Oculomotor Function at Low Temperature: Antarctic Versus Temperate Fish

For several million years antarctic fish have inhabited the world’s coldest marine environment, where the mean annual sea temperature is as low as — 1·9 °C (Littlepage, 1965). For a temperate fish, cooling to this degree would be lethal; even if the body fluids remained supercooled and did not freeze, CNS motor programmes would cease (respiration: Friedlander, Kotchabhakdi & Prosser, 1976; eye movement: Montgomery, McVean & McCarthy, 1983), peripheral nerve conduction would fail (Macdonald, 1981) as would muscle function (McCarthy, 1982), and the activity of many enzyme systems would be drastically reduced (Clarke, 1983). The ability of antarctic fish to survive at — 1 · 9 °C depends on a range of adaptations which enable their vital systems to function. For example, their body fluids are prevented from freezing by an antifreeze component (DeVries, 1980) and the activity of many of their biochemical systems is partially temperature compensated. That is: the activity of these systems exceeds their activity in temperate species lowered to near freezing, but does not match that of temperate fishes at warm temperature (see Clarke, 1983).

Cold adaptation of enzymes and membranes occurs in the nervous system, allowing adequate function of the neuronal elements (Prosser & Nelson, 1981). In antarctic fish, there is temperature compensation of both peripheral nerve conduction (Macdonald, 1981) and neuromuscular function (Macdonald & Montgomery, 1982), though in comparison with temperate fish, obvious functional deficits remain. For example, nerve conduction velocity in antarctic fish is less than half that of temperate fish, and the refractory period of peripheral nerves is around 15-25 ms (Macdonald & Montgomery, 1982). The implications of these differences within the context of a functioning motor control system are explored in this study. The problem addressed is how fish maintain integrated activity at low temperature.

Choice of the oculomotor system for study offers considerable advantages: it is one of the best understood vertebrate motor control systems, eye movements include components of accuracy (visual stabilization) and speed (saccades), and detailed mechanical analyses can be made. It is a system in which the potent techniques of control systems analysis have proved to be particularly useful, and where despite its apparent complexity, a single first order model of the peripheral oculomotor system provides a satisfactory description for most purposes (Robinson, 1981). The parameters of the model can be derived from a study of eye movements elicited by abducens nerve stimulation (Montgomery, 1983), and this approach has been used to quantify the effects of acute temperature change on the oculomotor system (Montgomery & Paulin, 1984). A comparison of the peripheral oculomotor system of antarctic and temperate fish provides the opportunity to study genetically controlled temperature compensation within a complete motor output pathway.

Experiments were performed on three species of teleost fish. The temperate eurythermal Girella tricuspidata was caught in hand nets at night by divers using SCUBA, and kept in tanks of circulating sea water at 24 °C. The two species of antarctic fish, Pagothenia borchgrevinki and Dissostichus mawsoni, were caught on hand lines in the vicinity of Scott Base (77°51’S, 166°48’E), and experiments were carried out in a fish hut on the sea ice.

In fish anaesthetized with Tricaine, the cranium was opened and the brain removed leaving the stumps of the cranial nerves accessible intracranially (Fig. 1). Bleeding was controlled by cautery, and the fish were maintained in a tank by passing sea water at the appropriate ambient temperature over the gills. The stump of the abducens nerve (cranial nerve VI) was secured in a suction electrode, and the cranial cavity filled with Ringer. Electrical stimuli were trains of supramaximal square wave pulses (0·5 ms, 1·5-2 V). Two types of pulse trains were used to stimulate the nerve: constant frequency trains in the range 0-50 Hz for antarctic fish and 0-200 Hz for G. tricuspidata, or trains in which the pulse frequency was sinusoidally modulated at modulation frequencies in the range 0·04-8 Hz.

Eye movements produced by electrical stimulation of the abducens nerve were recorded by an opto-electronic movement detector (Helversen & Elsner, 1977), in which a reflected light spot was focused on a Schott barrier photodiode mounted in a single lens reflex camera body. Further details of the method are described elsewhere (Montgomery et al. 1983).

The force required to produce and maintain a given eye deviation was measured with a calibrated strain gauge connected to the surface of the eye. Force was applied tangentially to the eye surface to produce a movement in the same plane as that produced by abducens nerve stimulation. Deflection of the eye was measured both with a protractor positioned above the eye and with the opto-electronic movement detector.

These teleost preparations did not show the remarkable stability reported for the carpet shark (Montgomery, 1983). Some preparations exhibited little or no drop in response over the course of an experiment (e.g. Fig. 4), whereas others declined during the long periods of stimulation required for the low frequency sine waves. Gain measurements from such declining preparations were adjusted in proportion to the response to a standard stimulus pulse train. At the conclusion of the experiment good responses were still obtained from other extraocular muscles in the same preparation, indicating that any drop in responsiveness was due to fatigue of the activated muscle.

The most dramatic difference between antarctic fish and Girella tricuspidata was the efficacy of low frequency stimulation at the colder temperature. Single supramaximal pulses in both Pagothenia borchgrevinki and Dissostichus mawsoni produced large eye movements (Fig. 2). Twitch contraction time, the rate of relaxation and response latency were all longer in D). mawsoni (Fig. 2; Table 1). Maximal eye deviation in the antarctic species P. borchgrevinki and D). mawsoni (about 15 ° and 20° respectively) was produced at 50 Hz stimulation. In the temperate fish however, single stimulus pulses produced no observable eye movement, and stimulus frequencies of around 50 Hz were required to produce a threshold response (Fig. 3). The eye movement response in this species was still increasing at impulse frequencies of 200 Hz.

In all three species of fish, when eye rotation was plotted as a function of impulse frequency, there was an approximately linear section of the stimulus response curve, below saturation (Fig. 3). This region corresponds to the natural operating range of the oculomotor system (Montgomery, 1983; Zuber, 1968). The ‘d.c.’ gain (output/ input) of the oculomotor system can be specified by the slope of the stimulus response curve, and was highest inZ). mawsoni and least in G. tricuspidata (Fig. 3; Table 1). A second, independent, estimate of gain can be obtained from the ratio of the amplitude of eye movement to the amplitude of sinusoidal modulation of the stimulus pulse train at low modulation frequencies (e.g. Fig. 5). Estimates of gain derived in this way agreed well with the values determined from the slopes of the stimulus response curves (Table 1).

Pulse trains, sinusoidally modulated between 0 and 40 Hz for antarctic fish and 0-160 Hz for G. tricuspidata, were used to determine the frequency response of the oculomotor systems. As modulation frequency was decreased, the resulting eye rotation increased in amplitude, as shown for G. tricuspidata (Fig. 4). Maximum and minimum stimulus pulse frequencies were unchanged. The response to a standard constant frequency pulse train preceding each sine wave stimulus remained constant throughout the stimulus sequence in this example.

The sinusoidal nature of the eye movement response to a modulated pulse train in the antarctic fish may be seen with the expanded time scale of Fig. 5. Responses to single stimulus pulses were evident, and the low frequency of the modulating input sine wave allowed direct comparison of the stimulus and response wave forms. Amplitude of the modulated response was determined by averaging the trough-to-peak excursion of a series of modulated cycles.

Relative gain at each modulation frequency was calculated as the ratio between gain at the chosen frequency and maximum (d.c.) gain. A Bode plot (log relative gain versus log modulation frequency, Fig. 6) shows that within this frequency range, the peripheral oculomotor system behaves very much as a linear first order system. The characteristic frequency (half power frequency; frequency at which response has dropped by 3 dB) of each group of fish was determined using a non-linear regression technique (Helwig & Council, 1979) to fit the best first order model of the form:

There was an approximately linear relationship between force (applied tangential to the eye surface) and angular deviation of the eye. The slope of this relationship was taken as an estimate of the elastic coefficient of the peripheral oculomotor system (Table 1). The elastic coefficient tended to increase with low temperature and with increased size, so that the tension required for a given eye deviation was greater in both antarctic fish, but considerably greater in the large D. mawsoni.

Preliminary experiments were performed on the effects of acute temperature change on the characteristic frequency of the peripheral oculomotor system of G. tricuspidata. The characteristic frequency was strongly temperature dependent, decreasing from 1·4 Hz at 24 °C to 0·8 Hz at 16 °C and 0·25 Hz at 12°C in a 24 °C-acclimated fish.

The approach adopted in this paper has been to study the relationship between supramaximal stimulation of the abducens nerve and resulting eye movement. It has been suggested that the response characteristics obtained in this way will be dominated by the fast motor units (Montgomery, 1983). Teleost fish present a unique opportunity to test this hypothesis, for, unlike other vertebrates, the extraocular muscles are composed of two discrete regions of red and white muscle fibres, and the abducens motoneurones are correspondingly divided into two subgroups (Sterling, 1977). The motoneurones of the caudal subgroup have a characteristic phasic-tonic activity during eye movements and are thought to innervate the white muscle, whereas the activity of rostral motoneurones is related only to eye position, and these units are thought to innervate the red fibres (Gestrin & Sterling, 1977).

Parameters of the model of the peripheral oculomotor system determined from supramaximal abducens nerve stimulation are in good agreement with the parameters determined from the firing patterns of phasic-tonic motoneurones. The slow motoneurones would not be expected to make a substantial contribution to the eye movement dynamics but would contribute to the low frequency gain, and may introduce a degree of phase lag in the response to low frequency sine wave stimulation (Montgomery, 1983).

The characteristic frequency of the peripheral oculomotor system is determined by the visco-elastic coupling of the globe to the orbit (Collins, 1971). Increasing viscosity will lower the characteristic frequency, as will decreasing elasticity. The combined effect of temperature on these two variables results in a very strong temperature dependence of characteristic frequency in the temperate fish. For G. tricuspidata acclimated to 24 °C the characteristic frequency measured at 12 °C is already considerably lower than that of the antarctic fish, indicating a degree of temperature compensation in the mechanical coupling of globe to orbit in antarctic fish. Connective tissue sheaths on the extraocular muscles of antarctic fish (J. C. Montgomery & J. A. Macdonald, unpublished observation) may provide the increased elasticity.

For a first order system, the lower the characteristic frequency the slower the response is to a given step input. Thus saccade velocity may be related to characteristic frequency, and for a given burst rate in the motoneurones a lower characteristic frequency would result in a reduced saccade velocity. The changes in characteristic frequency with temperature may thus explain some of the observed effects of temperature on saccade velocity. Saccade velocity decreases with decreasing temperature for G. tricuspidata (Montgomery et al. 1983), but is clearly temperature compensated in antarctic fish (Montgomery & Macdonald, 1983). Presumably changes in burst rates of oculomotor neurones with temperature will also contribute to the observed effects of temperature on rates of rapid eye movement.

Changes in the characteristic frequency of the peripheral oculomotor system will also affect the degree of central processing required to produce slow compensatory eye movements. Over the working range of the vestibulo-ocular reflex, afferent input from semicircular canals is in phase with head velocity. This signal must be integrated to produce eye movements in phase with head position in order to stabilize the visual field (Carpenter, 1977). For frequencies of head movement above the characteristic frequency of the peripheral oculomotor system, this integration step can be performed by the sluggish response of the motor pathway, while at lower frequencies, this integration must be performed by the central nervous system. At present insufficient is known about the frequency match between natural head movements, vestibular input and frequency response characteristics of the oculomotor system to assess adequately the relative importance of the contribution of oculomotor dynamics to the production of accurate compensatory eye movements.

The gain of the peripheral oculomotor system is inversely related to temperature (Montgomery, 1984), both for acute temperature change within one species and for fish adapted to different temperatures. The gain of the peripheral oculomotor system in the two antarctic species at -1·5 °C is considerably higher than the gain of the temperate fish. The increase in gain at low temperature can be explained in terms of the known effects of temperature on twitch contraction. Cooling decreases the rate of muscle relaxation and hence each muscle twitch is increased in size and prolonged (Hill, 1951; Wardle, 1980; Montgomery & Macdonald, 1984). The result of these changes is to produce summation at lower frequencies of stimulation, thereby increasing the slope of the stimulus-response curve; i.e. increasing gain at low temperature.

Large muscles have a slower twitch response (Wardle, 1980), which explains why the oculomotor responses of D. mawsoni (Fig. 2) exhibit a larger gain than those of the smaller antarctic fish P. borchgrevinki.

The large increase in gain observed in the antarctic fish at low temperature is likely to be of biological significance in offsetting reduced rates of CNS output, and lower sensory gain expected at low temperature (Montgomery & Paulin, 1984).

The authors wish to acknowledge grants from the Medical Research Council, the University Grants Committee, and the Auckland University Research Committee. We are grateful to the staff of New Zealand’s Scott Base, Antarctica, and to the staff of the Leigh Marine laboratory.