Local interleukin-18system in the basolateral amygdala regulates susceptibility to chronic stress

Tae-Kyung Kim1, Ji-Eun Kim1, Juli Choi1, Jin-Young Park1, Jung-Eun Lee1, Eun-Hwa Lee1, Yunjin Lee1, Bo Yeon Kim2, and Young J Oh3, #, Pyung-Lim Han1,4,#

Supplemental METHODS

Animals

Male C57BL/6 mice, 7 weeks of age, were purchased from DaehanBioLink (Eumsung, Chungbuk, Republic of Korea). IL18 KO mice [31] and NF-kB (p50) KO mice [32] were obtained from JAX, and werebackcrossed to C57BL/6J for11 and 19 generations, respectively. Genotyping was performed using PCR with thefollowing primer set: 5’-AATTCGCCAATGACAAGACG-3’, 5’-GGCAGCAAGCACTCTTAACC-3’ and 5’-ACAAACCCTCCCCACCTAAC-3’ for IL18KO mice; 5’-GCAAACCTGGGAATACTTCATGTGACTAAG-3’, 5’-ATAGGCAAGGTCAGAATGCACCAGAAGTCC-3’and 5’-AAATGTGTCAGTTTCATAGCCTGAAGAACG-3’ for NF-kB KO mice. Mice were housed with the same sexin a standard plasticcagewith free access to food and waterin a temperature (22 - 23°C)-and humidity (50-60%)-controlled roomat 250 lux with a 12-h light:12-h dark cycle. All animals were handled in accordance with the animal care guidelines of Ewha Womans University(IACUC 15-012).

Restraint and foot-shock stress treatments

Mice were restrained for 2 h dailyfor 14 days as previously described [27, 33].In brief, experimental groups of mice were prepared and housed in pairs for 5 days for acclimation. Mice were individually placed in a 50-ml polypropylene conical tube with holes for ventilation and were restrained within this tubefor 2 h daily. After each session of restraint, mice were returned to their home cages. This procedure was repeated for 14consecutivedays.

Footshock stress treatment was performed as previously described[34, 35] with minor modifications. In brief, the test apparatus consisted of a Plexiglas box (15 cm x15 cm x 45 cm)equipped with a shockgrid floor. Mice wererandomlydividedinto two groups; naïve control and mice treated with electrical foot-shock (shocked mice). All mice in the foot shock regime were individually exposed to 0.8-mA electrical shock 2sec in duration through a shock-grid floor five days before receiving the foot shock regime, and shock responses including jumping, high pitched sounds, and/or saccadic movement responses were monitored. From this foot shock sensitivity test, non-responders were excluded from the experiment. Mice subjected to the footshock regime were placedin pairs in the shockchamber for 5 minforhabituation. Then, mice in the shock chamberwere exposed to 0.8-mAelectrical shocksdelivered for 2sec in duration in30-sec intervals for 60 min through a shockgrid floor.After each 60-min session, shockedmice were returned to their home cages. This footshock treatment was repeated for seven consecutive days. In response to footshocks, mice normally exhibited jumping, high pitched sounds,saccadic movementsand/or freezing behavior.

IL18 KO mice and NF-kB KO mice were evaluated for shock escaping behaviors before being subjected to the footshock regime. An escaping block made of white Foamex PVC board(6.5 x 6.5 x 4.5 cm) was placed in a corner of the shock chamber. In brief, one day before beginning the footshock regime, mice were individually placed on the top of the escaping block for 5 sec, and then placed onto the floor of the shock chamber. After another 5 sec, mice were subjected to a single0.8 mA electrical shock 2sec in duration through theshockgrid floor. Most shocked mice successfully jumped onto the escaping block shortly after saccadic movements, and the success of escaping was recorded.The mice that failed to escape were subjected to a second round of electrical shock 10 sec after the first trial, and successful escapes were recorded.

The stress treatment room was controlled at 250 lux. Restraint and foot shock stresstreatments were started at 10 am. For IL18 and NF-kB KO mice, male and female mice were used as indicated unless indicated otherwise. Other handling procedures and conditions were similar to those described in the behavioral tests section.

Real-time PCR analyses

Quantitative real-time PCR wasperformed as previously described [27]. Total RNA from the amygdala or cell cultures was purified using TRIzol reagent (15596-018, Invitrogen, CA, USA). Isolated RNA was treated with DNase I to remove genomic DNA contamination. Reverse transcription was performedto convert tocDNA using a reverse transcription system (Promega, MO, USA). Real-time PCR was performed with 10 l of 2X iQTM SYBR Green Supermix (Bio-Rad Laboratories, Foster City, CA, USA), 1 l of cDNA (1/8 dilution of the convert) in a volume of 20l with a set of primers using the CFX 96 Real-Time PCR System Detector (Bio-Rad Laboratories). The primer sets used were as follows:5’- ACAACTTTGGCCGACTTCAC-3’ and 5’-GGTCACAGCCAGTCCTCTTA-3’ for IL18, 5’-TGCACAGGAATGAAACAGCC-3’ and 5’-ACGTTCCCTCATCCTCCATC-3’ for IL18R1, 5’-TTGGACCACTGCACTGAAGA-3’ and 5’-CCCAGGGAACCTTTGTAGAAG-3’ for Hcrt, 5’-TGAACGATGATGACAATAAGAA-3’ and 5’-TCAGAGCGAGGTAAGGTT-3’ for MCH, 5’-CCTACAGCGGATCTCAGAC-3’ and 5’-CCGAGGTCAGAGCCAGTA-3’ for OXT, 5’-CGGGTCAGTAGTGTCAAGCT-3’ and 5’-TAGATCCATGGGTTGCAGCA-3’ for OXTR1, 5’-GCTCAACACTACGCTCTC-3’ and 5’-CTTGGGCAGTTCTGGAAG-3’ for AVP, 5’-CCAAGATCCGCACAGTGAAG-3’ and 5’-TGGGAAGCTCTGGACACAAT-3’ for AVPR1a, 5’-CTGGATTCTGGAGCCTTG-3’ and 5’-GTTAGCACGAAGATCAAAGC-3’ for TRH, 5’-CGCAACATCACCCATCTG-3’ and 5’-TCATACCAGCATCGGATACT-3’ for G9a, 5’-AATGGAAATTGCCCGGATCG-3’ and 5’-TCCTGAAGATGCTGCTCCAA-3’ for STAT3, 5’-ACAGAGGCGTGTATTAGGGG -3’ and 5’-GGAGGAAGGCTGTGAACATG-3’ for NF-kB p50,5’-TTGTCTGACATGGGTCTCCA-3’and 5’-GCACTGTCTTCTTTCACCTCTGT-3’ for NF-kB p65, 5’-TCCTGAGCTCCGAGACTTTC-3’ and 5’-CAGGTAGCCGTGGATAGAGG-3’ for IkB, 5’-CTAGTGGTGCCAGCCGAT-3’ and 5’-CTTGACGGCAGAGAGGAGG-3’ for TNFα, 5’-GAAAATTTCCTCTGGTCTTCTGG-3’ and 5’-TCTCTGAAGGACTCTGGCTT-3’ for IL6, 5’-TCAGGCAGGCAGTATCACTC-3’ and 5’-AGCTCATATGGGTCCGACAG-3’ for IL1β,5’-AGAAGGTGGTGAAGCAGGCATC-3’ and 5’-CGAAGGTGGAAGAGTGGGAGTTG-3’ for GAPDH and5’-GCTGCCATCTGTTTTACGG-3’ and 5’-TGACTGGTGCCTGATGAACT-3’ for L32.

Immunohistochemical analyses

Immunohistochemical analyses were performed as previously described [29]. Briefly, isolated brains were perfused with 4% paraformaldehyde via a transcardiac method and post-fixed further in the same solution overnight at 4°C. Brains were coronally cut in 40-μm thickness using a vibratome (Leica VT 1000S; Leica Instruments, Germany). Free-floating sections were reacted with primary antibody, followed by secondary biotinylated antibody; anti-rabbit IgG (BA-1000; 1:200, Vector Laboratories, Burlingame, CA, USA) and anti-goat IgG (BA-9500; 1:200, Vector Laboratories). Signals were visualized using an ABC Elite kit (PK-6200, Vector Laboratories) for DAB staining. For immunofluorescent staining, anti-goat FITC (sc-2024; 1:100, Santa CruzBiotechnology, CA, USA), anti-goat TRITC (sc-2490; 1:100, Santa CruzBiotechnology, Santa Cruz, CA, USA), anti-mouse Dylight488 (DI-2488; 1:1,000, Vector Laboratories), anti-mouse Texas Red (TI-2000; 1:100, Vector Laboratories), anti-rabbit FITC (sc-2012; 1:100; Santa CruzBiotechnology), or anti-rabbit Dylight594 (DI-1094; 1:1,000, Vector Laboratories) was used.Stained images were analyzed using an Olympus BX 51 microscope equipped with a DP71 camera and MetaMorph Microscopy Automation & Image Analysis software (Molecular Devices, Sunnyvale, CA, USA).Quantitative analyses of co-localization signals were performed by manually counting the numbers of cells expressing fluorescence signals in double‐stained BLA sections after capturing stained images. Brain sections were prepared from 6-8 animals that were subjected to the 2h x 14d RST or naïve control mice.

Anti-IL18 (sc-7954, sc-6179), anti-NF-kB (sc-114),anti-TRH (sc-366754), and anti-c-Fos (sc-52) were purchased from Santa Cruz Biotechnology. Anti-IL18R(AF856) was purchased from R&D Systems(Minneapolis, MN, USA). Anti-phospho-STAT3 (9145s), anti-phospho-NF-kB p65 (3033), anti-STAT3 (12640)and anti-GFAP (12389) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-NeuN (MAB377), anti-GAD67 (MAB5406), anti-Hcrt (PC362), anti-oxytocin (AB911) and anti-vasopressin (AB1565) were purchased from Merck Millipore (Billerica, MA, USA).Anti-parvalbumin (P3088) and anti-Glu4 (G9282) were purchased from Sigma-Aldrich (St Louis, MO, USA). Anti-Iba1 (019-19741) was purchased from Wako (Osaka, Japan). Anti-MCH (H-070-47) was purchased from Phoenix Pharmaceuticals (Belmont, CA, USA).The specificity of anti-proIL18, anti-IL18R, anti-phospho-NF-kB p65and anti-phospho-STAT3antibodies was verified using BLA sections of IL18 KO mice for anti-proIL18, and BLA sections with siRNA-mediated knockdown of respective genes.

Stereotaxic injections of siRNA and drugsinto the BLA

Stereotaxic injection of siRNA was performed as previously described [27]. In brief, mice were anesthetized with a mixture (3.5:1) of ketamine hydrochloride (50 mg/ml) and xylazine hydrochloride (23.3 mg/ml) at a dose of 2.5 μl/g body weight. One volume of diluted (50 ng/l) siRNA-control, siRNA-IL18, siRNA-IL18R, siRNA-STAT3,or siRNA-NFkB-p50plus siGLO Green (1/19of siRNA-target gene)was mixed with 2.5 volumes of Neurofect transfection reagent (T800075; Genlantis, San Diego, CA, USA) and 0.5 volumes of 50% sucrose and incubated for 20 min before stereotaxic injection. A total 0.5 lof the mix containing 5.94ng of siRNAwas injected into each BLA(AP, -1.4; ML, ±3.0; DV, -4.8mm) at the speed of 0.2μl/min using a 30-G needle.Injected mice were placed on a warming-pad at 37°C until awakened from anesthesia and returned to home cages in pairs. Between 48 h and 52 h after siRNA injection, behavioral tests were performed.The injection sites were histologically examined for correct anatomical targeting under a microscope using the needle tract or the fluorescence emitted from the tracer siGLO Green, and mice with erroneousinjections were excluded from behavioral analyses.siRNA-control (SN-1012),siRNA-IL18(1372751, NM_008360.1), siRNA-IL18R1 (1372778, NM_008365.1), siRNA-STAT3 (1432038, NM_011486.4) and siRNA-NF-kB(p50) (1391602, NM_008689.1)were purchased from Bioneer Corporation (Deajon, Korea). The FAM-labeled RISK-independent siRNA transfection control siGLO Green (D-001630-01-05) was purchased from Dharmacon Incorporated (Chicago, IL, USA).

Behavioral assessments of emotional states

Behavioral tests of emotional states were performed as described previously [28, 33].The behavior testing room was maintained at 22°C–23°Cwith 50% - 60% humidity and lit with indirect illumination by 20 lux for the sociability test and novelty-suppressed feeding (NSF) test,and 250 lux for the TST and FST. Mice in home cages were placed for 20 min in the behavior testing room prior to the behavioral tests. All behavioral tests were performed during the light cycle (10 A.M. ~ 4 P.M.). The background sound in the testing room was masked with 65 dB of white noise during habituation and behavioral tests. All behavioral tests were conducted in a randomized fashion and/or in an alternative manner with respect to genotype, treatment (e.g., vehicle vs. IL18; con-siRNA vs. target gene-siRNA), and position within testing equipment or field (e.g., left vs. right; between positions in the test room). All animals were housed in pairs, and no bedding change was made the day before behavioral tests. All parts of the apparatus that were exposed to mice after each behavioral test were sprayed with 70% ethanol and cleaned with paper towels.

Sociability test

The sociability test was performed as previously described[27, 33]. In brief, the U-shaped fieldwas prepared by partitioning an open field (40 x 40 cm2) with a partitioningwall (20cm in height) to the central point, so that each U-shaped field had closed and open squares on each side. On test day, each subject mousewas allowed to freely explore the U-shaped field for 5 min, and itslocomotion was recorded.While the subject mouse was returned to its home cage for 2 min, acirculargrid cage (12 cm in diameter x 33 cm in height)loaded with a social target wasplaced in the target zone, and an unanimated grid cage was placed on the other side. Then, the subject mouse wasallowed to explore the U-field for 5min, and the time and trajectory of the subject mouse were recorded. The closed square positioned with a social target was defined as the target-zone, whereas the closed square positioned with an unanimated cagewas defined as the non-targetzone.For each session, the social target was replaced with other mice. The mice that were used as social targets were 2-4 weeks older than subject mice. For the social interaction test of IL18 and NF-kB KO mice, male mice were used.

Sucrose preference test

Sucrose preference test was performed as previously described [27].In brief, mice were housed in pairin home cages and habituated to drinksucrose solution from two 120-ml bottles containing 1% sucrosefor two days.On day 3, the two bottles were replaced with new two 120-ml bottles containing water.On day 4, mice were offered a 1% sucrose solution in a 120ml-bottle versus water in another bottle. Thepositions of the bottles were switched on the next day tocontrol for possible position preference. The amounts of water and sucrose solution intake were measured on day 4 and day 5 (pre-test period). During the footshock stress regime described in the above section, mice were given access to water in two bottles continuously. Twenty-four hours after the last day of footshock, the body weight of each mouse was measured, and then mice were offered 1% sucrose solution in a 120ml-bottle versus water in another. On the next day, thepositions of the bottle were switched tocontrol for position preference, and the body weight of each mouse was measured.Intakes of water and sucrose solution were measured for these two days (post-test period). Sucrose preferencewas calculated as the percentage of sucrose solution consumed relative to the total sucrose plus water ingested per body weight.

Forced swim test

The forced swim test was performed as previously described[28, 33]. Mice were individually placed in a clear Plexiglas cylinder (height: 27 cm, diameter: 15 cm) containing water at 24°C and a depth of 14 cm. Duringthe 6 min test, latency to the first immobility and summed immobility time for the final 5 min were measured. Immobility was defined when animals remained floating and motionless. Behavioral performances in the FST were recorded and analyzed.

Tail suspension test

The tail suspension test was performed as previously described[28, 33].Mice were suspended for 6 min by fixing the tail with adhesive tape to the ceiling of a rectangular box50 cm above the surface of a table. Duringthe 6 min period, latency to the first immobility and the cumulative immobility time were counted.Behavioral performances were recorded and analyzed.

Novelty Suppressed Feeding test

The novelty-suppressed feeding (NSF) test was performed as previously described [36]. Briefly, mice were deprived of food but not water in home cages for 24 h. On the test day, subject mice were individually placed in a corner of the open field (45 x 45 x 40 cm) with a single 2 x 2.5 cm food pellet placed on round filter paper (15 cm in diameter) in the center the open field. The latency to first contact withthe food pellet was recorded. Immediately after the mouse began to eat, the subject animal was placed in its home cage with a single food pelletfor 5 min, and the amount of food consumed was determined.

Statistical analyses

Two-sample comparisons were performed using Student’s t-test, and multiple comparisons were made using one-way or two-way ANOVA followed by the Newman-Keuls multiple comparison test. GraphpadPrism 6 software (GraphPad Software. Inc., CA, USA) was used to perform statistical analyses.All data are presented as means ± S.E.M. and statistical difference was accepted at the 5% level unless otherwise indicated.

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