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Scientific reports have indicated that the nature of the 30 litters were used during this study in three stages,
offspring’s neuropathology can depend on the nature each of which included 10 litters. Only one mouse per
and severity of insult and the pup’s age at the time dam was selected for each of the groups to avoid the
of insult. This argument is supported by previous litter effect. All procedures of the study were performed
studies in which we and others showed that the time in accordance with the ethical guidelines set by the
points of prenatal and neonatal immune activation, Research and Ethics Committee of the Tabriz University
the offspring’s age, and the dose of immunogen can of Medical Sciences, which completely adhere with
be important factors for assessing neuropsychological the National Institutes of Health NIH Guide for the
alterations such as anxiety- and depression-related Care and Use of Laboratory Animals (NIH; Publication
behaviors that persist until adulthood in mice and No. 85–23, revised 1985).
rats. [3,6,10,11] For instance, a study conducted by Walker
et al. [11] indicated that postnatal immune challenge had Neonatal immune activation
no impact on anxiety levels in adolescence, but did A summary of the experimental design is shown in
lead to increased levels of anxiety in adulthood and Figure 1. The pups were divided based on treatment
senescence in male rats. Furthermore, previous studies conditions into two clusters: group 1 – saline-injected
concluded that older age correlates with an increased mice and group 2 – Poly I:C-injected mice (each group
level of depression behavior in humans and rodents. [10,12] only used for two tests with a 5-day interval between
However, limited information is available regarding the each test; n = 8 per group). The dams were removed
relationship between the timing of neonatal immune from their pups for approximately 5 min, and the pups
challenge and age in development of depression-related were weighed and received a subcutaneous (in the
behaviors in adulthood. interscapular region) injection of Poly I:C (Sigma
Aldrich, St Louis, MO, USA; 1 mg/kg and 4 mg/kg)
Therefore, the aim of the current study was to investigate or saline (1 mL/kg) during the early neonatal phase
the impacts of neonatal immune activation with equal (PND 3–5, which corresponds to the third trimester
Poly I:C doses at two time points, postnatal days (PND) of human pregnancy when major brain growth
3–5 (early neonatal phase) or PND 14–16 (late neonatal occurs) or the late neonatal phase (PND 14–16,
phase), on depression-related behaviors in adolescent which corresponds to 1–2 years old humans). [6,14]
and adult male mice. The doses and timing of Poly I:C treatment were
chosen based on previous studies. [6,8,15] The Poly
METHODS I:C was dissolved in sterile saline (0.9% NaCl), and
injections were performed between 10:00 and 11:00.
Animals Each injection was performed through a 27-gauge
Male and female C57BL/6 mice (70–80 days) needle connected by polyethylene tubing to a 10-µL
were obtained from the animal house of Pasteur Hamilton syringe. Newborn mice were returned to
Institute (Tehran, Iran). Mice were housed in standard their housing immediately after injections.
polycarbonate cages in a room with a 12:12 h
light/dark cycle (lights on 08:00–20:00), controlled Behavioral tests
temperature (23 ± 1°C), and had free access to food
and water. These conditions were kept as a standard
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[6]
birth, all litters were culled to four pups per mother. On Figure 1: Experimental design: The effects of early and late neonatal immune
day 21, litters were weaned by removal of the mother, activation with Poly I:C on depression-related behaviors during adolescence
and adulthood in the tail suspension test (TST) and forced swimming test (FST)
and only male pups were used in this study. A total of in C57BL/6 mice

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