Food-induced reinforcement is abrogated by the genetic deletion of the MT1 or MT2 melatonin receptor in C3H/HeN mice

Abstract

Palatable food is known for its ability to enhance reinforcing responses. Studies have suggested a circadian variation in both drug and natural reinforcement, with each following its own time course. The goal of this study was to determine the role of the MT₁ and MT₂ melatonin receptors in palatable snack food-induced reinforcement, as measured by the conditioned place preference (CPP) paradigm during the light and dark phases. C3H/HeN wild-type mice were trained for snack food-induced CPP at either ZT 6 - 8 (ZT: Zeitgeber time; ZT 0 = lights on), when endogenous melatonin levels are low, or ZT 19 - 21, when melatonin levels are high. These time points also correspond to the high and low points for expression of the circadian gene Period1, respectively. The amount of snack food (chow, Cheetos®, Froot Loops® and Oreos®) consumed was of similar magnitude at both times, however only C3H/HeN mice conditioned to snack food at ZT 6 - 8 developed a place preference. C3H/HeN mice with a genetic deletion of either the MT₁ (MT₁KO) or MT₂ (MT₂KO) receptor tested at ZT 6 - 8 did not develop a place preference for snack food. Although the MT₂KO mice showed a similar amount of snack food consumed when compared to wild-type mice, the MT₁KO mice consumed significantly less than either genotype. We conclude that in our mouse model snack food-induced CPP is dependent on time of day and the presence of the MT₁ or MT₂ receptors, suggesting a role for melatonin and its receptors in snack food-induced reinforcement.

Keywords: C3H/HeN mouse; Conditioned place preference; MT(1) or MT(2) knockout mice; Melatonin; Melatonin receptors.

Figure 1. Experimental Design

A: Experiments for wild-type mice were conducted at two time points during the light/dark cycle, ZT 6 – 8 during the light phase and ZT 19 – 21 during the dark phase. These two time points correspond to the lowest and highest levels of melatonin, respectively [39]. Experiments for MT₁KO and MT₂KO mice were conducted only at ZT 6 – 8. B – D: For each experiment a new cohort of mice was used. All mice were subjected to a general experimental time line consisting of three days of handling prior to the onset of the experiment. Day 1 (Habituation) and Day 2 (Pre-CPP) consisted of free access to the entire test chamber for 20 min in order to eliminate novelty. Time spent in each choice compartment during the Pre-CPP was used to determine the initial compartment preference. Days 3 – 8 consisted of 60 min daily conditioning sessions during which mice were confined to one choice compartment. On Days 3, 5, and 7 mice were confined to their initially least-preferred compartment and received either no food, chow, or snack based on their group assignment. On Days 4, 6, and 8 mice were confined to their initially most-preferred compartment and received no food. Day 9 (Post-CPP) consisted of free access to the entire test chamber for a 20 min test session to determine final compartment preference.

Figure 2. Effect of Chow or “Snack” on Food-Induced Place Preference During the Light (ZT 6 – 8) Phase

Time spent in each compartment during Post-CPP was measured across the whole 20-minute test session for the wild-type mice following treatment with no food (A: n = 14), chow (B: n = 14) or snack food (C: n = 13). CPP scores were calculated by subtracting time spent in the initially preferred compartment (no food presented) from time spent in the initially non-preferred compartment (food presented in chow and snack groups) (D). Data represent mean ± S.E.M. of time (s) spent in each compartment or as mean ± S.E.M of CPP score (s). Compartment duration was compared using Student t-test. *p<0.05 when compared to other compartment. CPP scores were compared using one-way ANOVA, with a main effect of treatment indicated by the p value in the upper left corner. *p<0.05 when compared to no food group (Bonferoni post-test). s: second; n.s.: non-significant.

Figure 3. Effect of Snack Food-Induced Place Preference During the Dark (ZT 19 – 21) Phase

Time spent in each compartment during Post-CPP was measured across the whole 20-minute test session for the wild-type mice following treatment with no food (A: n = 10), or snack food (B: n = 9). CPP scores were calculated by subtracting time spent in the initially preferred compartment (no food presented) from time spent in the initially non-preferred compartment (food presented in snack group) (C). Data represent mean ± S.E.M. of time (s) spent in each compartment or as mean ± S.E.M of CPP score (s). Compartment duration and CPP scores were compared using Student t-test. s: second; n.s.: non-significant.

Figure 4. Effect of MT₁ or MT₂ Receptor Deletion on Snack Food-Induced Place Preference During the Light (ZT 6 – 8) Phase

Time spent in each compartment during Post-CPP was measured across the whole 20-minute test session for the MT₁KO mice following treatment with no food (A: n = 5) or snack food (B: n = 7). CPP scores were calculated by subtracting time spent in the initially preferred compartment from time spent in the initially non-preferred compartment (C). The same comparisons were performed for the MT₂KO mice including compartment duration for the no food group (D: n = 8) and snack food group (E: n = 8). CPP scores were also calculated for MT₂KO mice (F). Data represent mean ± S.E.M. of time (s) spent in each compartment or as mean ± S.E.M of CPP score (s). Compartment duration and CPP scores were compared using Student t-test. *p<0.05, when compared to other compartment or other treatment group. s: second; n.s.: non-significant.

Figure 5. Food Consumed During Conditioning Days During the Light (ZT 6–8) and Dark (19–21) Phases

Food consumed was measured for the chow and snack food treated groups during each conditioning day on which food was presented (Days 3, 5, and 7). Wild-type mice tested at ZT 6 – 8 were divided into a chow group receiving chow only (Harlan Teklad 2018sx) and a snack group receiving a mixture of chow, Oreos, Cheetos, and Froot Loops. Wild-type mice tested at ZT 19 – 21 as well as MT₁KO and MT₂KO mice tested at ZT 6 – 8 received the snack mixture. Effect of treatment was compared using two-way ANOVA for the wild-type mice at ZT 6 – 8 (A). One-way ANOVA was used to compare effect of treatment for wild-type mice at ZT 19 – 21 (B) as well as the MT₁KO (C) and MT₂KO (D) mice at ZT 6 – 8. Bars represent the mean ± S.E.M. of food consumed in grams. *p<0.05 when comparing food consumed between treatment days; ^@p<0.05 when comparing between treatment groups