PKM-ζ is not required for hippocampal synaptic plasticity, learning and memory

Abstract

Long-term potentiation (LTP), a well-characterized form of synaptic plasticity, has long been postulated as a cellular correlate of learning and memory. Although LTP can persist for long periods of time, the mechanisms underlying LTP maintenance, in the midst of ongoing protein turnover and synaptic activity, remain elusive. Sustained activation of the brain-specific protein kinase C (PKC) isoform protein kinase M-ζ (PKM-ζ) has been reported to be necessary for both LTP maintenance and long-term memory. Inhibiting PKM-ζ activity using a synthetic zeta inhibitory peptide (ZIP) based on the PKC-ζ pseudosubstrate sequence reverses established LTP in vitro and in vivo. More notably, infusion of ZIP eliminates memories for a growing list of experience-dependent behaviours, including active place avoidance, conditioned taste aversion, fear conditioning and spatial learning. However, most of the evidence supporting a role for PKM-ζ in LTP and memory relies heavily on pharmacological inhibition of PKM-ζ by ZIP. To further investigate the involvement of PKM-ζ in the maintenance of LTP and memory, we generated transgenic mice lacking PKC-ζ and PKM-ζ. We find that both conventional and conditional PKC-ζ/PKM-ζ knockout mice show normal synaptic transmission and LTP at Schaffer collateral-CA1 synapses, and have no deficits in several hippocampal-dependent learning and memory tasks. Notably, ZIP still reverses LTP in PKC-ζ/PKM-ζ knockout mice, indicating that the effects of ZIP are independent of PKM-ζ.

Figure 1. Normal LTP, gross brain morphology, and PKC isoform expression in conventional PKC-ζ/PKM-ζ KO mice

a, Hippocampal regions stained with DAPI. b, Southern blot analysis of wild-type, heterozygous (Het) and homozygous (KO) PKC-ζ/PKM-ζ mice. c, Protein expression of PKC isoforms from whole-brain tissue. d, e, TBS-LTP (d; WT, n=11, 156±6% at 175–180 min; KO, n=9, 149±8%; P>0.5) and HFS-LTP (e; WT, n=14, 155±11% at 175–180 min; KO, n=13, 148±6%; P>0.5) are intact and maintained for 3 h in mice lacking PKM-ζ. Data represent mean±s.e.m. Scale bars, 0.5 mV (vertical), 5 ms (horizontal).

Figure 2. LTP is intact in conditional PKC-ζ/PKM-ζ knockout mice

a, Western blot (left) and quantification (right) of PKM-ζ protein reduction in conditional KO mice normalized to tubulin (WT, n=17, 101±6.5%; KO, n=20, 22±3.6%). b, c, TBS-LTP (b; WT, n=16, 180±10% at 175–180 min; KO, n=16, 177±9%; P>0.8) and HFS-LTP (c; WT, n=6, 154±11% at 175–180 min; KO, n=9, 148±10%; P>0.7) are intact and maintained for 3 h in mice with PKM-ζ conditionally deleted in adulthood. Data represent mean±s.e.m. Scale bars, 0.5 mV (vertical), 5 ms (horizontal).

Figure 3. ZIP is not specific for PKM-ζ

a, ZIP is equally effective at reversing established TBS-LTP in WT mice and mice lacking PKM-ζ. Before ZIP application (40 min post-LTP): WT, n=8, 166±12%; KO, n=6, 167±10%, P>0.9. 140 min after ZIP application: WT, 48±7%; KO, 60±18%, P>0.5. b, c, ZIP decreases both tetanized (HFS) and non-tetanized synaptic responses in WT mice (b) and mice lacking PKM-ζ (c). Before ZIP application (60 min post-LTP): WT, n=8, tetanized=181±8%, non-tetanized=102±4%; KO, n=8, tetanized=203±12%, non-tetanized=100±4%, P>0.15 WT vs KO tetanized or non-tetanized. 120 min after ZIP application: WT, tetanized=60±18%, non-tetanized=50±9%; KO, tetanized=67±14%, non-tetanized=52±11%, P>0.6 WT vs KO tetanized or non-tetanized. d, Myristoylated PKI peptide does not affect LTP or basal transmission (tetanized n=5, non-tetanized n=4). Data represent mean±s.e.m. Scale bars, 0.5 mV (vertical), 5 ms (horizontal).

Figure 4. Hippocampal-dependent learning and memory are intact in PKC-ζ/PKM-ζ KO mice

a, Trace fear conditioning elicits freezing in PKC-ζ/PKM-ζ KO (n=13) and WT (n=15) mice with no significant main effect of genotype (F_(1,546)=0.50, P>0.48). ITI, inter-trial interval. b, WT and PKC-ζ/PKM-ζ KO mice exhibit similar contextual freezing behaviour (24 h post-training, WT=51.5±5.7%, KO=39.3±5.7%, P_{WT vs} KO>0.16; significant elevation in post-training vs pre-training freezing, P<0.05 for WT and KO), but show little freezing in a novel context (48 h post-training, WT±9.3±1.8%, KO=8.5±1.9%, WT and KO P>0.05 vs pre-training freezing). c, Trace fear conditioning is unaffected in PKC-ζ/PKM-ζ KO mice 48 h after training (no significant main effect of genotype, F_{(1, 78)}=0.33, P>0.57). d, Mean escape latencies during Morris water maze training (WT n=21, KO n=17, no significant main effect of genotype, F_(1,252)=0.00, P>0.98). e, Representative swim paths during probe trials for WT and PKC-ζ/PKM-ζ KO mice. f, Percentage of time spent in each quadrant during probe trials. Both genotypes showed a significant preference for the target quadrant at 24 (WT n=21, KO n=17) and 72 h (WT n=17, KO n=15); P<0.001 (target quadrant vs Q2, Q3 or Q4). There was no significant main effect of genotype at 24 (F_(1,108)=0.79, P>0.38) or 72 h (F_(1,90)=0.26, P>0.61). g, Number of platform crossings during probe trials (24 h: WT=4.14±0.52, KO=4.64±0.59, P>0.5, 72 h: WT=5.47±0.8, KO=4.80±0.54, P>0.5). Data represent mean±s.e.m.