Animal
C57BL/6 male mice (7 weeks old) were purchased from the Experimental Animals Center of Tongji Medical College, Huazhong University of Science and Technology. All mice experiments in this research were carried out in accordance with the recommendations of, and were approved by Animal Welfare Committee of Huazhong University of Science and Technology.
The mouse strains used included wild-type C57BL/6, PV-Cre mice. C57BL/6 male mice (7-weeks-old) were purchased from the Experimental Animals Center of Tongji Medical College, Huazhong University of Science and Technology. PV-Cre mice were described previously [14], PV-Cre mouse line used was kindly provided by Dr. Lin Mei (Case Western Reserve University). Tail genomic DNA was used for genotyping by PCR. A 300-bp fragment was detected for the PV-Cre allele.
Mice aged between 7 and 13 weeks were used for all experiments. Animals were housed less than 5 mice per cage at 22–24°C with 40–70% humidity, on a schedule of 12h/12h light/dark cycle, with water and food available ad libitum. Mice were all backcrossed with C57BL/6 mice for more than 10 generations.
Experimental Design
A schematic experimental design is shown in Fig. 1. AG1478 (HY-13524, MCE) or saline, at a dose of 50 mg/kg body weight, were chronically administered intraperitoneally (i.p.) every other day for four weeks. The timing and dosage of AG1478 administration was based on a previous study [15].
The procedures have been described previously[6]. Mice in running groups underwent adaptive run-training sessions in individual lanes of a treadmill (FT-200,Taimeng, China) (5 m/min for 45 min) for 5 days, and running sessions (5 m/min for 10 min, 8 m/min for 30 min, 5 m/min for 10 min) for the next 4 weeks to prevent stress-induced inhibition of hippocampal neurogenesis [16]. Mice in the static groups were left for the same duration on the treadmill without running.
BrdU Injections
5′-bromo-2′-deoxyuridine (BrdU; Sigma) in saline were administered i.p. every four hours (100 mg/kg), twice one day, for six days. Animals were sacrificed four weeks later after last BrdU injection.
Behavioral Analysis
No body weight, whisker number, and motor coordination difference was found in any groups during behavioral analysis. All behavioral tests were performed during light period, all mice were handled for at least 5 minutes twice a day for three days prior to the behavioral test [17]. Behavioral analysis was carried out with 12-week-old mice by investigators unaware of their genotype and groups.
Animals were tested in a sequential order of least disruptive (absence of noxious stimulation: open field and T-maze) to most disruptive (eg, MWM, fear conditioning test, and contextual fear discrimination learning, which involved physical stimuli, such as loud noise, foot shock or cool water).
Open field test (OFT)
To provide measures of locomotor activity and general anxiety-like behavior, locomotor activity was recorded in the open-field, made of a rectangular chamber (45×45×45 cm). The mouse was gently placed on the center square and allowed to freely explore the arena for 5min. The total distance travelled during a session, the time spent moving and total duration spent in the center were measured by an automated video tracking system (TMV-100S, TaiMeng, China) above the open field. After each trial, the apparatus was swept out with 75% alcohol to avoid the presence of olfactory cues. General activity and anxiety were assessed by calculating the total distance traveled, total movement time and time spent in the center of the field, respectively.
Forced-choice Spontaneous Alternation in T-Maze Test
A forced-choice paradigm was employed to encourage a higher level of alternation [18].The apparatus was an enclosed maze with 3 arms, a start arm (38 × 7 cm), a central choice area (7 × 7 cm), and two symmetrically choice arms (30 × 7 cm). In brief, each trial consisted of a 5 min acquisition phase, an intertrial-interval (ITI; 2 min) and a final 5 min test phase. ITIs of 2 min were included to assess the persistence of short-term spatial memory [19]. During the acquisition phase, the mouse was placed in the start arm facing the wall and allowed to explore the apparatus. As soon as the animal entered (with all four paws) one of the two choice arms, the door of that compartment was closed for 30 seconds. Then the mouse was removed gently from the maze to homecage for the ITI. At test, the block was removed and the mouse was placed back in the start arm to perform a second choice trial. The novel arm in the second trial was the right choice After each trial, the arena was cleaned thoroughly using 70% ethanol to remove any scent cues, which might identify the novel arm. The test was performed in the room where the animals were housed and comprised 10 trials. Correct percentage (%) = total novel arm in the second trials/ total trials × 100%.
Contextual and Cued Fear Conditioning Test
The contextual and cued fear conditioning test is the behavioral paradigm used to assess associative fear learning, hippocampus-dependent and hippocampus-independent memory function memory in rodents [20]. Briefly, the mouse was placed in the conditioning chamber using the Freeze Monitor system (San Diego Instruments, San Diego, CA, USA) for 3 min as an accommodation period and then one tone-foot-shock pairing (tone, 30 s, 65 dB, 1 kHz; foot-shock, 2 s, 0.75 mA) was delivered. The mouse was allowed to explore the chamber for another 30 s after the shock to study postshock freezing.
The contextual fear conditioning test was assessed 24 h after the training by placing the mice back in the same test chamber for 3 min to assess short-term memory.
The cued-fear conditioning test was assessed 2 h after the contextual fear conditioning test in a novel chamber changed in the shape, color, and smell and the training tone was delivered for 3 min.
Freezing behavior, defined as the absence of all visible movement of the body except the movement necessitated by respiration, was scored by an observe software. At the end of testing, the chamber was cleaned with 75% alcohol to avoid the presence of olfactory cues.
Morris Water Maze
To assess hippocampal-dependent spatial learning and memory, mice were tested in the Morris water maze (MWM,XR-XM101; Shanghai Softmaze Information Technology Co., Ltd., Shanghai, China) (130 cm diameter, 45 cm high) containing opaque water (24–26°C) and a circular platform (10 cm diameter, approximately 1 cm below the water surface) located in the center of the target quadrant. The MWM was virtually divided in four equal imaginary quadrants by the AnyMaze software. The test was executed as described previously [21]. Briefly, the test consisted of a four-day hidden platform training test and a one-day single probe test. In the training test, the mice were allowed to face to the pool wall in a randomized starting place in the pool to find the hidden platform. The mouse was allowed 120 s to find the platform upon which they sat for 20 s. If the mouse did not find the platform within 120 s, it was gently guided there and allowed to stay for 30 s. Twenty-four hours after the last training session, the platform was removed from the pool and the mouse was placed in the opposite quadrant, and a 60-s probe trial was performed. The escape latency, the percentage of time spent in the target and the number of platform crossings were recorded.
Contextual Fear Discrimination Learning
This paradigm tests the animal's ability to distinguish between two similar contexts [22]. Pattern separation is a fundamental computational function of the DG [23, 24], which depends on normal adult neurogenesis.
The shock-associated training context A (shock) and the similar (no-shock) context B shared many features, including an exposed stainless steel grid floor and roof. The similar context differed from the training context in that four plastic inserts were used to cover the walls. A non-alcoholic antiseptic solution was used to clean the grids between trials. In pilot experiments, mice were exposed to the training context where they received a single 2-s 0.75 mA foot shock, 185 s following placement in the sound proof chamber (29 × 29 × 24 cm; Coulbourn instruments, Allentown, PA, USA, model H10-11M-7C-SF). For discrimination learning, mice were again exposed to training context A. One hour later, mice were placed in the similar context and left for 180 s, and were never shocked. Freezing levels were measured by video camera each day and computed as a Discrimination ratio: (Freezing training context - Freezing similar context) / (Freezing training context + Freezing similar context). A score of 0 indicated complete lack of discrimination, i.e., freezing levels were the same in the similar and training contexts (Freezing similar context = Freezing training context).
In intro and in vivo calcium imaging
To assess the effect of AG1478 on intracellular Ca2 + levels in PV neurons, calcium signals of cells were observe in brain slices imaging. First, the mouse brains were quickly removed and placed in 4°C artificial cerebrospinal fluid (ACSF) composed of (in mM) choline chloride 110, KCl 2.5, NaH2PO4 1.25, NaHCO3 26.0, CaCl2 0.5, MgCl2 7, d-glucose 10, Na-ascorbate 11.6, Na-pyruvate 3.1, and atropine sulfate 0.01. Coronal slices (300 µm thick) were cut using a Leica VT1000S vibratome and then incubated in standard ACSF containing (in mM) NaCl 126, KCl 3, NaH2PO4 1.25, NaHCO3 26.0, CaCl2 2, MgSO4 2, d-glucose 11 saturated with 95% O2 and 5% CO2. Next, hippocampus slices from the mice were kept at 32°C approximately 30 min before recording. The Ca2 + signals were visualized by an inversed fluorescent microscope (Olympus,) with a water-immersion objective lens (40X, LUMPlanFL, 0.80 numerical aperture; OLYMPUS). Brain slices from one animal were divided into two groups. One group was incubated for 30 minutes at 32°C in ACSF as control group whereas another one in 10 µmol/LAG1478 as experiment group. Upon excitation at 488 nm, calciumion complexed GCaMP6 was collected at 10 frames per second using the Image-pro plus 7.0 software. The Ca2 + levels in slices was calculated as the following formula using calcium imaging in vivo assay
To further assess the effect of AG1478 on intracellular Ca2 + levels in PV neurons of running mice, calcium signals of cells in awake animals with head-fixed animals were observe by in vivo calcium imaging. Firstly, 300 nL of AAV-EF1α-DIO-GCaMP6m-WPRE-hGH-pA viruses (BrainVTA, catalog#PT-0283) were injected into the DG of PV-Cre mice using a stereotaxic instrument. The stereotactic technology procedures have been described previously[6]. Adult PV-Cre mice were anesthetized with chloral hydrate (400 mg/kg, i.p.) and head-fixed in a stereotaxic device (RWD life science; 68025). Viruses were unilaterally injected (0.3 mL per side, 0.05 mL/min) with a glass pipette [25] (tip size, ~ 20 mm) at the following coordinates relative to bregma: anteroposterior, -1.94 mm; dorsoventral, -2.14 mm; and mediolateral, ± 1.5 mm. After injection, the glass pipette was left in place for 15 min before slowly removing it. The titers of AAV-EF1α-DIO-GCaMP6m-WPRE-hGH-pA (BrainVTA, catalog #PT-0283) were 2x1012 genome copies per mL. Secondly, four weeks after virus injection, gradient-index (GRIN) lens (0.5 mm in diameter and 5.901 mm in length, Gofoton, USA, GRIN Tech) was firmly mounted to a stereotaxic holder. Then, the GRIN lens was inserted into the DG (from either the left or the right side) and positioned at 0.10 mm to 0.20mm above the highest viral injection site. Next, dental cement (New Century Dental Materials Co. Ltd., Shanghai, China) was applied to secure the GRIN lens. During recording, the distance of the microscope to the GRIN lens was adjusted by a micro-manipulator until the field of view was in focus. The gain was set at 16 and the LED (470 nm) power was maintained at 100%. Images were collected at 30 frames per second using the MiniScope V2.0 software.
Images were processed using MATLAB R2020b (Mathworks, Natick, MA) with customized code (https://github.com/thinkertech333/analysisforminiscope, Thinkertech, Nanjing, China), and Cellsort 4.0 software. After motion correction (set to 10) and denoising (set to 100), the region of interest was manually selected according to the fluorescence intensity. The Ca2+ signals in the first 5 seconds were considered as baseline and the average Ca2 + signal in this period was used as a reference (F0) to normalize the fluorescence signal (DF/F). The formula is as following:
$$\text{F/F=}\frac{{\text{F}}_{\text{signal}}\text{-F0}}{\text{F0}}$$
Fsignal is the real-time Ca2+ signal intensity of the cells of interest, F0 is the average Ca2+ signal intensity in the baseline period
Immunofluorescence
Mice were anesthetized with chloral hydrate and perfused transcardially with 4% paraformaldehyde (PFA) in phosphatebuffered saline (PBS, pH7.4), and tissues were fixed overnight in 4% PFA at 4°C. After cryoprotected in 30% sucrose, brain tissues were frozen in OCT and cut into 40 µm by a cryostat (Thermo Scientific, HM550).
The free-floating sections were rinsed three times in PBS, and blocked in PBS with 0.3% Triton and 10% goat serum and 0.1% Triton-X 100 (PBST) for 60 min at room temperature. Sections were then incubated with primary antibodies in PBST at 4°C overnight. After washing with PBS for 3 times, samples were incubated with second antibodies in PBST for 1 hr at room temperature. Samples were mounted with mounting medium, antifading (with DAPI) (S2110, Beijing Solarbio Science & Technology), and images were taken by Zeiss Axioplan light microscope. Quantification of labeling was determined by counting all fluorescent cells in every sixth coronal section.
For BrdU staining, sections were incubated with 2 N HCl for 30 min at 37°C to denature the DNA, followed by neutralization with 0.1 M borate buffer (pH 8.5) for 10 min at room temperature. After neutralization, sections were rinsed with PBS several times before incubation with primary antibodies.
Immunocytochemistry used following primary antibodies: rabbit anti-KI67 (ab15580, abcam, 1:500), rat anti-BrdU (FITC conjugated; ab74545, Abcam; 1:300), mouse anti-NeuN (ab104224, Abcam, 1: 500), rabbit anti-PV (A2791, Abclonal; 1:100), donkey anti-rabbit IgG conjugated with Alexa Fluor 594 (711-585-152, 1: 200, Jackson ImmunoResearch), goat anti-rabbit IgG conjugated with Alexa Fluor 488 (SA00006-2, 1: 250, Proteintech), goat anti-mouse 488༈SA00006-2, Proteintech, 1༚250༉
Western Blot
One day after the behavioral tests, mice were anesthetized with 5% chloral hydrate (8 ml/kg) and the tissues were rapidly collected. The tissue homogenates were prepared on ice in RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5% sodium deoxycholate, 1% NP40, 1 mM PMSF, and 1 µg/ml protease inhibitor cocktail. For immunoblotting p-ErbB4, homogenates subjected to immunoprecipitation with ErbB4 antibody and protein-A (Roche) at 4°C overnight. Homogenates or bound proteins were resolved on SDS/PAGE and transferred to PVDF membranes, which were incubated in Tris-Phosphate buffer (TBS) containing 0.1% Tween-20 and 5% milk (TBST) for 1 hr at room temperature before the addition of primary antibodies for incubation overnight at 4°C. After wash, the membranes were incubated with HRP-conjugated secondary antibodies (BL003A, 1: 30000, Biosharp) in TBS for 1 hr at room temperature. Primary antibodies used and blotting conditions were: rabbit anti-phosphate-ErbB4 (Ab76132, 1: 1000, Abcam), rabbit anti-PVALB (A2791, 1: 1000, Abclonal), rabbit polyclonal anti-a-Tubulin (AC003, 1: 500, abclonal). Immunoreactive bands were visualized using enhanced chemiluminescence (1705060, Biorad, United Kingdom), scanned with MicroChemi 4.2 (DNR Bio-imaging Systems, ISRAEL) and semiquantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Statistical Analysis
All Statistical analysis was performed by GraphPad Prism 6.0 Software(GraphPad Software, San Diego, CA). Data were analyzed by unpaired two-tailed Student’s t-test or analysis of variance (ANOVA). Significant main effects or interactions were followed up with Tukey’s post hoc test. Data were presented as mean ± SEM. Statistical differences were considered to be significant when P < 0.05.