Novel Wheel Running Blocks the Preovulatory Luteinizing Hormone Surge and Advances the Hamster Circadian Pacemaker

Transcription

1 Novel Wheel Running Blocks the Preovulatory Luteinizing Hormone Surge and Advances the Hamster Circadian Pacemaker S. J. Legan,*,1 K. M. Franklin, X.-L. Peng,* and M. J. Duncan *Department of Physiology, University of Kentucky, Lexington, KY, and Department of Anatomy and Neurobiology, University of Kentucky, Lexington, KY Abstract In rodents, the preovulatory luteinizing hormone (LH) surge is timed by a circadian rhythm. We recently reported that a phenobarbital-induced delay of the estrous cycle in Syrian hamsters is associated with an approximately 2-h phase advance in both the circadian locomotor activity rhythm and the timing of the LH surge. The following study tests the hypothesis that a >2-h nonpharmacological phase advance in the circadian pacemaker that delays the estrous cycle by a day will also phase advance the LH surge by approximately 2 h. Activity rhythms were continuously monitored in regularly cycling hamsters using running wheels or infrared detectors for about 10 days prior to jugular cannulation. The next day, on proestrus, hamsters were transferred to the laboratory for 1 of 3 treatments: transfer to a new cage (and wheel) from zeitgeber time (ZT) 4 to 8 (with ZT12 defined as time of lights-off), or exposure to a novel wheel at ZT5 or ZT1. All animals were then placed in constant dark (DD). Blood samples were obtained just before onset of DD and hourly for the next 6 h, on that day and the next day for determination of plasma LH concentrations. Running activity was monitored in DD for about 10 more days. Transfer to a novel wheel at either ZT5 or ZT1 delayed the LH surge to day 2 in most hamsters, whereas exposure to a new cage did not. Only the delayed LH surges were phase advanced at least 2.5 h on average in all 3 groups. However, wheel-running activity was similarly phase advanced in all 3 groups regardless of the timing of the LH surge; thus, the phase advances in circadian activity rhythms were not associated with the 1-day delay of the LH surge. Interestingly, the number of wheel revolutions was closely associated with the 1-day delay of LH surges following exposure to a novel wheel at either ZT1 or ZT5. These results suggest that the intensity of wheel running (or an associated stimulus) plays an important role in the circadian timing mechanism for the LH surge. Key words activity rhythm, nonphotic, phase shift, LH surge, female The preovulatory luteinizing hormone (LH) surge and its resulting ovulation occur at the same time every fourth day (proestrus) in rats, mice, and hamsters housed in long day photoperiods. This 4-day rhythm free-runs in constant light (Alleva et al., 1971; McCormack and Sridaran, 1978), is entrained by the photoperiod (Finkelstein et al., 1978; Moline et al., 1981), and is ablated by destruction of the suprachiasmatic nucleus (SCN), the site of the master circadian pacemaker (Palm et al., 1999; Weigand et al., 1980). These 1. To whom all correspondence should be addressed: Sandra J. Legan, Department of Physiology, University of Kentucky Medical Center, Lexington, KY ; sjlegan@uky.edu. JOURNAL OF BIOLOGICAL RHYTHMS, Vol. 25 No. 6, December DOI: / Sage Publications 450

2 Legan et al / NONPHOTIC PHASE ADVANCE OF HAMSTER LH SURGE 451 findings, in conjunction with the observations that LH surges occur daily in estrogen-treated ovariectomized hamsters (Norman et al., 1973), rats (Legan and Karsch, 1975) and mice (Christian et al., 2005), and in anestrous and lactating hamsters (Bridges and Goldman, 1975; Seegal and Goldman, 1975), indicate that the LH surge is controlled by the circadian timing system. Circadian control of the LH surge was first suggested by the finding that administration of a barbiturate, for example, sodium pentobarbital, to rats on proestrous afternoon delays the LH surge 1 day at a time for up to 3 successive days (Everett and Sawyer, 1950) and was later confirmed in hamsters using phenobarbital (Stetson and Watson-Whitmyre, 1977). In rats, barbiturate blockade of the LH surge is only successful when the barbiturate is administered during a specific period approximately 7 h before lights-off, referred to as the critical period, the time when the neural signal for the LH surge is presumed to occur (Everett and Sawyer, 1950). Hamsters have a similar critical period for phenobarbital blockade of the LH surge, but its duration, especially its time of onset, has been less well characterized (Greenwald, 1971). The mechanism whereby barbiturates cause a delay in the LH surge remains to be determined. One possible mechanism was suggested by the observation that presentation of a nonphotic circadian timing signal, or zeitgeber (ZT), at the beginning of the critical period on proestrus causes a 1-day delay in the estrous cycle (Janik and Janik, 2003). The nonphotic zeitgebers used were confinement to a novel wheel or transfer to a new cage and new room, manipulations known to induce phase advances in male rodents when presented during the midsubjective day (Janik and Mrosovsky, 1993; Mrosovsky et al., 1989; Wickland and Turek, 1991). Only female hamsters whose activity rhythms were phase advanced more than approximately 1.5 h by these nonphotic zeitgebers exhibited a 1-day delay in the estrous cycle, as indicated by vaginal discharge and receptivity to a male (Janik and Janik, 2003). These results strongly suggest that a phase advance in the circadian pacemaker greater than or equal to the duration of the critical period (~2 h) also blocks the LH surge on that day. Indeed, we recently demonstrated that the 1-day delay in the estrous cycle following phenobarbital administration to proestrous Syrian hamsters is associated with phase advances in both the circadian locomotor activity rhythm and the timing of the LH surge and with a suppression of Per1 mrna expression in the SCN (Legan et al., 2009). Although the foregoing findings strongly suggest that a phase advance in the circadian pacemaker can block the LH surge, barbiturates are central nervous system depressants and are known to have many other effects that could block the LH surge. In order to determine whether a 2-h phase advance in the circadian pacemaker is sufficient to block the LH surge, it is necessary to induce a phase advance by means of a nonpharmacological stimulus, such as exposure to a novel wheel and/ or a new cage. Therefore, the following experiments were designed to test the hypothesis that a nonpharmacological stimulus that causes a 2-h phase advance, when administered at the beginning of the critical period on proestrus, will block the LH surge for 1 day and cause it to peak at an earlier time on the next day. Animals MATERIALS AND METHODS All procedures were approved by the University of Kentucky's IACUC and conformed to the NIH Guide for the Care and Use of Laboratory Animals. Adult female hamsters 3 to 4 months old were housed in individual cages inside light-tight compartments under a 14L:10D photoperiod at 21 C with food and water available ad libitum. Each compartment held 4 individual cages and a packet of soiled male bedding. Activity rhythms were recorded continuously by means of either an infrared motion detector mounted on top of each cage or a running wheel. In order to monitor estrous cycles, vaginal discharges were checked daily (Orsini, 1961). After at least 2 consecutive 4-day cycles, hamsters were fitted with a right atrial cannula under isoflurane anesthesia on the morning of diestrus-2 and returned to the animal facility. On the next day, proestrus, they were transported to the laboratory at a specific time, described below. Within the next 10 min, a blood sample was obtained after which the animals were exposed to one of the following phase advancing paradigms. New cage experiment. Animals that had been housed with wheels for at least 10 days were transferred to a new room and placed in a new cage with a new wheel from zeitgeber time (ZT) 4 to 8 (ZT12 equivalent to time of lights-off). At ZT8, they were returned to their home cages and wheels. Novel wheel, ZT5 experiment. Eight of 11 animals that had never had access to a wheel were transferred to a new room into a new cage with a novel wheel at ZT5; the other 3 animals had not been exposed to a wheel for 1 week. Novel wheel, ZT1 experiment. Animals were treated the same as the previous novel wheel

3 452 JOURNAL OF BIOLOGICAL RHYTHMS / December 2010 Table 1. Number (%) of animals with delay of estrous discharge and/or LH surge. group, except that they were transferred to a new room and cage and exposed to a novel wheel at ZT1. Immediately following transfer to a new cage, all 3 groups of animals were exposed to continuous darkness (DD) for the remainder of the experiment. Hourly blood samples were obtained under dim red light for the next 6 h and at the same times on the next day for determination of plasma LH concentrations. During the 2 days of sampling, when the animals were housed in cages that were not interfaced with the ClockLab equipment (Coulbourn Instruments, Whitehall, PA) in the animal facility, the number of wheel revolutions were tracked by an event counter and recorded hourly at each sampling time in both novel wheel experiments. After the last blood sample, the animals were anesthetized lightly with isoflurane. Next, the cannulae were knotted as close to the skin as possible and cut off just distal to the knot, which was secured subcutaneously with a wound clip. The animals were then returned to the animal facility where they remained in DD, and daily vaginal discharges and running activity rhythms were monitored for at least 10 more days for determination of phase shifts. Radioimmunoassay (RIA) Plasma LH concentrations were determined by RIA as described previously (Legan et al., 2009). The samples from all experiments were run in 8 RIAs, for which the sensitivity averaged 0.02 ng per tube. Phase Shifts Phase shifts in running activity rhythms were calculated with ClockLab software (Coulbourn Instruments, Whitehall, PA). Statistics Data were analyzed by the Student t test. The significance level was p < New Cage Novel Wheel ZT5 Novel Wheel ZT1 Delayed estrous discharge only 0 2 (14%) 0 Delayed LH surge only 0 6 (43%) 4 (40%) Neither 6 (86%) 1 (7%) 4 (40%) Both 1 (14%) 5 (36%) 2 (20%) Total 7 (100%) 14 (100%) 10 (100%) % delayed estrous discharge 1/7 (14%) 7/14 (50%) 2/10 (20%) % delayed LH surge 1/7 (14%) 11/14 (79%) 6/10 (60%) RESULTS Experiment 1: New Cage Exposure to a new cage and wheel and DD from ZT4 to ZT8 on proestrus delayed the LH surge until day 2 in only 1 hamster, while the LH surge in the remaining 6 hamsters occurred on day 1, that is, proestrus (Table 1 and Fig. 1A). The estrous discharge was also delayed 1 day in the hamster whose LH surge was delayed until day 2, but not in the other hamsters (Table 1). The next 3 vaginal discharges after the delayed cycle occurred at regular 4-day intervals and reflected the 1-day delay. The day 1 LH peak either occurred at ZT9 in 2 of the 6 hamsters or at ZT10 in 3 other animals based on a large decrease in the slope of the LH curve (Fig. 1A). In the remaining hamster, the slope had not yet decreased, and her peak occurred at either ZT10 or later. Therefore, her data were eliminated from analysis of peak LH concentrations or timing (Fig. 1B). When the LH surge occurred on day 2, the peak was approximately 3-fold greater than the average peak of the LH surges on day 1 (Fig. 1A and 1B, left panel). Further, with regard to phase of the LH surges, the day 1 surges peaked on average at ZT9.6 (n = 5). In contrast, the LH surge that was delayed until day 2 peaked at ZT7 and thus was phase advanced at least 2.6 h compared to the LH surges on day 1 (Fig. 1B, right panel, and Fig. 2, right panel, black bars). Regardless of whether the estrous discharge and LH surge were delayed or not, the circadian rhythm in wheel-running activity was phase advanced 1.5 ± 0.1 h (mean ± SE, n = 6) on average in all animals (Fig. 2, right panel, gray bars). The time that animals began running on the first day of wheel availability varied. However, all of the animals were fully entrained to the LD cycle for at least 7 to 10 days immediately before presentation of the stimulus (new cage or novel wheel in subsequent experiments), and all the test subjects were at the same phase angle on day 1. Further, it should be noted that because the experimental design did not include a group transferred to DD without a new cage, the possibility that the phase of the free-running rhythm was determined by the prior entrained phase angle cannot be ruled out. However, it seems highly unlikely that apparent phase shifts of 90 min on average were solely the result of unusually large entrained phase angles.

4 Legan et al / NONPHOTIC PHASE ADVANCE OF HAMSTER LH SURGE 453 Figure 1. Plasma LH levels in hamsters transferred to a new cage (and wheel) from ZT4 to ZT8 on proestrus. (A) Profiles of plasma LH concentrations (mean ± SE) on expected proestrus (day 1) and estrus (day 2) from ZT4 to ZT10. The shaded area indicates exposure to constant darkness. (B) Peak LH concentrations (mean ± SE) and time of occurrence (mean ± SE) of peak plasma LH concentrations on days 1 (black bars) and 2 (open bars). Numbers at the base of the bars indicate the number of animals. Figure 2. Circadian phase advances in the activity rhythm and time of the LH surge in hamsters transferred to a new cage and wheel from ZT4 to ZT8. Left panel: A representative actogram of a hamster whose activity rhythm was phase advanced 1.49 h. The 24-h day is represented horizontally, from left to right, with the time of day indicated by the numbers shown at the top. The vertical axis represents successive days, from top to bottom. The days of exposure to 14L:10D are indicated on the right by the cross-hatched vertical bar. The asterisk indicates day and time of transfer to a new cage and onset of DD (shown by the black vertical bar on the right). Right panel: Phase advances (mean ± SE) in the wheel-running activity rhythm (shaded bars) or time of LH surges relative to day 1 (black bars) are depicted. Numbers at the base of the bars indicate the number of animals. Open circles depict values for individual animals. and DD beginning at ZT5 on proestrus delayed the LH surge to day 2 in 11 of 14 hamsters, only 5 of which also had a delayed estrous discharge (Table 1 and Fig. 3A). Two hamsters that had an LH surge on day 1 also exhibited a 1-day delay in their estrous discharge. Thus, delay of the estrous discharge was not associated with delay of the LH surge. The subsequent 1 or 2 estrous discharges following the initial delayed cycle occurred 4 and 8 days later, thereby reflecting the 1-day delay. Similar to the new cage experiment, whenever LH surges occurred on day 2, peak plasma LH concentrations averaged about 3-fold higher than those in day 1 LH surges, although this difference did not reach statistical significance (p = 0.08) (Fig. 3B, left panel). In addition, the average time of the LH peak was about 2.5 h earlier for LH surges that occurred on day 2 (~ZT8) than for those occurring on day 1 (~ZT10.5) (Fig. 3B, right panel, and Fig. 4, right panel, black bars). The circadian locomotor activity rhythm exhibited a phase advance of 2.8 ± 0.4 h (mean ± SE, n = 11), regardless of whether the LH surge occurred on day 1 (2.9 ± 1.0 h, n = 3) or was delayed until day 2 (2.7 ± 0.4 h, n = 8) (Fig. 4, gray bars). Thus, the magnitude of the phase advance in the activity rhythm was not correlated with a 1-day delay in the LH surge. The occurrence of the LH surge on day 2 was associated with increased running, indicated by the number of wheel revolutions (Fig. 5, left panel). Indeed, by ZT7, the earliest time that wheel revolutions were recorded after introduction of the wheel, the average number of wheel revolutions was several-fold greater in hamsters in whom the LH surge occurred on day 2 than in those whose LH surge occurred on day 1 (Fig. 5, right panel). Experiment 2: Novel Wheel ZT5 The results from 3 hamsters that had been exposed to a wheel previously were no different from those that had never had a wheel; therefore, the data were combined for analysis of results. Exposure to a novel wheel Experiment 3: Novel Wheel ZT1 Interestingly, LH surges were delayed in 6 of 10 hamsters when the nonphotic novel wheel stimulus was introduced at ZT1, 4 h prior to the time when the neural signal for the LH surge supposedly occurs

5 454 JOURNAL OF BIOLOGICAL RHYTHMS / December 2010 Figure 3. Plasma LH levels in hamsters exposed to a novel wheel at ZT5 on proestrus. (A) Plasma LH concentrations on expected proestrus (day 1) and estrus (day 2) from ZT5 to ZT11. The shaded area represents time of exposure to DD. (B) Peak LH concentrations and time of occurrence of peak plasma LH concentrations on days 1 (black bars) and 2 (open bars). All values are shown as mean ± SE. Figure 4. Circadian phase advances in the activity rhythm and time of the LH surge in hamsters exposed to a novel wheel at ZT5 on proestrus. Left panel: A representative actogram of a hamster whose activity rhythm was phase advanced 3.12 h. The 24-h day is represented horizontally, from left to right, with the time of day indicated by the numbers shown at the top. The vertical axis represents successive days, from top to bottom. The days of exposure to 14L:10D are indicated on the right by the cross-hatched vertical bar. The asterisk indicates day and time of transfer to a new cage and onset of DD (shown by the black vertical bar on the right). Right panel: Phase advances (mean ± SE) in the wheel-running activity rhythm (shaded bars) or time of LH surges relative to day 1 (black bars) are depicted. Numbers at the base of the bars indicate the number of animals. Open circles depict values for individual animals. (Fig. 6A). Day 2 LH surges tended to be greater in amplitude than those on day 1, but the average peak levels were not different between these groups (Fig. 6B, left panel). In addition, the day 2 LH surges were also phase advanced by about 2.5 h on average, compared to the LH surges occurring on day 1 (Fig. 6B, right panel, and Fig. 7, right panel, black bars). Similar to the results of the novel wheel ZT5 experiment, there was no correlation between the delay in estrous discharge and delay of the LH surge. The estrous discharge was delayed 1 day in only 2 of the 6 hamsters in which the LH surge occurred on day 2 (Table 1). Thereafter, the subsequent 2 or 3 cycles occurred at regular 4-day intervals and reflected the 1-day delay. Exposure to a novel wheel and DD at ZT1 on proestrus resulted in similar phase advances in the locomotor activity rhythm as in the novel wheel ZT5 experiment, averaging 2.6 ± 0.4 h (n = 10). The phase advances in wheel running were the same regardless of whether the LH surge occurred on day 2 (2.5 ± 0.5 h, n = 6) or on day 1 (2.6 ± 0.5 h, n = 4) (Fig. 7, right panel, gray bars); thus, they were not correlated with delayed LH surges. As in the novel wheel ZT5 experiment, the amount of wheel-running activity was associated with the delayed LH surges (Fig. 8, left panel). The number of wheel revolutions during the first hour after novel wheel exposure was very similar regardless of whether LH surges were delayed or not. Thereafter, however, the rate of increase in wheel running was greater in hamsters whose LH surge occurred on day 2. Thus, the average number of wheel revolutions attained at ZT4 by those hamsters with day 2 LH surges was not attained until about ZT8 in the hamsters with day 1 LH surges. Hamsters whose LH surge was delayed until day 2 ran twice as much on the average within the first 6 h after receiving a wheel, that is, by ZT7, which is the approximate time of onset of a day 1 LH surge (Fig. 8, right panel). However, this difference in mean activity between animals surging on day 1 or 2 was not statistically significant. This may have been due to the large variation among animals in both groups. In this regard, it is interesting to note that there was one animal who ran a great deal, but whose LH surge was not delayed, and another animal that hardly ran at all, whose LH surge was delayed (Fig. 8, asterisks, right panel).

6 Legan et al / NONPHOTIC PHASE ADVANCE OF HAMSTER LH SURGE 455 Figure 5. Running activity on proestrus following exposure to a novel wheel at ZT5. Left panel: Profile of running activity (cumulative wheel revolutions, mean ± SE) from ZT5 to ZT11 in relation to day of occurrence of LH surge. Shaded area depicts time of exposure to DD. Right panel: Cumulative wheel revolutions (mean ± SE) at ZT7 on day 1 are shown for animals whose LH surge was delayed (day 2) or not (day 1). The closed circles aligned over the bars indicate the wheel revolutions of individual animals. The numbers of animals are indicated at the base of the bars. DISCUSSION The foregoing results demonstrate that only exposure to a novel wheel, and not merely introduction to a clean cage and wheel, at the beginning of the critical period blocked the preovulatory LH surge by 1 day in most hamsters. Further, the 1-day delayed LH surges were always phase advanced, occurring about 2.5 h earlier, and were always larger in amplitude. The increased amplitude of the 1-day delayed LH surges is most likely due to an increase in the releasable pool of LH in the anterior pituitary, which remains under the stimulatory action of the sustained elevation in circulating estradiol levels that are generated by the unovulated preovulatory ovarian follicles. Because the phase advances in the circadian locomotor activity rhythm following exposure to a clean cage were typically <2 h, whereas those following exposure to a novel wheel were usually >2 h, this result appears at first to support our hypothesis that a 2-h phase advance plays a role in blocking the LH surge. However, phase advances in the locomotor activity rhythm averaging >2 h in duration were observed regardless of whether the LH surge occurred on day 1 or day 2. Also, in each novel wheel experiment, 2 animals exhibited a 1-day delay in the LH surge in spite of exhibiting a <2-h phase shift in the activity rhythm. These findings suggest that occurrence of a large circadian phase shift during the critical period is neither necessary nor sufficient for novel wheel blockade of the LH surge on proestrus and the consequent lengthening of the estrous cycle in hamsters. Therefore, the mechanism responsible for these phenomena does not appear to include a >2-h circadian phase shift. Alternatively, it should be considered that the indirect method used to determine phase shifts on day 2, which employs linear regression of activity onsets for at least 7 days, may not have sufficient resolution or precision to determine the phase of the master pacemaker during the critical period on day 1. Two of our results after introduction of a novel wheel at ZT1 contrast with previous findings. First, most previously published phase response curves for locomotor activity rhythms in male hamsters generated with nonphotic signals (e.g., induced running in a novel wheel, cage change, or social interaction) exhibit a small phase delay at CT1 (or ZT1) (Mrosovsky et al., 1989; Reebs and Mrosovsky, 1989a; Mrosovsky et al., 1992; Mrosovsky, 1996). In contrast, our findings showed that presentation of a novel wheel at ZT1 results in a robust phase advance rather than a small phase delay. This difference in results between most of the former studies and ours may be due to one or more differences in their experimental designs, such as sex of the animals, prior lighting condition (DD), and duration of confinement to the novel wheel. Unlike many studies, novel wheel access in our study was not limited to 2 to 3 h but was continuous and thus extended over a large portion of the phase response curve. Interestingly, phase advances were also observed in male hamsters given continuous access to novel wheels beginning at ZT2 (Gannon and Rea, 1995), supporting the concept that duration of novel wheel exposure influences the direction of the phase shift. The second finding in the current study that contrasts with previous reports is that novel wheel exposure initiated at either ZT1 or ZT5 was similarly effective in blocking the LH surge, in spite of the fact that ZT1 is 4 h earlier than the onset of the approximately 1.5-h critical period for barbiturate blockade of the LH surge described in hamsters (Greenwald, 1971; Norman et al., 1972). In contrast, in rats, administration of pentobarbital at ZT1, that is, 4 h before onset of their 4-h long critical period (Everett and Sawyer,

7 456 JOURNAL OF BIOLOGICAL RHYTHMS / December 2010 Figure 6. Plasma LH levels in hamsters exposed to a novel wheel at ZT1 on proestrus. (A) Plasma LH concentrations on expected proestrus (day 1) and estrus (day 2) from ZT5 to ZT11. The shaded area represents time of exposure to DD. (B) Peak LH concentrations and time of occurrence of peak plasma LH concentrations (right panel) on days 1 (black bars) and 2 (open bars). All values are shown as mean ± SE. Figure 7. Circadian phase advances in the activity rhythm and time of the LH surge in hamsters exposed to a novel wheel at ZT1 on proestrus. Left panel: A representative actogram of a hamster whose activity rhythm was phase advanced 2.17 h. The 24-h day is represented horizontally, from left to right, with the time of day indicated by the numbers shown at the top. The vertical axis represents successive days, from top to bottom. The days of exposure to 14L:10D are indicated on the right by the cross-hatched vertical bar. The asterisk indicates day and time of transfer to a new cage and onset of DD (shown by the black vertical bar on the right). Right panel: Phase advances (mean ± SE) in the wheel-running activity rhythm (shaded bars) or time of LH surges relative to day 1 (black bars) are depicted. Numbers at the base of the bars indicate the number of animals. Open circles depict values for individual animals. 1950; Everett and Tejasen, 1967), does not block the proestrous LH surge (Everett and Sawyer, 1950). However, in hamsters, phenobarbital was not tested any earlier than 1 h before the beginning of the critical period (Greenwald, 1971); therefore, whether the critical period in hamsters includes ZT1 remains to be determined. Furthermore, the effectiveness of the novel wheel presented at ZT1 in blocking the LH surge may be related to the fact that the induced running persisted for many hours and thus extended into the critical period, regardless of the precise time of its onset. Although exposure to a novel room, wheel, and DD at ZT5 or ZT1 on proestrus causes a phase advance in wheel-running activity >2 h, this phase advance is not correlated with a 1-day delay of the LH surge or with the phase advance in the time of the LH peak. This result contrasts with our previous finding that LH surges following phenobarbital administration at ZT5 on proestrus are only delayed in animals whose activity rhythm is phase advanced 2 h or more (Legan et al., 2009). Thus, these findings suggest that barbiturates and novel wheel exposure may block the LH surge by different mechanisms. Only one previous study (to our knowledge) reported the effect of nonphotic phase shifts on hamster estrous cycles (Janik and Janik, 2003), and there are some similarities as well as some differences between our results. For example, we found that exposure to a novel wheel was more effective in inducing phase shifts in activity rhythms than transfer to a new cage, whereas Janik and Janik (2003) reported that moving the animals to a new room or transferring them to a new cage was more effective than confinement of proestrous hamsters to a new wheel. Their animals had prior experience with wheels and were confined to a different wheel for 3 h beginning at ZT4.5 on proestrus. Our animals were first exposed to a wheel in a clean cage at ZT5 or ZT1 on proestrus and were housed in this cage for the duration of the experiment. Thus, exposure to a wheel for the first time constitutes an even stronger zeitgeber for proestrous hamsters than transfer to a clean cage. In both studies, nonphotic phase shifts induced at about ZT5 on proestrus delayed the vaginal estrous discharge by 1 day in approximately half of the hamsters. In the Janik and Janik (2003) study, this blockade of the estrous discharge

8 Legan et al / NONPHOTIC PHASE ADVANCE OF HAMSTER LH SURGE 457 Figure 8. Running activity on proestrus following exposure to a novel wheel at ZT1. Left panel: Profile of running activity (cumulative wheel revolutions, mean ± SE) from ZT2 to ZT11 in relation to day of occurrence of LH surge. Shaded area depicts time of exposure to DD. Right panel: Cumulative wheel revolutions (mean ± SE) at ZT7 on day 1 are shown for animals whose LH surge was delayed (day 2) or not (day 1). The circles or stars aligned over the bars indicate the wheel revolutions of individual animals. The stars represent the data from one animal in each group whose running activity was not associated with the delay of the LH surge. The numbers of animals are indicated at the base of the bars. was associated with the magnitude of phase shifts in the locomotor activity rhythm. In our study, however, the LH surge, a more salient feature of the estrous cycle, was delayed in approximately 80% of the hamsters, and this blockade of the LH surge was not well correlated with either the delay in estrous discharges or the phase shift in the locomotor activity rhythm. Perhaps the most important finding from both studies was that the amount of exercise was related to the delay of the estrous cycle and/or blockade of the LH surge. The results herein demonstrate that blockade of the LH surge was closely correlated with the number of wheel revolutions, suggesting that exercise intensity or perhaps an associated stimulus may be mechanistically important for this endocrine phenomenon. It is interesting to note that chronic intense exercise interferes with menstrual cycles in women (DeSouza et al., 2010). In contrast, chronic wheel running does not interfere with estrous cycles in hamsters, while intense, acute exercise on the afternoon of proestrus does. Many previous studies conducted extensively in male hamsters have shown that acute exercise can serve as a nonphotic zeitgeber that resets the circadian clock (Reebs and Mrosovsky, 1989b; Wickland and Turek, 1991; Bobrzynska and Mrosovsky, 1998). However, in a variety of paradigms, exercise intensity is not always associated with large phase shifts, which suggests that some other stimuli associated with increased running in a novel wheel such as increased arousal and/or motivation may constitute the critical variable for nonphotic phase shifts (for review, see Mrosovsky, 1996). Indeed, some arousal-inducing stimuli (e.g., gentle handling), but not all such stimuli, can induce nonphotic-like phase shifts even in the absence of exercise (Antle and Mistlberger, 2000; Mistlberger et al., 2003). It remains to be determined whether induced exercise is essential for novel wheel blockade of LH surges or whether some other associated stimulus, such as increased arousal, is the key factor. Although the association between exercise intensity and delay of the LH surge is strong in most animals in our study, there were some notable exceptions, namely, the few animals that ran a great deal but did not have a delayed LH surge and those that ran very little but did exhibit a delayed LH surge. Similar exceptions have been reported previously (Reebs and Mrosovsky, 1989a, 1989b). These outliers support the possibility that some stimulus associated with wheel running, and not wheel running per se, causes the 1-day delay of the LH surge. Earlier studies have shown that the timing of the LH surge is closely associated with the onset of running activity and that this phase relationship is maintained under a variety of photoperiods (Moline and Albers, 1988) including chronic exposure to constant light (Swann and Turek, 1985). Under constant light, the locomotor activity rhythm splits into 2 components within a 24-h period, and each of these components is associated with an LH surge (Swann and Turek, 1985). In the former studies, the photoperiod remained constant for several weeks. In the present study, which investigated the acute response to a change in housing and photoperiod conditions, phase advances in the locomotor activity rhythm were not always associated with phase advances in the LH surge. Thus, the circadian rhythms of LH release and locomotor activity can be uncoupled at least temporarily, suggesting that they may be regulated by separate oscillators.

9 458 JOURNAL OF BIOLOGICAL RHYTHMS / December 2010 In summary, these studies demonstrate that novel wheel exposure affects not only the phase of the circadian clock but also the day and time of occurrence of the LH surge, in a manner that is associated with running but not with the circadian phase shift in the locomotor activity rhythm. It is likely that exposure of proestrous hamsters to a novel wheel activates a variety of brain neurons, including but not limited to hypothalamic orexin neurons and thalamic neurons in the intergeniculate leaflet, as shown previously in male hamsters and mice (Webb et al., 2008; Anaclet et al., 2009; Mikkelsen et al., 1988). Neurons activated by novel wheel exposure may communicate, either directly or through intermediate targets, with the circadian clock and/or the GnRH neurons controlling the LH surge. Since a running-related signal may be transmitted by multisynaptic pathways, it may also be modulated at various levels, thereby enabling it to have differential effects on circadian rhythms and the LH surge. ACKNOWLEDGMENTS The authors gratefully acknowledge Dr. A.F. Parlow at the National Hormone and Peptide Program for providing reagents for RIA, Mr. Matthew Congleton for technical assistance, and Dr. David Glass for his generous assistance with and consultation on activity monitoring. This study was funded by NIH R01 NS (to M.J.D. and S.J.L.). REFERENCES Alleva JJ, Waleski MV, and Alleva FR (1971) A biological clock controlling the estrous cycle of the hamster. Endocrinology 88: Anaclet C, Parmentier R, Ouk K, Guidon G, Buda C, Sastre JP, Akaoka H, Sergeeva OA, Yanagisawa M, Ohtsu H, et al. (2009) Orexin/hypocretin and histamine: distinct roles in the control of wakefulness demonstrated using knock-out mouse models. 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