Experimental Techniques In Neurophysiology

Question 1

What does an electro physiologist study

Answer 1

Electrical and biophysical properties of organs, tissues, cells

Question 2

What is electrophysiology

Answer 2

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

Question 3

Role of intracellular electrode

Answer 3

Applies current or voltage command

Question 4

Role of extra cellular electrode

Answer 4

Reference electrode

Question 5

Role of amplifier

Answer 5

Increase small voltage (mV) or current (pA) changes so they can be detected by low impedance electronics

Question 6

Role signal generators

Answer 6

Initiate the voltage or current injections

Question 7

Role feedback amplifiers

Answer 7

Ensure supplied command matches the requested command

Question 8

What is an electrophysiology rig

Answer 8

All equipment needed for experiment using small scale materials

Question 9

Conditions of voltage clamp

Answer 9

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

Question 10

Conditions of current clamp

Answer 10

Experimenter injects current and measures resulting changes in membrane potential (voltage)- usually AP but could be EPSP

Question 11

When is voltage clamp useful

Answer 11

• 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

Question 12

When is current clamp useful

Answer 12

• 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)

Question 13

What technique does patch clamp utilize

Answer 13

Type of voltage clamp- measure current

Question 14

Who developed patch clamp electrophysiology and what was their achievement

Answer 14

Dr. Erwin Neyer
Dr. Bert Sakmann
Nobel prize- first to record currents from a single channel

Question 15

How does patch clamp physiology work

Answer 15

Form a tight (high resistance) seal between a glass micropipette containing an electrode and the plasma membrane of a cell
- channel trapped in pipette

Question 16

What does the patch clamp technique allow for

Answer 16

• 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

Question 17

What are the different patch clamp configurations

Answer 17

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

Question 18

What type of channels can be analyzed using inside-out recording vs outside out

Answer 18

Inside out- Nav
Control cytoplasmic contents

Outside out- nACh
ACh bind extracellular, have access to this side of channel

Question 19

Why kinds of current are there

Answer 19

Single channel current
Macroscopic current- through numerous channels

Question 20

What does depolarizing voltage look like on a recording

Answer 20

Upward rectangle

Question 21

What does a single channel current look like on recording

Answer 21

Square waves
Measured in pA

Question 22

What does a macroscopic current look like on recording

Answer 22

Sum of all individual currents
Large peaked mountain (up or down)
Down = inward current
Up = outward current

Question 23

Why will macroscopic current return to baseline

Answer 23

Channel inactivation

Question 24

Advantages to in vivo and in vitro recordings

Answer 24

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

Question 25

What are some disadvantages to in vivo and in vitro

Answer 25

In vivo
- technically difficult
- little to no control of intracellular and extracellular fluids

In vitro
- may be less physiologically relevant
- molecular techniques required

Question 26

What animals to use for in vivo and in vitro

Answer 26

In vivo- mouse
In vitro- isolated squid giant axon, cultured cells

Question 27

Advantages of heterozygous expression

Answer 27

• higher expression levels result in larger currents
• control over solution composition
• ability to modify channel structure using molecular biology techniques

Question 28

What is heterologous expression

Answer 28

Used to study a single type of channel
Express something where it isn’t usually expressed

Question 29

What kinds of cells can be used for heterologous expression

Answer 29

HEK293 cells
Xenopus oocytes
- because they don’t express many channels

Question 30

Disadvantages of heterologous expression

Answer 30

• less physiological relevance
• channel may require accessory component not expressed in system
• cloning, sub cloning, transfection, RNA purification
• endogenous channels may interfere with recording

Question 31

Ohms law

Answer 31

V = IR

Question 32

Plot I vs V curve for a single ion (ex. From patch clamp experiment)
What is the slope? (experimental design- no concentration gradient)

Answer 32

Linear relationship
Slope = conductance
Intersect at 0,0 because no gradient through channel (since one ion)
Pg 12

Question 33

What kind of channel opens at -130 mV

Answer 33

Hyperpolarizing channel