Effects of 5-HT and insulin on learning and memory formation in food-deprived snails

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Highlights

  • One-day food-deprived snails that exhibit the best CTA learning had a low 5-HT content in their CNS.

  • Five-day food-deprived snails, which did not learn, had a high level of 5-HT content in their CNS.

  • Insulin rescues the CTA deficit and this may be due to a decrease in the 5-HT content in the CNS.

Abstract

The pond snail Lymnaea stagnalis learns conditioned taste aversion (CTA) and consolidates it into long-term memory (LTM). How well they learn and form memory depends on the degree of food deprivation. Serotonin (5-HT) plays an important role in mediating feeding, and insulin enhances the memory consolidation process following CTA training. However, the relationship between these two signaling pathways has not been addressed. We measured the 5-HT content in the central nervous system (CNS) of snails subjected to different durations of food deprivation. One-day food-deprived snails, which exhibit the best learning and memory, had the lowest 5-HT content in the CNS, whereas 5-day food-deprived snails, which do not learn, had a high 5-HT content. Immersing 1-day food-deprived snails in 5-HT impaired learning and memory by causing an increase in 5-HT content, and that the injection of insulin into these snails reversed this impairment. We conclude that insulin rescues the CTA deficit and this may be due to a decrease in the 5-HT content in the CNS of Lymnaea.

Introduction

Serotonin (5-hydroxytryptamine: 5-HT) drives both feeding behavior and food satiety in the pond snail Lymnaea stagnalis (Dyakonova et al., 2015, Hatakeyama and Ito, 1999, Kawai et al., 2011, Kemenes et al., 1989, Kemenes et al., 1990, Yamagishi et al., 2015, Yamanaka et al., 2000, Yeoman et al., 2008). The cerebral giant cells (CGC) in Lymnaea, which are 5-HTergic, play key roles in mediating various aspects of feeding (Yeoman et al., 2008). Interestingly and consistent with the proposed role played by 5-HT in feeding and food satiation in Lymnaea are the findings that the activity of 5-HT-containing neurons in the central nervous system (CNS) are sensitive to the concentration of glucose in the hemolymph (Dyakonova et al., 2015, Dyakonova et al., 2015). Thus, the biophysical properties (e.g., membrane potential and spontaneous firing frequency) of the 5-HT containing neurons involved with the mediation of locomotion are altered by changes in hemolymph glucose concentration in vitro. Furthermore, the exogenous application of glucose to the isolated CNS causes a decrease in excitatory input to these 5-HTergic neurons (Chistopolsky and Sakharov, 2003, Dyakonova and Sakharov, 2001).

5-HT has long been proposed to play a key role in the mediation of learning and memory in molluscs. The best known example of this is dishabituation and sensitization of the gill withdrawal reflex in Aplysia is mediated by the release of 5-HT from interneurons (Kandel, 2001). In addition, 5-HT likely plays an important role in the reconsolidation of memory in Aplysia as well as in other forms of neuronal plasticity (Cai et al., 2012, Hawkins, 2013). In the terrestrial snail Helix lucolum, 5-HT signaling alters the defensive conditioning (Andrianov et al., 2015). As regards 5-HT and memory in Lymnaea, 5-HT plays a key role in the enhancement of LTM following operant conditioning of aerial respiration with exposure of the snail to a predator scent (Il-Han, Janes, & Lukowiak, 2010). In a taste-aversive learning in Lymnaea, administration of a 5-HT receptor antagonist before retention tests brings about the phenomenon of reversible amnesia (Nikitin & Solntseva, 2013). Acting as a neurotransmitter/neuromodulator, 5-HT in Lymnaea also plays important roles in the mediation of both locomotion and cardiovascular activity (Buckett et al., 1990, Tsyganov and Sakharov, 2000).

Previously, we have also shown that another neuromodulator/neurotransmitter, an insulin-like molecule, plays an important role in conditioned taste aversion (CTA) and its subsequent LTM formation (Murakami, Okada, Sadamoto, et al., 2013). There is an up-regulation of molluscan insulin-related peptides (MIPs), following CTA training (Azami et al., 2006). In addition, application of MIPs or mammalian insulin to the isolated CNS caused long-lasting changes in synaptic enhancement at sites thought to play key roles in CTA learning and LTM formation. This synaptic enhancement was blocked by an application of an insulin receptor antibody (Murakami, Okada, Sadamoto, et al., 2013). An injection of the same antibody into snails before CTA training, while not blocking learning, did block CTA-LTM formation (Murakami, Okada, Sadamoto, et al., 2013). Thus, insulin is thought to function in the long-term maintenance of synaptic enhancement in CTA learning-related circuits. The mechanisms by which insulin in snails acts in the formation of CTA-LTM have also been examined (Hatakeyama et al., 2013, Kojima et al., 2015, Mita et al., 2014, Mita et al., 2014, Murakami et al., 2013).

Insulin plays an important role in cognitive function across species. For example, human clinical studies suggest a link between type 2 diabetes mellitus (i.e., insulin resistance) and cognitive dysfunction (Biessels and Reagan, 2015, Heni et al., 2015, Mainardi et al., 2015). The role played by 5-HT in cognitive functioning is complicated and difficult to parse (González-Burgos and Feria-Velasco, 2008, Olivier, 2015, Olvera-Cortés et al., 2013, Voigt and Fink, 2015). Even more complicated is the relationship between the insulin signaling pathway and the 5-HT signaling pathway in mammals (Gerozissis, 2008). Thus, we posit that it may be easier to determine in Lymnaea how the two systems interact to mediate cognitive processes.

With CTA training, Lymnaea learn to associate their innate response to a substance such as sucrose (i.e., an increase in the number of feeding bites) with an aversive stimulus (e.g., electric shock) that suppresses biting behavior such that following CTA conditioning sucrose now no longer signals an increased feeding response. This CTA learning then is consolidated into CTA-LTM (Ito et al., 1999, Ito et al., 2013, Ito et al., 2015, Kojima et al., 1996, Kojima et al., 1998, Sunada et al., 2017). To produce CTA in snails, an appetitive stimulus (e.g., sucrose) is used as the conditioned stimulus (CS), and an aversive stimulus (e.g., electric shock) is used as the unconditioned stimulus (US). The US causes the snails immediately inhibit feeding (Kojima et al., 2001, Kojima et al., 1997). In the CTA training procedure, the CS is paired with the US. After repeated CS-US temporal pairings, the CS no longer elicits feeding, and this learned CTA response persists for at least a month (Kojima et al., 1996).

Furthermore, competency to learn and form CTA-LTM is dependent on the level of food satiation of the snail. Thus, 1-day food-deprived snails learn and form CTA-LTM better than food-satiated snails or 5-day food-deprived snails (Mita et al., 2014, Mita et al., 2014). Correlated with these aforementioned behavioral data are measurements of dopamine and octopamine in the CNS in snails subjected to the different lengths of food deprivation (Aonuma et al., 2016, Aonuma et al., 2017).

In the present study, we examined the relationship between the 5-HT content and insulin signaling in the snail CNS following CTA training. We found that there was a small range of 5-HT concentrations where feeding occurred. On either end of this range feeding behavior was compromised. In typically competent snails (i.e., 1-day food deprived), poorer CTA-LTM was coincident with altered 5-HT levels. Injection of insulin in these snails recused the snails’ ability to learn and form CTA-LTM.

Section snippets

Snails with various food-deprivation states

Specimens of Lymnaea stagnalis (Linnaeus, 1758) with a 15–25 mm shell obtained from our snail-rearing facility (original stocks from Vrije Universiteit Amsterdam, The Netherlands) were used. The rearing methods have previously been described (Sunada et al., 2017, Wagatsuma et al., 2004). To examine the effects of food-deprivation on CTA learning and subsequent LTM formation, we employed four cohorts of snails: (1) Snails that were in the act of eating just before the commencement of the CTA

Contents of 5-HT and related molecules in the CNS

Four separate cohorts of snails in four different satiated/hungry states were used (Fig. 1A): (1) Snails that were in the act of eating just before the commencement of the 5-HT measurement (i.e., Eating snails); (2) Snails that had been given ad libitum access to food but were not eating just prior to the measurement (Day −1 snails); (3) Snails that had been food-deprived for 1 day (Day 1 snails); and (4) Snails that had been food-deprived for 5 days (Day 5 snails). We measured the contents of

Discussion

Day 1 snails that exhibit the best CTA learning and memory formation were found to have the lowest CNS contents of 5-HTP, 5-HT, Nac-5-HT and 5-HIAA (Fig. 2, Fig. 3). Moreover, when the CNS 5-HT content was increased in the Day 1 snails, their response to the CS was decreased and they exhibited poorer learning and memory formation (Figs. 4B and 7). Further, when the 5-HT signaling cascade was blocked by the two 5-HT receptor antagonists, the feeding response was also decreased. These results

Conflict of interest

The authors declare no competing or financial interests.

Author contributions

H.A. and E.I. planned the experiments. V.E.D. and K.L. helped to design the experiments. H.A., Y.T., M.K., R.N. and E.I. performed the experiments. H.A., Y.T., M.K., W.T., D.H. and E.I. analyzed the data. Y.T. prepared the figures. H.A., W.T., D.H., V.E.D., K.L. and E.I. wrote the paper.

Funding

This work was supported by a grant from Network Joint Research Center for Materials and Devices in Japan [2015001 to H.A. and E.I.], KAKENHI grants from the Japanese Society for the Promotion of Science [24657055 and 25291074 to E.I.], Waseda University grants for Specific Research Projects in Japan [2016B-068, 2016B-069 and 2016S-037 to E.I.], and a grant from the Natural Sciences and Engineering Research Council of Canada [227993-2013 to K.L.].

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