Ionic Mechanism of Thermoreception in Paramecium

Thermoreception is found in many living organisms (Hensel, 1974; Jennings, 1906; Maeda & Imae, 1979), but its molecular mechanism remains unknown. The protozoan, Paramecium, responds to warming above the temperature to which it has been adapted, and to cooling below this temperature, by producing transient changes in the frequency of directional changes in swimming (Nakaoka & Oosawa, 1977). It has been shown that both types of temperature change induce a slow transient depolarization of the membrane potential which triggers spike-like depolarization and in turn causes the directional changes in swimming (Hennessey, Saimi & Kung, 1983; Toyotama, 1981).

In this study, to elucidate the ionic process of thermoreception in Paramecium, we first examined whether the thermosensitive response at one end of the cell was different from that at the other end since it is known that mechanoreceptors produce different responses at each end (Naitoh & Eckert, 1969; Ogura & Machemer, 1980). Application of thermal stimulation to discrete areas of Paramecium membrane is technically difficult, so the cell was transversely dissected with a glass micro-needle into anterior and posterior fragments, and the thermosensitive responses of each fragment were examined.

Paramecium multimicronucleatum (supplied by Professor Dr K. Hiwatashi) was cultured in a hay infusion inoculated with Klebsiella pneumoniae. Paramecium cells at stationary phase were collected by low speed centrifugation and suspended in a solution of 2 mmol I⁻¹ KC1, 0·25 mmol I⁻¹ CaC12, 0·5 mmol I⁻¹ MgCl₂ and 2mmoll⁻¹ Tris-HCl (pH 7·2). This solution was used as a standard solution in all experiments. For experiments investigating ionic dependence, potassium or calcium concentration of the standard solution was varied. The culture and suspension solution were maintained at 22°C.

The cell was observed under an inverted microscope (80×), and then transversely dissected, approximately at the centre, with a glass micro-needle, into anterior and posterior fragments. Fragments which were actively swimming a few minutes after the dissection were used for experiments. Phase contrast images of a whole cell, and anterior and posterior fragments are shown in Fig. 1.

A whole cell or a cell fragment was placed in a glass vessel whose temperature was controlled by water flowing beneath it (Nakaoka & Oosawa, 1977). The temperature was monitored by a thermistor probe submerged in the vessel. Swimming behaviour was recorded with a camera mounted just above the vessel.

The method of recording from cells and cell fragments was similar to that described by Naitoh & Eckert (1972). The electrodes were filled with 1 moll⁻¹ KC1 and their resistances were 50–80 MΩ. The cells were placed in a glass vessel mounted on an inverted microscope. The temperature was changed by switching water flow beneath the vessel, and was monitored with a thermistor probe submerged in the vessel.

The reversal potential of the thermoreceptor response was studied using a current clamp. The membrane potential was set at various levels by injection of constant current of the order of 10⁻¹⁰A. One or two minutes after the potential had been set, and when it had become relatively stable, a temperature change was applied. Reversal potential was determined as the potential at which the electrical response changed its polarity.

A temperature change away from the culture temperature produced an increase in the frequency of directional changes in swimming in whole cells, as reported previously (Nakaoka & Oosawa, 1977), and in cell fragments (Fig. 2). In the whole cell (Fig. 2A), both lowering and raising from the culture temperature (22°C) increased the frequency of directional changes, and returning the temperature towards 22°C decreased the frequency of directional changes. In anterior fragments (Fig. 2B), there was a transient increase in the frequency of directional changes upon cooling below 22°C, but warming above 22°C had little effect on directional changes. However, in posterior fragments (Fig. 2C) there was a transient increase in the frequency of directional changes upon warming above 22°C, whereas cooling induced little change in the swimming behaviour. When only the anterior quarter of the cell was removed, the remainder of the cell still did not show the avoiding response upon cooling, but showed it upon heating.

These results show that sensitivity to warming is concentrated in the posterior part of the cell, whereas sensitivity to cooling is apparently restricted to a small portion of the anterior.

Both cooling and warming elicited a slow, transient depolarization in the whole ceil, together with fast depolarizing spikes (Fig. 3A). During warming a sustained depolarization was sometimes observed. Similar changes in membrane potential have been reported previously (Hennessey et al. 1983; Toyotama, 1981). In anterior fragments (Fig. 3B), a transient depolarization was induced upon cooling and a small transient hyperpolarization was induced upon warming. However, in posterior fragments (Fig. 3C), a fast depolarization was induced upon warming and only a small, steady hyperpolarization upon cooling. The changes in the membrane potential of the whole cell can be approximated by the sum of the changes in the two fragments.

In the absence of any thermal stimulus, anterior fragments showed spontaneous depolarizations, posterior fragments showed spontaneous hyperpolarizations, and whole cells showed potential changes in both directions.

The polarity and amplitude of the potential response to a thermal stimulus cou be changed by the injection of a constant current (Fig. 4). To allow repetitive stimulation during each recording, the thermal stimulus was removed after the initial phase of the response (about 1 min) and the potential was then restored. In the anterior fragment (Fig. 4A), the depolarization that was induced by cooling became larger when the membrane potential was made more positive than the resting potential. When the membrane potential was made more negative, the response to cooling became smaller until a potential was reached at which the response changed polarity. In the posterior fragment (Fig. 4B), the hyperpolarizing response to cooling became larger as the membrane was hyperpolarized, and became smaller and reversed in polarity at more positive values than the resting potential.

The reversal potentials obtained upon cooling were almost the same as those obtained upon heating, in both anterior and posterior fragments.

The membrane potential of Paramecium is mainly determined by membrane permeabilities for potassium and calcium ions (Naitoh & Eckert, 1974). The roles of these ions in the thermal response were therefore examined by measuring the reversal potential at various concentrations of K⁺ and Ca²⁺. The reversal potential of the anterior fragment showed a slope of 52 mV for a 10-fold change in the concentration of K⁺ (Fig. 5A) and was slightly dependent on Ca²⁺ (5 mV/log[Ca²⁺]₀) (Fig. 5B). The reversal potential of the posterior fragment showed a slope of 28 mV for a 10-fold change in the concentration of Ca²⁺ (Fig. 5B) and was almost independent of K⁺ (Fig. 5A).

The present results show that the thermal response of Paramecium to cooling can be ascribed to receptors on the anterior membrane, while the response to warming is mediated by receptors on the posterior part: the anterior membrane is responsible for transient depolarization upon cooling, whereas the posterior membrane is responsible for depolarization upon warming (Fig. 3). These depolarizations trigger the spike-like depolarizations which cause directional changes in swimming.

Depolarization of the anterior membrane upon cooling can be explained by a decrease in K⁺ permeability, since the reversal potential was dependent on [K⁺]_o and more negative than the resting potential. Depolarization of the posterior fragment upon warming can be explained by an increase in Ca²⁺ permeability since the reversal potential was dependent on [Ca²⁺]_o and was more positive than the resting potential.

The behavioural response to thermal stimulation is given after 2–3 h adaptation to a new temperature (Nakaoka et al. 1982). The present results suggest that this adaptation requires a modulation of membrane permeability to K⁺ and Ca²⁺. This may be related to the change in lipid composition that occurs in Paramecium following a change in temperature (Hennessey & Nelson, 1983).

The sites of the K⁺ and Ca²⁺ channels involved in thermoreception are opposite to those observed for the channels involved in mechanoreception, for which Ca²⁺ permeability is located on the anterior membrane and K⁺ permeability is mainly on the posterior part (Naitoh & Eckert, 1969; Ogura & Machemer, 1980). Such opposite sites suggest that the channels for thermoreception are different from those for mechanoreception.

Although the ionic mechanism of thermoreception in higher organisms is unknown, it has been reported that in afferent fibres of the rat the removal of extracellular K⁺ results in an increased frequency of cold receptor discharge (Pierau, Torrey & Carpenter, 1975) and that in the cat an increase in extracellular [Ca²⁺] causes a marked increase in warm receptor discharge (Hensel & Schafer, 1974). These features of afferent fibres agree well with the present results obtained on Paramecium, because such changes in [K⁺] and [Ca²⁺] increase the electromotive forces for the respective ions, which tend to increase the amplitudes of depolarizations induced by cold and warm stimuli. The ionic mechanism of thermoreception in Paramecium may thus be similar to that in higher organisms.

We thank Professor Fumio Oosawa and Dr Eva Prochniewincz-Nakayama for critical reading of the manuscript.