INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

IN THE SUPRACHIASMATIC NUCLEUS (SCN), Clock is considered an essential transcription factor for cellular rhythmic processes. In the Clock¹⁹ mutant mouse (32), an A  T transversion in the splice donor site downstream from exon 19 leads to the skipping of this exon in the Clock mRNA and elimination of 51 amino acids in the COOH-terminal glutamine-rich region of the CLOCK protein (17). The BMAL1/CLOCK¹⁹ heterodimer lacks the ability to promote per transcription (9); consequently, per1, (13) BMAL1 (26), cry1 and cry2 (19), and arginine vasopressin (AVP) genes (13, 29) are arrhythmic in the SCN. Nevertheless, homozygous Clock¹⁹ mutants can express daily behavioral rhythms, and the rhythms may persist for 1 wk in constant darkness, with a period of ~27 h, before they become arrhythmic (20). Behavioral rhythms, however, can be masked by exogenous influences, and it can be quite challenging to distinguish between a masked and a genuine circadian rhythm.

The robust rhythm of pineal gland melatonin secretion is driven by the SCN in the anterior hypothalamus. The presence of melatonin receptors in the SCN (31) and their effects on SCN rhythmicity (21) suggest that the hormone may play an important role in the maintenance and entrainment of rhythms. The monitoring of melatonin rhythmicity is considered the "gold standard" for human studies, because it provides noise-free insight into SCN rhythmicity and is particularly useful in studies of rhythm disorders. In animals, it has proved useful in monitoring alterations in the melatonin rhythm in studies of the role of neurotransmitters in the control of rhythmicity by light (14).

Mice are widely used in circadian rhythm research because of the growth in our understanding of mouse genetics, particularly the ability to disrupt genes. They exhibit a robust rhythm of wheel-running activity, which allows simple, noninvasive quantitation. Unfortunately, the strains of laboratory mice that are so valuable for gene knockout studies, including the growing number of clock gene knockouts, are melatonin deficient because of mutations in arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) (8). The Clock¹⁹ mutant mouse was developed in melatonin-deficient C57Bl mice (32). The use of this background strain has seriously restricted the usefulness of the mutants for understanding the control of rhythmicity, because the SCN is a major target site for melatonin as well as the generator of rhythmicity. The CBA strain has a robust melatonin rhythm (12), which can be manipulated by light pulses (15).

We therefore set out to produce homozygous Clock^19/19 mutant animals with normal AA-NAT and HIOMT genes using selective crossing with CBA mice. The study addressed whether mice with a Clock¹⁹ mutation can synthesize melatonin. On the affirmative answer to this question, we then asked whether melatonin production and secretion are rhythmic in a 12:12-h light-dark photoperiod, whether the rhythm is endogenously generated, and whether light can suppress melatonin production and entrain the melatonin rhythm. Finally, we examined whether the wheel-running rhythms of Clock¹⁹ mutants that were melatonin proficient or deficient could be entrained with equal efficiency under conditions that minimized the effects of behavioral masking by light (skeleton photoperiods).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. On arrival in Adelaide, mice heterozygous for the Clock¹⁹ mutation (Clock^+/19) on a BALB/c background were paired to produce Clock^+/+, Clock^+/19, and Clock^19/19 lines. CBA/6CaH mice, a strain derived from stock originally provided by the Animal Resources Centre of the University of Western Australia (http://members.iinet.net.au/~arcwa/), were obtained from the University of Adelaide Central Animal House. Animals were housed in our specific pathogen-free facility in a 12:12-h light-dark photoperiod and fed standard laboratory chow ad libitum.

Genotyping. Inheritance of the Clock¹⁹ mutation was monitored in the breeding program by PCR of tail DNA (27). Briefly, mouse genomic DNA was extracted from tail biopsies and subjected to PCR analysis using primers reported previously (27). The products were digested with HincII and electrophoresed on a 1.5% agarose gel. The mutant allele produced a 460-bp product; the wild-type allele gave a 398-bp product.

Melatonin assays. Plasma was assayed by RIA using reagents obtained from Buhlmann Laboratories (Allschwil, Switzerland), as previously described (15). Briefly, plasma (100 µl) was added to the prewashed columns and sequentially washed with 10% methanol; hexane and melatonin were finally eluted with pure methanol. After evaporation of the solvent, the residue was reconstituted in 1 ml of buffer, and two 400-µl aliquots were subjected to RIA. Sensitivity of the assay was 10 pM. Intra- and interassay coefficients of variation were <10% and <15%, respectively, across the range of the standard curve.

Phenotyping for melatonin production. Because the mutations that disable AA-NAT vary between strains (23) or have not been characterized (HIOMT), we developed an in vivo phenotyping procedure for melatonin production based on the observation that early-morning administration of the -adrenergic agonist isoproterenol increased plasma melatonin levels in CBA, but not C57Bl or BALB/c, mice (15). Briefly, the test involves administration of isoproterenol (20 mg/kg sc) 2 and 4 h after lights on. Under light halothane anesthesia, blood (~300 µl) was collected from the retroorbital sinus 30 min after the last injection, and plasma was assayed for melatonin by RIA. Preliminary evidence indicated that mice carrying one mutant set of AA-NAT and HIOMT alleles produced half as much melatonin as those carrying two functional alleles (8). Thus we selected the highest melatonin producers at all stages of the breeding program to produce our target genotype, which we have designated Clock^19/19-MEL (see RESULTS).

Melatonin rhythmicity. To monitor the endogenous production and rhythmicity of melatonin in mice with a disabled Clock gene, Clock^+/+-MEL and Clock^19/19-MEL mice previously maintained in a 12:12-h light-dark photoperiod were released into continuous darkness, and blood and pineal glands were collected 4-64 h later from groups of 4-11 animals. Sampling times were concentrated around the time of subjective dawn during the first 2 days of continuous darkness. Tissue and blood were collected after brief exposure to dim red light.

Effects of a light pulse on melatonin. Exposure of animals to an unexpected light pulse during the night causes an immediate cessation of melatonin synthesis through inhibition of AA-NAT activity (18). To test whether melatonin synthesis in Clock^19/19-MEL mice could be suppressed by light, groups of five animals were killed in the dark around the expected time of peak melatonin production, specifically at ZT23.5, at ZT24, or 15 min after 15 min of exposure to a 100-lux light pulse that started at ZT23.5. As a control, Clock^+/+-MEL mice were killed around the time of their peak melatonin production, specifically at ZT21.5, at ZT22, or 15 min after a 15-min light pulse that started at ZT21.5.

Phase shifting of melatonin rhythms by light. To address whether Clock^19/19-MEL mice could be phase advanced or prevented from phase delaying (i.e., entrained) by exposure to light, groups of five mice were exposed to light (100 lux for 15 min) around the time of peak melatonin production, ZT21.5 and ZT23.5 for Clock^+/+-MEL and Clock^19/19-MEL mice, respectively, and kept in darkness until they were killed 21-28 h later. As a control, groups of mice from each line (n = 3-5 per time point) were not exposed to light. Potential phase shifting of the rhythm was analyzed by ANOVA from CT20 to CT23 and from CT2 to CT5 for Clock^+/+- MEL and Clock^19/19-MEL mice, respectively, with significance set at P < 0.05. The degree of shift was determined by changes in the time of the peak.

Wheel running. Mice were housed individually in cages equipped with 11.5-cm-diameter running wheels. A data acquisition system (LabPro, Data Sciences, St. Paul, MN) was used to record the number of wheel rotations in 10-min bins. After the data were downloaded, the Actiview software package (MiniMitter, Bend, OR) was used to display the actograms and to perform the ² method of period analysis.

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	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Breeding program for Clock^19/19-MEL mice. Male Clock^19/19 mice were crossed with five female CBA mice to produce heterozygotes for the Clock¹⁹, AA-NAT, and HIOMT mutations (3 litters of 7, 5, and 6 pups). The female offspring from each of the litters (n = 3, 3, and 2) were then backcrossed to Clock^19/19 male mice (not fathers) to produce the first backcross (BC1) line. This cross produced a total of 71 pups from 7 pregnancies, of which 37 were heterozygous and 32 were homozygous for the Clock¹⁹ mutation (2 died before genotyping). A second independent BC1 line produced 35 Clock^+/19 and 54 Clock^19/19 mice. Figure 1 shows the frequency distributions for the plasma melatonin levels after the isoproterenol phenotyping for the BC1 mice. On the basis of the expectation that the AA-NAT and HIOMT genes are not linked (8), we expected that 25% of the BC1 animals would be heterozygous for the enzyme. Thus animals with melatonin levels in the 75th-100th percentile could be assigned this genotype (Fig. 1). Clock^+/19 and Clock^19/19 genotypes had similar distributions. Indeed, the top 25% secretors had melatonin levels of 29.5 ± 4.4 pM (n = 24) and 28.4 ± 3 pM (n = 30), respectively, after isoproterenol administration.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Distribution of plasma melatonin levels of 1st backcross (BC1) mice after isoproterenol administration. Data are shown as frequency and percentage of mice with <10-140 pM melatonin in 10-pM bins. A: all BC1 mice. Horizontal line is drawn at 75th percentile to indicate undetectable levels in 75% of the mice, as expected. B: mice heterozygous or homozygous for Clock19 mutation.

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View larger version (17K): [in this window] [in a new window]   Fig. 2.   Distribution of plasma melatonin levels in intercross mice after isoproterenol administration. Data are shown as frequency and percentage of mice with <10-140 pM melatonin in 10-pM bins. Horizontal lines are drawn at 44th and 94th percentiles to identify mice presumed to have 0-1 functional arylaklylamine-N-acetyltransferase (AA-NAT) or hydroxyindole-O-methyltransferase (HIOMT) alleles (bottom 44%), 1 AA-NAT and 1 HIOMT allele (44-94%), or 2 functional AA-NAT and 2 functional HIOMT alleles (>94%). Mice with 78 and 135 pM melatonin were males and were used to establish the Clock19/19-MEL mouse line.

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Melatonin rhythms in Clock^19/19-MEL mice. The animals produced after the backcross and intercrossing provided an opportunity to conduct preliminary investigations on the endogenous production of melatonin in Clock¹⁹ mutants. Figure 3 shows the mean plasma melatonin levels after the isoproterenol test in those BC1 animals in which levels were higher than the 75th percentile; at these levels, the animals were likely to be carrying at least one functional allele of AA-NAT and HIOMT. Nighttime (ZT22) plasma melatonin concentration and pineal content are also shown in Fig. 3. The stimulated daytime levels were almost identical in the two genotypes, but 2 h before lights on (the time of peak melatonin production in the founder CBA strain), plasma and pineal melatonin levels were 50% lower in the Clock^19/19 mice than in the heterozygotes. Low, but detectable, plasma and pineal melatonin levels were recorded in some of the presumptive melatonin-deficient mice at ZT22, but even in this group, Clock^19/19 mice had lower levels at ZT22. We presume that the measurement of endogenous melatonin production in some animals categorized as deficient reflects a low false-negative assignment for the isoproterenol test.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Plasma melatonin levels and pineal gland melatonin content of presumptive melatonin-proficient (A) and -deficient mice (B) from 1st backcross (BC1) at 22 h zeitgeber time (ZT22). Values are means ± SE. Filled bars, Clock+/19 mice; open bars, Clock19/19 mice. Plasma melatonin levels measured after isoproterenol injection to phenotype the mice as melatonin-proficient or -deficient are also shown. All animals were killed 2 h before lights on (ZT22). Melatonin-proficient mice had identical isoproterenol responses regardless of Clock genotype, but levels were lower in Clock19/19 mice at ZT22 than in heterozygotes.

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View larger version (24K): [in this window] [in a new window]   Fig. 4.   Plasma melatonin levels and pineal gland melatonin content in Clock+/+-MEL and Clock19/19-MEL mice maintained in a 12:12-h light-dark photoperiod and released into continuous darkness. , Clock+/+-MEL mice; , Clock19/19-MEL mice. Values are means ± SE. CT, circadian time. A: plasma melatonin in 4-11 mice killed at each time point. Black horizontal bars, time of actual and expected darkness; gray bars, subjective day. ZT12 is the normal time of lights off, and CT12 is expected time of lights off in constant darkness. B: pineal gland melatonin content for animals in A. Melatonin rhythm peaks in Clock19/19-MEL mice 2 h later than in Clock+/+-MEL mice on the 1st night and shifts a further 2 h each day on release into constant darkness.

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View larger version (20K): [in this window] [in a new window]   Fig. 5.   Plasma melatonin levels (A) and pineal gland melatonin content (B) in Clock+/+-MEL and Clock19/19-MEL mice before and after exposure to 15-min 100-lux light pulse around the time of peak melatonin production. Because the 2 lines differed in the time of the peak, Clock+/+-MEL mice were pulsed or kept in darkness from ZT21.5 to ZT21.75 and killed at ZT22, whereas Clock19/19-MEL mice were pulsed from ZT23.5 to ZT23.75 and killed at ZT0. Values are means ± SE (n = 5) for each group. Exposure to light pulse decreased plasma melatonin levels and pineal melatonin content (open bars) to detection limit of assay and basal levels, respectively.

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View larger version (33K): [in this window] [in a new window]   Fig. 6.   Plasma melatonin levels and pineal gland melatonin content in Clock+/+-MEL and Clock19/19-MEL mice after a 15-min 100-lux light pulse. Clock+/+-MEL mice received the light pulse (arrows) at ZT21.5, whereas Clock19/19-MEL mice were pulsed at ZT23.5, the times of peak melatonin production in the 2 lines. Stippled areas represent melatonin levels from the 1st experiment (see Fig. 5). Values are means ± SE. A and C: Clock+/+-MEL mice. , No light exposure; , light pulse. B and D: Clock19/19-MEL mice. Alternating black and gray horizontal lines indicate subjective dark and light periods, respectively. Light pulse resulted in significantly reduced melatonin levels between CT20 and CT23 in Clock+/+-MEL mice and between CT2 and CT3 in Clock19/19-MEL mice.

Wheel-running rhythms. Wheel running in all four mouse lines in a 12:12-h light-dark photoperiod was characterized by intense running during the dark phase, with the greatest concentration of running early in the night and decreasing steadily toward lights on (Fig. 7). In the Clock^+/+ and Clock^+/+-MEL mouse lines, the onset of running occurred 6 ± 29 (SD) min after darkness and 11 ± 25 min before lights off, respectively. In the Clock^19/19 and Clock^19/19-MEL lines, the mean onset times were 6 ± 42 and 8 ± 42 min before lights off. The intra- and interanimal variance was higher in the mutants than in the wild-type animals. When the average number of wheel revolutions during the last 6 h of light was compared between genotypes, Clock^+/+ and Clock^+/+-MEL mice accumulated 4% and 3.7%, respectively, of their total wheel revolutions per day during this time. By contrast, 13.7% of the total daily running activity of Clock^19/19 mice and 14% of the activity of Clock^19/19-MEL mice occurred during this period. The melatonin-deficient Clock^+/+ and Clock^19/19 mice also accumulated 12.3% and 11.6%, respectively, of their total running during the first 6 h of light, in contrast to their melatonin-proficient genotypes (2.3% and 6.3%). In Fig. 7, the differences in the time of offset of running on light onset in the melatonin-deficient and -proficient Clock^+/+ lines are clearly evident.

View larger version (57K): [in this window] [in a new window]   Fig. 7.   Wheel-running activity rhythms of Clock19/19 and Clock19/19-MEL mice and their wild-type controls before and after release into constant darkness. Data are averages of the number of wheel revolutions per 10 min. A: Clock+/+ and Clock19/19 mice (light and darkness, n = 77 and 61, respectively, with 17 of each genotype continuing into constant darkness). B: Clock+/+-MEL and Clock19/19-MEL mice (light and darkness, n = 71 and 83, respectively, with 5 mutant and 11 wild-type mice continuing into constant darkness). Stippled areas, data from mutant lines; lines, data from wild-type controls. Vertical arrows, actual and expected times of lights off; square wave, light-dark transitions. Representative double-plotted actograms of 1 wild-type mouse, 2 free-running mutants, and 1 arrhythmic mutant for melatonin-deficient and -proficient lines are shown below A and B. DD, constant darkness.

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Entrainment of running activity by light pulses. When melatonin-deficient and -proficient Clock^+/+ mice were released into the skeleton photoperiod with the pulse occurring at the time of the previous lights on (ZT0), both lines free ran with a period of 23.5 ± 0.04 and 23.4 ± 0.25 h, respectively, over the duration of the experiment (Fig. 8). Over the duration of the experiment, the pulses never coincided with wheel-running activity in these mice. Therefore a double-pulse skeleton photoperiod (0.25:23.75:0.25:11.5-h light-darkness-light-darkness) was utilized (with the pulses occurring at the previous lights on and lights off). Under these conditions, it was hypothesized that the Clock^+/+ mice would be entrained to the "evening" pulse. As expected, the Clock^+/+ lines had periods of 24.0 ± 0.01 and 24.0 ± 0.03 h, showing that the pulses were sufficient to entrain the mice.

View larger version (41K): [in this window] [in a new window]   Fig. 8.   Wheel-running activity rhythm periods of Clock19/19 (A) and Clock19/19-MEL (B) mice and their wild-type controls before (open bars) and after (filled bars) release into a 0.25:23.75-h light-dark or 0.25:12:0.25:11.5-h light-dark-light-dark skeleton photoperiod. Values are means ± SE (hours). Results are grouped into mice that entrained (period 24 ± 0.5 h), entrained late (free ran and then had a period of 24 ± 0.5 h), free ran (period > 24.5 h), or became arrhythmic. Wild-type mice are shown as 2 groups, because all wild-type mice free ran in the 0.25:23.75-h light-dark photoperiod, where the pulse occurred at the subjective dawn, or all entrained to the double-pulse regimen, i.e., 0.25:12:0.25:11.5-h light-dark-light-dark photoperiod, where pulses occurred at subjective dusk and dawn. Double-plotted actograms show data from individual mice that are representative of the various categories in A and B. Vertical arrows, time of daily light pulses.

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View larger version (11K): [in this window] [in a new window]   Fig. 9.   Proportions of Clock19/19 and Clock19/19-MEL mice that entrained, free ran, or became arrhythmic in 0.25:23.75-h light-dark or 0.25:12:0.25:11.5-h light-dark-light-dark photoperiod. Values are percentages of total number of mice of each phenotype. Note high proportion of Clock19/19-MEL mice that were entrained compared with melatonin-deficient mutants.

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View larger version (68K): [in this window] [in a new window]   Fig. 10.   Double-plotted actograms of wheel running in Clock19/19-MEL mice exposed to a 0.25:23.75-h light-dark skeleton photoperiod, where daily pulses occurred at 3 h (A) or 6 h (B) after expected time of lights off. Top left actograms show timing of darkness (black) during experiments and time of daily single light pulses. e, Entrained; f, free running, a, arrhythmic.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A principal aim of the experiments was to investigate hormonal (melatonin) rhythmicity for the first time in mice carrying the Clock¹⁹ mutation. Unfortunately, the original Clock¹⁹ mutants were produced in C57Bl/6J mice and maintained on a BALB/c background and, therefore, are melatonin deficient. The CBA mouse strain produces melatonin rhythmically (12, 33), and the rhythm shifts in response to light pulses (15); therefore, we chose to introduce functional AA-NAT and HIOMT genes into Clock¹⁹ mice by selective breeding. It was possible that the mutants would not synthesize melatonin, because BMAL1/CLOCK heterodimer binding in the promoter region of AA-NAT is required for induction of the enzyme in the chicken pineal gland (7) and rat retina (5). Although there appears to be no such requirement in the rat pineal gland in vitro (5), this had not been tested in vivo. Our program showed that Clock is not required for AA-NAT induction in vivo and that Clock^19/19-MEL mice produced and secreted melatonin in amounts comparable to wild-type mice.

The selective breeding program was based on the assumption that the three genes segregated independently. AA-NAT has been mapped to mouse chromosome 11 (11) and Clock to mouse chromosome 5 (16), and AA-NAT and HIOMT genes are known to segregate independently (8). To our knowledge, mouse HIOMT has not been mapped; therefore, we had to assume that it was not close to Clock on chromosome 5. Therefore, a conservative selection procedure was used, with only top-ranking mice being used for breeding, especially after the intercross stage. The two putative Clock^19/19-MEL male mice were crossed again with CBA female mice to rapidly expand the colony size and reduce inbreeding and the chances of transmitting a mutant enzyme allele. After five generations, there has been no evidence of melatonin deficiency in the colony.

Melatonin-proficient mice carrying the Clock¹⁹ mutation showed a rhythm in melatonin production and secretion, with the peak occurring at the time of lights on, 2 h later than the wild-type strain. The melatonin production decreased to baseline within 1 h, even in the absence of light. This, together with the persistence of the melatonin rhythm in the Clock^19/19-MEL mice in constant darkness and the delay in the timing of the peak each cycle by 2 h, confirmed the endogenous nature of the melatonin rhythm.

To test whether control of the melatonin rhythm in the Clock^19/19-MEL mice was normal, these mice were subjected to unexpected exposure to light at night (10). When exposed to a brief light pulse at the time of the peak melatonin production, Clock^+/+-MEL and Clock^19/19-MEL mice responded by suppressing melatonin levels to baseline within 15 min. When Clock^+/+- MEL mice were exposed to a light pulse at the time of the peak of melatonin production and followed over the next cycle in constant darkness, the melatonin production ceased significantly earlier than nonpulsed mice, as shown previously in CBA mice (15). Exposure of Clock^19/19-MEL mice to light at the time of the melatonin peak resulted in significantly decreased pineal melatonin content and plasma melatonin levels at CT3 and CT4 compared with the nonpulsed animals. The fact that the peak at CT1 was still 1 h later than the time of the peak in a light-dark environment and 1 h earlier than in the control mutant animals suggests that the response to the light pulse was weak or that only a portion of the population responded. Nevertheless, we conclude that a light pulse can shorten the period of the melatonin rhythm and potentially entrain it. These responses to light are particularly interesting in view of the reported poor responsiveness of Clock^19/19 mutants to light pulses, at least with respect to induction of the immediate-early gene c-fos and per1 and per2 (27).

Using the more traditional method of wheel running to monitor rhythmicity, we confirmed the entrainment of Clock^19/19 mice (32) to a 12:12-h light-dark photoperiod and report similar results for Clock^19/19-MEL mice. The precision of the entrainment in both mutant lines was poor compared with the wild-type animals, and running during the light period was common. Because Clock¹⁹ mutants have a period of 26-27 h, the onset of running activity should be 2-3 h after lights off (1), but in the Clock^19/19 and Clock^19/19-MEL mice it occurred within ±10 min of lights off, similar to the wild-type mice. By contrast, the melatonin rhythm peak occurred 2 h later than in wild-type animals, which is expected from Aschoff's predictions (1). A similar lack of correlation between phase angle difference and period of the wheel running rhythm is evident in other Clock gene knockouts: per1 (2, 4), per2 (2, 34), per3 (25), per1/per2, per2/per3, and per1/per3 (2), cry1, cry2, and cry1/cry2 (30), and BMAL1 (3). Furthermore, when Clock¹⁹ mutants were released into constant darkness, there was a lag of one to two cycles before the onset shifted (so-called transients), a phenomenon not observed with the melatonin rhythm. By contrast, Clock^19/19 maintained on a Jcl:ICR genetic background have activity and body temperature rhythm acrophases in 12:12-h light-darkness consistent with the long endogenous period (24). This raises the question whether wheel running is a universally suitable output measure of the circadian clock in mice.

Given that the melatonin and wheel-running rhythms of Clock^19/19-MEL mice were strongly entrained to a 12:12-h light-dark photoperiod, we were interested in the extent to which rhythmicity could be entrained by short light pulses in a skeleton photoperiod. Vitaterna et al. (32) reported that a single 6-h light "pulse" could restore a long free-running period in Clock^19/19 mice that had become arrhythmic in constant darkness. We found that daily 15-min light pulses presented at the time of the previous lights on or 3 or 6 h later prevented animals from free running or eventually entrained them with a period close to 24 h. We observed that the Clock^19/19-MEL mice were more likely to be entrained than the melatonin-deficient mutants, although 40% still failed to entrain or became arrhythmic. Because we have introduced genes other than AA-NAT and HIOMT from the CBA strain, we cannot attribute the increased proportion of entrained animals solely to melatonin.

How can these results of robust hormone rhythmicity and entrainment in Clock^19/19-MEL mutant mice be reconciled with what is known about the molecular biology of the "essential" Clock gene? The Clock¹⁹ mutation is an antimorph that inhibits wild-type function (16). Indeed, not only is the BMAL/CLOCK¹⁹ heterodimer incapable of driving transcription of an mper1-luciferase reporter, it is less active than BMAL1 alone (9). As a result, Clock¹⁹ mutant mice should have decreased transcription of mper1 and other clock genes. This has been shown to be the case, with major disturbances in the rhythm of per1, per2, and per3 (13), cry1 and cry2 (19), BMAL1 (26), AVP (13, 29), and, more recently, prokineticin2 (6). One clock gene, mper2, appears to retain significant rhythmicity (13), and per1 and per2 have been shown to be induced, albeit at a reduced level, by a 15-min light pulse at ZT16 (27). The BMAL1/CLOCK heterodimer is the key driver of cellular rhythmicity; yet the Clock^19/19-MEL mutant mice show hormonal and behavioral circadian rhythmicity and entrainment to very brief light pulses. How can these hormonal and behavioral rhythms be sustained, especially at the unchanged amplitudes maintained by the mutants? One possibility is that the BMAL1/CLOCK heterodimer does have some intrinsic transcriptional activity at the E-boxes of some of the clock genes, but, as indicated earlier, at least for per1, there is no evidence for this (9). We are aware of no similar studies on per2. Another possibility is that another partner exists for BMAL1 that may drive transcription with decreased efficiency in the SCN or in centers that project to the SCN, resulting in the relatively poor precision of entrainment and long period length. NPAS2 would appear to be such a candidate protein, because it has been shown to be a functional analog of CLOCK and can partner with BMAL1 and drive transcription of appropriate reporter genes (22). However, npas2 is not expressed in the normal mouse SCN (28); instead, it is widely expressed in the forebrain. Although it is possible that npas2 is expressed in the SCN of Clock¹⁹ mice, and not normal mice, it is more likely that other parts of the brain that maintain rhythmicity via BMAL1/NPAS2 drive, project to the SCN, and maintain essential sleep-wakefulness and hormonal rhythms.

In conclusion, we have produced Clock¹⁹ mutant mice that can synthesize melatonin. We have shown that, despite producing a protein that fails to initiate transcription of Clock and clock-controlled genes, the animals have a normal endogenous melatonin rhythm than can be altered by light pulses. The timing of the melatonin rhythm is consistent with the period expressed in constant darkness, unlike the case with locomotor activity. Finally, we have shown that the Clock^19/19-MEL mice can be entrained to skeleton photoperiods, where the duration of the light period is 15 min.