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Browsing by Subject "Zebrafish"

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  • Enckell, Louise (2023)
    VMAT2 is an integral membrane protein with a crucial role in the monoaminergic system. It transports cytosolic dopamine and other neurotransmitters into synaptic vesicles for storage and later exocytotic release. VMAT2 has been found to have a neuroprotective effect, for instance by preventing oxidation of free cytosolic dopamine. Research has suggested that increased VMAT2 expression or function could have a protective effect against Parkinson’s disease. Rapid embryonic development, transparency during larval stage, and high homology with the human genome are some of the factors that makes the zebrafish a popular model organism. The aim of this research was to optimize the protocol for immunohistochemistry with an anti-dopamine antibody, and to find out how the absence of a functioning vmat2 gene in zebrafish mutants (vmat2-/-) affects dopamine immunostaining, compared to embryos with normal vmat2 function (vmat2+/+). Zebrafish larvae were fixed in a fixation agent, whereafter brains were dissected and immunostaining was performed. Individual methods in the protocol were adjusted for each experiment until the result of the dopamine staining was considered optimal. We found that the choice of fixative affected dopamine staining strongly. A clear reduction in dopamine immunoreactive cells was observed in zebrafish larvae lacking a functioning vmat2 gene compared to zebrafish with normal vmat2 function. The results also suggested that the localization of dopamine-storing cells in zebrafish larvae resembles more tyrosine hydroxylase 2 than tyrosine hydroxylase 1 expression pattern, especially in the hypothalamus. Vmat2-/- zebrafish could be useful when investigating how monoamine transport affects different diseases and their pharmacological treatment.
  • Lewis, Serena (2021)
    Histamine receptors are known to be expressed throughout the peripheral nervous system and are involved in regulating the gut and immune system. The gut-brain axis, which consists of bidirectional signaling between the central nervous system and gastrointestinal tract, links gut functions to emotional and cognitive controls in the brain. Many animal models are known to express histamine receptors in their gut and brain tissue which can be altered by a compromised gut-brain axis like stress. Histamine receptors also play an important role in many gastric and intestinal disorders. However, the precise expression pattern of histamine receptors in zebrafish gut tissue is unknown, as is whether their expression levels also change with stress. Here, I show that zebrafish gut contains several histamine receptors, but their role involving stress within the gut remains unknown. I found that histamine receptors hrh1 and hrh3 as well as the enzyme that synthesizes histamine, histidine decarboxylase (hdc), are expressed in zebrafish gut and brain in wildtype and hdc knockout adult zebrafish using in situ hybridization. Stress induction on wildtype male zebrafish through chronic social defeat and analysis of histamine receptor and hdc mRNA levels using quantitative real time PCR showed no differences in subordinate, dominate, or control fish. However, it did provide quantitative data that hrh1, hrh2, and hdc mRNA expresses in the adult gut. My results demonstrate the first data to suggest histamine receptors are expressed in zebrafish gut, and that even though stress can alter the gut-brain axis, it may not do so through the regulation of these receptors.
  • Lehto, Anna (2023)
    The histamine system in the brain has many important functions, including regulating the sleep-wake cycle, locomotor activity, cognition and memory. Gamma-Aminobutyric acid (GABA) on the other hand is the most important inhibitory transmitter in the brain, thus important for example in inducing sleep. Disturbances in these systems are present in various neurological diseases. Of the various histamine receptors, the histamine receptor 3 (HRH3) has a unique role in restricting the synthesis and release of both histamine and other transmitters, including glutamate, acetylcholine, dopamine and noradrenaline. GABA is synthesized from glutamic acid by glutamic acid decarboxylase (GAD), which has two different isoforms, GAD65 and GAD67. NKCC1 and KCC2 are ion transporter molecules essential for the development of the GABA system. The aim of this study was to examine the effect the HRH3 has on four essential genes regarding the GABAergic system GAD65, GAD67, KCC2, and NKCC1. This was accomplished using a HRH3 knockout zebrafish strain. The expression patterns of the genes were visualized by whole-mount in situ hybridization. The GAD67 and KCC2 gene expression patterns were also visualized in zebrafish brains treated with HRH3 antagonists ciproxifan and thioperamide. The results of the study showed that knocking out of the HRH3 did not seem to have an effect on the expression of the studied GABAergic genes. Samples treated with thioperamide or ciproxifan on the other hand showed diminished gene expression. This indicates that these pharmaceutical agents decrease the expression of KCC2, revealing new information about their effect on brain function. This result still needs to be confirmed using quantitative methods. New information about the genes was also acquired regarding their expression in the wild type zebrafish. For GAD65 and GAD67 new information was gained about the changes in expression during the zebrafish development. For KCC2 and NKCC1 the expression patterns in the zebrafish brain are completely new, previously unpublished information.
  • Stenbäck, Andreas; Stenbäck, Andreas (2022)
    proSAAS är en neuropeptid som fungerar som prekursor för diverse andra peptider. Neuropeptider som exempelvis bigLEN och PEN har visat sig ha inverkan på matintag hos försöksdjur. proSAAS har även undersökts för dess neuroprotektiva egenskap i exempelvis Parkinsons sjukdom. Experiment har visat att proSAAS förhindrar bildningen av amyloida plack. Zebrafisk (Danio rerio) är ett försöksdjur som under de senaste årtiondena vuxit i popularitet på grund av sitt breda användningsområde, lätta underhåll och förvaring och likhet till det mänskliga genomet. Zebrafiskens hjärna har flera neurokemiska och anatomiska likheter med människohjärnan. Här har undersökningsmålet varit att undersöka huruvida skillnad förekommer mellan embryon med normal förekomst av proSAAS och genmodifierade embryon utan en fungerande gen för proSAAS. Embryon av olika genetiska varianter har dissekerats och deras hjärnor immunofärgats och undersökts med hjälp av mikroskop. Förekomst av cellkärnor och signalvägar för neuropeptiderna dopamin, orexin, gonadotropinutsöndrande hormon, galanin och neuropeptid Y har undersökts. I studien kunde man inte finna variation mellan de olika genotyperna gällande förekomsten av orexin, gonadotropinutsöndrande hormon, galanin och neuropeptid Y. Man kunde upptäcka mera tyrosin hydroxylas, ett enzym ämne som deltar i bildningen av dopaminerga celler, i vissa delar av diencephalon hos de embryon som saknar proSAAS. Variationen av mängden tyrosinhydroxylaspositiva cellerkärnor mellan embryon med normal proSAAS-produktion och genmodifierade embryon öppnar dörrar för nya forskningsområden. Intressanta områden för vidare studier är att se huruvida variation också förekommer hos vuxna individer samt om skillnad i rörelsemönster även förekommer. (234 ord)