Home › Cell Biology › Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons
Cell Biology JoVE (Open Access) Citable · DOI

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

DOI: 10.3791/52361-v
What you'll learn
  • ✓ Differentiate mouse ES cells into pure neuronal populations with functional synapses
  • ✓ Perform electrophysiological measurement of synaptic transmission in neuronal networks
  • ✓ Quantify neurotoxin effects on synaptic function using the MIST assay
  • ✓ Evaluate multiple botulinum and tetanus neurotoxin serotypes on living neural cultures
Protocol

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Difficulty
advanced
Total time
~3–4 weeks (ES cell differentiation and network maturation); ~2–4 hours per electrophysiology experiment
Model organism
Mouse embryonic stem cells (ES cells)
Biosafety
BSL-2

Steps

1
Passage and maintain embryonic stem cells

Culture and serially passage mouse ES cells under standard conditions to maintain pluripotency and viability prior to differentiation.

▶ 01:49
2
Differentiate ES cells into mature neurons

Apply defined differentiation protocol to convert mouse ES cells into highly pure populations of central nervous system neurons exhibiting functional synapses and network behavior.

▶ 03:42
3
Perform MIST assay on neuronal networks

Conduct electrophysiological recordings to measure miniature excitatory post-synaptic currents and quantify baseline synaptic transmission in differentiated neuronal cultures.

▶ 08:26
4
Evaluate neurotoxin effects on synaptic transmission

Expose differentiated neurons to botulinum neurotoxin serotypes A–G and tetanus neurotoxin, then measure loss of synaptic transmission using electrophysiological analysis.

▶ 12:02
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