Animals and housing

The experiments were performed on male C57BL/6J mice (Charles River, Germany). Animals were maintained under standard laboratory conditions in terms of temperature (22 ± 2 °C), humidity (55 ± 10%), and lighting (light phase 7:00–19:00) with free access to food and tap water. The mice were seven weeks of age at the beginning of the experiment and were divided into two groups: control mice (non-stressed, designated NS) and mice subjected to CUMS. Each experimental group consisted of ten to twelve animals. The tests were performed by a person unaware of the individual’s affiliation to the experimental group. All procedures were conducted following the National Institutes of Health Animal Care and Use Committee guidelines and were approved by the Second Local Ethics Committee in Kraków, Poland (permission number: 154/2023). The three Rs principles were applied in the planning and execution of the experiments. Every effort was made to reduce the number of animals used and to avoid and minimize animal suffering.

Compounds and treatment

M-5MPEP (2-[2-(3-methoxyphenyl)ethynyl]-5-methylpyridine) was synthesized in the Department of Medicinal Chemistry, Maj Institute of Pharmacology Polish Academy of Sciences, by K.K. (compound synthesis and structure confirmation can be found in the Supplementary file). M-5MPEP and (S)-(+)-ketamine hydrochloride (Tocris Cookson, Ltd., Bristol, UK), UK) were diluted in a suspension of 0.5% methylcellulose/0.9% NaCl, which was used as a vehicle. All compounds and vehicles were injected intraperitoneally (i.p.) at a constant 10 ml/kg volume. Doses and times of drug administration were determined based on our own previous research and literature data.

CUMS and behavioral tests

The CUMS procedure was performed based on our experience and previously published experimental schedules with the necessary modifications (Pałucha-Poniewiera et al. 2020, 2021). The detailed experimental schedule, including the adaptation stage, chronic unpredictable mild stress, administration of test compounds, and subsequent behavioral experiments, is presented in Fig. 1.

Fig. 1

The schedule of the CUMS experiments. After ten days of adaptation, the animals were subjected to the CUMS procedure. Two stressors from those given in the scheme were applied daily, with a two-hour break between stressors. The NS group was not subjected to any procedures during this time. On the 18th day from the beginning of CUMS, drug administration started in the 4-day application group. On the 21st day, the animals were given the last of four doses of M-5MPEP or a single dose. In a reference group, a single dose of (S)-ketamine was given on the 21st day from the beginning of CUMS. After twenty-four hours, the splash test was performed, and the SPT started 2 h later. The next day, the TST was applied, and on the subsequent day, the locomotor activity was measured, after which the animals were handed over for tissue collection

The following procedures were used in the behavioral testing phase:

Splash test

The splash test was performed as described previously (Pałucha-Poniewiera et al. 2021) with minor modifications. Briefly, animals were adapted for the experimental room for 30 min. The test was performed under dimmed lighting. A high-viscosity 10% sucrose solution (approximately 0.2 ml) was sprayed on the dorsal coat of the mice to stimulate self-grooming behavior. Then, the duration of grooming was manually recorded for five minutes by an experimenter blind to the treatment.

Sucrose preference test (SPT)

The SPT was performed as previously described (Pałucha-Poniewiera et al. 2021). For 24 h, the animals could choose between one of the two identical bottles. The first bottle contained a 1% sucrose solution, and the second contained tap water. The position of the bottles was switched 12 h after the start of the experiment. At the beginning and end of the test, the bottles were weighed, and the liquid consumption was calculated. The preference for sucrose consumption was calculated as a ratio of the consumed sucrose solution to the total amount of liquid consumed.

Tail suspension test

The experiments were carried out as previously described (Pałucha-Poniewiera et al. 2020). The mice were habituated to the testing room for 30 min before the experiments. Each mouse was attached by its tail to the table’s edge with adhesive tape. The total duration of immobility was manually measured for six minutes by an experimenter blind to the treatment.

Locomotor activity

12-station photobeam activity system (Opto Varimex 4, Auto Track System 4.41, Columbus Instruments, Columbus, OH, USA) equipped with Plexiglas locomotor activity chambers (40 × 40 × 40 cm) was used to measure the locomotor activity of the mice. After placing the animals individually in the locomotor activity chambers, the total distance traveled during a 30-minute experimental session was measured and stored every 3 min.

Tissue preparation

After completing the behavioral tests, the animals were sacrificed by dislocating the cervical vertebrae. Then, the mice were decapitated, and the tissue was collected for further research (Western blot and Elisa). The animals intended for electrophysiological studies were anesthetized using isoflurane (Aerrane, Baxter, Deerfield, IL, USA).

Synaptosome preparation and Western blotting

Tissue samples were dissected from the PFC and hippocampus and frozen on dry ice. After thawing on ice, the samples were homogenized in ice-cold lysis buffer [0.32 M sucrose, 20 mM HEPES (pH 7.4), 1 mM EDTA; 1 × protease inhibitor cocktail, 5 mM NaF, and 1 mM NaVO3]. Homogenates were centrifuged at 2800 rpm for 10 min at 4 °C, and the resulting supernatant was centrifuged at 12,000 rpm for 10 min at 4 °C. The obtained pellets were then sonicated in protein lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 1 mM NaVO3, 5 mM NaF, and protease inhibitor cocktail). BCA kit (Thermo Scientific, USA) was used to measure protein concentrations. The proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. 1% of the blocking solution (BM Chemiluminescence Western Blotting Kit (Mouse/Rabbit) made by Roche, Switzerland) was used to block for 1 h. Then, the membranes were incubated overnight at 4 °C with the primary antibodies: Anti-mTOR (mTOR 1:1000; Cell Signaling Technology, USA), Anti-phospho-mTOR (pmTOR, S2481, 1:1000; Abcam, USA), Anti-eEF2 (eEF2 1:1000; Abcam, USA), Anti- phospho-eEF2 (pheEF2 (phospho T56) 1:1000; Abcam, USA), Anti-SERT (SERT 1:1000; Sigma-Aldrich, Germany), Anti-TrkB (TrkB 1:1000; Cell signaling Technology, USA). Afterward, the membranes were washed three times for 10 min using Tris-buffered saline with Tween (TBS-T) and incubated for 60 min with secondary antibodies (anti-mouse or anti-rabbit-IgG-peroxidase-conjugated antibodies Vector Laboratories, USA). After incubation, the membranes were washed thrice for 10 min with TBS-T and incubated with a detection reagent (Bio-Rad, USA). Fuji-Las 1000 system, equipped with Fuji Image Gauge v.4.0 software, was used to visualize and measure the signal. A primary monoclonal antibody, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:500; Millipore, Germany), was used to check the transfer and loading. The final result is the ratio of particular proteins’ optical density to GAPDH’s optical density.

Measurement of BDNF concentration

ELISA kit purchased from R&D Systems, Inc. Minneapolis, MN, USA (Catalog Number SBNT00) was used to measure the total BDNF concentrations. The samples were thawed on ice and 100-fold diluted in Calibrator Diluent RD5K. A 96-well microtiter plate containing 50 µl of Assay Diluent RD1-123 and 50 µl of each sample or standard was incubated for two hours at room temperature with vigorous mixing (550 rpm, ThermoMixer C, Eppendorf, Hamburg, Germany). After rinsing each well four times with 400 µl of wash buffer, an enzyme-linked monoclonal antibody specific for BDNF was added (200 µl) to the wells and incubated at room temperature for one hour. Then, the wells were washed four times with 400 µl of wash buffer. Then, 200 µl of substrate solution was added and incubated in the dark for one hour. The plates were read at 450 and 540 nm using a spectrophotometer (Synergy HTX multimode reader machine; BioTek Instruments Inc., Winooski, VT, USA). The data were linearized by plotting the total BDNF concentrations log versus the O.D log. The total BDNF concentration was normalized to the protein concentration of each sample.

Electrophysiological experimentSlice preparation

Under anesthesia conditions, the brains were rapidly removed from the skulls and prepared in NMDG-based, cold artificial cerebrospinal fluid according to the procedure described by Ting et al. (2018). Cortical slices (380 μm thick) obtained from the medial prefrontal cortex (mPFC) were cut in the coronal plane using a Leica VT 1000s vibrating microtome. The slices were incubated in carbogen bubbled ACSF containing (in mM) NaCl (132), NaHCO3 (26), CaCl2 (2.5), D-glucose (10), KCl (5), MgSO4 (1.3), and KH2PO4 (1.25) at 32 ± 0.5 °C, then transferred to the recording chamber (interface type) and superfused (2.5 mL/min) with ACSF containing (in mM) NaCl (132), NaHCO3 (26), CaCl2 (2.5), D-glucose (10), KCl (2), MgSO4 (1.3), and KH2PO4 (1.25).

Field potential recording and long-term potentiation (LTP) induction

A stimulating electrode (concentric, Pt-Ir; FHC, USA) was placed in cortical layer V. Basic stimulation (0.016 Hz frequency, duration of 0.2 ms) was applied using a constant-current stimulus isolation unit (WPI). Field potential (FP) recordings were performed using ACSF-filled glass micropipettes (1–2 MΩ). Glass electrodes were placed in cortical layer II/III. The recorded responses were amplified (EXT 10–2 F amplifier, NPI), then filtered (1 Hz-1 kHz), A/D converted (10 kHz sampling rate), and collected on a commercial personal computer with Micro1401 interface and Signal 4 software (CED). An input-output (stimulus-response) curve was made for every single slice. Stimulation intensity was increased stepwise from 0 to 100 µA with 5 µA steps to obtain the curve. One response was recorded at each stimulation intensity. Then, the stimulation intensity was adjusted to evoke a response of 30% of the maximum amplitude.

For LTP induction, theta burst stimulation (TBS) was used. TBS was composed of 10 stimuli trains at 5 Hz, repeated 5 times with 15-s gaps. The single train consisted of five 100 Hz pulses. The single pulse duration was increased from 0.2 to 0.3 ms.

For each slice, the stimulus-response data were fit with the Boltzmann equation: Vi = Vmax/(1 + exp((u − uh)/−S), where Vmax is the maximum FP amplitude; u is the stimulation intensity; uh is the stimulation intensity evoking FP of half-maximum amplitude; and S is the factor proportional to the slope of the curve.

Data analysis

All the results obtained were expressed as the mean ± standard error of the mean (SEM). GraphPad Prism 7.00 (GraphPad Software, San Diego, CA, USA) was used to analyze behavioral data. The effects obtained in the splash test, SPT, and TST were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Locomotor activity data were evaluated by repeated-measures ANOVA. The results obtained using Western blot and the ELISA method were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons tests using GraphPad Prism version 9.2.0 for Windows (GraphPad Software, San Diego, CA, USA). Statistical analysis of the electrophysiological data was done using two-way ANOVA followed by Tukey’s post hoc test using GraphPad Prism 4.00 (GraphPad Software, San Diego, CA, USA). The results were considered to be significant if the p-values were below 0.05.