Nucleus Infundibularis Ventralis Research Articles

Reproductive phase related variations in the expression of neuropeptide, cocaine- and amphetamine- regulated transcript (CART) in the brain and pituitary gland of adult male Microhyla ornata

Cocaine- and amphetamine-regulated transcript (CART) peptide is a multifaceted neuropeptide involved in several physiological functions including appetite and reproduction. While studies in mammals, aves and fishes suggest evolutionary conserved role of CART, the information in amphibian is scanty. We have investigated the reproductive phase related variations of CART in the brain of adult male Microhyla ornata. Seasonal changes in the expression of CART peptide were noticed in the brain and pituitary of M. ornata. Significant differences were observed in the nucleus infundibularis ventralis (NIV), epiphysis (E), anteroventral tegmental region (AV), raphe nucleus (Ra) of the brain and pars intermedia (PI), pars distalis (PD) of the pituitary. Compared to the pre-breeding and post-breeding seasons, increase in CART immunoreactivity was seen in E, NIV, AV, Ra of brain and PI, PD of pituitary gland of animals collected during breeding season. Similarly, highest mRNA levels of CART were also observed in the breeding season in the middle region of brain that includes hypothalamus and pituitary gland. Variation in the levels of CART peptide and mRNA in the brain of M. ornata suggests its conserved role in seasonal control of appetite and reproduction.

Brain profiling of endogenous Neuropeptide Y (NPY) in distinct reproductive phases of adult male Microhyla ornata

Neuropeptide Y(NPY) is known to play a pivotal role in various physiological functions including appetite and reproduction. While studies in mammals, fishes and reptiles suggest a temporal and evolutionary conserved role of NPY, the information in amphibian is scanty. We have investigated the reproductive phase related variations of NPY in the brain of Microhyla ornata (M. ornata), using immunohistochemistry and reverse transcription quantitative PCR (RT-qPCR). The highest expression of NPY peptide was observed in the preoptic area (Poa), nucleus infundibularis ventralis (NIV) and nucleus reticularis isthmi (NRIS) of M. ornata in breeding season compared to pre-breeding as well as post-breeding season. In parallel, highest mRNA levels of NPY were also observed in the breeding season in the middle region of brain that includes hypothalamus of M. ornata. Variation in the levels of NPY peptide and mRNA levels in the brain of M. ornata point towards seasonal control of appetite and reproduction.

Sex-specific distribution of Neuropeptide Y (NPY) in the brain of the frog, Microhyla ornata

Neuropeptide Y (NPY) is involved in sex-specific behavioural processes in vertebrates. NPY integrates energy balance and reproduction in mammals. However, the relevance of NPY in reproduction of lower vertebrates is understudied. In the present study, we have investigated neuroanatomical distribution and sex-specific differences of NPY in the brain of Microhyla ornata using immunohistochemistry and quantitative real time PCR. NPY is widely distributed throughout the brain of M. ornata. We observed NPY immunoreactivity in the cells of the nucleus accumbens, striatum pars dorsalis, dorsal pallium, medial pallium, ventral pallium, bed nucleus of stria terminalis, preoptic nucleus, infundibular region, median eminence and pituitary gland of adult M. ornata. A higher number of NPY- immunoreactive cells were observed in the preoptic nucleus (p < .01), nucleus infundibularis ventralis (p < .001) and anteroventral tegmental nucleus (p < .001) of the female as compared to that of the male frog. Real-Time PCR revealed higher mRNA levels of NPY in the female as compared to male frogs in the mid-brain region that largely contains the hypothalamus. Sexual dimorphism of NPY expression in M. ornata suggests that NPY may be involved in the reproductive physiology of anurans.

Temperature dependent changes in cocaine- and amphetamine regulated transcript (CART) peptide in the brain of tadpole, Sylvirana temporalis

Cocaine- and amphetamine-regulated transcript peptide (CARTp) has emerged as a novel neurotransmitter in the brain. Although the physiological role of the peptide has been intensely investigated in mammals, its role in amphibians has not been investigated. In the present study, an attempt has been undertaken to study the expression of CART in the tadpole brain of frog Sylvirana temporalis, subjected to thermal stress. Cells with strong CART-immunoreactivity were observed in the nucleus preoptic area (NPO) of tadpoles exposed to high temperature (37±2°C) as compared to those in the tadpoles exposed to low (12±2°C) and normal (24±2°C) temperatures. In the ventromedial thalamic nucleus (VM) and nucleus posterocentralis thalami (NPC), moderate CART-ir cells were observed in the control groups while number of cells and intensity of immunoreactivity was increased in tadpoles at low and high temperatures. In the nucleus infundibularis ventralis (NIV) and raphe nucleus (RA), CART immunoreactivity increased in the low as well as high temperature treated groups. Intensely stained CART cells were observed in the pituitary of tadpoles exposed to high temperature as compared to low temperature and control groups. We suggest that CART system in the brain and pituitary of tadpole may play a very important role in mediating responses to temperature variations in the environment.

Nesfatin-1/nucleobindin-2 like immunoreactivity in the olfactory system, brain and pituitary of frog, Microhyla ornata

Nesfatin-1 is a recently discovered anorectic protein derived from the precursor nucleobindin-2 (NUCB2). While nesfatin-1 has been widely studied in mammals, and goldfish, there are no reports of nesfatin-1 in amphibians. Using immunohistochemistry and Western blot analysis, this study assessed the distribution of NUCB2/nesfatin-1 in the brain of frog Microhyla ornata. NUCB2/nesfatin-1 like immunoreactivity was found in the olfactory receptor neurons, Bowman’s glands and in the olfactory epithelium of medial diverticulum. In the brain, immunoreactive perikarya were seen in the anterior preoptic area, magnocellular nucleus, suprachiasmatic nucleus, ventromedial thalamic nucleus, posterior thalamic nucleus, nucleus infundibularis ventralis and dorsalis, and the cerebellar nucleus. NUCB2/nesfatin-1like immunoreactivity was also detected in the pineal and pituitary glands of frog. The presence of NUCB2/nesfatin-1 in the key brain regions suggest possible roles for this protein in the regulation of physiological processes in frogs.

Localization and Enzyme Activity of Iodotyrosine Dehalogenase 1 during Metamorphosis of Frog Microhyla ornata

The distribution and activity of iodotyrosine dehalogenase 1 (DEHAL1) enzyme was determined during metamorphosis of the frog Microhyla ornata. Immunohistochemical and enzymatic studies in the thyroid, olfactory system, brain, skin, and tail of frog were performed. DEHAL1-positive cells were seen in the olfactory epithelium, the nucleus infundibularis ventralis, the ventricular lining, cerebellum in the brain, in the pituitary gland, in the mucus glands of skin, and in epithelial cells of tadpole tail fin. During metamorphosis, DEHAL1 enzyme activity was also detected in the above-mentioned frog tissues with the enzyme exhibiting higher activity on diiodotyrosine (DIT) as compared to monoiodotyrosine (MIT). Using DIT as a substrate, maximal activity was detected at stage 44 in the brain (157.7 U/mg protein), while it was higher at stage 42 in the tail (138.5 U/mg protein). With MIT as a substrate, highest activity was detected in the brain at stage 42 (65.8 U/mg protein). Thus, the localization, relative levels, and specificity of DEHAL1 suggest that it might be involved in tissue-specific deiodination of MIT and DIT, thus making free iodine available, and therefore plays an essential role during metamorphosis in M. ornata.

Amphibians as a model for studying xenoestrogen‐dependent neuroedocrine disruption

Several environmental chemicals are known to interact with functions related with the neuroendocrine system, and many of the endocrine disruptors present in the aquatic environment have estrogenic activity and can be responsible for feminization processes in wild fish and amphibians. The estrogenic effects of such disruptors include several levels of investigation: biological significance ‘in vivo’, and ‘in vitro’ by use of estrogenic biomarkers, and effects on the brain‐pituitary‐gonadal axis. Amphibians spend a great part of their life in the water, and can be considered ‘sentinel species'; therefore, the effects of an estrogenic compound, 4‐nonylphenol, were studied by ‘in vivo’ and ‘in vitro’ experiments. As a biomarker of estrogenic effects, vitellogenin changes were assayed in male frogs and newts; the plasma sex steroid trend was evaluated, and brain aromatase‐like‐immunoreactivity was described. 4‐Nonylphenol treatment was able to induce plasma vitellogenin in male frogs and newts, changes in the secondary sexual characteristics, and increase of androgen plasma levels in the treated male newts. Moreover, a reduction in both the number of ARO‐LI containing neurons and their immunola‐beling intensity was seemingly observed in the more caudal cluster of aromatase containing cells or ventral infundibular nucleus. In conclusion, 4‐nonylphenol seems to affect different targets variously involved in amphibian reproduction.

Immunocytochemical Localization of Growth Hormone-Releasing Hormone-like Peptide in the Brain of the Tiger Frog, Rana tigrina

Using antisera directed against carp growth hormone-releasing hormone (cGHRH), we found more extensive brain distribution of GHRH-like immunoreactive (ir) neurons in the tiger frog, Rana tigrina, than reported in previous studies, which employed mammalian GHRH antibodies. In the telencephalon, GHRH-ir perikarya were present in the pallium dorsale, pallium laterale (pars dorsalis and pars ventralis), pallium mediale, nucleus entopeduncularis, amygdala pars medialis, and in the ventral portion of the lateral preoptic area. Most GHRH somata were present in the nucleus infundibularis ventralis located around the third ventricle, extending from the region posterior to the optic chiasma to the caudal end of infundibulum. In the thalamic region, GHRH-ir perikarya occurred in the area ventrolateralis thalami, the nuclei posterocentralis thalami, and the posterolateralis thalami. The ir cell bodies in the nucleus posteroventralis tegmenti mesencephali represented the caudal-most brain GHRH perikarya. Extensive GHRH-ir fibers occurred around the nonreactive cells in the ventral preoptic area and ventral area of the infundibulum. GHRH-ir fibers were present in the outer layers of the median eminence, but not in the neural lobe or pars distalis of the pituitary gland. This wider neuroanatomical distribution of GHRH-like peptide in the brain of R. tigrina should, provide the basis for future studies to establish the exact role of GHRH-like peptides in anuran brain.

Ontogeny of pituitary adenylate cyclase-activating polypeptide (PACAP) in the frog (Rana ridibunda) tadpole brain: immunohistochemical localization and biochemical characterization.

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.

Distribution of vasoactive intestinal peptide-like immunoreactivity in the brain and pituitary of the frog ( Rana esculenta) during development

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.

Distribution of somatostatin-like immunoreactivity in the brain of the frog, Rana esculenta, during development

The anatomical distribution of somatostatin-like immunoreactivity in the central nervous system of the frog, Rana esculenta, during development and in juvenile specimens was investigated by indirect immunofluorescence. Soon after hatching, at stages II-III, somatostatin-like immunoreactive structures were found in the preoptic-median eminence complex. In stage VI tadpoles, new groups of immunopositive perikarya and nerve fibers appeared in the diencephalon, within the ventral infundibular nucleus and in the ventral area of the thalamus, as well as in the medial pallium. In stages XII-XIV of development, immunopositive perikarya were also present in the dorsal infundibular nucleus of the hypothalamus and ventrolateral area of the thalamus. A small group of somatostatin-like immunoreactive neurons appeared in the posteroventral nucleus of the rhombencephalon. However, these neurons were not seen in later stages of development. Tadpoles in stages XVIII, XXI-XXII and in juveniles were characterized by a wider distribution of immunoreactive cell bodies and fibers in the pallium. New groups of immunoreactive neurons were found in the dorsal and lateral pallium. The presence of positive perikarya in the lateral pallium is a transient expression found only in these stages. The organization of the somatostatinergic system was most complex during the metamorphic climax, with the appearance of positive cell bodies in the posterocentralis area of the thalamus, and in juvenile animals with the presence of perikarya in the ventral part of the medial pallium and within the central grey rhombencephali. In contrast to the adult frog, somatostatin neurons were not observed in the mesencephalon of tadpoles and juveniles.

Distribution of NADPH-diaphorase activity in the hypothalamo-hypophysial system of the frog, Rana esculenta

Using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase (ND) histochemistry, this study reports a wide distribution of ND activity in the hypothalamus, and for the first time in the median eminence (ME), the neural lobe (NL) and in the pars distalis (PD) of the frog, Rana esculenta. Perikarya are ND-active within the nucleus preopticus (NPO), the nucleus preopticus periventricularis (NPP), located around the preoptic recess (PR), the suprachiasmatic nucleus (SCN) and within five infundibular nuclei. Several ND-positive neurons of the nucleus infundibularis ventralis are cerebrospinal fluid-contacting in nature, while a few occupy a subependymal region. The infundibulum shows a thick sheet-like fiber plexus which receives fibers not only from its ND-active neurons, but also from the anterior and central thalamic nuclei. The ME, NL and most cells of the posterodorsal PD are ND-positive. The pituitary function may be mediated by nitric oxide through modulating the secretion of hormone-releasing factors of the hypothalamus. Possible functional significance of the ND-stained hypothalamic areas is discussed.

Immunocytochemical localization and biological activity of 3 beta- hydroxysteroid dehydrogenase in the central nervous system of the frog

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyzes biosynthesis of progesterone (P) and all precursors of glucocorticoids, mineralocorticoids, androgens, and estrogens. Despite the broad interest raised by neurosteroids, the cellular localization of 3 beta-HSD has never been investigated in the brain. We took advantage of the availability of an antiserum raised against human placental 3 beta-HSD to determine the distribution of 3 beta-HSD-immunoreactive structures in the brain of the frog Rana ridibunda by the indirect immunofluorescence technique. Three populations of 3 beta-HSD-immunoreactive cell bodies were observed in the hypothalamus, namely, in the rostral region of the preoptic nucleus, the dorsal infundibular nucleus, and the dorsal part of the ventral infundibular nucleus. A dense network of 3 beta-HSD-immunoreactive nerve fibers was visualized in the dorsal area of the diencephalon, that is, in the lateral neuropil, the corpus geniculatus lateralis, and the nucleus posterolateralis thalami. Reversed-phase HPLC analysis of frog hypothalamic extracts combined with RIA detection showed the presence of substantial amounts of immunoreactive steroids coeluting with P and 17-hydroxyprogesterone (17OH-P). The synthesis of delta 4-3-keto-steroids in the frog hypothalamus was investigated using the pulse-chase technique with 3H-pregnenolone (3H-delta 5P) as a precursor. The formation of five tritiated metabolites of 3H-delta 5P was observed, one of which coeluted with 17OH-P. Conversion of 3H-delta 5P into this radioactive metabolite was significantly reduced by trilostane, a specific inhibitor of 3 beta-HSD. Immunodetection of newly synthesized steroids in HPLC fractions of hypothalamic extracts, using 17OH-P antibodies, revealed the existence of an immunoreactive steroid that exhibited the same retention time as synthetic 17OH-P. The present study provides the first immunocytochemical mapping of 3 beta-HSD, a key enzyme of the steroid biosynthetic pathway, in the CNS of a vertebrate. The data also demonstrate for the first time biosynthesis of neurosteroids in the brain of a nonmammalian vertebrate.

Biosynthesis of Steroids in the Central Nervous System of Amphibians

Distribution and activity of steroid producing enzymes have been studied in the central nervous system (CNS) of the frog Rana ridibunda. Cell bodies containing 3β-hydroxysteroid dehydrogenase (3β-HSD)-like immunoreactivity were exclusively observed in the preoptic nucleus, the dorsal infundibular nucleus and the dorsal aspect of the ventral infundibular nucleus. Identification of astrocytes with antisera raised against GFAP, and oligodendrocytes with antisera against galactocerebroside revealed that 3β-HSD is expressed in frog neurones. Biochemistry showed that hypothalamic slices can convert pregnenolone into Δ-3-ketosteroids including progesterone (P) and 17-hydroxyprogesterone (17OH-P); these steroids likely serve as precursors for other neurosteroids. This is the first immunocytochemical localisation of 3Β-HSD in the brain and the first evidence for neurosteroid biosynthesis in a nonmammalian vertebrate.

Immunocytochemistry and in situ hybridization of neuropeptide Y in the hypothalamus of Xenopus laevis in relation to background adaptation

The amphibian Xenopus laevis is able to adapt to a dark background by releasing melanophores-timulating hormone from the pars intermedia of the pituitary gland. The inhibition of melanophore-stimulating hormone release is accomplished by neuropeptide Y-containing axons innervating the pars intermedia. To determine the production site of neuropeptide Y involved in this inhibitory control, the distribution of neuropeptide Y in the brain has been investigated by immunocytochemistry and in situ hybridization. Immunoreactive cell bodies were visualized in, among others, the ventromedial and posterior thalamic nuclei, and the suprachiasmatic and ventral infundibular hypothalamic nuclei. A positive hybridization signal with a Xenopus-specific probe for preproneuropeptide Y-RNA was found in the diencephalic ventromedial thalamic nucleus and in the suprachiasmatic nucleus. With both immunocytochemistry and in situ hybridization, suprachiasmatic neurons appeared to be stained only in animals adapted to a white background; animals adapted to a black background showed no staining. Quantitative image analysis revealed that this effect of background adaptation is specific for suprachiasmatic neurons because no effect could be demonstrated of the background light condition on the ventral infundibular nucleus (immunocytochemistry) or the ventromedial thalamic nucleus ( in situ hybridization). These results indicate that neurons in the suprachiasmatic nucleus enable the adaptation of X. laevis to a white background, by producing and releasing neuropeptide Y that inhibits the release of melanophore-stimulating hormone from the melanotrope cells in the pars intermedia of the pituitary gland.

Neuroanatomical and physiological evidence for the involvement of pituitary adenylate cyclase-activating polypeptide in the regulation of the distal lobe of the frog pituitary.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38 amino-acid peptide which belongs to the glucagon/secretin/vasoactive intestinal peptide superfamily. The sequence of PACAP is identical in all mammalian species studied so far but frog PACAP differs by one amino-acid from mammalian PACAP. The aim of the present study was to investigate the presence of PACAP in the hypothalamo-pituitary complex of the frog Rana ribibunda and to determine the biological activity of frog PACAP on homologous pituitary cells. The distribution of PACAP-containing neurons and fibers was examined by the indirect immunofluorescence method using an antiserum raised against the N-terminal region of the peptide. In the hypothalamus, PACAP-immunoreactive perikarya were localized in the preoptic nucleus and the dorsal and ventral infundibular nuclei. Beaded nerve fibers were observed coursing from the ventral infundibular nucleus to the external vascular layer of the median eminence. A dense network of immunoreactive axons terminated in the vicinity of the capillaries of the hypophysial portal system. The neurointermediate lobe and the distal lobe of the pituitary were devoid of immunoreactive elements. The amount of PACAP-like immunoreactive material in hypothalamus extracts was measured by radioimmunoassay; the apparent concentration of PACAP was 4.5 ng/mg protein. Synthetic frog PACAP38 and PACAP27 induced a similar dose-dependent stimulation of cAMP production in isolated frog distal lobe pituitary fragments (ED50 = 2 x 10(-8) M). At the maximum dose tested (5 x 10(-6) M), both frog PACAP38 and PACAP27 produced a 4-fold increase in cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of corticotropin-releasing factor immunoreactive neurons in the brain of the tigerfrog, Rana tigrina

The distribution of immunoreactive (ir) neurons containing corticotropin-releasing factor (CRF) is described in the brain of the tigerfrog, Rana tigrina. The olfactory bulb, medial pallium, nucleus of the diagonal band of Broca and medial area of the amygdala of the telencephalon showed ir-CRF perikarya. The anterior and ventromedial thalamic nuclei, and the magnocellular nucleus preopticus (NPO) revealed several ir cells; a few NPO neurons were cerebrospinal fluid contacting in nature. The nucleus hypothalamicus ventromedialis contained a few cells, but the nucleus infundibularis ventralis of the infundibulum revealed several diffusely distributed perikarya. Individual ir-CRF perikarya were visualized in the optic tectum and interpeduncular nucleus. Extensive fiber terminals were present in the median eminence, but no fibers were discerned either in the neural lobe or in the pituitary gland.

Opioids and testicular activity in the frog, Rana esculenta.

The presence and activity of brain, pituitary and testicular beta-endorphin (beta-EP)-like material have been studied in the frog, Rana esculenta, using reverse-phase high-pressure liquid chromatography, coupled with radioimmunoassay and immunocytochemistry. In-vivo and in-vitro treatments with naltrexone were carried out to assess the putative physiological activity of opioid peptides. beta-EP(1-31) and (1-27), together with their acetylated forms, have been identified in brain, pituitary and testis. In particular, beta-EP(1-31) concentrations peaked during July in the brain and pituitary, whilst in testes maximum concentrations were found in April and November. beta-EP immunoreactivity was present in the brain within the nucleus preopticus and nucleus infundibularis ventralis while positive fibres in the retrochiasmatic regions projected to the median eminence. In the testis, interstitial cells, canaliculi of the efferent system, spermatogonia and spermatocytes showed positive immunostaining for beta-EP. In intact animals, naltrexone treatment increased plasma and testicular androgen levels and this effect was confirmed in in-vitro incubations of minced testes. Naltrexone also induced a significant increase in germ cell degeneration. Our results indicated that an opioid system modulates the hypothalamus-pituitary-gonadal axis in the frog, Rana esculenta and, for the first time, we have shown that the testicular activity of a non-mammalian species may be regulated by opiates locally.

A Quantitative Autoradiographic Study of GABA&lt;sub&gt;A&lt;/sub&gt; and Benzodiazepine Receptors in the Brain of the Frog, &lt;i&gt;Rana esculenta&lt;/i&gt;

Specific binding sites for GABAA and benzodiazepines were detected in the brain of the frog Rana esculenta after the in vitro incubation of tissue sections with their respective specific agonists [3H] muscimol and [3H] flunitrazepam. Conditions for the binding assay were optimized and as a result binding was saturable and specific. Quantitative autoradiographic receptor measurements in the different brain sections showed that elevated levels of [3H] muscimol binding sites were found in the two layers of the cerebellum (periventricular and external) with the highest binding densities being detected in the periventricular layer. Relatively high densities of [3H] muscimol binding sites were also observed in the torus semicircularis of the mesencephalon and in the thalamic nucleus rotundus and posterolateral nucleus, plus the mitral cell layer of the olfactory bulb, the amygdala pars lateralis and the striatum of the telencephalon. Intermediate to low binding levels were obtained in the remaining brain areas such as the external layer of the optic tectum, the dorsomedial and dorsolateral anterior thalamic nuclei, the medial and lateral pallium, the medial septal nucleus, the preoptic nucleus, the dorsal and ventral infundibular nuclei of the hypothalamus and the interpeduncular nucleus. Autoradiographic evaluation of benzodiazepine receptors revealed that binding levels of [3H] flunitrazepam were overall lower than those of the GABAA sites. In fact the highest [3H] flunitrazepam binding levels were observed in the striatum, external layer of the optic tectum and the torus semicircularis.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of infundibular lesions on GnRH and LH release in the frog, Rana temporaria, during hibernation

The effects of lesions and deafferentations on accelerated hibernatory ovulation and on the concentration of GnRH in blood collected from the hypothalamo-pituitary area along with the blood and pituitary levels of luteinizing hormone (LH) in hibernating female frogs, Rana temporaria, were studied. The lesions to the caudal portion of the nucleus infundibularis ventralis resulted in: (1) an elevation in GnRH concentration in blood collected from the hypothalamo-pituitary area ( x ± SEM = 51 ± 9.5 and 100 ± 15 pg/ml in control and lesioned females, respectively), (2) an increase in plasma LH (from 11 ± 1.3 to 14.7 ± 2.5 ng/ml in controls to 73.1 ± 12.0 and 74.2 ± 15.8 ng/ml in lesioned females at 3 and 7 days), and (3) accelerated ovulations whose onset advanced as the hibernation season progressed. Histological and immunohistochemical analyses of the brains showed that GnRH seems to be stored during hibernation in the median eminence and suggested that the complete disruption of projections to the median eminence is prerequisite for accelerated ovulation. The influence of the hypothalamic inhibitory action on the release of GnRH and the way in which GnRH is transferred to the pituitary gland are discussed.