Temperature acclimation of the caudal photoreceptor response in the crayfish Orconectes rusticus (Girard)

JIM H. BELANGER Department of Zoology, University of Toronto, Toronto, Ont., Canada M5S 1Al

Received July 15, 1987

BELANGER, J. H. 1988. Temperature acclimation of the caudal photoreceptor response in the crayfish Orconectes rusticus (Girard). Can. J. Zool. 66: 1168-1171.

Crayfish (Orconectes rusticus (Girard)) were acclimated for 3 weeks at 5 and 25°C. The effects of temperature and tempera- ture acclimation on the latency, maximum frequency, and sensitivity to stimulus intensity of the caudal photoreceptor response were examined in isolated abdominal nerve cords. Each of these components was temperature dependent. The maximum fre- quency of the response showed thermal capacity acclimation but latency did not. Caudal photoreceptor response was insensi- tive to stimulus intensity at low temperatures but acclimation improved sensitivity.

BELANGER, J. H. 1988. Temperature acclimation of the caudal photoreceptor response in the crayfish Orconectes rusticus (Girard). Can. J. Zool. 66 : 1168- 1171.

Des ecrevisses (Orconectes rusticus (Girard)) ont ete acclimatees durant 3 semaines B 5 et B 25 "C. Les effets de la tempera- ture et de l'acclimatation B la temperature sur le temps de latence, sur la frequence maximale et sur la sensibilite B l'intensite du stimulus de la reaction du photorecepteur caudal ont ete examines dans des cordons nerveux abdominaux isoles. Les trois com- posantes de la reaction dependent de la temperature. La capacite d'acclimatation thermique se manifeste par une modification de la frequence maximale de la reaction, mais le temps de latence ne demontre aucune capacite d'acclimatation thermique. La reaction du photorecepteur caudal reste insensible B l'intensite du stimulus aux temperatures plus basses, mais l'acclimatation augmente la sensibilite.

[Traduit par la revue]

Introduction Methods and materials Poikilotherms inhabiting environments characterized by

stable temperatures have been shown to be highly intolerant of temperature fluctuations. Most organisms, however, come from more heterogeneous environments and have thus been forced to evolve mechanisms to cope with variable tempera- tures. One such mechanism, thermal capacity acclimation, is the ability to shift the optimal temperature for a given physio- logical process in response to chronic temperature changes (Prosser and Nelson 198 1).

There is evidence for thermal acclimation at several loci within the nervous system. Lagerspetz and Talo (1967) showed acclimatory changes in the functional parameters of earthworm axons, while Stephens and Atwood (1982) found alterations in both synaptic and muscle membrane properties in a crab. In vertebrates, Roots and Prosser (1962) have sug- gested synapses as sites of thermal acclimation.

The caudal photoreceptor of crayfish is interesting because it functions both as a central nervous system (CNS) inter- neuron (Kennedy 1963) and as a primary sensory neuron (Wilkens and Larimer 1972). First described by Prosser (1934), it consists of a single photosensitive cell in each half of the sixth abdominal ganglion (Wilkens and Larimer 1972). The caudal photoreceptor response (CPR) has been well characterized (Galeano 1976) and shown to mediate a negative photokinetic response (Harris and Stark 1972). It also displays a marked temperature sensitivity. Larimer (1967) reported very high (> 30) Qlo values for both maximum response fre- quency and latency of CPR.

This combination of accumulated information and behav- ioural relevance makes the crayfish CPR a useful preparation in which to study temperature acclimation and, indeed, Kivivuori (1982) has already demonstrated that acclimation does occur. The present study replicates some of her findings and further demonstrates that acclimation significantly improves the sensitivity of CPR.

Crayfish (Orconectes rusticus (Girard)) were obtained commer- cially and maintained in dechlorinated tap water under a 9L:15D photoperiod. To induce temperature acclimation, animals were main- tained at 5 + 1 "C or 25 & 1 "C for a minimum of 3 weeks. As no sex- ual differences in acclimation were noted during the study (see also Spoor 1955), animals of both sexes were used. The crayfish were in intermoult stage C (Drach 1939).

For experiments, isolated abdominal nerve cords were pinned out, ventral side up, under crayfish solution of pH 7.4 (van Harreveld 1936). The preparations were then held in darkness for 1 h at the acclimation temperature to stabilize. The temperature of the recording chamber was maintained by a water bath and was not allowed to vary at a rate greater than 0.2"C/min. It was also held constant for 10 min before any experiment to prevent effects due to temperature transients (e.g., see Kerkut and Taylor 1958).

Light stimuli of about 10 s duration were obtained from a Jena SM XX microscope lamp (approx. 5000 Ix) directed at the preparation. To vary the stimulus intensity, neutral optical density filters were placed in the light path. Except when being stimulated, each prepara- tion was kept in darkness. A minimum of 5 min was allowed between stimuli. (Preliminary experiments demonstrated that this was suffi- cient for the response to recover to base-line levels.)

Nervous activity was recorded extracellularly by platinum hook electrodes placed on the connectives of the fourth abdominal gan- glion. The signals were amplified conventionally and stored on magnetic tape. For analysis, data were run through a window dis- criminator set to pick out the large spikes of the CPR and were then digitized to produce frequency histograms. From these, latency was measured from the end point of the time bin displaying the stimulus onset artefact to the midpoint of the time bin displaying the highest response frequency.

Statistical analyses used Student's t-test, and differences were con- sidered significant at the P < 0.01 level.

Results In response to a brief pulse of light, the spiking activity of

the CPR neurons increases to a maximum and then exponen- Printed in Canada 1 Impnme au Canada

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BELANGER

2 light response \ 1

spontaneous

001 * Time a f t e r d issect ion (h)

FIG. 1. Activity of the crayfish caudal photoreceptor neurons in the isolated ventral nerve cord over the time course of these experiments. Circles, animals acclimated at 25OC; squares, animals acclimated at 5°C. Measurements were made at 22OC and are the means + SEM of four experiments.

tially decays back to spontaneous firing levels. Both the latency and maximum frequency of the response are dependent on the intensity of the stimulus (see below, latency data not shown).

Figure 1 illustrates the parameters measured in these experi- ments as a function of time after dissection. Latency and maxi- mum response frequency of CPR, as well as the level of spontaneous activity, all maintained their initial values over the time course of the experiments. One preparation, tested after 10 h, still showed no significant change in CPR.

In warm-acclimated crayfish, cold block for CPR occurred at 9.1 f 1.1 "C (n = 6), while the response was still present in cold-acclimated animals at 3 "C (n = 5). Heat block for CPR in cold- and warm-acclimated groups occurred at 34.3 f 0.8"C (n = 6) and 38.1 f l.O°C (n = 6), respectively. Both these differences are statistically significant.

Variations in the latency of CPR with temperature are shown in Fig. 2. There are at least two clear divisions in each curve. The cold-acclimated group showed a highly temperature- dependent response below 15 "C and a temperature-indepen- dent response from 15 to 25 "C. Similar divisions were present in the warm-acclimated group from 10 to 20°C and from 20 to 30°C. There were no significant differences between the two groups in latency as a function of temperature.

Tempera ture ( O C ) FIG. 2. The effect of temperature on latency of CPR. The points are

means f SEM of 10 experiments, except for warm-acclimated at 5OC (4 only; 6 failed because of cold block) and cold-acclimated at 35OC (5 only; 5 failed because of heat block). Circles, animals acclimated at 25°C; squares; animals acclimated at 5OC.

In the middle range of temperatures (20 -25 "C) there were no significant differences in maximum response frequency between the two groups (Fig. 3). Above this region, however, there was a significantly greater response from the warm- acclimated group. Conversely, below this range there was a significantly greater response from the cold acclimated group. Figure 3 also shows that the temperature-response curve is shifted horizontally by acclimation.

The effect of temperature on the sensitivity of CPR to stim- ulus intensity is shown in Fig. 4. At 5°C there was little change in the response frequency of either group over a 100- fold range of stimulus intensity. In fact, with the exception of the point for the cold-acclimated group at the maximum stim- ulus intensity, none of the responses were significantly above the level of spontaneous activity for their respective groups. At 15"C, the warm-acclimated animals still did not respond significantly above base-line levels, while the cold-acclimated animals produced a typical stimulus -response curve. Both groups did so at 25°C.

Discussion

The data presented show that thermal capacity acclimation of the maximum response frequency of CPR occurs in cray- fish. This finding is similar to that of Kivivuori (1982). Fur- ther, the data demonstrate that acclimation can improve the sensitivity of CPR over a range of temperatures, which has not been previously reported.

To date, the extreme temperature sensitivity of CPR has not been explained. Since the CPR neurons are primary sensory cells (Wilkens and Larimer 1972), the temperature sensitivity is not due to synaptic effects. Temperature has been shown to affect the coupling mechanism between sensory generator potentials and axonal spike firing (Ottoson 1965), so this is a likely location for at least part of the temperature effect. The

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1170 CAN. J . ZOOL. VOL. 66, 1988

Temperature ( O C ) FIG. 3. The effect of temperature on the maximum response fre-

quency of CPR. The points are from the same experiments as in Fig. 2 (sample sizes the same as in Fig. 2). Asterisks indicate pairs of points that are significantly different. Points are means f SEM. Circles, animals acclimated at 25°C; squares, animals acclimated at 5°C.

finding that spontaneous activity has a low Qlo relative to CPR led Larimer (1967) to suggest that the temperature effect involves intermediate steps having large Qlo values in the photochemical react ion.

From the present study, it is not clear which of the possible factors is responsible for the temperature effects. It is possible, however, to draw some conclusions about the locus of the acclimatory effects. Since the latency was not significantly affected by acclimation, then the transduction process (includ- ing the photochemical reactions) is probably not a site of major acclimatory changes. In contrast, the maximum frequency of the response is a direct consequence of membrane properties governing the kinetics of action potentials (e.g., duration of rising phase, hyperpolarization, channel inactivation), some of which have been shown to be affected by temperature and by acclimation (Lagerspetz and Talo 1967). These changes are probably a result of changing membrane lipid composition (Cossins 1977). (Such changes in membrane composition have been shown in crayfish (Farkas and Nevenzel 1981), but the results are not conclusive because the different experimental groups were held under different photoperiods and crayfish show seasonal variation in acclimatory abilities (Kivivuori 1980).) Such an alteration in the membrane properties could explain the changes in CPR sensitivity. For example, as the duration of the action potential increases, the maximum possible response frequency decreases. If a low-intensity stim- ulus is sufficient to reach this maximum frequency, then the in- tensity-response curve will be relatively flat, as observed here. Acclimatory changes that returned the action potential duration to a "normal" value would increase the frequency maximum and hence possibly "unflatten" the response curve.

Given the presumed biological significance of CPR (media- tion of a negative photokinetic response (Harris and Stark 1972)) there are interesting consequences of the stimulus

Log stimulus intensity

FIG. 4. The effect of temperature on the sensitivity of CPR to stim- ulus intensity. Each graph illustrates the effect of stimuli of varying intensity (arbitrary units) on maximum response frequency of CPR at the temperature indicated. Points are means f SEM of 4 - 10 experi- ments. Circles, animals acclimated at 25°C; squares, animals accli- mated at 5°C.

intensity - response data shown in Fig. 4. A decrease of 10°C in environmental temperature is apparently enough to abolish the negative photokinesis. This would probably result in increased risk of predation for the animal (due to its failure to remain concealed under rocks, etc.). However, because accli- mation allows the animal to compensate for the decreased temperature, slow environmental changes are easily accommo- dated. It would be intersting to see if the crayfish has other mechanisms that have evolved to deal with rapid temperature changes .

Acknowledgements

I thank Dr. Anne-Jane Tierney for identifying the crayfish for me, Mr. Jim Stanners for his excellent technical assistance, and Dr. Ian Orchard for reading a draft of this manuscript.

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