Husbandry
All procedures and protocols involving the care and use of laboratory animals were reviewed and approved by the Animal Ethics Committee of the Technological Unit of Nutrition and Health of EURECAT (Reus, Spain) and the Generalitat de Catalunya (DAAM 10026). All sections of this report adhere to the ARRIVE Guidelines for reporting animal research.
Zebrafish.
Adult (6–8 months old; 3–4 cm long; wild-type) male and female short-finned Danio rerio zebrafish (0.4–1 g) of heterogeneous genetic background were obtained from ZF Biolabs (Madrid, Spain). Fish were kept at 28.5 ± 0.5 °C on a 14:10-h light/dark cycles (25) in a standalone aquatic flow-through system (Dohse Aquaristik GmbH & Co. KG, Grafschaft-Gelsdorf, Germany), provided with constant filtration and aeration, at a density of 2 fish per liter. Water used in the system consisted of reverse osmosis water supplemented with 0.8 g/L sea salts (OSMO FIT, Hobby, Germany). Adult fish were fed daily with commercially available flake fish food (Ocean Nutrition, Newark, U.S.A.) and brine shrimps. Fish were acclimated to the aquatic system for 2 weeks before experiment begins. Behavioral tests took place during the light phase between 08:00 and 18:00 hours. All efforts were made to minimize the number of animals used and their discomfort. After experiments, zebrafish were euthanized by hypothermia.
Rats.
Male and female (3 weeks old; 80 g) Sprague-Dawley rats (Envigo RMS Spain, Barcelona, Spain) were maintained in the local animal unit. Food and water were available ad libitum and rats were maintained on a 12:12-h light/dark cycles with temperature at 22 ± 1 °C. Male and female rats were housed in separate animal rooms to avoid hormonal effects on the cognitive tests (26). Rats were single housed in plastic cages with sawdust bedding in an enriched environment with shredded paper and a cardboard roll. Rats were left undisturbed except for routine cage cleaning, twice per week, and body weight were measured weekly until treatments. After experiments, adult rats were fastened for 5 h (from 09.00 to 14.00 h) without restriction of water and killed by decapitation.
Treatments
MNE.
GGRE (6.2% glycyrrhizic acid) and RE (6% rosmarinic acid) were obtained by Ebro Regaliz (Barcelona, Spain) and Nutrafur (Murcia, Spain), respectively.
Zebrafish.
Combined males and females of equal proportion were randomly selected for exposure to GGRE, or RE at a concentration of either 100 or 250 mg/L of water (N = 10/group). Treatments were administered by immersion into a novel water tank for 30 min to minimize stress (27). After treatment, fish were transferred to another water tank system for 5 min before behavioral tests. To minimize the number of animals used, each fish performed a behavior test battery. Each fish was exposed to the assigned treatment before each behavioral test (twice per fish) (Fig. 1). Control fish were kept under the same conditions used for the treated fish, but without exposure to any treatment.
Rats.
Two sets of animals were used for two parallel experiments (Fig. 1).
(1) Healthy rats. Adult male rats were randomly allocated to the following experimental groups: control, GGRE (150 mg/Kg) or RE (150 mg/Kg), (n = 10/ group).
(2) LPS-treated rats. Adult male and female rats were randomly allocated to the following experimental groups: control, LPS, LPS + RE (n = 10/ group).
Once animals were four weeks old underwent oral administration of either GGRE (150 mg/Kg) or RE (150 mg/Kg) for six weeks. Treatments were dissolved in low-fat condensed milk:water (1:10 v/v) and administered by micropipette. Controls were treated with low-fat condensed milk:water (1:10 v/v), only. As for the LPS-treated rats’ study, LPS (300 ug/kg, i.p.) was administered for seven days, during the fourth week of the study, 2 hours after RE or vehicle treatments, when animals were seven weeks old. Controls for the LPS group were i.p. injected with 0.9% sterile saline water for the same period. All treatments were administered every day, between 09.00 and 10.00 h. Animals underwent behavioral testing immediately after LPS treatment (Fig. 1).
Behavioral testing
Zebrafish.
Zebrafish underwent a behavior test battery spread over 5 days: T-maze on days one and two; the novel object preference (NOP) on day five. All behavioral tests were performed in the morning between 9.00 and 12.00 h.
T-maze.
T-maze was used to measure spatial memory as described previously (28). The apparatus consisted of a white plexiglass maze with a T shape, filled with 10 cm of tank water. The long arm (50 × 10 cm) was intersected at one of its ends by a short arm (50 × 10 cm). One of the ends of the short arm opens to a larger reservoir (30 cm square) 5 cm deeper than the rest of the maze. The reservoir was enriched with artificial plants and color stones to offer a favorable habitat to the fish. By nature zebrafish would spend more time in the reservoir than in the rest of the maze (28). The other end of the long arm was connected to a starting zone (30 × 10 cm) by a white removable door (Fig. 2). The protocol consists in three trials [T-Maze T1 (TMT1), TMT2 and TMT3]. During each trial, fish were individually placed in the starting zone for 1 min with the door closed. Then, the start box was raised and lowered after the fish exited. During the TMT1, fish were given a maximum of 4 min to fully explore the maze and encounter the reservoir where they were left for at least 20 s before the end of the trial. Each fish repeated the trial 3 h (TMT2) and 24 h (TMT3) later. After each trial, fish were returned to their home tank. All trials were video-recorded, and the time needed to find the reservoir [transfer latency time (TLT)] was quantified using a digital tracking system (ANY-Maze, version 4.82).
NOP.
NOP was performed as previously described (29). Briefly, zebrafish were subjected to 5 min of acclimation to the novel tank (29 × 14 × 18 cm tank filled with 14 L of tank water) followed by 10 min of habituation to two identical objects (training phase). After a recovery period of 10 min, one of the two original objects was randomly replaced with a novel object and interactions were monitored for an additional 10 min (testing phase). To avoid thigmotaxis influence, the objects were placed one in front of the other in an equal distance between the center of the tank and the walls. An 8.4 cm × 8.4 cm box was superimposed over each object and the time the fish spent within the boxes was recorded and scored automatically (ANY-Maze, version 4.82).
Rats.
Behavioral tests were carried out during the fifth [EPM, NOR and open field (OF)] and sixth (MWM) week of MNE administration, when the animals were eight and nine weeks of age, respectively (Fig. 1). Before each behavioral test, rats were habituated to the test room for 30 min. All experiments were carried out between 9:00 am and 2:00 pm.
EPM.
EPM was performed to assess retention memory as described previously (30) with some modification. The maze consisted of two open arms (50 × 10 com) and two enclosed arms (50 × 10 × 40 cm) that all extended from a common central platform (10 × 10 cm). The apparatus was raised 60 cm above the floor on a central pedestal. On the training phase, rats were placed individually at the end of the open arm, facing it away from the central platform. The time that the animal took to move from the open arm to one of the two enclosed arms (4 paws in) was recorded. The same procedure was repeated 60 min (acquisition) and 24 h (retention) after training. To become acquainted with the EPM, if the rat failed to enter the enclosed arm within 120 s, it was guided to one of them and let it to explore the maze for further 60 s before to be returned to its home cage (training phase only). Animal behavior was recorded and TLT (acquisition and retention phases) was analyzed using a tracking system (ANY-Maze, version 4.82). The apparatus was wiped clean with 70% ethanol before testing each animal.
NOR.
NOR was used to assess recognition memory as described previously (31) with some modifications. Briefly, rats were placed in a grey plywood rectangular box (70 × 45 × 45 cm) for 5 min for habituation to the novel arena. The day after, rats were habituated to two identical objects [(A), acquisition phase)] placed in the back left and right corners of the experimental box for 3 min. After a delay of 1 h, one of the two familiar objects was replaced with a novel object (B) and rats were replaced in the middle of the box at the mid-point of the wall opposite the sample objects for a total time of 3 min (retention phase). Box and objects were cleaned with alcohol 70% to avoid any cue smell between each trial. Direct contacts with the objects, include any contact with mouth, nose or paw or minimal defined distance (< 2 cm), were recorded and scored automatically (ANY-Maze, version 4.82). Memory discrimination index (MDI), [(B exploration time – A exploration time)/ (B exploration time + A exploration time)], was used to express recognition memory in rats (32).
OF.
To assess anxiety-like behavior rats were individually placed in a wooden grey box (70 × 45 × 45 cm) for 5 min. A central area (20 × 40 cm) was drawn in the floor of the apparatus to score time and number of entries in the inner zone. Distance moved, velocity, percentage of time spent in inner zone and frequency of inner zone entries were recorded and analyzed using a tracking system (ANY-Maze, version 4.82). The apparatus was wiped clean with 70% ethanol before testing each animal.
MWM.
The MWM is a test of spatial learning and reference memory for rodents that relies on distal cues to navigate from start locations around the perimeter of an open swimming arena to locate a submerged escape platform (33). The swimming arena consisted of a circular metal pool (150 cm diameter x 58 cm tall) filled with tap water (23–25ºC, 40-cm deep). The maze was divided geographically into four quadrants: Northeast (NE), Northwest (NW), Southeast (SE) and Southwest (SW), and four starting positions, North (N), East (E), West (W), South (S), were located at equal distances along the pool rim. A circular white platform (10 cm diameter) was placed in the center of the NW quadrant and submerged 1.5 cm below the water surface. Water was white colored with a non-toxic white paint to hide the platform. Distal cues were arranged around the maze to provide landmarks that the animals could use to navigate to the platform.
Acquisition training: Rats underwent five days of training that consisted of four trials/day. At the beginning of each trial rats were individually placed in one of the starting positions (N, E, S, or W) facing the wall of the tank and allowed to explore the maze for 60 s. A different starting position was used for each of the 4 trials on a given day arranged in a semi-random pattern. Once rats reached the platform, they were held there for 20 s. If the platform was not found within the given time, rats were gently assisted to the platform by the experimenter and detained there for 20 s.
Removal phase: On day six (removal) the platform was removed, and the rats were allowed to freely explore the maze for 60 s. Rats were individually placed into the quadrant diagonally opposite to the one originally hosting the platform. The amount of time spent in the quadrant where the platform was previously hidden was video recorded and automatically analyzed using a tracking system (ANY-Maze, version 4.82).