Mechanical Stimulation of Tibial Companiform Sensilla of a Periplaneta americana Leading to Varied...
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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.