BACKGROUND: Previously has been shown that fish kisspeptin 1 (Kiss1) acts on the brains serotonin system to reduce anxiety-phobic reactions in Danio rerio. The kissspeptin gene (kiss1) of teleost fish is also a conservative orthologue of the kissspeptin gene (KISS1/Kiss1) of mammals. AIM: In this work we investigated the possible anxiolytic effect of mammalian kisspeptin analogs Kiss1 in Danio rerio in comparison with antidepressants of the serotonin-type of action. MATERIALS AND METHODS: A novelty test was used: the fish was first placed in a beaker with a dissolved pharmacological substance (or H2O), and then in a viewing tank for 6 min, where the trajectory of movement, the length of the path, the number of movements to the upper part of the tank, the time spent in the lower part of the tank, number and time of the freezing were automatically recorded. RESULTS: It is shown that, in response to the novelty of being placed in a viewing tank, fish react by moving to the bottom, increasing friezing, and reducing the number of movements to the upper half of the tank. Against the background of antidepressants clomipramine, paroxetine or trazodone (0.51 mg per 1 l of water), the fish were not only in the lower, but also in the upper part of the viewing tank. The average path length did not change significantly. The time in the lower part of the tank decreased by more than 2 times compared with the control group of animals and showed a dose-dependent effect. The number of movements to the upper part of the tank per experience increased significantly. Mammalian kisspeptin analogues Cloud Clone (USA) in a dose 0.011 mg per 1 l of water caused a similar patterns of behavior in fish in response to novelty. At the same time, the effects of kisspeptin analogs were lower than those of antidepressants. The most effective dose for the action of the studied kisspeptin analogs was 0.1 mg per 1 l of water. CONCLUSIONS: Thus, mammalian kisspeptin analogs Kiss1 reduce anxiety-phobic responses to novelty in Danio rerio. Data on the unidirectional effects of mammalian kisspeptin analogs and serotonin-type antidepressants support the potential role of Kiss1 in modulating serotonin-dependent behaviours in Danio rerio. The data obtained support the hypothesis that kisspeptin may be involved in the regulation of anxiety-phobic states, apparently to maintain the emotional aspects of reproductive behavior, such as sexual motivation and arousal.

The effect of benzodiazepine anxiolytic diazepam in Danio rerio was investigated. Methods. A stress test on novelty situation was used: a fish was placed first in a beaker with a dissolved pharmacological substance (or water) and then into a novel tank for 6 min, where the trajectory, the path length, the number of movements to the upper part of the novel tank, the number and time of the pattern of freezing of the experiment were measured. Results. In response to the novelty of tank, the fish was shown to react by submerging to the bottom, increasing the freezing, and reducing the number of movements to the upper half of the novel tank. After diazepam exposure (administration), the fish was not only in the lower, but also in the upper part of the novel tank. A pharmacological analysis of diazepam effect in Danio rerio showed that in a certain dose range of 110 mg/l anxiolytic reduces (in comparison with the control group of fish) the number and time of freezing, increases the number of movements in the upper half of the tank and the swimming time in upper part of the tank. Diazepam causes a disinhibition of motor activity at doses of 1 and 5 mg/l, which may be explained by the effect of small doses of tranquilizers on presynaptic GABA-A autoreceptors. Diazepam 20 mg/l has a depriving effect. Conclusion. Diazepam acts in a higher dose range (110 mg/l) than phenazepam (0.11 mg/l) in Danio rerio. At the same time, diazepam is characterized by a domed dose-dependent effect, in contrast to the action of phenazepam. The prospect of using Danio rerio as an animal model in behavioral pharmacology is not inferior to studies in rodents.

BACKGROUND: Literature data indicated that ghrelin may be a stress neuropeptide in some animals, including Danio rerio. AIM: The aim of the study was to measure the levels of ghrelin in Danio rerio brain (forebrain, midbrain, and hindbrain) after predator exposure (predator stress test) and administration of ghrelin and oxytocin antagonists to assess the role of ghrelin in stress response. MATERIALS AND METHODS: The study used 68 Danio rerio, one predator of Cichlasoma nicaraguensis. Ghrelin levels were measured by enzyme-linked immunosorbent assay. The following pharmacological agents were used: 1) ghrelin antagonist hexapeptide [D-Lys3]-GHRP-6 (Tocris, UK); 2) recombinant peptide analog of ghrelin agrelax with a molecular weight of 3.5 kDa, developed at the IEM (both compounds in equal dosages of 0.333 mg/L); corticoliberin (Tocris) at a dosage of 0.4 mg/L; and oxytocin (Gedeon Richter, Hungary) at a dosage of 3.8 L (0.005 IU/L) per 50 mL of aquarium water (0.019 IU/L). RESULTS: In the control group, ghrelin level was determined only in the hindbrain, and ghrelin levels in the forebrain and midbrain were 4 pg/mg of total protein. Contact with a predator led to a significant increase in ghrelin levels in the forebrain and midbrain, but not in the hindbrain. Thus, in fish forebrain, ghrelin levels increased to 966 12 pg/mg of protein, which is almost a 250-fold increase in the indicator compared with the level in intact animals. In the presence of agrelax, [D-Lys3]-GHRP-6, and oxytocin, ghrelin levels in the forebrain and midbrain of stressed fish decreased approximately equally to 88 3, 97 1, and 115 1 pg/mg protein, respectively. CONCLUSION: Thus, stress exposure (contact with a predator) significantly increases ghrelin levels in the forebrain and midbrain but decreases in the hindbrain. Stress-protective peptides (oxytocin, [D-Lys3]-GHRP-6, and agrelax) decreased ghrelin levels in the forebrain and midbrain, and to a lesser extent, in the hindbrain when exposed to stress.

The aim: of the study was to investigate the level of ghrelin in various brain structures during a stress response in Zebrafish to a predator, to evaluate this indicator as a potential biomarker of stress, and the effect of a benzodiazepine tranquilizer (phenazepam) on stress-induced changes Materials and methods: The object of the study was Zebrafish, or Danio rerio wild type, which was subjected to stress by exposure to a predator Hypsophrys nicaraguensis from the cichlid family. In the tail tissue, the level of cortisol was determined, in the brain – the level of total (acylated and non-acylated) ghrelin by the method of enzyme-linked immunosorbent assay. The benzodiazepine anxiolytic phenazepam (1 mg/L), a ghrelin antagonist [D-Lys3]-GHRP-6 (0.333 mg/l) and corticotropin-releasing hormone (CRF; 0.4 mg/L) were used as the pharmacological agents. Results and discussion: Exposure to a predator, just as administering CRF, more than doubled the level of cortisol in the tail tissue. [D-Lys3]-GHRP-6 and phenazepam prevented an increase in a tissue cortisol level. Simultaneously, in the medulla oblongata and cerebellum, the phylogenetically most ancient structures, rather than in the forebrain (telencephalon) or in the midbrain (corpora bigemia), the level of ghrelin was recorded about 500 pg/g of total protein. In response to exposure to a predator, the level of ghrelin increased in the forebrain and midbrain to nanogram concentrations and moderately decreased in the cerebellum. The effect was prevented by phenazepam and [D-Lys3]-GHRP-6. Conclusion: Increases in ghrelin in the brain in response to stressful situations can be seen as a functional brain biomarker of stress, along with increased levels of tissue cortisol levels. Both of these effects are prevented by both the ghrelin antagonist and the benzodiazepine tranquilizer. The mechanism of action of the tranquilizer is a functional antagonism between the GABAergic system of the brain and the ghrelin system.

Danio reriohas firmly established itself as a successful model for research in many areas of biology and medicine, first of all for developing new medicines. The aimof our study was to evaluate ghrelin level in zebrafish brain after stress and after phenazepam usage on stressed fish. Methods.In our study 96Danio rerio, predatorCichlasoma nicaraguensishave been used. The fish have been kept at anormal room temperature (2223C) with standard feeding time (twice per day). The level of neuropeptides has been tested by ELISA test. During experiment a fish has been firstly placed in a beaker with a dissolved pharmacological substance, then has been transferred into a tank with predator. In the end of experiment, it has been put into a novel tank for 6 min. The decapitation has been made. Thebrain has been divided into three anatomical parts:telencephalonjust behind the olfactory bulb, the middle partcorpora bigemiaandcerebellum, which is situated behind thecorpora bigemia. After that the material for ELISA test was made using GhrelinFISH, MyBioSource ELISA kit. Results.In the control group ghrelin has been determined only in thecerebellumin 57.14% of all fish. In the experiment with predator ghrelin has been found in all tested brain parts of fish, but in thetelencephalonthere was the highest level. Inthe experiment with phenazepam usage only and phenazepam administration after predator stress, the ghrelin value has not been determined atall. Conclusion.Thus we have found out that the ghrelin value increases after predator stress and the drug phenazepam eliminated it completely after its administration. We may suppose that the administration of anxiolytics such as phenazepam can reduce the anxiety inDanio rerio.

In our previous work, we suggested that analogs of mammalian kisspeptin Kiss1 reduce anxiety and phobic reactions novel in Danio rerio. The most effective dose for the action of the studied analogs of kisspeptin corresponded to 0.1 mg per 1000 mL of water. In this study, other analogs of mammalian Kiss1 at a dose of 0.1 mg per 1000 mL of water also reduced the anxious behavior of Danio fish. The effect of Kiss1 and Kiss2 kisspeptins on the behavior of Danio rerio was also evaluated. In the novel test, the number of freezing decreased by two times with the introduction of kisspeptin 10 and by three times after the introduction of the kisspeptin analog. An analog of mammalian kisspeptin reduced the freezing time by two times. The length of the trajectory decreased by two times under the influence of the mammalian Kiss1 kisspeptin analog. With the action of kisspeptin 10, the number of transitions to the upper part of the tank increased by two times. After the introduction of the kisspeptin analog, the number of transitions to the upper part of the aquarium increased by three times. In the predator test, the number and time of freezing decreased by 1.5 times with the action of mammalian kisspeptins. The length of the trajectory after the introduction of kisspeptin bony fish and kisspeptin 10 mammals increased. The length of the trajectory after the introduction of Kiss1 increased by 1.5 times. The length of the trajectory after the introduction of Kiss2 increased by three times. After the introduction of kisspeptin 10, the trajectory increased by two times, and the time spent in the lower part of the tank decreased by two times. Kisspeptins of bony fish also reduced the anxiety and phobic reactions in fish, but to a lesser extent. Thus, kisspeptin 10 and an analog of mammalian kisspeptin in response to the presentation of a predator had more significant effects on anxiety in Danio rerio compared with the action of kisspeptin bony fish Kiss1 and Kiss2. Thus, bony fish kisspeptins and mammalian kisspeptins can reduce anxiety and phobic reactions in Danio rerio; however, mammalian kisspeptins are the most effective. Bony fish kisspeptin Kiss1 has an anxiolytic effect in contrast to Kiss2, which suggests that it affects fear reduction, and Kiss2 appears to be responsible for social and sexual behavior. The results support the hypothesis that kisspeptins may be involved in the regulation of anxiety and phobic states, apparently to maintain the emotional aspects of reproductive behavior, such as sexual motivation and arousal.