Disrupted seasonal biology impacts health, food security and ecosystems.

The rhythm of life on earth is shaped by seasonal changes in the environment. Plants and animals show profound annual cycles in physiology, health, morphology, behaviour and demography in response to environmental cues. Seasonal biology impacts ecosystems and agriculture, with consequences for humans and biodiversity. Human populations show robust annual rhythms in health and well-being, and the birth month can have lasting effects that persist throughout life. This review emphasizes the need for a better understanding of seasonal biology against the backdrop of its rapidly progressing disruption through climate change, human lifestyles and other anthropogenic impact. Climate change is modifying annual rhythms to which numerous organisms have adapted, with potential consequences for industries relating to health, ecosystems and food security. Disconcertingly, human lifestyles under artificial conditions of eternal summer provide the most extreme example for disconnect from natural seasons, making humans vulnerable to increased morbidity and mortality. In this review, we introduce scenarios of seasonal disruption, highlight key aspects of seasonal biology and summarize from biomedical, anthropological, veterinary, agricultural and environmental perspectives the recent evidence for seasonal desynchronization between environmental factors and internal rhythms. Because annual rhythms are pervasive across biological systems, they provide a common framework for trans-disciplinary research.

Pregnancy modulates precursor cell proliferation in a murine model of focal demyelination.

In mice, pregnancy has been shown to have a beneficial effect on the endogenous repair of focal lysolecithin-induced CNS demyelinative lesions, enhancing the genesis of new oligodendrocytes and the degree of remyelination. To identify local cells undergoing mitosis in response to such lesions, we examined the time course of phospho-histone H3 (PH3) and myelin basic protein (MBP) expression by immunohistochemistry. After lysolecithin injection into the corpus callosum of virgin female mice, the number of dividing cells peaked about 48 h after injection and declined gradually to baseline by day 7; in pregnant mice, this initial peak was unchanged, but a new delayed peak on day 4 was induced. Colocalization data using PH3 and NG2 proteoglycan, or bromodeoxyuridine (BrdU) and oligodendrocyte transcription factor 1 (Olig1), suggested that about 75% of the proliferating cells on day 2, and about 40% of the cells on day 4, were likely of oligodendrocyte lineage; these differential percentages were of the same magnitude in both virgin and pregnant animals. Notably, the heightened proliferative response to focal lysolecithin injection during pregnancy was specific to gestational stage (early, but not late) and to lesion location (in the corpus callosum of the periventricular forebrain, but not in the caudal cerebellar peduncle of the hindbrain).

On the chronobiology of cohabitation.

Social regulation of animal circadian rhythms may enable individuals in a population to temporally synchronize or segregate their activities within the community. Relatively little is known about the mechanisms for such interindividual temporal adaptations or how the circadian system might be involved. The literature suggests that actual prolonged cohabitation might lead to robust effects on the rhythmicity of cohoused individuals but that these effects are not easily reproduced by indirect or pulsatile social contacts. We have begun to study the conditions under which such cohabitation effects might be revealed in the laboratory, and we present and discuss initial data that cohousing pairs of golden hamsters can result in a persistent change in the free-running circadian period of one of the two hamsters of the pair. We believe that analyzing the societal level of temporal organization, and ultimately dissecting its underlying mechanisms, will enrich our understanding of the circadian clock and its role in establishing ecological communities.

Behavioral arousal blocks light-induced phase advances in locomotor rhythmicity but not light-induced Per1 and Fos expression in the hamster suprachiasmatic nucleus.

Both photic and nonphotic stimuli entrain circadian rhythms. Although the adaptive significance of nonphotic clock resetting is unknown, one possibility is that nonphotic cues modulate circadian responses to light. Results of studies on the interaction between photic and nonphotic stimuli support this idea. During the day, light blocks the effects of nonphotic stimuli on the phase of locomotor rhythms and on expression of clock genes in suprachiasmatic nucleus (SCN) neurons. At night, novelty-induced activity prior to and during exposure to light attenuates the phase-shifting response to that light, but the effects of this manipulation on clock gene expression are unknown. The present experiments explore the interaction between behavioral state and response to light at the molecular level. We show that confining hamsters to novel wheels immediately after a light pulse during the late subjective night attenuates light-induced phase advances of wheel-running rhythms and the transient effects on circadian period. In contrast to the striking effect of novelty-induced activity on behavioral responses to light, Fos protein and Per1 mRNA were robustly expressed in the SCN of all light-pulsed animals, regardless of behavioral treatment. Our results are inconsistent with the idea that light and nonphotic stimuli block each other's effects on phase shifts by inducing or attenuating transcription of Per1. Photic regulation of clock genes and spontaneous rhythmic expression of clock genes are probably mediated by different mechanisms.

Towards the reconstruction of central nervous system white matter using neural precursor cells.

Epidermal growth factor-responsive neural precursor cells were used as donor cells for transplantation into wild-type and myelin-deficient shiverer (shi) mice. The cells engrafted robustly within the CNS following intracerebroventricular and cisternal transplantation in neonatal mice. The cells adopted glial phenotypes, and some functioned as oligodendrocytes, producing myelin basic protein and morphologically normal internodal myelin sheaths. When individual shi mice received two transplants (on post-natal days 1 and 3), donor-derived cells disseminated widely and expressed myelin basic protein in central white matter tracts throughout the brain.

Encoding le quattro stagioni within the mammalian brain: photoperiodic orchestration through the suprachiasmatic nucleus.

Within the suprachiasmatic nucleus (SCN) is a pacemaker that not only drives circadian rhythmicity but also directs the circadian organization of photoperiodic (seasonal) timekeeping. Recent evidence using electrophysiological, molecular, and genetic tools now strongly supports this conclusion. Important questions remain regarding the SCN's precise role(s) in the brain's photoperiodic circuits, especially among different species, and the cellular and molecular mechanisms for its photoperiodic "memory." New data suggesting that SCN "clock" genes may also function as "calendar" genes are a first step toward understanding how a photoperiodic clock is built from cycling molecules.

Neural precursor cells form rudimentary tissue-like structures in a rotating-wall vessel bioreactor.

We have analyzed the biology of embryonic, epidermal growth factor-responsive murine neural precursor cells cultured in the high-aspect ratio vessel (HARV). Within 2-3 d of rotary-cell culture, such cells formed multiple, macroscopic, three-dimensional structures that were orders of magnitude larger than the cellular clusters ("neurospheres") formed by these cells in conventional stationary-flask cultures. Each HARV structure was composed of a multilayered cellular shell surrounding one or more central cavities that were bordered by pyknotic cell nuclei. Although the cells in the HARV structures were more pleomorphic than those in neurospheres, the structures did not appear to represent primitive neural tumors: the formation of HARV structures by precursor cells was not an irreversible phenotypic change, and the structures did not originate from the clonal expansion of single-progenitor cells; the growth rate and invasiveness of the cells in HARVs were less than those in flasks; and HARV-cultured cells did not form tumors after subcutaneous inoculation into the flanks of NOD-scid/scid mice. Immunohistochemical analysis suggested that HARV structures might be novel "prototissues" characterized by a crude, but organized, architecture, with a surface layer of immature proliferating cells (nestin- and proliferating cell nuclear antigen-positive) that enclosed strata of more differentiated cells (beta-tubulin III- and glial fibrillary acidic protein-positive) within. Rotary-cell culture may have significant implications for the eventual utility of neural precursors for clinical neurotransplantation.

Assembling a clock for all seasons: are there M and E oscillators in the genes?

The hypothesis is advanced that the circadian pacemaker in the mammalian suprachiasmatic nucleus (SCN) is composed at the molecular level of a nonredundant double complex of circadian genes (per1, cry1, and per2, cry2). Each one of these sets would be sufficient for the maintenance of endogenous rhythmicity and thus constitute an oscillator. Each would have slightly different temporal dynamics and light responses. The per1/cry1 oscillator is accelerated by light and decelerated by darkness and thereby tracks dawn when day length changes. The per2 /cry2 oscillator is decelerated by light and accelerated by darkness and thereby tracks dusk. These M (morning) and E (evening) oscillators would give rise to the SCN's neuronal activity in an M and an E component. Suppression of behavioral activity by SCN activity in nocturnal mammals would give rise to adaptive tuning of the endogenous behavioral program to day length. The proposition-which is a specification of Pittendrigh and Daan's E-M oscillator model-yields specific nonintuitive predictions amenable to experimental testing in animals with mutations of circadian genes.

Antiphase oscillation of the left and right suprachiasmatic nuclei.

An unusual property of the circadian timekeeping systems of animals is rhythm "splitting," in which a single daily period of physical activity (usually measured as wheel running) dissociates into two stably coupled components about 12 hours apart; this behavior has been ascribed to a clock composed of two circadian oscillators cycling in antiphase. We analyzed gene expression in the hypothalamic circadian clock, the suprachiasmatic nucleus (SCN), of behaviorally "split" hamsters housed in constant light. The results show that the two oscillators underlying the split condition correspond to the left and right sides of the bilaterally paired SCN.

Differential regulation of fos family genes in the ventrolateral and dorsomedial subdivisions of the rat suprachiasmatic nucleus.

Extensive studies have established that light regulates c-fos gene expression in the suprachiasmatic nucleus, the site of an endogenous circadian clock, but relatively little is known about the expression of genes structurally related to c-fos, including fra-1, fra-2 and fosB. We analysed the photic and temporal regulation of these genes at the messenger RNA and immunoreactive protein levels in rat suprachiasmatic nucleus, and we found different expression patterns after photic stimulation and depending on location in the ventrolateral or dorsomedial subdivisions. In the ventrolateral suprachiasmatic nucleus, c-fos, fra-2 and fosB expression was stimulated after a subjective-night (but not subjective-day) light pulse. Expression of the fra-2 gene was prolonged following photic stimulation, with elevated messenger RNA and protein levels that appeared unchanged for at least a few hours beyond the c-fos peak. Unlike c-fos and fra-2, the fosB gene appeared to be expressed constitutively in the ventrolateral suprachiasmatic nucleus throughout the circadian cycle; immunohistochemical analysis suggested that delta FosB was the protein product accounting for this constitutive expression, while FosB was induced by the subjective-night light pulse. In the dorsomedial suprachiasmatic nucleus, c-fos and fra-2 expression exhibited an endogenous circadian rhythm, with higher levels during the early subjective day, although the relative abundance was much lower than that measured after light pulses in the ventrolateral suprachiasmatic nucleus. Double-label immunohistochemistry suggested that some of the dorsomedial cells responsible for the circadian expression of c-Fos also synthesized arginine vasopressin. No evidence of suprachiasmatic nucleus fra-1 expression was found. In summary, fos family genes exhibit differences in their specific expression patterns in the suprachiasmatic nucleus, including their photic and circadian regulation in separate cell populations in the ventrolateral and dorsomedial subdivisions. The data, in combination with our previous results [Takeuchi J. et al. (1993) Neuron 11, 825-836], suggest that activator protein-1 binding sites on ventrolateral suprachiasmatic nucleus target genes are constitutively occupied by DeltaFosB/JunD complexes, and that c-Fos, Fra-2, FosB and JunB compete for binding after photic stimulation. The differential regulation of fos family genes in the ventrolateral and dorsomedial suprachiasmatic nucleus suggests that their circadian function(s) and downstream target(s) are likely to be cell specific.

Rhythmic multiunit neural activity in slices of hamster suprachiasmatic nucleus reflect prior photoperiod.

The suprachiasmatic nucleus (SCN) is an endogenous circadian pacemaker, and SCN neurons exhibit circadian rhythms of electrophysiological activity in vitro. In vivo, the functional state of the pacemaker depends on changes in day length (photoperiod), but it is not known if this property persists in SCN tissue isolated in vitro. To address this issue, we prepared brain slices from hamsters previously entrained to light-dark (LD) cycles of different photoperiods and analyzed rhythms of SCN multiunit neuronal activity using single electrodes. Rhythms in SCN slices from hamsters entrained to 8:16-, 12:12-, and 14:10-h LD cycles were characterized by peak discharge rates relatively higher during subjective day than subjective night. The mean duration of high neuronal activity was photoperiod dependent, compressed in slices from the short (8:16 and 12:12 LD) photoperiods, and decompressed (approximately doubled) in slices from the long (14:10 LD) photoperiod. In slices from all photoperiods, the mean phase of onset of high neuronal activity appeared to be anchored to subjective dawn. Our results show that the electrophysiological activity of the SCN pacemaker depends on day length, extending previous in vivo data, and demonstrate that this capacity is sustained in vitro.

Morning and evening circadian oscillations in the suprachiasmatic nucleus in vitro.

Daily biological rhythms are governed by an innate timekeeping mechanism, or 'circadian clock'. In mammals, a clock in the suprachiasmatic nucleus (SCN) comprises multiple autonomous single-cell oscillators, but it is unclear how SCN cells interact to form a tissue with coherent metabolic and electrical rhythms that might account for circadian animal behaviors. Here we demonstrate that the circadian rhythm of SCN electrophysiological activity, recorded as a single daytime peak in hamster hypothalamic coronal slices, shows two distinct peaks when slices are cut in the horizontal plane. Substantiating an idea initially derived from behavioral observations, the properties of these two peaks indicate functional organization of SCN tissue as a clock with two oscillating components.

Altered circadian rhythmicity is an early sign of murine dietary thiamine deficiency.

To determine if circadian clock function is affected by thiamine deficiency, the rhythm of locomotor (wheel-running) activity was measured in a murine model of dietary thiamine deficiency. About 1 month before the expected onset of overt neurological illness, locomotor rhythmicity in deficient animals exhibited a shortened free-running period without a change in amplitude. This effect was fully reversible by thiamine administration. Disordered circadian timekeeping may contribute to altered physiological responses in Wernicke's encephalopathy.

Bioaccumulation of radionuclides in fertilized Canadian Shield lake basins.

Radionuclide tracers of heavy metals (59Fe, 60Co, 65Zn, 75Se, 85Sr, 134Cs and 203Hg) representing potential contamination from nuclear power plants, industry and agriculture were added to separate basins of Lake 226, Experimental Lakes Area, northwestern Ontario. The two basins were part of a eutrophication experiment and differed in their trophic status; the north basin (L226N) was eutrophic whereas the south basin (L226S) was mesotrophic. Our objective was to determine the uptake of the radionuclides by biota and the effect of lake trophic status on their bioaccumulation. The trophic status of the lakes did not appear to have a marked effect on the accumulation of radionuclides by the biota. This may have been because of a mid-summer leakage of nutrients between the basins which enhanced primary production in L226S, because there is a time lag between primary production and the availability of the radionuclides to the fishes or because trophic status does not affect the uptake of at least some of these radionuclides. However, there was a tendency for faster uptake of the radionuclides in L226N by fish than L226S, but the differences were not significant. Concentrations in the biota generally decreased in the order: fathead minnow > pearl dace > tadpoles > slimy sculpin > leeches. Concentrations in biota generally decreased in the order. 65Zn > 203Hg > 75Se > 134Cs > 60Co > 85Sr = 59Fe. Cobalt-60 concentrations in tadpoles were greater than in the other biota. Radionuclide concentrations in the tissues of lake whitefish indicated that uptake was predominantly from food. Radionuclide concentrations were usually higher in the posterior gut, liver and kidney than in other tissues, whereas body burdens were generally high in the muscle for 75Se, 134Cs and 203Hg; kidney and gut for 60Co; and bone for 65Zn and 75Se. Mercury-203 burdens were also high in the bone and gut.

Behavior of 60Co and 134Cs in a Canadian Shield lake over 5 years.

Radionuclides were added to the anoxic hypolimnion of a Canadian Shield lake to simulate the nuclear fuel waste disposal scenario where radionuclides might enter the bottom waters of a lake. The radionuclides remained in the hypolimnion until lake mixing at autumn turnover after which 60Co was rapidly lost and 134Cs was slowly lost from the water. Only 0.4% of the 60Co and 0.6% of the 134Cs remained in the water at year 5. Highest concentrations occurred in periphyton and filter feeders, Holopedium gibberum and clams (Anodonata grandis grandis). From maximum annual concentrations in clam tissues, it was estimated that the availability of 60Co for uptake had a half-time (t1/2) of 835 days in the lake, whereas that for 134Cs was 780 days. Loss rate coefficients, k, for the radionuclides from taxa ranged from 0.0008 to 0.0043 day-1 (t1/2 = 161-866 days) for 60Co and from 0.0009 to 0.005 day-1 (t1/2 = 139-770 days) for 134Cs. Cobalt-60 concentrations in forage fish were low, whereas 134Cs concentrations increased over the first year or two, then slowly declined. On the basis of k values measured for forage fish, the biological half-time of 134Cs in forage fish ranged from 428 to 630 days. Maximum 134Cs concentrations in forage fish were higher following hypolimnetic addition than epilimnetic addition. Relatively high 134Cs concentrations in periphyton at year 5 point to the importance of benthic pathways in the recycling of contaminants to higher trophic levels. The presence of 134Cs in biota 5 years after the addition, long after concentrations were no longer detectable in surface waters, is evidence of the persistence of Cs in aquatic systems. The k values (or t1/2 values) for the loss of 60Co and 134Cs from water and their uptake and loss from biota can be used to establish parameter values for assessment models. The results demonstrate that assessment models should account for the release of radionuclides from sediment and their subsequent recycling in the food chain when modeling over the long term after the end of a radionuclide release to the environment.

Flank-marking behavior and the neural distribution of vasopressin innervation in golden hamsters with suprachiasmatic lesions.

In golden hamsters, microinjections of arginine-vasopressin (AVP) within the anterior hypothalamus trigger a stereotyped scent-marking behavior, flank marking. Our experiment was carried out to test the contribution of AVP neurons within the suprachiasmatic nucleus (SCN) in the control of this behavior. Our results suggest that the SCN does not contribute to flank-marking behavior. Whereas SCN lesions disrupted circadian rhythms of wheel running, the same lesions did not disrupt flank-marking. The results also suggest that neurons located outside the SCN contribute significantly to the vasopressinergic innervation of the brain and the expression of AVP-dependent behaviors, such as flank-marking behavior. Although AVP-immunoreactive fibers were severely (ca. 95%) depleted from several forebrain areas in SCN-lesioned hamsters, the effect of the lesions was much more limited within the forebrain areas involved in flank-marking behavior as well as within the midbrain and hindbrain.

Light-induced c-Fos expression in the mouse suprachiasmatic nucleus: immunoelectron microscopy reveals co-localization in multiple cell types.

Although light is known to regulate the level of c-fos gene expression in the suprachiasmatic nucleus (SCN), the site of an endogenous circadian clock, little is known about the identities of the photically activated cells. We used light-microscopic immunocytochemistry and immunoelectron microscopy to detect c-Fos protein in the SCN of Sabra mice exposed to brief nocturnal light pulses at zeitgeber time 15-16. Stimulation with light pulses that saturated the phase-shifting response of the circadian locomotor rhythm revealed an upper limit to the number of photo-inducible c-Fos cells at about one-fifth of the estimated total SCN cell population. This functionally defined set was morphologically and phenotypically heterogeneous. About 24% could be labelled for vasoactive intestinal polypeptide, 13% for vasopressin-neurophysin, and 7% for glial fibrillary acidic protein. The remaining 56% of c-Fos-positive cells were largely of unknown phenotype, although many were presumptive interneurons, some of which were immunoreactive for nitric oxide synthase.