Vibroslice nvsl

Primary mouse astrocytes were prepared from the cortices of newborn C57BL/6 mice (postnatal day (PD) 3–5) as mixed cultures with microglia in Dulbecco’s modified Eagle’s medium (high glutamate) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The growth medium was supplemented with 10% heat-inactivated fetal calf serum (FCS) (PAN Biotech GmbH, Aidenbach, Germany) and 1% penicillin/streptomycin (Gibco). Fourteen days after seeding in 75 cm² cell culture flasks (Sarstedt AG & Co. KG, Nuembrecht, Germany), the cells were harvested. Culture treatment with PLY and LLO was performed in serum-free medium.
Acute brain slices were prepared from infant (PD 10–14) C57Bl/6 mice via decapitation and vibratome sectioning (Vibroslice NVSL, World Precision Instruments, Berlin, Germany) in artificial CSF continuously oxygenized with carbogen gas (95% O₂, 5% CO₂) at 4 °C. The slices were allowed to adapt in carbogenated Basal Medium of Eagle (Gibco) with 1% penicillin/streptavidin and 1% glucose at 37 °C for 1 h before being challenged with PLY or with LLO in the 5% CO₂-buffered medium (pH = 7.3). In these acute slices, cell lysis never exceeded 7% within 12 h.

Neonatal P0–P4 medullary brainstem slices were obtained following the same initial steps as those used for en bloc preparations^{62 (link)}. After the cerebellum was removed, the brainstem-spinal cord preparation was glued to an agar block with the rostral end up, and the block was mounted in a Vibroslice NVSL (World Precision Instruments, Sarasota, FL, USA). The brainstem was serially sectioned from the rostral to the caudal area in 200 µm steps until the facial nuclei were observed. Then, a section of 500 µm was discarded, and the next 700 µm slice was collected and immersed for 30 min in aCSF with a high concentration of K⁺ (9 mM) to improve the robustness and stability of the respiratory rhythm. Finally, the slice was transferred to a 250 µl recording chamber (RC-26G model, Warner Instruments, Hamden, CT, USA) mounted on the stage of an Eclipse FN1 microscope (Nikon, Tokyo, Japan) and superfused with aCSF containing 5 mM K⁺ (2 ml min⁻¹) at 29–30 °C.
The bath temperature was monitored with a thermistor probe placed at the outflow end of the en bloc or slice chamber and maintained at the desired temperature with an in-line heater operated by a temperature controller (TC-324C, Warner Instruments, Hamden, CT, USA).

All experimental procedures were performed on Wistar rats according to the guidelines provided by the National Institutes of Health for the humane treatment of animals and approved by the Animal Care Committee of Bogomoletz Institute of Physiology of National Academy of Science of Ukraine. Postnatal day 10–14 rats were deeply anesthetized using sevoflurane and decapitated. Transverse brain slices were prepared according to previously described techniques [18 (link)]. Briefly, brains were removed and placed in the ice-cold aCSF of the following composition (in mM): 126 NaCl, 3.5 KCl, 2 CaCl₂, 1.3 MgCl₂, 1.25 NaH₂PO₄, 24 NaHCO₃, 11 D-glucose, pH = 7.35–7.4, the pH of all aCSF solutions was adjusted with carbogen gas. Hippocampal slices were cut at 500 μm using a Vibroslice NVSL (World Precision Instruments, Sarasota, FL). Slices equilibrated at room temperature and constantly carbogenated aCSF for at least two hours before the experiment. Transverse hippocampal slices with a clearly visible pyramidal cell layer in CA3-CA1 zones were selected for electrophysiological experiments. Under microscopic control, the recording electrodes were positioned in the pyramidal cell layer in CA3 and CA1 zones.