BMS248 Lecture 2 - Methods in Neuroscience Flashcards
What are sharp electrode (intracellular) recordings?
What do they allow you to record and from what
Current is injected into the cell through a glass pipette (e.g. photoreceptor).
This allows you to record changes in membrane potential - i.e. voltage (e.g. an AP) in response to a stimulus.
This method cannot measure individual channels.
Sharp electrode recording, also known as “sharp microelectrode recording,” involves inserting a glass micropipette filled with an electrolyte solution into a neuron. The tip of the electrode tapers to a fine point, allowing it to penetrate the cell membrane without disrupting it. This technique is commonly used for extracellular recordings, where the electrode measures the electrical activity of the neuron near its surface.
What is the patch clamp technique?
What can you record?
Inhibitors?
A glass electrode filled with electrolyte is applied to the cell, part of the membrane forms a tight junction with the electrode when negative pressure is applied.
The voltage is clamped and ion currents are recorded either through single channels or whole cell when the membrane is broken.
Ion channel inhibitors can be added to see the effect on function
What are the 4 different configurations of the patch clamp technique?
On-cell: individual
Inside-out: individual
Outside-out: individual
Whole cell
What are the drawbacks of electrophysiology?
Cannot label many cells.
Limited labelling of specific cell types.
Limited labelling of cellular compartments.
Limited live labelling.
What is GFP?
What light is it stimulated by?
What light does it emit?
Green Fluorescent Protein.
Stimulated by blue light - 480nm (excitation).
Emits green light - 510nm (emission).
Fluorescent proteins can have different what?
What does this mean for labelling?
Different excitation and emission wavelengths.
You can combine them to label different cell types.
What is GCaMP?
What is it composed of?
What occurs when calcium is present?
Where is this useful to use?
A GFP based calcium indicator.
GFP and two calcium binding proteins - CaM and M13.
The two proteins interact - this changes the conformation of the protein - GFP becomes brighter.
In neurons - as when a neuron is active its calcium level rises - visualisation of where calcium is
What is the difference between wide field and confocal miscroscopy?
What is the benefit of confocal microscopy?
Wide field - collects light from, below and above the focal plane.
Confocal - rejects light not coming from the focal plane + decreases area of excitation.
Much higher spatial resolution.
What is a channelrhodopsin?
What happens when it is stimulated by blue light?
What is the channels resting state?
Non-selective cation channel.
The channel is activated + changes conformation - allowing cations through and depolarising the cell - this leads to depolarisation and an AP.
Closed in the dark, open in response to blue light.
What is halorhodopsin?
What happens when it is stimulated by yellow light? (570nm)
What is the channels resting state?
Selective Cl- channel.
Channel opens allowing chloride ions into the cell - this leads to hyperpolarisation.
In resting state, the chloride ions are unable to flow through the channel, maintaining the normal physiological conditions of the cell or intracellular compartment.
WHat does the dichroic mirror do
Only reflects light with a certain wavelength and allows other wavelengths to pass through
How does the patch clamp work
Uses an electrode with a bigger hole
Bring electrode to touching the membrane
Apply a negative pressure and the membrane will be sucked into the electrode forming a tight connection with no holes so the ions cna move freely
What are the problems with confocal microscopy
Animal is sedated using Na+ channel blockers
Animal can be stressed causing ireegular behavious
Animal isnt performing actions while awake as they are sedated
Solution - use VR, have the animal freely moving with a microscope attached to head
What does sharp electrode recordings measure
Membrane potentials in neurons
How do you do sharp electrode recordings
Glass electrode with very thin tip –> Insert electrode inside the soma and compare the difference of the potential of the membrane of the neurone
When you stimulate the neuron there are changes in the membrane potential and you can detect the APs of the soma
