On the edge: pharmacological evidence for anxiety-related behavior in zebrafish larvae.

Zebrafish larvae are ideally suited for high-throughput analyses of vertebrate behavior. The larvae can be examined in multiwell plates and display a range of behaviors during early development. Previous studies have shown that zebrafish larvae display a preference for the edge of the well and several lines of evidence suggest this edge preference (thigmotaxis) may be a measure of anxiety. In the present study, we further examined the relation between edge preference and anxiety by imaging zebrafish larvae exposed to three psychoactive drugs diazepam (Valium), fluoxetine (Prozac), and caffeine. The edge preference was first examined in a five-fish assay, with and without visual stimuli. Diazepam, a benzodiazepine that binds to GABA receptors, reduced the larval edge preference, with or without visual stimuli. In contrast, fluoxetine, a selective serotonin reuptake inhibitor, did not affect the edge preference. Caffeine increased the preference for the edge in response to visual stimuli. Similar effects were observed in a two-fish assay; diazepam-exposed larvae showed a reduced edge preference and caffeine exposed larvae showed an increased edge preference. These results suggest that the edge preference in zebrafish larvae is a measure of anxiety and further illustrate that the pharmaceuticals used in the study have different mechanisms of action. Although there are substantial differences between zebrafish and human brains, our results indicate that the signals that regulate anxiety are similar on a molecular level. We propose that high-throughput assays in zebrafish may be used to uncover genetic or environmental factors that cause anxiety disorders and may contribute to the development of novel strategies to prevent or treat such disorders.

Chemiluminescence microscopy as a tool in biomedical research.

Chemiluminescence has become a standard tool in biomedical research. Chemiluminescent probes are used for immunoassays, nucleic acid identification, reporter gene assays, measuring enzyme activity, and the detection of ions and small molecules such as Ca2+, ATP, NO, O2- and H2O2. Along with the development of new chemiluminescent probes, significant progress has been made in techniques to measure chemiluminescence. Ultra-sensitive photometers or luminometers have become widely available and can be obtained with automatic injectors and microplate readers. In addition, imaging photon detectors have been developed that allow the imaging of chemiluminescence from gels, blots, and microplates. Imaging photon detectors have also been attached to microscopes and allow imaging of chemiluminescent probes and reporter genes in cells and tissues. Specific methods of photon collection, storage, and analysis have been developed for microscopic imaging of chemiluminescence. Two of these methods are discussed in detail. The first is a method of data storage that allows days of continuous imaging without creating oversized files. The second is a method for calibrating photon imaging microscopes using a low-light standard. Such calibration will be helpful for comparing the performance of various photon imaging systems and for comparing data obtained in different laboratories.

Periodic increases in elongation rate precede increases in cytosolic Ca2+ during pollen tube growth.

Pollen tubes grown in vitro require an intracellular tip-high gradient of Ca2+ in order to elongate. Moreover, after about 2 h in vitro both the tip Ca2+ and the elongation rate of lily tubes begin to oscillate regularly with large amplitudes. This raises the question of the phase relation between these two oscillations. Previous studies lacked the temporal resolution to accurately establish this relationship. We have studied these oscillations with a newly developed, high temporal resolution system and the complementary use of both luminescent and fluorescent calcium reporters. We hereby show that the periodic increases in elongation rate during oscillatory growth of Lilium longiflorum pollen tubes clearly precede those in subtip calcium and do so by 4.1 +/- 0.2 s out of average periods of 38.7 +/- 1.8 s. Also, by collecting images of the light output of aequorin, we find that the magnitude of the [Ca2+] at the tip oscillates between 3 and 10 microM, which is considerably greater than that reported by fluorescent indicators. We propose an explanatory model that features cyclic growth and secretion in which growth oscillations give rise to secretion that is essential for the subsequent growth oscillation. We also critically compile data on L. longiflorum stylar growth rates, which show little variation from in vitro rates of pollen tubes grown in optimal medium.

Presence and roles of calcium gradients along the dorsal-ventral axis in Drosophila embryos.

Dorsal-ventral specification of the Drosophila embryo is mediated by signaling pathways which have been very well described in genetic terms. However, little is known about the physiology of Drosophila development. By imaging patterns of free cytosolic calcium in Drosophila embryos, we found that several calcium gradients are generated along the dorsal-ventral axis. The most pronounced gradient is formed during stage 5, in which calcium levels are high dorsally. Manipulation of the stage 5 calcium gradient affects specification of the amnioserosa, the dorsal-most region of the embryo. We further show that this calcium gradient is inhibited in pipe, Toll, and dorsal mutants, but is unaltered in decapentaplegic (dpp) or punt mutants, suggesting that the stage 5 calcium gradient is formed by a suppression of ventral calcium concentrations. We conclude that calcium plays a role in specification of the dorsal embryonic region.

Calcium imaging with chemiluminescence.

This review updates the imaging of free cytosolic calcium with the chemiluminescent aequorins. Basic principles of chemiluminescence are discussed and the biochemistry of aequorins is briefly described. The review provides practical tips on handling and microinjecting aequorins and describes available ultra low light imaging systems. It is argued that aequorin-based calcium imaging is the method of choice for exploratory studies, since it is extremely sensitive, can detect a broad range of calcium concentrations, and allows for continuous recording during long periods of time. However, fluorescent methods are needed to attain high spatial resolution.

On the conservation of calcium wave speeds.

Most long distance calcium signals are believed to take the form of actively propagated calcium waves. In 1991, when this proposal was first advanced, all such waves were thought to belong to one class, for which fertilization waves were the prototype. Moreover, the speeds of such waves were found to be conserved at about 10 microns/s for primary fertilization waves and 30 microns/s for waves through fully active systems at 20 degrees C. In 1993, preliminary evidence for a second class of such waves was published and the prototype for these were ones which drive cell cleavage. These move at only about 1 micron/s at 20 degrees C and were, therefore, called slow calcium waves as opposed to the fast ones first considered. Here we compile compelling evidence that slow waves comprise a second distinct class of actively propagated calcium waves. This is based on 30 papers which yield evidence of slow calcium waves in organisms ranging from Dictyostelium to mammals and phenomena ranging from the surface contraction waves seen long ago in axolotl eggs to embryonic cleavage and mitotic waves and to ones recently seen to accompany primary neural induction in axolotls. Ultraslow and ultrafast calcium waves are also considered.

Patterns of free calcium in zebrafish embryos.

Direct knowledge of Ca2+ patterns in vertebrate development is largely restricted to early stages, in which they control fertilization, ooplasmic segregation and cleavage. To explore new roles of Ca2+ in vertebrate development, we injected the Ca2+ indicator aequorin into zebrafish eggs and imaged Ca2+ throughout the first day of development. During early cleavages, a high Ca2+ zone is seen in the cleavage furrows. The high Ca2+ zone during first cleavage spreads as a slow wave (0.5 microm/second) and is preceded by three Ca2+ pulses within the animal pole region of the egg. When Ca2+ concentrations are clamped at the resting level by BAPTA buffer injection into the zygote, all signs of development are blocked. In later development, Ca2+ patterns are associated with cell movements during gastrulation, with neural induction, with brain regionalization, with formation of the somites and neural keel, with otic placode formation, with muscle movements and with formation of the heart. Particularly remarkable is a sharp boundary between high Ca2+ in the presumptive forebrain and midbrain versus low Ca2+ in the presumptive hindbrain starting at 10 hours of development. When Ca2+ changes are damped by injection of low concentrations of BAPTA, fish form with greatly reduced eyes and hearts. The present study provides a first overview of Ca2+ patterns during prolonged periods of vertebrate development and points to new roles of Ca2+ in cellular differentiation and pattern formation.

Expression of apo-aequorin during embryonic development; how much is needed for calcium imaging?

Aequorin is a bioluminescent calcium indicator consisting of a 21 kDa protein (apo-aequorin) that is covalently linked to a lipophilic cofactor (coelenterazine). The aequorin gene can be expressed in a variety of cell lines and tissues, allowing non-invasive calcium imaging of specific cell types. In the present paper, we describe the possibilities and limitations of calcium imaging with genetically introduced apo-aequorin during embryonic development. By injecting aequorin into sea urchin, Drosophila and zebrafish eggs, we found that higher aequorin concentrations are needed in smaller eggs. Our results suggest that for measuring resting levels of free cytosolic calcium, one needs aequorin concentrations of at least 40 microM in sea urchin eggs, 2 microM in Drosophila eggs, and only 0.11 microM in zebrafish eggs. A simple assay was used to determine the absolute concentrations of expressed apo-aequorin and the percentage of aequorin formation in vivo. The use of this assay is illustrated by expression of the aequorin gene in Drosophila oocytes. These oocytes form up to 1 microM apo-aequorin. In our hands, only 0.3% of this apo-aequorin combined with coelenterazine entering from the medium to form aequorin, which was not enough for calcium imaging of the oocytes, but did allow in vivo imaging of the ovaries. From these studies, we conclude that coelenterazine entry into the cell is the rate limiting step in aequorin formation. Based on the rate of coelenterazine uptake in Drosophila, we estimate that complete conversion of 1 microM apo-aequorin would take 50 days in zebrafish eggs, 2 days [corrected] in Drosophila eggs, 7 days in sea urchin eggs or 18 h in a 10 microm tissue culture cell. Our results suggest that work based on genetically introduced apo-aequorin will be most successful when large amounts of small cells can be incubated in coelenterazine. During embryonic development this would involve introducing coelenterazine into the circulatory system of late stage embryos. Calcium imaging in early stage embryos may be best done by injecting aequorin, which circumvents the slow process of coelenterazine entry.

Analysis of the calcium transient at NEB during the first cell cycle in dividing sea urchin eggs.

Accumulating evidence from several systems suggests that nuclear envelope breakdown (NEB) is triggered by an endogenous transient of free calcium. Using h- and f-semisynthetic aequorins as cytosolic calcium indicators, we have clearly and regularly visualized a single large, global calcium transient just before first NEB in normally developing, monospermic Lytechinus eggs. Although similar transients were not observed at NEB in subsequent cell cycles, microinjection of the calcium buffer BAPTA into one blastomere of the two-celled embryo resulted in the inhibition of NEB. The NEB transient in the first cell cycle was some five-fold smaller than the one associated with egg activation. Our data suggest that this transient takes the form of a calcium wave that spreads inwards from the periphery of the egg toward the nucleus. We confirmed that these NEB transients did not require extracellular Ca2+. In polyspermic eggs, NEB-associated transients were four-fold larger than in monospermic eggs and were periodically repeated. Examination of the distribution of fluorescein-conjugated aequorins with a laser scanning confocal microscope indicated that aequorin both enters the nucleus and is evenly distributed within the cytosol of the egg. The use of h- and f-aequorins did not reveal any NEB transients during subsequent cell cycles, nor did we detect transients associated with other cell cycle events. However, a complex train of calcium transients in the form of both localized pulses and propagated waves was detected from embryos beginning at about the morula-to-blastula transition and continuing through to hatching.

High pH prevents retinoic acid-induced teratogenesis.

Exogenously applied retinoic acid is known to cause teratogenic effects in a variety of animal systems. We examined whether the formation of teratogenic effects may be influenced by the electrical charge of retinoic acid. The pKa of retinoic acid ranges from 6 to 8, indicating that it is electrically neutral in a pH5 medium and is negatively charged in a pH9 medium. With this idea in mind, embryos of the pond snail Lymnaea stagnalis were pulse-treated with retinoic acid and cultured in media of different pH. The percentage of embryos with retinoic acid-induced eye defects was 6-fold lower in the pH9 medium as compared to the pH5 medium. In contrast, the apical plate defects induced by retinoic acid were not pH-dependent. The observation that high pH prevents eye defects but not apical plate defects can be explained by taking into account an electrophoretic redistribution of retinoic acid resulting from the voltage gradients that are generated by the Lymnaea embryo.

Polar ionic currents around embryos of Lymnaea stagnalis during gastrulation and organogenesis.

Embryos of Lymnaea stagnalis generate ionic currents which can be measured with the vibrating probe. Here we investigated the presence and origin of the currents during late embryonic development. During gastrulation the current pattern correlates with the animal-vegetal polarity and during organogenesis it is correlated to the newly formed antero-posterior axis. The origin of the ionic currents was studied by inhibition of the Na+/K(+)-pump with ouabain and by enzyme-cytochemical detection of the Ca(2+)-pump. Ouabain treatment resulted in a reduced current density around the embryo, indicating that the Na+/K(+)-pump contributes significantly to the net current. The Ca(2+)-pump was found to be localized in the vegetal blastomeres during gastrulation and in the larval kidney during organogenesis. It seems likely that this Ca(2+)-pump renders only a minor contribution to the net current in late embryonic development. Ionic currents have now been described in Lymnaea from the uncleaved egg up to the juvenile snail. During this period the overall current pattern changes only twice, demonstrating that the voltage gradient generated by the embryo remains stable during prolonged periods in development.