Electrophysiology techniques in neuroscience (Salmen) Flashcards
Types of electrodes and use
- Extracellular electrodes
- Intracellular electrodes (path clamp and sharp microelectrodes)
- Suction electrodes (recordings from nerves and fiber tracts)
- Electrode arrays for extracellular recordings (ex: for slices, drugs, etc.)
- Ion sensitive microelectrodes
- Carbon-fiber electrodes
- Patch-clamp electrodes
Ohm’s law
V = IR = 1/G
Kirchoff’s laws
- 2 Parallel resistors slip the current inversely proportional to their resistance → sum of currents stays constant at all times
- 2 resistances following each other split the voltage according to their resistances
Electrophysiology recording modes
- Extracellular recordings
- Used to record field potentials and for measuring ions - Intracellular recordings
- Current clamp
- Voltage clamp - Patch-clamp recordings
- Current clamp & Voltage clamp
What kinds of structures should be used for measuring field potentials
Highly laminar structures (i.e. hippocampus or cerebellum)
- Still possible to get decent recordings from cortex and others, but not as high quality (ex: can record from amygdala but is very unclear and low quality)
What can electrophysiology experiments record? (structures/cells/setting)
In vivo cells (would do surgery)
Acute slices
Slice culture (100-500 um thickness, using vibratome, chopper, or cut by hand)
Isolated cells
Expression systems like Oocytes, HEK-cells, etc.
What is a sink?
= Positive charges entering cell (moving away from the observer/electrode), or negative charge moving toward observer/exiting cell
What is a source?
Positive charge to observer/extracellular space or negative charge away from observer
What is the difference between an active and a passive sink/source?
Active means there’s ion movement through the membrane due to channel opening
Passive means there’s ion movement due to an active sink/source, which induces redistribution of ions away from the active site
Components of evoked field
- Excitatory postsynaptic potential (fEPSP)
- dendritic fEPSP is indicative for input; changes in are indicative for changes in synaptic transmission
- Somatic fEPSP and fPop-spike are indicative for how soma computes dendritic input and if it reacts with a spike or not - Stimulation artefact (basically just a sign that you are recording something; is a super quick and drastic negative current)
- Fiber volley (FV)
= a measurement of compound APs you’re activating from axons - fPop-spike represents the synchronous firing of APs from many neurons
Why record field potential from two positions ?
Somatic and dendritic fEPSP together are indicative for computation of inputs and corresponding output of the cell
Advantages of studying field potentials
Relatively simple
Leaves intracellular milieu intact
Disadvantages of field recordings?
No intracellular manipulation possible
Not possible to do detailed analysis of neuron subtypes of subtle changes in membrane properties
What is used for intracellular recordings?
Sharp microelectrodes and patch pipettes
- Comparison of sharp microelectrode and whole-cell patch shows results are not really the same, but the spike threshold is similar
Current clamp vs voltage clamp
Current clamp
- Apply constant current
- Record Voltage
- = traditional method for recording membrane potential
Voltage clamp - Membrane potential kept constant - Measures current - Does not mimic natural process (unlike current clamp), but allows us to study VG channels and see that/if current is linearly proportional to conductance ( V = IR, G = 1/R → I = VG → I = kG)
Advantages of intracellular voltage clamp
- Access to intracellular environment
- Allows for recording from cells too small for impalement
- Allows for recording of currents through single channels (= unitary currents)
Patch-clamp configurations
- Cell-attached
- Recording pipette touched to membrane, formation of gigaseal - Whole-cell: basically cell-attached + strong suction applied
- Cytoplasm continuous with pipette interior
- The intracellular solution is replaced by the pipette solution within 15 minutes - Inside-out configuration = cell-attached + pull
- Leads to vesicle formation
- Expose vesicle to air → cytoplasmic domain is accessible - Outside-out configuration: whole-cell + pull
- Ends of membrane anneal
- Extracellular domain is accessible
Response in cell attached
Will have 0 resistance of membrane when pulse starts
Basically, get artefact (200pA) that is approximately inverse of loading artefact
Response in whole cell when applying -5mV
Membrane acts as/is huge capacitor
Large current spikes (500pA) outward at start and inward at end of pulse
What resistance are we most interested in when doing a patch clamp recording?
Restiance of the membrane (Rm)
What are the four critical resistances in whole-cell patch clamping?
- Resistance of the pipette (resistance is inversely proportional to the size, so while a small pipette gives an easy seal, it also gives a high series resistance)
Rpip = Vcommand/I - Resistance of seal (Rseal)
- Generally in the 1-5 GOhm range
- Bad seal formation → bad signal - Series resistance (Rs)
- Is excess to cell
- If it is too high, too much voltage is “lost” (flow resisted) instead of at the cell membrane
- Rs = Vcommand/Imax - Membrane resistance
- Resistance of cell
- Inversely proportional to cell size
- Should be at least 10x Rs
- Rm = Vcommand/Ioffset
VG ion channel recordings show us
Differences between open and closed states
Let us see if there’s intermediate states
Expression systems
Oocytes
HEK cells
etc.
Are the ideal system to study pure receptor properties and recpetor interactions
Which is stronger, the somatic or the dendritic AP?
Dendritic much weaker, but when normalized, they’re about equal, except dendritic lasts longer
