Compensatory increases of atypical PKCι and conventional, but not novel PKCs, in conditional PKMζ-KO mouse hippocampus.

(A, B) Immunoblots of hippocampal extracts from Camk2a-CreERT2Prkczfl/flmice receiving tamoxifen (2 mg/200 µl i.p., 5 daily doses) to activate Cre recombinase selectively in excitatory neurons. Mice are sacrificed 7 days after the last dose. Left, representative immunoblots with Mr markers. Right, mean ± SEM. Significance by two sample Student t tests with Bonferroni correction denoted by *; not significant, n.s.; statistics in Table S1A,B. Tamoxifen is a partial PKC antagonist and may still be present after a week (O’Brian et al., 1985); therefore, 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. For clarity, the actin shown is for PKCs α, ι, and PKMζ immunoblots. (C) Immunohistochemistry shows ζ-cKO reduces PKMζ and increases PKCι in CA1 st. pyramidale (p) and radiatum (r), but not lacunosum-moleculare (lm) 1 week after training. Inset above, schematic of experimental protocol. PKMζ is deleted using Camk2a-CreERT2Prkczfl/flmice. Cre is activated using 4-OH tamoxifen (OH-TAM, 2 mg/200 µl i.p., 3 doses every other day). 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. Left above, immunohistochemistry reveals reduced PKMζ expression in cell bodies and dendritic compartments of the PKMζ-cKO. Left below, PKCι increases in cell bodies as well as in dendritic compartments where it is ordinarily expressed at low levels. Right, mean ± SEM. Student t tests with Bonferroni corrections compared differences in PKMζ and PKCι expression separately in strata of CA1 (Table S1C). DAPI staining of nuclei shown in blue. Bar = 50 µm.

Compensatory increases of PKCι during hippocampal late-LTP maintenance in Prkcifl/fl-Prkcz−/− mice.

(A) Left, schematic of experimental protocol shows AAV expressing Cre by CMV promoter injected into ipsilateral hippocampus of a Prkcifl/fl-Prkcz−/−mouse, and control AAV expressing eGFP in contralateral hippocampus. Hippocampal slices are prepared 3 weeks later. Right, representative images of PKCι-immunohistochemistry from adjacent slices in AAV-eGFP-injected (ζ-KO) hippocampus show PKCι persistently increases 3 h post-tetanization (top row), and low, unchanging levels of PKCι in the AAV-Cre-injected (ι/ζ-dKO) hippocampus (bottom row). White boxes show st. radiatum regions of interest. (B) Mean ± SEM. The two-way ANOVA reveals the main effects of treatment (AAV-Cre [ι/ζ-dKO] vs. AAV-eGFP [ζ-KO], F1,68 = 83.58, P < 0.00001, η2p = 0.55), and stimulation (HFS vs. test, F1,68 = 20.47, P = 0.00003, η2p = 0.23), and an interaction of treatment x stimulation (F1,68 = 9.09, P = 0.004, η2p = 0.12). 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 < 0.002 for both control and LTP in ι/ζ-dKO), and increased in ζ-KO after HFS (P = 0.00011, ζ-KO, n’s = 16, ι/ζ-dKO, n’s = 20). Intensity of PKCι immunoreactivity did not change in the ι/ζ-dKO between the control and HFS groups (P = 0.3). Bar = 100 µm.

ι/ζ-dKO hippocampus shows transient LTP, but not persistent LTP.

(A) Late-LTP is absent in ι/ζ-dKO hippocampus. Above left inset, schematic of intrahippocampal injections of AAC-Cre and AAV-eGFP. Middle inset, representative fEPSPs correspond to numbered times in time-course below. Below, filled red circles, AAC-Cre expressed by CMV promoter and HFS with 2 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 with HFS; open grey 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 when 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 recombinase 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 the stronger tetanization. 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). The ι/ζ-dKO, n = 5; control, n = 4.

Impaired spatial long-term memory and intact spatial short-term memory in mice with bilateral hippocampalι/ζ-dKO.

(A) Above, schematic of active place avoidance training apparatus shows a slowly rotating arena containing a nonrotating shock zone sector (delineated in red). Visual cues located on the walls of the room are needed to avoid the shock zone. Below, protocol for active place avoidance. Prkcifl/fl-Prkcz−/− mice are injected bilaterally in hippocampus with AAV-Cre (ι/ζ-dKO) or AAV-eGFP (ζ-KO, control). (B) 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), indicating both groups of mice successfully established short-term memory. However, 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. 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 their performance between trial 1 and trial 3 (P = 0.0002), whereas the AAV-Cre 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 group 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).

ζ-cKO increases activation-loop-phosphorylation state of atypical PKCι and conventional PKCs, but not novel PKCε or CaMKIIα autophosphorylation.

(A) Left, above, representative immunoblots show increases in activation-loop phosphorylated PKCι (p-PKCι) and conventional-PKCs (p-cPKCs) in PKMζ-cKO mice. Red, phospho-PKCs; green, total PKCs from the same samples. Mr’s shown at right. Below, 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 — table supplement 2.

Compensation for spatial memory in ζ-cKO mice.

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). Further 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). Moreover, comparison also 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.

(A) Representative immunohistochemistry of AAV expressing Cre-recombinase by CaMKIIα promoter in Prkcifl/fl-Prkcz−/−mice shows loss of PKCι in ι/ζ-cKO hippocampus and compensatory increase in PKCι during LTP maintenance in contralateral control eGFP-injected hippocampus. Left, schematic of sites of injection; right, PKCι immunohistochemistry. DAPI stains nuclei in CA1. Bar = 100 µm. (B) ι-cKO hippocampus shows compensated LTP. AAV expressing Cre-recombinase by CaMKIIα promoter was injected into Prkcifl/fl-Prkcz+/+mice. Loss of early-LTP is compensated in the PKCι-cKO as in previous reports (Sheng et al., 2017). Paired t-test reveals early-LTP is established at 20 min post-tetanization (t3 = 4.76, P = 0.02, Cohen’s d = 3.41, n = 4).

Statistics for data presented in Figure 1.

Significant differences with Bonferroni correction are in bold.

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

Significant differences with Bonferroni correction are in bold.