L8 & 9- Measuring Neural Activity

Question 1

How are properties measured at a single cell level?

Answer 1

Via voltage because neurons use electrical signals to communicate.
Via current because current flow produces voltage changes.

Question 2

Single Unit Recording – What is it? Strengths & Weaknesses?

Answer 2

Extracellular recording from single neuron or axon using a metal or glass capillary microelectrode where it detects currents produced by AP firing in individual neurons.

STRENGTHS:

• Can be used readily in vivo
• Can be used in any part of the NS
• Multiple recordings can be made in several sites allowing correlation of activity

WEAKNESSES:

• Does not identify membrane potential changes (synaptic potentials) that cause changes in firing
• Does not detect neurons that do not fire AP

Question 3

Intracellular Recording – What is it? Strengths & Weaknesses?

Answer 3

Sharp glass capillary microelectrode with 0.05 – 0.5 um tip, filled with conducting solution (3m KCL) is inserted into individual neuron. It records membrane potential, so can measure AP, synaptic potentials, passive membrane properties.

• Commonly used in vitro but can be used in vivo under right conditions
• Allows injection of intracellular markers to identify neuron morphology & neurochemistry
• It allows cell shape & chemistry to be correlated with electrical activity

STRENGTHS:

• Allows long term recording (3h) of membrane potential & subthreshold changes in membrane potential
• Identifies some membrane mechanisms that determine neuron excitability
• Suitable for pharmacological studies with drugs & mechanisms that last several 10s of minutes

WEAKNESSES:

• Does not allow underlying currents to be measured
• Does not identify locations from which signals are generated

Question 4

Voltage Clamp – What is it? Strengths & weaknesses?

Answer 4

• Records membrane potential with one electrode & passes current with other
• Measures current needed to keep membrane potential (voltage) constant – determines current passing through membrane after a stimulus
• First version – used squid giant axon

STRENGTHS:

• Very good time resolution
• Measures underlying currents
• Strong data for construction of math models

WEAKNESSES:

• Only works in large cells(uses wires rather than microelectrodes)
• Does not provide good control of dendrites, even in large cells
• Heavily dependent on properties of electrodes(electrical properties of dendrites distort their current passing signal)

Question 5

Patch Clamp – What is it? Strength?

Answer 5

Instead of impaling neuron (causing big hole), used electrode with 2 – 5 um tip that is fire polished which leads to the formation of a tight seal (called GigOhm – basically covalent bond) with membrane. This allows for the isolation of a single ion channel in intact cell membrane.

STRENGTHS:
As tip is large, you are able to use the patch pipette for “single electrode” voltage clamp – amplifier switches b/n voltage & current mode at high frequency (what is measured depends on patch configuration).
*If electrode has high resistance, won’t work as well

Question 6

Whole cell patch recording - What is it? Strengths? Weaknesses?

Answer 6

Making a large hole in membrane & brings inside of electrode into contact with cell cytoplasm (cytoplasm is continuous with pipette interior). Allows current or voltage clamp.

STRENGTHS:

• In vitro (in vivo under right conditions)
• Voltage-clamp mode to measure membrane currents – especially synaptic currents (EPSCs & IPSCs)
• Low resistance electrodes = low noise
• Seal to membrane gives stable recordings

WEAKNESSES:

• Main drawback: dialyses cytoplasm so recording duration can be limited: 30 mins (partially overcome with “perforated” patches which places a filter on tip of electrode)
• Needs clean cell (not good when there is connective tissue)
• Not useful for analysis of large networks but powerful when used to record 2-3 interconnected neurons or 2 parts of the same neuron

Question 7

How can whole cell patch recording drawbacks be useful?

Answer 7

• Dialysis of cell cytoplasm allows sampling of mRNA (using RT-PCR can get expression profile of selected proteins within the cell)
• As electrode filling solution replaces cell cytoplasm, it can manipulate cytoplasmic composition
• Can selectively inject cell with Ca2+ indicator & then correlate membrane potential or currents with Ca2+ transients

Question 8

Whole cell patch recording: what can you do with Ca2+ indicators?

Answer 8

e. g. Dendritic spine visualised with two photon microscope
- Ca2+ conc increases due to synaptic activity – increasing strength of fluorescent marker, showing what is happening in that location in real time, much higher spatial resolution than electrical recording
- Loss of temporal resolution or breadth of field as you can’t get enough data in nor can you record large areas
- Cytoplasmic Ca2+ responds to membrane potential, but is a qualitatively different signal