All procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Zhejiang University Animal Experimentation Committee (Protocol AIRB-2021-948). Fmr1tm1Cgr transgenic male mice (RRID: IMSR_JAX:002700) were used and maintained by heterozygous mating. Mice were housed under a 12h light/dark cycle and provided food and water ad libitum. Heterozygous (Fmr1+/−) female mice and wild-type (WT) male (Fmr1+/y) mice were used for breeding to generate male hemizygous full-mutant mice (Fmr1−/y) and WT littermate controls (Fmr1+/y). Only male mice aged P7 to P16 were then used in experiments.

Genotypes were determined by PCR using the following three primers: 5′-CAC-GAG-ACT-AGT-GAG-ACG-TG-3′ (mutant forward), 5′-TGT-GAT-AGA-ATA-TGC-AGC-ATG-TGA-3′ (WT forward), and 5′-CTT-CTG-GCA-CCT-CCA-GCT-T-3′ (common), which amplified 131 and 400 bp fragments from the WT and mutated alleles, respectively.

Brain slices were prepared as previously described [21]. Mice were anesthetized with sodium pentobarbital and then killed by decapitation. The brain was removed rapidly and immersed in ice-cold oxygenated slicing solution containing (in mmol/L): 110 choline chloride, 7 MgCl2·6H2O, 2.5 KCl, 0.5 CaCl2·2H2O, 1.3 NaH2PO4, 25 NaHCO3, and 20 D-glucose, saturated with 95% O2 and 5% CO2. Sagittal slices 300 μm thick were cut on a vibratome (Leica VT1200S, Weztlar, Germany). Slices were allowed to recover for 30 min at 32 °C and then at room temperature in artificial cerebrospinal fluid (aCSF) containing (in mmol/L): 125 NaCl, 2.5 KCl, 2 CaCl2·2H2O, 1.3 MgCl2·6H2O, 1.3 NaH2PO4, 25 NaHCO3, and 10 D-glucose. Oxygen was continuously supplied during recovery and recording. To block inhibitory synaptic transmission, picrotoxin (100 μmol/L) was added to the bath.

Whole-cell patch-clamp recordings were made from the soma of VPm neurons at room temperature using a Multiclamp 700B amplifier and Digidata 1440A with pCLAMP 10.4 software (Molecular Devices, Sunnyvale, USA). Picrotoxin (100 µmol/L) was added to the bath to block GABAergic transmission. For voltage-clamp recording, the pipette was filled with an internal solution containing (in mmol/L): 110 Cs methane sulfonate, 20 TEA-Cl, 15 CsCl, 4 ATP-Mg, 0.3 GTP-Na, 0.5 EGTA, 10 HEPES, 4 QX-314, and 1 spermine (pH 7.2, 290–300 mOsm with sucrose). To record evoked quantal events, strontium (Sr2+) was applied to replace Ca2+ in the ACSF, which reduces the peak amplitude of EPSCs at −70 mV and causes a large number of asynchronous miniature EPSCs (Sr-EPSCs). Patch electrodes had a resistance of 2–4 MΩ. The series resistance (Rs) was usually 8–18 MΩ with no compensation. When the Rs had changed by >20%, the data were discarded. Signals were filtered at 2 kHz and digitized at 10 kHz.

Data of evoked quantal events were initially processed using Clampfit (Molecular Devices). The amplitude and frequency of Sr-EPSCs were analyzed using MiniAnalysis software (Synaptosoft, Decatur, USA) with manual post hoc verification.

To determine the number of inputs to each VPm neuron, we recorded evoked AMPAR-/NMDAR- EPSCs from the same cells at holding potentials of −70 /+40 mV over a wide range of stimulus intensity. A concentric electrode (World Precision Instruments, Sarasota, USA) was placed on the medial lemniscus, and stimuli (usually between 0.02 and 1.0 mA, 100 µs) were delivered at 0.1 Hz via a Master-8 stimulator (A.M.P.I., Jerusalem, Israel). We first used paired-pulse stimulation with an interval of 100 ms to distinguish lemniscal synaptic responses from corticothalamic responses. We then searched for step numbers roughly by increments of 50–100 µA and used small increments of 1–10 µA near each transition point to verify that this was indeed a single step. Finally, we used a stronger stimulus of at least twice the intensity that was evoked in the previous step. We carried out two or more trials at each stimulus intensity.

Animals were anesthetized with sodium pentobarbital, and perfused with saline followed by 4% paraformaldehyde (PFA) in 0.1 mol/L phosphate buffer (PBS). Brains were removed, post-fixed for 5–6 h in 4% PFA, and then transferred to 30% sucrose and kept at 4 °C for 2 days. Sagittal sections were cut at 40 µm on a microtome (Cryostar NX50, Thermo, Waltham, USA). After washing three times with 0.01M PBS, rinsing with frozen methanol (10 min at -20 °C), and blocking with 10% bovine serum albumin (BSA) for 1 h at room temperature, the sections were incubated with primary antibodies as follows: anti-VGluT2 (guinea-pig polyclonal, 1:600, Millipore, Billerica, USA, Cat# AB2251-I, RRID: AB_2665454) and anti-NeuN (rabbit monoclonal, 1:500; Millipore Cat# MABN140, RRID: AB_2571567) at 4 °C for 12–24 h. After rinsing, secondary fluorophore-conjugated antibodies (Alexa Fluor 488, donkey anti-rabbit, 1:1000, Thermo Fisher Scientific Cat# A-21206, RRID: AB_2535792; Alexa Fluor Cy3, donkey anti-guinea pig, 1:1000, Jackson, West Grove, USA, Cat# 706-165-148, RRID: AB_2340460) were applied for 2 h at room temperature. The antibodies were diluted in PBS containing 5% BSA. Images were captured using a 60× objective on an Olympus FV-1000 or FV-1200 inverted confocal microscope. 10–12 consecutive images at 0.5 mm intervals were captured in Z-stacks.

Images were analyzed blindly using ImageJ (NIH, Bethesda, USA, version 1.51). Several VPm neurons were randomly selected from each slice. By using the Cell Counter plugin, the VGluT2 puncta on the soma were manually counted. The number of puncta in contact with the NeuN was determined as the puncta/soma. To quantify VGluT2 puncta/neuron, the Analyze Particles command of ImageJ was used to analyze the density of VGluT2 puncta in each image. The number of thalamic VPm neurons was then counted manually by the Cell Counter plugin. The puncta/neuron was calculated by dividing the total number of puncta by the total number of neurons.

The VPm of Fmr1-/y mice and WT littermates (P7–8, P12–13, and P15–16) was dissected, trimmed into ~1 mm3 pieces, and fixed overnight with 2.5% glutaraldehyde in PBS buffer. After rinsing in 0.1M PBS (10 min, 3 times), the specimens were post-fixed with 1% OsO4 for 1 h, stained with 2% uranyl acetate for 30 min, dehydrated by ethyl alcohol and acetone, and then embedded. An ultrathin microtome (UC7, Leica, Germany) was used to cut 400 nm sections for electron tomography. For the 2D imaging of synaptic vesicles, electron micrographs were acquired using a Tecnai G2 Spirit 120 kV (Thermo FEI). The 2D images were then analyzed using ImageJ software (NIH, version 1.51).

All data are presented as the mean ± SEM. Statistical analysis was applied with GraphPad Prism 6 (GraphPad Software, La Jolla, CA). Student’s t-tests or Mann-Whitney tests were used as indicated for differences between the two groups. Frequency distributions were analyzed using a χ2 test. For all statistical analyses, significance was set at *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001.