1. Neuroscience

PKMζ-PKCι/λ double-knockout demonstrates atypical PKC is crucial for the persistence of hippocampal LTP and spatial memory

  1. Panayiotis Tsokas
  2. Changchi Hsieh
  3. Alejandro Grau-Perales
  4. Andrew Tcherepanov
  5. Leo Kwok
  6. Laura Rodriguez-Valencia
  7. David A Cano
  8. Kim Allen
  9. Hannah J Smith
  10. Sabina Kubayeva
  11. Benson J Wei
  12. Samuel Sabzanov
  13. Rafael Flores-Obando
  14. Sourav Ghosh
  15. Peter John Bergold
  16. Jerry Rudy
  17. James Cottrell
  18. André Fenton  Is a corresponding author
  19. Todd Charlton Sacktor  Is a corresponding author
  1. Department of Physiology and Pharmacology, The Robert F. Furchgott Center for Neural and Behavioral Science, State University of New York Downstate Health Sciences University, United States
  2. Department of Anesthesiology, State University of New York Downstate Health Sciences University, United States
  3. Department of Pathology, State University of New York Downstate Health Sciences University, United States
  4. Center for Neural Science, New York University, United States
  5. College of Medicine, State University of New York Downstate Health Sciences University, United States
  6. Department of Biology, City University of New York-Medgar Evers College, United States
  7. Departments of Neurology and Pharmacology, Yale University, United States
  8. Department of Neurology, State University of New York Downstate Health Sciences University, United States
  9. Department of Psychology and Neuroscience, University of Colorado at Boulder, United States
  10. Neuroscience Institute at NYU Langone Medical Center, United States
https://doi.org/10.7554/eLife.110499.3
Share this article
Cite this article
  1. Panayiotis Tsokas
  2. Changchi Hsieh
  3. Alejandro Grau-Perales
  4. Andrew Tcherepanov
  5. Leo Kwok
  6. Laura Rodriguez-Valencia
  7. David A Cano
  8. Kim Allen
  9. Hannah J Smith
  10. Sabina Kubayeva
  11. Benson J Wei
  12. Samuel Sabzanov
  13. Rafael Flores-Obando
  14. Sourav Ghosh
  15. Peter John Bergold
  16. Jerry Rudy
  17. James Cottrell
  18. André Fenton
  19. Todd Charlton Sacktor
(2026)
PKMζ-PKCι/λ double-knockout demonstrates atypical PKC is crucial for the persistence of hippocampal LTP and spatial memory
eLife 15:RP110499.
https://doi.org/10.7554/eLife.110499.3
  2. Download BibTeX
  3. Download .RIS
5 figures, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements
Download asset Open asset
Compensatory increases of atypical PKCι and conventional, but not novel PKCs, in conditional PKMζ-knockout (ζ-cKO) mouse hippocampus.

(A, B) Immunoblots of hippocampal extracts from Camk2a-CreERT2; Prkczfl/fl mice that received tamoxifen (2 mg/200 µl i.p., five daily doses) to activate Cre recombinase selectively in excitatory neurons. Mice were sacrificed 7 days after the last dose. Left, representative immunoblots with Mr markers shown in kDa. Right, mean± SEM. Significance by two sample Student t-tests with Bonferroni correction denoted by *; not significant, n.s. Tamoxifen is a partial PKC antagonist and may still be present after a week (O’Brian et al., 1985); therefore, wild-type (WT) mice that also received tamoxifen are non-transgenic controls (NTC). (A) PKMζ decreases and PKCι increases in PKMζ-cKO mice. (B) Conventional PKCs increase and novel PKCs do not change. Actin loading controls shown in Figure 1—figure supplement 2. Statistics for (A) and (B) in Figure 1—source data 1.

Figure 1—source data 1

Statistics for data presented in Figure 1.

https://cdn.elifesciences.org/articles/110499/elife-110499-fig1-data1-v1.docx
Figure 1—source data 2

Labeled immunoblots of Figure 1.

https://cdn.elifesciences.org/articles/110499/elife-110499-fig1-data2-v1.zip
Figure 1—source data 3

Unlabeled immunoblots of Figure 1.

https://cdn.elifesciences.org/articles/110499/elife-110499-fig1-data3-v1.zip
Figure 1—figure supplement 1
Download asset Open asset
ζ-cKO increases the amounts of activation-loop-phosphorylated forms of atypical PKCι and conventional PKC, but not phosphorylated novel PKCε or T286-autophosphorylated CaMKIIα.

(A) Left, above, representative immunoblots show increased amounts of activation-loop-phosphorylated PKCι (p-PKCι) and activation-loop-phosphorylated conventional PKC (p-cPKC) in PKMζ-cKO mice, compared to non-transgenic controls (NTC). Red, phospho-PKCs; green, total PKCs from the same samples. Mr markers shown at right. Below, activation-loop-phosphorylated p-PKCε (p-PKCε) recognized by its higher Mr, does not change. Right, mean± SEM (B) Levels of total CaMKIIα and T286-autophosphorylated CaMKIIα do not change in ζ-cKO hippocampus. Statistics in Figure 1—figure supplement 1—source data 1.

Figure 1—figure supplement 1—source data 1

Statistics for data presented in Figure 1—figure supplement 1.

https://cdn.elifesciences.org/articles/110499/elife-110499-fig1-figsupp1-data1-v1.docx
Figure 1—figure supplement 1—source data 2

Labeled immunoblots of Figure 1—figure supplement 1.

https://cdn.elifesciences.org/articles/110499/elife-110499-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 1—source data 3

Unlabeled immunoblots of Figure 1—figure supplement 1.

https://cdn.elifesciences.org/articles/110499/elife-110499-fig1-figsupp1-data3-v1.zip
Figure 1—figure supplement 2
Download asset Open asset
Actin loading controls for immunoblots shown in Figure 1B.

The two left columns are from a pair of samples (non-transgenic control, NTC and ζ-cKO) processed on two mini-blots run in parallel. The third column is from a different pair of samples processed on two mini-blots run in parallel. The fourth column is from a third pair of samples processed on three mini-blots run in parallel.

Figure 2
Download asset Open asset
Compensatory increases of PKCι during spatial memory in conditional PKMζ-knockout (ζ-cKO) mouse hippocampus.

(A) Left, schematic of active place avoidance training apparatus with a slowly rotating arena containing a nonrotating shock zone sector (shown in red). Visual cues located on the walls of the room are needed to avoid the shock zone. Right, experimental protocol. PKMζ is genetically ablated in Camk2a-CreERT2; Prkczfl/fl mice. Cre is activated using 4-OH tamoxifen (OH-TAM, 2 mg/200 µl i.p., one injection every other day for three doses). Control mice receive vehicle injections. Active place avoidance training begins 3 weeks later, and 1 week after training memory retention is tested in the absence of shock followed immediately by sacrifice and immunohistochemistry. (B) Immunohistochemistry shows ζ-cKO reduces PKMζ and increases PKCι in CA1 str. pyramidale (p) and radiatum (r), but not lacunosum-moleculare (lm) 1 week after training. Left above, PKMζ expression decreases in cell bodies and dendritic compartments of the PKMζ-cKO. Left below, PKCι expression increases in cell bodies as well as in dendritic compartments where it is ordinarily expressed at relatively low levels. DAPI staining of nuclei shown in blue. Bar = 50 µm. Right, mean± SEM. Student t-tests with Bonferroni corrections compared differences in PKMζ and PKCι expression separately in the CA1 strata (Figure 2—source data 1). (C) Compensatory spatial memory in ζ-cKO. Left, representative paths during first 10 min of pretraining, training trial 3, and 1 day memory retention. Right, mean± SEM. Two-way ANOVA (treatment X training) revealed a significant effect of training (F1.662, 9.972 = 41.93, p<0.0001), but not an effect of treatment (F1, 6 < 0.001, p=1.0) or their interaction (F2, 12 = 0.48, p=0.6). Comparisons using Bonferroni-corrected tests revealed significant differences between pretraining and training (Trial 3) (p=0.002) and pretraining and retention (p=0.001), but no differences between training (Trial 3) and retention (p=0.1). Further comparisons of training in each treatment group separately revealed significant differences between pretraining and training (Trial 3) in both vehicle and 4-OH tamoxifen groups (p=0.02 and p=0.01, respectively) and between pretraining and retention in both vehicle and 4-OH tamoxifen groups (p=0.03 and p=0.05, respectively), confirming the treatment groups did not behave differently.

Figure 2—source data 1

Statistics for data presented in Figure 2B.

https://cdn.elifesciences.org/articles/110499/elife-110499-fig2-data1-v1.docx
Figure 3
Download asset Open asset
Compensatory increases of PKCι during hippocampal long-term potentiation (LTP) maintenance in Prkcifl/fl; Prkcz–/– mice.

(A) Left, schematic shows adeno-associated virus (AAV) expressing Cre by cytomegalovirus (CMV) promoter injected into one hippocampus of a Prkcifl/fl; Prkcz–/– mouse, and control AAV expressing eGFP by CMV promoter injected into the contralateral hippocampus. Hippocampal slices are prepared 3 weeks later. Right, representative images of PKCι-immunohistochemistry. Top row, adjacent slices from AAV-eGFP-injected (ζ-KO) hippocampus show PKCι persistently increases 3 h post-tetanization. Bottom row, adjacent slices from the AAV-Cre-injected (ι/ζ-dKO) hippocampus show low levels of PKCι that do not change post-tetanization. White boxes show str. radiatum regions of interest. DAPI staining of nuclei shown in blue. Bar = 100 µm. (B) Mean± SEM. The two-way ANOVA reveals the main effects of treatment (AAV-Cre [ι/ζ-dKO] vs. AAV-eGFP [ζ-KO], F1,13 = 154.61, p<0.0001, η2p = 0.92), and stimulation (high-frequency stimulation [HFS] vs. test stimulation, F1,13 = 26.61, p=0.0002, η2p = 0.67), and an interaction of treatment X stimulation (F1,13 = 24.78, p=0.0003, η2p = 0.66). Post-hoc analysis confirms that, compared to the ζ-KO control group, the intensity of PKCι immunoreactivity was significantly decreased in ι/ζ-dKO (p’s<0.0006 for both control and LTP), and increased in ζ-KO after HFS (p=0.0002). Intensity of residual PKCι immunoreactivity did not change in the ι/ζ-dKO between the control and HFS groups (p=0.9). ζ-KO, n’s=4; ι/ζ-dKO test, n=5; ι/ζ-dKO HFS, n=4.

Figure 4 with 1 supplement
Download asset Open asset
Impaired late-long-term potentiation (late-LTP) in ι/ζ-dKO hippocampus.

(A) Late-LTP is absent in ι/ζ-dKO hippocampus. Above left inset, schematic of intracranial injections of adeno-associated virus (AAV)-Cre recombinase and AAV-eGFP into separate hippocampi of a Prkcifl/fl; Prkcz–/– mouse. Middle inset, color-coded representative field excitatory postsynaptic potentials (fEPSPs) correspond to numbered times in the time course below. Below, filled red circles, AAV expressing Cre by CMV promoter and high-frequency stimulation (HFS) with two tetanic trains; open red circles, test stimulation of a second synaptic pathway within the hippocampal slice. HFS tetani shown at arrows. Open black circles, AAV expressing eGFP by CMV promoter with HFS; open gray circles, with test stimulation. Three-way mixed-design ANOVA reveals main effects of treatment (hippocampal injections of AAV-Cre [ι/ζ-dKO] vs. AAV-eGFP [ζ-KO], F1,20 = 8.45, p=0.0009, η2p = 0.30), and stimulation (HFS vs. test stimulation, F1,20 = 5.90, p=0.025, η2p = 0.23), as well as a 3-way interaction among treatment X stimulation X time (5 min average of pre-HFS and 3 h post-HFS, F1,20 = 12.68, p=0.002, η2p = 0.39). Post-hoc analysis confirms established LTP is not maintained in ι/ζ-dKO 3 h after HFS as compared to pre-HFS basal responses (p=0.7). Post-hoc analysis also confirms the control hippocampus maintains established LTP (p=0.0002). Test stimulation was unaffected by AAV-Cre or AAV-eGFP injections (p=0.9 and p=0.7, respectively). N’s=6. Right inset, ι/ζ-dKO by CaMKIIα promoter expression of Cre eliminates late-LTP. Three-way mixed-designed ANOVA reveals interaction between treatment (ζ-KO vs. ι/ζ-dKO) and stimulation (HFS vs. test stimulation, F1,14 = 6.62, p=0.02, η2p = 0.32), and a 3-way interaction among treatment, stimulation, and time (5 min pre-HFS and 3 h post-HFS, F1,14 = 8.56, p=0.01, η2p = 0.38). Post-hoc analysis confirms that compared to pre-HFS basal responses, LTP is not maintained in ι/ζ-dKO hippocampus 3 h post-HFS (p=0.8) and is maintained in the control hippocampus (p=0.003). Test stimulation was unaffected by AAV-Cre or AAV-eGFP injections (p=0.4 and p=0.9, respectively). ι/ζ-dKO HFS, n=5; ι/ζ-dKO test, n=4; ζ-KO HFS, n=5; ζ-KO test, n=4. (B) LTP does not persist in ι/ζ-dKO mice after stronger afferent stimulation with four tetanic trains. ANOVA with repeated measurements reveals main effects of time (5 min pre-HFS, 20 min post-HFS, and 3 h post-HFS, F2,14 = 20.51, p<0.0001, η2p = 0.75). Post-hoc analysis confirms that early-LTP is established in both ι/ζ-dKO and control groups (5 min pre-HFS vs. 20 min post-HFS, p=0.005 and 0.002, respectively), and no difference between these two groups at 20 min post-HFS (p=0.6). However, LTP in ι/ζ-dKO did not persist 3 h (5 min pre-HFS vs. 3 h post-HFS, p=0.4), whereas LTP is intact in control (p=0.008). ι/ζ-dKO, n=5; control, n=4.

Figure 4—figure supplement 1
Download asset Open asset
Effects of adeno-associated virus (AAV) expression of Cre recombinase by the CaMKIIα promoter in Prkcifl/fl; Prkcz–/– and Prkcifl/fl; Prkcz+/+ mice.

(A) Representative immunohistochemistry of AAV expressing Cre by CaMKIIα promoter in Prkcifl/fl; Prkcz–/– mice shows loss of PKCι in ι/ζ-dKO hippocampus and compensatory increase in PKCι during long-term potentiation (LTP) maintenance in control eGFP-injected hippocampus. Left, schematic of sites of injection; right, PKCι immunohistochemistry. DAPI staining of nuclei shown in blue. Bar = 100 µm. (B) ι-cKO (AAV with CaMKIIα promoter expressing Cre in hippocampus of Prkcifl/fl; Prkcz+/+ mice) shows compensation of early-LTP, as previously described (Sheng et al., 2017). The repeated measurement ANOVA reveals the main effect of LTP (5 min average of pre-high-frequency stimulation (pre-HFS), 20 min post-HFS, and 2 h post-HFS, F2,6 = 14.03, p=0.005, η2p = 0.82) in ι-cKO mice. Post-hoc tests confirm that LTP was established at 20 min post-tetanization (5 min pre-HFS vs. 20 min post-HFS, p=0.006) and maintained for 2 h (5 min pre-HFS vs. 120 min post-HFS, p=0.009); n=4.

Figure 5
Download asset Open asset
Impaired long-term memory and intact short-term memory for spatial information in mice with bilateral hippocampal ι/ζ-dKO.

(A) Experimental protocol. Prkcifl/fl; Prkcz–/– mice are injected bilaterally in hippocampus with adeno-associated virus (AAV)-Cre (ι/ζ-dKO) or AAV-eGFP (ζ-KO, control), and 3 weeks later they received pretraining and, after 1 day, a single 30 min trial repeated daily for a total of three trials. Long-term retention is tested without shock 1 day after the last training trial. (B) ι/ζ-dKO does not affect short-term memory in the first training trial. Left, ι/ζ-dKO does not affect short-term memory as assessed by the time to enter the shock zone for the first eight entries (all animals had up to at least eight entries in trial 1). ANOVA with repeated measurements finds the main effects of training (pretraining and trial 1, F1,28 = 35.19, p<0.00001, η2p = 0.56) indicating trial 1 learning, time to entry (the first to eighth entry within a trial, F7,196 = 145.80, p<0.00001, η2p = 0.84), and their interaction (F7,196 = 37.68, p<0.00001, η2p = 0.57). However, there is no group effect (AAV-eGFP- and AAV-Cre-injected, F1,28 = 0.19, p=0.7, η2p = 0.007) nor interaction with either training or time to each entry (F’s<0.66, p’s>0.60, η2p’s<0.02). Right, ι/ζ-dKO does not affect short-term memory as assessed by maximum avoidance time during the first training trial. The contrast analysis reveals that the increases of maximum avoidance time from pretraining to trial 1 are not different between AAV-eGFP-injected and AAV-Cre-injected groups (t14=1.91, p=0.08, d=1.91). Paired t-tests reveal trial 1 is greater than pretraining in each genotype (t’s>3.10, p’s<0.018, Cohen’s d’s>1.62), indicating both groups of mice successfully established short-term memory. In contrast, the improvement of maximum avoidance time from trial 1 to trial 3 are different between the groups (t14=2.93, p=0.01, d=2.88), suggesting the two groups performed differently between daily training sessions when between-day memory influences avoidance. In addition, the ANOVA with repeated measurement discovers no group effect (AAV-eGFP-injected vs. AAV-Cre-injected, F1,14 = 0.56, p=0.47, η2p = 0.04), but significant effects of trial (F3,42 = 30.37, p<0.0001, η2p = 0.68), and interaction (F3,42 = 2.93, p=0.04, η2p = 0.17). Post-hoc tests confirm that the maximum avoidance time in trial 1 is not different between the two groups (p=0.14). The AAV-eGFP-injected group improved its performance between trial 1 and trial 3 (p=0.0002), whereas the AAV-Cre-injected group showed no improvement (p=0.2; n’s=8). These data indicate no differences in short-term memory between AAV-eGFP- and AAV-Cre-injected groups, but only the AAV-Cre-injected failed to improve between daily trials, suggesting inability to retain avoidance memory across days. (C) PKCι gene ablation impairs long-term memory in Prkcifl/fl; Prkcz–/– mice. Left, representative paths during 10 min of pretraining, at end of training trial 3, and 1 day memory retention. Right, mean± SEM. The ANOVA with repeated measurement finds main effects of group (AAV-eGFP vs. AAV-Cre, F1,14 = 10.53, p=0.006, η2p = 0.43) and training phase (pretraining, trial 3 of training, retention, F2,28 = 7.65, p=0.002, η2p = 0.35). Post-hoc analysis reveals that the mice with AAV-Cre-injected ι/ζ-dKO hippocampus perform poorer during the memory retention test, compared to AAV-eGFP-injected littermates (p=0.02). The mice with ι/ζ-dKO hippocampus show no difference between the memory retention test and pretraining trial (p=0.9), whereas the AAV-eGFP-injected mice show long-term memory is maintained (p=0.02; n’s=8). In addition, pretraining vs. training trial 3 was significantly different in ζ-KO (p=0.006), but not in ι/ζ-dKO (p=0.4).

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)C57BL/6 JThe Jackson Laboratory000664
Strain, strain background (Mus musculus, C57BL/6)Prkcz-/-Messing Lab (Lee et al., 2013)
Strain, strain background (Mus musculus, C57BL/6)Prkcifl/flGhosh Lab (El Ellam et al., 2024)
Strain, strain background (Mus musculus, C57BL/6)Prkczfl/flGhosh Lab (Mercau et al., 2024)
Strain, strain background (Mus musculus, C57BL/6)Prkcifl/fl; Prkcz-/-Ghosh Lab (Scott et al., 2019)
Strain, strain background (Mus musculus)B6;129S6-Tg(Camk2a-cre/ERT2)1Aibs/JThe Jackson Laboratory012362
Genetic reagent (AAV2.9)pENN.AAV.CMVs.PI.Cre.rBGAddgene105537
Genetic reagent (AAV2.9)pAAV.CMV.PI.EGFP.WPRE.bGHAddgene105530
Genetic reagent (AAV2.9)pENN.AAV.CamKII 0.4.Cre.SV40Addgene105558
AntibodyPKMζ (C2) (Rabbit polyclonal)Sacktor Lab (Hernandez et al., 2003)WB (1:20,000)
IHC (1:1000, Figure 2; 1:8000 to confirm ζ-null in other Figures)
AntibodyPKCι (E-7) (Mouse monoclonal)Santa Cruzsc-376344WB (1:200)
IHC (1:500)
AntibodyPKCι (C83H11) (Rabbit monoclonal)Cell Signaling Technology2998WB (1:200)
IHC (1:1000)
AntibodyPKCι (Mouse monoclonal)BD Transduction Laboratories610207WB (1:200)
AntibodyPhospho-T410 PKCζ (H2) (Mouse monoclonal)Santa Cruzsc-271962WB (1:200)
AntibodyPhospho-PKC (pan) (ζ Thr410) (190D10) (Rabbit monoclonal)Cell Signaling Technology2060WB (1:100)
AntibodyPhospho-PKCζ (Thr410)/λ (Thr412) (Rabbit polyclonal)Cell Signaling Technology9378WB (1:200)
AntibodyPKCα (H-7) (Mouse monoclonal)Santa Cruzsc-8393WB (1:200)
AntibodyPKCα (Rabbit polyclonal)Gibco3191SAWB (1:200)
AntibodyPKCβI (E-3) (Mouse monoclonal)Santa Cruzsc-8049WB (1:500)
AntibodyPKCβII (F-7) (Mouse monoclonal)Santa Cruzsc-13149WB (1:100)
AntibodyPKCβII (Rabbit polyclonal)Sacktor Lab (Sacktor et al., 1993)WB (1:100)
AntibodyPKCγ (C-4) (Mouse monoclonal)Santa Cruzsc-166385WB (1:200)
AntibodyPKCγ (C-19) (Rabbit polyclonal)Santa Cruzsc-211WB (1:500)
AntibodyPKCδ [EPR17075] (Rabbit monoclonal)Abcamab182126WB (1:100)
AntibodyPKCδ (G-9) (Mouse monoclonal)Santa Cruzsc-8402WB (1:50)
AntibodyPKCε (E-5) (Mouse monoclonal)Santa Cruzsc-1681WB (1:1000)
AntibodyPKCε (Rabbit polyclonal)Messing Lab (Lee et al., 2013)WB (1:1000)
AntibodyPKCη [EPR18513] (Rabbit monoclonal)Abcamab179524WB (1:200)
AntibodyPKCθ (E-7) (Mouse monoclonal)Santa Cruzsc-1680WB (1:100)
AntibodyCaMKIIα (Mouse monoclonal)ThermoFisher (Invitrogen)137300WB (1:100)
Antibodyp-CaMKII [T286] (Rabbit monoclonal)Cell Signaling Technology3361WB (1:100)
AntibodyActin (Mouse monoclonal)SigmaA4700WB (1:5000)

Additional files

MDAR checklist
https://cdn.elifesciences.org/articles/110499/elife-110499-mdarchecklist1-v1.pdf
Source data 1

Numerical data for Figures 1—5, Figure 1—figure supplement 1, Figure 4—figure supplement 1B.

https://cdn.elifesciences.org/articles/110499/elife-110499-data1-v1.xlsx

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Panayiotis Tsokas
  2. Changchi Hsieh
  3. Alejandro Grau-Perales
  4. Andrew Tcherepanov
  5. Leo Kwok
  6. Laura Rodriguez-Valencia
  7. David A Cano
  8. Kim Allen
  9. Hannah J Smith
  10. Sabina Kubayeva
  11. Benson J Wei
  12. Samuel Sabzanov
  13. Rafael Flores-Obando
  14. Sourav Ghosh
  15. Peter John Bergold
  16. Jerry Rudy
  17. James Cottrell
  18. André Fenton
  19. Todd Charlton Sacktor
(2026)
PKMζ-PKCι/λ double-knockout demonstrates atypical PKC is crucial for the persistence of hippocampal LTP and spatial memory
eLife 15:RP110499.
https://doi.org/10.7554/eLife.110499.3