Cytochemical demonstration of acid phosphatase activity in the pineal organ of the rainbow trout,Salmo gairdneri

Summary Activity of acid phosphatase (ACP) was investigated cytochemically in the pineal organ of the rainbow trout, Salmo gairdneri. Intense reaction product for ACP activity was observed (1) in lysosomes varying in size and shape and (2) in endoplasmic reticulum associated with the Golgi complex of (i) the pineal photoreceptor and supporting cells, (ii) vascular endothelial cells, and (iii) macrophages inhabiting pineal lumen, parenchymal epithelium and perivascular spaces. This localization of ACP is discussed with particular reference to the capacity for lysosomal digestion in a pineal organ combining photoreceptive and secretory functions, and lacking a blood-brain barrier, as holds true for the pineal of the rainbow trout. Taking advantage of its capacity for endocytotic uptake and lysosomal digestion, the pineal organ of the rainbow trout may serve as a barrier between the blood circulation and the cerebrospinal-fluid compartment. Furthermore, the macrophages may be considered as an essential component in pineal function of fish.Fellow of the Alexander von Humboldt Foundation.Fellow of the Alexander von Humboldt Foundation.     

Microvasculature of the pineal organ of the rainbow trout (Salmo gairdneri)

Summary The angioarchitecture of the pineal organ of the rainbow trout (Salmo gairdneri) was investigated by means of the corrosion-cast preparation method and scanning electron microscopy. Two main arteries (aa. epiphyseales) supply the pineal parenchyma. They emerge from the aa. cerebri anteriores and run in the fissure between the prosencephalon and the mesencephalon. After entering the pineal stalk, the aa. epiphyseales branch off into several arterioles, most of which extend to the pineal end-vesicle where they give rise to a lobular, bilaterally symmetric capillary network. Capillaries establishing the main portion of the pineal vessels appear widened in comparison to those supplying other portions of the brain and resemble capillaries in other endocrine organs. In Salmo gairdneri, no specialized system of portal vessels appears to exist between the pineal organ and other portions of the brain.     

GABA-immunoreactive neurons in the photosensory pineal organ of the rainbow trout: two distinct neuronal populations

Summary The distribution of putative GABA-ergic neurons in the photosensory pineal organ of the rainbow trout was investigated by use of a specific antiserum against -aminobutyric acid (GABA). GABA-immunoreactive (GABA-IR) neurons were located in the rostral portion of the pineal end-vesicle, presumably constituting a population of interneurons. GABA-IR neurons were also found in the pineal stalk. The axons of these neurons were traced along the pineal stalk toward the brain. The terminal areas of these axons could not be established. GABA-IR glial cells were observed in the pineal end-vesicle, but not in the pineal stalk.     

Responses of pineal photoreceptors in the brook and rainbow trout

Summary Electron microscopy of the pineal receptor cells in light- and dark-adapted brook trout, Salvelinus fontinalis and the rainbow trout, Salmo gairdneri, revealed no significant differences in the tubular and filamentous elements of the inner segment, neck and supranuclear regions. However, changes in synaptic relations between the photoreceptor and nerve cell were induced by light and darkness. In the light-adapted state, the synaptic relationship between axon terminals and photoreceptor basal processes predominates, while in darkness the synapses between photoreceptor basal processes and ganglion cell dendrites are more prominent. Further, in darkness, the photoreceptor basal processes show a number of synaptic vesicles and synaptic ribbons. These findings suggest that the sensory function of the fish pineal is enhanced during darkness but inhibited by light, and that the synaptic relationships are involved in the control of sensory activity in the pineal photoreceptor and ganglion cells. These results corroborate those of electrophysiological studies in that the maximal spontaneous discharge frequency of the ganglion cells occurs in the dark, and it also shows a burst when light is removed. The typical chemical synapse between the axon terminal and the photoreceptor basal process in light seems to function as an inhibitor.The authors thank Dr. Mary Ann Klyne for her assistance in several aspects of this work. Financial assistance was provided by the NSERC of Canada and the Ministry of Education of Québec     

Substance P-like-immunoreactive neurons in the photosensory pineal organ of the rainbow trout,Salmo gairdneri Richardson (Teleostei)

Summary Restricted numbers of substance P-like-immuno-reactive (SPL-IR) neurons were demonstrated in the photosensory pineal organ of the rainbow trout. The small parapineal organ of this teleost species receives a distinct SPL-IR innervation via the habenular nuclei, but displays no intrinsic SPL-IR neurons. Intrapineal SPL-IR neurons were located in the rostral portion of the pineal end-vesicle. Neuronal somata were found in a lateral position with smooth axonal processes extending mediad. Immunoreactive somata and axonal processes were observed intraparenchymally as well as in the pineal lumen. The pattern of immunoreactivity was not changed in excised pineal organs that had been incubated in tissue culture medium in the dark for 18 h. The possibility that the intrapineal SPL-IR neurons are not part of the neural circuitry involved in the transduction of photic information, but may have other functions, is discussed.Fellow of the Alexander von Humboldt-Stiftung, Bonn, Federal Republic of GermanySupported by research funds from the Deutsche Forschungsge-meinschaft (Ko 758/2-4)     

Melatonin modulates the neural activity in the photosensory pineal organ of the trout: Evidence for endocrine-neuronal interactions

Summary Hormonal and neural signals transmitted from the pineal organ to the brain in cold-blooded vertebrates presumably convert information about the ambient illumination into signals which may be used to mediate photoperiodic and circadian responses. The possible intrapineal function of melatonin was investigated by recording intra- and extracellularly from photoreceptors and second-order neurons in the isolated superfused pineal organ of the trout (Salmo gairdneri). Melatonin added through the perfusion bath to the explanted pineal organ caused a dose-related and reversible inhibition of ganglion cells of the luminance type whereas the hormone did not significantly affect the membrane potential of photoreceptors and their light-evoked response. The observed effects seem to be independent from photoperiod and adaptation conditions. These results suggest that melatonin provides a feedforward signal to intrapineal neurons regulating the neural output of the organ.Laboratory of Fish Biology, School of Agriculture, Nagoya University, Chikusa, Nagoya 464 Japan     

Vascular permeability (problem of the blood-brain barrier) in the pineal organ of the rainbow trout,Salmo gairdneri

Summary The problem of the blood-brain barrier in the pineal organ of the rainbow trout, Salmo gairdneri, was investigated following intraperitoneal or intracardial injections of several tracers and dyes with different molecular weights. As demonstrated at the light-microscopic level, repeated injections of trypan blue or horseradish peroxidase (HRP) resulted in an accumulation of these substances in the pineal epithelium (parenchyma). By use of the electron microscope, HRP was found in electron-dense bodies, probably lysosomes, in (i) the endothelial cells and perivascular macrophages 4 h after intraperitoneal injection, (ii) the supporting cells and intrapineal or luminal macrophages 8 h after injection, and (iii) the receptor cells 24 h after injection of the tracer. Ferritin particles penetrated the fenestrated endothelium of pineal capillaries. They were confined to vesicles, vacuoles and the smooth endoplasmic reticulum of the supporting cells as well as to the synaptic vesicles and the smooth endoplasmic reticulum of the pineal photoreceptors. The intercellular passage of tannic acid mixed with the fixative was blocked at the luminal junctional complex separating the pineal lumen from the basal portion of the pineal epithelium. The passive intercellular transport of substances with high molecular weight from the bloodstream to the cerebrospinal-fluid compartment is thus prevented. However, no blood-brain barrier exists for exogenously administered proteins, which are rapidly taken up by pineal cells and actively transported in a transcellular manner.The findings on the blood-brain barrier of the pineal organ of the rainbow trout are discussed with particular reference to the endocrine capacity of pineal sensory organs.Fellow of the Alexander von Humboldt Foundation, Federal Republic of Germany.     

Intracellular responses from the photosensitive pineal organ of the teleost,Phoxinus phoxinus

Summary Intracellular potentials from the isolated dark-adapted pineal organ ofPhoxinus phoxinus were recorded by using glass microelectrodes. The majority of cells had resting potentials of 20 to 35 mV and responded to light with intensitygraded hyperpolarizations. Voltage intensity curves of responses to brief flashes followed the hyperbolic tangent functionV/V _max=Iⁿ/(I ⁿ + ⁿ ).The latency of onset for responses to light stimuli near threshold was 400 ms and decreased with saturating flashes to about 50 ms. The membrane resistance decreased during the hyperpolarization. Spectral sensitivity measurements for these cells exhibited curves with _max=530 nm. Intracellular dye injection unequivocally identified this cell type as a photoreceptor cell.A second cell type with resting potentials between 30 to 40 mV exhibited a biphasic response pattern to light stimulation. The cell depolarized with dim light flashes and hyperpolarized with bright flashes. The amplitude of the hyperpolarizing component showed no saturation over an intensity range of 5 log units. Latencies and rise times were comparable to those of photoreceptor potentials. Spectral sensitivity curves peaked at longer wavelengths ( _max=550 nm) than the action spectra of photoreceptors ( _max=530 nm). It is assumed that this rare cell type represents a small class of pineal interneurons.     

Signal processing in a simple vertebrate photoreceptor system: the teleost pineal organ

The integrating circuitry and efferent pathways for neural signals evoked in the photosensory pineal organ by changes in ambient illumination have been investigated by a multidisciplinary approach. Intrapineal efferent neurons were identified by means of retrograde filling with horseradish peroxidase (HRP). In addition to several types of neurons, photoreceptor cells that emitted axons to the brain via the pineal tract were observed. The presence of several populations of local interneurons (putatively cholinergic, GABAergic and substance P-containing) and possible afferent (putatively noradrenergic and peptidergic) central innervations were established by means of immunocytochemistry. The anatomical substrate for processing of neural signals thus delineated, the responses of pineal sensory and neural elements to photic stimulation were investigated by means of intracellular recording. Successful recordings were followed by intracellular injection with HRP or Lucifer Yellow CH, for subsequent light or electron microscopical investigation. The recordings indicate the presence of at least two types of photoreceptor cells, that display morphological and physiological features of both retinal rods and cones. In addition, one type of (sign-conserving) interneuron was identified. The photosensory pineal organ thus possess an integrative neural circuitry that may be involved in the elaboration of neural signals to the brain, and/or in the local control of intrapineal functions, e.g. indoleamine synthesis.     

Modulation of catecholamine storage and release by the pituitary-interrenal axis in the rainbow trout,Oncorhynchus mykiss

This study examined the effects of pituitary-interrenal hormones on catecholamine storage and release in the rainbow trout Oncorhynchus mykiss. An extract of trout pituitary elicited the release of adrenaline, but not noradrenaline, using an in situ perfusion preparation. A variety of doses of adrenocorticotropic hormone (2–2000 mU) caused the release of both catecholamines in situ which was unaffected by pre-treatment with the ganglion blocker, hexamethonium, or the serotonergic receptor antagonist, methysergide, but was abolished in calcium-free media. Intra-arterial injections of adrenocorticotrophic hormone in vivo caused an elevation of plasma adrenaline but not noradrenaline levels. Injections of cortisol in situ did not elicit catecholamine release. Trout given an intraperitoneal implant of cortisol (50 mg·kg^-1 body weight) had significantly higher plasma cortisol concentrations when compared to controls after 7 days of implantation. Increases in the levels of stored catecholamines were observed in various regions of the kidney and posterior cardinal vein following 3 and 7 days of cortisol treatment. The ability of the chromaffin cells to release catecholamines in response to cholinergic stimulation was assessed in situ after 7 days of treatment. Basal (non-stimulated) adrenaline outflowing perfusate levels were greater in the cortisol-treated fish. Cortisol treatment increased the responsiveness of the catecholamine release process to low doses of the cholinoceptor agonist carbachol. Three or 7 days of cortisol treatment did not alter the in vitro activity of the enzyme phenylethanolamine-N-methyl-transferase. The results of this study demonstrate that interactions within the pituitary-adrenal axis can influence both catecholamine storage and release in the rainbow trout.Abbreviations ACTH adrenocorticotropic hormone - AK anterior third of the kidney - APCV anterior third of the PCV - HPLC high performance liquid chromatography - MK middle third of the kidney - M1 maximum value - MPCV middle third of the PCV - MS222 ethyl-aminobenzoate - P1 pre value - PCA perchloric acid - PCV posterior cardinal vein - PK posterior third of the kidney - PNMT phenylethanolamine-N-methyltransferase - PPCV posterior third of the PCV - rbc red blood cells - SEM standard error of the mean - TK total kidney (i.e. the sum of the AK, MK, and PK) - TPCV total PCV (i.e. the sum of the APCV, MPCV and PPCV)     

Germ cell sex prior to meiosis in the rainbow trout

Germ cells make two major decisions when they move from an indeterminate state to their final stage of gamete production. One decision is sexual commitment for sperm or egg production, and the other is to maintain mitotic division or entry into meiosis. It is unclear whether the two decisions are made as a single event or separate events, because there has been no evidence for the presence of germ cell sex prior to meiosis. Here we report direct evidence in the fish rainbow trout that gonia have distinct sexuality. We show that dazl expression occurs in both male and female gonia but exhibits differential intracellular distribution. More strikingly, we show that boule is highly expressed in male gonia but absent in female gonia. Therefore, mitotic gonia possess sex, sperm/egg decision and mitosis/meiosis decision are two independent events, and sperm/egg decision precedes mitosis/meiosis decision in rainbow trout, making this organism a unique vertebrate model for mechanistic understanding of germ cell sex differentiation and relationship between the two decisions.     

Structure of the parapineal organ of the adult rainbow trout,Salmo gairdneri Richardson

Summary The parapineal organ of the teleost Salmo gairdneri Richardsonsu1 was investigated with the light and electron microscopes. It is a small cell mass, 0.1–0.3 mm in diameter, containing a narrow lumen and consistently situated to the left of the pineal stalk and dorsal to the left habenular nucleus. It is connected with the habenular nucleus through a conspicuous parapineal tract. The parapineal organ continues to grow at least until the fish reaches sexual maturity and shows no sign of cellular degeneration at the age of two years.The parapineal tissue consists of supporting cells and nerve cells; the latter give rise to the axons of the parapineal tract. Furthermore, a small number of receptor cells of the type existing in the pineal organ is present. No morphological evidence was obtained to suggest a sensory or secretory function of the parapineal organ.The existence of the parapineal organ in the adult pike, Esox lucius, L., and of a connection between the pineal tract and the habenular commissure in Salmo gairdneri is briefly reported. The results are discussed in the light of existing literature.Work done with the aid of a research scholarship from the Alexander von Humboldt Foundation, Bad Godesberg, Germany. —The electron microscope used in this study was placed at the disposal of Prof. Oksche by the Deutsche Forschungsgemeinschaft. —I wish to thank Prof. Oksche for the facilities made available at his institute and for his helpful interest in my work.     

Autoradiographic localization of 3H-5-HTP and 3H-5-HT in the pineal organ and circumventricular areas in the rainbow trout,Salmo gairdneri Richardson

Summary The time course of incorporation and cellular localization of ³H-5-hydroxytryptophan (³H-5-HTP) and ³H-5-hydroxytryptamine (³H-5-HT) in the pineal and some brain regions in rainbow trout, Salmo gairdneri, were studied by quantitative and qualitative autoradiography.Among the tissues examined, the pineal shows the highest and most rapid uptake of the two isotopes. Maximum incorporation of ³H-5-HTP is achieved by 2 h and that of ³H-5-HT by 20 minutes post injection. At the end of the six-hour experimental period, a significantly high amount of radioactivity is still detectable in the pineal. The results indicate a much slower turnover of the two indoles, especially 5-HTP, in the trout than is known for mammalian tissues.Both the ependymal supporting cells and the receptor cells of the pineal localize these isotopes. In contrast, the intrapineal neurons remain unlabeled. This is taken to suggest lack of capacity of these cells to metabolize 5-HTP and 5-HT.In the circumventricular regions, the two indoles occur in the ependyma of the recessus lateralis and the recessus praeopticus. The label is also localized in the neuropil and the neurons of the nuclei recessus lateralis and praeopticus. Semiquantitative estimates reveal a significant labeling of these areas only 20 minutes after injection, although a weak but inconsistent labeling of the ependyma is evident at 5 minutes. The significance of these results is discussed in regard to (a) normal capacity of circumventricular areas to metabolize indoles and (b) a possible chemical interaction between the pineal and the brain involving a direct pineal-cerebrospinal fluid pathway.Supported in part by a grant to the senior author from the University of Kentucky Research FoundationThe authors wish to thank the Department of Fish and Game, Kentucky, for supplying rainbow trout. The technical assistance of Mrs. Munira Nasser is gratefully acknowledged     

Modulation of Ah receptor and CYP1A1 expression by alpha-naphthoflavone in rainbow trout hepatocytes

The objective of this study was to evaluate whether alpha-naphthoflavone (ANF) modulates aryl hydrocarbon receptor (AhR) signaling in rainbow trout (Oncorhynchus mykiss). AhR and cytochrome P450 1A1 (CYP1A1) protein and mRNA content were used as indictors of AhR signaling. Primary culture of rainbow trout hepatocytes were exposed to different concentrations of ANF (10(-9)-10(-5) M), while beta-naphthoflavone (BNF 10(-10)-10(-6) M) and a combination of ANF and BNF were used to elucidate the impact of ANF on AhR signaling. ANF increased AhR and CYP1A1 protein expression in a concentration-related manner; the maximal induction was about 50% that of BNF. Despite the differences in protein content between ANF and BNF stimulation, the maximal AhR and CYP1A1 mRNA abundance seen with the high concentrations of ANF and BNF were similar. ANF significantly decreased ( approximately 50%) BNF-induced AhR protein expression (only at 10(-9) M), but not CYP1A1 protein and gene expression. In addition, ANF at a sub-maximal concentration (10(-7) M) did not affect BNF-induced AhR protein content, but increased the sensitivity of hepatocytes to BNF-mediated CYP1A1 protein expression. Taken together, the mode of action of ANF appears similar to BNF, including modulation of AhR expression and activation of AhR-mediated signaling in rainbow trout hepatocytes. Overall, ANF is not only a partial AhR agonist, but may also modify BNF-mediated AhR signaling in trout hepatocytes.     

Detection and transmission of infectious hematopoietic necrosis virus in rainbow trout.

Detection and transmission of Infectious Hematopoietic Necrosis Virus in rainbow trout (Salmo gairdneri) was studied at a commercial trout hatchery. Transmission of virus was demonstrated via water, feed and contaminated eggs. If eggs from carrier females were incubated several weeks in virus-free water, the resulting fry did not become infected. However, if fry subsequently became infected they were lifetime carriers. Infectious virus was readily detectable in most tissues of moribund fish; in carriers it was detected in sex products of spawning fish, and in samples from the intestine of post-spawning fish, but not in samples from blood, feces, kidney, or liver. The carrier rate was not significantly different between sexes. It was concluded that adult carriers are the reservoir of infection and that transmission occurs primarily when carriers shed virus and expose susceptable fish or eggs.