Ultrastructural and immunohistochemical characteristics of the olfactory organ cardinal tetra, Paracheirodon axelrodi (Characiformes: Characidae).

The cardinal tetra Paracheirodon axelrodi belongs to the family Characidae, an economically important and morphologically diverse family of fishes. Information on the olfactory system of this species is scattered and scarce. Among teleost fishes, differences exist in the shape, number, and arrangement of the olfactory lamellae, in the distribution of the sensory and nonsensory epithelium, as well as in the abundance of various receptor cell types. Here, an anatomical and morphological description of the olfactory system was carried out using light microscopic histology, immunohistochemistry, scanning electron microscopy, and transmission electron microscopy. P. axelrodi is a ditremous and isosmat species. It has an arrow-shaped olfactory rosette arrangement. The olfactory epithelium is covering the 12-14 lamellae of the olfactory rosette and, using scanning electron microscopy, we observed that the apical surface of the olfactory epithelium carries a dense layer of mucus. Based on the histological, immunohistochemical, and ultrastructural descriptions, all characteristic sensory and nonsensory cell types of the olfactory epithelium of teleost fish were identified. Three types of olfactory receptor neurons were identified: ciliated, microvilli, and crypt cells. The distribution of sensory and nonsensory cell types is like that described in Aphyocharax anisitsi, another species of the Characidae family. A. anisitsi inhabits slow-flowing water bodies with high-density vegetation such as P. axelrodi.

Conservation of Glomerular Organization in the Main Olfactory Bulb of Anuran Larvae.

The glomerular array in the olfactory bulb of many vertebrates is segregated into molecularly and anatomically distinct clusters linked to different olfactory functions. In anurans, glomerular clustering is so far only described in Xenopus laevis. We traced olfactory projections to the bulb in tadpoles belonging to six distantly related anuran species in four families (Pipidae, Hylidae, Bufonidae, Dendrobatidae) and found that glomerular clustering is remarkably conserved. The general bauplan consists of four unequally sized glomerular clusters with minor inter-species variation. During metamorphosis, the olfactory system undergoes extensive remodeling. Tracings in metamorphotic and juvenile Dendrobates tinctorius and Xenopus tropicalis suggest a higher degree of variation in the glomerular organization after metamorphosis is complete. Our study highlights, that the anatomical organization of glomeruli in the main olfactory bulb (MOB) is highly conserved, despite an extensive ecomorphological diversification among anuran tadpoles, which suggests underlying developmental constraints.

Multi-glomerular projection of single olfactory receptor neurons is conserved among amphibians.

Individual receptor neurons in the peripheral olfactory organ extend long axons into the olfactory bulb forming synapses with projection neurons in spherical neuropil regions, called glomeruli. Generally, odor map formation and odor processing in all vertebrates is based on the assumption that receptor neuron axons exclusively connect to a single glomerulus without any axonal branching. We comparatively tested this hypothesis in multiple fish and amphibian species (both sexes) by applying sparse cell electroporation to trace single olfactory receptor neuron axons. Sea lamprey (jawless fish) and zebrafish (bony fish) support the unbranched axon concept, with 94% of axons terminating in single glomeruli. Contrastingly, axonal projections of the axolotl (salamander) branch extensively before entering up to six distinct glomeruli. Receptor neuron axons labeled in frog species (Pipidae, Bufonidae, Hylidae, and Dendrobatidae) predominantly bifurcate before entering a glomerulus and 59 and 50% connect to multiple glomeruli in larval and postmetamorphotic animals, respectively. Independent of developmental stage, lifestyle, and adaptations to specific habitats, it seems to be a common feature of amphibian olfactory receptor neuron axons to frequently bifurcate and connect to multiple glomeruli. Our study challenges the unbranched axon concept as a universal vertebrate feature and it is conceivable that also later diverging vertebrates deviate from it. We propose that this unusual wiring logic evolved around the divergence of the terrestrial tetrapod lineage from its aquatic ancestors and could be the basis of an alternative way of odor processing.

Amphibian larvae and zinc sulphate: a suitable model to study the role of brain-derived neurotrophic factor (BDNF) in the neuronal turnover of the olfactory epithelium.

The vertebrate olfactory system has fascinated neurobiologists over the last six decades because of its ability to replace its neurons and synaptic connections continuously throughout adult life, under both physiological and pathological conditions. Among the factors that are proposed to be involved in this regenerative potential, brain-derived neurotrophic factor (BDNF) is a candidate for having an important role in the neuronal turnover in the olfactory epithelium (OE) because of its well-documented neurogenic and trophic effects throughout the nervous system. The aim of the present study was to generate a suitable model to study the participation of BDNF in the recovery of the OE after injury in vivo. We developed an experimental design in which the OE of Rhinella arenarum tadpoles could be easily and selectively damaged by immersing the animals in ZnSO(4) solutions of various concentrations for differing time periods. Image analysis of histological sections showed that different combinations of each of these conditions produced statistically different degrees of injury to the olfactory tissue. We also observed that the morphology of the OE was restored within a few days of recovery after ZnSO(4) treatment. Immunohistochemical analysis of BDNF was performed with an antiserum whose specificity was confirmed by Western blotting, and which showed drastic changes in the abundance and distribution pattern of this neurotrophin in the damaged olfactory system. Our results thus suggest that BDNF is involved in the regeneration of the OE of amphibian larvae, and that our approach is suitable for further investigations of this topic.

Heterogeneous distribution of G protein alpha subunits in the main olfactory and vomeronasal systems of Rhinella (Bufo) arenarum tadpoles.

We evaluated the presence of G protein subtypes Galpha(o), Galpha(i2), and Galpha(olf) in the main olfactory system (MOS) and accessory or vomeronasal system (VNS) of Rhinella (Bufo) arenarum tadpoles, and here describe the fine structure of the sensory cells in the olfactory epithelium (OE) and vomeronasal organ (VNO). The OE shows olfactory receptor neurons (ORNs) with cilia in the apical surface, and the vomeronasal receptor neurons (VRNs) of the VNO are covered with microvilli. Immunohistochemistry detected the presence of at least two segregated populations of ORNs throughout the OE, coupled to Galpha(olf) and Galpha(o). An antiserum against Galpha(i2) was ineffective in staining the ORNs. In the VNO, Galpha(o) neurons stained strongly but lacked immunoreactivity to any other Galpha subunit in all larval stages analyzed. Western blot analyses and preabsorption experiments confirmed the specificity of the commercial antisera used. The functional significance of the heterogeneous G-protein distribution in R. arenarum tadpoles is not clear, but the study of G- protein distributions in various amphibian species is important, since this vertebrate group played a key role in the evolution of tetrapods. A more complete knowledge of the amphibian MOS and VNS would help to understand the functional organization and evolution of vertebrate chemosensory systems. This work demonstrates, for the first time, the existence of a segregated distribution of G-proteins in the OE of R. arenarum tadpoles.

MALDI-MS argininyl bufadienolide esters fingerprint from parotoid gland secretions of Rhinella arenarum: Age, gender, and seasonal variation.

In many amphibians, the granular glands can be grouped in special regions forming macroglands. This is the case of toads, characterized by the presence of a pair of parotoid macroglands, strategically located to give protection by poison release in case of attacks. The product secreted consists of a wide variety of chemical compounds including proteins, peptides, biogenic amines, toxic steroidal bufadienolides, and various alkaloids, depending on the species. In this work, using Rhinella arenarum, we have performed, for the first time, the matrix assisted-ultraviolet laser desorption/ionization mass spectrometry and tandem mass spectrometry characterization of the components of the secretion used as crude material, just suspended in MeOH (or MeCN). The crude sample as a whole (whole suspension) was spotted on the matrix assisted-ultraviolet laser desorption plate for analysis. Electrospray ionization-Orbitrap was used for cross-checking experiments. The pattern of signals obtained at m/z ranges 600 to 800 and 1200 to 1600 could be assigned as the argininyl bufadienolide esters fingerprint characteristic of female and male. Variation patterns for gender (female, male), age (non-reproductive, reproductive), and season (non-reproductive, reproductive) are described.

Overcrowding-mediated stress alters cell proliferation in key neuroendocrine areas during larval development in Rhinella arenarum.

Exposure to adverse environmental conditions can elicit a stress response, which results in an increase in endogenous corticosterone levels. In early life stages, it has been thoroughly demonstrated that amphibian larval growth and development is altered as a consequence of chronic stress by interfering with the metamorphic process, however, the underlying mechanisms involved have only been partially disentangled. We examined the effect of intraspecific competition on corticosterone levels during larval development of the toad Rhinella arenarum and its ultimate effects on cell proliferation in particular brain areas as well as the pituitary gland. While overcrowding altered the number of proliferating cells in the pituitary gland, hypothalamus, and third ventricle of the brain, no differences were observed in areas which are less associated with neuroendocrine processes, such as the first ventricle of the brain. Apoptosis was increased in hypothalamic regions but not in the pituitary. With regards to pituitary cell populations, thyrotrophs but not somatoatrophs and corticotrophs showed a decrease in the cell number in overcrowded larvae. Our study shows that alterations in growth and development, produced by stress, results from an imbalance in the neuroendocrine systems implicated in orchestrating the timing of metamorphosis.