Drug-seeking behavior in an invertebrate system: evidence of morphine-induced reward, extinction and reinstatement in crayfish

Abstract

Several lines of evidence suggest that exploring the neurochemical basis of reward in invertebrate species may provide clues for the fundamental behavioral and neurobiology underpinnings of drug addiction. How the presence of drug-sensitive reward relates to a decrease in drug-seeking behavior and reinstatement of drug-seeking behavior in invertebrate systems is not known. The present study of a conditioned place preference (CPP) paradigm in crayfish (Orconectes rusticus) explores morphine-induced reward, extinction and reinstatement. Repeated intra-circulatory infusions of 2.5 microg/g, 5.0 microg/g and 10.0 microg/g doses of morphine over 5 days serve as a reward when paired with a distinct visual or tactile environment. Morphine-induced CPP was extinguished after repeated saline injections for 5 days in the previously morphine-paired compartment. After the previously established CPP had been eliminated during the extinction phase, morphine-experienced crayfish were challenged with 2.5 microg/g, 5.0 microg/g and 10.0 microg/g, respectively. The priming injections of morphine reinstated CPP in all training doses, suggesting that morphine-induced CPP is unrelenting, and that with time, it can be reinstated by morphine following extinction in an invertebrate model just like in mammals. Together with other recent studies, this work demonstrates the advantage of using crayfish as an invertebrate animal model to investigate the basic biological processes that underline exposure to mammalian drugs of abuse.

Fig 1

Illustration of the experimental arena for the CPP, Extinction and Reinstatements tests. In Fig 1A, an aquarium was constructed with Plexiglas. Tiles cut to 5cm by 11cm dimensions were placed on the floor. The thick ties represent the hard texture cue. For the soft texture, we used soft woolen materials cut to 5cm by 11cm dimensions and glued to the floor of the compartment. The hard texture and soft texture compartments represent the tactile environment. In Fig 1B, visual environments were created by maintaining the uniform color of the Plexiglas for the white compartment. For the striped compartment, we lined the inner walls of the compartments with plastic transparencies with continuous and alternating arrays of black or white stripes of about 10mm wide. Each compartment was divided into two compartments of equal sizes such that distinct visual or textured environments were always present in the opposite compartments.

Fig 2

Schematic representation of the experimental design used in the present study. Testing for initial preference was carried out in days 1 and 2. The conditioning consists of 10 alternate days (3-12) of drug and saline injections using the unbiased balanced protocol. During conditioning, the compartment in which morphine was administered was assigned randomly. Crayfish were treated for 10 consecutive days with alternate injections of morphine 2.5μg/g, 5.0μg/g and 10.0μg/g). After conditioning, animals were immediately confined to the conditioning compartment for 25 mins. On day 13, the partition separating the compartment was removed, and crayfish were placed at the center and allowed to move freely for 60 mins in a drug-free state to test for the expression of CPP. Following conditioning and the initial CPP test, each crayfish was given pairing of saline with each compartment, one per day, for 5 days (days14-18). Crayfish did not receive morphine during this period. Thereafter, crayfish were given a test for CPP on day 19. The next day (day 20), all crayfish received priming injections of morphine (2.50μg/g, 5.00μg/g, and 10.0μg/g) before the final test for CPP, also on day 20.

Fig 3

The spatial activities of crayfish in three different experiments, following our hypothesis that crayfish will spend equal amount of time in each of the tactile and visual environments. However, it turns out that crayfish showed preference for the uniform compartment following repeated measures of the spatial activities for 1 hour each day. During the first day, the mean time preference for the uniform compartment was 59.02% ±2.34 (SEM) and 40.91±2.33% (SEM) for the striped compartment. The preference was significant (T-test (μ =50.0%); t [₆] = 3.76, P= <0.05). In the second day, crayfish maintained their preference for the uniform compartment (62.91%±5.2) while 37.07% ± 5.28 (SEM) of its time was spent in the striped compartment and 62.91% ±5.2 (SEM) inside the uniform compartment. The preference for the uniform compartment was significant (T-test (μ =50.0%); t [₆] = 2.48, P= 0.05). During the CPP test, the preference shifted to the striped environment, though such preference was not significant (T-test (μ =50.0%); t [₆] = 0.20, P= 0.84). In the tactile environment, crayfish seem to prefer the soft compartment following two days of repeated measures. The mean time spent in the soft compartment was 52.9% ±2.71 (SEM) while crayfish spent an average of 47.71±2.85% of its time in the hard textured compartment. The preference for the soft texture compartment was not significant in the first day (T-test (μ =50.0%); t[₆]= 0.93, P=0.38. However, the preference for the soft texture compartment (meantime; 62.29%±4.5 SEM) in the second day when compared with the hard compartment (mean;=37.69±4.53% SEM) was significant (T-test (μ =50.0%); t[₆]= 2.66, P= 0.04). During the CPP test, the preference for the soft compartment was maintained, but not statistically significant (T-test (μ =50.0%); t[₆]= 0.84, P= 0.043).

Fig 4

Fig 4A: Repeated infusions of morphine induced CPP in crayfish in the textured environment. Five days of 2.5 μg/g produces 53.56% ± 2.3(SEM), while 5.0 μg/g and 10.0μg/g promote spatial activity of 62.55 % ± 2.6(SEM) and 69.13% ±3.6 respectively, of time preference for the hard textured compartment (F [_{2, 12}] = 20.88; P < 0.01). Post hoc test comparison indicates that crayfish treated with 5.0 μg/g and 10.0μg/g (**P<0.05) were higher and different from crayfish treated with 2.5 μg/g of morphine (*, P<0.05) and saline-paired crayfish. Fig 4B: Infusions of morphine produced CPP in the visual environment, such that repeated infusions of morphine for 5 days of 2.5 μg/g produces 53.79% ± 1.9 (SEM), while 5.0 μg/g and 10.0μg/g promote spatial activity of 58.20 % ± 2.8(SEM) and 63.59% ±2.8(SEM) respectively of time preference for the striped compartment (F [_{2, 10}] = 13.71; P = <0.01). Bonferroni post hoc test comparison indicates that morphine-conditioned crayfish (*, **P<0.05) were different from saline-paired crayfish. Crayfish treated with 5.0μg/g and 10.0μg/g were significantly different (**P<0.05) from 2.5μg/g (*P<0.05) treated crayfish.

Fig 5

Extinction by saline pairing and reinstatement in the tactile compartments. Mean (± SEM) percentage of time spent in the morphine-paired and saline-paired compartments in pre- and post extinction of 60mins test for CPP. The 60mins test for reinstatement of CPP following a priming injection of 2.5μg/g, 5.0μg/g and 10.0μg/g) is shown on the far right of each panel.

Fig 6

Fig 6A: Extinction by saline pairing and reinstatement in the visual compartments. Mean (± SEM) percentage of time spent in the morphine-paired striped compartment, and saline-paired compartments in pre and post extinction 60mins test for CPP. The 60mins test for reinstatement of CPP following a priming injection of 2.5μg/g, 5.0μg/g and 10.0μg/g) is shown on the far right of each panel.

Fig 7

Locomotion responses of crayfish pretreated with 2.5μg/g, 5.0μg/g and 10.0μg/g doses of morphine (n=7) or saline (n= 7) during CPP test in the textured and visual compartments. Data are expressed as mean traveled distances (pixels) within the test textured or visual compartments. In the textured compartment, ANOVA reveals that injection of morphine significantly (F [5, 41] = 2.5; P = 0.05) increased locomotion when compared with saline injection. Locomotion was high at 2.5μg/g and 5.0μg/g doses; *P<0.05, and low 10.0μg/g dose of morphine (**P<0.05). Whereas in the visual compartment, although locomotion was higher at lower doses of 2.5 μg and 5.0μg/g when compared with high dose of 10.0μg/g, however, such effect was not statistically significant (F [_{5, 41}] =0.93; P = 0.47).