3.2.1. NfuNT-6 mRNA Expression in Young versus Old Animals

We analyzed NfuNT-6 mRNA levels, by qPCR analysis, in brain homogenates of 5 and 27 wph animals. Comparable levels of NfuNT-6 mRNA were found in the brains of young and old animals (*p*-value: 0.70344) (Figure 2). For each time point, NfuNT-6 mRNA was normalized to the reference gene (TBP) and expression levels were compared using the relative delta curve threshold (ΔΔCT) method (*p*-value5wph = 0.00387; *p*-value27wph = 0.001085).

**Figure 2.** Expression levels of NfuNT-6 mRNA in the brain of young and old *N. furzeri*. Comparable expression levels of NfuNT-6 mRNA in the whole brain of young and old animals (*p*-value: 0.70344). For 5 and 27 wph, NfuNT-6 mRNA was normalized to TBP and expression levels were compared using ΔΔCT method (*p*-value5wph = 0.00387; *p*-value27wph = 0.001085).

3.2.2. Neuroanatomical Expression of NT-6 mRNA

LNA and riboprobes (Figure 3a,b) were used to localize the expression of NT-6 mRNA and revealed overlapping distribution patterns. No specific hybridization signal was observed in sections hybridized with the mismatch probe (Figure 3c). Due to overlapping pattern of expression of riboprobe and LNA, the results refer to NT-6 mRNA. The nomenclature follows the *N. furzeri* brain atlas [30]. Recognition of labeled neurons and/or glial cells was based on morphological criteria and by means of different markers: S100β [9,33], HuC/HuD [34] and MAP2 [35]. Purkinje neurons were identified by using parvalbumin as marker.

**Figure 3.** Comparison of riboprobe and LNA and negative control: (**a**) LNA probe and (**b**) Riboprobe to detect NT-6 mRNA expression (red) in the OT, at the margin with TL; and (**c**) mismatch LNA probe in the OT not revealing staining. Scale bar: (**a**,**c**) 25 μm; and (**b**) 12 μm. Abbreviations: PGZ, periventricular grey zone.

#### 3.2.3. NT-6 mRNA Expression in Mature Neurons

Before analyzing the pattern of expression of NT-6 over different brain areas, we combined in situ hybridization with immunohistochemistry to identify the phenotype of NT-6 mRNA expressing cells. We conducted the experiment on serial sections of the optic tectum, and we employed markers to identify glial and neuronal populations. S100β was used as marker of glial cells [9,33], HuC/HuD as marker of early-differentiated neurons [34,35], and MAP2 as marker of mature neurons [36,37]. NT-6 mRNA was not expressed in S100β immunoreactive cells (Figure 4a,a1,a2) or in HuC/HuD cells (Figure 4b,b1,b2). NT-6 mRNA signal probe was observed in MAP2 immunoreactive cells in the most posterior part of periventricular grey zone of the optic tectum and in some scarce cells in the most superficial layers of the same brain area (Figure 4c,c1,c2).

**Figure 4.** Immunohistochemical characterization of NT-6 mRNA expressing cells in transverse section of optic tectum: (**a**,**a1**,**a2**) single images and merge of NT-6 mRNA and S100β positive cells, showing any co-staining in the rostral part of optic tectum; (**b**,**b1**,**b2**) single images and merge of NT-6 mRNA and HuC/HuD showing any co-staining in the rostral part of optic tectum; and (**c**,**c1**,**c2**) single images and merge of NT-6 mRNA and MAP2 showing that NT-6 is expressed in numerous MAP2 immunopositive. Scale bar: (**a**,**c**) 50 μm; and (**b**) 25 μm. Abbreviations: PGZ, periventricular grey zone.
