Cell culture

Primary hippocampal neurons were prepared as previously described by Niere et al.43. Briefly, hippocampi were extracted from postnatal day 1–3 Sprague–Dawley rat pups, WT C57BL/6 mouse pups, or Fmr1-knockout (Fmr1 KO) mouse pups on a C57BL/6 background. The tissue was dissociated and plated in neurobasal A medium supplemented with B27, glutamine, and 1% fetal bovine serum. Cultures were plated at a density of ∼100,000 cells per 12 mm on glass coverslips that had been coated overnight with 50 μg ml−1 poly-D-lysine and 25 μg ml−1 laminin in borate buffer. Cultures were fed after 1 day in vitro (DIV), and media was replaced approximately once a week with either fresh rat culture media (neurobasal A supplemented with B27, glutamine and 3 μM AraC) or fresh mouse culture media (glial-conditioned media with 3 μM AraC) until cultures were used at DIV 14–21.

In vitro pharmacology

Primary hippocampal neurons were treated in ethanol vapour chambers according to a method adapted from Chandler et al.59. Ethanol vapour chambers were prepared by placing a reservoir of 31.5 mM ethanol (105% of the desired ethanol concentration, that is, 30 mM) in a plastic container with 24-well culture plates containing neuronal cultures in which 30 mM ethanol was added to the culture media. Chambers were filled with 95% O 2 /5% CO 2 and cultures were incubated for 2 h at 37 °C. Cultures treated with vehicle (H 2 O) were incubated in the same manner but in the absence of ethanol. For calcium imaging, ethanol was added directly to HEPES-based artificial cerebral spinal fluid (ACSF in mM: 100 NaCl, 10 HEPES (pH 7.4), 3 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 glucose) that was adjusted to match the osmolarity of cell culture media for live imaging. For GABA B R activation neurons were treated with (R)-baclofen (50 μM, Tocris). For Figs 5 and 6, cultured hippocampal neurons were pre-treated with cycloheximide (50 μM, Tocris) for 10 min before ethanol treatment. For Supplementary Fig. 3, neurons were treated with Ro-25-6981 (10 μM, Tocris) or Veh (H 2 O) for 2 h. All cultures were treated at 14–21 DIV. Following treatment, cultures were immediately fixed or live imaged.

In vivo pharmacology

Male C57BL/6 mice (Charles Rivers) or Fmr1-knockout (Fmr1 KO) mice on a C57BL/6 background (at least 7 weeks old) were given i.p. injections of either 200 μl saline or 2.5 g kg−1 ethanol (in a volume of 200 μl saline)23. For Fig. 8, CGP-35348 (100 mg kg−1) was i.p. injected with or without ethanol (in a volume of 200 μl saline). All animals were housed four mice per cage according to genotype. All treatments were administered to one mouse per home cage. At the time of drug treatment, animals were coded by number. During the behavioural tasks, animal performance was video recorded, and then later blindly scored. In certain tasks (for example, open field), the animals were scored by a computer program and blinding was not necessary during that process. For western blots the hippocampi were isolated 30 min post injection and flash-frozen. All experiments were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and approved by the UT-Austin Institutional Animal Care and Use Committee (IACUC).

Immunofluorescence

Primary neuronal cultures on glass coverslips were fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min, washed three times with phosphate-buffered saline (PBS) and permeabilized in 0.25% Triton X-100 in PBS for 5 min. For FMRP staining, neurons were fixed and permeabilized in 100% methanol at −20 °C for 10 min. Neurons were washed three times in PBS and then blocked (10% normal goat serum in PBS) for 30 min at room temperature. Primary antibodies were incubated in blocking buffer at 4 °C overnight. Neurons were washed three times for 10 min with PBS, and then incubated in secondary antibody in blocking buffer for 1 h at room temperature, and washed three times for 10 min with PBS before mounting in Fluoromount with DAPI (SouthernBiotech, 0100-20) as outlined in Sosanya et al.60. Surface staining was performed similarly to Workman et al.6. Neurons were first fixed in 4% PFA for 10 min on ice, washed three times in PBS, blocked with 3% normal goat serum, and then incubated in primary surface antibody in 3% blocking buffer overnight at 4 °C. Following primary surface antibody incubation, neurons were washed six times for 10 min each in PBS, then permeabilized with 0.25% Triton X-100 in PBS for 5 min, followed by three washes for 10 min each in PBS, and again incubated in primary total antibody in 3% blocking buffer overnight at 4 °C. Neurons were washed three times for 10 min each with PBS, and then incubated in secondary antibody in 3% blocking buffer for 1 h at room temperature, and finally washed four times for 10 min with PBS before mounting in Fluoromount with DAPI to slides. The primary antibodies used were: Total GABA B R1 (1/50 dilution; Santa Cruz, sc-14006), Surface GABA B R1 (1/200 dilution; Abcam, ab55051), GABA B R2 (1/100; Neuromab 75-124), FMRP (1/500 dilution; Abcam ab17722), MAP2 (1/2000 dilution; Abcam ab5392), GFP (1/1,000 dilution; Aves, GFP-1020). Secondary antibodies included: Alexa488, 555 and 647 developed in goat (1/500 dilution; Life Technologies, A-11039, A-11017, A-31621, A-21430, A-21449, A-21240 and A-21237).

Adeno-associated viral vectors

The FMRP and tdTomato coding sequences were cloned into separate adeno-associated viral (AAV) vectors containing a mouse synapsin promoter, a woodchuck post-transcriptional regulatory element and SV40 poly-adenylation sequence between flanking AAV2 inverted terminal repeats. rAAVs were assembled using a modified helper-free system (Stratagene) as serotype 2/1 (rep/cap genes) viruses, and collected and purified over sequential caesium chloride gradients as previously described61. Viral titres were >1 × 109 infectious particles per microlitre. For FMRP and tdTomato co-infections, rAAV:mSYN-FMRP and rAAV:mSYN-tdTomato were mixed at a ratio of 4:1. One microlitre of the resulting rAAV mix was used per coverslip of primary cultured neurons. Imaging was performed ∼1 week after infection.

Live calcium imaging

Dissociated hippocampal cultures were prepared from WT and Fmr1 KO mice as described43. Neurons at 14–21 DIV were used for live calcium imaging. Neurons were treated as outlined in in vitro pharmacology above. Before imaging, cells were incubated in ACSF with Oregon Green 488 BAPTA-1 AM (OGB, 200 μM; 30 min; 37 °C; ThermoFisher) as described6. After OGB incubation, cells were transferred to fresh ACSF (37 °C) for imaging (1 frame per 20 s). Baseline calcium signal was imaged (1 min), after which (R)-baclofen (50 μM, Tocris) or vehicle (H 2 O) was added. For ethanol-treated cells, the neurons were incubated with OGB and imaged in ACSF containing ethanol (30 mM). Neurons were imaged for 800 s at room temperature. Quantification of the calcium signal was performed using Metamorph (Molecular Devices) as described6. Briefly, dendritic regions of interest (ROI) that were at least 5 μm from the soma were analysed. The mean intensity values for each ROI at each time were averaged as baseline (F 0 ). The ROI intensity values obtained at each time point after the addition of baclofen or vehicle were averaged (F). The equation, ΔF/F=((F−F 0 )/F 0 ), was used to measure the change in signal and data were plotted as a percentage of the baseline.

BONCAT-PLA

BONCAT-PLA was performed using Click-it Metabolic Labeling azidohomoalaine (AHA), Biotin-Alkyne and Click-iT Reaction buffer kit (Life Technologies). Proximity ligation assay (PLA) was performed using Duolink kit (Duolink, Sigma)38. Briefly, primary hippocampal neuronal cultures were incubated in a methionine-free ACSF media for 30 min. AHA was then added to the media just before neurons were treated with ethanol for 2 h as previously described. Neurons were fixed in 4% PFA for 15 min, washed two times for 5 min with 3% bovine serum albumin (BSA) in PBS, followed by permeabilization with 0.25% Triton X-100 in PBS for 15 min, and washed as before. Neurons were incubated for 30 min at room temperature in Cell Buffer Additive/Click-it Cocktail according to manufacturer directions. Neurons were washed as before and then blocked and incubated with primary antibody as previously described. Next, neurons were incubated in the appropriate PLA probes diluted in blocking buffer and secondary antibody at 37 °C for 1 h. Neurons were washed in RT Buffer A two times for 5 min, and incubated in ligation solution at 37 °C for 30 min, and washed again in Buffer A. Neurons were incubated in amplification solution at 37 °C for 2–3 h, followed by washing in RT Buffer B two times for 10 min and 1% buffer B for 1 min. Last, neurons were mounted to slides in Duolink mounting media for imaging. Primary and secondary antibodies included: GABA B R1 (1/50 dilution; Santa Cruz, sc-14006), GABA B R2 (1/100; Neuromab 75-124), MAP2 (1/2,000 dilution; Abcam, ab5392), biotin/α-rabbit (1/500; Sigma, SAB3700857), Alexa488 (1/500; Life Technologies, A-11039). PLA probes used: Rabbit Minus (1/5; Duolink, 82005), Mouse Plus (1/5; Duolink, 82001).

Microscopy and analysis

Images were acquired with a Leica SP5 confocal microscope under a × 63 oil immersion lens for fixed tissue or a × 63 water immersion lens for live imaging. Max projected images were used for immunostaining analysis from 10 μm Z-stacks of 1,024 × 1,024 pixels obtained using a 400-Hz scan rate60. For each experiment, all images were collected using the same settings. Fixed images were analysed using NIH imaging software ImageJ, and live imaging quantification was performed with Metamorph (Molecular Devices, Sunnyvale, CA). Background signal was determined by shifting the ROI adjacent to the dendrite being traced, but void of all processes. Dendritic signal was background subtracted and averaged every 10 μm using a customized R script.

Western blot analysis

Protein was isolated from hippocampal synaptoneurosomes prepared from male mice age 7–8 weeks treated with ethanol or vehicle as previously described. synaptoneurosomes were prepared by homogenizing hippocampal tissue in homogenization buffer (20 mM HEPES pH 7.4, 5 μM EDTA pH 8.0, and protease inhibitor cocktail). Homogenate was filtered through a 100-μm nylon filter followed by a 5-μm filter, and centrifuged at 14,000g for 20 min at 4 °C (ref. 62). The pellet was resuspended in RIPA buffer (150 mM NaCl; 10 mM Tris pH 7.4, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EDTA and protease inhibitor cocktail) and centrifuged for 10 min. The supernatant was used for western blot analysis. Protein was separated on a 4–20% gradient SDS–polyacrylamide gel. The gel was then transferred to a nitrocellulose membrane, blocked in 5% non-fat dry milk in tris-buffered saline and 0.1% Tween20 (TBST) for 1 h, and incubated with primary antibody in blocking buffer overnight at 4 °C. The blot was washed in TBST three times for 10 min each, incubated in secondary antibody for 1 h, and washed as before. Blots were imaged using a LICOR Odyssey imaging system, and ImageJ was used for densitometry analysis. Representative images are pseudocoloured with black (lowest intensity at 0 pixels) to red (highest intensity at 255 pixels) using LICOR Image Studio software. Primary antibodies used consisted of: GABA B R1 (1/400 dilution; Santa Cruz, sc-14006), GABA B R2 (1/800; Neuromab 75-124), alpha-Tubulin (1/2,000 dilution; Sigma, T6074). Secondary antibodies included: anti-mouse-IR-Dye 800 (1/5,000 dilution excluding tubulin at 1/10,000 dilution; LICOR, 96-32210) and anti-rabbit Alexa680 (1/5,000 dilution; Invitrogen, A-21084).

RNA immunoprecipitations

Cortices from 6-week-old C57BL/6 and Fmr1 KO male mice were collected and flash-frozen on dry ice. RIP was performed by modified method of Jain et al. and Keene et al.63,64. Tissue was homogenized and lysed with a cordless pestle motor and disposable pellet mixers (VWR) in polysome lysis buffer (10 mM HEPES pH 7.0, 100 mM KCl, 25 mM EDTA, 5 mM MgCl 2 , 1 mM DTT, 0.5% NP-40) in a 1:1 tissue-buffer ratio. RNaseOUT (Thermo) and protease/phosphatase inhibitors (Halt Protease and Phosphatase Cocktail, Pierce Biotechnology) were freshly added to samples. Samples were rotated for 10 min at 4 °C to induce swelling and then flash-frozen on dry ice. Samples were thawed by holding between fingers at room temperature to lyse and nuclei were pelleted at 3,000g for 10 min. Lysates obtained above were pre-cleared by adding 50 μl of washed magnetic bead slurry (Protein A Dynabeads, Thermo) and rotating for 30 min at 4 °C. To bind the antibody to the beads, 50 μl of magnetic beads slurry was washed and then resuspended in eight volumes of NT-2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl 2 , 1 mM DTT, 0.05% NP-40 with RNaseOUT/protease and phosphatase inhibitors added fresh)+5% BSA. Ten micrograms of either FMRP (Abcam, ab17722) or IgG (Santa Cruz Biotechnologies, sc-2027) antibodies was added to the beads and rotated for 10 min at room temperature. Antibody-bound beads were washed four times with ice-cold NT-2 buffer. For the immunoprecipitation, 4.5 mg of protein from pre-cleared lysates was added to an RNase-free microcentrifuge tube containing the antibody-bound beads. Input collected at this step for downstream analysis was either 1% of the final pre-cleared lysate volume in the immunoprecipitation reaction (for immunoblotting) or 10% of the final pre-cleared lysate volume (to normalize in qPCR). The antibody–bead-lysate mixture was then diluted at a ratio of 1:5 with NET-2 buffer (20 mM EDTA pH 8.0, and 1 mM DTT in NT-2 buffer; RNaseOUT and protease/phosphatase inhibitors added fresh) and rotated for 1 h at room temperature. Beads were quickly washed six times in ice-cold NT-2 buffer and immediately resuspended in 350 μl TRI Reagent Solution (Ambion) for 10 min at room temperature. Beads were pelleted and the supernatant was removed and resuspended in 350 μl of absolute ethanol. RNA was extracted by applying ethanol-resuspended samples to spin column from the Direct-zol RNA MiniPrep Kit (Zymogen) according to the manufacturer’s instructions. Eluted RNA (25 μl) was DNase treated using the TURBO DNA-free kit (Thermo).

cDNA synthesis and quantitative real-time PCR

DNase-treated RNA samples were reverse-transcribed to complementary DNA using the iScript cDNA Synthesis Kit (Bio-Rad) in a 20 μl volume according to the manufacturer’s instructions. Quantitative real-time PCR (qRT–PCR) was performed in 20 μl reaction volume using the iQ SYBR Green Supermix (Bio-Rad) and primers for GABA B R1 (GeneCopoeia, MQP031832), GABA B R2 (GeneCopoeia, MQP026008), CaMKIIα (GeneCopoeia, MQP028785), and Cacna2δ2 (GeneCopoeia, MQP032309). qRT–PCR was run with the following protocol: 95 °C for 10 min, 40 cycles of 95 °C for 15 sec followed by 60 °C for 1 min, 95 °C for 1 min, and 55 °C for 1 min. Relative fold-enrichment was determined by the equation ΔΔCt=2−(Ct FMRP RIP−Ct IgG RIP)−(Ct FMRP input−Ct IgG input) (ref. 65).

Forced swim test

Male mice were tested in the forced swim test 24 h post injection as described previously6,8. Mice were individually placed into a cylinder containing 3 l of water (25 °C) for 6 min. Each session was video recorded and the last 4 min of the sessions were later scored blindly for immobility. Animals were scored for escape-directed behaviours. The water was replaced between animals. Experiments were repeated by three independent experimenters. Data were normalized by experimenter. Power analysis was performed in R Programming66 to predict sample size for all behavioural tests. This sample size was used as a guideline for the WT animals, however since transgenic animals were used, the exact sample size for each group may not have been possible. Transgenic animal sample size was as close as possible to that which was calculated, due to limitations of litter size.

Open field

Twenty-four hours after animals were injected i.p. with either saline or ethanol, they were studied in the open field test similar to Treit et al.31. Mice were individually placed in a 40 cm × 40 cm × 35 cm arena with opaque walls. Each test session lasted 30 min under 85 lux illumination. Sessions were video recorded and analysed via ANY-Maze (Stoelting, Wood Dale, IL). Mice were considered to be in the centre of the maze if they entered a 18.5 cm × 18.5 cm area in the centre of the apparatus. Mice were returned to their home cage at the end of the test session, and the arena was wiped down with 70% ethanol before the start of each run.

Splash test

Two and a half hours after open field testing, animals underwent the splash test27,28. Cage mates were moved from their home cage to a holding cage, and each animal was individually tested in its home cage. Two-hundred microlitres of 10% sucrose was applied to the dorsal fur of the mouse. Mice were monitored and video recorded for 5 min and then moved to a different holding cage. Videos were later scored blindly for latency to initiate grooming and for total time spent grooming. Grooming behaviour included licking, grooming with forepaws, and scratching.

Statistical analysis

Power analysis was performed in R Programming66 to predict sample size. Prism software (GraphPad) was used for all statistical analyses. Statistical comparisons were made using one-way analysis of variance (ANOVA), two-way ANOVA, Student’s t-test or χ2-test with Yates. Outliers were determined using Grubbs’ test (α=0.05). All data are expressed as mean±s.e.m.

Data availability

All relevant data are available from the authors upon request. Accession numbers for deposited date used in Fig. 4a: GSE26809 (ref. 35) and SRA029279 (ref. 37).