The Reprimo gene family member, reprimo-like (rprml), is required for blood development in embryonic zebrafish.

The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood. Heretofore, mammalian Reprimo (RPRM) has been described as a putative p53-dependent tumor suppressor gene that functions at the G2/M cell cycle checkpoint. Another family member, Reprimo-like (RPRML), has not yet an established role in physiology or pathology. Importantly, RPRML expression pattern is conserved between zebrafish and human species. Here, using CRISPR-Cas9 and antisense morpholino oligonucleotides, we disrupt the expression of rprml in zebrafish and demonstrate that its loss leads to impaired definitive hematopoiesis. The formation of hemangioblasts and the primitive wave of hematopoiesis occur normally in absence of rprml. Later in development there is a significant reduction in erythroid-myeloid precursors (EMP) at the posterior blood island (PBI) and a significant decline of definitive hematopoietic stem/progenitor cells (HSPCs). Furthermore, loss of rprml also increases the activity of caspase-3 in endothelial cells within the caudal hematopoietic tissue (CHT), the first perivascular niche where HSPCs reside during zebrafish embryonic development. Herein, we report an essential role for rprml during hematovascular development in zebrafish embryos, specifically during the definitive waves of hematopoiesis, indicating for the first time a physiological role for the rprml gene.

Just Keep Swimming: Neuroendocrine, Metabolic, and Behavioral Changes After a Forced Swimming Test in Zebrafish.

In this study, we show that an adaptation of the spinning test can be used as a model to study the exercise-exhaustion-recovery paradigm in fish. This forced swimming test promotes a wide range of changes in the hypothalamus-pituitary-interrenal axis functioning, intermediary metabolism, as well in fish behavior at both exercise and recovery periods. Our results pointed that this adapted spinning test can be considered a valuable tool for evaluating drugs and contaminant effects on exercised fish. This can be a suitable protocol both to environmental-to evaluate contaminants that act in fish energy mobilization and recovery after stressors-and translational perspectives-effects of drugs on exercised or stressed humans.

Effects of Lactobacillus rhamnosus GG on hepatic and serum lipid profiles in zebrafish exposed to ethanol.

Zebrafish is a powerful tool in pharmacological research and useful to identify new therapies. Probiotics can offer therapeutic options in alcoholic liver disease. This study was done in two independent experiments: first, we confirmed the intestinal colonization of probiotic Lactobacillus rhamnosus GG (LGG) after ethanol exposure. Second, four groups were performed: control (C), probiotic (P), ethanol (E), and probiotic+ethanol (P+E). Liver histology, hepatocytes morphometry, hepatic and serum lipid quantifications were conducted in second experiment. During 4 weeks, P and P+E groups were fed with LGG supplemented feed; E and C unsupplemented. E and P+E groups received 0.5% of ethanol added into tank water. Zebrafish exposed to ethanol (E group) presented intense liver steatosis after 28 days in contrast to the almost normalized liver histology of P+E group at the same period. Liver morphometry showed a significant enlargement of hepatocytes of E group after 4 weeks (p<0.0001). Serum triglycerides decreased in P+E group compared with C, P (p<0.001), and E (p=0.004), after 14 and 28 days similarly. Serum cholesterol was also decreased by LGG; P group decreased compared with C and E after 14 days (p=0.002 and p=0.007, respectively) and P+E group decreased significantly compared with E and C groups (p<0.0001) after 28 days. Hepatic triglycerides were reduced in P+E group after 28 days compared to E (p=0.006). The persistence of LGG in zebrafish intestines was demonstrated. LGG decreased serum levels of triglycerides and cholesterol and improved hepatic steatosis.

One for all and all for one: the importance of shoaling on behavioral and stress responses in zebrafish.

Zebrafish has been increasingly used in behavioral studies, but data can present high variability. Most studies have been performed using isolated zebrafish, despite their interactive nature and shoaling behavior. We compared adult zebrafish behavior and cortisol levels after exposure to novelty as well as sensitivity to Pentylenetetrazole (PTZ)-induced seizures in animals tested individually or in groups of three (triplets). In the exploratory behavior task, data from single fish and triplets were not significantly different, but single fish data were more disperse in latency, to enter and time spent in the tank upper part, and crossings. In the light-dark task, time in the light zone and crossings were not different between groups, but latency to enter the dark zone and data variability were. We also observed that the latency to reach stage III seizures induced by PTZ was higher in triplets, but data dispersion was not different from single fish. Finally, cortisol levels of fish individually exposed to a novel environment were higher and more variable than triplets, while both groups had higher levels than unmanipulated animals. Thus, when tested individually, zebrafish are more stressed and present more variable behavior due to disruption of their natural shoal strategies. These features can be beneficial or detrimental depending on study aims and should be considered when designing, analyzing, and interpreting zebrafish behavioral data.

Blood collection for biochemical analysis in adult zebrafish.

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies. There are relatively few publications related to adult zebrafish physiology of organs and systems, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. Recently, Gupta et al. described how to dissect adult zebrafish organs, Kinkle et al. described how to perform intraperitoneal injections, and Pugach et al. described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish. The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

L-histidine enhances learning in stressed zebrafish.

The aim of the present study was to determine the effect of the histaminergic precursor L-histidine and the H3 receptor antagonist thioperamide on the learning process of zebrafish submitted or not to confinement stress. On each of the 5 consecutive days of experiment (D1, D2, D3, D4, D5), animals had to associate an interruption of the aquarium air supply with food offering. Non-stressed zebrafish received an intraperitoneal injection of 100 mg/kg L-histidine, 10 mg/kg thioperamide or saline after training. Stressed animals received drug treatment and then were submitted to confinement stress for 1 h before the learning procedure. Time to approach the feeder was measured (in seconds) and was considered to be indicative of learning. A decrease in time to approach the feeder was observed in the saline-treated group (D1 = 141.92 +/- 13.57; D3 = 55 +/- 13.54), indicating learning. A delay in learning of stressed animals treated with saline was observed (D1 = 217.5 +/- 25.66). L-histidine facilitated learning in stressed (D1 = 118.68 +/- 13.9; D2 = 45.88 +/- 8.2) and non-stressed (D1 = 151.11 +/- 19.20; D5 = 62 +/- 14.68) animals. Thioperamide inhibited learning in non-stressed (D1 = 110.38 +/- 9.49; D4 = 58.79 +/- 16.83) and stressed animals (D1 = 167.3 +/- 26.39; D5 = 172.15 +/- 27.35). L-histidine prevented the increase in blood glucose after one session of confinement (L-histidine = 65.88 +/- 4.50; control = 53 +/- 3.50 mg/dL). These results suggest that the histaminergic system enhances learning and modulates stress responses in zebrafish.

L-histidine enhances learning in stressed zebrafish

The aim of the present study was to determine the effect of the histaminergic precursor L-histidine and the H3 receptor antagonist thioperamide on the learning process of zebrafish submitted or not to confinement stress. On each of the 5 consecutive days of experiment (D1, D2, D3, D4, D5), animals had to associate an interruption of the aquarium air supply with food offering. Non-stressed zebrafish received an intraperitoneal injection of 100 mg/kg L-histidine, 10 mg/kg thioperamide or saline after training. Stressed animals received drug treatment and then were submitted to confinement stress for 1 h before the learning procedure. Time to approach the feeder was measured (in seconds) and was considered to be indicative of learning. A decrease in time to approach the feeder was observed in the saline-treated group (D1 = 141.92 ± 13.57; D3 = 55 ± 13.54), indicating learning. A delay in learning of stressed animals treated with saline was observed (D1 = 217.5 ± 25.66). L-histidine facilitated learning in stressed (D1 = 118.68 ± 13.9; D2 = 45.88 ± 8.2) and non-stressed (D1 = 151.11 ± 19.20; D5 = 62 ± 14.68) animals. Thioperamide inhibited learning in non-stressed (D1 = 110.38 ± 9.49; D4 = 58.79 ± 16.83) and stressed animals (D1 = 167.3 ± 26.39; D5 = 172.15 ± 27.35). L-histidine prevented the increase in blood glucose after one session of confinement (L-histidine = 65.88 ± 4.50; control = 53 ± 3.50 mg/dL). These results suggest that the histaminergic system enhances learning and modulates stress responses in zebrafish.