Animal models

The study was approved by the Norwegian Animal Research Authority, and conducted in accordance with the laws and regulations controlling experiments on live animals in Norway. Spontaneously Hypertensive Rodents (SHR/NCrl) purchased from Charles River, Sulzfeld, Germany, were used as breeding subjects. During the first three weeks of life, the rats were under veterinarian care at the Norwegian Defence Research Establishment (Kjeller, Norway). Dams were caged singly under standard conditions (temperature ~22°C, humidity ~55%, 12 h light/dark cycle), with free access to water. The veterinarian administered feeding of either n-3 PUFA-enriched diet or chow to dams before breeding, under pregnancy and after birth. At postnatal day (PND) 25, 36 rats were shipped to the University of Oslo for behavioral testing. Thereafter, offspring of both sexes were fed similar diets as their parents, until sacrifice at PND 55–60. The weight was measured continously on SHR-rats from each experimantal group troughout the study. In order to obtain the growth rate, body weight was measured continuously on SHR-rats from each experimental group throughout the study. In the males, the n-3 PUFA diet increased the growth rate by 7.7% compared to the control-diet (with p = 0.01 and n = 8 and n= 11 respectively). Whereas n-3 PUFA diet had no effect on the female growth rate (p = 0.1, control diet; n = 10 and n-3 PUFA diet; n = 7).

Experimental diet

The experimental group was given a semi-synthetic n-3 enriched feed, as described in Rokling-Andersen et al. 2009[40]. This feed was high on the essential n-3 PUFAs EPA and DHA with 9.1% (w/w) lard and 10.4% Triomar (EPAX5500) delivered by Pronova Biocare, Lysaker, Norway. Triomar contained 55% of total n-3 FA as TAG including EPA, 300 mg/g; DHA, 190 mg/g; total n-3 FA, 580 mg/g (made up of EPA, DHA, ALA, stearidonic acid, eicosatetraenoic acid, heneicosapentaenoic acid, and docosapentaenoic acid). This dose represents about 3.6% of total energy intake of the rats and is comparable to traditional Inuit intake of marine FA[15]. In addition, 1.5% of soybean oil (Mills Soyaolje; Denofa Lilleborg, Fredrikstad, Norway) was provided to avoid essential n-6 FA deficiency.

The dietary composition (g/100 g food) was: total fat, 21; sucrose, 20; starch, 31.5; protein, 20; cellulose, 1; vitamin mixture, 1.5 and salt mixture, 5. Vitamin (Cat No: 904654) and salt mixture (Cat No: USP XVII) was bought from MP Biochemicals LLC. The feed was kept at −20 °C and given to the rats in portions sufficient for 1 day supply.

Control-feed

The control SHRs were given lab chow (RM3 (E) from Special Diet Services, Witham, Essex CM8 3 AD, UK,http://www.sdsdiets.com/pdfs/RM3-E-FG.pdf). This feed is low on EPA and DHA, and has a n-6 to n-3 ratio of about 7:1. The fatty acid composition of this feed (w/w) were as follows: saturated fatty acids: lauric acid, 0.5 mg/g; myristic acid, 2 mg/g; palmitic acid, 3.6 mg/g; stearic acid, 0.9 mg/g and monounsaturated fatty acids: myristoleic acid, 0.1 mg/g; palmitoleic acid, 1.3 mg/g; oleic acid, 10.3 mg/g and PUFAs: LA, 11.5 mg/g; ALA, 1.7 mg/g; AA, 2.2 mg/g; docosapentaenoic acid, 0.4 mg/g. The dietary composition (g/100 g food) was: total fat, 4.2; sucrose, 5.7; starch, 33.9; protein, 22.4; cellulose, 3.9; hemicellulose, 9.2; dietary fibre, 15.4; vitamin mixture, 0.5 and salt mixture, 4.6. The diet was kept at −20 °C until fed to the animals.

Behavioural analyses

At PND 21 the offsprings were moved to a separate location for behavioural testing. All 41 sessions included habituation, training, shaping and testing with reinforcers. The duration and reinforcement scedule of the different sessions is described in the summary of the experimental procedure (table1). Sessions used in the analyses lasted for 90 min.

Table 1 The reinforcement was given using either a fixed time schedule of reinforcement (FT), continuous reinforcement schedule (CRF) or a variable interval schedule of reinforcement (VI) Full size table

A total of 36 animals were tested using a two-lever visual discrimination task[39] with one session each day for 34 days (the last 28 sessions were used in the analyses). The animals were also video-recorded during the operant task in sessions 13, 24, and 34 to monitor spontaneous locomotion, and the results were averaged for each rat. The n-3 PUFA-fed group included 8 males and 7 females, whereas 11 males and 10 females served as control-fed reference animals. Testing of the experimental and control groups took place at different time points. During habituation and response acquisition, two offsprings were housed in one transparent cage 41 × 25 × 25 cm (height), whereas during response acquisition and throughout the rest of the study, the animals were housed individually in the same type of cages. The rats had free access to water during the habituation sessions, following which they were deprived of water for 21 h a day throughout the rest of the study. The behavioural testing took place between 0900 and 1400 h.

Table1: Summary of the behavioural procedures and reinforcement schedule.

Apparatus

The animals were tested in 16 operant chambers (Campden Instruments) enclosed in sound-resistant outer housings[39, 41]. The chambers were ventilated and equipped with a grid floor, and the animals’ working space was 25 × 25 × 30 (height) cm. The chambers were equipped with two retractable levers requiring a dead weight of at least 3 g to activate a micro-switch. A 2.8 W cue light was located above each lever. The reinforcer (0.05 mL tap water) was delivered by a liquid dipper located in a small recessed cubicle, where a 2.8 W cue light lit up. A 7 × 5 cm transparent plastic top-hinged flap separated the cubicle from the animal’s working space. The computer program LabVIEW 7.1 (National Instruments LabVIEW, Austin, Texas, USA, 2004) recorded the behaviour and scheduled reinforcements and lights. Each operant chamber was equipped with a video camera (Mini Color Hidden Cameras (420TVL, 0,1lux) from Tracer Technology Co. Ltd, Taiwan) positioned to capture the entire working space, in the upper rear corner of the ceiling at an angle of 45 o. The DVR Live Capture computer program (Novus Security, 2009, Warsaw, Poland) controlled the cameras and saved the video files for analyses.

Operant testing

Prior to behavioural testing, the rats were semi-randomly assigned an operant chamber. Following habituation in the operant chamber, the animals were trained to lever-press and then run for additional sessions to strengthen the newly learned behaviour. In the operant task, two levers were used. Pressing the lever, which was signalled by a lit cue light located above the lever, produced reinforcements according to a variable interval 180 s schedules of reinforcement (VI 180 s). During this period, the cue light above the alternative lever was off, and lever press had no consequences. During reinforcer delivery, the cue light above the lever was turned off, and a 2.8 W cue light was lit in the water cubicle. The lever producing reinforcers alternated unpredictably between the two lever-alternatives, but stayed the same until a reinforcer was produced by a lever-press. Following reinforcer delivery, the computer program semi-randomly selected which lever would produce the next reinforcer. To avoid development of lever-preferences, the program allowed a maximum of four consecutive reinforcers on the same lever.

Measure of reinforcement-controlled behaviour

Recording was made of number of presses on the reinforcer-producing lever and on the alternative lever, number of reinforcers produced and collected, and the time of the events. Percentage of responses on the lever producing reinforcers (for all responses, and for the first response following reinforcer delivery), and the time between two responses (inter-response time, IRT), were calculated. Attention was operationalized as the percentage of responses on that lever which produced reinforcers (the animal had to pay attention to and press the lever signalled by the lit cue light above the lever, i.e. stimulus control). Hyperactivity was operationalized as the total number of lever-presses on the two levers combined. The IRTs were split into responses with IRTs longer than 0.67 s and shorter than 0.67 s. Number of responses with short IRTs (< 0.67 s) was used as a measure of impulsivity (“premature responding” or “inability to wait”).

Video recordings

The animals were video-recorded during the operant task in 3 sessions, which were chosen to represent the spontaneous activity early, in the middle of and late in the experimental testing period. The cameras recorded 15 frames per second, and frame-to-frame analyses of changes in pixels, which occurred whenever the animal moved, were performed by using the computer program Musical Gestures Toolbox for audio and video analysis[42]. The total number of pixel-changes was used to quantitate movements. Each of the 3 sessions was divided into 5 segments in order to analyse within-session changes in locomotion in the experimental and control groups. To reduce noise, pixel-changes were averaged across 15 frames and a noise reduction threshold (0.25) and filter (8) were used.

Analysis of behavioural data

All statistical analyses were performed in Statistica 6.0 (StatSoft. Statistica for Windows, StatSoft Inc., Tulsa, OK, 2005). Data were evaluated either by multivariate analyses using Wilks lambda (MANOVAs) when the degrees of freedom relative to the number of levels of the repeated factor permitted this approach, or by univariate analyses of variance (ANOVAs), adjusting the degrees of freedom with the Huynh–Feldt epsilon[43]. Sessions were used as the within-individual factor in the operant task, whereas sessions and segments were used as the within-individual factor in the video analyses. n-3 PUFA-feeding was used as the between-individuals factor in both analyses. Session were included as a factor to look for changes in behaviour across development. Post-hoc tests on main effects were performed using the Unequal N HSD test. Preparation of graphs was performed using Prism (GraphPad Software Inc.) and the graphs represent means ± SEM for each group (n = 7–11).

Biochemical analyses

Chemicals

Monoamine and amino acid analyses were done with high performance liquid chromatography (HPLC). L-amino acid standards, including aspartic acid (Asp), glutamic acid (Glu), serine (Ser), glutamine (Gln) and glycine (Gly) were obtained from Pierce (Rockford, Ill., USA), whereas taurine (Tau), γ-amino butyric acid (GABA), and α-amino adipic acid were from Sigma (Sigma-Aldrich, Steinheim, Germany). The monoamine standards dopamine (DA), homovanillic acid (HVA), serotonin (5-HT), 5-hydroxyindole-3-acetic acid (HIAA) and 3,4-hydroxybenzylamine (DHBA), as well as HClO 4 and ascorbic acid were also obtained from Sigma. The BCA-assay kit (Thermo-Scientific, Rockford, USA), and n-hexane (Merck, Darmstadt, Germany) were bought from VWR. Solutions were made with purified distilled water (Milli-Q Advantage A10, Millipore).

Sample collection

Following behavioural testing, the SHR rats as well as age-matched Wistar Kyoto rats (WKY/NHsd)[37] were stunned and rapidly decapitated at PND 55–60 (the WKY/NHsd were used as additional reference strain in the biochemical analyses). The neostriata were removed, frozen in liquid N 2, and stored at −70°C until sample preparation and analyses.

Extract preparation and protein assay

The tissues were rapidly weighed and homogenized by hand with 20 strokes in 500 μL ice cold 0.2 M HClO 4, using a glass/teflon Potter-Elvehjem homogenizer. This suspension was mixed with an equal volume of DHBA (used as internal standard for monoamines) in 0.12 mM ascorbic acid to a final concentration of 0.227 μM DHBA. The homogenates were centrifuged for 20 min at 15000 × g at 2 °C in a Sorvall RMC-14-microcentrifuge. The pellets were frozen at −40 °C for later protein determination, performed by dissolving pellets in 0.1 M NaOH and measuring protein content by the BCA assay[44]. The supernatants were extracted with equal volumes of n-hexane to reduce lipid contamination, and the top layer was discharged. For amino acid analyses, parts of the delipidized HClO 4 extracts were mixed with a solution of α-amino adipic acid (used as internal standard) to a final concentration of 10 μM in a total volume of 1.9 mL. These extracts were neutralized to pH 7.2 with ice cold KOH and centrifuged for 20 min at 15000 × g at 2 °C as described above. The supernatants were stored at −70 °C until analyses. The samples were then transferred into glass-vials by filtering through Nylon-66 micro filters (0.22 μm) from Nalgene (Rochester, New York, USA), before HPLC analysis.

Monoamine analysis

Column and mobile phases were selected for analysis of catecholamine content in plasma and was supplied by Chromsystems (Germany). The frozen extracts were used directly for analyses of total DA and 5-HT, as well as their metabolites HVA and 5-HIAA, using a reversed-phase HPLC (Shimadzu, Kyoto, Japan) with electrochemical detection (ECD; Decade II with Sencell flow electrode, Antec Leyden) set to a working potential of 0.6 V. Each sample was eluted for 40 min with a flow rate at 1.3 mL/min and a representative chromatogram is shown in Figure1b. External standard solutions of DA, HVA, 5-HT, 5-HIAA and DHBA were analysed on the same day. The chromatograms were analysed using the software Lab Solutions (Shimadzu). Monoamine concentrations are expressed in pmol/mg total neostriatal protein.

Figure 1 Chromatographic profiles of rat neostriatal extracts. (a) Amino acids in a WKY control-fed female, and (b) monoamines in a control-fed SHR male. Panel a) shows the peaks of aspartic acid (asp), glutamate (Glu), serine (Ser), glutamine (Gln), α-amino adipic acid (AAA), glycine (Gly), taurine (Tau) and γ-amino butyric acid (GABA). Panel b) shows the peaks of 5-hydroxyindol-3-acetic acid (5-HIAA), homovanillic acid (HVA), 3,4-hydroxybenzylamine (DHBA), dopamine (DA) and serotonin (5-HT). Full size image

Amino acid analysis

Using a Chromspher 5 C18 column of 25 cm length and 4.6 mm inner diameter (Varian), total amino acids in the neostriatum extracts were analysed[45], using reversed-phase HPLC fitted with a fluorescence detector (Shimadzu, Kyoto, Japan) after derivatization with o-phthaldialdehyde (OPA; Sigma). The mobile phase comprised 75% 50 mM phosphate buffer, pH 5.25, and 25% methanol (v/v), changing linearly to 25% phosphate buffer and 75% methanol during 26.5 min, after which the methanol concentration was linearly reduced to 15%. Each sample was eluted for 45 min with a flow rate at 0.4 mL/min and a representative chromatogram is shown in Figure1a. A mixture of the amino acids of interest was used as external standards in a concentration of 100 μM. The chromatograms were analysed using the software Lab Solutions (Shimadzu). Amino acid concentrations are expressed in nmol/mg total neostriatal protein.

Statistical analysis

Statistical significance of biochemical differences between samples was determined by unpaired, two-tailed Student’s t-test, where p < 0.05 is defined as significant. Level of significance is symbolized with * (p-value ≤ 0.05), ** (p-value ≤ 0.01 and *** (p-value ≤ 0.001). Statistical analyses as well as preparation of graphs were performed using Prism (GraphPad Software Inc), and the graphs display the means ± SEM for each group (n = 4–8).