Animals

Adult Sprague Dawley rats were procured from Charles River Laboratories. All animals were kept under standard lighting conditions (12 h lights on; 12 h lights off) and fed with rodent chow and water ad libitum. Adult animals were bred to generate neonatal animals, which were used in this study. Animal care and treatment were performed in accordance with institutional guidelines, and protocols were approved by the Rutgers Institutional Animal Care and Facilities Committee (approval number 999900286) and complied with National Institutes of Health policy.

In-vivo studies

Postnatal rat pups (PND2; both sexes) were fed by gavage a milk formula containing 11.34% ethanol (v/v; 0.1–0.2 ml/animal; during a period of 1 min), yielding a total daily ethanol dose of 2.5 g/kg (AF), or isocaloric control (PF), or they were left in the litter with their mother (AD) as described by us previously [5, 18, 24]. Gavage feeding was conducted at 10:00 A.M. and 12:00 P.M. from PND2 to PND6. After feeding, these pups were immediately returned to the litter. RS504393 (Abcam, USA) treatment was given subcutaneously (1 mg/kg; 1 h before the first feeding) daily between PND2-6 [11]. Two hours after the last feeding on PND6, pups (both sexes) were sacrificed and brain tissues were collected and fixed with 4% PFA (2 h), cryoprotected in 30% sucrose (overnight), and cut into 30 μm coronal sections for immunohistochemistry. Another set of pups (with and without RS504393 treatment) were kept with the litters until PND21, weaned, and kept in sex specific cages until PND60. These animals were then used for stress response followed by behavioral studies. Following the behavioral studies these animals were transcardially perfused with ice cold 1X PBS, brains were collected, fixed, cryo-protected in 30% sucrose and cut into 30 μm coronal sections for immunohistochemistry.

Primary microglia culture

Microglia cell culture was prepared from hypothalami of PND 2 male and female rat pups using the method published by us previously [4, 24]. Cells were maintained in DMEM/F12 media with 10%FBS and an antibiotic solution (100 U/ml penicillin, 100 mg/ml streptomycin, and 250 ng/ml amphotericin B). After 12 days, a rotary shaker was used to suspend the cells (175 rpm for 2 h), and the suspended cells (1 × 106 cells/flask) were then plated on poly-ornithine coated T25 flask and fed with DMEM/F12 media with 10% FBS. The purity of the microglial cell culture was confirmed by IBA-1 + staining. Prior to alcohol treatment, microglial cells were fed with DMEM/F12 containing serum supplement (30 nM selenium, 20 nM progesterone, 1 µM iron-free human transferrin, 100 µM putrescine, and 5 µg/ml insulin). To assess the effect of acute alcohol treatment on microglia cell cultures, microglia cells were treated with 50 mM ethanol (ETOH) for 24 h. The conditioning medium was harvested for exosome isolation.

Primary POMC neuronal culture

Primary cultures of POMC (β-endorphin) neurons were prepared using the methods described by us previously [18, 25]. Pregnant rats of the Sprague Dawley strain at 16–18 d of gestation were sacrificed, and the fetuses were removed by aseptic surgical procedure. Hypothalami from the brains of these fetuses were dissected out and placed in ice-cold Dulbecco′s Modified Eagle′s Medium - high glucose (HDMEM) media containing an antibiotic solution, 0.1% BSA, HEPES (15 mM) and Sodium Bicarbonate (44 mM) (all from Sigma Millipore). The hypothalamic were washed with HDMEM media at room temperature and mechanically dissociated with 18-gauge sterile needle 3–4 times and strained through 70 µM nylon mesh. The cell suspension was seeded to poly-ornithine (100 µg/ml) coated T-75 flask in HDMEM media with 10% FBS. Media was changed after 48 h, and flask was further incubated for 24 h at 37oC with 7.5% CO2. On day 4, the medium was replaced with HDMEM-containing 10% FBS, 33.6 mg/ml uridine (Sigma Millipore) to reduce the growth of glial cells. On day 5, the culture medium was replaced with HDMEM containing serum supplement with antibiotics. Cells were maintained for the next 2 d with this medium. Cells were passaged three times until the third week when neurosheres started to develop. Cells were then trypsinized and cultured for two weeks in neuroshere formation media (DMEM-F-12, LIF, 0.1 mg/ml; L-glutamine, 10 mM; rat bFGF, 20 ng/ml; MEM amino acid solution, 0.5%; all the chemicals were from Sigma Millipore, except bFGF, which was obtained from R&D Systems). These neurospheres were then differentiated by treating them for two weeks with pituitary adenylate cyclase activating peptide (10 µM; SynPep) and dibutyryl cAMP (10 µM; Sigma Millipore). The differentiated cells were stained for POMC-derived peptide β-endorphin.

Exosome isolation from primary microglial cell culture medium

Exosome isolation from microglial cells was carried out using a procedure described by us previously [18]. Microglial cell culture media was collected after the treatment and centrifuged at 2000 g for 30 min to remove cells and debris. The supernatant containing the cell-free culture media was then transferred to a new tube and passed through a 0.2 μm syringe filter. Cell-free culture media were further centrifuged again at 300 g and 3000 g for 10 min and then at 10,000 g for 30 min. The exosome isolation reagent (500 ul) from Invitrogen (Catalog #4478359) was then added to 1 ml of cell-free culture media. This mixture was thoroughly mixed by vertexing and pipetting until became a homogeneous solution. The tubes were then incubated at 4 °C overnight. Next day, the tubes were centrifuged at 10,000 g for 1 h at 4 °C. The supernatant was discarded from the tube. The exosomes pellet was re-suspended in 100 µl of 1XPBS and used for biochemical measurements.

Exosome preparation from mediobasal hypothalamus (MBH) microglia

Microglial cells were prepared from mediobasal hypothalami of three neonatal pups using Optiprep density gradient method [24, 26]. The purified microglia were resuspended in DMEM/F12 media (1 ml) and gentle triturated multiple times. Gentle trituration of purified microglia release exosomes in the extracellular space. Cells were pelleted by centrifugation at 2000 g for 15 min and the supernatant was used for the exosome isolation as described previously [18].

Nanosight tracking analysis (NTA) of exosomes

The Nanosight LM10 was used to analyze the size distribution and concentration of exosomes as described previously [18]. The exosome samples were diluted at 1/1000 with PBS and were injected in the sample chamber with 1 ml sterile syringes (BD, USA) until the liquid reached the tip of the nozzle. This unique technology utilizes the properties of both light scattering and Brownian motion to obtain the size distribution and concentration measurement of particles in liquid suspension. A laser beam is passed through the sample chamber, and the particles in suspension in the path of this beam scatter light in such a manner that they can easily be visualized via a 20 X magnification microscope onto which a camera is mounted. The camera operates at 30 frames per second, capturing a video file of the particles moving under Brownian motion. The software tracks many particles individually and, using the Stokes-Einstein equation, calculates their hydrodynamic diameters. All measurements were performed at room temperature.

Western blot analysis for MCPIP1 protein level

The exosomes lysate (20 µg) and β-endorphin neuronal lysate (25 µg) samples prepared in RIPA buffer (Sigma) were separated by 4-20% SDS-PAGE and transferred overnight to immobilon-P PVDF membranes. Membranes were blocked with 5% milk for 1 h and incubated with primary antibody for Tsg101 (1:1000; ab125011, Abcam), Alix (1:1000; ab275377, Abcam), Iba1 (1:1000; ab178846, Abcam), Cytochrome C (1:1000, A4912, ABclonal Technology), Calnexin (1:1000, PA5-34754, Invitrogen), GM130 (1:1000, A11408, ABclonal Technology) and MCPIP1 (1:1000,ab97910, Abcam) for 18 h at 4 °C in blocking buffer. Membranes were then washed and incubated with peroxidase-conjugated secondary antibody (1: 5000) for 1 h. Afterward membranes were washed and then incubated with ECL Western blot chemiluminescence reagent (Pierce). The membrane was imaged with iBright 1500 (Invitrogen).

Transmission electron microscopy

Exosome samples were prepared for inspection as described previously [18]Briefly, exosomes were mounted on copper grids (FCF400-Cu-50 Formvar/Carbon 400 Mesh, Cu from Electron Microscopy Sciences) and then fixed by 1% glutaraldehyde in cold DPBS for 5 min to stabilize the immunoreaction. It was then washed in sterile distilled water and contrasted with uranyl-oxalate solution at pH 7 for 5 min. Finally, it was embedded by methyl cellulose-UA for 10 min on ice. A JEOL 1010 TEM was used to image exosome samples at a voltage of 80 kV.

Chemokine multiplex immunoassay

Exosome pellet were resuspended in ProcartaPlex™ Cell Lysis Buffer (EPX-99999-000) and used for ProcartaPlex multiplex immunoassay using Luminex® xMAP® (multianalyte profiling) technology. The Rat Chemokine 8-Plex ProcartaPlex Panel (EPX080-30121-901) enables the study of the immune response by analyzing 8 protein targets in a single well. Protocols described by the manufacturer were followed and data were analyzed with the LuminexTM instrument (MAGPIX® xPONENT®, Life Technologies Corporation, Grand Island, New York, NY, USA). Median fluorescence index (MFI) and presented.

MCP1 quantification with ELISA

Rat JE/MCP-1 DuoSet ELISA (R&D Biosystems, Minneapolis, MN, USA) was used to estimate MCP1 levels in exosome lysate prepared from primary microglia cells and microglia isolated from MBH tissue. Briefly, exosome pellet was lysed in ProcartaPlex™ Cell Lysis Buffer (EPX-99999-000) and supernatant (20 µl) was used for ELISA analysis for MCP1 as per the manufacturer instructions.

Nucleosome assay for neuronal apoptosis

β-Endorphin cells were cultured (1 × 104/well) in a 24-well plate in a neuronal medium. The cells were then exposed to medium containing exosomes (∼ 1 × 107 particles/well) from microglia (treated with or without 50 mM ethanol +/- 10 µM RS504393) for a period of 24 h. For in-vitro study, we evaluated toxicity of RS504393 on β -endorphin neurons for 10 µM, 20 µM, 50 µM and 100 µM (data not provided). We found that 10 µM of RS504393 cause no toxicity and death of β -endorphin neurons and therefore selected 10 µM of RS504393 as dose for in-vitro studies. The cells were then lysed with nucleosome lysis buffer and used for nucleosome assay using the ELISA kit (Sigma Millipore, catalog #11774425001) for determination of β-endorphin neuronal apoptosis.

qPCR

Real-time RT-PCR was carried out in 384-well plates in a final volume of 10 µL with 1:20 dilution of cDNA (25 ng/µL) in Power SYBR® Green PCR Master Mix (Applied Biosystems™) using the comparative CT (ΔΔCT) standard run on a the QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems™, Wilmington, DL). The primers were designed using Primer 3 (https://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). Data were analyzed using QuantStudio™ Real-Time PCR (Applied Biosystems™, Wilmington, DL) software to calculate relative quantification (RQ values). The primers used are listed in Supplementary Table S1; three genes were used as housekeeping control genes: GAPDH, ATCB, and HPRT1.

Immunofluorescence for β-endorphin of primary neuronal cultures

Neurosheres (1 × 104) were seeded and differentiated to β-endorphin neurons in 8-well chamber slides. Two weeks after differentiation, neurons were added with exosomes (∼ 1 × 107 particles/well) isolated from microglia and activated with or without ethanol or vehicle and with and without pre-treatment (3 h prior to exosome addition) with RS504393 (10 μm/ml) for a period of 24 h. The cells were then washed twice with 1X PBS and fixed for 15 min with 4% paraformaldehyde, washed and permeabilized for 10 min with 1X PBS with 0.2% Triton X-100, washed and blocked for 1 h at room temperature with 5% BSA in 1X PBS with 0.05% Tween-20. Cells were then incubated overnight at 4°C with the rabbit anti-β-endorphin in 1% BSA in PBS-T (0.05% Tween-20) (1:1000; Peninsula Laboratories). Next days, cells were washed three times with 1X PBS and incubated for 1 h with anti-rabbit Alexa-488 secondary antibody (1:500, Abcam). Cells were then with DAPI (1:1000) for 5 min, washed with 1X PBS and mounted with ProLong™ Gold Antifade Mountant (Thermofisher Scientific, USA). Fluorescent images were captured with EVOS M5000 Imaging System (Invitrogen, USA). Fluorescent intensity was measured using ImageJ software (National Institutes of Health).

Immunohistochemistry localization of β-endorphin neurons in tissue sections

Serial coronal sections of OCT embedded brains (PND6 and PND60-PND80) were made using a Leica cryostat at 30 μm in thickness from stereotaxic plates 19 to plates 23 (bregma-2.3 to -4.3 mm) spanning the arcuate nucleus. These sections were mounted on Superfrost Plus glass slides (VWR). The sections were washed in PBS twice followed incubation in blocking buffer (2.5% normal horse serum in PBS with 0.4% Triton X-100) at room temperature for 1 h. The sections were subsequently washed in PBST (0.05% Triton X-100) and incubated overnight at 4 °C with the rabbit anti-β-endorphin (1:1000; Peninsula Laboratories). After the primary antibody incubation, samples were washed in PBST, and then sections were incubated with an AlexaFluor-488 donkey anti-rabbit secondary antibody (1:500; Thermo Fisher Scientific). Sections were stained with DAPI (1:1000) for 5 min, washed with 1XPBS and mounted with ProLong™ Gold Antifade Mountant. These sections were then imaged with EVOS M5000 Imaging System. Cell counting was performed using ImageJ software (National Institutes of Health).

Corticosterone response to restraint stress

Corticosterone response to restraint stress at various time points were determined in male and female rats (on day of diestrus) at PND 60–80. Animals were restrained by placing them in a transparent plexiglass restrainer for 1 h (between 12:00 and 14:00 h). Time-dependent changes of cortisol before (0 min), during (30 min, 60 min), and after restraint (120 min) were determined. Female rats were restrained on the day of diestrus to avoid the influence of the fluctuating levels of steroid on the corticosterone response. Plasma corticosterone levels were measured using corticosterone ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA) following manufacturer’s instructions.

Elevated plus maze

Elevated plus maze (EPM) test was conducted to assess anxiety-related behaviors as described previously [27, 28]. The EPM consisted of four arms (50 cm L × 10 cm W), two arms closed by non-transparent walls (∼ 40 cm high) and two open arms without walls that were joined by a central square (10 cm × 10 cm). Rats were transported to the behavioral testing room under cover 1 h prior to testing to allow acclimation to the testing environment. Rats placed on the junction of the open and closed arms were allowed to freely explore the maze for 300 s. The time spent in the open and closed arms was recorded using ANY-Maze video tracking software. Proper care was taken to avoid any sudden noise or disturbance. The apparatus was cleaned with 75% ethanol after each trial.

Open field test

Open field test was conducted to determine exploratory and anxiety-related behaviors in the adult rats as described by us previously [28]. The open field comprises of a black plastic board measuring 90 cm (L) × 90 cm (W) × 40 cm (H) surrounded by black plastic walls with a height of 50 cm. Rats were transported to the room under cover 1 h prior to testing to allow acclimation to the testing environment. The rats were placed in the arena and allowed to explore it for 300 s. The movements of animals were recorded by ANY-Maze video tracking software and stored on a computer. Proper care was taken to avoid any sudden noise or disturbance. The apparatus was cleaned with 75% ethanol after each trial.

Statistical analysis

Results are expressed as mean ± SEM (n = 6–8). t-test was used to analyze the differences between two groups, and one-way ANOVA with Newman Keuls post hoc analysis was used to analyze the differences between multiple groups. Two-way analysis of variance with Tukey’s multiple comparison post hoc test was used when two variables were compared. p < 0.05 was considered significant. Data were analyzed using Prism 9.0 (GraphPad Software).