REGULATION OF HIBERNATING PERIODS BY TEMPERATURE.

1058 PHYSIOLOGY: TWENTE AND TWENTE PROC. N. A. S. The author wishes to thank Mr. Robert A. Brookshire for his excellent assistance during the course of this work, and H. Rickenberg and G. Williams for valuable suggestions. * Supported by grants G 24052 and GE 3612 from the National Science Foundation. 1 Beauchesne, G., M. Leboeuf, and R. Goutarel, in Regulateurs Naturels de la Croissance Vegtale (Paris: Centre National de la Recherche Scientifique, 1964), p. 119. 2 Letham, D. A., in Regulateurs Naturels de la Croissance Vegetale (Paris: Centre National de la Recherche Scientifique, 1964), p. 109; and ref. 6 (below). 3Miller, C. O., these PROCEEDINGS, 47, 170 (1961); and refs. 13 and 16 (below). 4Letham, D. S., and C. 0. Miller, Plant Cell Physiol., in press. 6 Letham, D. S., J. S. Shannon, and I. R. McDonald, Proc. Chem. Soc., 1964, 230. 6 Letham, D. S., Life Sci., 2, 569 (1963). 7 Kefford, K. P., Science, 142, 1495 (1963). 8 Miller, C. O., in Modern Methods of Plant Analysis (Berlin: Springer-Verlag, 1963), vol. 6, p. 194. 9 McCalla, D. R., D. J. Moore, and D. Osborne, Biochim. Biophys. Acta, 55, 522 (1962). 10 Hurlbert, R. B., H. Schmitz, A. F. Brumm, and V. R. Potter, J. Biol. Chem., 209, 23 (1954). 11 Jacobson, K. B., Science, 138, 515 (1962). 12 Khym, J. X., and W. E. Cohn, J. Am. Chem. Soc., 76, 1818 (1954). 13 Miller, C. O., Plant Physiol., 37, xxxv (1962). 14 Loeffier, J. E., and J. Van Overbeek, in Regulateurs Naturels de la Croissance V~ggtale (Paris: Centre National de la Recherche Scientifique, 1964), p. 77. 15 Fox, J. E., Plant Physiol., 39, xxxi (1964). 16 Miller, C. O., and F. H. Witham, in Rlgulateurs Naturels de la Croissance V~ggtale (Paris: Centre National de la Recherche Scientifique, 1964), p. I (erratum). REGULATION OF HIBERNATING PERIODS BY TEMPERATURE* BY JOHN W. TWENTE AND JANET A. TWENTE UNIVERSITY OF UTAH, SALT LAKE CITY Communicated by Henry Eyring, August 5, 1965 Hibernation of golden-mantled ground squirrels (Citellus lateralis) is characterized by periods of dormancy which are interrupted at intervals by arousal with a return to the homeothermic state.1 We have shown2 that the duration of hibernating periods of this species is related to the body temperature achieved; hibernating periods at 40C averaged approximately twice as long as those at 11 'C. These observations suggested that the following should be investigated: over what temperature range is hibernation possible; is the temperature dependency of the duration of hibernating periods continuous over the possible range; and if the temperature dependency is continuous, what function does the time/temperature relationship best approximate. Methods.-Adult golden-mantled ground squirrels of both sexes were caged singly and maintained undisturbed in two dark, relatively soundproof, constant-temperature rooms. Maximum temperature fluctuations at the specific places in cages where hibernation occurred (the microenvironmental temperature) were less than ±0.50C. Thirty-gauge, iron-constantan, Teflon-coated thermocouples (±0.050C) were implanted subdermally 1-2 months prior to experimentation with the sensing element tied to a lower rib in the midventral region.2 When ground squirrels were curled in the hibernating posture, temperatures recorded from the rib area were not different from heart and liver (core) temperatures as recorded by a hypodermic thermocouple probe. Temperature records were printed at 6-min intervals on VOL. 54, 1965 PHYSIOLOGY: TWENTE AND TWENTE 1059 time-stamped chart paper by two Leeds and Northrup Speedomax G 16-point recorders calibrated from 0 to 50'C ( ±0.080C). Continuous recordings of body temperature were measured during two hibernating seasons at 11 different temperature levels ranging from 2 to 250C. Experiments involving hibernation at microenvironmental temperatures above 250C were not attempted. Different groups of ground squirrels were utilized for the two hibernating seasons and for each temperature level, thereby allowing for the determination of variation within the laboratory population. The duration of the hibernating period was measured in hours from the time the body temperature reached 20C above the microenvironinental temperature during entrance into hibernation until the initiation of arousal. Thermocouples were placed in the nest beneath ground squirrels with surgically implanted thermocouples and control ground squirrels. The durations of the hibernating periods for a temperature level did not differ between the two groups, thereby indicating that normal hibernating behavior was not altered by the surgical procedures or the presence of permanently implanted thermocouples. The average duration of the hibernating period for each ground squirrel at a temperature level was calculated from recordings of two to six successive hibernating periods. The average duration of the hibernating period for each group at a temperature level was calculated from individual averages. 300 200 150 100 50\ 3 0 o F-129 _ £ : F-501 20- * Other Squirrels I0 2 4 6 8 10 12 19 25 30 36 Temperature (c) FIG. 1.-The average durations of hibernating periods for individual ground squirrels are plotted against the body temperatures. Averages for the population sampled at each temperature level are indicated by lines. Patterns of hibernating behavior of three females (F-94, F-501, and F-129) illustrate long-period, average-period, and short-period hibernators. The data reported in Figure 1 regarding the duration of hibernating periods were obtained from 74 undisturbed ground squirrels with surgically implanted thermocouples which had exhibited all established pattern of winter hibernating behavior. Temperature records obtained from the thermocouples of three active, nonhibernating ground squirrels maintained at 250C showed periods of 7-11 hr daily at which time the body temperature fell from the mean temperature of activity (approximately 380C) to 360C. These records represent periods of quiescence and may be indicative of sleep. Results and Discussion.-The durations of hibernating periods for individuals at each temperature level were relatively consistent; successive periods were usually close to the average duration at each temperature level. Individual variation seldom exceeded 20 per cent. Differences between individuals, however, sometimes exceeded 100 per cent (ref. 2 and Fig. 1). No sexual differences were demonstrated in regard to the duration of hibernating periods of undisturbed individuals. Generally, ground squirrels which hibernated longer than the population average for a temperature level behaved similarly at all temperatures studied as illustrated 1060 PHYSIOLOGY: TWENTE AND TWENTE PROC. N. A. S. by Female 94 (F-94, Fig. 1). Average-period hibernators (F-501) tended to be near the population average at different temperatures, and short-period hibernators (F-129) were usually below the population average. A line between 2 and 250C was fitted to the population averages (Fig. 1) by the least-squares analysis (log hours = 2.425 -0.0405 body temperature). The difference between the average duration for the population and the computed average for each temperature level was less than 1 7 per cent of the expected duration at each point except for 7°C (- 16%) and 130C (+12%). These discontinuities in the linear relationship can be explained on the basis that the group represented at 70C had a preponderance of short-period hibernators, whereas the group at 130C consisted primarily of long-period hibernators as determined by the patterns of hibernating behavior exhibited by these individuals at other temperatures. The data obtained at a body temperature of 360C which were interpreted as possibly representing sleep fit the extrapolation of this line. .051 o .01 - .005 325 335 345 355 365 IO5/K 36 25 19 13 9 2 C Temperatures FIG. 2.-The reciprocals of the average durations of the hibernating periods of each population are plotted against the temperatures expressed as 10/0K. Corresponding temperature levels are indicated in 'C. We have assumed that the rate of the process limiting the duration of hibernating periods is proportional to the reciprocal of the duration of the hibernating period in order to construct a graph which is equivalent to an Arrhenius plot. This graph (Fig. 2) shows the relationship between the log of the reciprocal of the average duration of the hibernating period and the reciprocal of the temperature in OK. The points on this graph between 2 and 190C appeared, by inspection, to represent a straight-line relationship. A line best fitting the data between these temperatures was calculated by the least-squares analysis. The data obtained from the relatively small number of animals at 25 and 360C fit this line. The apparent energy of activation3 represented by the slope of this line has a value of 15.5 kcal/mol. Because of the linearity of the data plotted in Figures 1 and 2, it is tempting to assume that the duration of the hibernating period may be limited by a single regulating process which operates similarly at all temperatures between 2 and 250C. Since the data obtained at the slightly depressed body temperature of 360C fit extrapolations of the lines in Figures 1 and 2, it is suggested that a relationship between the duration of hibernating periods and the duration of sleep may exist. VOL. 54, 1965 PHYSIOLOGY: LORENTE DE N(6 AND HONRUBIA 1061 Summary.-The duration of hibernating periods of golden-mantled ground squirrels was measured at body temperatures ranging from 2 to 250C. The log of the duration of hibernating periods was shown to be a function of body temperature throughout this range with no apparent discontinuities. It was postulated that a single process operating between 2 and 250C may limit the duration of hibernating periods. * These studies were supported by research grant AM-05942 from the National Institutes of Health, USPHS, and by the University of Utah Research Committee. I Pengelley, E. T., and K. C. Fisher, Can. J. Zool., 39, 105 (1961). 2 Twente, J. W., and J. A. Twente, J. Appl. Physiol., 20, 411 (1965). 3Glasstone, S., K. J. Laidler, and H. Eyring, in The Theory of Rate Processes (New York and London: McGraw-Hill, 1941), p. 1. THEORY OF THE FLOW OF ACTION CURRENTS IN ISOLATED MYELINATED NERVE FIBERS, VI* BY R. LORENTE DE N6 AND V. HONRUBIAt THE ROCKEFELLER UNIVERSITY Communicated June 10, 1965 We continue the presentation of the theory of the isolated fiber.' Forward and Backward Jumps of the Impulse.-When, with node N, in the central pool, the stimulating device is placed across the first gap and the amplifier across the second, poliphasic action potentials are often recorded (ref. la, Fig. 2, 1-8; if, Fig. 2, 1-4) which may be interpreted as the result of successive jumps of the impulse, first forward and then backward. However, since the late upward peaks may remain after anesthesia of the segment of fiber in the distal pool (la, Fig. 2, 9-15), the poliphasic spikes may also be interpreted in this manner. All the active zones are created by the applied current, the temporal order of their appearance being determined by the distributions of the electrotonus and of the stimulation threshold in the internodes. Figure 1 illustrates a situation for which there is only one possible interpretation. The situation is exceptional because it can appear only when, owing to differential injury done during the isolation process, the stimulation threshold is lower in the distal pool than in the neighborhood of the central pool (cf. ref. ic). Since node N, was located at the center of the 270-u-wide central pool, a zero-resistance shunt placed across the first gap blocked propagation of the impulse (Fig. 1, 2; cf. ref. lb). Propagation, however, was restored when in addition a 1-megohm shunt was placed across the second gap. Records 3-8 (Fig. 1) belong to a series obtained at 3-sec intervals during a 3-min period; they present the extreme types of action potentials. The variability of the response was referable to oscillatory changes in the flow of the demarcation currents (ref. la) which caused small up and down displacements of the base line. At times propagation failed to occur and only a small upward deflection was recorded (Fig. 1, 7), but as a rule the initial upward peak was interrupted by asharp downward peak, which was followed by two upward peaks (Fig. 1, 4 and 6), and at times the two late upward peaks became fused into a large