Hindbrain

Strong labeling was observed in the most rostral region of cerebellum, with scattered neurons largely diffused in the lateral nucleus of cerebellar valvula (Figure 7a). In the most caudal part of the inferior lobe of hypothalamus, probe signal was seen in numerous small neurons of the diffuse nucleus and in large neurons of the central nucleus (Figure 7b).

NT-6 mRNA was moderately localized in neurons of Purkinje layer of the lateral region of the cerebellar valvula (Figure 7a). Positive neurons in the Purkinje layer were also observed in the ventro-lateral and ventro-ventral subdivisions of cerebellar body (Figure 7c,d). The positive neurons were identified as Purkinje cells by double labeling with Parvalbumin (Figure 8a–b2). Strong labeling was seen also in elongated cells of the dorsal cerebellar subdivision (Figure 7c,d). Few positive neurons were labeled in the cerebellar crista (Figure 7c). In medulla oblongata, the expression pattern was seen in scattered neurons of octavolateral area, and in neurons of superior (Figure 7c) and intermediate reticular formation. Sense probe staining is shown in Figure 7e,f.

**Figure 7.** Expression of NT-6 mRNA in transverse section of hindbrain of adult *N. furzeri.* On the left side, schematic drawings of *N. furzeri* brain, with red dots indicating NT-6 mRNA distribution over the different brain areas. (**a**) Intense staining in the cells of caudal part of PGZ, and along the margin between OT and TL. Positivity in neurons of Purkinje layer of Va and in neurons of LV. (**b**) Positive neurons widespread over the central nucleus and dorsal hypothalamus of the most caudal part of DIL. (**c**) Sense probe staining in the cerebellar crista. (**d**) Intense staining in neurons of Purkinje, in neurons of RS. (**e**) Sense probe staining in the OT and CCe. (**f**) Intense staining labeling in the most upper portion of CCe (arrow) and in neurons of Purkinje layer of CCe. Scale bars: (**a**,**b**,**d**) 50 μm; and (**c**,**e**,**f**) 100 μm. Abbreviations: CCe, corpus of cerebellum; cc, cerebellar crista, DIL, diffuse inferior lobe of hypothalamus; LV, nucleus of lateral valvula; OT, optic tectum; PGZ, periventricular grey zone; RS, superior reticular formation; TL, longitudinal tori; Va, valvula of cerebellum.

**Figure 8.** Immunohistochemical characterization of NT-6 mRNA expression in Purkinje cells in sagittal section of cerebellum. (**a**,**a1**,**a2**) single images and merge of NT-6 mRNA and Parvalbumin positive cells, showing co-staining in the gl and in Purkinje cells (arrow). (**b**,**b1**,**b2**) higher magnification of single images and merge of NT-6 mRNA and Parvalbumin positive cells showing co-staining in the gl and in Purkinje cells (arrow). Scale bar: a = 50 μm; b = 25 μm. Abbreviation: gl, granular layer; ml, molecular layer.

#### **4. Discussion**

The accumulated evidence documents an age-associated dysregulation of neurotrophins in the brain of mammals [38,39]. This is the first study reporting both the age-related expression of a neurotrophin in a non-mammalian vertebrate, and a comprehensive description of NT-6 mRNA in the adult brain of a fish species.

NT-6 has been identified in very few fish species: *Xiphophorus maculatus* [21], *Danio rerio* (zebrafish) [22], and *Cyprinus carpio* [23]. Phylogenetic analysis on neurotrophins, carried out on mature amino acid sequences [40,41], supports the hypothesis that during chordate/vertebrate lineage two rounds of duplication events of an ancestral neurotrophin gene occurred. Studies have shown that the genomic organization and transcript structure of NGF and NT-6 in the teleost zebrafish share a high similarity with the mouse NGF [24] and suggest that teleost NT-6 has evolved from a common ancestor after a single "fish specific" duplication of NGF [40,41]. Our phylogenetic studies on nucleotides sequences further confirm the hypothesis of NT-6 originated from NGF/NT-3 ancestors. However, the role of NT-6 needs to be clarified in teleosts fish: despite the degree of functional overlapping among the different neurotrophins, individual neurotrophins display a specific activity [42,43].

According to the few data available in literature, we hypothesize that this fish-specific neurotrophin might have a peculiar function among different fish species. Indeed, NT-6 persists in the brain of *N. furzeri* and the closest relative *Xiphophorus* [21] beyond early stages, whereas in zebrafish its expression is strictly linked to the embryonic stages [25]. The observation that NT-6 mRNA expression displayed comparable levels in the brain of young and old animals suggests us that, in our model species, this molecule plays a role in brain development as well as in its maintenance in adults. However, we cannot exclude that the expression levels, despite appearing very similar, can derive from a modulation of the NT-6 neuronal synthesis. For instance, it could be possible that few neurons express high levels of NT-6, or at the same time, that a high number of neurons express low levels of NT-6, in an age-dependent manner. Most interestingly, the synthesis of NT-6 in the aged brain could be a consequence of microglia activation, as compensatory mechanism of physiological aging process [44]. Further experiments are necessary to test these hypotheses and thus to better understand the role of neurotrophins in aging process. This is the first time we explored the age regulation of a neurotrophin in the brain of *N. furzeri*, while our previous studies had been addressed to investigate the morphological distribution of neurotrophins and their receptors in the adult brain.

Herein, we also provide a complete neuroanatomical description of NT-6 mRNA in the brain of *N. furzeri*. In this respect, we employed two different digoxigenin modified probes: an LNA probe and a riboprobe. LNA probes are generally used to detect short DNA oligonucleotides for microRNA and mRNA detection [30,45–48]. LNA containing DNA probes have been previously employed for in situ hybridization detection of mRNAs [45–48] in whole mount embryos of chicken and on tissue sections in *N. furzeri* [2]. The LNA probes revealed an enhanced hybridization efficiency, hybridization specificity and duplex stability [49]. Remarkably, our in situ experiments showed an overlapping neuroanatomical distribution for both probes.

Briefly, our results demonstrate that NT-6 mRNA is expressed in the forebrain (dorsal and ventral telencephalon, and in several diencephalic nuclei), in the midbrain (optic tectum, longitudinal tori, semicircular tori), and in the hindbrain (valvula and body of cerebellum, reticular formation and octavolateral area of medulla oblongata). NT-6 mRNA has been documented during the developing stages of *Xiphophorus* [21] and zebrafish [25], respectively, expressed in the valvula cerebelli and optic vescicle. In adult *Xiphophorus*, although NT-6 is expressed, a neuroanatomical description has not been reported yet [21]. Overall, our findings document a wide NT-6 mRNA localization throughout the whole brain of *N. furzeri*. In addition, other neurotrophins (NGF, BDNF and NT-4), at either mRNAs or protein level, were observed in the adult brain of *N. furzeri* [48–50]. Remarkably, NT-6 mRNA is expressed in mature neurons, similar to other neurotrophins, such as BDNF, NGF, and NT-4 [50–52], which have been already documented in the brain of this model species. Most interestingly, neurotrophins display a peculiar neuronal expression also in zebrafish and the European eel [35,53–55]. These observations reinforce the hypothesis that the expression of neurotrophins in teleost fish is primarily linked to mature neurons. In adult mammalian brain, the neuronal mRNA levels of NGF and BDNF are tightly regulated by neural activity and influence the modulation of several key events such as synthesis, metabolism and release of neurotransmitters, postsynaptic ion channel fluxes, neuronal firing rates as well as long-term synaptic potentiation of neurons [56]. In this context, further studies are mandatory to explore the evolutionary conserved neuronal role of neurotrophins in the brain of fish species.

In conclusion, our findings document: (1) the identification and molecular characterization of NT-6 coding sequence of *N. furzeri* (NfuNT-6) and the nucleotide degree of conservation in *Xiphophorus*, *D. rerio* and mammalian NGF and BDNF; (2) the efficiency of using a sensitive LNA probe to detect NT-6 mRNA; (3) the unchanged expression levels of NT-6 in the brain of young and old animals; and (4) the expression of NT-6 mRNA in mature neurons of forebrain, midbrain and hindbrain in *N. furzeri*. These results provide a basis for future research on evolutionary function of neurotrophins, which are currently perceived as one of the primary factors underlying the complexity of vertebrate nervous systems. Therefore, their involvement in higher brain functions and aging is undoubtedly a relevant topic.

Further experimental work is noticeably needed both to characterize more in-depth NT-6 in this model and to confirm its importance in brain development and architecture. In addition, functional studies are required to explore and feature the potential role played by NT-6 in aging.

**Author Contributions:** Conceptualization, C.A. and A.C.; Data curation, A.P.; Funding acquisition, P.d.G.; Methodology, A.L.; Software, E.T.T.; Writing—original draft, L.D.A.; and Writing—review and editing, C.L. and M.P.

**Funding:** The project was supported by University of Naples Federico II (DR/2017/409–Project F.I.A.T.).

**Acknowledgments:** The authors are thankful to Antonio Calamo for technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.
