Vt1000s vibrating microtome

For embryonic tissue collection pregnant dams were killed by isoflurane inhalation, embryos were harvested and whole heads were immersion-fixed in 4% paraformaldehyde (PFA) at 4°C overnight, followed by brain dissection and additional immersion postfixation in 4% PFA for 4 h at 4°C. All postnatal tissue was fixed by transcardial perfusion with PBS followed by 4% PFA. Dissected brains were postfixed in 4% PFA at 4°C for 1 h. All brains were stored at 4°C in 0.2% sodium azide PBS (PBS-Az) and sectioned at 70 μm using a Leica VT 1000S vibrating microtome.

Acute coronal hippocampal slices were prepared from C57BL/6 wild-type mice, postnatal day (P)30–P43. Mice were anesthetized with isoflurane and decapitated. The brain was removed from the skull and rapidly placed in an ice-cold cutting solution containing the following: 215 mm sucrose, 20 mm glucose, 26 mm NaHCO₃, 4 mm MgCl₂, 4 mm MgSO₄, 1.6 mm NaH₂PO₄, 1 mm CaCl_2, and 2.5 mm KCl. Coronal slices (300 μm thick) were prepared using a VT1000S vibrating microtome (Leica). Slices were incubated at 32°C for 30 min in a holding chamber containing 50% cutting solution and 50% artificial CSF (ACSF; 127 mm NaCl, 25 mm NaHCO₃, 1.25 mm NaH₂PO₄, 2.5 mm KCl, 25 mm D-glucose, 2 mm CaCl₂, and 1 mm MgCl₂). After 30 min, this solution was replaced with ACSF at room temperature. Slices were allowed to recover for >1 h in ACSF before imaging and recording. All solutions were equilibrated for at least 30 min with 95%O₂/5%CO₂. Organotypic hippocampal slice cultures were prepared from P3 mice, following the guidelines set by the institutional animal care and use committee at the authors’ institutions.

Male Wistar rats (Charles River, Harlow, UK) were used for the optical imaging experiments at postnatal day 21, a developmental stage within the range previously shown to exhibit the conspicuous assemblies that indicate large-scale neuronal cohesion [37 (link),45 (link)]. All animal procedures were carried out according to the Home Office UK regulations, in compliance with the requirements of the UK Animals (Scientific Procedures) Act 1986.
Brain slice preparation was carried out as follows: consecutive 400 μm thick coronal rat brain slices containing the SN (coordinates −4.80 to −6.20mm from Bregma [64 ]) or striatum (1.20 to −0.20 mm from Bregma) were cut with a Leica VT1000S vibrating microtome in oxygenated (95% O₂, 5% CO₂) ice-cold cutting solution containing the following (in mmol): 120 NaCl, 5 KCl, 20 NaHCO₃, 2.4 CaCl₂, 2 MgSO₄, 1.2 KH₂PO₄, 10 glucose, 6.7 HEPES salt and 3.3 HEPES acid; pH: 7.1. Each slice was divided along the midline into hemisections; these were transferred to a bubbler pot with artificial cerebrospinal fluid (ACSF) containing (in mmol): 124 NaCl, 3.7 KCl, 26 NaHCO₃, 2 CaCl₂, 1.3 MgSO₄, 1.3 KH₂PO₄ and 10 glucose; pH: 7.1. They were incubated at 34 °C for 30 min. The hemisections were then kept at RT (22 ± 1.5 °C) for 30 min in oxygenated ACSF before VSD staining.

After recording, mice were deeply anesthetized by an additional i.p. injection of urethane and transcardially perfused with 4% paraformaldehdye (PFA). The brain was removed, fixed in 4% PFA overnight and stored in PB at 4 °C before histological processing for all brains. Next, 100 μm thick tangential slices were made using a Leica VT1000 S vibrating microtome. We stained for cytochrome oxidase to reveal the barrel cortex map and for biocytin, with a standard ABC kit (Vectastain), with DAB enhancement, to reveal the recorded neurons. Slices were mounted in Moviol, stored at 4 °C and reconstructed using NeuroLucida software (MicroBrightField).

Acute coronal cortical slices containing medial prefrontal cortex (mPFC) were prepared from C57BL/6 wild type and Gpr158^-/- mice, P50-60. Mice were anesthetized with isoflurane and decapitated. The brain was removed from the skull and rapidly placed in ice-cold cutting solution containing (in mM): 215 sucrose, 20 glucose, 26 NaHCO₃, 4 MgCl₂, 4 MgSO₄, 1.6 NaH₂PO₄, 1 CaCl₂ and 2.5 KCl. Cortical slices (400 μm thick) were prepared using a VT1000S vibrating microtome (Leica). Slices were incubated at 32°C for 30 min in a holding chamber containing 50% cutting solution and 50% artificial cerebrospinal fluid (ACSF; in mM: 127 NaCl, 25 NaHCO₃, 1.25 NaH₂PO₄, 2.5 KCl, 25 D-glucose, 2 CaCl₂, and 1 MgCl₂). After 30 min, this solution was replaced with ACSF at room temperature. Slices were allowed to recover for more than 1 hr in ACSF before recording. All solutions were equilibrated for at least 30 min with 95%O₂/5%CO₂.