Acute disruption of the NMDA receptor subunit NR1 in the honeybee brain selectively impairs memory formation

Abstract

Memory formation is a continuous process composed of multiple phases that can develop independently from each other. These phases depend on signaling pathways initiated after the activation of receptors in different brain regions. The NMDA receptor acts as a sensor of coincident activity between neural inputs, and, as such, its activation during learning is thought to be crucial for various forms of memory. In this study, we inhibited the expression of the NR1 subunit of the NMDA receptor in the honeybee brain using RNA interference. We show that the disruption of the subunit expression in the mushroom body region of the honeybee brain during and shortly after appetitive learning selectively impaired memory. Although the formation of mid-term memory and early long-term memory was impaired, late long-term memory was left intact. This indicates that late long-term memory formation differs in its dependence on NMDA receptor activity from earlier memory phases.

Figure 1.

Detection of the NR1 subunit by Western blot. A, Detection of the NR1 subunit in the MB and the AL regions of the honeybee brain and in rat brain with NR1-pan. B, The specificity of NR1-pan was evaluated in a peptide competition assay, on a honeybee brain extract. The membrane was cut between ∼79 and ∼47 kDa (dashed lines). In the control experiment, the membranes were incubated with NR1-pan or α-tubulin antibodies (C). In the peptide competition assay, the NR1-pan and α-tubulin antibodies were preincubated with the antigenic peptide (Ab/P) before the incubation on the membranes. C, Detection of the NR1 subunit in the honeybee brain with AmNR1. The NR1 subunit was detected with NR1-pan, with the AmNR1 preimmune serum (PIS), and with the AmNR1 serum (AmNR1). Arrows indicate proteins detected with AmNR1 that were not detected with PIS. mw, Molecular weight in kilodaltons.

Figure 2.

Evaluation of the RNAi effect. Quantification of relative NR1 subunit levels in the MB and AL regions 1 or 2 d after injection of dsRNA (gray bars, dsNEG; white bars, dsNR1) or siRNA (gray bars, siNEG; white bars, siNR1) is shown. The expression levels were evaluated by quantitative Western blot, using NR1-pan (A, D) or AmNR1 (B, C, E, F). Each column represents the mean ± SEM of n measurements as indicated by the numbers on the bars. Asterisks indicate a significant reduction (*p < 0.05, Student's t test).

Figure 3.

Evaluation of the RNAi effect in conditioned honeybees. A, One day after the injection of dsRNA (gray squares, dsNEG; white squares, dsNR1), animals were subjected to three CS–US pairings (paired, A1–A3) or to three unpaired CS–US presentations (unpaired, A1–A3). n represents the number of animals that participated in the experiment. Brains were dissected 2 h later. PER, Percentage of animals that showed a PER during the CS presentation. B, The relative NR1 subunit levels were quantified in the MB region after injection of dsRNA (gray bars, dsNEG; white bars, dsNR1). The expression levels were evaluated by quantitative Western blot, using AmNR1. Each column represents the mean ± SEM of n measurements as indicated by the numbers on the bars. Asterisks indicate significant differences between groups (**p < 0.01, χ² test; *p < 0.05, Student's t test).

Figure 4.

The inhibition of the NR1 subunit selectively affects memory formation. One day after the injection of dsRNA (A; gray, dsNEG; white, dsNR1) or siRNA (B; gray, siNEG; white, siNR1), animals were subjected to three CS–US pairings (A1–A3). Memory was retrieved 2 h, 2 and 3 d after conditioning. In the dsRNA experiment, the animals were tested only once. Data from the acquisition phase were pooled for all subgroups (inset, n). The numbers on the bars represent the number of animals tested for each time point. In the siRNA experiment, n animals received multiple posttraining tests. Asterisks indicate significant differences between groups (*p < 0.05; **p < 0.01; ***p < 0.001; χ² test). PER, Percentage of animals that showed a PER during the CS presentation.

Figure 5.

Evaluation of the behavioral effect by considering only learner honeybees. The behavioral data of honeybees conditioned 1 d after the injection of dsRNA (A; gray, dsNEG; white, dsNR1) or siRNA (B; gray, siNEG; white, siNR1) were analyzed by considering only animals that showed a PER during the CS presentation of the third acquisition trial (A3). Their memory performances were evaluated 2 h after conditioning. The number of considered animals is indicated in the inset. Asterisks indicate significant differences between groups (*p < 0.05, χ² test). PER, Percentage of animals that showed a PER during the CS presentation.

Figure 6.

The RNAi effect is reversible. Two days after the injection of dsRNA (A; gray, dsNEG; white, dsNR1) or siRNA (B; gray, siNEG; white, siNR1), animals were subjected to three CS–US pairings (A1–A3). Memory was retrieved 2 h, 2 and 3 d after conditioning. In the dsRNA experiment, the animals were tested only once. Data from the acquisition phase were pooled for all subgroups (inset, n). The numbers on the bars represent the number of animals tested for each time point. In the siRNA experiment, n animals received multiple posttraining tests. PER, Percentage of animals that showed a PER during the CS presentation.

Figure 7.

The inhibition of the NR1 subunit does not affect the recall of memory. A, Two days after conditioning, animals were given injections of dsRNA (gray, dsNEG; white, dsNR1). One day later, the NR1 expression levels were evaluated in the MB and AL regions. The NR1 levels were evaluated by quantitative Western blot, using AmNR1. Each column represents the mean ± SEM of n measurements as indicated by the numbers on the bars. The asterisk indicates a significant reduction (*p < 0.05, Student's t test). B, Animals were subjected to three CS–US pairings (A1–A3). Two days after conditioning, they were given injections of dsRNA, and memory was retrieved on day 3. The number of animals is indicated in the inset. PER, Percentage of animals that showed a PER during the CS presentation.