Electrophysiology Flashcards
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
