Intracardial perfusion of the African turquoise killifish v1

This perfusion protocol is essential for preserving tissue morphology in order to perform good quality immunohistochemical stainings. Here, we show you how we perform our perfusions on the African turquoise killifish. This protocol was already used in the following publications: Aging impairs the essential contributions of non-glial progenitors to neurorepair in the dorsal telencephalon of the Killifish Nothobranchius furzeri - PubMed (nih.gov) Single-cell sequencing of the adult killifish (N. furzeri) brain identifies an atypical progenitor, glial and neuronal heterogeneity | bioRxiv

Hormonal Regulation of Diapause and Development in Nematodes, Insects, and Fishes

Diapause is a state of developmental arrest adopted in response to or in anticipation of environmental conditions that are unfavorable for growth. In many cases, diapause is facultative, such that animals may undergo either a diapause or a non-diapause developmental trajectory, depending on environmental cues. Diapause is characterized by enhanced stress resistance, reduced metabolism, and increased longevity. The ability to postpone reproduction until suitable conditions are found is important to the survival of many animals, and both vertebrate and invertebrate species can undergo diapause. The decision to enter diapause occurs at the level of the whole animal, and thus hormonal signaling pathways are common regulators of the diapause decision. Unlike other types of developmental arrest, diapause is programmed, such that the diapause developmental trajectory includes a pre-diapause preparatory phase, diapause itself, recovery from diapause, and post-diapause development. Therefore, developmental pathways are profoundly affected by diapause. Here, I review two conserved hormonal pathways, insulin/IGF signaling (IIS) and nuclear hormone receptor signaling (NHR), and their role in regulating diapause across three animal phyla. Specifically, the species reviewed are Austrofundulus limnaeus and Nothobranchius furzeri annual killifishes, Caenorhabditis elegans nematodes, and insect species including Drosophila melanogaster, Culex pipiens, and Bombyx mori. In addition, the developmental changes that occur as a result of diapause are discussed, with a focus on how IIS and NHR pathways interact with core developmental pathways in C. elegans larvae that undergo diapause.

Age-Related Alterations in the Behavior and Serotonin-Related Gene mRNA Levels in the Brain of Males and Females of Short-Lived Turquoise Killifish (Nothobranchius furzeri)

Short-lived turquoise killifish (Nothobranchius furzeri) have become a popular model organism for neuroscience. In the present paper we study for the first time their behavior in the novel tank diving test and the levels of mRNA of various 5-HT-related genes in brains of 2-, 4- and 6-month-old males and females of N. furzeri. The marked effect of age on body mass, locomotor activity and the mRNA level of Tph1b, Tph2, Slc6a4b, Mao, Htr1aa, Htr2a, Htr3a, Htr3b, Htr4, Htr6 genes in the brains of N. furzeri males was shown. Locomotor activity and expression of the Mao gene increased, while expression of Tph1b, Tph2, Slc6a4b, Htr1aa, Htr2a, Htr3a, Htr3b, Htr4, Htr6 genes decreased in 6-month-old killifish. Significant effects of sex on body mass as well as on mRNA level of Tph1a, Tph1b, Tph2, Slc6a4b, Htr1aa, 5-HT2a, Htr3a, Htr3b, Htr4, and Htr6 genes were revealed: in general both the body mass and the expression of these genes were higher in males. N. furzeri is a suitable model with which to study the fundamental problems of age-related alterations in various mRNA levels related with the brains 5-HT system.

Organization of the Catecholaminergic System in the Short-Lived Fish Nothobranchius furzeri

The catecholaminergic system has received much attention based on its regulatory role in a wide range of brain functions and its relevance in aging and neurodegenerative diseases. In the present study, we analyzed the neuroanatomical distribution of catecholaminergic neurons based on tyrosine hydroxylase (TH) immunoreactivity in the brain of adult Nothobranchius furzeri. In the telencephalon, numerous TH+ neurons were observed in the olfactory bulbs and the ventral telencephalic area, arranged as strips extending through the rostrocaudal axis. We found the largest TH+ groups in the diencephalon at the preoptic region level, the ventral thalamus, the pretectal region, the posterior tuberculum, and the caudal hypothalamus. In the dorsal mesencephalic tegmentum, we identified a particular catecholaminergic group. The rostral rhombencephalon housed TH+ cells in the locus coeruleus and the medulla oblongata, distributing in a region dorsal to the inferior reticular formation, the vagal lobe, and the area postrema. Finally, scattered TH+ neurons were present in the ventral spinal cord and the retina. From a comparative perspective, the overall organization of catecholaminergic neurons is consistent with the general pattern reported for other teleosts. However, N. furzeri shows some particular features, including the presence of catecholaminergic cells in the midbrain. This work provides a detailed neuroanatomical map of the catecholaminergic system of N. furzeri, a powerful aging model, also contributing to the phylogenetic understanding of one of the most ancient neurochemical systems.

Mass spectrometric characterization of cyclic dinucleotides (CDNs) in vivo

Cyclic dinucleotides (CDNs) are key secondary messenger molecules produced by cyclic dinucleotide synthases that trigger various cellular signaling cascades from bacteria to vertebrates. In mammals, cyclic GMP-AMP synthase (cGAS) has been shown to bind to intracellular DNA and catalyze the production of the dinucleotide 2′3′ cGAMP, which signals downstream effectors to regulate immune function, interferon signaling, and the antiviral response. Despite the importance of CDNs, sensitive and accurate methods to measure their levels in vivo are lacking. Here, we report a novel LC-MS/MS method to quantify CDNs in vivo. We characterized the mass spectrometric behavior of four different biologically relevant CDNs (c-di-AMP, c-di-GMP, 3′3′ cGAMP, 2′3′ cGAMP) and provided a means of visually representing fragmentation resulting from collision-induced dissociation at different energies using collision energy breakdown graphs. We then validated the method and quantified CDNs in two in vivo systems, the bacteria Escherichia coli OP50 and the killifish Nothobranchius furzeri. We found that optimization of LC-MS/MS parameters is crucial to sensitivity and accuracy. These technical advances should help illuminate physiological and pathological roles of these CDNs in in vivo settings.

Sperm cryopreservation and in vitro fertilization techniques for the African turquoise killifish Nothobranchius furzeri

Over the last decade, the African turquoise killifish, Nothobranchius furzeri, has emerged as an important model system for the study of vertebrate biology and ageing. Propagation of laboratory inbred strains of Nothobranchius furzeri, such as GRZ, however, can pose challenges due to the short window of fertility, the efforts and space requirements involved in continuous strain maintenance, and the risks of further inbreeding. The current method for long term strain preservation relies on arrest of embryos in diapause. To create an alternative for long term maintenance, we developed a robust protocol to cryopreserve and revive sperm for in vitro fertilization (IVF). We tested a variety of extender and activator buffers for sperm IVF, as well as cryoprotectants to achieve practical long-term storage and fertilization conditions tailored to this species. Our protocol enabled sperm to be preserved in a cryogenic condition for months and to be revived with an average of 40% viability upon thawing. Thawed sperm were able to fertilize nearly the same number of eggs as natural fertilization, with an average of ~ 25% and peaks of ~ 55% fertilization. This technical advance will greatly facilitate the use of N. furzeri as a model organism.

Age-Dependent Regulation of Notch Family Members in the Neuronal Stem Cell Niches of the Short-Lived Killifish Nothobranchius furzeri

Background: The annual killifish Nothobranchius furzeri is a new experimental model organism in biology, since it represents the vertebrate species with the shortest captive life span and also shows the fastest maturation and senescence recorded in the laboratory. Here, we use this model to investigate the age-dependent decay of neurogenesis in the telencephalon (brain region sharing the same embryonic origin with the mammalian adult niches), focusing on the expression of the Notch pathway genes.Results: We observed that the major ligands/receptors of the pathway showed a negative correlation with age, indicating age-dependent downregulation of the Notch pathway. Moreover, expression of notch1a was clearly limited to active neurogenic niches and declined during aging, without changing its regional patterning. Expression of notch3 is not visibly influenced by aging.Conclusion: Both expression pattern and regulation differ between notch1a and notch3, with the former being limited to mitotically active regions and reduced by aging and the latter being present in all cells with a neurogenic potential, regardless of the level of their actual mitotic activity, and so is less influenced by age. This finally suggests a possible differential role of the two receptors in the regulation of the niche proliferative potential throughout the entire fish life.