A comprehensive structural, lectin and immunohistochemical characterization of the zebrafish olfactory system

1 Vol.:(0123456789) Scientific Reports | (2021) 11:8865 | https://doi.org/10.1038/s41598-021-88317-1 www.nature.com/scientificreports A comprehensive structural, lectin and immunohistochemical characterization of the zebrafish olfactory system Paula R. Villamayor 1,2, Álvaro. J. Arana2, Carlos Coppel2, Irene Ortiz‑Leal 1, Mateo V. Torres 1, Pablo Sanchez‑Quinteiro 1,4* & Laura Sánchez 2,3,4 Fish chemosensory olfactory receptors allow them to detect a wide range of water‑soluble chemicals, that mediate fundamental behaviours. Zebrafish possess a well‑developed sense of smell which governs reproduction, appetite, and fear responses. The spatial organization of functional properties within the olfactory epithelium and bulb are comparable to those of mammals, making this species suitable for studies of olfactory differentiation and regeneration and neuronal representation of olfactory information. The advent of genomic techniques has been decisive for the discovery of specific olfactory cell types and the identification of cell populations expressing vomeronasal receptors. These advances have marched ahead of morphological and neurochemical studies. This study aims to fill the existing gap in specific histological, lectin‑histochemical and immunohistochemical studies on the olfactory rosette and the olfactory bulb of the zebrafish. Tissue dissection and microdissection techniques were employed, followed by histological staining techniques, lectin‑histochemical labelling (UEA, LEA, BSI‑B4) and immunohistochemistry using antibodies against G proteins subunits αo and αi2, growth‑associated protein‑43, calbindin, calretinin, glial‑fibrillary‑acidic‑protein and luteinizing‑hormone‑releasing‑hormone. The results obtained enrich the available information on the neurochemical patterns of the zebrafish olfactory system, pointing to a greater complexity than the one currently considered, especially when taking into account the peculiarities of the nonsensory epithelium. The olfactory subsystems play a fundamental role in the daily life of all animal species1, having been extensively studied in mammals, with a fundamental difference between a main olfactory system (MOS) and a vomeronasal or accessory olfactory system (AOS)2. While the first one is known for its role in associative behaviours mediated by odorants sensed in the olfactory mucosa3, the AOS process innate capabilities mediated by pheromones detected by the vomeronasal organ4. Regarding fish, extensive information has been accumulated in recent decades about their olfactory capabilities5,6. Their chemosensory receptors allow them to detect a wide range of water-soluble chemicals, that mediate fundamental behaviours. For instance, nucleotides reveal the freshness of the food7, bile acids are implicated in migration to spawning sites8, steroids and prostaglandins excreted in urine, trigger reproductive behaviours9,10, and injured skin releases alarm pheromones11. In recent decades, zebrafish has become one of the most fruitful model organisms in the field of neurobiology12,13. General aspects of its physiology such as external fertilization or rapid development, together with its rapidly accumulating genome sequence data, have made them a suitable model to deepen into genetic engineering and transcriptomic analyses14–16. Zebrafish possess a well-developed sense of smell, which governs a variety of behaviours involved in reproduction, appetite, and fear17. Moreover, the functional properties within the sensory epithelium and the olfactory bulb (OB) are comparable to those of mammals. Major aspects OPEN 1Department of Anatomy, Animal Production and Clinical Veterinary Sciences, Faculty of Veterinary, University of Santiago de Compostela, Av Carballo Calero s/n, 27002 Lugo, Spain. 2Department of Zoology, Genetics and Physical Anthropology, Faculty of Veterinary, University of Santiago de Compostela, Lugo, Spain. 3Preclinical Animal Models Group, Health Research Institute of Santiago de Compostela (IDIS), Santiago de Compostela, Spain. 4 These authors jointly supervised this work: Pablo Sanchez-Quinteiro and Laura Sánchez. *email: [email protected] 2 Vol:.(1234567890) Scientific Reports | (2021) 11:8865 | https://doi.org/10.1038/s41598-021-88317-1 www.nature.com/scientificreports/ determined in mammals, as the so-called rule of one receptor-one neuron and the convergence of similar axons in the same glomerulus18, are basically preserved in zebrafish19. All this makes the zebrafish a model of vertebrate very suitable for studies of olfactory differentiation and regeneration and neuronal representation of olfactory information20,21. The paired nasal cavity of zebrafish is located at the extremity of the snout, between both eyes. Each cavity is composed of an anterior nostril, through which water enters the cavity, and a posterior nostril, through which water exits the nose. The olfactory epithelium (OE) lies between these two nostrils, arranged in several lamellae that converge in a central raphe, forming a cup-shaped structure known as the rosette22,23. Lamellae are composed of a continuous sensory area, found in the central and medial region of the rosette, as well as a lateral nonsensory epithelium. The sensory area comprises a characteristic pseudostratified columnar epithelium formed primarily by olfactory sensory neurons (OSNs), as well as basal and supporting cells24–26. Regarding the accessory olfactory system, apart from the isolated case of the Dipnoi27, all fishes, zebrafish included, lack of a chamber or vomeronasal organ and an accessory OB comparable to those present in amphibians, reptiles or mammals28. This led to the conclusion that there is no accessory olfactory system in fish. However, studies of morphological features of the olfactory rosette done in zebrafish29 have revealed a complexity that really corresponds to the overlapping and integrated presence of both the main and olfactory systems. Transgenic lines studied by Sato etal.30 have unravelled the existence of two segregated neural circuits that originate in the sensory neurons of the OE, each of them featuring specific cell morphology, molecular signatures, and axonal terminations in the OB. Both pathways probably transmit different types of olfactory information (pheromones versus odorants) to higher olfactory centres31–33. In the epithelium of the zebrafish olfactory rosette, five main receptor cell types are differentiated: ciliated, microvillous, crypt, kappe, and pear cells. Ciliated and microvillous are the most numerous neurons and differ from one another for their morphology and relative positions in the OE. The ciliated OSNs are located in the deep layers of the OE, project a long dendrite, and extend several long cilia into the lumen of the rosette cavity. The microvillous OSNs are situated in more superficial layers, bear a short dendrite, and produce short microvilli34. The crypt cells are located in the most superficial layer of the OE, have ovoid-shaped cell bodies bearing microvilli and short cilia within the same cell23,26,35. The complexity of the zebrafish peripheral OS was proven with the finding of a fourth olfactory sensory neuron population in this species, named kappe neurons for its characteristic shape. These neurons possess microvilli and show a distinct spatial distribution within the OE, similar to, but significantly different from that of crypt neurons36. Recently, a small population of OSNs was identified with a pear-shaped morphology and extremely short dendrites, located in the superficial layer of the OE37. Finally, scattered among the olfactory sensory neurons are ciliated nonsensory cells, which help to move the mucus covering the OE and basal cells in charge of regenerating the sensory cells38. In addition to these morphological differences, a discrimination among cell types can be clearly made according to their molecular expression profiles39. In the zebrafish genome there have been identified 140 OR-type genes40,41. The expression of these ORs has previously been observed in the ciliated OSNs42, associated with the transduction chain of the G protein-subunit Golf2, which is a direct ortholog of the mammalian Golf protein. Golf has been widely used as a marker of the zebrafish olfactory system29,43. 54 V2R-like olfactory C family receptor genes were identified by Alioto and Ngai44 and later on the number has been increased up to 6045. These receptors are found in the microvillous OSNs45. Using genome database mining, Saraiva and Korsching46 identified a new family of 6 V1R-type. Whereas half of the fish V1R genes show a multi-exon structure, all mammalian V1R genes possess a single exon structure47–49. It has not been clearly demonstrated which type(s) of OSNs express V1R receptors, although the zV1R1 (ORA1) receptor has been characterized in cells belonging to the apical side of the OE50 and Oka etal.51 found that crypt neurons express a single V1R-type receptor, the ORA4 receptor. Finally, 112 trace-amine associated receptors (TAAR) have been identified in zebrafish52. A comparative analysis between the olfactory transcriptomes of zebrafish and mouse53 revealed a high degree of molecular conservation, with orthologs of mouse olfactory cell-specific markers, and all but one of their chemosensory receptor classes expressed in the single zebrafish olfactory organ. All seems to indicate that, despite the remarkable morphological differences between the two classes, Actinopterygii and Mammalia, the molecular mechanisms supporting olfaction in teleost and mammals have similarities despite more than 400 million years of evolutionary divergence. The large size of zebrafish chemosensory gene families, combined with the high degree of nucleotide identity among their members, make it very difficult to perform comprehensive expression analysis by in-situ hybridization (ISH), Real-time-PCR, or microarray. Although the olfactory system of zebrafish has been subject of sequencing, transcriptomic, and ISH studies, there is a gap regarding immunohistochemical studies that would allow a more comprehensive and structurally precise assessment of its morphofunctional characteristics. The present study describes the histology, and the lectin-histochemical and immunohistochemical features of the adult zebrafish olfactory rosette and bulb. Three lectins were studied: Ulex europaeus agglutinin (UEA), Bandeiraea simplicifolia isolectin B4 (BSI-B4), and Lycopersicon esculentum agglutinin (LEA). The immunohistochemical study covered a range of antibodies against the G proteins, Gαi2 and Gαo, the calcium-binding proteins, calbindin (CB) and calretinin (CR), growth-associated protein 43 (GAP-43) glial fibrillary acidic protein (GFAP) and luteinizing hormone-releasing hormone (LHRH). To our knowledge, 7 of the 10 markers employed in this study have not been previously studied in the zebrafish olfactory system, including the lectins BSI-B4, UEA, LEA and the antibodies against Gαi2, CB, GAP-43, and LHRH. These markers have all played key roles in the understanding of the olfactory systems of amphibians, reptiles, and mammals. Therefore, our first aim is to fill the existing gap in the neurochemical characterisation of the olfactory system in zebrafish, which has become a model organism for the study of fields as diverse as developmental biology, cancer, toxicology, and neural regeneration. 3 Vol.:(0123456789) Scientific Reports | (2021) 11:8865 | https://doi.org/10.1038/s41598-021-88317-1 www.nature.com/scientificreports/ Furthermore, our study aimed to address two specific issues. First, in the light of existing studies comparing the olfactory transcriptomes between zebrafish and mouse, which have revealed a high degree of molecular conservation, we aimed to phenotypically characterize two markers of the olfactory sensory transduction chain that are characteristic of the mammalian vomeronasal system, Gαi2 and Gαo, to examine their expression pattern in zebrafish mirrors that in mammals. Second, we aimed to investigate the possible involvement of the nonsensory zone of the olfactory rosette epithelium in chemoreception. Material and methods Ten wild-type 1-year-old zebrafish (Danio rerio, wild-type) were used in this study. They were maintained at 28.5°C in 30 L aquaria at a rate of 1 fish per liter of dechlorinated water, with reverse osmosis purified, and under a light–dark cycle of 14:10. Fishes were euthanized by tricaine overdose (MS-222, Sigma, St. Louis, MO). We followed the ARRIVE guidelinesto ensure that all experiments were performed under good conditions. Whole heads were promptly immersed in modified Bouin’s fixative solution. After 24h, the samples were transferred into 70% ethanol. The samples were not decalcified. In all cases paraffin embedding was used to perform the histological procedures. All samples were cut with a Leica Reichert Jung microtome with a thickness of 4–8μm. To highlight the different tissue components, we used the following stainings: 1% Alcian Blue (AB) for acid mucopolysaccharides, Nissl staining (1% cresyl violet for 30min), and Gallego’s Trichrome. Gallego’s trichrome. This stain allows for the differentiation of components of the connective tissue. It stains erythrocytes green, muscle fibers and collagen light blue, epithelium and glandular tissue red, bone dark blue and cartilage purple. The protocol used was described in detail in54 as follows: sections were first stained with Ziehl acetic fuchsin for 2min. After several washes they were introduced into formalin-acetic acid solution for 5min. After two more washes, the sections were finally introduced into picroindigocarmine for 3–5min. Histochemical and immunohistochemical staining. The histochemical and immunohistochemical protocols followed by the authors have been fully described in previous contributions54,55. Briefly: Histochemical labelling (HQ) with lectins. We have used (1) a lectin that comes from gorse, the Ulex europaeus agglutinin (UEA), α-l-fucose specific, (2) the α-galactose-specific BSI-B4 that comes from Bandeiraea simplicifolia, and (3) Lycopersicon esculentum agglutinin (LEA), coming from tomato with a high affinity for N-acetyl-β-d-glucosamine oligomers (Table1). These stains selectively recognise the different components of the olfactory and vomeronasal pathways in some species55. The protocol for the UEA is as follows. (i) blocking the endogenous peroxidase activity of the sample by incubation in 3% H2O2 solution for 10min; (ii) incubation for 30min in 2% bovine serum albumin (BSA), to prevent nonspecific binding; (iii) incubation with the UEA lectin for 1h; (iv) 3 × 5min washes in 0.1M phosphate buffer (PB, pH 7.2), and (v) incubating for 12h in a peroxidase-conjugated immunoglobulin against the UEA. Finally, (vi) the sections were washed with PB and developed by (vii) incubation in 0.05% diaminobenzidine (DAB) and 0.003% H2O2 for 5min. The protocol for the LEA and BSI-B4 begins with the same two steps. Next, (iii) the incubation of the sections was done overnight in biotinylated lectins diluted in 0.5% BSA. The next day, the samples were (iv) 1.5h Table 1. Antibodies and lectins used, with species of elaboration, dilution, manufacturer, and catalogue number. Gαo: subunit αo of G protein; Gαi2: subunit αi2 of G protein; OMP: olfactory marker protein; MAP-2: microtubule associated protein-2; GAP-43: growth-associated protein 43; GFAP: glial fibrillary acidic protein; CB: calbindin; CR: calretinin; LHRH: luteinizing hormone-releasing hormone; UEA: Ulex europaeus agglutinin; LEA: Lycopersicum esculentum agglutinin; BSI-B4: Bandeiraea simplicifolia isolectin B4; HRP: horseradish peroxidase; IgG: Immunoglobulin G; ABC: avidin–biotin-complex. The lectins employed are indicated by an asterisk (*). Ab/lectin* 1st Ab/lectin species and dilution 1st Ab/lectin catalogue number 2nd Ab kit (catalogue number) Anti-Gαo Rabbit 1:100 Sta Cruz Biotechnology SC-387 ImmPRESS VR HRP Anti-Rabbit IgG Reagent MP-6401-15 Anti-Gαi2 Rabbit 1:100 Sta Cruz Biotechnology SC-7276 ImmPRESS VR HRP Anti-Rabbit IgG Reagent MP-6401-15 Anti-GFAP Rabbit 1:400 Dako Z0334 ImmPRESS VR HRP Anti-Rabbit IgG Reagent MP-6401-15 Anti-Calbindin Rabbit 1:5000 Swant CB38 ImmPRESS VR HRP Anti-Rabbit IgG Reagent MP-6401-15 Anti-GAP-43 Mouse 1:400–1:4000 Sigma G9264 ImmPRESS VR HRP Anti-Mouse IgG Reagent MP-6402-15 Anti-Calretinin Rabbit 1:5000 Swant 7697 ImmPRESS VR HRP Anti-Rabbit IgG Reagent MP-6401-15 Anti-LHRH Rabbit 1:500 Fisher Scientific A235481 ImmPRESS VR HRP Anti-Rabbit IgG Reagent MP-6401-15 UEA-I* 1:10 Vector L-1060 Rabbit 1:50 DAKO P289 LEA* 20μg/ml Vector B-1175 Vectastain ABC reagent PK-4000 BSI-B4* 100μg/ml Sigma L-2140 Vectastain ABC reagent PK-4000 4 Vol:.(1234567890) Scientific Reports | (2021) 11:8865 | https://doi.org/10.1038/s41598-021-88317-1 www.nature.com/scientificreports/ incubation in Vectastain ABC reagent (Vector Laboratories, Burlingame, CA, USA). The samples were finally (v) developed in the same DAB solution as the UEA54. Immunohistochemistry (IHQ) techniques. This protocol also began by (i) blocking the endogenous peroxidase. Then, (ii) non-specific binding was blocked with 2.5% horse normal serum from the ImmPRESS reagent kit Anti-mouse IgG/Anti-rabbit IgG (Vector Laboratories, CA, USA) for 30min. (iii) The primary antibody was added at the corresponding dilution (Table1) and incubated overnight. The next day, (iv) the samples were incubated for 20min with the ImmPRESS VR Polymer HRP Anti-Rabbit IgG Reagent. (v) After rinsing in Tris-buffer (pH 7.61) for 10min, (vi) the samples were finally developed using DAB in the same way as for the lectins54,55. All immunohistochemical protocols were checked with the appropriate controls. Samples for which the primary antibody was omitted were used as negative controls. Table2 gives references to previously published use in zebrafish and other fishes of the antibodies here employed against the same antigens. Acquisition of images and digital treatment. Digital images were captured using the Karl Zeiss MRc5 digital camera attached to a Zeiss Axiophot microscope. Adobe Photoshop CS4 (Adobe Systems, San Jose, CA, USA) was used to adjust parameters such as brightness, contrast and balance light levels for presentation in this work. No features of the image were enhanced in any way, moved, or introduced. Some photomicrographs were formed as a mosaic of several photographs merged with an image-stitching software (PTGui Pro, The Netherlands). In no case was this software used to generate images that do not correspond to the actual images presented in the manuscript. Ethical approval. The care, use and treatment of zebrafish were performed in agreement with the Animal Care and Use Committee of the University of Santiago de Compostela and the standard protocols of Spain (Directive 2012-63-UE). The protocol was approved by the Animal Care and Use Committee of the University of Santiago de Compostela. Xunta de Galicia Code AE-LU003. Table 2. Previously published use in fishes olfactory system studies of the antibodies employed in this study against the same proteins. Antigen Host Type, clone Source Code Fish studied References Calbindin Mouse Monoclonal Swant 300 Acipenser baeri 56 Calbindin Mouse Monoclonal Swant 300 Polypterus senegalus 57 Calbindin Mouse Monoclonal Swant 300 Polypterus senegalus Erpetoichthys calabaricus 58 Calretinin Rabbit Polyclonal Chemicon AB5054 Poecilia reticulata 59 Calretinin Rabbit Polyclonal Santa Cruz Biotech SC-11644 Danio rerio 60 Calretinin Rabbit Polyclonal Swant 7697 Danio rerio 61 Calretinin Rabbit Polyclonal Swant 7697 Danio rerio 62 Calretinin Rabbit Polyclonal Swant 7697 Salmo trutta fario 62 Calretinin Rabbit Polyclonal Swant 7697 Psetta máxima 63 Calretinin Rabbit Polyclonal Swant 7697 Danio rerio 29 Calretinin Rabbit Polyclonal Swant 7697 Danio rerio 64 GAP-43 Mouse Monoclonal Sigma G9264 Tilapia mariae 65 GFAP Mouse Monoclonal Sigma G3893 Danio rerio 66 GFAP Mouse Monoclonal ZIRC Zrf -1 Danio rerio 67 GFAP Rabbit Polyclonal Dako Z0334 Oryzias latipes 68 GFAP Rabbit Polyclonal Dako Z0334 Danio rerio 69 GFAP Rabbit Polyclonal Dako Z0334 Nothobranchius guentheri 70 GFAP Rabbit Polyclonal Dako Z0334 Poecilia reticulata Carassius auratus 71 GFAP Rabbit Polyclonal Dako Z0334 Danio rerio 72 GFAP Rabbit Polyclonal Dako Z0334 Astatotilapia burtoni 73 GFAP Rabbit Polyclonal Sigma G9269 Danio rerio 74 Gαo Rabbit Polyclonal Santa Cruz Biotech SC-387 Tenualosa ilisha 75 Gαo Rabbit Polyclonal Santa Cruz Biotech SC-387 Tenualosa ilisha 75 Gαo Rabbit Polyclonal Santa Cruz Biotech SC-387 Scyliorhinus canicula 76 Gαo Rabbit Polyclonal Santa Cruz Biotech SC-387 Chimaera monstrosa 77 Gαo Rabbit Polyclonal Santa Cruz Biotech SC-387 Carassius auratus 78 Gαo Rabbit Polyclonal Santa Cruz Biotech SC-387 Scyliorhinus canicula 79 LHRH Rabbit Polyclonal Immuno Nuclear U-705 Carassius auratus 80 5 Vol.:(0123456789) Scientific Reports | (2021) 11:8865 | https://doi.org/10.1038/s41598-021-88317-1 www.nature.com/scientificreports/ Informed consent. No human subject was used in this study. Results The zebrafish olfactory rosette occupies an anterodorsal position, slightly rostral to the orbits (Figs.1 and 2A, Suppl. Fig.S1A–C). In a parasagittal section at the orbital level, the outlet nostril coincides with a zone of lesser development of the lamellae (Suppl. Fig.S1C). In a sagittal section, which includes the central part of the OB (Fig.2B, Suppl. Fig.S1B), the rosette can be observed at its maximum expression, divided into two symmetrical zones by the presence of a wide raphe that serves to support the lamellae. The olfactory nerve associated with each lamella constitutes a single branch that reaches the OB from the ventromedial side (Fig.2G, Suppl. Fig.S1D). The staining of the sensory pseudostratified epithelium with Gallego’s trichrome reveals the organization and allows for the differentiation of basal cells from the olfactory sensory cells. The three primary olfactory cell types are densely intermingled but can be distinguished by their characteristic shapes and spatial positions: a slender dendrite and a basal soma for ciliated neurons, a rounded cell body and an intermediate soma position for microvillous neurons, and a large globose soma in an apical position for crypt neurons (Fig.2C). The luminal surface of the nonsensory epithelium is covered by cilia (Fig.2D,E). Alcian Blue staining shows the presence of acidic mucopolysaccharides on the epithelial surface of the lamella, mainly in the luminal surface of the sensory area, whereas the nonsensory epithelium border is free of acid mucins (Fig.2F). The zebrafish OB is diffusely laminated, but its three layers can be identified from the periphery toward the center: olfactory nerve layer, glomerular layer, and granular layer (Fig.2G,H). The olfactory nerve layer is formed by the axonal endings of olfactory receptor neurons (ORNs) (Fig.2G). The glomeruli do not resemble the distinct spheres observed in mammals due to the few numbers of periglomerular cells and glial elements found in zebrafish. The cell bodies of the mitral cells are intermingled in the boundaries between the glomerular and granular layers (Fig.2H). The latest is the deeper layer, which is primarily formed by granule cells (Fig.2G). Immunohistochemical staining of the olfactory rosette. The immunohistochemical study with anti-Gαi2 and anti-Gαo produce two differentiated patterns (Fig.3). Intense immunoreactivity was noticed in the central and medial portions of the OE when employing Anti-Gαi2 (Fig.3A–C,E,F). The immunopositive cells are distributed across the entire thickness of the epithelium, but appear mostly concentrated in its superficial half (Fig.3F). Contrastly, anti-Gαo produces a diffuse immunoreactivity circumscribed to cells present on the apical surface of the OE (Fig.3D,G–I). Interestingly, we also found large isolated cells with stronger anti-Gαo immunopositivity in the OE (Fig.3H). Additionally, the anti-Gαo labelling also stains the apical surface of the nonsensory area of the hair cells (Fig.3G). Both markers, anti-Gαi2 and anti-Gαo label the olfactory nerves (Fig.3E,I). Figure1. Macroscopic anatomy of the zebrafish olfactory organ. The olfactory rosette occupies in the head an anterodorsal position. The nostrils lie slightly rostral to the eyes and close to the mouth. Five neurosensorial olfactory cell types (OSNs) have been described: microvillous (mv); ciliated (cl); crypt (cr); kappe (kp); and pear (pr) OSNs. Drawing by Helena Reino Piñeiro published under a CC BY open access license. 6 Vol:.(1234567890) Scientific Reports | (2021) 11:8865 | https://doi.org/10.1038/s41598-021-88317-1 www.nature.com/scientificreports/ The results of the immunohistochemical study with anti-calbindin (anti-CB) and anti-calretinin (anti-CR) are depicted in Fig.4A,B,D,E and C,F respectively. Both markers label a subpopulation of olfactory sensory neurons. Sagittal and transverse sections of the rosette show how the anti-CB inmmunolabelling is mostly located in the deeper part of the neurosensorial epithelium (Fig.4D), whereas the nonsensory epithelium is Anti-CB immunonegative (Fig.4B). Anti-CR immunolabelling produces a stronger labelling than anti-CB in neuroepithelial cells, which is mostly concentrated in the medial part of the lamellae (Fig.4C), and their deeper neuroepithelial layers (Fig.4F). Very rarely superficial cells are immunolabelled. The nonsensory cripts do not show anti-CR labelling. Figure2. Microscopic anatomy of the zebrafish olfactory system. (A) Low power sagittal section of the anterior zebrafish stained with Gallego’s trichrome. (B) Higher magnification of the inset in (A), showing the olfactory rosette and the olfactory bulb (OB). (C) Histological section of the olfactory sensory epithelium stained with Gallego’s trichrome. Black arrowhead, crypt cell; open arrowhead: microvillous cell; back arrow, ciliated cell; white arrow: basal cell. (D) Histological section of the medial side of the lamellae. The dotted line demarcates the nonsensory epithelium (NS) of the olfactory epithelium (S). Arrowhead, ciliated cells in the nonsensory epithelium. (E) The lateral rim of the lamellae-forming channel-like system (asterisk). Arrowhead, ciliated nonsensory cells. (F) Histological section of the lamellae stained by Alcian Blue. The luminal mucociliary complex is restricted to the sensory area (black arrowheads). The nonsensory epithelium border is free from acid mucins (white arrowheads), but Alcian Blue-stained secretions are concentrated inside the channel. (G) Sagittal section of the olfactory bulb. (H) Inset from (G) showing the olfactory nerve layer (ONL) and the glomerular layer (GlL). Black arrowhead, mitral cells; open arrowhead, periglomerular cells. Stains: (A–E) Gallego’s trichrome; (F) Alcian Blue; (G,H) Nissl stain. Ai, anterior intestine; Ak, anterior kidney; Br, Brain; Es, esophagus; Gi, gilts; GrL, granular layer; He, heart; Hy, hypophysis; Li, liver; Oc, oral cavity; OR, Olfactory rosette; Te, telencephalon; cd, caudal; d, dorsal; r, rostral; v, ventral. Scale bars: 100µm (B,G,H); 50µm (C–F). 7 Vol.:(0123456789) Scientific Reports | (2021) 11:8865 | https://doi.org/10.1038/s41598-021-88317-1 www.nature.com/scientificreports/ Anti-GFAP immunolabels isolated big cell bodies in the apical part of the whole OE (Fig.4G–J). These cell bodies are present in both sagittal and transverse sections. Anti-GAP-43 immunolabelling is located in the apical part of the nonsensory epithelium and in individual cell bodies in the crypts. (Fig.4K,L). Anti-LHRH produces a light immunolabelling, mainly located in the cell processes of both the sensory and nonsensory epithelia (Fig.4M,N). Lectin histochemical staining of the olfactory rosette. The three lectins employed in this study labelled the olfactory system following individual patterns (Fig.5). The labelling with LEA of the whole OE clearly delineates the limits between the olfactory and the nonsensory epithelium -this latter unstained (Fig.5A,B). Additionally, big oval neuron-like cell bodies in the crypts of the nonsensory epithelium are also stained, and in some of them thin dendritic processes can be clearly appreciated (Fig.5C,D,G). UEA only labelled secretory material, mainly in the crypts, but also in the sensory epithelium but in lesser extent (Fig.5E,F). BSI-B4 marks Figure3. Immunohistochemical study of the olfactory rosette of zebrafish with antibodies against G-proteins. (A–C,E,F) Anti-Gαi2 immunolabelling. (A) Sagittal sections show a higher number of immunopositive neurons in the internal parts of the rosette, at both sides of the median raphe. (B) Higher magnification of the inset in (A). The transition between both the nonsensory and sensory epithelium is straight (arrows). (C) Transverse sections show how the lateral rim of the lamella (asterisks) lacks of anti-Gαi2 immunolabelling. (E) Inset in (B) showing the immunopositive olfactory nerves (white arrowheads). (F) Inset in C shows how the basal part of the neuroepithelium lacks immunopositive cells. (D,G–I) Anti-Gαo immunolabelling. (D) The immunoreactivity was present diffusely in apical neurons, as it is shown by open arrowheads in the inset (G). Additionally, more isolated big and oval receptor neurons showed immunoreactivity (black arrowheads) (H, inset in G). (I) The processes in the nonsensory epithelium (arrowheads) and the branches of the olfactory nerves (asterisks) are also immunopositive. Scale bar: 100μm (A,C,D); 50μm (B,E–I). 8 Vol:.(1234567890) Scientific Reports | (2021) 11:8865 | https://doi.org/10.1038/s41598-021-88317-1 www.nature.com/scientificreports/ 9 Vol.:(0123456789) Scientific Reports | (2021) 11:8865 | https://doi.org/10.1038/s41598-021-88317-1 www.nature.com/scientificreports/ individual cells in the crypts and in the nonsensory part of the lamella and very occasionally cells belonging to the sensory part of the OE. In all cases they are scattered cells, smaller than those marked by LEA (Fig.5H–J). Immunohistochemical and lectin histochemical staining of the olfactory bulb. The immunohistochemical labelling of the olfactory bulb (Fig.6) primarily reveals two patterns. The labelling for the calcium-binding proteins (CB and CR) is concentrated in the dorsal and lateral regions. Using the terminology established by Braubach etal.61,81, calcium-binding proteins were identified in the dorsal glomeruli (dG), clusters of the dorsolateral (dlG) and lateral glomeruli (lGx), and the large lateral glomerulus 1 (lG1). Additionally, anticalbindin stained glomeruli belonging to the mediodorsal (mdG) and the ventromedial parts of the bulb were stained with reduced intensity (ventral posterior glomeruli, VpG). Both G protein antibodies showed similar patterns, with a higher concentration of labelling in the ventromedial glomerular cluster 1–6 (VmG1-6) and the large ventromedial glomerulus 7 (VmG7), which appeared stronger for anti-Gαo. Both G proteins faintly stained the mediodorsal glomeruli (mdG), and Gαi2 marks glomeruli belonging to the ventral posterior cluster (VpG). Finally, LEA exemplifies a second pattern, in which the labelling was mainly circumscribed to the ventral area. This lectin also labels the dorsolateral area but with less intensity. LHRH is expressed primarily in the dorsolateral glomeruli (dlG). Anti-GAP-43 and the other two lectins examined in this study did not show significant results in the OB. Discussion 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 receptors42,44, thus putting an end to the longstanding controversy about the existence of an AOS in fish32. 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 systems82,83. 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 lamprey84. Taking into account that each vomeronasal type receptor, V1R and V2R, is associated in mammals to a unique G protein, Gαi2 and Gαo 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 OB85. For this reason, we have extended the study of these molecules of the chain transduction to the olfactory bulb. Regarding Gαo, Hansen etal.78 correlated in goldfish Carassius auratus the receptor cell morphology and the cell types distribution, with the expression of G-proteins, demonstrating that anti-Gαo immunoreactivity was present on microvillar ORNs located in the upper half of the OE. This happens similarly in the Gαo neurons identified by us in the zebrafish, pointing to the reliability of anti-Gαo 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 V2Rs76,77. In our case, additionally to the profuse microvillar Gαo-positive neurons, we have also found a subpopulation of large, oval-shaped Gαo 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.36 in zebrafish demonstrated that these cells constitute a new olfactory cell type, the kappe cells. Their immunofluorescence study showed that kappe neurons Figure4. Immunohistochemical study of the olfactory rosette of zebrafish. (A,B,D,E) Anti-Calbindin labelling. (A) A sagittal section of the olfactory rosette shows labelling in a neuronal subpopulation distributed widely in the rosettes. Insets in (B) and (D) show the immunolabelling mostly located in the deeper part of the epithelium. In (B), neuroephitelial cells in the superficial layer (open arrowhead) and branches of olfactory nerves (black arrowheads) are intensely labelled. A transverse section (E) shows a similar pattern. The nonsensory epithelium (asterisk) is immunonegative. (C,F) Anti-Calretinin immunolabelling produces a stronger labelling of neuroepithelial cells, mostly concentrated in the medial part of the lamellae, and their deeper layers, whereas the nonsensory cripts (asterisk) are not immunolabelled. The neuroepithelial cells are mainly distributed in deeper layers (black arrowheads in F), and very rarely the superficial cells are lightly immunolabelled. (G–J) Anti-GFAP immunolabels isolated big cell bodies in the apical part of the epithelium. They appear in both sagittal (G) and transverse (I) sections. Insets are shown in (H) and (J), respectively. Additionally, in the olfactory bulb the antibody anti-GFAP labels the whole glomerular layer (OB). (K,L) AntiGAP-43 immunolabelling is located in the apical part of the nonsensory epithelium (black arrowheads in K) and in individual cell bodies (black arrowheads in L) in the crypts (asterisk). (M,N) Anti-LHRH produces a light immunolabelling, mainly located in the cell processes of the nonsensory epithelium. Scale bars: 100μm (A,C–E,G,I–M); 50μm (B,F,H,N). ◂ 16 Vol:.(1234567890) Scientific Reports | (2021) 11:8865 | https://doi.org/10.1038/s41598-021-88317-1 www.nature.com/scientificreports/ 95. Scheib, J. & Byrd-Jacobs, C. Zebrafish Astroglial morphology in the olfactory bulb is altered with repetitive peripheral damage. Front. Neuroanat. 14, 4 (2020). 96. Peña-Melián, A. et al. 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Acknowledgements The authors thank to Helena Reino Piñeiro DVM for his artistic drawing of the zebrafish olfactory system. Author contributions P.R.V., P.S.Q., L.S. designed the research. P.R.V., A.J.A., C.C., I.O.L., M.V.T., P.S.Q., L.S. performed the work. P.R.V., P.S.Q., L.S. analyzed and discussed the results and wrote the paper. Competing interests The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1038/ s4159802188317-1. Correspondence and requests for materials should be addressed to P.S.-Q. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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