Animals

Tph2−/− mice24 and control Tph2+/+ male littermates were housed in standard Plexiglas cages at constant temperature/humidity (22 ± 1 °C, 50–60%) and maintained on a 12/12 h light/dark cycle, with food and water ad libitum. All animals used in each experiment were on a C57BL/6 genetic background. Experimental protocols were conducted in accordance with the Ethic Committee of the University of Pisa and approved by the Veterinary Department of the Italian Ministry of Health.

Experimental design and statistical analysis

Tph2−/− (KO) and control Tph2+/+ (WT) mice were individually housed after weaning, due to their high aggressive behaviour. For each behavioural test, independent cohorts were used to avoid potential confounding effects due to behavioural tests (Forced Swim Test WT n = 13, KO n = 12; Tail Suspension Test WT n = 10, KO n = 9; Novelty-Suppressed Feeding WT n = 14, KO n = 11; Neutral Arena Aggression Test n = 12). For locomotion analysis (Home Cage Locomotion n = 11), 5-minutes interval were scored for both square entries and rearings and the difference between mutants and controls were calculated for each time point separately. Independent cohorts of 10–15 weeks old male mice were used for functional Magnetic Resonance Imaging (n = 10), RNA-seq experiment (n = 3), BDNF protein level measurements (n = 8), electrophysiological recordings (WT n = 8; KO n = 9), dendritic spine analysis (WT n = 7; KO n = 8). FST experiment after unpredictable Chronic Mild Stress (uCMS) protocol was performed on independent cohorts composed by KO (n = 18) and control WT (n = 21) subjected to uCMS and KO (n = 17) and WT (n = 20) left undisturbed in their home cages. Immediately after the FST, the hippocampus of these animals was dissected and used for either BDNF/TrkB protein level measurement (WT n = 9, KO n = 8, WT-S n = 10, KO-S n = 9) or RNA-seq (n = 3). TST experiment after unpredictable Chronic Mild Stress (uCMS) protocol was performed on independent cohorts composed by KO (n = 14) and control WT (n = 15) subjected to uCMS and KO (n = 12) and WT (n = 10) left undisturbed in their home cages.

All values are expressed as mean ± s.e.m. unless stated otherwise. One- or two-way ANOVA tests with Fisher’s post hoc tests were used. For electrophysiological recordings, data were analysed by one-way repeated measures ANOVA (RM1W) for comparisons within a group; post-hoc analysis (Tukey’s) was performed only when ANOVA yielded a significant main effect. Two groups were tested for statistical significance using a Student’s t test. Statistically significant differences were considered at p < 0.05. Statistical analysis was performed using Statview 5.0.1 and GraphPad Prism 6.

Behavioural testing

All behavioural procedures were performed following standard protocols during the light phase of the light/dark cycle (11:00–13:00 h). Depression-like behaviours were assessed in the Forced Swim Test (FST) and Tail Suspension Test (TST) that were performed following standard protocols. Briefly, in the FST mice were placed in a 5L Plexiglas Beaker containing 4L of 26 °C water and video-recorded for 6 min. Minutes from 2 to 6 were analysed for immobility time. In the TST, mice were hanged by their tail from a bar 50 cm from the ground with a piece of autoclave tape and were recorded in a 6 min session. Minutes from 2 to 6 were analysed for immobility time. For both tests, immobility was considered as absence of any active movement of the paws. Anxiety-like behaviour was analysed in the Novelty-Suppressed Feeding (NSF). Food was removed from the cages of mice 24 h before testing. The next day, mice were placed for 10 min in a bright white arena (38 × 35 × 20 cm) without bedding with a food pellet at the centre. Mice were video-recorded and latency to feed was assessed offline. After the test, to avoid confounding effects of feeding behaviour on anxiety, hunger was measured by weighting before and after 5 min a single pellet of food placed in the home cage. Aggressive behaviours were measured in a Neutral Arena Aggression Test (NAAT). Two mice of the same genotype were placed in a novel standard cage (42.5 × 26.5 × 18.5 cm) with bedding and video-recorded for 10 min. Lateral threats and clinch attacks were considered as sign of aggression. Latency to the first attack, attack duration and number of attacks in 10 min were measured. Locomotor habituation to novelty was measured in the Novel Home-Cage (NHC) paradigm. Briefly, mice were individually placed into a novel standard cage (42.5 × 26.5 × 18.5 cm) with bedding and video-recorded for 60 min. The area of the cage was virtually subdivided in squares and the times the testing mouse crossed one of the grid lines with all four paws (i.e. square entry) was scored, as well as the times it stood on its hind legs (i.e. vertical activity, also known as rearing). Hedonic behaviour was assessed by sucrose preference test. Briefly, single-housed male mice were habituated to the presence of two bottles containing water for two days. On day 0, bottles were replaced by one containing water and the other containing 1% sucrose solution in drinking water. The two bottles were switched every 12 hours to reduce side bias and weighed every 24 hours.

Valproate treatment

Valproate sub-chronic treatment was performed according to Flaisher-Grinberg and Einat25. Briefly, Valproate (Sigma) was dissolved in saline solution to obtain a dose of 100 mg/kg in 10 ml/kg injection volume. Mice were injected intraperitoneally twice a day (12 h interval) for two consecutive days. On the third day, mice received the last injection 30 min before the behavioural test. Control mice received an equivalent volume of saline solution.

In vivo functional Magnetic Resonance Imaging (fMRI)

Animal preparation

Magnetic Resonance Imaging experiments were performed on adult Tph2+/+ (n = 10) and Tph2−/− (n = 10) littermate male mice. Briefly, mice were anaesthetized with isoflurane (5%), intubated and artificially ventilated. The left femoral artery was cannulated for contrast agent administration, continuous blood pressure monitoring and blood sampling. At the end of surgery, isoflurane was discontinued and substituted with halothane. Experiments were carried out at a maintenance anesthesia level of 0.8%. Arterial blood gases (paCO 2 and paO 2 ) were measured at the end of the functional time series. The values recorded were 16 ± 4 mmHg (paCO 2 ), 287 ± 95 mmHg (paO 2 ) and 17 ± 4 mmHg (paCO2), 272 ± 86 mmHg (paO 2 ) for Tph2−/− and control, respectively. No significant inter-group difference in paCO 2 or (paO 2 ) levels was observed between groups (p > 0.65, Student’s t test). Functional data acquisition commenced 30 min after isoflurane cessation.

Image Data Acquisition

All experiments were performed using a 7.0 Tesla MRI scanner (Bruker Biospin, Milan). Transmission and reception were achieved using a 72 mm birdcage transmit coil and a custom-built saddle-shaped solenoid coil for signal reception. Shimming was performed on a 6 mm × 6 mm × 6 mm region, using a FASTMAP protocol. For each session, high-resolution anatomical images were acquired with a fast spin echo sequence (RARE) with the following parameters: repetition time (TR)/echo time (TE) 3550/40 ms, matrix 192 × 192, field of view 2 × 2 cm2, 28 coronal slices, slice thickness 0.50 mm. Co-centered Cerebral Blood Volume (CBV) weighted fMRI times series were acquired using a Fast Low-Angle Shot (FLASH) MRI sequence with the following imaging parameters: FLASH TReff = 283.023 ms, TE = 3.1 ms, α = 30°; FOV 2 × 2 cm2, 156 × 156 × 500 µm resolution, dt = 60 s, Nr = 60, corresponding to 60 min total acquisition time. Images were sensitized to reflect alterations in CBV by injecting 5 µl/g of superparamagnetic iron oxide (Molday Ion, Biopal) intra-arterially after 5 baseline images.

Basal CBV mapping

To calculate basal CBV (bCBV), CBV-weighted time series were spatially normalized to a study-based anatomical template, and signal intensity was converted into basal cerebral blood volume (bCBV(t)) pixel-wise. bCBV time-series were calculated over a 5 minute time-window starting 15 min after contrast agent injection. Voxel-wise group statistics was carried out using FSL using multi-level Bayesian inference and a T threshold >2.1, and corrected cluster significance threshold of p = 0.01.

RNA extraction and whole transcriptome RNA analysis

Whole hippocampal tissue from n = 3 mice was rapidly dissected and quickly frozen in liquid nitrogen. Total RNA was extracted using the automated Maxwell 16 LEV RNA FFPE Purification Kit with the Maxwell 16 Instrument (Promega, Madison, WI, USA). We followed the manufacturer’s instructions protocol starting from the Lysis Buffer and Proteinase K step excluding the Mineral Oil procedure. Hippocampus tissue was homogenized in Lysis Buffer using a pestle.

RNA-seq was performed using NextSeq. 500 (Illumina, San Diego, CA, US) for Next Generation Sequencing. The library was prepared following the protocol TruSeq Stranded mRNA LT kit (Illumina). Libraries were quantified using Qubit 2.0 Fluorometer (Invitrogen, Life Technologies, Grand Island, NY) and the size profile was analyzed on the 2200 TapeStation instrument (Agilent Technologies, Santa Clara, CA).

Raw data were converted to FASTQ format using bcl2fastq (Illumina). We used the FastQC quality control tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to perform quality assessment. In addition, we evaluated raw data contamination from different organisms (bacteria, fungi, virus) by applying FastqScreen (http://www.bioinformatics.babraham.ac.uk/projects/fastq_screen/). RNA-Seq reads were aligned to the mouse genome (mm10; UCSC) with STAR aligner 2.5.1 (https://github.com/alexdobin/STAR). Differential expression between conditions was calculated using Cuffdiff (http://cole-trapnell-lab.github.io/cufflinks/cuffdiff/). All RNA-Seq analyses were performed in the cloud using the Seven Bridges Genomics platform (www.sbgenomics.com). Gene Set Enrichment Analysis was performed using GSEA (http://software.broadinstitute.org/gsea/). Hierarchical gene clustering on differentially expressed genes was performed using Bioconductor ctc package on R software 3.0.1 (https://www.bioconductor.org/packages/release/bioc/html/ctc.html).

Electrophysiological recordings

Extracellular recordings of field postsynaptic potentials (fPSP) were obtained in the CA1 stratum radiatum, using glass micropipettes filled with artificial Cerebral Spinal Fluid (aCSF). Stimuli (50–160 μA, 50 μs) to excite Shaffer collaterals were delivered through a bipolar twisted tungsten electrode placed 400 μm from the recording electrode. Long-Term Potentiation (LTP) was induced using the following theta burst stimulation protocol (TBS): 10 trains (4 pulses at 100 Hz) at 5 Hz, repeated twice with a 2-min interval. The magnitude of LTP was evaluated by comparing the fPSP normalized slopes from the last 5 min of baseline recordings with those 40–50 min after TBS.

For patch-clamp recordings, whole-cell recordings were made under direct IR-DIC (infrared-differential interference contrast) visualization of neurons in the hippocampal CA1 stratum pyramidale region. Excitatory postsynaptic currents (EPSCs) were evoked in the presence of the GABA A receptor antagonist gabazine (10 μM) by stimulation of stratum radiatum by using a theta glass electrode (20 µsc–80 µsc, 0.02 mA–0.1 mA) connected to a constant-current isolation unit (Digitimer LTD, Model DS3) and acquired every 5 seconds. The glass of theta electrode, composed by two isolated channels, was pulled to produce the tip for microstimulation and each channel of the glass tip was filled with a normal aCSF used during recordings. Voltage clamp experiments were performed on CA1 pyramidal neurons using borosilicate patch pipettes (3–4 MΩ) filled with a solution containing (in mM): 135 CsMeSO 3 , 5 CsCl, 5 NaCl, 2 MgCl 2 , 0.1 EGTA, 10 HEPES, 0.05 CaCl 2 , 2 Na2-ATP, 0.4 Na 3 -GTP (pH 7.3, 280–290 mOsm/kg). Each CA1 pyramidal neuron was voltage-clamped at −70 mV and at +40 mV to evoke AMPA and NMDA receptor-mediated EPSCs respectively. AMPA and NMDA EPSCs were recorded before and after blocking AMPA mediated currents by bath applying 20 µM NBQX disodium salt. Access resistance was monitored throughout the experiment. Signals were sampled at 10 kHz filtered at 2.8 kHz. Series resistance (range 15–20 MΩ) was monitored at regular intervals throughout the recording and presented minimal variations (≤20%) in the analyzed cells. Data are reported without corrections for liquid junction potentials. Data were acquired using a Multiclamp 700B amplifier controlled by pClamp 10 software (Molecular Device), with a Digidata 1322 (Molecular Device). AMPA/NMDA ratio of each neuron was calculated as the ratio between AMPA EPSC peak amplitude (pA) of the subtracted current and the NMDA EPSC peak amplitude (pA).

Immunohistochemistry and 3D modelling analysis

Animals were perfused transcardially with 4% paraformaldehyde (PFA), brains were dissected, post-fixed o/n at 4 °C and coronal sections (50 μm thick) were obtained with a vibratome (Leica Microsystems). Immunohistochemistry was performed following standard protocols. Briefly, free-floating sections were permeabilized with 0.5% Triton-X100 (Sigma) in PBS. Sections were then blocked in 5% horse serum (Gibco, Life Technologies), 0.5% Triton-X100 in PBS for 1 h followed by overnight incubation with the primary antibody (rabbit anti-GFP antibody, 1:2000, Molecular Probes) at 4 °C. After six washes with 0.5% Triton-X100 in PBS, sections were incubated overnight with the secondary antibody (Rhodamine Red-X goat anti-rabbit IgG, 1:500, Molecular Probes) at 4 °C. After three washes with 0.5% Triton-X100 in PBS, section were incubated with DAPI (0.1 μg/ml, Sigma), washed three times with PBS and then mounted onto glass slides and coverslipped with Aqua Poly/Mount (PolyScience).

For dendritic spine analysis, adult Tph2−/− mice and control mice carrying the Thy1-YFP-M allele were processed for immunohistochemistry using anti-GFP antibody and a rhodamine-conjugated secondary antibody to avoid interference of endogenous YFP fluorescence. A number of 6 confocal fields (35 Z-steps at 0.15μ interval) on consecutive coronal sections of both CA1 and CA3 fields in the dorsal hippocampus were imaged using a Nikon A1 confocal microscope equipped with a 60x PlanApo oil objective at 1024 × 1024 pixel resolution. Images were analyzed using the Filament Tracer semi-automated method for dendrites and spine properties quantifications (Imaris 7.2.3, Bitplane). For each animal, 20 to 30 dendrites in the apical region were reconstructed for each hippocampal field.

Unpredictable Chronic Mild Stress (uCMS)

uCMS paradigm was modified from Tye et al.26. Age-matched Tph2−/− and control mice were randomly subdivided in the uCMS and control group. Control mice of both genotypes were housed in standard conditions. uCMS protocol consisted in two stressors per day (one during the day, one during the night) for 8 weeks. Cage tilt on a 45° angle for 16 h, food deprivation for 6 h, white noise (http://www.simplynoise.com) for 16 h, continuous illumination for 36 h, 3 h darkness during the light cycle, continuous darkness for 36 h, water deprivation for 6 h, wet bedding (150 mL water into sawdust bedding) for 16 h, rat feces exposure in the cage for 16 h, cage switching between mice, restraint stress in 50 mL tube for 2 h, overcrowded housing for 3 h were the stressors randomly applied to be unpredictable for mice. Except for overcrowding, as well as for water and food deprivation sessions, water and food were available ad libitum. Tph2−/− and WT mice from uCMS and control groups were behaviorally tested with the Forced Swim Test 24 h after the last uCMS session, and then immediately sacrificed. Brains were rapidly removed, hippocampal tissue dissected and quickly frozen in liquid nitrogen for Western blot and RNA-seq analyses.

Western Blotting

Biochemical studies were performed as reported in Napolitano et al.27. The hippocampi were sonicated in a lysis buffer (320 mM sucrose, 50 mM Tris HCl pH 7.5, 50 mM NaCl, 1% Triton X-100, 5 mM β-glycerol phosphate, 1 mM Na3VO4, 5 mM NaF, protease inhibitor cocktail) and incubated on ice for 30 min. Samples were spin at 12,000 g × 10 min and the supernatant transferred to fresh microfuge tube. Aliquots of the homogenate were used for protein determination using Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA). Equal amounts of total proteins (30 μg) for each sample were loaded onto 15% (for BDNF detection) or 10% (for TrkB detection) polyacrylamide gels. Proteins were separated by SDS-PAGE and transferred overnight to membranes (PVDF; Amersham Pharmacia Biotech, Uppsala, Sweden). The membranes were immunoblotted overnight using selective antibodies against BDNF and TrkB (each diluted 1:1000, Santa Cruz Biotechnology). Both BDNF and TrkB optical density values were normalized using antibodies against GAPDH (1:1000, Santa Cruz Biotechnology) and α-Tubulin (1:50000, Sigma Aldrich), respectively. Blots were then incubated in horseradish peroxidase-conjugated secondary antibodies and target proteins visualized by ECL detection (Pierce, Rockford, IL), followed by quantification by Quantity One software (Biorad). Normalized values were averaged and used for statistical analysis performed by two-way ANOVA followed by post-hoc comparison, when required.