The Effects of Acclimation Temperature on A Neuromuscular System of the Crayfish, Astacus Leptodactylus

In a preceding publication (Harri & Florey, 1977) we described the effects of temperature on neuromuscular transmission in the closer muscle of walking legs of the crayfish Astacus leptodactylus. It was found that amplitude and time course of excitatory junction potentials (e.j.p.s) as well as membrane potential and tension development of the muscle fibres were greatly affected, so as to permit adequate functioning of the nerve-muscle system over a wide range of temperatures (from 6 to 30°C).

These findings, obtained with this eurytherm species, were in striking contrast to those obtained earlier with a similar nerve-muscle system from a stenotherm crustacean, the Hawaiian ghost crab, Ocypode ceratophthalma (Florey & Hoyle, 1976). In this preparation e.j.p. amplitude and muscle tension showed a narrow temperature optimum near the acclimation temperature (26°C) and declined sharply when the temperature was lowered or raised. As in the crayfish, the membrane potential increased with temperature and e.j.p. half-decay time (time constant of decay) decreased when the temperature was raised from the lower limit towards the acclimation temperature. In the temperature range above the acclimation temperature the decay time of the e.j.p.s showed a minimal temperature dependence in both Ocypode and Astacus. In the case of the crayfish preparation the temperature dependence of the membrane potential showed a steeper slope in the lower temperature range; the inflexion point of the curve was about 5°C above the acclimation temperature (12°C).

The results already reported suggested that the characteristic temperature dependence of several parameters of synaptic function is determined or affected by the acclimation temperature. We therefore investigated this relationship using two groups of crayfish; one acclimated to 12°C and another to 25°C.

Mature specimens (body length about 15 cm) of commerically obtained Astacus leptodactylus were maintained in filtered and aerated lake water at constant temperature, one group at 12°C and the other at 25°C. Before use the animals were allowed to acclimate for at least 2 weeks. The experimental techniques used were the same as those described earlier (Harri & Florey, 1977). The nerve bundle containing the two motor axons of the closer muscle was exposed in the opened meropodite and after transfer to the muscle bath it was lifted onto a pair of platinum electrodes and raised to the saline-air interphase. The muscle bath itself was set in a thermostated container. Temperature at or near the exposed closer muscle was monitored with a small thermocouple connected to an electronic temperature measuring device (Bailey Instruments, Model BAT-8). Muscle tension was measured using an RCA 5734 transducer tube whose movable anode-pin was attached to the muscle tendon. Membrane potentials were recorded with the aid of intracellular glass microelectrodes (3 M-KCI). The microelectrodes were always inserted into muscle fibres at the surface of the anterior portion of the muscle, about one-quarter muscle length from the distal end.

The signals from the transducer and from the microelectrode were displayed on a dual channel oscilloscope (Tektronix R 5103) and registered on a two-channel rectilinear chart recorder (Gould-Brush, Mark 220). To avoid effects of temperature on the potential of the Ag-AgCl reference electrode it was placed in a beaker containing saline which was connected with the muscle bath by a saline-agar bridge.

Nerve stimulation was accomplished with an electronic stimulator controlled by a programmable quartz-clock which was designed and built by the Electronics Division of the University of Konstanz. Separation of the axon giving rise to slow contractions (‘slow axon’) from that causing fast contractions (‘fast axon’) was not possible. Separate stimulation was therefore achieved by carefully adjusting the stimulus strength and duration. The slow axon usually had the lower threshold but in several preparations the reverse situation occurred, which permitted selective stimulation of the fast axon. Threshold shifts in the course of an experiment made it very difficult to obtain uninterrupted series of fast responses throughout the full temperature range tested. Contamination of fast responses with slow ones appeared to be minimal, but cannot be excluded in all measurements. However, the observation that fast contractions recorded from warm-acclimated preparations were generally weaker than the corresponding slow contractions (see Fig. 6) clearly argues against a summation of fast and slow responses.

The experiments were carried out from June to September 1976 and overlapped those already reported (Harri & Florey, 1977).

Table 1 lists several electrophysiological parameters of the slow and fast responses of muscle fibres from animals acclimated to 12°C (cold-acclimated preparations) and from those acclimated to 25°C (warm-acclimated preparations). The temperature dependences of the parameters listed in Table 1 are shown in Figs. 2–5. To facilitate interpretation, the electrophysiological data (with the exception of those of the resting potentials) were normalized by setting the values obtained at 12°C (see Table 1) at i 0. This procedure permitted statistical evaluation of the data.

As is shown in Fig. 1 the resting potential of the muscle fibres increases with increasing temperature. The temperature dependence in both cold-acclimated and warm-acclimated preparations was usually greater in the lower part of the temperature range. At these lower temperatures the membrane potential increased by 0·34–2·0 mV/°C, at higher temperatures it increased by 0·1–0·38 mV/°C.

The corresponding two slopes of the respective curves of cold- and warm-acclimated preparations were not significantly different, but in the case of cold-acclimated preparations the inflexion of the curves occurred at 17 ± 1·34°C (n = 13) as compared with 24·6 ± 1·6°C in warm-acclimated ones (n = 10). This difference is statistically significant at the level of P < 0·005.

In most experiments the preparation was first cooled to about 5·6°C and measurements made while the preparation was slowly warmed to 32°C at rates that were ways slower than 0·3°C/min. The strong variations of the thresholds for excitation of slow and fast axon that occurred when the temperature was altered, and the time required to make the necessary adjustments are responsible for the omission of some of the measurements in many serial observations. Table 1 which summarizes the data, therefore, shows different N-values for the different parameters measured.

The excitatory junction potentials (e.j.p.s) were generally small and never exceeded 4 mV. The temperature dependence of e.j.p. amplitudes of cold- and warm-acclimated preparations is shown in Fig. 2. Stimulation at 10 pulses s⁻¹ for 2 s was used routinely for these measurements. Where relative values are given, Table 1 should be consulted for absolute values.

The amplitude of the unfacilitated slow e.j.p.s (first e.j.p. in a given train) of cold-acclimated preparations remained rather constant up to 28°C and declined sharply thereafter with increasing temperature, while that of warm-acclimated preparations increased and reached a maximum around 20–26°C.

Facilitation of the e.j.p.s (20th e.j.p./ist e.j.p.) increased slightly with increasing temperature. In cold-acclimated preparations the extent of facilitation then declined, while that in warm-acclimated preparations stayed at this higher level over the higher part of the temperature range tested.

The fully facilitated slow e.j.p.s of cold-acclimated preparations were of nearly constant amplitude between 6 and 20°C and declined with a further rise of temperature. In warm-acclimated preparations the fully facilitated slow e.j.p.s measured in the lower temperature range were generally much smaller than those seen in cold-acclimated preparations. They increased with the temperature and at about 20°C reached the amplitudes of the slow e.j.p.s measured in cold-acclimated preparations. With a further rise in temperature slow e.j.p. amplitude stayed nearly constant up to 28°C and then declined.

Typically, unfacilitated fast e.j.p.s are much larger than unfacilitated slow e.j.p.s. Those of cold-acclimated preparations are generally larger than those of warm-acclimated ones.

The unfacilitated fast e.j.p.s increased with temperature towards a maximum around 20°C in cold-acclimated, and around 25°C in warm-acclimated preparations. In the median temperature range facilitation is slight or absent (see examples shown in Fig. 3) but becomes more conspicuous at higher temperatures (above 25°C), particularly in cold-acclimated preparations.

Facilitated fast e.j.p.s of cold-acclimated preparations are conspicuously larger than those of warm-acclimated preparations but both increase with temperature, reaching a maximum around 20° in cold-acclimated and 25°C in warm-acclimated preparations.

The time course of fast and slow e.j.p.s is clearly temperature dependent. This is particularly obvious when the decay of the e.j.p.s is considered. The decay time changes more drastically in the lower temperature range and becomes nearly independent of temperature at higher temperatures (Fig. 4). When the data are plotted on semi-logarithmic coordinates (also shown in Fig. 4) two slopes become evident. The transition to the temperature-independent phase of e.j.p. decay time occurs around 16–18°C in cold-acclimated and 21–23°C in warm-acclimated preparations.

The increased decay times at lower temperatures result in a conspicuous summation of the e.j.p.s: the individual e.j.p.s do not decay completely before the next one takes off. When maximal facilitation has been reached the e.j.p.s start from a steady value of depolarization, here referred to as plateau depolarization (see Fig. 3). Peak depolarization reached by the e.j.p.s is, therefore, the sum of this plateau depolarization and the e.j.p. amplitude. Fig. 5 shows the temperature dependence of both plateau depolarization and peak depolarization reached during 2 s trains of stimulation (at 10⁻¹ s) of fast and slow axons. A comparison of Figs. 5 and 4 reveals that the temperature dependence of the plateau depolarization more or less parallels that of the time constant of e.j.p. decay. Peak depolarization, in addition, follows the temperature-dependent change of e.j.p. amplitude. In the case of stimulation of the slow motor axon, peak depolarization in cold-acclimated preparations declines with increasing temperature while that observed in warm-acclimated preparations first drops (between 8 and about 15°C) and then rises to a new maximum between 20 and 25°C. When the fast axon is stimulated, similar but much less pronounced biphasic curves are obtained. The deviations from the values measured at 12°C are statistically not significant over the range from 12 to 27°C in cold-acclimated preparations, and over the range from 8 to 32°C in the warm-acclimated preparations.

In Fig. 6 the muscle tensions reached at the end of 2 s periods of stimulation at different frequencies are plotted against temperatures. The temperature dependence of tension development, elicited by stimulation of the slow axon, corresponds to the electrophysiological data. In cold-acclimated preparations the contractions decrease with rising temperature while in warm-acclimated ones contractions first increase and reach maximal values in the temperature range of 15–20°C. In the case of fast contractions the correlation with electrophysiological data is less obvious. A slight acclimation effect can be seen in the observation that in warm-acclimated animals tension first increased as the temperature was raised from 8 to 10°C and then fell, while in cold-acclimated animals there was a decline throughout the temperature range from 8 to 32°C with a clear maximum below 8°C. The contractions of the warm-acclimated preparations were markedly weaker than those of the cold-acclimated ones, except at higher stimulation frequencies (20 and 30 s⁻¹) in the lower temperature range (8–12°C).

Our previous investigation (Harri & Florey, 1977) has already shown that the maximal (peak) depolarization reached by the e.j.p.s during a given stimulus train is much more independent of temperature than are the single parameters which contribute to e.j.p. depolarization. In the case of slow e.j.p.s this is due to the increase in the facilitation with temperature even though e.j.p. amplitude and summation decrease with increasing temperature. Peak depolarization reached by fast e.j.p.s remains constant because the amplitude of the unfacilitated e.j.p.s increases even though facilitation is not significantly altered. Peak depolarization reached during slow or fast axon stimulation was thus found to be nearly constant over a temperature range extending from about 12 to 25°C. The results of the current investigation show that warm acclimation shifts this range towards higher temperatures. In the case of the fast e.j.p.s the range extends to 32°C and in the case of slow e.j.p.s to 30°C with statistically significantly larger peak depolarizations between 20 and 25°C.

Thermal acclimation clearly affects all membrane parameters investigated: the slope transition of the membrane potential (Fig. 2), e.j.p. amplitude (both slow and fast) and the slope transition of the time constant of e.j.p. decay (Fig. 6) are shifted towards higher temperatures after warm acclimation. The reduction of the amplitude of unfacilitated e.j.p.s that occurs with warm acclimation is compensated by the greater rate of facilitation.

The diminished amplitude (slow and fast e.j.p.s) and a corresponding reduction of developed muscle tension (slow response) at lower temperatures in the preparations from warm-acclimated animals indicate that warm acclimation is, therefore, similar to type IV A of Prosser & Brown (1961) and is reminescent of the temperature acclimation of the heart rate in the shore crab, Carcinus maenas (Ahsanullah & Newell, 1971).

While the peak depolarizations show a remarkable temperature independence, muscle tension is clearly and strongly temperature dependent. The results obtained with stimulation of the fast axon must be interpreted with caution because of the notorious lability of the fast contractions in crayfish nerve-muscle preparations (see for instance Wiersma & van Harreveld, 1938). The reduction of muscle tension that occurs with warm acclimation may be the result of an increased lability of excitation–contraction coupling in the isolated nerve-muscle preparation. In cold-acclimated preparations, however, the pattern of the temperature dependence of the fast contraction is very similar to that of the slow contraction. The tension developed during a given train of impulses at a given frequency declines with increasing temperature. In warm-acclimated preparations tension development at low temperatures (8–15°C) due to slow axon stimulation is clearly much diminished compared with those obtained in cold-acclimated preparations.

This is in striking contrast to the fact that peak depolarization improves as the temperature is lowered from 15 to 8°C and that plateau depolarization and e.j.p. duration increase throughout the temperature range from 32 down to 8°C. Clearly, the reduced tension development at temperatures below 15°C, in warm-acclimated preparations, must be due to changes within the muscle fibres, possibly a reduction of Ca mobilization.

The change of the slope of the curves which relate membrane potential to temperature as described in our investigation (see Fig. 2) is similar to that described by Dalton & Hendrix (1962) for the giant axons of Homarus. It may well be related to the slope transition of the curves relating the time constant of e.j.p. decay to temperature (see Fig. 4). The immediate cause of the changing time constant is, presumably, the parallel change of membrane resistance of the muscle fibres. Fatt & Katz (1953) and Colton & Freeman (1975) have shown for both crayfish (Astacus) and lobster (Homarus) muscle fibres that membrane resistance increases as the temperature is lowered. The systematic analysis made by Colton & Freeman (1975) has particularly demonstrated that the slope of the resistance-temperature curve increases drastically when the temperature is lowered below a certain median temperature. Direct measurements of membrane time constant and membrane resistance in crayfish muscle fibres (K. L. Fischer & E. Florey, unpublished observations) have since shown that the resistance changes can fully account for the changing time constant of e.j.p. decay.

The shifting of the inflexion point of the curves that relate membrane potential and decay time to temperature during acclimation from a lower (12°C) to a higher (25°C) temperature implies a change in membrane properties that are involved in the changing membrane behaviour that occurs at a critical temperature. It is quite possible that a phase-transition of membrane lipids is responsible for the inflexion of the curves. If this is so, acclimation must involve a change in the lipid composition of the muscle fibre membranes.

Such changes in membrane lipids have been described for goldfish brain synaptosomes; they involve increased membrane fluidity and unsaturation with acclimation to lower temperature (Roots & Johnston, 1968; Driedzic, Selivonchick & Roots, 1976; Cossins, Friedlander & Prosser, 1977).

Our investigation of temperature-dependent parameters of neuromuscular transmission has been concerned only with one aspect of the motor control in an eurythermal poikilotherm, the temperature dependence of the peripheral mechanisms. It is equally important, however, to consider the effect of temperature on the central nervous system, especially on the impulse pattern generated in the motoneurones. If crayfish are capable of normal coordinated movements over a temperature span of 30°C the motor output from the central nervous system must be matched to the temperature-dependent performance of the periphery. In the final analysis we might expect that the whole system is temperature-compensated to a large degree. How this is accomplished must be the subject of further investigation.

This investigation was supported by the Sonderforschungsbereich 138 of the Deutsche Forschungsgemeinschaft.

M. H. was supported by a fellowship of the Alexander von Humboldt-Stiftung.