Immunohistochemical analysis of the hypothalamic-pituitary-adrenal axis in dogs: Sex-linked and seasonal variation.

This study evaluated sexual dimorphism and seasonal variations in corticotrophs and adrenal zona fasciculata in dogs, as well as the expression of oestrogen receptor alpha (ERα). An immunohistochemical analysis was conducted in pituitaries for ACTH and in adrenal glands for ERα and for the melanocortin-2-receptor (MC2R) in winter and summer. Double immunofluorescence was performed to identify ERα in corticotrophs. Females had a greater proportion of corticotrophs per field (p<0.01), with a greater cellular area and optical density (p<0.001) than males. Optical density of corticotrophs was greater in winter for both sexes (p<0.001). In zona fasciculata, ERα and MC2R expression was greater in females (p<0.001) and was greater in winter (p<0.001). ERα was identified in corticotrophs. This study is the first to demonstrate ERα expression in corticotrophs and the adrenal cortex in dogs, providing evidence for sexual dimorphism and seasonal variations.

Gonadotrophin-Inhibitory Hormone in the Cichlid Fish Cichlasoma dimerus: Structure, Brain Distribution and Differential Effects on the Secretion of Gonadotrophins and Growth Hormone.

The role of gonadotrophin-inhibitory hormone (GnIH) in the inhibition of the reproductive axis has been well-established in birds and mammals. However, its role in other vertebrates, such as the teleost fish, remains controversial. In this context, the present study aimed to evaluate whether GnIH modulates the release of gonadotrophins and growth hormone (GH) in the cichlid fish Cichlasoma dimerus. First, we partially sequenced the precursor polypeptide for GnIH and identified three putative GnIH peptides. Next, we analysed the expression of this precursor polypeptide via a polymerase chain reaction in the reproductive axis of both sexes. We found a high expression of the polypeptide in the hypothalamus and gonads of males. Immunocytochemistry allowed the observation of GnIH-immunoreactive somata in the nucleus posterioris periventricularis and the nucleus olfacto-retinalis, with no differences between the sexes. GnIH-immunoreactive fibres were present in all brain regions, with a high density in the nucleus lateralis tuberis and at both sides of the third ventricle. Finally, we performed in vitro studies on intact pituitary cultures to evaluate the effect of two doses (10(-6)  m and 10(-8)  m) of synthetic C. dimerus (cd-) LPQRFa-1 and LPQRFa-2 on the release of gonadotrophins and GH. We observed that cd-LPQRFa-1 decreased ß-luteinising hormone (LH) and ß-follicle-stimulating hormone (FSH) and also increased GH release to the culture medium. The release of ß-FSH was increased only when it was stimulated with the higher cd-LPQRFa-2 dose. The results of the present study indicate that cd-LPQRFa-1, the cichlid fish GnIH, inhibits ß-LH and ß-FSH release and stimulates GH release in intact pituitary cultures of C. dimerus. The results also show that cd-LPQRF-2 could act as an ß-FSH-releasing factor in this fish species.

Orexin and neuropeptide Y: tissue specific expression and immunoreactivity in the hypothalamus and preoptic area of the cichlid fish Cichlasoma dimerus.

Neuropeptide Y (NPY) and orexin are neuropeptides involved in the regulation of feeding in vertebrates. In this study we determined the NPY and orexin mRNA tissue expression and their immunoreactivity distribution in both preoptic area and hypothalamus, regions involved in the regulation of feeding behavior. Both peptides presented a wide expression in all tissues examined. The NPY-immunoreactive (ir) cells were localized in the ventral nucleus posterioris periventricularis (NPPv) and numerous ir-NPY fibers were found in the nucleus lateralis tuberis (NLT), the nucleus recess lateralis (NRL) and the neurohypophysis. Ir-orexin cells were observed in the NPPv, dorsal NLT, ventral NLT, lateral NLT (NLTl) and the lateral NRL. Ir-orexin fibers were widespread distributed along all the hypothalamus, especially in the NLTl. Additionally, we observed the presence of ir-orexin immunostaining in adenohypophyseal cells, especially in somatotroph cells and the presence of a few ir-orexin-A fibers in the neurohypophysis. In conclusion, both peptides have an ubiquitous mRNA tissue expression and are similarly distributed in the hypothalamus and preoptic area of Cichlasoma dimerus. The presence of ir-orexin in adenohypohyseal cells and the presence of ir-orexin and NPY fibers in the neurohypophysis suggest that both peptides may play an important neuroendocrine role in anterior pituitary.

Anandamide injected into the lateral ventricle of the brain inhibits submandibular salivary secretion by attenuating parasympathetic neurotransmission

Our objective was to determine the effect of arachidonylethanolamide (anandamide, AEA) injected intracerebroventricularly (icv) into the lateral ventricle of the rat brain on submandibular gland (SMG) salivary secretion. Parasympathetic decentralization (PSD) produced by cutting the chorda tympani nerve strongly inhibited methacholine (MC)-induced salivary secretion while sympathetic denervation (SD) produced by removing the superior cervical ganglia reduced it slightly. Also, AEA (50 ng/5 µL, icv) significantly decreased MC-induced salivary secretion in intact rats (MC 1 µg/kg: control (C), 5.3 ± 0.6 vs AEA, 2.7 ± 0.6 mg; MC 3 µg/kg: C, 17.6 ± 1.0 vs AEA, 8.7 ± 0.9 mg; MC 10 µg/kg: C, 37.4 ± 1.2 vs AEA, 22.9 ± 2.6 mg). However, AEA did not alter the significantly reduced salivary secretion in rats with PSD, but decreased the slightly reduced salivary secretion in rats with SD (MC 1 µg/kg: C, 3.8 ± 0.8 vs AEA, 1.4 ± 0.6 mg; MC 3 µg/kg: C, 14.7 ± 2.4 vs AEA, 6.9 ± 1.2 mg; P < 0.05; MC 10 µg/kg: C, 39.5 ± 1.0 vs AEA, 22.3 ± 0.5 mg; P < 0.001). We showed that the inhibitory effect of AEA is mediated by cannabinoid type 1 CB1 receptors and involves GABAergic neurotransmission, since it was blocked by previous injection of the CB1 receptor antagonist AM251 (500 ng/5 µL, icv) or of the GABA A receptor antagonist, bicuculline (25 ng/5 µL, icv). Our results suggest that parasympathetic neurotransmission from the central nervous system to the SMG can be inhibited by endocannabinoid and GABAergic systems.

Localization of Neuropeptide Y-like immunoreactive structures in the brain of the pejerrey, Odontesthes bonariensis (Teleostei, Atheriniformes).

The aim of this study was to determine the distribution of Neuropeptide-Y (NPY) immunoreactive neurons and fibres in the brain and pituitary of Odontesthes bonariensis by immunohistochemical methods. A wide distribution of immunoreactive NPY (ir-NPY) cells and fibres in the forebrain and midbrain was observed. A prominent ir-NPY nucleus was found in the ventral telencephalon and other ir-NPY cells groups were recognized at the dorso-medial telencephalon. The diencephalon showed ir-NPY cells in the Nucleus entopeduncularis, the Nucleus preopticus periventricularis and in the Nucleus lateralis tuberis. Ir-NPY fibres were conspicuous in the preoptic region and the hypothalamus. There were also numerous ir-NPY fibres at the epithalamic level running ventrally to the hypothalamus and the pituitary stalk. At the rhomboencephalic level, the ir-NPY neurons were observed in the Locus coeruleus. Double-labelled immunostaining showed a close association between ir-NPY fibres that reach the adenohypophysis and growth hormone (GH)- and gonadotropin (GtH)-expressing cells. Although our results exhibit some relevant differences when compared to other fish groups, they support the existence of a conserved NPY system in teleosts.

Melanin-concentrating hormone system in the brain and skin of the cichlid fish Cichlasoma dimerus: anatomical localization, ontogeny and distribution in comparison to alpha-melanocyte-stimulating hormone-expressing cells.

Distribution and development of the melanin-concentrating hormone (MCH) system were examined by immunocytochemistry of the brain, pituitary gland and skin of the South American cichlid fish Cichlasoma dimerus. In adults, the most prominent group of MCH-ir perikarya was located in the nucleus lateralis tuberis (NLT). Outside the NLT, in the posterior hypothalamic region, a group of small neurons was found between the third ventricle and the lateral ventricular recess with delicate immunoreactive fibers that did not seem to contribute to the pituitary innervation. MCH-ir perikarya were identified at day 4 after hatching (AH) in a proliferating zone of the hypothalamic floor. Pituitary innervation could be detected at this stage. Another group of small MCH-ir neurons, only detected in pre-juvenile stages, originated close to the third ventricle in the medial hypothalamic region by day 6 AH. alphaMSH-ir neurons were localized in similar regions of the NLT and in the nucleus periventricularis posterior (NPP). Free MCH-ir neuromasts were detected in the ventral and dorsal skin of larval heads. These epidermal sensory organs were in close association with blood vessels and dermal melanocytes, suggesting that MCH synthesized in larval skin might act in an endocrine way reaching different targets and/or in a paracrine mode regulating melanin concentration in dermal melanocytes.

Ontogeny of immunoreactive somatolactin, prolactin and growth hormone secretory cells in the developing pituitary gland of Cichlasoma dimerus (Teleostei, Perciformes).

Prolactin, growth hormone and somatolactin constitute a hormone family because they are structurally related and are secreted by acidophilic cells of different regions of the adenohypohyisis. In this work, we report the ontogeny of ir-prolactin, ir-growth hormone and ir-somatolactin cells in the developing pituitary gland of the cichlid fish Cichlasoma dimerus (Teleostei; Perciformes). Antisera raised against fish pituitary hormones were used. In this species hatching occurs 54 hs after fertilization and the three different cell types were recognized two days later. The neurohypophysis was recognized on day 14 after hatching and in later stages it began to show the characteristic deep interdigitations of the adults. On day 42 (juvenile stage) the distribution of ir-PRL, ir-GH and ir-SL showed the pattern described for adults of this species. The ir-SL cells were not PAS-positive in larvae as they are in adults. This would suggest the presence of a nonglycosilated form of somatolactin in early stages of development which may coexist in adults with a glycosilated form. The appearence of these hormones so early in development suggest their importance in the survival of fish larvae but further studies focused on the ontogeny of hypothalamic factors that regulate their synthesis and secretion must be performed.

Immunocytochemical localization of different cell types in the adenohypophysis of the cichlid fish Cichlasoma dimerus (Heckel, 1840).

The adenohypophysis of the cichlid fish Cichlasoma dimerus was studied using the avidin-biotin-peroxidase method with antisera raised against piscine pituitary hormones and heterologous antisera against mammalian pituitary hormones. Antiserum raised against rabbit ACTH recognized a group of cells bordering the neurohypophysis (NH) in the rostral pars distalis (RPD). Anti-chum salmon prolactin (PRL) identified a compact group of cells in the periphery of the RPD. Gonadotropin II (GTH II), thyrotropin (TSH) and growth hormone (GH)-ir cells were localized in the proximal pars distalis. Ir-GTH II cells were also observed in the dorsal area of the pars intermedia (PI). Ir-GTH I cells could not be identified using anti-chum salmon GTH I, this may be due either to a failure of the antisera to recognize the gonadotropin or to a low expression of the hormone in adults of this species. PAS positive cells from the PI bound specifically with three different antisera raised against somatolactin (SL) of four different fish species. These cells surrounded deep branches of the NH in the PI.

The adenohypophysis of the swamp eel, Synbranchus marmoratus, an immunocytochemical analysis.

The adenohypophyseal cell types of the protogynous fish Synbranchus marmoratus were studied by histochemical and immunocytochemical staining with antisera raised against piscine and human pituitary hormones to ascertain their distribution. The prolactin (PRL) cells were distributed in the rostral pars distalis and showed specific binding to antisera to carp and chum salmon prolactin. No reaction was observed with antiserum to human prolactin. The corticotrops showed strong immunoreactivity with anti-human ACTH, these cells bordered the neurohypophysis and islets between PRL cells in the rostral pars distalis. Growth hormone (GH) cells were densely distributed and associated with the neurohypophysis only in pars distalis proximal. They reacted with antisera to piscine GH but not with antisera to human growth hormone. The thyrotrops were scattered in the proximal pars distalis and showed strong immunoreactivity to the human thyrotropin Beta subunit antiserum. Gonadotrops were located in the central area of the proximal pars distalis and in the external border of the pars intermedia. These cells were alcian blue and PAS positive, and reacted with anti-croaker GTH and anti-coho GTH I and GTH II. The PAS positive cells from the pars intermedia bound specifically to anti-chum somatolactin.