Adolescent cannabinoid exposure effects on natural reward seeking and learning in rats

Abstract

Rationale: Adolescence is characterized by endocannabinoid (ECB)-dependent refinement of neural circuits underlying emotion, learning, and motivation. As a result, adolescent cannabinoid receptor stimulation (ACRS) with phytocannabinoids or synthetic agonists like "Spice" cause robust and persistent changes in both behavior and circuit architecture in rodents, including in reward-related regions like medial prefrontal cortex and nucleus accumbens (NAc).

Objectives and methods: Here, we examine persistent effects of ACRS with the cannabinoid receptor 1/2 specific agonist WIN55-212,2 (WIN; 1.2 mg/kg/day, postnatal day (PD) 30-43), on natural reward-seeking behaviors and ECB system function in adult male Long Evans rats (PD 60+).

Results: WIN ACRS increased palatable food intake, and altered attribution of incentive salience to food cues in a sign-/goal-tracking paradigm. ACRS also blunted hunger-induced sucrose intake, and resulted in increased anandamide and oleoylethanolamide levels in NAc after acute food restriction not seen in controls. ACRS did not affect food neophobia or locomotor response to a novel environment, but did increase preference for exploring a novel environment.

Conclusions: These results demonstrate that ACRS causes long-term increases in natural reward-seeking behaviors and ECB system function that persist into adulthood, potentially increasing liability to excessive natural reward seeking later in life.

Keywords: Autoshaping; Endocannabinoid; Novelty; Nucleus accumbens; Palatable food; Reward.

Figure 1. Adolescent Cannabinoid Receptor Stimulation Procedure

a) Timeline of experimental procedures is shown, with age (postnatal day) shown at bottom. b) List of behavioral tests, number of animals tested in each, and the order in which they were conducted. c) Body weights during WIN ACRS period (shaded box), and for 3 weeks thereafter. Group m+SEM displayed with solid black (ACRS) and grey (control) lines, and individual rats shown with semi-transparent lines of the same colors. d) Chow intake in the 2.5h after the 14^th WIN injection (PD 43) is shown for vehicle (white bar) or WIN (grey bar) groups (m+SEM). * p<0.05 vehicle vs. WIN cages.

Figure 2. Acquisition of Sign and Goal Tracking to Food-Predictive Cues

a) the percentage of rats in control (top row) and ACRS (bottom row) that interacted with the lever cue only (white), cup only (black), both lever and cup (light grey), or neither stimulus (dark grey) on the first ever lever extension on sign/goal tracking training day 1 (left column), or the first cue presentation on the 8^th training day (right column). b) In each panel, m+SEM hand-scored interactions with the food cup (top left), cue lever (top right), or inactive lever (bottom right), or rears (bottom left) are shown during the first 5, or last 5 cue presentations on sign/goal tracking training days 1, 2, and 3. *p<0.05, interaction of ACRS × cue block. c) m+SEM rate of CS+ lever depressions (depressions/sec) on each training day in control (black line) and ACRS rats (grey line). d) m+SEM rate of food cup entries during cues (solid black, grey lines) or in non-cue periods (dashed black, grey lines). *p<0.05, ACRS group × day interaction.

Figure 3. Cue Preference in ACRS and Control Rats

a) Pavlovian Conditioned Approach (PCA) score across training days. Black line=control, grey line=ACRS. Positive values indicate bias toward the CS+ lever (sign tracking), negative values indicated bias toward food cup (goal tracking) *p<0.05, group × day interaction. b) Mean+SEM PCA Score on training days 7&8 show with black (control) or grey (ACRS) horizontal lines and error bars. Individual rats’ data are represented with white or grey circles. c) Rat-by-rat PCA scores, ranked from most negative to most positive. Control rats=black bars, ACRS rats=grey bars, ACRS rats defined as intermediates (PCA<0.3 and >−0.3)=white bars). d) CS+ lever (solid black, grey lines) and during cue food cup entries (dashed black, grey lines) emitted during the first, second, or third 25-cue blocks on sign/goal tracking extinction training test. * p<0.05, Main effect of ACRS on food cup entries.

Figure 4. ACRS Effects on Palatable Food Intake

a) In 30min intake tests held separate days, consumption of familiar banana-flavored pellets, a novel chocolate reward, or the same chocolate reward, now habituated, is shown for each group (black bars=control m+SEM), grey bars=ACRS m+SEM, circles represent individual control (black) or ACRS rats (grey). *p<0.05, control versus ACRS. b) Intake of water (left) or 15% sucrose solution (right) after acute food restriction in a 2-bottle choice test. *p<0.05, control versus ACRS. c) intake of water or sucrose after acute sucrose satiety. Group ns shown below bars.

Figure 5. ACRS Effects on Novelty Induced Locomotion and Novel Environment Preference

a) m+SEM Distance traveled on a 1h novel environment locomotion test is shown in 15min bins for control (black line; individual rats shown with faded black lines) and ACRS rats (m+SEM=dashed line, individual rats shown with faded white lines). b) In a novel environment preference test, ACRS rats (grey bar; circles represent individual rats) spent a greater percentage of the session time on the novel side than controls (black bar; circles represent individual rats). c) In an elevated plus maze task, percent of session time spent on the open arms of the apparatus, an index of anxiety, is shown. Group size shown in bars. *p<0.05, vehicle versus ACRS. Group ns shown in bars.

Figure 6. ACRS and Food Restriction Effects on Brain Endocannabinoid Levels

m+SEM levels of a–c) 2-AG, d–f) AEA, or g–i) OEA observed in a,d,g) medial prefrontal cortex, b,e,h) nucleus accumbens, or c,f,i) cerebellum are shown in ad libitum fed rats (left panels; white bars; circles represent individual rats) or food restricted rats (right panels; solid bars; circles represent individual rats). Control rats are represented with black borders or filled bars, ACRS rats represented with grey bordered or filled bars. ECB levels in dissected samples normalized by protein content in sample. Sample size shown in bars. *p<0.05, main effect of ACRS/control. #p<0.05, main effect of restriction