Rats

Adult male Sprague Dawley rats (n = 183; 350–400 g at arrival; Charles River, Quebec, Canada) were single-housed in temperature (24 ± 2 °C) and humidity controlled (55 ± 10%) animal facility rooms. The light:dark cycle was 12:12 h (light on at 7:00 a.m.). Food and water were available ad libitum unless stated otherwise. All procedures were approved by the Institutional Animal Care Committee and complied with the Canadian and National Institute of Health Guides for the Care and Use of Laboratory Rats (NIH Publication #20-23). All rats were behaviorally naïve prior to beginning the set-shift procedure. Following set-shifting testing, rats were allowed to return to their pre-set-shift body weights, prior to undergoing additional behavioral tests.

Surgery

Rats were anesthetized with a ketamine (80 mg/kg; Vetoquinol)/xylazine (6 mg/kg; Bayer) mixture (i.p.) and positioned in a stereotaxic apparatus (David Kopf Instruments). Post-surgical analgesia was ascertained with meloxicam (subcutaneous; 1 mg/kg; Boehringer Ingelheim). The scalp was incised, and burr holes were prepared above the PFC skull region. Two stainless steel guide cannulae (22 G, PlasticsOne) were implanted into the prelimbic PFC (coordinates: 3.1 mm anterior, 1.4 mm lateral from Bregma (10˚ angle), and 3.0 mm ventral to dura; [25]), and secured with anchoring screws and dental cement. Rats were allowed one-week recovery prior to commencement of experiments.

Drugs and injection procedure

(-)-Cannabidiol, NAD299 hydrochloride, AM251 and (+)-MK801 maleate were from Tocris and THC from Cayman Chemical (Ann Arbor, MI, USA). All drugs were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and diluted to final concentrations (final DMSO 5%) in saline containing 5% cremophor EL (Sigma-Aldrich). Before testing rats were handled for 5 min per day. For microinfusions, rats were gently restrained prior to insertion of the microinjectors. All microinfusions were 500 nl/hemisphere. Injectors were removed after 1 min and behavioral testing began 5 min later. The following drug concentrations (indicated later with subscript i.e. THC 100 ) were used (in ng/500 nl): THC (10, 50, 100 or 500), CBD (10, 100 or 500), NAD299 (100), AM251 (100 or 200) and (in μg/500 nl) MK801 (3 or 6). When two drugs were tested simultaneously they were injected as a co-mixture. To avoid possible sub-chronic effects of infused substances, any given treatment was administered only once. A seven-day rest period was allowed between the different behavioral tests to allow proper drug washout between tests.

Attentional set-shifting

Prior to training, rats were food restricted to ~85% of their free feeding body weight and familiarized with the reward cue (45 mg sucrose pellets; banana flavor; BioServ, USA). Set-shifting was conducted in an operant chamber (Med-associates, St Albans VT, USA) enclosed in a sound-attenuating box. The test chamber (30.5 × 24 × 21 cm) was equipped with a house light, two retractable levers, pellet receptacle and two cue lights (Fig. 1c). The chamber was computer controlled with customized software procedures (MED-PC IV, Med-Associates) adapted from [26] and [16].

Fig. 1 Histology and set-shift procedure. a Microphotograph of Cresyl violet stained coronal section containing guide canulae tracks for injections directly in the prefrontal cortex (PFC). b Schematic representation of the injection sites (circles) superimposed on the coronal plane of a rat brain (modified from [25]). Numbers on the schemes indicate distance from Bregma point. c Experimental timeline for the attentional set-shift procedure. The grey rectangles represent front panel of the operant box, circles correspond to the light cues (gray = light off; white = light on), white rectangles correspond to extended levers, the lever rewarded with sucrose pellet is indicated with plus sign above it; D1-D4 indicate different days of the procedure. Note that rats were injected with the drug solution on the last day of the procedure Full size image

Briefly, rats were placed in the chamber (house light on, one lever extended) and learned to associate each lever press with a food pellet reward. After 15 presses the lever was retracted, the alternate lever was inserted, and the rat needed to press it 15 times. Subsequently, the levers were randomly inserted into the chamber (15× each) until pressed by the rat. Next, the house light was switched off and a timed lever-pressing trial started (trial duration: 20 s). Trials began with illumination of the chamber and insertion of one lever for 10 s. Pressing the lever resulted in reward and a 4 s light cue. A failed trial (omission trial) resulted in lever retraction, no reward and turning off the house light. After 30 trials the rat was removed from the chamber. The next day the rat was exposed to the timed trials until a performance criterion of ≥85 presses/90 trials was achieved.

Following this, the rats’ preferred lever was determined based on seven sets of trials (set: initial trial + secondary trial/s). All trials began with chamber illumination and insertion of both levers for 10 s. During the initial trial, pressing any lever was rewarded while during the secondary trial/s only pressing the lever other than during initial trial. A set was considered completed when both levers were pressed. The lever with more presses on the initial trials was considered as ‘preferred’.

The next day rats were exposed to 100 visual-cue discrimination trials beginning with illumination of one cue light, which indicated the correct lever position. After 3 s the house light was switched on and both levers inserted for 10 s. Only a correct response was rewarded. The successful training criterion was 10 successive correct presses.

The next day the rat received a specific intra-PFC drug treatment and after 5 min 20 visual-cue discrimination trials were presented to test long-term memory retrieval and motoric functions. Immediately after, the response discrimination procedure began (set-shift = 10 successive correct presses; max 120 trials). Trials were similar to visual discrimination trials, except that the correct response was set to the non-preferred lever (see above) irrespectively of the cue light position.

The total number of trials to set-shift (excluding omissions) and the total number of errors were calculated. Perseverative (>5 errors/16 successive trials) and regressive errors (≤5 errors/16 successive trials) were counted when an error was made by following the light cue and never-reinforced errors when an error was made but the cue light indicated the correct lever.

Spontaneous alternation behavior

The Y-maze (black nonreflecting acrylic, 3 arms at 120˚ from each other; arm length: 50 cm; wall height: 40 cm) was located on the floor and dimly illuminated (40 lux). Following room acclimatization (1 h), intra-PFC-injected rats were placed at the end of one arm facing the wall, and allowed to explore the maze freely for 10 min. Alternation behavior was video recorded and the sequence and total number of arm entries (all paws in) was scored off-line. The alternation score was calculated as unique triplets/(total arm entries-2), where unique triplets = a consecutive entry to all three arms (i.e. ABC; Fig. 5a). Re-entries to the same arm (i.e. AA) and returns to previously visited arm (i.e. ABA) were scored separately.

Elevated plus maze test

The elevated plus maze (EPM) apparatus (black acrylic, 4 arms (10 × 50 cm) stemming from a 10 × 10 cm platform and forming a plus shape) was raised above the floor by 50 cm and was dimly illuminated (40 lux). Two opposite arms were enclosed with 40 cm high walls while other two arms were opened (except for 1 cm high ledge). Intra-PFC-injected rats were placed on the central platform (facing closed arm) and explored the maze for 10 min. Behavior was video recorded and analyzed offline (Behaview software; www.pmbogusz.net). The number of entries (all paws in) and the time spent in closed and open arms was scored.

The three-chambered social approach test

The social interaction apparatus consisted of a transparent acrylic chamber divided into three equal compartments separated with guillotine doors [15]. One day before testing rats were room acclimated for 30 min and subsequently habituated to the apparatus (5 min center + 8 min entire apparatus). Social interaction testing consisted of 2 phases. In phase 1 (social motivation test), following a 30 min acclimatization, rats received assigned intra-PFC microinfusions and placed in the central compartment (5 min; guillotine doors in place). Subsequently, wire enclosures were placed in the side compartments (one contained a stranger male rat) and the tested rat could explore the entire apparatus for 8 min. In phase 2 (social memory test), a new, novel unfamiliar rat was placed in the previously empty enclosure cage and the test rat could explore both chambers (containing the previously encountered rat or the new stranger rat) for 8 min. Behaviors were video recorded and analyzed offline. The total duration of exploratory bouts that the rat spent with a stranger vs. the empty enclosure (phase 1) was calculated as a sociability score = t stranger /(t stranger + t empty ). The time spent with the previously encountered vs. novel stranger rat in phase 2, was calculated as a social recognition score = t novel /(t novel + t familiar ).

Histology

At the end of experiments rats were overdosed with pentobarbital (Euthanyl) and decapitated. Brains were removed, fixed in 10% formalin and cryoprotected in 30% sucrose solution. Coronal sections (50 μm thick) were cut, mounted on glass slides and stained with Cresyl violet. Sections with the cannula tip locations were microphotographed and referred to a rat brain atlas [25].

Statistical analysis