Zebrafish Learning & Memory
Age-related impairments in cognitive functions represent a growing clinical and social issue. Genetic and behavioral characterization of animal models can provide critical information on the intrinsic and environmental factors that determine the deterioration or preservation of cognitive abilities throughout life.
Behavior of wild-type, mutant and gamma-irradiated zebrafish (Danio rerio) was documented using image-analysis technique. Conditioned responses to spatial, visual and temporal cues were investigated in young, middle-aged and old animals. The results demonstrate that zebrafish aging is associated with changes in cognitive responses to emotionally positive and negative experiences, reduced generalization of adaptive associations, increased stereotypic and reduced exploratory behavior and altered temporal entrainment. Genetic upregulation of cholinergic transmission attenuates cognitive decline in middle-aged achesb55/+ mutants, compared to wild-type siblings. In contrast, the genotoxic stress of gamma-irradiation accelerates the onset of cognitive impairment in young zebrafish.
Locomotor activity recordings
Zebrafish locomotor activity in the experimental paradigms described below was documented using VideoTrack image analysis software (View Point, Lyon, France).
In order to avoid potential entrainment of the fish to the visual cues in the distant environment, the recording apparatuses were shielded from the rest of the room with the all-white enclosure. Continuous tracking of each fish documents distance traversed and time spent in fast or slow motion in multiple areas of individual tanks or test apparatus. “Inactivity” was defined as less than 0.1 cm per min, to eliminate a possibility of camera noise or small movements of the fins interfering with the activity analysis. The speed of the fast movement was defined as 14 cm/min, or above. The range between 0.1 and 14 cm/min was considered to be a slow movement. The program conducts this distinction automatically, based on the pre-set thresholds, and the raw data obtained at the end of the experiment presents the results for each type of activity.
Conclusions/SignificanceThese findings would allow the use of powerful molecular biological resources accumulated in the zebrafish field to address the mechanisms of cognitive senescence, and promote the search for therapeutic strategies which may attenuate age-related cognitive decline.
Fish are an important model for the pharmacological and toxicological characterization of human pharmaceuticals in drug discovery, drug safety assessment and environmental toxicology. However, do fish respond to pharmaceuticals as humans do? To address this question, we provide a novel quantitative cross-species extrapolation approach (qCSE) based on the hypothesis that similar plasma concentrations of pharmaceuticals cause comparable target-mediated effects in both humans and fish at similar level of biological organization (Read-Across Hypothesis). To validate this hypothesis, the behavioural effects of the anti-depressant drug fluoxetine on the fish model fathead minnow (Pimephales promelas) were used as test case. Fish were exposed for 28 days to a range of measured water concentrations of fluoxetine (0.1, 1.0, 8.0, 16, 32, 64 µg/L) to produce plasma concentrations below, equal and above the range of Human Therapeutic Plasma Concentrations (HTPCs). Fluoxetine and its metabolite, norfluoxetine, were quantified in the plasma of individual fish and linked to behavioural anxiety-related endpoints. The minimum drug plasma concentrations that elicited anxiolytic responses in fish were above the upper value of the HTPC range, whereas no effects were observed at plasma concentrations below the HTPCs. In vivo metabolism of fluoxetine in humans and fish was similar, and displayed bi-phasic concentration-dependent kinetics driven by the auto-inhibitory dynamics and saturation of the enzymes that convert fluoxetine into norfluoxetine. The sensitivity of fish to fluoxetine was not so dissimilar from that of patients affected by general anxiety disorders. These results represent the first direct evidence of measured internal dose response effect of a pharmaceutical in fish, hence validating the Read-Across hypothesis applied to fluoxetine. Overall, this study demonstrates that the qCSE approach, anchored to internal drug concentrations, is a powerful tool to guide the assessment of the sensitivity of fish to pharmaceuticals, and strengthens the translational power of the cross-species extrapolation.
OptoGluNAM4.1, a negative allosteric modulator (NAM) of metabotropic glutamate receptor 4 (mGlu4) contains a reactive group that covalently binds to the receptor and a blue-light-activated, fast-relaxing azobenzene group that allows reversible receptor activity photocontrol in vitro and in vivo. OptoGluNAM4.1 induces light-dependent behavior in zebrafish and reverses the activity of the mGlu4 agonist LSP4-2022 in a mice model of chronic pain, defining a photopharmacological tool to better elucidate the physiological roles of the mGlu4 receptor in the nervous system.
Nanomaterials hold significant potential for industrial and biomedical application these years. Therefore, the relationship between nanoparticles and neurodegenerative disease is of enormous interest. In this contribution, zebrafish embryos and PC12 cell lines were selected for studying neurotoxicity of titanium dioxide nanoparticles (TiO2 NPs). After exposure of different concentrations of TiO2 NPs to embryos from fertilization to 96 hpf, the hatching time of zebrafish was decreased, accompanied by an increase in malformation rate. However, no significant increases in mortality relative to control were observed. These results indicated that TiO2 NPs exposure hold a risk for premature of zebrafish embryos, but not fatal. The further investigation confirmed that TiO2 NPs could accumulate in the brain of zebrafish larvae, resulting in reactive oxygen species (ROS) generation and cell death of hypothalamus. Meanwhile, q-PCR analysis showed that TiO2 NPs exposure increased the pink1, parkin, α-syn and uchl1 gene expression, which are related with the formation of Lewy bodies. We also observed loss of dopaminergic neurons in zebrafish and in vitro. These remarkable hallmarks are all linked to these Parkinson's disease (PD) symptoms. Our results indicate that TiO2NPs exposure induces neurotoxicity in vivo and in vitro, which poses a significant risk factor for the development of PD.
The company of biologists
Zebrafish can regenerate brain injury, and the regenerative process is driven by resident stem cells. Stem cells are heterogeneous in the vertebrate brain, but the significance of having heterogeneous stem cells in regeneration is not understood. Limited availability of specific stem cells might impair the regeneration of particular cell lineages. We studied regeneration ability of the adult zebrafish cerebellum. The zebrafish cerebellum contains two major stem and progenitor cell types, ventricular zone and neuroepithelial cells. Using conditional lineage tracing, we demonstrate that cerebellar regeneration depends on availability of specific stem cells. Radial glia-like cells are thought to be the predominating stem cell type in homeostasis and after injury. However, we find that radial glia-like cells play a minor part in adult cerebellar neurogenesis and in recovery after injury. Instead, we find that neuroepithelial cells are the predominant stem cell type supporting cerebellar regeneration after injury. Zebrafish are able to regenerate many, but not all cell types in the cerebellum, which emphasizes the need to understand the contribution of different adult neural stem and progenitor cell subtypes in the vertebrate CNS.
Light affects sleep and wake behaviors by providing an indirect cue that entrains circadian rhythms and also by inducing a direct and rapid regulation of behavior. While circadian entrainment by light is well characterized at the molecular level, mechanisms that underlie the direct effect of light on behavior are largely unknown. In zebrafish, a diurnal vertebrate, we found that both overexpression and mutation of the neuropeptide prokineticin 2 (Prok2) affect sleep and wake behaviors in a light-dependent but circadian-independent manner. In light, Prok2 overexpression increases sleep and induces expression of galanin (galn), a hypothalamic sleep-inducing peptide. We also found that light-dependent, Prok2-induced sedation requires prokineticin receptor 2 (prokr2) and is strongly suppressed in galn mutants. These results suggest that Prok2 antagonizes the direct wake-promoting effect of light in zebrafish, in part through the induction of galn expression in the hypothalamus.
The mineralocorticoid receptor (MR) in mammals mediates the effects of aldosterone in regulating fluid balance and potassium homeostasis. While MR signalling is essential for survival in mammals, there is no evidence that MR has any physiological role in ray-finned fish. Teleosts lack aldosterone and emerging evidence suggest that cortisol mediates ion and fluid regulation by activating glucocorticoid receptor (GR) signalling. Consequently, a physiological role for MR signalling, despite its conserved and ancient origin, is still lacking. We tested the hypothesis that a key physiological role for MR signalling in fish is the regulation of stress axis activation and function. Using either MR or GR knockout zebrafish, our results reveal distinct and complementary role for these receptors in stress axis function. GR−/− mutants were hypercortisolemic and failed to elicit a cortisol stress response, while MR−/− mutants showed a delayed, but sustained cortisol response post-stressor. Both these receptors are involved in stress-related behaviour, as the loss of either receptors abolished the glucocorticoid-mediated larval hyperactivity to a light stimulus. Overall, the results underscore a key physiological role for MR signalling in ray-finned fishes, and we propose that the regulation of the highly conserved stress axis as the original function of this receptor.
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