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Adult neurogenesis is a highly dynamic process, decreasing exponentially with aging in many vertebrate species. In mammalian brain, it is restricted to the subventricular and subgranular zones, while in teleost fish it occurs in approximately sixteen regions distributed along the entire rostro-caudal brain axis. Among teleost fish, Nothobranchius furzeri is a well-established model for aging studies, due to its very short lifespan together with anatomical and physiological aging features. In vertebrates, Col4a1 is mainly located in brain ventricular areas, is involved in axonal growth and guidance, and participates in nerve repair; Col25a1 is specifically expressed in neurons and encodes for a protein named collagenous Alzheimer amyloid plaque component (CLAC), which binds to Alzheimer plaques reducing Aβ growth. Id3 is another gene required for embryonic neurogenesis and angiogenesis, and it persists in adult brain, where it regulates adult neural stem cells maintenance and function. Quantitative and qualitative analysis of Col4a1, Col25a1 and Id3 was assessed in N. furzeri young (5 weeks post hatching (wph)) and old (27 wph) brains, showing all the three genes are upregulated during aging and are expressed in recognizable neurogenic areas. Furthermore, double labeling of fluorescence in situ hybridization (FISH), for genes' mRNAs, and immunofluorescence (IF), for PCNA, confirms that Col4a1, Col25a1 and Id3 are expressed in numerous PCNA containing cells, confirming that their expression in nerogenic areas is related to cells proliferation and / or differentiation. The phenotype of Col4a1, Col25a1 and Id3 expressing cells was assessed by FISH/IF double labeling with neural markers S100, DCX, and HuC/D. Our data confirm that, as for other vertebrates, also in N. furzeri Col4a1, Col25a1 and Id3 are involved in adult neurogenesis. Furthermore, they colocalize mainly with DCX and HuC/D expressing cells, suggesting that they are involved in proliferation and differentiation of neural cells more than radial glial ones. These data lay the foundations to further studies on the pathways regulating Col4a1, Col25a1 and Id3, to better understand their specific role in neural proliferation and differentiation pathways and identifying possible interactors.

Inhibitors of DNA (Id) are key transcription factors (TFs) regulating neurogenic processes. They belong to the helix-loop-helix (HLH) TF family and are dominant negative regulators of basic HLH proteins (bHLHs). Specifically, they inhibit cell differentiation and enhance cell proliferation and motility. The Id family includes four members, Id1, Id2, Id3, and Id4, which have been identified in nearly all vertebrates. The transcript catalog of the African turquoise killifish, Nothobranchius furzeri, contains all four TFs and has evolved showing positive selection for Id3. N. furzeri, a teleost, is the short-lived vertebrate and is gaining increasing scientific interest as a new model organism in aging research. It is characterized by embryonic diapause, explosive sexual maturation, and rapid aging. In this study, we investigated both the expression and the role of Id3 in the brain of this model organism. Interestingly, Id3 was upregulated age-dependently along with a distribution pattern resembling that of other vertebrates. Additionally, the gene has undergone positive selection during evolution and shows a high degree of conservation relative to that of other vertebrates. These features make N. furzeri a valid tool for aging studies and a potential model in translational research.

Nesfatin-1 (Nesf-1) was identified as an anorexigenic and well conserved molecule in rodents and fish. While tissue distribution of NUCB2 (Nucleobindin 2)/Nesf-1 is discretely known in vertebrates, reports on ontogenetic expression are scarce. Here, we examine the age-related central and peripheral expression of NUCB2/Nesf-1 in the teleost African turquoise killifish Nothobranchius furzeri, a consolidated model organism for aging research. We focused our analysis on brain areas responsible for the regulation of food intake and the rostral intestinal bulb, which is analogous of the mammalian stomach. We hypothesize that in our model, the stomach equivalent structure is the main source of NUCB2 mRNA, displaying higher expression levels than those observed in the brain, mainly during aging. Remarkably, its expression significantly increased in the rostral intestinal bulb compared to the brain, which is likely due to the typical anorexia of aging. When analyzing the pattern of expression, we confirmed the distribution in diencephalic areas involved in food intake regulation at all age stages. Interestingly, in the rostral bulb, NUCB2 mRNA was localized in the lining epithelium of young and old animals, while Nesf-1 immunoreactive cells were distributed in the submucosae. Taken together, our results represent a useful basis for gaining deeper knowledge regarding the mechanisms that regulate food intake during vertebrate aging.

Neurotrophins contribute to the complexity of vertebrate nervous system, being involved in cognition and memory. Abnormalities associated with neurotrophin synthesis may lead to neuropathies, neurodegenerative disorders and age-associated cognitive decline. The genome of teleost fishes contains homologs of some mammalian neurotrophins as well as a gene coding for an additional neurotrophin (NT-6). In this study, we characterized this specific neurotrophin in the short-lived fish Nothobranchius furzeri, a relatively new model for aging studies. Thus, we report herein for the first time the age-related expression of a neurotrophin in a non-mammalian vertebrate. Interestingly, we found comparable expression levels of NT-6 in the brain of both young and old animals. More in detail, we used a locked nucleic acid probe and a riboprobe to investigate the neuroanatomical distribution of NT-6 mRNA revealing a significant expression of the neurotrophin in neurons of the forebrain (olfactory bulbs, dorsal and ventral telencephalon, and several diencephalic nuclei), midbrain (optic tectum, longitudinal tori, and semicircular tori), and hindbrain (valvula and body of cerebellum, reticular formation and octavolateral area of medulla oblongata). By combining in situ hybridization and immunohistochemistry, we showed that NT-6 mRNA is synthesized in mature neurons. These results contribute to better understanding the evolutionary history of neurotrophins in vertebrates, and their role in the adult brain.

In our previous study, the presence and distribution of nerve growth factor (NGF) were described in adult zebrafish brain [1]. In mammals, the signaling of mature NGF (mNGF) is transduced by the tropomyosin receptor kinase A (TrkA) and acts as mediator in many essential functions in the central nervous system. Since TrkA expression was described in developing zebrafish [2], the aim of the present study is to describe the pattern of distribution of TrkA mRNA in the brain of adult zebrafish. The investigation was conducted on brain fixed in paraformaldehyde. Sections were firstly incubated overnight with digoxigenin-labeled riboprobes for TrkA [2] and then with anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. The chromogenic reaction was carried out by using Fast Red substrate. After counterstaining with DAPI, sections were visualized in fluorescence microscopy. In the telencephalon, TrkA mRNA was found in the olfactory bulbs, specifically in the glomerular layer and in the external cellular layer. In addition, some positive round small neurons were seen in the regions of the dorsal area and rare positive neurons in the ventral area. In the diencephalon, TrkA mRNA was seen in some neurons of the hypothalamus, in few neurons of mammillary body and synencephalon. In the mesencephalon, TrkA mRNA was detected in neurons of the optic tectum, specifically in the periventricular grey zone and superficial grey and white zone, and in neurons of the semicircular tori. Concerning rhombencephalon, TrkA mRNA was highly present in neurons of all regions of the cerebellum, and in neurons of cerebellar crest. Our study highlighted the wide expression of TrkA receptor throughout all brain regions of adult zebrafish. In mammals, this receptor mediates the action of mNGF [3]. However, in zebrafish brain extracts, only the immature form of NGF (pro NGF) was found. These findings suggest a different NGF signaling in zebrafish or the presence of low and fleeting undetectable quantities of mNGF. Cacialli P et al. Nerve growth factor is expressed and stored in central neurons of adult zebrafish. J Anat. Apr 4. doi: 10.1111/joa.12986, 2019. [2] Nittoli V. et al. A comprehensive analysis of neurotrophins and neurotrophin tyrosine kinase receptors expression during development of zebrafish. J Comp Neurol, 526:1057–1073, 2018. [3] Bucci C et al. The Role of Rab Proteins in Neuronal Cells and in the Trafficking of Neurotrophin Receptors. Membranes 4(4), 642-677; 2014.

Nerve growth factor (NGF) receptors are evolutionary conserved molecules, and in mammals are considered necessary for ensuring the survival of cholinergic neurons. The age-dependent regulation of NTRK1/NTRKA and p75/NGFR in mammalian brain results in a reduced response of the cholinergic neurons to neurotrophic factors and is thought to play a role in the pathogenesis of neurodegenerative diseases. Here, we study the age-dependent expression of NGF receptors (NTRK1/NTRKA and p75/NGFR) in the brain of the short-lived teleost fish Nothobranchius furzeri. We observed that NTRK1/NTRKA is more expressed than p75/NGFR in young and old animals, although both receptors do not show a significant age-dependent change. We then study the neuroanatomical organization of the cholinergic system, observing that cholinergic fibers project over the entire neuroaxis while cholinergic neurons appear restricted to few nuclei situated in the equivalent of mammalian subpallium, preoptic area and rostral reticular formation. Finally, our experiments do not confirm that NTRK1/NTRKA and p75/NGFR are expressed in cholinergic neuronal populations in the adult brain of N. furzeri. To our knowledge, this is the first study where NGF receptors have been analyzed in relation to the cholinergic system in a fish species along with their age-dependent modulation. We observed differences between mammals and fish, which make the African turquoise killifish an attractive model to further investigate the fish specific NGF receptors regulation.

Nesfatin-1 (Nesf-1) is an anorexigenic peptide involved in the regulation of homeostatic feeding. Nesf-1 is expressed in the central nervous system and other organs, including pancreas, where it promotes the release of insulin from β-cells. This raises the possibility that Nesf-1 dysfunction could be involved in metabolic disorders, particularly in type 2 diabetes mellitus (T2D). Recently, it has been discovered that dolphins can be a natural animal model that fully replicates human T2D, due to its prolonged glucose tolerance curve and maintenance of a state of hyperglycemia similar to human T2D during fasting. This correspondence suggests that dolphins may be a suitable model for investigating physiological and pathological metabolic disorders. Here, we have characterized Nesf-1 distribution in the pancreas of the common bottlenose dolphin (Tursiops truncatus) and measured plasmatic levels of Nesf-1 and glucose during fasting and post-prandial states. The Mediterranean Marine Mammal Tissue Bank (MMMTB) of the University of Padova provided us with pancreas samples, derived from four animals, and plasma samples, collected before and after the main meal. Interestingly, our results showed that Nesf-1-immunoreactive cells were distributed in Langerhans islets, co-localized with glucagon in α-cells. Similar to humans, dolphin plasma Nesf-1 concentration doesn’t show a statistically significant difference when comparing fasting and post-prandial states. On the other hand, blood glucose levels were significantly higher before than after the main meal. Our data provide a comparative analysis for further studies on the involvement of Nesf-1 in mammalian metabolic disorders.

RBFOX1 is a highly pleiotropic gene that contributes to several psychiatric and neurodevelopmental disorders. Both rare and common variants in RBFOX1 have been associated with several psychiatric conditions, but the mechanisms underlying the pleiotropic effects of RBFOX1 are not yet understood. Here we found that, in zebrafish, rbfox1 is expressed in spinal cord, mid- and hindbrain during developmental stages. In adults, expression is restricted to specific areas of the brain, including telencephalic and diencephalic regions with an important role in receiving and processing sensory information and in directing behaviour. To investigate the effect of rbfox1 deficiency on behaviour, we used rbfox1 sa15940 , a rbfox1 loss-of-function line. We found that rbfox1 sa15940 mutants present hyperactivity, thigmotaxis, decreased freezing behaviour and altered social behaviour. We repeated these behavioural tests in a second rbfox1 loss-of-function line with a different genetic background, rbfox1 del19 , and found that rbfox1 deficiency affects behaviour similarly in this line, although there were some differences. rbfox1 del19 mutants present similar thigmotaxis, but stronger alterations in social behaviour and lower levels of hyperactivity than rbfox1 sa15940 fish. Taken together, these results suggest that rbfox1 deficiency leads to multiple behavioural changes in zebrafish that might be modulated by environmental, epigenetic and genetic background effects, and that resemble phenotypic alterations present in Rbfox1 -deficient mice and in patients with different psychiatric conditions. Our study thus highlights the evolutionary conservation of rbfox1 function in behaviour and paves the way to further investigate the mechanisms underlying rbfox1 pleiotropy on the onset of neurodevelopmental and psychiatric disorders., Competing Interests: CONFLICT OF INTEREST The authors declare no conflict of interest.