CaMKII Autophosphorylation Is Necessary for Optimal Integration of Ca2+ Signals during LTP Induction, but Not Maintenance

Abstract

CaMKII plays a critical role in decoding calcium (Ca²⁺) signals to initiate long-lasting synaptic plasticity. However, the properties of CaMKII that mediate Ca²⁺ signals in spines remain elusive. Here, we measured CaMKII activity in spines using fast-framing two-photon fluorescence lifetime imaging. Following each pulse during repetitive Ca²⁺ elevations, CaMKII activity increased in a stepwise manner. Thr286 phosphorylation slows the decay of CaMKII and thus lowers the frequency required to induce spine plasticity by several fold. In the absence of Thr286 phosphorylation, increasing the stimulation frequency results in high peak mutant CaMKII^T286A activity that is sufficient for inducing plasticity. Our findings demonstrate that Thr286 phosphorylation plays an important role in induction of LTP by integrating Ca²⁺ signals, and it greatly promotes, but is dispensable for, the activation of CaMKII and LTP.

Keywords: CA1 pyramidal neurons; Schaffer collateral; calcium signaling; calmodulin; dendritic spines; hippocampus; signal transduction; synaptic plasticity.

Figure 1. CaMKII Activation Measured with Millisecond Temporal Resolution

(A) Representative fluorescence lifetime images of Camuiα during glutamate uncaging at 0.49 Hz. Warmer colors indicate higher fluorescence lifetime of Camuiα, corresponding to the active, open conformation of Camuiα. (B) Time course of fluorescence lifetime of Camuiα in (A) of the stimulated spine (black) and dendritic region (blue). Inset is expanded view of the rising phase of Camuiα activation. Black dots represent uncaging pulses. (C) Averaged change in fluorescence lifetime of Camuiα (n = 36 spines/14 neurons). Left panel is expanded view of the rising phase of right panel. The orange curve indicates the decay kinetics of fluorescence lifetime signal obtained by curve fitting of a double-exponential function: F(t) = F₀ ▪ [P_fast ▪ e^−t/τ_fast + P_slow ▪ e^−t/τ_slow], where F₀ is the initial fluorescence lifetime, τ_fast and τ_slow are the fast and slow decay time constants and P_fast and P_slow are the respective populations. The time constants are obtained as τ_fast = 6.4 ± 0.7 s (P_fast = 74%) and τ_slow = 92.6 ± 50.7 s (P_slow = 26%). (D) Representative fluorescence lifetime images of Camuiα in response to a single glutamate uncaging pulse. (E) Time course of fluorescence lifetime of Camuiα in (D). (F) Averaged change in fluorescence lifetime of Camuiα (n = 35 spines/8 neurons) in response to a single glutamate uncaging pulse. The orange curve indicates the kinetics of fluorescence lifetime signal obtained by curve fitting of a function: F(t) = [a ▪ e^−t/τ_fast + c] ▪ [1- e^−t/τ_rise], where c is a constant which represents the slow decay component as described in (C). The time constants are obtained as: τ_rise = 0.3 ± 0.1 s and τ_fast = 8.2 ± 1.7 s. All data are shown in mean ± s.e.m., and s.e.m. of time constants is obtained by bootstrapping. Scale bar, 1 µm.

Figure 2. Activation of Camuiα^T286A and Camuiα^T286A During sLTP Induction

(A) Activation of Camuiα^T286A (green) and Camuiα^WT (black) in response to a single glutamate uncaging pulse (black dot). The data and fitted curve for Camuiα^WT are from Figure 1F for comparison. The orange curve on Camuiα^T286A indicates the decay kinetics obtained by curve fitting of a function: F(t) = C ▪ [1 − e^−t/τ_rise] ▪ e^{−t/τ_decay}, where τ_rise is adapted from Figure 1F and is fixed during curve fitting (τ_rise = 0.3 s). The decay time constant for Camuiα^T286A is 1.9 ± 0.3 s (n = 30 spines/9 neurons). Inset is expanded view of the rising phase of Camuiα^T286A activation. (B) Averaged fluorescence lifetime of Camuiα^WT , Camuiα^T286A , Camuiα^T286D , and Camuiα^{T286D/T305A/T306A} during sLTP induction. Right panel is fluorescence lifetime averaged over −4–0 s: Camuiα^WT: 1.75 ± 0.02 ns; Camuiα^T286A: 1.69 ± 0.03 ns; Camuiα^T286D: 2.06 ± 0.01 ns; Camuiα^{T286/T305A/T306A}: 2.10 ± 0.01 ns. Asterisks denote the statistical significance (p < 0.05; ANOVA followed by post hoc Bonferroni test). (C–E) Averaged change in fluorescence lifetime of Camuiα^T286A (C; green), Camuiα^T286D (D; navy), Camuiα^{T286D/T305A/T306A} (E; light blue) and Camuiα^WT (black) in the stimulated spine during glutamate uncaging. The data and fitted curve for Camuiα^WT are from Figure 1C for comparison. Left panel is expanded view of the right panel. The orange curve on Camuiα^T286A is obtained by curve fitting of a function: F(t) = C ▪ e^{−t/τ_decay}. The decay time constant is obtained as 1.9 ± 1.2 s (26 spines/12 neurons). The orange curve on Camuiα^T286D and Camuiα^{T286D/T305A/T306A} are obtained by curve fitting of a function: F(t) = C ▪ e^{−t/τ_decay} + d₀, where d₀ is a constant. The decay time constant is obtained as 3.0 ± 0.6 s for Camuiα^T286D (33 spines/10 neurons) and 5.7 ± 0.5 s for Camuiα^{T286D/T305A/T306A} (32 spines/7 neurons). All data are shown in mean ± s.e.m., and s.e.m. of time constants is obtained by bootstrapping.

Figure 3. CaMKII Activation in Dendritic Spines in Response to High-frequency Glutamate Uncaging

(A–C) Averaged change in fluorescence lifetime of Camuiα^WT (black) and Camuiα^T286A (green) in response to high frequency glutamate uncaging at 1.9 Hz (A, B) or 7.8 Hz (C) for 30 pulses (A) or 120 pulses (B, C) in the dendritic spines. Insets are expanded view. The orange curves indicate the decay kinetics obtained by a double-exponential fitting for Camuiα^WT (see details in Figure 1C) and mono-exponential fitting for Camuiα^T286A (see details in Figure 2C). The obtained decay time constants are as follows (number of samples: spines/neurons): (A) Camuiα^WT: τ_fast = 6.9 ± 0.5 s (81%) and τ_slow = 127 ± 84 s (19%) (21/14). Camuiα^T286A: τ_decay = 1.9 ± 0.7 s (22/16). (B) Camuiα^WT: τ_fast = 7.8 ± 0.9 s (69%) and τ_slow = 192 ± 132 s (31%) (18/11). Camuiα^T286A: τ_decay = 1.5 ± 1.0 s (17/10). (C) Camuiα^WT: τ_fast = 7.2 ± 1.0 s (59%) and τ_slow = 103 ± 31 s (41%) (25/13). Camuiα^T286A: τ_decay = 3.1 ± 1.0 s (15/8). (D–F) Averaged change in fluorescence lifetime in dendritic region as shown in (A–C), respectively. All data are shown in mean ± s.e.m., and s.e.m. of time constants is obtained by bootstrapping.

Figure 4. Structural and Electrophysiological Plasticity Induced by High Frequency Stimulation

(A) Fluorescence intensity images (mEGFP) of spine structural plasticity during sLTP. The arrowhead indicates the spot of two-photon glutamate uncaging. Scale bar, 1 µm. (B–E) Structural LTP of neurons from Camka^T286A wild-type (WT/WT), heterozygous (WT/T286A) or homozygous (T286A/T286A) mice induced by glutamate uncaging at 0.5 Hz for 30 pulses (B), 2 Hz for 30 pulses (C), 2 Hz for 120 pulses (D) or 7.8 Hz for 120 pulses (E). Number of samples (spines/neurons) are 22/22 for WT, 27/26 for Het, and 25/25 for Homo in (B); 17/16 for WT, 18/18 for Het, and 12/12 for Homo in (C); 15/15 for WT, 13/13 for Het, and 14/14 for Homo in (D), and 15/15 for Homo, 20/19 for Het, and 16/16 for Homo in (E). (F–G) Quantifications of spine volume change during the transient phase (F; peak value recorded at 1 min) or the sustained phase (G; averaged over 25–30 min). Asterisks denote the statistical significance (p < 0.05; ANOVA followed by post hoc Bonferroni test). (H) Whole-cell recording of LTP induced at the Schaffer collateral in CA1 neurons from Camk2a^T286A and litter-mate control mice. LTP is induced by electrical stimulations at 2 Hz for 60 s or 40 Hz for 15 s with depolarization to 0 mV. Number of neurons is 19 for WT, 20 for T286A (2 Hz) and 27 for T286A (40 Hz). Typical EPSC traces before (-5–0 min) and after (43–63 min) are presented on top of the panel. (I) Quantification of EPSC potentiation averaged over 40 – 63 min. Asterisks denote the statistical significance (p < 0.05; ANOVA followed by post hoc Bonferroni test). All data are shown in mean ± s.e.m.