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Melissa Chan
Dr. Michael Kennedy
Neurobiology 301 Lab AD
Lab Report 4
February 18, 2016

The Leech Neuron Lab

Introduction

Intracellular recordings enable the acquisition of real-time electrical activity from within

a cell, rather than secondary measurements from extracellular representations of this activity.

Consequently, instead of recording only changes in voltage across the neuronal membrane,

intracellular recording allows us to record actual resting potentials and both synaptic potential

and action potential waveforms. Additionally, under ideal conditions, the intracellular recording

pipette is so fine that the impaled neuronal membrane seals around it and recordings can be made

with minimal damage to the cell, resulting in highly accurate recordings.

In this lab, we dissected leech ganglia and used an intracellular amplifier to apply stimuli

and record responses from individual nerve cells. A glass microelectrode filled with 3M KCl

solution was attached to the headstage of the intracellular amplifier in order to record responses

to stimuli, and maneuvered to impale nerve cells via a micromanipulator mounted on an air table.

The intracellular amplifier recordings were also filtered through a Humbug in order to eliminate

noise frequencies around 60 Hz. Using this set up, we varied the frequency and magnitude of

stimuli to targeted nerve cells, and obtained recordings describing action potential threshold and

firing properties, as well as characterizations of the neuronal membrane RC properties.

Methods

All methods used were as given in the protocol.

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Results
Note: The Initial Recording and Exercise 1 were done on a different cell (Cell A) than
Exercise 2 (Cell B) and Exercises 3-4 (Cell C). A total of three cells were used for recording.

Initial Recording of Spontaneous Activity

After initial impalement, we recorded the spontaneous activity of Cell A, as shown below

in Figure 1.1. The inset illustrates a magnified view of these spontaneous action potentials,

where the measured potential had a 9 mV amplitude, a 3.2 mV undershoot, and no overshoot.

We applied hyperpolarizing DC current and subsequently saw spontaneous activity stop.

Figure 1.1 Initial intracellular recording of leech neuron Cell A spontaneous activity.
Current is shown in Channel 2 (nA) and voltage in Channel 1 on the y-axis. Time is on the
x-axis (seconds). The black arrow marks the application of hyperpolarizing DC current,
which stopped spontaneous action potentials. The inset image illustrates a magnification of
the spontaneous potentials. Amplitude and undershoot measurements are indicated, and
were similar for all potentials. None had overshoot.
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However, after an 8.5-second period of no spontaneous activity at this hyperpolarized state, Cell

A began to fire spontaneously again, as shown in Figure 1.2. Correspondingly, we applied

hyperpolarizing DC current for the second time in order to completely eliminate spontaneous

activity.

Figure 1.2 Initial intracellular recording of leech neuron Cell A spontaneous activity:
second hyperpolarization. Current is shown in Channel 2 (nA) and voltage in Channel 1 on
the y-axis. Time is on the x-axis (seconds). The black arrow marks the second application of
hyperpolarizing DC current, which completely stopped spontaneous action potentials.

Short Pulse Experiment Exercise 1

We stimulated Cell A with single, short pulses of varying strengths to obtain the

recording illustrated in Figure 2. Stimuli of 5 mV and below resulted in responses just below -30

mV and failed to generate action potentials, but stimuli of 10 mV and above resulted in
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responses above -30 mV and each generated at least one action potential. Therefore, we

concluded that threshold for Cell A was -30 mV. The time constant of decay for the response to

the sub-threshold 5 mV stimulus (bright green markers) was between 0.0020 and 0.0025

seconds. This decay value became smaller and less exponential as the pulse got closer to

threshold. We took measurements from the action potential response to the 35 mV stimulus (dark

green markers) and found a 42.14 mV amplitude, 5.38 mV undershoot, no overshoot, and

duration of 0.016 seconds.

Figure 2 Response of leech neuron Cell A to single, short pulses. Current is shown in
Channel 2 (nA) and voltage in Channel 1 on the y-axis. Time is on the x-axis (seconds). The
application and termination of stimuli are indicated by the black arrows. The peak voltage
of each response is also indicated by a colored marker and described in the legend. Note
that threshold is marked by the horizontal, grey dotted line at -30 mV, and that only
responses to single, short stimuli of 10 mV or more generated action potentials.
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Summated Short Pulses Experiment Exercise 2

Expanding on Exercise 1, we then applied a series of short pulses at various strengths to a

new Cell B in order to obtain the results shown in Figure 3. A series of 10 mV stimuli failed to

generate action potentials in all of its responses, which remained below -40 mV. However, a

series of 11 mV stimuli generated an action potential in its last response, reaching above -40 mV.

Therefore, threshold of Cell B was determined to be -40 mV.

Figure 3 Response of leech neuron Cell B to series of short pulses. Current is shown in
Channel 2 (nA) and voltage in Channel 1 on the y-axis. Time is on the x-axis (seconds).
The application and termination of stimuli are indicated by the black arrows. The peak
voltage of each response is indicated by a colored marker and described in the legend.
Note that threshold is marked by the grey dotted line at -40 mV. The response to a series
of 10 mV stimuli (green markers) did not reach threshold. The response to a series of 11
mV stimuli (red markers) reached threshold only at the last stimulus, and generated an
action potential. An overlay of additional responses to series of stimuli greater than 11
mV are shown, unmarked, in light blue. All of these generated action potentials as well,
with responses to stronger stimuli generating at earlier stimuli in the series.
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Responses to Long Pulses Experiment Exercises 3-4

Finally, for comparison to Exercises 1 and 2, we stimulated a new Cell C with long

pulses of various strengths. In Exercise 3, we applied long, depolarizing stimuli to obtain the

recordings in Figure 4.1, which illustrate a correlation between increasing stimuli strength,

higher frequency of action potentials, and the appearance of phasic response patterns.

Figure 4.1 Response of leech neuron Cell C to long, depolarizing stimuli. Current is
shown in Channel 2 (nA) and voltage in Channel 1 on the y-axis. Time is on the x-axis
(seconds). The application and termination of stimuli are indicated by the black arrows. The
number of action potential peaks per given stimulus strength is indicated by the colored
markers and lines, and is described in the legend. Long, depolarizing stimuli were applied
over a period of 1.0 seconds. Note that as stimulus strength increases, action potential
frequency also increases and begins to exhibit phasic response properties.
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This relationship is also clearly evident in histogram Figure 4.2, which plots frequency of action

potentials against stimuli strength, and shows an exponential-like increase in frequency that

exhibits saturation behavior at 9-10 potentials.

Figure 4.2 Histogram of leech neuron Cell C response to long, depolarizing stimuli.
Number of action potentials recorded is on the y-axis. Stimulus amplitude is on the x-
axis (mV). Note that as stimulus strength increases, action potential frequency
increases in an exponential-like pattern, with saturation at 9-10 potentials.

In Exercise 4, we applied long, hyperpolarizing stimuli in order to obtain the recordings

in Figure 4.3, describing the RC properties of the Cell C neuronal membrane. As illustrated in

Figure 4.3, the charging curves for each response look like a single exponentials, each reaching

saturation at different points. The discharging curves after the end of each pulse also look like

single exponentials, except these all follow the same path. The response to the -16 mV stimulus

was used to calculate these properties. Final voltage for this curve was V = 38.86 mV, and

applied stimulus was I = 0.8 nA. Therefore, using Ohms Law, resistance was calculated via R =

V/I = 48.6 MOhms. Time constant tau is equal to the time taken to reach 63 percent of final
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voltage, which for this curve was 11.5 msec at (38.86 mV)(0.63) = 24.48 mV. Finally, tau is

equal to resistance times capacitance, and therefore capacitance was calculated by C = tau/R =

0.025 nF.

Figure 4.3 Response of leech neuron Cell C to long, hyperpolarizing stimuli. Current is
shown in Channel 2 (nA) and voltage in Channel 1 on the y-axis. Time is on the x-axis
(seconds). The application and termination of stimuli are indicated by the black arrows. Each
voltage response curve is labeled with its corresponding stimulus strength. Note that the
charging curves look like a single exponentials, each reaching saturation at a different point.
The discharging curves after the end of each pulse also look like single exponentials, except
these all follow the same path. The response to the -16 mV stimulus was used to calculate the
RC properties of the Cell C neuronal membrane.

Cell C was not able to demonstrate anode-break excitation. Figure 4.4 below is an edited

image of Figure 4.3, describing our theoretical prediction of what anode-break excitation at the
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response to -16 mV stimulus would have looked like. As illustrated in the figure, anode-break

excitation is characterized by the generation of an action potential once a hyperpolarized curve

returns to rest the neuron is over-excitable in that it can fire an action potential at the resting

potential, which is a sub-threshold voltage.

Figure 4.4 Response of leech neuron Cell C to long, hyperpolarizing stimuli: theoretical
illustration of anode-break excitation. Current is shown in Channel 2 (nA) and voltage in
Channel 1 on the y-axis. Time is on the x-axis (seconds). The application and termination of
stimuli are indicated by the black arrows. Anode-break excitation is illustrated with the
response to -16 mV stimulus (red line), and is characterized by the generation of an action
potential once the hyperpolarized curve returns to resting potential, as labeled with the
horizontal dashed grey line.
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Discussion

During initial recording of spontaneous activity, the appearance of transient spontaneous

potentials that disappeared within a few minutes would indicate that the activity was caused by

damage due to impalement by the microelectrode, and not spontaneously generated by the cell.

In our initial recording after impalement of Cell A, we observed constant spontaneous potentials

that did not disappear over time, and therefore concluded that the activity we were observing was

real and spontaneously generated by our cell (see Figure 1.1).

The subsequent experiments in Exercises 1-4 explored the frequencies and magnitudes of

stimuli necessary for neuronal membrane voltage to pass threshold and generate an action

potential. Threshold is defined as the membrane voltage at which current through the resting

channels is equal and opposite to current through the voltage-gated sodium channels, and

therefore there is a steady-state of zero net current. In Exercise 1, it was evident that potential

temporarily held constant at threshold after the stimulus was turned off, and then either

hyperpolarized again or fired an action potential (see Figure 2). This holding period can be

explained on the basis of probability. If a stimulus results in a depolarization that brings the

membrane potential just to threshold, after the stimulus is terminated, there is a 50:50 chance that

an action potential could occur. Either one more voltage-gated sodium channel inactivation gate

could open and result in an action potential, or one more inactivation gate could close and result

in hyperpolarization with no action potential. The holding period is the small time window in

which these inactivation gates are either opening or closing.

In Exercise 2, a series of pulses was shown to stimulate firing of an action potential at the

last pulse, but not at any earlier pulses (see Figure 3). This illustrates the effect of summation

with high frequency application of stimuli. Pulse 1 of the response to -11 mV stimuli in Figure 3
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did not reach threshold, and therefore could not generate an action potential. However, over the

course of eight consecutive pulses at high frequency, the repeat -11 mV pulses began before their

preceding pulses had completely decayed to rest, and therefore each successive repeat pulse

started from a higher resting voltage, allowing them to reach higher final voltage in the given

period of time the pulse was applied. Therefore, in the last Pulse 8, the response had summed to

be large enough to reach threshold and was able to fire an action potential. In living organisms,

summation fine tunes synaptic transmission by enabling selective response only to stimuli

applied with the right intensity and frequency.

In Exercise 3, our histogram describing action potential frequency vs. stimulus strength in

Figure 4.2 showed that frequency increased exponentially with stronger stimuli. This stimulus

strength-dependent pattern of response could equip an organism to distinguish between stimuli

that are potentially more dangerous, and then respond accordingly. Figure 4.2 also shows

saturation behavior at 9-10 action potentials, which describes the maximum firing rate of Cell C

according to its time constant. Additionally, the phasic response illustrated Figure 4.1 allows for

sensory adaptation to constant stimuli, so an organism could selectively filter its responses and

conserve energy.

Finally, Cell C in Exercise 4 did not exhibit anode-break excitation; however, the results

if it had are described in Figure 4.4. With the neuron initially at rest, half of the voltage-gated

sodium inactivation gates are open. After a period of hyperpolarization, more inactivation gates

open so that when the membrane potential returns to rest, more than half are now open and able

to pass current. Therefore, an increased conductance through the voltage-gated sodium channels

lowers threshold, and the neuron becomes over-excitable and is able to fire action potentials at

resting membrane potential.