C57BL/6 (wild type) mice (8 weeks old) purchased from the Animal Core Facility of Nanjing Medical University were used for behavioral test. NLRP3 KO mice were a gift from Qiulun Lv’s lab at Nanjing Medical University (Nanjing, China). Animals were housed individually, under a 12-h light:dark cycle, with lights on at 7:00 a.m. Food and water were provided ad libitum. Mouse body weight (BW) was recorded weekly. All animal care and experimental procedures were approved by Nanjing Medical University Institutional Animal Care and Use Committee (IACUC-2103028) and were conducted in accordance with the Chinese National Institute of Guiding Principles for the Care and Use of Laboratory Animals.
MCC950 was purchased from Selleck (Selleck, USA). Polyclonal rabbit Caspase-1 (1:1000 dilution; 22915-1-AP, Proteintech), polyclonal mouse NLRP3 (1:1000 dilution; AG-20B-0014, Adipogen), monoclonal mouse GAPDH (1:10,000 dilution; 60004-1-lg, Proteintech). The blots were developed with horseradish peroxidase-conjugated secondary antibodies (anti-mouse IgG, 1:10,000 dilution, 7076S, CST; anti-rabbit IgG, 1:10,000 dilution, 7074S, CST). rAAV-hSyn-ChrimsonR-tdTomato-WPRE-hGH pA (4.93 × 1012 vg/mL) and rAAV-hSyn-tdTomato-WPRE-hGH pA (5.55 × 1012 vg/mL) were purchased from BrainVTA (Shumi Company, China). Nissl staining kits were purchased from Solarbio (Solarbio, China). ELISA kits were purchased from Meimian (Meimian, China). The other reagents were obtained from Sigma.
Drinking in the dark
Mice were weighed on the first day of each week that a new fluid was offered. In session 1, on Days 1, 2, and 3, the water bottle was replaced with a tube containing 20% alcohol in tap water (v/v) and volume was recorded in the dark cycle. 2 h later, the volume consumed was recorded and the alcohol tube was replaced with the water bottle. On Day 4, mice were offered alcohol for 4 h. Mice then had free access to water for the next 3 days. In session 2 and session 3, the same procedure was followed.
2 h/4 h binge drinking
2 h/4 h binge drinking was performed in dark cycle. The water bottle was replaced with a tube containing 20% alcohol in tap water (v/v). The volume consumed was recorded for 2 h and 4 h drinking during the dark cycle.
Two-bottle choice for 2 h/4 h/24 h voluntary drinking
Eight-week-old male mice were single-housed under a 12:12 h light-dark cycle. Mice were given 2 h, 4 h, and 24 h voluntary drinking at 2 and 4 days into withdrawal. 24 h drinking was determined similarly as a two-bottle choice drinking paradigm. On the test day, mice were singly housed and given access to one bottle of 20% v/v ethanol and one bottle of water. The bottles were weighed before and after the test, respectively, to record the water and alcohol intake. The mice were weighed at the beginning. The position (left or right) of each solution was alternated as a control for side preference. 2 h/4–h voluntary drinking was performed in light cycle.
Mice were administered 25% alcohol for 3 days directly to the stomach via oral gavage at a dose of 5 g/kg after DID training. Control mice were treated with water.
Open field test
The open field test was used to evaluate the level of anxiety in binge drinking model mice. The open field consisted of a box (40 × 40 × 40 cm); the mice were placed in the middle of the box at the beginning of the test. Then, their behavior was recorded on video for 15 min. The box was cleaned with 75% alcohol and dried between each experiment to remove odor. Recording and analysis of behavior was performed using Trackermaster software (Zongshi, Beijing, China).
Elevated plus maze
The elevated plus maze (EPM) comprised two open arms (30 × 5 cm), and two closed arms (30 × 5 × 25 cm) extending from the intersection zone (5 × 5 cm) and elevated to a height of 50 cm. Animals were transported to and habituated for 3 h in a preparation room. Animals were individually transported to the plus maze and placed on the center of the platform facing an open arm then allowed to freely explore the maze for 5 min. The plus maze was thoroughly cleaned with 75% alcohol and dried between each experiment to remove odor. Recording and analysis of behavior were performed using Trackermaster software (Zongshi, Beijing, China). Parameters scored manually from video recordings included: number of closed arm entries, number of open-arm entries, time spent on the open arm, time spent on the closed arm and velocity. For these parameters, an arm entry was recorded when all 4 paws had entered a single arm. Open arm entries expressed as a percentage of total entries, and the time spent on the open arms expressed as a percentage of total time spent on either the open or closed arms were used as measures of anxiety.
Following the behavior test, brain tissue was removed after killing and preserved in 4% formalin overnight before paraffin embedding. The striatum and the mPFC serial sections from each group were cut, mounted on plexiglass, and single-immunostained using cresyl fast violet for the histochemical demonstration of Nissl substances. For analysis, the minimal pixel was set at 50 pixels, and the average optical density values of Nissl bodies were analyzed. To calculate the relative optical density, the optical density of the other groups was divided by that of the WT group.
Enzyme-linked immunosorbent assay
First, we extracted the mPFC and striatum from the mice brain tissue after the behavior test (Fig. 3) and after the last alcohol exposure (Fig. 6). Then, the mPFC and striatum were centrifuged at 12,000 r/min for 15 min and the clear supernatant was collected. Levels of IL-1β and TNF-α were detected using commercially available ELISA kits.
The brains were removed, and the mPFC and striatum were carefully dissected. The proteins were separated by SDS-polyacrylamide gel electrophoresis, probed with antibodies and visualized by an enhanced chemiluminescence substrate system (Tanon, China). The protein bands were quantitatively analyzed by Image J.
Stereotaxic viral infusions were performed as follows. Mice were anesthetized using isoflurane and mounted in a rodent stereotaxic frame. The skin was opened to uncover the skull and expose Bregma and Lambda, and the location of the desired injection site. Small drill holes were made in the skull at the appropriate coordinates, according to the Paxinos atlas . A microinjector was loaded with 0.4 µL of virus, and then lowered into the mPFC (AP: 1.94 mm, ML: ± 0.40 mm, DV: − 2.20 mm). This virus was infused into the brain at a rate of 0.08 µL/min. To avoid backflow of the virus, microinjectors were left in place for 10 min after the infusion was complete. After virus injections, bilateral optical fiber implants (300-μm core fiber secured to a 1.25-cm ceramic ferrule with 3 mm of fiber extending past the end of the ferrule) were lowered into the striatum right on the top of virus injection sites. Coordinates: AP, 1.00 mm; ML, ± 1.80 mm; and DV, − 3.00 mm. Implants were secured on the skull using metal screws and dental cement (Henry Schein) and covered with denture acrylic (Lang Dental). The incision was closed around the head cap and the skin Vet-bonded to the head cap. Mice were monitored for 1 week or until they resumed normal activity.
In vivo LTP and LTD induction and in vitro optogenetic electrophysiology
One week after stereotaxic viral injection, mice were treated with 3 weeks of DID and 3 days of gavage alochol drinking. In vivo optogenetic stimulation were performed after 24 h withdrawal from DID + gavage model. An LTP/LTD-inducing protocol was delivered by optogenetic stimulation system (Thinker Tech Nanjing Biotech, China) 30 min before the open field test. LTP induction using the following protocol: 100 pulses at 50 Hz of 590-nm light (2 ms, 3 mW), repeated four times with 18-s intervals. The protocol was repeated three times with 5-min intervals. LTD induction employed the following protocol: 900 pulses at 1 Hz of 590-nm light (2 ms, 3 mW).
After behaviral tests, mice were killed and the brain slices from the striatum were prepared for in vitro optogenetic electrophysiology. For optogenetic stimualtion, light stimulation (Thorlabs at 625 nm, 2 mW) through the objective lens evoked glutamatergic transmission from cortex to striatum (mPFC-striatum). The paired-pulse ratios (PPRs) of AMPAR-mediated excitatory postsynaptic currents (EPSCs) were obtained using optogenetic stimulation at an interval of 100 ms. For measurement of the NMDAR/AMPAR ratio, the peak currents of AMPAR-mediated EPSCs were measured at a holding potential of − 70 mV and the NMDAR-mediated EPSCs were estimated as the EPSCs at + 40 mV, 50 ms after the peak AMPAR-EPSCs. The NMDA/AMPA ratio was calculated by dividing the NMDAR-EPSC by AMPAR-EPSC. The input–output relationships for AMPAR-EPSCs were measured at 5 different stimulating intensities. All the experiments were conducted in the presence of the GABAA receptor antagonist, bicuculline (10 µM).
The histology procedure was performed as follows. Mice were anesthetized and perfused intracardially with 4% paraformaldehyde in phosphate-buffered saline (PBS). Whole brains were taken out and put into 4% paraformaldehyde in PBS for post-fixation overnight (4 °C), then placed to 30% sucrose in PBS (4 °C) and allowed to sink to the bottom of the container before preparing for sectioning. Frozen brains were cut into 30-μm coronal sections on a cryostat. A fluorescence microscope (Nikon, Japan) was used to image these sections with a 590-nm laser (to excite tdT). All images were processed using Image J.
Slice electrophysiology was performed as previously described [19, 24, 25]. Animals were anesthetized with isoflurane and killed after their last alcohol (or control water) consumption. 250-µm coronal sections containing the striatum were prepared in an ice-cold cutting solution containing (in mM): 40 NaCl, 148.5 sucrose, 4 KCl, 1.25 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 10 glucose, 1 sodium ascorbate, 3 sodium pyruvate, and 3 myoinositol, saturated with 95% O2 and 5% CO2. Slices were then incubated in a 1:1 mixture of cutting solution and external solution at 32 °C for 45 min. The external solution contained the following (in mM): 125 NaCl, 4.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 15 sucrose, and 15 glucose, saturated with 95% O2 and 5% CO2. Slices were then maintained in an external solution at room temperature until use.
Slices were perfused with the external solution at a flow rate of 3–4 mL/min at 32 °C. The striatal neurons, mainly the DMSs, were identified and patched. The data were recorded using an IPA-2 integrated patch amplifier controlled with SutterPatch software (Sutter Instrument, Novato, CA, USA). For whole-cell voltage-clamp recordings, we used a Cs-based solution, containing (in mM): 119 CsMeSO4, 8 TEA.Cl, 15 HEPES, 0.6 EGTA, 0.3 Na3GTP, 4 MgATP, 5 QX-314.Cl, 7 phosphocreatine. The pH was adjusted to 7.3 with CsOH, with an osmolarity of 270–280 mOsm.
For electrical stimulation, bipolar stimulating electrodes were positioned 100–150 μm away from the recording neurons to elicit glutamatergic transmission in striatal neurons. The PPRs of AMPAR-mediated EPSCs were obtained using two electrical stimuli at an interval of 50 ms. All the experiments were conducted in the presence of the GABAA receptor antagonist, bicuculline (10 µM).
MCC950, an NLRP3 inflammasome inhibitor, at 10 mg/kg was given to WT + alcohol mice on days 19–21, 0.9% saline was used for other groups through intraperitoneal injection.
All data are expressed as the mean ± SEM. Statistical significance was assessed using the unpaired or paired t test, one-way ANOVA, and two-way RM ANOVA followed by Student–Newman–Keuls (SNK). Statistical significance was set at p < 0.05.