*2.1. Animal Experiments*

All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the European Union Directives 86/609/EEC and 2010/63/EU, and were approved by Bioethics Committee of Lomonosov Moscow State University (protocol number 69-o from 09.06.2016). Animals were kept at 21 ± 2 ◦C and relative humidity 53 ± 5% with the 12/12 h light/dark cycle (lights on 9:00 = ZT 0, lights o ff 21:00 = ZT 12).

Wistar female rats, pregnan<sup>t</sup> and non-pregnan<sup>t</sup> ones, of about 250–300 g were used in the experiment. To obtain pregnan<sup>t</sup> rats, two virgin female rats were located in a cage with one male. After 24 h, vaginal smears were examined. When sperm was found in the vaginal smear, it was considered as the first day of pregnancy, and the male rat was separated. The rats were purchased from the State Research Center of the Russian Federation—Institute for Biomedical Problems, Russian Academy of Sciences, and were kept at our conditions for two weeks prior to the experiments. T/4K cages (555/4K, 580 × 375 × 200 mm) were used. Five to six animals were kept in each cage. Standard rodent pellet food (laboratorkorm.ru) and tap water were available ad libitum.

Four groups of female rats were used: (1) normoxic control non-pregnan<sup>t</sup> group; (2) hypoxic treatment non-pregnan<sup>t</sup> group; (3) normoxic control pregnan<sup>t</sup> group; (4) pregnan<sup>t</sup> group exposed to hypoxia at the 9–10th day of pregnancy, which in rats is roughly correspondent to the first trimester of human pregnancy [24]. Total number of animals in an experimental group, n, comprised animals from several independent experiments and is indicated in the figures and tables. On the second day after the treatment, when the physiological assessment was completed, animals were sacrificed for further biochemical analyses by decapitation. At this stage, the number of pups (10 ± 2) did not significantly di ffer between the control and hypoxic rats, although independent long-term physiological monitoring indicated that the pregnancy failures occurred more often in the latter than former rats.

Cerebella were quickly excised from animal brains on ice, frozen in liquid nitrogen, and stored at −70 ◦C prior to biochemical analyses. A half of each thawed cerebellum was used to prepare the homogenates for enzymatic assays, the other half—to make extracts for the amino acid quantification.

#### *2.2. Acute Hypobaric Hypoxia*

Female rats were exposed to hypobaric hypoxia at 5% O2 (11500 m altitude, 145 mm Hg) in a decompression (altitude) chamber "Mez Mohelnice" (Mohelnice, Czech Republic) of 3.3 L volume, as described previously [25,26]. Briefly, after closing the chamber, air pressure inside the chamber was progressively decreased during 1 min (200 m/sec) by the vacuum pump connected to the chamber. The pressure was controlled using the pressure gauge. Overall response of an organism to hypoxia at subcritical lack of oxygen is defined by the lifetime (LT) which is evaluated from the moment of reaching the target height (i.e., 11,500 m altitude, 5% O2, 145 mm Hg) to the second agonal breath. LT characterizes the ability of animals to mobilize their protective mechanisms for survival under life-incompatible extreme conditions. The pressure and oxygen in the chamber returned to the nominal values after the second agonal breath. According to LT, the rats were divided into groups of high resistance (HR, LT ≥ 10 min), middle resistance (MR, LT within 5–10 min), and low resistance (LR, LT within 1–5 min). These types of animals are known to demonstrate di fferent functional and metabolic patterns, including di fferences in the CNS activity and neurohumoral regulation, stress-responsive systems, oxygen transport by the blood, and tissue respiration [27–29].

## *2.3. Enzyme Assays*

Homogenization of cerebella and assays of the overall OGDHC activity of brain homogenates were performed as described earlier [30] with 20% glycerol added to the homogenization bu ffer and sonication as in [12].

#### *2.4. Ninhydrine Quantification of Amino Acids*

Preparation of the acetic acid/methanol extracts of cerebella and quantification of their amino acids was done as described in [31], using L-8800 amino acid analyzer (Hitachi, Tokyo, Japan) in the standard mode according to the manufacturers User Manual (Hitachi High-Technologies Corporation, Tokyo, Japan, 1998). The samples were stored at −70 ◦C. Briefly, the extracts were subjected to an ion-exchange column 2622SC (PH) (Hitachi, Ltd., Tokyo, Japan, P/N 855-3508, 4.6 × 80 mm), eluted by step gradient of four sodium-acetate bu ffers at a flow rate of 0.4 mL/min at 57 ◦C. A total of 20 μL of a 25-fold diluted amino acid standard mix (AA-S-18 −5 ML analytical standard, SIGMA, or standard of basic amino acids, Type B, Hitachi, 016-08641) or 50 μL of cerebellar extracts were injected to the column. Fifteen amino acids eluted as separate peaks in this procedure were quantified. Post-column derivatization (136 ◦C, flow rate 0.35 mL/min) was performed using the mix of equal volumes of ninhydrin bu ffer R2 and ninhydrin solution R1 (Wako Pure Chemical Industries, P/N 298-69601). Colored products were detected by absorption at 570 nm. Data were processed using MultiChrom for Windows software (Ampersand Ltd., Moscow, Russia).

#### *2.5. Data Acquisition and Statistics*

Statistical analysis was performed using Statistica 10.0 (StatSoft Inc., Tulsa, OK, USA). Averaged values are presented as means ± SEM. Comparison between the two experimental groups was done using non-parametric Mann–Whitney U-test. The Pearsons correlations between the levels of di fferent amino acids or between the levels of amino acids and OGDHC activity were characterized by the correlation coe fficients and *p*-values of the correlation. Statistical significance of di fferences in the parameters characterizing metabolic interactions between the levels of OGDHC activity and/or amino acids were assessed by the Wilcoxon signed rank test. Di fferences with *p* ≤ 0.05 were considered significant.
