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
Why aren’t intracellular recordings made in larger species?
respiration artefacts, blood pressure waves, motions
Are recordings easier on older or younger animals? Why?
The older the animal, the more difficult the technique becomes (maybe due to a thicker extracellular matrix) => mice/rats should be younger than 2/3 months
Is it possible to make intracellular recordings in awake moving animals?
Yes but insanely difficult; only one or two labs worldwide capable or even attempting
What equipment is required for intracellular recordings?
• Glass pipettes: glass capillaries, pipette
puller
• Artificial intracellular solution
• Recording set-up: anti-vibration table, head fixation/stereotaxic device, micromanipulator
• Electrophysiology equipment: electrodes, headstage, amplifier, software
• Histology (optional)
• Imaging setup (optional)
Is an Artificial intracellular solution required?
Yes or the cell will die
How can you obtain a glass capillary?
Buy a glass pipe and shape them yourself. Place in the centre of high heat coils then slowly pull apart around 10-12 mm; Recentre back around 4-6 mm then make a second pull on low heat to pull them apart.
Describe the standard solution of a glass pipette
Standard solution: 135 mM K-gluconate, 10 mM HEPES, 10 mM Na-phosphocreatine, 4 mM KCl, 4 mM ATP-Mg, 0.3 mM GTP (mimics cytoplasm)
Important: pH: 7.2 (pH has to be the exact same or cell dies)
Osmolarity: 291 mOsm/l
Optional: Biocytin 3mg/ml (for histological reconstruction)
Key features:
-ionic concentrations (chemical and electrical equilibrium)
-controlled pH
-key components for normal cell functionings
List three other solutions/ components
EGTA: blocker of Ca channels
QX-314: blocker of voltage-dependent channels
Ce-methanesulphonate: replaces K-gluconate and blocks potassium channels
Name and give the function of 5 pieces of recording set-up equipment (besides actual recording equipment)
Anti vibration table: Eliminates any sources of vibratio from the external environment; required when parameters are so small and tiny movements results in loss of the cell
Faraday cage: Mentioned previous
Head fixation/ stereotaxic system (for in-vivo)
Bath + microscope (ex-vivo or in vitro)
micromanipulator: Can move pipette 0.1mm
Name 6 pieces of equipment for actually recording (besides pipette)
Headstage ( tethered to the head of a laboratory animal, that allows multiple channels of brain activity to be monitored and recorded)
Pipette holder (with pressure outlet; needs to control pressure or cell can ‘explode’)
Amplifier: amplifies signal obviously
Analog-digital interface: convert process variables into equivalent signals and provide corresponding continuous outputs
Software
Ag0AgCl electrode (in the pipette)
Describe the procedure of carrying out a whole cell patch clamp recording (Blind approach)
Search around with pipette for a cell:
a) The tip has a RESISTANCE (easily measured with voltage pulses)
b) Approach: small steps with slight positive pressure
c) Touching the membrane: the resistance increases suddenly
d) Releasing pressure makes the membranes stick to the pipette tip
e) Sucking increases the seal (often with mouth), «Gigaohm seal» is obtained
f) More sucking ruptures the membrane: you are INSIDE the cell!
Why can’t you just push inside the cell?
The cell will just move
When is this glass pipette method often applied and when is it rarely applied anymore?
Not so much in vivo anymore; very difficult. Often used in vitro
How can you know when you are inside the cell?
there will be a sudden drop in voltage and spikes will be recorded (see docs)
Aside from whole cell recordings, describe two mistaken deviations in procedure during patch clamp recordings
Inside-out patch: When gigaohm seal, pulling back causes the sytoplasmic bridge to collapse and air exposure breaks out the cell patch. A ruptured piece of the membrane hangs outside the pipette and the uruptured side inside the pipette
Outside-out patch: When gigaohm seal is obtained and inside the cell, pulling back causes the sytoplasmic bridge to collapse and an outside-out patch is collected when a piece of the membrane hangs outside the pipette and the ruptured side inside the pipette
How else can an inside-out patch occur?
If you pull in a low Ca+ medium
How does this procedure change with a targeted approach?
The electrode is positioned onto a cell under visual guidance (often with calcium imaging)
Monitoring the tip resistance is still vital!
For each of the following symbols give the parameter they represent, their Unit and their unit abbreviation:
Q g C E R I
E; Potential/ voltage; volt; V
I: Current; ampere; A
R; Resistance; ohm; Ω
g; Conductance ; siemens; S
C; Capacitance; farad; F
Q; Charge; Coulomb; C
Define an electric potential
The amount of work energy needed to move a unit of electric charge from a reference point to the specific point in an electric field. More precisely, it is the energy per unit charge for a test charge that is so small that the disturbance of the field under consideration is negligible.
Define an electric current
Electric current refers to the flow of electricity in an electronic circuit, and to the amount of electricity flowing through a circuit.
Define resistance
Resistance is a measure of the opposition to current flow in an electrical circuit.
Define electrical conductance
the ability of a solution to conduct an electrical current
Define electrical capacitance
Capacitance is the ratio of the amount of electric charge stored on a conductor to a difference in electric potential ( to the potential difference (i.e., voltage) between them). [Capacitance is a measure of an object’s ability to store electric charge.]
There are two closely closely related notions of capacitance. Name and describe them
The method of measuring changes in capacitance with respect to earth ground is commonly referred to as self capacitance measurement.
Mutual capacitance involves measuring a change in capacitance just like self-capacitance, with one big difference: we define both plates of the capacitor, instead of utilising earth ground as the second plate.
Define electrical charge
A characteristic of a unit of matter that expresses the extent to which it has more or fewer electrons than protons.
Name what the following (badly drawn) symbols represents in a technical circuit
=|>- ⍉
-|=|- -○_○-
⏚ -+| |–
-|>- -| |-
- |>- : amplifier
=|>- : Differential amplifier - |=|- : Resistor
⏚ : Earth (usually bath electrode)
⍉ : input or output
-○_○- : switch
-+| |– : voltage source - | |- : capacitor
Define an amplifier
An amplifier, electronic amplifier or (informally) amp is an electronic device that can increase the power of a signal (a time-varying voltage or current).
Define a differential amplifier and explain the output
A differential amplifier is a type of electronic amplifier that amplifies the difference between two input voltages but suppresses any voltage common to the two inputs. It is an analog circuit with two inputs (Vin- and Vin+) and one output Vout. in which the output is ideally proportional to the difference between the two voltages:
Vout = A(Vin+ - Vin-)
where A is the gain of the amplifier
What is meant by earth in a technical circuit?
A reference point in an electrical circuit from which voltages are measured, a common return path for electric current, or a direct physical connection to the Earth.
What is meant by a capacitor?
A capacitor is a device that stores electrical energy in an electric field. It is a passive electronic component with two terminals.
What is meant by input/ output in a circuit?
I/O (input/output), pronounced “eye-oh,” describes any operation, program, or device that transfers data to or from a computer. Typical I/O devices are printers, hard disks, keyboards, and mouses.
What is meant by a resistor in a circuit?
A resistor is a passive two-terminal electrical component that implements electrical resistance as a circuit element. In electronic circuits, resistors are used to reduce current flow, adjust signal levels, to divide voltages, bias active elements, and terminate transmission lines, among other uses.
What is meant by a voltage source in a circuit
Supplies potential difference across two terminals; A voltage source is a two-terminal device which can maintain a fixed voltage. An ideal voltage source can maintain the fixed voltage independent of the load resistance or the output current.
What is meant by a switch in a circuit?
An electrical component that can disconnect or connect the conducting path in an electrical circuit, interrupting the electric current or diverting it from one conductor to another.
What does ohms law state?
Voltage is equal to the current * resistance:
V = I * R
When describing neurons, what does a resistor describe?
“user of electrical energy” =>
channels, leaks, conductive materials
When describing neurons, what does a capacitor describe?
“storage of electrical energy” =>
membrane, glass
When describing neurons, what does a generator describe?
Generator: “source of electrical energy” =>
ionic pumps, external interventions (amplifier)
Thus, describe the circuit we have formed through making a seal with the cell still attached
There is resistance from a leak (Rleak) from the puncture made by the electrode, additionally there is resistance from the pipette (Rpipette). The fluid in the pipette also acts as a capacitor (Cpipette). The bath electrode acts as the earth.
The current travels up from the electrode creating a current, this and a current from the reference electrode meets a differential amplifier which travels through it and resistance to the output.
What does Rleak examplify the importance of?
The “giga-seal”
What do you need to do if you have this Rleak? Why?
The injected current ( I(pA) ) forms more of a square wave while the recorded potential looks more like an actual wave ( E(mV) ). You are interested in what happens across the membrane, but for this you need to minimalise
or compensate for all other factors
Once inside a cell you have the option of studying two things, what are they?
Once inside a cell, one can measure either current or voltage fluctuations.
How can you measure either voltage or clamp?
Using two types of electrical feedback circuits, one can «clamp» the membrane in either «voltage-clamp» or «current-clamp».
Describe voltage-clamping and why it is useful
•The cell potential is kept fixed
•This allows measurement of currents flowing
into a neuron: EPSC and IPSC
•Useful because many channels are voltage-
dependent.
Describe current-clamping and why it is useful
•Usually this means clamped at 0, which means that
the cell potential is free to vary
•This allows measurement of voltage
variations: EPSP and IPSP and action potentials
•Useful because this is what is really going
on in neurons
