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 [ 5 ].
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.
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. A schematic representation of the chemotaxis assay system is shown in Figure 1 A. Two ml of water containing 0. The liver extract was divided into aliquots and frozen until use. A planarian was placed in the quadrant at the opposite end Zone 1 of the chamber Figure 1 A. A The assay chamber used to test chemotaxis.
The cross indicates the peak of the chemoattractant gradient. The circle indicates the start region. Planarian behavior was quantified using the time spent in the target quadrant Zone 4. The 2 opposing quadrants shaded gray indicate the textured regions.
The white quadrants indicate smooth regions. Planarian behavior was quantified using the time spent in the two smooth regions. C Planarian behavior was recorded using a digital video camera and was analyzed using a computer and behavior analysis software. A schematic representation of the thigmotaxis assay system is shown in Figure 1 B. The thermotaxis assay was performed as described previously [ 7 ]. 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.
One ml of water containing 0. Each planarian behavior was captured using a video recorder Sony placed above the container Figure 1 C for the time indicated in the Results section. All behavioral experiments were performed in a dark room with only a red light of a wavelength that cannot be sensed by planarians Figure 1 C [ 18 ]. Calculations based on the data obtained were performed using static ggplot2 package of R software [ 19 ].
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.
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. Whole-mount immunostaining was performed as described previously [ 10 ]. All images were obtained using the same photography conditions to allow direct comparison between experimental animals and controls. 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 1 A. 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. 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.
Soon thereafter, a planarian was placed in Zone 1, and its behavior was observed. Figure 2 B shows the time spent in Zone 1 during the 1-min assay period. In contrast, planarians did not stay in Zone 1 even after 5 min However, after 10 min, they did stay in Zone 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. Persistence assay for assessing the formation of a chemoattractant concentration gradient. A Schematic illustration of the method of the persistence assay. Planarians normally move around continuously after transfer to an assay chamber or culture dishes, and therefore the time spent in Zone 1 would normally gradually decrease.
However, they remain in the initial region Zone 1 if a sufficiently high concentration of chemoattractant is present there. B Time spent in Zone 1 during 1-min assay period. The chemoattractant diffused from Zone 4 to Zone 1 within 10 min after it was added.
Next, we tested the behavior of intact planarians in a normal field uniform-field assay chamber without chemoattractant. Figure 3 A 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 3 B. 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 3 C. 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 Chemotaxis of intact and headless planarians. A Heat map view with contour lines of the averaged behavior of 11 individually assayed intact animals in a uniform field.
Planarian showed a preference for moving along the edge of a uniform chamber. B Heat map view with contour lines of chemotaxis of intact planarians in chemoattractant concentration gradient field. C Heat map view with contour lines of chemotaxis of headless planarians in chemoattractant concentration gradient field.
Planarians showed a preference for moving along the edge of the uniform-field dish. In contrast, intact planarians moved to and stayed in the region with the highest concentration of chemoattractant in the chemoattractant-gradient field.
Headless planarians did not show such chemotaxis. D Rose plots show orientation of movement of intact and headless planarians in Zones 2 and 3 in a uniform-field and chemoattractant gradient-field.
The movement of intact planarians in a uniform-field, and that of headless animals in a chemoattractant gradient-field, showed no particular orientation, whereas the movement of intact planarians was biased toward being oriented toward the highest concentration of chemoattractant.
F Mean velocity of intact and headless planarians during assay. Next, the average time during a 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 In contrast, headless animals showed a much lower thermotaxis score 8.
There was no difference in the speed of movement of planarians in the chemoattractant-gradient field 2. These results indicate that this assay method is useful for quantitatively evaluating planarian chemotactic behavior, and that planarian chemotaxis is dependent on the head.
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 4 A.
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 4 D. 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 4 E. 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 4 F , without causing any defect in locomotor activity Figure 4 G.
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. Chemotaxis of planarians that had lost of brain neural activity by Readyknock of synaptotagmin gene. A Schematic illustration of experimental design of Readyknock of synaptotagmin gene. After injection of double-stranded RNA of Djsyt , planarians were amputated, and then allowed to regenerate their heads for 7 days.
Red-colored portion indicates newly regenerated head. B, C Control and Readyknock of Djsyt. Samples were stained with Hoechst for nuclei, shown in blue to visualize planarian tissues, including brain.
The dashed boxes indicate the border between the newly formed head region magnified in the right panels. D Heat map view with contour lines of chemotaxis of control and Djsyt RNAi planarians in chemoattractant concentration gradient field. Djsyt RNAi planarians showed random movement, whereas control planarians moved to and stayed in the region having the highest concentration of chemoattractant in the chemoattractant-gradient field.
E Rose plots show orientation of movement of control and Djsyt RNAi planarians in Zones 2 and 3 in chemoattractant gradient-field. The movement of control planarians was biased toward being orientated toward the highest concentration of chemoattractant, whereas Djsyt RNAi animals in the chemoattractant gradient-field showed no particular orientation of movement.
Planarians show reactions through mechanical-tactile sensing to such stimuli as water flow, touch, and contact with objects [ 5 , 25 ]. Figure 5 A 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 5 A.
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 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 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 5 C.
These analyses clearly indicate that intact animals spend a large fraction of their time in the target zone after reaching it This finding is consistent with the findings in our chemotaxis, phototaxis, and thermotaxis assays Figure 4 [ 7 , 11 ].
Intact planarians tended to move to the smooth-surface region after starting from the textured surface region indicated by the white circle.
In contrast, headless planarians continued to move around in the assay field independent of whether the bottom surface was smooth or textured. Djsyt RNAi planarian showed random movement, like headless planarians, whereas control planarians moved to and stayed in the smooth-surface region.
D The number of re-entries into a textured region from a smooth-surface region of intact, headless, control, and Djsyt RNAi planarians during the assay. 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.
When we compared behaviors in this combinatorial assay using planarians presented with both chemoattractant and lux of light, planarians preferred to move toward the chemoattractant rather than escaping from the light, even though they received strong enough light lux to induce phototaxis in a single-stimulus assay Figure 6 A [ 6 ]. The preference index for chemotaxis 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 6 G.
These data suggest that planarians may have the ability to integrate various different external kinds of information in the brain. Binary competitive behavior analyses. A-F The order of predominance of the four tested behaviors.
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 7 A.
When a planarian was placed into the center of the chamber Zone 3 , it moved randomly Figure 7 B. 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 3 A. When we assayed planarian phototaxis and chemotaxis using this chamber, planarians moved away from the lux light source Figure 7 C , as previously described [ 6 ], and planarians moved toward Zone 1, where the chemoattractant C.
In addition, when planarians were exposed to lux of light and chemoattractant simultaneously, they moved toward the region of the chemoattractant source Zone 1 Figure 7 E. This result was also consistent with the above results Figure 6 A.
All of these were mounted on an adjustable mounting pole as shown in fig. To identify opsin homologs in planarians, we used opsin sequences from Porter et al. To make a nonredundant planarian transcriptome data set, we used CD-HIT 57 to cluster multiple transcriptomes 58 — We identified putative opsin-like candidates.
We built a hidden Markov model HMM for opsin sequences and compared it with the transcript profiles. We used TmHMM www. We only considered transcripts with seven TM helices with optimum loop length for further analysis. Further, 66 transcripts with seven TM helices were selected.
From previous reports on opsin sequences from other organisms, we know that Lys K residue in the seventh last TM helix is the amino acid residue to which retinal binds via a Schiff base linkage We took the seventh last TM helix for 66 S.
Squid opsin 2z73 was obtained as a closest hit to our modeled three-dimensional 3D structure of six opsins. In addition, we obtained a root mean square deviation of 0. We did phylogenetic analysis of the identified six putative opsins from S.
The obtained phylogenetic tree was visualized using TreeDyn www. Seven TRP channels were identified by Inoue et al. We identified the homologs of these sequences in S.
Statistical analyses were performed using a nonparametric Wilcoxon signed-rank test. We thank V. Lakshmanan for his help in bioinformatics analysis. We are grateful to M. Mathew, M. Panicker, A. Ramesh, S. Sane, T. Mukherjee, R. Muddashetty, R.
Sambasivan, S. Mayor, and U. Bhalla for their discussions and advice. We thank S. Ramaswamy and the Technologies for the Advancement of Science. Work in A. Author contributions: N. Competing interests: The authors declare that they have no competing interests. Additional data related to this paper may be requested from the authors. Planarian eye opsin knockdown attenuates light sensing across the color spectrum.
Recovery of single-input light sensing and response during head regeneration. Aggregate time taken for eye-mediated phototaxis is insensitive to light intensity.
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Find articles by Siddharth Patnaik. Find articles by Manoj Mathew. Find articles by Dasaradhi Palakodeti. Find articles by Akash Gulyani. Author information Article notes Copyright and License information Disclaimer. Email: ni. Received Dec 1; Accepted Jun No claim to original U. Government Works. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license , which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.
This article has been cited by other articles in PMC. Planarian light sensing apparatus. Planarians are aversive to light across a broad wavelength range. Wavelength dependence of extraocular photoreception. Lack of wavelength discrimination in extraocular photoresponse. Phylogenetic tree maximum likelihood of planarian opsin sequence. Light setup for phototaxis experiments and recording. Planarian worm shows wavelength discrimination. Planarians show robust wavelength discrimination group response.
Imaging Smed eye opsin expression. Smed eye opsin is expressed in all photoreceptor cells. Regenerating cephalic ganglion day 3. Regenerating cephalic ganglion day 4. Regenerating cephalic ganglion day 5. Regenerating cephalic ganglion day 6. Regenerating cephalic ganglion day 7. Extraocular photoreception and phototaxis in planarians. Light avoidance extraocular response to long UV is directional. Abstract Light sensing has independently evolved multiple times under diverse selective pressures but has been examined only in a handful among the millions of light-responsive organisms.
RESULTS Behavioral photoswitching in wavelength choice assays Planarians are known to be light-aversive, with broad sensitivity to visible light 6 , 10 , 31 , Open in a separate window. Planarians show the ability to resolve light inputs of distinct wavelengths. Acute light intensity sensing in planarians Apparent wavelength discrimination by planarians in binary choice assays was surprising.
Smed eye opsin expression is restricted only to eye and is expressed in all PRNs. Hierarchical light processing revealed through regeneration Our data so far suggest that the organisms are able to resolve small differences in effective intensities of light sensed at the eye and convert these differences into binary behavioral outputs. Mapping recovery of planarian phototactic abilities during regeneration.
Eye-independent, directional light avoidance in planarians Planarians are highly light-aversive and show complex light discrimination through their cerebral eyes. Planarian extraocular photoresponse and hierarchical relationship with ocular light response. Dynamic interplay among ocular and extraocular sensing networks Planarians appear to have separate ocular and extraocular sensing systems.
Hierarchical light sensing and processing in planarians revealed through recovery of function with regeneration. Planarian maintenance S. Planarian negative phototaxis single-input sensing Earlier work shows that planarians respond broadly to visible light, with subtle differences in response to colors Wavelength discrimination choice assays All planarian choice assays were performed according to the schematic shown in Fig.
Extraocular photoreception and phototaxis For determining wavelengths that affect extraocular phototaxis, planarian tail pieces 24 hours after decapitation; fig. Fluorescence in situ hybridization The heads of asexual planarians 5 to 10 mm in length were cut and processed for FISH as per the protocol described by Pearson et al. Whole-body immunofluorescence and imaging Confocal immunofluorescence microscopy was performed to visualize changes in the planarian nervous system over regeneration.
RNAi-mediated knockdown Planarian eye opsin Smed eye opsin sequence was obtained from the published planarian eye transcriptome Bioinformatic analysis To identify opsin homologs in planarians, we used opsin sequences from Porter et al.
Statistical analysis Statistical analyses were performed using a nonparametric Wilcoxon signed-rank test. Acknowledgments We thank V. Nilsson D. B Biol. Randel N. Arendt D. Batistoni, chap. Tsonis, Ed. Elsevier, , pp. Brown H. Carpenter K. Cell Tissue Res. Kishida Y. Fine structures of the normal eye. Kanazawa Univ. Paskin T. Pineda D. Lapan S. Cell Rep. Sarnat H. Agata K. Tessmar-Raible K. Simakov O. Genomics 12 , — Reddien P. Cell Dev.
Bioessays 28 , — Growth Differ. Inoue T. Takano T. Cronin T. Backfisch B. Porter M. Steven D. Yau K. Cell , — Xiang Y. Nature , — Ward A. Pennisi E. Evo-devo devotees eye ocular origins and more. Science , — Yamaguchi S. Fukshansky, W. Shropshire Jr. Govardovskii V. Goldsmith T. Kelber A. Pichaud F. Rister J. Sandmann T. Genome Biol. Medved R. Kane E. Child C. Genes Evol. Bellono N. PLOS Genet. Ross K. BMC Dev. Purschke G.
Arthropod Struct. Oviedo N. There was also a half circle at the origin, with its apex midway through Q1, for directing light placement.
LED wands were secured above the testing dish with a clamp attached to a ring stand, while a second clamp secured the battery pack to prevent unintended movement of the wand. The end of the LED wand was positioned about 5 cm above the top of the testing dish with the light directed into the half circle in Q1.
An SLR camera was positioned over the testing dish using a tripod. On each experimental day, batteries were replaced in both flashlights and the LED wand.
The testing dish was filled to a depth of 0. In a single day, one wavelength was applied to total of 60 worms 10 groups of 6 worms, or 10 trials , repeated 3 times. For each trial, all worms were placed into Q1 before the camera was turned on. Except for controls, the light was switched on time 0 at 5 seconds after recording started.
Behavior was recorded for 2 minutes. Animals were allowed to rest at least overnight before the next wavelength. Filters used were A holder was designed from stiff foam pipe insulation to position the LED wand above the filter such that all emitted light passed through the filter. White paper was placed on the microscope stage so that laser light could be seen.
Individual worms were transferred to the middle of the dish and recording was started when the worm began traveling on the bottom of the dish. The laser beam was directed in front of the animal at a distance equal to one diameter of the circle of light approximately 2.
Only a single wavelength was tested each day with 30 worms repeated twice, for a total of 60 trials , and animals were allowed to rest at least overnight before the next wavelength in the following order: red, green, and UV. Recordings from all behavioral trials were examined using Windows Media Player. For the photophobia assay, the three repeat trials for each group were first averaged to compensate for individual animal variability. When determining location, at least 50 percent of the worm had to be in the quadrant.
A Bonferroni post hoc multiple comparisons test was conducted to examine differences between means. However, from available data, it is unclear whether planarians have a single general photophobic response or if their behavioral responses actually vary by wavelength as has been shown in other animals [29] — [33] , [39]. To distinguish between these possibilities, we developed a novel behavioral assay Materials and Methods.
Because the LED wand was exchangeable, our setup allowed not only for testing behavioral responses to different visible wavelengths, but provided a means to investigate planarian responses to ultraviolet UV and infrared IR wavelengths as well. One objective was to establish an easily reproducible photophobia assay with standardized testing parameters in order to improve comparability.
Therefore, each LED wand was clamped above the testing dish at a fixed distance of about 5 cm Figure 2A. Additionally, a sheet of white paper was placed beneath the testing dish, with four equal quadrants Q1 to Q4 demarked Figure 2B.
To verify that the amount of light gradually decreased from Q1 to Q4, the intensity of light in each quadrant was estimated with a phototransistor. Finally, the assay used easily-constructed LED wands powered by 9 volt batteries, as previously described [30] , [31] , which allowed for some control of the ranges of wavelengths tested. For our experiments, the nominal wavelengths used were Figure 2C : near IR — nm , red — nm , green — nm , blue — nm , and two distinct wavelengths of near UV light — nm and — nm.
In addition, we also tested worm responses to white light using a standard LED fiber optic illuminator with goosenecks as typically used with a dissecting scope. The use of white light, even though there are certainly different spectra involved using LED or halogen sources, allowed us to compare responses from more restricted and narrow ranges of wavelengths with the non-specific white light typically used in planarian photophobia studies.
A The imaging setup. B Close-up of testing dish. B1 The labeled guide placed underneath the dish marks the 4 quadrants Q1—Q4 and the semi-circle where the LED light will be directed. B2 Image of testing dish during a trial, showing the resulting light-to dark gradient. C The spectral composition of the LEDs used, and their location on the electromagnetic spectrum.
For the assay, the behavioral responses of 60 worms were tested in 10 groups of 6 worms for each wavelength a single trial. Trials were repeated 3 times and the data averaged, to compensate for variability in individual worm responses. Trial parameters were as follows: camera recording was turned on, a group of 6 worms was placed in Q1, after 5 seconds the LED wand was turned on, and behavior was recorded for 2 minutes the initial time was scored as when the light was first turned on.
Because of the remote possibility that the brief exposure to very weak UV light might cause damage, UV trials were performed last. Generally, worms were tested in order from longest to shortest wavelengths. Using the above parameters, we performed our photophobia assay with control ambient light only , IR, red, green, blue, and UV nm and nm wavelengths, as well as with white light Figure 3. Worm location by quadrant was scored at 30 second intervals Figure 3A , with photophobia being assessed after 2 minutes Figure 3B.
Statistical significance asterisks in Figure 3B was assayed for the overall pattern of worm location throughout the entire dish across all four quadrants , rather than for individual quadrants. Control groups explored the dish in an apparently random manner Figure 3A and Video S1 , such that by 1 minute animals were evenly distributed between all quadrants and remained so for the duration of the trial with an average of This random exploration is consistent with initial exploratory behavior in new environments previously noted in planarians [40] — [42].
All worms begin in quadrant 1 Q1, red circles. While control worms randomly explore the dish, in UV trials worms move rapidly away from the light white circles.
Images enhanced for visualization. B Graph showing overall photophobic responses for each wavelength, as measured by worm location in each of the four quadrants Q1—Q4 after 2 minutes. Photophobic responses are indicated by increased presence in Q4 black bars which is farthest from the light.
In most of the UV trials, the worms congregated on the wall of the dish furthest from the light Figure 3A and Video S2. As expected, worms exposed to white light also displayed strong negative phototaxis, with a striking correlation across all quadrants between white light Q1: 1. On the other hand, neither of the IR or red wavelength responses were statistically different from controls by the end of the trial Figure 3B. Overall, these results suggest that our novel planarian photophobia assay is able to recapitulate the strong photophobia previously demonstrated by other methods.
To confirm that the observed behavioral responses resulted from visual detection of specific wavelengths and not other variables such as heat or nociception, we repeated our photophobic assay with neutral density filters. If responses to light are in fact a result of visual detection, we would expect worm responses to diminish in a predictable fashion as light attenuation increases and the behaviorally relevant stimulus decreases.
The results confirmed that the number of worms displaying photophobia steadily decreased with increased light attenuation, suggesting that the behavioral responses were the result of visual responses to specific ranges of wavelengths and not uncontrolled variables. Graph showing behavioral responses over increasingly attenuated light, as measured by the number of worms in Q4 at 2 minutes.
The trend shows that phototactic responses decreased along with diminished behavioral stimuli light. Although our data revealed that green, blue and UV light all resulted in robust photophobic responses Figure 3B , we observed that worms exposed to near UV light appeared to move away from the light faster than for other wavelengths tested. This suggested that more complex differences exist between the photophobic responses than our scoring for photophobia at 2 minutes revealed.
Thus, we next examined the rate at which worms escaped direct light in Q1 by tracking both the number of worms that left Q1, and the number that returned, throughout the trial Figure 5. To do this, we calculated an escape index , where 0 indicated all worms remained in Q1 while 1 indicated all worms had left Q1. Therefore, higher values represented stronger photophobic responses. It should be noted that an important difference exists between the analyses in Figure 3 and the analyses here in Figure 5 that represent how fast worms escape from direct light exposure.
Because of this, the escape index as used here is a measure of the initial intensity of the response rather than a measure of overall strength of the response. Graph showing escape responses as a measure of the severity of phototactic behavior. The escape index is based on the number of worms that leave Q1 direct light , where a value of 1 indicates all worms have left Q1.
Thus, higher values indicate stronger photophobic responses. Note the latter data indicate by 2 minutes worms have returned to the direct red light source in Q1. Interestingly, white light was more similar to though not statistically different from blue escape responses Figure 5 , in contrast to overall photophobic response Figure 3 where white light was more similar to green.
This may be related to the spectral composition of white light LEDs that typically contain several broad peaks, including notable amounts of energy in the blue range. For IR light, the escape index Figure 5 at all time points was significantly different from controls as well as all other wavelengths.
This is in contrast to the overall photophobic response to IR light Figure 3 , which was not statistically different from controls even at the earlier 30 second time point. In particular, the escape index showed that IR wavelengths produced an opposite phototactic response, where worms were initially more likely to remain under direct light Q1 than controls.
This suggests the possibility that planarian responses to IR might be slightly photopositive, a hypothesis that would first need to be investigated in much greater detail. These data also indicate that the planarian visual system may be able to respond to IR wavelengths in some as yet unknown manner. This was particularly unexpected given that the overall photophobic response to red at 2 minutes was not different than controls Figure 5.
This reflects the observation that at 2 minutes, worms that previously left Q1 returned, despite the continued presence of the red light exposure. These data suggest that after an initial photophobic response worms subsequently stopped responding to red wavelengths. The overall photophobic response data, combined with escape index analyses, suggested that while planarians displayed different responses to different wavelengths with UV causing the most robust responses , there may also exist a separate, wavelength-independent photophobic response to being placed under direct light such as might be expected with broadly-tuned visual pigments.
In order to test this idea, we examined avoidance responses to different wavelengths Figure 6. Whereas previously we examined whether or not planarians would move away from light exposure, our avoidance assay tested the reverse behavior: whether or not worms would choose to enter a light source. However, the LED wands we used in our previous assay produced a field of light that was too large to record worm movement from outside the field into the light.
Therefore, we switched to the use of tiny spots of laser light under high magnification under a stereomicroscope. We covered the end of a laser pointer with a piece of tape that had a single pinhole in the center, thus obtaining a much smaller coherent circle of light. For illustration, compare the relative size of the light field versus a single worm in our photophobia assay UV panels in Figure 3A and in our avoidance assay Figure 6.
We chose red, green and UV laser lights as representative of the range used in our photophobia assay. We expected that if wavelength-specific responses existed, worms would respond with increasing severity to avoid entering regions lit by red, green and UV wavelengths, respectively.
Avoidance assay to test worm responses when approaching areas of direct light. This ensured that worms began the assay outside the direct light source but was close enough that worms continued moving in the direction of the light.
Three distinct behaviors were observed. As worms approached the light source they either 1 did not respond and continued moving directly into the light, 2 moved around the light by making a slight directional change to one side without crossing into the light, or 3 abruptly made a 90— degree turn in the opposite direction of the light photos in Figure 6. When confronted with the green light, the majority Furthermore, not a single worm chose to travel into the UV light, even though These results are consistent with our previous data showing that planarians exhibited differential responses to different ranges of wavelengths of light.
They also confirmed that not only did UV light produce the strongest photophobic responses and most robust initial responses, but that an intermediate and less severe photophobic response occurs with wavelengths within the visible spectrum such as green.
Furthermore, these results demonstrated that planarians lack a red wavelength-specific behavioral response, suggesting that the escape response we observed to red light reflects instead an initial wavelength-independent photophobic response Figure 7A.
A Graph showing the likely relationship between the two types of photophobic responses uncovered by our data: the general photophobic response, which occurs immediately after exposure to any wavelength, and the wavelength-specific responses. B Graph depicting the inverse relationship between photophobic responses and wavelength.
Our results support the hypothesis that planarians do possess differential behavioral responses to light across a range of wavelengths.
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