A mutation of the circadian system in golden hamsters.

A mutation has been found that dramatically shortens the period of the circadian locomotor rhythm of golden hamsters. The pattern of inheritance of this mutation suggests that it occurred at a single, autosomal locus (tau). Wild-type animals have rhythms with free-running periods averaging about 24 hours; animals heterozygous for the mutation have periods of about 22 hours, whereas homozygous animals have rhythms with periods close to 20 hours. Animals that carry the mutant alleles exhibit abnormal entrainment to 24-hour light:dark cycles or are unable to entrain.

Photoperiodic control of hamster testis.

The response of the testes of juvenile and adult hamsters to various photoperiods was examined. The testes of juvenile animals reached maturity regardless of the light cycle on which the animals were raised. However, the testes of adult hamsters required at least 12.5 hours of light per day to maintain spermatogenesis and prevent degeneration. This is one of the few demonstrations of a response suitable for study in investigations of the photoperiodic control of testicular function in a laboratory mammal.

Circadian clock in photoperiodic time measurement: a test of the Bünning hypothesis.

A technique has been developed for effectively separating the direct inductive effect of a light signal from its effect on the phase of the rhythm of sensitivity to photoperiodic induction. With this technique it has been shown that a 75-minute pulse of light per day, when appropriately positioned with respect to the circadian activity cycle of the sparrow Passer domesticus, is sufficient to produce a response normally produced only by long days. The results cannot be interpreted in terms of a requirement of an absolute amount of either darkness or light and offer strong confirmation of Bünning's hypothesis concerning the mechanism of photoperiodic time measurement.

Entrainment of circadian rhythms by sound in Passer domesticus.

The circadian locomotor rhythm of house sparrows was entrained by a sound stimulus. The birds were maintained at a constant temperature in, dim green light. The entraining agent was 4 (1/2) 12 hours of tape-recorded bird song ,played each day. Variations in the response to this stimulus have been correlated with individual variations in free-running period. This is the first clear demonstration that a biological clock can be influenced by sound stimuli.

Pineal function: the biological clock in the sparrow?

The pineal organ of the house sparrow, Passer domesticus, is essential for persistence of the circadian locomotor rhythm in constant conditions. Upon removal of the pineal body, activity becomes arrhythmic. However, pinealectomy does not abolish the rhythm of locomotor activity in birds exposed to light-dark cycles. Pinealectomized birds are entrained by light cycles in much the same manner as are normal birds. Our data demonstrate that the pineal organ is a crucial component of the endogenous time-measuring system of the sparrow.

Neural connections of sparrow pineal: role in circadian control of activity.

Surgical and chemical interference with the neural connections of the house sparrow (Passer domesticus) pineal does not abolish the free-running rhythm in constant darkness, unlike pinealectomy. Pineals transplanted to the anterior chamber of the eye are capable of restoring rhythmicity to pinealectomized birds in constant darkness. The avian pineal does not appear to be neurally coupled to other components of the circadian system.

Photoperiodically significant photoreception in sparrows: is the retina involved?

Blind house sparrows (150 birds) and normal ones (199 birds) were subjected to various photoperiodic treatments consisting of cycles of 12 hours light, 12 hours dark and 16 hours light, 8 hours dark at intensities ranging from 20 to 500 lux. The testicular, response of the blind birds was found to be indistinguishable from the response of normal birds under these conditions. The data show that a functional extraretinal photoreceptor exists in this species which is fully capable of mediating the gonadal response to photoperiodic stimuli.

Extraretinal light perception: entrainment of the biological clock controlling lizard locomotor activity.

The circadian activity rhythm of the iguanid lizard Sceloporus olivaceus can be entrained by light cycles whether or not the animals have eyes. Removal of the pineal organ and parietal eye in blinded lizards does not prevent entrainment. Our data demonstrate the existence of an extraretinal photoreceptor which can mediate entrainment of a biological clock in reptiles.

Circadian rhythms in cultured mammalian retina.

Many retinal functions are circadian, but in most instances the location of the clock that drives the rhythm is not known. Cultured neural retinas of the golden hamster (Mesocricetus auratus) exhibited circadian rhythms of melatonin synthesis for at least 5 days at 27 degrees celsius. The rhythms were entrained by light cycles applied in vitro and were free-running in constant darkness. Retinas from hamsters homozygous for the circadian mutation tau, which shortens the free-running period of the circadian activity rhythm by 4 hours, showed a shortened free-running period of melatonin synthesis. The mammalian retina contains a genetically programmed circadian oscillator that regulates its synthesis of melatonin.

Melatonin: effects on the circadian locomotor rhythm of sparrows.

The continuous administration of low levels of melatonin via intraperitoneally placed Silastic capsules either (i) shortened the free-running period of activity or (ii) induced continuous activity in house sparrows (Passer domesticus) maintained in constant darkness. After the melatonin-filled capsules were removed, the period of the circadian rhythm of activity lengthened in rhythmic birds and normal rhythmicity was restored in continuously active birds. The results suggest that melatonin is involved in the physiological control of circadian rhythmicity in sparrows.

Melatonin: antigonadal and progonadal effects in male golden hamsters.

Melatonin induced marked testicular regression in hamsters maintained on photostimulatory long days (light-dark 14 : 10). In animals maintained on nonstimulatory short days (light-dark 6 : 18), small amounts of melatonin (50 micrograms per day; 100 millimeters capsule length) prevented testicular regression; but testicular atrophy occurred in hamsters that received larger amounts of melatonin (75 to 100 micrograms per day; 150 to 200 millimeters capsule length) and in control hamsters that received none. The results demonstrate that melatonin can exert either pro- or antigonadal effects and emphasize that the effects of melatonin on the testis cannot be properly assessed unless account is taken of the dosage and mode of melatonin administration and the photoperiod on which experimental animals are maintained.

Pineal function in sparrows: circadian rhythms and body temperature.

Deep body temperature of the house sparrow, Passer domesticus, was monitored continuously by radio telemetry. Pinealectomy abolished the normal circadian rhythm of body temperature in constant darkness, and significantly altered the amplitude of body temperature rhythms entrained to light cycles. The body temperature minima of pinealectomized birds never fell as low as those of unoperated birds regardless of the light conditions; the temperature maxima of both normal and pinealectomized birds were higher in light than in darkness. In sparrows the pineal organ is essential to the normal function of the biological clock controlling both activity and body temperature rhythms and may be directly involved in thermoregulation.

Regulation of testis function in golden hamsters: a circadian clock measures photoperiodic time.

The photoperiodic testicular response of adult golden hamsters was examined by the use of a 6-hour light period coupled with dark periods of 18, 30, 42, and 54 hours. Cycle lengths of 24 and 48 hours resulted in testicular regression, whereas testicular weight was maintained by cycle lengths of 36 and 60 hours. Our data demonstrate a circadian rhythm of sensitivity to the effects of light on the photoperiodic testicular response of the hamster. The position of light relative to the circadian system (as measured by the locomotor rhythm) is critical in the response.

Genetic control of melatonin synthesis in the pineal gland of the mouse.

Pineal melatonin may play an important role in regulation of vertebrate circadian rhythms and in human affective disorders. In some mammals, such as hamsters and sheep, melatonin is involved in photoperiodic time measurement and in control of reproduction. Although wild mice (Mus domesticus) and some wild-derived inbred strains of mice have melatonin in their pineal glands, several inbred strains of laboratory mice (for example, C57BL/6J) were found not to have detectable melatonin in their pineal glands. Genetic analysis suggests that melatonin deficiency in C57BL/6J mice results from mutations in two independently segregating, autosomal recessive genes. Synthesis of melatonin from serotonin in the pineal gland requires the enzymes N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). Pineal glands from C57BL/6J mice have neither NAT nor HIOMT activity. These results suggest that the two genes involved in melatonin deficiency are responsible for the absence of normal NAT and HIOMT enzyme activity.

Circadian clock in culture: N-acetyltransferase activity of chick pineal glands oscillates in vitro.

N-Acetyltransferase activity was measured in organ-cultured chick pineal glands. A circadian rhythm of enzyme activity persisted in cultured glands for up to 4 days. The phase of the rhythm in vitro closely approximates its phase in vivo. These observations demonstrate that the pineal gland of chicks contains (or is) a self-sustained circadian oscillator.

Entrainment of the circadian clock in the liver by feeding.

Circadian rhythms of behavior are driven by oscillators in the brain that are coupled to the environmental light cycle. Circadian rhythms of gene expression occur widely in peripheral organs. It is unclear how these multiple rhythms are coupled together to form a coherent system. To study such coupling, we investigated the effects of cycles of food availability (which exert powerful entraining effects on behavior) on the rhythms of gene expression in the liver, lung, and suprachiasmatic nucleus (SCN). We used a transgenic rat model whose tissues express luciferase in vitro. Although rhythmicity in the SCN remained phase-locked to the light-dark cycle, restricted feeding rapidly entrained the liver, shifting its rhythm by 10 hours within 2 days. Our results demonstrate that feeding cycles can entrain the liver independently of the SCN and the light cycle, and they suggest the need to reexamine the mammalian circadian hierarchy. They also raise the possibility that peripheral circadian oscillators like those in the liver may be coupled to the SCN primarily through rhythmic behavior, such as feeding.

Transplanted suprachiasmatic nucleus determines circadian period.

The pacemaker role of the suprachiasmatic nucleus in a mammalian circadian system was tested by neural transplantation by using a mutant strain of hamster that shows a short circadian period. Small neural grafts from the suprachiasmatic region restored circadian rhythms to arrhythmic animals whose own nucleus had been ablated. The restored rhythms always exhibited the period of the donor genotype regardless of the direction of the transplant or genotype of the host. The basic period of the overt circadian rhythm therefore is determined by cells of the suprachiasmatic region.

Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau.

The tau mutation is a semidominant autosomal allele that dramatically shortens period length of circadian rhythms in Syrian hamsters. We report the molecular identification of the tau locus using genetically directed representational difference analysis to define a region of conserved synteny in hamsters with both the mouse and human genomes. The tau locus is encoded by casein kinase I epsilon (CKIepsilon), a homolog of the Drosophila circadian gene double-time. In vitro expression and functional studies of wild-type and tau mutant CKIepsilon enzyme reveal that the mutant enzyme has a markedly reduced maximal velocity and autophosphorylation state. In addition, in vitro CKIepsilon can interact with mammalian PERIOD proteins, and the mutant enzyme is deficient in its ability to phosphorylate PERIOD. We conclude that tau is an allele of hamster CKIepsilon and propose a mechanism by which the mutation leads to the observed aberrant circadian phenotype in mutant animals.

Resetting central and peripheral circadian oscillators in transgenic rats.

In multicellular organisms, circadian oscillators are organized into multitissue systems which function as biological clocks that regulate the activities of the organism in relation to environmental cycles and provide an internal temporal framework. To investigate the organization of a mammalian circadian system, we constructed a transgenic rat line in which luciferase is rhythmically expressed under the control of the mouse Per1 promoter. Light emission from cultured suprachiasmatic nuclei (SCN) of these rats was invariably and robustly rhythmic and persisted for up to 32 days in vitro. Liver, lung, and skeletal muscle also expressed circadian rhythms, which damped after two to seven cycles in vitro. In response to advances and delays of the environmental light cycle, the circadian rhythm of light emission from the SCN shifted more rapidly than did the rhythm of locomotor behavior or the rhythms in peripheral tissues. We hypothesize that a self-sustained circadian pacemaker in the SCN entrains circadian oscillators in the periphery to maintain adaptive phase control, which is temporarily lost following large, abrupt shifts in the environmental light cycle.

Light-induced phase shifts in tau mutant hamsters.

Phase shifts produced by single 1-hr light pulses were compared in homozygous tau mutant and wild-type hamsters after several different kinds of pretreatment regimens. There was a dramatic increase in the magnitude of phase delays in the mutant hamsters as they were kept for progressively longer times in constant darkness (DD), and a smaller increase in the magnitude of phase advances. Under the same conditions a small increase in the magnitude of phase delays and no significant increase in phase advances occurred in the wild-type hamsters. After only 7 days in DD the phase response curves (PRCs) of mutant and wild-type hamsters were both type 1 and were indistinguishable from each other, whereas after 49 days in DD the PRCs of mutant hamsters had become type O. Mutant hamsters were entrained to eight different T-cycles (1 hr of light per cycle), released into DD, and given a phase delaying light pulse 7 days later. T-cycles which entrained the animals so that the 1 hr of light fell between 6 and 9 hours after the onset of activity suppressed the amplitude of phase delays, whereas T-cycles which entrained the animals so that the 1 hr of light fell at other times did not suppress phase delays. The implications of the data for entrainment theory and the mechanism of action of the tau gene are discussed.