Supplementary data
Animals and housing
Male C57BL6N mice, aged 3 months, were purchased from Charles River (4-week dosing
study: Sulzfeld, Germany, 2-week dosing study: L’Arbresle, France).14 days before the
behavioral experiments, mice were single housed under a reverse 12h : 12h light-dark cycle
(lights on: 21:00 h) in standard laboratory conditions (22 ± 1°C, 55% humidity, food and
water ad libitum). All experiments were carried out in accordance with the European
Committees Council Directives and had been approved by the state governmental bodies of
animal care and welfare.
Design of experiment
In the first study, we investigated the effects of a 4-week administration of imipramine or
citalopram on basic physiological and behavioural variables in mice. The duration of dosing
was chosen in accordance with the most commonly used dosing regimes in this type of preclinical experiment. Naïve 3-month-old male mice were treated daily via drinking water either
with imipramine (15 mg/kg/day, n=10), citalopram (15 mg/kg/day, n=10) or they were left
untreated and were used as controls (n=10). Animals were assigned to these groups upon
their initial values of body weight and sucrose preference, so their means of baseline
characteristics were similar before the onset of the drug administration. Six mice of each
group were placed for eight weeks in SAMAB apparatus in standard Plexiglas cages (21 cm
x 42 cm x 15 cm); including four weeks of drug administration and the same period after a
discontinuation of dosing. Horizontal home cage activity was continuously monitored for the
duration. The remaining mice (n=4 per each group) were kept in identical cages, housing
and dosing conditions to those animals used in the SAMAB monitoring system. Starting from
the 5th day of dosing, sucrose preference test, novel cage exploration and body weight were
evaluated on a weekly basis in all animals on the same day, and in the same order.
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In a separate experiment, we addressed the question of whether imipramine interferes with
learning related to the memory of a novel environment, as suggested by the outcome from
repeated exposure of imipramine-treated mice to a novel cage. Mice were single housed in
standard laboratory cages (21 cm x 21 cm x 15 cm) under the conditions described above.
For each treatment group, 10 animals were used; the doses and the method of drug
administration of imipramine and citalopram were identical to those used in a previous study.
Starting from the day after the termination of a fourteen-day treatment period, all mice were
tested in a two-day object recognition task. The citalopram-treated animals displayed
marked suppression of exploratory behaviour prior to the training which precluded an
assessment of their learning abilities and this group was discarded from the subsequent
experiment.
Sucrose test
In order to assess the effects of the treatment regimes on sucrose preference, mice were
given free choice for 8 h (between 9.00 – 17.00 h) of two drinking bottles; one with 1%sucrose solution, and another with tap water. To prevent possible effects of side-preference
in drinking behavior, the position of the bottles in the cage was switched after 4 h. Special
precautions have been made in order to minimize the spillage of liquids and error of
measurement during sucrose test. The consumption of water, sucrose solution and total
intake of liquids was estimated simultaneously in the control and experimental groups by
weighing the bottles. Percentage preference for sucrose was calculated using the following
formula: Sucrose Preference = (Volume of Sucrose solution/Volume of Sucrose solution +
Volume of Water) x 100.
Novel cage test
The novel cage test was performed to assess exploration of a new environment. Mice were
introduced into a standard plastic cage (21 cm x 21 cm x 15 cm) filled with fresh sawdust.
The number of exploratory rears was counted under red light during a 5-minute period.
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Home cage activity
Home-cage activity was monitored in animals using the SAMAB (System for Automated
Measurement of Behaviour) system. Infrared beams placed 2 cm and 7 cm above the cage
bottom monitored horizontal activity in the home-cage environment, which allowed animals
access to food and water ad libitum and also permits normal behaviour to be monitored over
a number of days in this customized apparatus which consists of 20 home cages (21 cm x
42 cm x 15 cm; Technoplast, Rome, Italy). Locomotor activity was scored with a spatial
resolution 1.18 x 1.37 cm. Mice were placed in the SAMAB cages at the first day of the
treatment period for 8 weeks and left undisturbed, except weekly change of cages. Data
were analyzed with specialized SAMAB software. Mean velocity per hour was averaged per
24 h and then one week for each animal.
Object Recognition Memory test
Mice were placed in a glass cylinder observation chamber (Ø 25 cm, height 35 cm) that was
situated on a stand of 1m high placed by two walls of the lab room. The cylinder was 5 cm
from the edge on two opposite sides and 30 cm from the edge on the other two sides. Using
sound-proof conditions with subtle illumination (5 Lux) animals were allowed to explore two
identical objects (taste-free and smell-free plastic toys of 6 cm x 3 cm x 2.5 cm) which were
located in either part of the cylinder for 15 min. Previous experiments have shown that object
placed by the edge is explored by C57BL6 mice in average 50% less than the same object
at the opposite location that is likely to be owing to their species-specific tegmotaxic traits
and fear of height. On the Day 1, animals were allowed to explore two objects placed either
close to the walls (“preferable” zone, the area in 30 cm from edge) or distanced from walls
(“non-preferable” zone, the area in 5 cm from edge) in the observation cylinder. On the Day
2, the test was repeated. The object from “non-preferred” zone was replaced with a new
object of a similar size (3 cm x 6 cm x 3 cm) and of exactly the same material. A placement
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of a new object to “non-preferred” area meant to contrast behavioral manifestation of novelty
exploration under competing motivations of animals of staying by the walls and the object
exploration. The duration of exploration of each object was scored manually from recorded
video off-line. Preference for the object exploration was calculated as a percentage of the
duration of exploration of a particular object from the total duration of exploration of both
objects: Object preference = [Object exploration /Total exploration] × 100%. An increased
preference in the exploration of a new object that has replaced the former one in the “nonpreferred” area from Day 1 to Day 2, was taken as recognition of a novel object and memory
for the former one.
Dosing with imipramine or citalopram
Mice were treated continuously with imipramine or citalopram for 4 or 2 weeks via drinking
water. Citalopram (Lundbeck, Copenhagen, Denmark) or imipramine (SIGMA-ALDRICH,
Cambridge Soft Corporation, Cambridge, MA, USA) were dissolved in tap water; the
solutions were freshly prepared every 3-5 days. Bottles with imipramine solution were
protected from the light with aluminum foil. The calculation of the concentration of citalopram
in drinking water was based on the previously evaluated mean volume of daily water
consumption in C57BL6N mice that was in average 3.0 ml, and on the desirable dosage of
treatment (15 mg/kg/day). The regime of dosing was validated on the basis of previous
experiments.
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