In vitro models

• We need in vitro systems for the study of neurophysiology and neuronal function
• In vitro systems


  • Can examine specific subsets of cells in isolation
  • Simplified system that allows examination of specific neuronal processes in isolation, in a highly controllable biological environment
  • Allows you to manipulate neuronal biology in a manner that is impossible, impractical or unethical in whole animal
  • Provide exquisite temporal and spatial resolution that is very challenging to achieve in complex biological systems
  • Comparatively higher throughput than using whole animal studies
• Ethical considerations of using in vitro systems

  • Avoids the need for animals entirely in some cases
  • Can be used in combination with animal studies to minimise harm to animals
  • In some cases, there is no compelling reason to use whole animals
• Common in vitro systems for studying neuronal function

  • Primary neuronal culture
  • iPSCs (neuron-derived from)
  • Organoids
• Primary neuronal culture


  Cells (neurons) taken from animal tissue and grown in supportive nutrient media in an incubator
• How to do primary neuronal culture

  1. Collect rodent embryos or early postnatal pups

  2. Remove the brain

  3. Microdissection - separate the cortical hemispheres

  4. Remove meninges, further microdissection to isolate the brain region of interest

  5. Enzymatic digestion of tissue and trituration to separate cells into single-cell suspension

  6. Cells plated down and grown in supportive nutrient media

  7. Within around 2 weeks mature neurons exist
• Strengths of primary neuronal culture

  • Relatively inexpensive
  • Can be used in a high-throughput manner
  • Neurons mature quickly – 2-3 weeks in culture
  • Highly tractable – easy to control + modify the biology of the system, introduce genes of interest
  • Can be cultured from transgenic rodents
  • Can culture cells from different brain regions
  • Easy to assess synaptic function
• Limitations of primary neuronal culture

  • Simplified system – generally a monolayer
  • Neurons lose their normal interconnectivity to different brain regions – normal circuits are disrupted
  • Cannot assess neural circuits (but can look at network activity)
• iPSC-derived neurons


  Human neurons in a dish, induced pluripotent stem cells (iPSCs) are differentiated into specific neuronal subtypes
• How iPSC-derived neurons are collected

  1. Tissue sample collected e.g. fibroblasts from individuals with a genetic neurological disorder

  2. Reprogrammed into iPSCs

  3. Addition of neuronal growth factors

  4. Directed to differentiate into specific neurons (e.g. cortical neurons; motor neurons)
• Strengths of iPSC-derived neurons

  • Human neurons in culture
  • Can be programmed into many different types of neurons
  • Amenable to high throughput screens
  • Can be used for modelling human disease – fibroblasts taken from patients
  • Can be gene-edited to study gene function, make disease-relevant models (or isogenic controls)
  • Can assess synaptic physiology
• Limitations of iPSC-derived neurons

  • Expensive
  • Takes a long time to mature (months in culture) and to develop mature synapses
  • Simplified system - Neurons lose their normal interconnectivity and circuitry
  • Cannot assess neural circuits (but can look at network activity)
• Organoids


  3D mini-brains derived from iPSCs that display structures that resemble defined brain regions
• How to generate brain organoids

  1. 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

  2. Can also be achieved by supplementing with small molecules and growth factors to promote iPSCs to form specific structures of the different brain regions
• Strengths of organoid neurons

  • 3D human "mini-brains" in culture
  • Can be used to develop distinct brain structures which possess some of the layering that is found in human brain
  • Huge potential for modelling human disease and neurodevelopment – fibroblasts taken from patients
  • Can be gene-edited to study gene function, make disease-relevant models (or isogenic controls)
  • Can assess network activity and simple circuits
• Limitations of organoid neurons

  • Very expensive
  • Takes a very long time to develop (several months in culture)
  • Have a limited maturation
  • Significant issues with cell death due to limited oxygen and nutrient diffusion
  • Batch effects and heterogeneity - variation in efficiency of differentiation, morphology and variability in cell composition across different batches of organoids
  • Cannot assess complex neural circuits
• How we use in vitro systems

  Neurons can be transfected or transduced to express genes/proteins of interest, fluorescent reporters of neuronal activity, receptors that enable modulation of neuronal activity
• Immunolabeling and fluorescence microscopy


  Can be fixed and immunolabeled to assess expression and localisation of proteins or visualize organelles
• Super-resolution imaging


  Imaging beyond the diffraction limit, allows us to resolve single particles e.g. movement of single proteins in different neuronal compartments
• Live cell imaging


  Allows us to measure neurotransmitter release and synaptic vesicle dynamics in individual synapses, or populations of synapses
• Biochemical studies and 'omics

  Can be lysed and used for biochemical analyses (proteomics, RNAseq and transcriptomics, metabolomics)
• In vitro systems


  • Easily modifiable
  • Can be studied in comparatively higher throughput than whole animal studies
  • Allow great temporal and spatial resolution
• In vitro systems offer significant ethical advantages in the study of neuronal biology
• Each in vitro system comes with its own strengths and limitations – you must choose the right system to suit your experimental needs
• Range of assays available that allow us to tease apart different aspects of neuronal biology
• Baker-Gordon Syndrome


  A rare neurodevelopmental disorder caused by mutations to the SYT1 gene
• Symptoms of Baker-Gordon Syndrome include: developmental delay, intellectual disability, movement disorder, behavioural outbursts, altered brain activity
• Synaptotagmin 1


  An essential presynaptic protein that plays a critical role in synaptic transmission
• Synaptic vesicle exocytosis triggered by calcium binding to synaptotagmin 1


  1. Calcium enters neuron via calcium channels

  2. Calcium binds to synaptotagmin 1

  3. Charge change in synaptotagmin 1 allows it to bind and embed into plasma membrane

  4. This helps SNARE proteins drive vesicle fusion

  5. Neurotransmitter release
• Tested 5 variants of synaptotagmin 1 in 11 individuals with Baker-Gordon Syndrome
• Demonstrated multiple similarities in symptoms but also varied in some symptoms such as motor delay, movement disorder, intellectual disability
• Phenotypic spectrum


  Showed the disease had some milder variations and then some more severe variations
• The 5 variants tested were: M303K, D366E, N371K, I368T, D304G
• The in vitro model system used was primary cultured neurons (hippocampal or cortical) transfected with WT or mutant GFP-tagged synaptotagmin
• Assessing synaptotagmin expression using immunolabelling

  1. Green fluorescence for transfected neurons

  2. Primary antibody recognizes all synaptotagmin

  3. Secondary antibody recognizes primary antibody and has red fluorescent tag
• The M303K mutation impaired the stability and expression of synaptotagmin 1, while the other mutations had no effect
• The other synaptotagmin variants (D366E, N371K, I368T, D304G) expressed at normal levels and were trafficked correctly to nerve terminals
• Assessing synaptotagmin 1 function using an assay for synaptic vesicle exocytosis

  1. Measured neurotransmitter release triggered by action potentials

  2. All variants slowed neurotransmitter release to different extents

  3. D366E had the mildest impact, N371K, D304G, I368T had the strongest impact
• The in vitro model system allowed screening of therapeutics to try to ameliorate the changes to neurotransmitter release caused by the synaptotagmin 1 variants