Integraslice

Adult male and female mice (PND 90–120, N = 16 and N = 12 respectively) were deeply anesthetized with isoflurane and sacrificed according to institutional regulations. The brain was sliced (300 μm) in the coronal plane with a vibratome (Integraslice, Campden Instruments) in a sucrose-based solution at 4 °C (87 mM NaCl, 75 mM sucrose, 25 mM glucose, 2.5 mM KCl, 4 mM MgCl₂, 0.5 mM CaCl₂, 23 mM NaHCO₃, and 1.25 mM NaH₂PO₄). Immediately after cutting, slices containing anterior or posterior IC were stored for 30 min at 32 °C in a low-calcium artificial CSF (ACSF) that contained the following: 130 mM NaCl, 11 mM glucose, 2.5 mM KCl, 2.4 mM MgCl₂, 1.2 mM CaCl₂, 23 mM NaHCO₃, and 1.2 mM NaH₂PO₄, and were equilibrated with 95% O2/5% CO₂ and then at room temperature until the time of recording. During the recording, slices were placed in the recording chamber and continuously perfused at 2 ml/min with warm (32°–34 °C) low Ca₂ + solution.

Adult male and female rats were anesthetized with isoflurane and killed as previously described (Martin and Manzoni, 2014 (link); Manduca et al., 2017 (link)). The brain was sliced (300 μm) in the coronal plane with a vibratome (Integraslice, Campden Instruments) in a sucrose-based solution at 4°C (in mm as follows: 87 NaCl, 75 sucrose, 25 glucose, 2.5 KCl, 4 MgCl2, 0.5 CaCl₂, 23 NaHCO₃ and 1.25 NaH₂PO₄). Immediately after cutting, slices containing the medial prefrontal cortex (PFC) or accumbens were stored for 1 hr at 32°C in a low-calcium ACSF that contained (in mm) as follows: 130 NaCl, 11 glucose, 2.5 KCl, 2.4 MgCl₂, 1.2 CaCl₂, 23 NaHCO₃, 1.2 NaH₂PO₄, and were equilibrated with 95% O2/5% CO2 and then at room temperature until the time of recording. During the recording, slices were placed in the recording chamber and superfused at 2 ml/min with either low Ca²⁺ ACSF for mPFC or normal ACSF for the accumbens. All experiments were done at 32°C or room temperature for mPFC and accumbens respectively. The superfusion medium contained picrotoxin (100 µM) to block gamma-aminobutyric acid types A (GABA-A) receptors. All drugs were added at the final concentration to the superfusion medium.

Animals were anesthetized with halothane (rats) or isoflurane (mice) and decapitated according to institutional regulations. The brain was sliced (300 μm) in the coronal plane with a vibratome (Integraslice, Campden Instruments, Loughborough, UK) in a sucrose-based solution at 4°C (in mM: 87 NaCl, 75 sucrose, 25 glucose, 2.5 KCl, 4 MgCl₂, 0.5 CaCl₂, 23 NaHCO₃, and 1.25 NaH₂PO₄). Immediately after cutting, slices were stored for 1 h at 32°C in a low calcium artificial cerebrospinal fluid (low Ca²⁺ ACSF) that contained in mM: 130 NaCl, 11 Glucose, 2.5 KCl, 2.4 MgCl₂, 1.2 CaCl₂, 23 NaHCO₃, 1.2 NaH₂PO₄, and was equilibrated with 95% O₂/5% CO₂ and then at room temperature until the time of recording.

Horizontal hippocampal slices (400 µm for extracellular recordings, 250 µm for patch-clamp recordings) were prepared from Ophn1^+/y and Ophn1^−/y age-matched mice (3–9 weeks old) anaesthetised by intraperitoneal injection of medetomidine (1 mg/kg) and ketamine (76 /kg). Animals were transcardially perfused with ∼10 ml of ice-cold cutting solution comprising (mM): 189 sucrose, 26 NaHCO₃, 1.2 NaH₂PO₄, 2.5 KCl, 0.1 CaCl₂, 5 MgCl₂ and 10 glucose (flow rate ∼2.7 ml/min). Slices were cut using an Integraslice (Campden Instruments, Loughborough, UK) and stored at room temperature in an interface chamber containing 95%O₂–5%CO₂ oxygenated artificial cerebrospinal fluid (aCSF)(in mM: 135 NaCl, 16 NaHCO₃, 1.25 NaH₂PO₄, 3 KCl, 2 CaCl₂, 1 MgCl₂ and 10 glucose, pH 7.4).

Twenty-four hours after WIN or vehicle administration, rats were anesthetized with isoflurane and decapitated according to institutional regulations. The brain was sliced (300 μm) in the coronal plane with a vibratome (Integraslice, Campden Instruments, Loughborough, UK) in a sucrose-based solution at 4°C (values in mM: 87 NaCl, 75 sucrose, 25 glucose, 5 KCl, 21 MgCl₂, 0.5 CaCl₂, and 1.25 NaH₂PO₄). Slices were allowed to recover for 60 min at ±32°C in a low calcium artificial cerebrospinal fluid (aCSF; in mM: 126 NaCl, 2.5 KCl, 2.4 MgCl₂, 1.2 CaCl₂, 18 NaHCO₃, 1.2 NaH₂PO₄, and 11 glucose, equilibrated with 95% O₂/5% CO₂. Slices were maintained at room temperature until recording.

Rats and mice were anesthetized with isoflurane and decapitated according to institutional regulations. Brains were sliced (250–300 μm) in the coronal plane with a vibratome (Integraslice, Campden Instruments, Loughborough, UK) in a sucrose-based solution at 4°C (in mM: 87 NaCl, 75 sucrose, 25 glucose, 5 KCl, 21 MgCl₂, 0.5 CaCl₂ and 1.25 NaH₂PO₄). Slices were allowed to recover for 60 min at 32–35°C in artificial cerebrospinal fluid (aCSF) containing 126 mM NaCl, 2.5 mM KCl, 2.4 mM MgCl₂, 1.2 mM CaCl₂, 18 mM NaHCO₃, 1.2 mM NaH₂PO₄ and 11 mM glucose, equilibrated with 95% O₂/5% CO₂. Slices were then maintained at 22 ± 2 °C until recording.

Adult male and female rats were anesthetized with isoflurane and killed (Bara et al., 2018 (link); Borsoi et al., 2019 (link)). The brain was sliced (300 μm) in the coronal plane with a vibratome (Integraslice, Campden Instruments) in a sucrose-based solution at 4°C (87 mm NaCl, 75 mm sucrose, 25 mm glucose, 2.5 mm KCl, 4 mm MgCl₂, 0.5 mm CaCl₂, 23 mm NaHCO₃, and 1.25 mm NaH₂PO₄). Immediately after cutting, slices containing the medial PFC or the NAc were stored for 1 h at 32°C in a low-calcium artificial CSF (ACSF) that contained the following: 130 mm NaCl, 11 mm glucose, 2.5 mm KCl, 2.4 mm MgCl₂, 1.2 mm CaCl₂, 23 mm NaHCO₃, and 1.2 mm NaH₂PO₄, and were equilibrated with 95% O₂/5% CO₂ and then at room temperature until the time of recording. During the recording, slices were placed in the recording chamber and superfused at 2 ml/min with low Ca²⁺ or normal Ca²⁺ ACSF (PFC and NAc, respectively). All experiments were done at 32°C (PFC) or 25°C (NAc). The superfusion medium contained picrotoxin (100 mm) to block GABA-A receptors. All drugs were added at the final concentration to the superfusion medium.