Metrics details The vomeronasal system (VNS) is responsible for the perception mainly of pheromones and kairomones it plays a crucial role in their socio-sexual behaviour the capybara offers a more objective and representative perspective to understand the significance of the system in the Rodentia avoiding the risk of extrapolating from laboratory rodent strains exposed to high levels of artificial selection pressure We have studied the main morphological and immunohistochemical features of the capybara vomeronasal organ (VNO) and accessory olfactory bulb (AOB) The study was done in newborn individuals to investigate the maturity of the system at this early stage We used techniques such as histological stains lectins-labelling and immunohistochemical characterization of a range of proteins we conclude that the VNS of the capybara at birth is capable of establishing the same function as that of the adult and that it presents unique features as the high degree of differentiation of the AOB and the active cellular migration in the vomeronasal epithelium All together makes the capybara a promising model for the study of chemical communication in the first days of life By performing an in-depth study of the macroscopic and microscopic morphological characteristics of the vomeronasal system in the newborn capybara we aimed to obtain general information regarding the vomeronasal system in a rodent model that is distinct from most studied laboratory rodents because the capybara is a precocial animal species we aimed to determine the degree to which the capybara vomeronasal system morphology at birth has adapted to the requirements of a demanding socio-cognitive environment The laboratory mouse (Mus musculus) and rat (Rattus norvegicus) may not be representative of all animals that make up this family Differences in the maturation of sensory systems between altricial and precocial species may provide insight regarding behavioral development patterns no studies have examined the morphological and functional maturity of the VNS in precocial rodents during the perinatal period the accessory olfactory bulb (AOB) in capybaras Hydrochoerus hydrochaeris particularly to the morphometry of the anteroposterior zonation which is determined by the expression of the G proteins These authors showed how the Gαo-positive AOB caudal subdomain in capybaras is larger than the rostral subdomain which presents a larger Gαi2 anterior region Whereas capybaras are semi-aquatic mammals whose chemocommunication relies mostly on the oily secretions associated with male-to-male pheromonal communications the degus lives in semiarid spaces and prevalently establishes male–female interactions this study suggests that ecological specialisations may play important roles in shaping the AOB a specific marker for both olfactory systems were labelled with antibodies against microtubule-associated protein 2 (MAP-2) especially important during the first stages of life was studied by employing anti-growth-associated protein 43 (GAP-43) and anti-Luteinizing hormone-releasing hormone (LHRH) The maturity of the system was determined using anti-olfactory marker protein (OMP) The calcium-binding proteins calbindin (CB) and calretinin (CR) were used to identify neuroactive substances Astrocytes and ensheathing cells were recognised by an antibody against glial fibrillary acidic protein (GFAP) Our study aimed to address current gaps in our understanding of the rodent vomeronasal development by providing essential information regarding the newborn capybara VNS showing that this species presents an advanced stage of structural maturity during the first days of life histological and immunohistochemical peculiarities and differences from the VNS of mice and rats demonstrate the wide diversity of the VNS between even closely related species supporting the necessity of studying each species individually to avoid making incorrect extrapolations Through a collaboration with Marcelle Nature Park (Outeiro de Rei we were provided with three one-day-old capybaras (Hydrochoerus hydrochaeris) for use in this study The heads were separated and introduced into the fixative after removing the jaws and extracting the skin muscular plane and other structures such as the tongue and eyes A window was opened dorsally in the skull in the proximity of the olfactory bulbs to facilitate the penetration of the fixative The fixatives used were 10% formol and freshly prepared Bouin’s fixative The latter is especially suitable for the study of the nervous system due to its superior penetration capacity and because it lends consistency to the tissues thus facilitating its subsequent processing the samples were transferred into 70% ethanol We focused the extraction of the samples on the following anatomical structures: the nasal cavity (NC) Nasal cavity The entire NC was separated by a transverse incision made rostrally to the ethmoidal fossa to prevent damage to the olfactory bulbs The resulting sample was used to study the macroscopic and microscopic changes in the topography of the VNO throughout the NC Vomeronasal organ and nerves After opening the NC using a rotating saw the dorsal and ventral turbinates were removed This allowed the visualisation of the nasal septum in its entirety over which the vomeronasal nerves were dissected Once the VNOs were identified on both sides of the base of the anterior portion of the nasal septum—and because of their small size and the close contact they have with the vomer bone—it was necessary to extract them with the help of a surgical microscope (Zeiss OPMI 1 Ent) Main and accessory olfactory bulbs The complete removal of the cranial vault was performed using a gouge forceps It was begun caudally to take advantage of the lower resistance presented by the bone at this level Special care was taken when approaching the OBs which laterally covers the bulbs was removed the dura mater and the olfactory nerves were dissected together since both structures hold the bulbs against the ethmoidal cribriform plate Paraffin embedding was used to perform the histological processing of all samples (VNOs and OBs) the complete NC was pre-decalcified; it was immersed in a decalcifying solution (Shandon TBD-1 Decalcifier USA) and continuously stirred for thirty hours The samples were then washed under running water for two hours and were cut into several blocks which were serially cut from the incisor papilla to the caudal end of the vomeronasal cartilage in order to obtain information on the changes in the VNO throughout its length Cutting The samples were cut with a Leica Reichert Jung microtome with a thickness of 4–8 μm We opted for thinner cuts in the study of the VNO and thicker cuts in the study of the AOB as these allow a better visualisation of the nerve and glial processes In order to highlight the different tissue components we used the following stainings: Haematoxylin–Eosin (HE) as a general staining periodic acid-Schiff (PAS) and Alcian Blue (AB) for neutral and acid mucopolysaccharides The protocol used was as follows: sections were deparaffinised and rehydrated to stain with Ziehl acetic fuchsin for 2 min (10 drops Ziehl fuchsin they were introduced into formalin–acetic acid solution for 5 min (2 drops formalin the sections were finally introduced into picroindigocarmine for 3–5 min (one part 1% indigocarmine aqueous solution two parts saturated aqueous picric acid solution) These stains selectively recognise the different components of the olfactory and vomeronasal pathways in some species They have been used in both VNO and AOB sections It begins by (i) blocking the endogenous peroxidase activity of the sample avoiding possible interference with the developing solution the sample is incubated in 3% H2O2 solution for 10 min and then (ii) incubated for 30 min in 2% bovine serum albumin (BSA) The next step is (iii) incubation with the UEA lectin for 1 h to visualise the lectin-carbohydrate junction followed by (iv) 3 × 5 min washes in 0.1 M phosphate buffer (PB and (v) incubating for 12 h in a peroxidase-conjugated immunoglobulin against the UEA (vi) the sections were washed with PB and developed by (vii) incubation of the sections in a solution of 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 we (iii) incubated the sections overnight in biotinylated lectins diluted in 0.5% BSA the samples were (iv) incubated for 1.5 h in Vectastain ABC reagent (Vector Laboratories The samples were finally (v) developed by incubation in the same DAB solution as the UEA (iv) the samples were incubated for 20 min with the corresponding 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 as a chromogen in the same way as for the lectins All immunohistochemical protocols were checked with the appropriate controls In the absence of a positive control specific to capybaras we replicated the entire histochemical procedure with mouse tissues known to express the proteins of interest Samples for which the primary antibody was omitted were used as negative controls Digital images were taken using the Karl Zeiss Axiocam MRc5 digital camera coupled to a Zeiss Axiophot microscope USA) was used as needed to adjust parameters such as brightness or contrast and crop or resize images for presentation in this work Some photomicrographs were formed as a mosaic of several photographs merged with an image-stitching software (PTGui Pro All the animals employed in this study dead by natural causes Dissection of the VNO and the incisive papilla (A–D) The adult capybara skull gives us the first information on the topographic features of the VNO (D) The dorsorostral view of the skull shows the bony structures that support the caudal third of both organs (B) VNO cross section after its extraction (C) Dissection of the deep plane of the left nasal cavity The VNO corresponds to the triangular area in the anteroventral part of the nasal cavity (E) Ventral view of an adult capybara skull showing the palatine fissures (PF) (F) Roof of the oral cavity of the neonate capybara showing the incisive papilla (IP) v: ventral; In: Incisive bone; IT: Incisor teeth; Mx: Maxillary bone; NS: Nasal septum Olfactory bulbs of the neonate capybara (A) Dorsal view of the right olfactory bulb showing the location of the AOB (asterisk) (B) Ventral view of the brain showing the topography of the olfactory pathway MOB: Main olfactory bulb; LOT: Lateral olfactory tract; Pi: Piriform lobe (C) Rostrolateral view of the brain where the MOB and the AOB (arrow) are differentiated a: Anterior; p: posterior; d: dorsal; v: ventral The VNO in the newborn capybaras (P0) presents a capsule Histological sections of the capybara VNO showing its main components (A,B) Transverse sections of the nasal septum exposing the nature of the vomeronasal capsule where the cartilage is replaced ventrally by the dorsal projection of the maxillary bone (white arrow) (B) corresponds to a caudal level where the bone capsule fully encapsulates both VNOs (C) Cross section of the VNO showing the main components in the parenchyma: Vomeronasal duct (VND) lined medially by sensory epithelium (SE) and laterally by respiratory epithelium (RE) vomeronasal cartilage (VNC) and veins (Vv) insets are magnified in figures (E) and (G) respectively The microvilli (asterisk) contact with the lumen of the vomeronasal duct (D) Enlargement of the dorsolateral area of the VNO showing the serous and AB + nature of the vomeronasal glands (F) Study of the VNO irrigation by confocal microscopy showing veins along the lateral part of parenchyma Elastin autofluorescence of a transversal section Aa: Artery; Mx: Maxillary bone; MR: Respiratory mucosa of the nasal cavity: Vm: Vomer bone; l: lateral; m: medial Stainings: (A) Hematoxylin–Eosin; (B,C,E,G) Gallego’s trichrome; (D) Alcian blue Histological study of the capybara vomeronasal nerves (VNN) (A,C) Large branches of the VNN in the dorsomedial (A) and medial (C) areas of the VNO immunostained by anti-GAP43 (B,E) Migratory stream of cells departing from the sensory epithelium (arrowheads) (D) Higher magnification of the inset showed in E SE: sensory epithelium; VNN: Vomeronasal nerves Scale bars: (A) 250 µm; (B,D) 50 µm; (C,E) 100 µm The laminar organisation is visible and showed at higher magnifications in (B): Inset from (A) Vomeronasal nervous layer (VNL) white matter (WM) and subventricular zone (SVZ) (C) Higher magnification of the inset 1 in (D) The Tolivia staining shows the polyhedric morphology of mitral cells (arrowheads) (1) GCL; (2) IPL; (3) MCL; (4) EPL; (5) GlL (D) Horizontal section of the complete olfactory bulb stained with Tolivia to identify the convergence of myelinic fibres in the lateral olfactory tract (LOT) The differences in size and lamination of MOB and AOB and the arrival of the vomeronasal nerve (VNN) from the medial side of the olfactory bulb are noticeable (E) Higher magnification of the inset 2 in (D) showing the GlL of the MOB (F) Higher magnification of the inset 3 in (D) showing the GlL of the AOB a: Anterior; p: posterior; l: lateral; m: medial Scale bars: (A) 500 µm; (B,C,E,F) 250 µm; (D) 1 mm Capybara VNO histochemical and immunohistochemical labelling (A,D) UEA lectin strongly marks both the entire sensory epithelium and vomeronasal nerves It also allows the identification of the migratory current (arrow) (B,E) IHC labelling with anti-Gαo stains the vomeronasal nerves (white arrows) and produces a focally diffuse pattern in the neuroepithelium (C,F) IHC labelling with anti-Gαi2 stains the nerve component and marks isolated receptor cells (arrowheads) (G,J) The LEA lectin produces a label similar to the UEA lectin A major part of the sensory epithelium and the vomeronasal nerves are marked (H,K) Anti-Calbindin (CB) produces a cellular labelling distributed in the central and basal areas of the epithelium (I,L) Anti-Calretinin (CR) produces a cellular labelling mainly concentrated in the basal area of the epithelium Immunohistochemical labelling in the capybara AOB (A) The IHC labelling with anti-Gαi2 stains the nervous and glomerular layers of the anterior area of the AOB the anti-Gαo marks all of the nervous tissue except the anterior part of the AOB resulting in a complementary expression pattern of both G proteins (C,D) Marking with anti-GFAP produces a more prominent diffuse pattern in the nervous and glomerular part of the AOB both anti-Calretinin (CR) and anti-Calbindin (CB) produce a complete label more intense in the glomerular layer (arrowheads) (F,H) MAP2 labelling focuses on the external plexiform and in the glomerular layers (arrowheads) (I,J) The anti-OMP is immunopositive in the MOB marking intensely the nervous and glomerular layers (GlL) whereas in the AOB the labelling is very faint Scale bars: (A–C,E–G and J) 500 µm; (D,H and I) 250 µm Lectin histochemical labelling in the capybara AOB (A–C) UEA lectin is positive in both the nervous and glomerular strata of the entire olfactory bulb it produces a slightly more intense labelling in the anterior area (D) LEA lectin is positive in the entire AOB without differentiating zones Given this huge diversity more morphofunctional studies of the VNS are needed to understand the basis of this genetic and behavioural multiplicity Studying a rodent species that has not undergone artificial selection by humans was another goal of this study because the capybara is a precocial species the use of newborn individuals allowed us to determine whether and how the VNS morphology had adapted to the requirements of a challenging environment as V1R neuroreceptor cells specifically possess the αi2 subunit of the G proteins in their sensory transduction chain being an useful marker of both olfactory systems invite the hypothesis that some structural features of the AOB reflect the species lifestyle and arise during an early stage of the ontogeny Only studies in P0 mice show an astrocytic development close to that of the capybara but it occurs in the intermediate superficial zone which corresponds to a primitive stage of the internal plexiform layer the early labelling of astrocytes in P0 capybaras reinforces the idea that it presents a VNS with a high degree of maturity at birth In addition to this neurochemical findings our study in the P0 capybara has provided evidence for certain morphological and immunohistochemical features unique to this species—for instance the nature of the capsule that protects both VNOs their dorsal location in the nasal cavity over the palatal process of the incisive bone the high degree of morphological differentiation of the AOB at that early stage and finally the presence of a migratory stream from the neuroepithelium of the VNO to the VNNs This notorious variation in gland characteristics within the same order may reflect an adaptation in capybaras to the aquatic nature of their habitat which might require a specific pheromone-receptor interaction milieu It is difficult to hypothesise about the significance of these cells since this is an unprecedented finding in both the olfactory and vomeronasal nerves Although further studies should clarify the nature and fate of these cells the immunopositivity for GAP-43 suggests to their neuronal nature It clearly shows the arrival of the VNN to the AOB from the medial side of the left hemi-brain the capybara VNS does possess: (1) A VNO that communicates directly with the nasal cavity and indirectly with the oral cavity; (2) A VNO and an AOB that are morphologically similar to those of the adult; (3) Active secretory vomeronasal glands; (4) The same Gαo and Gαi2 sensitivity of the neurosensory epithelium and nervous and glomerular AOB layers as has been described in adult capybara; and (5) Almost 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study analyzed and discussed the results and wrote the paper The authors declare no competing interests Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-020-69994-w Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science