Reduced dorsal CA1 activity limits retention of the temporal component of declarative memory in the Cntnap2 knockout mouse model of autism
- Eccles Institute of Neuroscience, John Curtin School of Medical Research, The Australian National University, Australia
- School of Computing, The Australian National University, Australia
- Neurocentre Magendie, Physiopathologie de la plasticité neuronale, U1215, INSERM, France
- Université de Bordeaux, France
- The Queensland Brain Institute, The University of Queensland, Australia
Figures
Figure 1 with 1 supplement
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 s. (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), 24 hr post-conditioning with distinct trace durations (Genotype X Trace 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, 24 hr post-conditioning. (E) Fiber photometry. Left: scheme depicting the fiber optic implantation targeting the dorsal CA1 subfield of the hippocampus (dCA1). Right: Representative image of the optic fiber placement, in the pyramidal cell layer of the dCA1, scale: 100 µm. Calcium activity was recorded using the calcium indicator GCaMP6f (green). (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 Materials and methods), 24 hr post-conditioning with distinct trace durations (T40: Genotype: F1, 19=50.143; p<0.0001). (H) Left: Peak amplitude of Ca2+ transients during the three tone presentations of the conditioning under a 20 s trace-interval (ns). Right: Peak amplitude of Ca2+ transients during the three trace intervals of the conditioning under a 20 s trace-interval (Genotype x Trace interaction: p=0.0476; WT n=8; KO n=8, trace 1: ns (p=0.0675)). (I) Left: Peak amplitude of Ca2+ transients during the three 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 three 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: 100 µm. 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 40 s trace duration. (L) Conditioned fear response during the tone test (i.e. ratio of freezing before the tone compared to during the tone and the trace, see Materials and methods), 24 hr 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, determined by student’s t-test. # represents genotype x variable interaction, determined by two-wayANOVA, followed by Bonferroni post-hoc.
Figure 1—figure supplement 1
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: 5 s trace interval, T20: 20 s trace interval; T40: 40 s 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, 24 hr 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), 24 hr 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 20 s (T20, n=8 WT and n=8 Cntnap2 KO, left panel) or a 40 s trace interval (T40, n=11 WT and n=10 Cntnap2 KO, right panel). (F) Left: Peak frequency of Ca2+ transients during the three tone presentations of the T20 conditioning (tone effect: p=0.0176). Right: peak frequency of Ca2+ transients during the three trace intervals of the T20 conditioning (trace effect: p=0.0287). (G) Left: peak frequency of Ca2+ transients during the three tone presentations of the T40 conditioning (tone effect: p=0.0018). Right: peak frequency of Ca2+ transients during the three 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, 24 hr 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, 24 hr 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 40 s 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, 24 hr 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). Data shown at mean ± SEM. *: p<0.05; **: p<0.01; ***: p<0.001; * represents genotype effect, determined by student’s t-test. # represents genotype x variable interaction, determined by two-wayANOVA, followed by Bonferroni post-hoc.
Figure 2
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 first 5 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, determined by two-wayANOVA, followed by Bonferroni post-hoc.
Figure 3 with 1 supplement
Learning-induced activity and functional connectivity in Cntnap2 KO mice.
(A) Experimental design: cFos levels were quantified 90 min after the 3rd training session of the Relational/Declarative memory task. ITI: Inter-trial interval; +represents reward location within each pair of arms (WT Naïve: n=5; WT Trained: n=7; Cntnap2 KO: n=5; Cntnap2 KO Trained: n=7). (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: 100 µm, 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; Naïve WT vs. KO: p=0.0189; KO Naïve vs Trained: p=0.0085) with representative images of the cFos staining (Magenta) in DMS, scale: 200 µm. (D) Percentage of total activity: activity of each structure over to the total activity measured (percentage; Trained WT vs. KO: Hippocampus: p=0.00194; PFC: p=0.00107; Striatum: p=0.0382). (E) Percentage of total activity: activity each subfield within the total network, in WT mice (naïve vs trained, left panel; dCA1: p=0.0242), in Cntnap2 KO mice (Naïve vs trained, middle panel), and in trained mice (WT vs Cntnap2 KO mice, dCA1: p=0.009; dCA2: p=0.0174; dCA3: p=0.0274; ACC: p=0.0438; DMS: p=0.0350; IL: p=0.0267; 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, ***: p<0.001. * represents genotype effect, determined by student’s t-test.
Figure 3—figure supplement 1
cFos density in the hippocampus, striatum, and prefrontal cortex.
(A) Number of cFos-positive neurons and representative images (cell density/mm2, scale: 100 µm) in the dorsal hippocampus subfields: dCA2, dCA3 (Naïve 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 (WT Naïve: n=5; WT Trained: n=7; Cntnap2 KO: n=5; Cntnap2 KO Trained: n=7). (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 Cingulate Cortex (PFC, IL, PrL, ACC, respectively). Data shown at mean ± SEM. *: p<0.05. Data shown at mean ± SEM. *: p<0.05; * represents genotype effect, determined by student’s t-test.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | B6.129(Cg)-Cntnap2tm1Pele/J | Jackson | #017482; RRID:IMSR_JAX:017482 | Males and females |
| Strain, strain background (Adeno-associated Virus) | AAV5-pCaMKIIα-ArchT-GFP | UNC Vector Core | RRID:Addgene_99039-AAV5 | |
| Strain, strain background (Adeno-associated Virus) | AAV5-pCaMKIIα-ChR2 (H134R)-EYFP | UNC Vector Core | RRID:Addgene_26969 | |
| Strain, strain background (Adeno-associated Virus) | AAV5-pCaMKIIα-GFP | UNC Vector Core | RRID:Addgene_50469 | |
| Strain, strain background (Adeno-associated Virus) | AAV1-hsynjGCaMP6f-WPRE | Addgene | RRID:Addgene_100837 | |
| Antibody | Mouse monoclonal anti-cFos | Santa Cruz | SC166940; RRID:AB_2106783 | 1:1000 |
| Antibody | Mouse polyclonal anti-Biotin | Vector Laboratories | BA-2000; RRID:AB_2313581 | 1:200 |
| Antibody, fluorophore | Streptavidin 647 | Jackson | 016-600-084; RRID:AB_2341101 | 1:200 |
| Software, algorithm | Statview | N/A | N/A | |
| Software, algorithm | Fiji | ImageJ | https://imagej.net/software/fiji/downloads | |
| Software, algorithm | Bonsai/Neurophotometrics | FP3002 | ||
| Software, algorithm | Qu Path | RRID:SCR_018257 | ||
| Software, algorithm | GraphPad Prism | RRID:SCR_002798 | ||
| Other | Fear Conditioning | FEAR-SHUTTLE | Fear-Shuttle - Imetronic | Behavioral apparatus |
| Other | Stereotactic frame | Kopf Instruments | N/A | Stereotaxic surgery apparatus |
| Other | Optical fiber stubs 2 mm | thorlabs | N/A | Optic fiber implants for fiberphotometry and optogenetic experiments |
| Other | LED driver system | Thorlabs | LEDD1B T-Cube | LED for optogenetic stimulation |
| Other | Fiber-Coupled LED | Thorlabs | M470F4 | LED for fiberphotometry recordings |
| Other | vertical P10 puller | Narishige, Japan | N/A | Puller for glass pipette preparation for viral injection |
| Other | Glass Capillary, Borosilicate | Clarke Electromedical | GC-150F-15 | Glass pipette for viral injection |
| Other | vibratome VT1200 | LEICA | N/A | Slicer for fixed brain tissue for immunohistochemistry |
| Other | Slide scanner Axioscan Z1 | Zeiss | N/A | Epifluorescence microscope for imaging |
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Source data 1
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- https://cdn.elifesciences.org/articles/111851/elife-111851-data1-v1.xlsx
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Reduced dorsal CA1 activity limits retention of the temporal component of declarative memory in the Cntnap2 knockout mouse model of autism
eLife 15:RP111851.
https://doi.org/10.7554/eLife.111851.3
