Sensory adaptation: extracellular
recording
locust wing hinge stretch receptor
R. MELDRUM
Department
ROBERTSON
Queen’s
of Biology,
University,
Kingston,
Robertson, R. Meldrum. Sensory adaptation: extracellular
recording from locust wing hinge stretch receptor. Am. J. Physiol. 263 (Adu. Physiol. Educ. 8): S7-Sll,
1992.-Good student
laboratory exercisesthat do not require much manipulative or
technical expertise of the student and that have minor equipment demandsare hard to find. One experiment that hasthese
desirablecharacteristics is the description of adaptation of the
firing frequency of the locust forewing stretch receptor after
elevation of the wing. Unambiguousrecordingsof the activity of
the stretch receptor can be madeusinga simplemonopolarhook
electrode inserted into the thoracic cavity of a decapitated locust. Elevation movements of the forewing are simple to perform and measure.The responseof the stretch receptor as a
function of time and the stimulus history is monitored. Within
a relatively short time it is possibleto collect enough data to
characterize thoroughly the adequatestimulus of a single sensory neuron. There is considerablescopefor student innovation,
and several important concepts of sensory physiology can be
discussed.
teaching; student laboratory; proprioception; mechanoreceptor;
insect
A BRIGHT AND DEDICATED
student can become
daunted and dispirited by laboratory exercises that require excellent manipulative
skills and/or the use of
much highly sophisticated
equipment
that can take
months to learn to operate effectively. Too often students have to stand and watch while a teaching assistant
fiddles with the setup and extols the virtues of the particular preparation
that has again failed to work. Yet
without such attributes of difficulty, many physiology
laboratories,
especially those investigating
phenomena
at the cellular level, can become trivial and boring. What
is required is an exercise that illustrates important physiological principles, requires little exotic equipment, can
readily be set up by the most ham-fisted individual, uses
a preparation that is cheap, robust, and long-lived, and
provides a good scope for extended data collection and
independent inquiry. Several of these requirements can
be met by using invertebrate
preparations
(4), and I
describe here a preparation
of the locust that investigates the sensory physiology of a single proprioceptive
neuron responsive to elevations of the forewing and
that, to my mind, provides an excellent student laboratory exercise.
The preparation
was first described to me by A. N.
Spencer (Department of Zoology, University of Alberta).
I subsequently incorporated it into a laboratory course
in Neurobiology
offered in the Department
of Biology,
McGill University, which I taught from 1984-1988, and
into the laboratory component of a course in Comparative Animal Physiology offered in the Department
of
Biology, Queen’s University,
which I have taught since
1990. It has been a successful addition to these courses.
It has the advantages of needing little equipment while
enabling the activity of a single proprioceptive
neuron to
be monitored with ease. Natural stimulation
of the neuEVEN
from
Ontario K7L 3N6, Canada
ron is straightforward
and easily quantified. Moreover,
the preparation is robust and can last for 12-24 h without apparent deterioration.
Indeed a single preparation
has been known to serve for three different 3-h laboratory sections (morning,
afternoon, and evening) of a
class.
I provide here a version of the student handout that I
have used, some examples of the results that can be
obtained, and some hints that may be useful.
THEHANDOUT
Introduction.
Movements and deformations of each of a locust’s wings are monitored by several senseorgans located either on the wing or at the wing hinge. The function of the sense
organsis to compensatefor externally imposedperturbations of
wing movement during flight and for internal variations in the
motor system (2). They are also involved in generating the
motor pattern by controlling the phaseof activity of different
musclegroups (10) and, more importantly, by controlling the
wingbeat frequency in a cycle-by-cycle fashion (11, 15, 17). One
of these senseorgansis the wing hinge stretch receptor (SR). At
each wing it comprisesa single multipolar sensory neuron innervating a connective tissuestrand (6). This strand spansskeletal elementsat the wing hinge that move relative to eachother
as the wing is elevated or depressed.The strand is stretched by
elevation of the wing, causingan increasein the rate of firing of
the sensoryneuron (6,9). This information is transmitted to the
central nervous systemvia the axon that runs in nerve 1 of the
mesothoracicganglion (forewing SR) or the metathoracic ganglion (hindwing SR) (16). The axon of the forewing SR has
extensive branching throughout the three thoracic ganglia
where it has monosynaptic connectionswith motor neurons(1,
12) and interneurons (14) in the flight system. Although the
dynamic responsecharacteristics of this senseorgan, i.e., to
sinusoidalmovements of the wing at approximately the wingbeat frequency (7, 8), are possibly of greater significanceto the
animal, in this experiment you will be investigating the static
responsecharacteristics of the senseorgan to passively imposed
elevations and depressionsof the wing (9).
Preparation.
You will be provided with a locust. First remove
the legsby cutting them off at the level of the coxae. Next cut off
the head. Grasp the gut with a pair of forcepsand pull as much
as possibleout of the animal. The aperture must be cleared of
obstructing air sacs.The animal is then set up as depicted in
Fig. 1. Fix the locust to the stand provided @/&in. rod on a
magnetic base) using Plasticene (or wax). Ground your preparation with a silver wire inserted into the abdomen and connected to the ground socket of the preamplifier. You are provided with a simpledevice for moving a forewing and measuring
the anglethrough which it has beenmoved. Clamp this onto a
stand and arrange it sothat the axis of the rotating arm is in a
straight line with the hinge of one of the forewings,the protractor is at right anglesto the longitudinal axis of the locust, and
the 90” line of the protractor is horizontal. Carefully extend the
forewing that you have chosento use. Place it over the narrow
wing support that protrudes from the arm, and fix it in place
with a narrow piece of Scotch tape.
It is important to ensurethat the locust and the device are
lined up correctly so that movementsof the arm smoothly elevate and depressthe wing without twisting it or bending it
1043-4046/92 $2.00 Copyright 0 1992 The American Physiological
Society
S7
S8
LOCUST
STRETCH
RECEPTOR
Fig. 2. Diagram illustrating recording position. The axon of stretch
receptor travels in nerve 1 of mesothoracic ganglion (meso Nl; see Ref.
3 for anatomy) from base of forewing to ventral nerve cord. Electrode
has to be placed close enough to this nerve to pick up the extracellular
field potentials of stretch receptor action potentials.
how exactly doesthe SR respond to such elevation? Does the
responseadapt? Does it habituate? Can you document this
graphically? What information is coded for in the response?
Fig. 1. Diagram of experimental setup. Decapitated locust is mounted on
a rod such that a silver wire electrode can be inserted into thoracic
cavity through neck. Forewing is extended laterally from thorax and
fixed to arm of device that can elevate the forewing through measured
angles. Animal is grounded via a silver wire inserted into abdomen.
unduly (this will excite other receptors causing reflex motor
activity that will obscurethe extracellularly recordedSR spike),
and so tha.t a reasonablyaccurate measureof the anglethrough
which the wing has been moved can be read off the protractor.
To record activity from the nerve trunk that contains the
axon of the forewing SR (mesothoracicnerve I), you will usea
monopolar silver wire hook electrode. The glasstubing around
the silver wire servesto support it and insulate it to someextent.
Try not to break it. The electrodeshouldbe connectedto the Gl
input of the preamplifier. Becauseyou are recording with a
monopolar electrode, the amplifier is used in its single-ended
configuration, and the G2 input is connectedto ground.‘A connecting loop is provided for you to do this. The output from the
amplifier is connected to one of the channels (AC coupled) of
the oscilloscopeso that the voltage signalscan be displayed.Fit
the rod holding the electrode into a micromanipulator, and arrange things so that you can extend the electrode horizontally
into the body cavity of the locust. The procedure now is to
search for SR activity using the electrode. Figure 2 will give
someidea of where you should direct your search.
Move the electrodelaterally onto the musclesof the wing- you
are working with. Is there activity? (The SR is usually firing
tonically at - 10 impulses/swhen the wing is extended straight
out from the body at OO.)If not, then move the electrode.Now
is there activity? If not, move the electrode again, etc. From
time to time you could elevate the wing to stimulate the SR in
caseit is not firing spontaneously(this is rare). Set the preamplifier to amplify at xl00 with the high- and low-frequency
cutoffs at 10 kHz and 300 Hz, respectively. This will give you a
better chance of seeingthe SR spike. The extracellularly recorded action potential (Fig. 3) is triphasic (Why?). Once you
have found the nerve and are recording large enough SR spikes
(i.e., about 3 times the amplitude of the backgroundnoise), you
can prevent the animal from desiccating by building a wall of
Vaseline-mineraloil mixture at the entrance to the body cavity.
This is done with a syringe and a hypodermic needle.Be careful
not to knock your electrode out of position. The preparation
should now last for as long as you need it. The wing hinge is
reasonably robust; however, you should not try to elevate or
depressthe wing much past the 45” from horizontal positions.
Procedure. You are now in a position to investigate the responsecharacteristics of the SR. Starting with the knowledge
that the adequatestimulus for the SR is elevation of the wing,
SAMPLE
RESULTS
If the electrode is close enough to nerve 1 it is possible
to record SR spikes with little or no ambiguity. It is the
largest sensory axon in the nerve trunk, thus having a
relatively large extracellularly recorded spike, and it usually fires at a fairly constant frequency (- 10 impulses/s).
Activity of the dorsal longitudi nal motor neurons, which
have axons in the same nerve, wil 1 have larger extracellular sp .kes, bu t these motor neurons are rarely spontaneously active. A convenient method of measuring the
frequency of firing is to trigger the oscilloscope sweep
internally from the SR spike and measure the interspike
interval (ISI) [ l,OOO/ISI (in ms) = instantaneous
frequency (in impulses/s)]. For this experiment the measure
is of spike frequency while the stimulus parameters are
elevation angle, time after stimulus, and stimulus history.
Elevation of the forewing causes an immediate and dramatic increase in the frequency of firing of the SR that is
dependent on the extent of elevation (Fig. 4). The initially high frequency adapts over 2-4 min (Fig. 5) to a
final frequency that is characteristic of the maintained
angle. These results are depicted graphically in Figs. 6
and 7. Pabst (9) describes two periods of adaptation: during the first second of activity and during the subsequent
few minutes. Without specialized equipment it is not possible to document the initial rapid adaptation. Depression
of the forewing from 0” (horizontal) has little effect on
firing frequency (not shown). More interesting is that a
return to 0” from elevated angles results in variable periods of silence of the SR (e.g., 2 min on return from 40”, 1
Fig. 3. Extracellular recording of an action potential of the stretch receptor. Note that it is triphasic with a major negative peak (-0.3 mV)
because of the nature of the monopolar recording in unrestricted extracellular space.
LOCUST
STRETCH
s9
RECEPTOR
Fig. 4. Activity of stretch receptor after
elevation of forewing. Each trace presents 200-ms sample of firing of stretch
receptor -2 s after elevating forewing
from 0” position (extended horizontally
from thorax) to 10,20, 30, and 40”. Each
trace has been triggered at position of the
only action potential recorded in bottom
trace. Note the increase in frequency as a
result of increasing the angle of elevation.
10" /
O.lmV
J
2Oms
min on return from 30”, etc.; not shown) followed by a
gradual return to the resting frequency. Enterprising students could investigate 1) hysteresis effects on the fully
adapted firing frequencies of different angles of elevation,
2) whether equal increments of elevation cause the equal
increments of initial firing frequency independent of position in the range, 3) whether the rate of elevation affects
final frequency, or 4) whether the SR continues to fire if
the wing is folded.
TIPS
AND
SUGGESTIONS
The major difficulty in setting up this experiment is
finding the correct position for the recording electrode. It
is essentially a blind search with little indication that one
is getting closer or further away. Nevertheless, it is my
experience that with some patience most students will get
a satisfactory recording within ~0.5 h. It is good to remember that the locust is bilaterally
symmetrical
and
that after searching for a while on one side it is a relatively simple matter to switch to the opposite side. I have
also found that a small proportion of animals yield nothing in spite of the most dedicated efforts. It is better to
discard these and dissect a new animal than to waste time
with recalcitrant specimens. The size of the signal can be
improved by gently drying the interior of the thorax with
a Kimwipe before inserting the electrode. This mitigates
the problem of shorting the electrode to ground through
the hemolymph.
Any motor activity picked up either in the nerve trunk
or as electromyographic
signals from active muscles will
mask the SR activity. This can be a problem in two ways.
First, some animals are particularly
sensitive and will
attempt to fly, especially in response to auditory stimulation (sudden, high-pitched
noises, such as hissing or
rustling, in the laboratory). This problem can be alleviated by ablating the ears. Second, in some preparations,
Fig. 5. Adaptation of activity of stretch
receptor after elevation of forewing from
0 through 40”. Each trace presents a
100-ms sample of firing of stretch receptor ~5, 10, 15, and 30 s after elevating
the forewing. Each trace has been triggered at position of the first action potential recorded in bottom trace.
0.1mV
10ms
SlO
LOCUST
v
l
v
cl
&
7
W
g
-
10
20
degrees
degrees
-
30
40
degrees
degrees
STRETCH
50
E
LLc-7
7E
25
0
0
1
I
I
I
I
I
I
1
30
60
90
120
150
180
210
240
TIME
(seconds)
Fig. 6. Adaptation
of firing frequency
(in Hz, or impulses/s)
of stretch
receptor
after elevating
forewing
through
different
angles. Adaptation
is
complete after -3 min. Note the initial high rate of firing, which decays
to a final value that is dependent
on angle through
which wing is
elevated.
XT
I
75
w
z
7
W
c-7
7-
E
RECEPTOR
slide over the support rather than deforming it or the
wing hinge.
In more than one-half of the preparations that I have
witnessed, maintained elevations ~40” will cause a rapid
diminution
in the firing rate of the SR to values around
the resting value or even to zero firing rate. It seems as if
the geometry of the strand and hinge is such that at
elevations MO” the strand suddenly becomes relaxed to
relieve all stretch in it. Doing this has never appeared to
damage the organ, and subsequent performance has been
normal.
I have always used specimens of Locusta migratoria.
However, I am sure that any species of large locust or
grasshopper will work equally wel1.l Crickets and cockroaches undoubtedly have SRs; however, I have not tried
to use them, and I would be hesitant to do so because of
the relative lack of open space in the thorax in which to
search with the electrode. Furthermore, I have no knowledge of whether the organs in these other insects have a
spontaneous firing rate; such tonic firing makes the
search quicker.
The setup can easily be made slightly more sophisticated in two ways. First, movements of the forewing
could be driven using a pen motor and a function generator. This would allow one to investigate the dynamic
properties of the SR in a controlled fashion, for example
by moving the forewing sinusoidally at about the wingbeat frequency. Second, it is easy to mount the protractor and elevating arm on the spindle of a variable
resistor. Wiring this to a battery allows the elevation
angle to be monitored as a voltage drop across the variable resistor.
Pfau et al. (13) have shown that the response of the SR
is temperature
dependent. Also it is well known that
locusts are poikilothermic
and have little control of thoracic temperature
during flight. Indeed, during flight
thoracic temperature
can often exceed ambient by as
much as lO”C, and this affects the wingbeat frequency
(5). Investigation
of the effect of temperature on firing
frequency and adaptation rates would be relatively simple
25
0
ELEVATION
(degrees)
Fig. 7. Initial
(after 5 s) and adapted
(after 2 min) firing frequency
of
stretch receptor
as a function
of angle through
which forewing
has just
been elevated.
Activity
of stretch receptor
contains
information
about
movement
and position
of forewing.
raising the wing causes reflex motor activity. This is usually because the wing and lifting device are not arranged
correctly and raising the wing is also bending, twisting,
pushing, or pulling it. Numerous other sense organs will
be stimulated by this and generate the reflexes. Proper
care and attention to the alignment of the preparation
usually avoids this problem. Another way is not to stick
the wing to the support with the tape but simply to use
the tape to provide a channel that the wing can slide
through. Thus slight misalignments
will cause the wing to
l The locusts I have used were obtained
from breeding
colonies
of Locusta
migratoria
maintained
for research
either by myself
at
McGill
University,
Montreal,
Quebec, or by Dr. G. Wyatt
at Queen’s
University,
Kingston,
Ontario.
I have no knowledge
of the difficulty
or
ease of obtaining
a supply of locusts or other large grasshoppers
outside of North
America.
Within
North
America,
as far as I am aware,
the usual biological
supply companies
do not supply live grasshoppers
or locusts. This leaves the options
of catching
wild specimens
of appropriate
species when they are available,
obtaining
specimens
from
established
breeding
colonies used for research,
or establishing
a small
breeding
colony for teaching
purposes.
In the area around
Kingston,
Ontario,
a suitable
endemic
alternative
would be the Carolina
locust,
Dissosteira
Carolina.
However,
this is available
only during
the late
summer
months,
and Canadians
with limited
needs would be better
advised to contact someone with a research
colony of either L. migratoria or one of the Schistocerca
species. L. migratoria
is not available
in the United
States and cannot
be imported.
However,
there are
numerous
research
colonies
of Schistocerca
scattered
throughout
the
United
States. The best way to find them may be to contact
the
authors
of publications
using the desired species. Modest
quantities
will usually be supplied
willingly
from these sources. For larger numbers it may be worthwhile
establishing
a small breeding
colony with
some seed stock, assuming
the proper
authorization
can be obtained.
Such a colony is relatively
easy and inexpensive
to maintain
and has
the advantage
that the locusts can be used for numerous
other laboratorv
exercises.
LOCUST
STRETCH
using a 250-W heat lamp (supplied for chick brooders)
and a small thermocouple inserted into the thorax.
CONCLUSION
In my opinion this is an excellent laboratory exercise
because it fulfills most of the criteria outlined in this
paper’s introduction.
Much can be done with it, and the
handout can be tailored to suit particular levels of student. I have tended to use limited direction of procedure
so that the students have to think more about doing an
experiment to discover how the organ works rather than
completing
a list of instructions.
The latter approach
could be used for less advanced students.
I thank Andy Spencer for suggesting
this laboratory
exercise to me
many years ago. I also thank Chris Gee and Jack Gray for their comments on a previous
version
of this manuscript.
The author’s
research
on locust flight is funded by the Natural
Sciences and Engineering
Research
Council of Canada and by the Faculty
of Graduate
Studies and Research at Queen’s University.
An abstract of this material
has been published
(Sot. Neurosci.
Abstr.
17: 516, 1991).
Address reprint
requests to R. M. Robertson.
Received
29 June
1992; accepted
in final
form
13 August
1992.
REFERENCES
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M. Monosynaptic
connexions
between
wing stretch
receptors
and flight motoneurones
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189-219,
1975.
2. Camhi,
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Sunderland,
MA: Sinauer
Associates,
1984, p. 345-353.
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J. I. The anatomy
of the nervous
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of Locusta migratoria
migratorioides
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RECEPTOR
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J. A., and R. M. Robertson.
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dependency
of wing-beat
frequency
in intact and deafferented
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II.
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and
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and
flight motor neurones
in the locust revealed
by double cobalt labelling
for electron
microscopy.
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and B. Mohl.
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in the locust. J. Comp. Physiol.
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D. N., and K. G. Pearson.
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