The PP2Aflox/flox wild-type mice were provided by Xiang Gao’s lab . The Tg (Camk2a-cre) T29–2Stl mice, which mediate Cre/loxP recombination predominantly in CA1 pyramidal cells, were a kind gift from Prof. Tsai L-H (Picower Institute for Learning and Memory, MIT, USA). We crossed the PP2Af/f control (Cont) mice with T29–2 Cre transgenic mice [12, 13] to generate the hippocampal CA1-specific PP2A conditional knockout (CKO) mice. We used 8- to 10-week-old mice in the behavioural experiments (male mice, N = 54/group), PCR (male and female mice, N = 3/group), western blot (male and female mice, N = 3/group) and immunohistochemistry (male and female mice, N = 4/group) and 4- to 6-week-old mice in the electrophysiology recording experiments (male and female mice, Cont group: N = 24, CKO group: N = 20). Separate animals were used for the behavioural tests, biochemical experiments and electrophysiology recordings. All animals were given ad libitum access to food and water and were housed in groups with males and females apart under a 12-h light/dark cycle. All animal experiments were performed in accordance with the recommendations of the Experimental Animal Ethics Committee at the Nanjing Medical University.
Polymerase chain reaction (PCR)
To identify the genotype of the mice, we collected toes form 7–9 days old mice. The protocol of PCR was performed as described previously . Briefly, to identify the genotype, the toe was collected from mice (before 10-day-old) to isolate DNA for PCR. The sequences of primers are listed as follows:
loxP-Forward primer: 5′ > TAGCCCATGCCTTTAATCTCAGAGC< 3′.
loxP-Reverse primer: 5′ > CACTCGTCGTAGAACCCATAAACC< 3′.
Cre-Forward primer: 5′ > TGCCACGACCAAGTGACAGCAATG< 3′.
Cre-Reverse primer: 5′ > ACCAGAGACGGAAATCCATCGCTC< 3′.
During the procedures of PCR, we first denatured DNA at 95 °C for 5 min, and then denaturing at 94 °C for 30 s. The step of annealing was at 58 °C for 30 s and extending at 72 °C for 1 min. After repeating for 35 more times, we extended DNA strands at 72 °C for 5 min. Then, the PCR products were analyzed by 1% agarose (BA0047, Nanjing best biological technology Co.,Ltd) gel electrophoresis and developed under ultraviolet light using Gel Image System (Tanon-2500, Shanghai, Tianneng Technology Corporation).
Western blot analysis
To confirm the specific PP2A knockout, the brain (except for the olfactory bulb and cerebellum), including the hippocampal CA1 area, was collected from Cont and CKO mice. Coronal hippocampal slices were prepared at 500-μm thickness using a Leica VT1000S vibratome (Leica Instruments Ltd., Wetzlar, Germany) in ice-cold oxygenated (95% O2/5% CO2) cutting ACSF containing (in mM) 75 sucrose, 87 NaCl, 2.5 KCL, 1.25 NaH2PO4, 21.4 NaHCO3, 0.5 CaCl2, 7 MgCl2, 1.3 ascorbic acid and 20 D-glucose (pH 7.2–7.4). The hippocampal CA1 area was dissected with surgical blades and forceps. Lysates (50 mM MOPS, 100 mM KCl, 50 mM NaF, 20 mM NaPPi, 20 mM Glycerd-P, 320 mM Sucrose, 0.2 mM DTT, 1 mM EDTA, 1 mM EGTA, 0.5 mM MgCl2, 1 mM NaVO4, half of a protease inhibitor tablet in 10 ml) were incubated on ice and cleared with an 8000-rpm spin for 15 min, and protein content was quantified (BCA protein assay, Thermo Scientific). Four hundred micrograms of protein was diluted with 5× loading buffer consisting of the following: 250 Mm Tris pH 6.8, 10% SDS (w/v), 0.5% bromophenol blue (w/v), 50% glycerol (v/v), 5% β-mercaptoethanol. Samples were boiled at 95 °C for 10 min and resolved on a 10% SDS-polyacrylamide gel with 8% stacking gels using Laemmli buffer. Proteins were transferred by electrophoresis using tris-glycine wet transfer onto PVDF membranes (Millipore, 0.45 μm) for 1 h on ice. After blocking with blocking buffer (5% non-fat dry milk/0.1% Tween-20/TBS) for 1 h, membranes were probed with the anti-PP2A C subunit antibody (#2038, Cell Signaling Technology, 1:3000) and Tubulin β polyclonal antibody (AP0064, Bioworld, 1:1000) at 4 °C overnight. Membranes were washed three times using 0.1% Tween-20/TBS and incubated with a goat anti-rabbit IgG (H + L) HRP-linked antibody (BS13278, Bioworld, 1:8000) for 1 h at room temperature. Membranes were washed again and developed using Western Lightning Gel Imaging System (Tanon 2500, Shanghai, Tianneng Technology Corporation).
The mice were perfused with 4% paraformaldehyde in phosphate-buffered saline, and the brain was dissected and placed in sucrose solution. After cryoprotection using a 15 and 30% sucrose gradient, coronal hippocampal slices were prepared at 25-μm thickness using a freezing microtome (CM-1950, LEICA). To confirm the efficiency of the specific PP2A knockout and the effect on the development of neurons and neurogliocytes in the hippocampal CA1 area, the slices were incubated in primary antibody overnight at 4 °C. After incubation with the secondary antibody for 2 h and DAPI (10,236,276,001, Roche, 1 μg/ml) for 15 min at room temperature, the samples were examined by using confocal laser microscopy (FV-1000, OLYMPUS). The antibodies and dilutions were as follows: PP2A C subunit antibody (#2038, Cell Signaling Technology, 1:250), anti-NeuN rabbit polyclonal antibody (ABN78, Millipore, 1:500), goat anti-rabbit IgG (H + L) Cy3 (BS10007, Bioworld Technology, 1:400) and anti-glial fibrillary acidic protein (GFAP) antibody, and clone GA5 (MAB3402, Millipore, 1:500).
Open field test
Locomotor activity and anxiety responses of rodents can be tested using an open field test [14, 15]. The open field apparatus (50 × 50 cm, Shanghai Xinruan Informatlon Technology Co. Ltd., Shanghai) was divided into 16 compartments in the ANY-Maze software (Stoelting, Illinois), and the 4 in the middle were defined as the centre area. The mice (N = 12/group) were individually placed in one corner of the open field apparatus and allowed to explore freely for 10 min. Mouse movement was tracked by ANY-Maze tracking software (Stoelting, Illinois). The distance moved in the apparatus every 2 min and total time spent in the centre area were recorded.
Forced swim test
The forced swim test was conducted according to the Porsolt protocol . Mice (Cont group: N = 14, CKO group: N = 11) were forced to swim for 6 min in a big glass cylinder filled with water at 25 ± 1 °C. The immobility time during last 4 min was recorded to evaluate depression-like behaviour.
Prepulse inhibition (PPI) of startle reflex test
The PPI of the acoustic startle response was tested as described previously . The mice (N = 12/group) were habituated to the chamber with a white-noise background (70 dB) for 5 min. Each test consisted of 80 trials with 6 null trials, 68 prepulse-pulse trials, and 6 pulse-alone trials. The average intertrial interval was 15 s (range from 10 to 20 s). Null trials consisted of a 40-ms burst of a 120-dB stimulus. Prepulse-pulse trials included 7 types of trials presented randomly, including a 40-ms burst of a 120-dB single stimulus, a 40-ms prepulse stimulus that was 74, 82, or 90 dB and three prepulse stimuli followed 100 ms later by a 120-dB stimulus. The test terminated with pulse-alone trials using the same protocol as used for the null trials. PPI responses were calculated as % PPI = [1– (prepulse trials/startle-only trials)] × 100%.
Object recognition test
The object recognition test was performed as described previously in the literature [18, 19]. Briefly, mice (Cont group: N = 8, CKO group: N = 10) were habituated in the empty open field for 5 min each day for 1 week. Two identical 150-ml bottles were placed in their cages to serve as “old objects”. After the habituation phase, two identical old objects were placed in the open field at an equal distance from the mice. The familiarization session lasted for 2 days during which mice were placed in the open field for 5 min four times a day. Then, the trained mice were divided into short-term memory (STM) and long-term memory (LTM) groups. At the beginning of the test session, all animals were allowed to explore the old objects for 5 min. One hour later in the STM group and 24 h later in the LTM group, the animals were placed in the open field with one old and one new object. The test session lasted 10 min. The time the mouse spent sniffing (sniff time) the old and new objects was recorded by using ANY-Maze tracking software. The memory index was used to evaluate the memory function of mice: Memory Index = (Sniff time of new object – Sniff time of old object)/(Sniff time of new object + Sniff time of old object) × 100%.
Contextual fear conditioning (cFC)
The experimental protocol was modified from work published previously . The animals (Cont group: N = 8, CKO group: N = 9) were placed in the chambers for 3 min. After habituation, three consecutive foot shocks of 0.7 mA lasting for 2 s at 2 min intervals were administered to form the conditioned fear memory. On the second day, all animals were returned to the same chamber, and freezing was automatically recorded using FRAMEFREEZE software (Coulbourn Instruments) for 3 min. They were then removed from the chamber and returned to their home cages. One hour later, the animals were put back into the chamber for 21 min without receiving foot shocks and then returned to their home cages for 21 min. This extinction phase process was repeated three times. Freezing was recorded for 3 min 24 h, 48 h, and 72 h after the extinction phase.
Electrophysiological recordings were performed as previously described in Yang et al  Horizontal hippocampal slices were prepared at 350-μm thickness using a Leica VT1000S vibratome (Leica Instruments Ltd., Wetzlar, Germany) in ice-cold oxygenated (95% O2/5% CO2) cutting artificial cerebrospinal fluid (ACSF) containing (in mM) 75 sucrose, 87 NaCl, 2.5 KCL, 1.25 NaH2PO4, 21.4 NaHCO3, 0.5 CaCl2, 7 MgCl2, 1.3 ascorbic acid and 20 D-glucose (pH 7.2–7.4). Slices were transferred to a holding chamber and incubated for 60 min at 32 °C submerged in oxygenated (95% O2/5% CO2) recording ACSF containing (in mM) 119 NaCl, 2.5 KCl, 1 NaH2PO4, 26.2 NaHCO3, 2.5 CaCl2, 1.3 MgSO4 and 11 D-glucose (pH 7.2–7.4). The slices were then incubated at room temperature for at least 1 hour before recording.
The stimulator was placed in the Schaffer collateral/commissural pathway. Recording electrodes (resistance, 1–4 MΩ) were pulled from borosilicate glass capillary tubes (1.5-mm outer diameter, 0.86-mm inner diameter, World Precision Instruments) using a Brown-Flaming micropipette puller (P-97; Sutter Instruments Company) and filled with recording ACSF. Field excitatory postsynaptic potentials (fEPSPs) in the hippocampal CA1 area were recorded. We chose the slices whose maximal fEPSP amplitude was at least 0.7 mV, and the stimulation intensity was adjusted so that baseline fEPSPs were recorded at 40% of the maximal amplitude. Input-output data were collected by varying the intensities of seven stimuli applied to the CA1 area. Paired-pulse facilitation induced by paired-pulse stimulation (inter-pulse intervals were 10 ms, 20 ms, 50 ms, 100 ms and 200 ms) were evoked every 30 s. After a 10-min stable baseline, LTP was induced by two theta burst stimulations (TBSs) separated by 20 s (5 trains at 5 Hz with each train including 4 pulses at 100 Hz) or 50-Hz high-frequency stimulation (HFS) (5 trains of 1-s stimulation at 50 Hz with 200-ms inter-train intervals) followed by 40 min of fEPSP recording. To investigate the changes in depotentiation in Cont and CKO mice, we first used four TBSs separated by 20 s to induce a saturated LTP. After 45 min, we used low-frequency stimulation (LFS) (900 trains of 15 min stimulation at 1 Hz) to induce depotentiation [22, 23].
Changes in LTD expression in Cont and CKO mice were also detected. After a 15-min stable baseline, LTD induced by LFS was recorded for 45 min. Pharmacological treatment was used to further verify the role of PP2A in LTD. A stock solution of PP2A inhibitor okadaic acid (OA, Sigma) dissolved in 0.1% dimethyl sulphoxide (DMSO) was prepared and stored at − 20 °C . The stock OA solution was mixed with freshly made recording ACSF to a final concentration of 25 nM . Before electrophysiological recording, we incubated the slices from Cont mice in OA solution for 30 min, and during LTD recording, the slices were maintained under OA treatment.
The LTP and LTD magnitude was calculated from the average of the last 10 min of recording and reported as the (%) Mean ± SEM of baseline fEPSP slope.
Data were analysed using SPSS 19.0 (SPSS, Inc., Chicago, IL, USA), and illustrations were created using Origin 8.5 (Electronic Arts Inc., California, USA). Differences in the behaviour tests between Cont and CKO mice were tested for statistical significance using an independent t test. To further assess the effect of PP2A on memory extinction, we analysed the cFC data, input-output curves, paired-pulse stimulation and the last 10 min of the fEPSP slope after LTP or LTD of the Schaffer Collateral-CA1 pathway using a repeated measures ANOVA. Data were reported as the mean ± SEM. The significance level for all tests was set at p < 0.05.