The thermal dependence of fast-start performance in fish

Many fish species use fast-starts to escape predators and capture prey. There is evidence for changes in fast-start behaviour with temperature, over acute, seasonal, developmental and evolutionary time scales. Maximum velocity often increases with acute temperature changes. Thermal acclimation can improve fast-start performance, although responses appear to be reduced in more eurythermal species. Changes in performance with thermal acclimation are often reflected at the molecular, biochemical and cellular levels of organisation. There appears to be little compensation in fast-start performance in Antarctic fish compared to warmer water species.     

Effects induced by an actin-polymerizing peptide in goldfish mauthner neurons

In goldfishes, we studied (i) manifestations of functional activity of Mauthner neurons (MNs) reflected in motor behavior and (ii) changes in 3-D morphometry (ratio of volumes) and ultrastructure of MNs after applications of an actin-polymerizing peptide obtained from scorpion venom and after vestibular rotational stimulations (trainings) inducing natural modification of functions of the MNs (adaptation). In MNs subjected to application of the peptide, the increase in the functional resistance and morphological stability caused by long-lasting stimulation directly depended on the dose of the applied peptide or on the effectiveness of trainings, whereas in intact and control MNs such stimulation resulted in significant decreases in the activity and volumes of these cells. At the ultrastructure level, both applications of the peptide and trainings caused the formation of extensive bundles of actin filaments (“stress-fibers”) in the cytoplasm of MNs and led to an increase in the dimension of desmosome-like contacts (DLCs) in afferent synapses. At chemical synapses, the effect looked like a reciprocal decrease in the length of the active zones (a structural sign of long-term depression, LTD), while at mixed synapses this was manifested in an increase in the number of fibrillar bridges in the gaps of DLCs (a structural sign of long-term potentiation, LTP). The data obtained allow us to hypothesize that LTD of the efficacy of transmission through chemical synapses is involved in the formation of the adaptation state of the MNs and that polymerization of actin in the cytoplasm and DLCs underlies the mechanism of LTD and adaptation. The development of ultrastructural manifestations of LTP at mixed synapses after polymerization of actin by the peptide, which is related to a reciprocal increase in the efficacy of “mixed” afferent inputs, explains the maintenance of a high integral level of activity of the MNs, despite a drop in the functional activity of “ chemical” afferent inputs. Therefore, the actin cytoskeleton plays a clearly significant role in the maintenance of the balance of excitation at the neuronal level. Neirofiziologiya/Neurophysiology, Vol. 38, Nos. 5/6, pp. 389–401, September–December, 2006.     

The influence of starvation on fast-start performance of Spinibarbus sinensis

Juvenile Spinibarbus sinensis (n = 48, body length, 5.86 ± 0.10 cm, 25 °C) were fasted for 0, 0.5, 1, 2, 4 and 6 weeks. The fast-start performances of the experimental fish were assessed using high-speed video photography and the locomotive kinematics analysis. The morphological parameters including tail height (H₂), tail length (L₂), lateral body area (S₁), median fin area (S₂), dorsal cross section area (S₃) and tail cross section area (S₄) were also measured using TpsDig and the Photoshop. The results showed that 6 week starvation resulted in significant decreases in the escape distance (d), maximum linear velocity (V_max) and maximum linear acceleration (a_max) of center of mass in Stage 1 and Stage 2 of fast-start process (P < 0.05), however there were two relatively sTable phases in the V_max and d, during the week 1–2 (V_max = 0.67 ± 0.06 mm/ms; d = 8.86 ± 0.73 mm) and week 4–6 (V_max = 0.31 ± 0.04 mm/ms; d = 3.70 ± 0.56 mm). When compared with the control group (0 week starvation group), only the 6 week starvation group showed the significantly different response time (t) with average t = 9.20 ± 0.37 ms in week 1–4. There were no significant difference in mass center turning angles at first stage (T_a1) , second stage (T_a2) and the sum of two stages (T_a(1+2)) was also not different (P > 0.05). The fish did not show any directional preference for left or right during escape turning, and all of the related parameters also remained unchanged among treatment group (P > 0.05). The areas of dorsal body cross-section decreased more acutely (P < 0.05) than caudal body cross-section (45.4% vs 38.0%) during the entire starvation period while no significant differences were observed in both the tail height and tail length among all treatment groups (P > 0.05). The results indicated that fast-start performance of juvenile S. sinensis is affected by the starvation; metabolic energy related traits such as the maximum linear velocity and the maximum linear acceleration decreased significantly after starvation; whereas traits with no direct link to metabolic energy such as the response time and turning angle remained unchanged during starvation. The lack of starvation induced change in the maneuverability of the fish suggests that fast-start ability related to escape strategy is relatively well conservative in juvenile S. sinensis.     

Morphological parameters of mauthner neurons of goldfishes with modified asymmetry of motor behavior

We examined the morphological peculiarities of Mauthner neurons, MNs, in goldfishes with a phenotypically different or an experimentally modified preference to perform rightward vs leftward turnings in the course of motor behavior; this preference was characterized by values of the motor asymmetry coefficient (MAC). 3D reconstruction of MNs was performed based on several histological sections; volumes of the soma, lateral and ventral dendrites (LD and VD, respectively), initial segment of the axon, as well as full volumes of the right and left neurons, were calculated. Differences between the above parameters were expressed as structural asymmetry coefficients (SACs). It was shown that clear orientation asymmetry of motor behavior of the fish is accompanied by differences in the dimensions of MNs and their compartments; MNs localized contralaterally with respect to the preferred turning side were considerably bigger than ipsilateral neurons. Experimental influences inducing inversion of the motor asymmetry of fishes inverted structural asymmetry of their MNs. In fishes with no phenotypical preference of the turning side and in individuals whose motor asymmetry was smoothed due to experimental influences (rotational stimulations), structural asymmetry of the MNs was also smoothed. Changes of the structural proportions developed, as a rule, due to decreases in the dimensions of one or both MNs and their compartments. The MAC value was in direct correlation with the value of SAC of the MNs and with values of this coefficient for the soma and the sum soma + LD. At the same time, reciprocal relations were found for the MAC and structural asymmetry of the VD; the decrease in the volume of VD was related to an increase in the preference of the contralateral turning side by the fish, and vice versa. In general, the results of our study demonstrate that both morphological and functional peculiarities of MNs correlate to a significant extent with such a form of motor behavior of fishes as realization of spontaneous turnings. Neirofiziologiya/Neurophysiology, Vol. 38, No. 1, pp. 18–31, January–February, 2006.     

Functional development in the mauthner cell system of embryos and larvae of the zebra fish

In the embryonic zebra fish as early as 40 hr after fertilization, the Mauthner cells (M-cells) initiate an escape response, elicited by tactile-vibrational stimulation. The initial part of this behavior is similar to the acoustic startle reflex seen during the larval stage which begins at 96 hr. The embryonic response is directional and is followed by a series of strong tail flexures which are more pronounced than those during swimming. In the embryo the M-cell fired at the beginning of the response and rarely fired again during subsequent contractions; in our experiments the M-cell did not mediate iterative movements of the tail. The M-cell system is probably involved in evoked hatching behavior, as the tactile response is sufficient to rupture the egg membrane and allow the animal to escape. The M-cell sometimes fired spontaneously, which suggests that it might function also in spontaneous hatching behavior which occurs in the absence of phasic stimulation. At 48 hr the M-cell has morphologically mature synapses on its soma and dendrites, but its cytoplasm is relatively undifferentiated; it has few oriented neurofilaments and no distinct axon hillock. During these stages the extracellular M-spike is longer in duration and smaller in amplitude than at later times when the cell is more mature morphologically. Our data suggest that long-term inhibitory control of the M-cell system begins to function at about the time of hatching. At this time the cell is morphologically mature and is richly supplied with synaptic endings over its soma and dendrites.     

Measuring the optokinetic response of zebrafish larvae

Our laboratory screens for visual mutants by examining larval eye movements in response to rotating illuminated stripes. This behavior, which is termed an optokinetic response (OKR), is a reflex that appears in zebrafish at the same time as the development of the visual system. The OKR can be accurately measured by 4 d post-fertilization, which is the age when larvae begin foraging for food. The OKR requires approximately 1 min per larva analyzed. After identifying fish with defective eye movements, we conduct secondary screens (such as histological analysis and electroretinography) to identify the subset of fish with disruptions in the function of the outer retina. This paper describes our protocol for the OKR. Our setup is simple to construct and the materials needed are inexpensive. This makes our system especially useful for new undergraduate and graduate students, as well as introductory science lecturers.     

Scaling of fast-start performance and its thermal dependence in mummichog Fundulus heteroclitus

Fast-start predator-escape performance and its sensitivity to temperature (24, 30, and 36°C) were evaluated in mummichog Fundulus heteroclitus across a range of body sizes spanning YOY to adult (35–68 mm standard length). Mummichogs exhibit isometry of body dimensions and areas of the dorsal and anal fins but negative allometry of the caudal fin area. These scaling relationships are consistent with observed decreases in fast-start angular velocities with increasing body size. Linear velocity, on the contrary, does not vary with size, and both large and small mummichogs are capable of traversing similar distances in a given amount of time. In addition, temperature influences fast-start performance in similar ways over the size range, though the magnitude of the effect varies with size for some performance measures. In general, fast-start performance increases with test temperature, but mummichogs acclimated to warmer temperatures exhibit lower performance at each test temperature. Altogether, our results suggest that mummichogs across the adult size range may suffer decreases in their predator-escape performance as increasing sea temperatures combine with short-term temperature fluctuations in the estuaries these fish occupy.     

Abnormal differentiation of dopaminergic neurons in zebrafish trpm7 mutant larvae impairs development of the motor pattern

Transient receptor potential, melastatin-like 7 (Trpm7) is a combined ion channel and kinase implicated in the differentiation or function of many cell types. Early lethality in mice and frogs depleted of the corresponding gene impedes investigation of the functions of this protein particularly during later stages of development. By contrast, zebrafish trpm7 mutant larvae undergo early morphogenesis normally and thus do not have this limitation. The mutant larvae are characterized by multiple defects including melanocyte cell death, transient paralysis, and an ion imbalance that leads to the development of kidney stones. Here we report a requirement for Trpm7 in differentiation or function of dopaminergic neurons in vivo. First, trpm7 mutant larvae are hypomotile and fail to make a dopamine-dependent developmental transition in swim-bout length. Both of these deficits are partially rescued by the application of levodopa or dopamine. Second, histological analysis reveals that in trpm7 mutants a significant fraction of dopaminergic neurons lack expression of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Third, trpm7 mutants are unusually sensitive to the neurotoxin 1-methyl-4-phenylpyridinium, an oxidative stressor, and their motility is partially rescued by application of the iron chelator deferoxamine, an anti-oxidant. Finally, in SH-SY5Y cells, which model aspects of human dopaminergic neurons, forced expression of a channel-dead variant of TRPM7 causes cell death. In summary, a forward genetic screen in zebrafish has revealed that both melanocytes and dopaminergic neurons depend on the ion channel Trpm7. The mechanistic underpinning of this dependence requires further investigation.     

Laser-scanning velocimetry: A confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae

Background  

The zebrafish Danio rerio is an important model system for drug discovery and to study cardiovascular development. Using a laser-scanning confocal microscope, we have developed a non-invasive method of measuring cardiac performance in zebrafish embryos and larvae that obtains cardiovascular parameters similar to those obtained using Doppler echocardiography in mammals. A laser scan line placed parallel to the path of blood in the dorsal aorta measures blood cell velocity, from which cardiac output and indices of vascular resistance and contractility are calculated.     

应用意愿价值评估法评价河流生态恢复的时间稳定性——以上海城市内河为例

意愿价值评估法 (CVM)的评估结果是否具有时间稳定性是其可靠性检验中的重要问题,决定其能否应用于我国的生态系统服务价值评估.本文以上海城市内河生态恢复为评估对象,设计相隔1个月和2年的3次意愿价值评估方案,分别对3次调查的426、498和200份问卷进行了对比分析.结果表明： 3次支付意愿均值分别为14.2、14.1和18.0元,中位数分别为5、5和10元.进一步对支付意愿分布和主要统计值、影响因素、模型时间变量的显著性分析结果表明,相隔1个月的CVM 结果具有时间上的稳定性,而相隔2年的CVM 结果表现出一定差异.     

Optokinetic behavior is reversed in achiasmatic mutant zebrafish larvae

The vertebrate optokinetic nystagmus (OKN) is a compensatory oculomotor behavior that is evoked by movement of the visual environment. It functions to stabilize visual images on the retina. The OKN can be experimentally evoked by rotating a drum fitted with stripes around the animal and has been studied extensively in many vertebrate species, including teleosts. This simple behavior has earlier been used to screen for mutations affecting visual system development in the vertebrate model organism zebrafish. In such a screen, we have found a significant number of homozygous belladonna (bel) mutant larvae to be defective in the correct execution of the OKN [1]. We now show that about 40% of homozygous bel larvae display a curious reversal of the OKN upon visual stimulation. Monocular stimulation leads to primary activation of ipsilateral eye movements in larvae that behave like the wild type. In contrast, affected larvae display contralateral activation of eye movements upon monocular stimulation. Anatomical analysis of retinal ganglion cell axon projections reveal a morphological basis for the observed behavioral defect. All animals with OKN reversal are achiasmatic. Further behavioral examination of affected larvae show that OKN-reversed animals execute this behavior in a stimulus-velocity-independent manner. Our data support a parsimonious model of optokinetic reversal by the opening of a controlling feedback loop at the level of the optic chiasm that is solely responsible for the observed behavioral abnormality in mutant belladonna larvae.     

Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae

Background

Mammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders) indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations.

Results

Cell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres.

Conclusions

Our results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with in vivo cell tracing experiments to better understand the regenerative mechanisms.     

Locomotor behaviors in zebrafish (Danio rerio) larvae

Locomotor behaviors were examined in two experiments using zebrafish (Danio rerio) larvae at 4, 5, 6 and 7 days post fertilization (dpf). Larvae were observed in individual wells of a 12-well plate for 1 h a day. In Experiment 1, the same larvae were observed for four consecutive days beginning on post-fertilization day 4; in Experiment 2, different groups of larvae from the same egg collection were observed at 4, 5, 6 and 7 dpf. Automated images collected every 6 s were analyzed for information about larval location, orientation and general activity. In both experiments, 4 dpf larvae rested significantly more, used a smaller area of the well more frequently, and were generally less active than older larvae. All larvae exhibited a preference for facing away from the center of the well and for the edge of the well. However, prolonged exposure to the well influenced overall activity, orientation, and preference for the edge region. The implications of these results for understanding the development of larval behavior and for the design of procedures to measure the effects of experience in zebrafish larvae are discussed.     

Structure and function of skeletal muscle in zebrafish early larvae

Zebrafish muscles were examined at an early developmental stage (larvae 5-7 d). Using aluminum clips, preparations (approximately 1.5 mm length, 150 microm diameter) were mounted for force registration and small angle x-ray diffraction. Sarcomeres were oriented mainly in parallel with the preparation long axis. Electrical stimulation elicited fast and reproducible single twitch contractions. Length-force relations showed an optimal sarcomere length of 2.15 microm. X-ray diffraction revealed clear equatorial 1.1/1.0 reflections, showing that myofilaments are predominantly arranged along the preparation long axis. In contrast, reflections from older (2 mo) zebrafish showed two main filament orientations each at an approximately 25 degrees angle relative to the preparation long axis. Electrical stimulation of larvae muscles increased the 1.1/1.0 intensity ratio, reflecting mass transfer to thin filaments during contraction. The apparent lattice volume was 3.42 x 10(-3) microm(3), which is smaller than that of mammalian striated muscle and more similar to that of frog muscles. The relation between force and stimulation frequency showed fusion of responses at a comparatively high frequency (approximately 186 Hz), reflecting a fast muscle phenotype. Inhibition of fast myosin with N-benzyl-p-toluene sulphonamide (BTS) showed that the later phase of the tetanus was less affected than the initial peak. This suggests that, although the main contractile phenotype is fast, slow twitch fibers can contribute to sustained contraction. A fatigue stimulation protocol with repeated 220 ms/186 Hz tetani showed that tetanic force decreased to 50% at a train rate of 0.1 s(-1). In conclusion, zebrafish larvae muscles can be examined in vitro using mechanical and x-ray methods. The muscles and myofilaments are mainly orientated in parallel with the larvae long axis and exhibit a significant fast contractile component. Sustained contractions can also involve a small contribution from slower muscle types.     

Proton pump-rich cell secretes acid in skin of zebrafish larvae

The mammalian kidney excretes its metabolic acid load through the proton-transporting cells, intercalated cells, in the distal nephron and collecting duct. Fish excrete acid through external organs, gill, or skin; however, the cellular function is still controversial. In this study, molecular and electrophysiological approaches were used to identify a novel cell type secreting acid in skin of zebrafish (Danio rerio) larvae. Among keratinocytes covering the larval surface, novel proton-secreting ionocytes, proton pump (H⁺-ATPase)-rich cells, were identified to generate strong outward H⁺ flux. The present work demonstrates for the first time, with a noninvasive technique, H⁺-secreting cells in an intact animal model, the zebrafish, showing it to be a suitable model in which to study the functions of vertebrate transporting epithelia in vivo. hydrogen-adenosinetriphosphatase; ionocytes; epithelial transport; ion-selective electrode