Lungfishes, like tetrapods, possess a vomeronasal system

Abstract

The vomeronasal system (VNS) is an accessory olfactory system that in tetrapod vertebrates is composed of specific receptor neurons in the nasal organ and a set of centers in the forebrain that receive and relay the information consecutively towards the hypothalamus. Thus, only in tetrapods the VNS comprises a discrete vomeronasal (Jacobson's) organ, which contains receptor cells that are morphologically distinct from those of the olfactory epithelium and use different transduction mechanisms. The axons of the vomeronasal receptors in tetrapods project to the accessory olfactory bulb (AOB) in the rostral telencephalon. Secondary vomeronasal connections exist through the medial amygdala to the hypothalamus. Currently, the lungfishes are considered the closest living relatives of tetrapods. Here we show that the African lungfish, Protopterus dolloi, has epithelial crypts at the base of the lamellae of the olfactory epithelium that express markers of the vomeronasal receptors in tetrapods. The projections of these crypts allow us to identify an AOB on the lateral margin of the main olfactory bulb. The projections of this AOB reach a region that is topologically, hodologically, and immunohistochemically identical to the medial amygdala and could represent its homolog. Neurons of this putative medial amygdala were demonstrated to project to the lateral hypothalamus, as they do in tetrapods. All these features that lungfishes share with tetrapods indicate that lungfishes have the complete set of brain centers and connections involved in processing vomeronasal information and that these features were already present in the last common ancestor of lungfishes and tetrapods.

Keywords: accessory olfactory bulb; evolution; homology; lobe-finned fish; medial amygdala.

Figure 1

The olfactory organ of Protopterus contains a series of suspended lamellae and epithelial crypts that lie at the base of the lamellae. (A) Histological sections of Masson's trichromic stain of the olfactory organ illustrating the olfactory lamellae with the sensory epithelium (arrowheads) and the crypts at the base (arrow). (B,C) High magnification of two basal crypts showing the two distinct parts that constitute the acini; the asterisks mark the distinct part of the crypt formed by elongated cells with basal nuclei and a narrow apical cytoplasm. Scale bars = 200 μm (A) and 50 μm (B,C).

Figure 2

The olfactory epithelium in the lamellae and the crypts contains cells immunoreactive for Goα. (A,B) Fluorescence microscopy (A) and Goα immunoreactivity combined with light microscopy (B) showing Goα immunoreactivity in the olfactory epithelium and in the epithelial crypts at the base of the lamellae (arrows). (C) Higher magnification of the area indicated in a. (D) Detail of receptor grooves in a lamella showing the particularly intense Goα immunoreactivity at the surface (arrowheads). (E) Detail of a crypt showing a characteristic mixed crypt with Goα immunoreactivity only in one part and mainly in the apical processes towards the surface (arrows; asterisk marks the non-reactive part of the crypt). Scale bars = 200 μm (A,B) and 100 μm (C–E).

Figure 3

The olfactory epithelium of the lamellae is Giα2- immunoreactive but the crypts only show Goα-immunoreactivity. (A) Giα2 immunoreactivity in the lamellar epithelium (arrowheads) and lack of reactivity in the basal crypts (asterisk). (B–D) The basal crypts are mixed structures with a Goα-positive part (B,C) that is Giα2-negative (D) and a distinct part that lacks both reactivities (asterisks; E, Masson's trichromic staining). Scale bars = 200 μm (A) and 100 μm (B–E).

Figure 4

The crypts are distinctly labeled with anti-calbindin antibodies. (A,B) As observed in the same doubly labeled section, only the crypts are CB-immunoreactive. (C) Axons from the CB-positive cells course separately from the olfactory organ and collect at the lateral aspect of the olfactory nerve. (D–D”), Higher magnification of a crypt double labeled for Goα (D), CB (d′) and the merge image (D′). (E) Image of numerous CB-immunoreactive crypts and the labeled axons (arrows). (F) High magnification of two labeled crypts. (G–I) Detail of a crypt stained for normal bright field microscopy (F) and CB-immunofluorescence (G), overlapped figure shows the location of the immunoreactive cells (H). Scale bars = 200 μm (A,B,E), 100 μm (C), 50 μm (D–D”,F) and 25 μm (G-I).

Figure 5

The AOB and the pathway to the medial amygdala. (A–C) The CB-positive axons in the lateral aspect of the olfactory nerve (on) reach a subset of glomeruli in the AOB, in the ventrolateral part of the bulb (A, transverse section, lateral is to the left; B, sagittal section, rostral is to he left). The glomerular layer (gl) on the surface of the olfactory bulb is completely labeled for NADPH-diaphorase (C). (D–F) Following CB-immunohistochemistry in the whole-mount brain (D) to localize the AOB, crystals of DiI were applied (E,F). (G-I) Transverse sections through the left hemisphere, lateral is to the left, in which the anterogradely labeled fibers from the application site in the left AOB were followed through the lateral pallium (G) and seen to form a tract that at caudal telencephalic levels forms a neuropil and ends as varicose fibers in the MeA (H,I). (J) After a small injection of DiI into the AOB, retrogradely labeled cells were only observed in the basal crypts (arrowheads). Abbreviations: AOB, accessory olfactory bulb; gl, glomerural layer; igl, inner granule cell layer; Lp, lateral pallium; MeA, medial amygdala; OB, main olfactory bulb; on, olfactory nerve. Scale bars = 200 μm (A–C), 400 μm (D–F), 100 μm (G–J).

Figure 6

Characterization of the MeA. (A,B) The MeA can be identified by its specific OTP-immunoreactivity. (C,D) The presence of ISL1-immunoreactivity and the virtual lack of NKX2.1 labeling characterize the MeA. (E,F) The distinct distribution of NOS and SP immunoreactive structures identifies the MeA. (G) Retrogradely labeled cells in the MeA after DiI application to the lateral hypothalamus. All photographs correspond to transverse sections and in (E–G) only the left side is shown. Abbreviations: ac, anterior commissure; MeA, medial amygdala; PO, preoptic area. Scale bars = 200 μm.

Figure 7

Summary diagram of the vomeronasal system in lungfishes. (A–C) Transverse Nissl stained sections at the levels indicated on the lateral view of the brain showing the location of its three relay centers in the accessory olfactory bulb (AOB), the medial amygdala (MeA) and the lateral hypothalamus (LH).