The Role of Serotonin in the Influence of Intense Locomotion on the Behavior Under Uncertainty in the Mollusk Lymnaea stagnalis

Abstract

The role of serotonin in the immediate and delayed influence of physical exercise on brain functions has been intensively studied in mammals. Recently, immediate effects of intense locomotion on the decision-making under uncertainty were reported in the Great Pond snail, Lymnaea stagnalis (Korshunova et al., 2016). In this animal, serotonergic neurons control locomotion, and serotonin modulates many processes underlying behavior, including cognitive ones (memory and learning). Whether serotonin mediates the behavioral effects of intense locomotion in mollusks, as it does in vertebrates, remains unknown. Here, the delayed facilitating effects of intense locomotion on the decision-making in the novel environment are described in Lymnaea. Past exercise was found to alter the metabolism of serotonin, namely the content of serotonin precursor and its catabolites in the cerebral and pedal ganglia, as measured by high-performance liquid chromatography. The immediate and delayed effects of exercise on serotonin metabolism were different. Moreover, serotonin metabolism was regulated differently in different ganglia. Pharmacological manipulations of the serotonin content and receptor availability suggests that serotonin is likely to be responsible for the locomotor acceleration in the test of decision-making under uncertainty performed after exercise. However, the exercise-induced facilitation of decision-making (manifested in a reduced number of turns during the orienting behavior) cannot be attributed to the effects of serotonin.

Keywords: decision-making; effect of exercise on the brain; goal-directed locomotion; mollusks; serotonin.

FIGURE 1

Schematic representation of the experimental procedure. (A) Procedure for the investigation of the effects of enhanced motor activity (exercise, E, EE) and rest after exercise (ER), redrawn after (Korshunova et al., 2016; Dyakonova et al., 2019). In double-blind experiments, snails were divided into control and experimental groups and resided in similar light conditions before being placed in the test arena. Control snails were kept in deep water so they could use ciliary locomotion for 2 h in similar light conditions. Two control groups were used: snails taken directly from their breeding colony (Ch, marked with green color) and snails that were kept for 2 h in 300 ml containers filled with the same water as in the breeding stock (Cn, marked with blue color). The last group was used to test whether novelty by itself produces locomotor arousal in snails. The experimental and control animals were investigated in parallel in one experiment. Snails of the ‘exercise’ experimental group (E, marked with red color) were kept in a tank (25 × 50 cm) filled with a thin (1 mm) layer of water which protected them from drying but forced them to use crawling (intense muscular locomotion) to compensate for the lack of water supporting the weight of the shell for 2 h (E, marked with light red color) or 4 h (EE, marked with dark red color). Snails of the “rest after exercise” experimental group (ER, marked with purple color) were placed into in a cylinder filled with water up to 9 cm to be able to use ciliary locomotion for 2 h after 2 h of exercise in low water. In different experimental series, the combination of control and experimental groups could vary, for details see Methods and Results. (B) Behavioral paradigm for the investigation of decision-making in a novel environment. One or four snails (in different experimental series) were placed on the same central points of a square arena on a flat and dry plastic surface with one-side asymmetric illumination. All snail movements were tracked and video-recorded for 15 min in each experiment. The camera was placed under the transparent plastic bottom of the arena. For the behavioral analysis, the virtual arena border (22 cm from the central point) was defined. C, example of one record from four simultaneously tracked snails. In their behavior, the two clearly distinct phases can be seen: (1) uncertain movements, characterized by comparatively low speeds of movement, stops and repeated changes of movement direction and (2) an intense fast locomotion in a chosen direction as previously reported by Korshunova et al. (2016).

FIGURE 2

Effects of novelty and “exercise then rest” on the behavior of snails on a dry surface. (A) The latency of the first movement in the new environment, in seconds. (B) The sum of turns prior to decision-making, in degrees. (C) The speed of locomotion, cm/min. (D) Time to the finish line (virtual arena borders), in seconds. Left to right in each diagram: green color indicates snails that were taken from the breeding stock (Ch, n = 30); blue color indicates snails that were kept for 2 h in a cylinder filled with water up to 9 cm (Cn, n = 30); purple color indicates snails that were placed for 2 h in low water to exercise and then rested for 2 h in a container filled with water up to 9 cm (ER, n = 31); The significance of differences was tested using the multiple comparisons Kruskal–Wallis test, followed by post hoc tests. All values are given as median with the lower and upper quartiles * indicates p < 0.05.

FIGURE 3

Overlay tracks of snails the locomotor arousal in control and “exercise then rest.” (A) Snails that were taken from the breeding colony (Ch). (B) Snails that were kept for 2 h in a cylinder filled with water up to 9 cm (Cn). (C) Snails that were placed for 2 h in low water to exercise and then rested for 2 h in a container filled with water up to 9cm (ER). Tracks presented in C show that ER snails spent less time in the central zone and made fewer turns than Ch and Cn snails although the dark/light choice was not different between Ch, Cn, and ER. In (B), the effect of novelty can be seen as well, the rotational behavior is weaker in (B) than in (A). (D) Locomotor arousal in the Ch, Cn, and ER groups: the mean speed of locomotion by equal time intervals (1–2 min, 2–4 min, 4–6 min, 6–8 min, 8–10 min).

FIGURE 4

Acute and delayed effects of intense locomotion on the content of serotonin and its catabolites within the pedal ganglia of Lymnaea measured with high-performance liquid chromatography with electrochemical detection (pmol). (A) Left to right: Serotonin (5-HT); a precursor of 5-HT (5-hydroxytryptophan, 5-HTP, see also below in a higher scale); N-acetylserotonin, Nac-5-HT; 5-hydroxyindole acetoaldehyde, 5-HIAA. (B) The magnified scale for 5-HTP content shown in (A) second to the left. Blue color indicates control snails that were kept in a new cylinder filled with water up to 9 cm for 2 h (Cn, n = 13); light red color indicates snails that were placed for 2 h in low water to exercise (E, n = 11); purple color indicates snails that were placed for 2 h in low water to exercise and then rested for 2 h in a container filled with water up to 9 cm (ER, n = 13); dark red color indicates snails that were placed for 4 h in low water to exercise (EE, n = 12). The significance of differences was tested using the multiple comparisons Kruskal-Wallis test, followed by post hoc tests. All values are given as median with the lower and upper quartiles *, **, *** indicate p < 0.05, 00.1 and 0.001, respectively.

FIGURE 5

Acute and delayed effects of intense locomotion on the content of serotonin and its catabolites within the cerebral ganglia of Lymnaea measured with high-performance liquid chromatography with electrochemical detection (pmol). Left to right: Serotonin (5-HT); a precursor of 5-HT (5-hydroxytryptophan, 5-HTP); N-acetylserotonin, Nac-5-HT; 5-hydroxyindole acetoaldehyde, 5-HIAA. Blue color indicates control snails that were kept in a cylinder filled with water up to 9 cm for 2 h (Cn, n = 13); light red color indicates snails that were placed for 2 h in low water to exercise (E, n = 14); purple color indicates snails that were placed for 2 h in low water to exercise and then rested for 2 h in a container filled with water up to 9 cm (ER, n = 13); dark red color indicates snails that were placed for 4 h in low water to exercise (EE, n = 12). The significance of differences was tested using the multiple comparisons Kruskal-Wallis test, followed by post hoc tests. Values are given as median with the lower and upper quartiles *, ** indicate p < 0.05 and 0.01, respectively.

FIGURE 6

Effects of serotonin precursor 5-HTP on the behavior of snails on a dry surface. (A) The latency (in seconds) of the first movement. (B) The sum of turns prior to decision-making, in degrees. (C) The mean speed of locomotion (in cm/min). (D) Time to the finish line (crossing of the virtual arena border) in seconds. The significance of differences was tested using the Mann–Whitney U-test. All values are given as median with the lower and upper quartiles * indicates p < 0.05.

FIGURE 7

Effects of serotonin (5-HT) on the behavior of snails on a dry surface. (A) The latency (in seconds) of the first movement. (B) Total rotation prior to decision-making, in degrees. (C) The mean speed of locomotion (in cm/min) measured during the phase of the directional crawl (6–8 min from the beginning of the experiment). (D) Time to the finish line (crossing of the virtual arena border) in seconds. The significance of differences was tested using the Mann–Whitney U-test. The significance of differences was tested using the Mann–Whitney U-test. All values are given as median with the lower and upper quartiles * indicates p < 0.05.

FIGURE 8

Effects of the serotonin receptor antagonist ketanserin 0.1 mM on the behavior of snails on a dry surface. (A) The latency (in seconds) of the first movement. (B) The sum of turns prior to decision-making, in degrees. (C) The mean speed of locomotion (in cm/min). (D) Time to the finish line (crossing of the virtual arena border) in seconds. The significance of differences was tested using the Mann–Whitney U-test. All values are given as median with the lower and upper quartiles * indicates p < 0.05.

FIGURE 9

Overlay tracks of snails from the control group injected with saline and the experimental group injected with 0.1 mM ketanserin. In the group injected with ketanserin (lower image, red tracks), the intermediate direction choices (semi-circular routes) can be seen which never occurred in any other groups. The tracks of experimental snails are also obviously shorter since the speed of locomotion was lower in ketanserin-treated animals.