Lecture 3: Experimental Techniques in Neuroelectrophysiology

Patch clamp recording can be used for..

(1) Smaller neurons (2) Direct observation of the ionic current through a single ion channel (3) Can be used to do either voltage or current clamp recordings

What was one of the earliest forms of neuronal recording techniques?

-Microelectrodes -Measured membrane voltage

Think through the content of the voltage clamp recordings through I-V plots.

1. Start of experiment the membrane potential is clamped at -70 mV 2. Voltage clamp equipment moves (steps) membrane potential to -40 mV for milliseconds. To move the membrane potential to -40 mV the equipment delivers current to the inside of the neuron to change it. 3. When membrane potential reaches -40 mV a portion of the available voltage gated K+ channels are open, and there is a flow of K+ ions out of the neuron (can be verified from the fractional activation curves) 4. Without the voltage clamp technique, the K+ current would hyperpolarize the membrane. But, the voltage clamp technique applies a current that balances the current caused by the flow of K+ ions.

What is kept constant in current clamp recording?

Amount of current

For this type of configuration, the electrode is placed against the neuronal membrane and a seal is made between the pipette tip and the membrane. Pipette is filled with EXTERNAL solution because this solution will only be touching the OUTSIDE of the neuron. This recording is able to trap a single ion channel. This allows the researcher to figure out how that single ion channels contributes to the flow of ions across the membrane

Cell-Attached Configuration

Explain ball and chain mechanism for na+ during an action potential.

Channel opens and Na+ flows through but very quickly the ball binds to the binding site preventing Na+ from moving through. This inactivation is what causes the dramatic increase in open sodium channels to last such a brief time during the rising phase of an action potential. When the membrane potential hyperpolarizes sufficiently, the ball moves out of the channel and closes in it's original resting state (called channel deactivation)

What is the "ball and chain mechanism" or the "N-type-inactivation"?

Channels use this method of inactivation by using a group of amino acids on the intracellular surface that form a ball which is tethered to the rest of the channel by a chain. When the channel is open, the ball hangs away from the binding site allowing ions through. When the channel inactivates, the ball binds to the binding site preventing movement.

In what type of experiments is the ionic current flowing across the membrane controlled by the experimenter, not by the voltage across the neuronal membrane. The change in voltage that is required to maintain the desired current is recorded and indicates how the membrane potential is affected by the current flowing across the membrane.

Current Clamp

What method would you use to determine how the membrane potential changes in response to current moving through ion channels in the membrane?

Current Clamp Recording

In a voltage clamp experiment, is the current depolarizes the neuron what would we see?

Excitatory Post synaptic current (EPSC)

In a current clamp experiment, if the current depolarizes the neuron what would we see?

Excitatory post-synaptic potenital (EPSP)

True or False: Patch clamp recording cannot use voltage or current clamp recording

False Patch clamp recording can be used to do either voltage clamp or current clamp recordings

What is the difference between deactivation and inactivation of a channel?

Inactivation occurs during stimulus for channels to be open and is a result of a ball blocking the binding site. Deactivation occurs after significant hyper polarization and is considered the resting state. Occurs when the stimulus that opens channels has been removed.

In a voltage clamp experiment, if the current hyperpolarizes the neuron what would we see?

Inhibitory post-synaptic current (IPSC)

In a current clamp experiment, if the current hyperpolarizes the neuron what would we see?

Inhibitory post-synaptic potential (IPSP)

Starting from whole cell configuration, the electrode is pulled away from the plasma membrane and moves the electrode and membrane patch out of the extracellular fluid and back into the fluid. This turns the membrane inside out so the inside surface of the membrane is exposed to the external solution.

Inside-out recording

Ion channels can inactive which means..

Ion channel closes despite the continued presence of stimulus that causes them to open.

The equipment for voltage clamp recordings is designed to...

Measure how much current needs to be applied with the electrode to keep the membrane potential at the chose voltage and this current is what is recorded in the experiment.

The equipment for current clamp recording is designed to ...

Measure how much the membrane potential needs to be adjusted to keep the desired amount of current flowing.

In voltage-clamp experiments, what is controlled by the experimenter?

Membrane potential -This is controlled by the experimenter and not by the currents moving through the neuronal membrane. The change in current that is required to maintain the desired voltage is recorded and indicates how much current flows through ion channels when the membrane potential is at that voltage

Which is faster, C-type or N-type inactivation?

N-type

Are both neuronal solutions the same?

No The internal solution and the external solutions are different from one another. Among these differences are the concentrations of ions (Na+, K+ Ca 2+, Cl-) and pH

Explain C-type inactivation

Occurs in the continued presence of stimuli that causes channels to open, but the channel collapses and crimps closed using conformation changes among the other transmembrane segments. -S4 segments remain in the active position

We say membrane voltage can change the ______ of a channel

Open probability

Starting from whole cell configuration, this type of recording can be done by pulling the electrode away from the plasma membrane and we see the membrane reseals and forms a patch with the outside of the membrane facing the solution surrounding the neuron

Outside-out recording

What is recorded during a voltage-clamp experiment?

The change in current required to maintain the desired voltage is recorded and indicates how much current flows through ion channels when the membrane potential is at that voltage.

What is the difference between whole-cell and cell-attached

Pipette is filled with external solution from cell-attached and looks at a single ion channels contribution to the flow of ions across a membrane whereas for whole-cell the pipette is filled with internal solution and can measure currents or voltage depending on which method is used.

How is the voltage clamp technique similar to a thermostat?

The voltage clamp technique measures membrane potential (the room temperature) and applies positive or negative current (hot or cold) to keep the membrane potential where the experimenter set it.

Why are current recordings usually smooth given that ion channels themselves are either completely open or completely closed?

Single-channel traces show the current flowing through only a single channel at a time but neurons did not have just a single channel in their membrane. When recording from the entire membrane of a neuron, the current value at any given point in time is the average of the current moving through all the channels that are open at the time. That is why EPSCs, IPSCs, EPSPs, and IPSPs all look like smooth curves. The recordings reflect the changes in current or voltage made up of many channels.

What allows patch clamp recording to be so precise?

The pipette suctions the membrane of the neuron forming a tight seal.

What is the relationship between current and voltage?

The relationship between current and voltage is that as current flows through ion channels the membrane potential is changed which will then alter the number of channels that open which affects the amount of current that will flow. Also if there are positive ions such as Na+ and K+ moving across the membrane in both directions it would not be possible to separately measure the amount of current for each ion only the collectively net current could be recorded

Why don't all channels in a membrane open at the same time?

This is because the opening of each ion channel is probabilistic. When membrane potential is at a certain value a given ion channel is more likely to be open than at a lower membrane potential. But the membrane voltage does not directly cause a channel to open, it rather increases the probability that a channel will open.

What method was developed to study ionic current in a controlled way?

Voltage Clamp -Allows us to measure ionic current in a controlled way while measuring the amount of time of the flow across the membrane. -Additionally they were able to control the concentrations of various ions both inside and outside of a neuron. -This allowed them to test how membrane potential changed when there were different ionic concentration gradients across the membrane. -This can also use compounds to selectively block certain ion channels which allowed them to study the flow of different ions independently

What is recorded during a current clamp experiment?

Voltage signal is recorded and can be used to determine how the membrane potential changes in response to change in current.

In what type of experiment is the membrane potential controlled by the experimenter, not by the currents moving through the neuronal membrane. The change in current that is required to maintain the desired voltage is recorded and indicates how much current flows through ion channels when the membrane potential is at that voltage

Voltage-Clamp

What is the problem with the voltage clamp and current clamp techniques?

Was not easy to use for small neurons -Developed patch clamp technique for this

For this type of configuration, the pipette tip is moved on the membrane with a small amount of suction. Pipette tip seals with the membrane and small bursts of suction are applied to acquire the small patch of tissue suctioned by the pipette. After this patch of neuronal tissue is removed, the electrode has direct ionic and electrical access to the inside of a neuron. Electrode must be filled with internal solution, experimenter can now control the membrane voltage (if using voltage clamp) or the currents moving across the membrane (if using current clamp)

Whole-cell configuration

It is possible to record the ionic current passed by a single ion channel through..

Whole-cell recording