PJ17

This protocol assesses in vitro neurotoxicity by combining neuronal viability, mitochondrial/metabolic activity, neurite outgrowth, and optional neuronal network function readouts. Calcein-AM or resazurin/PrestoBlue readouts estimate viable or metabolically active cells; βIII-tubulin immunofluorescence detects neuronal morphology and neurite networks; TMRE detects mitochondrial membrane potential; and MEA recordings detect functional changes in neuronal network activity.

Cytotoxicity assays are usually based on the assessment of cell membrane damage, which can also be indirectly detected by measuring cell viability. Detection methods include MTT assay, CKK-8 assay, LDH assay and ATP assay, etc.

Cerebellar granule neuron culture is a primary neuronal culture method in which postnatal rodent cerebella are dissected, meninges are removed, tissue is enzymatically and mechanically dissociated, and neurons are plated on poly-cation-coated surfaces in medium that supports neuronal attachment, maturation, neurite-network formation, and biochemical or imaging analysis. Cultured CGNs are used to study neuronal development, survival, apoptosis, differentiation, neurotransmitter release, and toxin-sensitive synaptic proteins; mature cultures develop dense neuritic networks, neuronal activity, glutamate release, and neuronal marker expression. A common survival paradigm uses depolarizing extracellular potassium: CGNs maintained in high potassium medium survive and differentiate, whereas switching mature cultures from 25 mM KCl to 5 mM KCl induces apoptotic death that can be used as a readout of activity-dependent neuronal survival.

Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by a progressive decline in cognitive functions and loss of specific types of neurons and synapses. Alzheimer's symptoms can be simulated in mice by injecting drugs (such as Aβ) or genetically modified.