Mice and slice preparation for electrophysiology. Experiments were performed on 4- to 6-week-old male C57BL/6J mice as described in (71). Mice were treated in accordance with the ethical guidelines for the use of animals in experiments; experiments were approved by the local animal care committee (Landesverwaltungsamt Sachsen-Anhalt). The animals were decapitated after cervical dislocation. The brain was rapidly removed and placed in ice-cold solution containing 230 mM SUC, 2.5 mM KCl, 7 mM MgCl2, 1.25 mM NaH2PO4, 26.6 mM NaHCO3, 0.5 mM CaCl2, and 10 mM d-glucose (all chemicals here and below were from Sigma-Aldrich, Steinheim, Germany). The frontal lobe was removed, and the brain was glued to a vibratome stage. Horizontal or transverse hippocampal slices were cut at 350 μm with a vibrating microtome (VT1200S, Leica). The slices were then incubated at room temperature (23° to 25°C) for at least 1 hour in a submerged chamber to recover in artificial cerebrospinal fluid (ACSF) containing 113 mM NaCl, 2.38 mM KCl, 1.24 mM MgSO4, 0.95 mM NaH2PO4, 24.9 mM NaHCO3, 1 mM CaCl2, 1.6 mM MgCl2, and 27.8 mM d-glucose. Subsequently, the slices were transferred to the recording chamber and were continuously perfused (2 to 3 ml/min) with carbogen-bubbled ACSF containing 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 1 mM NaH2PO4, 26.2 mM NaHCO3, 2.5 mM CaCl2, and 11 mM d-glucose. All solutions were saturated by 95% O2/5% CO2 and adjusted to a pH of 7.4 and an osmolarity of 295 ± 5 mosM. Before whole-cell patch-clamp recording, Rhodiola extractcrude (0.28 μg/ml) or FAE-20 (0.1, 1.0, and 4 μM) was bath applied. The vehicle (80% ethanol) was applied in the same way.

Whole-cell patch-clamp recordings and data analysis. Recordings were obtained using an EPC-10 amplifier (HEKA Elektronik, Lambrecht, Germany). The data were digitized at 20 kHz and filtered at 2 to 3 kHz. All recordings were made at room temperature in whole-cell configuration from CA1 pyramidal cells visually identified with an infrared differential interference contrast microscope (SliceScope, Scientifica, Kings Grove, UK). Whole-cell patch-clamp recordings were performed with glass pipettes (4 to 5 megohms; Hilgenberg, Malsfeld, Germany) filled with internal solution containing 140 mM K-gluconate, 8 mM NaCl, 0.2 mM CaCl2, 10 mM Hepes, 2 mM EGTA, 0.5 mM NaGTP, and 2 mM MgATP (pH 7.2 with KOH; 290 mosM). To evoke action potentials, current pulses were applied using a patch amplifier in current-clamp mode. A series of 14 current pulses (500-ms duration, from −80 to 440 pA in 40-pA increments) were applied. Membrane potential was held at −70 mV during interpulse intervals by injecting direct current using the Patchmaster software (HEKA Elektronik). The numbers of action potentials at each current step were counted.

Excel (Microsoft, USA) and SigmaPlot 12.3 (Systat Software Inc., Erkrath, Germany) were used for statistical analyses and graphical presentation of electrophysiological data, presented as the means ± SEM. Statistical significance was determined using two-way rmANOVA.

Contextual fear conditioning in mice. For the contextual fear conditioning experiments, young adult (3 months old) or aged (2.4 to 2.8 years old) C57BL/6J male mice (Charles River, Sulzfeld, Germany) were used as mentioned in Results. They were housed in groups of three to four animals and had free access to food and water. All experiments took place during the light phase of the 12-hour light/12-hour dark cycle. The experiments were carried out in accordance with the European Committee Council Directive (86/609/EEC) and were approved by the local animal care committee (Landesverwaltungsamt Sachsen-Anhalt 42502-2-1191).

We used an automated system (TSE Systems, Bad Homburg, Germany) for fear conditioning, located in a sound-attenuating chamber. We used cubic test boxes (23 cm by 23 cm), which were surrounded by an array of infrared light beams to detect the movements of the animals. The floor consisted of a grid, by which the unconditioned stimulus (1-s, 0.5-mA scrambled foot shock) could be delivered. To provide distinct contexts, the color of the test boxes (black or transparent), the floor (grid or plastic floor), the odor [70% alcohol or Deskosept (Dr. Schumacher GmbH, Melsungen)], and the background noise (provided by a fan) could be changed. To avoid any bias, these different contextual stimuli were randomly changed.

On the first day, the animals were placed into the conditioning chamber for a total of 10 min. After a 2-min habituation period, they received three scrambled foot shocks (1 s, 0.5 mA) at random intervals (1.5 to 4 min). After the last foot shock, the animals remained in the chamber for the last 2 min. Twenty-four hours after the conditioning, the animals were first tested for unspecific fear by exposing them for 5 min to a neutral novel context and scoring their freezing behavior. One hour later, we exposed the animals for 10 min to the context in which the training had been carried out (training context) and scored their freezing behavior.

To apply FAE-20 to the animals, 3 ml of a 4.06 mM FAE-20 solution [10% (v/v) ethanol in phosphate-buffered saline (PBS)] per kilogram of body weight (i.e., 6 mg of FAE-20 per kilogram of body weight) or, for the control, 3 ml of an ethanol solution [10% (v/v) ethanol in PBS] per kilogram of body weight was injected intraperitoneally 30 min before the fear conditioning started. To test an FAE-20 concentration that was twice as high, 6 ml of the abovementioned 4.06 mM FAE-20 solution per kilogram of body weight (i.e., 12 mg of FAE-20 per kilogram of body weight) or of an ethanol solution [10% (v/v) ethanol in PBS] per kilogram of body weight for the control was injected.

The behavioral data of the mice were analyzed by rmANOVA using the intraperitoneally injected “drug” as the between-subject factor and the “context” of testing as the within-subject factor. For the detailed group comparisons, the ANOVA was followed by Fisher’s LSD post hoc comparisons. P < 0.05 was considered a statistically significant difference.

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