Comparison of expression patterns of tac1 mRNA in the forebrain regions of male (A−D) and female (A’−D’) zebrafish. In the top panel, a schematic drawing of an adult zebrafish brain (lateral view) showing levels of cross-sections presenting in Figures 1−3. Alphabets represent respective figure numbers (A−D: Figure 1, E−H: Figure 2, and I−L: Figure 3). In the most-right panels (A”−D”), a schematic drawing of half of the brain section (left side) processed for tac1 staining indicating major brain structures at this level. There was a sexually dimorphic expression of tac1 in the anterior part of the parvocellular preoptic nucleus (A, A’), posterior part of the parvocellular preoptic nucleus (B, B’), and suprachiasmatic nucleus (C, C’). In contrast, no sexually dimorphic expression of tac1 was observed in the dorsal habenula (C, C’) and the ventral region of the periventricular hypothalamus (D, D’). Scale bars: (A–C), (A’–C’), 100 µm; (D, D’), 200 µm. See Glossary for abbreviation.

Comparison of expression patterns of tac1 mRNA in the midbrain regions of male (E−H) and female (E’−H’) zebrafish. In the most-right panels (E”−H”), a schematic drawing of half of the brain section (left side) processed for tac1 staining indicating major brain structures at this level. There was no sex difference in expression of tac1 in the diencephalic and mesencephalic regions including the torus semicircularis, dorsal tegmental nucleus, posterior tuberal nucleus, caudal and dorsal zones of the periventricular hypothalamus, nucleus of medial longitudinal fascicle, and oculomotor nucleus. Scale bars: 200 µm. See Glossary for abbreviation.

Comparison of expression patterns of tac1 mRNA in the hindbrain regions of male (I−L) and female (I’−L’) zebrafish. In the most-right panels (I”−L”), a schematic drawing of half of the brain section (left side) processed for tac1 staining indicating major brain structures at this level. There was no sex difference in expression of tac1 in the mesencephalic and rhombencephalic regions including the interpeduncular nucleus, dorsal tegmental nucleus, central gray, and trigeminal motor nucleus, while in the superior raphe, there was more tac1 expression in males as compared to females (J, J’). Scale bars: 200 µm. See glossary for abbreviation.

Sexually dimorphic expression of tac1 mRNA and Tac1 immunoreactivity in the brain. Photomicrographs in the left (A−H), middle (A’−H’) and the right (A”−H”) columns for each gender are tac1 mRNA, Tac1-immunoreactivity and a higher magnification of respective Tac1 immunoreactivities observed in the middle panels, respectively. Bar graphs in the bottom panel (I) represent the number of cells expressing tac1 counted in different brain regions of males (blue columns) and female (red columns) fish. In the anterior part of the parvocellular preoptic nucleus (PPa), there were male-dominant tac1 mRNA expression (A and B) and Tac1 immunoreactivity (A’ and B’). In the posterior part of the parvocellular preoptic nucleus (PPp) and suprachiasmatic nucleus (SC), tac1 mRNA expression was significantly higher in males (C and D), while no apparent sexual dimorphism was observed in Tac1 immunoreactivity (C’ and D’). In the ventral region of the periventricular hypothalamus (Hv), numbers of cells expressing tac1 mRNA were higher in females as compared to males (E, F, and I, P<0.01), while there was no apparent sexual morphism in Tac1 immunoreactivity (E’ and F’). In the superior raphe, more number of cells expressing tac1 mRNA were observed in males (G and G’) as compared to females (H and H’), but there was no significant difference (ns, I). Scale bars: A−H and A’−H’: 50 µm; A”−H”: 20 µm. **P<0.01. See Glossary for abbreviation.

Sexual dimorphisms in Tac1-immunoreactive processes in the telencephalic and diencephalic regions. Left (A−D) and middle (A’−D’) columns represent photomicrographs of Tac1-immunoreactive processes (arrows) in the brain of males and females, respectively. Boxes with a dotted line in the schematic drawing of coronal view in the right column indicate the localization of the boundaries of the respective immunohistochemical illustrations. In the ventral part of the ventral telencephalon, more Tac1-immunoreactive processes are seen in male brains (A−C) as compared to females (A’−C’). Similarly, more Tac1-immunoreactive processes are observed in the habenula nuclei in males as compared to female (D, D’). Scale bars: 20 µm. See Glossary for abbreviation.

Comparison of Tac1-immunoreactive processes in the diencephalic and mesencephalic regions. Left (E−H) and middle (E’−H’) columns represent photomicrographs of Tac1-immunoreactive processes (arrows) in the brain of males and females, respectively. Boxes with a dotted line in the schematic drawing of coronal view in the right column indicate the localization of the boundaries of the respective immunohistochemical illustrations. In some diencephalic nuclei, more Tac1-immunoreactive processes are seen in male brains (E, G) as compared to females (E’, G’). In the mesencephalic nucleus, densities of Tac1-immunoreactive processes were slightly higher in males (F, H) as compared to females (F’, H’). Scale bars: 20 µm. See Glossary for abbreviation.

Neuronal association between Tac1-immunoreactive processes and GnRH3 cell soma in the male and female zebrafish. Photomicrographs for immunofluorescence of GnRH3 (1^st column, A−D), and Tac1 (2^nd column, A’−D’) were merged (3^rd column, A”−D”) to elucidate the possible neuronal association (arrows) between Tac1 and GnRH3 neurons. A representative GnRH3 cell soma accompanied by Tac1 processes was further examined by two-dimensional confocal scanning in XY, XZ, and YZ plane (4^th column, A’”−D’”). Bar graphs in the bottom panel (E) show the percentage of GnRH3 cells accompanied by Tac1 processes in males (blue columns) and females (red columns) in the ventral nucleus of ventral telencephalic area (Vv) and olfactory-terminal nerve (OB-TN) region. Numbers in each columns indicate the number of GnRH3 cells accompanied by Tac1 processes and total GnRH3 numbers counted. In the Vv, more Tac1-immunoreactive processes (magenta) are in proximities of GnRH3 cells (green) in males (A−A’”) as compared to females (C−C’”), but there was no significant difference in the percentage of GnRH3 cells between males and females (E). In the olfactory bulb (OB), OB-GnRH3 neurons are accompanied by Tac1 processes in both males (B−B’”) and females (D−D’”), and there was no significant sex difference in the percentage of GnRH3 cells (E). ns, non-significant. Scale bars: 10 µm.

Neuronal association between Tac1-immunoreactive processes and kiss2 cell soma in the male and female zebrafish. Photomicrographs for mRNA expression of kiss2 (1^st column), and Tac1-immunoreactivity (2^nd column) were merged (3^rd column) to elucidate the possible neuronal association between Tac1 and kiss2 neurons. Occasionally, some kiss2 mRNA containing cells (green) are accompanied by Tac1 processes (magenta, arrows) in the dorsal zones of periventricular hypothalamus (Hd). However, they do not seem to form close associations based on the results of confocal scanning (data not shown). Scale bars: 10µm.

Expression of tacr1a mRNA in the forebrain regions containing GnRH3 cells. In the forebrain, tacr1a mRNA is expressed in the olfactory bulb (OB), dorsal (Vd) and ventral (Vd) regions of the ventral telencephalon, and anterior part of the parvocellular preoptic nucleus (PPa) (A). In the OB, weak expression of tacr1a mRNA was seen in the glomerular layer (b, black arrowheads), and in the primary olfactory fiber layer of the olfactory bulb where GnRH3 neurons are present (B’, red arrowheads). In the Vv, where another GnRH3 cell population is present (C’, red arrowheads), tacr1a mRNA is broadly expressed (C, black arrowheads). Doted box indicates the location of the inset. Scale bars: (A), 100 µm; (B, B’, C, C’), 50 µm; insets, 10 µm.

Expression of tacr1a mRNA in the hypothalamic regions containing kiss2 cells. In the hypothalamic regions, tacr1a mRNA is expressed in several regions (A, C) including the ventral nucleus of the ventral telencephalic area and the periventricular nucleus of the posterior tuberculum (dotted box), where kiss2 neurons are present (D, E). In addition, tacr1a expression was also seen in the lateral hypothalamic nucleus towards the dorsal zone of the periventricular hypothalamus (B, F), where another group of kiss2 neurons is present (G). Scale bars: (A, B), 200 µm; (C, F, G), 100 µm; (D, E), 50 µm. See Glossary for abbreviation.