Overview

1
The resting membrane potential of Drosophila
melanogaster larval muscle depends strongly on
the calcium gradient.
Jacob Krans1, Karen Parfitt2, Patricia Rivlin3,
David Deitcher3, Ron Hoy3 1Biological Sciences,
Mount Holyoke College, South Hadley, MA.
2Department of Biology, Pomona College,
Claremont, CA. 3Department of Neurobiology and
Behavior, Cornell University, Ithaca, NY.
650.13
Chemistry
Physiology
Overview Scope
The neuromuscular junction (NMJ) of larval fruit
fly, Drosophila melanogaster, has the unique
qualities of being a recognized model of synaptic
transmission. The preparation illustrates
concepts across several disciplines and gives
investigators unprecedented control of the
expression of a myriad of well-understood genes
and their products. Over the course of unrelated
studies, we noticed a trend of surprisingly
depolarized resting membrane potentials (RMPs)
when using salines containing low calcium. This
was noted in earlier work by Jan and Jan (1976),
but has not been addressed methodically since.
Moreover, although the trend is present in many
preparations, its mechanism is widely variable
and its magnitude rarely comparable to that in
fruit fly. We have characterized the dependency
of RMP upon calcium concentration (Ca). As
saline Ca increases, so does the absolute
magnitude of the RMP, yielding more
hyperpolarized potentials. The strength of the
relationship between RMP and Ca led us to
question the natural extracellular environment at
the NMJ. We thus analyzed, and report here, the
ionic composition of larval hemolymph with
particular emphasis on our use of voltammetric
techniques to measure divalent cations (Ca and
Mg).
The effect of calcium on membrane potential has
been documented in a diverse phylogeny of
preparations, but the extreme magnitude of the
effect is somewhat unique in D. melanogaster. In
fruit fly and many organisms (some are listed
below), the sign of this effect is opposite of
that predicted by permeability equations (e.g.
Goldman, 1943 Hodgkin and Katz, 1949).
The various effects of divalents on membrane
physiology underscore the importance of accurate
measures of their presence in fruit fly
hemolymph. Physiologists have exploited the
anesthetic effect of high extracellular / saline
magnesium (Mg) in crustacean preparations
for decades. This is particularly interesting in
light of the high divalent concentrations typical
of many insects including several Lepidoptera,
Coleoptera, and Hymenoptera. Duchateau et. al.
1953 offers an exhaustive review of hemolymph ion
concentrations. It has been reported that
laboratory strains of D. melanogaster which
consume primarily yeast have high divalent ion
concentrations (Begg and Cruickshank, 1963).
Additionally, the ratio of Ca to Mg has
also been implicated in membrane stabilizing
effects on resting membrane potential (Stefani
and Steinbach, 1969). The ionic composition of
larval hemolymph has never been measured in
non-diluted / pure form, and only rarely in
dilution. Also, we have measured Mg in
larval hemolymph, which has not been reported
using modern voltammetric techniques.
Muscle Rat Thelsleff and Ward, 1975 Rabbit
Guinea-Pig Pholpramool and Korppaibool, 1977
Bolton, 1972 Sheep and Cow Reuter and Schultz,
1967 Frog Bulbring et. al. 1956 Luttgau, 1963
Apter and Koketsu, 1960 Worm Brading and
Caldwell, 1971 Neurons Squid Frankenhauser and
Hodgkin, 1957
Method 1 Centrifugation of lesioned larvae
(left, top). A superficial cut was made in the
bodywall of the third abdominal segment. Lesioned
larvae were placed on a filter within a
centrifuge tube. Samples were spun at 3000 G.
Filtered (10 nm CO) hemolymph was analyzed using
ion selective, voltammetric equipment.
Introduction
The larval NMJ preparation of fruit fly is
simple, anatomically repetitive, and provides
large EJPs suitable for quantitative analysis.
ABOVE RMP vs. saline Ca. Data are
equivalent on both sides, but are plotted on a
linear scale on the left and logarithmic scale on
the right. Data collected in SSA A logarithmic
model is used to fit the data and gives an r2
value of 0.97. The slope of the relationship,
when using either a logarithmic or linear model
(r2 0.88), is 7.9 mV RMP / 1 mM Ca. Use of
HL-3 saline (open squares) yields more
hyperpolarized potentials than SSA and an
increased slope (9.5 mV / mM). The relationship
between Ca and RMP, in HL-3, is not as well
fit by a logarithm (r2 0.91).
Method 2 Microcapillary collection (left,
bottom) has been described (Stewart et. al.,
1994). Briefly, microcapillary tubes were placed
against small incisions made in the bodywall of
3rd instar larvae, as above. Between 0.5 and 1
ul of hemolymph was gathered from each larva
using this technique, which was immediately
ejected into an iso-osmotic volume of dilutant.
RIGHT The bodywall musculature of a larva.
Anterior is shown at top, nerves are black
FAR RIGHT A single segment showing the segmental
nerves and their specific innervation. By
severing the nerves, spontaneous activity is
abolished and single potentials can be evoked by
electrical stimulation of the nerves via suction
electrode. The muscles recorded from in this
study are 6, 7, 12, and 13.
We analyzed two types of hemolymph sample (1)
Non-diluted, pure, samples were gathered from
thousands of larvae. (2) Hemolymph was diluted
to final concentrations between 110 to 120.
In many preparations, a RMP in the range of -50
to -70 mV is one of several metrics used to
determine the viability of a particular
recording. Though this is somewhat of an
unspoken axiom, the occasional reference is made
in reports (Xing et. al. 2005). An RMP of -35 mV
might be considered too depolarized in most
common NMJ preparations. In larval fruit fly,
however, data from these muscles in very low
calcium may be physiologically normal.
Discussion
ABOVE EJP amplitude (voltage difference) is
plotted as a function of saline Ca. Once
again, the left panel provides a linear scale,
and the right, a logarithmic scale. Data are
well fit by a sigmoid (not shown) function based
on a 3 variable Hill equation. SSA 50 EJP
amplitude Ca is achieved using this model
at 0.51 mM. Although the 50 saturation point
observed in HL-3 (open squares) is unchanged, the
slope of the fit is greater, suggesting increased
sensitivity to Ca.
Increasing available extracellular Ca yields
a substantial hyperpolarization of RMP (in SSA
8 mV / 1 mM Ca). This effect appears to be
mediated by the muscle, rather than the
presynaptic motoneurons. Dr. Robin Coopers
laboratory has reported that the pathological
change in presynaptic Ca that is
characteristic of cacophony mutants (cacTS2) has
little or no effect on muscle RMP (Xing et al.
2005). Non-voltage clamp recordings of larval
muscle RMP and EJP amplitude should be considered
relative to saline Ca, regardless of the
saline chosen. One means for such consideration
for EJPs comes from the McLachlan and Martin
equation (1981). Results from this study suggest
that saline divalent concentration must be
considered carefully when characterizing the
physiology of Drosophila mutants, particularly
those with neuromuscular excitability phenotypes.
As RMP shifts with Ca, so does EJP amplitude.
However, the functions these relationships
follow are different, which has important
implications to analyses of evoked potentials.
RIGHT Membrane potential is recorded from a
single muscle as saline Ca increases from 0.5
to 1.5 mM. RMP hyperpolarizes about 5 mV, but
the peak of the EJP shifts less than half that
2.3 mV. This phenomenon supports the use of
scaling equations such as McLachlan and Martins
(1981).
ABOVE Membrane potential of larval muscle 6 is
plotted versus time. Saline containing 0.5 mM
Ca is replaced with 3.0 mM Ca saline at
25 s. At 150 s, the preparation is returned to
3.0 mM Ca saline. Two commonly used salines
are shown BLUE Standard Saline A (SSA Jan and
Jan 1976), and RED Hemolymph-Like saline 3
(HL-3 Stewart et. al. 1994). Mechanical
artifacts indicate the exchange of solution.
Each exchange was accomplished over several
washouts.
This work was supported by a Professor Award to
RRH from HHMI and faculty support from Mt.
Holyoke College (JLK) and Pomona College (KDP).