Vt1000

In vitro local field potential (LFP) recordings from hippocampus slices were performed as previously described (Leao et al. 2009 (link)). Following decapitation under isoflurane anaesthesia, brains of P18-P25 cKO and littermate control mice were removed from the skull and placed in ice-cold high-sucrose artificial cerebrospinal fluid (ACSF) (in mM: KCl, 2.49; NaH₂PO₄, 1.43; NaHCO₃, 26; glucose, 10; sucrose, 252; CaCl₂, 1; MgCl₂, 4). A vibratome (VT1000, Leica Microsystems) was used to obtain horizontal hippocampal slices that were moved to a submerged recording chamber containing ACSF (in mM: NaCl, 124; KCl, 3.5; NaH₂PO₄, 1.25; MgCl₂, 1.5; CaCl₂, 1.5; NaHCO₃, 30; glucose, 10), constantly bubbled with 95 % O₂ and 5 % CO₂ and kept at 35 °C for 1 h then maintained at room temperature. For LFP recordings, slices were transferred to an interface-type chamber and kept at 35 °C (Zelano et al. 2013 (link)). A recording glass pipette filled with ACSF was placed in the stratum radiatum of CA3. LFP signals were amplified 100× using custom-made amplifier (John Curtin School of Medical Research, Australian National University), low-pass filtered at 3 kHz and digitized at 10 kHz by a National Instruments DAQ card. Data were analysed using Matlab (Mathoworks).

The mouse was anesthetized deeply with isoflurane on day 7 (D7) PO and the brain was quickly removed to icy cold artificial cerebral spinal fluid (ACSF) oxygenated with 95% O₂ and 5% CO₂. The brain slices in 300 mm containing the mPFC were obtained by a vibratome (VT1000, Leica Instruments, Germany). The slices were then incubated for at least 30 min at 33 °C and another 1 h at RT in oxygenated ACSF. Then the slices were transferred to a recording chamber and were continuously perfused with oxygenated ASCF at a rate of 3–4 ml/min before electrophysiological recordings at RT. For action potential recordings, the ACSF contained (in mM): 120 NaCl, 11 Dextrose, 2.5 KCl, 1.28 MgSO₄, 3.3 CaCl₂, 1 NaH₂PO₄, and 14.3 NaHCO₃, with pH at 7.4 and osmolarity at 310.5 mOsm. For spontaneous post-synaptic current recordings, a low divalent ion ACSF containing (in mM): 125 NaCl, 3.5 KCl, 1.25 NaH₂PO₄, 0.5 MgCl₂, 26 NaHCO₃, 25 Dextrose, and 1 CaCl₂, with pH at 7.4 and osmolarity at 310.5 mOsm was used.

Anesthetized mice were perfused with 0.01 mol/L phosphate-buffered saline (PBS, Sigma-Aldrich) and then with 4% paraformaldehyde (PFA, Sigma-Aldrich). The brain was removed from the skull, and post-fixed in 4% PFA at 4℃. Each brain was rinsed 3 times with 0.01 mol/L PBS and embedded in 5% agarose (Sigma-Aldrich). Coronal sections (50 µm) were cut on a vibratome (VT1000, Leica Microsystems). Every second section was used for whole-brain imaging with an automated slide scanner (VS120 Virtual Slide, Olympus).

The protocol was according to the method described previously (Kitahara et al., 2016 (link)). Briefly, mice were deeply anesthetized and then perfused with 2% paraformaldehyde and 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.4). Three to 4 h after perfusion, the brains were removed, and coronal slices of the dorsal dentate gyrus (DG; 300 μm) were cut with a vibrating blade microtome (VT1000S, Leica Microsystems, Nussloch, Germany). The slices were subsequently postfixed and en bloc stained for FIB/SEM. The slices were treated with 1% thiocarbohydrazide and were then immersed in a solution of 2% OsO₄. For en bloc staining, the slices were immersed in a solution of 4% uranyl acetate solution overnight and washed with double distilled water. The slices were further stained with Walton’s lead aspartate solution, dehydrated in an ethanol series, placed in ice-cold dry acetone, subjected to infiltration of an epoxy resin (Epon 812, TAAB, England) mixture, and polymerized for 72 h at 60°C.

Acute coronal Anterior Cingulate Cortex (ACC) slices were prepared as previously described (Li et al., 2019 (link)). Briefly, mice were deeply anesthetized using 10% chloral hydrate with intubation and ventilation and then decapitated; brains were quickly removed and immersed in prechilled well-oxygenated (95% O₂/5% CO₂) artificial cerebrospinal fluid (aCSF) containing (in mM): 125 NaCl, 2.5 KCl, 12.5 D-glucose, 1 MgCl₂, 2 CaCl₂, 1.25 NaH₂PO4, and 25 NaHCO₃, pH 7.35–7.45. Forebrain was cut and glued onto the cutting slab and sectioned using a vibratome (Leica Microsystems VT 1000S) into 300-µm-thick coronal slices. For recovery, slices were incubated at 31°C in aCSF for at least 1 h. Then a piece of ACC containing slice was placed in a recording chamber and planked by a nylon mesh. The slice was observed with a microscope (Olympus, BX51WI) equipped with an infrared differential interference contrast video monitor. The recording bath temperature was maintained at 29–30°C using a heat circulator and exchanger for aCSF perfusion. Excitatory postsynaptic currents (EPSCs) were recorded from layer II/III pyramidal neurons and the stimulations were delivered at an intensity of 40 μA by a bipolar tungsten stimulating electrode placed in layer V/VI of the ACC.