Background

Neuronal tissue slices provide unique advantages in basic neuroscience research as it allows direct investigation of neuronal network activity and the influence of pharmacological compounds on the central nervous system. Hence, it is imperative to prepare and maintain slices viability at the highest quality for experimental purposes. The viability of neuronal slices relies on several intrinsic and extrinsic factors (Buskila et al., 2014), which can be divided into three periods according to the slicing procedure: before (pre-slicing), during (slicing) and after (post-slicing) slicing procedure. The main factors that decrease slice viability in the pre-slicing period include ischemia and hypoxia occurring due to elimination of continuous blood supply (Buskila et al., 2020). Both hypoxia and ischemia, starting seconds after decapitation, lead to excitotoxic processes such as mitochondrial dysfunction and Ca²⁺ influx, which contribute to neuronal hyperexcitability and decrease in slice viability. The slicing procedure itself leads to physical damage of the semi-permeable membrane of neurons, resulting in neurotransmitter spill-over and inflow of compounds (i.e., Ca²⁺, Na²⁺, etc.) into the cell, which enhances neuronal cytotoxicity (Reid et al., 1988). The main factors affecting the slice viability during the post-slicing episode includes the temperature of the incubation solution and the stability of its pH, as these factors affect processes which impact the cells deterioration rate, including redox metabolism and the reduction in the oxygen transfer into the deep layers of the tissue (Reid et al., 1988). For long-term incubation of the slices (> 4 h), the level of bacteria is also an essential factor, as it contributes to slice deterioration via releasing of endotoxins, glial activation and neurodegeneration (Qin et al., 2007; Breen and Buskila, 2014).

To reduce the impact of the factors affecting the viability of the slices, we capitalize on previous protocols that found effective in increasing slice viability by reducing excitotoxicity during the pre-slicing and slicing steps and additionally use an active recovery (post-slicing) incubation chamber, the Braincubator^TM, which controls several parameters, such as temperature, pH and bacteria levels. In this system, slices are kept in a temperature-controlled chamber, while the aCSF flows through a secondary tank exposed to an ultraviolet C (UVC) light source that eradicates bacteria. The design and use of the Braincubator^TM have been described previously (Breen and Buskila, 2014; Buskila et al., 2014; Cameron et al., 2016 and 2017). Hypothermia has been proven highly efficient in reducing the metabolic rate and post-slicing cellular damage, hence, we have incubated brain slices at 15-16 °C. The combination of hypothermia with the UVC filtration system can extend slice viability for > 24 h (Buskila et al., 2014).