3.3.1. In Situ Hybridization

In the diencephalon of young fish, NUCB2B mRNA was detected in neurons of the cortical nucleus (Figure 3E–G). Positive neurons were displayed in the ventro-medial thalamic nucleus (Figures 3E–H and 4C), in the paraventricular organ (Figure 3G–J), and in the periventricular nucleus of the posterior tubercle (Figures 3G–J and 5C–E) along the ventricle. Labelled neurons were found in the dorsal (Figures 3I,J and 4C), central, and ventral part of the hypothalamus (Figure 3G–J), close to the ventricle. Small and intensely positive neurons were detected in the diffuse inferior lobe of the hypothalamus (Figures 3L,M and 4E). In the diencephalon of old fish, some positive neurons were detected in the periventricular nucleus of the posterior tuberculum. Many packed positive neurons were observed in the hypothalamic region, particularly in the dorsal, ventral, lateral, and caudal parts (Figure 5A). Moreover, many neurons were positively stained in the nucleus of posterior recess (Figure 5C–E), unlike in the young subjects.

In addition, in young fish, positive neurons were detected in non-diencephalic areas: along the margin of the dorsal telencephalon (Figures 3A–C and 4A) and in the periventricular grey zone of the optic tectum (Figures 3G and 6A). Conversely, in old fish, only scarcely positive neurons were detected along the margin of the dorsal telencephalon (Figures 5E and 6C).

**Figure 3.** Atlas of NUCB2B gene and Nesf-1 protein distribution: schematic drawings of transversal sections of *N. furzeri* brain [46], specifically referred to (**A**–**F**) forebrain; (**G**–**M**) midbrain. White triangles indicate sites of mRNA positive neurons (ISH); black triangles indicate sites of immunopositive neurons (IHC); small dots indicate immunoreactive fibres (IHC). Scale bar: 200 μm.

3.3.2. Immunohistochemistry

In young fish, immunoreactivity to Nesf-1 protein was found in the ventral telencephalon and diencephalon. In the telencephalic ventral areas, especially in the supracommissural zone, abundant positive ir-fibers were observed (Figures 3C and 4B). In the diencephalon, positive neurons were detected in the magno- and parvo-cellular parts of the preoptic nucleus (Figure 3D–F). Also, ir-neurons enveloped in a tight net of projections were visible in the cortical nucleus (Figure 3E–G), preglomerular nucleus, mainly in its medial part (Figure 3G), pretectal nucleus, as well as in the paraventricular organ (Figure 3G–J). Numerous immune-labelled neurons, with a notable quantity of fibers surrounding, was identified in the ventral, dorsal, and caudal parts of the tuberal hypothalamus. Several stained neurons and fibers were recognized in the thalamic nucleus, both dorsal posterior and ventro-medial parts (Figures 3G–J and 4D). Ir-perikarya were located around the margin of the periventricular nucleus of the posterior tuberculum (Figure 3E–G). Many ir-projections were found in the medial part of the glomerular nucleus (Figure 3J,K). Positive ir-neurons were disseminated in the diffuse inferior lobe of the hypothalamus, especially on the external margin (Figures 3K–M and 4F), with some fibers spread in the medial zone. In non-diencephalic regions, several ir-positive neurons were detected in the optic tectum (Figure 6B), especially packed in the periglomerular grey zone (Figure 6B), with projections towards the external margin (Figure 3G–M). Moreover, weakly ir-neuronal cells were widespread in the different layers of semicircular tori (Figure 3J,K).

**Figure 4.** Transversal section showing localization of NUCB2B mRNA and Nesf-1 protein in the young brain of *N. furzeri*. (**A**) NUCB2B expressing neurons in Dc, Dld, Dll, Dlv, Dm, PPa, Vp, and Vs; (**B**) Nesf-1 immunoreactivity (ir) in neurons of Dm, DIL, PPa, Vp, and Vs; (**C**) NUCB2B expressing neurons in VM and Hd; (**D**) Nesf-1 ir in neurons of VM and Hd; (**E**) NUCB2B expressing neurons in DIL; (**F**) Nesf-1 ir in neurons of DIL. Scale bars: A-B-F 100 μm; C-E 200 μm; D 300 μm.

Nesf-1 protein distribution did not show notable differences between the young and elderly subjects (Figures 5B,D,F and 6D).

**Figure 5.** Transversal section showing localization of NUCB2B mRNA and Nesf-1 protein in the brain of old *N. furzeri*. (**A**) NUCB2B expressing neurons in HI and Hv; (**B**) Nesf-1 immunoreactivity (ir) in neurons of HI and Hv; (**C**) NUCB2B expressing neurons in TNp and NRP; (**D**) Nesf-1 ir in neurons of NRP and TNp; (**E**) NUCB2B expressing neurons in DIL, NG, NRP, PGZ, and TNp; (**F**) Nesf-1 ir-neurons of DIL and NRP. Scale bars: A-C-E 200 μm, B-D-F 100 μm.

**Figure 6.** Transversal section showing localization of NUCB2B mRNA and Nesf-1 protein in non-diencephalic regions of *N. furzeri* brain. (**A**,**B**) young fish: (**A**) NUCB2B expressing neurons in PGZ; (**B**) Nesf-1 immunoreactivity (ir) in neurons of PGZ and OT; (**C**,**D**) old fish: (**C**) NUCB2B expressing neurons in PGZ and TS; (**D**) Nesf-1 ir-neurons of PGZ. Scale bars: 200 μm.
