Light-dark Stimulus Research Articles

Integrating morphological, biochemical, behavioural, and molecular approaches to investigate developmental toxicity triggered by tebuthiuron in zebrafish (Danio rerio)

Tebuthiuron (TBU), a phenylurea herbicide, is widely applied in agricultural and non-agricultural soils. Because TBU resists degradation, it can contaminate water and reach the biota once it is released into the environment. However, the potential toxic effects of TBU on aquatic developing organisms have been poorly studied. By taking advantage of the early-life stages of zebrafish (Danio rerio), we have combined morphological, biochemical, behavioural, and molecular approaches to investigate the developmental toxicity triggered by environmentally relevant concentrations (from 0.1 to 1000 μg/L) of TBU. Exposure to TBU did not elicit morphological abnormalities but it significantly delayed hatching. In addition, TBU altered the frequency of tail coils in one-day post-fertilization (dpf) old embryos. Moreover, TBU exposure during four days significantly inhibited the whole body AChE activity of larvae. At the molecular level, TBU did not significantly affect the mRNA levels of four genes (elavl3, gfap, gap43, and shha) that play key roles during the neurodevelopment of zebrafish. By assessing the motor responses to repeated light-dark stimuli, 6 dpf larvae exposed to TBU displayed hyperactivity, showing greater travelling distance during the dark periods. Our categorization of swimming speed revealed an interesting finding – after the light was turned off, the exposed larvae abandoned the freezing mode (<2 mm/s) and travelled mainly at cruising speed (2–20 mm/s), showing that the larval hyperactivity did not translate into higher swimming velocity. Overall, our results offer new insights into the TBU toxicity to developing organisms, namely effects in AChE activity and hyperactivity, providing support data for future studies considering environmental risk assessment of this herbicide.

Standardized Method for the Assessment of Behavioral Responses of Zebrafish Larvae.

Zebrafish are easy to breed in a laboratory setting as they are extremely fertile and produce dozens of eggs per set. Because zebrafish eggs and the skin of the early-stage larvae are transparent, their embryos and the hearts and muscles of their larvae can be easily observed. Multiple rapid analyses of heart rate and behavior can be performed on these larvae simultaneously, enabling investigation of the influence of neuroactive substances on abnormal behavior, death, and associated pathogenetic mechanisms. Zebrafish larvae are becoming increasingly popular among researchers and are used in laboratories worldwide to study various vertebrate life phenomena; more experimental systems using zebrafish will undoubtedly be developed in the future. However, based on the available literature, we believe that the conceptualization of a protocol based on scientific evidence is necessary to achieve standardization. We exposed zebrafish larvae at 6–7 days post-fertilization to 50 repeated light–dark stimuli at either 15-min or 5-min intervals. We measured the traveled distance and habituation time through a video tracking apparatus. The traveled distance stabilized after the 16th repetition when the zebrafish were exposed to light–dark stimuli at 15-min intervals and after the 5th repetition when exposed at 5-min intervals. Additionally, at 15-min intervals, the peak of the traveled distance was reached within the first minute in a dark environment, whereas at 5-min intervals, it did not reach the peak even after 5 min. The traveled distance was more stable at 5-min intervals of light/dark stimuli than at 15-min intervals. Therefore, if one acclimatizes zebrafish larvae for 1 h and collects data from the 5th repetition of light/dark stimuli at intervals of 5 min in the light/dark test, a stable traveled distance result can be obtained. The establishment of this standardized method would be beneficial for investigating substances of unknown lethal concentration.

Influence of light–dark cycle on delayed recovery from isoflurane anesthesia induced by hypnotics in mice

We previously reported that brotizolam, but not suvorexant, delayed recovery from isoflurane anesthesia in mice. However, the effects of hypnotics may be altered by the circadian rhythm. Locomotor activity was measured using sighted (ICR and C57BL/6J) and blind (FVB/N and C3H/HeN) mice, and the effects of hypnotics on isoflurane anesthesia were compared during the light and dark periods. In sighted mice, recovery induced by brotizolam was delayed in the light period, while that by suvorexant was delayed in the dark period. In C57BL/6J mice, delayed recovery induced by brotizolam was marked, and that by suvorexant was observed in the light and dark periods. Locomotor activity was low in the last 6 h of the dark period in blind mice, and was similar to that in the light period. In blind mice, delayed recovery induced by brotizolam was identical in both periods, while suvorexant did not influence recovery from isoflurane anesthesia. These results suggest that the effects of hypnotics on isoflurane anesthesia are altered by the circadian rhythm and that daily light–dark stimuli may be required for the chronopharmacological effects of hypnotics.

Fish behavior: A promising model for aquatic toxicology research

Fish behaviors have great potential as models for the study of pharmacology, genetics, and neuroscience. Zebrafish (Danio rerio), Japanese medaka (Oryzias latipes) and Chinese rare minnow (Gobiocypris rarus) are popular freshwater animal models. However, their behavioral use in aquatic toxicology research is generally hampered by oversimplified behavioral tasks and the fact that they are not well-developed animal models for toxicology. Here, this study presented a comparative analysis of multiple behavioral traits (i.e., anxiety-like behavior, novel object recognition, social preferences, habituation to light-dark stimulus and noise stimulus, and spatial learning and memory). We found that only medaka (d-rR) presented a weak or no response to repeated light-dark stimulus and noise stimulus. In addition, no significant behavioral changes were observed for the three species of juvenile fish models after 7 days of exposure to 0.01% v/v carrier solvents (i.e., ethanol, acetone, and DMSO). In contrast to zebrafish and Chinese rare minnow, medaka showed no significant changes in spatial memory after subacute exposure to 1 mg/L imidacloprid or 2.5 μg/L chlorpyrifos (cpf); instead, a hyperactivity response in the open field test and reduced social time were induced by cpf and imidacloprid, respectively. Our results suggest that: (1) behavioral effects are negligible when using <0.01% v/v carrier solvents for behavioral assessment; (2) given the differences in sensitivities of behavioral responses, a single behavior used alone as an endpoint may be insufficient for estimating the toxic impacts of pesticides or other environmental contaminants. In conclusion, these results could have major implications for aquatic toxicology research and water quality monitoring and ecotoxicological risk assessment.

Ferroptosis, Mechanism Of Cell Death In Vitamin E Deficiency During Embryogenesis?

Vitamin E (α-tocopherol), discovered in 1922, prevents rodent fetal resorption, but the mechanisms are unknown. Adult zebrafish maintained on vitamin E-deficient (E–) or sufficient (E+) diets were spawned to obtained E– and E+ embryos, respectively, which we evaluated up to 12 days post-fertilization (dpf). Initially, we investigated mechanisms causing lethality using metabolomic analyses of E– embryos daily from 1 to 5 dpf. By 5 dpf, 80% of E– embryos experienced increased morbidity or mortality; even normally appearing E– embryos at 4 dpf were 84% less responsive to light-dark stimuli in a locomotor assay. We found that vitamin E deficiency not only caused behavioral abnormalities, it caused: 1) lipid peroxidation of docosahexaenoic acid (DHA), depletion of DHA-containing phospholipids, especially phosphatidylcholine, and choline depletion; 2) glucose depletion, which was potentiated by shunting it to the pentose phosphate pathway for NADPH production; and 3) impairment of oxygen consumption and energy homeostasis. The observed morbidity and behavioral outcomes could be attenuated by glucose injection into E– embryos at 1 dpf. In subsequent experiments, normally appearing E– and E+ embryos at 5 dpf were fed an adequate diet (containing DHA and vitamin E) from 5-12 dpf. Both the E– and E+ groups survived and grew normally; however, by 12 dpf E– larvae continued to have behavioral defects. These defects coincided with sustained vitamin E deficiency despite adequate dietary supplementation. We also found in E– vs. E+ larvae DHA depletion with increased lipid peroxidation, along with defective energy metabolism. Thus, embryonic vitamin E deficiency in zebrafish leads to a metabolic reprogramming that adversely affects the neurologic status of embryos that is not readily repaired by an adequate diet suggesting neuronal cell death in the developing brain, possibly by ferroptotic mechanisms.

Glucose tolerance in mice exposed to light\u2013dark stimulus patterns mirroring dayshift and rotating shift schedules

Glucose tolerance was measured in (nocturnal) mice exposed to light–dark stimulus patterns simulating those that (diurnal) humans would experience while working dayshift (DSS) and 2 rotating night shift patterns (1 rotating night shift per week [RSS1] and 3 rotating night shifts per week [RSS3]). Oral glucose tolerance tests were administered at the same time and light phase during the third week of each experimental session. In contrast to the RSS1 and RSS3 conditions, glucose levels reduced more quickly for the DSS condition. Glucose area-under-the-curve measured for the DSS condition was also significantly less than that for the RSS1 and RSS3 conditions. Circadian disruption for the 3 light–dark patterns was quantified using phasor magnitude based on the 24-h light–dark patterns and their associated activity–rest patterns. Circadian disruption for mice in the DSS condition was significantly less than that for the RSS1 and RSS3 conditions. This study extends previous studies showing that even 1 night of shift work decreases glucose tolerance and that circadian disruption is linked to glucose tolerance in mice.

Entrainment of circadian clocks in mammals by arousal and food

Circadian rhythms in mammals are regulated by a system of endogenous circadian oscillators (clock cells) in the brain and in most peripheral organs and tissues. One group of clock cells in the hypothalamic SCN (suprachiasmatic nuclei) functions as a pacemaker for co-ordinating the timing of oscillators elsewhere in the brain and body. This master clock can be reset and entrained by daily LD (light-dark) cycles and thereby also serves to interface internal with external time, ensuring an appropriate alignment of behavioural and physiological rhythms with the solar day. Two features of the mammalian circadian system provide flexibility in circadian programming to exploit temporal regularities of social stimuli or food availability. One feature is the sensitivity of the SCN pacemaker to behavioural arousal stimulated during the usual sleep period, which can reset its phase and modulate its response to LD stimuli. Neural pathways from the brainstem and thalamus mediate these effects by releasing neurochemicals that inhibit retinal inputs to the SCN clock or that alter clock-gene expression in SCN clock cells. A second feature is the sensitivity of circadian oscillators outside of the SCN to stimuli associated with food intake, which enables animals to uncouple rhythms of behaviour and physiology from LD cycles and align these with predictable daily mealtimes. The location of oscillators necessary for food-entrained behavioural rhythms is not yet certain. Persistence of these rhythms in mice with clock-gene mutations that disable the SCN pacemaker suggests diversity in the molecular basis of light- and food-entrainable clocks.

Diurnally Entrained Anticipatory Behavior in Archaea

By sensing changes in one or few environmental factors biological systems can anticipate future changes in multiple factors over a wide range of time scales (daily to seasonal). This anticipatory behavior is important to the fitness of diverse species, and in context of the diurnal cycle it is overall typical of eukaryotes and some photoautotrophic bacteria but is yet to be observed in archaea. Here, we report the first observation of light-dark (LD)-entrained diurnal oscillatory transcription in up to 12% of all genes of a halophilic archaeon Halobacterium salinarum NRC-1. Significantly, the diurnally entrained transcription was observed under constant darkness after removal of the LD stimulus (free-running rhythms). The memory of diurnal entrainment was also associated with the synchronization of oxic and anoxic physiologies to the LD cycle. Our results suggest that under nutrient limited conditions halophilic archaea take advantage of the causal influence of sunlight (via temperature) on O2 diffusivity in a closed hypersaline environment to streamline their physiology and operate oxically during nighttime and anoxically during daytime.

Daily growth lines in some living Pectens (Mollusca: Bivalvia), and some applications in a fossil relative: Time and tide will tell

Laboratory experiments on two species from the Pecten (Pecten) subgroup of pectinids indicated that they form growth lines with a true (solar) daily periodicity. Experimental subjects that formed growth lines in numbers equal to their days of growth were consistently those with uniform, apparently continuous shell growth, and subjects that formed fewer lines than days were those with stress marks, or disturbance lines. No subjects formed more growth lines than days. Furthermore, those specimens forming stress marks had those marks spaced out in very similar patterns, apparently in response to stressful events in their aquarium, such that the patterns could be correlated, not only between the specimens with interrupted growth, but with the others as well. In the original experiments, a natural light–dark stimulus had been present (transparent aquaria near an outside window). To test whether the subjects were responding to this stimulus, another experiment was conducted in which several individuals were subjected to an accelerated light–dark regime. These subjects responded by increasing production of their growth lines to approach the number of cycles to which they had been exposed. An opportunity to extend this work into the fossil record came with the collection of a number of Pliocene pectinids, closely related to one of the species used in the experiments, from a thin horizon that almost certainly represents a catastrophic event. Measurements of the intervals between growth lines were made across hundreds of increments on many paired (and presumably untransported) valves. Comparison of these growth rate records suggested that many, and perhaps all of these individuals died at the same time. Moreover, frequency analysis of these records revealed a regular fluctuation of about 15 days, suggesting a tidal cycle marked off by solar increments. When the growth rate patterns were compared with nearby tidal regimes, the best fit was for a regime matching the currently accepted paleogeography of the collecting site in Pliocene time. This strongly supports the experiments on the living species, for tidal patterns in the fossil group could not have been revealed without an accurate solar marker.

2P192ホヤ幼生の連続明暗刺激に対する鋭敏化と慣れ : (2)刺激光強度依存性

2P192ホヤ幼生の連続明暗刺激に対する鋭敏化と慣れ : (2)刺激光強度依存性

2I1545 動画像解析装置によるホヤ幼生の連続明暗刺激に対する鋭敏化と慣れ

2I1545 動画像解析装置によるホヤ幼生の連続明暗刺激に対する鋭敏化と慣れ

1PA100 ホヤオタマジャクシ型幼生の明暗刺激に対する学習行動

1PA100 ホヤオタマジャクシ型幼生の明暗刺激に対する学習行動

Involvement of Ca2+and cGMP in bacterial taxis

The level of cGMP in a suspension of Escherichia coli cells increased transiently upon the addition of chemoattractants. Ca2+ (1 mM), but not Mg2+, produced constant tumbling of cells in the presence of the ionophore A23187. The effect was observed either in stationary-state cells, or in a logarithmic culture treated with EDTA to increase permeability by A23187. Under the same conditions, Ca2+ decreased the cytoplasmic level of cGMP. In Phormidium uncinatum, rapid 45Ca2+ accumulation followed a light-dark stimulus, or the addition of tetramethylquinone (TMQ), a chemorepellent. La3+, which increases the reversal rate, also enhanced the level of cytoplasmic Ca2+, presumably by blocking the outward Ca2+ flux. In both E. coli and P. uncinatum Ca2+ inhibited methylaccepting chemotaxis protein (MCP) methylation. It is concluded that cGMP and Ca2+ are secondary messengers in taxis information-processing.

Electrical nature of the taxis signal in cyanobacteria.

Electrical events after a light-dark stimulus were studied in the multicellular organism Phormidium uncinatum. Normally, such a stimulus causes the gliding trichome to reverse direction. By directing a large light spot on the end of a batch of trichomes and then switching it off, we achieved synchronization of the trichomes, since the "head" is much more sensitive than the "tail." The abrupt disappearance of a uniform light produced a depolarization wave which initiated at the head, as registered by externally applied electrodes. The second stimulus produced a depolarization of the opposite direction, reflecting the reorientation of the trichomes. No electrical response was observed at Ca2+ concentrations less than or equal to 10(-8) M. Factors causing oscillatory reversals, i.e., a combination of Ca2+ and A23187, or a viscous environment also abolished the electrical signal. Changes in an externally applied electrical field (4 V/cm2) had little effect on the motile behavior of P. uncinatum or Oscillatoria princeps. However, in the presence of 5 microM Ca2+-1 microM A23187, all the trichomes reversed synchronously to the anode after a change in polarity of an externally applied electrical field. We suggest that an increased Ca2+ concentration together with a change in delta psi (or delta mu H+) represents the taxis signal in cyanobacteria.

Light-dark discrimination and reversal learning in early postnatal kittens.

During the early postnatal period kittens were trained in a visual discrimination task involving light-dark stimuli in order to determine the age of onset of visual control of learned behavior. A Y water maze was used with escape from water as the reinforcer. The results indicate that kittens are able to master the discrimination within the 5th week of life. The relevance of this result to electrophysiological measures of maturation is discussed.