Formation of long-term memory without short-term memory revealed by CaMKII inhibition

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Data availability

Source data are provided with this paper.

Code availability

The code used for data acquisition and analysis in our electrophysiological experiments is available at https://github.com/ryoheiyasuda/FLIMage_Matlab_phys.git.

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Acknowledgements

We thank K. Peter-Giese (King’s College London) for providing Camk2aT286A mice, Y. Hayano, K. Liu, Y. Nakahata, I. Sponitsky-Kroyter and P. Rusina for technical support, L. Colgan for critical reading and Y. Hayashi (Kyoto University) for discussion. This study was supported by NIH (R01MH080047 and R35NS116804 to R.Y.).

Author information

Authors and Affiliations

  1. Max Planck Florida Institute for Neuroscience, Jupiter, FL, USA

    Myung Eun Shin, Paula Parra-Bueno & Ryohei Yasuda

Authors

  1. Myung Eun Shin
  2. Paula Parra-Bueno
  3. Ryohei Yasuda

Contributions

M.E.S. and R.Y. conceptualized and designed the experiments, and prepared the manuscript. M.E.S. planned and performed behavioral experiments and analysis. P.P.-B. planned and carried out electrophysiological experiments and analysis.

Corresponding author

Correspondence to Ryohei Yasuda.

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Competing interests

The authors declare no competing interests.

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Nature Neuroscience thanks Karl Giese, Tommaso Patriarchi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Expression of mEGFP-P2A-paAIP2 in vivo.

a, The expression of mEGFP-P2A-paAIP2 in amygdala. The white and blue triangles indicate the area of transgene expression and the fiber track above amygdala, respectively. White dotted line indicates basolateral amygdala (BLA). Scale bar = 500 μm. Animals expressing detectable transgene expression in BLA in at least one hemisphere were included in the behavioral analyses. b, Examples of mEGFP-P2A-paAIP2 expression in vivo following AAV injection. White dotted line indicates BLA. Scale bar = 100 μm. In some animals, the transgene expression was also found in the brain regions adjacent to the BLA, such as entorhinal cortex, piriform cortex, endopiriform nucleus and central and medial amygdala. This experiment was repeated across four independent cohorts with similar results. The quantification of the transgene expression for all samples can be found in the Extended Data Table 1.

Extended Data Fig. 2 Acquisition of IA memory for mice in which CaMKII is transiently inhibited in amygdala.

Cross-latency during IA training for mice injected with paAIP2neg (left) or paAIP2 (right). Median is indicated by the horizontal bar. Two-sided Wilcoxon rank sum test was performed between the two trials of training in each group. Two-sided Mann–Whitney test was conducted between paAIP2neg and paAIP2 groups at each trials. n = 27 for paAIP2neg and 33 for paAIP2 group. ****p < 0.0001. Data points with maximum latency values (120 s) have been randomized between 121 and 140 s.

Source data

Extended Data Fig. 3 Acquisition of IA memory for transgenic mice with inhibited CaMKII autophosphorylation.

a–c, Cross-latency during multi-trial IA training for Camk2aT286A mice (right) or their littermate nontransgenic control (left). Median is indicated by the horizontal bar. Two-sided Wilcoxon rank sum test was performed between the first and subsequent trials of training in each group. Two-sided Mann–Whitney test was conducted between control and Camk2aT286A mice at each trials. n = 29 for control and 28 for Camk2aT286A mice (a), n = 40 for control and 41 for Camk2aT286A mice (b) and n = 47 for control and 45 for Camk2aT286A mice (c). **p < 0.01, ***p < 0.001 and ****p < 0.0001. Data points with maximum latency values (120 s) have been randomized between 121 and 132 s.

Source data

Extended Data Fig. 4 Performance of IA task after 2 trials of training for transgenic mice with inhibited CaMKII autophosphorylation.

a, Performance of IA task immediately, 1 h, 1 d, 7 d and 4 w after 2 trials of training for Camk2aT286A mice (right) or their littermate nontransgenic control (left). b, Cross-latencies at two selected time points (1 h and 1 d) from a are shown. Median is indicated by the horizontal bar. Two-sided Wilcoxon rank sum test was performed between 1 h and the other respective time points in each group. Two-sided Mann–Whitney test was conducted between control and transgenic mice at each time points. n = 29 for control and 28 for Camk2aT286A mice (a,b). *p < 0.05 and ****p < 0.0001. Data points with maximum latency values (300 s) are displayed as random numbers between 301 and 330 s (a,b).

Source data

Extended Data Fig. 5 Sex analysis on the performance of IA task for transgenic mice with inhibited CaMKII autophosphorylation.

a,b, Performance of IA task immediately, 1 h, 1 d, 7 d and 4 w after 3 trials (a) or 4 trials (b) of training for male (left) or female (right) Camk2aT286A transgenic and their littermate control mice. The same set of animals shown in Fig. 2a,c were further analyzed to assess if there are sex differences in performance of IA task. Median is indicated by the horizontal bar. Two-sided Wilcoxon rank sum test was performed between 1 h and the other time points, respectively in each group. Two-sided Mann–Whitney test was conducted between control and transgenic mice at each testing sessions. In addition, two-sided Mann–Whitney test was performed between males and females of each genotype at each time point. The resulting P values are shown in the light orange (control) and light purple (mutant) boxes, respectively. No statistically significant difference was found from the comparison between the two sexes. ncontrol, nCamk2aT286A = 22, 19 for males and 18, 22 for females in 3× IA (a), 27, 19 for males and 20, 26 for females in 4× IA (b). *p < 0.05 and **p < 0.01. Data points with maximum latency values (300 s) are displayed as random numbers between 301 and 330 s.

Source data

Extended Data Fig. 6 Performance of IA task for transgenic mice with inhibited CaMKII autophosphorylation with a maximum cross-latency of 600 s.

a, Performance of IA task immediately, 1 h, 1 d, 7 d and 4 w after 4 trials of training for Camk2aT286A mice (right) or their littermate nontransgenic control (left). The experiment is similar to that in Fig. 2c, but with a longer maximum cross-latency (600 s). b, Cross-latencies at two selected time points (1 h and 1 d) from a are shown. c, Acquisition of IA memory for transgenic and control mice that were shown in a,b. Median is indicated by the horizontal bar. Two-sided Wilcoxon rank sum test was performed between 1 h and the other time points (a,b) or between the first and subsequent trials of training (c), respectively in each group. Two-sided Mann–Whitney test was conducted between control and transgenic mice at each testing (a,b) or training sessions (c). n = 35 for control and 42 for Camk2aT286A mice (a–c). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Data points with maximum latency values (600 s for a,b, 120 s for c) are displayed as random numbers between 601 and 640 (a) or 631 (b), or between 121 and 132 s (c).

Source data

Extended Data Fig. 7 Performance of IA task for transgenic mice with inhibited CaMKII autophosphorylation in which the memory test 1 h after the IA training has been omitted.

a,b, Performance of IA task 1 d (left) and 7 d (right) after 4 trials of training for Camk2aT286A mice (b) or their littermate nontransgenic control (a). Each genotype was divided into two groups, and for each group, a memory test was either conducted (‘1 h’ group) or omitted (‘no 1 h’ group) at 1 h from the training. c, Performance of IA task immediately, 1 h, 1 d, and 7 d after 4 trials of training for the ‘1 h’ group mice in both genotypes shown in a,b. Median is indicated by the horizontal bar. Two-sided Mann–Whitney test was conducted between ‘1 h’ and ‘No 1 h’ groups (a,b) or between the ‘1 h’ control and transgenic mice at each time points (c). Two-sided Wilcoxon rank sum test was performed between 1 h and the other respective time points in each group (c). n = 31 for ‘1 h’ control, 30 for ‘No 1 h’ control, 35 for ‘1 h’ Camk2aT286A, 33 for ‘No 1 h’ Camk2aT286A (a–c). *p < 0.05, **p < 0.01 and ***p < 0.001. Data points with maximum latency values (300 s, a–c) are displayed as random numbers between 301 and 330.

Source data

Extended Data Fig. 8 Correlation between AMPA and NMDA current amplitudes in acute brain slices obtained from transgenic mice with inhibited CaMKII autophosphorylation.

Correlation between AMPA and NMDA current amplitudes in brain slices acquired from control and transgenic mice after 2-trial and 4-trial-training IA. Values used in analysis were from the same set of cells shown in AMPA/NMDA ratio graphs in Fig. 3b,d. The square of the correlation coefficients (R2) and slopes from the nonlinear regression fit (solid line) are indicated. ncells/animals for 2× training IA = 16/6 (control naive), 21/5 (control 1 h), 15/5 (control 1 d), 22/11 (mutant naive), 21/8 (mutant 1 h), 17/6 (mutant 1 d). ncells/animals for 4× training IA = 29/6 (control naive), 28/8 (control 1 h), 23/7 (control 1 d), 30/9 (mutant naive), 37/11 (mutant 1 h), 34/13 (mutant 1 d).

Source data

Extended Data Table 1 Quantification of mEGFP-P2A-paAIP2 expression in basolateral amygdala (BLA) complex in amygdala

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Supplementary information

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Shin, M.E., Parra-Bueno, P. & Yasuda, R. Formation of long-term memory without short-term memory revealed by CaMKII inhibition. Nat Neurosci 28, 35–39 (2025). https://doi.org/10.1038/s41593-024-01831-z

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