Lecture 11 – ELECTROPHYSIOLOGICAL TECHNIQUES Flashcards
What is Electrophysiology:
- Ions are charged (Ca2+, K+, Na+)
- Flow of ions (electrons) is known as the current
- Can measure currents using electrical recording equipment (AMP, Volts and resistance)
- Measurements are made on physiological samples (charges in nano/picto range)
Why are electrophysiological recordings made?
¥ Extremely fast events – sub-millisecond timescale upwards (action potentials are 2ms)
¥ Extremely sensitive – as little as one ion channel can be detected
¥ Spatial resolution – good – record from a single synaptic terminal/ channel and upwards
¥ Dissect details of individual channels
– activation, inactivation, pore properties (cation/anions?), synaptic/neuronal integration
Extracellular
(needle is an electrode) – tip can be sub-micron sizes
filled with solution, silver wire is added to the electrode which extends down to the tip
- sees a change in potential to –ve
- as potassium comes out a +ve charge builds up
- easy
- not specific
- suited for the clinic
- EEG used in clinics
- measure areas of activity of the brain, directly under each electrode
- measuring electrical activity of ions
- can embed electrodes under the brain but it is not as easy
used in drug-targeted epilepsy
Intracellular
wire is inside the cell
- ion channels open they enter the cell
- action potentials form as Na+ goes in making it +ve
- as K+ leaves, becomes –ve
- does not tell how individuals ions work
- difficult
- specific
- suited for the lab to measure neuron activity
- brain slice shown
- input into the CA3
- lots of action potentials fire
- pharmacology can be used to inhibit or trigger action potentials
SNARE proteins (genetics) have an effect
Patch
- this is the best one to give an insight on how channels work
a blunt pipette is used which cannot pass membrane
a tight seal is made in the membrane which increases the resistance, no leakage of current within the pipette
aim is to isolate a single receptor molecule and channel under the patch which is done by creating a round seal under the membrane and then opening it up to see any changes compared to the current one shown by using the other tests - very difficult
- very specific
- suited for the labs to measure channel activity
Patch Clamp measurements of ion channel activity:
Glass tube pulled to give fine tipped electrode
- Measuring channel currents
- Neher and Sakmann
- Patch clamp methodology – glass tube pulled to give a very fine tipped electrode (right)
- Configurations - Ion fluxes (currents) through single channels
- On or Off responses
- Channel lifetime
- Channel currents - Ion fluxes through many channels
- Single channel currents add up
Fine tipped electrodes:
¥ pipette (electrode) is filled with electrical conducting solution – pipette solution
¥ pipette is then connected to very fast amplifier and recording equipment
- piece of tissue would be in bath solution (ECM)
- solution has wire and mimics the inside of the cell
- circuit is between the two ends of the wire that leads into the solution
- air tables dampen everything out and makes sure there’s no ‘bouncing’ around
- amplifier needs a interphase with a computer as it is analogue and needs to be converted to digital
Whole-Cell Configuration:
- used to get over complications with small cells
- negative pressure is applied to inside of pipette – suck on tube
as you rupture from sucking the tube it removes it from the membrane
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
- Patching and lining the hole of the patch
- Intracellular for small cell configuration – most brain cells too small
Cell-Attached Configuration:
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 onto cell and isolate 1 ion channel underneath the membrane
- Movement comes in as action potentials are fired which can record how much Na+ is coming in by that action potential detected
- Channels group together in different channels of lipid wrap
Inside-out configuration:
Use:
- To record currents through a single active channel away from cells
- Good for looking at agents that modulate channel by working at its intracellular face
- Once patch is made, rip away the piece of membrane that is attached to the electrode, isolating it further
- External face of membrane is inside the patch, internal face is exposed to the saline which can now be experimented on
- Kinase phosphorylated?
G-protein coupled receptor?
Outside-Out Configuration:
Use:
- To record currents through a single active channel away from cell
- Good for looking at agents that modulate channel by working at its extracellular face
- This looks at how ligands bind
- Do the exact same as before, isolating the patch
- The difference in this is you remove the patch from the solution for a second which inverts the membrane
- This means the internal face is now facing the inside and the external is exposed to saline
- You then add ligands to activate the channels
Helps to study kinetics
Ion Channels open in an ‘on or off’ way:
- As the K+ channel open, we want to measure the amount going through a single channel
- Voltage activated so when depolarised it will open
When channel opens ions flow – a ‘blip’ of current:
- A blip of positive current
- K+ is moving out of the patch
- If Na+ was leaving and going into the cell then it would be –ve
- Time on x-axis, as channel opens there’s a blip
- Can measure time of opening and magnitude of current
- Activation is almost instantaneous
- Only one open state
- Channels are either on or off
Anatomy of a single ion channel opening event:
- Can measure how big the current is from the channel
- Record how long cells stay open for
- Blip is usually 0.1ms
