Identification of accessory olfactory system and medial amygdala in the zebrafish

Abstract

Zebrafish larvae imprint on visual and olfactory cues of their kin on day 5 and 6 postfertilization, respectively. Only imprinted (but not non-imprinted) larvae show strongly activated crypt (and some microvillous) cells demonstrated by pERK levels after subsequent exposure to kin odor. Here, we investigate the olfactory bulb of zebrafish larvae for activated neurons located at the sole glomerulus mdG2 which receives crypt cell input. Imprinted larvae show a significantly increased activation of olfactory bulb cells compared to non-imprinted larvae after exposure to kin odor. Surprisingly, pERK activated Orthopedia-positive cell numbers in the intermediate ventral telencephalic nucleus were higher in non-imprinted, kin odor stimulated larvae compared to control and to kin-odor stimulated imprinted larvae and control. Moreover, DiI tracing experiments in adult zebrafish show a neuronal circuit from crypt/microvillous olfactory sensory neurons via dorsomedial olfactory bulb and intermediate ventral telencephalic nucleus (thus, arguably the teleostean medial amygdala) to tuberal hypothalamus, demonstrating for the first time an accessory olfactory system in teleosts.

Figure 1

(A) The distribution of the transcription factor Otpa (shown in green) in the preoptic region and a posterior ventral telencephalic region (the latter indicated by a question mark) shown in a sagittal view of a 5 dpf zebrafish larva (drawn after29). Major telencephalic regions treated in the Introduction are schematically indicated. (B) Comparison of receptor molecules and associated G proteins on olfactory sensory neurons in zebrafish and mouse. See text for more details and references. Abbreviations: ac: anterior commissure; Ce: Cerebellum; Dm: medial zone of dorsal telencephalic area (pallium); Ha: Habenula; Po: preoptic region; poc/oc: postoptic commissure/optic chiasma; OB: olfactory bulb; PT: posterior tuberculum; T: midbrain tegmentum; TH: tuberal hypothalamus; Tel: telencephalon; TeO: tectum opticum; Vd, Vp, Vs; Vv: dorsal nucleus, postcommissural nucleus, supracommissural nucleus and ventral nucleus of ventral telencephalic area (subpallium).

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5. Experimental set-up: Schema shows how imprinted (red) and non-imprinted (blue) larval zebrafish were created and subsequently tested for kin odor activation.

Zebrafish larvae were either exposed to kin odor or E3 medium at day 6 and both groups were subsequently tested either for kin odor or E3 medium at 9 days. Then, the larvae were sacrificed immediately after olfactory stimulation and further processed. Previous experiments had established that 7 minutes allow for optimal assay for pERK. E3 medium is a commonly used medium for raising zebrafish eggs.

Figure 6. Example 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).

Figure 7. Number of pERK-positive cells in imprinted and non-imprinted zebrafish larvae, stimulated with either kin or control odor, in different olfactory bulb fields.

Box plots show median (Mdn), upper and lower quartile and whiskers (maximum interquartile range: 1.5). *Indicates statistical significance p: *p < 0.05; **p < 0.01. kin = kin odor stimulus; ctr = control stimulus. n_{imprinted kin} = 7; n_{imprinted ctr} = 8; n_{non-imprinted kin} = 7; n_{non-imprinted ctr} = 9. (A) Total cell quantity of pERK-positive cells in entire olfactory bulb section at level of mdG2. Number of activated cells is significantly higher in imprinted larvae exposed to kin compared to imprinted larvae exposed to control stimulus (Mann-Whitney U = 6, p = 0.001, Mdn_{impr kin} = 21, Mdn_{impr ctr} = 4).Significant difference in cell number were detected between imprinted and non-imprinted larvae, exposed to kin (U = 6, p = 0.018, Mdn_{impr kin} = 21, Mdn_{non-impr kin} = 11). (B) pERK + cells around mdG2 (see Fig. 6) show a difference in activation between imprinted larvae stimulated with kin odor compared to imprinted larvae exposed to control stimuli (U = 4.5, p = 0.003, Mdn_{impr kin} = 4, Mdn_{impr ctr} = 0) or compared to non-imprinted larvae stimulated with kin odor (U = 6, p = 0.015, Mdn_{impr kin} = 4, Mdn_{non-impr kin} = 0). The same picture of neuronal activity arises in cells of inner cellular layer (C). Cell number differs significantly in imprinted larvae exposed to kin compared to imprinted larvae exposed to control stimulus (U = 0.5, p = 0.001, Mdn_{impr kin} = 11, Mdn_{impr ctr} = 3). pERK + cell number differs between imprinted larvae and non-imprinted larvae exposed to kin (U = 3, p = 0.006, Mdn_{non-impr kin} = 5). (D) shows the number of pERK + cells in glomerular layer of the olfactory bulb. A significant difference in pERK-positive cell number was found (Kruskall-Wallis test: H(2) = 9.357, p = 0.025); but after Mann-Whitney U correction for multiple comparisons (Bonferroni correction; α = 0.017) no significant differences could be detected between treatments for glomerular layer pERK + cell numbers (U = 9, p = 0.024, Mdn_{impr kin} = 4, Mdn_{non-impr kin} = 3).

Figure 8. 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).

Figure 9. Analysis of pERK activated cell number in a restricted area of telencephalon, defined by Otpa staining.

Both, pERK + cells, as well as double-labeled cells for Otpa and pERK were counted and analyzed in imprinted and non-imprinted zebrafish larvae, stimulated with either kin or control odor. Box plots show median (Mdn), upper and lower quartile and whiskers (maximum interquartile range: 1.5). *Indicates statistical significance p: *p < 0.05. kin = kin odor stimulus; ctr = control stimulus. n_{imprinted kin}: 10; n_{imprinted ctr}: 8; n_{non-imprinted kin}: 10; n_{non-imprinted ctr}: 9. Total cell quantity of single-labeled pERK-positive cells does not differ significantly between imprinted and non-imprinting larvae, stimulated with either kin odor or control stimulus (Kruskall-Wallis test: H(2) = 3.78, p = 0.295). Furthermore, pERK-positive and Otpa-positive double-labelled cell quantity was analyzed. A significant difference in cell number was found (H(2) = 8.579, p = 0.035) between treatments. A Mann-Whitney U test was performed to determine significant differences between two treatments. No significant differences could be detected after performing the Bonferroni correction (U = 28.5, p = 0.044, Mdn_{non-impr kin} = 0.5, Mdn_{impr kin} = 0; U = 22.5, p = 0.018, Mdn_{non-impr kin} = 0.5, Mdn_{non-impr ctr} = 0).

Figure 10. Schema of primary and secondary olfactory pathways in the adult zebrafish.

(A) Neuronal activity quantified with pERK at three successive synaptic levels from peripheral sensory olfactory sensory neurons to central nervous targets (mdG2, which is immunohistochemically identified with S100 antibody because the projections of S100 immunopositive crypt cells terminate there; Vi, which is immunohistochemically identified with Otpa antibody for many of its cell bodies) after kin odor stimulation of imprinted and non-imprinted larvae. The counted pERK activated cells were located around the mdG2 and within Vi. Red tickmarks indicate significant changes in activated cell numbers seen at each level (see text for details). Higher order (i.e. secondary) olfactory projections of mediodorsal bulb area are indicated with solid black lines (targets shared with projections of entire olfactory bulb) and dashed black lines (targets specifically attributed to mediodorsal bulbar area; see literature below and text for more details). Crypt cells are widely distributed over the entire olfactory epithelium. (B) Projections of adult zebrafish mediodorsal olfactory bulb area (incl. mdG2) as shown in the present paper using the lipophilic tracing substance DiI (red lines). Tracer injections into tuberal hypothalamus (TH) demonstrate also a teleostean accessory olfactory pathway via Vp/Vi. Arrowheads indicate where a projection terminates. Olfactory bulb projections shown as dashed lines to telencephalic targets are selective for mediodorsal olfactory bulb (present paper and additional data from9101314151644). Abbreviations: Cr: crypt cells; Dp: posterior zone of dorsal telencephalon; Ha: habenula; Had: dorsal part of Ha; Hav: ventral part of Ha; OB: olfactory bulb; OE: olfactory epithelium; oc: optic chiasma; on: olfactory nerve; Po: preoptic region; Tel: telencephalon; TeO: optic tectum; TH: tuberal hypothalamus; Vd, Vp, Vs, Vv: dorsal, postcommissural, supracommissural, ventral nucleus of ventral telencephalon.