Mice lacking GPR120 are described previously . The established mixed C57BL/6/129 background GPR120 KO mice were backcrossed into the C57BL/6J background using a marker-assisted breeding approach . Genotyping of the GPR120 KO mice was performed using the primers, Forward: 5′-aagtcaatcgcacccacttc-3′ Reverse: 5′-caagctcagcgtaagcctct-3′. We confirmed that GPR120 gene was knockout in GPR120 KO mice (Additional file 1: Fig. S1A). Male WT C57BL/6J (Tokyo Laboratory Animals Science, Tokyo, Japan) and GPR120 KO mice were maintained on a 12 h/12 h light/dark cycle with free access to a powdered diet (CLEA Japan, Tokyo, Japan) and tap water. 16-weeks old male WT and GPR120 KO mice were used for all experiments.
5-weeks old mice were placed on a powdered diet containing 0.01% indomethacin (Nacalai tesque, Tokyo, Japan) for a total period of 11 weeks for indomethacin treatment studies. For liraglutide studies, 5-weeks old mice were inserted subcutaneously an Alzet osmotic pump (Muromachi, Tokyo, Japan) filled saline dissolved liraglutide (Novo Nordisk, Bagsværd, Danmark) in the abdomen. The pumps delivered saline or 200 mg/kg of liraglutide per day for 11 weeks. Sitagliptin phosphate monohydrate (SPM, 50 mg/kg per day, ApexBio, Boston, MA, USA) was orally administrated to 15-weeks old mice for a week. The dose of liraglutide and SPM was based on previous reports [21, 22].
We used the following antibodies: anti-Ionized calcium binding adapter molecule 1 (Iba-1, 019-19741, Wako, Osaka, Japan), anti-Glial fibrillary acidic protein (GFAP, ab68428, Abcam, Cambridge, MA), anti-cyclooxygenase-1 (COX-1, sc-19998, Santa Cruz Biotechnology, CA), anti-COX-2 (sc-376861, Santa Cruz Biotechnology, CA), anti-L-PGDS (PA1-46023, Thermo Fisher Scientific, Tokyo, Japan), anti-H-PGDS (PA5-24347, Thermo Fisher Scientific, Tokyo, Japan), anti-doublecortin (DCX, ab18723, Abcam, Cambridge, MA), anti-Ki67 (NB500-170, Novus Biologicals, Inc., Littleton, CO), anti-superoxide dismutase 2 (SOD2, 13194, Cell Signaling Technology, Beverly, MA), anti-14-3-3ζ (7413, Cell Signaling Technology, Beverly, MA), anti-synaptophysin (ab32127, Abcam, Cambridge, MA), anti-postsynaptic density protein 95 (PSD95, 610495, BD Biosciences, San Diego, CA), anti-α-tubulin (T5168, Sigma-Aldrich, Deisen-hofen, Germany), and anti-GAPDH (ABS16, Millipore, Billerica, MA).
Mice were intracardially perfused with 4% paraformaldehyde in phosphate buffered saline (PBS). Brains were removed and postfixed overnight in 4% paraformaldehyde in PBS and subsequently cryoprotected in 30% sucrose solution in PBS, snap frozen and stored at -80 °C until required. Coronal brain sections (25 µm thick) were cut on a cryostat (LEICA CM1900, Wetzlar, Germany) and mounted on gelatin-coated glass slides. Nissl staining was performed according to standard protocols. Sections were cover slipped using Poly-Mount (Polysciences Inc. Boston, MA). Fluoro Jade C (FJC) staining was performed according to the manufacturer's instruction [Ready-to-Dilute (RTD) Fluoro-Jade C Staining Kit, Biosensis, CA] . Slides were incubated in sodium hydroxide for 5 min, then washed with 70% EtOH followed by distilled water. Slides were then incubated in potassium permanganate for 10 min. Next, slides were washed with distilled water and moved to low-light for staining with FJC and 4, 6-diamidino-2-phenylindole (DAPI) for 15 min. Slides were rinsed with distilled water, and cleared by brief immersion in xylenes. Slides were then coverslipped using DPX (Merck KGaA, Darmstadt, Germany).
For immunohistochemistry, sections were incubated for 1 h in a blocking buffer (PBS 5% BSA, 0.1% Polyoxyethylene Sorbitan Monolaurate) and incubated with the primary antibody (anti-Ki67), at 4 °C overnight, followed by incubation for 1 h with secondary antibody polymer solution conjugated with anti-rabbit IgG secondary antibodies and horse-radish peroxidase (Envision + System, Dako, Glostrup, Denmark). Ki67 positive cells in dentate gyrus were counted. For immunofluorescence, sections were incubated with the primary antibodies (anti-Iba-1 and anti-DCX) after blocking, at 4 °C overnight, followed by incubation for 1 h with secondary antibody (Cy3-conjugated AffiniPure goat anti-Rabbit IgG; 1 : 500, Jackson ImmunoReseach, inc. PA) in the dark at 25 °C. Sections were cover slipped using DPX. Sections were photographed at 40 × magnification and images were captures using a KEYENCE BZ-X710 microscope (Keyence Corporation, Osaka, Japan). Iba-1 positive cell in the hippocampus, CA1, and CA3, and DCX positive cells in the dentate gyrus were counted, and densities (counts/mm2) calculated.
Measurements of hippocampal and cortical volume
Serial coronal brain slices were cut at a thickness of 25 µm using a cryostat. H&E staining was performed according to standard protocols. Areas of the left hippocampus and cortex (primary somatosensory cortex, motor cortex, and insular cortex) were measured in every 100 µm that contained whole hippocampus and cortex using a KEYENCE BZ-X710 microscope. These area (mm2) × 0.1 (mm) from all sections were summed and recorded as a unilateral hippocampal and cortical volume (mm3) . Relative values of hippocampal and cortical volume were represented as a percentage of average volume of control mice.
Brain tissues and primary cell cultures were homogenized on ice in RIPA [50 mM Tris–HCl pH 8.0, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate (DOC)] buffer containing 1 : 1000 dilution of a protease inhibitor cocktail (CalBiochem, San Diego, CA, USA) with a tissue homogenizer (Brinkmann Instruments, Westbury, NY, USA). Protein concentrations were determined using a BCA protein assay kit (Nacalai tesque, Tokyo, Japan). 10 µg protein/lane of lysates was subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad, Redmond, WA). After blocking with 5% skim milk (MEGMILK SNOW BRAND Co Ltd, Tokyo, Japan) in PBS containing 0.05% Tween 20 (PBS-T), the membranes were incubated with the primary antibodies overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Beverly, MA) and washing with PBS-T three times. The membranes were treated with reagent for exposure (Chemi-Lumi One Super, Nacalai tesque, Tokyo, Japan; ImmunoStar LD, Wako, Japan). Image of the membranes was captured using a C-DiGit blot scanner (LI-COR, Lincoln, NE) and subjected to ImageJ analysis. Each membrane was probed with only 1 antibody, with α-tubulin or GAPDH used as a loading control.
Total lipids were extracted using n-hexane/2-propanol (3:2, by vol, HIP). The HIP was added to hippocampal tissue. Samples were homogenized at maximum speed. The homogenate was centrifuged at 1500 × g for 10 min at room temperature. The supernatant fraction was decanted and being dried down using an integrated SpeedVac® concentrator (SPD111V, Thermo Scientific, Rockford, IL, USA). The fraction was diluted 500 µl in assay buffer. The PGD2, PGE2, and PGF2α concentration was assayed with each EIA kit (Cayman Chemicals, Ann Arbor, MI).
The WT and GPR120 KO mice were fasted overnight. For plasma GLP-1 quantification, blood samples were collected in test tubes containing a sitagliptin (100 µM) and then centrifuged for 20 min at 1,200 × g at 4 °C. Intestinal and intracerebral GLP-1 was extracted according to the method by Cani et al  and McClean et al , respectively. The acid ethanol (75% ethanol + 0.15 mol/L hydrochloric acid) was added to intestinal and brain tissue. Samples were homogenized at maximum speed and placed at 4 °C for 24 h. The homogenate was centrifuged at 5000 × g for 20 min at 4 °C. The supernatant fraction was decanted and being dried down using an integrated SpeedVac® concentrator. The active GLP-1 concentration was assayed with Active GLP-1 ELISA Kit (FUJIFILM, Gunma, Japan). Results were measured in a Benchmark Microplate Reader (Bio-Rad, Redmond, WA).
RNA extraction and quantitative real-time PCR (Q-PCR)
Tissue samples and neuronal and glial primary cell cultures were processed for RNA extraction using ISOGEN (NIPPON GENE, Tokyo, Japan) following the manufacturer's instructions. RNA was reverse transcribed using PrimeScript RT reagent kit (TAKARA BIO INC, Shiga, Japan) reverse transcriptase. Q-PCR was performed using the Quant Studio 12K Flex (Applied Biosystems, CA). The following primer sequences were used: Phosphoglycerate kinase 1 (PGK1; Forward: 5′-tgctgttccaagcatcaaa-3′ Reverse: 5′-gcatcttttcccttcccttc-3′); GPR120 (Forward: 5′-gtcgtctgccacctgctctt-3′ Reverse: 5′-tttctcctatgcggttgggc-3′); NeuN (Forward: 5′-agcagcccaaacgactacat-3′ Reverse: 5′- acaagagagtggtgggaacg-3′); GFAP (Forward: 5′-gcttcctggaacagcaaaac-3′ Reverse: 5′-cggcgatagtcgttagcttc-3′); Iba-1 (Forward: 5′-gaagcgaatgctggagaaac-3′ Reverse: 5′-gaccagttggcctcttgtgt-3′); SOD2 (Forward: 5′-ggccaagggagatgttacaa-3′ Reverse: 5′-gaaccttggactcccacaga-3′); 14-3-3ζ (Forward: 5′-cccattcgtttaggtcttgc-3′ Reverse: 5′-cctgcagcgcttctttattc-3′); COX-1 (Forward: 5′-cagtgcctcaaccccatagt-3′ Reverse: 5′-gtggctatttcctgcagctc-3′); COX-2 (Forward: 5′-ccccaaagatagcatctgga-3′ Reverse: 5′-gctgtacaagcatggcaaa-3′); L-PGDS (Forward: 5′-catagttggccaccact-3′ Reverse: 5′-tccgggagaagaaagctgta-3′); H-PGDS (Forward: 5′-cgaggtgcttgatgtgtgag-3′ Reverse: 5′-tgttttggaggtggaaggac-3′); GLP-1 receptor (Forward: 5′-ccaggttccttcgtgaatgt-3′ Reverse: 5′-caaggcggagaaagaaagtg).
The Y maze apparatus (Hazai-ya, Tokyo, Japan) was a 3-arm radial maze with equal angles between all arms (8 cm width) and a bottom with 40 cm (length) and 15 cm height. Mice were tested individually by placing them in an arm of the maze and allowing them to move freely throughout the 3 different arms for 10 min. The sequence and entries into each arm were recorded. An alternation was determined from successive consecutive entries into the 3 different arms on overlapping triads in which all arms were represented. For example, ACBABCABAB, a sequence of entries to the 3 arms A, B, or C, would generate 5 ‘successful’ alternations, ACB, CBA, ABC, BCA, and CAB; the total number of possible alternations corresponded to the number of the total arm entries minus 2 (in this example, the total number would equal 8). The percentage alternation was calculated as (the number of ‘successful’ alternations divided by the number of the total arm entries minus 2) × 100. We analyzed the percentage alternation and the total number of arm entries.
Morris water maze consisted of circular pool (diameter, 150 cm; height, 40 cm) was divided into four quadrants (north, east, west, and south) and at the center of the north quadrant, a platform was placed. The geometric shapes were pasted at the walls for visual cues. A 10 cm transparent platform was placed 1 cm beneath the surface of the water and 40 cm from the wall in the South-West quadrant of the pool. Mice were placed in a quadrant and given time to find the platform in 90 seconds during the first five days (escape latency). If the animal did not find the platform at the set time, it handler directed to the platform in training. The next five days, the platform was removed, the amount of time the mice spends in proximity to its former location is gauged (known as a probe trial) to assess memory. The mice were allowed 300 seconds to swim in order to evaluate their reference memory (cross-platform time). Mice were video tracked and analyzed behavioral parameters.
Primary cell cultures were separately following the method . Primary neurons were prepared from cerebral cortex of embryonic day 18 mouse embryo. Brains were stripped of meninges and dissected from diencephalon, were dispersed and incubated at 37 °C in Hank’s balanced salt solution (HBSS) containing 0.25% trypsin (Life Technologies, CA) and 0.001% DNase I (Roche Diagnostics, Mannheim, Germany). After inhibiting the trypsin with fetal bovine serum, the suspension was again disrupted with a pipette and filtered through a 70 µm nylon mesh (BD Falcon, MA). The filtered cell suspension was placed in poly-L-lysine-coated 75 cm2 flasks and kept at 37 °C in a humidified incubator with 5% CO2 in air. Neuronal cells were cultured in Neurobasal Medium (Life Technologies, CA) with B27 supplement. After 1 day, the medium was replaced with Neurobasal Medium. The culture medium was subsequently changed twice a week. Cells were harvested after 14 days in vitro.
Primary mixed glial cultures (astrocytes and microglia) were prepared from forebrains of postnatal 2 days old mice using a differential detachment method [26, 27]. Briefly, forebrains free of meninges were digested with HBSS containing 0.25% trypsin and 0.001% DNase I and triturated with DMEM containing 10% heat-inactivated fetal bovine serum and 1% penicillin–streptomycin. Dissociated cells were plated in poly-L-lysine-coated 75 cm2 flasks. The culture medium was changed twice a week. Astrocytes were detached from the 75 cm2 flasks by trypsinization. Individual glial cells were used for the experiments. After 14 days in vitro, when cultures reached a confluence, microglia were isolated by shaking the mixed glia-containing flasks for 1 h at 200 rpm and plated with 500,000 cells/ well in 6-well plates. After resting for 24 h, microglia were stimulated with 100 ng/ml Lipopolysaccharide (LPS, Sigma, Deisen-hofen, Germany) 1 h after pretreatment with 10 ng/ml liraglutide. In the PGD2 addition experiment, microglia were incubated with the medium containing rat recombinant GM-CSF (20 ng/mL, Pepro Tech, London, UK) after plated with 500,000 cells/well. After resting for 24 h, cells stimulated with 1 µM PGD2 (Cayman CHEMICAL, Ann Arbor, MI) 1 h after pretreatment with 1 µΜ MK-0524 (DP1 antagonist, Axon MEDCHEM, Groningen, Netherlands), 1 µM OC459 (DP2 antagonist, Axon MEDCHEM, Groningen, Netherlands). The isolated primary cells with each isolation method were determined by PCR using NeuN, GFAP, and Iba-1 primers (Additional file 1: Fig. S1B).
Two-sample comparisons were carried out using a student’s t-test. Multiple comparisons were performed by one-way ANOVA followed by Newman-Keuls post-hoc test or two-way ANOVA followed by post-hoc Tukey test. Graph Pad Prism Ver. 5.01 (Graph Pad Software, Inc., San Diego, CA) and expressed as mean ± SEM. p values < 0.05 were considered statistically significant.