BrainPhys Neuronal Media Support Physiological Function of Mitochondria in Mouse Primary Neuronal Cultures

Abstract

In vitro neuronal cultures are extensively used in the field of neurosciences as they represent an accessible experimental tool for neuronal genetic manipulation, time-lapse imaging, and drug screening. Optimizing the cultivation of rodent primary neuronal cultures led to the development of defined media that support the growth and maintenance of different neuronal types. Recently, a new neuronal medium, BrainPhys (BP), was formulated envisioning the mimicry of brain physiological conditions and suitability for cultured human iPSC-derived neurons and rat primary neurons. However, its advantages in mouse primary neuronal cultures and its effects in neuronal bioenergetics are yet to be demonstrated. In this study, we validated the beneficial use of BP in mouse primary neuronal cultures based on the observation that neuronal cultures in BP media showed enhanced ATP levels, which increased throughout neuronal maturation, a finding that correlates with higher mitochondrial activity and ATP production at later maturation stages, as well as an increased glycolysis response on mitochondrial inhibition and increased mitochondrial fuel flexibility. Taken together, our data demonstrate that BP medium promotes mitochondrial activity along with neuronal maturation of in vitro cultures.

Local mitochondrial replication in the periphery of neurons requires the eEF1A1 protein and the translation of nuclear-encoded proteins

“…We treated neurons with cycloheximide and chloramphenicol either at DIV7 - where neurons are still maturating, but synapses are already present to some extent – or at DIV14 – where neurons are mature and a higher number of synapses are established. 34 …”

“…Mouse primary neuronal cultures were obtained from whole brain of E18 embryonic C57BL/6 mice, as previously described. 34 Whole-brains were collected in Hank’s Balanced Salt Solution supplemented with 1M HEPES (HBSS-HEPES). Meninges were removed, and brains were homogenised and dissociated with 0.25% trypsin + 10μl/ml DNaseI in HBSS-HEPES for 15min at 37°C.…”

Age‐dependent NMDA receptor function is regulated by the amyloid precursor protein

“…Hippocampal neurons were cultured from 18‐day Sprague–Dawley rat embryos, adapting the protocol from (Faria‐Pereira et al, 2022). Cells were plated on poly‐D‐lysine‐coated glass coverslips in 24‐multiwell plates at a final density of 70,000 cells/coverslip, in neuronal plating media ((Minimum Essential Medium) supplemented with 10% horse serum, 0.6% glucose, and 100 U/ml Pen‐Strep) and maintained at 37°C in a 5% CO 2 ‐humidified incubator.…”

“…Hippocampal neurons were cultured from 18-day Sprague-Dawley rat embryos, adapting the protocol from (Faria-Pereira et al, 2022).…”

“…Cultures were maintained in the humidified incubator for 2 weeks, feeding the cells once per week with neuronal culture medium by replacing half of the medium per well. This protocol is associated with an enrichment in neuronal cells (NeuN-positive cells ≈80%) and a low number of glial cells (GFAP-positive cells ≈6%) at DIVs 11-14 (Faria-Pereira et al 2022).…”