Thermal Acclimation of A Central Neurone of Helix Aspersa: II. Electrophysiological Recordings

The basic mechanisms underlying long-term temperature acclimation in ectotherms are not well defined (Hazel & Prosser, 1974). Molluscs provide ideal subjects for investigations of the electrophysiological correlates of thermal acclimation, due to the large size of central neurones and the wealth of literature on the electrophysiology of these cells: furthermore, the available studies of the haemolymph of molluscs permit a more comprehensive picture to be constructed of the acclimation process. In recent years electrophysiological research upon temperature adaptation of neuronal membranes has been largely directed at short-term adaptation (Airapetyan, 1969 a, b; Carpenter, 1967, 1970; Chalazonitis, 1961; Chalazonitis, Romey & Arvanitaki, 1967; Gorman & Marmor, 1970; Kerkut & Ridge, 1962; Marchiafava, 1970; Magura, Valeyev & Zamekhovsky, 1975; Marmor, 1971) although there has been a notable study of long-term adaptation in Helisoma trivolvis (Merickel & Kater, 1974).

In the preceding paper (Langley, 1979a) it was shown that thermal acclimation of the snail Helix aspersa is accompanied by changes in the composition of the haemolymph, most especially in Na and K concentrations. Neuronal excitability is vitally dependent upon the ionic gradients across the cell membrane, and it is, therefore, conceivable that the charges in the chemistry of the haemolymph could be involved in the thermal adaptation of the neuronal membrane.

In the present study, recordings were made from a large, identifiable autoactive neurone of the parietal ganglia the F1 cell of Kerkut et al. (1975). This cell has been implicated by previous workers in neurosecretion, osmoregulation and possibly hibernation. Chronic and acute aspects and the temporal stability of thermal adaptation of this neurone were investigated.

Adult specimens of Helix were kept under four acclimation regimes, as previously described (Langley, 1979a), with control animals being maintained at 19°C and the others being acclimated for 4 weeks at 4°, 12° or 30°C. Dissection and preparation of the Fi cell (see Fig. 1) was carried out according to the technique of Walker (1968), usually at 20°C.

The experiments reported in this paper were carried out using Ringer solutions of the following composition: NaCl, 80 mm; KC1, 4 mm; CaCl₂, 7 mm; MgCl₂, 5 mm; Hepes 5 mm. Hepes was chosen for its lack of effect on thermoacclimated neurones (Langley, 1979b). The pH of all solutions was adjusted with Hepes to 7·5 at 20°C.

Conventional single-barrelled microelectrodes were used and filled with either 1·5 M potassium acetate or 3 M potassium chloride. Tip potentials (less than 2 mV) and resistance (1–10MΩ) had only a small temperature sensitivity in the 0°–30°C range. Signals were fed into a 4 channel Telequipment DM63 storage oscilloscope via a microelectrode amplifier with provision for current injection. Permanent records were obtained either with a Polaroid camera or Servoscribe pen-recorder.

Preparations were maintained in a constantly flowing Ringer solution with continuous aeration.

The temperature control device utilized a Pelcool MSE Cryostat stage with a thermister-controlled regulator unit. The temperature of the bathing solution was monitored using a thermistor probe linked to a Philips GM 6020 voltmeter.

F 1 cells from control (19°C) animals typically spiked with a regular beat of 1·9–2·1 Hz at 20°C (‘bursting’ was never encountered in starved snails from the four acclimation regimes).

Compared to the resting potential of control snails (Table 1) the R.P. was depolarized in the warm-acclimated (30°C) snails, unaffected in the cold-acclimated groups (Table 1). As noted above, dissection was usually at 20°C. In some experiments the cell was kept at the acclimation temperature until the recordings were made, but the R.P. was no different.

The magnitude of the A.P. did not significantly vary between the four groups (Table 1). However, the rise and fall times of the spike were inversely proportional to acclimation temperature, with the fall time being most affected (Table 1).

F1, like most autoactive cells, had small after-potentials, their magnitude being affected by acclimation temperature, such that there was a maximum at 19°C with a decrease at higher and lower temperatures (Table 1).

Spike duration, when measured at 20°C, showed an increase with a decrease in the temperature of acclimation (Table 1).

No observable accommodation of the various spike parameters occurred if the ganglia were maintained for up to 2 h in Ringer solution at temperatures other than those of acclimation.

At least 3 weeks acclimation was required before spike parameters were affected. This proved to be the same time as that required to change block temperature (see below).

Autonomous spike production showed marked acclimation alteration (Fig. 2a-c). These changes were not modifiable by incubation in normal Ringer at 20°C for up to 2 h.

Table 1 shows the effect of acclimation upon block temperature. (In this paper, block temperature refers to the cold-induced cessation of spontaneous activity - the cells were still excitable, as indicated by the injection of short pulses of depolarizing current.) The temperature was lowered by 5°C/min; upon rewarming the preparations at the same rate there was a marked hysteresis in spike production. The events preceding the cessation of spontaneous activity showed variations between the acclimated groups. In Fi cells from^- warm-acclimated individuals the cessation was abrupt (Figs. 2c, 5), with a steep change in input resistance, whilst in the two cold groups cold-block was preceded by a gradual change in frequency, even with steep changes of temperature, and involved a slower increase in input resistance (Figs. 2 a, b, 5).

The apparent upper-lethal temperature (Table 1) also showed a significant modification as a result of thermal acclimation, ranging from 33°C in warm-acclimated snails to only 22°C in the 4°C-acclimated group. Since the lethal limit depended on both the temperatures recently presented to the cell, and the duration of the exposure to these temperatures, the warming procedure was made as comparable between groups as possible by a gradual approach to the lethal limit.

To investigate the acute effects of thermal change on the F1 cells of the four groups, first, recordings were made as temperature was stepped progressively down from 20°C, and then, after return to 20°C and the recovery of original data values, recordings were made as temperature was progressively stepped up. Each recording was made after temperature had been held constant for up to 5 min. (The step changes were either completed in 10 s or made at a rate of 10°C/20–35 s, but no differences were observed between the effects produced with the different rates.)

In all groups there was a depolarization of resting potential as temperature was raised from a few degrees below the lethal limit to the limit. Fig. 3 shows the effects of temperature upon R.P. but because of the temperature employed, the above depolarization can only be seen for the 4°C group (Fig. 3 a). In cold-acclimated and control snails, there was also a hyperpolarization as temperature was brought below 6°C (Fig. 3,a-c). In the warm-acclimated group, there was a depolarization with increasing temperature in the range I6° –30°C.

A.P. amplitude was not observed to be affected by temperature in the control group (Fig. 4c). In all other groups, however, there was a decrease in amplitude as temperature was lowered from 20°C. In the 4° and 12° groups there was also a fall as temperature was raised above this point

Rise and fall times of spikes (especially fall times) showed an inverse relationship to temperature in all four acclimation groups, the effect on the fall phase being most marked in the 30°C-acclimated group. The effect of these changes was to lengthen the spike as temperature was decreased, with a greater effect in the 4° and 12°C groups than the other two.

The input resistance was inversely proportional to temperature in the 4°, 12°, and 19°C groups, with higher acclimation temperature resulting in a greater sensitivity (Fig. 5). In the 30°C group, input resistance did not vary much with temperature (Fig. 5)

Such variation in input resistance cannot be attributed to differences in the shape of the F i cell, since the diameter of cells in the warm- and cold-acclimated groups fell within the 175-200 μm range obtaining in the control group.

Fig. 6 shows the relationship between temperature and spontaneous spike frequency for all four groups. In general, the data showed that the lower the acclimation temperature, the lower was the temperature at which a given frequency was observed: acclimation had led to a compensatory change.

The relationship between ambient temperature and the electrical parameters of the F1 cell discussed in Sections A and B could not be modified by incubation of the ganglia at 20°C for periods up to 4 h.

Some idea of the time course of acclimation is given in Fig. 7 by the change in block temperature during acclimation. Before 12 days acclimation, there was little change in block temperature, which was reversible by incubation for 30–45 min at the original temperature. After this period of time block temperature changed rapidly and became irreversible.

It was also found that ganglia from snails immediately out of hibernation (March-April) were least able to withstand elevated bath temperatures, and such animals showed higher mortality rates with increasing environmental temperatures than snails collected in mid-summer. Conversely ganglia from summer-aestivated snails showed the best survival at bath temperatures above 19°C, and readily survived 30°C environmental temperatures, whereas winter hibernation snails had low tolerance of high temperature.

Shorter acclimation times were required for animals collected in late summer or early autumn months, when placed at 4° or 12°C than was the case with summer or winter-quiescent snails, as revealed by cold-block. The former, however, required longer periods at 30°C. As can be seen in Fig. 7, a longer period was required for acclimation to higher than lower temperatures.

The membrane potential of cells from all four groups varies with temperature in. a fashion not predicted by the constant field equation, since if temperature affected only the physico-chemical aspects of the neuronal membrane one would expect an indirect relationship with a change of – 0·2 mV/°C (compare Fig. 2a-c). Such a thermally induced change in the electrical parameters of excitable cells has been reported previously in the giant nerve fibres of Lumbricus terrestris by Lagerspetz & Talo (1967), Talo & Lagerspetz (1967) and also in the crayfish CNS by Gladwell, Bowler & Duncan (1975).

In view of the data from both chronic and acute thermal change, it appears that acclimation leads to a permanent alteration in the permeability of the cell membrane. Such an irreversible permeability change induced by temperature manipulation has been elucidated before in Helix (Gerasimov, Yanishevskii & Skubalyanka, 1967), and in Aplysia (Carpenter, 1967; Chalazonitis et al. 1964; Marchiafava, 1970; Murray, 1966). Also, Merickel & Kater (1974) showed that the compensatory thermal acclimation of the resting potential of the lateral giant neurone of Helisoma trivolvis involved modification of the sodium pump.

Thermal change has a more pronounced effect upon spike characteristics than resting potential, partly because there will be an indirect effect through a change in the resting potential. The effects of temperature upon spike frequency is that which is to be expected from the model of repetitive firing in a central neurone of Connor & Stevens (1971 a-c). In this model, interspike interval is dominated by a potassium conductance, g_A, which has a marked thermal sensitivity (Magura, 1967; Magura & Zamekhovsky, 1973; Magura et al. 1975). The effect of acclimation could be to modify this conductance in a permanent fashion. Similarly, moderate thermal modulation of the conventional Hodgkin-Huxley g_K and g_Na would lead to the observed changes in spike profile (see Magura & Zamekhovsky, 1973). Such acclimatory changes are also suggested in the present work by measurement of the input resistance (Fig. 5). The different thermal sensitivities of the rise and fall times of the spike would be explained by different thermal sensitivities of g_Na and g_K, as found in other systems, including eel-electroplaque (Ruiz-Manresa & Grundfest, 1976), squid giant axon (Hodgkin & Katz, 1949) and the giant axons of Lumbricus terrestris (Dierolf & McDonald, 1969; Dierolf & Brink, 1973).

The F 1 cell could well be important in a number of physiological process including hibernation, osmoregulation and excretion control (by indirect comparison with analogous cells in other molluscs; Gainer, 1972a-c; Kerkut et al. 1970; Krause, 1960). It would seem likely that thermal variation will influence such processes by means of a change in the rate of release of neurosecretory material from F1, a relationship between spike frequency and neurosecretory release being well known (Cooke, 1977). Chronic and acute changes in the thermal sensitivity of F1 could permit such control to continue over a wide thermal spectrum.

The mechanisms underlying acclimation-induced alteration of ionic permeability are investigated further in the following paper (Langley, 1979b).

I wish to thank the M.R.C. for financial assistance.