Visualization of Calcium Influx in Ventral Midbrain Neurons Derived from Mouse Embryos

Take a coverslip with a culture of ventral midbrain neurons derived from mouse embryos.

The cells are infected with a viral vector to express a calcium indicator.

Transfer the coverslip to the recording chamber of a confocal microscope perfused with a recording buffer.

Using visible light, identify a region containing multiple neurons, then switch to fluorescence imaging.

At low intracellular calcium levels, the indicator remains unbound, exhibiting low fluorescence intensity.

Intrinsic properties of the neurons generate spontaneous action potentials, triggering voltage-gated calcium channels to open and allow calcium influx.

The excess calcium binds to the indicator, inducing a conformational change that increases fluorescence intensity.

Record the fluorescence intensity over time to track the spontaneous calcium activity.

Introduce a neurotransmitter at a high concentration, which binds to receptors on the neurons, generating continuous action potentials.  

The sustained voltage-gated calcium channel activation prolongs calcium influx, causing a sustained increase in fluorescence intensity.