Animals
Generation of App-KI (AppNL−G−F/NL−G−F) mice was described previously [13]. Mice were obtained from the RIKEN Center for Brain Science (Wako, Japan). Male AppNL−G−F/NL−G−F, littermate, and wild-type (C57BL/6 J, CLEA Japan, Inc., Japan, RRID:IMSR_JAX:000,664) mice were 4 months old at the start of touchscreen experiments and 6 months old for the Morris water maze test. All mice were housed in plastic cages and kept in a regulated environment (23 ± 1 °C; 50 ± 5% humidity) with a 12-h light/dark cycle (lights on at 8:00 AM). Food (CE-2; CLEA Japan, Inc.) and tap water were available ad libitum. All experiments were performed in accordance with the Guidelines for Animal Experiments of Nagoya University, the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society and the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Research Institute of Environmental Medicine, Nagoya University (Permit Number: RIEM19273). One week before starting behavioral experiments, mice were food-restricted to achieve approximately 85% of their ad libitum bodyweight. Mice that exhibited severe bodily injury due to fighting were excluded from behavioral analysis.
Touchscreen apparatus
Touchscreen tests were performed as previously described with slight modifications [19,20,21]. Testing was conducted with a touchscreen-based automated operant system for mice housed within a sound- and light-attenuating box (87 × 50 × 79 cm, TOP-M1, O’hara & Co., Ltd., Tokyo, Japan). The sound-attenuating box contained a house light, a ventilation fan that also provided white noise, and a pair of tone generators. The operant system contained a 15-inch touch panel unit and a 10-mg food pellet dispenser on the opposite side, fitted with a photocell head entry detector and a camera directly on top of the chamber. To decrease the frequency of unintended responses to the touchscreen due to contact with the tail or other body parts, a black plastic “mask” with task-specific response windows was placed in front of the screen: six windows for LD, two windows for visual discrimination and reversal learning, and three windows for different object–location paired-associate learning (dPAL) touchscreen-based behavioral tasks.
Pretraining
Before performing any of the touchscreen tests described in this study, mice had to go through pre-training steps, which consisted of the following: (1) Magazine training (1 day, 30 min), in which mice received food (10-mg pellet, AIN-76A Rodent Tablet 10 mg, #1811213 (5TUL), TestDiet, USA) for head entry into the food receptacle. (2) Autoshaping (1 day, 30 min), in which food was delivered after contingent disappearance of presentation of a white stimulus in all windows. (3) Must touch (2 days, 60 min/100 trials), in which mice had to touch the screen to receive a food reward; all windows presented white stimuli; (4) Correct touch (2 days, 60 min/100 trials), in which mice had to touch the stimulus presented randomly in only one window to receive a food pellet; incorrect response had no effect. (5) Correct touch error (2 days, 60 min/100 trials, at least 80% correct response), in which mice had to correctly touch the randomly presented white stimulus to receive a food reward. Pretraining required 10 sessions and data from pretraining sessions are not shown.
In all pretraining steps and touchscreen tests, the trial was automatically started followed by a 3-s inter-trial interval (ITI), after which the mouse was required to enter its head into the food receptacle to start the trial, as described previously [21]. Head entry into the magazine during a session resulted in stimuli being displayed. A stimulus remained on the screen until the mouse responded to it, after which (if the choice was correct) the mouse was rewarded with a pellet accompanied by a tone, the magazine light was illuminated, and the trial was ended. This was followed by a 3-s ITI before starting the next trial. The house light was on during the trial. After a choice was made, the first head entry into the magazine after the ITI resulted in the stimuli being displayed for the next trial. This meant that on every trial, the mouse was situated at the back of the testing chamber when the stimuli were displayed.
Location discrimination (LD) task
The LD task was performed as described previously, with slight modification [22]. We used mask and stimulus dimensions as follows: number of windows, 6; window size, 25 × 25 mm2, window gaps, 10 mm; floor gap, 25 mm; stimulus size, 25 × 25 mm2. Following pretraining sessions, the LD task included an additional training step in which the mice were presented with two square white stimuli separated by an intermediate degree of separation (LDmedium: LDm). One square was designated as correct, and the other as incorrect (Phase 1). Responses at the correct location resulted in a reward delivery followed by 3-s ITI as described above. Responses at the incorrect location resulted in a 5-s timeout period with the house light off. Seven correct responses out of eight consecutive trials resulted in reversal of reward contingences, in which the previous incorrect location now became correct (Phase 2) (Fig. 1a). The initial correct location was counter balanced between animals in each genotype. Mice were given a maximum of 62 trials/session/d for 10 days. Following LDm training, pattern separation was assessed by presenting stimuli with either an LDlarge (LDl) task with a high degree of separation (four empty windows between two stimuli) or an LDsmall (LDs) task with a low degree of separation (no empty windows between the two stimuli). Mice received a 30-min once daily session. Mice were subjected to each task for 4 days. The same degree of separation was presented for two consecutive days. The order of separation was counterbalanced between animals in each group across days. The average number of changes between phases during the LDl and LDs tasks were calculated. The LD task required 20 training sessions: 10 sessions for the LDm task and 10 sessions for the LDs and LDl tasks.
Different object–location paired-associate learning (dPAL) task
We used mask and stimulus dimensions as follows: number of windows, 3; window size, 57 × 57 mm2; window gaps, 10 mm; floor gap, 25 mm; stimulus size, 53 × 53 mm2. Following pretraining sessions, dPAL tasks also had one additional training step in which place-associated stimuli, were presented but the incorrect response had no effect. The dPAL experiment was performed as previously described [23]. Briefly, six different combinations were designed using three lined stimuli. Each stimulus was considered S+ in a specific location. For each trial type, one visual stimulus was presented in its correct location and a second visual stimulus was presented in an incorrect location, leaving one window blank (Fig. 2a). Each combination of stimuli was presented an equal number of times. A correct choice was followed by reward delivery with tone, illumination of the magazine light, a 3-s ITI, and the next trial. An incorrect choice was followed by a 5-s timeout and a 3-s ITI, after which correction trials started in which the same set of stimuli are presented until the correct choice was made. Correction trials were not included in the calculation of percent correct response. The session finished either when the 120 trials were completed or when 60 min had passed. The dPAL task required 40 training sessions.
Visual discrimination, and reversal learning task
Acquisition Visual discrimination and reversal learning tests were performed as described previously [24] with slight modifications. We used mask and stimulus dimensions as follows: number of windows, 2; window size, 60 × 60 mm2; window gap, 30 mm; floor gap, 25 mm; stimulus size, 60 × 60 mm2. Mice were presented with a pair of black-and-white, brightness-matched stimuli on the touchscreen, one of which was correct (S+) and the other incorrect (S−) (Fig. 3a). Response to the S+ resulted in a tone, magazine illumination, and delivery of a single reward pellet. After incorrect responses, the house light was extinguished. Both correct and incorrect responses were followed by a 3-s ITI. Each daily session consisted of 100 trials in 60 min. Percent correct response per 100-trial session was calculated to evaluate performance. In this stage, although the animal reached an 80% correct response rate by the 5th training session, we continued training to ensure that both groups of animals reached the highest level of learning.
Reversal After reaching the acquisition criteria, in following sessions, reward contingencies of S+ and S− were reversed (Fig. 3a). In this phase, the previously unrewarded stimulus provided reward. Each training session had parameters similar to those in acquisition phase. The reversal phase continued until one or both groups reached ~ 80% correct responses for 2 consecutive days, which required 8 sessions.
Morris water maze test
The Morris water maze test was performed as previously described [8] with minor modifications. Briefly, a circular pool 1.2 m in diameter was filled with water at a temperature of 22 ± 1 °C. A transparent platform (7 cm in diameter) was submerged inside the pool. Objects of different shapes were placed on the surrounding walls. The mice were trained in three 60 s sessions for 9 days, during which the platform and the objects on the walls were fixed in the same position. Twenty-four hours after the last training trial, the mice were given a probe test without the platform and were allowed to search the platform for 60 s. Mice that did not swim were excluded from all behavioral experiments. The time taken to locate the escape platform (escape latency) and the distance moved was determined in each trial using the SMART system (SMARTBASIC / SMART 3.0 BASIC PACK, Panlab, Barcelona, Spain). Nine training sessions were required to reach stable performance. Mice that exhibited odd behaviors such as spinning, lack of swimming, staying close to the periphery, or being unable to find the platform before time ran out after 6 days of training in the Morris water maze test were excluded from all behavioral experiments.
Immunohistochemistry
Mice were deeply anesthetized by high-concentration isoflurane for animal (MSD Animal Health K.K., Tokyo, Japan) and perfused with 4% paraformaldehyde in phosphate buffer (4% PFA). Brains were dissected and post-fixed with 4% PFA for 24 h, and then cryoprotected in 30% sucrose in PBS for 24 h. Twelve-micron sagittal cryosections were prepared and treated with HistoVT One (Nacalai Tesque, Japan) at 70 °C for 20 min. The sections were then pre-incubated with 5% normal donkey serum/0.3% Triton-X-100 in PBS for 1 h and immunostained with primary antibodies against Iba-1 (1:250, Novus Biologicals, USA, RRID:AB_521594), GFAP (1:500, DAKO, Denmark, RRID:AB_10013382), and the N-terminal region of human Aβ conjugated with biotin (1:200, IBL, Japan, RRID:AB_10705565) followed by Alexa Fluor 488–conjugated anti-goat IgGs (1:1000, Invitrogen, USA, RRID:AB_2534102), Alexa Fluor 647–conjugated anti-rabbit IgGs (1:500, Jackson ImmunoResearch Laboratories, USA, RRID:AB_2492288), and Alexa Fluor 546–conjugated streptavidin (1:500, Invitrogen, USA, RRID:AB_2532130). Images were obtained on a confocal laser microscope (LSM700, Carl-Zeiss, Germany).
Immunostaining for analysis of hippocampal neurogenesis was performed as described previously with slight modifications [25]. Twenty-five–micron coronal frozen sections were post fixed with 4% PFA for 20 min and washed three times with PBS. They were incubated with methanol for 30 min, and then with 0.3% Triton-X/PBS buffer for 30 min at 37 °C. They were then autoclaved with Antigen Unmasking Solution (H-3300, Sigma-Aldrich, RRID:AB_2336226) at 105 °C for 2 min, followed by three washes with PBS. They were incubated for 30 min in blocking serum (10% normal goat serum in 0.3% Triton-X 100/PBS) and then for 24 h at 4 °C in the presence of a primary antibody against doublecortin (DCX) (E-6) (a neuronal lineage marker) (1:100, sc-271390, Santa Cruz Biotechnology, Dallas, TX, USA, RRID:AB_10610966) and Ki-67 (SP6) (a proliferating cell marker) (1:100, ab16667, Abcam, Cambridge, MA, USA, RRID:AB_302459). Sections were then washed three times with 0.05% tween in PBS, incubated in a secondary antibody (Alexa 488, RRID:AB_143165 and Alexa 546, RRID:AB_144695) (1:1000, Invitrogen, USA) for 2 h, and washed three times with 0.05% Tween in PBS. Antibodies were diluted in the Can Get Signal® immunostain Solution A (NKB-501, TOYOBO, Japan).
The dentate gyrus (DG) was segregated into dorsal regions (approximately − 1.8 to − 2.3 mm from bregma) [26], and cells in each segregation were quantified to determine any difference in neurogenesis between groups. Samples were observed with a microscope (BZ-9000, KEYENCE Corp., Osaka, Japan) and analyzed at 40 × magnification. The number and density of cells positive for immunoreactivities were analyzed using ImageJ. The values were summed and divided by the number of slices analyzed for each animal. Four areas of interest (362.99 µm × 273.31 µm), two each in the right and left DG, were imaged on one slice, and averages of at least five slices (20 areas) in each mouse were counted within areas of interest and used for statistical analysis.
Statistical analysis
All data are expressed as means ± SEM. Statistical analyses were performed with GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA, RRID:SCR_002798). Statistical significance (p < 0.05) was determined using Student’s t-test for comparisons between two groups; two-way analysis of variance (ANOVA) for multigroup comparisons; or repeated-measures ANOVA. Bonferroni test and Tukey test were used for post hoc comparison when the F value was significant. The sample size for each experiment was determined based on our previous studies using the relevant type of experiment [7, 8, 27].