Maria Di Meglio

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.

Distribution of FMRFamide-like Immunoreactivity in the Amphibian Brain: Comparative Analysis

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 vertebrates. J. Comp. Neurol. 414:275–305, 1999.

Distribution of FMRFamide-like immunoreactivity in the brain and pituitary of Rana esculenta during development

Developmental aspects of the distribution of FMRFamide (Phe-Met-Arg-Phe-NH2) immunoreactivity (ir) were investigated by indirect immunofluorescence in the brain, pituitary and terminal nerve of the frog, Rana esculenta. Soon after hatching. FMRFamide neurons were found in the proximal terminal nerve, mediobasal olfactory bulb, caudal dorsolateral pallium, diagonal band of Broca, anterior preoptic area, suprachiasmatic area, thalamus, infundibulum, and developing pituitary. FMRFamide fibers were present in the olfactory epithelium, terminal nerve, olfactory bulbs, dorsal and midventral telencephalon, epiphysis, mediolateral thalamus, pretectal gray, optic tectum, infundibulum, posterior interpeduncular nucleus-tegmentum area, and rostral rhombencephalon. During successive developmental stages, ir neurons were no longer observed in the dorsal telencephalon and pituitary. In late larval stages, ir neurons appeared in the medial septal area, and ir fibers in the cerebellum and torus semicircularis. At the same time, the frequency of ir neurons increased progressively in the anterior preoptic area, suprachiasmatic area and infundibulum. FMRFamide-ir neurons were never revealed in mesencephalon and rhombencephalon. Numerous ir fibers terminated in the median eminence and intermediate lobe of the pituitary. The adult pattern of distribution of FMRFamide-ir elements in the brain was achieved during the postmetamorphic development. In light of the existing literature, the possible placodal origin of forebrain-located FMRFamide neurons is briefly discussed.

Immunohistochemical localization of GnRH in the crested newt (Triturus carnifex) brain and terminal nerve: a developmental study

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.

Immunohistochemical Localization of Multiple Forms of Gonadotropin‐Releasing Hormone in the Brain of the Adult Frog

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.

Circannual testicular rhythm in the green frog, Rana esculenta

Abstract We followed testis weight, seminiferous tubule diameter, spermatogenetic activity, pituitary gonadotropin secretory activity and spermiation response in Rana esculenta, under day length 12:12. On this lighting schedule, groups of frogs were studied at 4°C, 15°C and 24°C. A group of frogs reared in the laboratory under natural temperature and lighting conditions served as captive controls, whereas freshly captured frogs were used as wild controls. In frogs exposed to 24°C the testicular growth was greatly speeded up compared to other groups, but long-term exposure to this temperature apparently caused regression of pituitary gonadotropic activity and testicular growth. Circannual testicular rhythm was evident in frogs reared at constant 15°C. In the 4°C group the testicular cycle proceeded for 120 days and then apparently “stopped” at a specific phase associated with winter stasis. The rhythm, in fact, was reinitiated by an appropriate environmental input (15°C). In summary, it was demonstrated th...

Circadian variation in mitotic index of primary spermatogonia in the adult frog (Rana esculenta)

Abstract Circadian rhythmicity in the average proliferation rate of the primary spermatogonial cell crop in the frog testis was studied by the stathmokinetic method. Colchicine was used to specifically block cells after they enter metaphase. The in vivo mitotic activity of primary spermatogonia was studied in three different periods of the year and was analyzed at 8 time points of the 24‐hour day. Circadian variations were suggested in all three periods of the year. Mitotic activity of the primary spermatogonia was also analyzed in vitro at 16 time points through a 48‐hour experimental period. Daily rhythmic fluctuations in the division rate of primary spermatogonia were observed. Thus, the proportion of cells in mitosis within the primary spermatogonial cell crop of the frog testis exhibits not only significant seasonal variations (as reported elsewhere), but also a daily rhythmic variation. The percent difference between low and high values of mitotic circadian rhythm varies with season.

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. Microsc. Res. Tech. 78:268–276, 2015.