Mechanical Stimulation of Tibial Companiform Sensilla of a

Periplaneta americana Leading to Varied Responses

Omar Hallouda

BIO409

March 2012

Abstract

The aim of the experiment was to study spines, campaniform sensilla, located on the tibia

of the American cockroach (Periplaneta americana ). More specifically to study the various

responses, in terms of amplitude and frequency, of the campaniform sensilla by stimulating

different spines in different locations, by stimulating spines in different directions, and by

stimulating spines with different displacement pressures. Additionally, the study aimed to

observe if the effects of adaptation occurred in the response of the spines with prolonged

stimulation. It was found that spines higher up the tibia and closer to the coxa had the greatest

responses to stimulation, spines stimulated against the grain resulted in the greatest responses,

and also stronger displacement pressures resulted in the greatest neural responses. The effects of

adaptation were also witnessed over a 5 second stimulation.

Introduction

Animals must always be prepared to escape attacks from predators or other foreign

attackers like humans in order to survive. For such a reason, many animals have developed

escape responses (Bullock & Horridge, 1965). American cockroaches (Periplaneta americana),

have such a response (Roeder, 1948). The escape response for these cockroaches consists of a

stereotyped turn away from where the displacement of air came from followed by a quick run in

any random direction (Camhi & Tom, 1978). The escape response appears to sacrifice control

over the direction of the escape in order to increase the speed of the escape response, a

significant adaptation for survival (Wine & Krasne, 1972).

Cockroaches are able to perform this escape response with the help of the campaniform

sensilla. Campaniform sensilla are a form of mechanoreceptors called proprioceptors (Moran &

Rowley, 1975). Proprioceptors are internal mechanoreceptors used for detecting internal changes

(Dethier, 1963). The campaniform sensilla are found on the tibia of the leg (See Appendix Fig. 6

for the structure of the cockroach leg). The campaniform sensilla are connected to structures

called domes and spines. The spines are extended outward in a uniform direction and send

electrical signals to the central nervous system in response to stimuli on the spines (Spinola &

Chapman, 1975). These electrical signals, action potentials, can be experimentally elicited to

observe different action potentials caused by different stimulations. By using mechanical

stimulation, as done in this lab, one can create such signals.

Neural receptors can be different types. They can be phasic, tonic, or phasi-tonic, a

combination of the two. Tonic receptors are sensory receptors that continuously fire action

potentials during the entire time of stimulation. (Randall, Burggren, & French, 2002). In contrast,

phasic receptors fire with the first and last part of a stimulus rather than firing during the entire

stimulus (Randall, Burggren, & French, 2002). Phasi-tonic receptors are a combination of the

two. The phasic part makes the receptor initially fire quickly when the stimulus is first applied.

Then the firing slows down and becomes more consistent, tonic part of the receptor. And again

the receptor fires very quickly as a part of the phasic part of the receptor, this time when the

stimulus is relieved (Randall, Burggren, & French, 2002).

An important aspect of neurons are their ability to adapt to prolonged stimuli. If there is a

constant stimulus then it will adapt, begin to ignore the stimulus, when it begins to realize that

the stimulus is no longer perceived as important.

In the four parts of this study, mechanical stimulation is used to obtain signals. In part 1a

of the lab, spines on different parts of the tibia varying from the higher part of tibia (towards the

coxa. “High spine”) to the middle part of the tibia (“Mid spine”) and to the lower part of the tibia

(“Low spine”) were manually and mechanically stimulated (See figure 5 in appendix). The

stimulations on spines at different areas of the tibia should lead to different responses. In part 1b,

the same spine would be manually and mechanically stimulated but this time in different

directions (See figure 5 in appendix). The spine that had shown the greatest amount of response

to mechanical stimulation in part 1a would be used for part 1b. It was predicted that going in the

opposite direction of the way the spine is facing, “against the grain”, would produce the greatest

response in contrast to “with the grain”. This is because going against the grain should cause the

most tension and thus creates a larger signal to be sent by neurons from the mechanoreceptor to

the central nervous system. In part 2, the same neuron that was chosen in part 1b would be

chosen again but this time to compare varying displacement pressures. The spine would be

moved in the direction that was found to again produce the strongest response in part 1b. The

displacement pressures would vary from “light”, “average”, and “heavy” and were all manually

determined. It was predicted that the heavier the displacement pressure the greater the response.

In part 3 of the experiment, adaptation of the neuron was studied by stimulation of the spine with

extended and continuous stimuli. It was predicted that with prolonged stimulation of a spine,

adaptation of the receptors can be seen in the response. This would be seen with responses

becoming less frequent and with smaller amplitudes for its action potentials.

Materials and Methods

Periplaneta

americana

PicoScope

Computer

Preamplifier (P15)

Audio amplifier

loud speaker

Tape

Glass probe

Faraday cage

Dissecting scope

Plasticine

Electrode leads

Various Electric

connection cables

Pexiglass plate

(holder) with three

electrodes

Micromanipulator

The cockroach metathoracic leg was obtained by Dr. Lange and Dr. Orchard. The leg was

placed onto the preparation holder with 2 electrode points from the holder going through the

femur and one electrode point through the coxa. Two small pieces of tape are placed onto the

bottom and top of the leg to hold it in place during stimulation. By this time all cell phones

should be off to not create distortions in the audio amplifier’s loud speaker. The preparation

holder leads should then be connected according to its proper wiring (for example the G lead to

the G cable) to the P15 preamplifier inside the Faraday cage. The Faraday cage reduces

interference of the preparation for more accurate results. The cable is then split from the

preamplifier to the audio amplifier loud speaker and to the Oscilliscope (the PicoScope and

computer). A micromanipulator is also set up in the Faraday cage to be used for Part 3 of the

study when a continuous and prolonged stimulus is needed. The micromanipulator makes this

more possible by giving a constant and equal stimulation. After that, the setup is complete and

testing may begin.

For part 1a of the experiment, different spines located in varying positions of the tibia

from “high spine” to “mid spine” to “low spine” are mechanically stimulated. For this part of the

experiment the mechanical stimulation of the spines were done in the same direction (against the

grain) and at equal (or as close to equal as possible) pressures. The stimulation was done

manually using a glass probe. It is important to note that during all the testing it was important to

keep a hand planted on the Faraday cage to ground one’s self and thus to further block

interference. 2 trials for each area were conducted by briefly holding the spine.

For part 1b of the experiment the spine that had produced the greatest response in part 1a

was used for testing. The same spine was used throughout the testing of parts 1b, 2, and 3. In part

1b of the experiment the spine was manually stimulated in two different directions using a glass

probe. One direction was “with the grain”, manually stimulating the spine in the direction it was

facing. The other direction was “against the grain”, manually stimulating the spine in the

opposite direction that the spine was facing. The direction that produced the greatest response

would be used for part 2 and 3. 2 trials were conducted for each direction.

For part 2 of the experiment the spine was again manually stimulated using a glass probe.

This time the spine was stimulated using different pressures as done by the person conducting

the stimulus, Omar Hallouda. The three relative displacements of pressures according to Omar

Hallouda were, “Light”, “Average”, and “Heavy”. 2 trials were conducted for each displacement

of pressure.

For part 3 of the experiment the spine (to repeat: the biggest response in part 1a and in the

direction that gave the biggest response in part 1b) was stimulated for 5 continuous seconds

using the micromanipulator to give a controlled response. 3 trials were conducted.

During the all the parts of the experiment while one person is stimulating, the other

partner must be watching the PicoScope readings on the computer and recording the approximate

times of each trial. This lab was conducted by Omar Hallouda and Christina____.

Results

In experiment 1a different spines in different areas of the tibia were tested. The traces

obtained had shown greater amplitudes and thus stronger responses from the spine when it was

higher up the tibia and closer to the coxa (Figure 1). The “high” spine had maximum peaks of

100.5 mV, 96.5 mV, and 93.3 mV and an approximated average of 46.8 mV (Figure 1A). The

“middle” spine had maximum peaks of 53.0 mV, 52.6 mV, and 51.9 mV and an approximated

average of 34.2 mV (Figure 1B). The “lower” spine had maximum peaks of 48.7 mV, 34.2 mV,

and 27.3 mV and an approximated average of 14.7 mV (Figure 1C).

In experiment 1b different directions of displacement were conducted on the “high”

spine. The traces obtained reveal that displacing the spine against the grain (against the way the

spine was facing) results in a greater response than moving the spine with the grain (with the

way the spine was facing; Figure 1). Going against the grain resulted in max peaks at 74.9 mV,

74.9 mV, and 63.0 mV and an approximated average of 41.3 mV (Figure 1D). Going with the

grain resulted in max peaks at 46.4 mV, 44.6 mV, and 43.2 mV and an approximated average of

24.7 mV (Figure 1E). Additionally, it appeared that going against the grain results in a greater

frequency of action potential firing (Number of peaks / time).

In experiment 2 different displacement pressures, “heavy”, “average”, and “light”,

compared on the “high” spine going against the grain (Figure 2). This resulted in the greater the

pressure condition the greater the response. Thus accordingly, the heavy condition had the

greatest response followed by the average condition and then the light condition. The light

pressure had max peaks at 59.8 mV, 55.5 mV, and 53.0 mV and an approximated average of

34.0 mV (Figure 2A). The average pressure condition had max peaks at 100.7 mV, 90.6 mV, and

81.2 mV and an approximated average of 37.1 mV (Figure 2B). The heavy pressure condition

had max peaks at 99.6 mV, 96.4 mV, and 96.4 mV and an approximated average of 60.1 mV

(Figure 2C).

In experiment 3 adaptation of the response was studied with the manipulation of a high

spine against the grain for 5.063 seconds (Figure 4). The original peak upon initial stimulation

was 69.6 mV. The final peak upon the very end of stimulation was 56.7. The difference between

the first peak and the last peak obtained over 5.063 seconds was 12.9 mV. The peaks show a

decreasing rate of both firing and size of amplitude throughout the stimulus. The interspike

interval plot also shows the decreasing frequency. The first 5 spontaneous periods between

spikes were 39 msec, 32 msec, 48 msec, 80 msec, and 88 msec. The last 5 spontaneous periods

between spikes were 294 msec, 49 msec, 1036 msec, 613 msec, and 172 msec.

Figures

Figure 1: Amplitude (mV) over time (seconds) traces of neural responses due to mechanical

stimulation of a tibial spine on an American cockroach (Periplaneta americana) in various

locations throughout the tibia and in different directions. Traces were obtained using PicoScope.

Stimulations were done by Omar Hallouda using a glass probe going “against the grain” for

traces 1A,B,C. For traces 1D,E the stimulations were done on the “high” spine. Trace A is for a

spine located at the top of tibia (closer to the coxa; “high spine”), trace B is for a spine located in

the middle of the tibia (“Mid spine”), and trace C is a spine located at the bottom of the tibia

(“low spine”). Trace D is for a spine that was stimulated against the grain, trace E is for a spine

that was stimulated with the grain. The scale bar displayed with a black bar represents one

second (on the x-axis).

A B

C

1 sec

D

E

Figure 2: Amplitude (mV) over time (seconds) traces of neural responses due to mechanical

stimulation of a tibial spine on an American cockroach (Periplaneta americana) at various

displacement pressures. Traces were obtained using PicoScope. Stimulations were done by Omar

Hallouda using a glass probe on a “high spine” and against the grain. Pressure determinations

were determined by Omar. Trace A is for a “light” mechanical stimulation, trace B is for an

“average” mechanical stimulation, and trace C is for a “heavy” mechanical stimulation. In trace

2A, the response displayed smaller amplitudes relative to the other traces. In trace 2B, the

response displayed amplitudes in between A and C. In trace 2C, the response displayed larger

amplitudes relative to the other traces. The traces displayed are one of two trials conducted. The

red boxes have been used to make it easier to see where the responses are .The scale bar

displayed with a black bar represents one second (on the x-axis).

A B

C

1 sec

Figure 3: Interspike interval plot (Spontaneous Period [msec] over Accumulated Time [msec]) of

neural responses due to mechanical stimulation of a tibial spine on an American cockroach

(Periplaneta americana) for an extended period of time to display the effects of adaptation. Time

period between spikes increases as accumulated time increases. The trace, from which the graph

was derived from, was obtained using PicoScope and the stimulations were done using a

micromanipulator for 5.063 seconds (5063 ms).

0

200

400

600

800

1000

1200

0 1000 2000 3000 4000 5000

Spo

nta

ne

ou

s P

eri

od

(m

sec)

Accumulated Time (msec)

Discussion

Upon testing different spines in different regions of the tibia it could be easily seen that

the higher the position of the spine on the tibia (In other words closer to the coxa) the greater the

response (Figure 1). The trace revealed greater amplitudes and a greater frequency of action

potentials for the spine located higher up on the tibia (Figure 1A). As the location of the spine

was moved lower, the response was seen to be reduced with it (Figure 1B,C). Thus it can be

determined that there was a positive correlation between height of the spine (in terms of location

on the tibia) and strength of the response (in terms of amplitude and frequency). Zill and Moran

(1981) had found similar results: that the greatest responses occurred at the proximal ends of

tibia.

In experiment 1b we had tested to see if the direction of the stimulation had any effect. It

became very clear from the traces that there was a difference in response with a difference in the

direction of stimulation (Figure 2). Deflecting the spine against the grain led to a greater

response than when deflecting the spine with the grain. The results were seen as an increased

size of amplitude and an increased rate of firing. These results were similarly found by several

other studies (Spinola and Chapman, 1975; Zill and Moran, 1981). Furthermore, Zill and Moran

(1981) had not only studied these two deflections but had also studied “torques” (twisting) and

the effects of different sized caps. These variables could be an interesting future addition for

testing (Zill and Moran, 1981).

Experiment 2 was studied the effects of different displacement pressures. The experiment

had predictable results: The greater the displacement pressure the greater the response (Figure 3).

This can possibly be used to help in the escape response. When there is a greater displacement of

air creating a greater pressure it could indicate that either something is close to it or is moving

fast towards it. Thus resulting in greater action potentials that may help it react quicker and thus

escape any danger. Dethier (1963) found that with increased stimulus voltage (displacement

pressure) there was an increased response in the form of more frequent action potentials. These

results coincide with the results found in this experiment.

In the final part of the lab, experiment 3, we studied the effects of adaptation on

prolonged stimulation. In this case the stimulation was for 5.063 seconds and during the duration

of the stimulation there was a decreasing size of action potential amplitudes and a reduced

frequency of action potentials up until the last .5 seconds (Figure 4). Using the Interspike

Interval plot it can be seen even more clearly that adaptation occurs. As accumulated time

increases action potential spikes occur less frequently after the previous spike (Figure 3). This is

clear evidence for adaptation occurring. It is possible if given enough time the response may

have completely adapted to the stimulus resulting in the receptors entirely ignoring the stimulus

and in the process producing no response. Zill and Moran (1983) found that when they produced

a small force (15mg), the response stopped completely (adapted) after only 100msec.

The increased amplitudes and frequencies at the beginning and the end indicate a phasic

receptor. But there were also continuous amplitudes and frequencies throughout the stimulus

between the phasic receptors indicating the existence of tonic receptors. Thus it can be deducted

that the spines (or that spine at least) was a phasi-tonic receptor.

Appendix

Figure 5: Amplitude (mV) over time (seconds) traces of neural responses due to mechanical

stimulation of a tibial spine on an American cockroach (Periplaneta americana) for an extended

period of time to display the effects of adaptation. The trace was obtained using PicoScope. The

stimulation was done using a micromanipulator. The red line displayed the amount of time of

stimulation (5.063 seconds). The response trace displayed a decreasing size of amplitude over

the duration of the stimulus demonstrating adaptation. The response trace also clearly displays

evidence for a phasi-tonic receptor. The trace displayed is one of three trials conducted. The

scale bar displayed with a black bar represents one second (on the x-axis).

1 sec

Figure 6: Image of the cockroach leg taken from University of Toronto at Mississauga BIO409:

Lab Physiology, lab manual. For part 1a: The red arrow points at the approximate location of a

“high spine”. The blue arrow points at the approximate location of a “mid spine”. The green

arrow points at the approximate location of a “low spine”. Note that the side of the arrow is not

reflective of the side of the spine used in the experiment. All the spines that were measured were

on the same side but at varying locations. For part 1b: The purple arrow demonstrates the

direction of the stimulation for “against the grain”. The black arrow demonstrates the direction of

the stimulation for “with the grain”.

Table 1: Interspike Interval (ISI) Plot Raw Data; Instantaneous period (msec) over Accumulated

time (msec) Data from Part 3 Trace

Accumulated time

(msec)

Instantaneous period

(msec) 39 39

64 32

111 48

143 80

222 88

380 167

553 190

656 112

736 81

1142 408

1331 179

1844 519

2601 747

2909 337

3197 294

3336 49

4286 1036

4889 613

5063 172

Sample Calculation for ISI

At accumulated time 1844 msec

Difference in time it took to reach the peak at 1844 from the previous spike give the

instantaneous period

2.991seconds – 2.472 seconds These are the times the peaks occurred at

= 0.519 seconds *1000 to get milliseconds

= 519 msecs.

References

Bullock TH, Horridge A. Structure and function in the nervous systems of invertebrates. San

Francisco: Freeman, 1965.

Camhi MC, Tom W. The escape behavior of the cockroach Periplaneta americana. J. Comp.

Physiol. 128: 193-201, 1978.

Dethier VG. The physiology of insect senses. London: Methuen, 1963.

Moran DT, Rowley JC. High voltage and scanning electron microscopy of site of stimulus

reception of an insect mechanoreceptor. J. Ultra. Res. 50: 38, 1975

Randall D, Burggren W, and French K. Animal physiology. New York: Freeman, 2002.

Spinola SM, Chapman KM. Proprioceptive indentation of the campaniform sensilla of cockroach

legs. J. Comp. Physiol. 96: 257-272, 1975.

Wine JJ, Krasne FB. Organization of escape behaviour in the crayfish. J. Exp. Biol. 56: 1-18,

1972.

Zill NS, Moran DT. The exoskeleton and insect proprioception. I Responses of campaniform

sensilla to external and muscle-generated forces in the American cockroach, Periplaneta

americana. J. Exp. Biol. 91: 1-24, 1981.