Animals
C57BL/6 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 (permit no. SYXK2020-0022) and were conducted in accordance with the Chinese National Institute of Guiding Principles for the Care and Use of Laboratory Animals.
Behavioral Procedures
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, starting 3 h after lights off, the water bottle was replaced with a tube containing 20% alcohol in tap water (v/v) and volume was recorded. Two hr 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.
Two bottle choice for 24h voluntary drinking
8 weeks old male mice were single-housed under a 12:12 h light-dark cycle. Mice were given 24h drinking 3 days after 2/4h drinking. 24h drinking was determined samely 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. The possible loss of solutions due to the handling of the bottles, was controlled by weighing bottles in empty cages.
Gavage
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 by 0.9% saline [22].
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), 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) [23].
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[24]. Animals were transported to and habituated for 3 h in a preparation room. Animals were individually trans ported to the plus maze and placed on the central platform facing an open arm then allowed to freely explore the maze for 5 min[25]. The plus maze was thoroughly 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). 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 parame ters, an arm entry occurred when all 4 paws were presentin 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.
Nissl’s staining
Following the behavior test, brain tissue was removed after sacrifice and preserved in 4% formalin overnight before paraffin embedding. The Striatum and the Medial prefrontal cortex serial sections from each group were cut, mounted on plexiglass, and single-immunostained using Cresyl fast violet (CFV) for the histochemical demonstration of Nissl substances [26].
Enzyme-linked immunosorbent assay
First, we extracted mPFC and striatum from the mice brain tissue. Then, the mPFC and striatum were centrifuged at 12000 r/min for 15min and the clear supernatant was collected. Levels of IL-1β and TNF-α were detected using commercially available ELISA kits (Meimian, Jiangsu, China).
Western blots
The brains were removed, and mPFC and striatum were carefully dissected. The proteins were separated by SDS-polyacrylamide gel electrophoresis and probed with antibodies against polyclonal rabbit Caspase-1 (1:1000 dilution; 22915-1-AP, Proteintech), polyclonal mouse NLRP3 (1:1000 dilution; AG-20B-0014, Adipogen), monoclonal mouse GAPDH (1:10000 dilution; 60004-1-lg, Proteintech). The blots were developed with horseradish peroxidase-conjugated secondary antibodies (Anti-mouse IgG, 1:10000 dilution, 7076S, CST; Anti-rabbit IgG, 1:10000 dilution, 7074S, CST) and visualized by an enhanced chemiluminescence substrate system (Tanon, China). The protein bands were quantitatively analyzed by Image J.
Stereotaxic surgery and in vivo LTP and LTD induction
The adeno-associated virus ( rAAV-hSyn-ChrimsonR-tdtomato-WPRE-hGH pA ) was purchased from BrainVTA (Sumi Company, Wuhan). Bilateral optical fiber implants were purchased from Thinker Tech Nanjing Biotech.
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 [27]. A microinjector was loaded with 0.4 µL of rAAV-hSyn-ChrimsonR-tdtomato, 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.
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), 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).
Histology
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.
Electrophysiology
Slice electrophysiology was performed as previously described[18, 28]. Animals were anesthetized with isoflurane and sacrificed 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. For optogenetic stimualtion, light stimulation (Thorlabs at 625 nm ) through the objective lens evoked striatal glutamatergic transmission from mPFC to striatum. The paired-pulse ratios (PPRs) of AMPAR-mediated excitatory postsynaptic currents (EPSCs) were obtained using two electrical stimuli at an interval of 50 ms, while 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).
Statistical analysis
All data are expressed as the mean ± SEM. Statistical significance was assessed using the unpaired or paired t test, one-way RM ANOVA, and two-way RM ANOVA followed by Student-Newman-Keuls (SNK). Statistical significance was set at p < 0.05.