Endurance exercise modifies the circadian clock in zebrafish (Danio rerio) temperature independently.

AIM: Several rodent and human studies revealed that physical exercise acts as a non-photic zeitgeber for the circadian clock. The intrinsic entraining mechanism is still unknown, although it was assumed that the exercise-mediated increase in core temperature could be the underlying zeitgeber. As the homoeostatic control of mammalian core temperature interferes strongly with the investigation of this hypothesis, the present study used the poikilotherm zebrafish to answer this question. METHODS: Gene transcription levels of the two circadian core clock genes period1 and clock1 were quantified using real-time qPCR of whole animal zebrafish larvae. RESULTS: Long-term endurance exercise of zebrafish larvae aged 9-15 days post-fertilization (dpf) or 21-32 dpf at a constant water temperature of 25 °C caused significantly altered transcription levels of the circadian genes period1 and clock1. Cosinor analysis of diurnal transcription profiles obtained after 3 days of swim training revealed significant differences regarding acrophase, mesor and amplitude of period1, resulting in a phase delay of the gene oscillation. After termination of the exercise bout, at 15 dpf, oscillation amplitudes of both circadian genes were significantly reduced. CONCLUSION: The results showed that physical exercise is able to affect the transcription of circadian genes in developing zebrafish larvae. Considering the poikilothermy of zebrafish, an exercise-mediated change in body core temperature could be excluded as the underlying intrinsic zeitgeber. However, the day-active zebrafish arises as a useful model to address the synchronizing effect of exercise on the circadian clock.

Late onset of NMDA receptor-mediated ventilatory control during early development in zebrafish (Danio rerio).

Increased ventilation frequency (fV) in response to hypoxia in adult fish depends on ionotropic N-methyl-D-aspartate (NMDA) receptors. Nonetheless, the ontogeny of central control mechanisms mediating hypoxic ventilatory chemoreflexes in lower vertebrates has not been studied. Therefore, the aim of this study was to determine when the hypoxic ventilatory response during zebrafish (Danio rerio) development is mediated via NMDA receptors, by performing physiological experiments and western blot analysis of NMDA receptor subunits. Zebrafish larvae at stages 4-16 days post-fertilisation (dpf) were exposed to an hypoxic pulse in control groups and in groups treated with MK801 (NMDA receptor antagonist). The hypoxic increase in fV was present at all larval stages, and it matured during development. The reflex became MK801 sensitive at 8 dpf, but did not completely rely on a glutamatergic transmission until 13 dpf. This, together with changing subunit composition during the different stages (increasing amounts of NMDAR1 subunits and appearance of NMDAR2A subunits in adults), suggests that the amount of functional NMDA receptors needed to achieve a fully developed reflex is not attained until later stages. Furthermore, our results suggest that other non-NMDA receptor mechanisms are responsible for the hypoxia-induced increase in fV during the earlier developmental stages.

Influence of swim training on cardiac activity, tissue capillarization, and mitochondrial density in muscle tissue of zebrafish larvae.

Larval zebrafish (Danio rerio) of two different age classes ("swim-up" larvae, 9 days old; "free-swimming" larvae, 21 days old) were exposed to either an endurance/continuous training or interval training. Control animals were kept in stagnant water. A comparison of cardiac activity of trained (either endurance or interval) and untrained animals at the end of the training regime revealed no differences in heart rate, end-diastolic and end-systolic ventricular volume, and cardiac output. Training also had no influence on the concentration of erythrocytes in the blood. Thus, at the level of total oxygen transport in the blood, training did not provoke any improvement during the first 32 days of development. Significant changes, however, were observed at the tissue level. In free-swimming larvae [i.e., between 21 and 32 days postfertilization (dpf)] endurance training increased the capillarization of both axial muscle caudal to the anus and the tail fin. In addition, mitochondrial density of red and intermediate muscle fibers increased significantly. In contrast to capillarization, even swim-up larvae, trained between 9 and 15 dpf, were affected. The observed increase in mitochondrial content indicates a high demand for oxygen and energy-rich metabolites for oxidative phosphorylation. In older larvae, this is met by the increase in capillarization that improves the blood supply and with it the required oxygen and metabolite supply of muscle tissue. Both of these adaptational changes result in a reduction of diffusion distances (between capillary and muscle fiber as well as mitochondria) and may contribute to a higher resistance toward oxygen deficiency. Furthermore, this study indicates that plasticity of muscle tissue is already established in early stages of development at both the tissue and cellular levels.

Influence of hypoxia and of hypoxemia on the development of cardiac activity in zebrafish larvae.

Cardiac activity and anaerobic metabolism were analyzed in zebrafish larvae raised under normoxia (PO(2) = 20 kPa) and under chronic hypoxia (PO(2) = 10 kPa) at three different temperatures (25, 28, and 31 degrees C). Heart rate increased with development and with temperature. Under normoxia, cardiac output increased significantly at high temperature (31 degrees C), but not at 28 or at 25 degrees C. Under chronic hypoxia, however, heart rate as well as cardiac output increased at all temperatures in larvae at about hatching time or shortly thereafter. Cardiac activity of larvae raised for 2 wk after fertilization with a reduced hemoglobin oxygen-carrying capacity in their blood (hypoxemia; due to the presence of CO or of phenylhydrazine in the incubation water) was not different from control animals. Whole body lactate content of these animals did not increase. Thus there was no indication of a stimulated anaerobic energy metabolism. The increase in cardiac activity observed during hypoxia suggests that at about hatching time receptors are present that sense hypoxic conditions, and this information can be used to induce a stimulation of convective oxygen transport to compensate for a reduction in bulk oxygen diffusion in the face of a reduced oxygen gradient between environmental water and tissues. Under normoxia, however, the PO(2) gradient between environmental water and tissues and diffusional oxygen transport assure sufficient oxygen supply even if hemoglobin oxygen transport in the blood is severely impaired. Thus, under normoxic conditions and with a normal metabolic rate of the tissues, convective oxygen transport is not required until approximately 2 wk after fertilization.

Nitric oxide and vascular reactivity in developing zebrafish, Danio rerio.

We used a newly developed digital motion analysis video technique to study the effects of nitric oxide (NO) and epinephrine on the early larval arterial and venous vasculature of zebrafish. Application of the NO donor sodium nitroprusside resulted in a significant increase in both the venous and arterial vessel diameters, whereas N(G)-nitro-L-arginine methyl ester caused a significant decrease in the same diameters. Thus our results show that both the venous and arterial vasculature of the 5- and 6-day-old zebrafish larvae are influenced by endogenously produced NO. By use of immunohistochemistry, NO synthase immunoreactivity was demonstrated in endothelial cells of the dorsal vein. Local application of epinephrine onto the dorsal artery had no effect on vessel diameter. However, if the embryos were preincubated with N(omega)-nitro-L-arginine methyl ester, addition of epinephrine resulted in a significant reduction in both arterial and venous vessel diameters. Thus this study provides increasing evidence that before a functional autonomic innervation of the peripheral vascular system, vascular tone in larval tissue is regulated by a complex interaction of vasoactive substances that are produced locally by vascular endothelial cells.

Temperature-dependent development of cardiac activity in unrestrained larvae of the minnow Phoxinus phoxinus.

The minnow (Phoxinus phoxinus) was raised up to the stage of swim bladder inflation at temperatures between 10 degrees C and 25 degrees C, and the time of development significantly decreased at higher temperatures. Accordingly, initiation of cardiac activity was observed at day 2 in 25 degrees C animals and at day 4 in 12.5 degrees C animals. Only a minor increase in body mass was observed during the incubation period, and, at the end of the incubation period, animals raised at 25 degrees C did not have a significantly lower body mass compared with animals raised at 15 degrees C. Metabolic activity, determined as the rate of oxygen consumption of a larva, increased from 3.3 to 19.5 nmol/h during development at 15 degrees C and from 5.6 to 47.6 nmol/h during development at 25 degrees C. Heart rate showed a clear correlation to developmental stage as well as to developmental temperature, but at the onset of cardiac activity, diastolic ventricular volume and also stroke volume were higher at the lower temperatures. Furthermore, stroke volume increased with development, except for the group incubated at 12.5 degrees C, in which stroke volume decreased with development. Initial cardiac output showed no correlation to incubation temperature. Although metabolic activity increased severalfold during development from egg to the stage of swim bladder inflation at 15 degrees C and at 25 degrees C, weight-specific cardiac output increased only by approximately 40% with proceeding development. At 12.5 degrees C, cardiac output remained almost constant until opening of the swim bladder. The data support the notion that oxygen transport is not the major function of the circulatory system at this stage of development. The changes in heart rate with temperature appear to be due to the intrinsic properties of the pacemaker; there was no indication for a regulated response.

Digital motion analysis as a tool for analysing the shape and performance of the circulatory system in transparent animals.

The analysis of perfusion parameters using the frame-to-frame technique and the observation of small blood vessels in transparent animals using video microscopy can be tedious and very difficult because of the poor contrast of the images. Injection of a fluorescent probe (fluorescein isothiocynate, FITC) bound to a high-molecular-mass dextran improved the visibility of blood vessels, but the gray-scale histogram showed blurring at the edges of the vessels. Furthermore, injection of the fluorescent probe into the ventricle of small zebrafish (Danio rerio) embryos (body mass approximately 1 mg) often resulted in reduced cardiac activity. Digital motion analysis, however, proved to be a very effective tool for analysing the shape and performance of the circulatory system in transparent animals and tissues. By subtracting the two fields of a video frame (the odd and the even frame), any movement that occurred within the 20 ms necessary for the acquisition of one field could be visualised. The length of the shifting vector generated by this subtraction, represented a direct measure of the velocity of a moving particle, i.e. an erythrocyte in the vascular system. By accumulating shifting vectors generated from several consecutive video frames, a complete trace of the routes over which erythrocytes moved could be obtained. Thus, a cast of the vascular system, except for those tiny vessels that are not entered by erythrocytes, could be obtained. Because the gray-scale value of any given pixel or any given group of pixels increased with the number of erythrocytes passing it, digital motion analysis could also be used to visualise the distribution of blood cells in transparent tissues. This method was used to describe the development of the peripheral vascular system in zebrafish larvae up to 8 days post-fertilisation. At this stage, food intake resulted in a clear redistribution of blood between muscle tissue and the gut, and alpha-adrenergic control of peripheral blood flow was established.

Vasodilation of swimbladder vessels in the european eel (Anguilla anguilla) induced by vasoactive intestinal polypeptide, nitric oxide, adenosine and protons

The effects of &bgr;-adrenergic stimulation, vasoactive intestinal polypeptide (VIP), adenosine, the nitric oxide (NO)-releasing agent sodium nitroprusside and of metabolic end-products of gas gland cell metabolism on swimbladder blood flow were investigated using saline- or blood-perfused swimbladder preparations of the freshwater European eel Anguilla anguilla. While &bgr;-adrenergic vasodilation was not detectable, a bolus injection of adenosine (100 microl, 10(-)7 mol l-1) and application of VIP (10(-)7 mol kg-1) caused a significant decrease in perfusion pressure in saline-perfused swimbladder preparations. Immunohistochemical analysis revealed the presence of VIP-immunoreactive nerve fibres in the swimbladder artery and in the swimbladder vein (seawater-adapted eels were used for immunohistochemical studies). Application of sodium nitroprusside also elicited a small, but significant, decrease in perfusion pressure in saline-perfused swimbladder preparations, while preincubation of swimbladder tissue with N(&ohgr;)nitro-l-arginine, a non-selective inhibitor of nitric oxide synthase, significantly enhanced the flow-induced increase in perfusion pressure. Lactate, the major metabolic end-product of gas gland cell metabolism, had no effect on perfusion pressure. In contrast, an increase in proton concentration in both saline- and blood-perfused preparations induced a vasodilation, as indicated by a significant decrease in perfusion pressure. The results demonstrate that VIP, NO, adenosine and protons may induce a vasodilation of swimbladder blood vessels. None of these effects, however, compares in time span with the previously described immediate, short-lasting vasodilation of swimbladder vessels elicited by pulse stimulation of the vagus nerve.

Effects of vagal stimulation on swimbladder blood flow in the european eel Anguilla anguilla.

The influence of the vagus nerve on swimbladder blood flow in the European eel (Anguilla anguilla) was characterized by recording the changes in blood flow rate and blood pressure following stimulation of the vagus nerve. After electrical stimulation, blood flow in the swimbladder artery increased from 0.9 ml min-1 to 2.1 ml min-1. Video recordings of small vessels on the caudal side of the rete mirabile revealed an increase in erythrocyte velocity combined with a small vasodilation. This effect could not be blocked by injection of the -adrenergic antagonist phentolamine, the ss-adrenergic antagonist propranolol or the muscarinic cholinoceptor antagonist atropine. In all preparations with a high initial flow rate (>1.9 ml min-1), vagotomy resulted in a marked decrease in blood flow (by approximately 80 %). This effect was not observed in preparations with a low initial swimbladder blood flow. Stimulation of the vagus nerve produced a decrease, and vagotomy produced an increase, in perfusion pressure in blood-perfused swimbladder preparations. Histological studies revealed the presence of a ganglion in the vagus nerve located on the anterior part of the resorbing section of the swimbladder close to the origin of the ductus pneumaticus, which is probably associated with swimbladder function. These results suggest that swimbladder blood flow, at least to some extent, is under vagal tonic control. The effects do not, however, appear to involve the classical - and ss-adrenergic or muscarinic cholinoceptor functions.