Immunocytochemistry of luteinizing hormone-releasing hormone during spontaneous and thyroxine-induced metamorphosis of bullfrogs

Double-bridge peroxidase-antiperoxidase immunocytochemistry was used to compare the developmental appearance of immunoreactive LH-RH (ir-LH-RH) in brains of bullfrog (Rana catesbeiana) tadpoles during either spontaneous or thyroxine-induced metamorphosis. During spontaneous metamorphosis, ir-LH-RH was localized in fibers of the external layer of the median eminence (ME) of stage XIII-XXV animals, while immunoreactive perikarya and other immunostained brain structures were absent. The extent and intensity of ME immunostaining increased concomitantly with measured ME morphological development. Tadpoles induced with thyroxine to metamorphic stages XIX-XXI exhibited ME structural development and neurohypophysial neurosecretory staining similar to spontaneously metamorphosed individuals of equal stages. However, comparable ME ir-LH-RH immunostaining and gonadal size were both less developed in thyroxine-treated animals, although increased relative to non-metamorphic vehicle-injected controls. These results indicate that the hypothalamic LH-RH system changes concurrently with ME structural development during spontaneous metamorphosis. Reduced ME ir-LH-RH staining and gonadal size in thyroxine-treated animals suggest that during prometamorphosis, factors other than thyroxine alone may coordinate the normal maturation of the hypothalamo-pituitary-gonadal axis of the bullfrog.     

The LH-RH system of the male European starling: photoperiod induces changes to a possible multifunctional peptide system

In many birds reproduction is triggered by long daylengths but, paradoxically, continued exposure to long days leads to photorefractoriness and a complete shut down of the reproductive system. As these effects are thought to be mediated through the secretion of LH-RH, immunocytochemical techniques were used to investigate changes in the LH-RH system when European starlings were exposed to different photoperiods. Starlings exposed to 11L:13D and with mature testes show strong immunostaining both of LH-RH perikarya and fibers. Photosensitive short-day (8L:16D) starlings with undeveloped testes show an almost identical distribution of strongly immunoreactive perikarya but with less dense fibre staining. However, long-day (18L:6D) photorefractory starlings with fully regressed testes, show a profound reduction in LH-RH immunostaining. Perikarya have the same distribution but show a much reduced intensity of staining and fibers had almost entirely disappeared from all regions of the brain. Preliminary observations on the ultrastructure of immunocytochemically identified LH-RH neurones are also reported.     

The paraventriculo-infundibular corticotropin releasing factor (CRF) pathway as revealed by immunocytochemistry in long-term hypophysectomized or adrenalectomized rats

The immunocytochemical localization of corticotropin releasing factor (CRF)-containing pathways projecting from the paraventricular nucleus (PVN) to the external layer of the median eminence (ME) in long-term hypophysectomized or adrenalectomized rats is described. Immunocytochemistry was followed by silver intensification of the diaminobenzidine end-product. In comparison with untreated control rats, both hypophysectomy and adrenalectomy resulted in a dramatic increase in immunostaining of the CRF-containing perikarya and fibers, particularly those originating from the PVN and terminating in the ME. The staining was more intense in adrenalectomized than in hypophysectomized rats. The CRF-positive fibers emerging from the PVN form a medial, an intermediate and a lateral fiber pathway. The lateral and intermediate CRF tracts leave the dorsolateral part of the PVN and course laterally and medially of the fornix, respectively, then ventrally toward the optic tract. Just dorsal to the optic tract they turn in caudal direction and run parallel with and very close to the basal surface of the hypothalamus; individual fibers then turn medially to terminate in the external layer of the ME. Only a few fibers originate from the medial-ventral part of the PVN (medial pathway). These fibers run in ventral direction along the walls of the 3rd ventricle and terminate in the ME. Thus the majority of CRF fibers, similarly to other peptidergic systems, reach the medial basal hypothalamus from the anterolateral direction.     

Immunocytochemical identification of an LHRH-producing system originating in the preoptic nucleus of the duck

Summary A fluorescent technique applying specific LHRH and vasotocin antisera was used for the immunocytochemical localization of the respective neurosecretory systems in the hypothalamus of gonadectomized, testosteronetreated and/or serotonin injected male domestic ducks. An immunoreactive (IR) LHRH-producing system, with perikarya located in the preoptic nucleus, could be traced through the ventral hypothalamus down to the external layer of the rostral and caudal ME, in close vicinity to the hypophysial portal system. An IR-vasotocin system originating in the paraventricular and supraoptic nuclei ran through the ventral hypothalamus, but terminated in (i) the external layer of the rostral ME, and (ii) in the posterior lobe of the hypophysis.Dr. B. Kerdelhué, Laboratoire des Hormones polypeptidiques du CNRS, F-91190 Gif-sur-Yvette, France     

Immunocytochemistry of luteinizing hormone-releasing hormone in brains of breeding eastern narrow-mouthed toads (Gastrophryne carolinensis)

Unlabeled antibody-enzyme immunocytochemistry was used to localize mammalian-like immunoreactive luteinizing hormone-releasing hormone in brains of breeding eastern narrow-mouthed toads (Gastrophryne carolinensis). Intense immunostaining occurred over the infundibular floor and the median eminence, yet staining of few perikarya in the anterior preoptic area was minimal. This pattern of markedly regional immunostaining differs from that reported for other anurans.     

Corticotropin-like immunoreactivity in the brain of intact,hypophysectomized, cortisol- and metopirone-treated eels

Summary An ACTH-like peptidergic system was demonstrated in the brain of three teleost species by immunocytochemistry. In order to investigate the origin of brain ACTH and factors modulating its synthesis, similar techniques were applied to the brain of eels (1) submitted to hypothysectomy in order to suppress pituitary ACTH and plasma cortisol, (2) injected with cortisol to inhibit pituitary ACTH synthesis and release, and (3) injected with metopirone to block cortisol synthesis and stimulate ACTH synthesis and release. Hypophysectomized eels showed a normal distribution of immunoreactive perikarya in the ventral hypothalamus and fibers in the brain, suggesting that brain ACTH does not arise from the pituitary. In cortisol-treated eels immunostaining was markedly reduced in brain perikarya and pituitary corticotropes, suggesting a reduced synthesis. In metopirone-injected eels, one third of the animals showed an increased immunostaining in perikarya and a dense network of immunoreactive fibers, suggesting that ACTH synthesis was increased. Brain ACTH was not affected in other animals. Pituitary corticotropes were rapidly degranulated. Responses of ACTH in the brain and pituitary occur independently when cortisol synthesis is inhibited. These responses are compared to those of the corticotropin-releasing factor system in the same eels.     

Short photoperiod-induced gonadal regression: effects on the gonadotropin-releasing hormone (GnRH) neuronal system of the white-footed mouse, Peromyscus leucopus

The peroxidase-antiperoxidase method was used to determine quantitatively the effect of short photoperiod-induced gonadal regression on the immunoreactive gonadotropin-releasing hormone (GnRH) neuronal system of female Peromyscus leucopus. In mice exposed to either long (16L:8D) or short (8L:16D) photoperiod, immunoreactive cell bodies were loosely organized into six groups: olfactory peduncle, diagonal band of Broca, septum, preoptic area (POA), anterior hypothalamus (AH), and basal hypothalamus. The POA and AH contain the largest number of cell bodies, which supply the major GnRH innervation to the median eminence (ME) and several extrahypothalamic brain sites. Exposure to short photoperiod increased the number of immunoreactive cell bodies within the anterior hypothalamus and preoptic area (AHPOA) and also increased the optical density for staining of immunoreactive cell bodies in the AHPOA and olfactory peduncle. The ME of mice exposed to short photoperiod had a higher density of GnRH fibers relative to that of mice exposed to long photoperiod, and the content of GnRH fibers in the rostral ME was correlated with the optical content for immunostaining of cell bodies in the AHPOA. These results are evidence that gonadal regression induced by short photoperiod (mediated by the pineal gland) involves alterations of GnRH neuronal activity. Notably, data from this study are consistent with the hypothesis that suppressed release of GnRH from neurovascular terminals in the ME, rather than lack of availability of the decapeptide, promotes gonadal regression.     

Immunocytochemical distribution of corticotropin-releasing factor in the brain and hypophysis of larval Bufo arenarum; effect of KClO4 during early development

The maturation of the corticotropin-releasing factor (CRF) neuronal system was evaluated by immunocytochemistry and morphometry in Bufo arenarum, during spontaneous metamorphosis and in tadpoles with inhibited thryroid function. The first appearance of CRF immunoreactive fibers was at the end of premetamorphosis (stage VIII). These fibers were found in small numbers and weakly stained in the median eminence and infundibular stalk. With the advance of larval development, CRF-like material stained intensely and tended to aggregate in the external zone of the median eminence. At climax stages, immunoreactive fibers and perikarya (weakly stained) were identified in the interpeduncular nucleus and in the dorsal infundibular nucleus. Morphometric and immunocytochemical results indicate that the maturation of the CRF neuronal system in Bufo arenarum occurs just before metamorphic climax, coinciding with high levels of thyroid and steroid hormones. We have also found that in larvae with inhibited thyroid function, the CRF neuronal system is able to develop, and that thyroid hormone could exert a negative feedback control on the synthesis of CRF.     

Quantitative changes of CRF-like immunoreactivity in eels treated with reserpine and cortisol

Immunocytochemical techniques were applied to brain and pituitary sections of European eels after experimental manipulation of the pituitary-interrenal activity. A corticotropin-releasing factor (CRF) antiserum allowed the identification of a CRF-like peptide in the preoptic nucleus (PON) and rostral and caudal neurohypophysis (NH). CRF-immunoreactivity (ir) was not affected in solvent-injected eels compared to noninjected eels. Reserpine induced a stimulation of the pituitary interrenal axis, decreased ir-CRF in the rostral NH, but did not affect hypothalamic ir-CRF. Cortisol reduced the immunostaining of hypothalamic CRF-ir perikarya and perikarya cross-sectional area. In the rostral NH, CRF-ir fibers decreased in number and almost disappeared in long-term treated eels. The immunostaining of ACTH cells with ACTH antiserum was greatly reduced. These data suggest that cortisol induces a marked reduction in the activity of the CRF-corticotrop axis. The intensity of the ir-CRF staining observed in the caudal NH, close to the intermediate lobe (IL) was not significantly affected in reserpine-treated eels, and only slightly reduced in long-term cortisol-treated eels. The intensity of ir-CRF staining in the caudal NH did not correlate with melanocorticotropic activity or plasma cortisol level. These data suggest that immunoreactive CRF fibers in the rostral and caudal NH are differently regulated.     

Identification of growth-hormone- and prolactin-containing neurons within the avian brain

Prolactin (PRL)- and growth-hormone (GH)-containing perikarya and fibers independent of the anterior pituitary gland have been reported to exist in the central nervous system of several mammalian species. The specific distributions of PRL- or GH-like neurons in the avian forebrain and midbrain, however, have not been reported. The objective of the study was to identify GH- and PRL-containing neurons in the hypothalamus and a few extrahypothalamic areas of two avian species. Brain and peripheral blood samples were collected from laying and broody turkey hens and ring doves. Broody turkey hens and doves had significantly higher plasma PRL concentrations compared with laying hens. Coronal brain sections were prepared and immunostained using anti-turkey GH and anti-chicken synthetic PRL antibodies. In turkey hens, the most dense GH-immunoreactive (ir) perikarya and fibers were found in hippocampus (Hp), periventricular hypothalamic nucleus, paraventricular nucleus, inferior hypothalamic nucleus, infundibular hypothalamic nucleus, medial and lateral septal area, and external zone of the median eminence (ME). In the ring dove, a similar pattern of distribution of GH-ir neurons was noticed at the brain sites listed above except that GH-ir fibers and granules were found only in the internal zone of ME and not in the external zone. In both turkeys and doves, the most immunoreactive PRL-ir perikarya and fibers were found in the medial and lateral septal area, Hp (turkey only), and bed nucleus of the stria terminalis pars magnocellularis. There were no apparent differences in the staining pattern of GH- or PRL-ir neurons between the laying and broody states in either species. However, the presence of GH-ir- and PRL-ir perikarya and fibers in several hypothalamic nuclei indicates that GH and PRL may influence parental behavior, food intake, autonomic nervous system function, and/or reproduction.     

Immunocytochemical localization of enkephalin in the lateral septum of the guinea-pig brain

By the use of antisera to met-enkephalin and leu-enkephalin, enkephalin-containing structures were visualized in the lateral septum of the guinea-pig brain. The present results do not reveal immunoreactive perikarya in this area. The immunostaining is exclusively located in numerous nerve fibers and endings mostly encompassing neuronal perikarya, which accounts for the fact that at the light-microscopic level cellular somata appear to be immunostained. The immunoreactive terminals and fibers contain granules approximately 110 nm in diameter and synaptic vesicles. The origin and the functional role of these numerous enkephalin terminals remain to be established.     

Immunocytochemical demonstration of peptidergic neurons in the central and peripheral nervous systems of the flatworm Microstomum lineare with antiserum to FMRF-amide

Summary The central nervous system (CNS) and the peripheral nervous system (PNS) of the flatworm Microstomum lineare were studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method, with the use of antisera to the molluscan cardioactive peptide FMRF-amide. FMRF-amide immunoreactive perikarya and nerve fibres are observed in the CNS and the PNS. In the CNS, immunoreactive perikarya and nerve fibres occur in the brain, in the epithelial lining and the mesenchymal surroundings of the ciliated pits, and positive fibres in the longitudinal nerve cords. In the PNS, immunoreactive fibre bundles with variocosities occur in the pharyngeal nerve ring, in symmetrical groups of perikarya on each side of the pharynx, and in the mouth area. Positive perikarya and meandering nerve fibres appear in the intestinal wall. A few immunoreactive cells and short nerve processes are observed at the male copulatory organ and on both sides of the vagina. Some immunoreactive peptidergic cells do not correspond to cells previously identified by histological techniques for neurosecretory cells. The distribution of immunoreactivity suggests that the FMRF-amide-like substance in CNS and PNS in this worm has roles similar to those of the brain-gut peptides in vertebrates. The status of FMRF-amide-like peptides as representatives of an evolutionarily old family of peptides is confirmed by the positive immunoreaction to anti-FMRF-amide in this primitive microturbellarian.     

Immunocytochemical markers revealing retinal and pineal but not hypothalamic photoreceptor systems in the Japanese quail

Summary The retinal proteins opsin,-transducin, S-antigen and interstitial retinol-binding protein (IRBP) are essential for the processes of vision. By use of immunocyto-chemistry we have employed antibodies directed against these photoreceptor proteins in an attempt to identify the photoreceptor systems (retina, pineal and deep brain) of the Japanese quail. Opsin immunostaining was identified within many outer (basal portion) and inner segments of retinal photoreceptor cells and limited numbers of photoreceptor perikarya. Opsin immunostaining was also demonstrated in limited numbers of pinealocytes with all parts of these cells being immunoreactive. These results differ from previous observations. In contrast to the results obtained with the antibody against opsin, S-antigen and-transducin immunostaining was seen throughout the entire outer segments and many photoreceptor perikarya of the retina. In the pineal organ immunostaining was seen in numerous pinealocytes in all follicles. These results conform to previous findings in birds. In addition, IRBP has been demonstrated for the first time in the avian retina and pineal organ. These findings underline the structural and functional similarities between the retina and pineal organ and provide additional support for a photoreceptive role of the avian pineal. No specific staining was detected in any other region of the brain in the Japanese quail; the hypothalamic photoreceptors of birds remain unidentified.     

Serotonin immunoreactivity in the nervous system of the free‐swimming larvae and sessile adult females of Stephanoceros fimbriatus (Rotifera: Gnesiotrocha)

Unlike most rotifers (Rotifera), which are planktonic and direct developers, many gnesiotrochan rotifers (Monogononta: Gnesiotrocha) are sessile and have indirect development. Few details exist on larval metamorphosis in most gnesiotrochans, and considering the drastic transformation that takes place at metamorphosis—the replacement of the ciliated corona with a new head that bears ciliated tentacles (the infundibulum)—it is perhaps surprising that there are limited data on the process. Here, we document part of this metamorphosis by examining the presence and distribution of neurons with serotonin immunoreactivity in the nervous system of both planktonic larvae and sessile adult females. Using antibodies against serotonin combined with confocal laser‐scanning microscopy (CLSM) and 3D reconstruction software, we mapped the immunoreactive cell bodies and neurites in both life stages and found that relatively few changes occurred during metamorphosis. The larvae possessed a total of eight perikarya with serotonergic immunoreactivity (5HT‐IR) in the brain, with at least two pairs of perikarya outside the brain in the region of the corona. Cells with 5HT‐IR in the brain innervated the larval corona and also sent neurites to the trunk via the nerve cords. During metamorphosis, the corona was replaced by the infundibulum, which emerged from the larval mouth to become the new functional head. This change led to a posterior displacement of the brain and also involved the loss of 5HT‐IR in the lateral brain perikarya and the gain of two perikarya with 5HT‐IR in the anterior brain region. The innervation of the anterior end was retained in the adult; neurites that extended anteriorly to the mouth of the larva formed a distinct neural ring that encircled the infundibulum after metamorphosis. Significantly, there was no innervation of the infundibular tentacles by neurites with 5HT‐IR, which suggests that ciliary control is unlikely to be modulated by serotonin within the tentacles themselves.     

Distribution of FMRFamide-like immunoreactivity in the brain,retina and nervus terminalis of the sockeye salmon parr,Oncorhynchus nerka

Summary Neurons displaying FMRFamide(Phe-Met-Arg-Phe-NH₂)-like immunoreactivity have recently been implicated in neural plasticity in salmon. We now extend these findings by describing the extent of the FMRF-like immunoreactive (FMRF-IR) system in the brain, retina and olfactory system of sockeye salmon parr using the indirect peroxidase anti-peroxidase technique. FMRF-IR perikarya were found in the periventricular hypothalamus, mesencephalic laminar nucleus, nucleus nervi terminalis and retina (presumed amacrine cells), and along the olfactory nerves. FMRF-IR fibers were distributed throughout the brain with highest densities in the ventral area of the telencephalon, in the medial forebrain bundle, and at the borders between layers III/IV and IV/V in the optic tectum. High densities of immunoreactive fibers were also observed in the area around the torus semicircularis, in the medial hypothalamus, median raphe, ventromedial tegmentum, and central gray. In the retina, immunopositive fibers were localized to the inner plexiform layer, but several fiber elements were also found in the outer plexiform layer. The olfactory system displayed FMRF-IR fibers in the epithelium and along the olfactory nerves. These findings differ from those reported in other species as follows: (i) FMRF-IR cells in the retina have not previously been reported in teleosts; (ii) the presence of FMRF-IR fibers in the outer plexiform layer of the retina is a new finding for any species; (iii) the occurrence of immunopositive cells in the mesencephalic laminar nucleus has to our knowledge not been demonstrated previously.     

Galanin-like immunoreactivity is increased in the brain of estradiol- and methyltestosterone-treated eels.

A galanin-like peptidergic system was demonstrated in the brain of Anguilla. A group of immunoreactive perikarya was located in the nucleus preopticus periventricularis close to the recessus preopticus. Galaninergic fibers occurred in various brain areas. Galanin identified in mammalian pituitary cells was undetectable in fish adenohypophysial cells. Estradiol increased the immunostaining of the rostral perikarya and brain fibers in both male and female European eels kept in fresh water and in female American eels in sea water. Methyltestosterone, an aromatizable androgen, increased galanin immunoreactivity in rostral perikarya and brain fibers of male European eels and female American eels. The cross-sectional area of these perikarya increased significantly after both treatments whereas cell bodies of the posteroventral hypothalamus were slightly affected. Dihydrotestosterone showed no clear effect. Fibers close to the corticotropes were sometime increased, but galanin synthesis was not induced in pituitary cells. In contrast, estradiol induced galanin synthesis in rat pituitary cells, but had a still controversed effect on hypothalamic galanin. A putative influence of galanin on the pituitary-gonadal axis is discussed as gonadal hormones diversely affect gonadotropes and gonosomatic indices in Anguilla.     

中华大蟾蜍多种组织内5—羟色胺免疫染色细胞的分布

The distribution of 5-hydroxytryptamine (5-HT) immunostaining cells in the digestive tracts (hibernation and nonhibernation), the brain and other various tissues of Bufo bufo gargarizans was studied by peroxidase anti-peroxidase immunocytochemical method. In the brain, 5-HT immunostaining cells were localized in the raphen nuclear area of brain stem and in the ependyma cell area of the ventriclus tertius of diencephalon. These immunostaining cells were round or oval. The cells usually possess processes which were filled with immunoreactive substance. Some of the processes were contact with the processes of other cells. A few 5-HT positive reactive nerve fibers were observed in the brain stem and the diencephalon. The density of 5-HT immunostaining cells in the digestive tubes were the highest in the pylorus, fundus, cardia of gaster, and moderate in the esophagus and duodenum and the lowest in the large intestine and the small intestine. The density of 5-HT immunostaining cells in the digestive tubes were higher in nonhibernant toads than in hibernant toads. By the statistical method, the difference of the density between the two sorts of toads were notable (P less than 0.05). The 5-HT immunostaining cells were visualized to distribute between the epithelium cells of the mucosa or the epithelium cells of gland. These positive cells usually had one or more processes which contained 5-HT immunoreactive substance. Some were reached into lumen surface of the gland or intestine. Some were extended into lamina propria through the basal membrane. These results indicate that the 5-HT immunostaining cells in digestive tubes could release 5-HT by both endocrine and exocrine ways.     

Ultrastructural immunohistochemical localization of vasopressin in the hypothalamic-neurohypophysial system of three murids

Vasopressin was immunohistochemically localized at the electron microscopic (EM) level in the hypothalamic-neurohypophysial system (HNS) of three murids. Antiserum to vasopressin was produced in rabbits injected with lysine vasopressin (LVP) conjugated to egg albumin (EA), anti-EA being precipitated prior to staining. Sternberger's unlabeled antibody peroxidase technique was employed, immunoreactivity being designated by peroxidase anti-peroxidase (PAP) molecules and electron opacity. Immunoreactive neurosecretory granules (NSG) were found in the perikarya of the supraoptic nucleus (SON) in all three murids investigated, although far more profusely in the two wild strains. Immunoreactive axonal NSG were observed in the inner and outer zones of the median eminence (ME), and within most of the axons and terminals in the neurohypophysis. The concentration of primary serum effective for staining the SON (110–150) was far higher than that required for the ME and the neurohypophysis (1:500–1:1,200). AntiLVP also induced electron opacity of granules in cells of the pars intermedia (PI). Discussion centers on the significance of immunoreactive NSG in the neurosecretory (NS) perikarya, on the possibility of an extragranular pool of hormone, and on speculation about the electron opacity of the PI granules.     

中华大蟾蜍多种组织内5-羟色胺免疫染色细胞的分布

用过氧化物酶-抗过氧化物酶(PAP)法,对中华大蟾蜍消化道(冬眠期与非冬眠期),脑及其他组织的5-HT分布进行了研究。5-HT免疫染色细胞位于脑干中缝核区和间脑的第Ⅲ脑室腹侧的室管膜细胞区。阳性神经元呈圆形或卵圆形,细胞常有突起与其他阳性细胞突起相连,上述部位中还有一些阳性神经纤维。消化道的免疫染色细胞密度在胃幽门、胃体和胃贲门处最高,食道和十二指肠次之,大肠和小肠最低。非冬眠期蟾蜍消化道内免疫染色细胞密度明显高于冬眠期的(P<0.05)。阳性细胞位于粘膜上皮或腺上皮细胞间,细胞有一个或一个以上呈阳性反应的突起,有的突起伸入肠腔面或腺腔面,有的穿过基膜到达固有层,表明这些细胞兼有内、外分泌的功能。在甲状旁腺的主细胞间,肺呼吸性细支气管上皮和肺泡管上皮细胞间都有5-HT免疫染色细胞,细胞呈立方形、圆形、卵圆形或不规则形,常有几个细胞成簇分布。     

Immunoidentification of CRF-like material in the intermaxillary glands during Bufo arenarum development

The presence of corticotropin-releasing factor-like material in the intermaxillary glands was studied by immunocytochemical techniques during the metamorphosis of Bufo arenarum. The intermaxillary glands appeared at stage XV (midprometamorphosis) with CRF-like material slightly immunoreactive. These glands are located posterior to the premaxillae and between the nasal capsules in the roof of the mouth and are formed of alveoli or tubules. During metamorphic climax, corticotropin-releasing factor-like material was identified strongly immunostained at the apices of the secretory cells. It was observed that collecting ducts of the gland open to the anterior palatal surface suggesting that the secretion could be ingested by tadpoles. Our results clearly showed that ir-CRF-like material present in the intermaxillary glands is ingested by tadpoles during metamorphosis and could play an important role during amphibian development.