/ . Embryol. exp. Morph. Vol. 23, 2, pp. 311-22,1970
Printed in Great Britain
311
Electrical properties of the slime mould grex
D. R. G A R R O D , 1 J. F. PALMER 2 AND L. W O L P E R T 3
From the Departments of Biology as Applied to Medicine and
Physiology, Middlesex Hospital Medical School
An electrophysiological investigation of the migrating grex of the slime mould,
Dictyostelium discoideum, has been carried out with two aims in view. It was
hoped to obtain information which would be relevant to, first, the formation
and regulation of cellular pattern in the grex, and secondly, the problem of
grex movement.
During migration the grex develops a simple, linear cellular pattern. The cells
at the front become the so-called 'prestalk' cells which will form the stalk of the
fruiting body while those at the back become 'prespore' cells and form spores
at culmination (Raper, 1940; Bonner, 1944; Bonner & Slifkin, 1949). Moreover,
this cellular pattern is capable of polarized regulation. Raper (1940) has shown
that portions isolated from the front or back of the grex are capable of forming
normally proportioned fruiting bodies.
A number of workers have suggested that bio-electric potentials may be involved in regulation of linear cellular pattern. For example, regeneration in the
hydroids Obelia (Lund, 1923) and Tubularia (Rose, 1966), and the turbellarian
Dugesia (Marsh & Beams, 1952), can be inhibited by externally applied electric
currents and it has been postulated that extracellularly maintained electric
currents may be involved in pattern formation in these organisms (see also
Barth, 1955). Moment (1946) has postulated a similar involvement of extracellular potentials for the control of segment number in the earthworm. Rose
(1957, 1967) has claimed that specific regional inhibitors may be involved in
regulation in Tubular ia and that these are transported from cell to cell in a
polarized bio-electric field. Since polarity would seem to be an extremely
important factor in pattern formation (Wolpert, 1968), it is essential to know
whether bio-electric potentials are involved in its maintenance. In general there
are very few reliable data to enable one to assess the role of bio-electric potentials
in developing systems. The cellular pattern in the grex is morphologically simple
1
Author's address: Department of Biology, Princeton University, New Jersey 08540,
U.S.A.
2
Author's address: Department of Physiology, Middlesex Hospital Medical School,
London, W. 1.
3
Author's address: Department of Biology as applied to Medicine, Middlesex
Hospital Medical School, London, W.l.
312
D. R. GARROD, J. F. PALMER AND L. WOLPERT
and we therefore considered this a very convenient organism for re-investigation of the problem of electrical potentials in pattern formation and regulation.
In addition it seemed possible that a potential difference might be involved in
controlling the polarity of grex movement.
Our observations have put an upper limit on the possible value of a potential
difference between the ends of the grex and have provided information which
may be relevant to the synthesis and properties of the slime sheath which
surrounds the grex and which may be important in controlling its movement
(Garrod, 1969).
MATERIALS AND METHODS
Grex were grown on 2 % agar containing 0-01 % glucose, 0-01 % bacterial
peptone, and buffered to pH 6-9 with 01 M phosphate buffer. Slime mould
spores and about four drops of a suspension of Escherichia coli were spread
together over the surface of the plates which were then incubated at 22 °C for
about 65 h.
n
JI>'
n
AAA
El,
/VWVWWVXA ^
R grex
Fig. .1. Circuit diagram showing method of measuring grex resistance, Rgrex. Square,
constant current pulses are delivered at one end of the grex through a stimulating
electrode EIx of resistance Rv The size of the pulse is measured at points along the
grex by means of recording electrode, El2. El3 is a low resistance electrode connected to earth through a current measuring resistance, R2, in this case 100 kD. The
voltage developed across R2 is measured by a recorder (not shown in the diagram)
connected in series with R2.
The experimental arrangement for measuring resistance involved the use of
three extracellular micro-electrodes. Square, constant current pulses were
delivered at one end of the grex, the other end being earthed. The size of the
pulse was measured at several equally spaced points along the grex with another
electrode so that the grex itself was effectively treated as a potentiometer
Slime mould electrophysiology
313
(Fig. 1). Conventional 3 M-KCl-filled glass micro-electrodes of resistance
ca. 20 MÛ were used for stimulation and recording. It was necessary to use an
earth electrode with as low a resistance as possible in order to obtain sufficiently
sensitive readings since the resistance of the earth electrode formed the lower
part of the potentiometer. Resistances of 1-2 MQ could be achieved by breaking
the tips from high-resistance KCl electrodes; these, however, tended to leak
KCl into the preparation. In order to overcome this difficulty glass electrodes
having a large tip diameter were filled with 2-5 % agar in Bonner's salt solution
(Bonner, 1947) and in this way low-resistance, non-leaky electrodes could be
consistently produced. (The main difficulty in preparing agar electrodes was to
ensure that the tip of the electrode remained full when the agar solidified.)
The resistance of the grex was determined in two ways.
(1) The difference in the size of the voltage pulses at each end of the grex
represents the voltage drop which is produced along the grex by a known
current. The resistance of the grex can then be calculated from Ohm's law. The
current was monitored by measuring the voltage developed across a standard
resistance in series with the earth electrode. In these experiments, a resistance of
100 kQ was used so that a voltage of 1 mV developed across this resistance
corresponds to a current of 10~8 amps.
(2) When the 100 kQ current measuring resistance is switched out, the voltage
pulse across the grex will be reduced by an amount equivalent to 100 kQ. The
resistance of the grex can, therefore, be measured by comparing the voltage drop
along the grex with the known increment equivalent to 100 kù, as this current
measuring resistance is in series with the grex and as the current flowing remains
constant throughout. This method is less sensitive than the first and was used as
a check only.
As will be shown later, the resistance of the grex on agar increases with
exposure to air. It was therefore necessary to prevent drying while resistance
measurements were being made and also to surround the grex with a high resistance medium to prevent any short circuiting of the preparation as might
occur on nutrient agar which has a resistance of ca. 30 k£l per mm. Grex were
therefore transferred into paraffin oil for these measurements: they are able to
continue migration for some time in this medium (Garrod, 1969). Later experiments were performed with grex on agar, specifically in order to investigate
changes in resistance with exposure to air.
Potential difference measurements were made both with grex in paraffin oil and
on agar. For these experiments two KCl filled micro-electrodes were used, one
to earth the preparation and the other as an exploratory recording electrode.
Potentials which were measured represent the potential difference between the
two electrodes.
Initially it was hoped to be able to measure the degree of electrical coupling
between grex cells as this may be of importance in development and intercellular communication (Loewenstein, 1966) but at no stage during this investi-
314
D. R. GARROD, J. F. PALMER AND L. W O L P E R T
gation were we able to penetrate the cells in order to measure potentials intracellular^. N o doubt this is partly due to the small size of grex cells (ca. 10 fi) but
also probably because they are not particularly firmly adherent to each other, so
that they are pushed aside by the advancing electrode tip.
RESULTS
Resistance
measurements
Measurements of the extracellular resistance of the grex in paraffin oil gave
an average value of 1 x 106 Q. per mm (range 0-5 x 106 Q to 1-6 x 106 Q. per mm).
The resistance of migrating grex on agar has been measured after a period of
exposure to the air. About 35-40 min after the lid was removed from the culture
dish the value of the resistance was found to be about 5 x 106 D/ram which is
five times the value found for migrating grex taken from a culture dish and
immediately placed in paraffin oil. Fig. 2 shows the increase of resistance with
time for three grex in which measurements were made at intervals after removing
the lid from the culture dish.
Minutes
Fig. 2
Distance from Earth in mm
Fig. 3
Fig. 2. Graph showing the increase in resistance with time of three grex exposed to
the air. Values for each grex are represented by points of a different shape.
Fig. 3. Graph showing the relationship between grex resistance and length. Each
point represents the resistance calculated from the voltage measured by the recording electrode placed in the grex at a different distance from earth. From the plotted values the correlation coefficient was calculated giving a value of 0-997 (10 degrees
of freedom; P < 0001). The straight line is plotted from the regression of length on
resistance.
Slime mould electrophysiology
315
As the recording electrode was moved along the grex in paraffin oil from
maximum to 'minimum, the recorded pulse was found to decrease approximately linearly with distance. In several cases the resistance of the grex has been
plotted against distance from earth and the regression coefficient calculated to
determine the probability of a linear relationship between resistance and length.
Fig. 3 shows a result of this treatment where a typically high probability of a
linear relationship has been found.
Potential measurements
Two different types of extracellular potential measurements have been made
on the grex.
The first type of measurement involved earthing one end of the grex while the
recording electrode was moved along the outside of the grex, being allowed to
Tip
Tail
^ — — — " — ^ — • • - • — ^ ^
Fig. 4. Record showing change in potential along the length of the grex recorded with
an electrode which was allowed to touch the outside of the grex at several points
along its length. With the electrode in position at each point the recorder was
allowed to run for a few seconds so that each horizontal line represents the potential
at one point on the grex surface. The potential at the left-hand end of the trace is that
for the grex tip and that at the right-hand end for the tail. It can be seen that the
potential is approximately linearly graded from one end of the grex to the other
except for a slight positive jump which corresponded morphologically to a position
about one-third of the length back from the grex tip. Calibration = 10 mV positive
in 1 mV steps. Vertical jumps on some records are marks made by the absolute zero
recorder (A.Z.).
touch the outside at several points along its length. All measurements of this type
were made with grex on agar. It was found that the potential changed approximately linearly along the length of the grex (Fig. 4), the front end of the grex
being negative with respect to the back. The average value of the potential
difference between the ends of the grex obtained by this method was 80 mV
(range 5-5—12 mV). With early culminating grex an average value of 15mV
between tip and base was obtained using the same electrodes.
Secondly, the extracellular potential difference between the anterior and
posterior ends of the grex were measured with grex on agar or in paraffin oil.
Again, one electrode was used to earth the preparation and the other moved
from one end to the other. A potential difference was found between the ends of
316
D. R. GARROD, J. F. PALMER A N D L. W O L P E R T
the grex. This had an average value of 2-7 mV (range 0-6 to 50 mV), the front of
the grex being negative with respect to the back. However, in making these
measurements no attempt was made to use micro-electrodes of similar resistance.
It was therefore considered doubtful whether the measurements represented a
true potential difference or merely a change in electrode tip potential brought
about by moving the recording electrode from front to back of the grex. We
therefore made an attempt to try to distinguish between these possibilities.
Electrodes were filled with 2 M-KC1. Electrode resistances were measured in
0-1 M-NaCl before the experiment and electrodes with very high and very low
resistances selected. Potential measurements were then made first with high
Tip
Tip
Tail
V 10mV
Tail
Tip
Tail
>
10 mV
yJ
Fig. 5. Records showing measurements of potential difference between the ends of
the same grex made with low resistance (A) and high resistance {B) recording electrodes positioned extracellularly inside the grex. As in Fig. 4, horizontal lines represent
the potential at one point (indicated infigure).Micro-electrode resistances were
84 MÜ (A) and 184 Mfl (B). Calibration = 10 mV negative. With the low resistance
electrode virtually no potential difference between the ends of the grex was recorded
but with the high resistance electrode a large potential difference of about 10 mV,
negative towards the tip, was obtained.
resistance electrodes, then with low resistance electrodes, on the same grex on
agar. With high resistance electrodes we were always able to record a potential
which was negative towards the front end of the grex. However, with low
resistance electrodes no consistency was obtained; potentials were very small and
varied in direction, the front end of the grex being sometimes negative and
sometimes positive (Fig. 5). Adrian (1956) has shown that micro-electrodes of
high resistance have a higher negative tip potential than those of low resistance.
Our results therefore seem consistent with the view that the potentials measured
Slime mould electrophysiology
317
in the grex are in fact changes in micro-electrode tip potential. We do not consider that we can detect a true potential difference of cellular origin between the
ends of the grex.
Measurements of electrode tip potential in different concentrations of
poly-L-glutamic acid
Since we believe that our potential measurements are in fact changes in
electrode tip potential we wanted to try to produce similar tip potential changes
in a defined system. We were guided in this by two considerations. Firstly, one of
us has found that the slime sheath surrounding the grex stains with Alcian
Blue at very low pH after fixation with a cationic fixative. Thus it probably
consists of a highly negatively charged, sulphated acid mucopolysaccharide.
W ^ A ^ A - ^ S ^
2mV {£\jf
\ hOmV
Fig. 6. Record showing the effect on the tip potential of a 3 M-KCI filled microelectrode in NaCl solution when 2 % PGA in saline is added and then the whole is
diluted with saline. From left to right the record shows (i) the stable potential record
obtained with the electrode tip in 001 M-NaCl solution; (ii) the artifact produced by
addition of 2 % PGA in 001 M-NaCl solution (arrow, a); (iii) the stable, less
negative potential with the electrode tip in PGA solution (final concentration = 1 % ) ;
(iv) a further artifact produced by dilution of the PGA solution to a concentration of
0-5 % with 0-01 M-NaCl (added at arrow b)\ and (v) the stable but more negative
potential obtained with the more dilute PGA solution. Calibrations = 7 mV
negative in 1 mV steps and 10 mV negative.
Secondly, it may be suggested that this mucopolysaccharide is more dilute at the
front end of the grex, where it is presumably formed, than at the back. Therefore
we determined to measure the tip potential of a micro-electrode in solutions
containing different concentrations of negatively charged polymers. Although
Adrian (1956) has shown that the tip potential of a micro-electrode is dependent
on the concentration of simple electrolyte in the solution surrounding the tip, it
does not necessarily follow that the same holds true for different concentrations
of large, polymeric molecules.
Fig. 6 shows the results obtained in experiments in which the tip potential of a
glass micro-electrode was measured in two different concentrations of PGA
318
D. R. GARROD, J. F. PALMER A N D L. W O L P E R T
(poly-L-glutamic acid) dissolved in 0 1 M-NaCl. A continuous record of the
potential was made while the PGA (2 % saline) was added to saline and then
diluted with saline. Since the saline concentration was kept constant throughout,
the changes in potential are due to the differences in concentration of PGA. The
potential was first measured with the electrode in saline (Fig. 6). On addition of
2 % poly-L-glutamic acid in saline a positive deflection of 6 mV was recorded.
Then on subsequent dilution of the PGA solution to half the previous concentration a negative deflection of 3 mV was observed (Fig. 6). This experiment has
been repeated several times with similar results so we may conclude that, for a
given micro-electrode, the tip potential is more negative the lower the concentration of PGA in the solution surrounding the tip.
Experiments with heparin, a sulphated muco-protein, were unsuccessful in
that after adding heparin to the solution surrounding the electrode tip we were
unable to obtain stable values of potential. This may have been because heparin
blocked the tip of the electrode.
DISCUSSION
Our results suggest that the potential differences found between the ends of
the slime mould grex are due to changes in micro-electrode tip potential. We
have reached this conclusion for two reasons. Firstly, the potential difference
varies with the resistance of the micro-electrode used and in a way which is
consistent with the finding of Adrian (1956). Using low resistance electrodes the
results were inconsistent which does not seem to be indicative of the existence of
a true potential, although it is possible that a potential, if present, may vary with
time. Secondly, Tasaki & Singer (1968) have pointed out that caution should
be exercised in interpreting measured potential differences in living organisms as
true potentials resulting in flow of current. Tasaki & Singer state, for example,
that the potentials measured between the ends of a moving amoeba (Bingley &
Thompson, 1962) may be due to 'local variations in the nature, ionization or
concentration of intracellular polyelectrolytes'. We do not feel that as yet we
can completely exclude the possibility that a potential difference exists in the
grex. In these experiments we were working at the limit of stability of our
recording technique so that we cannot exclude the possibility that there is a
small extracellular potential difference of say 1 mV or less between the ends of
the grex. The fact that it proved possible to measure the resistance of grex on agar
(nutrient agar has a resistance of about 30 kQ per mm compared with 1 MQ for
the grex) would seem to indicate that the grex cells are to some extent insulated
from their environment, perhaps by the slime sheath. It therefore might be possible for the cells to maintain a small potential difference, if they could generate
it ! In general, however, it is difficult to understand how an extracellular potential
difference can be maintained in a tissue where the extracellular fluid is highly
conducting. The specific resistance of the extracellular space in the grex may be
roughly estimated as follows. The average diameter of grex used was about
Slime mould electrophysiologic
319
0 1 mm. Assuming that about 10 % of the cross-sectional area of the grex is
extracellular space, the area of the extracellular space would be approximately
3 x 10~5 cm2. For a linear resistance of 106 Q per millimetre, this would give a
value for specific resistance of about 300 Ücm which is relatively low and similar
to values obtained with crustacean, decapod and amphibian systems (Cole,
1942; Katz, 1948).
Table 1
Experiment
Current
density
Author
2
Inhibition of regeneration in Obelia
6600/*A/cm
Reversal of polarity of regeneration
in Dugesia
Suppression of distal regeneration by
extracts of distal regions in
Tubularia
2162 /tA/cm 2
Marsh & Beams (1952)
2200/*A/cm2
Rose (1966)
Lund (1923)
It is useful to consider the current required to maintain a small potential
difference of, say, 1 mV between the ends of the grex. From Ohm's law, the
current given by a potential difference of 1 mV across the grex resistance of
106 Q. would be 10~9 amps. This would give a current density in the grex of
about 30 juA/cm2. Such a current density is well below the levels found necessary
to inhibit regeneration or bring about polarity reversal in other organisms (see
Table 1). A current of similar magnitude to those shown in Table 1 (say 2 mA/
cm2) would necessitate a potential difference between the ends of the grex of
about 60 mV. We would certainly have been able to detect such a potential
difference, so that we can be fairly confident that no potential of this magnitude is present. However, Jaffe (1966) has measured an intracellular current
of about 6 /<A/cm2 in developing Fucus eggs and claims that this could be
developmentally significant as a current of this density could stratify charged
particles. As we have seen above, a potential of 1 mV between the ends of the
grex would give a current density of this order, which might be of some developmental significance. This voltage may be regarded as a possible upper limit to
any bio-electric mechanism which might be invoked to explain grex behaviour.
The higher negative tip potential found when a micro-electrode was placed at
the front end of the grex may suggest that the slime sheath contains more water at
the front of the grex than at the back. Adrian (1956) showed that the tippotential of a micro-electrode becomes more negative as the concentration of electrolyte (KCl or NaCl) in the solution surrounding the tip is decreased. We have
shown that the same is true when a highly negatively charged polymer, poly-Lglutamic acid (PGA) is used. This result shows that the effect of dilution on tip
potential is not confined to solutions of simple salts, though PGA is probably a
very poor slime analogue. The most interesting observation in this context is the
320
D. R. GARROD, J. F. PALMER A N D L. W O L P E R T
graded decrease in the tip potential of a micro-electrode allowed to touch the
outside of the grex at several points along its length, beginning at the front end.
This may indicate a graded decrease in water content of the slime sheath from
front to back, which, in turn, suggests two possibilities; firstly, that the slime
sheath may increase in rigidity from front to back and, secondly, that the
slime sheath may be synthesized mainly at the front of the grex. (Previous
observations in which particles have been placed on the slime sheath (Bonner,
1966; Shaffer, 1965) have shown that the slime sheath must be synthesized at the
tip of the grex but do not indicate whether it is synthesized everywhere else as
well.) As pointed out by Garrod (1969), each of these possibilities could be
important with regard to the likely role of the slime sheath in partly controlling
the polarity of grex movement. We would emphasize, however, that owing to the
present inadequate knowledge of the mechanism of tip potentials, these conclusions must be regarded as tentative.
The large decrease in absolute grex resistance, possibly due to a decrease in the
availability of water and diffusible ions, which occurs when grex are exposed to
the air may be important in relation to the observation that a reduction in
relative humidity of the grex environment brings about culmination (Raper,
1940; Whittingham & Raper, 1957; Bonner & Shaw, 1957). Further, the change
in electrode tip potential between base and tip seems to be somewhat larger in
the culminating grex than from end to end of the migrating grex, which seems to
indicate that the difference in properties between the ends of the grex is exaggerated at culmination.
SUMMARY
1. Measurements of electrical potential and resistance have been carried out
on the slime mould grex.
2. No evidence could be found for an extracellular bio-electric potential
which might be involved in pattern formation and regulation or in controlling
the polarity of grex movement.
3. Using changes in tip potential of glass micro-electrodes as a tool for analysis, we find evidence which suggests that the slime sheath may contain more
water at the front end of the grex than at the back. This in turn suggests that
the slime sheath may be more deformable at the front of the grex and may be
synthesized at the tip only.
4. Exposing migrating grex to the air, which brings about fruiting body
formation, markedly increases grex resistance.
RÉSUMÉ
Propriétés électriques dupseudoplasmode ('grex') d'un Acrasié
1. Des mesures de potentiel et de résistance électriques ont été effectuées sur
le pseudoplasmode d'Acrasié.
Slime mould electrophysiology
321
2. Il ne semble pas qu'un potentiel bio-électrique extra-cellulaire soit impliqué dans la formation et la régulation du 'pattern', ni dans le contrôle de la
polarité du mouvement du pseudoplasmode.
3. Utilisant comme moyen d'analyse des variations de potentiel de pointe de
micro-électrodes en verre, nous mettons en évidence des faits qui suggèrent
que la nappe cellulaire contiendrait plus d'eau à l'avant du pseudoplasmode
qu'a l'arrière. Cette constatation suggère à son tour que la nappe cellulaire
serait plus déformable à la partie antérieure du pseudoplasmode et qu'elle ne
pourrait se synthétiser qu'à cette extrémité.
4. L'exposition à l'air de l'agrégat, qui provoque la formation de sorocarpes,
augmente nettement la résistance du pseudoplasmode.
This work was supported by the Nuffield Foundation and D. G. would like to thank the
Science Research Council for a research studentship. Our thanks are due to Dr Anne Warner
for reading the manuscript.
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(Manuscript
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1969)