The protein kinase C (PKC) enzymes have long been established as critical for synaptic plasticity. However, it is unknown whether Ca2+-dependent PKC isozymes are activated in dendritic spines during plasticity and, if so, how this synaptic activity is encoded by PKC. Here, using newly developed, isozyme-specific sensors, we demonstrate that classical isozymes are activated to varying degrees and with distinct kinetics. PKCα is activated robustly and rapidly in stimulated spines and is the only isozyme required for structural plasticity. This specificity depends on a PDZ-binding motif present only in PKCα. The activation of PKCα during plasticity requires both NMDA receptor Ca2+ flux and autocrine brain-derived neurotrophic factor (BDNF)–TrkB signaling, two pathways that differ vastly in their spatiotemporal scales of signaling. Our results suggest that, by integrating these signals, PKCα combines a measure of recent, nearby synaptic plasticity with local synaptic input, enabling complex cellular computations such as heterosynaptic facilitation of plasticity necessary for efficient hippocampus-dependent learning.
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We would like to thank A. F. Brantley and the Scripps Florida behavior core, who performed animal behavioral studies; L. Parada (Memorial Sloan Kettering) for Bdnf fl/fl mice; D. Ginty (Harvard) for TrkBF616A mice; M. Dowdy and the MPFI ARC for animal care; members of the Yasuda laboratory; L. Yan; and D. Kloetzer. This work was funded by F32MH101954 (L.A.C.), R01MH080047 (R.Y.), 1DP1NS096787 (R.Y.) and the Max Planck Florida Institute for Neuroscience.
Integrated supplementary information
A,B) Schematic of ITRACKneg (A) and IDOCKneg (B). C,D) Overlay of mean time courses of PKCα, PKCβ, and PKCγ activity as measured by ITRACK (C; n[neurons/spines]: PKCα=32/103 (same as Figure 2D), PKCβ =13/31, PKCγ = 10/27) or IDOCKS (D; n[neurons/spines]: PKCα = 6/28(same as in Figure 2K), PKCβ = 6/24, PKCγ= 8/29). Insets: Quantification of area under the curve (mean and SEM) of PKC isozymes to each glutamate uncaging pulse (as in Figure 2C,L). One way ANOVA with Sidak’s multiple comparison compared to PKCα. E) Average translocation in response to each glutamate uncaging pulse (uncaging triggered average) of ITRACKα (same data as in C), ITRACKα in the absence of glutamate (n[neurons/spines]= 6/15) or the control sensor ITRACKα neg (n[neurons/spines]= 6/19). Mean and SEM (shaded) are shown. F) Average translocation in response to each glutamate uncaging pulse (uncaging triggered average) of IDOCKSα (same data as in D), IDOCKSα in the absence of glutamate (n[neurons/spines]= 5/12) or the control sensor IDOCKSα neg (n[neurons/spines]= 7/20). Mean and SEM (shaded) are shown.
A) Change in ITRACKα activity as a function of the expression level of the ITRACKα donor measured by intensity of eGFP at the primary dendrite (n[neurons/spines]= 19/55) and compared to standards of purified eGFP of known concentration. Pearson’s correlation coefficient (r2) indicated. B) Change in ITRACKα activity as a function of the relative expression level of the donor to the acceptor as measured by intensity of eGFP and mCh excited with 920nm at the primary dendrite (n[neurons/spines] = 19/55). Pearson’s correlation coefficient (r2) indicated. C) Change in basal lifetime of eGFP (n[neurons/spines] 1/63), ITRACKα (n[neurons/spines]=1/60) or IDOCKSα (n[neurons/spines]=1/47) as a function of spine size. Pearson’s correlation coefficient (r2) indicated. D) Mean basal lifetime of eGFP (n: spines=63, dendrites=16), ITRACKα (n: spines=60, dendrites=15) or IDOCKSα (n: spines=47, dendrites=15) measured in spines or dendrites of a single neuron. Two-tailed, unpaired t-test with Welch’s correction not significant. E) Average timecourse of fluorescence decay in spines after photoactivation of paGFP (τ =0.91, n[neurons/spines]=4/78) or paGFP-PKCα (τ =2.19, n[neurons/spines]=3/40). F) Average timecourse of fluorescence decay in spines after photoactivation paGFP-PKCα (same as in E) or paGFP-PKCα and ITRACK acceptor construct mCh-CAAX (n[neurons/spines]=3/48) or paGFP-PKCα and IDOCKS acceptor construct 2mCh-PSα (n[neurons/spines]=2/29). G) Quantification of mean sustained sLTP (25-30 min) in neurons expressing eGFP (n[neurons/spines] = 7/11), ITRACKα (n[neurons/spines] = 5/7), or IDOCKSα (n[neurons/spines] = 5/8). One way ANOVA non-significant. H) Average change in PKCα activity to each uncaging pulse (uncaging triggered average) measured with ITRACKα (same as Figure 3D) or a modified ITRACKα in which the acceptor fluorophore was targeted to the membrane with an H-Ras derived CAAX domain (n[neurons/spines] = 3/12). Two way ANOVA not significant by sensor design (F (1, 1872) = 2.769, p=0.0963). For correlation analysis (panels A-C) lines represent linear regression and 95% confidence intervals (dotted). For averaged data (panels D-H) mean and SEM are shown.
A) Mean + SEM sustained sLTP (25-30 min) in the presence of Gö6983 added 10 minutes (n [neurons/spines] = 5/5) or 45 minutes (n [neurons/spines] = 7/7) before the induction of plasticity. Unpaired two-tailed t- test not significant. B) Expression level of rescue constructs eGFP-tagged PKCα, PKCβ, and PKCγ (for experiments in Figure 3E, F; n[neurons/spines]: KO+PKCα = 9/21, KO+PKCβ = 6/16, KO+PKCγ = 6/16). Line represents median value of expression and error is 95% CI of median. C) Overlay of mean time courses of sLTP and SEM of neurons from WT and PKCα KO littermate mice (shown in Figure 3E; n[neurons/spines]: WT = 10/21, KO = 12/26) and WT and PKC α,β,γ TKO mice (as quantified in Figure 3G; n[neurons/spines]: WT=8/21, TKO=7/18). Two way ANOVA significant by genotype (F (3, 1480) = 44.05, p<0.0001). D) Average time courses of sLTP and SEM in eGFP expressing neurons from WT and TKO mice and neurons from TKO mice overexpressing eGFP-PKCα (as quantified in Figure 3G; n[neurons/spines]: WT=8/21, TKO=7/18, TKO +PKCα=7/17). Two way ANOVA significant by genotype (F (1, 560) = 51.08, p<0.0001).
Performance of male PKCα KO (n[animals] = 13) and WT (n[animals] = 15) littermate mice in open field (A, Mean and SEM shown, two-tailed unpaired t test), spontaneous alternation with and without delay (B, Mean shown, two-sided Fisher’s exact test) and hot plate test (C, Mean and SEM shown, two-tailed unpaired t-test).
A) Quantification of mean sustained structural plasticity of neurons treated with indicated pharmacologic agents. One way ANOVA with Dunnett’s multiple comparison test against controls (n[neurons/spines]: CTL=11/13, APV (50 µM) = 5/9, Edel (50 µM) = 6/13, CTL= 13/15, MCPG (250 µM) = 5/9, NPS (20 µM) = 8/13, CTL=9/9, Veh= 4/7, 1NMPP1 (1 µM) = 7/12. B) Average change in PKCα activity (measured with IDOCKSα) in response to each glutamate uncaging pulse (uncaging triggered average) before (n[neurons/spines] = 5/14) and after edelfosine application (50 µM, n[neurons/spines] = 5/17). Two way ANOVA significant by drug (F (1, 464) = 30.94, p<0.0001). C) Average change in PKCα activity (measured by ITRACKα) in response to each glutamate uncaging pulse (uncaging triggered average) before (n[neurons/spines] = 7/24) and after U73122 application (10 µM, n[neurons/spines]= 3/9) or application of the inactive analog U73343 (10 µM, n[neurons/spines]= 4/13). Two way ANOVA by drug is significant by drug (F (1, 31) = 10.54, p=0.0028). U73343 is not significant compared to CTL (F (1, 35) = 0.5582, p=0.46). D) Average change in PKCα activity (measured by ITRACKα) in response to each glutamate uncaging pulse (uncaging triggered average) before (n[neurons/spines] = 9/25) and after NPS application (20 µM, n[neurons/spines]= 9/28). Two way ANOVA by drug is non-significant (F (1, 816) = 0.0009298, p=0.9757). E) Average change in PKCα activity (measured with IDOCKSα) in response to each glutamate uncaging pulse (uncaging triggered average) in neurons from TrkBF616A mice before (n[neurons/spines] = 5/17) and after 1NMPP1 application (1 µM, n[neurons/spines]= 5/18). Two way ANOVA significant by drug (F (1, 528) = 37.84, p<0.0001). For all panels data shown is mean and SEM.
PKCα activation (measured by ITRACKα) in individual spines receiving subthreshold stimulation after induction of sLTP in a nearby spine plotted as a function of distance to the nearby spine (same data as in Figure 7 F, G (paired), n [neurons/spines= 8/15). Pearson’s correlation is non-significant (p=0.956, r2=0.0026).
Supplementary Figures 1–6 and Supplementary Note
Video of PKCα activity (Fig. 1b) during the induction of sLTP by two-photon glutamate uncaging. Fluorescence lifetime images of the FRET sensor ITRACKα were acquired at 8 Hz and uncaging pulses were delivered at 0.5 Hz (indicated by arrowheads). Only the first eight uncaging pulses are shown. Fluorescence lifetime lookup table spans 2.65 ns (blue) to 2.3 ns (red), with PKCα activation corresponding to warm (red) colors
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Nature Neuroscience (2018)