Experimental Techniques In Neurophysiology Flashcards
What does an electro physiologist study
Electrical and biophysical properties of organs, tissues, cells
What is electrophysiology
Branch of physiology pertaining to movement of ions in tissue and their flow across cellular membranes. Electrical recording techniques that enable measurement of ionic movement
Role of intracellular electrode
Applies current or voltage command
Role of extra cellular electrode
Reference electrode
Role of amplifier
Increase small voltage (mV) or current (pA) changes so they can be detected by low impedance electronics
Role signal generators
Initiate the voltage or current injections
Role feedback amplifiers
Ensure supplied command matches the requested command
What is an electrophysiology rig
All equipment needed for experiment using small scale materials
Conditions of voltage clamp
Experimenter specifies the voltage and measures the resulting current
Ex. Step the voltage from -70 to -50
Note- if want to look at Nav you can block other channels
Conditions of current clamp
Experimenter injects current and measures resulting changes in membrane potential (voltage)- usually AP but could be EPSP
When is voltage clamp useful
- study single channels or one specific type
- investigate speed of opening in closing of channel
- investigate which ions are permeable to the channel
- test if particular chemical alters any of the biophysical properties of channel
When is current clamp useful
- study excitable cells like neurons
- find out which ions are important for AP (which ions are needed)
- test if particular drug blocks AP
Ex. 1- see if cell can make AP. 2- remove specific ions in solution to see if AP stops (indicating the ions are needed for AP)
What technique does patch clamp utilize
Type of voltage clamp- measure current
Who developed patch clamp electrophysiology and what was their achievement
Dr. Erwin Neyer
Dr. Bert Sakmann
Nobel prize- first to record currents from a single channel
How does patch clamp physiology work
Form a tight (high resistance) seal between a glass micropipette containing an electrode and the plasma membrane of a cell
- channel trapped in pipette
What does the patch clamp technique allow for
- experimenter can record movement of ions or changes through single channels or a population of channels
- control extracellular and intracellular composition
- control voltage and measure current
What are the different patch clamp configurations
Cell-attached recording
- tight contact between pipette and membrane creates seal
Inside-out recording
- membrane detached. Cytoplasmic domain is accessible because it faces outside solution (think stuck in pipette inside now faces outward while outside of channel sucked in first)
- can control cytoplasmic contents
Whole-cell recording
- cytoplasm is continuous with pipette interior
- can record for all channels in the plasma membrane
Outside-out recording
- extracellular domain accessible
- extracellular face put in
What type of channels can be analyzed using inside-out recording vs outside out
Inside out- Nav
Control cytoplasmic contents
Outside out- nACh
ACh bind extracellular, have access to this side of channel
Why kinds of current are there
Single channel current
Macroscopic current- through numerous channels
What does depolarizing voltage look like on a recording
Upward rectangle
What does a single channel current look like on recording
Square waves
Measured in pA
What does a macroscopic current look like on recording
Sum of all individual currents
Large peaked mountain (up or down)
Down = inward current
Up = outward current
Why will macroscopic current return to baseline
Channel inactivation
Advantages to in vivo and in vitro recordings
In vivo
- real time recordings in live animals
- high physiological relevance
In vitro
- isolated tissues or cells are easier to work with
- control over solutions
What are some disadvantages to in vivo and in vitro
In vivo
- technically difficult
- little to no control of intracellular and extracellular fluids
In vitro
- may be less physiologically relevant
- molecular techniques required
What animals to use for in vivo and in vitro
In vivo- mouse
In vitro- isolated squid giant axon, cultured cells
Advantages of heterozygous expression
- higher expression levels result in larger currents
- control over solution composition
- ability to modify channel structure using molecular biology techniques
What is heterologous expression
Used to study a single type of channel
Express something where it isn’t usually expressed
What kinds of cells can be used for heterologous expression
HEK293 cells
Xenopus oocytes
- because they don’t express many channels
Disadvantages of heterologous expression
- less physiological relevance
- channel may require accessory component not expressed in system
- cloning, sub cloning, transfection, RNA purification
- endogenous channels may interfere with recording
Ohms law
V = IR
Plot I vs V curve for a single ion (ex. From patch clamp experiment)
What is the slope? (experimental design- no concentration gradient)
Linear relationship
Slope = conductance
Intersect at 0,0 because no gradient through channel (since one ion)
Pg 12
What kind of channel opens at -130 mV
Hyperpolarizing channel
