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 (0.2mA/1s) 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 168hours 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.
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 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.
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.
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.
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.
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).
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.
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.
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
PBS / N / NPS / NTraining / 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 / N / KO / NNovel Object / 18 / 14
Training / 25.56 ± 4.148 / 22.64 ± 2.879
Test / 21.17 ± 2.58 / 23.99 ± 3.622
Novel Place / 11 / 11
Training / 28.88 ± 3.702 / 34.55 ± 4.27
Test / 24.20 ± 2.072 / 26.73 ± 6.562
Novel Context / 11 / 7
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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