Body temperature patterns before, during, and after ... - Springer Link

J Comp Physiol B (2002) 172: 47±58 DOI 10.1007/s003600100226

O R I GI N A L P A P E R

R.A. Hut á B.M. Barnes á S. Daan

Body temperature patterns before, during, and after semi-natural hibernation in the European ground squirrel

Accepted: 31 July 2001 / Published online: 20 September 2001 Ó Springer-Verlag 2001

Abstract Ground squirrels undergo extreme body temperature ¯uctuations during hibernation. The e€ect of low body temperatures on the mammalian circadian system is still under debate. Using implanted temperature loggers, we recorded body temperature patterns in European ground squirrels kept in an enclosure under natural conditions. Although hibernation onset was delayed, hibernation end corresponded closely to that measured in a ®eld population. Circadian body temperature ¯uctuations were not detected during deep torpor, but indications of circadian timing of arousal episodes at higher temperatures were found at the beginning and end of hibernation. One male exhibited synchronised arousals to a relatively constant phase of the day throughout hibernation. All animals ®rst entered torpor in the afternoon. Daily body temperature ¯uctuations were decreased or distorted during the ®rst days after hibernation. We hypothesise that hibernation may a€ect the circadian system by either decreasing the expression of the circadian oscillator, or by decreasing the amplitude of the circadian oscillator itself, possibly due to gradual, temperature dependent, internal desynchronisation. The latter mechanism may be bene®cial because it might facilitate post-hibernation re-entrainment rates. Communicated by G. Heldmaier R.A. Hut (&) á S. Daan Zoological Laboratory, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands E-mail: [email protected] Tel.: +33-4-72913478 Fax: +33-4-72913461 B.M. Barnes Institute of Arctic Biology, University of Alaska, Fairbanks, Alaska USA 99775-7000 Present address: R.A. Hut INSERM U371 `Cerveau et Vision', 18, Avenue du Doyen LeÂpine, 69675 Bron, France

Keywords Circadian rhythmicity á Torpor á Euthermia á Arrhythmicity á Implantable temperature loggers Abbreviations DD continuous darkness á LD light-dark cycle á SCN suprachiasmatic nuclei

Introduction Two important topics in research on biological rhythms during hibernation are the regulation of circannual rhythms and the e€ect of low body temperature on circadian rhythms. Both topics are still debated, and possible di€erences between genera and species of hibernators have obscured a general picture (KoÈrtner and Geiser 2000). Several studies have reported circadian patterns in the timing of torpor bouts or arousal intervals under light-dark cycle (LD) or continuous darkness (DD) conditions or entrained patterns under LD conditions (Daan 1973b; Pohl 1987, 1996; Twente and Twente 1987; Canguilhem et al. 1994; Grahn et al. 1994; Wollnik and Schmidt 1995; Waûmer and Wollnik 1997; KoÈrtner et al. 1998). Such patterns are more easily detected and indeed better documented when torpor bouts are short (Daan 1973b; Waûmer and Wollnik 1997; Waûmer 1998). When torpor bouts are long (>5 days) it is often unclear whether the timing of the subsequent arousal is timed by a circadian system. Persisting circadian oscillations in body temperature during torpor have only rarely been documented (Menaker 1959, 1961) and appear to be highly variable (Grahn et al. 1994). Although the suprachiasmatic nuclei (SCN) are among the few brain structures maintaining relatively elevated glucose uptake during hibernation (Kildu€ et al. 1989), the evidence for persistent circadian functioning is not overwhelming. If circadian oscillations persist in deep torpor, their functional role is obscure. Most hibernators remain underground during euthermic phases. It is unclear both as to how circadian timing of these arousals would be entrained in the

48

absence of light cues and which selection pressures would favour daily timing. Such questions call for data obtained under naturalistic hibernation conditions. Hibernation patterns have frequently been measured under laboratory conditions (for review see: KoÈrtner and Geiser 2000). Such conditions are characterised by unnatural ambient temperature patterns and di€erent thermal insulation properties of the nest site from those in natural hibernacula (Strijkstra 1999). Body temperature data collected during hibernation under natural conditions are scarce (Wang 1979; Barnes 1989; Barnes and Ritter 1993; Waûmer 1998), and do not focus on circadian rhythms of euthermic body temperature patterns before and after the hibernation season. Field data recorded with temperature-sensitive collar transmitters may re¯ect timing of arousal episodes, but are not accurate or collected frequently enough to detect possible circadian body temperature ¯uctuations (Young 1990; Michener 1992). Laboratory studies indicate a gradual loss of internal synchronisation of the circadian oscillator at temperatures below 15 °C (Pohl 1981) and persistence of circadian patterns may depend on the duration and the level of minimum brain temperatures reached during torpor. In this study we record body temperature patterns before, during, and after deep hibernation and the time of year and the time of day when hibernation starts and ends. Data are analysed to detect possible daily or circadian patterns during hibernation in torpor body temperature and timing of arousals. Several lines of evidence indicate that oscillations of the circadian system are decreased, if not lost, at brain temperatures below 15 °C as they occur during deep hibernation. In the Turkish hamster, circadian rhythmicity in the ®rst few days after hibernation at 8 °C is disturbed, and Pohl (1981) suggested ``either arrhythmicity or internal desynchronisation of components of the circadian system during natural hypothermia''. In addition, circadian ¯uctuations in deoxyglucose uptake in the SCN of euthermic mammals (Schwartz et al. 1983) appears to be lost in golden-mantled ground squirrels hibernating at 5 °C (Kildu€ et al. 1989). In vitro experiments showed a temperature e€ect on the period of the SCN in rats and, to a lesser extent, also in ground squirrels (Ruby and Heller 1996). SCN neurons in brain slices of golden-mantled ground squirrels cease electrical activity below temperatures of 15 °C (Miller et al. 1994). On the other hand, circadian body temperature ¯uctuations in bats (Menaker 1959, 1961) and ground squirrels (Grahn et al. 1994) are reported to persist at ambient temperatures of 8±10 °C. In this study, we recorded body temperature patterns of European ground squirrels kept in outdoor enclosures under natural conditions before, during and after hibernation. Body temperatures were recorded using temperature data loggers implanted in the abdominal cavity. This proved to be a reliable technique which has not been used in hibernation research before. Light-dependent telemetry before hibernation and individual observations after

hibernation gave additional information on the timing of above-ground activity before and after hibernation. Data during hibernation were analysed to assess circadian or daily rhythmicity. We also compared euthermic body temperature rhythms before and after hibernation since these may shed light on the functioning of the circadian pacemaker during hibernation.

Materials and methods Animals, housing and data acquisition Animals used in this study were either caught from a population near Vienna, Austria (Millesi et al. 1999b) or born under laboratory conditions from females caught pregnant from the same population. Body temperature patterns were recorded every 48 min for 7±8 months by implanted loggers (Onset Computer Corporation, Pocasset, Mass., USA; customised Stowaway Tidbit temperature loggers; 12.8 g; range ±4 °C to 44 °C; 0.18 °C accuracy). Soil temperature was recorded by a single temperature logger (Orion Components, Chichester, UK; Tinytalk; range ±39 °C to 123 °C; 0.4 °C accuracy) at 0.5 m depth, which was the expected depth of the hibernacula (Hut and Schar€ 1998). Three male and three female European ground squirrels (Spermophilus citellus) were implanted intraperitoneally with body temperature loggers between September 4 and September 25 1996. During implantation and removal of the loggers the animals were anaesthetised with halothane. After implantation, the animals were kept individually in cages in a room without temperature control for recovery (2±10 days). An open window provided a natural LD and ambient temperature ¯uctuations. Animals were released during September 14±26 1996 into two 11´22 m outside enclosures in Haren, The Netherlands (53°10¢N, 6°36¢E). The animals were recaptured from March 5 to April 7 1997 after which they were housed in the room described above. Loggers were removed between May 1±13, 1997. Water and food (rabbit breeding chow, 3 mm diameter, Teurlings, Waalwijk, The Netherlands) were supplied ad libitum outside the hibernation season, both in the housing room and in the enclosures. Animals could be individually recognised from a distance by black hair dye marks on their backs and during handling by subcutaneously injected passive identi®cation transponders (Trovan, PIT tags, EID Aalten bu, Aalten, The Netherlands). Observations were made from an elevated blind to record the above-ground presence of the animals in the enclosure. Several 2±4-h observations were made around noon before hibernation (September 20, 24, 26, 27, 30) and after hibernation (March 4±6, 10, 13, 17±20, 25; April 1, 7). In addition, the animals received light-sensitive collar transmitters (TXH-2/L, weight 10 g, Televilt International AB, Lindesberg, Sweden) which gave information on the timing of above-ground activity once every 2 min for 1±2 months after release (for details see: Hut et al. 1999a). Localisation of the transmitters with a specialised ground search antenna (Televilt) in combination with the individual observations enabled us to determine the position of the individual hibernacula within a radius of 20 cm. All burrows were dug by the animals and had one or two entrances per burrow. There was no indication that burrows were inhabited by more than one animal. Inspection of the entrances before and during hibernation provided additional information on the onset of hibernation. Burrow entrances of active European ground squirrels are di€erent from hibernating animals because this species plugs its burrow entrances at the surface before going into hibernation (RuzicÂ1978; Hut and Schar€ 1998). De®nitions of di€erent phases in the hibernation cycle We de®ne hibernation as the phase of the yearly cycle where the (heterothermic) body temperature of the animal decreases to

49 hypothermic values (torpor) with periodic interruptions of relatively short arousal episodes (Barnes and Ritter 1993), consisting of a rewarming phase and a euthermic phase. Thus, possible short torpor bouts [shorter than a day; sometimes referred to as ``test drops'' (Strumwasser 1959) or daily torpor (Barnes and Ritter 1993)] before or after hibernation are not included. Timing of hibernation and timing of arousal episodes were established according to Daan (1973b) and Strijkstra (1999). Onset of the arousal episode (o€set of the torpor bout) was determined visually as the ®rst data point on the rewarming curve followed by a complete arousal to euthermic body temperature. O€set of the arousal episode (onset of the torpor bout) was determined visually as the ®rst point of the cooling curve (Fig. 1). Both onset and o€set of the arousal episode, as de®ned above, coincide with major changes in the metabolic rate of the hibernating animal (Pohl 1961; Snapp and Heller 1981; Heldmaier and Ruf 1992; Heldmaier et al.1993; Boyer and Barnes 1999). We did not include the cooling phase in our de®nition of the arousal episode. Cooling after a period of actively terminated euthermia (Heldmaier and Ruf 1992; Heldmaier et al. 1993) is a passive heat exchange process of the animal with its environment (Strijkstra 1999). Over time, this process asymptotically approaches a level of 1±2 °C above ambient temperature and thus there is no clearly de®nable end of the cooling curve. Statistics In general, comparisons within the individual were tested using a paired t-test and comparisons between the sexes were performed by a two-sample t-test. Rhythmicity of body temperature data was tested by v2-periodogram analyses (Sokolove and Bushell 1978) using peak DQp-values (di€erence between the peak Qp-value and its signi®cance threshold value) as an indicator for the presence of rhythmicity (Gerkema et al. 1994). Angular-angular correlations (spherical correlations) were used to test the correlation between the timing in hours of the day of two events in the hibernation cycle. For this purpose we modi®ed the angular-angular correlation test as presented in Zar (1999) by replacing the erratically presented jack-kni®ng method by a resampling technique (bootstrapping

method) creating a null distribution based upon 10,000 simulations (Efron and Tibshirani 1993). Raleigh's test was used to test for uniformity of the circular distribution of time of day of torpor onsets and the Hotelling test for paired angular data was used to test for a signi®cant change in time of day for the ®rst two consecutive torpor onsets (Zar 1999).

Results Body temperature patterns and timing of hibernation The complete data set of body temperature patterns used in this study is presented in Fig. 2. Temperature ranges show that during torpor body temperatures below 0 °C occurred in half of the animals (Table 1). Arousal episodes were clearly detectable in the body temperature records and were suciently long compared to the signal resolution (48 min) to never have passed unobserved. Soil temperature was measured at approximately the same depth as the hibernacula of the ground squirrels. Therefore, it corresponds closely with the minimal body temperatures during deep torpor (Fig. 2). However, since the location was not exactly the same as the di€erent hibernacula, the minimum body temperatures of the ground squirrels might have been sometimes slightly below recorded soil temperature. Duration of hibernation was found to be on average 43.5 days shorter in males than in females (Table 1; two sample t-test: t=3.11, df=4, P=0.04). Number of arousals was on average 5.3 less in males (Table 1; two sample t-test: t=3.58, df=4, P=0.02) which should be attributed mainly to the shorter duration of hibernation since neither torpor bout duration nor arousal duration di€ered between the sexes (Table 1; two sample t-test, respectively: t=0.46, df=4, P=0.67 and t=0.6, df=4, P=0.58). Circadian rhythmicity during torpor

Fig. 1 Example of the temporal pattern of an arousal episode (male no. 67; January 5, 1997) during deep hibernation at minimal soil temperatures. The timing of onset and o€set of euthermia, based upon underlying physiological processes as mentioned in the introduction, are indicated in the graph (see Materials and methods)

To address the issue of persistence of circadian rhythmicity during phases of deep prolonged torpor, we analysed body temperature patterns during the longest torpor bout for each individual by v2-periogram analyses. In all individuals the longest torpor bout occurred during January 1997. During this phase the environmental soil temperature was found to be most stable (Fig. 2), which increased the probability of detecting circadian body temperature ¯uctuations. Elevated body temperatures due to the decreasing cooling curve after an arousal episode were excluded from the analyses. Body temperature patterns during deep torpor did not reveal any obvious daily or circadian rhythms (Fig. 3, left panels) and the v2-periogram analyses failed to detect signi®cant periodicity for periods between 12 h and 36 h (Fig. 3, right panels). To summarise the results of the v2periogram analyses per individual we calculated DQppeak values (Table 1). A negative DQp-value indicates that the v2-periogram analysis failed to detect signi®cant periodicity in the data.

50 Fig. 2 Body temperature during hibernation in three female and three male European ground squirrels in an outdoor enclosure at the Biological Centre (Haren, the Netherlands). Soil temperature was measured at approximately the same depth as the locations of the hibernacula (0.5 m below the soil surface). All data sets start immediately after implantation of the body temperature loggers and end just before removal of the loggers. Release into, and recapture from the outside enclosures are indicated by open triangles; start and end of hibernation are indicated by black triangles

Pre-hibernation and post-hibernation euthermia The animals exposed themselves to light until 0±5 days before the onset of the ®rst multi-day torpor bout (Fig. 4). The timing of light exposure is con®ned to the hours of full day light and thus corroborate earlier results on light perception in this species (Hut et al. 1999a). Furthermore, light exposure was always associated with high values of body temperature, indicating locomotor activity of the animal. Light-sensitive

transmitters were functional well after the onset of hibernation (until November/December, except in male no. 28 where the transmitter failed before the onset of hibernation) but never showed light exposure during hibernation, neither during torpor nor during arousal episodes. The euthermic body temperatures 10 days before and 10 days after hibernation are shown in more detail in Fig. 4. These data were used to calculate the amplitude of daily euthermic body temperature rhythms. Short

±47.4 ±48.3 ±47.9 ±48.4 ±47.6 ±48.8 [±0.1, 11.8] [±0.9, 14.2] [0.3, 15.2] [±0.7, 6.5] [0.8, 12.7] [0.1, 13.0] (3.5) (4.0) (4.7) (2.3) (3.7) (4.1) 36.3 36.7 36.4 36.5 36.5 37.5 12.8 16.6 15.3 14.0 13.4 15.1

(4.7) (5.3) (3.4) (5.3) (2.2) (4.3)

(0.6) (0.8) (0.4) (0.4) (0.3) (0.8)

Maximum temperature (°C)

19 21 19 13 13 17 162.5 171.1 180.3 110.4 119.8 153.2 28 March 1 April 26 March 4 March 24 Feb 11 Mar Female Female Female Male Male Male 69 70 84 28 67 99

16 October 29 September 27 September 13 November 27 Oct 9 Oct

Number

Sex

End

Duration Start

Duration (h)

[35.1, [36.0, [35.8, [35.8, [36.0, [36.6,

37.5] 38.1] 37.1] 37.2] 37.1] 39.8]

7.6 7.7 8.4 7.3 8.0 7.9

(4.1) (4.4) (4.6) (3.1) (3.9) (4.2)

[2.3, [2.8, [2.3, [1.6, [3.8, [2.9,

16.8] 18.5] 17.3] 12.1] 16.2] 16.7]

5.7 6.4 7.4 2.9 5.7 6.5

DQp Minimum temp. (°C) Duration (days)

Torpor Euthermic episodes Hibernation

phases of hypothermia, occurred both before and after hibernation. The distinction between such short daily torpor episodes and normal low body temperatures at night is not very clear in this data set. We identi®ed these short hypothermic periods by combining the depth of the temperature drop and the rate of temperature change during cooling and rewarming. Such periods are indicated in Fig. 4 with an asterisk, and were omitted from the data set to calculate the amplitude of daily euthermic oscillations during the last 5 days before hibernation and the ®rst 5 days after hibernation. During the ®rst 5 days after hibernation, mean amplitude was found to be 1.69 °C (SD=0.15 °C) smaller than in the last 5 days before hibernation (Fig. 5 upper panel; paired t-test: after-before: t=±11.04, df=5, P=0.0001). Periodogram analyses on these data (after de-trending by subtracting a 24-h running mean from the original data values after which the original grand mean was added to the residuals) also showed a decrease in rhythmicity of the daily body temperature pattern (Fig. 5 lower panel; paired t-test after-before: t=±33.38, df=5, P=0.012). Average temperature levels during euthermic phase of arousal episodes (36.0 °C; rewarming curve and cooling curve not included) were slightly lower than average euthermic temperature before (36.8 °C) and after hibernation (37.3 °C) and compared best with pre-hibernation body temperatures during the nocturnal rest phase (Table 2; paired t-tests: during-before: t=±3.26, P=0.02; after-during: t=5.00, P=0.004; after-before: t=2.97, P=0.03; during-rest: t=±1.00, P=0.36). Timing of torpor and arousal episodes

Id

Table 1 Individual summaries of the timing of hibernation (start, end, and duration), number and duration (h) of arousal episodes, average maximal temperature during arousal episodes (°C), average duration of torpor bouts (days), average minimal body temperature (°C) during a torpor bout and DQp-peak values calculated from v2-periodogram analyses on torpor body temperatures during longest individual torpor bouts (negative values indicate the lack of signi®cant rhythmicity for periods between 12 h and 36 h). Standard deviations are enclosed in parentheses; ranges are enclosed in brackets

51

Circadian in¯uences on arousal timing can potentially be detected when the arousal episodes are double plotted on a 24-h basis (Fig. 6). Consistency in the circadian timing of arousal episodes is recognised in all animals either at the beginning or at the end of hibernation. Male no. 67 also showed synchronisation of arousal times around 0800 hours starting in the middle of the hibernation season (after day 350) and lasting until the timing of the ®nal arousal which ends hibernation. Possible circadian patterns in the timing of arousal episodes may exist also around the middle of hibernation, but these are impossible to detect due to long torpor bout durations and the lack of a circadian phase reference during torpor. Timing of hibernation onset was synchronised in all individuals to the afternoon between 13:00 hours and 16:08 hours (Fig. 6; Raleigh's test: Z=5.51, P0.5; Z®nal arousal=0.39, P>0.5). However, within individuals the time of day for the onset of the ®rst arousal episode correlated with the time of day for the end of hibernation (Fig. 6; angular-angular correlation: r=0.56, P=0.045).

Discussion Timing of hibernation In general, hibernation started later than reported from ®eld observations (Millesi et al. 1999b) on the same species (Females: enclosure 27 September±16 October, ®eld 6±19 August; males: enclosure 9 October±13 November, ®eld 27 August±26 September). This di€erence may partly re¯ect the release dates of 14±26 September, but does not explain why four animals delayed hibernation onset until October or even November. Although arti®cial burrows were available in the enclosures, all animals dug their own burrows. Preparation of these self-dug hibernacula may have delayed hibernation onsets. Healing of the surgery wounds and long term e€ects of anaesthesia, possibly inducing body mass loss, may have played additional roles. The tendency of males to start hibernation later and stop earlier than females corresponds with ®eld observations (Millesi et al. 1999b). The di€erences between hibernation onsets in the ®eld and enclosure sharply contrasts with the close resemblance of hibernation end between free-ranging (Millesi et al. 1999b) and enclosed females presented here (Females: enclosure 26 March±1 April, ®eld 28 March±6 April; males: enclosure 24 February± 11 March, ®eld 5 March±19 March). This indicates the presence of an accurate annual timing mechanism during 6±7 months of hibernation, which persisted in the animals that participated in this study.

Some ground squirrel species may remain continuously in their burrows for several days or weeks during post-hibernation euthermia. During this pre-emergence euthermic interval (PEEI) re-growth of the reproductive apparatus and spermatogenesis occurs and these ground squirrel species may emerge from their hibernacula with a fully developed reproductive system (Barnes et al. 1986, 1988). We have no indication that a PEEI occurs in male European ground squirrels. Males in this study were observed within 0±8 days after their ®nal arousal (Fig. 4) and those males which were ®rst caught within 3±4 days after their ®nal arousal had no scrotal pigmentation or measurable testis size (Millesi et al. 1999b). Females were observed and caught 4±6 days after their ®nal arousal without indications that they had fully entered the reproductive state (Millesi et al. 1999a). Body temperatures before, during, and after hibernation Average euthermic body temperatures were highest after hibernation and lowest during hibernation, while the values obtained before hibernation were intermediate (Table 2). These results are consistent with those obtained in the European hamster (Wollnik and Schmidt 1995) and may re¯ect an annual cycle in set points of euthermic body temperature. Body temperatures during euthermic episodes were similar to those during the night-time rest phase of the activity cycle before hibernation. This may be explained by the ®nding that European ground squirrels sleep for 71.5% of the time during a euthermic episode (Strijkstra and Daan 1997) and is thus in a similar physiological state as during its daily rest phase before hibernation. Lowest body temperatures during torpor may reach sub-zero temperatures (Table 1). The lowest abdominal body temperature measured in this study was ±0.94 °C. In the arctic ground squirrel body temperatures may drop to ±2.9 °C, and it has been shown that a supercooling mechanism protects these animals from freezing (Barnes 1989). Blood plasma solute concentrations of hibernating arctic ground squirrels provide protection

53 Fig. 4 Body temperature patterns for each animal during the last 10 days of euthermia before hibernation and the 1st 10 days of euthermia after hibernation. Short periods of possible hypothermia are marked with an asterisk and excluded from calculation of the amplitude of the euthermic rhythm (Fig. 5). Individual pre-hibernation above ground activity phases, measured with light-sensitive radio collar transmitters, are indicated with a thick horizontal line; rest phases are indicated with a thin horizontal line. Days when the animal was observed above ground are indicated with a ®lled triangle; an open triangle indicates when the animal was ®rst caught after which the animals stayed in the housing room in the laboratory

from freezing only to temperatures of ±0.6 °C and there was no evidence that freezing points were depressed by antifreeze molecules. Although lowest body temperatures in this study were not as extreme as in the arctic ground squirrel (Table 1), we cannot exclude that the European ground squirrel also avoids freezing during torpor by a supercooling mechanism. Circadian oscillations during torpor Circadian body temperature oscillations during torpor would indicate directly continuous functioning of the

circadian system at low body temperatures. We were not able to detect such oscillations in the European ground squirrel (Fig. 3; Table 1). Grahn et al. (1994) did detect circadian body temperature ¯uctuations of 0.2±0.6 °C in the golden-mantled ground squirrel at an ambient temperature of 10 °C and Menaker (1959, 1961) showed circadian free-running body temperature patterns with ¯uctuations of 0.5±1.0 °C in torpid bats at an ambient temperature of 8±10 °C. The golden-mantled ground squirrels kept at 10 °C maintain a torpor body temperature around 12.2 °C, while torpor body temperatures in our study dropped to levels around 0 °C during mid-winter. At higher torpor body temperatures in the

54

Fig. 5 Amplitude of the euthermic body temperature rhythm during the last 5 days of euthermia before hibernation and the ®rst 5 days of euthermia after hibernation. Points of the same animal are connected (females open circles, males closed circles)

beginning of hibernation, the torpor bouts were too short to detect possible circadian oscillations added to the environmental ¯uctuations and cooling curves of the animals. These comparisons may re¯ect a temperature dependency of the functioning of the SCN, with no apparent expression at body temperatures below 10±15 °C. Circadian timing of arousals The timing of the ®rst arousal from torpor is not related to a certain phase of the day (Fig. 6), although the ®rst torpor onset is restricted to the afternoon. This contrasts with the onset of torpor around midnight in nocturnal mammals (Daan 1973b). The second torpor onset is Table 2 Comparison between average euthermic body temperatures before, during and after hibernation. Mean euthermic body temperatures were calculated over the following periods: (A) before hibernation: from the day after release to the enclosure until several days before hibernation onset, in order to avoid interference with pre-hibernation ``test drops'' and set point decrease, as evident for some individuals (Fig. 4), (B) during arousal episodes: from the Id

69 70 84 28 67 99 Average

delayed by 1.7 h/day on average, possibly indicating a free-running circadian timing mechanism with an average period of 25.7 h. This circadian period is rather long when compared to the circadian period of this species outside the hibernation season (24.31 h; Hut et al. 1999b). This may re¯ect slowing down of the circadian clock during torpor. Thus, an e€ect of low body temperatures on circadian period cannot be excluded. Such an e€ect may obscure the detectability of circadian patterns in the double plots presented in Fig. 6. In this data set we also detected an unexpected correlation between the timing of the ®rst and the ®nal arousal from torpor. It is highly unlikely that this correlation re¯ects an entrained circadian oscillator since there in no evidence that intermediate arousals within the individual are also correlated with the phase of the ®rst arousal. A possible explanation may be that the animals reacted to slight daily temperature changes undetected by the limited sensitivity of the loggers used in this study. The timing of these daily temperature changes may vary in the course of the year and with the location of the hibernaculum. An increased sensitivity to arousing stimuli (Twente and Twente 1968; Beckman and Stanton 1976) towards the end of a torpor bout may result in a triggered arousal at a certain time of day. This time of day may di€er between di€erent locations of hibernacula, but may be similar for each hibernaculum at the beginning and at the end of the hibernation season. Circadian in¯uences on the timing of arousals are only detectable when torpor bouts are within the range of several days. In our data, circadian in¯uences are sometimes clear at the beginning and at the end of hibernation where torpor bouts were in the range of 2±10 days. When torpor bout length increases to 16±18 days, circadian in¯uences become increasingly hard to detect. Male no. 67 was exceptional in showing a daily pattern of arousal timing around noon after day 350. This behaviour could not be explained by the location of its hibernaculum, that was located using its collar transmitter signal. The possibility exists, however, that this animal hibernated close to the soil surface and was thus exposed to environmental zeitgebers. ®rst point (Ti) after the rewarming curve where Ti £ Ti±1 until the point before the ®rst point of the cooling curve (Fig. 1) and (C) after hibernation: from the day after hibernation end until the day before transport from the enclosures (Fig. 4). Standard deviations are given in parentheses for individual means; the SEM is given in brackets for the column averages

Before hibernation Mean

Activity

Rest

36.7 36.8 36.4 37.1 37.3 36.3 36.8

37.9 37.9 38.0 38.2 38.4 37.4 38.0

36.1 36.3 35.8 36.6 36.8 35.9 36.2

(1.2) (1.0) (1.3) (1.0) (0.9) (1.4) [0.16]

(1.0) (0.8) (0.9) (0.8) (0.7) (1.3) [0.14]

(0.8) (0.6) (0.7) (0.7) (0.6) (1.2) [0.17]

During hibernation

After hibernation

Mean

Mean

35.9 36.0 35.6 35.9 36.0 36.6 36.0

37.5 37.7 37.4 37.3 37.2 36.7 37.3

(0.6) (0.6) (0.5) (0.3) (0.2) (0.5) [0.14]

(0.8) (0.6) (0.8) (0.4) (0.9) (1.0) [0.13]

55 Fig. 6 Timing of individual patterns of arousal episodes presented as double plots. Euthermia is indicated with a horizontal line and onsets of euthermia are indicated with a dot

The occurrence of circadian rhythmicity during hibernation in the timing of arousals has been emphasised by several authors (Strumwasser 1959; Pohl 1961, 1967, 1987, 1996; Daan 1973b; Twente and Twente 1987; Canguilhem et al. 1994; Grahn et al. 1994; Wollnik and Schmidt 1995; Waûmer and Wollnik 1997; KoÈrtner et al. 1998), but in the presented data sets disturbed circadian rhythmicity or even arrhythmicity is often seen and studies using di€erent methods may lead to di€erent results (Thomas 1992; Waûmer and Wollnik 1997). The timing of arousal episodes under daily LDs was found to be arrhythmic in four out of seven Turkish hamsters (Pohl 1987) and in European hamsters after deep hibernation bouts (Waûmer and Wollnik 1997). Two studies in the European hamster (Canguilhem et al. 1994; Waûmer and Wollnik 1997) claimed circadian timing in torpor-arousal cycles. In addition, Wollnik and Schmidt (1995) found freerunning circadian patterns in arousals from torpor in enclosed European hamsters with body temperatures of 4±9 °C, while torpor onsets seemed to be synchronised to a certain phase of the day. European hamsters typically have torpor bouts of 2±6 days, and other studies showing clear circadian timing in torpor-arousal cycles were also performed in species which typically show relative short torpor bouts of several days (Pohl 1967; Daan 1973b). Taken together, the available studies demonstrate that spontaneous arousals can be controlled by circadian oscillations persisting during brief (2±5 days) torpor bouts at body temperatures above 10±15 °C. On the other hand, there is little evidence for

such timing at lower body temperatures and corresponding longer torpor bouts. Thus, the results appear consistent with the proposition that the circadian system damps out in a few days at body temperatures below 10±15 °C. Alternatively, the circadian system may still be functioning, but only the oscillation it drives is damped out. Gradual damping of oscillations in the circadian system does not necessarily imply that the intra-cellular molecular clock work is a€ected. Due to the loss of electrical activity in SCN neurons at body temperatures below 10±15 °C (Krilowicz et al. 1988, 1989; Miller et al. 1994), the output of the SCN may be weak and synchronisation of SCN neurons may be less or even lost after a number of circadian cycles during torpor. Other neuronal communication signals, such as gapjunctions and nonsynaptic neuropeptide release, could allow for some persistence of synchronisation between SCN neurons. These mechanisms may be functional in some species and may explain why circadian body temperature oscillations were found in bats at 8±10 °C (Menaker 1959, 1961) and in ground squirrels at 12.0± 12.8 °C (Grahn et al. 1994). Such persistence is also made plausible by the apparent circadian patterns in arousals during the beginning and the end of hibernation in our data set (Fig. 6), as well as the correlation between the time of day of the ®rst and the second torpor onset, even though body temperatures during the ®rst torpor bout dropped to 10±15 °C (Fig. 6). Loss of di€erent synchronisation mechanisms may result in a gradual, temperature-dependent loss of circadian

56

rhythmicity during torpor bouts longer than 2±5 days (Fig. 6). The potential bene®ts of a circadian system which remains functional during hibernation and is involved in timing of arousal episodes may depend on the behaviour of the species during hibernation (KoÈrtner and Geiser 2000). Bats often hibernate in open caves, ¯y around during arousal episodes, may feed and drink, and even occasionally leave their hibernacula during winter (Daan 1973a). The European hamster (Waûmer 1998) and the pygmy possum (KoÈrtner and Geiser 1998; KoÈrtner et al. 1998) have also been reported to leave their hibernaculum for above-ground activity. A functional circadian system may well be useful to synchronise such exposures to outside conditions at a preferred phase of the day. In contrast, most rodent hibernators remain sequestered in their burrows during arousal episodes and would not bene®t much from precise daily timing of their arousals (Barnes and Ritter 1993; KoÈrtner and Geiser 2000). Daily body temperature oscillations before and after hibernation Daily euthermic body temperature ¯uctuations in this study are pronounced and, on average, are higher than the maximal individual daily ¯uctuation of 3.5 °C measured in Richardson's ground squirrels in the ®eld (Wang 1972). Di€erences between daily body temperature ¯uctuations before and after hibernation may be a€ected by the loss in body mass over hibernation. Using the last measurement (2±7 weeks) before hibernation and the ®rst measurement (1±8 days) after hibernation, the mean body mass loss over hibernation was 93.7 g (SD 42.1 g), which is within the normal range for this species (Strijkstra 1999). Body mass di€erences between species can in¯uence the expression of circadian body temperature ¯uctuations: smaller mammals have larger circadian body temperature ¯uctuations (Ascho€ 1982). Thus, on the basis of their reduced body mass, we would expect ground squirrels to show an increased circadian temperature amplitude after hibernation. This prediction sharply contrasts with the observed decrease. Dampening out of the circadian system during deep torpor might a€ect circadian body temperature ¯uctuations during post-hibernation euthermia. Internal desynchronisation of SCN neurons may require internal synchronisation to be re-established, and cause temporary post-hibernation arrhythmicity. After hibernation the Turkish hamster shows a gradual re-appearance of circadian rhythmicity under both LD and dim light conditions (Pohl 1981). Waûmer (1998) found circadian arrhythmicity in body temperatures in the European hamster under natural conditions during arousal episodes of several days only after relatively long periods of deep torpor in which body temperature dropped below 10±15 °C. The decreased amplitude of daily body temperature rhythm during post-hibernation euthermia in our data set may re¯ect a similar process of internal

desynchronisation of the circadian system after deep hibernation. Alternatively, it may re¯ect an interaction between direct environmental e€ects of the environment on body temperature that mask the circadian component in body temperature when the circadian system is out of phase with the environment after hibernation. In a follow-up study we also observed post-hibernation arrhythmicity for 1±3 weeks under DD conditions in the laboratory (Hut et al. 2001). This lead to the conclusion that post-hibernation arrhythmicity re¯ects a property of the circadian system rather than masking e€ects of transient days during re-entrainment after hibernation. Decreased ¯uctuations in circadian body temperature patterns could re¯ect a smaller amplitude of the circadian system. While this may be merely a direct physiological consequence of the low body temperature during hibernation, it also may be bene®cial to end hibernation while the circadian system oscillates with a low amplitude. European ground squirrels remain in the complete darkness of their closed burrows during the 6±7 months of hibernation and they appear to loose synchrony with the day-night cycle. Fast re-entrainment to this daynight cycle after hibernation can be expected to be pro®table. A low amplitude of the circadian system will enhance its sensitivity to environmental zeitgebers (Hau and Gwinner 1995; Gwinner 1996) and therefore facilitate post-hibernation re-entrainment rate. Acknowledgements We thank M. Oklejewicz for helpful comments on the paper and H. Berkhof for his statistical advice. This study was supported by the Human Frontiers Science Program and approved by the Animal Experiments committee of the University of Groningen (BG02196).

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