SUPPLEMENTARY INFORMATION
Legends to supplementary figures.
Supplementary Figure 1. Spines length is similar in both juvenile HRM and wild-type
mice.
Cumulative frequency plots of spine length measured on oblique dendrites of layer V/VI
PrPFC pyramidal neurons in wild-type (n=11 cells) and HRM (n=15 cells). Error bars
represent SEM.
Supplementary Figure 2. Effect of rapamycin on the restorative properties of ketamine
on synaptic AMPAR contents.
(a) Cumulative histograms of the distribution of AMPAR-spEPSCs amplitudes measured in
juvenile HRM injected with saline (black triangles), ketamine 30 or 100 mg/kg (keta, gray
triangles) or rapamycin prior ketamine (rapa+keta, open gray triangles). Ketamine caused a
dramatic shift of AMPAR-spEPSCs towards larger amplitudes compared to HRM saline (p =
0.008, Kolmogorov-Smirnov test). This effect was prevented by pretratment with rapamycin
(p = 0.965, Kolmogorov-Smirnov test, rapa+keta compared to HRM saline).
(b) Cumulative histograms of the distribution of AMPAR-spEPSCs inter-event intervals
measured in the same groups as in panel a. Inter-events were significantly modified in
ketamine-injected HRM compared to HRM saline (p = 0.039, Kolmogorov-Smirnov test) and
this effect was prevented by rapamycin pretreatment (p = 0.999, Kolmogorov-Smirnov test
compared to HRM saline). Data are ensemble average ± SEM of cumulative distributions.
Supplementary Figure 3. Ketamine injection and pretreatment with rapamycin in HRM
had no effect on NMDAR-spEPSCs.
Cumulative histograms of the distribution of NMDAR-spEPSCs amplitudes (a) or inter-event
intervals (b) measured in juvenile HRM injected with saline (black triangles), ketamine 30 or
100 mg/kg (keta, gray triangles) or rapamycin prior ketamine (rapa+keta, open gray
triangles). (a) Amplitudes were not different in ketamine-injected HRM (p = 0.998,
Kolmogorov-Smirnov test) or in rapa+keta-injected HRM (p = 0.220, Kolmogorov-Smirnov
test) compared to saline HRM. (b) The distribution of inter-event intervals was similar in all
animal groups (Kolmogorov-Smirnov test: p = 0.125 saline versus keta and p = 0.064 saline
versus rapa+keta). Spontaneous NMDAR-EPSCs were recorded at +40 mV in the presence
of DNQX (20µM).
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Supplementary Figure 4. In juvenile wild-type mice, theta-burst LTP is neither affected
by ketamine or rapamycin treatment.
(a) Grouped time courses of fEPSP responses expressed as percentage of baseline before
and after theta-burst (indicated by arrow) in wild-type mice treated either with saline (saline),
ketamine (keta; 100mg/kg) or rapamycin (rapa; 3mg/kg).
(b) The percentage of potentiation measured between 20 and 30 min after TBS was 29.3 ±
8.1% in saline (n=8), 28.5 ± 3.6% in ketamine (n=8) and 26.8 ± 4.9% in rapamycin (n=6).
Error bars represent SEM and n is the number of animals.
Supplementary Figure 5. Fear conditioning and extinction in HRM injected with either
saline, ketamine or rapamycin prior ketamine.
(a) During fear conditioning, HRM injected with either saline (black squares, n=9), ketamine
(gray squares, n = 9) or rapamycin prior ketamine (rapa+keta, open gray squares, n = 7)
displayed similar freezing levels. All groups showed a significant increase in the percentage
of freezing time after each paired CS-US presentation compared to their respective baseline
(BL; *** P <0.001).
(b) During extinction, all groups showed a significant decrease of freezing levels between the
first 2 and the last 2 trials of extinction sessions (*** P<0.001).
Supplementary Figure 6. Fear conditioning and extinction in Ro25-6981-injected HRM
compared to saline treated littermates.
(a) Similar freezing levels were measured during fear conditioning in Ro25-6981-injected
HRM (n = 8) and saline-injected HRM (n = 9). Freezing progressively increases in both
groups after each pair of tone-shock presentation. *** P < 0.001, significantly different when
compared to their respective baseline (BL).
(b) Freezing behavior evoked by the first (CS 1-2) and last pair (CS 23-24) of CS
presentation during extinction. Both groups of HRM pretreated with either Ro25-6981 (n = 8)
or saline (n = 9) show significant auditory fear extinction in CS 23-24 when compared to the
initial 2 trials of extinction session (*** P < 0.001).
Supplementary Figure 7. Fear conditioning and extinction in adolescent HRM injected
with ketamine at juvenile age.
(a) Freezing behavior of HRM (n=8) and ketamine-injected HRM (n=7) during fear
conditioning. Freezing progressively increases in both groups after each pair of tone-shock
presentation. *** P < 0.001, significantly different when compared to their respective baseline
(BL). The freezing percentage in CS2 is higher in ketamine-injected HRM (# P < 0.05).
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(b) During extinction both groups showed a significant decrease of freezing levels between
the first 2 and the last 2 trials of extinction sessions (*** P<0.001).
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Statistical analysis.
Figure 1.
(a) Conditioning: Two-way repeated-measure ANOVA (genotype x time) revealed a
significant main effect of time (F(5,75) = 82.54, P <0.0001), a non significant effect of genotype
(F(1,15) = 2.51, P> 0.05) and a non significant interaction between factors (F(5,75) = 0.54, P
>0.05). *** P <0.001, significantly different from BL (post-hoc pairwise comparisons with
Bonferroni correction).
(b) Extinction:Two-way repeated-measure ANOVA (genotype x time) showed a significant
main effect of time (F(1,15) = 42.94, P <0.0001), a non-significant effect of genotype (F(1,15) =
0.1, P >0.05) and a non significant interaction between factors (F(1,15) = 0.01, P >0.05). *** P
<0.001 significantly different from the initial 2 trials of the first extinction session (post-hoc
pairwise comparisons with Bonferroni correction).
(c) Renewal: Two-way repeated-measure ANOVA (genotype x time) revealed a significant
main effect of genotype (F(1,15) = 8.80, P <0.01) and time (F(5,75) = 3.28, P <0.01) and a
significant interaction between factors (F(5,75) = 3.08, P <0.05), followed by Sidak’s multiple
comparisons test for each CS presentation (*** P <0.001 and ** P <0.01, wild-type versus
HRM).
(d) Mann-Whitney t-test revealed significant higher freezing levels in no extinction HRM
compared to extinction HRM both in the conditioning context (*** P <0.001) and during
exposition to the CS (*** P <0.001).
Figure 4.
(a) Conditioning: Two-way repeated-measure ANOVA (group x time) revealed a significant
main effect of time (F(5,130) = 100.4, P < 0.0001) and a non-significant effect of group (F(2,26) =
0.08, P >0.05) and a non significant interaction between factors (F(10,130) = 1.58, P >0.05), ).
*** P <0.001, significantly different from BL (post-hoc pairwise comparisons with Bonferroni
correction).
(b) Extinction: Two-way repeated-measure ANOVA (group x time) showed a significant
main effect of time (F(1,26) = 87.0, P <0.0001) and a non significant effect of group (F(2,26) =
1.57, P> 0.05) and a non significant interaction between factors (F(2,26) =0.44, P >0.05). *** P
<0.001 significantly different from the initial 2 trials of the first extinction session (post-hoc
Sidak’s multiple comparison test).
(c) Renewal: Two-way repeated-measure ANOVA (group x time) revealed a significant main
effect of group (F(2,26) = 3.56, P < 0.05) and time (F(5,130) = 3.52, P < 0.01) and a non
significant interaction between factors (F(10,130) = 0.69, P >0.05). Post-hoc Bonferroni t-test
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revealed
##
P <0.01 HRM saline versus wild-type saline and * P <0.05 HRM saline versus
HRM Keta.
(d) One-way ANOVA comparing the percentage of freezing behavior between groups during
the first two CS presentations revealed a significant effect of treatment (F(2,22) = 5.45, P<
0.01). Post-Hoc Newman-Keuls analysis revealed significant differences in HRM saline
versus HRM Keta (** P <0.01) and HRM Keta versus HRM Rapa+Keta (* P <0.05).
Figure 5.
(d) Renewal: Two-way repeated-measure ANOVA (treatment x time) revealed a significant
main effect of treatment (F(1,15) = 5.36, P <0.05) and a non significant effect of time (F(5,75) =
1.86, P >0.05) and a non significant interaction between factors (F(5,75) = 0.82, P >0.05),
followed by Sidak’s multiple comparisons test for each CS presentation (* P < 0.05 HRM
saline versus HRM Ro 25-6981).
Figure 6.
(a) Conditioning: Two-way repeated-measure ANOVA (genotype x time) revealed a
significant main effect of time (F(5,70) = 80.90, P <0.0001) and a non-significant effect of
genotype (F(1,14) = 1.11, P >0.05) and a non-significant interaction between factors (F(50,70) =
0.82, P >0.05). *** P <0.001, significantly different from BL (post-hoc pairwise comparisons
with Bonferroni correction).
(b) Extinction: Two-way repeated-measure ANOVA (genotype x time) revealed a significant
main effect of time (F(1,14) = 107.0, P <0.0001) and a non-significant effect of genotype (F(1,14)
= 2.86, P >0.05) and a non-significant interaction between factors (F(2,14) = 0.82, P >0.05). #
P <0.05 versus HRM saline (Tukey post-hoc test). *** P<0.001 significantly different from the
initial 2 trials of the first extinction session (post-hoc pairwise comparisons with Bonferroni
correction).
(c) Renewal: Two-way repeated-measure ANOVA (group x time) showed a significant main
effect of group (F(2,20) = 4.44, P <0.01) and time (F(5,100) = 2.32, P <0.05) and a non-significant
interaction between factors (F(10,100) = 0.46, P >0.05). # P <0.05,
##
P <0.01 HRM versus wild-
type and * P <0.05, ** P <0.01 HRM versus HRM keta (Tukey post-hoc test).
(d) Mann-Whitney t-test revealed significant higher freezing levels in no extinction HRM
compared to extinction HRM both in the conditioning context (*** P <0.001) and during
exposition to the CS (*** P <0.001).
Supplementary Figure 5.
(a) Conditioning: Two-way repeated-measure ANOVA (treatment x time) showed a
significant main effect of time (F(5,110) = 107.5, P <0.0001) and non significant effect of
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treatment (F(2,22) = 0.25, P >0.05) or interaction between factors (F(10,110) = 0.98, P >0.05).
***P <0.001, significantly different from baseline (BL, pairwise comparisons with Bonferroni
correction).
(b) Extinction: Two-way repeated-measure ANOVA (treatment x time) showed a significant
main effect of time (F(1,22) = 129.3, P <0.0001) and a non significant effect of treatment (F(2,22)
= 0.69, P >0.05) and a non significant interaction between factors (F(2,22) = 0.58, P >0.05). ***
P <0.001, significantly different from the initial 2 trials of the first extinction session (post-hoc
pairwise comparisons with Bonferroni correction).
Supplementary Figure 6.
(a) Conditioning: Two-way repeated-measure ANOVA (treatment x time) revealed a
significant main effect of time (F(5,75) = 76.36, P <0.0001), a non significant effect of treatment
(F(1,15) = 0.45, P >0.05) or interaction between factors (F(5,75) = 0.81, P >0.05). *** P <0.001,
significantly different from BL (post-hoc pairwise comparisons with Bonferroni correction).
(b) Extinction: CS-evoked freezing during early and late extinction trials. Two-way repeatedmeasure ANOVA (treatment x time) showed a significant main effect of time (F(1,15) = 90.6, P
<0.0001), a non significant effect of treatment (F(1,15) = 0.89, P >0.05) and a non significant
interaction between factors (F(1,15) = 0.32, P >0.05). *** P <0.001, ** P <0.01 significantly
different from the initial 2 trials of the first extinction session (post-hoc pairwise comparisons
with Bonferroni correction).
Supplementary Figure 7.
(a) Conditioning: Two-way repeated-measure ANOVA (treatment x time) revealed a
significant main effect of time (F(5,65) = 67.9, P <0.0001) and a non-significant effect of
treatment (F(1,13) = 4.4, P >0.05) and a non-significant interaction between factors (F(5,65) =
1.53, P >0.05). *** P <0.001, significantly different from BL (post-hoc pairwise comparisons
with Bonferroni correction). Ketamine-injected HRM showed a higher level of freezing in CS2
(# P < 0.05, post-hoc pairwise comparisons with Bonferroni correction).
(b) Extinction: Two-way repeated-measure ANOVA (treatment x time) revealed a significant
main effect of time (F(1,12) = 81.6, P <0.0001) and a non-significant effect of treatment (F(2,12) =
2.0, P >0.05) and a non-significant interaction between factors (F(2,12) = 0.06, P >0.05). *** P
<0.001 significantly different from the initial 2 trials of the first extinction session (post-hoc
pairwise comparisons with Bonferroni correction).
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