Under an appearance of simplicity, in recent decades the olfactory system of fish has revealed a very high structural complexity, according to cell types and receptors involved. The advent of genomic techniques such as RT-PCR, ISH, genomic and transcriptomic analyses, has been decisive for the discovery in fish of specific olfactory sensorial cell types such as kappe or crypt cells. These techniques have also played a significant role in the identification and characterization of large cell populations expressing vomeronasal receptors [40, 42], thus putting an end to the longstanding controversy about the existence of an AOS in fish [32].
In zebrafish, genomic advances have been so rapid that they have unavoidably marched ahead of morphological and neurochemical studies. Instead, studies in mammals, have formed a solid basis on which to build knowledge of the olfactory and vomeronasal systems [78, 79]. For this reason, there is a lack of specific histological, lectin-histochemical and immunohistochemical studies of the olfactory rosette and OB of the zebrafish. To discuss our results, we must therefore contextualize them in a higher taxonomic order including other fish families.
Although the presence of a vomeronasal organ is a tetrapod evolutive innovation, the vomeronasal receptor genes have been identified in fish and even in the lamprey [80]. Taking into account that each vomeronasal type receptor, V1R and V2R, is associated in mammals to a unique G protein, Gai2 and Gao respectively, the present study aimed to determine whether there is a correlation between such G-proteins and the zebrafish olfactory cell morphology. It is known from the literature that the receptor molecules and the G-protein specific for each receptor are detectable not only in the dendritic process of the neuroreceptor cell, but also along the axons and their termination in the glomeruli of the OB [75]. For this reason, we have extended the study of these molecules of the chain transduction to the olfactory bulb.
Regarding Gao, Hansen et al. [75] correlated in goldfish Carassius auratus the receptor cell morphology and the cell types distribution, with the expression of G-proteins, demonstrating that anti-Gao immunoreactivity was present on microvillar ORNs located in the upper half of the OE. This happens similarly in the Gao neurons identified by us in the zebrafish, pointing to the reliability of anti-Gao as a marker of microvillar V2R-like cells in this species. Other studies in different fish species, such is the case of Chondrichthyes, support this view. Thus, immunohistochemical studies of G protein α subunits in the olfactory organ of Scyliorhinus canicular (Elasmobranch) and Chimaera monstrosa (Holocephali) found the presence of Gαo in virtually all ORNs, which was consistent with the presence of V2Rs [73, 74].
In our case, additionally to the profuse microvillar Gao-positive neurons, we have also found a subpopulation of large, oval-shaped Gao positive cells, always located on the apical surface of the OE. Although their morphology is reminiscent of crypt cells, the study by Ahuja et al. [35] in zebrafish demonstrated that these cells constitute a new olfactory cell type, the kappe cells. Their immunofluorescence study showed that kappe neurons are identified by their anti-Gao immunoreactivity, demonstrating a scattered spatial distribution within the OE, similar to, but significantly different from that of crypt neurons. Our study is the first immunohistochemical report that confirms the presence of kappe cells in fish.
Ahuja et al. [35] also found that kappe neurons project to a single identified target glomerulus within the OB, belonging to the mediodorsal cluster. This observation coincides with the anti-Gao pattern of labelling found by us in the OB, showing immunopositivity to Gao in the mediodorsal part of the bulb. Additionally, we have also found an intense immunoreactivity to Gao in the ventrolateral glomeruli, an area which has been attributed to an important projection of fibers from microvillar cells [30, 81].
Our observations confirm the validity of the anti-Gao antibody as a reliable marker of V2R-like receptor cells in zebrafish, pointing to the presence in zebrafish of a large population of microvillar and kappe cells whose transduction chain is analogous to that present in mammals V2R vomeronasal cells. Therefore, this appears to be an ancient trait conserved through the vertebrate evolution [75].
Regarding crypt olfactory receptor cells, none of the antibodies and lectins employed by us have labelled specifically these cells. Nonetheless, the study by Catania et al. [82] characterized immunohistochemically these crypt cells in zebrafish employing an antibody against the neurotrophin receptor Trk-A.
To our knowledge, the inhibitory subunit Gai2 has not been studied in the fish olfactory system. This fact is surprising since Gai2 is part of the vomeronasal V1R receptor transduction chain in mammals. Mammalian Vmn1r genes show a rather dynamic evolution, in striking contrast to the highly conserved fish orthologous, the ORA gene family. In zebrafish, six ORA receptors have been identified [43], and only in one of them, the ORA4, the precise location of its expression has been studied. Thus, ORA4 receptor was found to be expressed in crypt neurons, but not associated with Gai2 but with the inhibitory G protein, Gi1b [48]. As for its ligands, it is only known that the ORA1 gene recognizes with high specificity and sensitivity the 4-hydroxyphenylacetic acid [83], which might function as a pheromone for reproductive behaviour in zebrafish. ORA1 is ancestral to mammalian V1Rs, and its putative function as pheromone receptor is reminiscent of the role of several mammalian V1Rs as pheromone receptors.
The anti-Gai2 pattern of labelling is very different from that found with anti-Gao, as it covers a wider thickness of the epithelium, although it rarely reaches the deepest cell layers. Moreover, unlike anti-Gao immunolabelling, it comprises the entire extension of the olfactory epithelium clearly demarcating it from the nonsensory epithelium. The immunohistochemistry of the olfactory bulb using anti-Gai2 results in the labelling of a glomeruli subpopulation confirming that the Gai2-positive cells in the olfactory rosette are sensory neurons that convey information to the brain. If ORA receptors coincide with the V1Rs in having the protein subunit Gai2 in their transduction chain, it is surprising that such a small number of receptors are expressed on such a high number of olfactory cells as those detected in our zebrafish olfactory rosette and bulb immunolabelling.
Calcium-binding proteins contribute to calcium homeostasis by buffering the intracellular free calcium concentration [84]. Both CR and CB protect sensory neurons against calcium increases during periods of high frequency discharge as well as in pathological conditions [85]. Moreover, calretinin and calbindin immunoreactive (CR-ir and CB-ir, respectively) neurons in the cerebral cortex are resistant to degenerative processes in Alzheimer’s disease [86].
The distribution of CR in the olfactory system of the zebrafish was investigated for the first time by Castro et al. [59], by using immunocytochemical techniques. Our CR immunoreactivity coincide essentially with their own observations. Accordingly, it is remarkable the presence of numerous CR-ir bipolar cells in the neuroepithelium and an intense immunopositivity in the olfactory nerve. Parisi et al. [61] performed immunofluorescence against CR on zebrafish crypt cells, finding immunolabelling in the OE, primarily in the intermediate cells, but also in the superficial layer. Morphologically, immunopositive cells resembled to them both microvillous and crypt cells. However, our light microscopy anti-CR immunopositive cells featured a slender dendrite and an elongated soma; a morphology closer to olfactory than to microvillous cells. Our observations agree with the immunoelectron microscopy investigation by Gayoso et al. [29] who found a consistent anti-CR immunopositivity in the ciliated cells and only very rarely in the microvillar cells. Additionally, neither Castro et al. [59], Gayoso et al. [29] nor us have found anti-CR immunopositive crypt cells. Studies in other fish species such as the one carried out in the olfactory rosette of guppy [56] have proved similar observations to those found by us in zebrafish.
Regarding the OB, Castro et al. [59] and Braubach et al. [58] in a comprehensive study of the whole population of olfactory glomeruli found that the dorsolateral, ventrolateral, and ventromedial glomerular fields exhibited strong anti-CR labelling. Instead, the dorsomedial area exhibited only faintly CR-ir fibers. Our observations are mostly comparable to those found by them, with the exception that we did not find immunopositive ventromedial glomeruli, likely due to a difference in the levels chosen for each study.
Regarding the expression of calbindin in the olfactory system of the zebrafish, there is a lack of information. However, there have been studies in other fish species such as the chondrostean, Acipenser baerii [53] and the cladistian fish Polypterus senegalus [54], in both cases finding a very faint expression in the olfactory rosette. Our study shows a very high expression of calbindin in the zebrafish olfactory organ, which is accompanied by a parallel expression in the OB. The immunolabelling is comparable to that produced by anti-calretinin, but wider in the case of calbindin, as it is extensible to the ventrolateral glomeruli.
The expression of GFAP has been very little studied in the olfactory system of fish. Notwithstanding, there is a specific study in zebrafish by Lazzari et al. [69] about olfactory ensheathing cells (OECs) with employed different markers (antibodies against GFAP, S100, NCAM, p75). OECs are unique glial cells with axonal growth-promoting properties, involved in the regenerating capability of ORNs throughout life. These cells sustain the continuous axon extension and successful topographic targeting of the olfactory receptor neurons. They are present in the OE and the OB and are also expressed along the entire length of the olfactory nerve. Lazzari et al. [69] found in zebrafish a slight immunostaining in the OE and moderate immunolabelling in the nerve layer of the bulb. In our case, we have found a stronger labelling in both structures. Although we have used the same fixative and commercial antibody as the one used by Lazzari et al. [69] their samples suffered decalcification with EDTA, whereas we did not subject the tissue to any kind of chelating treatment. This fact might explain the differences in the immunolabellig intensity found between the two studies. Remarkably, we found the presence of immunopositive cells in the apical surface of the epithelium, which vary in shape and size, but they are predominantly globose. The meaning of such immunopositive cell bodies, previously undescribed, should be further studied.
Anti-LHRH has been used in fish and mammals to characterize the terminal nerve [77]; a ganglionated extrabulbar nerve, independent of the olfactory nerve, but close enough to be identified by classical histological methods. Its function is uncertain, although its fibres facilitate migration of LHRH cells to the hypothalamus, thus participating in the development of the hypothalamic-gonadal axis [87]. Although extrabulbar elements have been characterized in the forebrain of the zebrafish [29], they differed from the terminal nerve as they were not immunoreactive to the most widely employed marker for this nerve, FMRFamide [88]. We were not able to identify LHRH fibers in the olfactory rosette, but we observed immunoreactivity in neuronal elements of the dorsolateral OB. This result is consistent with that observed in fish by Münz & Class [89] and mammals by Witkin & Silverman [90].
Histochemical labelling with lectins has been widely used in fish, but to a lesser extent in the olfactory system, and with only a few references to the specific case of zebrafish. UEA-I, specific for L-fucose, has been studied in trout [91] finding intense labelling in cell processes located in the apical region of the OE, in some elements of the basal layer and a few cells in the nonsensory epithelium. However, regarding the OB the authors only found positive glomerular fields in five of seven trouts, and in a heterogeneous shape. Pastor et al. [92], studied two Teleostei, Sparus auratus and Dicentrachus labrax finding negative reaction in the olfactory rosette. Our own findings highlight the interspecific diversity in UEA marking, as we have found a positive reaction on the luminal surface of the neuroepithelium and a high concentration of L-fucose in the crypts of nonsensory epithelium, probably due to the mucosal secretion of these cells. Regarding the OB we did not find labelling in our specimens.
LEA has been more widely used to characterize the olfactory system of fish than UEA, but surprisingly there are no specific studies in zebrafish, where we have seen that it is an excellent marker of the sensory epithelium. LEA labels all its cellular elements, establishing a clear border with the unlabelled nonsensory epithelium. The striking presence in the crypts of individualized cells with a clear neuronal morphology, oval shape and fine dendritic processes, has not previously described and should be object of future studies. Interestingly, the neuronal features of these cells, would coincide with the observations by Amato et al. [93] who found TRPV4 immunoreactive “unknown cells” in the nonsensory epithelium of the zebrafish olfactory rosette. TRPV4 is a nonselective cation channel that belongs to the vanilloid subfamily of transient receptor potential ion channels. These authors suggest that these TRPV4 cells might be involved in olfactory sensation. Moreover, Parisi et al. [61] verified that TRPV4 cells did not colocalized with calretinin, which is consistent with the lack of calretinin immunopositive cells in the nonsensory crypts, reported by us. Moreover, our immunolabelling with anti-GAP-43 produced a similar pattern in the crypt epithelium when compared to LEA. All these results together point for the first time to a chemosensory nature of the crypts LEA positive cells.
All mammal and fish species studied till date have shown positivity to LEA in their olfactory sensory epithelia, apart from the case of Pleuronectiformes [94], in which surprisingly and as an exception, LEA negative staining in the OE was reported. Regarding the OB, our results are consistent with those observed in the lungfish Protopterus annectens [95] where LEA positivity was found in the ventral part of the OB, a region associated with reproductive behaviour [96].
Our marking with BSI-B4 stained both the olfactory and the nonsensory epithelium, respectively producing a striking pattern containing scattered cells in the olfactory sensory epithelium, and a widespread labelling of neuronal-like cells similar to those identified with LEA in the nonsensory epithelium. There are no references to compare this striking result in zebrafish, since studies with this lectin existing in other fish species, such as eels and sharks [97, 98] have been restricted to the sensory epithelium, where these authors described a diffuse labelling.
As a whole, we have for the first time exhaustively characterized the olfactory system of the zebrafish at a morphological and immunohistochemical level expanding the results obtained by other authors in specific studies with some of the antibodies and lectins employed by us. These results enrich the information available on the neurochemical profile of the olfactory system of zebrafish and point to a greater complexity than the one currently considered, especially if the peculiarities of the nonsensory epithelium are taken into account.