Background: Acute brain slices represent a powerful tool for analysis of brain function in physiology and pathology. Commercial systems and custom-build solutions with carbogen (95% O2/5% CO2) aeration, but they are expensive, have a high working volume requiring large amount of substances, and only limited options for treatment in parallel are possible.
New method: We developed a novel cost-effective incubation system using materials available in every laboratory, allowing parallel incubation of several treatment conditions, thus also reducing the number of experimental animals. Our system incubation parameters were optimized for cortical neuron observation.
Results: We tested several different options using 6, 12 or 24 standard culture well plates, combining them with cell strainer baskets inside. The system was placed in a pre-warmed incubator at 37 °C. Carbogen was injected through a 22 gauge needle, positioned between the basket and the wall of the well. Best results were achieved in a 6-well plate. In 12 and 24-well plates bubbles accumulated beneath the basket, displacing it upwards, making it unsuitable for our purposes. The gas oxygenized the medium without mechanically disturbing the slices, protected within the strainer basket, but still allowing optimal diffusion through the 100 µm pores. In a 6-well plate, six simultaneous treatments were possible in parallel. LDH/Cytotoxicity tests showed an acute toxicity of less than 7%. The system lost about 2.5% per hour of the fluid through evaporation, which was replenished every 2 h. Up to 6 h after treatment, however, this evaporation was excellently tolerated by the neurons even without fluid replenishment, most probably due to the anti-swelling effect of the mildly hypertonic medium. We performed two staining procedures, working excellently with this experimental setup, namely - a modified DiI staining and a slice silver impregnation method, both confirming the intact neuronal morphology. Preserved CA3 calcium influx and removal response following KCl depolarization confirmed the normal physiology of the pyramidal neurons 6 h after exposure in the system.
Comparison to existing methods: The proposed system is much cheaper than the commercial solutions, can be constructed in any lab, allows up to 6 different treatments in parallel, which none of the existing systems allows. Antibiotic presence in the incubation medium and adequate evaporation control is required if longer incubation (> 6 h) is needed. Lower incubation volumes (3-6 ml) allow sparing expensive reagents. Our procedure was optimized for cortical neurons, further fine tuning to meet other specific requirements is possible.
Conclusions: The system we propose allows filling the gap for budget solutions for short to mid-term incubation of acute brain slices.
Keywords: Acute brain slice; Cost-effective; DiI staining; Incubation system; Silver impregnation.
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