Animals
All animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals and were approved by the institutional review committee of Kanazawa Medical University (Approval No. 2018-21) and by the Animal Care and Use Committee of Kanazawa University (Approval No. AP-184013). Experiments were conducted in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, and in compliance with ARRIVE guidelines. Atf6b+/- mice were generated as previously described [20], and backcrossed to the C57BL/6N strain for eight or more generations at the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University. Atf6b+/- mice were intercrossed to obtain Atf6b+/+, Atf6b+/-, and Atf6b-/- mice. Atf6b+/+ mice were used as WT controls. The mice were each housed in a standard cage on a 12-h light/dark cycle (lights on at 7:00 AM) with food and water available ad libitum in the Institute of Experimental Animal Sciences, Kanazawa Medical University. A total of 61 male WT and 53 male Atf6b-/- mice (age 11–16 weeks) were used for experiments.
In situ hybridization
In situ hybridization was performed as described previously [21]. Briefly, a − 600 bp mouse ATF6β cDNA fragment was polymerase chain reaction (PCR)-amplified using primer sets (Table 1) and inserted into a pGEM-T vector (Promega, Madison, WI, USA). The plasmid was linearized using EcoRV and ApaI to synthesize the antisense and sense probe, respectively. Brains were isolated from mice after perfusion with phosphate-buffered saline (PBS) and immediately placed at -80℃. Serial 14-µm sagittal sections were generated using a cryostat and hybridized with a digoxigenin-labeled ATF6β RNA probe. Imaging was performed using a light and fluorescence microscope (BZ-X700, Keyence, Osaka, Japan).
Quantitative real-time PCR (RT-qPCR)
One mm thick brain slices were prepared using a coronal mouse brain slicer (Visikol, Hampton, NJ, USA), and collected in the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala based on the mouse brain atlas. Total RNA was extracted using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. RNA was then reverse transcribed to synthesize first-strand cDNA using a ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). RT-qPCR was then performed using oligonucleotide primer sets (Table 2). Individual cDNA samples were preheated at 98°C for 2 min and subjected to 40 amplification cycles (denaturation at 98°C for 10 sec, annealing at 60°C for 10 sec, extension at 68°C for 30 sec) using Thunderbird™ SYBR qPCR Mix (Toyobo) or KOD SYBR® qPCR Mix (Toyobo), and a QuantStudio™ 12K Flex Real-Time PCR System (Thermo Fisher Scientific, Tokyo, Japan). Cycle threshold values were calculated using the ΔΔ cycle threshold method to obtain fold differences. Relative expression of each gene was normalized against Gapdh mRNA expression level.
General health and neurological screen
A general health check and neurological screen was conducted as described previously [22]. Body weight was measured, and neuromuscular strength was assessed using wire hang tests. In the wire hang test, mice were individually inverted, placed on a wire mesh with 5-mm square holes, and the latency to fall from the wire was recorded with a 60 sec cutoff time.
Light/dark transition test
A light/dark transition test was performed as previously described in two chambers [23], a blight arena (25 × 25 × 24 cm, 485 lux) and a dark arena (16 × 25 × 24 cm, 6 lux). Individual mice were placed in the dark chamber and allowed to move freely between the two chambers for 10 min. The total number of transitions between chambers, the time spent in the light chamber (%), and the latency to first entrance into the light chamber (sec) were recorded automatically by a floor-pressure sensor and analyzed using software (PPCWIN v2.0, Panlab Harvard Apparatus, Barcelona, Spain).
Open field test
An open field test was used to evaluate locomotor activity and emotional response [24]. The open field test was measured with a circle field apparatus (45 cm height × 79 cm diameter), a central field (50%), and an outer field (periphery). Individual mice were placed in the periphery of the field at the start of the test and allowed to freely move about the apparatus for 10 min. The distance traveled and time spent in the center field (%) were recorded automatically by video tracking software (SMART v3.0, Panlab Harvard Apparatus).
Elevated plus maze test
An elevated plus maze test was conducted as previously described [25]. The elevated plus maze consisted of two open arms (30 × 6 cm) and two enclosed arms with walls (30 × 6 × 13.5 cm). The arms and a central platform (6 × 6 cm) were made of gray plastic plates elevated 42 cm from the floor. Arms of the same type were arranged opposite each other. Mice were individually placed in the central area facing the enclosed arm. The total number of transitions between each arm and the central platform, the number of open arm entries, and the time spent in the open arms (%) were recorded automatically for 10 min with video tracking software (SMART v3.0, Panlab Harvard Apparatus).
Social interaction test
A social interaction test was conducted as described previously [26]. The social interaction test was performed in a gray plastic box (40 × 40 × 40 cm), and a wire cage (9 cm height × 9 cm diameter) with 6-mm square holes was placed in one corner of the arena for the test. The interaction zone was defined as a 20-cm quadrant area surrounding the interaction apparatus, and the area diagonal to the interaction zone was defined as the evacuation zone. The area between the interaction and evacuation zones was defined as the interference zone. The day before testing, test mice were individually placed in the box without a stranger mouse (C57BL/6N male) in the wire cage and allowed to freely explore the box for 10 min. In the social interaction test, a stranger mouse that had no prior contact with the test mouse was placed in the wire cage. The test mice were individually placed in the interference zone of the arena and allowed to explore freely for 10 min. The number of the contact zone entries, time spent in the contact zone (%), and the mean duration of time in the contact zone (sec) were measured automatically by video tracking software (SMART v3.0, Panlab Harvard Apparatus).
Hot plate test
The hot plate test was performed to evaluate sensitivity to a painful stimulus. In the hot plate test, mice were placed on a 55.0°C hot plate (UGO Basile, Comerio, Italy) into an acrylic cylinder (24.5 cm height × 19 cm diameter), and latency to the first paw response was recorded with a 15 sec cutoff time [27]. The paw response was defined as either a foot shake or a paw lick.
Rotarod test
The rotarod test was performed to evaluate motor coordination and balance ability using an accelerating rotarod (Panlab Harvard Apparatus). Mice were placed on a rotating rod (3 cm diameter) that accelerated from 4–40 rpm over a 5-min period, and the time individual mice were able to maintain balance on the rotating rod was recording. Timing stopped when the mouse fell off the rotating rod (sec).
Porsolt forced swim test
The Porsolt forced swim test [28] was performed as previously described [29]. The apparatus, consisted of an acrylic cylinder (50 cm height × 24 cm diameter), filled with water at 23°C up to a height of 20 cm. Individual mice were placed into the cylinders, and total distance and immobility time were recorded for 10 min (days 1 and 2) by video tracking software (SMART v3.0, Panlab Harvard Apparatus).
Drug treatment
CRHR1 antagonism was conducted as described previously [30]. Antalarmin, a CRHR1 antagonist (A8727, Sigma-Aldrich, Saint Louis, MO, USA), was dissolved in 4% dimethylsulfoxide (DMSO) with 0.9% saline as a stock solution (5 mg/ml). The solution was brought to a final concentration of 2 mg/ml with 4% DMSO immediately prior to behavioral testing. Mice received intraperitoneal injections of 4% DMSO control vehicle (WT n = 11; Atf6b-/- n = 8) or Antalarmin (10 mg/kg; WT n = 10; Atf6b-/- n = 6) 40–50 min prior to the light/dark transition test.
Immunohistochemistry
Mice (WT and Atf6b-/-) were deeply anesthetized with medetomidine (0.75 mg/kg), midazolam (4.0 mg/kg), and butorphanol (5.0 mg/kg). Mice were transcardially perfused with 4% paraformaldehyde in PBS. The brains were dissected, postfixed overnight at 4°C in 4% paraformaldehyde in PBS, and subsequently immersed overnight at 4°C in PBS containing 30% sucrose. The brain specimens were then embedded in Tissue-Tek OCT compound (Sakura Finetek USA, Torrance, CA, USA) and kept frozen at -80°C until use. Cortical sections (20 µm) were cut on a cryostat (Leica Biosystems, Wetzler, Germany). Sections were processed for immunohistochemistry with rabbit anti-CRT (1:1,000, Proteintech, 10292-1-AP, RRID: AB_513777 or 1:2,000, Enzo Life Sciences, ADI-SPA-600, RRID: AB_10618853) and guinea pig anti-CRH (1:2,000, Peninsula Laboratories, T-5007.0050, RRID: AB_518256). Anti-rabbit Alexa Fluor 488 (1:1,000, Molecular Probes, A-21206, RRID: AB_141708), and anti-guinea pig Alexa Fluor 488 (1:1,000, Molecular Probes, A-11073, RRID: AB_142018) secondary antibodies were used to visualize immunolabeling. Imaging was performed using a laser scanning confocal microscope (Eclipse TE200U, Nikon, Tokyo, Japan) at 20X magnification with Nikon EZ-C1 software.
For quantification of immunohistochemical results, eight serial brain sections were selected per mouse. The intensities of CRT and CRH signals were calculated using ImageJ software (National Institutes of Health). Analyses were conducted blindly.
ELISA
To measure plasma corticosterone concentrations, we used a Corticosterone Enzyme Immunoassay Kit (K014: Arbor Assays, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Blood was collected from the tail veins of WT and Atf6b-/- mice (14–16 weeks of age) under basal conditions, immediately combined with EDTA (1.4 mg/ml), and centrifuged at 1,000 g for 15 min. The supernatant was stored at -80°C until use. The dissociation reagent was allowed to warm completely to room temperature prior to use. Plasma was added to an equal amount of the dissociation reagent, vortexed gently, and incubated at room temperature for 5 min. The mixture was then diluted with assay buffer (1:100). Standard solutions with known corticosterone concentrations and plasma samples were pipetted into wells on a plate. DetectX® corticosterone conjugate was added to each well, and then DetectX® corticosterone antibody was added to each well except for the non-specific binding wells. The plate was covered with a plate sealer and placed on a shaker at room temperature for 1 h. After washing each well four times with wash buffer, the TMB substrate was added, and the plate was incubated at room temperature for 30 min without shaking. Subsequently, stop solution was added to each well, and absorbance values were read at 450 nm in a microplate reader (Multiskan GO, Thermo Fisher Scientific). The corticosterone concentration for each sample was calculated using the microplate reader software (Thermo Fisher Scientific).
Statistical analyses
All data are presented as means ± SEM. In the test between two groups, Kolmogorov-Smirnov test or Shapiro-Wilk test was performed for normality test. Mann-Whitney U-test was performed when there was no normality, Student’s t-test was used when there was normality and equal variances, and Welch’s t-test was used when there was no equal variances. One-way analysis of variance followed by the Tukey-Kramer test were used for multigroup testing. SPSS version 25 was used for statistical analyses, P-values < 0.05 were considered statistically significant.