Lab 4: Intracellular recordings from leech

neurons
Author: Mackenzie Andrews

INTRODUCTION
Intracellular recording from neurons allows the recorder to observe the resting potential, action

potential waveform, and smaller events such as post-synaptic potentials from presynaptic

neurotransmitter release in a specific cell. All cells have a resting potential which corresponds to

the potential achieved by the open resting channels in the cell and maintained by the ion pumps

in the cell membrane. All cells have a resting potential negative to their extracellular

environment.

Most neurons also exhibit spontaneous activity mediated either by specialized ion channels

within the cell or by excitatory synaptic input from other cells. The spontaneous activity of a cell

is dependant on the voltage at which the cell is held at. If the cell is held at a low enough voltage,

all spontaneous activity will cease because the cell will be unable to depolarize enough to initiate

an action potential.

The voltage at which a cell is able to generate an action potential is called threshold. The

threshold potential of a cell is dependant on the population and behavior of the open ion channels

within that cell. For example, when a cell is hyperpolarized, an increased number of sodium

inactivation gates will open, effectively lowering threshold for that cell because more

voltage-gated sodium channels are available to pass current.

Another property of neurons mediated by their specific population of ion channels is the RC

behavior of their membrane. All neuronal membranes act as a capacitor able to hold charge
across the insulative membrane. Typically, the capacitance of a membrane is a constant value

dependant on the dielectric constant of lipids and the thickness of the bilayer, both of which are

negligibly varying under normal conditions. In contrast, the resistance property of the membrane

is dependant on the properties of the ion channels spanning the membrane and is highly variable

both between cells and within any one cell. The more ion channels that are open within the cell,

the less resistance there is to current flow. The combination of the resistive and capacitive

properties of a neuron allows the cell to behave like an RC charging circuit in which the response

of the cell outlasts the input. This property of neuronal membranes allows for summation of

synaptic inputs.

In this lab, we used the leech as our model animal to observe the various properties of neuronal

behavior. Leeches are segmented worms that have ganglia containing relatively large nerve cell

bodies ideal for intracellular recordings. Using a glass microelectrode filled with Leech Ringer

solution (a saline solution isotonic to leech body fluids), inserted across the plasma membrane of

a leech neuron, we were able to both stimulate and record from the cell simultaneously. Using

various intracellular stimulating and recording techniques we explored the spontaneous activity,

resting potential, action potential threshold, firing properties, and the RC properties of the

neuronal membrane.

METHODS
All methods used were as given in the protocol in the ​Neurobiology 301 Course Manual (Winter

Quarter, 2017)​.

RESULTS
Set Up

A leech was dissected and a single ganglion was pinned to a dish and placed under a dissecting

microscope. A glass microelectrode filled with Leech Ringer’s solution was attached to a
micromanipulator. The micromanipulator was then used to insert the tip of the microelectrode

into a cell body of one of the neurons in the ganglion.

Inserting the Microelectrode - Observing Spontaneous Activity

When the microelectrode was inserted into the cell, the recorded voltage immediately dropped to

a negative value corresponding to the negative resting potential of the cell it was inserted into

(Figure 1). In some cases, the cell would have spontaneous activity without any hyperpolarizing

or depolarizing stimulus. As shown in Figure 1, when the microelectrode came close to the cell

membrane, recorded voltage decreased from 0mV to -5mV. At this point we stimulated with 1nA

of current to see if we could generate an action potential. Since no action potential was

generated, we continued to move the microelectrode into the cell. As soon as the microelectrode

entered the cell, the voltage reading dropped to approximately -10m V and the cell exhibited

spontaneous action potentials with a frequency of approximately 7 Hz and amplitude of 4 mV.

After about 1 minute of being in the cell, the spontaneous action potentials slowed to a frequency

of about 4 Hz and remained at this frequency without any current being injected into the cell

(Figure 2). It appears the the action potentials are resulting from the summation of synaptic

input. As shown in figure 2, the amplitude of the signal increases in a stepwise fashion between

action potentials. This phenomenon is clearly observed between the second and third action

potentials in figure 2 where there are 3 jumps in voltage that behave like a summating RC curve.

Once the inputs summated to approximately -7.3 mV, an action potential was fired indicating

that the threshold of this cell was approximately -7.3mV.

Note: This cell was used for the “Responses of Cell to Long Stimuli” and “RC Properties of the

Membrane” sections. Due to the small amplitude of the action potentials, other cells were used

for the “Response of Cell to Brief Stimuli” and “Response of Cell to Multiple Stimuli” sections.
Response of Cell to Brief Stimuli - Finding Threshold

Note: Data for this section and the following section (“Response of Cell to Multiple Stimuli”)

were obtained by William Palmer and Mackenzy Isaacson due to the fact that we ran out of time

to collect this data.

After the electrode was inserted into a cell, a hyperpolarizing current was delivered to the cell to

stop spontaneous activity. For this exercise, the cell was held at -35mV. A depolarizing current

of 3.4 nA was then delivered to the cell for a duration of 10 ms. Figure 3 shows an overlay of 8

responses of the same cell to the same current pulse of 3.4 nA. In 4 of the 8 cases, the cell

depolarized to -10 mV and then returned to rest. In the other 4 cases, the cell depolarized to -10

mV then generated an action potential approximately 5 to 10 ms after the end of the stimulus.

The action potential had an amplitude of 45 mV from the resting potential of -35 mV. After the

action potential, the cell undershot the resting potential by 5 mV before returning to rest. The

duration of the action potential from peak to undershoot was 10 ms. The cell then took an

additional 90 ms to return to rest from its hyperpolarized state.

Due to the fact that the cell had approximately a 50 percent chance of generating an action

potential at -10 mV, then we concluded that -10 mV was the threshold of this cell. In the

instances when the cell did not generate an action potential, the RC charging curve as the cell

recovered from the simulated membrane potential could be used to calculate the time constant of

the membrane. The membrane remained above its resting potential for 90ms after the end of the

stimulus. The cell had returned 63% of the way back to its resting potential after approximately

15 ms after the stimulus was over meaning that the time constant of the membrane was 15 ms.

Response of Cell to Multiple Stimuli - Observing Summation

After finding the cell’s threshold and RC charging behavior, a series of 10 ms pulses with a delay

of 10 ms between pulses were delivered to the cell. The darker lines in figure 4 show the

response of the cell to 5 consecutive 2.0 nA pulses. The cell’s responses summate and the cell
reaches a membrane potential of -15 mV (from a resting potential of -37 mV). The pulse

stimulus amplitude was then increased to 2.25 nA. The lighter lines in figure 4 show that the

cell’s response summates until it reaches -10 mV (threshold) where the cell generates an action

potential. The action potential had a peak amplitude of 45 mV with a hyperpolarizing undershoot

of 5 mV after the action potential.

Responses of Cell to Long Stimuli

We next observed the behavior of a neuron to a long (1 second) pulse of current of various

amplitudes. We began stimulating with .4 nA of current and stepped the current up by

approximately .2 nA per trial. Figure 5 shows the response of the cell to various current stimuli

between .4nA and 1nA. As the current increased, the action potential response happened closer

to the start of the stimulus. As we continued stepping up the current, the number of action
potentials fired by the cell during the 1 second interval increased (Figure 6). Graph 1 shows a

plot of the number of action potentials versus the amount of current injected into the cell. As

current is increased, the frequency of firing increased.

RC Properties of the Membrane

Finally, we sought out to find the RC properties of this cell. We stimulated the cell with a 6 nA

hyperpolarizing current. This current brought the cell from a resting potential of -26 mV to -44

mV (Figure 7). The charge curve appears to be the sum of a constant value (-11 mV) most likely

due to the electrode resistance, and an exponential due the the membrane RC circuit. Subtracting

out the constant due to the electrode and normalizing the resting potential to zero, the |Vmax| of

the membrane was 7 mV. Using Ohm’s Law with a known current of 6 nA, the resistance of the

membrane can be calculated to be 1.17 MOhms. Next, using the graph to determine the time

constant (63% charged), we found RC = 0.2 seconds. Therefore, the capacitance of the

membrane was 0.17 microFarads.

We did not observe an anode-break excitation, but if we had, we predict it would have looked

like the drawing in Figure 8. This excitation is due to the fact that under hyperpolarization, more

voltage-gated sodium channel inactivation gates are opened which lowers the resistance of the

cell membrane which allows more current to pass at the same membrane potential, effectively

lowering threshold. If the new threshold is below the resting membrane potential, the cell will

generate an action potential.

DISCUSSION
As observed in figures 1 and 2, when the electrode enters the cell, the firing frequency of the cell

increases and then settles back to a stable spontaneous firing frequency after a minute or two.

This phenomenon is probably due the the fact that inserting the microelectrode into the

membrane temporarily disrupts the cell. The Leech Ringer’s solution is surrounding the cells and

is the fluid in the microelectrode. When the electrode is placed in the cell, the membrane aorund
the electrode becomes damaged and likely lets some of the Ringer’s solution into the cytosol of

the neuron. Since the Ringer’s solution is electrically positive to the intracellular fluid, the entry

of the solution causes a depolarization of the cell causing rapid firing of action potentials. The

cell is able to repair itself and restore its typical resting membrane potential after a few minutes.

Under the stimulation of short pulses, the cell behaves like an RC circuit with a charging and

discharging curve. As the pulse of the cell get’s closer to threshold, the decay remains

exponential. The time constant of decay also remains constant so the exponential curve actually

appears steeper as the pulse approached threshold.

Threshold is the value at which net current in the cell is zero, i.e. the current passing through the

voltage-gated sodium channels exactly balances that of the current flowing through the resting

channels. Once the pulse reaches threshold, the potential will temporarily hold constant due to

the fact that the currents are balanced. This state is an unstable equilibrium where a slight

depolarization will cause a rapid increase in the number of open voltage-gated Na channels

causing an action potential or a slight repolarization will cause the channels to close and the cell

to return to rest. This is why the cell fires an action potential only half the time that it is brought

to threshold. This behavior is entirely due to a perfectly balanced probability of one more sodium

channel opening.

When multiple short current pulses are used to stimulate the cell, the cell’s response will

summate. If the pulses are delivered with a large enough current and frequency, the cell’s

membrane potential will eventually summate to threshold and fire an action potential. The cell is

able to summate the input due to the RC behavior of the membrane. Since the cell’s response

outlasts the stimulus (due to the membrane capacitance), another stimulus within the discharging

time of the cell will generate a new depolarization starting from a higher baseline. This allows

cells to summate synaptic inputs that occur in rapid succession and respond to various

combinations of input in non-trivial ways.

When the current delivered to a cell is increased, the cell fires more rapidly. This allows a cell to

encode the magnitude of a signal in the frequency of firing. The larger the signal, the greater the

current and the higher the frequency. This is biologically beneficial because frequency is a

digital encoding of an analog signal. Digital signals are able to travel long distance without

decrement whereas analog signals are susceptible to interference and decrement. Therefor,

transmitting magnitude as a frequency allows the nervous system to transfer information a long

distance without loss of information.