Modern methods in neuroscience II Flashcards
What did Cajal and Golgi develop?
Ways to label cells
What was the difference between labelling cells in Cajal and Golgi techniques?
Golgi:
- Spare labelling (not all of the cell)
Cajal:
- Fine details
With Golgi labelling, what can be seen?
The MORPHOLOGY of the NERUONS
With Cajal labelling, what can be seen?
Individual DENDRITES
What are the problems with Golgi and Cajal methods of looking at the morphology of neurons?
- Can’t label INDIVIDUAL neurons
- Cannot combine ELECTOPHYSIOLOGY and MORPHOLOGY assesment in the same cell (neurons are dead)
What do we want to study in order to understand about new neuronal types?
1) Morphology of the neurons
2) Map connections
3) Activity of the neurons (what stimuli activate them)
4) Theoretical study
What is the electrophysiochemical method used to study neurons?
Patch clamp technique
What can be used to measure morphology and electrophysiology in one experiment?How?
What is the advantage of this?
Patch clamp technique with flurorescence in the electrode
- Fluorescence will diffuse into the cell
Advantage:
- The neuron is still alive
- Can see how the morphology/electrophysiology changes?
What are the problems with patch clamp with fluorescence to study neurons?
1) Cannot label may cells
2) Limited ability to label a specific cell type
3) Limited ability to label cellular compartments (eg. dendrites, specific receptors)
4) Still LIMITED ability for live labelling
Why is there limited ability to label cellular compartments (eg. receptors) in the cell with patch clamp/fluorescence?
Use a SYNTHETIC DYE - goes everywhere in the cell
Using the patch clamp/fluorescence technique, why is there still limited ability for live labelling?
In most cases, method is applied to SLICES of tissue
Complicated with live animals
How are the problems with patch clamp/fluorescence techniques overcome?
Using GFP
What is GFP stimulated by?
What does it emit?
Blue light
Emits green light
What are RFP, YFP and CFP?
Modifications of GFP that have different colours
different excitatory and emission wavelengths of light
What can GFP be used for?
- To label cells (morphology)
- Follow MIGRATION of cells and the PROJECTIONS of the neurons
How can GFP be used to study the activity of neurons?
Use a modification of GFP (GCaMP)
What is GCaMP?
What is the structure?
GFP based calcium indicator
GFP fused to 2 calcium binding proteins (M13 and Calmodulin)
What happens in GCaMP in the presence of calcium?
What does this cause?
M13 and Calmodulin interact and STABILSE the GFP
Causes the GFP to become brighter
What happens when the neuron is active when using GCaMP and why>
Calcium channels open
Calcium levels in the cell rise
Causes more M13 and calmodulin interaction
Causes GFP to be brighter
What is the difference between wide field and confocal microscopy?
Wild field - collects light from above and below the focal plane
Confocal - Pinhole rejects light NOT coming FROM the FOCAL plane (reject light from above and below the FP)
What does confocal microscopy result in?
A large increase in SPATIAL resolution
What is the problem with using GCaMP to study function of neurons?
How are these problems overcome?
- Animal is sedated using Na channel blockers
- Neurons spike less
OR
- Animal is stressed
- Release of other molecules in the brain
- Animal doesn’t perform the behaviour that is normally does
Overcome by using:
- Virtual reality
- Freely moving animal
Describe the virtuality technique of studying the function of neurons
- Mouse on a ball that moves in a certain direction when the mouse moved
- Head is fixed
- Turn of the ball leads to a turn of the field of view in that direction
Describe the freely moving mice teqnique of studying the function of neurons
- Tiny fluorescent microscope placed directly into the skull of the mouse
- Image is delivered via light guide
How are channel-rhodopsin and halorhodopsin used?
Introduced into a cell
Used to modify neuronal activity
How is channel-rhodopsin activated?
Using blue light
What happens when channel-rhodopsin is activated?
Depolarisation of the membrane
What type of channel is channel-rhodopsin?
Non-specific cation channel
What activates halorhodopsin?
Yellow light
What ions pass through halorhodopsin?
Cl
What happens what halorhodopsin is activated?
The cell is hyperpolarised
