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Anoxia induces thermotolerance in the locust flight system

B. S. Wu, J. K. Lee, K. M. Thompson, V. K. Walker, C. D. Moyes and R. M. Robertson^*

Department of Biology, Queen’s University, Kingston, Ontario, Canada K7L 3N6

View larger version (20K): [in a new window]   Fig. 1. Diagram of an isolated locust thoracic ganglia preparation for intracellular recording from motoneurons. (A) The meso- (I) and meta- (II) thoracic ganglia with a flight muscle motoneuron. In this preparation, the motoneuron is stimulated extracellularly and recorded from intracellularly from the neuropil. (B) Sample trace of an action potential recorded from a motoneuron (Mn) at 25°C. Note the stimulus artefact preceding the action potential. (C) Sample trace of an action potential trace showing the parameters that were measured. Note the stimulus artefact preceding the action potential.

View larger version (15K): [in a new window]   Fig. 2. The effect of prior heat shock (3 h at 45°C) and anoxia (2 h in nitrogen) treatment on the survival of locusts at the normally lethal temperature of 53°C. Heat shock and anoxia pre-treatment significantly altered the thermotolerance of locusts by increasing the number of locusts alive after 0.5 h (G-test, G=10.09, d.f.=2, P<0.05) and 1 h (G-test, G=8.02, d.f.=2, P<0.05) at 53°C. Asterisks indicate a significant difference between control and experimental groups (P<0.05).

View larger version (22K): [in a new window]   Fig. 3. Effects of prior exposure to anoxic conditions on whole-cell K+ currents in locust neurons. Outward K+ currents obtained with voltage steps made from a holding potential of –60 mV in 10 mV increments from –80 mV to 40 mV. (A) Representative whole-cell voltage-clamp recordings from neurons in locust metathoracic ganglion slices of a control and an anoxia-treated animal at 25°C. (B) Mean I/V plots of control (N=9) and anoxia-treated (N=10) neurons at 25°C. Prior anoxia treatment significantly affected the I/V relationship (two-way ANOVA with Tukey pairwise multiple-comparison test, F=2.98, d.f.=12, P<0.001). Following anoxia, less current was evoked with each voltage step compared with control recordings. Values are means ± S.E.M. (C) Mean activation curves of whole-cell K+ currents recorded from control and anoxia-treated neurons at 25°C. G is the whole-cell conductance; Gmax is the maximal conductance at a voltage step to 40 mV. V1/2, the test potential at which there is half-maximal conductance, of control cells was –10.55±1.27 mV and did not differ from the V1/2 of –7.27±2.01 mV for anoxia-treated cells (t-test, t=1.34, P=0.20, d.f.=17). Asterisks indicate a significant difference between control and experimental groups (P<0.05).

View larger version (36K): [in a new window]   Fig. 4. The effect of prior anoxia treatment on whole-cell outward K+ currents of neurons in locust metathoracic ganglion slices with increasing temperature. Outward currents were obtained using voltage steps from a holding potential (Vh) of –60 mV made in 10 mV increments from –80 mV to 40 mV. (A) Representative traces of whole-cell outward K+ current recorded from neurons in locust metathoracic ganglion slices at different temperatures (25, 30 and 35°C) showing the current amplitude for both control and anoxia-treated animals. (B) Peak currents after anoxia treatment. When the temperature was held at 25°C, peak currents from neurons of control animals (N=9) were significantly greater than peak currents from cells of anoxia-treated animals (N=10) when Vh=–60 mV (two-way ANOVA with Tukey pairwise multiple-comparison test, F=4.58, P=0.04, d.f.=1). Anoxia treatment did not significantly alter the peak current evoked when Vh=–60 mV at 30°C (control, N=6; anoxia, N=6) or at 35°C (control, N=5; anoxia, N=6). Values are means + S.E.M. (C) Histograms of peak currents normalized to peak currents at 25°C. Control and anoxia currents were equally reduced when the temperature was increased from 25 to 30°C and to 35°C. Asterisks indicate a significant difference between control and experimental groups (P<0.05).

View larger version (27K): [in a new window]   Fig. 5. The effects of heat shock and anoxia on the thermosensitivity of action potentials recorded from locust forewing motoneurons. (A) Thermosensitivity of action potential latency in control (filled circles) (N=6), heat-shock (stippled circles) (N=9) and anoxia (open circles) (N=7) neurons. Latency in heat-shock neurons is significantly shorter than latency in control and anoxic neurons (two-way ANOVA with Tukey multiple pairwise comparisons, F=6.74, d.f.=2, P<0.05). No value for anoxia neurons at 45°C is shown because action potentials remaining at this temperature occurred spontaneously. (B) Thermosensitivity of action potential time to peak in control (filled circles) (N=6), heat-shock (stippled circles) (N=9) and anoxia (open circles) neurons (N=8). Time to peak in heat-shock neurons is significantly different from that of both control and anoxia neurons (two-way ANOVA with Tukey multiple pairwise comparisons, F=7.02, d.f.=2, P<0.05). (C) Thermosensitivity of action potential duration in control (filled circles) (N=6), heat-shock (stippled circles) (N=9) and anoxia (open circles) (N=9) neurons. Action potentials in anoxia neurons have significantly longer durations than those of both control and heat-shock neurons (two-way ANOVA with Tukey multiple pairwise comparisons, F=5.99, d.f.=2, P<0.05). (D) Thermosensitivity of action potential amplitude in control (filled circles) (N=6), heat-shock (stippled circles) (N=9) and anoxia (open circles) (N=6) neurons. Amplitude in heat-shock and anoxia neurons is significantly smaller than that in control neurons (two-way ANOVA with Tukey multiple pairwise comparisons, F=9.15, d.f.=2, P<0.05). Statistical tests include data only from temperatures between 22 and 35°C. At higher temperatures, the majority of control action potentials failed and, consequently, there were not enough data points for statistical comparisons at temperatures above 35°C. Values are means ± S.E.M.

View larger version (18K): [in a new window]   Fig. 6. The effect of prior heat shock on time to recover (i.e. time to self-right, see text for details) of whole locusts after exposure to anoxia. Heat shock significantly altered locust sensitivity to anoxia (two-way ANOVA, F=12.82, d.f.=1, P<0.001). Values are means + S.E.M. Exposure time of locusts to anoxia had a significant effect on time to recover (two-way ANOVA, F=64.42, d.f.=5, P<0.001). There was also a significant interaction between experimental treatment (i.e. control or heat shock) and time under anoxic conditions (two-way ANOVA, F=4.58, d.f.=5, P=0.001). Compared with controls, heat-shocked locusts took longer to recover after 4 h and 5 h under anoxic conditions (two-way ANOVA with Tukey pairwise multiple-comparison, F=4.58, d.f.=5, P=0.001). All columns represent data from seven animals with the exception of heat-shock animals at 6 h, where N=5 because two animals died.

View larger version (26K): [in a new window]   Fig. 7. The effects of prior heat shock, anoxia and exercise on the bioenergetic status of locust flight muscle as measured by the ratio [ArgP]/([ArgP]+[Arg]). (A) Heat shock alone did not disrupt [ArgP]/([ArgP]+[Arg]) (t-test, t=–2.05, P=0.09, d.f.=6). [ArgP]/([ArgP]+[Arg]) ratios were perturbed after 1 h under anoxic conditions in control (t-test, t=2.79, P=0.03, d.f.=6), control + recovery and heat-shock animals (three-way ANOVA with Tukey multiple pairwise comparison, F=17.22, P<0.001, d.f.=4). [ArgP]/([ArgP]+[Arg]) ratios indicated that energy reserves in all groups were restored after 2 h under anoxia. [ArgP]/([ArgP]+[Arg]) ratios increased after 1 h under anoxia despite animals being under constant anoxic conditions. The line through the control value at 0 h indicates the basal level of [ArgP]/([ArgP]+[Arg]). (B) I: heat shock alone did not significantly affect [ArgP]/([ArgP]+[Arg]). II: there was no significant difference between control animals kept at room temperature for 4 h and heat-shocked animals that had a recovery period of 1 h at room temperature. III: exercise resulted in a significant reduction in the [ArgP]/([ArgP]+[Arg]) ratio in the flight muscle of animals that had been previously subjected to heat shock. Values are means + S.E.M. (N=7–10). Asterisks indicate a significant difference between control and heat-shocked animals (P<0.05). A horizontal bar indicates no significant difference.