Ethics and Animal Use Statement

Male C57bl/6 mice (30–45 day-old) were used and randomly assigned to two main groups: (i) tDCS-treated and (ii) sham-stimulated animals (controls). Different groups of mice were used for each experimental test and time point from tDCS.

All animal procedures were approved by the Ethics Committee of the Catholic University and were fully compliant with Italian (Ministry of Health guidelines, Legislative Decree No. 116/1992) and European Union (Directive No. 86/609/EEC) legislations on animal research.

The methods were carried out in strict accordance with the approved guidelines.

The animals were housed under a 12-h light-dark cycle at room temperature (RT: 19–22 °C). Efforts were made to limit the number of animals used and to minimize their suffering.

Electrode implantation and transcranial direct current stimulation

For tDCS stimulation we adopted an electrode montage widely used and well validated in both rat and mouse models consisting in an unilateral epicranial electrode arrangement48,49. Specifically, the active electrode consisted of an epicranial implanted tubular plastic jack (inner area = 6.25 mm2; model GB3305-00, Star electronic SPA, Italy) filled with saline solution (0.9% NaCl) just prior to stimulation; the counter electrode was a conventional rubber-plate electrode surrounded by a wet sponge (5.2 cm2; Physiomed Elektromedizin AG, Germany) applied over the ventral thorax by a custom corset. According to the literature this unipolar arrangement prevents currents from bypassing between two juxtaposed epicranial electrodes.

For the epicranial electrode implant, animals were anesthetized with a cocktail of ketamine (70 mg/Kg, intramuscular injection [i.m.]) and medetomidine (1 mg/Kg, i.m.) and temperature during surgery was maintained at 37 °C. The scalp and underlying tissues were removed and the electrode was implanted using a carboxylate cement (3 M ESPE, Durelon, 3 M Deutschland GmbH, Germany). The centre of the active electrode was positioned on the skull over the left hippocampal formation 1 mm left and 1 mm posterior to bregma26. After surgery, all animals were allowed to recover for 3–5 days before undergoing tDCS.

TDCS was applied to awake and freely moving mice at a current intensity of 350 μA for 20 min using a battery-driven, constant current stimulator (Stimulus Isolator, model: A385, World Precision Instruments, USA). This intensity corresponded to a current density of 56 μA/mm2 (0.35 mA/0.0625 cm2) that was within the range of that used previously in rats48 (57.1 μA/mm2) or mice38,49 (55.5 μA/mm2). The current intensity was ramped for 10 s instead of switching it on and off directly to avoid a stimulation break effect. The animals were observed during tDCS and in vivo experiments to determine possible abnormal behaviors related to the stimulation. Control animals received sham stimulation in which no current was applied but the animal underwent the same manipulations as in the stimulation condition. We chose to apply tDCS over the left hippocampus given that, experimental evidences suggest that long-term memory processing is strictly dependent on the left hemisphere65.

Experimental design and protocol timeline of experiments are summarized in Figs 1, 7a and 8a. Electrophysiological, molecular and behavioral analyses were performed either at short-term (2–24 h) and long-term (7 days) intervals from sham stimulation or tDCS, to assess the duration of effects elicited by a single tDCS session.

Investigators were blinded to the identity of the groups during experiments and analysis.

Histological processing

Histological evaluation was carried out to detect possible current-induced neurotrauma (e.g., oedema, necrosis, haematoma, cellular alterations). At the end of the stimulation session animals were deeply anesthetized with a cocktail of ketamine (80 mg/Kg, i.m.) and medetomidine (1 mg/Kg, i.m.) and perfused transcardially with saline followed by a fixative containing 4% paraformaldehyde in 0.1 M PBS. After post-fixation, brains were removed from the skulls and stored at 4 °C in a high sucrose solution (30% sucrose in 0.1 M PBS) for 2 days. A vibratome (VT1000S, Leica Microsystems) was used to collect serial coronal 40-μm thick sections containing the hippocampus. All sections were further processed for hematoxylin-eosin staining. Images were acquired with Olympus BX3-CBH microscope.

Electrophysiology

Whole-cell patch-clamp and field recordings were performed on hippocampal coronal slices (300- and 400-μm-thick, respectively) as previously described28,66,67. Briefly, mice were anesthetized by halothane inhalation (Sigma) and decapitated. The brain was rapidly removed and put in ice-cold cutting solution (in mM: 124 NaCl, 3.2 KCl, 1 NaH 2 PO 4 , 26 NaHCO 3 , 2 MgCl 2 , 1 CaCl 2 , 10 glucose, 2 sodium pyruvate and 0.6 ascorbic acid, bubbled with 95% O 2 -5% CO 2 ; pH 7.4). Slices were cut with a vibratome (VT1000S) and incubated in artificial cerebrospinalfluid (aCSF; in mM: 124 NaCl; 3.2 KCl; 1 NaH 2 PO 4 , 1 MgCl 2 , 2 CaCl 2 ; 26 NaHCO 3 ; 10 glucose; pH 7.4; 95% O 2 -5% CO 2 ) at 32 °C for 60 min and then at RT until use. Slices containing the left (i.e., stimulated) hippocampus were used for subsequent analyses.

Slices were transferred to a submerged recording chamber and continuously perfused with aCSF (flow rate: 1.5 ml/min). The bath temperature was maintained at 30–32 °C with an in-line solution heater and temperature controller (TC-344B, Warner Instruments). Identification of slice subfields and electrode positioning were done with 4× and 40× water immersion objectives on an upright microscope equipped with differential interference contrast and fluorescence optics under infrared illumination (BX5IWI, Olympus) and video observation (C3077-71 CCD camera, Hamamatsu Photonics).

All recordings were made using MultiClamp 700 A amplifier (Molecular Devices). Data acquisition and stimulation protocols were performed with the Digidata 1440 Series interface and pClamp 10 software (Molecular Devices). Data were filtered at 1 kHz, digitized at 10 kHz and analyzed both online and offline.

Field recordings were made using glass pipettes filled with aCSF (tip resistance 2–5 MΩ) and placed in the stratum radiatum of the CA1 region. FEPSPs were evoked by stimulation of the Schaffer collaterals with a bipolar tungsten electrode (FHC, USA) connected to a S11 Grass stimulator (Grass Instruments).

The stimulation intensity that produced one-third of the maximal response was used for the test pulses, LTP induction and paired-pulse facilitation protocols. The fEPSP amplitude was measured from baseline to peak.

Before the LTP induction protocol, to check for a possible effect of tDCS on the basal synaptic transmission, I/O curves were obtained i) by recording fEPSPs induced by presynaptic stimulation at intensities ranging from 0 to 50 V (in 5 V-steps); ii) by plotting fEPSP amplitudes against presynaptic fiber volley amplitudes and then comparing the angular coefficients of I/O slopes. On the same synapses paired-pulse facilitation was also examined at 50 ms interpulse intervals.

For LTP recordings, stable baseline responses to test stimulations (once every 20 s for 10 min) were recorded and then HFS was delivered (4 trains of 50 stimuli at 100 Hz, 500 ms each, repeated every 20 s)19,68. Responses to test pulse were recorded every 20 s for 60 min to assess LTP. LTP magnitude was expressed as the percentage change in the mean fEPSP peak amplitude normalized to baseline values (i.e., mean values for the last 5 minutes of recording before HFS, taken as 100%). The occurrence of LTP was statistically verified (paired Student’s t-test) by comparing mean fEPSP amplitude measured at 55–60 min after HFS relative to baseline responses (Supplementary Table 1). Slices were prepared soon after the delivery of stimulation protocol and LTP recordings started ~2 hours after tDCS or sham stimulation.

Whole-cell voltage-clamp recordings were performed to measure the AMPA/NMDA ratio, expressed as the peak AMPA evoked post-synaptic currents (EPSCs, mean values of 30–50 events) at −70 mV divided by the peak NMDA EPSCs (mean values of 20–40 events) measured at 50 ms after the onset of the dual EPSC component at +40 mV.

For this set of recordings the electrodes were filled with internal solution containing (in mM): 135 CsMeSO 3 , 8 NaCl, 10 HEPES, 0.25 EGTA, 2 Mg 2 ATP, 0.3 Na 3 GTP, 0.1 spermine, 7 phosphocreatine and 5 QX-314 (pH: 7.25–7.30; 294–298 mOsm/l). We monitored the access resistance and membrane capacity before and at the end of the experiments to ensure recording stability and the health of studied cells. Recordings were considered stable when the series and input resistances, resting membrane potential and stimulus artifact duration did not change > 20%.

Behavioral tests

A set of animals was tested in the MWM and NOR that are the most widely used and standardized tests to investigate hippocampal-dependent learning and memory51,52. To better link changes of learning and memory performances to tDCS we avoided challenging the animals in more than one behavioral test. Therefore different groups of mice were used for each behavioral test and time point from tDCS (Fig. 1).

Mice were trained in the MWM to find a platform hidden 1 cm below the surface of a pool (127 cm in diameter) filled with water made opaque with white nontoxic paint. The acquisition training session started 4 days before the test session (probe test) and consisted of six trials a day for 4 consecutive days, during which the animals were allowed to reach the platform within 40 s. Starting points were changed daily and different starting points were used for each trial. A trial lasted either until the mouse had found the platform or for a maximum of 40 s. Mice rested on the platform for 10 s after each trial. Time (latency) needed to navigate to the platform and swim path length were recorded by an automated video tracking system (Panlab Harvard Apparatus). The probe test session was performed 24 h after the last day of the training. In this session, the platform was removed and each mouse was allowed to swim for 60 s; the time spent in each quadrant was measured69.

The NOR protocol lasted three consecutive days including a familiarization phase, a training phase and a test phase. On the 1st day, mice were individually submitted to a single familiarization session of 10 min, during which they were introduced into the empty arena (45 × 45 cm). On the 2nd day, animals were submitted to a single 10 min session (training phase) during which two identical objects were placed in a symmetric position from the centre of the arena. An explorative behavior was scored when the head of the animal was facing close (>2 cm away) to the object or any part of the body except the tail was touching the object. The time spent exploring each object was recorded. The animals were returned to their home cages immediately after training. On the 3rd day, during the test phase, one of the familiar objects used during the training was replaced by a novel object and the animals were allowed to explore freely for 10 min. All objects were balanced in terms of physical complexity and were emotionally neutral. The open-field and the objects were cleaned by 70% alcohol after each session to avoid possible odorant cues. Preference index, i.e., the ratio of the amount of time spent exploring any one of the two items or the novel object over the total time spent exploring both objects, was used to measure recognition memory70.

RNA extraction and cDNA synthesis

Total RNA was extracted from the left hippocampi of control and tDCS-mice using QIAzol Lysis reagent (Qiagen) according to the manufacturer’s instructions. RNA sample integrity and concentrations were evaluated with the BioPhotometer plus (Eppendorf, Germany). Reverse transcription reactions were performed on equal amounts of RNA (2 μg) with a high capacity cDNA reverse transcription kit (Applied Biosystems, USA).

Semiquantitative RT-PCR and quantitative Real-Time PCR

Semiquantitative PCR of the cDNA was performed using Taq Polymerase (Fischer) and primers described by Aid et al.23 (Supplementary Table 3). The values of the control samples were set to 1.0 and the others were expressed as fold changes relative to the controls. Analyses were performed in triplicate on the left hippocampi obtained from controls and tDCS-mice.

Quantitative Real-Time PCR (qRT-PCR) amplifications were performed using Power SYBR® Master Mix on AB7500 instrument (Life Technologies) according to the manufacturer’s instructions. The thermal cycling profile featured a pre-incubation step of 94 °C for 10 min, followed by 40 cycles of denaturation (94 °C, 15 s), annealing (55–57 °C, 30 s) and elongation (72 °C, 20 s). Melting curves were subsequently generated by heating amplified products at 94 °C for 15 s, cooling to 50 °C for 30 s, followed by slow heating to 94 °C in increments of 0.5 °C. Melting-curve analyses confirmed that only single products had been amplified. The primer sequences coding the Bdnf exons I, IV and IX are shown in Supplementary Table 4. All data were normalized by reference to the amplification levels of the glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene; a reference dye was included in the SYBR master mix. The thresholds calculated by the software were used to calculate specific mRNA expression levels using the cycle-at-threshold (Ct) method and all results are expressed as fold changes (compared to control) for each transcript, employing the 2-ΔΔCt approach.

Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) assays were performed as previously described with minor modifications69. Animals were sacrificed 24 h or 1 week after the end of the stimulation protocol (Fig. 1). Mice were anesthetized with a cocktail of ketamine (80 mg/Kg, i.m.) and medetomidine (1 mg/Kg, i.m.) and transcardially perfused with an oxygenated Ringer’s solution (pH: 7.3), followed by 4% freshly depolymerized paraformaldehyde in 0.1 M PBS (pH:7.4). The brain was post-fixed overnight at 4 °C and then transferred to a solution of 30% sucrose in PBS for 2 days. Coronal brain sections (45-μm-thick) containing hippocampi were then cut with a vibratome (VT1000S) and floated in ice-cold PBS.

Left hippocampi were isolated under optic microscope and minced through a 10 ml-syringe with decreasing needle size (18 to 22 gauge). Tissue lysate was resuspended in 200 μl lysis buffer containing SDS (1%), Tris-HCl (50 mM, pH 8.1) and EDTA (10 mM) and sonicated on ice with six 10-s pulses with a 20-s interpulse interval. Sample debris was removed by centrifugation and supernatants were precleared by incubation with protein-G Sepharose 4B beads (Sigma) for 1 h at 4 °C. Beads were collected by centrifugation and supernatants were subjected to immunoprecipitation. Two μg of specific antibody (anti-pCREBSer133, anti-acetyl histone H3K9 from Millipore, anti-CBP from Abcam) or control IgG were added overnight at 4 °C. Immune complexes were collected by incubation with protein-G Sepharose 4B beads for 2 h at 4 °C and subjected to a series of seven sequential washes. Immune complexes were eluted from beads by vortexing in elution buffer (1% SDS and NaHCO 3 0.1 M; pH 8.0). NaCl was added (final concentration 0.33 M) and cross-linking was reversed by incubation overnight at 65 °C. DNA fragments were purified by using the PCR DNA fragments purification kit (Geneaid). The primer sequences for promoter I were designed on the basis of mouse Bdnf structure described by Aid et al.23 (Supplementary Table 2).

PCR conditions and cycle numbers were determined empirically and each PCR reaction was performed in triplicate. Data are expressed as percentage of input calculated by the “Adjusted input value” method according to the manufacturer’s instructions (ThermoFisher Scientific ChIP Analysis). In particular to calculate the Adjusted input the Ct value of input was subtracted by 6.644 (i.e., log2 of 100). Next, the percent input of control and tDCS samples was calculated using the formula: 100*2^(Adjusted input – Ct(ChIP). In the same way, the percent input of IgG samples was calculated using the formula 100*2^(Adjusted input – Ct(IgG).

Semiquantitative PCR amplification of Bdnf regulatory sequences performed on chromatin immunocomplexes showed that tDCS induced enhancement of H3K9 acetylation at promoter I but not at promoters II, III and IV, thus supporting specificity of tDCS effects (Supplementary Fig. 3b and Supplementary Table 3; same samples used for ChIP assays showed in Fig. 5d).

Western Immunoblot

Total proteins were extracted from the left hippocampi of control and tDCS-mice sacrificed 2 h after stimulation, by using ice cold RIPA buffer (Pierce; 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% DOC, 1% Triton X-100, 0.1% SDS and 1 × protease, phosphatase-1 and phosphatase-2 inhibitor cocktails [Sigma]). The lysate was centrifuged (20,000 × g, 30 min, 4 °C) and a 5-μl aliquot of the supernatant was assayed to determine the protein concentration (microBCA kit, Pierce). SDS-PAGE reducing sample buffer was added to the supernatant and samples were heated to 95 °C for 5 min. Protein lysates (40–60 μg) were loaded onto 10% Tris-glycine polyacrylamide gels for electrophoretic separation. ColorburstTM Electrophoresis markers (Sigma) were used as molecular mass standards. Proteins were then transferred onto nitrocellulose membranes at 100 V for 2 h at 4 °C in transfer buffer containing 25 mM Tris, 192 mM glycine, 0.1% SDS and 20% methanol. Membranes were incubated for 1 h with blocking buffer (5% skim milk in TBST) and then incubated overnight at 4 °C with primary antibodies directed against one of the following proteins: pCREBSer133; CREB; pGSK-3βSer9; GSK-3β; panH3ac; NeuN; actin and tubulin (1:1,000). After three 10-min rinses in TBST, membranes were incubated for 1 h at RT with HRP-conjugated secondary antibodies (1:2,500). The membranes were then washed and the bands were visualized with an enhanced chemiluminescence detection kit (GE Healthcare, UK).

Protein expression was evaluated and documented by using UVItec Cambridge Alliance. Experiments were performed in triplicate.

Images of Western blots have been cropped for presentation and full-size images are shown in Supplementary Figs 5 and 8. Antibodies are listed in Supplementary Table 5.

Curcumin and ANA-12 administration

Curcumin (Sigma) was diluted in DMSO40 (i.e., vehicle). Mice received curcumin 50 mg/kg or vehicle via intraperitoneal (i.p.) injection. Mice were randomly assigned to sham or tDCS-group. Curcumin or vehicle was given 24 h prior to tDCS or sham stimulation, soon before stimulation and the following day (Fig. 7a). Two hours later some animals were engaged in: i) the habituation session of the NOR test; ii) the 1st training session of the MWM; iii) tissue explant for ELISA assay. A week after stimulation (i.e., 6 days after the last curcumin injection) the other mice were used for: i) slice preparation for LTP recordings; ii) tissue explant for ELISA assay (Fig. 7a).

For experiment with ANA-12, mice were divided in two groups receiving sham stimulation or anodal tDCS and subjected to NOR or MWM tests. A volume of 10 μl/g body weight was i.p. injected for vehicle and ANA-12 (0.5 mg/kg body weight) solutions43. TDCS was applied 24 h before the 1st day of either NOR or MWM test (Fig. 8a).

Mice subjected to the NOR test were further distributed into ANA-12 and vehicle-injected groups (1% DMSO dissolved in 0.9% NaCl solution). ANA-12 was injected 4 h before and soon after the training day of the NOR test as well as 4 h before the test phase.

Animals subjected to the MWM received ANA-12 injection 4 h before: i) each of the 4 training sessions and ii) the probe test session.

With regard to behavioral tests, given that statistical analysis revealed no significant effect of vehicle administration on the NOR test (P = 1 vs. naïve, unpaired Student’s t-test), we enrolled in the MWM test only two groups of animals, including tDCS-mice and sham-stimulated mice all injected with curcumin or ANA-12.

ELISA measurements

Brain tissues were obtained as described for Western blot and stored at −80 °C. Prior to analysis, samples were thawed and then weighed. Lysis buffer (100 mM PIPES pH 7, 500 mM NaCl, 0.2% Triton X-100, 2% BSA, 2 mM EDTA, 200 μM PMSF, 1 × protease, phosphatase-1 and phosphatase-2 inhibitor cocktails from Sigma) was then pipetted into each tube (100 μl per mg of tissue for each left hippocampus). Samples were homogenized, sonicated and centrifuged for 30 min at 16,000 × g at 4 °C. Supernatants were then removed and frozen at −80 °C until analysis. The concentration of Bdnf was determined using the E-Max ImmunoAssay system (Promega) according to the manufacturer’s instructions.

Statistical analysis

Sample sizes were chosen with adequate statistical power (0.8) according to results of prior pilot data sets or studies, including our own, that used similar methods or paradigms. Sample estimation and statistical analysis were performed using the SigmaPlot 12 software. Data were first tested for equal variance and normality (Shapiro-Wilk test) and then the appropriate statistical tests were chosen. The statistical tests used (i.e., Student’s t-test, Mann-Whitney test, one-way ANOVA, two-way ANOVA, two-way RM ANOVA) are indicated in the main text and in the corresponding figure legends for each experiment. Post-hoc multiple comparisons were performed with Bonferroni correction.

All statistical tests were two-tailed and the level of significance was set at 0.05. Results are presented as mean ± s.e.m. In the graphs, y-axis error bars represent s.e.m. Analyses were performed in blind.

Criteria of animal exclusion/inclusion were pre-established according to Ethics Committee guidelines but no data were excluded from analysis.