Spontaneous neuronal activity of cultivated cells is one of the physiologically most relevant read-outs related to neuronal health. Calcium concentration is widely used in neuroscience as a competent indirect reporter of neural activity, as action potential (AP) firing triggers large influxes of Ca2+ through voltage-gated calcium channels.
Utilizing IncuCyte® NeuroBurst, a genetically-encoded calcium indicator (GECI), in combination with the IncuCyte® live-cell imaging system allows to monitor morphological changes and measure neuronal activity of thousands of functional neurons inside a culture over an extended period of time.
To analyze spontaneous neuronal activity, rat primary neurons are transduced with NeuroBurst and cultivated until DIV8. Thereafter, cells are analyzed for spontaneous baseline activity in the IncuCyte® live-cell imaging system before treatments are applied. Cells can be treated with:
- Picrotoxin, a non-competitive channel blocker for GABA receptor chloride channels, described to act as neuronal stimulant leading to seizure-genic activities
- MK-801, a NMDA antagonist, acting as anti-convulsant, reducing neuronal activity
- Forskolin, a cAMP elevating agent, described to increase neuronal differentiation, health, and activity.
The response of cells is again monitored via IncuCyte® live-cell imaging system 6, 12, 24, 48 and 72 h after application of the treatments.
All three items present the expected results compared to the respective vehicle control (VC). While MK-801 treatment leads to a reduction of active neurons, reflecting its anti-excitatory activity, picrotoxin and forskolin increase the number of active neurons (Figure 1A). Forskolin additionally impacts on the burst rate up to 72 h after treatment start (Figure 1B), while picrotoxin shows a strong and fast effect on correlation and synchronicity of neuronal activity (Figure 1C).
Figure 1: Monitoring neuronal activity in response to treatments with time using IncuCyte® NeuroBurst. A: Number of active neurons per well (objects that burst at least once per recording time), B: Burst rate presented as total number of bursts per minute, C: Correlation of neuronal activity as mean of all objects in relation to each other, 0 being completely random and 1 being highly synchronized. Data are generated via the integrated IncuCyte® Neuronal Activity Analysis Software Module.
Videos: Primary rat cortical neurons seeded at 30,000 cells/well in a 96-well microplate were infected with IncuCyte® NeuroBurst Orange Reagent to progressively monitor neuronal activity. Movies captured on DIV10 (48 h after treatment start; treatment with picrotoxin, forskolin, MK801 or vehicle control (VC)) show changes in spontaneous neuronal activity (calcium oscillations) due to different treatments.
Rat primary neurons can therefore be used to evaluate spontaneous neuronal activity. The compounds Picrotoxin, MK-801, and Forskolin can be used as reference compounds.