L3 - Methods in Modern Neuroscience Flashcards
What are the 4 methodological approaches to study new neuronal types?
Describe morphology – where projections are sent, size of dendritic tree
Map connections – which cells it connects with
Describe activity – which stimuli activate neurons
Theoretical study
Morphological studies - Cajal and Golgi
Development a method to label cells – don’t label every cell
Allowed them to characterise the morphology of the neurons
Sparse labelling was discovered by?
Golgi
Fine details were discovered by?
Cajal
What are the problems with staining morphology?
Does not allow for staining of individual neurons
Cannot combine electrophysiology and morphology assessment of same cell
- Staining kills the neuron
What was the solution to the problems with staining morphology?
Patch clamp
Patch clamp method - electrophysiology
- Use glass electrode filled with electrolyte - similar to intra- or extracellular solution
- Touch the cell and apply negative pressure
- Part of the membrane will form tight junction with the membrane
- Can record currents through this patch
How can you record currents through the whole cell?
Can break the membrane
How do you carry out morphology and electrophysiology in one?
Adding fluorescent dye in the pipette solution will give information about the morphology of recorded neuron
What are the problems with electrophysiology and morphology?
Cannot label many cells
Limited ability to label specific cell type
Limited ability to label cellular compartments
Limited ability for live labelling- mostly uses slices of tissue
What is the solution to the problems with electrophysiology and morphology?
GFP
What is GFP?
Fluorescent protein
Stimulated by blue light
Emits green light
What can GFP be used for?
Used to understand morphology and neuron function
What are the differences between fluorescent proteins?
Can have different excitation and emission wavelengths
All have different colours
How does a
simple fluorescent microscope work?
Need an excitation light – use a dichroic mirror
- Reflects light of just one wavelength
- Every other wavelength passes though – light we see
What is GCaMP?
GFP based calcium indicator
What is the structure of GCaMP?
Fusion of GFP and two calcium binding proteins
- M13 and Calmodulin
How does GCaMP work?
In presence of calcium the two proteins interact
GFP becomes much brighter
Used to understand the function of neurons
What is GCaMP role in active neurons?
Expressing GCaMP means can observe the activity in the neurons
When neuron is active lots of Ca channels on the membrane opens and Ca in cytoplasm rises
Neurons then become brighter
Can image the entire brain with a single neuron resolution
What are the two types of high resolution microscopes?
Wide field microscopy
Confocal microscopy
Allows for massive increase in spatial resolution
Wide field microscopy
Collects light from, below and above the focal plane
Confocal microscopy
Rejects light coming not from the focal plane
Decreases the area of excitation
Higher resolution
What are the problems with in-vivo methods?
Animal is sedated using Na+ channel blockers
Animal is stressed
Animal does not perform behaviour that it usually does
What are the solutions to the problems with in-vivo methods?
Virtual reality
Freely moving animal
Virtual reality with mouse
Mouse is placed on a ball that moves in certain direction when mouse moves
Turn of the ball leads to turn of the field of view
Head is still fixed
Objective is placed on top as usual
Free moving mice
Tiny fluorescent microscope placed directly in the skull
Image delivered via light guide
Neuronal stimulation by light - channelrhodopsin
Non-specific ion channel activated by light
When channel opens, cell depolarizes
Activated by blue light – channel opens
What is the problem with blockers?
They are not very specific – diffuse
Hyperpolarisation by light - halorhodopsin
Specific for chloride ions
Activated by yellow light – channel opens
When channel opens, cell is hyperpolarized
