Complex circadian regulation of pineal melatonin and wheel-running in Syrian hamsters

Circadian regulation of pineal melatonin content was studied in Syrian hamsters (Mesocricetus auratus), especially melatonin peak width and the temporal correlation to wheel-running activity. Melatonin was measured by radioimmunoassay in glands removed at different circadian times with respect to activity onset (= CT 12). Pineal melatonin peak width (h; for mean 125 pg/gland) and activity duration () were both 4–5 h longer after 12 or 27 weeks than after 5 or 6 days in continuous darkness (DD). Increased peak width was associated with a delay in the morning decline (M) of melatonin to baseline, correlated with a similar delay in wheel-running offset. In contrast, the evening rise (E) in melatonin occurred at approximately the same circadian phase regardless of the length of DD. Fifteen min light pulses produced similar phase-shifts in melatonin and activity. In a phase advance shift, M advanced at once, while E advanced only after several days of adjustment. Independent timing of shifts in the E and M components of the melatonin rhythm suggest that these events are controlled separately by at least two circadian oscillators whose mutual phase relationship determines melatonin peak width. This two-oscillator control of melatonin peak width is integral to the circadian mechanism of hamster photoperiodic time measurement.Abbreviations CT circadian time - DD continuous dark - L: D light: dark cycle - PMEL pineal melatonin - PRC phase response curve - RIA radioimmunoassay; , duration (h) of the active phase of the circadian wheel-running rhythm; , free-running period     

Dim nocturnal illumination alters coupling of circadian pacemakers in Siberian hamsters,<Emphasis Type="Italic"> Phodopus sungorus</Emphasis>

The circadian pacemaker of mammals comprises multiple oscillators that may adopt different phase relationships to determine properties of the coupled system. The effect of nocturnal illumination comparable to dim moonlight was assessed in male Siberian hamsters exposed to two re-entrainment paradigms believed to require changes in the phase relationship of underlying component oscillators. In experiment 1, hamsters were exposed to a 24-h light-dark-light-dark cycle previously shown to split circadian rhythms into two components such that activity is divided between the two daily dark periods. Hamsters exposed to dim illumination (<0.020 lx) during each scotophase were more likely to exhibit split rhythms compared to hamsters exposed to completely dark scotophases. In experiment 2, hamsters were transferred to winter photoperiods (10 h light, 14 h dark) from two different longer daylengths (14 h or 18 h light daily) in the presence or absence of dim nighttime lighting. Dim nocturnal illumination markedly accelerated adoption of the winter phenotype as reflected in the expansion of activity duration, gonadal regression and weight loss. The two experiments demonstrate substantial efficacy of light intensities generally viewed as below the threshold of circadian systems. Light may act on oscillator coupling through rod-dependent mechanisms.Abbreviations activity duration - DD constant dark or dim - E evening oscillator - ETV estimated testis volume - LDLD light-dark-light-dark cycle - LED light emitting diode - M morning oscillator - SCN suprachiasmatic nuclei - free-running period     

Circadian rhythms of corneal mitotic rate,retinal melatonin and immunoreactive visual pigments,and the effects of melatonin on the rhythms in the Japanese quail

We investigated circadian ocular rhythms in the Japanese quail, Coturnix coturnix japonica. The birds were placed under light-dark cycles (LD 1212), constant light (LL) and constant darkness (DD), and the retinas were dissected out at four-hour intervals throughout 24 h. Following measurements were performed. (1) Melatonin content in the retina was measured by radioimmunoassay. It was low in light and several folds higher in darkness under LD 1212. The rhythm continued in DD, but disappeared in LL. (2) Mitotic figures in the corneal epithelium were counted. Similar rhythms to the melatonin content were observed in the corneal mitotic rate with a slight phase delay. (3) The retinas were fixed at 4-h intervals and immunostained with anti-bovine rhodopsin serum and anti-chicken iodopsin monoclonal antibodies. The outer segments of photoreceptor cells were stained intensively throughout 24 h in LD 1212, LL and DD. In contrast, the stainability of the locus close to the outer limiting membrane where the Golgi apparatus exists changed diurnally. Scores showing the ratio of cells with positive staining indicated high values from 4 h after the onset of light to the beginning of dark phase under LD 1212. The values were high throughout 24 h in LL and intermediate or low in DD. (4) To investigate the effect of melatonin on the corneal mitotic rate and visual pigments at the Golgi region, melatonin was injected into one eye and saline into the contralateral eye. Melatonin induced a phase advance in the corneal mitotic rate under LD 1212, but did not induce a rhythm under LL. The ratio of photoreceptor cells with positive staining to anti-visual pigment antibodies at the Golgi region was not affected by melatonin injection.Abbreviations ANOVA analysis of variance - BSA bovine serum albumin - DD constant darkness - Io-mAb monoclonal antibodies against chicken iodospin - LD light-dark - LL constant light - mRNA messenger ribonucleic acid - PBS phosphate buffer solution - Rh-As antiserum against bovine rhodopsin - SCN suprachiasmatic nucleus - T transducin - T transducin      

Circadian activity of the red squirrel,Tamiasciurus hudsonicus,in continuous darkness and continuous illumination

In order to examine the effects of constant conditions on wheelrunning activity, 58 red squirrels (Tamiasciurus hudsonicus ) were taken directly from the field and placed in either total darkness (DD) or continuous illumination (LL) 500 lux, for up to 70 days. The squirrels were sampled at various times of the year so that seasonal changes in the endogenous rhythm could be examined. The initial phasing of activity correlated with the photoperiod in the field and had a free-running period () close to 24 hours. However, increased in a nearly linear fashion, averaging about 0.19 min/day for squirrels in DD and about 0.87 min/day for squirrels in LL. All animals in LL had rhythms which eventually became dissociated into two or more components. On the other hand, squirrels kept in DD had running patterns which usually remained intact. After being in constant conditions for between 20 and 70 days, 28 squirrels were subjected to step transitions in illumination from DD to LL (500, 350 or 200 lux) or vice versa. An increase in illumination level generally resulted in a phase advance and a decreased. A decrease in illumination level generally resulted in a phase delay and increased. The maximum amount of activity occurred at some critical intensity between 200 and 350 lux. The free-running period was directly correlated with the entrainment phase-angle difference. This relationship was compared to the red squirrel's light-pulse response curve. These data support Pittendrigh's (1965) entrainment model.Presented during the Seventh International Biometeorological Congress, 17–23 August 1975, College Park, Maryland, USA.     

Expression of circadian rhythmicity in Djungarian hamsters under constant light: Effects of light intensity and the circadian system's state

Summary Djungarian hamsters (Phodopus sungorus), were exposed to constant light with increasing intensities (20, 60, 350 lux), and wheel running activity was recorded. With increasing light intensity the percentage of hamsters showing a split in their daily activity pattern increased and the free running period was lengthened for both the unsplit and the split state. The fact that the free running period of both states depended on the light intensity together with the observation that the highest incidence of acircadian activity occurred under 350 lux, provoked the idea that the emergence of splitting or acircadian rhythmicity is a direct consequence of the light induced lengthening of the free running period. However, analysis of the data failed to support the idea that emergence of a split or acircadian activity is a threshold phenomenon with respect to the free running period.Due to differences in circadian function some Djungarian hamsters do not exhibit photoinduction following short day exposure. In these individuals splitting also occurred but required exposure to a higher light intensity than in photo-responsive hamsters. This observation is in accordance with the idea that the two phenotypes differ in the interaction of the two component oscillators underlying circadian rhythmicity.Abbreviations LD long day photoperiod - LL constant light - SD short day photoperiod - free running period     

The effect of photoperiod on daily rhythms of Oxygen consumption in the tropical toad,Bufo marinus

Summary Oxygen consumption was measured with an automatic continuously recording electrolysis system in toads acclimated at 15° C and under photoperiods of LD 816, 1212, 168, 66, 231 and LL and DD. All groups exposed to LD had pronounced significant daily rhythms with a trough near the onset of the photophase and a peak at the beginning of the scotophase (Figs. 1–3, 9, 10), while no rhythms were detected in animals exposed under free-running conditions of constant light (Figs. 6–7) or constant darkness (Fig. 8), even on the first day under LL or DD. These rhythms are thus shown to be non-circadian, since they do not persist in freerunning conditions of up to 45 days. Some preliminary evidence from studies on locomotor activity (Figs. 12, 13) indicates that the daily rhythm in metabolic rate may be independent of the locomotor activity cycle. The difference in the rate of oxygen consumption during peak and low hours of each daily cycle is defined as the daily routine metabolic scope, which may be more useful in ecological studies of animal energetics than the difference between the minimum and a forced maximum metabolic rate. No correlations were found between daily changes in atmospheric pressure and cycles of oxygen consumption.This work was supported by a grant from the National Science Foundation (GB-3574) and a University of Rhode Island Research Committee Grant-in-aid. We are grateful to Robert Cubert for aid in designing and constructing the electrolysis system and for assistance with computer programs.     

Entrainment and phase shifting of the circadian rhythm of cell division by light in cultures of the achlorophyllous ZC mutant ofEuglena gracilis

The photosynthesis-deficient ZC mutant ofEuglena gracilis Klebs (strain Z) was cultured at 16°C on an aerated, magnetically stirred, mineral medium containing 0.1% ethanol (pH 7.0). Cell division could be entrained by a 12: 12 light: dark cycle (LD: 12, 12) or even by a one-pulse skeleton photoperiod (LD: 1,23) The rhythm free-ran in DD for at least 8 days with a circadian period (=25.5 h) in populations that had been previously entrained by LD. The freerunning rhythm could be phase-shifted by a single 1-h light pulse (3000 lx). The strong (Type 0) phase-response curve derived from the resetting effects of such signals given at different circadian times was similar to that for the photosynthetic wild-type strain. These results demonstrate that the presence of a functional chloroplast compartment is not necessary for the circadian clock to function inEuglena and suggest that phase resetting of the circadian clock by light occurs via a similar pathway in both photosynthetic and non-photosynthetic cell types.     

Effect of melatonin on changes in locomotor activity rhythm of Syrian hamsters injected with beta amyloid peptide 25-35 in the suprachiasmatic nuclei

1. Alzheimer's disease is associated with circadian rhythm disturbances, probably because of beta amyloid-induced neuronal damage of hypothalamic suprachiasmatic nuclei (SCN).2. Since there is no published study on the circadian consequences of injecting beta amyloid peptide in experimental animals, one objective of the present study was to examine circadian locomotor activity in Syrian hamsters injected with beta amyloid peptide 25–35 into both SCN.3. Because one of the proposed therapies for circadian alterations in dementia is the administration of melatonin, a chronobiotic agent with antioxidant properties, the preventive effect of melatonin on the circadian changes produced by beta amyloid microinjection into SCN was also assessed.4. Wheel running activity was recorded by using the Dataquest III system in male golden hamsters kept under 14:10 light–dark photoperiods. Animals received microinjections of beta amyloid peptide 25–35 (100 M solution, 1 L) or saline in each SCN. Only those animals with neuronal lesions larger than 10% of SCN after beta amyloid injection were considered for further analysis.5. To assess the effect of melatonin on beta-amyloid peptide activity, melatonin was given in the drinking water (25 g/mL) starting 15 days in advance to the microinjection of beta amyloid peptide into SCN.6. Beta amyloid-treated hamsters exhibited a significant phase advance of onset of running activity of about 22 min as compared to saline-injected animals. They also showed a significantly greater variability in onset time of wheel running activity, mainly evident from 6 to 15 days of treatment.7. Melatonin administration in the drinking water prevented the phase advance of onset time and the increased variability of onset time brought about by beta amyloid peptide.8. The results support the existence of a neuroprotective effect of melatonin on beta amyloid-induced circadian changes in hamsters.     

Mosquito circadian and circa-bi-dian flight rhythms: a two-oscillator model

Summary Circadian rhythmicity was found in the flight activity ofCuliseta incidens recorded in constant darkness for up to 14 weeks. The first nonhuman circa-bi-dian (about-two-day) rhythms were also found (Figs. 6–7). Circadian periods were either stable, remaining <24h (Fig. 1), or labile, with a change from <24h to >24 h (Fig. 2). Inactivity phenomena (day-skipping) were common in the latter group only (Fig. 5). The period at activity onset was much more labile than the period at offset (Fig. 4). The activity patterns of some period-lengthened animals suggested control by two oscillators which could temporarily or permanently uncouple (Figs. 8–9).A pacemaker model consisting of a labile evening (E) oscillator mutually coupled to a stable morning (M) oscillator is the most economical proposal which can account for these results. The view that E and M uncouple and run with different periods can account for many records in which the period was labile. Circa-bi-dian rhythms can be explained by the period of E lengthening to where it synchronizes with M in a 21 mode. Thus, E and M are proposed to behave similarly to the human activity and temperature oscillators. It is speculated that day-skipping might indicate that E oscillates between circadian and circa-bi-dian ranges without overt activity being expressed.Abbreviations LD1212 alternating 12h light, 12h dark - DD constant dark - LL constant light - period of rhythm - _on period at activity onset - _off period at activity offset - activity time - mean activity time     

Running activity mediates the phase-advancing effects of dark pulses on hamster circadian rhythms

Summary Pulses of darkness can phase-shift the circadian activity rhythms of hamsters,Mesocricetus auratus, kept in constant light. Dark pulses under these conditions alter photic input to the circadian system, but they also commonly trigger wheel-running activity. This paper investigates the contribution of running activity to the phase-shifting effects of dark pulses. A first experiment showed that running activity by itself can phaseshift rhythms in constant light. Hamsters were induced to run by being confined to a novel wheel for 3–5 h. When this was done at circadian times (CT) 0, 6, and 9, the mean steady-state phase-shifts were 0.6 h, 3.5 h, and 2.3 h, respectively. The latter two values are at least as large as those previously obtained with dark pulses of similar durations and circadian phases. A second experiment showed that restricting the activity of hamsters during 3-h dark pulses at CT 9 reduces the amplitude of the phase-shifts. Unrestrained animals phase-advanced by 1.1 h, but this shift was halved in animals whose wheel was locked, and completely abolished in animals confined to nest boxes during the dark pulse. Activity restriction in itself (without dark pulses) had only minimal phase-delaying effects on free-running rhythms when given between ca. CT 10 and CT 13. These results support the idea that, in hamsters at least, dark pulses affect the circadian system mostly by altering behavioural states rather than by altering photic input to the internal clock.Abbreviations CT circadian time - DD constant darkness - LD light-dark - LL constant light - PRC phase response curve - period of rhythm     

Ambient temperature cycles entrain the free-running circadian rhythms of the stripe-faced dunnart,Sminthopsis macroura

Summary We examined the effect of cycles of 12 h warm (35 ± 2 °C) and 12 h (21 ± 2 °C) ambient temperature (Ta) upon the circadian activity rhythms of stripe-faced dunnarts, Sminthopsis macroura, free-running in conditions of constant dark (DD) or constant light (LL). It was hypothesized that dunnarts would entrain to the temperature cycles (TaHLs) or show perturbations of period, and that LL would act synergistically with the TaHLs in these effects. Under DD, 2 of 6 animals showed clear entrainment to the TaHLs. Other animals exhibited changes of period () and heavy negative masking of activity during the warm fraction of the TaHLs. Under LL, 3 of 12 animals entrained to the TaHLs. It was concluded that Ta is a significant though weak Zeitgeber for S. macroura compared to light. It is possible that TaHLs entrain homeotherm activity rhythms by altering the rhythm of body temperature, which is usually tightly coupled to activity.Abbreviations TaHL a cycle of Higher and Lower ambient temperature - TaC Constant Ta - Tb body temperature     

Effect of Aging on Circadian Activity in Gray Mouse Lemurs

Microcebus murinus s a very photoperiod-dependent primate with a potentially extended longevity (13 years). Reduction of artificial seasonal cycles allows acceleration of the aging process. Under these conditions, age is defined according to the number of seasonal cycles. We conducted experiments in order to assess the effects of aging upon (1) the main parameters (period: duration: ) of the circadian activity–rest rhythm; and (2) the plasticity of the response to light, which is the main entraining factor of the internal clock. We studied the evolution of and through two types of experiments: a transverse one comparing 36 males of various ages (1–13 seasonal cycles) and a longitudinal one following 2 pairs of males from the same litter (one from each pair was maintained under natural cycle while the other was submitted to a shortened cycle) over 54 months. Results from transverse experiments demonstrated no statistical difference in and with age except in 4 senescent (>10 cycles) subjects in which these two parameters were decreased. Longitudinal experiments confirmed this tendency. The plasticity of responses to light, resynchronization after a shift of the day–night cycle, or shift of activity onset after presentation of a light pulse at various circadian times was unaffected by aging. Taken together, the data demonstrate that the parameters of the circadian activity–rest rhythm remain stable over a long span of life and/or that light remains a powerful entraining parameter even in very old individuals.     

Indigogenic methods for glycosidases

Summary 4-Cl-5-Br-3-indolyl--D-fucoside (IbF) was tested in histochemical and bio-chemical experiments. Sections from differently treated parts of the small intestine of suckling and adult rats, of jejunum of adult hamsters, guinea pigs, cooks, monkeys, of human jejunal biopsies, of kidney of suckling and adult rats, adult monkeys, guinea pigs and hamsters, and of some other rat organs were used in histochemical experiments. Neutral and acid -D-galactosidases prepared from homogenates of the small intestine of suckling rats by chromatography on Sephadex G 200 were used in biochemical experiments.The recommended medium for histochemical studies consists of 0.1 M citrate phosphate buffer pH 6 (brush border enzyme) and pH 4 (lysosomal enzyme), 3.6·10^–4M IbF and 3.1·10^–3 M potassium ferri- and ferrocyanide.IbF is split quite efficiently by the brush border -D-galactosidase (=lactase) and the recommended histochemical method with IbF at pH 6 is the method of choice in studies concerned with the localization of intestinal lactase. In unfixed cold microtome sections the brush border activity can be easily detected within 15–240 minutes, depending on the lactase activity of the studied sample. In this study this activity was shown to be present in the brush border of enterocytes in the upper part of crypts reaching its maximum in differentiated enterocytes covering the sides and tops of villi in the small intestine of suckling (the highest activity) and adult rats (3–4x lower activity according to the time of appearance of the staining), and of human, monkey and hamster jejunum. In the cock jejunum traces of brush border staining were seen only after 24 hours of incubation. In enterocytes of patients with celiac sprue the brush border activity was very much reduced in dependence on the stage of the disease. Brush border staining along with a diffuse cytoplasmic reaction was found in the proximal convoluted tubules of the monkey kidney. The question of localization of enzyme activity splitting IbF in the monkey kidney deserves further investigation.IbF is also split by isolated intestinal acid -D-galactosidase and histochemically a positive reaction was found in lysosomes of many cells displaying a high activity of -D-galactosidase when IbF was used in the recommended medium of pH 4. The use of aldehyde fixation (glutaraldehyde is to be preferred) is a prerequisite for the assessment of this lysosomal localization. The lysosomal activity splitting IbF is not firmly bound structurally and escapes from cold microtome sections prepared from unfixed tissue samples into the incubation solution.The recommended method is also very suitable for processing zymograms and immunoprecipitation lines of lactase with antisera obtained by Ouchterlony's technic and by immunoelectrophoresis.     

Changes in brain components during the development of mice homozygous for the locus “dwarf” (dw)1,5

Brain composition and developmental changes were investigated in mice homozygous for the locus dwarf, and characterized by a reduced level of growth hormone, thyroid stimulating hormone, and prolactin, and by secondary hypothyroidism. The difference in adult brain weight (–32%) between the dwarf and the normal mice was not found to parallel the difference in body weight (–71%), whereas the differences in the weight of the liver (–79%) and that of the kidney (–75%) did. Several biochemical parameters of brain development were assayed in dwarf and normal mice between the ages of 15 and 210 days. Levels of cerebrosides, sulfatides, gangliosides, phospholipids, cholesterol, protein, and RNA (per gram wet weight) were the same for the dwarf and the controls, but the net difference in total brain DNA was less than the net total brain RNA difference (–11% vs. –27%). Total brain lipids (absolute quantities) were the same at 15 days. The difference was –37% by the 50th day, and remained constant thereafter. No change in the specific activity of 2,3-cyclic nucleotide 3-phosphohydrolase or 3-phosphoadenosine-5-phosphosulfate: galactocerebroside sulfotransferase was observed. These data suggest that the regulation of the development of brain structures is maintained, but the level of the synthesis of the various brain constituents is reduced in proportion to the brain weight. The development of the dwarf brain seems to proceed harmoniously.Abbreviations used PAPS 3-phosphoadenosine-5-phosphosulfate - PAPS-CST 3-phosphoadenosine-5-phosphosulfate:galactocerebroside sulfotransferase - CNP 2, 3-cyclic nucleotide 3-phosphohydrolase - Neu NAc N-acetylneuraminic acid This paper is part of the Doctorat d'Etat thesis of L. L. Sarliève.     

Circadiane Periodik von Finkenvögeln unter dem Einfluß eines selbstgewählten Licht-Dunkel-Wechsels

Zusammenfassung In zwei Versuchsserien wurden einzeln in Drahtkäfigen gehaltene Finkenvögel (Buchfink, Grünfink und Bergfink) in lichtdichten Kammern zwei Arten von Wahl-Bedingungen mit entweder räumlich oder zeitlich variabler Beleuchtungsstärke ausgesetzt. In der ersten Serie war ein Käfig mit drei Sitzstangen zur Hälfte hell und zur Hälfte dämmrig ausgeleuchtet. Der Vogel hatte damit die Möglichkeit, durch Wahl entsprechender Sitzstangen die von ihm im circadianen Rhythmus bevorzugte Helligkeitsstufe aufzusuchen. Als Folge davon war der Vogel einem selbstgewählten (circadianen) Licht-Dunkel-Wechsel (sLD_r) ausgesetzt. Vorher oder nachher wurde jeder Vogel außerdem im gleichmäßig ausgeleuchteten Käfig bei konstant heller Beleuchtung (LL) und bei konstant dämmriger Beleuchtung (DD) gehalten. In der zweiten Serie saß der ruhende Vogel (in einem Käfig mit zwei Sitzstangen) im Dämmerlicht. Wurde der Vogel aktiv, so ging helles Licht an, wenn eine bestimmte Zahl von Hüpfern je halbe Stunde überschritten war; kurz nach Ende der Aktivität des Vogels ging das helle Licht aus und es herrschte wieder Dämmerlicht. Der Vogel war somit einem selbstgesteuerten (circadianen) Licht-Dunkel-Wechsel (sLD_z) ausgesetzt. Wie in der ersten Serie, wurde jeder Vogel außerdem in konstant hellem Licht (LL) und in konstant dämmrigem Licht (DD) gehalten. Die Aktivität der Tiere wurde auf einem Zeitmarkenschreiber registriert.Da in beiden Versuchsserien der Vogel selbst die jeweiligen Änderungen in der Beleuchtungsstärke wählte bzw. steuerte, wirkte der Licht-Dunkel-Wechsel nicht als Zeitgeber. Die Tiere verhielten sich deshalb wie autonome Systeme und zeigten freilaufende circadiane Perioden in allen Bedingungen (LL, DD und sLD). Bei allen Individuen waren die Werte für die Aktivitätsmenge, für das Verhältnis zwischen Aktivitätszeit und Ruhezeit (-Verhältnis) und für die Frequenz (Kehrwert der Periode) im LL größer als im DD. Im Versuch mit räumlich variabler Beleuchtungsstärke (sLD_r) lagen die Werte für alle drei Parameter zwischen den im LL und im DD gemessenen Werten. Im Versuch mit zeitlich variabler Beleuchtungsstärke (sLD_z) galt dies nur für die Aktivitätsmenge und das -Verhältnis; der unter diesen Bedingungen gemessene Wert für die Periode lag jedoch nicht nur über dem im LL, sondern auch beträchtlich über dem im DD gemessenen Wert. Diese Rückkopplungswirkung des selbstgesteuerten Licht-Dunkel-Wechsels auf das circadiane System, die sich in einer Verlängerung der freilaufenden circadianen Periode äußert, ist nach schwingungstheoretischen Überlegungen zu erwarten. Sie kehrt die positive Korrelation zwischen Frequenz und Beleuchtungsstärke, wie sie für tagaktive Vögel die Regel ist, in ihr Gegenteil. Derselbe Effekt könnte auch bei subjektiv bedingtem Wechsel der Beleuchtungsstärke bedeutungsvoll sein.

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Circadian period of finches as influenced by self-selected light-dark cycles

Summary In two series of experiments, single caged finches (Chaffinch, Greenfinch and Brambling) have been exposed in light-proof boxes to two types of conditions with either a spatial or a temporal choice for light and dark. In series 1, a cage with three perches was illuminated half with bright light and half with dim light, giving the bird a chance to follow its circadian rhythm of preference for bright and dim light by selecting the proper perches. By doing so, the bird was exposed to a self-selected (circadian) light-dark cycle (sLD_r). Before or afterwards, the bird was also kept in constant bright light (LL) or constant dim light (DD) throughout the whole cage. In series 2, the resting bird was sitting (in a cage with two perches) in dim light, and when it became active, bright light was turned on by means of an electronic device after a certain number of hoppings had been accumulated; the light was turned off a short time after the bird went to rest. The bird therefore was exposed to a self-triggered (circadian) light-dark cycle (sLD_z). As in series 1, the bird was also tested in constant bright light (LL) as well as in constant dim light (DD). Activity was recorded on an event-recorder.Since in both series, changes in the intensity of illumination were selected or triggered, respectively, by the bird itself, the light-dark cycle did not act as a Zeitgeber but allowed the bird to free-run as an autonomous system. The bird, therefore, showed clear circadian rhythms in all conditions (LL, DD, and sLD). In all individuals, the values for amount of activity, for ratio between activity-time and rest-time (-ratio), and for frequency (reciprocal of circadian period) were greater in LL than in DD. In the choice experiments with spatial variation of light intensity (sLD_r), the values for all three parameters were inbetween those measured in LL and in DD, indicating that the birds averaged the light intensities of the two compartments of the cage. In the second choice situation (sLD_z), only the values for -ratio and amount of activity were inbetween those measured in LL and in DD; the period, however, was longer even than in DD. This feedback-effect of a self-triggered light-dark cycle on the circadian system which results in a lengthening of the free-running period, is to be expected from oscillation theory; it reverses the positive correlation between frequency and light intensity which is the rule for day-active birds in constant conditions. Possible implications of this effect for subjective instead of objective changes in light intensity are discussed.

     

A study of the relationship between photoperiod and pinealocyte granulated vesicles in the golden Syrian hamster

Summary Previous studies in rabbits and mice have revealed distinct circadian rhythms in the number of pinealocyte granulated vesicles (PGVs) and control of their synthesis and/or secretion by sympathetic nerves. The present study demonstrates the absence of a circadian rhythm in PGV content in hamsters functionally pinealectomized by maintenance under long photoperiod (L/D=14/10 h). On the other hand, a highly significant rhythm of low amplitude was noted in PGVs of hamsters placed in photoperiods (<12.5 h) which are known to initiate pineal antigonadotropic activity. Bilateral optic enucleation, which also leads to pineal-mediated gonadal atrophy in the hamster, produced a significant decrease in the number of perivascular PGVs when compared to intact control animals. Daily late afternoon injections of melatonin produced no significant difference in the number of PGVs between treated and control animals at any sample time examined.Supported in part by N.I.H. Grant#HD08759     

Chemical induction of interleukin-8, a proinflammatory chemokine,in human epidermal keratinocyte cultures and its relation to cytogenetic toxicity

Tumor promoters, proinflammatory cytokines, endotoxins, and protein synthesis inhibitors can modulate cell cycle kinetics of various cell types, stimulate production of reactive oxygen species, and induce keratinocytes to produce interleukin-8 (IL-8), a potent chemotactant for polymorphonuclear neutrophils and T lymphocytes. The aim of this study was to determine whether perturbations of cytogenetic responses correlated with the induction of IL-8 expression. Cultures of primary human keratinocytes were grown in serum-free medium with 5 mol/L bromodeoxyuridine to label DNA and exposed either to phorbol-13-myristate-12-acetate (PMA) (0.0001–100 ng/ml), cycloheximice (CHX) (0.01–50 g), lipopolysaccharide (0.1–100 g/ml), tumor necrosis factor- (TNF) (3.13–50 ng/ml), or interleukin-1 (IL-1) (1–182 pg/ml). Metaphase chromosome preparations were stained by a fluorescence-plus-Giemsa technique to differentiate sister chromatids. For IL-8 production, keratinocytes were grown to 70% confluency and then exposed to chemicals for 24 h. Immunoreactive IL-8 was quantitated from the supernatants by ELISA. With the exception of benzo(a)pyrene used as a positive control, none of the agents induced sister chromatid exchanges. However, PMA and TNF induced IL-8 production that coincided with significant cell cycle inhibition. IL-1 had no effect on cytogenetic endpoints, yet stimulated a 6.3-fold increase in IL-8. CHX inhibited cell cycle progression and mitotic activity at concentrations that were 200 times lower than required for IL-8 induction; however, puromycin (0.31–10 g/ml), another protein synthesis inhibitor, did not induce IL-8. At all concentrations tested, TNF reduced the mitotic index by 45%, slowed cell cycle progression by 3.5 h, and induced a flat, albeit large, IL-8 response at concentrations 12.5 ng/ml. These agent-specific response patterns suggest that induction of IL-8 production is not always the inevitable result of cell cycle perturbations or genetic damage.Abbreviations B(a)P benzo(a)pyrene - BrdU 5-bromo-2-deoxyuridine - CHX cycloheximide - ICAM intercellular adhesion molecules - IL-1 interleukin-1 - IL-8 interleukin-8 - KGM keratinocyte growth medium - LPS lipopolysaccharide - PKC protein kinase C - PMA phorbol-13-myristate-12-acetate - PMN polymorphonuclear neutrophil - ROS reactive oxygen species - SCE sister chromatid exchange - TNF tumor necrosis factor      

Inhibition of DNA-ethidium bromide intercalation due to free radical attack upon DNA

Summary The fluorescent intercalation complex of ethidium bromide (ETB) with DNA was used as a probe to compare the effects of various radicals with respect to impairment of the DNA base-pair region.OH radicals inhibit up to 0.7 dye intercalations perOH at low salt concentration, and for various oxidizing species the effect decreases in the orderOH > Br ₂ ^– > N ₃ > $$ " align="middle" border="0"> (SCN) ₂ ^–. DNA impairment by theOH product of Met-Gly is comparable to that of N ₃ , but no effect was found due to the interaction between DNA and Lys-Tyr-Lys phenoxyl radicals. The reducing speciese _aq ^– , H, O ₂ ^–, and CO ₂ ^– hardly affect the DNA-ETB intercalation.     

Variable thermal sensitivity as output of a circadian clock controlling the bimodal rhythm of temperature choice in the ant Camponotus mus

Along a thermal gradient and under a LD 1212 h cycle, nurse workers of the ant Camponotus mus select for the brood two different temperatures daily: 30.8°C at the middle of the light period (circadian phase = 90°), and 27.5°C 8 h later, during the dark period (CP = 210°). Brood-carrying activity proved to be self-sustained, running its two daily bursts free with a similar period of 23.5 h, under both LL and DD. The LD alternation acted as a strong Zeitgeber. A phase-delay of the LD 1212 h cycle reset the overt rhythm at once, being both daily events locked-on to the delayed light: dark transition. However, changes in expression, non-occurrence, or even splitting of the two daily brood-carrying events during resetting depended on the phase of the delayed DL transition. By comparing the occurrence of activity with predictions based on a threshold curve of thermal sensitivity, results indicated that an immediate resetting of the involved pacemaker actually takes place. Nurse workers do not directly control the total time spent by the brood at the selected temperature. Instead, the endogenously-driven thermal sensitivity triggers their thermal-searching behavior at two critical times of the day, when environmental temperature is expected to reach its maximum and minimum.     

Effects of cAMP,theophylline, imidazole,and 4-(3,4-dimethoxybenzyl)-2-imidazolidone on the leaf movement rhythm of Trifolium repens—a test of the cAMP-hypothesis of circadian rhythms

The period length of the leaf movement rhythm of Trifolium repens L. is lengthened by continuously offered cAMP (0.5–1.0 mol m^-3) and theophylline (0.5–4 mol m^-3). At the higher concentrations this effect is more pronounced and the rhythm damps out faster. Imidazole (0.5 and 1 mol m^-3) has no effect on the period length; however, after 5 mol m^-3 the rhythm is abolished. Offered as 4 h pulses the resulting phase response curves for cAMP and imidazole are similar and show delays of up to 4 h during the day position of the leaves. Theophylline pulses lead to delays of up to 5 h during closure and advances of up to 3 h during opening. No phase shift is brought about by 4-(3,4-dimethoxybenzyl)-2-imidazolidone. The results do not support the cAMP-model of the circadian clock which has been proposed by Cummings (J. theor. Biol. 55, 455–470; 1975). The effect of the substances tested could, however, be based upon influences on the transport of Ca²⁺.Abbreviations ATP adenosine triphosphate - cAMP cyclic adenosine 35 monophosphate - AMP adenosine 5 monophosphate - AC adenyl cyclase - PDE phosphodiesterase - LL continuous light