0.9665; 3 independent experiments). Significance compared to PN17 or PN24 naive rats: *P < 0.05, **P < 0.01, ***P < 0.001; # symbol indicates significance levels comparing PN19 naive to 48 h trained groups (GluN2A, unpaired two-tailed Student's t-test, t = 3.113; d.f. = 10, #P = 0.0110). (d) Ifenprodil (3 μM) depressed the amplitude of NMDA EPSCs recorded at Vm = +40 mV in PN17 naive rats (n = 6 rats, 12 cells) when compared to PN24 animals (n = 10,17; P < 0.05), but not in PN17 trained animals (n = 6, 10) (One-way ANOVA followed by Bonferroni's post hoc test, F2,36 = 3.298, P = 0.0484). Representative sample traces from before (color) and 20 min after ifenprodil (gray) are shown on the right. Error bars, s.e.m. Scale bars, x-axis = 200 ms, y-axis = 10 pA (top), 40 pA (middle), and 25 pA (bottom). (e) Correlation of western blot (percentage of naive adult GluN2A/GluN2B ratio) and electrophysiology data (percentage of control peak). r, Pearson correlation. Full-length blots and gels are presented in Supplementary Figure 9.

  4. BDNF is required for the formation of the latent infantile memory and for the GluN2B/GluN2A switch.
    Figure 4: BDNF is required for the formation of the latent infantile memory and for the GluN2B/GluN2A switch.

    Experimental schedule is shown above each panel. (a,b) Mean latency ± s.e.m. of rats injected (↑) in the dorsal hippocampus with (a) IgG or anti-BDNF (n = 9, 9, two-way ANOVA followed by Bonferroni post hoc test, treatment F1,32 = 9.021, P = 0.0051; testing F1,32 = 21.72, P < 0.0001; interaction F1,32 = 8.234, P = 0.0072; 3 independent experiments) or (b) IgG or TrkB-Fc (n = 6, 6; two-way ANOVA followed by Bonferroni post hoc test, treatment F1,20 = 25.48, P < 0.0001; testing F1,20 = 59.34, P < 0.0001; interaction F1,20 = 33.81, P < 0.0001; 2 independent experiments) 30 min before training (Tr) at PN17. Rats were tested 7 d after training (T1), received a reminder shock (RS) 2 d later, and were tested again 1 d after that (T2). At T2, upon entering the shock compartment, rats were trained again (Tr) and tested 1 d later (T3). (c) Representative examples and densitometric western blot analyses of dorsal hippocampal extracts obtained from naive and trained rats given hippocampal injections of IgG or anti-BDNF 30 min before Tr at PN17 and euthanized 24 h after training. Data are expressed as mean percentage ± s.e.m. of naive rats injected with IgG and euthanized at the matched time point (i.e., PN18) (n = 8,8,8; one-way ANOVA followed by Newman-Keuls multiple comparison test, pTrkB F2,15 = 8.858, P = 0.0029; GluN2A F2,21 = 6.864, P = 0.0051; GluN2B F2,21 = 6.731, P = 0.0055; GluN2A/GluN2B F2,21 = 7.632, P = 0.0032, 3 independent experiments). *P < 0.05, **P < 0.01. Full-length blots and gels are presented in Supplementary Figure 10.

  5. GluN2B- and mGluR5-dependent switch of GluN2B/GluN2A is required to form the latent infantile memory.
    Figure 5: GluN2B- and mGluR5-dependent switch of GluN2B/GluN2A is required to form the latent infantile memory.

    Experimental schedule is shown above each panel. (a,b) Mean latency ± s.e.m. of rat injected (↑) in the dorsal hippocampus with vehicle, Ro 25-6981 or PEAQX 30 min before training at (a) PN17 (n = 9, 10, 9, two-way ANOVA followed by Bonferroni post hoc test, treatment F2,75 = 6.639, P = 0.0022; testing F2,75 = 58.35, P < 0.0001; interaction F4,75 = 6.496, P = 0.0022; 3 independent experiments) or (b) PN24 (n = 9, 7, 9, two-way ANOVA followed by Bonferroni post hoc test, treatment F2,66 = 43.55, P < 0.0001; testing F2,66 = 32.29, P < 0.0001; interaction F4,66 = 12.81, P < 0.0001; 3 independent experiments). At T2, upon entering the shock compartment, rats were trained again (Tr) and tested 1 d later (T3). (c,d) Representative examples and densitometric western blot analyses of dorsal hippocampal total extracts obtained from (c) naive rats euthanized at PN17, PN24 or PN80 (adult; n = 8 rats per group, one-way ANOVA followed by Newman-Keuls multiple comparison test F2,21 = 33.89, P < 0.0001; 3 independent experiments); (d) naive and trained rats injected in the dorsal hippocampus with either vehicle or MTEP 30 min before Tr at PN17 and euthanized 24 h after training (n = 5, 4, 4, one-way ANOVA followed by Newman-Keuls multiple comparison test, GluN2A F2,12 = 18.64, P = 0.0004; GluN2B F2,12 = 6.314, P = 0.0169; GluN2A/GluN2B F2,12 = 4.481, P = 0.0408; 2 independent experiments). Data are expressed as mean percentage ± s.e.m. of (c) adult naive rats or (d) naive rats injected with vehicle and euthanized at the matched time point (i.e., PN18). (e,f) Mean latency ± s.e.m. of rats injected (↑) in the dorsal hippocampus with either vehicle or MTEP 30 min before training at (e) PN17 (n = 6, 6, two-way ANOVA followed by Bonferroni post hoc test, treatment F1,30 = 41.18, P < 0.0001; testing F2,30 = 134.9, P < 0.0001; interaction F2,30 = 25.02 P < 0.0001; 2 independent experiment) or (f) PN24 (n = 7, 8, two-way ANOVA followed by Bonferroni post hoc test, treatment F1,39 = 0.003504, P < 0.9531; testing F2,39 = 19.68, P < 0.0001; interaction F2,39 = 0.05990, P = 0.9419; 3 independent experiments). At T2, upon entering the shock compartment, rats were trained again (Tr) and tested 1 d later (T3). (g) Representative examples and densitometric western blot analyses of dorsal hippocampal total extracts obtained from naive and trained rats given hippocampal injections of vehicle or MTEP 30 min before Tr at PN17 and euthanized 24 h later (n = 7, 6, 6, One-way ANOVA followed by Newman-Keuls multiple comparison test, pTrkB F2,18 = 9.162, P = 0.0022; 3 independent experiments). Data are expressed as mean percentage ± s.e.m. of naive rats injected with vehicle and euthanized at the matched time point (i.e., PN18). *P < 0.05, **P < 0.01, ***P < 0.001. Full-length blots and gels are presented in Supplementary Figure 10.

  6. BDNF closes the infantile amnesia period.
    Figure 6: BDNF closes the infantile amnesia period.

    Experimental schedule is shown above each panel. Acquisition (Acq) and memory retention are expressed as mean latency ± s.e.m. (a) Mean latency ± s.e.m. of rats that received hippocampal injections (↑) of vehicle or BDNF immediately after IA training at PN17. Memory retention was tested 1 d (T1) and 7 d (T2) after training and, 4 d after T2, in a new context (NC) (n = 7, 7, two-way ANOVA followed by Bonferroni post hoc test, treatment F1,36 = 18.49, P < 0.0001; testing F2,36 = 22.57, P < 0.0001; interaction F2,36 = 14.36, P < 0.0001; 3 independent experiments). (b,c) Representative examples and densitometric western blot analyses of dorsal hippocampal total extracts obtained from naive or trained rats given hippocampal injections of either vehicle or BDNF immediately after IA training (Tr) at PN17 and euthanized 2 h later (n = 6, 6, 6; one-way ANOVA followed by Newman-Keuls multiple comparison test, pTrkB F2,17 = 10.35, P = 0.0015; GluN2A F2,17 = 14.66, P = 0.0003; GluN2B F2,17 = 19.17, P < 0.0001; GluN2A/GluN2B F2,17 = 11.82 P = 0.0008; 3 independent experiments). Data are expressed as mean percentage ± s.e.m. of naive rats injected with vehicle and euthanized at the matched time point. (d) Mean latency ± s.e.m. (expressed in seconds, s) of rats that received a bilateral hippocampal injection (↑) of either vehicle or DHPG immediately after IA training at PN17. Memory retention was tested at 1d (T1) and 7 d (T2) after training and, 4d after T2, in a new context (NC) (n = 8, 8, two-way ANOVA followed by Bonferroni post hoc test, treatment F1,42 = 157.2, P < 0.0001; testing F1,42 = 61.70, P < 0.0001; interaction F2,42 = 57.4, P < 0.0001). *P < 0.05, **P < 0.01, ***P < 0.001. Full-length blots/gels are presented in Supplementary Figure 10.

  7. Short- and long-term memory retentions of adult rats.
    Supplementary Fig. 1: Short- and long-term memory retentions of adult rats.

    Experimental schedule is shown above each panel. Acquisition (Acq.) and memory retention are expressed as mean latency ± s.e.m. (in seconds, s). (a) Mean latency ± s.e.m. of naïve and rats trained at PN80 (adults) and tested: (a) 30min (T1) and 1d after training (T2) [n= 11, 10, Two–way ANOVA followed by Bonferroni post hoc, Treatment F(1,38)= 98.74, P< 0.0001. Testing F(1,38)= 3.718, P=0.0613, Interaction F(1,38)=3.894, P=0.0558; 3 independent experiments], and (b) 1d (T1) and 7d after training (T2) [n=5, 6, Two–way ANOVA followed by Bonferroni post hoc, Treatment F(1,18)= 121.1, P< 0.0001,Testing F(1,18)= 0.2594, P= 0.6167, Interaction F(1,18)= 0.2921, P= 0.5955; 2 independent experiments]. ***P < 0.001. Latency score in Supplementary Table 10.

  8. Nociception of naive PN17 and PN24 rats.
    Supplementary Fig. 2: Nociception of naive PN17 and PN24 rats.

    Escape latency is expressed as mean ± s.e.m. (in seconds, s). Pain threshold in infant rats was measured by the escape latency to withdrawal from a 53 ± 1°C hotplate. The escape latency was averaged from four sessions, with an inter-session interval of 15-min. Student’s t-test revealed no significant difference between PN17 (10.1±0.8 seconds, n=6) and PN24 rats (8.2±0.9 seconds, n=6) (two-tailed t test, t =1.562, df=10, P=0.7028).

  9. Effect of training or shock-only on rat weight gain.
    Supplementary Fig. 3: Effect of training or shock-only on rat weight gain.

    Gain weight compared to the weight taken immediately before training is expressed as mean ± s.e.m. (in grams, g). Training and shock-only at (a) PN17 [n= 8, 8, 8, Two–way ANOVA followed by Bonferroni post hoc, Treatment F(2,42)= 1.332, P>0.05, Testing F(1,42)= 2515, P<0.0001, Interaction F(1,42)= 1.656, P>0.05; 3 independent experiments] or (b) PN24 [n= 8, 8, 8, Two–way ANOVA followed by Bonferroni post hoc, Treatment F(2,42)= 0.4406, P>0.05, Testing F(1,42)= 801.7, P<0.0001, Interaction F(1,42)= 0.3349, P>0.05; 3 independent experiments] did not alter the average gain weight measured 1 d and 7 d later. Numeric values in Supplementary Table 11.

  10. The unpaired context-shock protocol failed to reinstate memory.
    Supplementary Fig. 4: The unpaired context–shock protocol failed to reinstate memory.

    Experimental schedule is shown above the panel. Acquisition (Acq.) and memory retention are expressed as mean latency ± s.e.m. (in seconds, s). In the unpaired protocol, rats were exposed to the IA context similarly to the trained rats but did not receive a footshock in the dark chamber. They were returned to their home cage and, one hour later, were placed directly onto the grid floor of the dark chamber and immediately shocked (1.0 mA). The unpaired protocol failed to reinstate memory (n=8, 8, Two–way ANOVA followed by Bonferroni post hoc, Condition F(1.56,)=1.901, P=0.1735, Testing F(3,56)=4.720, P=0.0053, Interaction F(3,56)=1.245, P=0.3021; 3 independent experiments). Latency score in Supplementary Table 12.

  11. Memory reinstatement following different time intervals between test and reminder shock.
    Supplementary Fig. 5: Memory reinstatement following different time intervals between test and reminder shock.

    Experimental schedule is shown above the panel. Acquisition (Acq.) and memory retention are expressed as mean latency ± s.e.m. (in seconds, s). Mean latency ± s.e.m. of naïve, shock-only and rats trained at PN17, tested 7d later (T1) and given a reminder shock (RS) 4h, 1d or 7d after the test (T1). Memory retention was tested 1d (T2) and 7d (T3) after RS, and, 4d later, in a new context (NC) [n= 11, 13, 11, 11, 11, Two–way ANOVA followed by Bonferroni post hoc, Treatment F(4,208)=18.27, P<0.0001, Testing F(3,208)=26.93, P<0.0001, Interaction F(12,208)=4.269, P<0.0001; 3 independent experiments]. Latency score in Supplementary Table 13.

  12. Hippocampal molecular changes either in untrained (naive) conditions or following IA training at PN17 or PN24.
    Supplementary Fig. 6: Hippocampal molecular changes either in untrained (naïve) conditions or following IA training at PN17 or PN24.

    (a) Representative examples and densitometric western blot analyses of dorsal hippocampal total extracts from naïve rats euthanized at PN17, PN24 or PN80 (adult) (n=8, 8, 8). Data are expressed as mean percentage ± s.e.m. of adult naïve rats [One–way ANOVA followed by Newman-Keuls Multiple Comparison Test, TrkB F(2,21)= 0.6080, P=0.5537; GluN1 F(2,21)= 0.1954, P=0.8240, 3 independent experiments]. (b-c) Representative examples and densitometric western blot analyses of dHC total extracts from rats trained in IA at (b) PN17 or (c) PN24, and euthanized 30min, 9h, 24h or 48h after training (n=6-10/group). To account for developmental differences, two groups of naïve were used [(b) PN17 and PN19 or (c) PN24 and PN26]. Data are expressed as mean percentage ± s.e.m. of (b) PN17 [n= 8, 6, 10, 7, 6, 6, One–way ANOVA followed by Dunnett's Multiple Comparison Test, TrkB F(3,27)= 0.1618, P= 0.9211; GluN1 F(3,27)= 0.08967, P= 0.9650; 3 independent experiments] or (c) PN24 naïve rats [n=8, 6, 6, 7, 7, 8, One–way ANOVA followed by Dunnett's Multiple Comparison Test, TrkB F(3,23)= 0.06099, P= 0.9798; GluN1 F(3,23)= 0.2067, P= 0.8907; 3 independent experiments]. The numeric values are reported in Supplementary Table 3.

  13. Hippocampal molecular changes after shock- or context-only.
    Supplementary Fig. 7: Hippocampal molecular changes after shock- or context-only.

    Densitometry of western blot analyses of dorsal hippocampal total extracts from rats euthanized 24 hours after (a) receiving a footshock immediately after being placed on a shock grid (shock-only) [n= 7, 7, Unpaired two-tailed Student’s t-test, pTrkB t=0.3263 df=12, P = 0.7498; GluN2A t=0.5967 df=12, P = 0.5618l; GluN2B t=0.1414 df=12, P = 0.8899; 2 independent experiments] or (b) exposed to the IA context without receiving the footshock (context-only) [n= 7, 7, Unpaired two-tailed Student’s t-test, pTrkB t=0.5199 df=12, P = 0.6126; GluN2A t=0.6308 df=12, P = 0.5400; GluN2B t=0.4679 df=12 P = 0.6482; 2 independent experiment]. Data are expressed as mean percentage ± s.e.m. of naive rats euthanized at the matched time point (i.e. PN18). The numeric values are reported in Supplementary Table 14.

  14. Hippocampal molecular changes after memory reinstatement.
    Supplementary Fig. 8: Hippocampal molecular changes after memory reinstatement.

    Experimental schedule is shown above the panel. Densitometric western blot analyses of dorsal hippocampal total extracts obtained from rats trained (Tr) at PN17, 7 days later exposed to the reinstatement protocol [test (T1) and 2d later reminder shock (RS)] and euthanized 30min or 24 hours later. Data are expressed as mean percentage ± s.e.m. of rats trained at PN17, tested (T1) but not exposed to RS and euthanized at the matched time point [n=6/group, One–way ANOVA followed by Newman-Keuls Multiple Comparison Test, pTrkB F(2,17)= 0.27, P=0.77; GluN2A F(2,17)= 0.72, P=0.50; GluN2B F(2,17)= 0.08, P=0.82; 2 independent experiment). The numeric values are reported in Supplementary Table 15.

  15. Full-length pictures of the blots presented in the main figures.
    Supplementary Fig. 9: Full-length pictures of the blots presented in the main figures.
  16. Full-length pictures of the blots presented in the main figures
    Supplementary Fig. 10: Full-length pictures of the blots presented in the main figures

Change history

Corrected online 29 August 2016
In the version of this article initially published, y-axis labels in Figures 3a,b, 4c, 5d and 6c report the “GluN2B/GluN2A ratio”; this should be changed to “GluN2A/GluN2B ratio.” The error has been corrected in the HTML and PDF versions of the article.

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Author information

Affiliations

  1. Center for Neural Science, New York University, New York, New York, USA.

    • Alessio Travaglia,
    • Reto Bisaz &
    • Cristina M Alberini
  2. Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

    • Eric S Sweet &
    • Robert D Blitzer
  3. Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

    • Eric S Sweet
  4. Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

    • Robert D Blitzer

Contributions

C.M.A. led the design and development of the study and the writing of the manuscript; R.D.B, designed the electrophysiology study; A.T., R.B., E.S.S., R.D.B. and C.M.A. designed experiments and analyzed data; A.T. carried out behavioral experiments and the majority of molecular and pharmacological experiments; R.B. carried out behavioral experiments and contributed to molecular and pharmacological experiments; E.S.S. carried out electrophysiology experiments; and A.T., E.S.S., R.D.B. and C.M.A. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Short- and long-term memory retentions of adult rats. (108 KB)

    Experimental schedule is shown above each panel. Acquisition (Acq.) and memory retention are expressed as mean latency ± s.e.m. (in seconds, s). (a) Mean latency ± s.e.m. of naïve and rats trained at PN80 (adults) and tested: (a) 30min (T1) and 1d after training (T2) [n= 11, 10, Two–way ANOVA followed by Bonferroni post hoc, Treatment F(1,38)= 98.74, P< 0.0001. Testing F(1,38)= 3.718, P=0.0613, Interaction F(1,38)=3.894, P=0.0558; 3 independent experiments], and (b) 1d (T1) and 7d after training (T2) [n=5, 6, Two–way ANOVA followed by Bonferroni post hoc, Treatment F(1,18)= 121.1, P< 0.0001,Testing F(1,18)= 0.2594, P= 0.6167, Interaction F(1,18)= 0.2921, P= 0.5955; 2 independent experiments]. ***P < 0.001. Latency score in Supplementary Table 10.

  2. Supplementary Figure 2: Nociception of naive PN17 and PN24 rats. (43 KB)

    Escape latency is expressed as mean ± s.e.m. (in seconds, s). Pain threshold in infant rats was measured by the escape latency to withdrawal from a 53 ± 1°C hotplate. The escape latency was averaged from four sessions, with an inter-session interval of 15-min. Student’s t-test revealed no significant difference between PN17 (10.1±0.8 seconds, n=6) and PN24 rats (8.2±0.9 seconds, n=6) (two-tailed t test, t =1.562, df=10, P=0.7028).

  3. Supplementary Figure 3: Effect of training or shock-only on rat weight gain. (87 KB)

    Gain weight compared to the weight taken immediately before training is expressed as mean ± s.e.m. (in grams, g). Training and shock-only at (a) PN17 [n= 8, 8, 8, Two–way ANOVA followed by Bonferroni post hoc, Treatment F(2,42)= 1.332, P>0.05, Testing F(1,42)= 2515, P<0.0001, Interaction F(1,42)= 1.656, P>0.05; 3 independent experiments] or (b) PN24 [n= 8, 8, 8, Two–way ANOVA followed by Bonferroni post hoc, Treatment F(2,42)= 0.4406, P>0.05, Testing F(1,42)= 801.7, P<0.0001, Interaction F(1,42)= 0.3349, P>0.05; 3 independent experiments] did not alter the average gain weight measured 1 d and 7 d later. Numeric values in Supplementary Table 11.

  4. Supplementary Figure 4: The unpaired context–shock protocol failed to reinstate memory. (71 KB)

    Experimental schedule is shown above the panel. Acquisition (Acq.) and memory retention are expressed as mean latency ± s.e.m. (in seconds, s). In the unpaired protocol, rats were exposed to the IA context similarly to the trained rats but did not receive a footshock in the dark chamber. They were returned to their home cage and, one hour later, were placed directly onto the grid floor of the dark chamber and immediately shocked (1.0 mA). The unpaired protocol failed to reinstate memory (n=8, 8, Two–way ANOVA followed by Bonferroni post hoc, Condition F(1.56,)=1.901, P=0.1735, Testing F(3,56)=4.720, P=0.0053, Interaction F(3,56)=1.245, P=0.3021; 3 independent experiments). Latency score in Supplementary Table 12.

  5. Supplementary Figure 5: Memory reinstatement following different time intervals between test and reminder shock. (155 KB)

    Experimental schedule is shown above the panel. Acquisition (Acq.) and memory retention are expressed as mean latency ± s.e.m. (in seconds, s). Mean latency ± s.e.m. of naïve, shock-only and rats trained at PN17, tested 7d later (T1) and given a reminder shock (RS) 4h, 1d or 7d after the test (T1). Memory retention was tested 1d (T2) and 7d (T3) after RS, and, 4d later, in a new context (NC) [n= 11, 13, 11, 11, 11, Two–way ANOVA followed by Bonferroni post hoc, Treatment F(4,208)=18.27, P<0.0001, Testing F(3,208)=26.93, P<0.0001, Interaction F(12,208)=4.269, P<0.0001; 3 independent experiments]. Latency score in Supplementary Table 13.

  6. Supplementary Figure 6: Hippocampal molecular changes either in untrained (naïve) conditions or following IA training at PN17 or PN24. (302 KB)

    (a) Representative examples and densitometric western blot analyses of dorsal hippocampal total extracts from naïve rats euthanized at PN17, PN24 or PN80 (adult) (n=8, 8, 8). Data are expressed as mean percentage ± s.e.m. of adult naïve rats [One–way ANOVA followed by Newman-Keuls Multiple Comparison Test, TrkB F(2,21)= 0.6080, P=0.5537; GluN1 F(2,21)= 0.1954, P=0.8240, 3 independent experiments]. (b-c) Representative examples and densitometric western blot analyses of dHC total extracts from rats trained in IA at (b) PN17 or (c) PN24, and euthanized 30min, 9h, 24h or 48h after training (n=6-10/group). To account for developmental differences, two groups of naïve were used [(b) PN17 and PN19 or (c) PN24 and PN26]. Data are expressed as mean percentage ± s.e.m. of (b) PN17 [n= 8, 6, 10, 7, 6, 6, One–way ANOVA followed by Dunnett's Multiple Comparison Test, TrkB F(3,27)= 0.1618, P= 0.9211; GluN1 F(3,27)= 0.08967, P= 0.9650; 3 independent experiments] or (c) PN24 naïve rats [n=8, 6, 6, 7, 7, 8, One–way ANOVA followed by Dunnett's Multiple Comparison Test, TrkB F(3,23)= 0.06099, P= 0.9798; GluN1 F(3,23)= 0.2067, P= 0.8907; 3 independent experiments]. The numeric values are reported in Supplementary Table 3.

  7. Supplementary Figure 7: Hippocampal molecular changes after shock- or context-only. (210 KB)

    Densitometry of western blot analyses of dorsal hippocampal total extracts from rats euthanized 24 hours after (a) receiving a footshock immediately after being placed on a shock grid (shock-only) [n= 7, 7, Unpaired two-tailed Student’s t-test, pTrkB t=0.3263 df=12, P = 0.7498; GluN2A t=0.5967 df=12, P = 0.5618l; GluN2B t=0.1414 df=12, P = 0.8899; 2 independent experiments] or (b) exposed to the IA context without receiving the footshock (context-only) [n= 7, 7, Unpaired two-tailed Student’s t-test, pTrkB t=0.5199 df=12, P = 0.6126; GluN2A t=0.6308 df=12, P = 0.5400; GluN2B t=0.4679 df=12 P = 0.6482; 2 independent experiment]. Data are expressed as mean percentage ± s.e.m. of naive rats euthanized at the matched time point (i.e. PN18). The numeric values are reported in Supplementary Table 14.

  8. Supplementary Figure 8: Hippocampal molecular changes after memory reinstatement. (193 KB)

    Experimental schedule is shown above the panel. Densitometric western blot analyses of dorsal hippocampal total extracts obtained from rats trained (Tr) at PN17, 7 days later exposed to the reinstatement protocol [test (T1) and 2d later reminder shock (RS)] and euthanized 30min or 24 hours later. Data are expressed as mean percentage ± s.e.m. of rats trained at PN17, tested (T1) but not exposed to RS and euthanized at the matched time point [n=6/group, One–way ANOVA followed by Newman-Keuls Multiple Comparison Test, pTrkB F(2,17)= 0.27, P=0.77; GluN2A F(2,17)= 0.72, P=0.50; GluN2B F(2,17)= 0.08, P=0.82; 2 independent experiment). The numeric values are reported in Supplementary Table 15.

  9. Supplementary Figure 9: Full-length pictures of the blots presented in the main figures. (384 KB)
  10. Supplementary Figure 10: Full-length pictures of the blots presented in the main figures (254 KB)

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