Article
3.3 Text
3.3.2 Materials and methods .1 Animals
Male Flinders sensitive line (FSL) rats, and a corresponding behavioural control line, the Flinders resistant line (FRL) rats were used for the current study. Pregnant female dams, as carriers of the unborn male foetuses, were used during the prenatal phases of the study for the administration of drug or vehicle. All animals were housed under conditions of constant temperature (22 ± 1°C) and humidity (50%) with a 12:12-h light/dark cycle (lights on 06:00 to 18:00). Food and water were provided ad libitum.
From the Animal Centre log books from 2009 and 2010, the number of litter per dam was calculated as 8.2 ± 0.3 for FRL rats and 8.5 ± 0.5 for FSL rats, with the nominal difference being statistically insignificant. Furthermore, the sex distribution of male and female pups for FRL rats was calculated as 53.0 ± 1.8% male and 47.6 ± 1.8%
female and for FSL as 52.4 ± 1.6% male and 48.2 ± 1.6 female, again with no statistically significant difference (data not shown). The data therefore suggest a birth rate of approximately 50% male pups per litter, but with a degree of variance per pregnancy.
The study and all animal procedures were approved by the Ethics Committee of the North-West University (approval number: NWU-00045-10S5) and were in accordance with the guidelines of the National Institutes of Health guide for the care and use of laboratory animals.
3.3.2.2 Drug treatment
Venlafaxine hydrochloride (a kind gift from Cipla Medpro, Cape Town, South Africa) was dissolved in saline and administered at doses that were used previously to investigate antidepressant-like effects or other central action. As such, pregnant dams were injected subcutaneously (s.c.) with either vehicle control (saline) or venlafaxine at a dose of 10 mg/kg/day (Folkessen et al., 2010; Larsen et al., 2010;
Scaini et al., 2010) between 08:00 to 12:00 each morning, for 14 consecutive days from natal days -15 to -1 (i.e. prenatal phase). New-born pups were injected with either vehicle control or venlafaxine at a dose of 3 mg/kg/day s.c. (Dawson et al.,
Page | 47 1999) between 08:00 to 12:00 each morning for 14 consecutive days from natal days +3 to +17 (i.e. postnatal phase). In both instances, pregnant dams or new-born pups were measured every day and injected according to the weight on the specific day.
Pregnant dams received up to 1.0 ml of either drug or saline, while the new-born pups received a total volume of up to 0.15 ml. Four treatment groups (usually consisting of 8 rats/group, but not less than 3 rats/group) for both FRL and FSL rats received injections prenatal + postnatal as follows: 1. (saline + saline), 2.
(venlafaxine + saline), 3. (saline + venlafaxine) and 4. (venlafaxine + venlafaxine).
The study aimed to employ 8 rats per treatment group, but due to lower than expected birth rate of male pups in some cases (natural variance), some groups had as little as 3 rats per group.
3.3.2.3 Behavioural tests
Following the prenatal and postnatal vehicle/drug treatments, the animals were housed under normal conditions until postnatal days 21, 35 or 60 (PostND21, 35 or 60), at which time the behavioural and memory tests described below were performed. All behavioural tests were performed sequentially between 1 to 4 hours after the start of the dark cycle (i.e. 19:00 – 22:00), implementing the novel object recognition test (nORT), locomotor assessment using the Digiscan® animal activity monitor (DAAM), the elevated plus maze (EPM) and the forced swim test (FST), in this order. The tests were carefully spaced to allow 30 minutes between each test for acclimatisation. The order of the tests was designed to start with the least stressful interventions and to end with the most stressful interventions. Importantly, a pilot study verified that preceding tests in the test battery do not significantly affect any of the subsequent tests (data not shown).
Following 30 minutes acclimatisation, cognition was evaluated by implementing the standard nORT test, as described previously (Abildgaard et al., 2011). Briefly, the nORT was performed in a square box (dimensions L x W x H = 50 x 50 x 40 cm) with opaque (black) walls and under low light intensity (20 lx). Each test consisted of an acquisition and a retention trial, spaced 90 minutes apart, and with the box wiped clean with 10% ethanol after each trial to eliminate any olfactory cues in subsequent tests. In both trials the animals were placed in the centre of the box facing the objects, with two immovable objects (see below) placed in two corners, 20 cm from
Page | 48 the walls. Animals were left to explore the objects for 5 minutes, during which time the exploration was video-recorded with a camera installed directly above the box.
Exploration was defined as sniffing, licking or physically touching an object, and scoring involved the measurement and calculation of the total time spent exploring the familiar or novel object in each trial. In the first (acquisition) trial, the animals were left to freely explore two identical objects (red metal balls, immovably mounted on a flat surface), followed 90 minutes later by the second (retention) trial, during which one of the objects used in the acquisition trial (now familiar object) was replaced with a novel object (green metal squares). Rats tend to spend more time exploring novel objects when memory is intact and therefore the time spent exploring the novel object, relative to the familiar one, is considered to be indicative of memory consolidation.
Following 30 minutes acclimatisation, locomotor activity was measured by implementing the DAAM apparatus under low light intensity (20 lx), as described previously (Korff et al., 2008). Briefly, each animal was placed in a Digiscan® box, with horizontal movement scored as the number of beam breaks as well as the total distance covered during a 5 minute period.
Following 30 minutes acclimatisation, anxiety-like behaviour was measured by implementing the standard EPM under a light intensity of 80 lx in open arms and 20 lx in closed arms, as described previously (Carola et al., 2002). The apparatus consisted of a plus-shaped maze (1 m x 1 m and 10 cm arm width), elevated 50 cm from the floor. Two opposing arms were enclosed with a 30 cm high black opaque wall (designated the closed arms) while the remaining two arms remained open (designated open arms), without ledges. As anxiety-like levels decrease, rats tend to spend more time exploring the open arms, whereas rats tend to spend more time in the closed arms (perceived as more protective) as anxiety levels increase.
The animals were placed in the centre of the maze, facing an open arm and left to explore the maze for 5 minutes. During this time the movement of the animals were recorded with a video recorder installed above the maze. The percentage time spent in the open arms was calculated, as well as the number of entries and full entries into the open arms counted. A full entry was defined as crossing into the open arm section with all four paws.
Page | 49 Following 30 minutes acclimatisation, depressive-like behaviour was measured by implementing the standard FST under higher light intensity of 200 lx, as described previously (Liebenberg et al., 2010). Rats were placed into a cylindrical tank (60 cm high and 30 cm in diameter) containing 30 cm of water maintained at 23°C. The rats were allowed to swim for 5 minutes, during which time swimming behaviour was video-recorded via a camera installed in front of the cylinders. It is important to note that no conditioning swim trial 24 hours prior to the scoring trial is necessary with FSL rats (Overstreet and Griebel, 2004; Porsolt, 1977). Rats were scored as immobile (as opposed to swimming) when only the necessary movements were made to keep their heads above water. Swimming was defined as horizontal movements throughout the swim cylinder that includes crossing into another quadrant, and climbing was defined as upward-directed movements of the forepaws along the side of the swim chamber (Cryan et al., 2002).
3.3.2.4 Statistical analysis
The unpaired Student‟s t-test (two-tailed) was performed to compare the data from two treatment groups. Multiple comparisons relative to control were analysed by one way analyses of variance (ANOVA) followed by the Dunnett‟s post-test. GraphPad Prism® (version 5.00, San Diego California, U.S.A.) was used for all statistical analyses and graphical representation. Data are presented as averages ± S.E.M.
with a value of p < 0.05 taken as statistically significant.