Transcript Development of a sensitive force transducer for use with larval

Development of a sensitive force transducer for use with larval Dipteran muscle
Julia Dettinger, Stephanie Albero, Jacob Krans
Department of Biology, Mount Holyoke College, South Hadley, MA 01075
Physiology
Electronics
Overview: We are interested in measuring forces in the larvae of flies.
The reduced size of muscles in these preparations required the
development of a particularly sensitive force transducer. Using a simple
Wheatstone Bridge circuit and semiconductors - variable resistance
elements - we achieved sufficient sensitivity for our investigations. Our
broad goal is to investigate hysteresis in the motoneuron (MN) rate –
force relationship of larval fly muscle as part of a comparative analysis.
It has been demonstrated that hysteresis can result in force generation
greater (or lesser) than that predicted by a monotonic relationship
between MN rate and force in several common arthropods (Blaschko et
al., 1931; Wilson and Larimer, 1968).
Adapted from Wilson and Larimer (1968,
PNAS), hysteresis is described in two
crustacean and one insect preparation(s). The
authors describe hysteresis in the following
passage:
If a brief high-frequency train of impulses is
added to the low-frequency background,
then the muscle may contract to a new
tension plateau from which it does not fully
relax for long periods after the return to low
frequency stimulation (1968).
Hysteresis: A muscle that is activated by equivalent stimulus rate and
strength may generate profoundly different forces under different
conditions. Factors that contribute to inequalities in the MN rate – muscle
force relationship include pre- and post-synaptic changes at the
neuromuscular synapse as well as intramuscular dynamics such as
hysteresis. The latter category of factors remains somewhat poorly
understood. Use of a model organism, one with a clear behavioral
context, may elucidate common principles of hysteretic activation and
control. The final stage of larval development in many flies is
characterized in part by increased locomotion, providing a useful context
for results.
Methods
Force measurements were made in the third
larval instar of two species, Sarcophagidae
and Calliphoridae. Flies were reared using
housefly medium supplemented with liver,
and were kept at room temperature. A
single, mid-line, longitudinal incision on the
dorsal side of the larvae exposed the guts,
which were subsequently removed to
expose the ventral ganglion and nerves.
Plexiglas tabs with hooks were glued to both
ends of the animal. The posterior hook was
attached to the transducer whereas the
anterior was immobile. The ventral ganglion
was severed, and a suction electrode was
used to gather segmental nerves. Brief
pulses from a Grass stimulator drove activity
in the nerves. Stimulus voltage was set to
~150% of threshold. The preparation was
maintained at 20 °C in standard insect
Ringers and data were collected within 45
minutes of dissection.
A
B
Silicon wafers. (A) semiconductor
production in a clean-room (after Apanius
et al., 2001). Silicon edges were etched
rather than cut for improved continuity in
molecular geometry. Semi-conductors
used in these experiments were printed in
one batch, to ensure equal gage factors
and temperature compensation. (B)
Wheatstone Bridge circuit diagram.
Physiological results indicate that Sarcophagid and Calliphorid larval
muscles behave like most arthropod slow muscle. Force production
increases with MN rate but saturates at relatively low stimulus rates.
Moreover, there is magnitude-dependent fatigue at rates above ~25 Hz.
Semiconductor Wheatstone Bridge: The force transducer is designed
from two pairs of silicon wafers (4 total semiconductors) mounted on
opposite sides of a cantilever. In our application, semiconductors act as
variable resistors in a Wheatstone Bridge circuit (Allen et al., 1980).
The resistance of silicon changes as a function of torsion. Thus, voltage
passed through the circuit is proportional to the torsion applied across
the cantilever. The final gauge factor of the device exceeds 400.
Coiled Foil Strain Gauge: We began with a simpler force transducer
design utilizing “coiled foil” strain gauges which are readily available.
Despite placing 15 foil strain gauges in series this design proved
insufficient for our purposes. The nature of the foil device’s gauge factor
(each ~1.4) necessitated extensive bending: several larval body lengths
more than was feasible. The characterization curves for both coiled foil
and semiconductor strain gauges are given below:
Images of the force transducer. (A) the placement of semiconductors (two per side; tension vs.
compression) in relation to the cantilever on the transducer. (B) an individual semiconductor,
flanked by two copper leads for soldering connections. (C) a close up of the semiconductor
itself showing the fine copper strand attachments to leads shown in (B). Scale bars in (A) and
(B) represent 3.30 mm and in (C), 1.65 mm.
(A) Raw data traces from stimulus rates: 1, 10, 25, and 50 Hz. (B) Signal averaged data (10
trials each) of force produced at varied stimulus rates (3, 5, and 40 Hz).
Force increases with stimulus
rate. Peak force during a threesecond stimulus train increases
with stimulus frequency and
plateaus at frequencies around
25 Hz (n = 6 animals). Values
are normalized to the peak force
observed from any one animal,
irrespective of stimulus rate or
trial. Mean +/- standard error is
shown.
Force (Normalized)
Introduction
Preliminary data illustrating hysteresis. In black, nerves
were stimulated at 10 Hz for two seconds. In contrast, the
blue trace represents trials during which nerves were
stimulated at 60 Hz for 500 ms followed by a 10 Hz train
identical to above (black). There was a decrease in
steady-state / plateau force produced during trials
containing a high frequency, hysteretic burst. Each trace is
an average generated from 10 repetitions.
Future Directions
Larval Sarcophagid anatomy. (A) a filettype dissection of the larvae (see text).
(B) a rough schematic of the connections
between segmental nerves and
musculature for visualization only.
Muscles not drawn to scale.
Characterization curves. (A) Calibration for a transducer consisting of 15 foil strain gauges in
series. The left axis shows displacement of the sensor required for resistance / voltage
change. The right axis gives the resultant voltage change. (B) shows similar data from the
semiconductor force transducer, which required nominal bending (<<1 mm, data not shown).
An averaged force trace (20 trials) in
Sarcophagid larvae muscle. A single
electrical impulse was delivered for
computation of rise and decay rate. In
this example, the time constant (tau) of
ascending force was 168.2 ms, and
descending tau was 772.1 ms.
- Two designs for improving signal to noise ratio of our transducer(s) are
in the works: (1) a Wheatstone Bridge consisting of eight
semiconductor wafers; four in compression and four in tension. (2) A
custom instrumentation amplifier with high gain and offset adjustment.
- We will continue characterization of the rate-force relationship in these
two species as well as in Musca and Drosophila.
- An essential component of this research is the measurement of
changes in voltage from the muscles. We have just begun this effort.
- Improved control of muscle activation may be gained by stimulation of
single segmental nerves as well as ablation of surrounding
musculature.
Acknowledgements
We thank Gretchen Sminkey for fly maintenance, Cole Gilbert for expert advise on rearing
media, and Bill Chapple for assistance with strain gauges. This work was supported by a Mount
Holyoke College Faculty Grant (JLK) and the Abby Howe Turner Award (both SJA and JCD)