Electrophysiology Flashcards
(22 cards)
What is the principle of electrophysiology?
Ions have charges
What is the flow of ions called, how do we measure it and what are the measurements made on?
• Flow of ions is current
- Can measure currents using electrical recording equipment
- Measurements made on physiological samples
Why are electrophysiological recordings made?
- Extremely fast events – sub-millisecond timescale upwards (microseconds)
- Extremely sensitive – as little as one ion channel can be detected
- Good spatial resolution
- Dissect details of individual channels – activation, inactivation, pore properties
What are the three different types of electrophysiological recordings?
- Extracellular: easy, not specific – electrode placed on outside of cell
- Intracellular: difficult, specific – electrode inserted into the intracellular solution (overcome the problem of specificity but causes damage to the neurones)
- Patch: very difficult, very specific
What are extracellular recordings, and what are their pros and cons?
- Called electrocenelphogram EEG
- Lots of electrodes placed on scalp
- Suited for the clinic
- Excellent for localising seizures
- Not good for studying individual neurones
- Cheap
What are intracellular recordings and give it’s pros and cons
- Put an electrode into the cell
- Find neuron of interest and impale with glass electrode
- Damages the neuron
- You can record action potential firing
- Suited for lab to measure neuron activity
What is patch clamp recording and what is it useful for?
- Very difficult
- Don’t break into the cell – patch onto the outside of neurons
- Suitable for smaller neurons 30 microns or below
- Suited for the lab to measure channel activity
What is patch clamping?
- Glass tube pulled to give aa very fine tipped (<1um wide) electrode
- The pipette tip can stick to the outside of the cell membrane very tightly
- Pipette (electrode) is filled with electrical conducting solution – pipette solution
- Pipette is then connected to a very fast amplifier and recording equipment and is patched onto the outside of the cell
What is the patch clamp rig?
- Fluorescence microscope sitting on an
- Air table (so won’t get other electrical signal)
- Faraday cage (gets rid of noise)
- Patch amplifier
- AD board
- PC
- Micromanipulator amplifier head stage
What is cell-attached configuration of patch clamping?
- Use: to record currents through a limited number (1-2) active channels at cell surface
- Good for looking at single channel currents in response to regulation of channels by cell
- Patch pipette sucked onto surface of cell – with a ion channel under it
- Channel opening stimulus (e.g. ligand or voltage)
- Current through single channel
What is inside-out configuration of patch clamping?
- Use: to record currents through a single active channel away from cell
- Good for looking at agents that modulate channel by working at its intracellular rate
- Patch pipette sucked onto surface of cell
- Rip off patch of membrane with channel in it. Stick it in bath
- Add agent that modulates channel by acting on its intracellular face
- You will get a current that is entering the cell
What is outside-out patch configuration?
- Use: to record currents through a single active channel away from cell (current leaves the cell)
- Good for looking at agents that modulate channel by working at its extracellular face
- Rip of patch of membrane with channel in it. Hold it in air – membrane flips over. Stick it in bath.
- Add agent that modulates channel by acting on its extracellular face
- Binds to exposed outer face and activates it.
What is whole cells patch clamping configuration?
- Use to record currents through active channels in whole cell
- Good for looking at cell currents in response to drugs added from outside
- Or regulation of channels by cell
- Apply negative suction to inside of pipette – rips small piece of membrane under patch open
- This gives us access to whole cell but doesn’t cause enough damage to kill whole cell
- Allows to record the sum of all the channels
How do voltage gated ion channels work?
• It’s in it’s closed state then opens then closes
- Opens due to change in voltage
- Voltage stimulus:
- Starts cell depolarisation
- Then channel inactivates even if cell is still depolarising
- Goes back to resting membrane potential
How can you measure voltage using patch?
- See current increase as cell opens and ions move through channel, stays open for a bit then closes
- Can measure the time and current of the channel
What can channels differ in?
- Channels can differ in opening time
- Channels can differ in how much current they allow to flow
What can activation of channels increase the probability of?
• Activation of Channels increases their probability of opening
- While the voltage stimulus is absent you still see ‘rare’ spontaneous opening events at negative potentials
- But more depolarised the cell becomes the higher the probability the cell will open
- The same is true with ligand gated channels
How do you work out the total of all the ion channel currents in a cell?
Add up all the single channel currents
Why is patch clamping so useful?
- Ohms law: current (I) – volts (V) x conductance
- Implies if we could hold the voltage constant (using voltage clamp) the resulting current would allow us to determine the conductance of the channels
- The conductance tells us:
how well the channels work
Properties of the pore – ion selectivity, open time, modification by drugs ect.
How does patch clamping work with slices of brain tissue or the whole brain?
- This type of recording is made by applying positive pressure to the fine glass recording-electrode. A jet of solution comes out of the tip of the electrode and is used like a pressure washer to ‘blast’ debris out of the way as the electrode is pushed into the tissue.
- In slices it is sometimes possible to use a microscope to see when the electrode is close to a neuron, but frequently the technique is done ‘blind’ with the neuron hunter relying on changes in electrical resistance to indicate when a cell is under the tip of the electrode. Suction is then applied and recordings can be made just as you would with a cultured cell
What does single cell RT PCR allow measuring of?
the levels of individual mRNA molecules and so infer the expression levels of particular proteins
How does patch clamp/ single cell RT PCR work?
- At the end of a patch clamp recording experiment the contents of the cell are sucked into the patch clamp
- The contents of the electrode are then transferred into a test tube for RT PCR
- The first step in RT PCR is the conversion of the mRNAs present in the sample into cDNAs using a reverse transcriptase enzyme. Primers are added for the protein of interest and the corresponding cDNA is amplified and measured using quantitative PCR (qPCR)
- The information this technique provides about the expression levels of particular proteins can then be related back to the results from the patch clamp experiment
