Pineal projections in the zebrafish (Danio rerio): overlap with retinal and cerebellar projections.

The pineal organ in fishes is a photoreceptive organ with dual outputs, neuroendocrine and neural. The neural projections of the zebrafish pineal were experimentally studied by means of tract-tracing with carbocyanine dyes (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO)). Double-labeling experiments were also performed in order to investigate the degree of overlapping of pineal, retinal or cerebellar projections in zebrafish. The pineal organ sends efferent fibers bilaterally to the rostral hypothalamus, thalamus, pretectum, posterior tubercle and the mesencephalic tegmentum. A few pinealofugal fibers could also be traced to the optic tectum. Most of the targets of the zebrafish pineal also receive retinal and/or cerebellar afferents, indicating a high degree of overlap with these projections. Since most of the targets of the pineal projections of zebrafish appear to be premotor and precerebellar centers, the neural output of the pineal organ is probably, because of its photoreceptive and circadian function, involved in photic and circadian modulation of these centers.

Orexin A modulates neuronal activity of the rodent suprachiasmatic nucleus in vitro.

In mammals, as in rats and mice used in the present study, the major internal timekeeping mechanism is located in the suprachiasmatic nucleus (SCN). It is composed of a complex tissue of multiple, individual oscillator cells that drive numerous physiological and endocrine processes via an electrical and humoral output. Several afferent input systems can interact with the clock mechanism and lead to phase-resetting actions. The recent discovery of orexin-containing fibers in the SCN region and the presence of orexin receptors in the SCN prompted us to investigate the possible role of orexin in the SCN. Multielectrode array recordings from dispersed SCN neurons revealed that orexin A dose-dependently enhanced the extracellularly recorded neuronal activity of many neurons (38%), whereas other neurons were inhibited (28%). The influence of orexin A on neuronal activity in the SCN was confirmed by whole-cell patch-clamp recordings from brain slices and dispersed cell cultures. Orexin A caused significant changes in the frequency but not mean amplitude or decay time constant of spontaneous inhibitory postsynaptic currents (sIPSCs). Low concentrations of orexin evoked an increase of sIPSCs, whereas the highest concentration predominantly caused a decrease of sIPSCs. The effects of orexin A on inhibitory postsynaptic currents were prevented by the orexin 1 receptor antagonist SB 334867 and also reduced in the presence of tetrodotoxin. Long-term recordings of the discharge rate of SCN neurons revealed that orexin A is able to induce phase shifts in cultured SCN neurons as well as in organotypic brain slices.

The pituitary adenylate cyclase-activating polypeptide modulates glutamatergic calcium signalling: investigations on rat suprachiasmatic nucleus neurons.

Circadian rhythms generated by the hypothalamic suprachiasmatic nucleus (SCN) are synchronized with the external light/dark cycle by photic information transmitted directly from the retina via the retinohypothalamic tract (RHT). The RHT contains the neurotransmitters glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP), which code chemically for 'light' or 'darkness' information, respectively. We investigated interactions of PACAP and glutamate by analysing effects on the second messenger calcium in individual SCN neurons using the Fura-2 technique. PACAP did not affect NMDA-mediated calcium increases, but influenced signalling cascades of non-NMDA glutamate receptors, which in turn can regulate NMDA receptors. On the one hand, PACAP amplified/induced glutamate-dependent calcium increases by interacting with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate signalling. This was not related to direct PACAPergic effects on the second messengers cAMP and calcium. On the other hand, PACAP reduced/inhibited calcium increases elicited by glutamate acting on metabotropic receptors. cAMP analogues mimicked this inhibition. Most neurons displaying PACAPergic neuromodulation were immunoreactive for vasoactive intestinal polypeptide, which is a marker for retinorecipient SCN neurons. The observed PACAPergic effects provide a broad range of interactions that allow a fine-tuning of the endogenous clock by the integration of 'light' and 'darkness' information on the level of single SCN neurons.

Analyses of signal transduction cascades reveal an essential role of calcium ions for regulation of melatonin biosynthesis in the light-sensitive pineal organ of the rainbow trout (Oncorhynchus mykiss).

Signal transduction processes regulating melatonin production in the light-sensitive trout pineal organ were investigated by immunocytochemical and immunochemical demonstration of phosphorylated cyclic AMP-responsive element-binding protein (pCREB) and measurements of cyclic AMP, melatonin, and calcium levels. Melatonin levels were tightly controlled by light and darkness. Elevation of cyclic AMP levels by 8-bromo-cyclic AMP, forskolin, and 3-isobutyl-1-methylxanthine increased the levels of pCREB and melatonin in light- or dark-adapted pineal organs in vitro. Without pharmacological treatment, the levels of pCREB and cyclic AMP remained constant for several hours before and after light onset. Inhibition of cyclic AMP-dependent proteasomal proteolysis by lactacystin, MG 132, and calpain inhibitor I did not prevent the rapid, light-induced suppression of melatonin biosynthesis. However, changes in the intracellular calcium concentration by drugs affecting voltage-gated calcium channels of the L type and intracellular calcium oscillations (cobalt chloride, nifedipine, Bay K 8644) had dramatic effects on the rapid, light-dependent changes in melatonin levels. These effects were not accompanied by changes in cyclic AMP levels. Thus, the rapid, light-dependent changes in melatonin levels in the trout pineal organ are regulated apparently by a novel calcium signaling pathway and do not involve changes in cyclic AMP levels, cyclic AMP-dependent proteasomal proteolysis, or phosphorylation of cyclic AMP-responsive element-binding protein.

Responses of neurones of the rat suprachiasmatic nucleus to retinal illumination under photopic and scotopic conditions.

1. We have examined the responses of neurones in the suprachiasmatic nuclei (SCN) of the rat to retinal illumination under photopic and scotopic conditions to identify the types of photoreceptor input to these nuclei. 2. The majority of visually responsive SCN neurones studied under dark adaptation received rod input (48 of 52, 92 %). The action spectrum conformed to the sensitivity of rhodopsin, with maximal sensitivity at around 505 nm. 3. When also studied under light adaptation, most visually responsive SCN neurones (20 out of 26, 77 %) responded to input from cones. The action spectra conformed to the spectrum of green cone opsin, with a main sensitivity peak at 510 nm and a significant secondary peak in the near-ultraviolet region of the spectrum. 4. The frequency of spontaneous activity was typically low under scotopic conditions (range 0.2-17.2 Hz) and higher under photopic conditions (range 0.6-40 Hz) for any given neurone. The most common response under scotopic conditions was an 'on-excitation' (32 of 48, 62.5 %), which changed under photopic conditions to an on-excitation followed by a more prominent off-inhibition. 5. Responses also changed due to endogenous ultradian cycles. Depending on the phase, responses could be altogether absent and even reverted from excitation to inhibition on opposite phases of a cycle. Ultradian cycles had a circadian dependence and were most common at around the light phase:dark phase (L:D) and D:L transition points of the circadian cycle. 6. Under photopic conditions, SCN neurones showed rhythmic electrical activity, with a preferred firing interval that had a value between 18 and 39 ms. This rhythmic activity was probably the result of endogenous subthreshold membrane potential oscillations. 7. In conclusion, light acting either via rod or cone pathways could have powerful, opposing actions on SCN neurones. These actions were state dependent. The presence of these neuronal responses suggests a role for rod and cone photoreceptors in SCN function.

Pituitary adenylate cyclase-activating polypeptide and melatonin in the suprachiasmatic nucleus: effects on the calcium signal transduction cascade.

The suprachiasmatic nucleus (SCN) harbors an endogenous oscillator generating circadian rhythms that are synchronized to the external light/dark cycle by photic information transmitted via the retinohypothalamic tract (RHT). The RHT has recently been shown to contain pituitary adenylate cyclase-activating polypeptide (PACAP) as neurotransmitter/neuromodulator. PACAPergic effects on cAMP-mediated signaling events in the SCN are restricted to distinct time windows and sensitive to melatonin. In neurons isolated from the SCN of neonatal rats we investigated by means of the fura-2 technique whether PACAP and melatonin also influence the intracellular calcium concentration ([Ca2+]i). PACAP elicited increases of [Ca2+]i in 27% of the analyzed neurons, many of which were also responsive to the RHT neurotransmitters glutamate and/or substance P. PACAP-induced changes of [Ca2+]i were independent of cAMP, because they were not mimicked by forskolin or 8-bromo-cAMP. PACAP caused G-protein- and phospholipase C-mediated calcium release from inositol-trisphosphate-sensitive stores and subsequent protein kinase C-mediated calcium influx, demonstrated by treatment with GDP-beta-S, neomycin, U-73122, calcium-free saline, thapsigargin, bisindolylmaleimide, and chelerythrine. The calcium influx was insensitive to antagonists of voltage-gated calcium channels of the L-, N-, P-, Q- and T-type (diltiazem, nifedipine, verapamil, omega-conotoxin, omega-agatoxin, amiloride). Immunocytochemical characterization of the analyzed cells revealed that >50% of the PACAP-sensitive neurons were GABA-immunopositive. Our data demonstrate that in the SCN PACAP affects the [Ca2+]i, suggesting that different signaling pathways (calcium as well as cAMP) are involved in PACAPergic neurotransmission or neuromodulation. Melatonin did not interfere with calcium signaling, indicating that in SCN neurons the hormone primarily affects the cAMP signaling pathway.

Photoendocrine signal transduction in pineal photoreceptors of the trout. Role of cGMP and nitric oxide.

This study describes the presence and distribution of cGMP-immunoreactivity and of the nitric oxide (NO) synthesizing enzyme, NO synthase (NOS), as demonstrated by use of the NADPH-diaphorase technique in directly light sensitive pineal organ of the trout. Cyclic GMP immunohistochemistry revealed immunoreactivity in pineal photoreceptor cells that were identified by double-labeling with S-antigen, whereas NADPH-positive structures were located adjacent to these photoreceptor cells. Since NO is known to stimulate synthesis of cGMP, these results indicate a role for NO in pineal function, e.g. in cGMP related events in the phototransduction process as well as in the light-dark control of melatonin synthesis.

The pituitary adenylate cyclase-activating polypeptide-induced phosphorylation of the transcription factor CREB (cAMP response element binding protein) in the rat suprachiasmatic nucleus is inhibited by melatonin.

The mammalian hypothalamic suprachiasmatic nucleus (SCN) is an endogenous pacemaker generating circadian rhythms. SCN activity is synchronized with environmental light/dark cycles by photic information primarily transmitted via the retinohypothalamic tract (RHT). The SCN controls synthesis and release of melatonin, the hormone of the pineal gland. Melatonin itself feeds back to the SCN. Using brain slice technique and immunocytochemistry we demonstrate that (1) pituitary adenylate cyclase-activating polypeptide (PACAP) induces the phosphorylation of the transcription factor cAMP response element binding protein (CREB) in the SCN during late subjective day and (2) melatonin inhibits this PACAP-induced phosphorylation. Our data suggest that PACAP is a neurotransmitter which affects gene expression in the SCN probably via the cAMP signaling pathway and that the antagonistic effect of melatonin mirrors a feed-back loop within the circadian system.

Photic regulation of pineal function. Analogies between retinal and pineal photoreception.

Light absorbed by a photopigment in a photoreceptor cell causes a photochemical reaction converting the 11-cis retinal chromophore into the all-trans configuration. These changes lead to a series of events that causes cGMP hydrolysis, a following decrease of cGMP in the cytoplasm of the photoreceptor outer segment and a closure of cGMP-gated cationic channels. As a consequence of these processes the membrane hyperpolarizes. In pineal photoreceptor cells of lower vertebrates these processes are only partly investigated. Molecules involved in the phototransduction process and the desensitization, like opsin, vitamin A, alpha-transducin and arrestin, have been immunocytochemically localized in pineal photoreceptors and also electrophysiological studies have shown that phototransduction mechanisms in pineal photoreceptors might be very similar to those found in retinal photoreceptors. This review will summarize some of the current knowledge on pineal photoreception and compare it with retinal processes.

Regulation of melatonin production and intracellular calcium concentrations in the trout pineal organ.

The present in vitro study correlates measurements of the melatonin production from trout pineal organs with those of the intracellular calcium concentration in pinealocytes. Melatonin production increases with decreasing irradiance and shows maximal values in darkness. Some pinealocytes exhibit spontaneous calcium oscillations, although most of them have a stable basal calcium concentration. Diminishing extracellular calcium and enhancing magnesium reduces melatonin release in the light-and dark-adapted state. The application of Co2+ decreases melatonin secretion in the mesopic and scotopic range, reversibly blocks spontaneous calcium oscillations, reduces the basal intracellular calcium concentration in non-oscillating pinealocytes, and inhibits the KCl-induced rise in intracellular calcium. Application of glutamate, norepinephrine, isoproterenol, or dopamine has no significant effect on melatonin secretion. Norepinephrine does not influence the calcium concentration in any of the trout pinealocytes. Treatment with the GABAA-receptor agonist muscimol causes a slight reduction of melatonin release in the mesopic and scotopic range of illumination, without affecting intracellular calcium concentrations. Thus, Co2+ and low calcium/high magnesium buffer reduce melatonin release through an action on the calcium concentration in trout pinealocytes and not through a blockade of synaptic transmission. All the data show that the trout pineal organ synthesizes and releases melatonin in relation to the irradiance of the incident light and that neuronal inputs have a minor, if any, influence on melatonin synthesis.

Central projections of the parapineal organ of the adult rainbow trout (Oncorhynchus mykiss).

Neural connections of the small parapineal organ of the adult rainbow trout were experimentally investigated by using a lipophilic carbocyanine dye as a tracer. The dye was applied to the parapineal organ, to the pineal organ, or to the left or right habenular ganglion. The parapineal organ mainly projected via a coarse parapineal tract to a conspicuous neuropil in the rostrodorsal part of the left habenular ganglion. A small accessory parapineal tract projecting to the right habenular ganglion was also found in some animals. No pineal afferents were observed in the parapineal, nor was any neuron in the brain seen to project to this organ. These results suggest a functional relationship of the parapineal organ to the limbic system.

Diazepam increases melatonin secretion of photosensitive pineal organs of trout in the photopic and mesopic range of illumination.

The pineal organ of teleost fish receives photic information directly through specialized photoreceptor cells that transmit their light response to second-order neurons and respond also with an endocrine light-dependent melatonin signal. In the present study we have analyzed the action of diazepam, a full agonist of the benzodiazepine receptor, on the photic regulation of the endocrine melatonin response of cultured trout pineal organs. Melatonin release of explanted pineal organs was clearly dependent on the irradiance of incident light with a maximum change during mesopic illuminations. Addition of diazepam to the superfusion medium significantly increased melatonin production in the mesopic and partly in the photopic range of illumination, without showing clear effects in the dark-adapted organ. Flumazenil, a central acting benzodiazepine antagonist, slightly reduced melatonin secretion. The action of diazepam appears to be comparable to a dark-pulse in the mesopic range of illuminations.

Secretion of the methoxyindoles melatonin, 5-methoxytryptophol, 5-methoxyindoleacetic acid, and 5-methoxytryptamine from trout pineal organs in superfusion culture: effects of light intensity.

The teleost pineal organ is a photoreceptive endocrine organ that synthesizes the hormone melatonin through specialized intrapineal photoreceptor cells in a light-dependent manner. The present study investigated whether the methoxyindoles 5-methoxytryptophol (5-MTOL), 5-methoxyindoleacetic acid (5-MIAA), and 5 methoxytryptamine (5-MT) correspond to melatonin secretion synthesized and released from explanted, superfused pineal organs in response to direct light stimulation and whether their release is correlated with the level of dark adaptation. Melatonin release in superfusion cultures using Hank's buffer was clearly dependent on irradiance of the incident light and increased with decreasing light intensity from an average release of 1 ng/pineal/hr in light-adapted pineal glands to about 9 ng/pineal/hr in dark-adapted pineal glands. The maximal release could be further enhanced if Medium 199 was used as the superfusion medium. Although the other methoxyindoles showed a less clear response to light and their production was clearly dependent on the external medium, their mean secretion rate in the light-adapted state was considerably higher than that of melatonin. In Hank's superfusion culture, 5-MTOL production decreased with decreasing light irradiances, but 5-MT and 5-MIAA concentrations remained almost constant. Using Medium 199 as the superfusion culture medium, concentrations of 5-MTOL and 5-MT showed no changes with light intensity, but 5-MIAA production clearly increased with decreasing irradiances. Pargyline, a monoamine oxidase inhibitor, significantly reduced the secretion of 5-MIAA and 5-MTOL, but caused a dramatic increase of 5-methoxytryptamine concentrations. The production of melatonin was not affected. A slight decrease of 5-MTOL secretion was also observed after the addition of 1 microM eserine, an inhibitor of the melatonin deacetylase, to the superfusion medium, whereas melatonin release remained constant. These results indicate that the pineal gland of the trout releases the methoxyindoles 5-MTOL, 5-MIAA, and 5-MT in addition to melatonin. The release appears to be only partly light dependent. Furthermore, the present results suggest that methoxyindoles released from the trout pineal gland are independent from melatonin deacetylation.

Secretion of methoxyindoles from trout pineal organs in vitro: indication for a paracrine melatonin feedback.

Synthesis and release of the pineal hormone melatonin is in all vertebrates primarily regulated by the light/dark cycle. In pineal organs of teleost fish, like in other non-mammalian vertebrates, melatonin formation is regulated by a direct photoreception of the pineal organ. We performed measurements in explanted, perifused pineal organs of the rainbow trout, Oncorhynchus mykiss, to examine whether melatonin can influence its own production. For this purpose we have continuously perifused isolated pineal organs under light- and dark-adapted conditions and measured the release of melatonin and other methoxy-indoles by HPLC with electrochemical detection. Addition of 2-iodomelatonin to the perifusate in a concentration of 2 ng/ml significantly inhibited melatonin release in light-, as well as in dark-adapted organs. The release of 5-methoxytryptamine and 5-methoxytryptophol was also significantly reduced in light-adapted organs. These results indicate that extracellular melatonin may act as a paracrine or autocrine feedback signal and may be important for the illumination-dependent melatonin production.

Benzodiazepines influence melatonin secretion of the pineal organ of the trout in vitro.

The effect of benzodiazepines (BZP) on melatonin release was investigated in the pineal gland of the rainbow trout, Oncorhynchus mykiss, maintained under in vitro perifusion culture conditions. Melatonin and the methoxyindoles 5-methoxytryptophol (5-MTOL), 5-methoxyindoleacetic acid (5-MIAA), and 5-methoxytryptamine (5-MT) were determined directly in samples of the superfusion medium by HPLC with electrochemical detection. Melatonin release was significantly increased by addition of diazepam and clonazepam in a dose-related and reversible manner. The effects of benzodiazepines were more pronounced in light-adapted pineal organs, when melatonin secretion is low, than under scotopic conditions. When the perifusion medium was replaced by a medium containing low calcium, high magnesium concentrations, melatonin release was considerably decreased by 70% in light-adapted and 20% in dark-adapted pineal organs. Addition of diazepam to low Ca2+, high Mg(2+)-medium reversed the decrease of melatonin release and produced a clear rise in its secretion rate. Addition of the BZP antagonist flumazenil to the perifusion medium slightly decreased melatonin release in the light- and dark-adapted state, whereas the peripheral receptor antagonist PK 11195 did not alter melatonin release. The effect of diazepam is reduced by simultaneous addition of flumazenil to the superfusion medium, suggesting that the effects of diazepam are receptor-mediated. The methoxyindoles 5-MTOL, 5-MIAA, and 5-MT showed no significant changes of their release pattern after diazepam application in light- and dark-adapted pineal organs. These results suggest that BZP can influence melatonin production and release by an intrapineal action possibly on the melatonin synthesizing photoreceptor cell.

Pineal photosensitivity. A comparison with retinal photoreception.

Pineal photoreceptors of poikilothermic vertebrates possess numerous anatomical, physiological and biochemical similarities to retinal photoreceptors, including the rhythmic melatonin biosynthesis, with nocturnal peaks and low day-time levels. This brief outline will survey the photoreceptor properties of the pineal organ of poikilothermic vertebrates, which suggest that the pineal is not only a simple light detector (that acts as a kind of photometer), but that it is capable of processing the light information and to discriminate it from informations that have no meaning for its assumed photoperiodic function.

Effects of dopaminergic and noradrenergic mechanisms on the neuronal activity of the isolated pineal organ of the trout, Oncorhynchus mykiss.

The effects of exogenous applied catecholamines on the neuronal activity of ganglion cells of the luminance type (achromatic cells) were investigated in the photosensitive pineal organ of the trout, Oncorhynchus mykiss. Extracellular recordings were performed on neurons of the superfused isolated pineal organ. Addition of dopamine to the superfusion medium increased the spontaneous activity of more than 60% of the achromatic neurons (n = 25). The D1-dopamine antagonist SCH-23390 and D2-dopamine antagonist spiperone reversed the dopamine-induced stimulation of ganglion cells and inhibited their maintained activity, which suggests that dopamine acts via both D1- and D2-receptors. Norepinephrine, the beta-adrenergic agonist isoproterenol, and DOPA enhanced the spontaneous activity of most of the ganglion cells, whereas the beta-antagonist propranolol depressed the discharge rate and reversed the action of isoproterenol. This suggests that catecholamines might play a modulatory role in the regulation of the neural activity of pineal luminance neurons.

Action of gamma-aminobutyric acid (GABA) in the isolated photosensory pineal organ.

The effect of GABA (gamma-aminobutyric acid), its agonists (muscimol, baclofen) and antagonist (bicuculline) on pineal ganglion cells of the luminosity type were studied in the isolated, superfused pineal organ of the rainbow trout, Oncorhynchus mykiss. Extracellular recordings revealed that GABA added through the superfusion medium caused a clear alteration of the activity of projecting neurons, which transmit luminosity responses to the brain. Spontaneous discharges of ganglion cells were predominantly suppressed by GABA (33 neurons out of 48), but 10 neurons were clearly excited. Similar effects were observed after addition of muscimol, but not of baclofen. Bicuculline reversed the GABA and muscimol induced inhibition or excitation. In 4 neurons of the luminosity type, GABA caused bidirectional, inhibitory and excitatory responses depending on the state of light- or dark-adaptation. These observations suggest a role of a GABAergic mechanism in the generation and transmission of luminosity responses in the trout pineal organ. It appears that GABA participates in the modulation of light sensitivity during light- and dark-adaptation processes and that this action is mediated by GABAA receptors.

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

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.

Neural elements in the pineal complex of the frog, Rana esculenta, I: Centrally projecting neurons.

The pineal complex of anuran amphibians is a directly photosensory organ, encompassing both an extracranial portion, the frontal organ, and an intracranial portion, the pineal organ proper. The projection neurons of the frontal organ respond differentially according to the wavelengths of the light stimuli. The pineal organ, on the other hand, functions mainly as a luminosity meter. Most of its centrally projecting neurons respond to all increases in ambient illumination with decreases in spontaneous firing of action potentials, although some neural units in the pineal organ may respond according to wavelength. This difference in responses to light stimulation may be reflected in the neural organization of the two parts of the pineal complex. In the present study, we have analyzed the morphology of the projection neurons of the frontal and pineal organs of the frog, Rana esculenta, by backfilling of the neurons with horseradish peroxidase through their cut axons. In the pineal organ, several types of centrally projecting neurons were observed: peripherally situated unipolar and multipolar neurons, the dendrites of which extend into a superficial axon plexus that surrounds the pineal epithelium; smaller unipolar, bipolar, or multipolar neurons situated close to the central pineal tract; and radially oriented bipolar neurons, with short dendritic processes oriented towards the lumen of the pineal organ.(ABSTRACT TRUNCATED AT 250 WORDS)