People addicted to drugs of abuse experience withdrawal effects as they try to become sober. This withdrawal behavior can be studied in a simple model system: planaria, a type of freshwater flatworm. Using planaria, this study measured the withdrawal effects of fentanyl, a potent opioid, and cocaine, a widespread psychostimulant. We hypothesized that longer exposure to these compounds would lead to greater withdrawal behaviors in the form of decreased locomotor activity. Planaria were exposed to cocaine (10 μM), fentanyl (10 nM or 100 nM), or spring water (control) for 15 minutes, 60 minutes, 180 minutes, or 24 hours. After each exposure period, their locomotor activity was measured by analyzing their video recorded movements for distance traveled. Although the results were statistically insignificant, qualitatively, planaria exposed to cocaine showed an overall increase in locomotor activity. Planaria exposed to a lower concentration of fentanyl also behaved similarly, but with a decrease in locomotor activity at 24 hours. However, planaria exposed to the higher concentration of fentanyl had an increase in locomotor activity only at 180 minutes of exposure, with an otherwise decreasing trend. These observations suggest that exposure to cocaine may increase locomotor activity, contrary to our hypothesis. Fentanyl’s effects are concentration-dependent, with low doses causing an acute increase in locomotor activity and high doses showing minimal change in locomotion. These data show that planaria are sensitive to the effects of drugs of abuse and are effective models to study behaviors related to drugs of abuse, including withdrawal.
Keywords Cocaine, fentanyl, planaria, withdrawal behavior, addiction, locomotor activity
Drugs of abuse and addiction have long been a social and medical problem. There have been several waves of substance abuse epidemics, with the current opioid crisis being declared as a public health emergency in 2017 by the United States Department of Health and Human Services.1 These epidemics lead to the development of pharmacotherapies, which help those who are affected attain the goal of abstinence from the drug(s) of abuse.. The vicious cycle of drug abuse starts with exposure or use of the drug that can ultimately lead to continuously seeking the drug. A culmination of diverse factors may force an individual to abstain – with an aim to fully recover – but the drug-taking habit can make it difficult to contain the urge and may push the individual to relapse into the drug-taking behavior. Abstinence from drugs of abuse can cause withdrawal effects that may push people from recovery to relapse, also disrupting therapeutic interventions. Thus, studying and understanding withdrawal behavior is important to allow for effective interventions (pharmacological or behavioral) to be developed.
Stopping drug-taking may cause various withdrawal symptoms, including reduced locomotor activity. These behavioral changes – which affect locomotor activity – are known to be mediated by neurotransmitter systems in the brain that are shared across species, including many vertebrates and invertebrates.2 Therefore, studying alterations in locomotor activity caused by drugs of abuse offers a unique way of testing pharmacological interventions for both drug addiction and drug withdrawal behavior. However, studying withdrawal behaviors in humans and rodents is also associated with limitations, such as accessibility, convenience, and cost. This creates a need for other model systems and an opportunity for the use of simple organism models, such as planaria, that have the potential to exhibit withdrawal-like behaviors analogous to those seen in mammalian models, such as rats and mice.
Planaria are free-living, freshwater flatworms with cephalic ganglia and peripheral nerve cords.3 They are referred commonly to as a group of invertebrates belonging to the phylum Platyhelminthes and order Tricladida, which are non-parasitic flatworms.4 Planaria are a well-established model to study developmental biology and regeneration, drug reward behavior5, anxiety-like effects6, and to measure changes in neurotransmitters7, among others.
In a previous study aimed at studying withdrawal effects of certain drugs of abuse on planaria, Sacavage et. al., (2008) demonstrated that following exposure to cocaine (10μM), methamphetamine (10μM), or caffeine (10μM) planaria show changes in locomotor activity when exposure to the drug is stopped. It was also shown that planaria exposed to cocaine and methamphetamine showed a greater decrease in distance traveled – i.e. shorter distance that corresponded to a greater withdrawal behavior.5 This suggested that locomotor activity could be used as an index of withdrawal. Furthermore, they showed that the magnitude of withdrawal-like behavior is dependent on the duration and specific drug of exposure. However, the withdrawal effects of fentanyl, which is at the center of the ongoing opioid crisis, were not studied.3
Fentanyl is a potent synthetic opioid that has received national attention for the overwhelming increase in the number of its related mortalities.8 On the other hand, fentanyl is used to treat pain and has other beneficial effects as well. As such, understanding withdrawal effects from fentanyl is very important for the development of effective treatment strategies for its dependence and withdrawal properties. Accordingly, the purpose of this study was to assess the time course of withdrawal-like behavior in planaria exposed to fentanyl.
Cocaine hydrochloride and fentanyl hydrochloride were obtained from the National Institute of Drug Abuse Drug Supply Program (Bethesda, MD). Planaria (Dugesia dorotocephala) were procured from Carolina Biological Supply Company (Burlington, NC). Upon arrival, all planaria were acclimated to a reversed light-dark cycle (i.e. lights off 6 am – 6 pm) in springwater at room temperature for 2 days before starting experiments.
Planaria exposure to drugs of abuse
Cocaine (10 μM) and fentanyl (10 nM and 100 nM) solutions were prepared in Petri dishes with spring water (Carolina Biological Reserve, Burlington, NC) mixed with AmQuel water conditioner (Kordon, Hayward, CA). Planaria were divided into groups of 8 and placed in Petri dishes containing either spring water (control), cocaine (10 μM), or fentanyl (10 nM or 100 nM). The duration of exposure to these conditions was monitored by timers set to 15 minutes, 60 minutes, 180 minutes, or 24 hours. All experiments were performed during the dark cycle of the planaria (between 6am to 6pm of the reversed light-dark cycle) under red light conditions to allow experimenters to work without disrupting the circadian cycle of planaria.9 Withdrawal-like behavior after exposure at each of these time points was video recorded. These data were scored for changes in locomotor activity as an indicator of withdrawal like behavior. At each time point, 2 planaria from each exposure condition were placed into individual wells of a 6-well plate each filled only with spring water with no drug. The locomotor activity of the planaria in the drug-free condition was recorded for 5 minutes by the video analysis software StCamWare. All recordings were performed in a room illuminated by a red light. Video recording and other observations were performed under red light illumination because planaria are generally described as photophobic and were shown to have an intense aversion to small ranges of visible length (blue and green) but not to red light. Additionally, red light also offered a viable contrast for better visualization of planaria while analyzing locomotor activity. Once the recording was complete, the planaria were frozen in dry ice and stored at -80oC for future neurochemical analysis.
Video measurement and analysis of locomotor activity:
Video analysis of planaria was performed to determine the distance traveled by each planarian following exposure to spring water, cocaine, and fentanyl at each time point. The total distance traveled (in centimeters) during the 5 min drug-free period was measured using the tracking tools of the EthoVision software (Wageningen, the Netherlands). Data were compared for overall effect and group-wise differences. Average locomotor activity and further comparisons were made where results were normalized to spring water and all values were expressed as a percentage of the mean of the spring water using Prism8 (GraphPad Software, San Diego, CA 92108).
Figure 1 shows the effect of cocaine and fentanyl on planaria withdrawal behavior (distance traveled). No statistically significant differences were found at any time point of exposure to any drug; however, qualitative results are presented here to reflect non- significant changes. Qualitatively, planaria exposed to 10 μM of cocaine showed a decrease in locomotor activity 15 minutes after exposure compared to controls. By 60 minutes and 180 minutes post-exposure, there appears to be an increase in distance traveled compared to controls which remained very close to that of controls by 24 hours of exposure (Figure 1).
Change of 10nm of fentanyl
Planaria exposed to 10 nM of fentanyl also showed a decrease in the locomotor activity by 15 minutes of exposure in comparison to controls. By 60 minutes and 180 minutes after exposure, there appeared to be an increase in distance traveled compared to control planaria. However, by 24 hours, planaria exposed to 10 nM of fentanyl showed a decrease in locomotor activity compared to controls, an observation that was different from cocaine (Figure 1).
Change of 100nm of fentanyl
The effects of 100 nm of fentanyl on planaria were the largest and most drastic changes compared to its cocaine and 10 nm fentanyl. While there is no statistical significance between the distances traveled throughout the timepoints, the increases (at 15 minutes and 180 minutes) and decreases (at 60 minutes and 24 hours later) in locomotor activity are greater than all other changes in locomotor activity for the other planaria groups.
Results of normalized data
To better visualize the changes in the planarian behavior, the results were normalized to spring water and all values were expressed as a percentage of the mean data for spring water (Figures 2, 3, and 4). When expressed as a percentage of the mean, many of the trends remained as in Figure 1 but offer an improved visual perspective of various changes.
Qualitatively, by 15 minutes exposure to cocaine or fentanyl (10 or 100 nM) resulted in a decrease in locomotor activity (Figure 2-4). By 60 minutes, exposure to cocaine (10μM) and fentanyl (10 nM) resulted in a 110% (Figure 2) and 109.7% (Figure 3) increase in locomotor activity, respectively versus controls. By 180 minutes, the changes in locomotor activity were striking. In the case of planaria exposed to cocaine, the locomotor activity with 110% of the control was very similar to that of activity from 60- minute exposure (Figure 2). However, fentanyl 10 nM exposure resulted in a 116% increase in locomotor activity (Figure 3), while 100 nM fentanyl exposure resulted in the most robust activity with 123% of the control (Figure 4). By 24 hours, all three compounds resulted in a decrease in locomotor activity compared to normalized control values set at 100%. Planaria exposed to cocaine only showed a decrease at a modest 98% of control, and those exposed to fentanyl 10 nM and 100 nM showed more pronounced decreases at 84% and 94%, respectively compared to normalized control.
Figure 1: Temporal effects of cocaine and fentanyl on planaria locomotor activity. Qualitatively, planaria exposed to cocaine, and the two concentrations of fentanyl showed a trend of decreasing locomotor activity by 24 hours of exposure. However, at acute exposure times, there appears to be a differential effect on the locomotor activity.
Figure 2: Effect of 10 μM of cocaine on locomotor activity of planaria. 2A. Although acutely (15 minutes or 0.25 hours) planaria showed a decrease in locomotor activity there was an increase in locomotor activity at subacute exposures (1 and 3 hours). By 24 hours, the activity was very close to control planaria. 2B: A spline of the mean distance traveled by planaria exposed to cocaine (10 μM) when normalized to the control.
Figure 3: Effect of 10 nM of fentanyl on locomotor activity of planaria. 2A. Similar to cocaine exposure, acutely (15 minutes or 0.25 hours) planaria showed a decrease in locomotor activity with a later increase in locomotor activity at subacute exposures (1 and 3 hours). By 24 hours, the activity decreased dramatically unlike those in cocaine exposure. 3B: A spline of the mean distance traveled by planaria exposed to fentanyl (10 nM) when normalized to the control.
Figure 4: Effect of 100 nM of fentanyl on locomotor activity of planaria. 4A. 100 nM of fentanyl exposure resulted in the most diverse changes in the locomotor activity with a trend of decrease in decrease locomotor activity between 15 min (0.25 hours) and 1 hour of exposure. By 3 hours, a robust increase in locomotor activity was followed by a modest decrease in activity by 24 hours of exposure. 4B: A spline of the mean distance traveled by planaria exposed to fentanyl (100 nM) when normalized to the control. The spline that best models the data shown in Figure 4A resembles a quadratic pattern, with locomotor activity being the least when planaria were exposed for 15 minutes and 24 hours and peaking at around 3 hours.
This study assessed the effect of different times of exposure to cocaine or fentanyl on the withdrawal-like behavior of planaria, as well as the effect of different concentrations of fentanyl on these withdrawal behaviors. Testing with 10 μM of cocaine resulted in planaria showing little change in their distances traveled between the timepoints. At 24 hours post-exposure, the planaria exposed to cocaine showed only a slight decrease in the distance traveled compared to those exposed to the control solution. In the case of the study performed by Sacavage et. al., planaria exposed to cocaine showed a clear trend of decreasing distance during withdrawal at all the exposure time points. Sacavage did not use a control substance in their experiment; however, unlike the present study, their study showed a greater decrease in distance traveled between the 15 minute and 24-hour timepoints.
In a study using mammalians, Fujii et. al, assessed the effects of fentanyl administration on behavioral characteristics of mice.10 The results of this study showed that mice exposed to high amounts of fentanyl showed a large decrease in locomotor activity compared to when they weren’t exposed. In the present study, the changes from fentanyl exposure can be described as fluctuating and increase in locomotor activity between acute and subacute exposures. However, by 24 hours, there appears to be a modest decrease in locomotor activity (albeit this change is not statistically significant). It is not understood why the results of the present study differed from those of Sacavage’s study even though we used extremely similar methods and setup. The different trends could be attributed to varying tracking methods of the planaria. In Sacavage’s study, locomotor activity was measured in terms of the number of gridlines crossed, with the petri dishes containing the planaria placed over grid lines spaced 0.5 cm apart. However, in this study, planaria were tracked using the software EthovisionTM, whose tracking methods may have resulted in discrepancies between Sacavage’s study’s results, and this study’s results. Other limitations of this study that could have contributed to the observed variations in the results could be attributed to the limited sample size of the planaria used.
Taken together, planaria exposed to cocaine and fentanyl (10 nM and 100 nM) show a very similar withdrawal behavior by 24 hours of exposure as evidenced by a decrease in locomotor activity. The dose dependent effects of fentanyl appeared to be limited to short exposure periods (15 to 60 min). At greater exposure periods, the decrease in locomotor activity from a low dose fentanyl exposure appears to be very similar to that of high dose exposure, which can be attributed to the highly potent nature of fentanyl. Future studies may be directed at replicating the study with a larger sample size for greater power in the results. Another consideration would be to increase the duration of exposure beyond 24 hours to understand the effects of chronic exposure and sufficiently determine differences between low and high dose fentanyl exposure.
The authors would like to extend their sincere thanks and gratitude for the invaluable mentorship and guidance of Dr. Shane Perrine, Ph.D., Department of Psychiatry and Behavioral Neurosciences (DPBN), Wayne State University School of Medicine, and John D Dingell Veterans Affair Medical Center, Detroit, MI 48201. The authors also thank Ms. Nareen Sadik, BS (DPBN), and Dr. Srinivasu Kallakuri, Ph.D. (DPBN) for their valuable guidance and technical help in enabling us to complete this research project. The research supplies were purchased using funds from an NIH/NIDA grant (DA042057) awarded to Dr. Shane Perrine. Fentanyl and cocaine were generously supplied free by the NIDA Drug Supply Program (Bethesda, MD).
A compulsive need to consistently use habit-forming substances such as nicotine, heroin, fentanyl, and cocaine.
A group of nerve cells forming a nerve center, typically outside the brain.
An increased effect of a drug on a subject after repeated use.
The movement or motion of a living being to move between places.
Chemical substances released at the end of nerve terminals to transmit messages.
Peripheral nerve cords
A cord of nerve fibers that run the length of the body of the animal and connect the ganglia.
The behavior exhibited when a subject abstains from a drug that they are addicted to.
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About the Author
Merwan Malakapalli is a junior at Lakeside High School in Seattle, Washington. He is especially interested in technology and computer science, which he’s been passionate about since middle school. He loves to read books and play tennis, both of which were started at a young age. In his spare time, he tutors young children in science and helps organize fundraisers to support children in India.