Neuronal Physiology and Electrophysiological Recording techniques

Question 1

Describe the structure of a neuron and briefly explain what the different parts do

Answer 1

Neurons have:

• A cell body or soma.
• Dendrites extend upwards and allow for neurons to receive information from other neurons.
• Axons transmit signals between distant neurons. These end in synaptic terminals, where NTs are released.

Question 2

What is a synaptic potential?

Answer 2

This is the input, and is the signal received by the dendrite.
They are analogue.

Question 3

What is an action potential?

Answer 3

This is the output, and is only seen when the threshold is reached.
They are digital as they are all or nothing.

Question 4

What determines neuronal excitability?

Answer 4

The membrane potential. This is determined by the hydrophobicity of the plasma membrane.

Question 5

What is summation?

Answer 5

The addition of multiple synaptic potentials, in order to cause a stronger depolarisation and thus, more action potentials.

Question 6

How is the membrane potential achieved?

Answer 6

The plasma membrane is hydrophobic, so only certain molecules can pass through.
There is a higher concentrations of anions (-ve) inside, which makes the inside negative relative to outside. This leads to a negative resting potential.

Question 7

What ions determine the resting potential of a membrane and what are their equilibrium potentials? What is the neuronal resting potential?

Answer 7

K+: Em = -94mV
Na+: Em = +60mV

The neuronal resting potential is -67mV, which is more shifted to Em(K+). This shows that when resting, the cell is more permeable to K+.

Question 8

What is equilibrium potential and what is it determined by?

Answer 8

Equilibrium potential is the voltage at which ions balance themselves. It is dependent on 2 driving forces:

• The chemical driving force (concentration gradient).
• The electrical driving force (voltage).

Question 9

What is the chemical and electrical driving force for K+ and Na+?

Answer 9

K+: Chemical = outside, Electrical = inside.

Na+: Chemical = inside, Electrical = inside

Question 10

How do neurons fire action potentials?

Answer 10

Excitation of the neuron causes voltage gated Na+ channels to open, causing depolarisation as Na+ ions move inside the cell.

Question 11

What is the threshold for an action potential?

Answer 11

-30mV

Question 12

How is the action potential propagated to another neuron?

Answer 12

1. The action potential arrives at the pre-synaptic membrane and causes depolarisation, which opens Ca2+ channels.
2. Calcium is released, which triggers vesicle fusion and release of NT.
3. The NT binds to receptors on the post-synaptic membrane, allowing depolarisation.
4. A post-synaptic potential is formed.

Question 13

What ions are excitatory channels permeable to? Give examples of excitatory ligands.

Answer 13

They are permeable to Na+ and Ca2+.

Ligands include: glutamate (CNS), acetylcholine (CNS & PNS)

Question 14

What ions are inhibitory channels permeable to? Give examples of inhibitory ligands.

Answer 14

They are permeable to Cl-. Chloride ions cause hyperpolarisation so are inhibitory.
Ligands include: GABA (CNS), glycine (CNS & PNS)

Question 15

How is extracellular electrophysiological recording done and what does it tell you?

Answer 15

1. A micropipette is put on the surface of the tissue.
2. The voltage is recorded and compared to that of a reference electrode.

Tells you about the properties of a population of neurons.

Question 16

How is intracellular electrophysiological recording done and what does it tell you?

Answer 16

1. A micropipette is put in or onto the surface of an individual cell.
2. The voltage and current is recorded.

Tells you about the properties of an individual cell.

Question 17

What does an extracellular recording look like and what does this show?

Answer 17

Shows a downward wave.
- This shows depolarisation, but is downwards because the positive charge is moving outwards relative to the electrode/micropipette.

Question 18

What does an intracellular recording look like and what does it show?

Answer 18

Shows an upwards wave.

- This shows depolarisation as the positive charge enters the cell. The electrode/micropipette directly measures this.

Question 19

What is the voltage clamp method?

Answer 19

• Uses 2 electrodes to tell us about the current, using the law V=IR. It uses a feedback circuit to keep Em at a set level.
  1. The first electrode records the membrane potential of the cell and relays this info to the 1st amplifier.
  2. The 1st amplifier relays this info to the 2nd amplifier, which has a command voltage set.
  3. If the measured membrane potential is different to the set voltage, the 2nd amplifier injects a certain amount of current into the cell via the 2nd electrode (current passing electrode) to ‘fix’ the actual voltage.

Question 20

How is the voltage clamp method used in research?

Answer 20

• Allows us to see what response ion channels have when the voltage is changed.
• Allows us to identify receptors on cells and which ions they are permeable to.
• Allows us to detect synaptic currents and analyse synaptic plasticity.

Question 21

What does patch clamp recording involve?

Answer 21

Patch clamping involves putting blunt tipped electrodes on top of cells.

• This is useful as it can be used in small cells such as glia and interneurons.
• This method only uses one electrode.

Question 22

What are the different types of patch clamp recording?

Answer 22

1. Cell attached recording
2. Whole cell recording
3. Outside-out patch recording

Question 23

Describe cell attached recording and what it measures.

Answer 23

• The electrode sucks a bit of the membrane in, allowing for a patch to be in the electrode.
• This allows for individual channels to be measured.

Question 24

Describe whole cell recording and what it measures.

Answer 24

• More pressure is applied so the electrode sucks the membrane harder, causing it to rupture.
• The whole membrane is now attached to the electrode like a circuit.
• This allows for whole cell measurements to be done.

Question 25

Describe outside-in patch recording and what it measures.

Answer 25

• After the electrode is attached to the whole cell, it is pulled up. This causes the membrane to be pulled off the cell with a patch still attached.
• This allows for the outside to now be on the inside.
• This allows us to expose the outside of the cell to chemicals and measure responses.

Question 26

Why can’t extracellular methods be used to measure neural circuits?

Answer 26

The brain has a high spatial and temporal precision, as the fibres are densely packed.

Traditional methods are not specific enough.

Question 27

What methods are used to measure neural circuits and why?

Answer 27

Optogentics is used. These involve the use of photoactivated ion channels and pumps.
They have spatial and temporal precision so are used.

Question 28

What are 2 examples of photoactivated ion channels?

Answer 28

• Channel Rhodopsin (ChR2)

- Halorhodopsin (Halo)

Question 29

How does the ion channel ChR2 work?

Answer 29

• ChR2s are permeable to Na+ and K+.
• Light stimulation opens this channel, allowing entry of Na+ and K+ into the cell, causing depolarisation and an action potential.

Question 30

How does the ion channel Halo work?

Answer 30

• Halos are permeable to Cl-.
• Light stimulation opens this channel, allowing entry of Cl- into the cell, causing hyperpolarisation and stopping action potentials occurring.

Question 31

How are these photoactivated ion channels delivered to animals?

Answer 31

Molecular biology techniques.

1. Recombinant vectors encoding a virus are made.
   - The vector has a promoter, actuator (ion channel) and a reporter.
2. The vector is injected in vivo into the brain.
3. Activating light is applied using fibre optics.
4. The reporter is looked for, which tells us where the channels have been expressed.

Question 32

How do Cre recombinase mice show spatial and cell specific expression?

Answer 32

• These mice have: promoter-LoxP-STOP-LoxP-actuator.
• Therefore, the actuator is blocked by the stop codon.

Cre recombinase cuts at LoxP sites, therefore, the STOP codon is cut out, and the actuator is expressed.

Question 33

What is the importance of thermogenetics?

Answer 33

There are naturally occurring channels which are sensitive to temperature.

• These are important as it avoids delivery of light to animals which can include invasive surgery.
• They also have large conductancies, which means that a weak expression of these channels is useful.

Question 34

What are requirements of thermo sensitive channels?

Answer 34

• They must be sensitive to small changes in temperature, as large changes affect neural function.