Rakesh K. Rastogi

Localization of FMRFamide-Like Immunoreactivity in the Brain of the Viviparous Skink (Chalcides chalcides)

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.

Reproduction in the Mexican leaf frog, Pachymedusa dacnicolor: III. The female

The female of the Mexican leaf frog, Pachymedusa dacnicolor, displays a marked annual ovarian cycle. This consists of a long period of vitellogenic stasis, starting in early fall, soon after the breeding season, and ending in early spring. Oogonial proliferation continues throughout the year and new previtellogenic follicles are formed continuously. During the period of vitellogenesis, from spring to early summer, early, advanced, and postvitellogenic follicles are all found together, a situation that continues through the breeding season. This is correlated with the fact that a breeding female can lay three or more clutches per season. Breeding can begin as early as June and end as late as early September, with peak spawning activity taking place during July and August. An examination of the ovarian hormone secretion pattern in P. dacnicolor during the year revealed that plasma levels of testosterone and estradiol correlated with ovarian growth and attained highest levels in amplectant and ovulating females. Both hormones showed quite similar plasma levels and patterns of change during the annual reproductive cycle. Lowest plasma levels of testosterone and estradiol were found during fall and winter, in females possessing exclusively previtellogenic ovarian follicles. Plasma progesterone levels were maintained at a very low level throughout the year, except for the ovulatory surge, when amplectant and ovulating females may show a three- to sixfold increase. Plasma androstenedione showed a low peak during this phase of the reproductive cycle. Plasma levels of 5 alpha-dihydrotestosterone were 13 to 30 times lower than plasma testosterone levels. The potential roles of these gonadal steroids in controlling ovarian activity and reproduction are discussed briefly.

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.

The effects of antiandrogens and antiestrogens in nonmammalian vertebrates

A number of compounds with antiandrogenic and antiestrogenic activity have been tested on representative species of fish, amphibians, reptiles, and birds. The results obtained so far show that cyproterone acetate (CPA) and ICI 46474 behave in a manner similar to that reported from mammalian studies. This applies particularly to their ability to inhibit testosterone- and estradiol-sensitive target tissues. It has also been shown in the frog that CPA competes specifically for the androgen receptor molecules in the thumb pads, but does not interfere with testosterone secretion. CPA did not, however, inhibit the masculinizing effects of testosterone propionate or of 11-ketotestosterone in the teleost Xiphophorus helleri. Earlier reports had found CPA to be effective in causing regression of the seminal vesicles in the catfish. In a frog oviduct test some steroids, like 6-chloro-17α-hydroxy-pregna-4,6-diene-3,20-dione, 2α,17α-dimethyl-dihydrotestosterone, and 2α-methyl-dihydrotestosterone, manifest antiestrogenic effects. ICI 46474 is estrogenic in high doses but antiestrogenic in low amounts. As far as the mechanism of action of these compounds is concerned it is only possible to claim for CPA that it competes against the steroid for the receptor sites on the target organ. Finally the challenging problem of the nature of embryonic sex inductors in frog tadpoles has been approached through the use of such antihormones. The mesculinizing effects of CPA on one hand and lack of any effect of ICI 46474 on the other strongly suggest that embryonic sex inductors are different from gonadal steroids.

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 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.

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.

Comparative immunocytochemical study of FMRFamide neuronal system in the brain of Danio rerio and Acipenser ruthenus during development.

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.

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.

Immunoreactive luteinizing hormone-releasing hormone in the frog (Rana esculenta) brain: Distribution pattern in the adult, seasonal changes, castration effects, and developmental aspects

The present work describes the neuroanatomical distribution of the immunoreactive luteinizing hormone-releasing hormone (ir-LHRH) system in the brain of adult male and female, castrated male and developing Rana esculenta. No obvious sex differences in the distribution pattern of ir-LHRH were observed. Immunoreactive neuronal cell bodies are not contained within a single anatomically defined area of the brain. They are present as distinct groups in the olfactory bulbs, medial septal area, anterior preoptic area (APOA), retrochiasmatic area of the infundibulum, and interpeduncular nucleus-tegmentum area. Of the entire brain, the medial septal-APOA region exhibits the highest frequency of ir-LHRH cell bodies in both sexes. ir-LHRH fiber projections are present in the olfactory bulbs, medial septal area, APOA, floor of the diencephalon, subhabenular-periventricular area in the epithalamus, lateral suprachiasmatic area, ventrolateral infundibulum, median eminence, pars nervosa, optic tectum, interpeduncular nucleus-tegmentum area, and rhombencephalon grey. Castration seems to bear no effect on the pattern of ir-LHRH system in the frog brain. The influence of castration consisted in decreased intensity of the immunostaining and frequency of occurrence of the septal-APOA neuronal cell bodies. In median eminence, castration also induced a sensible decrease in the immunoreactivity, whereas in the pars nervosa of 50-day castrates ir-LHRH fibers totally disappeared. During ontogenesis, ir-LHRH elements first become evident in stage 31 tadpoles (beginning of metamorphic climax); LHRH immunoreaction is restricted to the cell bodies and fibers in the APOA and some fibers in the ventral hypothalamus and a few in median eminence. This condition remains unaltered until stage 33 when the tail is almost totally resorbed. The possible implications of the ir-LHRH-containing brain areas in the different aspects of reproduction in the frog are briefly discussed.

Development and distribution of gonadotropin-releasing hormone neuronal systems in the frog (Rana esculenta) brain: immunohistochemical analysis

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 [His5,Trp7,Tyr8]GnRH (cGnRH-II). Later in development, mammalian [Tyr5,Leu7,Arg8] 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.