Lecture 8- Measuring neural activity I Flashcards
What are the levels at which you can measure neural activity?
• Behaviour (the highest level= everything we do requires some neural activity) • Activity in specific regions or networks associated with a behaviour • Network activity in networks resulting from a defined stimulus – In vivo or in vitro • Activity in single cells • Activity in cellular sub-compartments
What is voltage?
- is the electric energy charge difference of electric potential energy -Voltage is equal to the work done per unit of charge against a static electric field to move the charge between two points
Can you masure voltage in neurons?
-yes, because they use electrical signals to communicate -cells are electrical circuits
Can you measure current in neurons?
-yes because current flow produces voltage changes
What is current?
-In electric circuits this charge is often carried by moving electrons in a wire. It can also be carried by ions in an electrolyte, or by both ions and electrons such as in a plasma
What is conductance?
-conductance of an electrical conductor is the opposition to the passage of an electric current through that conductor. The inverse quantity is electrical conductance, the ease with which an electric current passes. Electrical resistance shares some conceptual parallels with the notion of mechanical friction.
What is Ohm’s law?
-Ohm’s law states that the current through a conductor between two points is directly proportional to the potential difference across the two points. Introducing the constant of proportionality, the resistance • R is resistance, G is conductance G = 1/R
What are the properties you can measure at single cell level?
-conductance is the inverse of resistance -resistance is what you actually measure -more recently people have developed methods in measuring the changes in ion flux = easiest to do with is calcium -calcium is an indirect measure of electric activity in neurons -you can measure calcium= get a dye, that responds to the presence of calcium by fluorescenec then measure light output of the essence -calcium changes will reflect the ability of the -can also do it with Cl, and pH indicators -cells are elctrical cicrcuits, have resitance, conductance -the number of ion channels will determine this -orhms law -conductance= measur eof how ion channels operate (open/close) but measure the resistance -new way of measuring the influx of ions= Ca= indirect measure of e activity in neurons -can also do it with Na or pH indicators, but Ca is most common to use
What can you measure at single cell level (short)?
-voltage, current, conductance, resistance -transient changes in intracellular ions (notably Ca2+)
What are the characteristics of the technique of measurement: single cell recording?
- Extracellular recording from single neuron or axon – Metalorglasscapillary microelectrode
- Detects action potential firing in individual neurons – Actuallydetectscurrents produced by these action potentials
- Workhorse of in vivo neurophysiology
- when people start trying to measure the activity neurons, sherrington = use electrodes to get electric signals from the neurons
- single sharpened and insulated bit of wire that you could bring into contact with a neuron = can detect the e activity of a neuron
- advantage is that you can discriminate the e signal produced by one neuron -still used, fully developed in 1950s
- electrodes meaure cureent that flows through the membrane as the AP goes= that causes a voltage change at the tip of the coelectrode, can discriminate if the insulators and amplifiers good aeenough to discriminate from the noise
What are the strengths of single unit recordings?
• Can be used in any part of nervous system • Multiple recordings can be made in several sites allowing correlation of activity • Can be readily used in vivo -can record from more neurons but difficult to extract beyond a number of neurons -very old technology
What are the weaknesses of single unit recordings?
-it doesn’t tell you what is driving the AP, is it an increase in membrane potential or due to removal of inhibitory membrane potential, don’t tell you when inhibition occurs= is it removal excitation or inhibited it? cannot get that information from this process -also doesn’t tell you which neuron you are recording from, if you have a mix of groups of neurons, which is often the case, which is active? • Does not indicate the membrane potential changes (synaptic potentials) that lead to changes in firing • Does not give information about neurons that do not fire action potentials
What are the characteristics of the technique of measurement: sharp electrode intracellular recording?
-put in a microelectrode and heats it, provide force, sharp tip about a twentieth of a micrometer -high potassium chloride concentration -can measure the voltage across the membrane -can emasure the firing, the synaptic potential, what the ion channels are doing -the experiment is very difficult to do! art form -• Sharp microelectrode with 0.05 – 0.5 μm tip inserted into individual neuron • Electrode glass capillary filled with conducting solution (usually KCl up to 3 M) • Records membrane potential,so can measure action potentials, 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 and neurochemistry -huge advantage= can put in compound that will allow you to visualise the cell! –once in the cell= an measur ethe membrane potential changes, can also measure firing, the properties of the embrane, info about ion channels etc.= more technically difficult -better in vitro= since tissue doesn’t move around, in vivo only if no moving! -can out into the electrode compunds that will allow you to visualise the cell
What is this?
- this is what you see in the intracellular recording technique
- insert the electrode and record the resting membrane potential= can record the passive properties of the cell
What does intracellular recording allow you to do?
-allows cell shape and chemistry to be correlated with electrical activity -eg.: • Neurons in gut injected with marker, biocytin, after electrophysiological characterisation • AH neurons are intrinsic sensory neurons • S neurons are motor neurons and interneurons • Also have characteristic neurochemical codes
What are the 2 types of the gut neurons used as he example in what you can do in an intracellular recording?
- AH cell and C cell
- real recordings, 2 types in neurons
- in tissue taken from guinea pig gut wall
- after an AP= a potassium conductance increase= that hyperpolarises the membrane after the depolarisation of the AP!!! know this
- these neurons have extra hyperpolarisation that can last up to 20s which is much longer! -AH neurons= big after hyperpolarisation
- distinct shape= long shape body
- another class of neuorn in the same system= single long process, short lamellar dendrite and different electrophysiological properties (no after hyperpolarisation)
- can use dye injection from these electrodes to get all this info
What is the voltage clamp technique?
- First version – Used squid giant axon
- Records membrane potential with one electrode and passes current with other
- Measures current needed to keep membrane potential constant – Determines current passing through membrane after a stimulus
- giant sqid axon= can put wires in = put 2 in, one across the wall, one to measure voltage differences the axon is so large= for escape response so it is quick and not brain involved -also can squeeze the contents in
- the wires in= pretty much an intracellular recording techniques
- feedback amplifier, not only measures the membrane potential= also possible tho dial in the membrane potential you wanted= so could make current go through= voltage clamp= holds the membrane potential constant and measures the current needed for this to happen
- not very useful technique for normal size neurons, need to be big!
What are the characteristics of the patch clamp?
-electrode with 2-5 μm tip
– fire polished
- Forms tight seal (called GigOhm) with membrane
- Can isolate single ion channel in intact cell membrane
- mid 1970s- uses voltage clamps circuitry but a different type of electrode
- take one of the microelectrode= break the tip of and polish it
- if brought against a membrane in the right way= forms a GigOhm seal= actually a covalent bond= sealed onto the membrane
- in its simplest form= mild suction, one ion channel in the tip of the electrode= can look through the ion channel and study the piece of membrane
- this is how they figured out how ion channels work
What is outside out patch (patch clamp) recording?
- Outside out patch
- Voltage-clamp mode
- A Voltage step
- B Successive current traces for 7 repetitions
- Downward deflections are inward sodium currents
- if you pull the membrane of, so the outside of the membrane is outside the electrode (can also be inside)
- outside out patch
- from -80 to -10 mV -the current measure through a series of traces= show current going downwards- -first step cause 2 pikoamp current, then broader the amount of charge going through -sodium channels open qickly and close by deactivation= this is the proof
What is the whole cell recording (patch clamp) like?
- Whole cell recording makes large hole in membrane and brings inside of electrode into contact with cell cytoplasm
- Like intracellular recording in current clamp, but also allows voltage clamp
- here have sharp suction and pull the membrane off and into the elctrode= now the whole cell is attached to the electrode= so coninuos fussion and electrically
- the cell is part of the electrode
- can use current clamp and measure voltage potential or current clamp and measure voltage
- can also stimulate other part of the cell and look at what happens
- no problems with movement= as have a covalent bond, so if cell moves= electrode does too
- allows multiple recordings from neurons
