Methods in Neuroscience Flashcards
Give some examples of model organisms used for neuroscience methods and why they are suitable/not
- Humans
- Primates (unethical)
- Cats and rodents (rodents have short lifespan)
- Zebrafish (transparent so can visualise)
- Drosophila (key mechanisms conserved but holistically v different)
Explain the Marilyn Einstein illusion
- Far away the picture looks like Marilyn Monroe due to low spatial resolution
- Closer looks like Einstein due to high spatial resolution
What experiments can be used to study where the behaviour of interest is initiated in the brain?
- Brain lesions (removal or dysfunction)
- Whole brain imaging techniques
3, Multi-electrode recordings in different brain areas
What are the two ways we can study brain lesions?
- Through direction (brain lesions for the purpose of the experiment in animals)
- Patients who already have lesions due to neurodegenerative diseases
How is patient LM an example of a brain lesion in studying behaviour related to brain localisation?
- Stroke patient
- Good with colour, face and object recognition
- But people tended to just appear, she didn’t see motion
How does fMRI work?
- Highlights parts of the brain that are metabolically active by looking at blood flow
- Glucose is carried in the blood, neurons need glucose for ATP and synaptic transmission
Can be used with visual system, getting a person to look at stimuli and observe which brain parts are active
What is the problem with fMRI?
Very low resolution, shows areas but not individual neurons or synapses
Once you have found the brain area what techniques do you use next?
- Describe morphology of individual neurons to split into different classes
- Map connections
- Describe activity (methods that tell you how and when the neuron is active during behaviour)
- Theoretical study
How did Cajal and Golgi investigate morphological studies of neurons/cells?
- Staining techniques, dye to label neurons
- Sparsely labelled so its not just one big blob, you can see the start and end of neuron
- Cajal labelled retinal neurons and looked at their spatial organisation
What is an enhancer trap and some of its drawbacks?
Enhancer = contain binding sites for specific transcription factors which when bind upregulate expression of gene
Enhancer trap= fluorescent reporter gene to see expression patterns of regulatory genes of transcription
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- But cannot stain individual neurons
- Cannot combine electrophysiological and morphology assessment for the same cell
What are sharp electrode recordings?
- Sharp tip injects current into the cell so you can image the membrane potential of the neuron
- On the x axis you have time and on the y is membrane potential
- You can then depolarise the neuron or activate a neighbouring neuron etc and see what happens
Give an example of where sharp electrodes have been used
- Juusola lab = sharp electrodes being used in drosophila
- Show that fly photoreceptor cells show high depolarisation when exposed to light
What are the disadvantages of sharp electrodes?
- No solution change inside or outside the cell
- Limited possibility for controlling the membrane potential
- Cannot measure single channels
How do patch clamp methods work?
-Tip is not as small as a sharp electrode - Contact with membrane and applies suction rather than puncture
- Can measure currents which go through that part of the membrane
What are the types of patch clamp methods?
Inside-Out Patch clamp= exposes the intracellular face of the membrane to the extracellular solution, direct manipulation of intracellular signaling pathways and ion channels
Outside- Out patch clamp= excising a patch of membrane from the cell such that the extracellular face of the membrane is exposed to the intracellular solution of the pipette
Whole cell = Suction applied to rupture membrane patch and establish electrical contact between the pipette solution and contents of cell
What is the Green Fluorescent Protein? (GFP)
- A flourescent protein stimulated by blue light which emits green light
- Used for cell part identification
- Gene encoding GFP is inserted into gene codes encoding for interested protein
Why is it good that fluorescent proteins can have different excitation and emission wavelength
Different colours can be with different neurons to identify many at once
What does a simple fluorescent microscope have that a normal one doesn’t and what do they do?
- Excitation filter
only passes light that excites the molecule (blue light) - Emission filter
passes light emitted by the GFP (green) - Dichroic mirror
reflects light of certain wavelength and passes light of different wavelength (reflect blue and passes green)
Give examples as to why GFP is useful
- Can identify promoters of genes that are expressed in neurons i.e. label all the neurons in the drosophila larvae
- Videos made to look at axons developing and moving to their specific target, shows molecules that express a certain protein (for e.g.) go in a certain developmental direction
- You can look at molecular mechanisms which regulate the pathways
What is GCaMP and what is it used for?
- To look at function of neuron rather than morphology
- Fusion of GFP and two calcium binding proteins , in the presence of calcium, GFP and the binding proteins interact and the GFP becomes much brighter
- So calcium = action potential= GFP becomes brighter due to rising Ca2+ levels during action potential
What is the difference between a fluorescent microscope and a confocal microscope?
- Introduce a tiny pinhole to reject most of the above and below focal light
- Better spatial resolution and quality
- Decreases area of excitation as it rejects light not coming from the focal plane
What are the problems in using animals in microscopy study?
- Animal is sedated using Ca2+ blockers or is stressed meaning it doesn’t perform behaviour like it usually would
- Sedated animal = unnatural (cant generalise)
- Instead: use virtual reality or a free moving animal instead (microscope would follow the animal via a tiny microscope attached directly to the skull but image quality is worse)
How does the ‘moving fish’ method better typical microscopy studies?
Uses a mechanism where the microscope predicts the fishes next move and moves to that place so the fish is always under the objective
How is neuronal stimulation stimulated by light using channelrhodopsin?
- Ion channel that is modified so that when stimulated by blue light the channel opens and it depolarises the membrane as its not selective
- When you switch off the light the channel closes and the membrane is repoalrised again
- Means you can make the animal move more/less
How is neuronal stimulation repressed using Harlorhodopsin?
- Light is turned on and it opens and transmits chloride causing hyperpolarisation of the membrane
- Can see modification of specific neurons and see how it effects processing as the action potential doesn’t occur
What is the difference in conducting large scale studies in C.elegans and other animals?
- C.elegans have only 302 neurons whereas animals such as drosophila have 25,000
- Animals have more neurons, connections and complexity and become less stereotypical
How can electron microscopy be used to identify each individual neuron?
Stack of thin slices and manual segmentation of different parts of the brain showing each neurons and each connection localised to where they are
What is RNA tomography?
- Slice the embryo and for each slice they perform RNA expression profiling
- Mathematical image reconstruction to determine genome-wide 3D expression patterns
