FIGURE SUMMARY
Title

Identification of accessory olfactory system and medial amygdala in the zebrafish

Authors
Biechl, D., Tietje, K., Ryu, S., Grothe, B., Gerlach, G., Wullimann, M.F.
Source
Full text @ Sci. Rep.

Neuroanatomical analysis and identification of the intermediate nucleus of ventral telencephalon using (A–C,D,D') nuclear stain (DAPI) and (A'–C',D,D'') immunohistochemistry for Otpa. (E) Lateral view of adult zebrafish brain shows level of sections shown in (A-C,A'–C'). (F) Dorsal view of adult zebrafish brain shows level of sections shown in (D–D''). Abbreviations: CC: crista cerebellaris; CCe: corpus cerebelli; Ctec: commissura tecti; D: dorsal telencephalic area; Dl: lateral zone of dorsal telencephalic area; Dm: medial zone of dorsal telencephalic area; EG: eminentia granularis; OB: olfactory bulb; LI: hypothalamic lobus inferior; LL: lateral line nerves; MO: medulla oblongata; MS: medulla spinalis; oc: optic chiasma; Pit: pituitary; Po: preoptic region; SC: spinal cord; TeO: optic tectum; TH: tuberal hypothalamus; TLa: torus lateralis; V: ventral telencephalon; Vi: intermediate nucleus of ventral telencephalon; VLo/LX: vagal lobe. I: olfactory nerve; II: optic nerve; IV: trochlear nerve; VII: facial nerve; VIII: octaval nerve; X: vagal nerve.

Projections after a unilateral DiI injection into the olfactory bulb of an adult zebrafish shown at four telencephalic levels from anterior (A,A') to posterior (E,E'), with corresponding DAPI and fluorescent photomicrographs demonstrating tracing results. (A'') is a confocal blow-up at anterior levels detailing terminal fields and retrogradely labeled cells in the posterior zone of the dorsal telencephalon (Dp) and the dorsal nucleus of the ventral telencephalon (Vd). (C) shows section levels of (A) and (E). Sections (B) and (D) are the immediate caudal and rostral sections, respectively, and are not separately indicated. (D'') confocal photomicrograph shows Otpa staining in the preoptic region and the intermediate nucleus of the ventral telencephalon (Vi; insert). (F,F') are confocal blow ups of E' showing Di terminals and Otpa-positive cells in Vi. Stippled double arrows indicate midline. Abbreviations: acd: dorsal part of anterior commissure; Dm: medial zone of dorsal telencephalic area; Dl: lateral zone of dorsal telencephalic area; Dp: posterior zone of dorsal telencephalic area; OB: olfactory bulb; ENv: ventral entopeduncular nucleus; LI: hypothalamic lobus inferior; lot: lateral olfactory tract; mot: medial olfactory tract; PG: preglomerular complex; Pit: pituitary; Po: preoptic region; PPa: anterior parvocellular preoptic nucleus; TeO: optic tectum; TH: tuberal hypothalamus; TLa: torus lateralis; Vd: dorsal nucleus of ventral telencephalic area; SY: sulcus ypsiloniformis; Vi: intermediate nucleus of ventral telencephalon; Vp: posterior nucleus of ventral telencephalic area; Vv: ventral nucleus of ventral telencephalic area. I: olfactory nerve; II: optic nerve.

Neuronal connections after a unilateral DiI injection into the tuberal hypothalamus in adult zebrafish shown at three levels from anterior (A,A') to posterior (C,C'; note yellow arrow at injection site) with corresponding DAPI and fluorescent photomicrographs demonstrating tracing results. (B'') Confocal photomicrograph shows retrograde tracing result in the telencephalon at the level of the intermediate nucleus of the ventral telencephalon (Vi). Note also that Dm, but not Dl, has retrogradely labeled cells (see text). (D,D') shows confocal blow-up of B'' (D) and corresponding Otpa stain (D'). (E–E'') details another injection site (yellow arrow) which is shown for DAPI, Otpa and DiI in confocal photomicrographs. (F) shows section levels of (A) and (E). Section (B) is immediately caudal to (A), and section (C) is at the same level as (E). Abbreviations: ATN: anterior tuberal nucleus; Dm: medial zone of dorsal telencephalic area; Dl: lateral zone of dorsal telencephalic area; Dp: posterior zone of dorsal telencephalic area; OB: olfactory bulb; E: epiphysis (pineal); ENv: ventral entopeduncular nucleus; Hd: dorsal zone of periventricular hypothalamus; Hv: ventral zone of periventricular hypothalamus; lfb: lateral forebrain bundle; LH: lateral hypothalamic nucleus; LI: hypothalamic lobus inferior; lot: lateral olfactory tract; mfb: medial forebrain bundle; mot: medial olfactory tract; PG: preglomerular complex; Pit: pituitary; Po: preoptic region; PPa: anterior parvocellular preoptic nucleus; PVO: paraventricular organ; Vd: dorsal nucleus of ventral telencephalic area; SY: sulcus ypsiloniformis; TeO: optic tectum; TH: tuberal hypothalamus; TLa: torus lateralis; TPp: periventricular part of posterior tuberculum; Vi: imtermediate nucleus of ventral telencephalon; Vp: posterior nucleus of ventral telencephalic area; Vv: ventral nucleus of ventral telencephalic area. I: olfactory nerve; II: optic nerve.

xample of pERK activation in olfactory bulb section containing mdG2 (frame) in imprinted and non-imprinted zebrafish larva.

(A–A''') confocal photomicrograph of a sectioned imprinted larva. (B–B''') confocal photomicrograph of a sectioned non-imprinted larva. Channels comprise in addition to pERK, the nuclear stain DAPI and the calcium-binding protein immunostain S100. (C) Shows DAPI (left) and a schema with olfactory bulb fields that were counted (mdG2, INL, GL). (D) Larval brain in lateral view shows section level. Abbreviations: ac: anterior commissure; CeP: cerebellar plate; DT: dorsal thalamus (thalamus); E: epiphysis; EmT: eminentia thalami; GL: glomerular layer; H: hypothalamus; Ha: habenula; INL: inner nuclear layer; mdG2: mediodorsal glomerulus 2; MO: medulla oblongata; N: nucleus of the medial longitudinal fascicle; OB: olfactory bulb; ON: olfactory nerve; P: pallium; Po: preoptic region; Pr: pretectum; PTd, PTv: dorsal, ventral part of posterior tuberculum; S: subpallium; T: tegmentum; TeO: optic tectum; TeVe: tectal ventricle; Va: valvula cerebelli; VT: ventral thalamus (prethalamus).

Example of pERK activation and Otpa-positive cells in the intermediate nucleus of the ventral telencephalon (Vi) and indication of the sector that was counted.

(A–A'') Confocal photomicrographs show in addition to pERK, the nuclear stain DAPI and Otpa. (B) Higher power details of insert show pERK and Otpa-positive cells in confocal photography in histological material of tested fish. (C) Larval brain in lateral view shows section level. Abbreviations: ac: anterior commissure; CeP: cerebellar plate; DT: dorsal thalamus (thalamus); E: epiphysis; EmT: eminentia thalami; GL: glomerular layer; H: hypothalamus; Ha: habenula; INL: inner nuclear layer; lfb: lateral forebrain bundle; mdG2: mediodorsal glomerulus 2; MO: medulla oblongata; N: nucleus of the medial longitudinal fascicle; OB: olfactory bulb; on: optic nerve; P: pallium; Po: preoptic region; Pr: pretectum; PTd, PTv: dorsal, ventral part of posterior tuberculum; S: subpallium; T: tegmentum; TeO: optic tectum; TeVe: tectal ventricle; Va: valvula cerebelli; VT: ventral thalamus (prethalamus).

Olfactory bulb projections in adult zebrafish (A) – (K) Series of cross-sections through an adult zebrafish telencephalon showing secondary olfactory projections following an olfactory bulb DiI tracer injection. (L) Levels of sections. For abbreviations: see main paper.

Acknowledgments
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