Adjusting the neuron to astrocyte ratio with cytostatics in hippocampal cell cultures from postnatal rats: A comparison of cytarabino furanoside (AraC) and 5-fluoro-2’-deoxyuridine (FUdR)

Abstract

Cell culture studies offer the unique possibility to investigate the influence of pharmacological treatments with quantified dosages applied for defined time durations on survival, morphological maturation, protein expression and function as well as the mutual interaction of various cell types. Cultures obtained from postnatal rat brain contain a substantial number of glial cells that further proliferate with time in culture leading to an overgrowth of neurons with glia, especially astrocytes and microglia. A well-established method to decrease glial proliferation in vitro is to apply low concentrations of cytosine arabinoside (AraC). While AraC primarily effects dividing cells, it has been reported repeatedly that it is also neurotoxic, which is the reason why most protocols limit its application to concentrations of up to 5 μM for a duration of 24 h. Here, we investigated 5-fluoro-2’-deoxyuridine (FUdR) as a possible substitute for AraC. We applied concentrations of both cytostatics ranging from 4 μM to 75 μM and compared cell composition and cell viability in cultures prepared from 0-2- and 3-4-day old rat pups. Using FUdR as proliferation inhibitor, higher ratios of neurons to glia cells were obtained with a maximal neuron to astrocyte ratio of up to 10:1, which could not be obtained using AraC in postnatal cultures. Patch-clamp recordings revealed no difference in the amplitudes of voltage-gated Na+ currents in neurons treated with FUdR compared with untreated control cells suggesting replacement of AraC by FUdR as glia proliferation inhibitor if highly neuron-enriched postnatal cultures are desired.

Oxidized linoleic acid metabolites regulate neuronal morphogenesis in vitro

“…Three to 4 h post-plating, culture medium was replaced with Neurobasal Plus basal medium supplemented with 2% B27 and 1% GlutaMAX. On day in vitro (DIV) 4, half of the conditioned medium was replaced by fresh Neurobasal Plus basal medium supplemented with 2% B27 and 1% GlutaMAX and 10 μM cytosine-arabinoside (AraC) at a final concentration of 5 μM AraC to inhibit glial proliferation ( Lesslich et al, 2022 ).…”

Analysis of neuronal injury transcriptional response identifies CTCF and YY1 as co-operating factors regulating axon regeneration

“…We observed that at days in vitro 1 (DIV1), FABP7 positive glial cells are present in high numbers ( Figures 1A,B ) but are nearly absent by DIV7 ( Figures 1A,B ), consistent with our previous results ( Avraham et al, 2020 ). FDU treatment eliminates non-neuronal proliferating cells from the culture, without affecting neuronal properties and viability ( Lesslich et al, 2022 ). Consistently, we did not observe pycnotic nuclei in our FDU-treated cultures compared to no FDU.…”

Pathological Bergmann glia alterations and disrupted calcium dynamics in ataxic Canavan disease mice

“…On the subsequent in vitro day, the medium was further enhanced with 1 μm of cytarabino furanoside (AraC, C998100, TRC) for a duration of 24 h, to attenuate glial proliferation. Subsequently, medium was replenished with a fresh batch of coculture medium, with half-media changes implemented daily thereafter ( Goshi et al, 2020 ; Hasan & Berdichevsky, 2021 ; Lesslich et al, 2022 ). On the seventh day, the coculture medium was augmented with varied concentrations of N -acetyl-L-asparate (NAA; 1, 2.5, and 10 mM) or a vehicle control, and this was maintained for an additional 2 days.…”