Quantifying Spontaneous Ca²⁺ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Abstract

Parkinson's disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca 2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca 2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca 2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Extracellular S100B alters spontaneous Ca2+fluxes in dopaminergic neurons via L-type voltage gated calcium channels: Implications for Parkinson’s disease

“…Immunostaining showed that midbrain cultures contain mature NeuN + neurons that are either TH + or TH - (Fig. 2B) (Bancroft & Srinivasan, 2020;Srinivasan et al, 2016;Zarate et al, 2020). These neurons are co-cultured with primary midbrain astrocytes expressing S100B (Fig.…”

“…Both TH + and THneurons exhibited robust spontaneous Ca 2+ fluxes with heterogenous frequencies and amplitudes ( Fig. 2D and supplementary movie 1) (Bancroft & Srinivasan, 2020).…”

“…To test our hypothesis, we cultured primary mouse ventral midbrain neurons from ED14 embryos, and maintained primary cultures for 19 days in vitro (DIV) prior to imaging ( Fig. 2A) (Bancroft & Srinivasan, 2020). Immunostaining showed that midbrain cultures contain mature NeuN + neurons that are either TH + or TH - (Fig.…”

“…Timed-pregnant mice (Texas A&M Institute for Genomic Medicine) were sacrificed via cervical dislocation and embryos were removed. Embryos were decapitated and ventral midbrain was dissected using previously described methods (Bancroft & Srinivasan, 2020;Henley et al, 2017). Following dissection, cells were digested in papain for 15 min at 37°C, then cells were separated using DNase treatment and mechanical trituration in a stop solution of 10% equine serum in PBS.…”

“…All AAV infections were performed at 14 days in vitro (DIV), as previously described (Bancroft & Srinivasan, 2020). For AAV infections, the culture medium was removed and 1 ml of serum-free DMEM + GluMAX medium containing 1 μl each of Syn-GCaMP6f and TH-tdTomato virus (titer = 1 x 10 13 genome copies/ml) was added to each dish and allowed to incubate at 37°C with 5% CO2 for 1 h, following which serum-free medium containing the AAVs were removed and replaced with 3 ml of supplemented neurobasal medium.…”