Single Unit Recordings Lecture Flashcards
What does it mean to say that electrophysiological recordings are always differential?
measure voltage at one point
(=the electrode)
relative to another point
(= the reference electrode)
How does the eAP relate to the iAP?
The extracellular action potential (or spike) relates to the intracellular action potential as:
- a scaled down version
(resistive component)
- and the first derivative
(capacitive component)
What are three important electrode features?
Size matters: tissue damage vs. Breaking
Number of contact points
Contact size (tip)
- larger tip: lower resistance/ larger range: more cells
- smaller tip: higher resistance/ smaller range: fewer cells
What metal are electrodes often made out of?
Tungsten (coated in glass / plastic); Classic electrode
What do tungsten electrodes record and where do they record from?
Records single units at the tip
Describe some advantages to tungsten electrodes (3)
•Cheap
•Strong
•Can be used chronically and acute
What is a disadvantage of tungsten electrodes?
Disadvantage: only 1 contact point
What equipment is commonly used for intracellular recordings? Are they useful for extracellular?
(Micro)Pipette: Typically used for intracellular recordings, but also very useful for extracellular recordings.
What is contained in the pipette?
Contains silver wire electrode and 2M NaCl solution which is conductive and mimics extracellular solution
Describe 3 pieces of equipment used to measure from multiple neurons
Tetrodes: constructed by bundling together four very small electrodes
Laminar: Silicon probe with up to 1,000 electrodes. Each works as a single electrode. Allows for comparison of single unit activity across cortical layers
Chronic Arrays: Electrode arrays for chronic implantation which can’t be moved once implanted. Quite superficial but multiple electrodes.
What is the benefit of multiple recording sites?
They can be used to classify extra-cellular action potentials into sets generated by the individual neurons, as each channel of the tetrode is usually close enough to a cell such that action potentials emitted by that cell are detected on each of the four channels, but because of the spatial distribution of the individual channels, the amplitude of the signal varies across the four channels.
How stable are chronic arrays?
Very; can last for months or years
What is the most common noise problem you’ll encounter while making these recordings?
50 Hz noise; Noise due to being embedded in an electrical field. Microvolts at the biological stage is very small
What solutions are there to this noise problem?
-Faraday cage: Protects against external electrical influence
-Local shielding; Usually aluminium foil around the electrode area and around the first amplification stage.
-Keep your wires short
-Avoid ground loops (a ground loop or earth loop occurs when two points of a circuit are intended to have the same ground reference potential but instead have a different potential between them; when enough current is flowing in the connection between the two ground points to produce a voltage drop and cause two points to be at different potentials)
-Get a good “real” ground
-Working at night when others have left (Especially in medical centre)
- ban cell-phones etc
Say you have 4 electrodes but there is quite a lot of noise and it is difficult to see the spikes. What is the most common solution?
Filtering; Depending on the type of noise (correlated, Gaussian, etc…), choose appropriate filtering technique
What filter is typically applied to extracellular units?
•High pass filter (>~300Hz)
•Low pass filter (<~5000 Hz)
Exact values vary between researchers/ labs/ conditions/ type of recordings; 1000 Hz is the ballpark range for AP.
How does finding cells work with recordings?
Chronic implants: hope you have plenty of nice units to record
How does finding cells work with moveable electrodes?
–Look for spontaneously active cells
–Stimulate cells (e.g. with sensory stimuli)
–NB this search method has a bias
–Pipette electrodes: find cells based on resistance
–Locate cells with microscopy
How can you isolate individual units?
APs are stereotyped; they are the same every time. Differences in amplitude and width are likely due to two different neurons at different distances. This can be quite difficult to spot in practice however, with all the noise. You can carry out waveform sorting, feature extraction and plot these differences and carry out a cluster analysis to assign these ‘clouds’ to different neurons
It was claimed that within a type of neuron, APs are stereotyped however this isn’t strictly true. Why is this the case?
While the duration and size of a spike depends on its distance from the recording site, its shape remains constant. Excitatory neurons and inhibitory fast-spiking cell typically have different ratios of duration vs half-width. People find that excitatory neurons are wider and inhibitory are more narrow (duration). This can be used to diagnose which an AP is
What can we analyse from a spike response of a single unit to a single stimulus presentation?
The spike response of a single unit to a single stimulus presentation is often not very informative (nor statistically valid).
What is the solution to this problem with single responses?
That is why we have to combine and average across trials. The Peri-Stimulus-Time-Histogram (PSTH) correlates spike responses according to stimulus onset.
What are some advantages of intracellular recordigs? (6)
• Direct access to membrane potential (Vm), and not only to action potentials (AP) => EPSPs and IPSPs can be recorded
• Possibility to manipulate the membrane potential
• Possibility to record currents related to single ionic and synaptic channels.
• Pharmacological manipulations of neuronal activity.
• In vivo recordings => integration with imaging, post-mortem identification of recorded cells
Describe three problems with intracellular recordings
• Mostly limited to one cell at a time (in vivo)
• Recordings only last few minutes (<30); pipette is 2um, neuron is about 10um
• It is a very difficult technique; takes months if not years of training
What is an alternative to a pipette?
A sharp electrode
List things you can observe through intracellular recordings of EPSPs and IPSPs
• Can quantify the threshold of an action potential
• Resting membrane potential
• Action potentials
• ISI; inter-spike interval
• PSP (post-synaptic potential)
What kind of neurons can have excitatory and inhibitory projections?
None:
Excitatory neurons have ONLY excitatory projections
Inhibitory neurons have ONLY inhibitory projections
Name the three levels in which intracellular recordings can be made in increasing difficulty
• In vitro (cell culture)
• Ex vivo (brain slices)
• In vivo (anaesthetised and awake animals)
What species are intracellular recordings commonly made in?
zebrafish, mice, rats, cats
