Gravity and light effects on the circadian clock of a desert beetle, Trigonoscelis gigas.

Circadian function is affected by exposure to altered ambient force environments. Under non-earth gravitational fields, both basic features of circadian rhythms and the expression of the clock responsible for these rhythms are altered. We examined the activity rhythm of the tenebrionid beetle, Trigonoscelis gigas, in conditions of microgravity (microG; spaceflight), earth's gravity (1 G) and 2 G (centrifugation). Data were recorded under a light-dark cycle (LD), constant light (LL), and constant darkness (DD). Free-running period (tau) was significantly affected by both the gravitational field and ambient light intensity. In DD, tau was longer under 2 G than under either 1 G or microG. In addition, tauLL was significantly different from tauDD under microG and 1 G, but not under 2 G.

Vasopressin-deficient suprachiasmatic nucleus grafts re-instate circadian rhythmicity in suprachiasmatic nucleus-lesioned arrhythmic rats.

It was investigated whether grafts of the suprachiasmatic nucleus could re-instate circadian rhythmicity in the absence of its endogenous vasopressin production and whether the restored rhythm would have the long period length of the donor. Grafts of 17-days-old vasopressin-deficient homozygous Brattleboro rat fetuses, homotopically placed in arrhythmic suprachiasmatic nucleus-lesioned Wistar rats, re-instated circadian drinking rhythm within 20-50 days similar as seen for grafts of heterozygous control fetuses. Period length of the recovered rhythm revealed a similar difference (average 24.3 vs. 23.8 h) as reported for the rhythm between the adult Brattleboro genotypes. In all transplants, also those of the two-third non-recovery rats, a surviving suprachiasmatic nucleus was visible as a vasoactive intestinal polypeptide-positive neuronal cell cluster, whereas heterozygous transplants also revealed the complementary vasopressinergic cell part. Explanation of the absence of recovery failed since no undisputable correlation emerged between recovery of rhythm and vasoactive intestinal polypeptide, vasopressin and/or somatostatin immunocytochemical characteristics of the suprachiasmatic nucleus of the transplant. Special focus on the somatostatinergic neurons revealed their presence only occasionally near or in between the vasoactive intestinal polypeptidergic and (in the case of heterozygous grafts) vasopressinergic cell cluster. However their aberrant cytoarchitectural position appeared not to have affected the possibility to restore drinking rhythm of the suprachiasmatic nucleus-lesioned arrhythmic rat. It was concluded that grafted Brattleboro fetal suprachiasmatic nucleus develop their intrinsic rhythm conform their genotype and that vasopressin is not a crucial component in the maintenance nor in the transfer of circadian activity of the biological clock for drinking activity. Vasopressin of the suprachiasmatic nucleus may instead serve modulation within the circadian system.

Circadian rhythmicity of vasopressin levels in the cerebrospinal fluid of suprachiasmatic nucleus-lesioned and -grafted rats.

Transplantation of the fetal suprachiasmatic nucleus (SCN) in arrhythmic SCN-lesioned rats can reinstate circadian drinking rhythms in 40% to 50% of the cases. In the current article, it was investigated whether the failure in the other rats could be due to the absence of a circadian rhythm in the grafted SCN, using a circadian vasopressin (VP) rhythm in the cerebrospinal fluid (CSF) as the indicator for a rhythmic SCN. CSF was sampled in continuous darkness from-intact control rats and SCN-lesioned and -grafted rats. VP could be detected in all samples, with concentrations of 15 to 30 pg/ml in the control rats and 5 to 15 pg/ml in the grafted rats. A circadian VP rhythm with a two- to threefold difference between peak and nadir values was found in all 7 control rats but in only 4 of 13 experimental rats, despite the presence of a VP-positive SCN in all grafts. A circadian VP rhythm was present in 2 drinking rhythm-recovered rats (6 of 13) and in 2 nonrecovery rats. Apparently, in these latter rats, the failure of the grafted SCN to restore a circadian drinking rhythm cannot be attributed to a lack of rhythmicity in the SCN itself. Thus, the presence of a rhythmic grafted SCN, as is deduced from a circadian CSF VP rhythm, appears not to be sufficient for restoration of a circadian drinking rhythm in SCN-lesioned arrhythmic rats.

Cellular requirements of suprachiasmatic nucleus transplants for restoration of circadian rhythm.

Fetal neurografts containing the suprachiasmatic nucleus (SCN) can restore the circadian locomotor and drinking rhythm of SCN-lesioned (SCNX) rat and hamster. This functional outcome finally proves that the endogenous biological clock autonomously resides in the SCN. Observations on the cellular requirements of the "new" SCN for restoration of the arrhythmic SCNX animals have led to some new insights and confirmed findings from other studies. A critical mass of SCN neurons appeared necessary for functional effects, whereas the temporal profile of reinstatement of rhythm correlated with the delayed maturation of the grafted SCN. Cytoarchitectonically, the grafted SCN does not seem to develop normally for all anatomical aspects. Complementary clusters of vasoactive intestinal polypeptide(VIP)- and vasopressin(VP)ergic neurons are formed, but somatostatin(SOM)ergic neurons do not always "join" this group, as is normally seen in situ. Nevertheless, these new SCNs can restore the ablated functions. As the period length of restored rhythms tends to vary, it might be that the grafted SCN underwent an altered or impaired maturation that resulted in a different setting of its clock mechanism. A prominent role of VIPergic neurons seems indicated by their presence in all functional grafts, but, although they may be required, these cells do not appear to be a sufficient condition for restoration of rhythm. Many grafts exhibit the presence of VIPergic cells without counteracting the arrhythmia, whereas VP- and SOMergic SCN neurons are usually present as well. Findings with VP-deficient Brattleboro rat grafts indicated that VP is not the primary obligatory signal of circadian activity. It is argued that perhaps the role of SOMergic neurons in the clock function of the (grafted) SCN has been insufficiently considered. However, one should keep in mind that the peptides of the various types of SCN neurons may function only as cofactors, mutually modulating molecular or bioelectrical cellular activities within the nucleus or the message of the main transmitter gamma-aminobutyric acid.

Effects of microgravity on circadian rhythms in insects.

NASA: The desert beetle Trigonoscelis gigas Reitt. was used as a biological model in studies that examined the effects of space flight on the circadian timing system. Results from studies aboard the Bion-10, Bion-11, and Photon-11 missions are reported. The control study is an ongoing Mir experiment. The studies indicate that the free-running period in beetles may be longer during space flight.^ieng

Seasonal fluctuations in the cervical smear detection rates for (pre)malignant changes and for infections.

The detection of diseases can exhibit seasonal fluctuations. This can be studied in cervical smears. Over a 9-year observation span (January 1983-January 1992) a series of 504,093 cervical smears obtained from a routine cytology laboratory in The Netherlands were examined for infections (monilia, trichomonas, actinomyces, human papilloma virus [HPV], chlamydia, and herpes) as well as for mild, moderate, and severe dysplasias, carcinoma in situ, and squamous carcinoma. Statistical analysis (principal component analysis) demonstrates clear seasonal rhythms in the detection of infections as well as in precursor lesions. These findings suggest that we are dealing with "true" detection rhythms. For the detection of (pre)malignancy and HPV, yearly fluctuations in women being screened might be the explanation for our observations.

General introduction to chronobiology.

Rhythmicity is part of our life. All living organisms exhibit in some way or another a form of rhythmicity. Some of these rhythms have a geophysical counterpart in the environment. These are the so-called biological rhythms. This paper gives an introduction to the circadian rhythms, i.e., those rhythms with a period length of about one day. General characteristics and properties are described as well as the control and disturbances of these rhythms.

General introduction to chronobiology

Rhythmicity is part of our life. All living organisms exhibit in some way or another a form of rhythmicity. Some of these rhythms have a geophysical counterpart in the environment. These are the so called biological rhythms. This paper gives an introduction to the.circadlan rhvthms. i.e., those rhythms with a period length of about one day. General characteristics and properties are described as well as the control and disturbances of these rhythms.

Ionic currents in cultured rat suprachiasmatic neurons.

Whole-cell voltage-clamp recordings were made from cultured neurons obtained by dissociation of the suprachiasmatic area of rat fetuses. Neurons were held for seven to 14 days in culture. These neurons possessed several voltage-dependent ionic currents. A transient inward Na+ current was present, which could be completely blocked by tetrodotoxin. No inward Ca2+ currents were detected. Three types of outward K+ currents were recorded, which could be separated to a reasonable extent by their differences in voltage sensitivity and pharmacology. These K+ currents corresponded to the transient current IA, the delayed rectifier current IKo and a calcium-dependent current IK(Ca) as described in other neurons. The A current activated at -50 mV, reached half-maximal conductance at about -30 mV and maximum conductance between 0 and 30 mV. During depolarizing steps it inactivated completely within 100 ms and steady-state inactivation was half-maximal at -66 mV. The outward rectifier activated at -30 mV, reached half-maximal conductance close to 0 mV and maximum conductance at about 70 mV. Slow inactivation of IKo occurred with 50% reduction in amplitude at the end of 2 s depolarizations above 0 mV. The K+ channel blocker 4-amino-pyridine (4 mM) reduced the amplitude of IA by 21% and of IKo by 32%, whereas tetraethylammonium (10 mM) decreased IA by 27% and IKo by 83%. The calcium-dependent K+ component was also voltage dependent and was present at voltages more positive than 0 mV. No inward rectifying K+ current was present. Considering its voltage dependence, IA must play a role in determining the excitability of these neurons, through its probable influence on the action potential threshold and interspike interval. Both IA and IKo should take part in membrane repolarization following an action potential. The Ca(2+)-dependent current should also contribute to repolarization following any event which gives rise to an increase in intracellular Ca2+. Apart from IA, which may make a slight contribution, none of these currents appear to be involved in determining the resting membrane potential. All three outward current components will act together in suprachiasmatic neurons to control their spontaneous firing frequency, which is the major feature of the output of these neurons in vivo. Variations in properties of these conductances could contribute to the circadian rhythm in firing frequency described in suprachiasmatic hypothalamic neurons.

Proliferation profile of benign and premalignant cervical epithelium as established by PCNA staining pattern.

Since immunohistochemical detection of proliferating cell nuclear antigen (PCNA) is closely related to the cell cycle, this method can be used to visualize proliferative activity in paraffin sections of benign and premalignant cervical lesions. This was done in two types of benign lesions, immature and mature metaplasia, and the three types of CIN (I, II, and III). PCNA staining was assessed as heavy, faint, or negative nuclei in the deep, middle, and superficial layers of the epithelium. In the analysis of variance, significant differences between the three layers and between the five groups were found. The PCNA values can be helpful to distinguish immature metaplasia from CIN I. The values for PCNA-positive (heavy+faint) for an individual lesion at the three layers constitute its proliferation profile. The PCNA profiles of the CIN lesions differ fundamentally from the metaplasia profiles. It is conceivable that for an individual CIN lesion, PCNA staining might be prognostically more valuable than its CIN grade.

Impact of microgravity and hypergravity on free-running circadian rhythm of the desert beetle Trigonoscelis gigas Reitt.

Free-running circadian rhythms of locomotor activity of Tenebrionid beetles Trigonoscelis gigas Reitt., taken from the Turkmenian sand desert, were monitored in DD. The effects of microgravity --11 days in space flight aboard the Russian BION-10 "COSMOS" satellite, and of 2G hypergravity--seven days on a centrifuge, were determined. Two kinds of effects were found. In stable 2-peak records, there was a moderate decrease of tau in microgravity and an increase of tau in 2G, both of about 0.3 hr. In unstable records, alterations of gravity caused drastic deviations of tau and phi. Remarkably, two peaks of the activity rhythm, which are supposed to be controlled by separate oscillators, responded to gravity transitions in different ways. Gravity effects on the circadian system could be explained from a direct effect on the oscillator(s) itself or from a feed-back by altered locomotion to the pacemaker. Thus, for the first time the gravity dependence of a free-running circadian rhythm was proved in a combination of real space flight and centrifuge experiments.

Suprachiasmatic lesions eliminate 24-h blood pressure variability in rats.

To evaluate whether the suprachiasmatic nuclei (SCN) control 24-h rhythmicity in cardiovascular function, rats were subjected to computerized recording of 24-h blood pressure (BP) and heart rate (HR) following SCN lesions. In sham- and nonlesioned rats, circadian patterns of BP and HR ran in phase with those in feeding behavior. In SCN-lesioned rats circadian rhythmicity in BP, HR and food intake were abolished. Minute-to-minute variation of BP and HR was similar as in sham- and nonlesioned rats. We conclude that long-term but not short-term variability of blood pressure and heart rate is largely determined by SCN-controlled activity levels.

Circadian rhythms and masking: an overview.

Masking, as is well known, enables an organism to act immediately and in an appropriate way to changes of the environment, integrating with internally produced rhythmicity. It now appears that masking can be used to cover a far wider range of problems than was originally intended. To separate masking effects from the effects due to an internal oscillator, several techniques have been used. Such protocols, however, like the constant routine protocol, often replace one form of masking by another. The situation becomes even more complex when one realizes that the output of an internal oscillator modifies the input. The question might be asked whether it is possible to study the properties of the internal oscillator in vivo at all. This article attempts to produce a framework for future discussions.

Functional development of fetal suprachiasmatic nucleus grafts in suprachiasmatic nucleus-lesioned rats.

Recovery of circadian drinking rhythms in suprachiasmatic nucleus (SCN)-lesioned rats after fetal SCN grafting was related to the immunocytochemical appearance and fiber outgrowth of vasopressin (VP)-, vasoactive intestinal polypeptide (VIP)-, and somatostatin (SOM)-containing neurons in the implants. At 4 weeks postgrafting, the first recovered animal was found. After longer survival times, 38% of the animals showed recovery. Immunocytochemical evaluation indicated that full maturation of the SCN grafts was not reached until 4 weeks postgrafting. Grafted VP and VIP cells were always located together, whereas SOM cells were clustered nearby but separate. Neuropeptide Y fibers were observed with an increasing fiber density between 2 and 5 weeks posttransplantation and were clustered particularly at the level of the SOM cells. In all rhythm-recovered animals transplants of VP and VIP fibers had grown laterally into the hypothalamus. A few nonrecovered animals also showed ingrowth of such fibers, though more caudally to the lesioned SCN. Many of the nonrecovered rats showed similar stainings but without these efferent outgrowth to the host. We conclude that neither a humoral factor nor the presence of VP and VIP efferents in the host brain alone are enough for the restoration of circadian drinking rhythms.

Chronobiology: neural pacemakers of biological rhythms.

Biological rhythms are ubiquitous being demonstrable of any level of organization in living matter. However, the myriad of biological oscillators in peripheral organs are organized and synchronized by special structure, i.e., biological clocks, mostly located in central nervous system. Neural pacemakers show intrinsic properties which are illustrated and discussed. Understanding the meaning and function of neural oscillators is fundamental for those who wants to know how the biological structure of time is processed in living systems.

Spontaneous and stimulated firing in cultured rat suprachiasmatic neurons.

Neurons from the suprachiasmatic nucleus (SCN) of the hypothalamus, the site of a circadian pacemaker in mammals, were isolated from embryonic rat. After mechanical dissociation neurons were brought into culture for 1-2 weeks, using a chemically defined medium. Recordings were made from 74 bipolar neurons using two different configurations of the patch-clamp technique. During cell attached patch recordings, 45% of neurons fired spontaneously. The mean firing rate was 0.7 +/- 0.6 Hz and the firing pattern was irregular. In whole cell recordings 73% of the investigated neurons showed spontaneous activity with an irregular firing pattern. The mean spontaneous firing rate with an intracellular Cl- concentration of 145 mM was 1.0 +/- 0.6 Hz. The resting membrane potential of the bipolar neurons was estimated to be -62 +/- 24 mV. An intracellular Cl- concentration of 145 mM depolarised the membrane potential. It also increased the probability of spontaneous firing. A depolarising current stimulus produced an action potential with a threshold voltage of -46 +/- 9 mV. Suprathreshold stimuli resulted in repetitive firing with a mean frequency of 12 +/- 4 Hz. The minimum interspike interval was 52 +/- 14 ms. All action potentials either occurring spontaneously or elicited by current stimuli were abolished by the Na(+)-channel blocker TTX. These results indicate that our cultured neurons have some electrophysiological properties in common with SCN neurons in brain slices and in vivo.

Prediction of various grades of cervical neoplasia on plastic-embedded cytobrush samples. Discriminant analysis with qualitative and quantitative predictors.

The purpose of this study was to investigate whether discrimination into five groups of various grades of cervical preneoplasia and neoplasia is possible using discriminant analysis models. Data were analyzed for 242 cases diagnosed as either slight dysplasia (n = 50), moderate dysplasia (n = 50), severe dysplasia (n = 50), carcinoma in situ (n = 50) or invasive carcinoma (n = 42) and consisted of qualitative and quantitative features of cells derived from a repeat sample taken from the ectocervix as well as the endocervix using Cytobrushes. The samples were embedded in plastic, and thin sections were prepared, resulting in a monolayer of cut nuclei. The percentages of expected correct prediction were obtained by using 10,000 double cross-validation samples; the mean percentage of correct prediction into five groups using cross-validation was 65% (in the original analysis, 72%) and into two groups (dysplasia versus carcinoma in situ and invasive carcinoma) was 91% (93%). The results reflect group discrimination potential; we do not claim reliability of prediction for an individual patient. The patients were not a representative sample of the population; to investigate whether groups of patients could be discriminated on the basis of both qualitative and quantitative features, the data analyzed contain an almost equal number of observations in each of the five groups. The results indicate that features do not classify the cases in the same way; the discriminant analyses suggest that quantitative features play an important role in the discrimination of dysplasia from carcinoma cases, while the majority of the qualitative features are important in discrimination within the three dysplasia groups.

Neurotransmitters and the pharmacology of the suprachiasmatic nuclei.

The suprachiasmatic nucleus is the major component of the biological clock responsible for the generation and the regulation of circadian rhythms in behavioral and physiological functions. The SCN acts as an endogenous circadian pacemaker that becomes entrained to the light/dark cycle. Although many neuropeptides and neurotransmitters have been localized within and around the SCN, their role in the generation of the circadian signal still has to be elucidated. There are some data about the transmitters that are involved in the mechanism of entrainment. The way the SCN modulates the homeostatic control systems that are involved in the control of behaviour, also needs more clarification.