Microlance needle

Microaerobic cultivation was performed in 250 mL airlock-capped Neubor infusion bottles (38 mm neck, Dijkstra, Lelystad, Netherlands) containing 200 mL 3-fold diluted wort supplemented with 0.4 mL L^-1 Pluronic antifoam (Sigma-Aldrich). Bottle caps were equipped with a 0.5 mm x 16 mm Microlance needle (BD Biosciences) sealed with cotton to prevent pressure build-up. Sampling was performed aseptically with 3.5 mL syringes using a 0.8 mm x 50 mm Microlance needle (BD Biosciences). Microaerobic cultures were inoculated at an OD₆₆₀ of 0.1 from stationary-phase precultures in 50 mL Bio-One Cellstar Cellreactor tubes (Sigma-Aldrich) containing 30 mL of the same medium, grown for 4 days at 12°C. Bottles were incubated at 12°C and shaken at 200 RPM in a New Brunswick Innova43/43R shaker. At regular intervals, 3.5 mL samples were collected in 24 deep-well plates (EnzyScreen BV, Heemstede, Netherlands) using a LiHa liquid handler (Tecan, Männedorf, Switzerland) to measure OD₆₆₀ and external metabolites. 30 μL of each sample was diluted 5 fold in demineralized water in a 96 well plate and OD₆₆₀ was measured with a Magellan Infinite 200 PRO spectrophotometer (Tecan, Männedorf, Switzerland). From the remaining sample, 150 μL was vacuum filter sterilized using 0.2 μm Multiscreen filter plates (Merck, Darmstadt, Germany) for HPLC measurements.

Whole cell lysates (WCLs) were prepared by lysing cells in RIPA lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and an in-house protease inhibitor cocktail) for 30 min on ice followed by centrifugation at 16,000 g for 10 min at 4°C in order to remove insoluble material. For preparation of the mitochondria-enriched fraction, cell fractionation was carried out on ice in MB buffer (10 mM HEPES pH 7.4, 210 mM mannitol, 70 mM sucrose, 1 mM EDTA, and the in-house protease inhibitor cocktail) following 30 passages through a 25G x 1" Microlance needle (BD, 300400). Nuclei and unbroken cells were discarded after centrifugation at 2,000 g. The mitochondria-enriched fraction was then recovered by centrifugation at 10,000 g. Protein content was determined using the BCA Protein Assay Kit (Pierce).

The rats were ∼ 3 months old at the time of surgery. All rats were anaesthetised with isoflurane gas. They were then placed in a stereotaxic frame with the incisor bar set at − 3.3 mm, and given analgesia in the form of 0.1 mg/kg Metacam (Boehringer Ingelheim Vetmedica, Germany) administered subcutaneously. To expose the skull, a midline sagittal incision was made in the scalp, and the skin was reflected. A craniotomy was made above the injection sites, and the dura was cut to expose the cortex. The hippocampal lesions (n = 22) were made with injections of ibotenic acid (Biosearch Technologies, San Rafael, CA, USA) diluted to 63 mm in phosphate-buffered saline (PBS; 0.1 m, pH 7.4). The ibotenic acid was administered via a 2-μm Hamilton syringe connected to a microinjector (Model 5000; Kopf Instruments) set at a rate of 0.1 μL/min, with a subsequent diffusion time of 2 min. The rats received 14 injections into each hemisphere [for coordinates and volumes, see Iordanova et al. (2009 (link))]. The surgical control rats (n = 20) were treated in the same way until the dura was exposed. While nothing was infused into the brain, the dura was pierced 14 times per hemisphere with a 25-gauge Microlance needle (Becton Dickinson, Drogheda, Ireland).