Techniques in pharmacology Flashcards
What is electrophysiology?
• Electrophysiology- the study of the electrical properties of cells or tissues
What information does electrophysiology provide?
o Tells us about function (but can also tell us about location)
Why is electrophysiology possible?
o All cells have a membrane potential that is maintained by control by movement of sodium, calcium and potassium
o Using electrophysiology, we can measure the movements of these (and other) ions
What does electrophysiology allow the exploration of?
o How drugs modify the way ion channels activate, open, inactivate and desensitize
o How drugs change neuronal excitability
o How drugs change synaptic transmission and synaptic plasticity
o Location of drug action
o Drug interactions with a mutated protein
What instrument can be used to measure bioelectricity at the amp range?
Fingers
What instrument can be used to measure bioelectricity at the milliamp range?
Wires
What instrument can be used to measure bioelectricity at the microamp range?
Extracellular cell recordings
Microelectrodes
What instrument can be used to measure bioelectricity at the nanoamp range?
Microelectrode/patch
What instrument can be used to measure bioelectricity at the picoamp/attoamp range?
Patch only
What is the advantage of extracellular recordings?
Can be done in vivo
What are the disadvantages of extracellular cell recordings?
- Very hard to control drug administration
- No control over the cell of interest
- No choice of cells
- Very general- not able to pinpoint specific effects
What is the process of extracellular cell recordings?
o As action potential propagates through a cell the electrical current flows in and out of the cell. This changes the voltage in and out of the cell
o If an electrode is inserted into the brain and near a neuron, this extracellular electrode will measure this voltage change
How are single-unit extracellular recordings performed?
o Small electrodes can record activity of one neuron (single-unit)
How are multi-unit extracellular recordings performed?
o Larger electrodes record the activity of several neurons (multi-unit)
Where are extracellular cell recordings mainly used?
o Main use in in vivo anaesthetized or awake behaving animals
How are microelectrode/intracellular cell recordings performed and what does it measure?
o Very fine glass electrode is inserted into a cell of interest
o Metal wire in the liquid filled glass electrode connects to amplifier
o Allows measurement of voltage or current across the membrane compared to a reference electrode
Where are microelectrode/intracellular cell recordings used and why?
o Mainly used in oocytes due to their large size, some neurons (but very few are able to put two electrodes in due to their small size, and hard to control voltage and measure current at the same time with a single electrode)
What are the advantages of using microelectrode/intracellular cell recordings with oocytes?
Advantages:
• Oocytes express proteins well (good for mutation studies)
• Technically easy
What are the disadvantages of using microelectrode/intracellular cell recordings with oocytes?
Disadvantages:
• They are frog oocytes
o Need to consider applicability to mammals
• Difficult to exchange drugs
• Difficult to get good electrical control of neurons as only one electrode
• Noisy
• Makes a hole in the cell
Who developed patch clamping, when and how?
• Patch clamping o 1976- patch clamping arrived Neher and Sackman made first patch-clamp recordings Did this using: • Used better amplifiers • Changed electrode type
How is patch clamping performed and what is the result of this?
o Patch electrode process
Patch electrode pushed up on edge of membrane
Apply small amount of suction so that membrane forms tight seal with glass
Can either
• Pull electrode back to pull out small piece of membrane with just one ion channel of interest
o Record from a single ion channel
• Whole cell- suck on piece of membrane and break it open
o Record from all ion channels/conductances all over the cell
What is the use of patch-clamp, what does it measure and what allows this measurement?
Allows for the voltage-clamp of a cell (set the voltage of a cell)
• Can control voltage-dependent processes
• Amplifier maintains a membrane potential and measures the currents required to maintain it
• This allows measurement of voltage-dependent ion channel responses
• Used to elucidate details of how a drug interacts with a receptor to change its function
What is the advantage of patch-clamp?
o Can control the intracellular milieu of the cells
Manipulate the signalling systems of one cell at a time
o Very low noise and high resolution-can measure the current flow through a single channel
No hole in the membrane around the electrode
o Measure/study activation of one or a few proteins
o Versatile
Permits measurement of several proteins in the same cell
Many different applications
Single cell, cultured cells with mutated proteins expressed, slices, in vivo
What is the disadvantage of patch-clamp recordings?
• Disadvantages- o Quite hard o Can be expensive o Not suitable for all cells o One cell at a time, low throughput o Can lose important components of cell when accessing the inside