Chemotactic behavior analysis in planarian
To observe and quantify planarian chemical-sensing behavior, a tractable assay method for tracking chemotaxis behavior was developed. For this assay, we used liver-extract solution, the food used for culturing planarians in our laboratory, as chemoattractant (Figure 1A). We reasoned that if planarian recognized the chemoattractant and showed chemotaxis toward it, the chemoattractant should be present with a concentration gradient in the assay field. However, because we could not visualize or measure the concentration gradient of the chemoattractant(s) sufficient to induce the planarian chemotaxis, we instead used a bioassay we named the “persistence assay” (Figure 2A). The rationale for the “persistence assay” is that whereas planarians normally move in various directions and away from the original region (Zone 1) after being placed there, they would remain in the original region (Zone 1) if a concentration of chemoattractant sufficient for them to sense were present there. We preliminarily measured the chemoattractant's diffusion rate in the assay field to determine when we should start the analysis after adding the chemoattractant as follows. Ten μl of chicken liver extract as chemoattractant were placed in the center of a quadrant (Zone 4) of the assay chamber. After 0, 5, or 10 min, 3 μl of agar solution was transferred from point 1 or point 2 into the center of a quadrant (Zone 1) of a fresh assay chamber. Soon thereafter, a planarian was placed in Zone 1, and its behavior was observed. Figure 2B shows the time spent in Zone 1 during the 1-min assay period. Although at 0 min after adding the solution transferred from point 1, planarians moved away from Zone 1 and showed a low score of time spent there (percent of time ± SEM, 26.7 ± 2.9%), after 5 min planarians continuously stayed in Zone 1, and thus showed a higher score of time spent there (100 ± 0.0%) (Figure 2B). In contrast, planarians did not stay in Zone 1 even after 5 min (47.8 ± 8.31%), when the solution had been transferred there from point 2. However, after 10 min, they did stay in Zone 1 (84.3 ± 3.1%). In addition, we did not find any differences in locomotion among these planarians (data not shown). These results suggest that the chemoattractant reproducibly diffused throughout the entire assay chamber within 10 min after it was added, and that this bioassay using planarians is useful for directly and efficiently detecting the diffusion of a chemoattractant that induced planarian chemotaxis. Therefore, we decided to use the chemoattractant gradient field in the assay chamber 10 min after adding the chemoattractant to it in subsequent experiments.
Next, we tested the behavior of intact planarians in a normal field (uniform-field assay chamber without chemoattractant). Figure 3A shows the averaged movements of 11 planarians together with a heat map for these movements (in which warm colors indicate locations where much time was spent, and cool colors those where little time was spent), and indicates that planarians tended to move near the edge of the field. In contrast, planarians placed at the start region indicated by an open circle in a chamber with chemoattractant showed a preference to move toward the region where the chemoattractant had been dropped (cross in Figure 3B). However, headless planarians did not move toward the chemoattractant, and instead showed random movements around the start region indicated by a white circle in Zone 1, indicating that the head is required for chemotaxis (Figure 3C). In order to investigate whether planarians orient their movement up a gradient of chemoattractant, we analyzed the overall orientation (angle) of their movement in Zones 2 and 3 (except for the start and target quadrants). Intact animals in a chemoattractant gradient field showed orientation biased toward the chemoattractant (80.2% of their movement was directed toward the chemoattractant), whereas planarians in a uniform field (49.2%) and headless planarians (47.6%) did not show linear movement directed toward the chemoattractant, and instead showed random movements (Figure 3D).
Next, the average time (during a 600-sec test interval) spent by the animals in the target quadrant region where the chemoattractant had been placed was measured to assess the ability of animals to recognize attractant chemicals and move to the region where those chemicals were concentrated. Intact animals spent a large fraction of their time in the target zone after reaching it (62.7 ± 4.1%) (Figure 3E). In contrast, headless animals showed a much lower thermotaxis score (8.9 ± 5.1%) (Figure 3E). There was no difference in the speed of movement of planarians in the chemoattractant-gradient field (2.69 ± 0.12 mm/sec) and uniform field (2.58 ± 0.11 mm/sec), indicating that the difference of chemotaxis score between animals on a chemoattractant-gradient, and thus the difference of planarian movement between a uniform-field chamber and a plate containing chemoattractant, was not the result of acceleration of the locomotor activity by the chemoattractant (Figure 3F). These results indicate that this assay method is useful for quantitatively evaluating planarian chemotactic behavior, and that planarian chemotaxis is dependent on the head. Although decapitation inhibited planarians’ movement, and they frequently stopped, resulting slower value of velocity (0.60 ± 0.07 mm/sec) (Figure 3F), the 600-sec assay time was thought to be sufficiently long for planarians to move to the target quadrant, suggesting that the headless planarians may have lost chemotaxis rather than that they spent less time in the target quadrant due to slowing of their movement.
Analysis of the brain neurons involved in chemotaxis with Readyknock of synaptotagmin
The above data showed that headless planarian showed slower movement, and therefore we next analyzed various brain neurons to test whether the brain was required for chemotaxis. In order to perturb the activity of brain neurons, we performed regeneration-dependent conditional gene knockdown (Readyknock), which knocks down protein expression more severely in the differentiating cells in the regeneration blastema than in the pre-existing terminally differentiated cells [7,11], using dsRNA of the gene encoding the planarian synaptotagmin (Djsyt), which is involved in synaptic transmission [22] (Figure 4A). Immunohistochemical analysis revealed the presence of DjSYT in the axons in the brain and VNCs (Figure 4B). Next, Readyknock using Djsyt(RNAi) treatment caused severe reduction of the level of DjSYT protein only within the newly formed brain in the head seven days after amputation, whereas strong signals of the DjSYT protein were detected in the pre-existing VNCs in the trunk region (Figure 4C). Previous reports indicated that Djsyt(RNAi) planarians cannot distinguish the direction of light or a thermal-gradient, and moved randomly when they were exposed to light or temperature stimuli [7,11]. To investigate the brain functions involved in chemotaxis, the chemotactic behavioral assay was carried out after Readyknock with Djsyt(RNAi) and revealed that Djsyt(RNAi) planarians did not preferentially move toward a chemoattractant, although control animals did (Figure 4D). In order to investigate whether a lack of the activity of the brain neurons would impair the linear movement toward chemoattractant that was seen in control planarians, we analyzed the overall direction (angle) of movement in Zones 2, and 3 (except in the start and target quadrants). In control animals, almost all movements were directed toward the chemoattractant, whereas Djsyt(RNAi) planarians were clearly less able to orient their movement in the correct direction toward the chemoattractant, and instead showed random movements (Figure 4E). When we calculated the fraction of movements directed toward the chemoattractant by dividing the overall direction of movement into two directions—the angle toward chemoattractant (+180°) and that in the opposite direction (–180°)—in the control animals, 91.3% of their movement was directed toward the chemoattractant, whereas Djsyt(RNAi) animals showed 40.1% of their movement directed away from the chemoattractant (Figure 4E). Quantitative analysis of time spent in the target quadrant (Zone 4), where the concentration of chemoattractant was highest, clearly demonstrated that the loss of DjSYT in the brain inhibited planarian chemotaxis (Figure 4F), without causing any defect in locomotor activity (Figure 4G). These results strongly suggest that neural activity in the brain is required for planarian chemotactic behavior, and that the chemotaxis assay system is useful for analyzing the function of the planarian brain and nervous-system-related genes.
Thigmotactic/kinetic behavior analysis in planarians
Planarians show reactions through mechanical-tactile sensing to such stimuli as water flow, touch, and contact with objects [5,25]. To study such sensing, we next established a thigmotaxis/kinesis assay (Figure 1B). Figure 5A shows the averaged movements of 10 planarians together with a heat map of these movements, and indicates that planarians placed at a start region with a textured surface region showed a preference to move away from the textured surface region and move to a region with a smooth surface, and then stopped on the smooth region (Figure 5A). Note that intact animals that started from a textured surface region little spent time in the textured surface region of the assay plate opposite to that of the start region. In contrast, headless planarians showed random movements, and stopped without regard to the start-site condition, indicating that the head is required for thigmotaxis/kinesis (Figure 5A). Next, to investigate brain functions involved in thigmotaxis/kinesis, we performed a thigmotaxis/kinesis behavioral assay after Readyknock with Djsyt(RNAi). The results revealed that Djsyt(RNAi) planarians did not preferentially move to the smooth-surface region, although control animals showed normal thigmotactic/kinetic behavior (Figure 5B). In order to better analyze the data, we quantified behaviors by calculating the average time spent by the animals in the smooth-surface region during a 600-sec test period to assess the ability of animals to recognize the physical properties of the surface and to move to a smooth-surface region, and plotted the results graphically (Figure 5C). These analyses clearly indicate that intact animals spend a large fraction of their time in the target zone after reaching it (81.4 ± 14.2%) (Figure 5C), whereas headless animals show a much lower thigmotaxis/kinesis score (32.8 ± 7.3%) (Figure 5C). Similarly, quantification of the time spent in the smooth-surface region by Djsyt(RNAi) planarians clearly showed that although control RNAi animals showed normal thigmotactic/kinetic behavior (75.3 ± 14.3%) (Figure 5C), Djsyt(RNAi) animals moved randomly and stopped in a random manner, like headless planarians (37.5 ± 5.0%) (Figure 5C). This finding is consistent with the findings in our chemotaxis, phototaxis, and thermotaxis assays (Figure 4) [7,11].
Next, comparison of intact and control RNAi animals at seven days of regeneration revealed that the average time they spent in the smooth-surface region was nearly the same, indicating that the thigmotactic/kinetic behavior in planarian was fully recovered within seven days after amputation (Figure 5C). Furthermore, when we measured the number of times of that a planarian re-entered the textured-surface region once it had exited from that region (Figure 5D), the data showed that intact and control RNAi animals rarely re-entered the textured-surface region (average number of times ± SEM; number of individuals that re-entered the textured-surface region, 0.1 ± 0.1, 1/10; 0.1 ± 0.1, 1/10), whereas headless and Djsyt(RNAi) planarians frequently re-entered the textured-surface region (1.3 ± 0.4, 7/9; 1.7 ± 0.3, 8/9), indicating that planarians avoid textured surfaces, and that this behavior may require brain neural activity (Figure 5D). These results suggest that the thigmotaxis/kinesis assay system is useful for analyzing the function of the planarian brain and nervous-system-related genes, and that neural activity in the brain is required for several planarian behaviors, including chemotaxis, thigmotaxis/kinesis, phototaxis, and thermotaxis [7,11].
Prioritization among behaviors
Next, by combining assays of different behaviors, namely chemotaxis, phototaxis, thermotaxis, and thigmotaxis/kinesis, we examined the ability of planarians to integrate various stimuli. To determine the order of predominance of these planarian behaviors under specific conditions in this study using constant strengths of stimuli, at first we performed combinatorial assays in which two distinct stimuli were presented simultaneously to planarians. For these assays, a planarian was placed in the center position of a 60 × 30 × 10 mm assay chamber, and was given different stimuli from the two different ends, and then the number of planarians at a given position was measured after 600 sec.
When we compared behaviors in this combinatorial assay using planarians presented with both chemoattractant and 400 lux of light, planarians preferred to move toward the chemoattractant rather than escaping from the light, even though they received strong enough light (400 lux) to induce phototaxis in a single-stimulus assay (Figure 6A) [6]. The preference index for chemotaxis (95.0 ± 2.0%) clearly indicated that planarians predominantly showed chemotaxis rather phototaxis behavior (Figure 6A). Moreover, planarian chemotaxis was dominant over thermotaxis and thigmotaxis/kinesis (Figure 6B,C). Next, when phototaxis was compared to thermotaxis and thigmotaxis/kinesis, phototaxis was predominant over thermotaxis and thigmotaxis/kinesis (Figure 6D, E). Finally, when thermotaxis and thigmotaxis/kinesis were compared, the preference index of thermotaxis (80.0 ± 4.9%) was higher than that of thigmotaxis/kinesis (Figure 6F). The results of our analyses using these combinatory assay systems (chemotaxis, phototaxis, thermotaxis, and thigmotaxis/kinesis) revealed that one behavior tends to predominate when planarians receive two different stimuli (Figure 6A-F). In these combinatory experiments, planarians gave top priority to a chemical stimulus and second-highest priority to a light stimulus, and gave the lowest priority to a mechanical stimulus (Figure 6G). These data suggest that planarians may have the ability to integrate various different external kinds of information in the brain.
Establishment of a behavioral integration assay method to analyze brain function using two distinct external stimuli
It is thought that animals integrate multiple signals, and show appropriate behaviors after integrating their responses. Next, to investigate in more detail whether planarians prioritize different stimuli, and whether the order of predominance of behaviors in planarian is absolute, we employed an integrative assay that we developed to perform combinatory assays using two distinct stimuli. Our combinatory assay system using two distinct stimuli, light and chemoattractant, is illustrated in Figure 7A. The 60 × 10 × 3 mm assay chamber was divided into five zones to enable quantitative measurement of the planarian behaviors. When a planarian was placed into the center of the chamber (Zone 3), it moved randomly (Figure 7B). The heat map of planarian movement showing the time spent in each zone did not indicate any particular tendency of movement in this control condition, which was consistent with the above data shown in Figure 3A. When we assayed planarian phototaxis and chemotaxis using this chamber, planarians moved away from the 400-lux light source (Figure 7C), as previously described [6], and planarians moved toward Zone 1, where the chemoattractant (C.A.) had been added (Figure 7D), as seen in Figures 3 and 4. In addition, when planarians were exposed to 400 lux of light and chemoattractant simultaneously, they moved toward the region of the chemoattractant source (Zone 1) (Figure 7E). This result was also consistent with the above results (Figure 6A). However, by exposing planarians to different intensities (800 or 1600 lux) of light and exposing them to a constant dose of chemoattractant at the same time, it was found that planarians changed their preference and moved toward the dark side of the chamber, depending on the level of the stimuli (Figure 7E). These results suggest that planarians can change their behavioral features in response to a stimulus to which they are repeatedly exposed, and that planarian behavior can be manipulated. Figure 7F shows the time spent in the darkest zone (Zone 5) and in the chemoattractant-source zone (Zone 1), and this graph clearly indicates that we successfully manipulated the outcome of the integration process by changing the signal strength. When planarians were exposed to 800 lux of light and chemoattractant simultaneously, they seemed to move in a random way, as seen in control planarians without stimulation (Figure 7B). Consistently, there was no significant difference in spent time in Zone 1 or 5 between planarians with no stimulation and combined exposure to 800 lux of light and chemoattractant (Figure 7F). However, careful analysis of their trajectories revealed that each control planarian moved extensively in the assay chamber, whereas each planarian exposed to 800 lux of light and chemoattractant moved toward the chemoattractant or moved toward the dark region (Figure 7G). This suggests that planarians might decide upon a behavior strategy from among several possible candidate strategies, and they may select different strategies depending on the individual. These results suggest that planarians may decide their behavioral strategies via their brain function when exposed to multiple stimuli.