Topographic organization of descending projection neurons to the spinal cord of the goldfish, Carassius auratus

Several neurons from different nuclei give rise to descending spinal tracts and project to various levels in the spinal cord of goldfish, Carassius auratus. These were visualized by retrograde transport of horseradish peroxidase (HRP) administered to the bilaterally transected spinal cord at 6 levels, corresponding to 1st, 5th, 10th, 15th, 20th and 25gh segments of the vertebral column. As many as 16 brain nuclei or neuronal aggregations and the Mauthner cells project posteriorly up to the 20th spinal segment. Restricted neurons of the dorsolateral area in the nucleus preopticus magnocellularis and those of the ventromedial nucleus of the thalamus projected up to the 20th and 25th segments respectively. In the mesencephalon, the nucleus ruber and the nucleus of the medial longitudinal fasciculus revealed retrogradely labeled somata; the former extended up to the 20th segment, while the latter projected up to the 25th segment. The remaining descending projected neurons of the brain belonged to the rhombencephalon. The nucleus of the lateral lemniscus; anterior, magnocellular, descending and posterior divisions of the octaval nucleus: raphe nucleus; Mauthner cell and the neurons located adjacent to the trigeminal tract and those in the vicinity of the secondary gustatory tract sent their processes up to 20th segmental level. However, somata of the superior, medial and inferior divisions of the reticular nucleus and restricted neurons of the facial lobe extended up to 25th segmental level. The pattern of neuronal projections into the spinal cord suggests a topographic organization.     

Telencephalic projections to the goldfish hypothalamus: An anterograde degeneration study

In this study, large areas of goldfish telencephalon were ablated including rostral nucleus preopticus periventriculare (rNPP), and degenerating axons were traced by a modified Fink and Heimer procedure. The lesioning procedure ablated large regions of area dorsalis telencephali pars medialis, centralis, and dorsolateral complex; and completely removed area ventralis telencephali pars dorsalis, ventralis, and lateralis. In addition, the supracommissural nucleus and rNPP were lesioned specifically because both nuclei have been thought to be involved in courtship behavior and endocrine control of reproduction. This investigation demonstrated extensive fiber projections from telencephalic nuclei and/or rNPP to the hypothalamus. Lesioned telencephalon and/or rNPP projected bilaterally to nucleus preopticus and the suprachiasmatic nucleus and unilaterally to the following tuberal nuclei: nucleus anterior tuberis, and the lateral hypothalamic nucleus. A much larger fiber projection to the inferior lobe nuclei was also observed with a large contralateral as well as ipsilateral input.     

Branchial ventilation rates of cataleptic goldfish (Carassius auratus) and the effects of light and electric shock

Inverted, cataleptic goldfish showed an increase in ventilation rate over a 20 min period. The time-dependent increase in respiratory movement was initially suppressed when a 10 sec light or a 0.1 sec shock was presented at 1.5 min intervals. Contingent presentation of the light and shock, in a classical conditioning paradigm, resulted in reduced ventilation rates during the session. Physiological changes accompanying conditioning may thus alter the cataleptic state.     

Myelination and remyelination in the regenerating visual system of the goldfish

Summary The remyelination of regenerated optic axons was investigated in goldfish following either optic nerve crush or ouabain retinal intoxication. Axons grown after nerve crushing acquire thinner myelin sheaths than axons originating from reconstituted ganglion cells. If axons of reconstituted ganglion cells are crushed and allowed to regenerate, the subsequent myelination is weaker than that of control axons not interrupted by crushing, but stronger than that of axons of preexisting retinal ganglion cells.The present results suggest that a neuron is capable of inducing a normally developed myelin sheath when its axon contacts an oligodendrocyte the first time, whereas a neuron whose axon contacts an oligodendrocyte the second time is not capable of forming a normal myelin sheath in the adult animal. The present results also support the notion that the oligodendrocyte requires a neuronal signal for myelin sheath formation.Supported by the Deutsche Forschungsgemeinschaft (Wo 215/5)     

Follistatin suppresses FSHbeta but increases LHbeta expression in the goldfish - evidence for an activin-mediated autocrine/paracrine system in fish pituitary

Our previous study demonstrated that recombinant goldfish activin B stimulated goldfish FSHbeta but inhibited LHbeta expression. Similar to activin B, activin A also exhibited the inverse effects on the expression of the two gonadotropins. The novel dual effects of activins on FSH and LH in the goldfish raise an interesting question as to where the activin comes from in vivo. In the present study, we first demonstrated the expression of activin, its receptors and binding protein follistatin in the goldfish pituitary, leading to a suggestion that an autocrine/paracrine regulatory system involving activin is operative in fish pituitary. To investigate the functionality of the pituitary-derived activin system in the regulation of gonadotropin biosynthesis, we further examined the effects of follistatin, an activin-binding protein, on goldfish FSHbeta and LHbeta expression. Follistatin not only reversed the effects of exogenous activin on FSHbeta and LHbeta expression but also had inverse effects on the basal expression of the genes; and its effects were opposite to those of activin. This suggests that the endogenous activin plays roles in controlling the expression of both FSHbeta and LHbeta genes. It is conceivable that any factors that influence the intrapituitary activin system in vivo will likely affect the biosynthesis of the two gonadotropins in the goldfish.     

Involvement of the histaminergic system on appetitive learning and its interaction with haloperidol in goldfish

This study investigated the actions of the histaminergic system on appetitive learning and memory, and its interaction with the dopaminergic system in goldfish. It consisted of nine sessions, in which fish were tested in a four-arm tank. On day 1, the animals were habituated for 10 min. On day 2, they were placed in one arm and had to find food at the left or the right arm. Time to begin feeding was recorded, and the procedure repeated for more 3 days (training phase). On training day 4, seven groups were injected with saline, seven with haloperidol (2.0 mg/kg) and one with DMSO solution before training and after feeding, three groups received saline, six chlorpheniramine (CPA) (1.0, 4.0 and 8.0 mg/kg), and six l-histidine (LH) (25, 50 and 100 mg/kg). Saline groups were considered as control of CPA and LH treated groups and DMSO as control of haloperidol. A non-injected group was also included. Testing occurred after 24 h. A reversal procedure was conducted 24h after testing and repeated for 3 days. The groups receiving CPA at 1.0 and 8.0 mg/kg and LH at 25, 50 and 100 mg/kg differed between Test and Reversal day 1. Pre-treatment with haloperidol plus 8.0 mg/kg of CPA and 25 and 50 mg/kg of LH reverted the treatment effect. However, in the groups treated with 1.0 mg/kg of CPA and 100 mg/kg of LH, the difference remained. This study confirmed the interaction between the histaminergic and the dopaminergic systems on memory process in goldfish.     

In vitro uptake and metabolism of testosterone by goldfish,Carassius auratus,mesenteric adipose tissue

Mesenteric adipose tissue was removed from adult goldfish, Carassius auratus, and incubated in vitro with (1alpha,2alpha)-3[H]testosterone (T). Total radioactivity in the medium decreased and tissue total radioactivity increased in a time-dependent fashion between 1 and 6 h of incubation with maximum uptake occurring between 4 and 6 h. After ether extraction and thin layer chromatography the amount of radioactivity comigrating with authentic T standard decreased over time in both medium and tissue samples. Radioactivity in the aqueous fraction remaining after ether extraction increased in a time-dependent manner, suggesting the presence of water-soluble conjugated steroids. Acid hydrolysis of the aqueous fraction yielded radioactivity that principally comigrated with T standard following a second ether extraction and TLC, along with a small, unidentified peak of poorly migrating radioactivity. Aromatization of T was assessed after charcoal stripping samples and measuring radioactivity remaining in the form of 3H(2)O. Small but significant amounts of 3H(2)O were present in both incubation medium and tissue samples. Incubation with the aromatase inhibitor 4-androsten-4-ol-3,17-dione acetate (ATD) decreased the formation of 3H(2)O in a dose-dependent manner. These results suggest that goldfish mesenteric adipose tissue is capable of converting T to several metabolites including water-soluble conjugates and estrogen.     

Relationship between α-melanocyte-stimulating hormone- and neuropeptide Y-containing neurons in the goldfish hypothalamus

Intracerebroventricular (ICV) injection of α-melanocyte-stimulating hormone (α-MSH) inhibits, whereas ICV injection of neuropeptide Y (NPY) stimulates food intake in the goldfish. However, there is little information about the functional relationship between α-MSH-induced anorexigenic and NPY-induced orexigenic actions in the goldfish. In this study we examined the relationship between α-MSH- and NPY-containing neurons in the goldfish hypothalamus to investigate whether these α-MSH- and NPY-containing neurons have direct mutual inputs. α-MSH- and NPY-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. In particular, α-MSH-containing nerve fibers or endings lay in close apposition to NPY-containing neurons in a specific region of the hypothalamus, the nucleus posterioris periventricularis (NPPv). NPY-containing nerve fibers or endings also lay in close apposition to α-MSH-containing neurons specifically in the interior part of the nucleus lateralis tuberis (NLTi). We also investigated the effect of ICV injection of melanocortin 4 receptor agonist (melanotan II) at 100 pmol/g body weight (BW), which is enough to suppress food intake, or NPY at 10 pmol/g BW, which is enough to enhance food intake, on expression levels of mRNA for NPY or proopiomelanocortin (POMC) in the hypothalamus. ICV injection of melanotan II and NPY induced a significant decrease in the expression levels for NPY and POMC mRNA, respectively. These observations suggest that α-MSH- and NPY-containing neurons share direct mutual inputs in the NPPv and the NLTi of the hypothalamus, and that α-MSH and NPY functionally interact or exhibit mutual inhibition to regulate feeding behavior in the goldfish.     

Possible disruption of pheromonal communication by humic acid in the goldfish,Carassius auratus

Humic acids are large, complex, organic molecules which are ubiquitous components of aquatic environments as products of degradation of plant material. In aqueous solution they form microvesicles. As many teleost pheromones are steroidal in nature, we hypothesised that they would preferentially dissolve in the organic, hydrophobic core of these vesicles instead of in water and therefore be unavailable for detection. This would have obvious and profound effects on many aspects of fish biology. To test this hypothesis we recorded electro-olfactogram (EOG) response of the goldfish (Carassius auratus) olfactory epithelium to the pheromones 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P), its sulphated conjugate (17,20β-P-SO₄) and prostaglandin F₂ (PGF₂), all at 10⁻¹¹ to 10⁻⁸ M, in the absence and presence of humic acids (1–1000 mg l⁻¹). At nearly all concentrations of humic acid tested, there was a significant attenuation of the amplitude of the initial (phasic) response to 17,20β-P compared to 17,20β-P alone. At higher concentrations of humic acid, the EOG response to 17,20β-P was often completely obliterated, suggesting that the concentration of the pheromone available to the olfactory epithelium was below the threshold of detection. Exposure of the olfactory epithelium to humic acid did not cause any short-term loss of sensitivity to 17,20β-P per se. Furthermore, simultaneous recording of electro-encephalograms from the olfactory bulb demonstrated that the nervous activity evoked by the same concentration of 17,20β-P was less intense in the presence of humic acid than its absence. PGF₂ is non-steroidal and much more soluble in water. In contrast to 17,20β-P, only the higher concentrations of humic acid (100 and 1000 mg l⁻¹) significantly diminished the EOG amplitude. 17,20β-P-SO₄ is detected via a distinct olfactory mechanism to the free form. Given that the sulphate group increases the water solubility, we predicted that the effect of humic acid would be reduced. However, the effect of humic acid on EOG amplitude in response to 17,20β-P-SO₄ was similar to that of the free form. We suggest that the steroid portion of the molecule adsorbs onto the surface of the humic acid microvesicles and is still effectively unavailable for olfactory detection. In conclusion, humic acid may significantly reduce the concentration of 17,20β-P and 17,20β-P-SO₄ available for detection by Carassius auratus in natural environments. Furthermore, as many teleost pheromones are steroid derivatives, this phenomenon may be applicable to chemical communication systems in teleosts in general.     

Role of leptin in the control of feeding of goldfish Carassius auratus: interactions with cholecystokinin,neuropeptide Y and orexin A,and modulation by fasting

To assess the role of leptin on food intake regulation in goldfish, we examined the effects of central (intracerebroventricular, ICV) and peripheral (intraperitoneal, IP) injections of recombinant murine leptin on feeding behavior. Centrally (100 ng/g) and peripherally (300 ng/g) injected leptin both caused a significant decrease in food intake, compared to the saline-treated controls. To test the hypothesis that leptin influenced orexigenic neuropeptide systems in goldfish, fish were co-injected with neuropeptide Y (NPY) or orexin A and leptin. Both NPY (5 ng/g) and orexin A (10 ng/g) significantly increased food intake. Fish co-injected ICV with NPY (5 ng/g) or orexin A (10 ng/g) and leptin (1 or 10 ng/g) had a food intake lower than that of fish treated with NPY or orexin A alone. NPY mRNA expression in goldfish brain was reduced 2 and 6 h following central injection of leptin. To test the hypothesis that the cholecystokinin (CCK) mediates the effects of leptin in goldfish, fish were simultaneously injected ICV with an ineffective dose of leptin (10 ng/g) and either ICV or IP with an ineffective doses of CCK (1 ng/g ICV or 25 ng/g IP). These fish had a food intake lower than vehicle-treated fish, suggesting that leptin potentiates the satiety actions of CCK. CCK hypothalamic mRNA expression was increased 2 h following central treatment with leptin. The CCK receptor antagonist proglumide blocked both central and peripheral CCK satiety effects. Blockade of CCK brain receptors by proglumide resulted in an inhibition of the leptin-induced decrease in food intake and an attenuation of the inhibiting action of leptin on both NPY- and orexin A-induced feeding. These data suggests that CCK has a role in mediating the effects of leptin on food intake. Fasting potentiated the actions of leptin and attenuated the effects of CCK. Whereas fasting had no effects on the brain mRNA expression of CCK, it increased the brain mRNA expression of NPY and decreased the expression of CART. These changes in neuropeptide expression were partially reversed when fish were treated ICV with leptin. These results provide strong evidence that, in goldfish, leptin influences food intake, in part by modulating the orexigenic effects of NPY and orexin and that its actions are mediated, at least in part, by CCK.