Brain and Behavior Student Manual 2022

ELECTRICAL POTENTIALS IN NEURONS I: A Model Dendrite

QUESTION 8:
Create a graph for each breadboard by plotting the distance (x-axis) vs. the voltage (y-axis).

100K BREADBOARD

VOLTAGE [V]

0
0

DISTANCE
30K BREADBOARD

VOLTAGE [V]

0
0

DISTANCE

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ELECTRICAL POTENTIALS IN NEURONS I: A Model Dendrite

PART II: SPATIAL SUMMATION

Using the 100K breadboard, you will now explore what happens when you connect more than
one battery. Different points on the 100K breadboard are labeled A, B, C, D and E.

QUESTION 9:
When we connect more than one battery, what are we simulating in terms of biology?

 Connect the probe (red lead from the DMM) and the ground (black lead from the DMM) to
the center position, marked C.

 Connect one 9V battery at the point marked A; record voltage at point C in DATA TABLE 3.

 Now disconnect the battery at A and connect a second 9V battery at the point marked B;
record voltage at point C in DATA TABLE 3.

 Reconnect the battery at A while keeping the battery at B connected; record voltage at C in
DATA TABLE 3.

DATA TABLE 3:

Voltage recorded at C

Synaptic input from A

Synaptic input from B

Synaptic inputs from A and B

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ELECTRICAL POTENTIALS IN NEURONS I: A Model Dendrite

You will now repeat the experiment using points D and E, which are much closer in distance
compared to points A and B:

 Connect the probe and the ground to the center position, marked C.

 Connect one 9V battery at the point marked D; record voltage at point C in DATA TABLE 4.

 Now disconnect the battery at D and connect a second 9V battery at the point marked E;
record voltage at point C in DATA TABLE 4.

 Reconnect the battery at D while keeping the battery at E connected; record voltage at C in
DATA TABLE 4.

DATA TABLE 4:

Voltage recorded at C

Synaptic input from D

Synaptic input from E

Synaptic inputs from D and E

QUESTION 10:
A. Since two 9V batteries were used, was the summation of synaptic inputs 18V?
B. Provide a brief summary of the results for each experiment (A+B and D+E respectively), and
explain why there are differences in recorded voltage output.

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ELECTRICAL POTENTIALS IN NEURONS I: A Model Dendrite

PART III: THRESHOLD

Summation of local, graded potentials is important for reaching the threshold membrane
potential needed to elicit an action potential in the dendrite. A single synaptic input is very rarely,
if ever, going to be strong enough to depolarize the membrane potential enough to reach
threshold.
QUESTION 11:
Assume that synaptic inputs measured at point C on your 100K breadboard need to reach at least
2V to reach the threshold for an action potential in the dendrite.
Based on the data you collected in Part II, which combination of synapses will cause an action
potential: A alone, B alone, and/or A and B together?

QUESTION 12:
A neuron receives both excitatory (EPSPs) and inhibitory inputs (IPSPs).
Suppose synaptic inputs at locations A and B are equally strong, but synapse A causes an EPSP
and synapse B causes an IPSP. Write a hypothesis that describes what voltage you expect to
measure at point C.
Be sure to explain why you make such a prediction.

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ELECTRICAL POTENTIALS IN NEURONS I: A Model Dendrite

Now test your hypothesis:

 Connect the battery at point A. Record the voltage at point C from A alone.

 Switch the red and black battery leads and connect them at point B. Record the voltage at C
from B alone. What do you notice about the measured voltage?

 Record voltage at point C when you have synaptic input from A alone, from B alone, and from
A and B together.

DATA TABLE 5:

Voltage recorded at C

EPSP from A

IPSP from B

Synaptic inputs from A and B

QUESTION 13:
A. Revisit your hypothesis from Question 12. Do the data support your hypothesis?
B. Why or why not?

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ELECTRICAL POTENTIALS IN NEURONS I: A Model Dendrite

…

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ELECTRICAL POTENTIALS IN NEURONS I: A Model Dendrite

CLEAN-UP PROCEDURES

When you are done with your work, you must clean up. You will not be permitted to leave the
lab until the lab instructor checks your lab bench.
 Disconnect both batteries and turn off the DMM before leaving the lab!!!!
 Please leave your lab station as clean and orderly as when you arrived.
 Before leaving the lab, double-check your area for your personal belongings.

THE LAB STAFF IS NOT RESPONSIBLE FOR ANY LOST OR STOLEN ITEMS.
Failure to properly clean up will result in a 5-point deduction from your laboratory assignment.

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ELECTRICAL POTENTIALS IN NEURONS I: A Model Dendrite
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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

LABORATORY 3

ELECTRICAL POTENTIALS IN NEURONS II:
Action Potentials in an Axon

SUMMARY
In our last lab session, A Model Dendrite, we examined passive and active conduction of action
potentials, calculated a dendrite’s length constant while we observed how it can be modulated
by certain properties of neurons, and came to understand how spatial summation relates to
action potential thresholds. These are all considered “passive potentials” and/or “graded
potentials” because the currents change gradually and arise from the fixed electrical properties
like voltage (concentration gradient) and resistance (=1/conductance). Connect these elements
into a circuit as you did and they happen “passively,” meaning without any element changing.
For the experts amongst you, for simplicity we left out capacitance, another so-called passive
electrical component.

For this session, we will zoom in to the membrane of an axon to examine some important details
of the action potential. These are not passive potentials because the currents are dynamic, and
propagating meaning they change and are persistent (moving) rapidly in time; they arise from
changing electrical properties of the membrane elements that create voltages and conductances
(=1/resistance). These dynamic properties of the protein channels that you will learn about, are
the result of the “gating” properties of the channels and that is what gives the action potential
its special electrical properties.

OBJECTIVES

Understand the changes that take place in a nerve cell during an action potential:
a. location and movement of ions
b. changes in electrical potential
c. membrane proteins involved

*Adapted from “Action Potential Simulation – Guided Inquiry Activity” by Kara Reichert, Chicago Public Schools, last updated Jan. 13, 2020, and
“Stimulating Neurons” by PocketLab, last updated Jun. 24, 2021.

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

INTRODUCTION

THE NERVE AXON
The image shown represents a cross-section of the nerve axon. (What’s a cross-section? Imagine
laying a nerve cell lengthwise along an x-axis and slicing through it along the y-axis: that’s how
you get a cross-section!) The yellow represents the plasma membrane of the axon. It is the
phospholipid bilayer that has been discussed in lecture. The myelin sheath is not shown here.

Membrane channels

Plasma membrane

Figure 1. Cross section of a nerve axon.

When a nerve cell is stimulated, it triggers an action potential. An action potential is the change
in electrical potential that propagates (travels) along the membrane of a nerve cell. This is how
information moves through our nerves. There are several different processes occurring,
simultaneously and within milliseconds, that require closer examination to fully understand and
appreciate these functions.
The simulation you will use in this lab session will let you explore how a neuron works.
Specifically, how membrane permeability and ion movement create potential differences across
the neuron membrane. The aim is for you to understand the mechanisms behind an action
potential and the sequence of membrane channel opening/closing that creates the potential.

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

To help you visualize diffusion, explore the Membrane Channels simulation, available at
https://phet.colorado.edu/en/simulation/legacy/membrane-channels. Here you can insert
leakage and/or gated channels in a membrane and see what happens to concentration, as well
as how these different types of channels allow particles to move through the membrane (or not).
NOTE: This simulation is not compatible with iPad.

Figure 2. Screenshot of Membrane Channels simulation.

 Click on red dots to add selected particles. The more clicks, the more particles.
 Selection buttons allow you to inject either green circles or blue diamonds.
 Leakage Channels and Gated Channels are added to the membrane with a click-and-drag

motion. You can add as many channels as you like.
 Check the box next to Show Concentrations to see the bar graph changing as concentrations

of particles change.
 The green and blue gated channels can be opened and closed by clicking on their respective

Open Channels button.
Using this simulation prior to your lab session should assist you in performing the tasks associated
with investigating the intricacies of action potentials in a timely manner.

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

Brain & Behavior: CORE-UA 306

LABORATORY COVER SHEET
Name: _____________________________ Lab Partner’s Name: _________________________
Date of Lab: ___________________________ Date Due: _______________________________
Lab Instructor: _____________________________ Lab Section Number: __________________

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

…

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

PROCEDURES and EXERCISES

Each student laptop should be set to the Neuron simulation [Figure 3]. If this is not set, let your
instructor know and they will provide access to you. Since you may need to utilize the simulation
after your lab session to complete the conclusion section, make sure you note the web address
while you are in the lab.

Figure 3. Screenshot of Neuron simulation, starting view (PhET).

When you click “Stimulate Neuron,” you will notice a purple and yellow thing moving down the
length of the axon: this represents the action potential. What you’ll be observing in this activity
is what happens when the action potential reaches this cross-section of axon. You are not
observing the entire nerve cell, just a tiny fraction of its membrane. The changes you observe
here are what happen down the entire length of the neuron.
In the box labeled “Show,” start by checking all the boxes. This will allow you to see everything
that is going on. Click the “Stimulate Neuron” button on the lower right corner of the simulation
to simulate an action potential. You can pause the simulation at any time, scroll back on the
potential chart to rewind, zoom in/out, and speed up or slow down the animation.
Take several minutes to play around with this simulation and get comfortable with it.
Observe what happens when you check or uncheck boxes or click different buttons. You cannot
break it, so go ahead and click everything! After you’re comfortable with the various settings, use
the simulation to answer the questions below.

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

HINTS:
- You will need to zoom in and change the speed to really understand what’s happening as
you work through the questions.
- When you are ready to start answering questions, check all the boxes in the Show section.

QUESTION 1:
Observe the membrane closely while the axon is at rest.
A. Which membrane channels are open?
B. Which channels are closed?
C. Are there more open or closed channels present while the axon is at rest?

QUESTION 2:
The concentrations of sodium and potassium ions are different inside and outside the membrane.
A. Which direction will sodium ions move as a result of facilitated diffusion through the leak
channels?
B. Which direction will potassium ions move?

QUESTION 3:
A. Which side of the neuron is negatively charged - inside the neuron or outside?
B. This simulation only shows positively charged ions. How can one side of the membrane have
a net negative charge in this scenario? What must be present but not shown in the image?

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

Although facilitated diffusion is clearly happening, the indicated ion concentrations are not
changing. That’s because this image is missing a very important protein called the sodium-
potassium pump also called the Na+/K+ ATPase. Think about what you know about the
concentration gradients.
QUESTION 4:
A. What must this pump be doing to maintain the concentration gradient? In other words, what
kind of transport is it doing?
B. Which direction must each ion be moving through the pump?

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

Now it’s time to stimulate your neurons.
- On the axes provided, sketch the graph that is generated when you click “Stimulate
Neuron.” Include ALL necessary titles, labels, and units. This does not need to be perfect,
so don’t bother writing every number or filling in grid lines.
- Then, complete Table 1 below by writing the answer or circling the correct word.
Pay close attention to +/- signs on the membrane potential values. The signs matter a lot!

Table 1. Features of an Action Potential. Peak of Immediately After
At Rest (0-2 ms) Action Potential (~2-7 ms) Action Potential (4 ms)

Membrane potential (mV)

Which side of the membrane has a net Inside / Outside Inside / Outside Inside / Outside
negative charge? Inside / Outside Inside / Outside Inside / Outside
Which side of the membrane has a greater Inside / Outside Inside / Outside Inside / Outside
concentration of sodium ions?
Which side of the membrane has a greater
concentration of potassium ions?

State of the gated channels? Open / Closed Open / Closed Open / Closed / Inactive

QUESTION 5:
A. When the membrane potential is negative, which side of the membrane is negatively charged?
B. What about when the membrane potential is positive?

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

If the membrane potential is at the equilibrium potential for a specific ion, there is no net
tendency for that ion to move in or out of the cell. But what if the membrane potential is not at
the equilibrium potential for a specific ion, and there are open ion channels? This means the
membrane conductance for that ion has increased and this conductance will cause the ion to
tend to move across the membrane. If the ion is moving across the membrane (i.e., the
membrane is permeable to it, or the channel is conducting the ion), then the membrane potential
should get closer and closer to the equilibrium potential for the ion.

So, if the cell is more permeable to sodium, the membrane potential will become closer to the
equilibrium potential of sodium (ENa). If the cell is more permeable to potassium, the membrane
potential will become closer to the equilibrium potential of potassium (EK).

Let’s see if our data confirms this.

QUESTION 6:
Plug in the concentrations of sodium and potassium into the Nernst equation below and calculate
the equilibrium potentials for sodium and potassium. Remember that the constant in the
equation depends on the exact temperature under consideration, and so can be larger if it is
warmer (e.g., 62 mV) or smaller if it is cooler (e.g., 58 mV).

= 58 � [ ] �
[ ]

ENa+ =

EK+ =

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

QUESTION 7:
A. When the cell is at rest, is the membrane potential closer to the equilibrium potential of
potassium or sodium ions?
B. What about at the peak of the action potential?

QUESTION 8:
Based on this information, predict the membrane’s relative permeability to sodium and
potassium at rest, during the upstroke of the action potential, and during the down stroke of the
action potential. Think about when the potential is approaching ENa+ and when it is approaching
EK+. Explain your predictions.

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

Observe the sodium and potassium ions passing across the membrane during the simulation.
You might want to slow down the simulation speed and use the zoom feature to get a closer
view.
QUESTION 9:
A. Which gated channel opens first (during the upstroke)?
B. Which opens second (during the peak/downstroke)?
C. What direction are sodium ions moving through the sodium gated channel?
D. What direction is potassium moving?

QUESTION 10:
A. What is happening to the membrane potential as each of these channels open?
B. Do these observations agree with your prediction in QUESTION 8? Explain.

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

QUESTION 11:
Look carefully at the graph of the membrane potential.
A. At the bottom of the downstroke, what is the approximate membrane potential?
B. Is this higher, lower, or the same as the resting membrane potential?

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

CONCLUSION QUESTIONS

Observe the “Stimulate Neuron” button throughout the course of the action potential. It turns
gray, indicating that it is not available to be clicked. This represents an important concept in
neuron function, the refractory period. This is a period after the action potential occurs during
which a second action potential cannot be propagated.
QUESTION 12:
What is the membrane potential when you can stimulate the neuron again? How does this
compare to the resting membrane potential?

QUESTION 13:
The drug ouabain inhibits the function of the sodium-potassium pump (Explained in question 5).
Predict the short-term and long-term effects of ouabain on the excitability (ability to be
stimulated) of a neuron. Think about the effect this would have on the resting membrane
potential.

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

CLEAN-UP PROCEDURES

 Reload the webpage to clear all data.
 Note the simulation’s web address to complete lab questions after lab.
 Please leave your lab station as clean and orderly as when you arrived.
 Before leaving the lab, double-check your area for your personal belongings.
THE LAB STAFF IS NOT RESPONSIBLE FOR ANY LOST OR STOLEN ITEMS.
Failure to properly clean up will result in a 5-point deduction from your laboratory assignment.

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ELECTRICAL POTENTIALS IN NEURONS II: Action Potentials in an Axon

…

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Recording Action Potentials from the Model Organism Periplaneta Americana (L.)

LABORATORY 4

Recording Action Potentials from the
Model Organism Periplaneta Americana (L.)

SUMMARY
In this lab, we will use a cockroach leg as a biological preparation to integrate many of the topics
we have covered thus far. By employing the method of extracellular recording, we can measure
action potentials (i.e. changes in voltage) which occur as a result of externally delivered stimuli.
With this technique in place, we will explore the topics of receptive fields and neural summation.

OBJECTIVES
 Understand the difference between extracellular and intracellular recording methods
 Understand the neural processing that occurs in a nerve (a bundle of axons, or ‘fibers’)
vs. a single axon (a single fiber)
 Apply the principles of summation and receptive fields to a biological model
 Interpret neural recording traces (e.g., noise vs. action potentials in a single fiber vs.
multiple fibers)

American Cockroach, Periplaneta americana (Linnaeus).
Average length is 35-41 mm (1.4-1.6”), sample size shown below.

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Recording Action Potentials from the Model Organism Periplaneta Americana (L.)

INTRODUCTION

Review of Membrane Physiology

The leg of a cockroach is a simple model that can be used to study how primary sensory receptors
process information from real world stimuli and communicate this information to the brain.
Recall from earlier labs and lectures that primary sensory receptors are activated by specific types
of physiological stimuli. When they are activated, ion channels open, allowing ions to move into
or outside of the cell. This causes EPSPs or IPSPs, which change the membrane potential of the
cell (a depolarization or hyperpolarization, respectively). Some primary sensory receptors can
communicate information to the next neuron simply by these local, graded potentials, while
other primary sensory receptors need to fire action potentials to communicate to the next
neuron. Recall that if an EPSP (or multiple EPSPs if there is summation) sufficiently depolarizes
the membrane potential to the threshold for voltage-gated sodium channels to open, an action
potential will occur in an “all-or-none” fashion.

In the Model Dendrite lab, we used two recording electrodes to measure voltage changes caused
by a 9V battery, which simulated a local, graded potential. Today, we will observe and measure
voltage changes which occur in the cockroach leg when biologically relevant stimuli (i.e. air or
touch) are delivered to the leg. If our stimulation is sufficient to activate sensory receptors, we
will be able to record the voltage ‘spikes’ that result from sensory receptors firing action
potentials.

Extracellular vs. Intracellular Recording

We have previously simulated local, graded potentials in lab and have covered the mechanism of
action potentials in lecture. Before we turn our attention to recording voltage spikes from our
model organism, there is one very important distinction to make: the difference between
recording action potentials (or any kind of voltage change) from inside of the cell (intracellularly)
versus from outside of the cell (extracellularly).

We can place a recording electrode inside of a neuron to measure the voltage changes which
occur for a specific neuron. We already know the principles of action potentials that apply to
intracellular recording:

1. Action potentials result in stereotyped changes in the membrane potential (e.g. Na+
enters the cell causing depolarization; K+ leaves the cell causing hyperpolarization).

2. Action potentials are all-or-none, so they do not ‘sum’ like local, graded potentials.

3. Increasing the strength of the stimulus can increase the frequency of action potentials.

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Recording Action Potentials from the Model Organism Periplaneta Americana (L.)

If we place a recording electrode into tissue in the general area of many neurons, we can measure
the average voltage changes that the population of neurons is experiencing. As such, the
principles of extracellular recordings differ from those of intracellular recordings:

1. The maximum voltage change measured will depend on how many neurons are firing
action potentials at the same time.

2. Neurons firing action potentials at the same time will be recorded as a single voltage spike
with a greater amplitude. For example, two neurons firing action potentials at the same
time will result in about two times the voltage change as a single neuron firing an action
potential, thus the recorded voltage spike will be about two times the amplitude.
By the same logic, three action potentials recorded at the same time will be a single
voltage spike with a tripled amplitude, and so on.

3. Increasing the stimulus strength can increase both the frequency of action potentials as
well as the number of neurons firing action potentials simultaneously. So, voltage spikes
can become both more frequent and larger in amplitude.

It is very important to understand the distinction between extracellular and intracellular
recordings before continuing with the lab. If you are struggling, please discuss this with your
lab partners and your laboratory instructor.

Biological Model: The Cockroach Leg

Along the cockroach leg are a series of spines and hairs (Figure 1), which contain many primary
sensory receptors that send axons up the leg to the nerve cord, and ultimately, to the brain.
Deflection of a spine induces local, graded potentials (often termed generator potentials in the
case of sensory receptors) in the sensory receptors that innervate that spine, and if the
stimulation is strong, action potentials will be generated.

Figure 1: Nerve cells of the cockroach “escape system” are organized so that the stimulus provided by a puff of air
evokes turning and running. Nerve impulses that originate in wind-receptor neurons are relayed at the terminal
ganglion to giant interneurons running up the central nerve cord to the metathoracic ganglion. There the signal
processing is completed that leads to the activation of motor neurons in the legs.
(Camhi, JM. 1980. The Escape System of the Cockroach. Scientific American. 243:6,158-172.)

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Recording Action Potentials from the Model Organism Periplaneta Americana (L.)

Those action potentials will travel up the axons of the sensory receptors towards the main nerve
cord. As you can see in Figure 2, our recording electrode is in the femur, an area of axons rather
than sensory neuron cell bodies. Therefore, we will be recording the propagation of action
potentials in the axons, rather than the local, graded potentials that occur in the cell body.

Figure 2: Cockroach leg anatomy and recording electrode set-up.
One electrode (black) is inserted into the coxa and will serve as the
“ground,” or zero voltage point, since it is not close to any nerves of
interest. The other electrode (white) is inserted into femur, the axonal
region of the cockroach leg.

PRE-LAB ASSIGNMENT:
To have an appreciation for the skill of listening to and observing action potentials requires time
and patience, even for the experienced neuroscientist. Therefore, in the interest of getting the
most out of your actual lab time, it is important that you take a few moments now to view two
similar experiments before your lab session*, because YOU will utilize the same equipment to
accomplish your lab tasks. Visit the following website to read about Backyard Brains’ experiment,
Recording and Manipulating Live Neurons:

https://backyardbrains.com/experiments/spikerbox
There are 2 short videos embedded on the site. View them from the same page or click on them
to view in a new tab.

1. Recording Action Potentials from Cockroach Nerves (1:59) NOTE: Before you arrive to
lab, the lab staff performs similar preparations seen at 0:14-0:48. In other words, you will
not be handling live cockroaches during your lab session.

2. Cricket Leg Electrode Placement for hearing spikes (1:44)
 There will be 2 additional quiz questions, one about each video. Therefore, it would be in your

best interest to view the videos before taking the quiz (at the very least, before lab).

Now that you have a visual of what you will be doing, continue reading this Introduction.

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Recording Action Potentials from the Model Organism Periplaneta Americana (L.)

It is important to realize that a single spine can be innervated by multiple sensory receptors, and
each is able to generate and propagate action potentials. The axons from each sensory receptor
serve as individual fibers, converging together to form a nerve (see Figure 3). This means that our
stimulation could induce activity in a single fiber, or multiple fibers of the nerve. Therefore, there
could be instances when we are recording a voltage spike that represents action potentials from
more than one sensory neuron. This is possible due to the recording method we are using -
extracellular recording.

Figure 3: A nerve is comprised of multiple nerve
fibers, or axons from individual sensory neurons.
When stimulating a nerve, the strength of the stimulus will dictate how many neurons are ‘recruited’, or firing action
potentials, simultaneously. Therefore, a stronger stimulus could increase both the frequency of action potentials as
well as the number of simultaneous action potentials in multiple neurons.
(Image: Milton S. Hershey Medical Center, Penn State)

A Brief Review of Important Topics

A. Sensory Receptive Fields: A receptive field is the area innervated by a sensory receptor;
if stimulation falls within this area the receptor will respond by producing local, graded
potentials and action potentials. If stimulation falls outside of this receptive field area, the
sensory receptor will not respond (instead, this other location will be in the receptive field
of a different sensory receptor).

In this lab, we will map out receptive fields in the cockroach leg using extracellular recording.
Recall that stimulation of spines will produce action potentials in the sensory receptors that
innervate it, which will be propagated along the nerve that runs between our two recording
electrodes. So, if we stimulate a spine and record voltage spikes, we can infer that there is one
or more sensory receptor innervating that spine that is sending its axon near our recording
electrodes. Importantly, if we move our recording electrodes, we would be sampling activity from
different axons, which would change our interpretation of the receptive fields.

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Recording Action Potentials from the Model Organism Periplaneta Americana (L.)

B. Neural Summation: Local, graded potentials can sum spatially (as we saw in the Model
Dendrite lab) as well as temporally. Action potentials from a single neuron cannot sum –
a stronger stimulus will yield a higher frequency of action potentials, not an action
potential with a greater voltage change because action potentials are all-or-none.
However, as introduced in this lab, when we record from an area outside of many cells,
we can detect voltage changes from the entire population of neurons surrounding the
electrode. In this case, we can record voltage spikes resulting from action potentials in
multiple neurons simultaneously, which will look like a single voltage spike with double,
or even triple, the amplitude.

We will explore neural summation by stimulating more than one spine at the same time. Because
we are recording extracellularly, you may record an increase in the frequency of voltage spikes,
voltage spikes with higher amplitudes, and/or no change in voltage spikes. Before you begin the
lab, consider why you might see each of those three outcomes.

Overview of the Recording Process

Students will work in groups of 4 (minimum 3) with one neuronal recording device (Figure 4),
which will be placed inside a small box (made primarily of metal mesh) known as a Faraday cage.
This hollow metal conductor will serve to shield our signal from exterior electric fields, thereby
reducing extraneous noise in our recordings.
Upon your arrival, you will notice the cockroach leg has already been prepared as in Figure 2.
Groups will record from one electrode (white) placed in the center of the femur, which lies along
the sensory nerves in the leg, The other electrode is black and is inserted into the coxa, where it
serves as a ‘ground’ or zero voltage point. Any voltage changes that occur between the two
recording electrodes will be compared to the ground electrode (which is sufficiently far enough
from the sensory nerve we will be stimulating), and then transmitted to the computer screen.
When a spine is stimulated on the tibia, the sensory receptors innervating it will send action
potentials into the femur. If the axons are sufficiently close to the recording electrodes, the
electrodes will detect this voltage change and it will be displayed as a voltage spike on your
computer screen. Remember, there are many sensory receptor neurons that can respond to
stimulation of a single spine, so you may see spikes that represent action potentials from multiple
neurons.

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Recording Action Potentials from the Model Organism Periplaneta Americana (L.)

Features of the Recording Device

The prepared cockroach leg has been placed on an examination stage made of cork which is
affixed to a recording and amplifying device called the Neuron SpikerBox Pro* (Figure 4).
It will allow you to hear and see spikes (i.e. action potentials) of live neurons, specifically those
within the leg of a cockroach for this exercise. Take several minutes to examine the
components of your unit.

A
A

B

CD

EF

CD
EF

Figure 4. The Neuron SpikerBox Pro (Left: top view, Right: side view).
Images available at https://backyardbrains.com/products/neuronspikerboxpro

A. Micro USB port [left] requires blue USB cable connection to PC laptop [right].
B. Cockroach leg mounted on cork stage [right].
C. Power/Volume switch: turn dial clockwise to power ON/increase volume;

counterclockwise to power OFF/decrease volume.
D. Channel selection switch (must remain on CH 1 for our exercises).
E. Channel 1 (CH1): receives recording electrode (white top) and ground (black top).
F. Channel 2 (CH2): receives secondary recording electrode

(red top electrode will not be utilized for our exercises, thus not included in lab materials).

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Division of Duties

o You will be working in groups of 4 (minimum of 3) for this experiment. Coordinating your
individual roles may require some practice. Please be assured – this practice time will payoff
later when you need to analyze the data obtained by your group.

o Discuss among your group which student will be assigned to each of the following 4 roles.
Write each member’s name or initials next to their role to keep track:

1. DIRECTOR: keeps group on task, reads directions aloud, and records observed data.

2. MATERIALS MANAGER: oversees operation of computer software and organizes cleanup
procedures at the completion of lab session.

3. TECHNICIAN: follows experimental directions as given by DIRECTOR, i.e. stimulating the
cockroach leg mounted on SpikerBox.

4. FACILITATOR: vocally keeps track of time and announces event markers (for groups with
only 3 students, the MATERIALS MANAGER can fulfill this role as well).

Director: ______________ Materials Manager: ___________

I WILL: I WILL:
- KEEP GROUP ON TASK - OVERSEE SOFTWARE OPERATION
- READ DIRECTIONS ALOUD - ORGANIZE CLEANUP PROCEDURES
- RECORD DATA

Technician: Facilitator: _____________
______________
I WILL:
I WILL: - VOCALLY KEEP TRACK OF TIME
- FOLLOW EXPERIMENTAL DIRECTIONS - ANNOUNCE EVENT MARKERS
AS GIVEN BY DIRECTOR

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Getting Started: From this point onward, directions given for a specific role will be prefaced

with a role title, as seen on the previous page.
DIRECTOR: read all instructions aloud while other group members read along.
MATERIALS MANAGER: The software you will use is called Spike Recorder*. If not already open,
click on the desktop shortcut shown below. When the software opens, enlarge the window to a
comfortable viewing and working size.

*NOTE - If SpikerBox or Spike Recorder ceases to work at any time during the lab session:
1. Immediately inform lab instructor so the occurrence can be documented and

corrected (e.g. SpikerBox may need a new 9V battery).
2. In case of a software “crash”, reboot Spike Recorder by closing the window and

reopening it from the desktop shortcut.

TECHNICIAN: Use the Power/Volume switch to Turn ON SpikerBox and adjust the volume so the
background “noise” is not loud. You should be able to hear some ‘static-like’ noise, but it should
be quiet enough to not disturb others working elsewhere in the lab. Look for a green light to
indicate the battery connection has been made when the power is ON (circled below).

Power/Volume switch CLOCKWISE:
Power ON/Volume UP

Power/Volume switch COUNTERCLOCKWISE:
Power OFF/Volume DOWN

*Equipment and software: Neuron SpikerBox Pro and Spike Recorder obtained from Backyard Brains.
Available at https://backyardbrains.com/

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MATERIALS MANAGER: Upon connection, a USB button will appear in the top left corner of the
start-up window shown below. Click on this button to connect your SpikerBox to the Spike
Recorder software; this will employ the recorder to start acquiring data from your SpikerBox.

Figure 5. Spike Recorder startup window.
Click on USB symbol to connect SpikerBox to Spike Recorder software.

Spike Recorder will switch to the Real-time View, where you will see a waveform (or two) of a
signal being recorded from your SpikerBox (Figures 6 and 7 demonstrate only 1 waveform).
On the next page are several useful features and functions of the Real-time view. You will need
to take a few minutes to acquaint yourself with this screen because some maneuvers may take a
little practice, so use this time wisely to configure some of the settings to prepare the program
for your experiments.
Additionally, each group will be supplied with an abridged booklet version of the Spike Recorder
- User Manual: a valuable reference to have on hand while using the software.

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Figure 6. Spike Recorder, Real-time View – default mode.
By default, waveform starts in Play mode and moves from right to left.

 Config button: configure various settings, such as changing the color of the waveform from
green to red. See User Manual booklet for more information.

 Threshold mode button: enter Threshold mode to get a snapshot of the signal whenever its
waveform crosses the threshold level you set.

 Record button: start/stop recording; waveform signals are automatically saved to an audio
file when a recording has been stopped.

 Browse Experiments button: open an experiment’s recording in order to analyze spikes.
 Play/Pause button: while reviewing a signal recording, clicking Pause allows the Scroller to

become active so the waveform can be reviewed at any desired time point.
 Level handle: see Axis handle.
 Zoom buttons: change the vertical scale of the signal to obtain details of the waveform; zoom

in (+) or zoom out (-)
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Time Scale

Figure 7. Spike Recorder, Real-time View - paused.
Waveform will stop moving when Play/Pause button is clicked.

 Axis handle: click and drag to move entire waveform vertically.
 Scroller: 2 functions –

1. Time Scale, controls time ranges of 0.1 ms to 1.0 s (white horizontal line, lower right);
2. Slide bar, controls movement of the waveform (gray horizontal slide bar spanning

lower portion of screen and only visible when waveform is paused or stopped);
To control either of the above, hover your mouse over the desired function and:

o Scroll up to shorten the time scale or to move the signal back in time,
o Scroll down to lengthen the time scale or to move the signal forward in time.
o Slide bar may also be controlled with a “click and drag” motion, instead of scrolling.
 Go back 5s: rewinds playback of a recording by 5 seconds.
 Return to Live Data: returns to the default screen, i.e. Real-time view.
Remember: refer to the Spike Recorder - User Manual for more information and imagery on
the features listed here, plus several other useful functions.
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Brain & Behavior: CORE-UA 306

LABORATORY COVER SHEET
Name: _____________________________ Lab Partner’s Name: _________________________
Date of Lab: ___________________________ Date Due: _______________________________
Lab Instructor: _____________________________ Lab Section Number: __________________

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…

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PROCEDURES and EXERCISES

 PART I: INTRODUCTION TO SPIKE RECORDER

A. Obtaining a Baseline Reading

Once your Spike Recorder has been configured to your desired settings, you are ready to begin.
DIRECTOR: “We will be mimicking stimuli that are biologically relevant to the cockroach.
But first, we must OBTAIN A BASELINE READING before the stimulus is delivered. We should use
this reading to also familiarize ourselves with the helpful features of the software listed on the
two preceding pages.”
1. MATERIALS MANAGER: Once the group is ready to begin, make a 10-second recording to

obtain the baseline measurement without stimulus.
a) Click the Record button [Figure 6] and watch the time lapse at the top of the screen.
b) At 10 seconds, click Record again to stop.
Once the recording is stopped, a pop-up message will display the full path of the recorded file
saved on your computer (there is no need to make note of this file path because all recordings
are stored in order of occurrence). Spike Recorder will revert back to the Real time view.
2. MATERIALS MANAGER: To review the baseline recording, click the Browse Experiments
button. A file folder named “BYB” should open to a chronological list of all recordings made
from the student laptop as .wav files. The most recent recording will always appear last on
the list [Figure 8].

Figure 8. BYB file folder example.
Displays a list of recordings as .wav files, as well as recordings of marked events as Text Documents.

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3. MATERIALS MANAGER:
a) Double-click on the last .wav file to open your baseline recording in Spike Recorder.
b) Click the Play button to see and listen to your recording.
c) Click the Spike Analysis button to observe any potential spikes in the signal and then filter
the spike set (see page 10: Spike Recorder - User Manual: Spike Analysis).
d) After the spike set has been filtered, click the Spike Analysis button again to return to the
playback of your recording to quantify important aspects of the signal in one second, such
as frequency, number of spikes, and root mean square or RMS (see page 11: Spike
Recorder - User Manual: Signal Measurement). Record your data in the spaces below.

o QUALITATIVE OBSERVATIONS – Baseline Reading (i.e. no stimulus):
 SpikerBox: hissing or static-like sounds, without “crackling” noises.
 Spike Recorder: waveform should show little variance in frequency and no spikes, as in
the example of a baseline recording below [Figure 9].

Figure 9. Spike Recorder, sample of a baseline recording. Note the absence of spikes in the waveform.

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4. DIRECTOR: “To quantify this data, we need to record two variables to report a baseline
reading: frequency and root mean square (RMS).

- Frequency is the number of spikes per any selected time interval. It is equal to the number
of cycles in one second and its standard unit of measurement is the Hertz (Hz).

- Root mean square is a measurement of the magnitude of a signal over time.
For example, if we analyze a one second window of our recording with no stimulation, we
will have a lower RMS value than if the leg is being stimulated and causing an increased rate
of the neuron's firing.”

o QUANTITATIVE OBSERVATION – Baseline Reading (i.e. no stimulus):

 Baseline Frequency: ______________Hz (this number will be a mixed decimal)
 Actual Number of Spikes in 1 second: _______ (this will be a whole number)
 Baseline RMS: _____________ (this will also be a mixed decimal)

Upon completion of reviewing the Baseline recording, the MATERIALS MANAGER must return to
Spike Recorder: Real Time view to begin the experiment.

B. Biologically Relevant Stimulus: WIND

DIRECTOR: “Now that we have a baseline to compare, we are ready to mimic a biologically
relevant stimulus: wind. To mimic wind, the TECHNICIAN will use a slow but steady breath as a
relatively weak stimulus, which should elicit spikes that represent activation of a small number
of sensory receptors firing action potentials. We can then analyze and report the frequency and
the root mean square of our recorded signal, using our baseline recording as a reference to
determine the difference between background noise and an action potential spike elicited from
the ‘wind stimulus.’”

1. FACILITATOR, TECHNICIAN, and MATERIALS MANAGER: you should coordinate your timing
and practice the following steps a few times if necessary:

a) FACILITATOR should announce to TECHNICIAN: “Begin to administer a continuous, gentle
and slow breath to the cockroach leg for 10 seconds, starting now.”

b) MATERIALS MANAGER: Click Record to begin recording when FACILITATOR says “now.”
c) TECHNICIAN: Inhale deeply, then with one continuous breath, blow gently and slowly

onto the cockroach leg for 10 seconds.
d) FACILITATOR must tell TECHNICIAN and MATERIALS MANAGER when to stop

administering the stimulus and stop recording. For example, at the end of 10 seconds say
“Ten seconds, Stop.”
e) MATERIALS MANAGER: after 10 seconds, click Record again to stop the recording.

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During the 10 seconds of recording, be sure to observe any changes in sounds coming from the
SpikerBox and changes in the waveform of the Spike Recorder.
o QUALITATIVE OBSERVATIONS – Weak Stimulus: Wind (long, slow breath):

 SpikerBox: crackling or hissing static sounds should seem louder than the “background
noise” of your baseline recording; these sounds represent action potentials (i.e. spikes) in
individual fibers of the sensory nerve.

 Spike Recorder: waveform should show an increase in fluctuation; the peaks in the
waveform demonstrate voltage changes associated with action potentials in the nerve.

2. DIRECTOR: “To quantify this data, we will again record the following variables: frequency,
number of spikes, and RMS.

o QUANTITATIVE OBSERVATIONS – Weak Stimulus: Wind (long, slow breath):
QUESTION 1:
A long, slow breath is a relatively weak stimulus and should elicit spikes representing activation a
small number of sensory receptors firing action potential.
For a single spike, quantify and report the below variables. Use your best judgement to determine
the difference between background noise and a spike elicited from a weak stimulus.

 Weak Stimulus Frequency: ______________Hz
 Actual Number of Spikes in 1 second: _______
 Weak Stimulus RMS: _____________

3. DIRECTOR: (To the MATERIALS MANAGER) “Please return to Spike Recorder: Real Time view
so we can begin recording for the Moderate Stimulus portion of the experiment.”

(To the TECHNICIAN) “This time, you will use several quick, short breaths to stimulate the
cockroach leg.

(To the FACILITATOR and MATERIALS MANAGER) “As we did for the baseline recording, keep
in mind to only Record for 10 seconds.”

(To the GROUP) “Then we will report our qualitative findings below. During the 10 seconds
of recording, we need to carefully observe any changes in sounds from the SpikerBox and
changes in the waveform of Spike Recorder.”

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o QUALITATIVE OBSERVATIONS – Moderate Stimulus: Wind (short, quick breaths)
QUESTION 2:
Based purely on visual inspection of your data (a qualitative rather than quantitative measure),
how do short, quick breaths differ from a long, slow breath?

o QUANTITATIVE OBSERVATIONS – Moderate Stimuli: Wind (short, quick breaths)
QUESTION 3:
A. Record your data from the recording of Moderate Stimuli administered to the cockroach leg.
B. Then, in sentence format, describe any changes in spike frequency and root mean square
between the two types of “wind” stimuli, if applicable.
A.

 Moderate Stimuli Frequency: ______________Hz
 Actual Number of Spikes in 1 second: _______
 Moderate Stimuli RMS: _____________
B.

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QUESTION 4:
Did each breath evoke spike trains with the same RMS value? What was the average RMS
amplitude of each successive breath?
Note: You do not need to measure every spike; just measure the spikes that are representative
of the different RMS values to find the range present in your data. Plot the RMS for each breath
in the graph below.

RMS

1 2 3 4 5 6 7 8 9 10

NUMBER OF BREATHS
QUESTION 5:
In some datasets, it is possible that there will be spikes with RMS values (i.e. amplitudes) that are
much higher than the individual spike RMS value you measured in QUESTION 1.
Hypothesize what might explain this observation.

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QUESTION 6:
Hypothesize why it might be adaptive for the cockroach to respond less to the light breath
stimulus compared to the strong, quick breath stimulus?

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 PART II: RECEPTIVE FIELD MAPPING

In this exercise, you will create a receptive field map which will illustrate the activity induced by
deflecting the spines along the tibia. While looking through a magnifying glass (preferably with
the light switched ‘ON’), you will use a fine metal probe to move each of the tibial spines, one at
a time, and record the approximate frequency of spikes elicited from deflecting each spine.

On the next page, you will be filling in the blank “map” of the cockroach leg with your data to
construct a “receptive field” profile.

1. Working from the top of the tibia to the bottom, use the metal probe to move each spine.
Remember to restrict your data recording to only 10 seconds, so record the activity
elicited from deflecting one spine at a time. You should probe at least 6 spines to get an
adequate record.

Helpful Hints:
 With a steady hand, the FACILITATOR should hold and aim the magnifying glass (with the

light ON) at the cockroach leg for the TECHNICIAN.

 The best way to achieve deflecting each spine is for the TECHNICIAN to announce to the
MATERIALS MANAGER when they are going to move a spine. To be consistent, count 2
seconds for each spine, i.e. announce to your partner, “Now, 1, 2, Stop.”

 TECHNICIAN: To help steady your hand while holding the probe, place your other hand
around your wrist to keep it from moving about. Use your thumb and forefinger to
manipulate the probe. This may require some practice before starting the activity.

2. Stop after each spine and transfer your data onto the Receptive Field Map on the next
page. Quantify the data by recording the approximate spike frequency in Hertz (don’t
worry about the RMS value of the spikes).

QUESTION 7:
Draw a line next to the spine on the cockroach leg diagram, in the approximate position of the
one you stimulated. Record your measure of the spike frequency in Hz next to the spine and
repeat steps for each spine.

3. Stimulate the next spine, repeating the quantification and mapping until you reach the
end of the tibia, going in a straight line down the length of the leg. Try to keep your hand
as steady as possible while performing this procedure and be sure to move each spine by
roughly an equal amount each time! This will allow you to compare the receptive fields
across the spines on the leg.

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Receptive Field Map:

COXA

FEMUR

TIBIA

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When you have finished quantifying the spike frequency for all the spines, use a 4-point scale to
quantify the strength of activation for each spine as: (1) absent, (2) weak, (3) medium, or (4)
strong. Weak spines are those which produce very few spikes. Strong spines are those that
produce a high frequency of voltage spikes. Use the following key scheme to color-code your
receptive field map of the cockroach leg:

1. Absent—Uncolored
2. Weak—Blue
3. Medium—Yellow
4. Strong—Red
QUESTION 8:
Do you notice a pattern in the receptive field map? For example, do spines in certain areas of the
tibia tend to exhibit similar spike frequencies when stimulated?

QUESTION 9:
When you deflect a spine and the result is a high frequency of spikes, what does that tell you
about the innervation of that spine?

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