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All behavioral parameters were recorded by observers for multiple comparisons. P < 0.05 was considered as
who were blind to the treatment. In addition, all statistically significant.
behavioral tests were conducted in a quiet room during
the light period (between 13:00 and 18:00) under RESULTS
bright and moderate illumination, and the mice were
kept in the room for at least 1 h before the assessment. Effects of early and late neonatal immune activation on
Depression-related behavior was separately studied in depression-related behaviors during adolescence and
adolescent (PND 35: tail suspension test [TST]; PND 40: adulthood in the TST
forced swimming test [FST]) and adult (PND 85: TST; The three-way analysis revealed the significant effects
PND 90: FST) male mice. of the time of neonatal immune activation (F 1,84 =5.65,

P < 0.03), age (F = 43.03, P < 0.001), and treatment
1,84
TST: The TST was performed according to the (F 2,84 = 11.57, P < 0.001) on the immobility time in

previously described procedure. At the beginning of the TST. Significant interactions existed between
[6]
the experiment, each mouse was individually suspended age × treatment (F 2,84 = 4.66, P < 0.02) and the time

by its tail using a clamp, 2 cm from the distal end, for of neonatal immune activation × age × treatment
5 min in a gray wooden box (40 cm high, 30 cm wide, (F 2,84 = 3.37, P < 0.04). However, there was no

and 20 cm deep), with the head about 25 cm above the significant interaction between the time of neonatal
floor. The total duration of immobility was recorded immune activation × age and the time of neonatal
(in seconds). All animals that climbed their tails during immune activation × treatment. These results indicate
the TST were excluded from the further analyses. that neonatal immune activation with Poly I:C can
Immobility was defined as the lack of whole-body influence depression-related behaviors in dose-, age-,
motion, whereas mobility was defined as hind leg and time-dependent manner in mice. Therefore, the
movement. [16] dose of immunogen, the timing of immune activation,
and age may be important factors for evaluating the
FST: The FST remains one of the most widely used tools consequences of neonatal immune activation on
for measuring behavioral despair in rodents. To describe affective disorders, like depression, later in life.
this behavioral model in mice, the following procedure
was adopted: mice were individually placed into the The data analysis indicated that early neonatal
transparent glass cylinders (height: 25 cm, diameter: immune activation with Poly I: C increased the total
10 cm) filled with water to a height of 15 cm and duration of immobility at the dose of 4 mg/kg in
maintained at 25 ± 1°C. The water was replaced between adolescence [Figure 2a; P = 0.037] and at both doses
each test. The total duration of immobility was recorded in adulthood [Figure 2b; P = 0.042 and P = 0.002],
during the last 4 min of the 6-min testing period. At the indicating high levels of depression-related behaviors
end of swimming session, the animals were removed in Poly I:C-treated mice in comparison with the
from the cylinder, dried with towels, and gently placed saline-treated group.
near an electric heater for 15–30 min. Each mouse was
judged to be immobile when it ceased struggling and As shown in Figure 3, late neonatal immune challenge
remained floating motionless in the water, making only with Poly I:C resulted in an increase in the total
those movements necessary to keep its head above duration of immobility time at the dose of 4 mg/kg in
water. A decrease in the duration of immobility time adulthood [P = 0.03], but not in adolescence [Figure 3].
is considered indicative of depression-like behavior in
mice. [6,10]

Statistical analysis
The statistical analysis was performed using Statistical
Package for Social Sciences software (Version 21,
IBM, Armonk, NY, USA). The depression results were
analyzed using three-way analysis of variance, with age,
treatment, and neonatal infection timing as the main a b
factors. All data are presented as the mean ± standard Figure 2: Effects of early neonatal immune activation on depression-like behavior
error of the mean. Further analysis was carried out during adolescence (a) and adulthood (b) in the tail suspension test. The data
are presented as mean ± standard error of the mean (n = 8). *P < 0.05 and
using Tukey’s honest significant different post‑hoc tests **P < 0.01 compared with the saline-treated group

Neuroimmunol Neuroinflammation | Volume 1 | Issue 1 | June 2014 37