Supplementary information
Behavioral analysis
Object-based attention test.
The object-based attention test was conducted to assess the attentional function as
described previously (Alkam et al, 2011). In the first habituation phase, mice were
individually subjected to a single habituation session of 10 min, during which they were
exposed to both empty chambers. In the second acquisition phase, mice were subjected
to a single 3-min session, during which five floor-fixed objects (A, B, C, D, and E) were
placed separately in the exploration chamber. Mice were allowed to explore the objects
in the exploration chamber. Two of the five objects were randomly chosen (e.g., object
A and object D) and the time the mice spent on exploring these objects was recorded as
the object-exploration time. Upon completion of the training session, all five objects,
but not the mouse, were immediately withdrawn from the exploration chamber. In the
third phase retention, which immediately followed the acquisition phase, object A was
replaced from the exploration chamber into the test chamber in the position analogous
with its original position in the exploration chamber. A novel object F was placed in the
test chamber in the position analogous with the original position of object D in the
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exploration chamber. The mouse, which had not been moved from the exploration
chamber, was immediately allowed to enter the test chamber and explore the two
objects: the familiar object A and the novel object F. A recognition index was expressed
as the ratio (TF × 100) / (TA + TF), where TA and TF were the times spent on objects A
and F during the retention phase, respectively.
Marble-burying behavior test.
The marble-burying behavior test was conducted to assess the anxiety and compulsive
behavior as described previously (Alkam et al, 2013; Deacon, 2006). On test day, 12
glass marbles were evenly spaced on flattened sawdust in one half of the cage in the
presence of the mouse. Marble burying behavior was expressed as the number of
marbles buried at least two-thirds deep in a 15-min test period.
Elevated plus-maze test.
The elevated plus-maze test was conducted to assess the anxiety level as described
previously (Alkam et al, 2013; Miyamoto et al, 2002). Recorded parameters consisted
of the frequency of entry into open and closed arms in a 5-min test period. These data
were used to calculate the percentage of open arms entries [i.e., (open arms entries /
open and closed arms entries) × 100].
Fear conditioning test.
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The auditory fear conditioning test was performed to assess the cognitive function and
flexibility as described previously (Mouri et al, 2010; Niwa et al, 2011). On day 1
(conditioning phase), the conditioned stimulus was a 30 s tone (85 dB), and the
unconditioned stimulus was a 5 s foot-shock (0.8 mA) that terminated simultaneously
with the tone. This procedure was repeated four times at 1 min intervals. On day 2
(extinction phase), mice were placed in neutral cages, and after a lapse of 3 min, a 1 min
tone (85 dB) was played six times at 1 min intervals without foot-shock. On day 3
(recall phase), the tone was played three times to test for recall of extinction learning
(Milad and Quirk, 2002). Some of the mice underwent the recall phase (day 3) without
undergoing the extinction phase (day 2) (No-Ext groups; No-Ext control and No-Ext
PNE). Time spent freezing was automatically assessed by fear-conditioning video
software (MED-VFC2-SCT-M; Med Associates Inc, USA) during the conditioning
phase (Anagnostaras et al, 2010).
Latent inhibition test.
The latent inhibition (LI) test was performed to assess the attentional shifting and
cognitive flexibility as previously reported (Kuroda et al, 2011; Vuillermot et al, 2012).
For the pre-exposure phase, the pre-exposed (PE) group received a 10-tone stimulus (85
dB, 30-sec tones with random intervals), whereas the non-pre-exposed (NPE) group
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received no stimulus during an equivalent period. In the conditioning phase commenced
immediately after the pre-exposure phase, a 30-sec tone stimulus was delivered to both
the PE and NPE groups, and during the last 1 sec of each tone stimulus, foot shocks of
0.3 mA were simultaneously delivered as unconditioned stimuli. This procedure was
repeated two or four times at 180-sec intervals. LI was measured after two or four
pairings of the conditioned stimulus with the unconditioned stimulus (2CS-US or
4CS-US, respectively). Twenty-four hr after the conditioning phase, the conditioned
tone stimulus was played continuously for 3 min, during which the time of conditioned
freezing was evaluated.
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Reference
Alkam T, Hiramatsu M, Mamiya T, Aoyama Y, Nitta A, Yamada K, et al (2011).
Evaluation of object-based attention in mice. Behav Brain Res 220: 185-193.
Alkam T, Kim HC, Hiramatsu M, Mamiya T, Aoyama Y, Nitta A, et al (2013).
Evaluation of emotional behaviors in young offspring of C57BL/6J mice after
gestational and/or perinatal exposure to nicotine in six different time-windows.
Behav Brain Res 239: 80-89.
Anagnostaras SG, Wood SC, Shuman T, Cai DJ, Leduc AD, Zurn KR, et al (2010).
Automated assessment of pavlovian conditioned freezing and shock reactivity
in mice using the video freeze system. Front Behav Neurosci 4.
Deacon RM (2006). Digging and marble burying in mice: simple methods for in vivo
identification of biological impacts. Nat Protoc 1: 122-124.
Kuroda K, Yamada S, Tanaka M, Iizuka M, Yano H, Mori D, et al (2011). Behavioral
alterations associated with targeted disruption of exons 2 and 3 of the Disc1
gene in the mouse. Hum Mol Genet 20: 4666-4683.
Milad MR, Quirk GJ (2002). Neurons in medial prefrontal cortex signal memory for
fear extinction. Nature 420: 70-74.
Miyamoto Y, Yamada K, Noda Y, Mori H, Mishina M, Nabeshima T (2002). Lower
5
sensitivity to stress and altered monoaminergic neuronal function in mice
lacking the NMDA receptor epsilon 4 subunit. J Neurosci 22: 2335-2342.
Mouri A, Noda Y, Shimizu S, Tsujimoto Y, Nabeshima T (2010). The role of cyclophilin
D in learning and memory. Hippocampus 20: 293-304.
Niwa M, Matsumoto Y, Mouri A, Ozaki N, Nabeshima T (2011). Vulnerability in early
life to changes in the rearing environment plays a crucial role in the
aetiopathology of psychiatric disorders. Int J Neuropsychopharmacol 14:
459-477.
Vuillermot S, Joodmardi E, Perlmann T, Ogren SO, Feldon J, Meyer U (2012). Prenatal
immune activation interacts with genetic Nurr1 deficiency in the development
of attentional impairments. J Neurosci 32: 436-451.
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