Electrophysiological Methods

Question 1

What are heterologous expression systems?

Give advantages and disadvantages of this technique.

Answer 1

• Heterologous protein expression means expressing a protein in a cell that does not normally express that protein.
• By expressing these proteins in different cells, they can be more easily studied.

Advantages include:

• Cells can be used that are easy to culture and to study.
• The protein can be studied in isolation (because the cell’s protein expression is being controlled).

Disadvantages include:

• It is not possible to analyse how the protein is involved in controlling cell function in its native cell.

Question 2

What is the main disadvantage of studying the function neurones in an isolated preparation?

Answer 2

• The main disadvantage of studying the function of neurones in an isolated preparation is that the network of input/output systems will have been cut off from the neurones being studied in the sample.
• This means that the function of the neurones in the sample might not reflect the function of the neurones in the brain.

Question 3

How can an individual cell be visualised in a brain slice preparation?

Answer 3

An individual cell can be visualised in a bran slice by using infrared or Nomarski microscopy.

Question 4

List two routes of administration of drugs into the brain for widespread delivery.

Answer 4

Routes of administration of drugs into the brain for widespread delivery include:

1 - Intravenous (IV).

2 - Intracerebroventricular (ICV).

Question 5

List 3 advantages and 4 disadvantages of in vivo neurone preparations.

Answer 5

Advantages:

1 - Physiological state of the sample is preserved.

2 - Stimulation of input and output pathways is possible.

3 - Impact of drugs on brain function can be assessed.

Disadvantages:

1 - Anaesthesia may be required.

2 - Control of the extracellular environment is difficult.

3 - Drug concentrations are difficult to measure because they are likely to diffuse away.

4 - Intracellular recordings are difficult.

Question 6

List 3 methods of drug administration to specific regions of the brain in vivo.

Answer 6

Methods of drug administration to specific regions of the brain in vivo include:

1 - Iontophoresis.

2 - Microdialysis.

3 - Nanodrop application (no explanation card for this one because it is what it sounds like).

Question 7

What is iontophoresis?

List one advantage and two disadvantages of iontophoresis for drug administration.

Answer 7

• Iontophoresis is the administration of an ionised form of a drug from an electrode by passing a current through the electrode, causing the drug to be ejected.

Advantage:

1 - Application of the drug by iontophoresis is fast.

Disadvantages:

1 - The concentration of applied drug is unknown in the tissue.

2 - The current used to eject the drug might cause electrical artefacts.

Question 8

What is microdialysis?

Answer 8

• Microdialysis is a technique used to administer and measure the concentration of a drug at a tissue using an electrode.
• One electrode has a semipermeable membrane at its tip.
• This electrode is infused with the drug, which is able to pass through the semipermeable membrane when the electrode is placed in a tissue.
• Another recording electrode is placed nearby to measure the concentration of the drug and other biochemicals in the tissue.

Question 9

List 2 methods of drug administration to specific regions of the brain in vitro.

Answer 9

Methods of drug administration to specific regions of the brain in vitro include:

1 - Bath perfusion.

2 - Pressure injection.

Question 10

What is bath perfusion?

Give an advantage and disadvantage of bath perfusion for drug administration.

Answer 10

• Bath perfusion involves administering a drug to a sample by bathing the sample in the drug.
• An advantage is that the concentration of the administered drug can be accurately predicted if left to reach equilibrium.
• A disadvantage is that the bath disturbs electrical recordings.

Question 11

What is pressure injection?

Give two advantages and two disadvantages of pressure injection for drug administration.

Answer 11

• Pressure injection involves squirting a drug from a micropipette at high pressure to a discrete area in the sample.

Advantages:

1 - The technique is simple.

2 - The drug can be administered to a discrete area on the sample.

Disadvantages:

1 - The high pressure can damage the tissue.

2 - The concentration of applied drug is unknown in the tissue.

Question 12

List 2 methods of recording electrical activity in tissue samples.

Answer 12

Electrical recordings can be done by:

1 - Microelectrodes.

2 - Patch clamps.

*Both are explained in more detail in later cards.

Question 13

How does the magnitude of intracellular electrical activity differ from that of extracellular electrical activity?

Answer 13

Intracellular electrical activity is much larger than extracellular electrical activity (millivolts compared to microvolts).

Question 14

What is the difference between AC and DC recordings of extracellular electrical activity?

Answer 14

• AC recordings are only able to tell whether or not there has been a change in voltage, and for how long - however they cannot measure the amplitude of this change.
• DC recordings are able to measure both the amplitude of voltage change and when the voltage change occurs.

Question 15

When are AC recordings useful in the extracellular space?

Answer 15

AC is useful for picking up action potentials, (whether or not they are present, and how long they last).

*Since action potentials are usually all the same amplitude under physiological conditions, measuring the amplitude is not necessarily always relevant.

Question 16

When are DC recordings useful in the extracellular space?

Answer 16

• For DC recordings in extracellular spaces, the voltage changes are very small. This means that a lot of synchronous activity is required to pick up any detectable signal.
• On the recordings, the synchronous activity is picked up as sharp waves on a field of background activity.

Therefore, DC recordings are suitable for:

1 - Patterned cell firing (bursting).

2 - Synaptically driven population discharge.

3 - Muscle activity, i.e. electromyography (EMGs) and electrocardiography (ECGs).

4 - Electroencephalography (EEGs).

Question 17

How is intracellular recording carried out?

List 1 advantage and 3 disadvantages of intracellular recording.

Answer 17

• Intracellular recording is carried out by inserting an intracellular (sharp) microelectrode into the cell, perforating the plasma membrane.
• The advantage of using this technique is that the contents of the microelectrode don’t mix with the cytoplasm because the tip of the micropipette is too small, so the cell’s cytoplasmic constituents aren’t disturbed.
• The disadvantage of using a microelectrode is that there are some practical challenges:

1 - The microelectrode is prone to becoming unstable, e.g. due to vibrations.

2 - Insertion of the microelectrode into the cell can cause damage to the cell.

3 - Intracellular recording is difficult in small cells.

Question 18

Give an example of a technique that measures the electrical activity of an entire cell.

What does this technique involve?

Give an advantage and disadvantage of using this technique.

Answer 18

• The electrical activity of an entire cell can be measure using a whole-cell patch clamp.
• This involves pushing a microelectrode against the cell surface and forming a high resistance (gigaohm) seal, preventing leak of current.
• Suction is then applied to the back of the microelectrode, rupturing the cell membrane but maintaining the gigaohm seal.
• This creates a direct electrical continuity between the cell and the electrode, meaning the summed current flowing through all the channels of the cell can be recorded.
• An advantage of this method is that it provides a low-resistance access to the cytoplasm.
• A disadvantage of this method is that it disturbs the physiological conditions of the cytoplasm, because there is exchange of solution between the microelectrode and the cell.

Question 19

What are the two recording configurations of a whole-cell patch clamp?

How do they differ?

How are these configurations useful?

Answer 19

• A whole-cell patch clamp can either be set up as a current clamp or a voltage clamp.

Current clamp (sets the current, measures the voltage):

• In a current clamp, an electrode injects a charged substance into the cell for a period of time.
• This charge causes the initiation of action potentials / depolarisations.
• The same electrode records the change in voltage over time.
• Current clamp configurations are useful for characterising neurones, because it can differentiate their diverse electrical behaviour in response to the injection of charge.

Voltage clamp (sets the voltage, measures the current):

• In a voltage clamp, an electrode sets the voltage across a cell at a particular value.
• The same electrode measures the flow of current through the all of the cell’s channel proteins in the membrane at that set voltage.
• Voltage clamp configurations are useful for assessing the kinetics of particular channel proteins (i.e. measuring the effect of different voltages on the function of the ion channels, such as whether they open at a particular voltage etc.), and investigating how these kinetics change with the application of different drugs.

Question 20

List 2 techniques for recording electrical activity at individual channels.

Briefly describe the techniques.

How are these methods useful?

Answer 20

• The current flow through individual channels can be recorded using inside-out or outside-out patch clamps.

Inside-out patch clamp:

• A microelectrode is pushed against a cell surface, and a high resistance (gigaohm) seal is created (as with a whole-cell patch clamp).
• The microelectrode is the pulled away from the cell, and the part of the membrane to which the microelectrode is attached separates from the cell and forms and inside-out cluster which is attached to the tip of the microelectrode.
• A voltage clamp or current clamp can then be applied to this sample using the electrode.
• This is useful for investigating the effects of modulators of channel function.

Outside-out patch clamp:

• The same gigaohm seal is created as above.
• The microelectrode applies suction to this area of the cell membrane in such a way that the membrane separates from the cell and forms and outside-out cluster which is attached to the tip of the microelectrode.
• A voltage clamp or current clamp can then be applied to this sample using the electrode.
• This is useful for enabling the application of drugs to channels found on the exterior surface of the cell membrane.