Critical roles of nicotinic acetylcholine receptors in olfactory memory formation and retrieval in crickets

Abstract

Acetylcholine (ACh) is a major excitatory neurotransmitter in the insect central nervous system, and insect neurons express several types of ACh receptors (AChRs). AChRs are classified into two subgroups, muscarinic AChRs and nicotinic AChRs (nAChRs). nAChRs are also divided into two subgroups by sensitivity to α-bungarotoxin (α-BGT). The cricket Gryllus bimaculatus is one of the useful insects for studying the molecular mechanisms in olfactory learning and memory. However, the roles of nAChRs in olfactory learning and memory of the cricket are still unknown. In the present study, to investigate whether nAChRs are involved in cricket olfactory learning and memory, we tested the effects of two different AChR antagonists on long-term memory (LTM) formation and retrieval in a behavioral assay. The two AChR antagonists that we used are mecamylamine (MEC), an α-BGT-insensitive nAChR antagonist, and methyllycaconitine (MLA), an α-BGT-sensitive nAChR antagonist. In crickets, multiple-trial olfactory conditioning induced 1-day memory (LTM), whereas single-trial olfactory conditioning induced 1-h memory (mid-term memory, MTM) but not 1-day memory. Crickets injected with MEC 20 min before the retention test at 1 day after the multiple-trial conditioning exhibited no memory retrieval. This indicates that α-BGT-insensitive nAChRs participate in memory retrieval. In addition, crickets injected with MLA before the multiple-trial conditioning exhibited MTM but not LTM, indicating that α-BGT-sensitive nAChRs participate in the formation of LTM. Moreover, injection of nicotine (an nAChR agonist) before the single-trial conditioning induced LTM. Finally, the nitric oxide (NO)-cGMP signaling pathway is known to participate in the formation of LTM in crickets, and we conducted co-injection experiments with an agonist or inhibitor of the nAChR and an activator or inhibitor of the NO-cGMP signaling pathway. The results suggest that nAChR works upstream of the NO-cGMP signaling system in the LTM formation process.

Keywords: cricket; long-term memory; mecamylamine; methyllycaconitine; nicotine; nicotinic acetylcholine receptors; olfactory learning.

FIGURE 1

MLA impairs LTM, while MEC impairs both MTM and LTM (A) Effects of pre-training application of methyllycaconitine (MLA) and mecamylamine (MEC) on long-term memory (LTM) retention. At 20 min prior to 3-trial conditioning to associate an odor with water reward, crickets in five groups were each injected with 3 µL of saline or saline containing 10 μM MLA, 100 μM MLA, 100 µM MEC or 1 mM MEC. The time schedule of the experiment is shown above the figure. Relative preference between the rewarded odor and control odor was tested before and at 1 day after training. (B) Effects of post-training application of MLA and MEC on LTM retention. At 20 min after 3-trial conditioning, crickets in three groups were each injected with 3 µL of saline or saline containing 100 µM MLA or 1 mM MEC. Relative preference between the rewarded odor and control odor was tested before and at 1 day after training. (C) Effects of pre-training application of MLA and MEC on medium-term memory (MTM) retention. At 20 min prior to 3-trial conditioning, crickets in three groups were each injected with 3 µL of saline or saline containing 100 µM MLA or 1 mM MEC. Relative preference between the rewarded odor and control odor was tested before and at 1 h after training. Preference indexes (PIs) for the rewarded odor before (white boxes) and after (grey boxes) training are shown as box and whisker diagrams. The individual data was color-coded according to the CS used for conditioning (apple: black dot, banana: open circle). Odor preferences before and after training were compared by the WCX test. The results of statistical comparisons are shown by asterisks (***p < 0.001, **p < 0.01, *p < 0.05, NS p > 0.05). The number of animals tested is shown at each data point in this figure and in subsequent figures.

FIGURE 2

MEC impairs acquisition, STM, and LTM of MER conditioning, while MLA impairs LTM At 20 min before appetitive conditioning, crickets in three groups were each injected with 3 µL of saline (saline group: black circles), saline containing 100 µM MLA (MLA group: gray diamonds) or 1 mM MEC (MEC group: open squares). In all groups, peppermint odor was applied as CS for approximately half of the crickets, and apple odor was applied as CS for the other half. Since the results from these two sub-groups did not significantly differ, datasets were pooled within each group (see Supplementary Figure S2). (A) Acquisition performance of appetitive conditioning. The percentage of MER (%MER) during a 3-s period of CS presentation prior to US presentation is shown. (B) Retention performance at around 10 min after conditioning. In the retention test, each cricket was tested with the CS and the novel odor separated by a 4-min interval. The saline and MLA groups exhibited a significantly higher %MER to the CS (black bars) than that to the novel odor (gray bars), indicating that the memory is CS-specific. In contrast, in the MEC group, %MER to the CS was as low as that to the novel odor, indicating no CS-specific short-term memory (STM). (C) Retention performance at 1 day after conditioning. The saline group exhibited significantly higher %MER to the CS (black bar) than that to the novel odor (gray bar), indicating that the memory is CS-specific. In contrast, in the MLA group and the MEC group, %MER to the CS was as low as that to the novel odor, indicating no CS-specific LTM. A repeated measures ANOVA was used for within-group comparison of %MER during acquisition. McNemar’s test was used for pairwise comparison of %MER between the CS and the novel odor in the retention test. Fisher’s exact test was used for pairwise comparison of %MER of different groups in each conditioning trial. The results of statistical comparisons are shown by asterisks (***p < 0.001, **p < 0.01, *p < 0.05, NS p > 0.05).

FIGURE 3

MEC impairs retrieval of LTM Crickets in three groups were each subjected to 3-trial appetitive conditioning. One day after the training, they were each injected with 3 µL of saline or saline containing 100 µM MLA or 1 mM MEC. Relative preference between the rewarded odor and control odor was tested before training (pre-training test), at 22 h after training (before the injection test) and then at 20 min after drug injection (after the injection test). Preference indexes (PIs) for the rewarded odor before training (white boxes), before injection (light gray boxes) and after injection (dark gray boxes) are shown as box and whisker diagrams. The individual data was color-coded according to the CS used for conditioning (apple: black dot, banana: open circle). Odor preferences before and after training were compared by the WCX test. The results of statistical comparisons are shown by asterisks (***p < 0.001, **p < 0.01, NS p > 0.05, adjusted by Holm’s method).

FIGURE 4

Nicotine application paired with single-trial conditioning induces LTM (A) Effects of nicotine injection prior to 1-trial conditioning on LTM formation. At 20 min prior to 1-trial conditioning, crickets in four groups were each injected with 3 µL of saline or saline containing 1 µM nicotine, 10 µM nicotine or 100 µM nicotine. Relative preference between the rewarded odor and control odor was tested before training and at 1 day after training. (B) Effects of nicotine injection after 1-trial conditioning on LTM. At 20 min after 1-trial conditioning, crickets were injected with 3 µL of saline containing 10 µM nicotine. (C) Effects of nicotine injection prior to the 1-day memory retention test. At 20 min before the retention test 1 day after single-trial conditioning, crickets were injected with 3 µL of saline containing 10 µM nicotine. Preference indexes (PIs) for the rewarded odor before (white boxes) and after (gray boxes) training are shown as box and whisker diagrams. The individual data was color-coded according to the CS used for conditioning (apple: black dot, banana: open circle). Odor preferences before and after training were compared by the WCX test. The results of statistical comparisons are shown by asterisks (**p < 0.01, NS p > 0.05).

FIGURE 5

The nAChRs act upstream of the NO-cGMP signaling in the LTM formation process (A) Effects of SNAP, NOR-3, and 8br-cGMP paired with 1-trial conditioning on 1-day retention. Crickets in three groups were each injected with 3 µL of saline containing SNAP (200 µM), NOR-3 (40 µM) or 8br-cGMP (200 µM) at 20 min prior to single-trial conditioning. Relative preference between the rewarded odor and control odor was tested before training and at 1 day after training. (B) Effects of co-injection of an agonist/antagonist of nAChR and an inhibitor/accelerator of NO-cGMP signaling on 1-day retention. Crickets in four groups were individually co-injected at 20 min before single-trial conditioning with 3 µL of saline containing one of the following pairs of chemicals: nicotine (10 µM) and L-NAME (400 µM), nicotine (10 µM) and ODQ (200 µM), NOR-3 (40 µM) and MLA (100 µM), and 8br-cGMP (200 µM) and MLA (100 µM). Relative preference between the rewarded odor and control odor was tested before and at 1 day after training. PIs for the rewarded odor before (white boxes) and after (grey boxes) training are shown as box and whisker diagrams. The individual data was color-coded according to the CS used for conditioning (apple: black dot, banana: open circle). Odor preferences before and after training were compared by the WCX test. The arrows in the graph (A) indicate the results of comparison by the M-W test, between that group and the saline group in Figure 1A, at 1 day after training. The results of statistical comparisons are shown by asterisks (***p < 0.001, **p < 0.01, *p < 0.05, NS p > 0.05).