Supplementary information PKMζ maintains memories by regulating GluR2- dependent AMPA receptor trafficking Paola Virginia Migues, Oliver Hardt, Dong Chuan Wu, Karine Gamache, Todd Charlton Sacktor, Yu Tian Wang and Karim Nader
5 45 Freezing response (%) 4 35 3 25 2 15 1 5 Scr-ZIP ZIP Supplementary Figure 1. ZIP does not impair the ability to reacquire auditory fear conditioned memories. ZIP or its inactive scrambled version (Scr-ZIP) was infused into the amygdala 7 days after fear conditioning. Memory was tested 1 day later. Fifteen days after the first test, rats were again fear conditioned and tested the following day. Values represent the mean percentage of the rats freezing time to the tone during the second memory test. Five rats were used for each treatment group. Error bars are s.e.m. Freezing times of rats infused with ZIP were similar to those injected with Scr-ZIP, as determined by One way ANOVA (F(1,8)=.466, p=.98).
PKMζ inhibition (%) 1 8 6 ZIP ZIP + GluR23Y 4 2 1 2 3 4 [ZIP] (μm) Supplementary Figure 2. Representative experiment showing the effect of ZIP alone and ZIP + GluR23Y (15 pmoles/1 µl) on the inhibition of PKMζ activity. The half-maximal inhibitory concentration (IC5) of ZIP on PKMζ activity (1.92 ±.34 µm), was not changed by GluR23Y (1.76 ±.6 µm) (p=.83 t-test, n=3).
GluR2 GluR1 PSD PSD PSD95 PSD Supplementary Figure 3. Separation of surface from intracellular proteins in subcellular fractions by biotinylation and neutravidin pull-down. Representative Western blots with molecular weight markers (MW) for GluR1, GluR2, and PSD95 in the biotin-bound (surface) and unbound (intracellular) fractions in PSDs and extrasynaptic membranes. The intracellular protein PSD95 was used as a control to validate that biotin did not bind intracellular proteins and to show PSD95 enrichment in the PSD fraction compared to the extrasynaptic fraction. Results are representative of 3 experiments.
Scr-GluR23Y +ZIP Scr-GluR23Y + Scr-ZIP Relative percentage 12 1 8 6 4 2 Scr-GluR23Y + Scr-ZIP Scr-GluR23Y +ZIP Supplementary Figure 4. Postsynaptic GluR2 levels in untrained rats after PKMζ inactivation. Scr-GluR23Y was infused into the amygdala 1 day after the rats were placed in the training context without receiving a shock. ZIP or its corresponding inactive scrambled (Scr-ZIP) was infused 1 h later. Rats were placed in the testing context 1 day after the infusions and killed 1 day later for BLA extraction (A, B). Representative Western blot (left) with molecular weight marker (MW) and quantification of GluR2 protein levels (right) in the postsynaptic densities fractions from the BLA. Data were normalized to the Scr-GluR23Y-Scr-ZIP group mean. Bars represent the group means and error bars are s.e.m. There was no significant difference between the groups (F(1,9)=1.165 p=.38) as determined by one-way ANOVA.
8 Freezing response (%) 7 6 5 4 3 2 1 Veh+Scr-ZIP Veh+ZIP AP5+ZIP AP5+Scr-ZIP Supplementary Figure 5. Blocking NMDA activation does not prevent the memory impairment induced by PKMζ inactivation. AP5 (.25 mg/ml) or vehicle (Veh) was infused into the amygdala 1 day after the training session. ZIP or its corresponding inactive scrambled peptide (Scr-ZIP) was infused 1 h later. Memory was tested 1 day after the infusions. Data represent the mean percentage of the freezing time during the tone. Error bars are s.e.m. Both Veh+ZIP and AP5+ZIP infusions decreased the freezing response (p=.34, p=.4, respectively). There was no difference between Veh+ZIP and AP5+ZIP (p=1.) as determined by the Kruskal Wallis analysis of ranks test.
GluR2 Postsynaptic GluR1 Supplementary Figure 6. Full length Western blots with molecular weight markers (MW) from which the blots shown in Fig. 2 were cropped. The membranes were cut below the kd band of the marker prior to antibody.