Lab 2: Extracellular recording from cockroach

leg sensory neurons

Author: Mackenzie Andrews

INTRODUCTION
Sensory stimulus is recognized by the nervous system with two properties: ​modality ​and

intensity. ​Modality is encoded by what neuron is active, telling the brain what type of signal is

being received and where it’s coming from. Intensity is encoded by the frequency of action

potentials where a higher frequency encodes a stronger stimulus. The pattern of action potential

frequency is also dependent on the type of sensory neuron that is encoding it: ​phasic ​vs. ​tonic

receptors. Phasic receptors encode ​changes ​in stimulus intensity by generating action potentials

at a higher frequency on the onset of a stimulus with decreasing frequency as the neuron adapts.

Tonic receptor neurons encode absolute amplitude of a stimulus by maintaining the same action

potential frequency through the duration of a sustained stimulus.

In this lab, we explored these neuronal properties by taking extracellular recordings from

cockroach leg sensory neurons. While taking extracellular recordings, multiple action potentials

from multiple neurons are recorded. The amplitude and frequency of these action potentials vary

depending on the type, size, and distance away an axon is to the electrodes. While complicating

the signal, this variance also allows us to distinguish between multiple types of neurons in a

single reading.

By stimulating the spines on a cockroach leg, receptor neurons responded by generating

action potentials which were detected by a pair of electrodes placed in the extracellular space of

the femur and tibia of the leg. We explored the effect on the signal of bending different spines
along the leg, bending a single spine in two different directions, performing a stepwise

stimulation of a spine, blowing on the spines, and tapping the micromanipulator while touching a

spine. Our results demonstrate the difference between phasic and tonic receptors as well as

amplitude differences dependent on the neuron population and type of stimulation.

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

Quarter, 2017)​.

RESULTS
Set Up

In order to begin recording, the cockroach leg was placed on two electrodes, held in place by a

cork mounted to the microscope platform (Figure 1). The electrode punctured the exoskeleton on

one side of the leg inorder to record the extracellular voltages within the leg. The stimulation

probe on the micromanipulator was then positioned near one of the spines on the leg by using the

course manual controls and viewing the leg through the microscope lens (Figure 2).

Spontaneous Activity

Before recording any stimulation response from the leg, a recording of the spontaneous activity

of the neurons within the leg was taken. Figure 3 shows 0.8 seconds of spontaneous activity. The

figure shows 2 descernable action potentials with additional action potentials getting lost in the
noise of the recording. The largest action potential occured approximately once every 0.35

seconds or at a frequency of 2.85 Hz. The smaller descernable action potential occured at a

frequency of approximately 50 Hz. The largest action potential had a recorded amplitude of

approximately 12mV (peak to valley). This recording was taken with a gain of 100 meaning that

the actual amplitude of the largest action potential was 120 microvolts.

Response to Spine Deflection

After recording the spontaneous activity of the neurons in the leg, we stimulated a single spine to

get a recording of the response to spine deflection. For this test, a spine on the tibia of the leg

was bent against it’s natural curvature. Figure 4 shows the response to 8 seconds of stimulation.

There are 2 easily desernible action potentials in the signal. The first is the large amplitude action

potential (approximately 20mV peak to valley). Immediately after stimulation, this action

potential increased to a frequency of aproximately 150 Hz; approximately 50 times the frequency

of the large amplitude action potential in the spontaneous activity signal. After 5 seconds of

stimulation, this action potential decreased to a frequency of 20 Hz. This behavior is a clear

example of a phasic response. The smaller amplitude action potential (approximately 5 mV peak

to valley) had a frequency of approximately 200 Hz which was maintained throughout the signal

(a 10 fold increase from the resting frequency of this action potential population of 20 Hz). This

behavior is indicative of a tonic response.

This recording was repeated over 6 separate spines on the tibia of the leg. In each recording, the

amplitude of the action potentials were fairly consistent, however the response frequency profile

changed for each spine. Some spines showed a clearly phasic response while other spines

showed mainly tonic responses.

Spike Discriminator - Rate Meter

Using the signal recorded in Figure 4, the spike discriminator rate meter function of LabChart

was applied to the data. The spike discriminator split the spikes into two separate populations

based on the ratio between width and amplitude of the action potential. Figure 5 shows the

discrimination of the spikes for a short segment of the signal (20 ms). The rate meter function

was then used to calculate the count per bin of the two signal populations. Using a 2 second bin,

Figure 6 shows the counts of each population. The counts per pin can be converted into a

frequency by dividing the count number by the bin size (2 seconds). The population on the top of

Figure 6 shows a decreasing frequency over the time course of the signal, representative of a

phasic response. The bottom population in Figure 6 shows a steady frequency throughout the

duration of the signal, indicating a tonic response.

Directional Sensitivity

In order to test the directional sensitivity of the spines, 2 recordings were made on the same

spine; the first against the natural band of the spine and the second with the natural bend of the

spine. Figure 7 shows the response of these 2 stimulations. The first set of dashed lines (before

the solid recording break line) signify the start and end of stimulation against the natural bend of

the spine. The second set of dashed lines show the start and end of stimulation with the natural

bend. While both signals show a phasic response to the stimulation, the response to stimulation

against the bend of the spine resulted in a higher frequency signal with a longer adaptation

period. It can be observed that the two signals have the same amplitude indicating that they are

most likely coming from the same neuron. This indicates that a singal neuron can encode

responses to multiple types of stimuli by changing the frequency and patern at which it generates

action potentials.

Additional Experiments
To develop an increased understanding to neuronal response to a variety of stimuli, we

conducted 3 additional experiments: stepwise stimulation, stimulation by blowing, and

stimulation by tapping. For stepwise stimulation, a spine with a strongly phasic response was

stimulated with the probe. As the frequency of the signal decreased, we bent the spine slightly

more. We repeated this for 4 steps (Figure 8.a). Each time the spine was bent, the signal

frequency increased initially before the spine readapted to the stimulation.

For stimulation by blowing, we removed the probe from the leg and simply blew in the spines

(Figure 8.b). During the blowing stimulation, the frequency of action potentials increased by

about 5 fold. Since we were using our mouth, it was hard to maintain a constant blowing force so

its difficult to tell whether the response was phasic or tonic.

For stimulation by tapping, the probe was placed on a phasically responding spine. We

stimulated the spine initially then tapped the micromanipulator after the neurons adapted to the

stimulation (Figure 8.c). Each time we tapped the micromanipulator, a new spike in action

potential frequency occured.

DISCUSSION
In this lab, we were taking extracellular recordings of neuronal action potentials. In addition to

the fact that this allowed us to observe multiple action potentials in one signal (desribed in

introduction), extracellular recordings greatly differ from intracellular recordings in their

amplitude. The extracellular recordings we were taking were on the scale of 100 microvolts

(with 100 fold amplification). In contrast, intracellular recordings are on the order of 100 mV (a

1000 fold amplitude increase from extracellular recordings). This small signal amplitude and the
fact that many action potentials are contained in a single signal results in a relatively poorly

resolved, noisy signal.

That being said, we were still clearly able to discern multiple action potentials, especially those

with a phasic response. Phasic responses (such as the ones recorded in this lab) allow the sensory

system to filter out important information from an extraneously noisy environment. Take for

example a student attempting to write a lab report in a house full of 20 year old college students.

In the room next door, loud trap music has been playing for 6 hours straight. Directly outside the

student’s room, the smell of ramen and burnt popcorn lingers. The ambient temperature of the

room is approximately 50 degrees because college students are broke and don’t want to pay to

heat the house. If the nervous system wasn’t able to adapt and phasically filter out all of these

distracting stimuli, the student would have an extraordinarily difficult time focusing on their

report. However, when the loud crash of someone shattering a wine glass in the hallway occured,

the student was still able to respond to the change in stimulus thanks to phasic responses.