Tract-tracing study of the extrabulbar olfactory projections in the brain of some teleosts.

The extrabulbar olfactory projections (EBOP) is a collection of nerve fibers that originate from primary olfactory receptor neurons. These fibers penetrate into the brain, bypassing the olfactory bulbs (OBs). While the presence of an EBOP has been well established in teleosts, here we morphologically characterize the EBOP structure in four species each with a different morphological relationship of OB with the ventral telencephalic area. Tract-tracing methods (carbocyanine DiI/DIA and biocytin) were used. FMRFamide immunoreactive nervus terminalis (NT) components were also visualized to define any neuroanatomical relationship between the NT and EBOP. Unilateral DiI/DiA application to the olfactory chamber stained the entire olfactory epithelium, olfactory nerve fibers, and ipsilateral olfactory bulb. Labeled primary olfactory fibers running ventromedially as extrabulbar primary olfactory projections reached various regions of the secondary prosencephalon. Only in Moenkhausia sanctaefilomenae (no olfactory peduncle) did lipophilic tracer-labeled fibers reach the ipsilateral mesencephalon. The combination of tracing techniques and FMRFamide immunohistochemistry revealed a substantial overlap of the label along the olfactory pathways as well as in the anterior secondary prosencephalon. However, FMRFamide immunoreactivity was never colocalized in the same cellular or fiber component as visualized using tracer molecules. Our results showed a certain uniformity in the neuroanatomy and extension of EBOP in all four species, independent of the pedunculate feature of the OBs. The present study also provided additional evidence to support the view that EBOP and FMRFamide immunoreactive components of the NT are separate anatomical entities.

A comparative cluster analysis of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry in the brains of amphibians.

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) is a key enzyme in the synthesis of the gaseous neurotransmitter nitric oxide. We compare the distribution of NADPH-d in the brain of four species of hylid frogs. NADPH-d-positive fibers are present throughout much of the brain, whereas stained cell groups are distributed in well-defined regions. Whereas most brain areas consistently show positive neurons in all species, in some areas species-specific differences occur. We analyzed our data and those available for other amphibian species to build a matrix on NADPH-d brain distribution for a multivariate analysis. Brain dissimilarities were quantified by using the Jaccard index in a hierarchical clustering procedure. The whole brain dendrogram was compared with that of its main subdivisions by applying the Fowlkes-Mallows index for dendrogram similarity, followed by bootstrap replications and a permutation test. Despite the differences in the distribution map of the NADPH-d system among species, cluster analysis of data from the whole brain and hindbrain faithfully reflected the evolutionary history (framework) of amphibians. Dendrograms from the secondary prosencephalon, diencephalon, mesencephalon, and isthmus showed some deviation from the main scheme. Thus, the present analysis supports the major evolutionary stability of the hindbrain. We provide evidence that the NADPH-d system in main brain subdivisions should be cautiously approached for comparative purposes because specific adaptations of a single species could occur and may affect the NADPH-d distribution pattern in a brain subdivision. The minor differences in staining pattern of particular subdivisions apparently do not affect the general patterns of staining across species.

Mast cell population in the frog brain: distribution and influence of thyroid status.

In the developing frog brain, the majority of mast cells (MC) are distributed in the pia mater, and some immature MC are located adjacent to the blood capillaries in and around the neuropil. In the adult brain, MC are more numerous than in pre- and pro-metamorphic tadpoles; they are mainly located within the pia mater and are particularly numerous in the choroid plexuses. Many MC are found within the brain ventricles juxtaposed to the ependymal lining. MC are rarely observed in the brain parenchyma. In the adult brain, MC number is much higher than in the brain of post-metamorphic froglets. In the latter, MC number is nearly 2-fold over that found in the pre-metamorphic brain. Treatment of pre- and pro-metamorphic tadpoles with 3,5,3'-triiodothyronine (T(3)) and thyroxine (T(4)) stimulates overall larval development but does not induce a significant change in MC population within the brain. By contrast, treatment with 6-n-propyl-2-thiouracil (PTU) delays larval development and leads to a significant numerical increase of brain MC. In the adult, PTU treatment also has a similar effect whereas hypophysectomy causes a drastic decrease of MC population. The negative effects of hypophysectomy are successfully counteracted by a two-week replacement therapy with homologous pars distalis homogenate. In the adult frog, MC population seems to be refractory to thyroid hormone treatment. The present study on frog brain suggests that pituitary-thyroid axis may be involved in the regulation of MC frequency.

Mast cells in the amphibian brain during development.

This is the first descriptive study of ontogenesis and anatomical distribution of mast cells in the developing brain of three different amphibian species. In the toad and the green frog, mast cells are preferentially located in: (i) the meningeal lining (pia mater), (ii) the choroid plexuses, both anterior and posterior, and (iii) the neuropil, in close association with the epithelial cell lining of blood vessels. It is only in the perennially aquatic African clawed frog that mast cells never appear inside brain ventricles and within the neuropil. Mast cells first become identifiable in brain of different species in different stages of development. While there are differences in the number of mast cells in different species at different stages of development, the number nearly doubles in all three species during the transition from pro-metamorphic stage of larval development to the peak of metamorphic climax. Furthermore, the number of mast cells is comparatively higher in the toad and remarkably lower in the fully aquatic Xenopus laevis, in which species the first appearance of identifiable mast cells during larval development occurs much later than in equivalent stages of development of the toad and the green frog. The secretory nature of mast cells can be assumed by the presence of cytoplasmic granules, which may show species-specific texture. Further experimental analyses are required to unveil the usefulness of mast cells in the amphibian brain.

Thyroid status can influence brain mast cell population.

Neuroanatomical mapping as well as the influence of thyroid status on brain mast cell distribution and detectable mast cell number in adult Rana esculenta is studied. Treatment with tizoxin (T4) does not modify number or activational state of brain mast cells, whereas administration of the antithyroid agent 6-n-propyl-2-thiouracil induces a significant increase (up to 40%) in the mast cell number within the telencephalon and diencephalon. Hypophysectomy induces a significant decrease (up to 65%) of mast cells in all brain regions, whereas the pituitary homogenate augments their number. The results suggest that the pituitary-thyroid axis may be involved in the regulation of brain mast cell population.

Morphological differentiation and genetic structure in island lizard populations.

Only some island populations of Podarcis sicula are hyperchromatic. The study of this phenomenon and its relationship with the lizards of the mainland and other islands, exhibiting a "normal" coloration, provides useful hints in our understanding of evolutionary mechanisms that have created the observed morphological variation. We performed a comparative morphological and genetic analysis of a hyperchromatic lizard population from Licosa Island, and compared the data with that obtained from normal-colored lizard populations both from Ustica and Cirella islands in the Tyrrhenian sea and from nearby mainland Italy. Morphological and microsatellite gene differentiation in the hyperchromatic Licosa population appears to have been much more rapid than the molecular evolution of the mtDNA. We discuss herein that the comparison of hyperchromatism and other types of morphological variation with molecular data in island populations of lizards may provide useful hints as to evolutionary mechanisms.

Proliferative activity in the frog brain: a PCNA-immunohistochemistry analysis.

By means proliferating cell nuclear antigen (PCNA) immunohistochemistry, we have provided a detailed neuroanatomical mapping of proliferative activity during development and adulthood in the frog (Rana esculenta) brain. Western blot analysis confirmed the presence of this protein in brain extracts from adults and tadpoles. Proliferative activity was observed in the ventricular and subventricular zones throughout the brain. The present study provides details as to which of the morphologically distinguishable brain region(s) has a long-lasting proliferative activity and in which region this activity undergoes a progressive decrease during development. In the subventricular zones of the third ventricle, PCNA-labeled cells were particularly abundant in the magnocellular preoptic nucleus and the ventromedial thalamic nucleus. It was observed that proliferation zones are present practically in all major subdivisions of the forebrain, midbrain and hindbrain, including the cerebellum in which PCNA-labeled cells were located in the outer granular layer and the inner molecular layer. The habenulae, epiphysis and isthmic nuclei never showed the presence of PCNA-immunoreactive nuclei. The widespread proliferative activity implies that the frog brain has a great potential for neurogenesis/gliogenesis not only during larval development but also in the adulthood.

D-aspartic acid in the nervous system of Aplysia limacina: possible role in neurotransmission.

In the marine mollusk Aplysia limacina, a substantial amount of endogenous D-aspartic acid (D-Asp) was found following its synthesis from L-aspartate by an aspartate racemase. Concentrations of D-Asp between 3.9 and 4.6 micromol/g tissue were found in the cerebral, abdominal, buccal, pleural, and pedal ganglia. In non nervous tissues, D-Asp occurred at a very low concentration compared to the nervous system. Immunohistochemical studies conducted on cultured Aplysia neurons using an anti-D-aspartate antibody demonstrated that D-Asp occurs in the soma, dendrites, and in synaptic varicosities. Synaptosomes and synaptic vesicles from cerebral ganglia were prepared and characterized by electron microscopy. HPLC analysis revealed high concentrations of D-Asp together with L-aspartate and L-glutamate in isolated synaptosomes In addition, D-Asp was released from synaptosomes by K+ depolarization or by ionomycin. D-Asp was one of the principal amino acids present in synaptic vesicles representing about the 25% of total amino acids present in these cellular organelles. Injection of D-Asp into live animals or addition to the incubation media of cultured neurons, caused an increase in cAMP content. Taken as a whole, these findings suggest a possible role of D-Asp in neurotransmission in the nervous system of Aplysia limacina.

Dehydration-regulated processing of late embryogenesis abundant protein in a desiccation-tolerant nematode.

Late embryogenesis abundant (LEA) proteins occur in desiccation-tolerant organisms, including the nematode Aphelenchus avenae, and are thought to protect other proteins from aggregation. Surprisingly, expression of the LEA protein AavLEA1 in A. avenae is partially discordant with that of its gene: protein is present in hydrated animals despite low cognate mRNA levels. Moreover, on desiccation, when its gene is upregulated, AavLEA1 is specifically cleaved to discrete, smaller polypeptides. A processing activity was found in protein extracts of dehydrated, but not hydrated, nematodes, and main cleavage sites were mapped to 11-mer repeated motifs in the AavLEA1 sequence. Processed polypeptides retain function as protein anti-aggregants and we hypothesise that the expression pattern and cleavage of LEA protein allow rapid, maximal availability of active molecules to the dehydrating animal.

Extrabulbar olfactory system and nervus terminalis FMRFamide immunoreactive components in Xenopus laevis ontogenesis.

The extrabulbar olfactory system (EBOS) is a collection of nerve fibers which originate from primary olfactory receptor-like neurons and penetrate into the brain bypassing the olfactory bulbs. Our description is based upon the application of two neuronal tracers (biocytin, carbocyanine DiI) in the olfactory sac, at the cut end of the olfactory nerve and in the telencephalon of the developing clawed frog. The extrabulbar olfactory system was observed already at stage 45, which is the first developmental stage compatible with our techniques; at this stage, the extrabulbar olfactory system fibers terminated diffusely in the preoptic area. A little later in development, i.e. at stage 50, the extrabulbar olfactory system was maximally developed, extending as far caudally as the rhombencephalon. In the metamorphosing specimens, the extrabulbar olfactory system appeared reduced in extension; caudally, the fiber terminals did not extend beyond the diencephalon. While a substantial overlapping of biocytin/FMRFamide immunoreactivity was observed along the olfactory pathways as well as in the telencephalon, FMRFamide immunoreactivity was never observed to be colocalized in the same cellular or fiber components visualized by tracer molecules. The question whether the extrabulbar olfactory system and the nervus terminalis (NT) are separate anatomical entities or represent an integrated system is discussed.

Occurrence and neuroendocrine role of D-aspartic acid and N-methyl-D-aspartic acid in Ciona intestinalis.

Probes for the occurrence of endogenous D-aspartic acid (D-Asp) and N-methyl-D-aspartic acid (NMDA) in the neural complex and gonads of a protochordate, the ascidian Ciona intestinalis, have confirmed the presence of these two excitatory amino acids and their involvement in hormonal activity. A hormonal pathway similar to that which occurs in vertebrates has been discovered. In the cerebral ganglion D-Asp is synthesized from L-Asp by an aspartate racemase. Then, D-Asp is transferred through the blood stream into the neural gland where it gives rise to NMDA by means of an NMDA synthase. NMDA, in turn, passes from the neuronal gland into the gonads where it induces the synthesis and release of a gonadotropin-releasing hormone (GnRH). The GnRH in turn modulates the release and synthesis of testosterone and progesterone in the gonads, which are implicated in reproduction.

Ontogenetic organization of the FMRFamide immunoreactivity in the nervus terminalis of the lungfish, Neoceratodus forsteri.

The development of the nervus terminalis system in the lungfish, Neoceratodus forsteri, was investigated by using FMRFamide as a marker. FMRFamide immunoreactivity appears first within the brain, in the dorsal hypothalamus at a stage around hatching. At a slightly later stage, immunoreactivity appears in the olfactory mucosa. These immunoreactive cells move outside the olfactory organ to form the ganglion of the nervus terminalis. Immunoreactive processes emerge from the ganglion of the nervus terminalis in two directions, one which joins the olfactory nerve to travel to the brain and the other which courses below the brain to enter at the level of the preoptic nucleus. Neither the ganglion of the nervus terminalis nor the two branches of the nervus terminalis form after surgical removal of the olfactory placode at a stage before the development of FMRFamide immunoreactivity external to the brain. Because this study has confirmed that the nervus terminalis in lungfish comprises both an anterior and a posterior branch, it forms the basis for discussion of homology between these branches and the nervus terminalis of other anamniote vertebrates.

Reproduction in the mexican leaf frog, Pachymedusa dacnicolor. IV. Spermatogenesis: A light and ultrasonic study.

Primary spermatogonia have highly lobate nuclei and can be distinguished as pale and dark types on the basis of nuclear and cytoplasmic features. Nuclei of secondary spermatogonia are also lobate. Primary spermatocytes have spherical nuclei and display synaptinemal complexes in late zygotene-pachytene. Spermatocytes are connected by intercellular bridges, which persist through spermiogenesis. During spermiogenesis no acrosomal granule is formed. The acrosomal vesicle is large and forms in the apical part of the cell. A helical system of perinuclear microtubules accompanies the phase of nuclear elongation. Microtubules disappear in late spermatids and there forms a compact bundle of filaments which extends into the subacrosomal area. These filaments probably derive from the breakdown of the microtubules. A mitochondrial sleeve is formed around the proximal portion of the tail and much of it is cast off in the mature spermatid. The tail is composed of a spirally coiled contractile element and a stiff fibrous axial rod connected together by an undulating membrane. The axial rod and the axoneme-associated rodlet derive from a dense, juxtacentriolar fibrous mass. Sertoli cells surrounding the spermatogonial and spermatocyte cysts are slender and have oblong nuclei. In contrast, those associated with spermatids are columnar and have deeply indented nuclei. They possess many Golgi complexes, elongated mitochondria, cisternae of smooth endoplasmic reticulum, lysosome-like bodies, masses of glycogen particles, few lipid droplets, and an array of microtubules running longitudinally around the elongating spermatid nuclei. Desmosomes are formed between adjacent Sertoli cells.