Search

Overexpression of the agouti-signaling protein ( asip1 ), an endogenous melanocortin antagonist, under the control of a constitutive promoter in zebrafish [Tg(Xla.Eef1a1:Cau.Asip1]iim4] ( asip1 -Tg) increases food intake by reducing sensitivity of the central satiety systems and abolish circadian activity rhythms. The phenotype also shows increased linear growth and body weight, yet no enhanced aggressiveness in dyadic fights is observed. In fact, asip1 -Tg animals choose to flee to safer areas rather than face a potential threat, thus suggesting a potential anxiety-like behavior (ALB). Standard behavioral tests, i.e., the open field test (OFT), the novel object test (NOT), and the novel tank dive test (NTDT), were used to investigate thigmotaxis and ALB in male and female zebrafish. Results showed that the asip1 -Tg strain exhibited severe ALB in every test, mainly characterized by pronounced freezing behavior and increased linear and angular swimming velocities. asip1 -Tg animals exhibited low central serotonin (5-HT) and dopamine (DA) levels and high turnover rates, thus suggesting that central monoaminergic pathways might mediate melanocortin antagonist-induced ALB. Accordingly, the treatment of asip1 -Tg animals with fluoxetine, a selective serotonin reuptake inhibitor (SSRI), reversed the ALB phenotype in NTDT as well as 5-HT turnover. Genomic and anatomical data further supported neuronal interaction between melanocortinergic and serotonergic systems. These results suggest that inhibition of the melanocortin system by ubiquitous overexpression of endogenous antagonist has an anxiogenic effect mediated by serotonergic transmission., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Over the last decade, the zebrafish has emerged as an important model organism for behavioural studies and neurological disorders, as well as for the study of metabolic diseases. This makes zebrafish an alternative model for studying the effects of energy disruption and nutritional quality on a wide range of behavioural aspects. Here, we used the zebrafish model to study how obesity induced by overfeeding regulates emotional and cognitive processes. Two groups of fish (n = 24 per group) were fed at 2% (CTRL) and 8% (overfeeding-induced obesity, OIO) for 8 weeks and tested for anxiety-like behaviour using the novel tank diving test (NTDT). Fish were first tested using a short-term memory test (STM) and then trained for four days for a long-term memory test (LTM). At the end of the experiment, fish were euthanised for biometric sampling, total lipid content, and triglyceride analysis. In addition, brains (eight per treatment) were dissected for HPLC determination of monoamines. Overfeeding induced faster growth and obesity, as indicated by increased total lipid content. OIO had no effect on anxiety-like behaviour. Animals were then tested for cognitive function (learning and memory) using the aversive learning test in Zantiks AD units. Results show that both OIO and CTRL animals were able to associate the aversive stimulus with the conditioned stimulus (conditioned learning), but OIO impaired STM regardless of fish sex, revealing the effects of obesity on cognitive processes in zebrafish. Obese fish did not show a deficiency in monoaminergic transmission, as revealed by quantification of total brain levels of dopamine and serotonin and their metabolites. This provides a reliable protocol for assessing the effect of metabolic disease on cognitive and behavioural function, supporting zebrafish as a model for behavioural and cognitive neuroscience.

The melanocortin system is a key structure in the regulation of energy balance. Overexpression of inverse agonists, agouti-signaling protein (ASIP), and agouti-related protein (AGRP) results in increased food intake, linear growth, and body weight. ASIP regulates dorsal-ventral pigment polarity through melanocortin 1 receptor (MC1R) and overexpression induces obesity in mice by binding to central MC4R. Asip1 overexpression in transgenic zebrafish ( asip1 -Tg) enhances growth, yet experiments show fish overexpressing Asip1 do not develop obesity even under severe feeding regimes. Asip1 -Tg fish do not need to eat more to grow larger and faster; thus, increased food efficiency can be observed. In addition, asip1 -Tg fish reared at high density are able to grow far more than wild-type (WT) fish reared at low density, although asip1 -Tg fish seem to be more sensitive to crowding stress than WT fish, thus making the melanocortin system a target for sustainable aquaculture, especially as the U.S. Food and Drug Association has recently approved transgenic fish trading.

Ghrelin (GRL) is a gut-brain hormone with a role in a wide variety of physiological functions in mammals and fish, which points out the ghrelinergic system as a key element for the appropriate biological functioning of the organism. However, many aspects of the multifunctional nature of GRL remain to be better explored, especially in fish. In this study, we used the CRISPR/Cas9 genome editing technique to generate F0 zebrafish in which the expression of grl is compromised. Then, we employed high-throughput mRNA sequencing (RNA-seq) to explore changes in the brain transcriptome landscape associated with the silencing of grl. The CRISPR/Cas9 technique successfully edited the genome of F0 zebrafish resulting in individuals with considerably lower levels of GRL mRNAs and protein and ghrelin O-acyl transferase (goat) mRNAs in the brain, intestine, and liver compared to wild-type (WT) zebrafish. Analysis of brain transcriptome revealed a total of 1360 differentially expressed genes (DEGs) between the grl knockdown (KD) and WT zebrafish, with 664 up- and 696 downregulated DEGs in the KD group. Functional enrichment analysis revealed that DEGs are highly enriched for terms related to morphogenesis, metabolism (especially of lipids), entrainment of circadian clocks, oxygen transport, apoptosis, and response to stimulus. The present study offers valuable information on the central genes and pathways implicated in functions of GRL, and points out the possible involvement of this peptide in some novel functions in fish, such as apoptosis and oxygen transport.

Dorso-ventral (DV) countershading is a highly-conserved pigmentary adaptation in vertebrates. In mammals, spatially regulated expression of agouti-signaling protein (ASIP) generates the difference in shading by driving a switch between the production of chemically-distinct melanins in melanocytes in dorsal and ventral regions. In contrast, fish countershading seemed to result from a patterned DV distribution of differently-coloured cell-types (chromatophores). Despite the cellular differences in the basis for counter-shading, previous observations suggested that Agouti signaling likely played a role in this patterning process in fish. To test the hypotheses that Agouti regulated counter-shading in fish, and that this depended upon spatial regulation of the numbers of each chromatophore type, we engineered asip1 homozygous knockout mutant zebrafish. We show that loss-of-function asip1 mutants lose DV countershading, and that this results from changed numbers of multiple pigment cell-types in the skin and on scales. Our findings identify asip1 as key in the establishment of DV countershading in fish, but show that the cellular mechanism for translating a conserved signaling gradient into a conserved pigmentary phenotype has been radically altered in the course of evolution.

Physiological mechanisms driving stress response in vertebrates are evolutionarily conserved. These mechanisms involve the activation of both the hypothalamic-sympathetic-chromaffin cell (HSC) and the hypothalamic-pituitary-adrenal (HPA) axes. In fish, the reduction of food intake levels is a common feature of the behavioral response to stress but the central mechanisms coordinating the energetic response are not well understood yet. In this work, we explore the effects of acute stress on key central systems regulating food intake in fish as well as on total body cortisol and glucose levels. We show that acute stress induced a rapid increase in total body cortisol with no changes in body glucose, at the same time promoting a prompt central response by activating neuronal pathways. All three orexigenic peptides examined, i.e., neuropeptide y (npy), agouti-related protein (agrp), and ghrelin, increased their central expression level suggesting that these neuronal systems are not involved in the short-term feeding inhibitory effects of acute stress. By contrast, the anorexigenic precursors tested, i.e., cart peptides and pomc, exhibited increased expression after acute stress, suggesting their involvement in the anorexigenic effects.

Zebrafish embryos are treated with anti-thyroidal compounds, such as phenylthiourea, to inhibit melanogenesis. However, the mechanism whereby the thyroidal system controls melanin synthesis has not been assessed in detail. In this work, we tested the effect of the administration of diets supplemented with T3 (500μg/g food) on the pigment pattern of adult zebrafish. Oral T3 induced a pronounced skin paling in both adult female and male zebrafish that was reversible upon cessation of treatment. The number of visible melanophores was significantly reduced in treated fish. Accordingly, treatment down-regulated expression of tyrosinase-related protein 1 in both sexes. We also found sexually dimorphic regulation of some melanogenic genes, such as Dct/Tyrp2 that was dramatically up-regulated in females after T3 treatment. Thus, we demonstrated that melanogenesis is reversibly inhibited by thyroid hormones in adult zebrafish and make the discovery of gender-specific differences in the response of melanogenic gene expression. Thus, fish gender is now shown to be an important variable that should be controlled in future studies of fish melanogenesis., Competing Interests: The authors have declared that no competing interests exist.

The melanocortin system is probably one of the most complex hormonal systems since it integrates agonist, encoded in the proopiomelanocortin precursor, endogenous antagonist, agouti signaling protein and agouti-related protein, five different G-protein coupled receptors and two accessory proteins. These accessory proteins interact with melanocortin receptors to allow traffic to the plasma membrane or to regulate the pharmacological profile. The MC1R fill the extension locus, which is primarily responsible for the regulation of pigmentation. In zebrafish, both MC1R and MRAP2 system are expressed in the skin. We demonstrate that zebrafish MC1R physically, or closely, interacts with the MRAP2 system, although this interaction did not result in modification of the studied pharmacological profile. However, progressive fasting induced skin darkening but also an upregulation of the MRAP2 expression in the skin, suggesting an unknown role for MRAP2a that could involve receptor desensitization processes. We also demonstrate that crowding stress induces skin darkening and a downregulation of MC1R expression in the skin., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In this paper, we identify three different MRAPs in zebrafish, zfMRAP1, zfMRAP2a and zfMRAP2b, and demonstrate that zfMC2R is not functional in the absence of MRAP expression. ZfMRAP1 expression was restricted to adipose tissue and the anterior kidney whereas MRAP2a and MRAP2b were expressed in all the tissues tested. Quantification of surface receptor and immunofluorescence studies indicated that the receptor is unable to translocate to membrane in the absence of MRAP isoforms. MRAP1 and MRAP2b are localized in the plasma membrane in the absence of zfMC2R expression but MRAP2b is retained in perinuclear position. MRAP1 and MRAP2a displayed an equivalent translocation capacity to the membrane of zfMC2R but only zfMRAP1 expression led to intracellular cAMP increases after ACTH stimulation. ZfMRAP2b had no effect on zfMC2R activity but both zfMRAP2 isoforms enhanced the zfMRAP1-assisted cAMP intracellular increase, suggesting an interaction between zfMRAP1 and zfMRAP2s when regulating zfMC2R activity., ((c) 2010 Elsevier Ireland Ltd. All rights reserved.)