Metabolomic analysis. Metabolomic analysis of standardized hippocampal specimens of ♂ Nxnl2-/- and Nxnl2+/+ aged of 2 months (Fig. 4) or treated ♂ and ♀ Nxnl2-/- and Nxnl2+/+ aged of 2 months (Fig. 5) were performed by the national infrastructure MetaToul https://www6.toulouse.inrae.fr/metatoul/. The brain is extracted from the cranium, making sure not to damage it, then rinsed in PBS. We removed the cerebellum, making sure not to damage the extremities of the 2 lobes and glued the brain on the support of the vibratome, posterior side up. Using a vibratome, we cut 0.5 mm slices lengthwise until reaching the hippocampus. We collected 0.5 mm slices with hippocampal tissue in PBS. Using a binocular magnifying glass, we selected slices with well visible hippocampus morphology (serrated gyrus of the hippocampus visible). We then made three standardized sections using a punch of 2 mm diameter, collected in an Eppendorf tube, and immediately frozen in liquid nitrogen while awaiting sample processing, then further stored at -80°C. On the day of extraction of the metabolites, we set-up the freeze-mill to cool. The tubes were taken out of -80°C and left them in liquid nitrogen while waiting. We added 3 steel balls to the tubes containing the hippocampus specimens and immediately placed them in the previously cooled ball mill. We performed 5 x 1 min 30 frequency/sec on a liquid nitrogen-refrigerated CryoMill (Retsch). When tissues were powdered, we added 1 ml per tube of methanol / H2O (80/20) previously cooled to -80°C plus 120 µl of 13C internal standard. We proceed for 1 min successively with 10 sec of vortex + 10 sec of sonicator + 10 sec on ice. The specimens were centrifuged for 5 min at 4°C 13,000 g. The supernatants were collected in a 2-ml Eppendorf tube to which we added 1 ml of cold methanol / H2O (80/20) mixed to the pellet and performed the same 1 min vortex / sonicator / ice cycle, as before. We centrifuged the tubes for 5 min at 4°C at 13,000 g, recovered the 2nd supernatant and pooled it with the 1st one. The resulting standardized hippocampal specimens were frozen by immersing in liquid nitrogen and stored at -80°C pending metabolomic analysis. The specimens were sent on dry-ice. For the first experiment (Supplementary Fig. 4a), intracellular metabolites were analyzed as described in89,90 Briefly, analysis was performed by high performance anion exchange chromatography (Dionex ICS 2000 system, Sunnyvale, USA) coupled to a triple quadrupole QTrap 4000 (AB Sciex, CA USA) mass spectrometer 90. This analytical technology allows the separation and analysis of numerous highly polar metabolites belonging to several chemical families in the same analytical run. All samples were analyzed in the negative mode by multiple reaction monitoring. The amounts of metabolites of glycolysis, pentose phosphate pathways, tricarboxylic acid cycle as well as nucleotides were determined. To ensure highly accurate quantification, the isotope dilution mass spectrometry (IDMS) method was used49. For quantification the addition of full 13C E. coli extract which contains a majority of the target metabolites was used, the internal standard. The quantification for each metabolite was first expressed as 13C/12C ratio or as 12C area if the internal 13C standard was not available. For metabolites for which a chemical standard was available, the absolute quantification was calculated from the corresponding calibration curve. For the second and the third experiment (Supplementary Fig. 4a and Supplementary Fig. 6a), we used a LTQ Orbitrap Velos™ / Liquid anion exchange chromatography Dionex™ ICS-5000+ Reagent-Free™ HPIC™ equipment. The analyses were carried out on an IC-MS platform of a liquid anion exchange chromatography Dionex™ ICS-5000+ Reagent-Free™ HPIC™ (Thermo Fisher Scientific™, Sunnyvale, CA, USA) system, coupled to a Thermo Scientific™ LTQ Orbitrap Velos™ mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a heated electrospray ionization probe. Liquid anion exchange chromatography was performed with the Thermo Scientific Dionex ICS-5000+ Reagent-Free HPIC system (Thermo Fisher Scientific) equipped with an eluent generator system (ICS-5000+EG, Dionex) for automatic base generation (KOH). Analytes were separated within 50 min, using a linear KOH gradient elution applied to an IonPac AS11 column (250 x 2 mm, Dionex) equipped with an AG11 guard column (50 x 2 mm, Dionex) at a flow rate of 0.35 ml/min. The gradient program was following: 0 min: 0.5 mM, 1 min: 0.5 mM, 9.5 min: 4.1 mM, 14.6 min: 4.1 mM, 24 min: 9.65 mM, 31.1 min: 90 mM and 43 min: 90 mM, then 43 to 48 min vat 0.5 mM. The column and autosampler temperatures were thermostated at 25°C and 4°C, respectively. The injected sample volume was 15 µl. Measures were performed in triplicates from separate specimens. Mass detection was carried out in a negative electrospray ionization (ESI) mode at a resolution of 60 000 (at 400 m/z) in full-scan mode, with the following source parameters: the capillary temperature was 350°C, the source heater temperature, 300°C, the sheath gas flow rate, 50 arbitrary units (a.u.), the auxiliary gas flow rate, 5 a.u., the S-Lens RF level, 60%, and the source voltage, 2.75 kV. Data acquisition was performed using Thermo Scientific Xcalibur software. Metabolites were determined by extracting the exact mass with a tolerance of 5-10 ppm. For quantification the addition of full 13C E. coli extract which contains a majority of the target metabolites was used, and quantified as above. Data were processed using TraceFinder 4.1 software. For the third experiment (Supplementary Fig. 6a), the gradient was modified as follows equilibration with 7 mM KOH during 1.0 min; then KOH ramp from 7 to 15 mM, 1–9.5 min; constant concentration 10.5 min; ramp to 45 mM in 10 min; ramp to 70 mM in 3 min; ramp to 100 mM in 0.1 min; constant concentration 8.9 min; drop to 7 mM in 0.5 min; and equilibration at 7 mM KOH for 7.5 min. Processed data are available at DOI 10.17632/yjmhvpp7rf.1. Row data are available upon request.
LTP recording. Recordings were performed on hippocampal slices of groups of ♂ Nxnl2-/- and Nxnl2+/+ aged of 2 months at E-Phy-Science https://www.e-phy-science.com/ (Fig. 2 and 5). After delivery, Mice were housed in standard ventilated cages (IVC, Sealsafe, Techniplast) coupled to an air-handling unit (TouchSLIMline, Exhaust, Techniplast), equipped with solid floors and a layer of bedding. The cages were cleaned at regular intervals to maintain hygiene. Environmental parameters were as follows: temperature: ~22°C, relative humidity: ~55%. Mice had ad libitum access to standard rodent chow. The food was stored under dry and cool conditions in a well-ventilated storage room. Mice had ad libitum access to pre‐filtered and sterile water. The amounts of food and water were checked daily, supplied when necessary and refreshed once a week. Mice were kept on a 12-h light/dark cycle. Mice were deeply anesthetized with isoflurane and decapitated. The brain was quickly removed and immersed in ice-cold pre-oxygenated artificial cerebrospinal fluid (aCSF). 400 µm-thick slices were prepared using a vibratome (VT 1000S; Leica Microsystems, Bannockburn, IL), and placed in a holding chamber in aCSF containing: 124 mM NaCl, 3.5 mM KCl, 1.5 mM MgSO4, 2.5 mM CaCl2, 26.2 mM NaHCO3, 1.2 mM NaH2PO4, 11 mM glucose, continuously oxygenated (pH = 7.4, 27°C). Slices were allowed to recover in these conditions from the slicing at least 1 h before recording. For electrophysiological recordings, a single slice was placed in the recording chamber, submerged and continuously superfused with gassed (95% O2, 5% CO2) aCSF (28–31°C) at a constant rate (2 ml min−1) for the reminder of the experiment. Extracellular fEPSPs were recorded in the CA1 stratum radiatum using a glass micropipette filled with aCSF. fEPSPs were evoked by the electric stimulation of Schaffer collaterals/commissural pathway at 0.1 Hz with a bipolar tungsten stimulating electrode placed in the stratum radiatum (100 μs duration). Stable baseline fEPSPs were recorded by stimulating at 30% maximal field amplitude for 20 min prior to beginning experiments [single stimulation every 20 s (3 Hz)]. Synaptic transmission (input / output) curves were constructed to assess basal synaptic transmission in groups of animals. LTP was induced by the following stimulation protocol: 3 trains of 100 stimulations at 100 Hz at the same stimulus intensity, with a 20 s interval between trains. Following this conditioning stimulus, a 1 h test period was recorded where responses were again elicited by a single stimulation every 20 s (3 Hz) at the same stimulus intensity. Signals were amplified with an Axopatch 200B amplifier (Molecular Devices, Union City, CA) digitized by a Digidata 1550 interface (Axon Instruments, Molecular Devices, US) and sampled at 10 kHz. Recordings were acquired using Clampex (Molecular Devices) and analyzed with Clampfit (Molecular Devices). Experimenters were blinded to genotype and treatment for all experiments. Data were analyzed by measuring the slope of individual fEPSPs at 0-1.5 ms from the top of the signal by linear fitting using Clampfit (Molecular Devices). LTP was quantified by comparing the mean fEPSP slope over the post-HFS period with the mean fEPSP slope during the baseline period. Group effects was assessed by changes in fEPSP slope, expressed as the percentage of the baseline value. For figure 5, only one hippocampal slide was used per animals. Recordings were also performed on hippocampal slices of groups of ♂ Nxnl2-/- and Nxnl2+/+ aged of 2 months at Institut du Fer à Moulin https://ifm-institute.org/en/home/ according to a protocol previously described35 (Supplementary Fig. 2a). Row data are available at https://data.mendeley.com/datasets/2rprjfnvk4/1.