The strategy of point mutation of the CLN1 gene to generate PPT-KI mice (Cln1 c.451C>T (p.R151X)) is shown in Fig. S1. C57BL/6N mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (animal license number: 2016-0006) and were used as the wildtype (wildtype) controls. All animals were housed and maintained in the specific pathogen-free animal facility of Animal Experiment Center of the Institute of Psychiatry and Neuroscience of Xinxiang Medical University (XXMU) with a 12-h light/dark cycle. Animals had ad libitum access to food and water, except during food or water deprivation. A previous study demonstrated that BuHA can easily pass the blood-brain barrier in the mouse brain . PPT1-KI mice and their littermates were orally administrated with 1 mM BuHA for 3 months before mating. After pregnancy, the young mice took food and water (containg 1 mM BuHA) freely during the whole period. All efforts were made to minimize animal suffering and reduce the number of animals used. All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.
In vivo electrophysiological recording
Stereotaxic surgery, electrode implantation and signal acquisition
Mice were deeply anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (0.45 mL~0.5 mL/100g). Under anesthesia, mice were secured in a stereotaxic frame with ear bars. The head was shaved with a razor, and a midline 5-mm incision was made with a sterile scalpel. The subcutaneous tissue was removed from the skull, a craniotomy (~1.5 x 0.5 mm) was drilled (AP: 1.82 mm, ML:1.25 mm, DV:1.5 mm, right hemisphere) at CA1 region. Two steel screws were anchored at the anterior and posterior edges of the surgical site to secure the implant in place. After endocranium was removed, a 4*2 micro wire electrode (KD-MWA-8, 25 μm nitinol wire, Kedou (Suzhou) Brain-Computer Technology Co., Ltd.) with 3 μm polyethylene glycol coating was implanted into pyramidal cell layer of CA1. The craniotomy site was then sealed with a sterile silicone elastomer (Kwik-Sil WPI) to alleviate brain injury. After surgery, the implanted electrodes and screws were cemented integrally to the skull using denture base resin type II (Shanghai Medical Instruments Co., LTD.). After surgery, animals were housed individually on a reversed 12/12 hr day/night schedule.
Following one week of recovery, wideband signals were recorded using OmniPlex Neural Recording Data Acquisition System (Plexon Inc., Dallas, TX, USA) with 8 kHz global low-pass filter. Continue spike was sampled at 4 kHz following with 300 Hz low-cut filter. Field potential (FP) was set at 200 low-pass filter and down-sampled to 1 kHz. After recording, the hippocampus was post-fixed to perform Nissl stain in order to verify the proper placement of the electrodes in the target region.
Spikes were sorted with the Offline Sorter (Plexon Inc., Dallas, TX, USA) to classify the electric activity of individual neurons, based on the first to third principal components. Spike units were excluded using Remove Short ISI Waveform Tools when the absolute refractory period of single unit autocorrelation was < 1 ms. Cross-channel artifacts identified by their time-concidence across channels were also invalidated.
The power spectrum density and spectrogram of continuous filed potential (FP) were computed using NeuroExplorer (Nex Technologies, Colorado Springs, CO, USA) with 1024 frequency values and 25% window overlap. Before this process, FP signal values were multiplied by the coefficients of the Hann window, and discrete fast Fourier transformations of the results were calculated using formulas defined previously. gamma waves were filtered by band-pass filtering of FP data using NeuroExplorer software with Digital Filtering of Continuous Variables function. The valley of γ wave timestamp was identified as a reference event using Find Oscillation function for plotting perievent raster.
In vitro electrophysical recording
The mice were anesthetized with urethane and perfused with ice-cold artificial cerebrospinal fluid (aCSF) through the left ventricle until the limbs turned white. The brain was then rapidly removed and immersed in ice-cold aCSF containing 225 mM sucrose, 3 mM KCl, 6 mM MgCl2, 1.25 mM NaH2PO4, 24 mM NaHCO3, 0.5 mM CaCl2, 10 mM glucose. Transverse slices (350 μM thickness) were prepared using a vibratome (Ci-7000SMZ2, Campden instrument, Loughborough, UK). Immediately after preparation, slices were transferred to a nylon net within a chamber, and two sides of the chamber were exposed to normal aCSF containing mM 126 NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 2 mM MgSO4, 24 mM NaHCO3, 2 mM CaCl2, and 10 mM glucose, bubbled with a mixture of 95 % O2 and 5 % CO2, and kept at pH 7.35-7.45 at room temperature (RT) for storage or 32 °C for recording. The perfusion rate was maintained at 1-2 mL/min .
Patch clamp recording
Miniature inhibitory post synaptic currents (mIPSCs) were measured in voltage-clamp mode at -70 mV in the presence of metabotropic glutamate receptors blocker, [250 μM (S)-α-methyl-4-carboxyphenylglycine (MCPG)], a glycine receptor blocker (1 μM strychnine), and GABAB receptor blocker (1 μM CGP55845). The pipette solution contained 140 mM CsCl, 10 mM Na-HEPES, 10 mM EGTA, 2 mM Mg-ATP and 5 mM QX-314 (pH 7.3). To block EPSCs, 25 μM D-(–)-2-amino-5-phosphonopentanoic acid (D-APV) and 5 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were added in the bath solution just before used[39, 40]. For evoked IPSCs recordings, the cells were clamped at +40 mV. All patch-clamp recordings were performed at RT.
To isolate AMPAR-mediated miniature excitatory postsynaptic currents (mEPSCs), 1 μM tetrodotoxin, 10 μM D-APV, 10 μM bicuculline, and 10 μM strychnine were added to the bath solution. Pipette (3-4 MΩ) solution contained 125 mM Cs-gluconate, 20 mM KCl, 4 mM Mg-ATP, 10 mM Na2-phosphocreatine, 0.3 mM Na2-GTP, 10 mM HEPES, 0.5 mM EGTA, and 5 mM QX314 (pH 7.3) adjusted with CsOH. For evoked EPSCs recording, neurons were voltage-clamped at -70 mV to record AMPAR- mediated EPSCs or at +40 mV to record dual-component EPSCs containing NMDAR-mediated EPSCs. AMPAR/NMDAR ratios were also calculated by dividing the peak of the AMPAR-mediated EPSC at -70 mV by the value of the NMDAR-mediated EPSC after a stimulation start time 50 ms at +40 mV. For current clamp recording, a K-gluconate-based intercellular solution were used, and cells were clamped at their resting potentials. A 300-pA current was injected into the cell to induce action potential by bath application with 10 μΜ CNQX, 5 μΜ L-AP5, 10 μΜ bicuculline and 10 μΜ strychnine.
Currents were amplified with a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA), low-pass filtered at 1 kHz, and digitised with a Digidata 1550B interface (Molecular Devices) at 5 kHz. The detection and analysis of synaptic currents were conducted using pClamp 10 software (Molecular Devices). Offline analysis of mEPSCs/IPSCs was performed using Clampfit 10.4.2.0 (Molecular Devices) software. Only recordings where series resistance remained below 16 MΩ and did not increase by more than 20 % during the experiment were included in the analysis.
Acyl biotin exchange (ABE) assay
ABE was performed as previously described [13, 17, 42]. Briefly, the lysates were incubated with 10 mM N-ethylmaleimide (NEM, E3876; Sigma) overnight at 4 °C. Then, NEM was removed by three sequential chloroform/methanol (CM) precipitations. After three CM precipitations, proteins were solubilized in solubilizing buffer with 1 M hydroxylamine hydrochloride (159417, Sigma), 1 mM HPDP-Biotin (A8008; APExBIO, Houston, TX), 0.2 % TritonX-100 (T8787, Sigma), and protease inhibitors (4693132001; Roche, Basel, Switzerland) in phosphate-buffered saline (pH 7.4). After incubation for 1 h at RT, proteins were precipitated by CM and then suspended with 200 μM HPDP-biotin and 0.2 % Triton X-100 for 1.5-2 h at RT. After 3 CM precipitations, the proteins were incubated with streptavidin-agarose (16-126; Millipore sigma, Burlington, MA) for one night at 4 °C. The beads were washed five times with wash buffer containing 150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA (pH 7.4), and 0.2 % Triton X-100. After washing, the proteins were eluted with wash buffer containing 1.5 % β-mercaptoethanol (Sigma-Aldrich, M3148) at 37 °C for 0.5 h with agitation (350 rpm), and then heated in a 100 °C water bath for 10 min. After centrifugation, the released proteins in the supernatant were denatured in sodium dodecyl sulfate sample buffer and processed for Western blotting with primary antibody ABE assays; all other biochemical experiments were performed at least three times. In each case, a representative experiment is presented.
The possible palmitoylation sites (C165, C179, C260, C319) of GABAAR α1 subunit was forecasted by CSS-Palm Online Service webset. All the mutants (GABAARα1 C165A, C179A, C165/179A) were purchased from Fenghui Biology (Hu’nan, China). ABE assay was performed as mentioned above.
Other experimental procedures are provided in the Supplementary Information:
S1 Behavioral studies
Morris water maze (MWM)
S2 Biochemical analysis
Cell membrane/cytoplasmic protein extraction
Quantitative polymerase chain reaction (qPCR)
All data were acquired and analyzed by experimenters who were blinded with respect to the genotype of the mice and acute slices. All data were analysed using the SPSS Statistics 20 (IBM, Armonk, NY). We confirmed homogeneity of variances by the Levene’s test and equality of mean by the Brown-Forsythe test. Electrophysiology data were analysed using t-test for two group comparisons and one-way ANOVA test for multi-group comparisons. Western blot results were analysed by t-test for two group comparisons and one-way ANOVA test for multi-group comparisons.