Protein synthesis required for long-term memory is induced by PKC activation on days before associative learning

Abstract

Protein synthesis has long been known to be required for associative learning to consolidate into long-term memory. Here we demonstrate that PKC isozyme activation on days before training can induce the synthesis of proteins necessary and sufficient for subsequent long-term memory consolidation. Bryostatin (Bryo), a macrolide lactone with efficacy in subnanomolar concentrations and a potential therapeutic for Alzheimer's disease, is a potent activator of PKC, some of whose isozymes undergo prolonged activation after associative learning. Under normal conditions, two training events with paired visual and vestibular stimuli cause short-term memory of the mollusc Hermissenda that lasts approximately 7 min. However, after 4-h exposures to Bryo (0.25 ng/ml) on two preceding days, the same two training events produced long-term conditioning that lasted >1 week and that was not blocked by anisomycin (1 mug/ml). Anisomycin, however, eliminated long-term memory lasting at least 1 week after nine training events. Both the nine training events alone and two Bryo exposures plus two training event regimens caused comparably increased levels of the PKC alpha-isozyme substrate calexcitin in identified type B neurons and enhanced PKC activity in the membrane fractions. Furthermore, Bryo increased overall protein synthesis in cultured mammalian neurons by up to 60% for >3 days. The specific PKC antagonist Ro-32-0432 blocked much of this Bryo-induced protein synthesis as well as the Bryo-induced enhancement of the behavioral conditioning. Thus, Bryo-induced PKC activation produces those proteins necessary and sufficient for long-term memory on days in advance of the training events themselves.

Fig. 1.

Effect of Bryo on long-term retention. Animals were trained with four and six paired conditioned stimulus/unconditioned stimulus TEs with Bryo (0.25 ng/ml). Bryo was added during dark adaptation (10 min) before training and continued for 4 h. All animals were tested with the conditioned stimulus alone at 4 h, then at 24-h intervals after training. Animals trained suboptimally but treated with Bryo all demonstrated long-term retention (n = 8-16, ANOVA, P < 0.01). Filled squares (dashed lines) are displaced slightly to the right for clarity. Lowest dashed line indicates previously observed effect of nine training trials.

Fig. 2.

Effects of blockers on retention. (A) Four-hour retention effects of Bryo under control and antagonist experimental regimes (NSW, normal sea water controls). R032, Ro-32-0432 blocks retention with six TE plus Bryo. (n = 4-8; ANOVA, P < 0.01.) (B) Effects of Bryo and anisomycin on retention after 4 h. Animals received two paired TEs in normal sea water (NSW), 0.25 ng/ml Bryo, or 0.25 ng/ml Bryo plus 1 ng/ml anisomycin. (n = 12, each group; two-way ANOVA, P < 0.01.)

Fig. 3.

Effect of two successive days of 4-h Bryo exposure (0.25 ng/ml) followed 1 day later by two TEs. Retention was measured up to 144 h after training. (n = 16; ANOVA, P < 0.01.)

Fig. 4.

Effect of three successive days of 4-h Bryo exposure (0.25 ng/ml) followed 1 day later by two TEs. Retention was measured over 96 h after training. Nonexposed animals (same as in Fig. 3) did not demonstrate any behavioral modification (no conditioned response to conditioned stimulus testing) (Upper). Anisomycin (1 μg/ml) administered immediately and remaining for 4 h after training to animals receiving the 3-day Bryo treatments did not prevent long-term retention (Lower). (n = 16; ANOVA, P < 0.01.) Note that the control data (Upper) are the same as in Fig. 3.

Fig. 5.

Dose-response curves for four and nine paired conditioned stimulus/unconditioned stimulus TEs. Bryo concentrations <0.50 ng/ml augmented acquisition and memory retention with suboptimal (four TE) training conditions. Those concentrations had no demonstrable effects on retention performance with nine paired TEs. However, with all training conditions tested, Bryo ≥1.0 ng/ml inhibited acquisition and behavioral retention (n = 16). Retention of four paired TE conditioning with 20-h preexposure to Bryo persisted for 24 h (n = 8; ANOVA at 48 h, P < 0.01).

Fig. 6.

Behavioral effects of Bryo and lactacystin. Animals were incubated simultaneously for 4 h with Bryo (0.25 ng/ml) with and without lactacystin (10 μM), and then 24 h later were conditioned with two paired conditioned stimulus/unconditioned stimulus TEs. Animals were subsequently tested with the conditioned stimulus alone at 4 h after training and then at 24-h intervals (n = 28, combined Bryo/lactacystin; n = 20, Bryo alone; n = 16, lactacystin alone).

Fig. 7.

Representative tissue sections from Hermissenda eyes immunolabeled with CE antibody. (A) Random presentations of the two stimuli (TEs) did not produce behavioral modifications or a rise in CE above normal background levels. Basement membrane and lens staining are artifacts associated with using vertebrate polyclonal antibodies. (B) Positive CE immunostaining occurred in B cell photoreceptors (^*B-Cell) of trained animals (two TEs) after 2 days' Bryo exposure intervals. (C) Intensity measures after nine random TEs or two exposures on successive days to Bryo (0.25 ng/ml), and then associatively followed by two paired TEs. Two exposures of Bryo coupled with two TEs significantly increased CE to levels associated with nine paired TEs and consolidated (long-term) memory (n = 4-8; t test, P < 0.01). CE immunostaining also resolved boutons within synaptic fields of photic-vestibular neurites (D). Arrows indicate arborization field between an interneuron (a), axon from a contralateral neuron (b), and terminal boutons of neurites from a putative photoreceptor (c). (Scale bars, 10 μm.) CPG, cerebropleural ganglion.

Fig. 8.

Effects of Bryo and training on CE immunostaining. (A) Immunointensity measurements (0-255) of CE antibody labeling. CE levels (0-255 relative units) increased >4.3 times with paired training (mean ± SE, n = 5 animals per treatment). 4RanTE, random control, four trials with random light and rotation; 6PTE, paired trials, six trials with paired light and rotation. 6PTE-0Bry vs. 6PTE-0.25Bry: P < 0.001; 4RTE-0.25Bry vs. 6PTE-0.25Bry: P < 0.001 (t test). (B) Effect of Bryo alone (without associative conditioning) administered for 4-h over each of 1, 2, and 3 days with and without two TEs on CE levels in the B photoreceptors of Hermissenda 24 h after each of the periods of Bryo exposures. (n = 16; ANOVA, P < 0.01.)

Fig. 9.

PKC activity in Hermissenda nervous systems after Bryo. Intact Hermissenda were exposed for 4-h intervals to Bryo (0.28 nM) on successive days as described in the text. PKC activity in isolated circumesophageal nervous systems was then measured in the cytosol fraction. (Left) Cytosol. (Right) Particulate fraction. (n = 6-12 for each measurement; ^*, P < 0.05; ^**, P < 0.01, two-tailed t test).

Fig. 10.

Effect of Bryo on membrane-bound PKC activity in hippocampal cultured IGF-IR cells after a single 30-min exposure (Upper) or two 30-min exposures separated by intervals of 30 min to 8 h (Lower) (n = 3-6 for each measurement; ^*, P < 0.05; ^**, P < 0.01, two-tailed t test). Two 30-min exposures separated by intervals of 2 or 3 h (but not 4 h) caused PKC down-regulation in the cytosol (n = 3-6 for each group; P < 0.05 and P < 0.01, respectively, two-tailed t test).

Fig. 11.

Effect of Bryo on protein synthesis. (Upper) Rat IGF-IR cells were incubated for 30 min with 0.28 nM Bryo for incubation times ranging from 1 to 79 h. [³⁵S]Methionine (9.1 μCi) was then added to the medium followed by analysis of radiolabel as described in Materials and Methods. (Lower) Rat IGF-IR cells were incubated as above in the presence or absence of 100 nM Ro-32-0432. (n = 6 for each measurement; ^*, P < 0.05; ^**, P < 0.01, two-tailed t test.)