Skip to main content
. Author manuscript; available in PMC: 2017 Jul 7.
Published in final edited form as: Neurobiol Learn Mem. 2016 Jul 11;138:135–144. doi: 10.1016/j.nlm.2016.07.008

Fig. 2.

Fig. 2

Antisense blockade of new PKMζ synthesis prevents late-LTP and spatial long-term memory. (A) PKMζ-antisense selectively blocks activity-dependent PKMζ synthesis. Inset above, diagram of PKMζ mRNA shows the 5′- and 3′-untranslated regions (UTRs), the open reading frame (ORF), and translation initiation site (AUG). PKMζ-antisense sequence is displayed hybridized with its complementary sense sequence, located between positions −3 and +15 from the translation start site. PKMζ-antisense or scrambled oligodeoxynucleotides are introduced into slices by intracranial hippocampal injection or in bath as described in Section 2. Immunoblots of CA1 extracts from hippocampal slices are probed with antisera to PKMζ, PKCι/λ, eEF1A, and actin as loading control. LTP slices are frozen 30 min after tetanization, and controls from adjacent slices within each hippocampus (set at 100%) receive only test stimulation. Left, representative immunoblots; right, mean ± SEM. PKMζ: scrambled, tetanized vs. untetanized, n’s = 18, t17 = 3.49, P < 0.005; PKMζ-antisense, tetanized vs. untetanized, n’s = 18, t17 = 1.07, P = 0.30; PKMζ-antisense vs. scrambled, t34 = 2.70, P < 0.02; PKCι/λ: scrambled, tetanized vs. untetanized, n’s = 14, t13 = 2.76, P < 0.02; PKMζ-antisense, tetanized vs. untetanized, n’s = 6, t5 = 3.41, P < 0.02; PKMζ-antisense vs. scrambled, t18 = 0.42, P = 0.68; eEF1A: scrambled, tetanized vs. untetanized, n’s = 14, t13 = 4.04, P< 0.002; PKMζ-antisense, tetanized vs. untetanized, n’s = 14, t13 = 2.52, P<0.05; PKMζ-antisense vs. scrambled, t26 = 0.37, P = 0.71. (B) Late-LTP is blocked by PKMζ-antisense, but not by scrambled oligodeoxynucleotide injected in the contralateral hippocampus. Above, numbered, color-coordinated representative field excitatory postsynaptic potential (fEPSP) traces correspond to time points noted below. Below, filled black circles, antisense (n = 7); filled green circles, scrambled (n = 6); color-coordinated open circles are untetanized control pathways recorded within each slice (for average of fEPSPs at 145–150 post-tetanization, two-way ANOVA, oligodeoxynucleotide: F1,28 = 16.2, P = 0.002; stimulation: F1,28 = 39.2, P< 0.0001; interaction: F1,28 = 10.0, P< 0.05). (C) PKMζ-antisense blocks long-term memory formation. Left, representative paths. Right, mean ± SEM of active place avoidance training and 1-day memory retention. There is a significant effect of training phase (pretraining, training, retention) (F2,64 = 38.26, P < 0.001), treatment (uninjected, scrambled, antisense) (F2,32 = 21.29, P < 0.001), as well as their interaction (F4,64 = 9.02, P< 0.001). Retention in the antisense group is worse than each of the other groups (uninjected control, n = 11, scrambled, n = 13, antisense, n = 11, * indicates significance by Tukey post hoc test, both P’s < 0.01). (D) PKMζ antisense blocks training-induced PKMζ synthesis measured 1 day after 8-trial training. Rats were treated in trained and untrained pairs, and PKMζ in each trained rat was normalized by the amount in the untrained animal. Above, representative immunoblots; below, mean ± SEM. Scrambled, untrained vs. trained, n’s = 11, t10 = 2.56, P<0.05; PKMζ-antisense, untrained vs. trained, n’s = 7, t6 = 0.65, P = 0.54; PKMζ-antisense vs. scrambled, t16 = 2.16, P < 0.05.