Lecture 36: Cortical Circuits 3 Flashcards
What are the advantages of in vitro systems over in vivo systems when it comes to the study of neurophysiology and neuronal function?
can examine specific subsets of cells in isolation, simplified system, allows you to manipulate neuronal biology, exquisite temporal and spatial resolution, comparatively higher throughput (allows you to directly compare many variables in parallel)
What are the common in vitro systems for studying neuronal function?
primary neuronal culture, iPSCs and organoids
What is primary neuronal culture?
cells (neurons) taken from animal tissue and grown in supportive nutrient media in an incubator
How is a primary neuronal culture made?
collect rodent embryos or early postnatal pups -> remove the brain -> separate the cortical hemispheres -> remove meninges, microdissection to isolate the brain region of interest -> enzymatic digestion of tissue and trituration to separate cells into single cell suspension -> cells plated down and grown in supportive nutrient media
What are the strengths of primary neuronal culture?
relatively inexpensive, can be used in a high throughput manner, neurons mature quickly, easy to control, can be cultured from transgenic rodents, can culture cells from different brain regions, easy to assess synaptic function
What are the limitations of primary neuronal culture?
simplified system (monolayer), neurons lose their normal interconnectivity to different brain regions, cannot assess neural circuits (but can look at network activity)
What are iPSC-derived neurons?
human neurons
induced pluripotent stem cells which are differentiate into specific neuronal subtypes
How is the differentiation of iPSCs directed?
specific neuronal growth factors are added
What are the strengths of iPSC-derived neurons?
can be programmed into many different types of neurons, amenable to high throughput screens, can be used for modelling human disease, can be gene-edited to study gene functions, can assess synaptic physiology
What are the limitations of iPSC-derived neurons?
expensive, takes a long time to mature and to develop mature synapses (months), neurons lose their normal interconnectivity and circuitry, cannot assess neural circuits (but can look at network activity)
What are organoids?
3D “mini-brains” derived from iPSCs
display structures that resemble defined brain regions
How are organoids generated?
embryoid bodies derived from iPSCs are generally embedded into an extracellular matrix and then cultivated in a rotating bioreactor to promote amplification and neural differentiation
What are the strengths of organoids?
can be used to develop distinct brain structures which possess some of the layering that is found in the human brain, huge potential for modelling human disease and neurodevelopment, can be gene-edited to study gene function, can assess network activity and simple circuits
What are the limitations of organoids?
very expensive, takes a very long time to develop (months), have a limited maturation, significant issues with cell death due to limited oxygen and nutrient diffusion, batch effects and heterogeneity, cannot assess complex neural circuits
How can neuronal cultures be used?
can be used for a variety of different assays to assess cellular function and synaptic physiology
