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LùorrloryotCcllulâr BioloBy. Dcpartment ot Elcctron MioroscoD ofth€ Univc(ilv ùd Lrborâtorv CNRS

No.29ll. Universny ol Poiticrs, Frrncc

KeyWords. Pike Pinealorgân Photoreccptor ' I ndole biosynl hesis PhotosensitivityPhotoDerbdicst imul i Photoneuroendocrinetransducers

Abstrrct. The pineûl orgân of thc pikc is a functional photoreceptive organ in which amultiplicity of thc cclls of the receptor line (CRL i.e. typic.rl and rnodified photoreceptors)was dcmonsrrâted. The CRL appe.rred to bc ( i ) regional ly distr ibutcd, ( i i ) pholoscnsit ive,and (iii) responsiblc lor thc storagc of vârious indoles (melatonin and precursors), theirlcvcls showing liSht-dark fluctuâtions. It is strongly suggested th.rt CRL ârc photoncuro-endocrine trânsducers in which photopcriodic stimuli are converted into nervous (lypicfllphotorcccptors) ând neurohumoral (all CRL) messâges, displâying circâdiân variâtions.CRL appcared to be one of the components by which cnvironmcntal lighting inTlucnccsphysiologicnl ând behavioral proccsses

'n fish.

Photosensitivity and Biosynthesis of Indole Compounds in theCells of the Receptor Line of the Pineal Organ ofthe Pike

Ophlhalmic Res. /6j 123 128(1984)

Introduction

Numerous histologicâl and cytologicalstudies have shown the structural adapta-t ions of the pincalorgan of f ish to thc pholo-rcccption lcf. references in 6l and its directphotosensitivity has bcen dcmonslrâtcdclcctrophysiologicâlly in various species byDodt er à1. 13,41. I t hâs been further sug-gested that pineal sense orgâns mây possessnot only the capacity of sending nervousmessages to the brain. but mây also be câp-ablc of a light-dependent synlhesis and

release of neurohumoral mediators [2].Somc indole compounds arc thought 1C) bcinvolved in thc ncurocndocrine function [cf.references in l7l. It has been emphasizedtbat indolc melabolism might be a commoncharâcteristic of all cells of the receptor Iine(CRL), including thc typicâl concl ikephotoreceptor cells (PC), the rudimentary(i.e. moclified) PC (RPC), the pineâlocyte(Pi), and intermediâte cell types [2. ând pre-vious works]. Moreover the existence ofphotoneuroendocrine trânsducers in thepineâl organ of lower vertebrates was

hypothesized [2]r in such cells a photic inputmight be convertcd inlo both nervous ândneurohormonal outputs. in a singlc PC.

]n ordcr to investigâte the possible exis-tence of photoneurocndocrine transducersin the pineal orgân of lower vertebrâtcs. wcundertook a ltw yeârs ago a study of thepineal orgân of the pike (Er(,.r lu.trs, L.)including structural and functionâl aspects.A survey ot our resulls tnd conclusions fol

Sfructure and Sensory F[nction

Among âll teleost pincal organs investi,galcd so far, that of the pike exhibih a uni-que ând hctcrogeneous orgânization. ltsepithelium can be dividcd into three tnor-phologically different regions (distâ1. mcdiâland proximal) I6l. Typical cone,like PCwere found in the distâl ând proximalregions. whilc ûore or less modified PC,characterized by an involution and remodel-ling of the polar terminals, wcrc mainlylocâlized in thc medial one [7]. The nervccells alsoexhibited ânhctcrogcneouspatternlthcy could be localized in the distâl rndproximâl regions only- Their .xons formedthe pineal tractwhich rùn dorsâlly in thepro-ximâl region towards the posterior commis-sure [l], el.

Electrical recordings âllowcd thc locali-zation of the photosensitive regions, and thecharâcteristics of the photosensory lunctionwere studied I12. l4l. Extrâcellular record-ings showed a maintained activity of nervefibers from the pireal trâct. ând ol singlcneurons from the distal region. Withincreasing levcls ofsteady exposure to whitelight. the impulse lrequency decrcascd-lllùminâlion of thc org n with white light, or

wavelengths betwcen 380 and 710 nmresulted in ân inhibition of the spikc {ctivit_\r(âchromatic rcsponse), associâted with slowgraded responses (clcctropinealogran orEPG). Sensitivity curves exhibilcd maximaât 530 and 620 nm in the light-adapted, and530 nm in the dark-adaplcd organ. On rareoccasions, inhibi tory ()" , , , . , ,3 i l0 nm) ândexcitâtory (lni, 620 nm) responses wererecorded Trom single nerve cclls (chromaticrcsponse). Slow potentials without spikcpotentials could be recorded from the iso-Iâted mediâl region. Flashcs ol vcry strongintensity elicited photoresponses appeâringwith extremely shorl la(cnc]. ,rnd identifiedrs early receptor potentials (ERP) Ifordelâi ls cf . l4 l . ERP spectralcomparable to thosc oblaincd with spikeând slow potential responses.

Indole Compounds in th€ Cells of th€R€ceptor Lin€

Attcntion was focused on melâtonin(aMT) which is a biologically active agentwith hormonal properties in poikilothermicvertebrâlcs [cf. rcfcrences in 171. and precursors. Let us recall that the main pâthwâylcading to the synthesis of aMT involvesrespectively lfor dclails. cf. il tryptophan(W), s-hydroxytryptophan (tIw). s-hy-droxytryptamine (serotonin, Hl) . and N-acetylserotonin (â[IT).

In the pineal organ of Éio-r, clRL wereexclusivcly or m.linly responsible for theuptake of [ ]Hl-w ând [ ' l l l - I Iw l ]1. A poolof HWHT wâs manrly evidenced in CRL bymcans of the formaldehyde-induced fluores-cence method I l0l . Thc levels of therluorophores vâried, depending (i) on thepineâl region (highcr in thc mcdi l rcgion

Photoreceptors of the Pineal Organ of the Pike 125

Fig. L. Immunocytochemical de-

monstration of serotonin and mela-

tonin in the pineal organ of the pike

(animals sacrificed at 10:00). a) b

Serotonin immunoreactivity is

localized in cells of the receptor line

(arrowheads) in the medial region

(immunofluorescence method bar:

100 pm (a) and 10 pm (b); pl :

pineal lumen). c Melatonin im-

munoreactivity in cells of the re-

ceptor line of the distal region; note

the accumulation of reaction prod-

ucts in the apical part of the inner

segment (large arrows) protuding in

the pineal lumen (pl), and in the

photoreceptor pedicles (small

arrows ; peroxidase-antiperoxidase

method; bar:10 pm).

than in the others), and (ii) on the time of

sacrifice (high during day, low at night).

After inhibition of the monoamine oxidase(MAO) activity, apparently all the cells of

the pineal parenchyma accumulated the

fluorophores. The fluorescence disappeared

after treatment with (i) inhibitors of the HT

synthesis, or (ii) a monoamine depletor. A

MAO activity was revealed light and elec-

tron microscopically which seemed to be

higher at the end than at the beginnittg of

the photophase; the sites of the oxidative

deamination pathways were mainly local-

ized in CRL [10].The cell types responsible for the storage

of endogenous HT, aFIT and aMT were

identified using light and electron im-

munocytochemical procedures lI3, 15]. In

winter HT and aFIT immunoreactivities,

when detected, were exclusively localized in

126 Falcôn

Fig.Z. Electron microscopic immunocytochemical

demonstration of serotonin (a) and melatonin (b) in the

cells of the receptor line (CRL) of the pineal organ of

the pike (animals sacrificed at 10:00, peroxidase-anti-

peroxidase method) ; el - ellipsoid; IC - interstitial

cell; is - inner segment; n - nucleus; os - outer seg-

ment (bars- L pm).

CRL (fig .I, 2), independently of the time ofthe light-dark cycle investigated. The levelsof the three indoles displayed circadian vari-ations, being high for HT during the photo-phase, and low during the scotophase. BothaHT and aMT could be detected only at thebeginning of the light and of the darkphases, their levels being higher during thescotophase. The aMT immunoreactivity wasrestricted to the CRL at the beginning of thescotophase, when an increased biosynthesisis presumed (radioimmunoassay data) ; itwas localized in CRL of the distal region andin interstitial cells of the medial and proxi-mal regions during the first hours of thephotophase (fig .I,2). After inhibit ion of theMAO activity only HT and aHT im-munoreactivities v/ere increased, and local-ized apparently in all cells of the pinealparenchyma.

Concluding Remarks : PhotoperiodicInformation and Elaboration of VariousMessages. Possible Existence of Photo-neuroendocrine Transducers

The pineal organ of the pike appears tobe a functional photoreceptive organ, whichmay act as a dosimeter of solar radiation andan indicator of day length [I2]. The absenceof nerve cells in the medial region is to berelated with the partial involution of theCRL, and explains why successful record-ings were extremely difficult to obtain fromthis region. Altogether, it seems that PCconvert photic information into rhythmicnervous messages transmitted from the dis-tal and proximal regions to the brain cen-ters, via the pinealofugal innervation.

According to present knowledge themodified PC appear incapable of converting

Phor.r.ceplors ol the Pin.âl Organ oI the Pikc 121

photic information into ncrvous messagcs.I Io$e\er. rhe) \c(m lo be \ t r l l phùtu\en\ i-r i \c \ rn(c s lu$ potenr i l l \ shich are gen( 'ured b) Pa I5. 12. l l l could b(

'egular l )recorded from isolatcd medial regions.PC as wellas modified PC are involvcd in

the storage and very probably biosynthesisand rclease of indolc compounds lfbr cxtensive discussion see f3 and l5l. As it is su-e-gcsted by the circâdian fluctuârions of H],âHT rnd aMT in CRL, we believe thal thesecells âre capable of converting photic infbr,matron into one (or several) neurohormonalmessâgc(t released inlo the bloodsrrcamand/or into thc cerebrospinal lluid [13, 15]-'Io our knowledge, our results bring the firststrong support to the hypothesis âccordingto which CRI- of lower vcrtebrâtes arcphotoneuroendocrine transducers J2l.

ln summary, the CRL of rhe pike sccmc,rpable of convcrting photic informationinto two kinds of messages: (i) a ncrvous(PC), ând (ii) one or several 5 methoxyin-dolic ncurohormonâl message(s) (all CRL)displaying circâdian varialions. They appearrcally to be one component in the pâthwâysby which lighfdârk cycles inf'luence phy-siologicâl ând behavioral processes.

Acknowledgments

I wish to exprcss ny respecr ârd gralilude to ProtDr ,oo., for tnc cônnrnt and hchrut ârlention hc pridta ne rnd my clccftophysiological ùort dùrirg my slây

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