Exposure assessment through realistic laboratory simulation of a soccer stadium fire.

On Sunday April 13, 2008 a fire broke out on a grandstand in the Euroborg soccer stadium in Groningen The Netherlands. The polyamide chairs on the grandstand were set on fire and supporters were exposed to the emitted smoke which induced mild health effects. The Dutch government was concerned about potential health risks that such fires could have to exposed fans. Especially the exposure to toxic fumes was considered a risk because prior research has proven that large amounts of chemical compounds are emitted during the burning of chemical substances such as polyamide. Among these emitted compounds are HCN, CO, NO(x), NH(3) and volatile organic compounds. To study if supporters were exposed to hazardous chemical compounds we designed a laboratory controlled replica of a part of the grandstand of the Euroborg stadium to perform fire-experiments. This simulation of the fire under controlled circumstances proved that a wide variety of chemicals were emitted. Especially the emission of CO and NO(x) were high, but also the emission of formaldehyde might be toxicologically relevant. The emission of HCN and NH(3) were less than expected. Exposure assessment suggests that the exposure to NO(x) is the main health risk for the supporters that were present at the Euroborg fire.

Bradykinin into amygdala induces thermal hyperalgesia in rats.

Bradykinin is one of the most potent endogenous algesic substances and its role in pain transmission has been intensively studied in the periphery. However, the action of this peptide in central structures involved in pain transmission remains unclear. Administration of bradykinin (0.25 nmol/site) into the right amygdala of adult male Wistar rats induced thermal hyperalgesia, evaluated in the paw-flick test. Bradykinin-induced hyperalgesia was abolished by co-administration with the B(2) receptor antagonist Hoe 140 (5 pmol/site), the NMDA antagonist MK-801 (5 nmol/site), the cyclooxygenase inhibitor indomethacin (10 nmol/site) and the glial metabolic inhibitor fluorocitrate (1 nmol/site). Since the intra-amygdala administration of bradykinin did not alter spontaneous locomotion in the open-field test, it is unlikely that the current described hyperalgesic effect of bradykinin is due to an unspecific action on motor activity. These findings provide evidence that bradykinin, through activation of amygdalar B(2) receptors induces hyperalgesia and that glutamatergic- and prostanoid-mediated mechanisms are involved in such effect.

Succinate causes oxidative damage through N-methyl-D-aspartate-mediated mechanisms.

In this study we investigated whether succinate, the accumulating substrate in succinate dehydrogenase (SDH) deficiencies and SDH inhibitor intoxication, causes lipoperoxidation and protein carbonylation, and if NMDA receptors are involved in the succinate-induced oxidative damage. Adult male mice (30-40 g) received an intracerebroventricular injection of succinic acid (0.7, 1.0 and 1.7 micromol/5 microl) or 0.9% NaCl (5 microl) and had their exploratory behavior assessed in an open field for 10 min. Succinate (0.7 and 1.0 micromol/5 microl) decreased locomotor activity behavior and increased thiobarbituric acid reactive substances (TBARS) and protein carbonylation in the forebrain. Conversely, 1.7 micromol of succinate did not alter locomotor activity or oxidative damage parameters. The involvement of NMDA receptors in the succinate-induced increase of total protein carbonylation content and exploratory behavior inhibition was assessed by co-administrating MK-801 (7 nmol/2.5 microl icv), a noncompetitive NMDA receptor antagonist, with succinate (1 micromol/2.5 microl icv). The co-administration of MK-801 protected against succinate-induced increase of total protein carbonylation and decrease of locomotor activity. These results suggest the involvement of NMDA receptors in these effects of succinate, which may of particular relevance for succinate-accumulating conditions, such as SDH inhibitors intoxication and inherited SDH deficiencies.

Dopaminergic inhibition of reproduction in teleost fishes: ecophysiological and evolutionary implications.

In many teleosts, dopamine (DA) exerts direct inhibitory control on gonadotropes, counteracting the stimulatory effect of gonadotropin-releasing hormone (GnRH) on gonadotropin release. This dual control by GnRH and DA has been demonstrated in various adult teleosts and has major implications for aquaculture. Because of its unique life cycle, the European eel has provided a powerful model for demonstrating the key role of DA in the control of puberty. Data from tetrapods suggest that the inhibitory role of DA on reproduction is not restricted to the teleosts. Thus, DA inhibitory control could represent an ancient evolutionary component in the neuroendocrine regulation of reproduction that may have been differentially maintained throughout vertebrate evolution. The intensity of DA inhibition, its main site of action, and its involvement in the control of puberty, seasonal reproduction, ovulation, spermiation, or even sex change may differ among classes of vertebrates, as well as within smaller phylogenetic units such as teleosts or mammals. An inhibitory role for DA has been reported also in some invertebrates, indicating that neuronal DA pathways may have been recruited in various groups of metazoa to participate in the control of reproduction. In addition to the incontestable GnRH neurons, the recruitment of DA neurons for the neuroendocrine control of reproduction provides an additional brain pathway for the integration of various species-specific, internal, and environmental cues. In teleosts, the plasticity of the DA neuroendocrine role may have contributed to their large diversity of biological cycles and to their successful adaptation to various environments.

Brain expression of tyrosine hydroxylase and its regulation by steroid hormones in the European eel quantified by real-time PCR.

In the eel, dopamine inhibits pubertal development. To investigate the regulatory mechanisms involved, we developed a quantitative real-time RT-PCR assay for measurement of brain expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine. TH expression was highest in the olfactory bulb, followed by the di-/mesencephalic areas and the telencephalon/preoptic area. TH expression in the optic lobes and hindbrain was low or below the detection limit. In vivo treatment with testosterone, but not estradiol, resulted in increased TH expression in the forebrain, except the optic tectum, but not in the hindbrain. The results were confirmed by in situ hybridization.

Evidence for a negative feedback in the control of eel growth hormone by thyroid hormones.

The regulation of growth hormone (GH) by thyroid hormones (THs) has been shown to present species variation. We investigated the regulation of GH in the eel, a representative of an ancient group of teleosts. In vivo administration of triiodothyronine (T(3)) or thyroxine (T(4)) significantly reduced pituitary and serum GH levels, as measured by homologous RIA. In order to investigate the ability of THs to regulate GH production directly at the pituitary level, we used a long-term, serum-free primary culture of eel pituitary cells. Both T(3) and T(4) inhibited GH release in a concentration-dependent manner, producing up to 50% inhibition at 10 nM, with an ED(50) of <0.2 nM, within the range of their physiological circulating levels. Other hormones also acting via the nuclear receptor superfamily, such as sex steroids (testosterone, estradiol and progesterone) and corticosteroid (cortisol), had no effect on GH release in vitro, underlining the specificity of the regulatory effect of THs on GH. Measurement of both GH release and cellular content for calculation of GH production in vitro indicated that THs not only inhibited GH release but also GH synthesis. Dot-blot assay of GH messenger RNA (mRNA) using an homologous eel cDNA probe showed a decrease in GH mRNA levels in cells cultured in the presence of T(3), as compared with control cells. This demonstrated that the inhibition of T(3) on GH synthesis was mediated by a decrease in GH mRNA steady state levels. In conclusion, we demonstrate inhibitory regulation of eel GH synthesis and release by THs, exerted directly at the pituitary level. These data contrast with the rat, where THs are known to have a stimulatory effect and suggest that the pattern observed here in an early vertebrate and also found in birds, reptiles and some mammals including humans, may represent an ancestral and more generalized vertebrate pattern of TH regulation of pituitary GH.

Pituitary growth hormone secretion in the turbot, a phylogenetically recent teleost, is regulated by a species-specific pattern of neuropeptides.

In mammals, growth hormone (GH) is under a dual hypothalamic control exerted by growth hormone-releasing hormone (GHRH) and somatostatin (SRIH). We investigated GH release in a pleuronectiform teleost, the turbot (Psetta maxima), using a serum-free primary culture of dispersed pituitary cells. Cells released GH for up to 12 days in culture, indicating that turbot somatotropes do not require releasing hormone for their regulation. SRIH dose-dependently inhibited GH release up to a maximal inhibitory effect of 95%. None of the potential stimulators tested induced any change in basal GH release. Also, neither forskolin, an activator of adenylate cyclase, nor phorbol ester (TPA), an activator of protein kinase C, were able to modify GH release, suggesting that spontaneous basal release already represents the maximal secretory capacity of turbot somatotropes. In contrast, forskolin and TPA were able to increase GH release in the presence of SRIH. In this condition (coincubation with SRIH), pituitary adenylate cyclase-activating polypeptide (PACAP) stimulated GH release, whereas none of the other neuropeptides tested (GHRHs; sea bream or salmon or chicken II GnRHs; TRH; CRH) had any significant effect. These data indicate that inhibitory control by SRIH may be the basic control of GH production in teleosts and lower vertebrates, while PACAP may represent the ancestral growth hormone-releasing factor in teleosts, a role taken over in higher vertebrates by GHRH.

Evidence that corticotropin-releasing hormone acts as a growth hormone-releasing factor in a primitive teleost, the European eel (Anguilla anguilla).

The inhibitory control of growth hormone (GH) release by somatostatin (SRIH) has been conserved throughout vertebrate evolution. In contrast, the neuropeptides involved in the stimulatory control of GH vary according to species and/or physiological situations. We investigated the direct pituitary regulation of GH release in a primitive teleost, the European eel (Anguilla anguilla L.) at the juvenile stage. Short-term serum-free primary cultures of dispersed pituitary cells were used, and GH release was measured by an homologous radioimmunoassay. Whereas growth hormone-releasing hormone (GHRH), gonadotropin-releasing hormone (GnRH), thyrotropin-releasing hormone (TRH), neuropeptide Y (NPY) and cholecystokinin (CCK) failed to induce any change in GH release, corticotropin-releasing hormone (CRH) dose-dependently stimulated GH release with a significant effect at 1 nM and a maximal effect (> or =400% of controls at 24 h) at 100 nM. In agreement with our previous studies, PACAP also stimulated GH release but its maximal effect was lower than that of CRH. Proopiomelanocortin (POMC)-peptides, corticotropin (ACTH), melanotropin (alpha-MSH), beta-endorphin) had no effect on GH release, at any dose tested (0.1-1000 nM), indicating that the stimulatory effect of CRH on GH release by somatotrophs was not mediated by CRH-induced release of POMC-peptides from corticotrophs and melanotrophs. The CRH antagonist, alpha-helical CRH(9-41), significantly inhibited the stimulatory effect of CRH on GH release, suggesting the implication of specific CRH receptors related to mammalian ones. The stimulatory effect of CRH on GH release was reduced after 24 h of incubation, indicating a desensitization. In contrast, no desensitization to the inhibitory effect of SRIH was observed. SRIH inhibited CRH action in a dose-dependent manner. The effect of SRIH was overriding, 1 nM SRIH being able to abolish the effect of 1000 nM CRH. In conclusion, in the eel, CRH stimulates GH release directly at the pituitary cell level. GH and cortisol secretions could interact in controlling several physiological functions such as metabolism and ion exchange. This study suggests that CRH may have played an important early role in vertebrates co-ordinating the activation of various endocrine axes involved in metamorphosis, osmoregulation, stress and fasting. The stimulatory role of CRH on GH release may have been partially conserved during evolution, as it is found in some human physio-pathological situations such as stress, fasting and depression.

Cortisol selectively stimulates pituitary gonadotropin beta-subunit in a primitive teleost, Anguilla anguilla.

It has been suggested that in mammals, glucocorticoids, beside their stress-related inhibitory effects on reproductive function, may also play a stimulatory role at the onset of puberty. Using the juvenile female eel as a model, we investigated the potential stimulatory role of cortisol (F) on pituitary gonadotropin (GtH-II). GtH-II levels were measured by RIA, and messenger RNA (mRNA) levels for alpha- and GtH-II beta-subunits were determined by dot blot using homologous probes. F treatment increased eel pituitary GtH-II content in vivo and in vitro. Using a long term, serum-free primary culture of pituitary cells, we studied the direct effect of F on GtH-II production. F increased the GtH-II cellular content in vitro in a dose- and time-dependent manner. The relative potencies of various corticosteroids on GtH-II were: triamcinolone acetonide > dexamethasone > F >> cortisone and aldosterone, indicating a glucocorticoid-specific receptor (GR). F stimulated GtH-II production through a selective increase in mRNA levels for GtH-II beta-subunit; no significant effect was observed on alpha-subunit mRNA levels. This stimulatory effect of F on GtH-II beta, played out directly at the pituitary cell level, recalls that of F on FSHbeta in the rat. The present study, performed in a primitive teleost at the juvenile stage, suggests that the role of F in the positive regulation of gonadotropins at puberty may have arisen early in vertebrate evolution.

Insulin-like growth factor-I stimulates gonadotrophin production from eel pituitary cells: a possible metabolic signal for induction of puberty.

Insulin-like growth factor (IGF)-I has been suggested as a potential signal linking growth and puberty in mammals. Using the juvenile European eel as a model, we employed a long-term, serum-free primary culture of pituitary cells to study the direct effect of IGF-I on gonadotrophin (GtH-II=LH) production. IGF-I increased both cell content and release of GtH-II in a time- and dose-dependent manner. IGF-I and IGF-II had similar potencies but insulin was 100-fold less effective, suggesting the implication of an IGF type 1 receptor. Other growth and metabolic factors, such as basic fibroblast growth factor and thyroid hormones, had no effect on GtH-II production. IGF-I did not significantly increase the number of GtH-II immunoreactive cells, indicating that its stimulatory effect on GtH-II production does not result from gonadotroph proliferation. Comparison of IGF-I and somatostatin (SRIH-14) effects showed that both factors inhibited growth hormone (GH) release but only IGF-I stimulated GtH-II production by eel pituitary cells. This indicates that the effect of IGF-I on gonadotrophs is not mediated by the reduction of GH released by somatotrophs into the culture medium. This study demonstrates a specific stimulatory effect of IGF-I on eel GtH-II production, played out directly at the pituitary level. These data obtained in a primitive teleost suggest that the role of IGF-I as a link between body growth and puberty may have been established early in the evolution of vertebrates.

Long-term inhibitory effects of somatostatin and insulin-like growth factor 1 on growth hormone release by serum-free primary culture of pituitary cells from European eel (Anguilla anguilla).

To investigate the ability of hypothalamic and peripheral factors to directly regulate growth hormone (GH) release in a primitive teleost, the European eel (Anguilla anguilla L.), we used primary cultures of dispersed pituitary cells. When cultured for 12 days in a serum-free medium, pituitary cells continuously released large amounts of GH, which exceeded the initial cellular content. Somatotropin-release inhibiting hormone (SRIH-14) dose-dependently inhibited GH release (EC50 0.75 nM) up to a maximal inhibitory effect of 95%. No desensitization of somatotropes to SRIH was observed over the 12 days of culture. Use of receptor subtype-selective SRIH agonists suggests the existence on eel somatotropes of SRIH receptor(s) related to the mammalian sst2/sst3/sst5 class rather than to the sst1/sst4 class. Insulin-like growth factor 1 (IGF1) dose-dependently inhibited GH release (EC50 0.03 nM) up to a maximal inhibitory effect of 85%, without desensitization. IGF1 and IGF2 were equipotent in inhibiting GH release, whereas insulin was 1,000 times less active, suggesting the implication of a receptor related to the mammalian IGF type 1 receptor. These results indicate that eel somatotropes are active in vitro without any specific additional factors, and suggest the existence of a dominant inhibitory control of GH release in vivo. Two potential candidates for this chronic negative regulation are a neurohormone, SRIH and a circulating factor, IGF1. These data underline the early evolutionary origin of the molecular and functional SRIH-GH-IGF1 neuroendocrine axis in vertebrates.

Primary structure of ovine fibroblast growth factor-1 deduced by protein and cDNA analysis.

The amino acid sequence of full-length ovine fibroblast growth factor-1 (FGF-1) was determined by a combination of protein and cDNA sequencing. FGF-1 cDNA analysis indicated that ovine kidney cells express mRNAs encoding both full-length FGF-1 and a truncated FGF-1 variant. An overall comparison of the ovine FGF-1 primary sequence to the eight species studied to date revealed a high degree of conservation, with ovine FGF-1 sharing 90 and 95% sequence identity with human FGF-1 and bovine FGF-1, respectively. Additionally, the FGF-1 proteins from the various species have conserved cysteine residues at positions 30 and 97 and contain acetylated amino-terminal alanine residues. Mass spectrometry analysis confirmed that the blocking group of ovine FGF-1 is also consistent with that of an acetyl-moiety. In contrast to the other FGF-1 proteins, the 154 residue primary sequence of ovine FGF-1 contains three unique amino acid differences: Arg9, Arg44, and Ile123. Ovine FGF-1, unlike human FGF-1, is a potent mitogenic factor for NIH 3T3 fibroblasts in the absence of heparin. In the presence of exogenous heparin, the mitogenic activity of ovine FGF-1 is potentiated slightly.

Androgens stimulate gonadotropin-II beta-subunit in eel pituitary cells in vitro.

Primary cultures of juvenile eel (Anguilla anguilla) pituitary cells were used to study the direct effects of sex steroids on gonadotropin (GtH-II) cell content and release (radioimmunoassay) as well as on mRNAs levels for alpha and GtH-II beta-subunits (dot-blot). Testosterone stimulated GtH-II production in a dose- and time-dependent manner by selectively increasing mRNAs for GtH-II beta-subunit but not alpha-subunit. This positive effect was also induced by non-aromatizable androgens (androstanediol and dihydrotestosterone) but not by estradiol, indicating an androgen-specific effect in the eel. The androgen-specific stimulation of eel GtH-II beta appears closer to the regulation of mammalian follicle stimulating hormone-beta (FSHbeta) than that of salmonid GtH-II beta or mammalian luteinizing hormone-beta (LHbeta)-subunits. Comparison with previous in vivo experiments suggests multiple sites of action of sex steroids on the brain-pituitary gonadotropic axis for the positive feedback on GtH-II synthesis in this juvenile fish.

Primary cultures of dispersed pituitary cells from estradiol-pretreated female silver eels (Anguilla anguilla L.): immunocytochemical characterization of gonadotropic cells and stimulation of gonadotropin release.

To analyze the multihormonal control mechanisms of GTH secretion in the eel, primary culture of pituitary cells from control or estradiol-treated female silver eels, a treatment known to stimulate in vivo GTH synthesis, was developed. Dispersed eel pituitary cells obtained by enzymatic (trypsin/DNAse) and mechanical dispersion were cultured in Earles M199, at 18 degrees. Immunoreactive GTH (ir-GTH) cells were characterized by the immunoperoxidase method, using antiserum to carp GTH beta subunit. Ir-GTH cells from control silver eels were small and represented 14% of the dispersed pituitary cells. In contrast, ir-GTH cells from estradiol-treated eels were larger (cell area x 2.5) and represented a higher proportion (21%) of the pituitary cells. Intracellular and medium contents of GTH were measured by radioimmunoassay for the GTH beta subunit. In vivo estradiol-treatment increased more than 100-fold the GTH content of cell cultures. GTH release, studied over 1 to 4 hr, was undetectable in cultures from normal eels. In contrast, GTH release was low (less than 2% of cell content) but measurable in cultures from estradiol-treated eels. Subsequent experiments examined effects of various secretagogues on GTH release from primary cultures of pituitary cells from estradiol-pretreated eels. GTH release was significantly increased (x1.5 to x3 basal release) by 10(-6) M GnRH-A as well as by both native GnRHs in the eel (mammalian GnRH, mGnRH, and chicken GnRH-II, cGnRH-II), at the same concentration. Lower GnRH concentrations had no significant effect, indicating a low sensitivity of gonadotrophs to GnRH, likely to be related to their immature state at the silver stage. The similar efficacy of mGnRH and cGnRH-II suggested that the pituitary GnRH receptor had a low specificity toward various molecular forms, in the eel as in the other nonmammalian species. The protein kinase C (PKC) activator (phorbol ester: PMA) also stimulated GTH secretion, with a maximal effect at 10(-8) M, indicating that the PKC pathway was functional. In contrast, a depolarizing agent (50 mM KCl) had no significant effect on GTH release, suggesting lack of a functional voltagesensitive calcium channel (VSCC) secretory pathway. Perifusion experiments on whole pituitary confirmed the lack of effect of KCl on gonadotrophs from E2-pretreated eels and indicated that an additional in vivo treatment with GnRH agonist and dopamine antagonist could induce the differentiation of a functional VSCC pathway. These characteristics of the transduction mechanisms may be related to the immature state of the eel gonadotrophs at the silver stage.

Development of a radioimmunoassay for European eel growth hormone and application to the study of silvering and experimental fasting.

A specific and sensitive homologous radioimmunoassay for eel (Anguilla anguilla L.) growth hormone (angGH) has been developed. The antiserum, raised against purified angGH and used at 1:20,000 final dilution, did not cross-react with eel prolactin or thyrotropin, carp gonadotropin II, bovine GH, or serum from hypophysectornized eel. The inhibition curves for eel pituitary extracts and serum were parallel to that of angGH standard. The ED50 value was between 1 and 2 ng/tube and the recovery of purified angGH added to the serum was about 100%. In immunocytochemical studies, the antiserum, used at 1:1000 dilution, specifically labeled the somatotrophs in the pituitaries of the glass, yellow, and silver eels. The GH contents were determined in the pituitaries of glass, yellow, and silver eels and in the serum at the yellow and silver stages. GH variations during the transformation of the yellow to silver eel were examined. The results indicated a decrease in GH production between the yellow and the silver eels, possibly related to the cessation of growth at the silver stage. In contrast to the situation in the naturally fasting silver eel, submitting yellow eels to 3 months of starvation (experimental fasting) greatly increased GH production. This suggests a variation in the regulation of GH according to the type of fasting (natural or experimental) and/or the stage of the fish (yellow or silver).

Differential regulation of the two forms of gonadotropin-releasing hormone (mGnRH and cGnRH-II) by sex steroids in the European female silver eel (Anguilla anguilla).

The effect of steroids on the two gonadotropin-releasing hormone (GnRH) forms present in the eel (mammalian GnRH, mGnRH and chicken GnRH-II, cGnRH-II), as well as on gonadotropin (GTH), was studied using specific radioimmunoassays. Female silver eels received chronic treatments with various steroids (estradiol, testosterone, androstenedione, 5 alpha-androstane-3 beta, 17 beta-diol). Estradiol or the combination of estradiol and androgens induced increases in brain and pituitary mGnRH levels and pituitary GTH level, whereas androgens given alone had no significant effect. In contrast, androgens or their combination with estradiol reduced brain cGnRH-II levels (this form remaining undetectable in the pituitary), estradiol given alone having no significant effect. This work demonstrates that the two forms of GnRH undergo a differential regulation by steroids, with a positive estrogen-dependent feedback on mGnRH (as well as on GTH) and a negative androgen-dependent feedback on cGnRH-II. These data are in agreement with previous results obtained in experimentally matured female eels (induced by a gonadotropic treatment which stimulates the production of both estrogens and androgens) showing increases in mGnRH and GTH levels, as well as a decrease in cGnRH-II [1]. The positive feedback of steroids on the mGnRH-GTH axis adds credence to the hypothesis according to which mGnRH would be the main form involved in the control of the gonadotropic function. This positive feedback would play an important role, amplifying pubertal stimulation of the gonadotropic axis, in this fish species.

The distribution of GABA-immunoreactive neurons in the brain of the silver eel (Anguilla anguilla L.).

The distribution of GABA-immunoreactivity was studied in the brain of the silver eel (Anguilla anguilla) by means of antibodies directed against GABA. Immunoreactive neuronal somata were distributed throughout the brain. Positive perikarya were detected in the internal cellular layer of the olfactory bulb, and in all divisions of the telencephalon, the highest density being observed along the midline. Numerous GABA-reactive cell bodies were found in the diencephalon, particularly in the preoptic and tuberal regions of the hypothalamus, and the dorsolateral, dorsomedial and ventromedial thalamic nuclei. In the optic tectum, the majority of GABA-positive cell bodies were located in the periventricular layer. A number of immunolabeled cell bodies were observed in different tegmental structures, notably the torus semicircularis. In the cerebellum, the Purkinje cells were either very intensely or very weakly immunoreactive. In the rhombencephalon, reactive cell bodies were observed in the eminentia granularis, the valvula cerebellaris, the octavolateral nucleus, the lobus vagus and in the vagal and glossopharyngeal motor nuclei. Intensely immunoreactive axons and terminals were observed in the external granular layer and internal cellular layer of the olfactory bulb. In the telencephalon, the highest density of reactive fibres and boutons was found in the fields of the medial wall. Many immunolabeled fibres were seen in the medial and lateral forebrain bundles. In the diencephalon, intense labelling of fibres and terminals were observed in the nuclei situated close to the midline. In the optic tectum the highest density of reactive fibres was seen in the sfgs, the layer to which the retina projects massively. Finally, in the rhombencephalon the strongest labelling of neurites was observed in the nuclei of the raphé, the nucleus octavocellularis magnocellularis and the nuclei of the IXth and Xth cranial nerves. The GABAergic system of the eel, which is well developed, appears to be generally comparable to that described in tetrapod vertebrates.

Differential distribution and response to experimental sexual maturation of two forms of brain gonadotropin-releasing hormone (GnRH) in the European eel, Anguilla anguilla.

Using specific radioimmunoassays for the two GnRH molecular forms present in the European eel, Anguilla anguilla, (mGnRH and cGnRH II), we compared their distributions in the pituitary and different parts of the brain of female silver eels, as well as the modifications of their levels in experimentally matured female eels (treated with carp pituitary extract). In control eels, mGnRH levels were higher than cGnRH II levels in the pituitary, olfactory lobes and telencephalon, di- and mesencephalon, while the opposite was found in the posterior part of the brain (met- and myelencephalon). Experimental sexual maturation of the gonads significantly increased mGnRH levels in the pituitary and anterior parts of the brain; such a positive effect was not observed on the low cGnRH II levels, which were, in contrast, reduced. These data indicate that the positive feedback of gonadal hormones on GnRH, that we previously demonstrated, would specifically affect the mGnRH form. The differential distribution and control of mGnRH and cGnRH II suggest that these two forms have different physiological roles in the eel. The large increase in mGnRH during sexual maturation suggests the prime implication of this form in the neuroendocrine control of reproduction.

An experimental study of the retinal projections of the European eel (Anguilla anguilla), carried out at the catadromic migratory silver stage.

A radioautographic study of the European eel (Anguilla anguilla) was carried out in ten female specimens at the catadromic migratory silver stage. Terminal arborizations of contralaterally projecting visual fibres were identified in ten hypothalamic structures (area optica preoptica ventralis and the nuclei suprachiasmaticus, opticus hypothalamicus ventromedialis, preopticus magnocellularis lateralis, posterioris lateralis, posterioris dorsalis periventricularis posterioris dorsalis lateralis, posterioris dorsalis medialis, posterioris ventralis lateralis, and posterioris ventralis periventricularis), ten thalamo-pretectal structures (Areas C1 and C2, area optica tractus opticus ventrolateralis and the nuclei dorsolateralis thalami, ventrolateralis thalami pars ventralis, opticus ventralis thalami, geniculatus lateralis, opticus pretectalis partes dorsalis et ventralis, and opticus commissurae posterioris), and in the tectal strata opticum partes externa et interna, fibrosum et griseum superficiale, griseum centrale and album centrale. An accessory optic system was identified, and a contralateral retinal projection to the anterior region of the anterior semicircular torus (n. opticus dorsolateralis mesencephali) was identified. Ipsilateral projections to hypothalamic and thalamopretectal structures were also observed. Apart from the retinal projection to the preoptic area, which is exceptionally important in the silver eel, the general plan of organization of the primary visual centres of this form is comparable to that described in other species of teleost. However, the architecture of some primary visual centres shows characteristics similar to those described in more primitive Actinopterygians.

Positive feedback control by the gonads on gonadotropin (GTH) and gonadoliberin (GnRH) levels in experimentally matured female silver eels,Anguilla anguilla.

Treatment of sham-operated female silver eels with carp pituitary extract stimulated ovarian development and induced increases in pituitary gonadotropin (GTH) and gonadoliberin (GnRH) contents. Both effects of carp pituitary extract were abolished in ovariectomized eels, indicating the involvement of the gonads. Endogenous sexual steroids, the secretion of which was increased during sexual maturation, should be responsible for the stimulation of GTH and GnRH levels. Ovariectomy itself had no significant effect on pituitary GTH and GnRH contents, reflecting the fact that, at the silver stage, sexual steroid levels are too low to exert any significant effect on pituitary GTH and GnRH. The positive feedback control exerted by the gonads on GTH and GnRH levels during sexual maturation, in the eel as well as in some other teleosts, would produce an amplification of the pubertal stimulation of the hypothalamo-pituitary-gonadal axis.