Rate Coding Lab – Teacher Guide
Learning Goals:
In this lab students will:
 Learn what rate coding is and about the rate coding model
 Understand how brains distinguish between different types of stimuli
Before doing this lab students should understand:
 You should know how to use the SpikerBox before beginning this lab
After doing this lab students should be able to:
 After this lesson plan, you should be ready to move onto some more intermediate SpikerBox
experiments.
Equipment:
 SpikerBox
 Toothpick


Smartphone Cable
Source of Compressed Air


Cockroaches
Computer with
Audacity installed
Educational Standards: Please see the back of the teacher’s guide for information on how this activity
meets the Next Generation Science Standardsand Common Core Standards.
Part 1: Observing Spikes
In this exercise, you will use several methods to record data from your cockroach leg, and how to
measure the spikes caused by different stimuli. Now that you have successfully witnessed spikes, you
are ready to record, quantify, and graphically present electrophysiology data. The method you will use
to compare differences in spiking from different stimuli is called “rate coding” and it is one of the most
reliable tools in neuroscience. Rate coding is simply measuring the number of spikes that occur during
a set period of time. However, even though it is simple, rate coding can be used to answer complex
questions about how neurons respond to stimuli.
Set-Up
1. You’ll need a cockroach leg set prepared in the same way as the first lab, Getting Started With
the SpikerBox. You will place one electrode in the coxa and one in the femur (see diagram
below).
2. Plug the audio adapter cable into the SpikerBox and your computer. Note: for the SpikerBox to
record properly, your computer’s audio input jack must be audio only, not a combined audio
In/Out jack.To make sure everything is working properly, listen to the sounds coming from the
SpikerBox speaker.
3. Turn on Audacity, open a New window, and ensure the setup described in the previous exercise
has been completed.
4. To record from your SpikerBox, simply click the Red Circle at the top of the screen. You can
stop recording at any time by pressing the Yellow Square. If you begin to record again, the new
track will appear below the previous recordings. You can rename each track by selecting the
Audio Track button next to your waveform. If you want to delete a track, select the X button in
the upper left of each track.
5. Turn off your cell phone and Wi-Fi. Signal interference from these devices is significant. Ask
your teacher permission to send a classmate a text. What does this tell you about how cell
phones work?
6. During each experiment, it is best to record without stopping. Noise created from cell-phones,
moving the electrodes, or a variety of other sources can be removed prior to analysis. However,
turning your recording on and off may become confusing. The easiest way to keep track of
what your data corresponds to is to keep good notes about the timing of the experimental
activities in your data log.
7. When you save, Audacity will save your project in two forms that may be confusing. The first
file saved is a folder ending in _data and the second is a file ending in .aup. The .aup file must be
in the folder holding the _data folder. In other words, keep the .aup file in the parent directory
of the _data folder.
Part 2: How the nerves of the femur respond to stimulation
You can now begin experimenting with your cockroach leg! With your cockroach leg, you will compare
how neurons in the leg communicate with no stimulation, a light touch from a toothpick, and a strong
blow of air through a straw.
Mini Experiment:
Have a lab partner brush your arm with a sheet of paper. Now have them give your arm a forceful
poke. Briefly describe how the sensations differ:
A variety of answers would work, here but students should be able to distinguish between the
“shallow” or “light” sensation of the paper, and the “deeper” or “solid” sensation of the finger poke.
There are several reasons for why you differentiate the two stimuli. First, the light touch likely
stimulates nerves from a very small area while the more forceful poke may stimulate neurons from
much more of your arm. Second, the light touch may only stimulate neurons that respond to being
compressed. One type of neurons calledMerkel cells, send action potentials to the spinal cord and brain
in response to light touch. These cells fire faster when stimulated. Therefore a light touch increases
their firing rate, which in kind is interpreted by your spinal cord and brain as a light touch. Additionally,
there may be other neurons stimulated for fire faster that convey another sensation such as pain.
Teacher Note: A Short Version of Rate Coding
If your schedule doesn’t allow you to do the full rate coding activity, you can use a shortened version of
the activity, which uses only a few types of stimuli and only measures the response to stimulation in the
femur.
1. Set up your leg for recording following the directions in the Getting Started With The SpikerBox
activity.
2. Ensure your setup is functional by poking the leg a few times. If you see no response, you may
need to adjust your electrodes or select another leg.
3. When you have stable recordings, isolate your leg from any wind and begin the actual
recording. Take notes when anything happens to the leg.
4. Record the spontaneous spiking patterns from the leg for 5 minutes. Note the beginning and
end time of your control recordings in Table 1.
5. Take a toothpick and stimulate the hairs on the tibia of the leg. Try several variations of
stimulation including constant pressure or repetitive poking, until you find one that gives you
consistent reactions. Write your stimulation method in Table 1.
Table For Shortened Activity:
Stimulation Method
Drawing of Spikes
Time
6. Follow the directions in section 4: The Basic Analysis of Spikes to quantify the differences
between your treatments
Part 2: Experimentally testing different strengths of stimuli on the Femur
Now you will do a controlled experiment to see how different strengths of stimulation affect the
cockroach leg. You will use the compressed air on your lab bench to stimulate the cockroach leg for this
experiment.
Teacher Note: No Compressed Air in your classroom? No Problem!
If you don’t have compressed air in your laboratory or classroom, there are still plenty of ways to do this
experiment. Here are some that have worked for us:

Use a drinking straw and have students blow on the cockroach leg from different heights. You
can tape straws at different heights to a ruler, to standardize the heights. Be sure to remind
students to blow consistently during each trial.

You can purchase compressed air in an aerosol canister at an office supply store. Compressed
air is often sold in the computer section because it is used to clean keyboards and computer
components. As with the drinking straw method, you can have the students hold the
compressed air canister at different heights to achieve different strengths of stimulation.
Be sure to modify the student worksheets to reflect your new experimental design!
7. First observe the spikes that occur without any stimulation. Record the start time and stop
time of your observation and write down any notes that you observe.
8. Give the cockroach leg at least a 30 second break. Next, stimulate the leg by turning on the
compressed air at the lowest pressure (5 psi). Use the hose to direct the compressed air at the
cockroach leg. Record the start and stop time and any other notes you might observe.
9. Now raise the air pressure to the other psi levels listed in the table. As always, note the start
and stop time for each observation in the table below.
Table 2. Stimulating the Femur
Condition
Method Notes
No Stimulation
5 psi
15 psi
25 psi
35 psi
Part 3: Basic Analysis of Spikes
Timing Notes
Start
Stop
To better analyze your recordings, you will want to isolate them into sections. Use the following
method to isolate sections of your recordings. You will begin with the 5 psitrial, and then repeat the
procedure the other air pressures that you used.
1. Using the recordings from your trial at 5 psi, find a representative section of spikes between 510 seconds long and select it with your cursor. Press the left click and drag across the waveform
to select. Select a region with clear spikes and as little noise as possible.
2. Copy the selection (Control-C), open a new Audacity window (Control-N), and paste the
selected 5-10 second waveform (Control-V).
3. In the Effect drop down menu, select Amplify. A dialogue box will appear which asks you to
select the amount of amplification. The default selection is the amplification that keeps the
audio from reaching beyond 1.0 or -1.0 on the scale.
4. Record the Amount of Amplification value from the dialogue box in Table 1. Use the default
settings and click OK.
5. One of the first things you may see is that spikes do not always occur in a regular fashion. You
may also notice that not all spikes look the same. As shown in Figure 9, spikes may have 2, 3, or
even 4 peaks to them. These are not special action potentials, but groups of action potentials.
In the next step you will quantify the relative frequency and size of action potentials produced by a
cockroach leg in response to your stimulations. You will be categorizing the spike peaks by amplitude
in a process called binning, or separating spikes into categories (bins) based on size. Figure 10 provides
a screen shot of Audacity that will help you in this process. The bins will be the scale used by Audacity
(Yellow Arrow). Keep in mind that you are measuring negative peaks as well as positive.
1. Isolate and amplify a 5 second section of recording from the No-Stimulation condition, using
the above method.
2. Be sure you have recorded the Amount of Amplification in Table 2.
3. Widen your Audacity window to fill the width of your screen using the Window Resize tool (Red
Arrow).
4. Use the Zoom In tool (Fig. 10A; Purple Arrow) so that you can visualize a small section of your
trace, and easily identify the peaks of your spikes. At this point it is easy to eyeball the
difference between peaks like those circled in Green and Red. However, for the purposes of
quantification, you want to have a consistent methodology that will allow you to reproducibly
determine if the peak circled in Blue is different than that in Red.
5. Look over your data briefly and decide on a threshold for what is noise and you will accept as
data. One way to do this is to look at your control recording (no-stimulation) and identify the
largest spike during the recording. You can take the height of this spike as your cut –off.
What was the height (or depth of the largest spike in the no stimulation condition?
Students should note the height of the spike
Does this seem like a reasonable threshold for determining what constitutes a spike in the other
treatments? (i.e. is it surprisingly large or small, or did it occur due to some accidental stimulus you
recorded in your notes?)
Students should note any unusual conditions that could have led to the spike and make a determination as
to whether this would be an appropriate cut off.
6.
Using the Window Resize tool (Red Arrow), shrink the Audacity window vertically, so that the scale
on the left of the screen shows only 0.1 of the total height of the waveform (Fig. 10B). This will allow
you more accurately determine whether the spikes you observe are under or over the threshold.
7.Select from 0.0 - 0.1 seconds of your 5psi recording to collect data. Count the number of peaks that
exceed the threshold you determined in question 5. Record these in the table below.
8. Now move to the 0.1 -0.2 second section of your recording, and record the number of spikes in the
row below. Do the same for the remaining sections of recording, until you reach 0.5
9. Find the section of recording for the other treatments, and repeat steps 7 and 8 for your trials at 5
psi, 15 psi, 25 psi, and 35 psi.
Table 3: Number of spikes recorded in the femur
Number of Spikes Recorded in the Femur
Bin
0.0 - 0.1 secs
0.1 - 0.2 secs
0.2 - 0.3 secs
0.3 - 0.4 secs
0.4 - 0.5 secs
TOTAL:
No Stimulation
5 psi
15 psi
25 psi
35 psi
Part 4: Comparing Spikes From the Femur and the Tibia
1. Remove the electrode from the femur and insert it into the tibia of the cockroach leg
1. Ensure your setup is functional by poking the leg a few times. If you see no response, you may
need to adjust your electrodes or select another leg.
2. Give the cockroach leg at least a 30 second break. Next, stimulate the leg by turning on the
compressed air at the lowest pressure (5 psi). Use the hose to direct the compressed air at the
cockroach leg. Record the start and stop time and any other notes you might observe.
3. Now raise the air pressure to the other psi levels listed in the table. As always, note the start
and stop time for each observation in the table below.
Table 4: Stimulating the tibia of the cockroach leg
Condition
Method Notes
Timing Notes
Start
Stop
No Stimulation
5 psi
15 psi
25 psi
35 psi
6. Using the directions from Part 3: Basic Analysis of Spikes, count the spikes for each treatment from
0.0-0.5 seconds, using 0.1 second bins. Record the results in the table below
Table 3: Number of Spikes Recorded in the tibia
Number of Spikes During Stimulation – Tibia
Bin
No Stimulation
5 psi
15 psi
25 psi
35 psi
0.0 - 0.1 secs
0.1 - 0.2 secs
0.2 - 0.3 secs
0.3 - 0.4 secs
0.4 - 0.5 secs
TOTAL:
Now construct two graphs that compare the spiking rate in the tibia and the femur at different levels of
stimulation. Think carefully about what the x and y axes will be.
Discussion Questions
1. At the beginning of the rate coding exercise you chose a threshold for which spikes you would count.
How, if at all do you think that your results would differ if you had used a higher threshold? A lower
threshold?
If you chose a higher threshold, fewer spikes will exceed the threshold, and therefore the counts for every
treatment will be lower. Choosing a lower threshold would mean more spikes would exceed the threshold,
and therefore counts would be higher. Choose too high a threshold, and you will have no data, choose too
low a threshold, and every spike will exceed it and you will have nothing but noise.
2. How did the neuron firing change as you stimulated the femur with increased air pressure? How did
neuron firing change when you stimulated the tibia with increased air pressure? Describe your findings
in the paragraph below?
For both treatments, the spike rate increased with greater stimulation. However, the spike rate increased
more when the tibia was stimulated, than the femur. Neurons in the tibia respond more strongly to
stimulation.
3. Did neuron firing change when you applied the same stimulus continuously? If it did, why might this
be the case?
Neurons fired rapidly when a stimulus began. However, when the stimulus was applied over time they did
began to respond less to it. This is because the neurons become accustomed to the stimulus and then
reduced their firing rate, a phenomena know as neural adaptation or sensory adaptation.
4. Neuron spikes are all the same size, and scientists have demonstrated that amplitude of a spike does
not change with the strength of the stimuli. Yet in some treatments you may have observed some
spikes that were smaller than the others. How might the set up of the Spikerbox cause some spikes to
appear smaller (hint: think about distance between a neuron firing and the recording electrode). Why
would this occur more in some treatments than others?
Spikes might appear smaller if the neuron firing was far away from the recording electrode. In lowstimulus treatments, fewer neurons fired over all. Therefore it was more common for far away neurons to
fire, and for these spikes to be registered as “smaller” when recorded by the SpikerBox.
5. Why might being able to discriminate different types of stimuli be advantageous to insects?
Different strengths of stimuli can indicate different types of environmental changes. For example a
predator rapidly approaching might create a strong air current, while a soft breeze would create only a
weak air current. Being able to discriminated between these types of air currents would be help insects
distinguish between real threats, and background air movement.