Okamura et al.
Supplementary Information
Materials and Methods
Step-through Inhibitory Avoidance
Mice were tested on a trough-shaped step-through inhibitory avoidance apparatus in
which a straight alley is divided into two compartments, one 7.5 cm long and the other 24
cm long, by a partition with a guillotine door. Both compartments were 14 cm high and
the floor was 2.5 cm wide. The walls of the smaller “start” compartment were made of
white plexiglass while the walls and cover of the larger “shock” compartment consisted
of black plexiglass. The floor of the larger compartment consisted of stainless steel plates
through which scrambled constant current could be delivered.
The step-through inhibitory avoidance test was divided into a training and a testing phase.
In the training trial, the mouse was placed in the white start compartment and the
guillotine door was opened. After the mouse crossed over into the dark chamber, the
latency was recorded, the door was closed and a mild (0.2 mA/1 s) foot shock was
delivered. The animals remained in the shock compartment for an additional 30 s to
associate spatial cues with the treatment. The initial latency to enter the dark (shock)
compartment served as the baseline measure. Mice were then placed again in the light
compartment at different post-training delays (48, 96 or 168 hours later) and their latency
to enter the dark compartment (previously associated with the foot shock) was measured
as an index of inhibitory avoidance. Cut-off latency was 10 min.
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NPS was administered by i.c.v. injection at different time points in order to investigate its
effects on acquisition, consolidation or retrieval of conditioned memory. Male NPSR KO,
HET and WT mice did not receive injections and were only tested 48 h post-training.
Novel Object Recognition
Novel object recognition was measured in single-housed mice tested in their home cage.
For C57Bl/6 mice, a total of four sets containing two different objects symmetrically
glued 8 cm apart on a clear plexiglass board (5 x 15 cm) which can be placed beneath the
cage bedding were used. All objects were made of plastic, similar in size (6-7 cm high)
but different in color and shape. A crossover design was used in all recognition memory
experiments, by using different sets of objects for training, placing objects in alternating
corners of the cage during the novel place recognition test or by randomly assigning mice
to either one of the two contexts that were used to train and then test recognition of the
non-matching object, in order to exclude potential confounding influences of preference
for particular objects, location or context of objects.
The novel object recognition test consisted of two sessions: a training session followed
by a retention trial 24, 48 or 96 hours later, respectively. Each mouse was individually
housed for at least 3 days before the training session. During the training session of
C57Bl/6 mice, a plexiglass board with two different objects (A and B) was placed in the
center of the cage and the board was covered with bedding. Each animal was allowed to
explore the objects for 5 min. The mouse was considered to be exploring the object when
the head of the animal was facing the object or the animal was touching or sniffing the
object. The total time spent exploring each object was recorded by a trained observer
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blind to treatment or genotype condition using ODLog 2.0 (Macropod Software). After
training, the board with objects was immediately removed from the home cage. NPS or
vehicle (PBS, 0.1 % BSA) was administered by i.c.v. injection 5 min after the training
and separate groups of mice were tested for retention 24, 48, 96 or 168 hours later,
respectively. During the retention session with C57Bl/6 mice, a new set of objects
containing one identical and one novel object (A and C) was used. The animal was again
allowed to explore the objects for 5 min and the time spent exploring each object was
recorded. Since absolute exploration times differed considerably between individual
animals, exploration times were normalized [(time exploring object A) + (time exploring
object B/C) = 100 %] and exploration of each object was expressed as percentage of total
exploration time. Increased exploration of the novel object was interpreted as successful
retention of memory for the familiar object.
The novel object recognition test was modified for NPSR KO and WT mice since pilot
studies with 129S6/SvEvTac mice indicated consistent preference for one object when
sets of two difference objects were presented during the training, regardless of
configuration. Therefore, NPSR KO or WT mice were presented with two identical
objects arranged as described above and allowed to explore for 5 min on the training day.
Two sets of two identical objects were used and mice were assigned randomly to either
one for training. 24 h after training, NPSR KO or WT mice were presented with one
novel and one familiar object and total time of exploration was recorded as described
above. Different novel objects were used in combination with the previously explored
familiar object in order to exclude object preferences.
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Novel place recognition was investigated by presenting two identical objects, placed in
distinct corners of the home cage, and allowing exploration for 5 min on the training day.
Alternating corners were chosen for object presentation during training in order to
exclude spatial preferences of object location and different pairs of objects were used for
training. 24 h later, one of the objects was moved to a different corner (randomly chosen)
and the time spent exploring both objects over 5 min was recorded as described above.
Increased time spent exploring the object in the novel location was interpreted as
successful retention of spatial memory for the object that had not been moved.
Novel context recognition was used to test the ability of mice to remember that an object
was previously encountered in a particular context. Mice were randomly assigned to
either one of the two contexts for object presentation and different pairs of objects were
used for training. The familiarization session was divided into two phases in which two
identical objects were placed in the arena. A 2 min interval separated the two
familiarization phases: In the first phase, two identical objects (A and A) were presented
in context 1. In the second phase two differently shaped identical objects (B and B) were
presented in context 2. After 24 h the animals were tested for context-specific object
recognition in either context 1 or 2 by presenting objects A and B. Thus during the test
phase, one object was in the same context as during the familiarization phase and the
other object was presented in a non-matching context. Increased exploration of the object
presented in a different context over the object previously encountered in the same
context was interpreted as increased formation of contextual memory.
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Exploration times were recorded and used to calculate a discrimination index [time spent
with object A - time spent with object B] / [total time exploring both objects] for training
and test sessions. Discrimination indices of 0 indicate equal exploration of both objects.
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Supplementary Figures
Figure S1: Effect of NPS on acquisition and retrieval of IA memory. (A) NPS does not
appear to modulate memory acquisition. Separate groups of male C57Bl/6 mice were
injected centrally with 1 nmole NPS (n = 10) or vehicle (n = 10) 15 min before training
and IA latencies were tested 48 h later. (B) NPS appears to attenuate memory retrieval,
but this effect may likely be confounded by the acute anxiolytic properties of NPS. Male
C57Bl/6 mice were injected i.c.v. with 1 nmole NPS (n = 10) or vehicle (n = 9) 15 min
before retrieval of IA memory. ** p < 0.01, two-way ANOVA followed by Bonferroni’s
post hoc test.
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Figure S2: Genetic inactivation of NPSR does not affect pain sensitivity in the hot plate
test. NPSR KO (n = 8), HET (n = 7), WT (n = 11).
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Figure S3: Novel object recognition training. Drug-naive groups of C57Bl/6 mice spent
equal amounts of time exploring the two objects A and B during training. Data from all
experimental groups were pooled. The dashed line indicates 50% exploration time if none
of the objects is preferred. Absolute exploration times were normalized and are shown as
means ± S.E.M. PBS-group, n = 30; NPS-group, n = 31.
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Figure S4: Discrimination index for novel object (A), novel place (B), and novel context
(C) recognition in NPSR WT and KO mice. Object discrimination was calculated as
described in the Supplementary Methods. See Fig. 5 for experimental details. *** p <
0.001 by two-way ANOVA with Bonferroni’s post hoc test, n.s., not significant.
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Supplementary Table
Table S1: Mean total exploration times (s ± SEM) and number of animals (N) in object
recognition tests.
C57Bl/6 mice injected with PBS or NPS
N
N
PBS
NPS
Training 21.50 ± 1.261 30 20.55 ± 1.531 31
24 h
18.94 ± 2.678 7 19.91 ± 2.862 7
48 h
19.53 ± 3.183 7 19.01 ± 1.207 8
96 h
22.93 ± 2.218 8 24.71 ± 2.804 8
168 h
22.45 ± 4.226 8 23.65 ± 2.291 8
Training latencies from all groups were pooled.
NPSR wildytpe and knockout mice (129S6/SvEvTac)
WT
Novel Object
Training
Test
Novel Place
Training
Test
N
18
25.56 ± 4.148
21.17 ± 2.58
KO
22.64 ± 2.879
23.99 ± 3.622
11
28.88 ± 3.702
24.20 ± 2.072
N
14
11
34.55 ± 4.27
26.73 ± 6.562
11
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Novel Context
Training context 1 36.35 ± 3.45
30.0 ± 2.399
Training context 2 31.07 ± 1.726
32.97 ± 2.953
Test
24.32 ± 1.807
26.92 ± 1.895
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