All the experiments were performed in accordance with the ARRIVE guidelines.
Animals
The animal experiments were approved by the Animal Care and Use Committee of Nagoya City University Graduate School of Medical Sciences (permit number 18149, 19-017H02) and conformed to the guidelines for the use of laboratory animals which was published by the Japanese government (Law No. 105, October 1973). The generation of homozygous HCNP-pp KO mice and littermate HCNP-pp floxed control mice was performed as previously reported[21]. The animals were housed in specific pathogen-free conditions with a 12 h light/dark cycle (with the lights on from 08:00 to 20:00) and were given free access to food and water.
We used 176 slices from 80 male mice for the electrophysiological experiments for LTP (young generation, 11–17 weeks old; control, n = 17, N = 10, HCNP-pp KO, n = 26, N = 10; adult generation, 24–35 weeks old; control, n = 65, N = 28, HCNP-pp KO, n = 68, N = 32), and 20 female mice were used for a Western blot analysis (adult generation, 24–27 weeks old, control, N = 5, HCNP-pp KO, N = 5; old generation, 54–55 weeks old, control, N = 5, HCNP-pp KO, N = 5). Then, paired-pulse facilitation was studied by selected slice samples used in the electrophysiological experiments for LTP (young generation, control n = 24, N = 5, HCNP-pp KO, n = 14, N = 5; adult generation, control, n = 41, N = 14, HCNP-pp KO, n = 38, N16)
Antibodies
The rabbit polyclonal anti-mouse/rat HCNP (HCNP-pp) antibody was generated as previously described[14]. Then, the anti-HCNP-pp antibody was purified with an HCNP affinity column prepared with a Hitrap NHS-activated HP Column (GE Healthcare, Waukesha, WI, USA). The following antibodies were obtained commercially: rabbit monoclonal anti-human/mouse synaptophysin antibody (Abcam, Cambridge, UK), mouse monoclonal anti-rat/mouse postsynaptic density protein 95 (PSD95) antibody (Merck-Millipore, Billerica, MA, USA), goat polyclonal anti-human/mouse ChAT antibody (Merk-Millipore), rabbit polyclonal anti-mouse VAchT/SLC18A3 antibody (NOVUS biological, Littleton, CO, USA), rabbit polyclonal anti-human/mouse CHT-1 antibody (Merck-Millipore), mouse monoclonal anti-β-actin antibody (Sigma, St. Louis, MO, USA), rabbit polyclonal anti-human/mouse muscarinic receptor 1 antibody (Sigma), rabbit polyclonal anti-rat/mouse NR-1 antibody (Sigma), rabbit polyclonal anti-rat/mouse NR-2A antibody (Merck-Millipore), rabbit polyclonal anti-mouse NR-2B antibody (Merck-Millipore), rabbit polyclonal anti-human/mouse GluA1 antibody (Merck-Millipore), and rabbit polyclonal anti-rat/mouse GluA2/3 antibody (Merck-Millipore) for the Western blots, and HRP-conjugated anti-rabbit (MP Biomedicals, Santa Ana, CA, USA), anti-mouse (MP Biomedicals), or anti-goat (Abcam) IgG antibodies were used as secondary antibodies.
Slice preparation
Slice preparation was performed following a previous study[10, 19]. Briefly, the mice were deeply anesthetized with halothane and decapitated. The brains were quickly removed and transverse hippocampal slices with a 400 µm thickness were prepared using a vibrating slice cutter (Linear Slicer Pro 7, Dosaka, Kyoto, Japan) in an ice-cold solution containing (mM): sucrose, 260; KCl, 3; NaH2PO4, 1.25; NaHCO3, 26; D-glucose, 10; and MgCl2, 1 (pH 7.4), which was continuously bubbled with 95% O2/5% CO2. One to four slices were obtained from each animal and one experiment was performed on each slice. The hippocampal slices were incubated for 30 min in artificial cerebrospinal fluid (ACSF) containing (mM): NaCl, 125; KCl, 2.5; CaCl2, 2.4; MgCl2, 1; NaH2PO4, 1.25; NaHCO3, 25; and D-glucose, 12.5, which was saturated with 95% O2/5% CO2 (pH adjusted to 7.4) at 32°C. The slices were kept at room temperature (25 ± 1°C) in the ACSF at least for 1 h until they were ready for recording.
Electrophysiology
The electrophysiological study was conducted as per previously published studies[10, 19]. Briefly, the slices were fixed in a recording chamber (~ 0.4 mL volume, RC-26GLP, Warner Instruments, Hamden, CT, USA) under a nylon mesh attached to a stainless-steel anchor, and then they were submerged in and continuously perfused with ACSF at a flow rate of 2 ml/min. All the experiments were performed at room temperature (25 ± 1 °C). The fEPSPs were recorded from the stratum radiatum (SR) in the CA1 field using borosilicate glass electrodes (3–5 MΩ) filled with perfusing ACSF. The recordings were made using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA) with a high-cut filter at 2 kHz.
A stainless steel concentric bipolar electrode (Unique Medical, Tokyo, Japan) was placed on the SR, and the SCs were stimulated with a 0.1 ms pulse every 30 s. At the beginning of each experiment, a stimulus-response curve was established, and the stimulus intensity was adjusted to evoke 30–50% of the maximal response, which fell within the stimulus intensity of 20–40 µA. Long-term potentiation in the CA1 region was induced from S-TS or D-TS (0.1 ms pulse duration, 100 Hz for 1 s) of the SCs. The fEPSPs were recorded in CA1 for 60 min after the induction of LTP. Then, paired-pulse facilitation was studied by applying a pair of stimulations at varying interpulse intervals (25–200 ms).
Drug application
The drugs Prz and CCh were commercially purchased (Sigma). All the drugs were dissolved in ACSF and applied by gravity-fed 60 mL reservoirs bubbled with 95% O2–5% CO2 for 10 min just before the induction of LTP.
Western blot analysis
The Western blot was performed following a previous study[17]. In brief, under deep pentobarbital anesthesia, each mouse was transcardially perfused with phosphate-buffered saline. After the brains were removed and placed on ice, the hippocampi were dissected and immediately frozen in liquid nitrogen and stored at -80°C until further use. The frozen hippocampi from five HCNP-pp KO mice and five control mice were homogenized in four volumes of lysis buffer containing 30 mM Tris-HCl (pH 8.5), 7 M urea, 2 M thiourea, 4% w/v CHAPS, and a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IND, USA). After incubation for 60 min on ice, the homogenates were centrifuged at 15,000 × g for 3 min at 4°C. After the protein content was measured using the Bradford assay (Pierce, Rockford, IL, USA), the supernatants were stored at -80°C until further use. Then, 10 µg of each supernatant fraction was loaded onto each lane of 10% gel for SDS-Polyacrylamide gel electrophoresis. After electrophoresis, the samples were transferred to Hybond-P membranes (GE Healthcare, Tokyo, Japan) using 25 mM Tris, 192 mM glycine, 0.1% SDS, and 10% methanol as a transfer buffer. Then, the membranes were incubated with 1:500 goat polyclonal anti-ChAT antibody, 1:5,000 rabbit polyclonal anti-VAchT antibody, 1:1,000 rabbit polyclonal anti-CHT-1 antibody, 1:200,000 rabbit polyclonal anti-synaptophysin antibody, 1:2,000 rabbit polyclonal anti-NR-1 antibody, 1:5,000 rabbit polyclonal anti-NR-2A antibody, 1:1,000 rabbit polyclonal anti-NR-2B antibody, 1:5,000 rabbit polyclonal anti-GluA1 antibody, 1:1,000 rabbit polyclonal anti-GluA2/3 antibody, 1:2,000 mouse monoclonal anti-PSD95 antibody, 1:2,000 rabbit polyclonal anti-muscarinic receptor 1 antibody, or 1:50,000 mouse monoclonal anti-β-actin antibody. The membranes were then probed with Horseradish peroxidase- conjugated anti-rabbit, anti-mouse, or anti-goat IgG antibodies. The immunoreactive bands were visualized using the ECL Advance Western Blotting Detection kit (GE Healthcare, Tokyo, Japan) and recorded using ImageQuant LAS 4000 (GE Healthcare, Tokyo, Japan). The Western blots were quantified using Amersham Imager 600 Analysis Software (GE Healthcare, Tokyo, Japan).
Data acquisition and statistical analysis
The data sampling and statistical analysis were performed following previous studies [10, 19]. In brief, all the data were included in the final analyses except for when the baseline response changed during the experiment. The fEPSPs were sampled online at 4 kHz (PowerLab, AD Instruments, Sydney, Australia), stored on a hard disk, and analyzed offline with Scope 4 and Chart 5 (AD Instruments). The maximal slope values of the initial rising phase of the fEPSPs were measured to avoid the contamination of the voltage-dependent components as much as possible. The magnitude of LTP was calculated as a percentage of the averaged fEPSP slope values from 50 to 60 min after each tetanic stimulation relative to the baseline fEPSP slope values. The derived parameters (n = number of slices, N = number of animals) were compared using a one-way analysis of variance, which was followed by multiple comparisons (Tukey test) or an unpaired t-test with the level of significance set at p < 0.05. The quantified bands in the Western blots were also calculated and compared using an unpaired t-test with the level of significance set at p < 0.05. Also, the parameters and bars were expressed as the mean ± the standard error of the mean.