Electrical properties of the slime mould grex

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 Tubularia 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 and we therefore considered this a very convenient organism for re-investiga-tion 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).

Grex were grown on 2 % agar containing 0·01 % glucose, 0·01 % bacterial peptone, and buffered to pH 6·9 with 01M 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.

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 (Fig. 1). Conventional 3 M-KCl-filled glass micro-electrodes of resistance ca. 20 MΩ 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 MΩ could be achieved by breaking the tips from high-resistance KC1 electrodes; these, however, tended to leak KC1 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 kΩ 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 kΩcurrent measuring resistance is switched out, the voltage pulse across the grex will be reduced by an amount equivalent to 100 kΩ. The resistance of the grex can, therefore, be measured by comparing the voltage drop along the grex with the known increment equivalent to 100 kΩ, 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 kΩ 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 KC1 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-gation were we able to penetrate the cells in order to measure potentials intracellularly. No doubt this is partly due to the small size of grex cells (ca. 10 μ) but also probably because they are not particularly firmly adherent to each other, so that they are pushed aside by the advancing electrode tip.

Measurements of the extracellular resistance of the grex in paraffin oil gave an average value of 1 x 10⁶ Ω per mm (range 0·5 x 10⁶ Ω to 1·6 x 10⁶ Ω 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 × 10⁶ Ω /mm 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.

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.

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 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 8 0 mV (range 5·5–12 mV). With early culminating grex an average value of 15 mV 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 the grex. This had an average value of 2·7 mV (range 0·6 to 5·0 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-KCI. 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 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 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.

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.

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 (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.

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 kΩ per mm compared with 1 MΩ 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 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 3×10⁻⁵ cm². For a linear resistance of 10⁶ Ω per millimetre, this would give a value for specific resistance of about 300 Ω cm which is relatively low and similar to values obtained with crustacean, decapod and amphibian systems (Cole, 1942; Katz, 1948).

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 Ω would be 10⁻⁹ amps. This would give a current density in the grex of about 30 μ) A/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 μ) 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 tip potential of a micro-electrode becomes more negative as the concentration of electrolyte (KC1 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-L-glutamic 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 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 ofIhe 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.

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.

1. Des mesures de potentiel et de résistance électriques ont été effectuées sur le pseudoplasmode d’Acrasié.

2. I1 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.