Reduced retention of temporal associations is associated with altered calcium responses during trace fear conditioning acquisition in Cntnap2 KO mice

A. Experimental design: Trace Fear Conditioning includes a conditioning session in which mice have to associate a tone and a shock, separated by a time interval (trace). Independent groups of mice were trained with trace intervals of 5, 20, or 40 seconds. B. Freezing levels (percent) during the third tone of the conditioning session for WT (black) and Cntnap2 KO (purple) groups, conditioned with distinct trace durations (T5: WT n=10; Cntnap2 KO n=11; T20: WT n=12; Cntnap2 KO n=11; T40: WT n=12; Cntnap2 KO n=14). C. Conditioned fear response during the tone test (i.e., ratio of freezing before the tone compared to during the tone and the trace, see methods), 24h post-conditioning with distinct trace durations (Trace X Genotype interaction: F2, 65=11.278; p<0.0001; T40: Genotype: F2, 24=6.944; p=0.0163). D.Freezing levels in the conditioning context (context test) for WT (black) and Cntnap2 KO (purple) groups, 24h post-conditioning. E. Fiber photometry. Left panel: scheme depicting the fiber optic implantation targeting the dorsal CA1 subfield of the hippocampus (dCA1). Right panel: Representative image of the optic fiber placement, in the pyramidal cell layer of the dCA1, scale: 100µm. Calcium activity was recorded using the calcium indicator GCaMP6f. F. Freezing levels (percent) during the third tone of the conditioning session for WT (black) and Cntnap2 KO (purple) groups, conditioned with distinct trace durations (T20: WT n=8; Cntnap2 KO n=8; T40: WT n=11; Cntnap2 KO n=10). G. Conditioned fear response during the tone test (i.e., ratio of freezing before the tone compared to during the tone and the trace, see methods), 24h post-conditioning with distinct trace durations (T40: Genotype: F1, 19=50.143; p<0.0001). H. Left: Peak amplitude of Ca2+ transients during the 3 tone presentations of the conditioning under a 20s trace-interval (ns). Right: Peak amplitude of Ca2+ transients during the 3 trace intervals of the conditioning under a 20s trace-interval (Genotype x Trace interaction: p=0.0476; WT n=8; KO n=8, trace 1: ns (p=0675)). I. Left: Peak amplitude of Ca2+ transients during the 3 tone presentations of the T40 conditioning session (Genotype x Tone interaction: p=0.0247; Tone: p=0.0053; WT n=11; KO n=10). Right: Peak amplitude of Ca2+ transients during the 3 trace intervals of the T40 conditioning session (Genotype: p=0.0408; WT n=11; KO n=10). J. Optogenetics: Left panel: scheme depicting the fiber optic implantations bilaterally targeting the dorsal CA1 subfield of the hippocampus (dCA1). Right panel: Representative image of the optic fiber placement, in the pyramidal cell layer of the dCA1, scale: 100µm. Pyramidal neurons activity was manipulated only during the trace interval using Channel Rhodopsin (ChR2). K. Freezing levels (percent) during the third tone of the conditioning session for GFP-injected WT (black, n=6), GFP-injected Cntnap2 KO (purple, n=7), and ChR2-injected Cntnap 2 KO (light purple/white, n=11) groups, conditioned with 40s trace duration. G. Conditioned fear response during the tone test (i.e., ratio of freezing before the tone compared to during the tone and the trace, see methods), 24h post-conditioning with distinct trace durations (Groups: F2, 21=15.576; p<0.0001; WT-GFP vs KO-GFP: p=0.0010; KO-GFP vs KO-ChR2: p<0.0001). Data shown at mean ± SEM. *: p<0.05; **: p<0.01; ***: p<0.001; * represents genotype effect. # represents genotype x variable interaction.

Cntnap2 KO mice display impaired relational/declarative memory.

A. Experimental design: Relational/Declarative memory task. ITI: Inter-trial interval; +: represent reward location within each pair of arms. B. Average correct answers for WT (black; n = 15) and Cntnap2 KO mice (purple; n = 14) for pairs A, B, and C during the 5 first days of acquisition (left panel; day (D); D4: Genotype: p=0.0149), and the last 5 days of acquisition (right panel; D-5: 5 days before they reach learning criterion; D-1: Genotype: p=0.0082). C. Comparison of the correct answers during the last day of training (D-1) averaged on pairs A and B, and during the flexibility test on the recombined pair AB (Genotype X Performance interaction: F1,27=12.639, p=0.0014; Test, Genotype: p=0.0239). Inset: D-1 vs. Test performance index on pairs AB (Genotype: p=0.0022). D. Comparison of the correct answers on pair C during the last day of training (D-1) and the flexibility test. Inset: D-1 vs. Test performance index for Pair C. E. Average performance on the new pair N during the flexibility test. F. Maximum lateralization index during stage 1 (absolute value of the average performance between pairs A and B (left baited) minus Pair C (right baited); Genotype: p=0.0153). G. Average decision latencies for correct (+) and incorrect (−) answers throughout training (Genotype X Response interaction: F1,31=7.960, p=0.0083). H. Average runtimes for correct (+) and incorrect (−) answers throughout training (Resp -, Genotype: p=0.0500). Dashed line illustrates chance level. Data shown at mean ± SEM. *: p<0.05; **: p<0.01; # shows genotype x performance interaction.

Learning-induced activity and functional connectivity in Cntnap2 KO mice.

A. Experimental design: cFos levels were quantified 90min after the 3rd training session of the Relational/Declarative memory task. ITI: Inter-trial interval; + represents reward location within each pair of arms. B. number of cFos-positive neurons (cell density/mm2) in the dorsal dCA1 (WT naïve vs Trained: p=0.0491; Trained WT vs. KO: p=0.0129) with representative images of the cFos staining (Magenta) in dCA1, scale: 100µm, for naïve (light colors) and trained (dark colors) WT (grey/black) and Cntnap2 KO (purple) groups. C. number of cFos-positive neurons (cell density/mm2) in the Dorso-Medial Striatum (DMS; Naive WT vs. KO: p=0.0189; KO Naïve vs Trained: p=0.0085) with representative images of the cFos staining (Magenta) in DMS, scale: 200µm. D. Percentage of total activity: activity of each structure over to the total activity measured (percentage; Trained WT vs. KO: Hippocampus: p=0.00023; PFC: p=0.00139; Striatum: p=0.02895). E. Percentage of total activity: activity each subfield within the total network, in WT mice (naïve vs trained, left panel; dCA1: p=0.0382), in Cntnap2KO mice (Naïve vs trained, middle panel), and in trained mice (WT vs Cntnap2 KO mice, dCA1: p=0.0183; dCA2: p=0.0201; dCA3: p=0.0307; ACC: p=0.0378; DMS: p=0.0303; IL: p=0.0060; PrL: p=0.0476; right panel, same data as in left and middle panels). F. Pairwise between-structure correlation strength of cFos cell density. Data shown as mean ± SEM. *: p<0.05; **: p<0.01; # shows genotype x performance interaction.

Freezing levels and calcium peak frequencies during the fiber photometry recordings (Figure 1).

A. Freezing levels (percent) during the conditioning session for WT (black) and Cntnap2 KO (purple) groups, conditioned with distinct trace durations (T5: 5s trace interval, T20: 20s trace interval; T40: 40s Trace interval). B. Freezing levels (percent time freezing) during the pre-tone, tone, trace and post-trace periods in the tone test for WT (black) and Cntnap2 KO (purple) groups, 24h post-conditioning with distinct trace durations (T40: Pre-tone: p=0.001; Tone: p=0.0177; Trace: p=0.261; Post-Tone: p=0.0064; Genotype x Freezing interaction: F3, 72=12.671; p<0.0001). C. Left: Freezing levels (percent) during the third tone of the conditioning session for littermate WT (black) and Cntnap2 KO (purple) T20 and T40 groups, (T20: Cntnap2 KO n=6; T40: WT n=8; Cntnap2 KO n=12). Right: Conditioned fear response during the tone test for the littermate groups (i.e., ratio of freezing before the tone compared to during the tone and the trace, see methods), 24h post-conditioning with distinct trace durations (Trace in Cntnap2 KO: p<0.0001; Genotype in T40: p<0.0001). D. Analysis of locomotion in an open field. Left: Total distance (m); genotype: p=0.046. Middle: Mean velocity (m/s); genotype: p<0.0001. Right: Resting time (s); genotype: ns; n= 14 WT, 15 Cntnap2 KO. E. Fiber photometry: Freezing levels (percent) during acquisition of trace fear conditioning for WT (black) and Cntnap2 KO (purple) groups, conditioned with a 20s (T20, n=8 WT and n=8 Cntnap2 KO, left panel) or a 40s trace interval (T40, n=11 WT and n=10 Cntnap2 KO, right panel). F. Left: Peak frequency of Ca2+ transients during the 3 tone presentations of the T20 conditioning (tone effect: p=0.0176). Right: peak frequency of Ca2+ transients during the 3 trace intervals of the T20 conditioning (trace effect: p=0.0287). G. Left: peak frequency of Ca2+ transients during the 3 tone presentations of the T40 conditioning (tone effect: p=0.0018). Right: peak frequency of Ca2+ transients during the 3 trace intervals of the T40 conditioning (trace effect: p=0.0229). H. Freezing levels (percent) during the tone test of the fiber photometry (T20) for WT (black) and Cntnap2 KO (purple) groups, 24h post-T20 conditioning (ns; WT n=8; KO n=8). I. Freezing levels (percent) during the tone test of the fiber photometry (T40) for WT (black) and Cntnap2 KO (purple) groups, 24h post-conditioning (Genotype: p=0.0009; PreTone: p=0.0258; Tone: p<0.0001; Post-Tone: p=0.0177; WT n=11; KO n=10). J. Optogenetic: Freezing levels (percent) during acquisition for GFP-injected WT (black; n=6), GFP-injected Cntnap2 KO (purple; n=7), and ChR2-injected Cntnap2 KO (light purple, white circles n=11) groups, conditioned with a 40s trace interval (Group: ns). K. Freezing levels (percent) during the tone test for GFP-injected WT (black; n=6), GFP-injected Cntnap2 KO (purple; n=7) and ChR2-injected Cntnap2 KO (light purple white circles n=11) groups, 24h post-T40 conditioning (Group x freezing: F6, 63 = 5.656; p<0.0001; Tone: p=0.0009; Trace: p=0.0224; Post-T: 0.0004).

cFos density in the hippocampus, striatum, and prefrontal cortex.

A. Number of cFos-positive neurons (cell density/mm2) in the dorsal hippocampus subfields: dCA2, dCA3 (Naive WT vs. KO: p=0.0400; Trained WT vs. KO: p=0.0454), dDG for naïve (light colors) and trained (dark colors) WT (grey/black) and Cntnap2 KO (purple) groups. B. Number of cFos-positive neurons (cell density/mm2) in the Dorso-Lateral Striatum (DLS; ns). C. Number of cFos-positive neurons (cell density/mm2) in the prefrontal cortex: Infra-Limbic, Pre-Limbic, Anterior Cingular Cortex (PFC, IL, PrL, ACC, respectively). Data shown at mean ± SEM. *: p<0.05.