Animals

The C57BL/6 N-A < tm1Brd > Ptprd < tm2a(KOMP)Wtsi>/WtsiOrl mice strain was purchased from Wellcome Trust Sanger Institute and maintained as heterozygous. The cross between heterozygous mice allowed the Ptprd+/+, Ptprd+/-, and Ptprd-/- mice to be obtained. The following primers were used to identify the three genotypes mentioned above: Ptprd_111547_F: 5’-TCACCTCGCTGTTCTTCCTG-3’; Ptprd_111547_R:5’-CTTCTCAGTGCCCAACCCTC-3’; CAS_R1_Term: 5’-TCGTGGTATCGT TATGCGCC-3’. In addition, for sociability and social novelty tests, BALB/c wild-type mice were used as “old mouse” and “new mouse”. All mice had free access to rodent chow and water in a 12-hour dark-light cycle room with temperature controlled at 20–22 °C.

Perfusion of mice and brain extraction

Ptprd+/+, Ptprd+/-, and Ptprd-/- mice were perfused transcardially at 3 months old. The mice were first placed in an isoflurane chamber to be lightly anesthetized. Once the animal was numb, the heart was exposed, and a saline solution (0.9% NaCl) was injected gradually through the right ventricle. Once the blood flow stopped, the brains were extracted and fixed in a 4% PFA solution overnight at 4 °C. The next day, the brains were dehydrated in 20%, 25%, and 30% sucrose (Merk, 107,651,100) solutions at 4 °C for 24 h each incubation. Subsequently, brains were embedded in the optimal cutting temperature compound (OCT; Sakura, 4583) and stored at -80 °C until cut. Next, 18 μm coronal slices were obtained by cryostat (Leica, CM1850) at a temperature of -20 °C and stored at -80 °C until use.

Immunofluorescence and quantification

For morphometric analysis, immunostaining of tissue sections was performed as described [11, 12, 20]. Briefly, brain sections were washed with 1X TBS for immunostaining. Then were permeabilized with TBS − 0.3% Triton X-100 for 30 min. The tissues were then incubated in TBS with 5% BSA 0.3% Triton X-100 for 1 h as a blocking solution. Brain slices were incubated overnight with primary antibodies in a blocking solution at 4 °C. The next day, the sections were washed with TBS and then incubated with secondary antibodies in a blocking solution for 1 h at room temperature. After TBS washes, sections were counterstained with Hoechst 33,258 for 10 min and mounted. To evaluate the number of GABAergic neurons in the somatosensory cortex and medial prefrontal cortex (mPFC), rabbit anti-parvalbumin antibody (1:100; Swant, PV 25) and mouse anti-somatostatin antibody (1:500; Santa Cruz, sc-55,565) were used. To evaluate the number of glutamatergic neurons in the somatosensory cortex and mPFC, rabbit anti-Tbr1 antibody (1:400; Abcam, ab31940) and rabbit anti-Satb2 antibody (1:500; Abcam, ab92446) were used. To quantify the specific neuronal types, the somatosensory cortex and mPFC boundaries were established using the mouse brain Atlas [21] and Adobe Photoshop CC 2015. Then, the images were quantified using the Cell Counter tool of the Fiji program.

Prefrontal cortex slice preparation

Acute coronal brain slices (300 μm thick) were obtained as previously described [22]. Briefly, the brain was removed and slices containing the mPFC were cut using a DTK-1000 Microslicer (Ted Pella, Inc.) in an ice-cold high-choline solution (110 mM Choline-Cl, 2.5 mM KCl, 1.25 mM NaH2PO4, 7 mM MgCl2, 25 mM NaHCO3, 15 mM glucose, 0.5 mM CaCl2, 11.6 mM ascorbate, 3.1 mM pyruvate (290–305 mmol/Kg)). Thirty minutes post-sectioning, coronal slices were gradually switched to artificial cerebral spinal fluid (aCSF) solution (124 mM NaCl, 2.69 mM KCl, 1.25 mM KH2PO4, 1.3 mM MgSO4, 26 mM NaHCO3, 10 mM glucose, 2.5 mM CaCl2, pH 7.4 (300–305 mmol/Kg)). All solutions were equilibrated with 95% O2 and 5% CO2 (pH 7.4), and slices were kept at room temperature for at least 30 min before recording.

Electrophysiology

All experiments, unless otherwise stated, were performed at 28 ± 1 °C in a submersion-type recording chamber perfused at ~ 2 mL/min with aCSF supplemented with either the GABAA receptor antagonist picrotoxin (PTX; 50 µM) or the AMPA/Kainate and NMDA receptor antagonists CNQX (25 µM) and D-APV (50 µM) to block fast inhibitory and excitatory transmission, respectively. Coronal slices were visualized using infrared differential interference contrast on a Nikon Eclipse FN1 microscope, and layer 2/3 pyramidal neurons from prelimbic (PrL) and infralimbic (IL) subdivisions of mPFC were morphologically identified. Whole-cell voltage-clamp recordings using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA) were made from 2/3 pyramidal neuron somas located ~ 200 mm from pia and voltage-clamped at -60 or 0 mV (unless otherwise stated) using patch-type pipette electrodes (~ 3.0–4.5 MΩ) containing 131 mM Cs-gluconate, 8 mM NaCl, 1 mM CaCl2, 10 mM EGTA, 10 mM glucose, 10 mM HEPES, 5 mM MgATP, and 0.4 mM Na3GTP; pH 7.2–7.4 (285 mmol/kg). To assess cell stability, series, and input resistances were monitored with test pulses (-4mV, 80 ms) throughout all experiments. Cells with > 20% change in series resistance were excluded from the analysis.

To elicit excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs, respectively), a bipolar-stimulating patch pipette filled with aCSF was placed in layer 2/3 around 100–150 μm from the recording neuron and voltage pulses (1–15 V, 100–200 ms pulse width) were delivered through a stimulus isolator (DS2A-Mk.II, Digitimer Ltd). Typically, stimulation was adjusted to obtain comparable magnitude synaptic response across experiments (e.g., 100–150 pA EPSC and IPSC). Two different protocols were used to evaluate short-term synaptic plasticity: First, two pulses at different interstimulus intervals (10, 30, 70, 100, and 300 ms) were used to calculate the paired-pulse ratio (PPR) that was defined as the ratio of the amplitude of the second response to the amplitude of the first response. Second, synaptic depression was evaluated using a burst of 25 (for EPSC) and 20 (for IPSC) stimuli at 14 and 10 Hz, respectively, and delivered every 60 s; 5 burst-evoked responses were averaged for each experiment. Miniature EPSCs (mEPSCs) were recorded at 32 ± 1 °C from neurons voltage-clamped at -60 mV in the continuous presence of tetrodotoxin (TTX, 500 nM). In contrast, isolated miniature IPSCs were recorded at 0 mV in the continuous presence of TTX, CNQX (25 µM), and D-APV (50 µM). mEPSCs and mIPSC were identified using a minimal threshold amplitude (≥ 5 pA) and analyzed using the mini-analysis software Synaptosoft (Synaptosoft). Spontaneous excitatory and inhibitory currents (sEPSCs/sIPSCs) were recorded without TTX, whereas AMPAR/NMDAR ratios were analyzed by recording AMPAR-mediated EPSCs at -60 mV. In contrast, NMDAR-mediated EPSCs were recorded at + 40mV in the continuous presence of CNQX (25 µM) to isolate NMDA-mediated EPSC. All drugs were obtained from Sigma and Tocris, prepared in stock solutions in milli-Q water or DMSO, and added to the ACSF as needed. Total DMSO in the aCSF was maintained at > 0.1%. Synaptic EPSCs and IPSCs were elicited at 10-sec intervals (0.16 Hz), filtered at 2.2 kHz, and acquired at 10 kHz using custom-made software written in Igor Pro 4.09 A (WaveMetrics).

Morris water maze test

Morris water maze navigation task was performed to evaluate spatial learning and memory as previously described [23]. Briefly, in the learning phase, the animals were trained to locate a hidden 9-cm-diameter white platform (1 cm below the surface of the water) for 4 consecutive days following 4 external cues, 4 times per day per animal in a 1.2-m-diameter circular pool filled with 23 ± 2 °C painted white water. Each trail ended when the animals found the platform and remained there for 10 s. If the animal did not find the platform at the end of 60 s, they were gently guided there for the remaining 10 s. Later in each trial, the animals were gently removed from the maze, dried with a tissue paper towel, and put in a similar housing cage with additional tissue paper towels to continue drying. On the fifth day, in the memory phase, the test was performed by removing the platform and allowing the free swimming for 60 s. The behavior was monitored during all tasks using an automatic tracking system (ANY-maze video tracking software, Stoelting Co, Wood Dale, IL, USA). The learning phase measured latency and path length to the platform, while the memory test measured the time spent in each quadrant and the target quadrant and the traveled distance within the target quadrant.

Y-Maze test

Memory was also evaluated with the Y-maze as previously described [24]. Briefly, Y-Maze tests were performed using a maze with 3 arms of 8 cm width x 35 cm long and walls of 20 cm height, connected at a 120° angle, shaping a Y and assigned as arms A, B, and C. Each animal was placed in the arm (A), looking to the center of the maze, and tracked for 8 min by the ANY-maze software. Four clues were put on the maze walls to guide the animals. Alternation was defined as a visit to 3 different arms (ABC, ACB, BCA, BAC, CAB, CBA), and % spontaneous alternation was determined by the following formula:

% Spontaneous alternation = (Total alternations / (Total arm entries-2) x 100.

Open field test

Anxiety was evaluated with the open field test as described previously [25]. Briefly, mice were exposed to a free exploration in an open field chamber of 40 cm (l) x 40 cm (w) x 40 cm (h). This arena is divided into two areas by a line: the peripheral and central regions. The time in each chamber area was recorded for 10 min by ANY-maze software.

Elevated plus maze test

Anxiety was also evaluated with the evaluated plus maze as described previously [26]. Briefly, the test was performed using a maze with 4 arms of 7.5 cm width x 35 cm long, shaping a cross with a 7.5 × 7.5 cm center and 50 cm from the floor. 2 arms (closed arms) had walls of 20 cm height, and the others (open arms) were uncovered. The animals were placed in the center of the maze and tracked for 5 min by ANY-maze software.

Self-grooming test

Repetitive behavior was measured by the self-grooming test as previously described [25, 27]. Briefly, mice were recorded for 10 min through a 20 × 20 cm transparent box to allow visualization of the animal. The cumulative time spent by the animals performing the self-grooming process was quantified.

Marble burying test

Repetitive behavior was also evaluated by the marble burying test as previously described [22, 25]. Briefly, the animals were exposed to a free exploration arena of 23 × 33 cm in the presence of 20 proportionally distributed black marbles. The marbles were placed in a 5 cm thick layer of dry sawdust to allow the animal to perform the digging behavior. After 30 min, the number of buried marbles was quantified. A marble was considered buried if 2/3 were covered.

Three-chamber test

The three-chamber social novelty and sociability test was used to evaluate deficits in social behavior as previously described [25, 27, 28]. First, the animals were subjected to 10 min of habituation in the three chambers. Then, to assess the sociability of the animals, mice were placed in a context in which they chose to interact with either a caged mouse (old mouse) or an inanimate object, each in a different chamber for 10 min. Subsequently, to assess the social novelty, the inanimate object was replaced by a second unfamiliar mouse (new mouse), and the experimental mouse was placed in a context in which it had to choose between interacting with a familiar or an unfamiliar mouse for 10 min. ANY-maze software recorded the time the experimental animals spent with each option.

Statistical analyses

Statistical analyses were performed using the one-way ANOVA test with unpaired data to compare data between Ptprd+/+, Ptprd+/-, and Ptprd-/- genotypes. Unless otherwise indicated, all electrophysiological results were statistically analyzed using paired and unpaired two-tailed Student’s t-test and One- or Two-way ANOVA at the p < 0.05 significance level in OriginPro 7.0 and 8.6 (OriginLab). In the figures illustrated, traces are averages of 25–30 responses. In the case of the statistical analysis used to evaluate the sociability and social novelty test, the 2-way ANOVA test was used. Corrections for multiple comparisons were performed using Tukey’s post hoc test. All statistical tests were performed with Prism 6 software. In all cases, the bars indicate the standard error of the mean.