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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.

The extrabulbar olfactory projections (EBOP) is a collection of nerve fibers that originate from primary olfactory receptor neurons. These fibers penetrate into the brain, bypass- ing the olfactory bulbs (OBs). While the presence of an EBOP has been well established in tele- osts, 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 pri- mary olfactory fibers running ventromedially as extrabulbar primary olfactory projections reached various regions of the secondary prosencephalon. Only in Moenkhausia sanctaefilome- nae (no olfactory peduncle) did lipophilic tracer-labeled fibers reach the ipsilateral mesencepha- lon. 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 cel- lular or fiber component as visualized using tracer molecules. Our results showed a certain uni- formity 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 ana- tomical entities. Microsc. Res. Tech. 78:268-276, 2015. V C 2015 Wiley Periodicals, Inc.

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. J. Comp. Neurol. 522:2980–3003, 2014. © 2014 Wiley Periodicals, Inc.

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. © 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Summary The influence of cyproterone acetate (CA) (0.1 mg/mouse/day) on the epididymis and male fertility was analyzed in a long-term study in the Swiss albino cc strain. CA inhibits epididymal function in all segments as measured by organ weights, protein content and s-glucuronidase activity. This antiandrogen also suppresses male fertility. It is important to note that the low dose of CA, used in this study, does not affect spermatogenesis. The effects of CA on epididymis and fertility are reversible and the magnitude of recovery or the period (in days) required for a significant recovery after the discontinuation of CA were seemingly directly proportional to the duration of antiandrogen treatment. Langzeit-Studie uber den Einflus von Cyproteronacetat auf den Nebenhoden und die Fruchtbarkeitsbeeinflussung bei der Maus Zusammenfassung In einer Langzeitstudie (180 Tage) wurde der Einflus von Cyproteronacetat (CPA) auf die Funktion des Nebenhodens und auf die Fruchtbarkeitsbeeinflussung bei Schweizer Albino-Mausen untersucht. Gepruft wurden die Proteinkonzentrationen im Kopf-, Korpus-und Cauda-Bereich des Nebenhodens sowie die β-Glukuronidaseaktivitat. Es wird festgestellt, das CPA die Nebenhodenfunktion in alien Segmenten hemmt. Ebenso unterdruckt CPA die Fertilitat der untersuchten Tiere. Als sehr wahrscheinlich wird herausgestellt, das dieses Antiandrogen in der angegebenen geringen Dosis keine Beeintrachtigung der Spermatogenese bedingt. Der Einflus von CPA auf die Nebenhodenfunktion und auf die Fruchtbarkeitsrate ist reversibel.

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.

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. J. Comp. Neurol. 522:2980–3003, 2014.

Neuroanatomical distribution of FMRFamide-like immunoreactivity was investigated in the brain and olfactory system of the viviparous skink, Chalcides chalcides. In the adult brain FMRFamide immunoreactive (ir) perikarya were observed in the diagonal band of Broca, medial septal nucleus, accumbens nucleus, bed nucleus of the anterior commissure, periventricular hypothalamic nucleus, lateral forebrain bundle, and lateral preoptic, subcommissural, suprachiasmatic and lateral hypothalamic areas. This pattern was seen in both male and female brains. Though all major brain areas showed FMRFamide-ir innervation, the densest ir fiber network was observed in the hypothalamus. During development, ir elements were observed for the first time in embryos at mid-pregnancy. FMRFamide perikarya were located along the ventral surface of the vomeronasal nerve, in the olfactory peduncle mediobasally, as well as in the anterior olfactory nucleus and olfactory tubercle. Furthermore, some ir neurons were observed in the rhombencephalic reticular substance; however, the ir fiber network was poorly developed. Later in development FMRFamide-ir neurons appeared also in the bed nucleus of the anterior commissure as well as the rhombencephalic nucleus of solitary tract and the dorsal motor nucleus of vagus nerve. In juveniles, the distribution profile of FMRFamide immunoreactivity was substantially similar to that of the adults, with a less widespread neuronal distribution and a more developed fiber network. Ontogenetic presence of FMRFamide immunoreactivity in the nasal area has been linked to the presence of a nervus terminalis in this reptile.

Daily s.c. injections of cyproterone acetate cause a time-related decrease in the weight and beta-glucuronidase activity of seminal vesicles in mouse. Protein content shows an initial increase and subsequently, after 120-180 days of treatment, a decline (reaching control levels). Acid phosphatase activity also decreases but only after long-term treatments. 5 alpha-DHT tends to bring the values of various parameters towards the control range. However, its effects are of a lesser magnitude in animals treated for 90 days with the antiandrogen. It is proposed that the antifertility action of cyproterone acetate is due mainly to its negative influence not only on the epididymis but also on seminal vesicles.

The distribution of FMRFamide (FMRFa)-like peptides in caiman (Caiman crocodilus) and turtle (Trachemys scripta elegans) brains was studied by immunohistochemistry. In both species, distinct groups of FMRFa-like immunoreactive (ir) perikarya were present in the medial septal nucleus, accumbens nucleus, nucleus of the diagonal band of Broca, suprachiasmatic area, lateral hypothalamic area, and periventricular hypothalamic nucleus. A few FMRFa-ir neurons in the hypothalamic area were located in the neuroepithelial cell lining of the third ventricle. FMRFa-ir fibers were scattered in all major areas of the brain, from the olfactory bulbs to the rhombencephalon. They formed dense aggregates in the medial septal area, basal telencephalon, median eminence, and infundibulum, and adjacent to the fourth ventricle. The most obvious difference between the FMRFa-ir systems in caimans and turtles concerned the number of nuclei that contained neurons with this immunoreactivity. Eight such clusters were present in the caiman brain, whereas thirteen clusters were found in the turtle brain. The turtle also displayed scattered FMRFa-ir somata in the anterior olfactory nucleus, striatum, lateral septal nucleus, medial and lateral cortex, medial forebrain bundle, lateral preoptic area, and lateral geniculate nucleus. In the caiman brain, a few FMRFa-ir neurons were noted in the ventrolateral area of the pallial commissure and an even smaller number of ir neurons was found dispersed in the optic tracts. Neither formed nuclear aggregates. The results are compared with those described for other vertebrates.

FMRFamide is a small neuropeptide present in particular neurons of the basal forebrain and midbrain of the vertebrate groups studied, especially fishes and mammals. In order to assess interspecies variation, the distribution of FMRFamide-like immunoreactivity was studied in the brains of 13 species of amphibian. Although FMRFamide-immunoreactive (IR) terminals occurred throughout much of the brain, IR cell groups were noted in circumscribed regions of the CNS. In the eight anuran species studied, two major populations of labeled perikarya were observed: one in the septopreoptic area and another one in the caudal portion of the diencephalon. The rostrocaudal extent of both and the number of labeled somata in each neuronal group displayed species-specific differences. In urodeles and gymnophiones, labeled perikarya were located in the diencephalon, but there were remarkable species differences in the number of such cells. It is discussed whether sex or season of collection may account for some of the differences observed. The distribution of FMRFamide-IR perikarya, fibers, and pathways in the brain of anurans, urodeles, and gymnophiones was compared. The existence of FMRFamide perikarya in the anterior preoptic neuropil and medial septum appeared to be a feature common to all anurans; labeled neurons in the dorsal thalamus, however, may be present only in the (viviparous) gymnophione Typhlonectes compressicauda. Cerebrospinal fluid contacting FMRFamide neuronal cell bodies and fibers were observed in each of the three taxonomic orders. The data are compared with those previously obtained for other groups of

From a comparative viewpoint, we have investigated the presence and neuroanatomical distribution of gonadotropin-releasing hormone (GnRH)-immunoreactive material in the brain of a gymnophione amphibian, Ichthyophis beddomei. Immunocytochemical analysis of the adult brain and terminal nerves in both sexes shows the presence of neurons and fibers containing mammalian GnRH (mGnRH)- and chicken GnRH-II (cGnRH-II)-like peptides. With respect to GnRH-immunoreactive material, there are two distinct neuronal systems in the brain: one containing mGnRH, which is located in the forebrain and terminal nerve, and the other containing cGnRH-II, which is restricted to the midbrain tegmentum. Basically, this distribution pattern parallels that of many species of anurans and a urodele. Whereas the presence of cGnRH-II-immunoreactive fibers in the dorsal pallium of I. beddomei is a feature in common with a urodele amphibian, the total absence of cGnRH-II-like material in the median eminence is unique to this species. It is suggested here that the distribution profile of GnRH-like material within the brain and terminal nerve ofI. beddomeirepresents a primitive pattern. J. Comp. Neurol. 384:283‐292, 1997. r 1997 Wiley-Liss, Inc. Indexing terms: caecilian brain; immunocytochemistry; gonadotropin-releasing hormone; terminal nerve

The ontogenetic pattern of immunohistochemically detectable FSH beta and LH beta cells was investigated in the pars distalis of the pituitary of the frog, Rana esculenta. The appearance, distribution, and percentage of these cells were examined in tadpoles from soon after hatching to the end of metamorphosis and in juveniles. We used monoclonal antibodies against bullfrog FSH beta and LH beta for single staining, and either mouse anti-bullfrog LH beta + guinea pig anti-rat FSH beta or rabbit polyclonal anti-bullfrog LH beta + mouse monoclonal anti-bullfrog FSH beta for double staining. The first appearance of gonadotropes, immunopositive for FSH beta, was revealed in stage 26 tadpoles. In successive stages of development the percentage of FSH beta-positive cells increased progressively and significantly. The mean percentage of these in the pars distalis cells increased from 0.7% in stage 26 to nearly 10% during the metamorphic climax (stages 31-33). In juveniles, the mean percentage of FSH beta-positive cells increased more than twofold compared to the climax value. The appearance of LH beta-positive cells was first recorded during the climax, and the mean percentage of LH beta-positive cells in juveniles reached levels as high as 30% or more, exceeding the number of FSH beta-positive cells. In climax, all LH beta-positive cells stained with anti-FSH beta as well. In juveniles, however, up to 80% of gonadotropes demonstrated colocalization of FSH beta and LH beta staining. We argue that both gonadotropins may be synthesized in all gonadotropes, and a small number of cells immunoreactive to either of the two gonadotropins may simply indicate that at that particular moment the cell contained detectable amounts of only one form of gonadotropin. These observations are discussed in relation to the possible involvement of hypothalamic influence in the differentiation of gonadotropes of the pituitary.

The presence and distribution of gonadotropin-releasing hormone (GnRH) has been investigated in the Mexican leaf frog,Pachymedusa dacnicolor,brain during development and in the adult. The ontogenetic pattern of GnRH neurons illustrates their extracranial as well as intracranial sites. Immunohistochemical analysis indicates that GnRH-immunoreactive neurons appear during the metamorphic climax. They are located in the mesencephalon and subsequently other GnRH neurons appear in the peripheral terminal nerve and anterior preoptic area of the brain. Use of specific antisera and homologous combined with heterologous preabsorption tests indicate that mammalian and chicken GnRH-II-like peptide-containing neurons are differentially located within the brain, the former in the anterior preoptic area and peripheral terminal nerve and the latter in the mesencephalon. HPLC and RIA data suggest the presence of three forms of immunoreactive GnRH in theP. dacnicolorbrain. A mammalian GnRH-like molecule and a chicken GnRH-II-like molecule are present. A third form, suspected to be [hydroxyproline9]mGnRH elutes before the mammalian GnRH.

The immunohistochemical localization of neuropeptide Y (NPY)-like peptide has been investigated in the peripheral terminal nerve, brain and pituitary of the frog, Rana esculenta, during development. Soon after hatching, a rather simple NPY-immunoreactive (-ir) neuronal system is present, with elements located mainly in the diencephalon. When hind limbs appear and develop, the NPY-neuronal system undergoes considerable elaboration and NPY-ir perikarya appear in several regions of the telencephalon (dorsal, medial, and lateral pallium; medial septum; medioventral telencephalon; anterior preoptic area), diencephalon (ventromedial, central and posterior thalamic nuclei; suprachiasmatic nucleus; infundibulum), mesencephalon (anteroventral mesencephalic tegmentum), and rhombencephalon (central grey; area of the cerebellar and vestibular nuclei). The frequency of NPY-ir neurons increases during larval development, and then decreases in the anterior preoptic area during the metamorphic climax. Dense plexuses of NPY-ir fibers are formed in several brain areas. NPY-ir fibers are found in the peripheral terminal nerve, and ir-neurons through its course along the ventromedial surface of the olfactory bulbs. NPY-ir fiber projections to the median eminence and pars intermedia derive mainly from the ventral infundibular group of NPY-ir neurons, with a contribution from the suprachiasmatic group of NPY neurons. NPY and carboxyl terminal flanking peptide of proneuropeptide Y coexist in the same neurons throughout the brain. The ontogenetic pattern of NPY-ir neuronal system in the brain of Rana esculenta is remarkably different than that reported for Xenopus laevis.

Two forms of gonadotropin-releasing hormone (mammalian, mGnRH and chicken-II, cGnRH-II) were measured by radioimmunoassay in the nasal area (containing peripheral terminal nerve), brain and pituitary of Rana esculenta during larval development, metamorphosis, and until prior to becoming reproductively active. Small amounts of both forms of GnRH were first detected in the brain extract of early tadpoles (stage 26–27, when hindlimbs begin to develop). Later, there was a gradual, but constant, stage-dependent increase in the brain content of GnRHs, with the most remarkable increase recorded at postclimax and in young frogs. In tadpoles, postclimax froglets, and young frogs, the brain concentration of mGnRH was higher than that of cGnRH-II, with a ratio of approximately 2:1 in favor of mGnRH. In juveniles, however, the brain extract contained more cGnRH-II than mGnRH. No GnRH immunoreactivity was detected in the nasal area until stage 31. In successive stages of development, however, only mGnRH was present in the nasal area, and this confirmed our previous immunohistochemical analysis which showed that the peripheral terminal nerve contains only mGnRH-immunoreactive neurons and fibers. Although both GnRH forms were detected in the anterior (telencephalon, diencephalon) and posterior (mesencephalon, rhombencephalon) brain halves from juveniles, mGnRH content predominated in the anterior half, whereas in the posterior half cGnRH-II was present in greater amounts. Pituitaries from male and female postclimax froglets and young frogs contained both forms of GnRH in a ratio of approximately 10:1 in favor of mGnRH. This finding may shed light on the question of which GnRH(s) regulate gonadotropin release from the pituitary. The developmental changes in GnRH-immunoreactive content of the brain and pituitary have been discussed in the light of functional maturation of the brain-pituitary-gonad axis.

The ontogenesis of the gonadotropin-releasing hormone (GnRH) neuronal systems was studied in the brain of the frog, Rana esculenta . Attention was also focussed on the differential distribution of molecular forms of GnRH during development. The first GnRH-immunoreactive neurons appear in the mesencephalon of posterior limb-stage tadpoles. These neurons are shown to contain only chicken [His 5 ,Trp 7 Tyr 8 ]GnRH (cGnRH-II). Later in development, mammalian [Tyr 5 ,Leu 7 ,Arg 8 ] GnRH (mGnRH)-like peptide-containing neurons appear simultaneously in the terminal nerve as well as in the anterior preoptic area of the telencephalon. Subsequently, only after metamorphosis, mGnRH-containing neurons appear in the medial septal area of the posterior telencephalon. It is here shown that neurons containing the two forms of GnRH are distributed in distinct brain areas during development and in the adult: mGnRH-immunoreactive neurons in the terminal nerve, olfactory bulb, mediobasal telencephalon, medial septal area, anterior preoptic area, ventrolateral thalamus and infundibulum, whereas cGnRH-II neurons are located in the mesencephalon. We hypothesize that the terminal nerve/forebrain-located GnRH neurons express immunohistochemically late in development and originate extracranially migrating centrally, along the terminal nerve, during development, whereas those located in the mesencephalon express earlier and may have an intracranial site of origin.

Developmental stages of Rana esculenta, starting with the posterior limb-bud stage (stage 26) up to a few days after metamorphosis, were examined immunohistochemically to localize cells and fibers producing some POMC-derived peptides, namely, alpha-MSH, ACTH and beta-END. Anti ACTH and anti alpha-MSH revealed a positive reaction in the pars intermedia during all stages of development included in this study, whereas no immunoreactivity in this pituitary zone was ever evidenced with anti beta-END. In the pars distalis strongly positive cells were seen with anti ACTH and anti beta-END, while anti alpha-MSH yielded weakly positive cells. Interestingly, these peptides were colocalized in the same cells. Immunoreactivity for alpha-MSH was no longer present in the pars distalis during metamorphic climax and postmetamorphosis. In the brain of premetamorphic tadpoles, belonging to stages 26 to 30, a few neurons in the posterior telencephalon showed a positive reaction only with anti alpha-MSH, but from stage 31 (prometamorphosis) onwards, ACTH and beta-endorphin-like peptide producing cells, together with alpha-MSH-immunoreactive cells, were seen in this region and in the anterior preoptic area and infundibulum. This situation persisted in the subsequent stages of development. Anti alpha-MSH also revealed weakly positive cells in the olfactory epithelium in premetamorphic tadpoles; strong immunoreactivity with anti alpha-MSH was seen in olfactory epithelium cells in animals during prometamorphosis, metamorphic climax and postmetamorphosis. The possible significance of these findings is briefly discussed.

Localization of GnRH-immunoreactive neuronal system was studied by immunohistochemistry in the nasal-brain area of the crested newt, Triturus carnifex. Besides adults, developmental stages were those from hatchlings up to complete metamorphosis. Neurons containing immunoreactive GnRH were first detected in the nasal area of larvae with yet undifferentiated gonads. Subsequently, in prometamorphic stages, GnRH-immunoreactive cell bodies and fibers were detected in the proximal part of the terminal nerve as well as along the ventromedial surface of the olfactory bulbs. In older larvae with sexually differentiated gonads and up to the metamorphic climax GnRH-neurons were detected, as a rostral to caudal continuum, along the ventromedial surface of the olfactory bulbs and midtelencephalon. This is exactly the route followed by the terminal nerve. In the adult brain, besides the presence of occasional GnRH-neurons and fibers in the terminal nerve proximal to olfactory bulbs, olfactory bulbs and the mid-basal telencephalon, another aggregate of immunoreactive neurons was present in the anterior preoptic area, and a greater number of fibers in the habenular area as well as in the infundibular floor, median eminence and pars nervosa. These data suggest the nasal area to forebrain migration (along the course of the terminal nerve) of GnRH-neurons during development in the crested newt.

Mast cells are best known as multifunctional entities that may confer a benefit on immune system. This review presents the known facts on mast-cell system and breakthroughs in mast-cell biology in fish, amphibians, reptiles, and birds. As compared to mammals, there are relatively few data available on mast cells in many nonmammalian vertebrates. Nevertheless, like in mammals, mast cells in nonmammalian vertebrates contain a wide range of bioactive compounds including histamine, heparin, neuropeptides, and neutral proteases. In bony fishes, these cells secrete antimicrobial peptides as well. Mast cells have a widespread distribution in the brain, endocrine glands, intestine, liver, kidney, skin, tongue, and lungs, the highest concentration occurring in different tissues in the different taxa. Currently, researchers are grappling with the nature of scientific support to substantiate the functional importance of mast cells in nonmammalian vertebrates. Ultimately, the origin and evolution of vertebrate mast cell is of great interest to comparative immunologists seeking an underlying trend in the phylogenetic development of immunity.

Anuran amphibians display a variety of reproductive modes including, in one genus, a non-placental viviparous species. In frogs and toads, reproduction is hormonally controlled and, in most species, environmentally mediated. The reproductive cycle includes several components: oogenesis and spermatogenesis, including cell maturation and vitellogenesis; courtship and mating; spermiation and oviposition; and the timing of these events in terms of seasonality or its absence. These topics are briefly summarized. In many species, breeding is synchronized within the population, whereas in others it is not. Some species of AQ1 anuran have predictable breeding season, whereas others are more flexible and some breed opportunistically in response to unpredictable humidity availability. Temperature is the principal environmental cue used to time and tune the pre-breeding stages of the reproductive cycle, allowing anurans to adapt their physiology in advance of predictable environmental changes. Endocrine and neuroendocrine plasticity allows non-thermal cues (photoperiod, food availability) to modulate timing to enable individuals to reproduce. Circumstantial evidence indicates that GnRH from the hypothalamus exerts its regulatory functions on the pituitary gonadotropins via blood circulation. The gonadotropins are responsible for the seasonal and/or annual developmental changes in gametogenesis and in the production of sex steroids that drive reproductive organ function, culminating in reproduction. There are many areas of uncertainly, and future integrated studies are required.

SUMMARY 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 (T3) and thyroxine (T4) 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.

Many DNA mutation-based diseases recognised in Campania have recently been related to toxic substances in illegal dumping areas. We performed a comet assay on edible frog erythrocytes to evaluate DNA damage. Differences in genotoxic parameters were observed among populations. We show that severe DNA damage occurred in the north Campania where the emergence of environmental waste exploded recently. Although a similar magnitude of genotoxic damage was observed in some southern populations, it is attributable to a massive pesticide pollution related to intensive farming. The frog species analysed seems to be a good bioindicator for detecting genotoxic effects of chemical environmental hazards.

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. © 2010 The Authors. Journal compilation © 2010 Anatomical Society of Great Britain and Ireland.

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.

During the last years, the population of Italian hare decreased significantly in central and south Italy. This is imputable to harvest, poaching, habitat fragmentation, and the probable competition with congeneric European hare introduced in the last decades by man for hunt. The goal of our work is to define the ecological characteristics of the two aforementioned species in order to understand how landscape facilitates or impedes movement. Spatially explicit models are used to identify a species ecological niche and to build a landscape model of suitability. To validate ecological modeling of landscape, we performed a population genetic analysis. Results suggest that the Italian hare shows an ecological requirement close to average of available resources in the considered landscape. The genetic structure of this autochthonous species validates the habitat suitability model and highlights the differences with European hare. This work analyzes for the first time the ecological relationship between those two sympatric species.

The presence and distribution of immunoreactive gonadotropin-releasing hormone (GnRH) in brains of adult male and female Pachymedusa dacnicolor has been studied immunohistochemically using antisera against mammalian, chicken-II, and salmon GnRHs. The distribution map of the immunoreactive-GnRH elements in the brain of P. dacnicolor is extremely simple, being limited to the anterior preoptic area-infundibulum-median eminence circuit. No sex- or reproductive status-related difference in either the distribution pattern or intensity of immunoreaction was revealed in this study. This is also the first immunohistochemical evidence of the presence of different structural forms of GnRH in the brain of an amphibian.

Immunohistochemical mapping with antibodies against four different types of gonadotropin-releasing hormone (GnRH)-like neuro-peptides has been studied in the brain of adult Rana esculenta. This study confirms the earlier described distribution pattern of the immunoreactive mammaiian GnRH system in the frog brain, as well as revealing that this system of neuronal cell bodies and fibres is immunopositive to antisera for mammalian, chicken-I, chicken-II and salmon GnRH-like molecules. The results also indicate coexistence of the four GnRH variants in the same anatomical areas. The presence of immunoreactive fibre endings in the cerebellum is also described, perhaps for the first time in the vertebrate brain. In addition, it was found that many immunoreactive GnRH fibres arising in the anterior preoptic area and thalamus-periventricular area project posteriorly to reach the interpeduncular nucleus-tegmentum area, thus connecting the diencephalon with the rhombencephalon. These data provide further information on the complex GnRH system in the frog brain. What role(s) in vivo the non-mammalian forms of GnRH-like peptides may play in amphibian reproduction is briefly discussed, and in the light of paucity of data it is here stressed that more amphibian species should be studied.

The distribution and density of cell bodies and fibers immunoreactive to GnRH-like peptides were investigated in the brain of male juvenile frogs (Rana esculenta) during postmetamorphic development. An immunohistochemical technique was used, involving antisera raised against 4 variants of GnRH: mammalian GnRH, chicken GnRH-I, chicken GnRH-II and salmon GnRH. A comparison of the immunohistochemical distribution at 8 different developmental stages shows that the maximum density of immunoreactive-GnRH elements, and the full distributional complexity of this system, is attained at the completion of spermatogenesis. Immunoreactive-GnRH cell bodies first appear in the anterior preoptic area during the metamorphic climax, and then appear sequentially in the medial septal area, tegmentum and, lastly, in the retrochiasmatic area and olfactory bulb when immunoreactive-fibers also reach the cerebellum. The GnRH system reacts positively to antisera for all 4 GnRH variants, but immunoreactivity for chicken GnRH-I is the weakest.

This study has examined the presence and distribution of extrabulbar olfactory projections (nerve fibers that originate from cells in the olfactory epithelium, bypass the olfactory bulbs and project caudally into the brain) in larvae and adult stages of a semi-aquatic frog, Rana esculenta. This species was chosen because previous work had suggested that the presence of an extrabulbar olfactory system (EBOS) may correlate with the detection of water-borne odorants. The main result is that this extrabulbar system is present in both larval and adult specimens of this frog species. In stage 26, the earliest one used in our study, the EBOS is well-developed and projects as far caudal as the rhombencephalon. During successive larval development and until the completion of metamorphosis, there occurs a progressive reduction in the caudal extent of the EBOS. This reduction is more dramatic during the metamorphic climax (stages 31-33). A reduction in the caudal extent of the extrabulbar projections in adult stages is similar to other species previously examined. We are inclined to believe that there is no correlation between a reduction in the caudal extent of the EBOS and transition from water to land.

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.

In order to study the regulation of primary spermatogonial (SPG) proliferation in the frog (Rana esculenta), mitotic and labelling indices of these cells were analysed, in vivo and in vitro, under different experimental conditions and periods of the year. Hypophysectomy, irrespective of the period of the year and independent of temperature, showed a remarkable negative influence on the mitotic or labelling index of the primary SPG. Replacement therapy with homologous pars distalis homogenate stimulated the proliferative activity, the stimulation being of significantly greater magnitude at 18°C than at 4°C. In parallel, mitotic index in vitro, in different periods of the year, after a 24-h incubation, was significantly higher at 20°C than at 8°C. At 2°C no 3H-thymidine labelling of the primary SPG was observed. Primary SPG labelling index in vitro increased with temperature, reaching the highest value at 15°C; it was, however, very low at 28°C. Under in vitro conditions FSH and LH stimulated primary SPG proliferation only when given together, but in vivo hypophysectomy stimulated SPG proliferation independently; GnRHa, thyroxine and prolactin were strongly stimulatory. The present in vitro data confirmed that testosterone acts synergistically with FSH-like substances to influence SPG proliferation. Unilateral castration rapidly increased the labelling index of the primary SPG in the remaining testis and this increased proliferative activity is assumed to be responsible for increased spermatogenetic activity and augmentation of testis mass later in time. It is suggested that temperature represents the constraint which controls the primary SPG responsiveness to hormonal factors.

This study demonstrates that the temporal pattern of incorporation of a labeled lipid precursor (Acetyl‐CoA (1‐C14)) in the liver, fat body and testis of the frog may vary during the reproductive cycle. The magnitude of this incorporation is greater in the intact autumn frogs in which the liver is the first organ to show peak lipid labeling, followed in time by the peak labeling of the fat body lipids and finally of the testis lipids. In other periods of the year this precise time sequence of incorporation is either absent or not so clear. The blood lipids are also labeled rapidly, but the amount of radiolabel incorporated usually declines significantly 3 hrs after tracer injection. Results obtained in the frogs deprived of both the fat bodies, or both the testes, or one fat body and/or testis showed that the fat bodies represent a necessary anatomical step for liver lipids en route to the testis, the blood being the main channel between the liver and the fat body.

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. © 2006 Elsevier B.V. All rights reserved.

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 μmol/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. © 2005 Wiley-Liss, Inc.

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. © 2004 Elsevier B.V. All rights reserved.

Publisher Summary This chapter provides an overview of GnRH in the invertebrates. The gonadotropin-releasing hormone (GnRH), which was previously called luteinizing hormone-releasing hormone (LHRH), represents a pivotal peptide in animal reproduction. GnRH is a decapeptide originally isolated from the porcine and ovine hypothalamus and is characterized for its ability to enhance the release of pituitary gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH). All known forms of GnRH peptides have in common a pyroglutamyl residue at the amino terminus and an amidated glycine at the carboxy terminus. Except for the octoGnRH, all other known forms are decapeptides and differ by one or more amino acids; amino acids 4 and 9 are conserved in all forms known to date. Studies involving an evolutionary approach to GnRH have adopted one particular strategy; attention is on understudied invertebrate taxonomic groups. Invertebrate species that vary in their phylogenetic relationships are likely to reveal differences, as well as similarities, in GnRH form and function. Studies on invertebrate species are beginning to provide further insights on the structural and functional similarities and differences. The perspective that evolution has acted to shape the GnRH molecule effectively leads to investigations in a comparative approach.

The development of the nervus terminalis system in the lunglish, 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. © 2002 Wiley-Liss, Inc.

Mapping of FMRFamidergic neural circuitry in the amphibian brain has been done by immunohistochemical methods. Comparative evidence suggests that there are similarities and differences in the overall pattern of distribution of FMRFamide-ir elements in the brain among the three amphibian orders and within each order. FMRFamide is expressed in neurons in some circumscribed areas of the brain. A part of these neurons is concentrated in classical neurosecretory areas of the hypothalamus in a bilaterally symmetrical fashion. Similar neurons occur occasionally in the midbrain, but are virtually absent from the hindbrain. Anurans are unique among amphibians to show FMRFamide neurons in the medial septum and diagonal band of Broca. A viviparous gymnophione is known to possess a small population of such neurons in the dorsal thalamus. Together, the FMRFamide neurons contribute to an extensive fiber network throughout the amphibian brain. Descriptive developmental studies suggest that the rostral forebrain-located FMRFamide neurons originate in the olfactory placode and then migrate into the brain along the route of the vomeronasal-olfactory-terminal nerve complex. Olfactory placodal ablation in an anuran and a urodele provide experimental support to this contention. Other FMRFamide neuronal cell groups, in the hypothalamus and dorsal thalamus, are supposed to arise from non-placodal precursors. The neuroanatomical distribution (projection of immunoreactive processes to areas of the fore-, mid-, and hindbrain as well as to cerebrospinal fluid, co-localization with other neuropeptides, and presence in the median eminence) has furnished morphological correlates of possible functions of FMRFamide in the amphibian CNS. While amphibian FMRFamide-like or structurally related peptides remain to be isolated and characterized, the sum of the distribution pattern of FMRFamide-like immunoreactivity suggests that it may act as a neurotransmitter or a neuromodulator, and also may have endocrine regulatory functions. © 2001 Wiley-Liss, Inc.

The anatomic distribution and biochemical characteristics of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) were investigated in the central nervous system of the frog, Rana ridibunda, during development. Three to four days after hatching, at stages IV-VII, PACAP-immunoreactive perikarya were detected in the dorsal thalamus within the anterior ventral area, and a few fibers were found in the medial pallium. Positive cell bodies were first observed in the hypothalamus at stages VIII-IX, at the, level of the dorsal and ventral infundibular nuclei. In these regions, the number of positive perikarya increased during ontogeny. In tadpoles, during the mid- and late premetamorphosis, a more complex organization of the PACAP-immunoreactive system was found in the thalamus with the appearance, at stages IX-XII, of two additional groups of positive neurons in the ventrolateral area and posterocentral nucleus. At stages XIII-XVIII of larval development and subsequent larval stages, PACAP-immunoreactive fibers were found in the median eminence. In newly metamorphosed animals, several additional groups of positive perikarya appeared in the medial pallium, the preoptic nucleus, the torus semicircularis, the tegmentum of the mesencephalon, and the cerebellum. The immunoreactive peptide contained in the tadpole brain was characterized by high performance liquid chromatography analysis combined with radioimmunoassay quantification. At all stages investigated, the predominant form of PACAP-immunoreactive material coeluted with synthetic frog PACAP38. The occurrence of PACAP soon after hatching indicates that the peptide may exert neurotrophic activities. The existence of immunoreactive elements in several thalamic regions at mid- and late premetamorphic stages suggests that PACAP may act as a neurotransmitter, neuromodulator, or both, during ontogenesis. Finally, the presence of PACAP-immunoreactive perikarya in hypothalamic nuclei and nerve fibers in the median eminence supports the view that PACAP may play a role in the control of pituitary hormone secretion during larval development. © 2001 Wiley-Liss, Inc.

By using immunohistochemistry, we studied the development and distribution of the FMRFamide-like immunoreactive (ir) neuronal system in the toad brain during the ontogeny. In addition to this, experimental evidence was provided to show that the rostral forebrain-located FMRFamide neurons originate in the olfactory placode and then migrate into the brain along the olfactory pathway. During early development, within the brain, FMRFamide-ir perikarya first appeared in the periventricular hypothalamus. Later in development, FMRFamide-ir cells were visualized in the rostralmost forebrain simultaneously with similar ir cells in the developing olfactory mucosa. Selective ablation of the olfactory placode(s), prior to the appearance of the first FMRFamide-ir cells in the brain, resulted in the total absence of ir cells in the telencephalon (medial septum and mediobasal telencephalon) of the operated sides(s). The preoptic-suprachiasmatic-infundibular hypothalamus-located FMRFamide-ir neurons were not affected by olfactory placodectomy, arguing that they do not originate in the placode. This result points to the placode as the sole source of such neurons in the rostral forebrain. © 2001 Elsevier Science B.V.

The distribution of FMRFamide-like immunoreactive (ir) neurons and fibers was investigated in the central nervous system of developing zebrafish and juvenile sturgeon (sterlet). Adult zebrafish was also studied. In zebrafish embryos FMRFamide-ir elements first appeared 30 h post- fertilization (PF). Ir somata were located in the olfactory placode and in the ventral diencephalon. FMRFamide-ir fibers originating from diencephalic neurons were found in the ventral telencephalon and in ventral portions of the brainstem. At 48 h PF, the ir perikarya in the olfactory placode displayed increased immunoreactivity and stained fibers emerged from the somata. At 60 h PF, bilaterally, clusters of FMRFamide-ir neurons were found along the rostro-caudal axis of the brain, from the olfactory placode to rostral regions of the ventro-lateral telencephalon. At 60 h PF, numerous ir fibers appeared in the dorsal telencephalon, optic lobes, optic nerves, and retina. Except for ir fibers in the hypophysis at the age of 72 h PF, and a few ir cells in the nucleus olfacto-retinalis (NOR) at the age of 2 months PF, no major re-organization was noted in subsequent ontogenetic stages. The number of stained NOR neurons increased markedly in sexually mature zebrafish. In adult zebrafish, other ir neurons were located in the dorsal zones of the periventricular hypothalamus and in components of the nervus terminalis. We are inclined to believe that neurons expressing FMRFamide originate in the olfactory placode and in the ventricular ependyma in the hypothalamus. On the same grounds, a dual origin of FMRFamide-ir neurons is inferred in the sturgeon, an ancestral bony fish: prior to the observation of ir cells in the nasal area and in the telencephalon stained neurons were noted in circumventricular hypothalamic regions. (C) 2000 Published by Elsevier Science B.V.

The localization of vasoactive intestinal peptide (VIP)-like immunoreactive (ir) elements was investigated in the brain of the anuran amphibian, Rana esculenta, during development. Using an antiserum raised against the porcine VIP, ir cell bodies and fibers were observed in the forebrain of tadpoles a few days after hatching. During early premetamorphosis, ir perikarya were distributed in the ventral infundibular nucleus of the hypothalamus and in the posterocentral nucleus of the thalamus. Labeled fibers were detected in the olfactory bulbs and in the hypothalamus. In these larvae, furthermore, several VIP-ir cells were found in the pars distalis of the pituitary and there were ir fibers in the pars nervosa. In tadpoles at stages VIII-IX, a new group of VIP-labeled neurons was observed in the dorsal part of the infundibular nucleus. In other brain regions, the distribution of the immunoreactivity was similar to that described in the earliest stages, i.e., IV-VII. During mid-premetamorphosis, stages X-XII of development, an additional set of ir perikarya appeared in the ventrolateral area of the thalamus. During late premetamorphosis, stages XIII-XVIII, the organization of VIP-like immunoreactivity was more complex and its distribution more widespread. Two new groups of ir cell bodies appeared, one in the preoptic nucleus and another in the anteroventral area of the thalamus, and for the first time, VIP immunoreactivity was observed in the median eminence. This distribution pattern persisted through to the prometamorphic, four-limb stage. Strikingly, no VIP-ir elements were observed anywhere in the mid- and hindbrain. The present results indicate that a VIP-like ir peptide may be involved in the processing of olfactory information or may act as a neurohormone, hypophysiotropic factor, and neuromodulator in the brain of R. esculenta during development. Copyright (C) 1999 Elsevier Science B.V.

Circulating levels and pituitary content of FSH and LH were determined by specific radioimmunoassays in Rana esculenta starting a few days after hatching until the completion of metamorphosis. Both gonadotropins were found in the pituitary as well as in the blood plasma at all stages of development examined here. The plasma concentrations of FSH and LH were more or less uniform during pre- and prometamorphosis, but increased significantly at the onset of metamorphic climax. The plasma levels of FSH and LH remained high at the completion of metamorphosis. The pituitary content of FSH and LH was low in early premetamorphosis. It increased slightly through prometamorphosis and metamorphic climax, following which a highly significant increase occurred. Whereas plasma concentrations of FSH and LH were essentially similar within a single stage of development, the pituitary FSH content was severalfold higher than pituitary LH. The significance of these results is discussed in relation to the functional maturation of the brain–pituitary–gonadal axis in the frog.