Planarian shows decision-making behavior in response to multiple stimuli by integrative brain function
- Takeshi Inoue†1Email author,
- Hajime Hoshino†1,
- Taiga Yamashita1,
- Seira Shimoyama1 and
- Kiyokazu Agata1
© Inoue et al.; licensee BioMed Central. 2015
Received: 26 September 2014
Accepted: 27 November 2014
Published: 1 February 2015
Planarians belong to an evolutionarily early group of organisms that possess a central nervous system including a well-organized brain with a simple architecture but many types of neurons. Planarians display a number of behaviors, such as phototaxis and thermotaxis, in response to external stimuli, and it has been shown that various molecules and neural pathways in the brain are involved in controlling these behaviors. However, due to the lack of combinatorial assay methods, it remains obscure whether planarians possess higher brain functions, including integration in the brain, in which multiple signals coming from outside are coordinated and used in determining behavioral strategies.
In the present study, we designed chemotaxis and thigmotaxis/kinesis tracking assays to measure several planarian behaviors in addition to those measured by phototaxis and thermotaxis assays previously established by our group, and used these tests to analyze planarian chemotactic and thigmotactic/kinetic behaviors. We found that headless planarian body fragments and planarians that had specifically lost neural activity following regeneration-dependent conditional gene knockdown (Readyknock) of synaptotagmin in the brain lost both chemotactic and thigmotactic behaviors, suggesting that neural activity in the brain is required for the planarian's chemotactic and thigmotactic behaviors. Furthermore, we compared the strength of phototaxis, chemotaxis, thigmotaxis/kinesis, and thermotaxis by presenting simultaneous binary stimuli to planarians. We found that planarians showed a clear order of predominance of these behaviors. For example, when planarians were simultaneously exposed to 400 lux of light and a chemoattractant, they showed chemoattractive behavior irrespective of the direction of the light source, although exposure to light of this intensity alone induces evasive behavior away from the light source. In contrast, when the light intensity was increased to 800 or 1600 lux and the same dose of chemoattractant was presented, planarian behaviors were gradually shifted to negative phototaxis rather than chemoattraction. These results suggest that planarians may be capable of selecting behavioral strategies via the integration of discrete brain functions when exposed to multiple stimuli.
The planarian brain processes external signals received through the respective sensory neurons, thereby resulting in the production of appropriate behaviors. In addition, planarians can adjust behavioral features in response to stimulus conditions by integrating multiple external signals in the brain.
KeywordsPlanarian Chemotaxis Thigmotaxis Thigmokinesis Higher brain function Decision-making Integration RNAi Readyknock
As an animal survives under exposure to many kinds of stimuli, its nervous system detects sensory cues and converts this information into adaptive movement. For behaviors in response to a simple stimulus, sensory neurons sometimes communicate directly with motor neurons; however, when animals are exposed to more complex stimuli, integration of sensory information should be necessary to decide the appropriate behavior. Furthermore, integration of sensory information in this neural machinery is essential for choosing an animal's behavioral strategy based on the context and on the animal's memory, and such integration enables animals to refine their behaviors. Although some of the specific neuronal processing activities that encode neuronal activation into a behavioral response have been extensively studied, much remains to be understood about how to these processing activities decide an adaptive behavioral strategy under multiple environmental signals.
Planarians are free-living platyhelminths, and belong to an evolutionarily early group possessing a CNS that includes a brain with simple architecture, i.e., a bi-lobed brain composed of around 2.0 ~ 3.0 x 104 neurons in a planarian of length about 8 mm [1-3]; and their brain consists of several functional and structural domains defined by the discrete expression of homeobox genes, with a surprisingly complex set of expressed genes, sophisticated neural networks, and neural modulators that are quite similar to those used by mammals . In addition, planarians can sense a variety of environmental signals, and rapidly display distinct responsive behaviors depending on the type of signal, such as light or temperature [5-7], conveyed through sensory neurons projecting to their brain [1,2]. Despite the increasing knowledge that has been gained recently about the morphogenesis of the planarian brain and its robust regenerative ability [4,8], examination of the function of the planarian brain at the molecular level has just begun [6,7,9-11]. Research during the past two decades using molecular and cellular techniques has shown that the planarian brain is divided into several functional and structural domains that are composed of several neural subtypes, and that it uses many neurotransmitters and neuronal modulators, such as glutamate, dopamine, serotonin, GABA, acetylcholine, and neuropeptides, that are quite similar to those used by mammals [12-16]. These findings indicate that analysis of the planarian brain with its structurally simple, but nevertheless well-organized, brain may provide a unique opportunity as an emerging good new model system to elucidate molecular mechanisms underlying the basis of brain function. However, it has been difficult until now to clarify the mechanisms of planarian higher brain function, including learning and memory, because of the lack of knowledge about the neural processing pathway(s) in the brain regulating the behavior in response to a particular stimulus or to multiple stimuli.
Planarians display stereotypical behaviors in response to external stimuli, for example, they display phototaxis, chemotaxis, thermotaxis, and thigmotaxis . Phototaxis and chemotaxis of planarians have been relatively well studied because of their association with morphologically well-characterized organs, namely the eyes and auricles, respectively [6,11,17]. The sensory organs of planarians are located in the head portion of the animal and send projections to the brain. The brain processes these signals and directs appropriate behavioral responses [6,11]. These findings clearly showed that planarian behavioral assays are useful for analyzing the CNS function. In this study, we focused on chemotaxis and thigmotaxis in addition to phototaxis and thermotaxis, and thereby assessed planarian behaviors that might reveal molecular and neural pathways in the brain involved in producing appropriate behavior in response to multiple signals.
Materials and methods
A clonal strain of planarian (Dugesia japonica), SSP, cultured at 23°C in tap water was used. Planarians were starved for at least one week prior to amputation, anesthetized by chilling on ice, and then cut. Planarians 7 mm in length were used for all experiments. All planarians were maintained and manipulated according to a protocol approved by the Animal Care and Use Committee of Kyoto University.
Assay for planarian behaviors
A schematic representation of the thigmotaxis assay system is shown in Figure 1B. The surface of two opposing quadrants of a 50-mm-diameter plastic Petri dish were sanded (“textured”) using #12 sandpaper, as shown in Figure 1B. Planarians were placed on these textured regions of the assay plate that had been covered with 5 ml of autoclaved tap water at 23°C. The thermotaxis assay was performed as described previously .
For assays to examine the priorities among the four planarian behaviors studied here (chemotaxis, phototaxis, thigmotaxis, and thermotaxis), two behaviors were tested simultaneously as follows. A planarian was placed in the center of the 60 × 30 × 10 mm assay chamber described above. A preference index was calculated as follows: preference index = (number of planarians in one particular region – number of planarians in the opposite region)/(total number of planarians in the assay chamber).
To assay for external stimuli integration, a planarian was placed in the middle of a 60 × 10 × 3 mm chamber constructed from glass . One ml of water containing 0.1% low melting point agarose was put in the container. Each planarian behavior was captured using a video recorder (Sony) placed above the container (Figure 1C) for the time indicated in the Results section. The trajectory of movement was analyzed with SMART software (Panlab) (Figure 1C) . All behavioral experiments were performed in a dark room with only a red light of a wavelength that cannot be sensed by planarians (Figure 1C) . Calculations based on the data obtained were performed using static ggplot2 package of R software .
Data were analyzed by determining the statistical significance of differences between test results as determined by Student's t test; p values greater than 0.05 were taken as not significant (ns).
Double-stranded RNA (dsRNA) was prepared as described previously [20,21]. For Readyknock , dsRNA was injected into the posterior intestinal duct of planarians using a Drummond Scientific Nanoject injector (Broomall, PA, USA). At four hours after injection, planarians were amputated posterior to the auricles and the resulting regenerants were used for analysis at seven days of regeneration. Control animals were injected with dsRNA for green fluorescent protein (GFP), a gene that is not found in planarians.
Whole-mount immunostaining was performed as described previously . Planarians were stained using the following dilutions of antibodies: 1/2000 anti-planarian synaptotagmin (anti-DjSYT) , 1:1000 anti-planarian arrestin , 1:2000 anti-G-protein β subunit (anti-DjGβ) , or 1:2000 anti-planarian tyramine beta-hydroxylase (anti-DjTBH) , in 10% goat serum in 0.1% Triton X-100-containing phosphate buffered saline (TPBS). After washing, the samples were incubated with fluorescently labeled goat secondary antibodies (Alexa488-labeled anti-mouse IgG(H + L) antibody and 10 μg/ml Hoechst 33342 (Life Technologies) in TPBS containing 10% goat serum overnight at 4°C. Fluorescence was detected with an FV10 confocal scanning microscope (Olympus) (10x/0.4 NA, or 60x/1.34 NA oil immersion objective lens). Images were processed with FV10-ASW (Olympus) and ImageJ software (NIH). All images were obtained using the same photography conditions to allow direct comparison between experimental animals and controls.
Chemotactic behavior analysis in planarian
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
Thigmotactic/kinetic behavior analysis in planarians
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.
Establishment of a behavioral integration assay method to analyze brain function using two distinct external stimuli
Planarian chemotaxis and thigmotaxis/kinesis
Chemosensing in addition to visual sensing and appropriate responses to various stimuli in order to find food, to escape from predators, and to communicate with other individuals, are among the most important primary functions of organisms. Planarian food-finding behavior is thought to be completely dependent on chemosensing , and the auricles and head margin are thought to be an important organ of chemosensing , although no chemosensory receptors or chemosensory neurons have been identified. Here, we established a new, tractable behavioral analysis system for chemotaxis to a food attractant, and used it to quantify the movements of planarians exposed to food as a chemoattractant. This behavioral assay system combined with RNAi may be a powerful tool for finding genes involved in chemotaxis, genes important in processing neurons in the brain, genes encoding chemosensory receptors, and other genes important in chemosensory neurons.
In this study, to evaluate the strength of a chemoattractant gradient that could be established by diffusion through a low-concentration agar gel, we made successive transfers of the chemoattractant solution, thereby successively diluting it, and directly examined whether the transferred solution was able to induce planarian chemotaxis (Figure 2). This bioassay may be useful for studying chemotaxis induced by substances for which we have no way of measuring the concentration gradient directly. When placed in this reproducible chemoattractant-gradient field, planarians spent the most time in the region containing the highest concentration of the chemoattractant (Figure 3B). Furthermore, the direction of the planarians’ movement clearly showed a tendency to move toward the region with the highest concentration of the chemoattractant (Figure 3D). These results clearly suggest that the planarian may recognize a concentration gradient of chemicals: they moved toward the higher concentration of the chemoattractant without showing increased kinesis.
In contrast, unfortunately we have not been able to prepare a texture-gradient field, and therefore we were unable to investigate how planarians recognize the difference between textured and smooth surfaces to reach a region with a preferred surface. In addition, we are unable to distinguish whether the thigmotaxis/kinesis we observed here was positive or negative (preference for a smooth surface or evasion of a textured surface), although the planarians showed a clear tendency to move to and stay for a long time in the smooth surface region (Figure 5). We speculate that planarians can attach more strongly to smooth surfaces with wider adhesion areas than to textured surfaces, and that this behavior may benefit planarians by enabling them to find safer and more stable surfaces. Consistently, in nature, planarians can usually be found under small stones or leaves, where they may avoid light, strong water flow, and predators. Moreover, we found that this feature of planarians could be utilized to arbitrarily restrict the regions where planarians moved in a field to regions without vertical walls (Additional file 1: Figure S1).
Planarian behaviors require neural activity of the brain
The robust regenerative ability of planarians makes them very useful for analyzing the phenotypes of amputated body fragments, such as headless animals, in order to investigate the organs regulating behaviors, since these fragments do not die. Head-amputated planarians completely lost both chemotaxis and thigmotaxis (Figures 3C, and 5A), suggesting that the head is required for both of these planarian behaviors. Furthermore, the planarian's strong regenerative ability enabled us to perform region-conditional gene knockdown by the procedure called Readyknock , and the resultant specific loss of neural activity in the brain by Readyknock of the synaptotagmin gene caused complete loss of both chemotaxis and thigmotaxis. These results suggest that neural activity in the brain is required for the sensing of chemical and mechanical stimuli, and/or for processing in the brain of the signals received through chemo- and thigmosensory neurons.
Note that decapitation caused a decrease of mean velocity of locomotion (Figure 3F), although the duration of the assay (600 sec) was sufficient for planarians to reach target regions from the start point in the assay chamber, and for us to judge their chemotactic behavior. Because of this decrease of mean velocity we cannot completely exclude the possibility that the decapitation-induced decrease of velocity led to the reduction of the chemotactic and thigmotactic scores. However, loss of synaptotagmin gene function in the brain did not affect their velocity of locomotion (Figure 4G), but did cause loss of these behaviors, suggesting that planarian chemotaxis and thigmotaxis are dependent on the brain in their head, and that our chemotaxis and thigmotaxis assay systems are useful for analyzing various functions of the planarian brain and of nervous-system-related genes. Previously, we reported that neural activity in the brain is required for both phototaxis and thermotaxis [7,11,28]. Our results in this study together with those previous results strongly suggest that planarian behaviors responsive to external stimuli are controlled by the brain.
Panel of behavioral assays may provide means to unravel neural networks of higher brain function in planarian
Planarian higher brain function was first described in the 1950s, when Thompson and McConnell used classical conditioning experiments using light and electrical shock to show planarian's ability to learn and remember . However, as this phenomenon has not been verified by other groups, McConnell's studies of planarian learning remain controversial [30,31].
The unique features of planarians’ higher brain functions have provided many insights into brain function in metazoans, and studies on planarian classical conditioning have attracted many researchers [32,33]. However, the neural pathways of planarian higher brain functions, including integration and learning, have not yet been unraveled, and even many of the original observations made decades ago have not been verified yet at the neuronal level. Moreover, it is difficult to analyze the neural mechanisms underlying higher brain function in planarians using the originally reported classical conditioning assay system utilizing light and electrical shock, because electrical stimulation might affect muscles directly. Previously, assay systems for planarian phototaxis and thermotaxis have been reported, and a number of genes and neuronal subtypes involved in sensing external stimuli and related characteristic behaviors have been identified [6,7,28]. Here we took another step forward by developing quantitative methods for chemotaxis and thigmotaxis/kinesis that enabled us to investigate the mechanisms underlying information processing and integration in the planarian brain. In order to acquire information about planarian behaviors in response to multiple simultaneous stimuli, we performed a binary competitive assay in which a planarian was given two distinct stimuli simultaneously under specific conditions in combinatory experiments. The results showed that planarians displayed an order of priority among their responses to these different stimuli (Figure 6), suggesting that the planarian brain has the ability to integrate signals coming from outside to decide appropriate planarian behaviors. Furthermore, we showed that planarian movements vary according to the stimulus level (Figure 7E), although we did not find any light-intensity dependence of planarian phototaxis (data not shown). These results suggest that planarians do not show a simple, direct response to a stimulus, but rather integrate external stimuli and then behave suitably in response to the overall conditions.
Neural networks predicted to be involved in controlling decision-making
It seems unlikely that individual neurotransmitters are involved in only single neural circuits or behaviors [24,28,37]; rather, neurons expressing a particular neurotransmitter may have different roles according to the particular cell or position in the brain, and may accordingly construct more complex neural networks. By accumulating further data from the kinds of experiments reported here and further refining the assay systems, we should be able to unravel the neural networks or systems involved in the higher brain function in planarian. Here we found that planarian behaviors in response to stimuli could be changed flexibly according to the level of stimulation. Axons of the sensory neurons in planarian, such as the axons of the optic neurons and chemosensory neurons, project to different regions in the brain (Figure 8B,C). For example, visual neurons project to the dorso-medial region (visual center) in the brain, whereas axons of chemosensory neurons in the lateral branch neurons project to the ventro-lateral region of the brain, as visualized by immunostaining with specific antibodies (Figure 8B,C), consistent with previous results obtained using fluorescent dye tracing . It has been suggested that external stimuli sensed by such organs are integrated in some region(s) of the brain, finally resulting in planarians’ behaviors. Ultimately, the information acquired by sensory neurons might be accumulated inside the brain, and then processed and integrated to transduce the signals into the activity of motor neurons (Figure 8).
The chemotaxis and thigmotaxis/kinesis assay methods established here are useful for quantitatively analyzing planarian chemotactic and thigmotactic/kinetic behaviors. Headless planarian body fragments and planarians treated by Readyknock of synaptotagmin in the brain lost both chemotactic and thigmotactic behaviors, suggesting that brain neural activity is required for the chemotactic and the thigmotactic behavior in planarian. When we tested the priority among four planarian behaviors (phototaxis, chemotaxis, thigmotaxis/kinesis, and thermotaxis) by presenting the respective stimuli to planarians simultaneously as binary stimuli, the results revealed that planarians showed a clear order of predominance of these behaviors, with chemotaxis being the strongest stimulus under our conditions. We also found that planarians showed predominance of their behavioral response to either of two simultaneously presented stimuli depending on the strengths of the two stimuli in a competitive binary stimuli assay using light and chemoattractant. Taken together, our results support the notion that external stimuli sensed by respective sensory organs are integrated in the brain and determine planarian behaviors.
Central nervous system
Ventral nerve cords
Regeneration-dependent conditional gene knockdown
G-protein β subunit
Dugesia japonica tyramine beta-hydroxylase
Dugesia japonica synaptotagmin
Dugesia japonica transient receptor potential ion channel melastatin family a
Dugesia japonica tryptophan hydroxylase
Glutamic acid decarboxylase; gamma-aminobutyric acid
Dugesia japonica tyrosine hydroxylase
Dugesia japonica choline acetyltransferase
Standard error of mean
We thank Dr. Elizabeth Nakajima for critical reading of the manuscript. This work was supported by a Grant-in-Aid for Young Scientists (B) to TI, The Kyoto University Research Funds for Young Scientists to TI, The Brain Science Foundation funds to TI, a Grant-in-Aid for Scientific Research on Innovative Areas to KA, a Grant-in-Aid for Creative Scientific Research to KA, and the Grants to Excellent Graduate Schools (MEXT) program of Kyoto University.
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