Mu Opioid Receptor Agonist DAMGO Produces Place Conditioning, Abstinence-induced Withdrawal, and Naltrexone-Dependent Locomotor Activation in Planarians

Abstract

Unlike the behavioral effects planarians display when exposed to cocaine, amphetamines, cathinones, ethanol and sucrose, effects of opioid receptor agonists, especially mu opioid receptor agonists, are poorly defined in these flatworms. Here, we tested the hypothesis that planarians exposed to a selective mu opioid receptor agonist, DAMGO (0.1, 1, 10 µM), would display a triad of opioid-like effects (place conditioning, abstinence-induced withdrawal, and motility changes). DAMGO was selected versus morphine because of its greater mu opioid receptor selectivity. In place conditioning and abstinence experiments, the planarian light/dark test (PLDT) was utilized (i.e., planarians are placed into a petri dish containing water that is split into light and dark compartments and time spent in the compartments is determined). Planarians conditioned with DAMGO (1 µM) spent more time on the drug-paired side compared to water controls. In abstinence experiments, planarians exposed to DAMGO for 30 min were removed and then placed into water, where light avoidance (e.g. defensive responding) and depressant-like effects (i.e., decreased motility) were quantified. Compared to water controls, DAMGO-withdrawn planarians spent less time in the light (10 µM) and displayed decreased motility (1, 10 µM). Acute DAMGO exposure (1 µM) produced hypermotility that was antagonized by naltrexone (1, 10, 100 µM). In contrast, acute exposure to the kappa opioid receptor agonist U50,488H (0.1, 1, 10 µM) resulted in decreased motility. Our results show that a mu opioid agonist produces mammalian-like behavioral responses in planarians that may be related to addiction and suggest opioid-like behavioral effects are conserved in invertebrates.

Keywords: Addiction; DAMGO; Mu opioid; Place preference; Planarians; Withdrawal.

Fig. 1. DAMGO produced environmental place conditioning (EPC)

Planarians were conditioned with DAMGO (0.1, 1, 10 μM) and time spent on the DAMGO-paired side was quantified and graphed (+ S.E.M.). N=11–12 planarians/group. ^**p < 0.01 compared to water control (0 μM). (Box) Planarians were conditioned with the highest concentration of DAMGO (10 μM) by itself or in combination with the kappa opioid antagonist nBNI (1 μM). Time spent on the DAMGO-paired side was quantified and graphed (+ S.E.M.). N=8 planarians/group.

Fig. 2. DAMGO-withdrawn planarians display abstinence-induced withdrawal responses. (A)

Abstinence from DAMGO exposure enhances light avoidance (defensive responding). Planarians were exposed to different concentrations of DAMGO (0, 0.1, 1, 10 μM) for 30 min and then removed and placed into a petri dish containing water that was split evenly into light and dark sides. Time spent in the light was measured over 10 min. Data was presented as a scatter plot showing the median percentage of time spent in the light (with interquartile range) over the 10-min interval. N=11–12 planarians/group. ^*p < 0.05 compared to water control (0 μM). (B) Abstinence from DAMGO exposure decreases motility. Planarians were exposed to different concentrations of DAMGO (0, 0.1, 1, 10 μM) for 30 min and them removed and placed into a petri dish containing water. Motility counts were quantified as the number of gridlines that planarians crossed in 5 min. Data were presented as percentage of water control (+S.E.M.) over the 5-min interval. N=18 planarians/group. ^*p < 0.05 compared to water control (0 μM).

Fig. 3. Acute DAMGO exposure produces hypermotility that is antagonized by NTX and the kappa opioid agonist U50,488 decreases motility. (A)

Planarians were exposed for 5 min to either DAMGO (1 μM) or water in combination with different concentrations of NTX (0, 1, 10, 100 μM). Motility was measured over the 5-min interval, and data were presented as mean number of motility counts (+ S.E.M.) over 5 min. N= 12 planarians/group. ^***p < 0.001 compared to DAMGO (1 μM) by itself. (B) Morphine does not significantly affect planarian motility. Planarians were exposed for 5 min to morphine (0.1, 1, 10 μM) and motility was measured over 5 min. Data were presented as mean number of motility counts (+ S.E.M.) over 5 min. N=8 planarians/group. (C) U50,488 reduces planarian motility. Planarians were exposed for 5 min to U50,488 (0.1, 1, 10 μM) and motility was measured over 5 min. Data were presented as mean number of motility counts (+ S.E.M.) over 5 min. N=8 planarians/group. ^***p < 0.001 compared to water control (0 μM).