Ethics statement

Experimental procedures with animals were performed according to German animal welfare legislation and the ARRIVE guidelines, and approved by the appropriate animal welfare committee and the animal welfare officer of the Albert-Ludwigs-Universität Freiburg (University of Freiburg), Faculty of Medicine (X-17/07K, X-21/01B; preparation of organotypic tissue cultures). Mice were obtained from the Center for Experimental Models and Transgenic Services (CEMT, University of Freiburg). Mice were maintained in a 12 h light/dark cycle with food and water available ad libitum. Every effort was made to minimize distress and pain of animals.

Preparation of tissue cultures

Organotypic entorhino-hippocampal tissue cultures were prepared from C57BL/6J mice of either sex at postnatal day 3–5 as previously described [20]. The cultivation medium contained 50% (v/v) MEM, 25% (v/v) basal medium eagle, 25% (v/v) heat-inactivated normal horse serum, 25 mM HEPES buffer solution, 0.15% (w/v) bicarbonate, 0.65% (w/v) glucose, 0.1 mg/ml streptomycin, 100 U/ml penicillin, and 2 mM glutamax. The pH was adjusted to 7.3 and the medium was replaced 3 times per week. Prior to experimental procedures, all tissue cultures were allowed to mature in a humidified atmosphere with 5% CO2 at 35 °C for at least 18 days.

Pharmacology

For some experiments, tissue cultures were treated with tetrodotoxin (TTX, 2 µM, 2 d; #ab120055, Abcam) while control cultures were only treated with vehicle (water, 1 µl).

Whole-cell patch-clamp recordings

Whole-cell patch-clamp recordings in organotypic tissue cultures were performed in a bath solution containing (in mM) 126 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 2 MgCl2, and 10 glucose. In order to record miniature excitatory postsynaptic currents (mEPSCs), the solution was substituted with TTX (0.5 µM; #ab120055, Abcam), D-AP5 (10 µM; #ab120003, Abcam) and (-)-bicuculline-methiodide (10 µM; #ab120108, Abcam). Recordings were carried out at 35° under continuous oxygenation (5% CO2/95% O2) and 3–6 cells were patched per culture. Cells were visually identified using an LN-Scope (Luigs and Neumann, Germany) equipped with infrared dot-contrast and a 40× water-immersion objective (numerical aperture [NA] 0.8; Olympus). Electrophysiological signals were amplified using a Multiclamp 700B amplifier, digitized with a Digidata 1550B digitizer, and visualized with the pClamp 11 software package. Patch pipettes contained (in mM) 126 K-gluconate, 4 KCl, 10 HEPES, 4 MgATP, 0.3 Na2GTP, 10 PO-creatine, and 0.3% (w/v) biocytin (pH = 7.25 with KOH; 285 mOsm/kg) and had a tip resistance of 3–5 MΩ. Cells were recorded in voltage-clamp mode at a holding potential of -70 mV. Series resistance was monitored before and after recording and recordings were discarded if the resistance reached ≥ 30 MΩ.

Post hoc staining and confocal visualization

After electrophysiological assessment, tissue cultures were fixed in 4% (w/v) paraformaldehyde (PFA; in PBS (0.1 M, pH 7.4) with 4% (w/v) sucrose) overnight. After fixation, they were washed in PBS (0.1 M, pH 7.4) and incubated with 10% (v/v) normal goat serum (NGS) in 0.5% (v/v) Triton X-100 containing PBS for 1 h, in order to reduce nonspecific staining. For post-hoc visualization of patched neurons, the tissue was incubated with Streptavidin Alexa Fluor 488 (Streptavidin A488, 1:1000; #S32354, Invitrogen) diluted in 10% (v/v) normal goat serum (NGS) in 0.1% (v/v) Triton X-100 containing PBS at 4° C overnight. After washing, the tissue was incubated with DAPI nuclear stain (1:5000 in PBS for 15 min; #62248, Thermo Scientific) in order to visualize cytoarchitecture, transferred onto glass slides and mounted with a fluorescence anti-fading mounting medium (DAKO Fluoromount; #S302380-2, Agilent). Confocal images were acquired using a Leica SP8 laser-scanning microscope equipped with a 20× multi-immersion (NA 0.75; Leica), a 40× oil-immersion (NA 1.30; Leica), and a 63× oil-immersion objective (NA 1.40; Leica). Confocal images were stored as .tif files.

Transcriptome analysis

For experiments in organotypic tissue cultures, 5–6 cultures of the same mouse were collected and used per sample. RNA was isolated using the Monarch® Total RNA Miniprep Kit (#T2010S, New England Biolabs) according to the manufacturer’s instructions. RNA quantity and quality were assessed using an Agilent RNA 6000 Pico Kit (#5067 − 1513; Agilent) with a 2100 Bioanalyzer (#G2939BA, Agilent). After RNA isolation from TTX-treated tissue cultures, library preparation and paired-end RNA sequencing (read length: 150 bp) was performed using the genome sequencer Illumina HiSeq technology in NovaSeq 6000 S4 PE150 XP sequencing mode (service provided by Eurofins). For further analysis, fastq files were provided. Data were analyzed at the Galaxy platform (usegalaxy.eu; [38]). All files contained more than 10 M high-quality reads (after mapping to the reference genome) with a phred quality of at least 30 (> 90% of total reads).

HPG-SILAC analysis of newly synthesized proteins

For the analysis of TTX-induced changes in de novo protein synthesis, we used a homopropargylglycine (HPG) based protocol in combination with strategy for stable isotope labeling with amino acid in cell culture (SILAC). Three weeks old tissue cultures were treated with TTX (2 µM, 2 days) or vehicle-only. From 24 to 48 h, cultures were depleted from methionine, arginine and lysine through the incubation with depletion medium consisting of Neurobasal A (#041-96642 M, Gibco), B27 supplement (#17504-044, Invitrogen), penicillin/streptomycin (#15140-122, Invitrogen) and Glutamax (#35050-038, Invitrogen). The medium was supplemented with HPG (4 mM, #CLK-016-100, Jena) and stable isotope labeled amino acid with either medium or heavy weights (L-arginine – 400 µM, L-lysine – 800 µM; medium weight amino acids: Arg-6 (#CLM2265, eurisotop) and Lys-4 (#DLM2640, eurisotop); heavy weight amino acids: Arg-10 (#CNLM539, eurisotop), Lys-8 (#CDNLM-6810, eurisotop)). At the end of the treatment period, 3–6 cultures were pooled for each condition (control-medium, control-heavy, TTX-medium, TTX-heavy) and flash frozen until further processing.

To release proteins for pulldown, slices were lysed in 400 µl of Pierce RIPA buffer (#89900, Thermo Scientific) supplemented with protease inhibitor cocktail III – EDTA-free at 20 µl/ml (#539134, Merck). Control culture material fed with “HPG-medium” amino acids was mixed with TTX-treated culture material fed with “HPG-heavy” amino acids, and vice versa for the reverse condition. The mixtures were then stored on ice before being homogenized 15 times using a tissue dounce homogenizer before being repeatedly vortexed (every 5 min) over a period of 20 min. Samples were then sonicated (3 × 3-second bursts at 80% continuous power). Sonicated samples were then spun at 10,000 xg for 2 min through a cell shredder (#1011711, QIAGEN). Lysed proteins were then incubated with 200 µl of azide-agarose beads (#1038-2, Click-Chemistry Tools), in a “click-solution” containing: 0.2 mM Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA, #762342, Sigma-Aldrich), 20 mM Sodium L-ascorbate (#A7631, Sigma-Aldrich) and 0.2 mM Copper(II) sulfate pentahydrate (#C8027, Sigma Aldrich) in injection grade water (AMPUWA). Azide-beads and proteins were incubated for 24 h, in the dark, on an orbital shaker (room temperature). After incubation, beads and proteins were then pelleted at 3000 x g for 3 min and washed with injection grade water. Beads and proteins were then transferred to “agarose wash buffer” (AWB; 100 mM Tris, 1% SDS, 250 mM NaCl, 5 mM EDTA, pH 8.0). To break disulfide bonds proteins were then treated for 30 min with 10 mM dithiothreitol (DTT) at 37 °C. After washing, proteins were treated for 40 min at room temperature with 400 mM Iodoacetamide (IAA), in AWB, in the dark. Following DTT and IAA treatments, beads were then repeatedly pelleted and washed (10 times with each solution) in Pierce 0.8 ml centrifuge columns (#89868, Thermo Fisher Scientific) in the following solutions: (1) agarose wash buffer, (2) 8 M Urea in 100 mM Tris, and (3) 70% acetonitrile solution (with 100 mM ammonium bicarbonate buffer; ABC). Following washing, beads were then resuspended in 10% acetonitrile in 50 mM ABC buffer. Cells were then taken for trypsin digestion and mass spectrometry analysis.

LC-MS/MS analysis was carried out using an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) coupled online with an Ultimate 3000 RSLCnano system (Dionex, Thermo Fisher Scientific). The sample was separated on a 50 cm EASY-Spray C18 column (Thermo Scientific) operating at 45 °C column temperature. Mobile phase A consisted of 0.1% (v/v) formic acid and mobile phase B of 80% v/v acetonitrile with 0.1% v/v formic acid. Samples for analysis were resuspended in 0.1% v/v formic acid, 1.6% v/v acetonitrile. Peptides were loaded and separated at a flow rate of 300 nl min− 1. Peptides were separated using a gradient with linear increases from 2% mobile phase B to 35% over 95 min then to 45% over 15 min, followed by an increase to 55% in 3 min and then a steep increase to 95% mobile phase B in 2 min.

Eluted peptides were ionized by an EASY-Spray source (Thermo Scientific) and introduced directly into the mass spectrometer. The MS data were acquired in data-dependent mode with a 3 s cycle time. For every cycle, the full scan mass spectrum was recorded in the Orbitrap at a resolution of 120 K. Ions with a precursor charge state between 2 + and 7 + were isolated and fragmented employing higher-energy collisional dissociation (HCD) with a normalized collision energy of 30% applied. The fragmentation spectra were then recorded in the ion trap with the scan rate set to “Normal”. Dynamic exclusion was enabled with a single repeat count and a 60 s exclusion duration.

Quantification and statistics

Electrophysiological data were assessed using the pClamp 11 software package (Axon Instruments). mEPSC properties were analyzed with an automated templated-based search tool for event detection. The template was previously created in the Clampfit11 software based on manually identified and selected EPSCs. The template match threshold was set at 2.5 and the time period for the allowed duration of a single event starting from the baseline was defined. Both parameters were not changed within the different recordings or groups.

For transcriptome analysis, sequencing data were uploaded to the Galaxy web platform (public server: usegalaxy.eu), and the analysis was performed based on the RNA-seq data analysis tutorial [39]. In short, the CUTADAPT tool was used to remove adapter sequencing, low quality and short reads. Remaining reads were subsequently mapped using the RNA STAR tool with the mm10 (Mus musculus) reference genome. The evidence-based annotation of the mouse genome (GRCm38), version M25 (Ensembl 100), served as a gene model (GENCODE). An unstranded FEATURECOUNT analysis of the RNA STAR output was performed for an initial assessment of gene expression. Only samples that contained > 60% uniquely mapping reads (feature: “exon”) were considered for further analysis. Statistical evaluation was performed using DESeq2 with “treatment” as the primary factor affecting gene expression. Genes with mean reads (base mean) < 150 were excluded from further analysis. Genes with an adjusted p-value of < 0.05 were considered as differentially expressed. Heatmaps were generated based on the z-scores of the normalized count tables. The g: Profiler (version e107_eg54_p17_bf42210) with g: SCS multiple testing correction method (significance threshold of 0.05 [40]) was used for functional enrichment analysis of both transcriptomic and proteomic data.

Protein quantification was performed using MaxQuant (v1.6.5.0) and output data manipulated using Perseus (1.6.2.3; [41,42,43]. The Uniprot mouse proteome SP_UP000000589 was used as a reference database (downloaded 26/03/2021). MaxQuant search parameters were set as previously described [44], including cysteine (C) carbamidomethyl, as fixed modification, together with methionine (M) oxidation, protein N-terminal acetylation and asparagine (N) or glutamine (Q) deamination, as variable modifications. Lys0 and Arg0, Lys4 and Arg6, or Lys8 and Arg10 were set as multiplicity labels. First search and main search peptide tolerances were left as 20 and 4.5 ppm, respectively. False discovery rate was 1% for both peptide spectrum match and protein levels. Minimum peptide count was 2. Before further analysis, predicted contaminants, reverse database hits and peptides only identified by modification were excluded. Proteins with a q-value < 0.05 (FDR < 5%) were considered to be differentially expressed.

Data were statistically analyzed using GraphPad Prism 9 (GraphPad Software, USA). All values represent mean ± standard error of the mean (s.e.m.). We used a non-parametric Mann-Whitney test for comparison of two experimental groups in electrophysiological experiments. P-values < 0.05 were considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001); results without statistical significance were indicated as ‘ns’. N-numbers are provided in the figure legends.

Digital illustrations

Confocal images were stored as .tif files and image brightness and contrast were adjusted. Figures were prepared using the ImageJ software package (https://imagej.nih.gov/ij/) and Photoshop graphics software (Adobe, San Jose, CA, USA).