Cell Tiss. Res. 182, 525-536 (1977)
Cell and Tissue Research 9 by Springer-Verlag 1977
Evidence for New Catecholamines or Related Amino Acids in Some Invertebrate Sensory Neurons R. Elofsson, B. Falck, O. Lindvall, and H. Myhrberg Department of Zoology and Department of Histology, University of Lund, Sweden
Summary. In certain sensory neurons of many different invertebrate species, including the sea anemones. Metridium senile and Tealiafelina and the crustacean Artemia salina, fluorophores are formed during the course of the fluorescent histochemical technique of Falck-Hillarp. The presumed catecholamine nature of the neuronal fluorogenic compound was investigated by microspectrofluorometry, and the spectral characteristics of the fluorescence in the taxonomically different species was found to be very similar (excitation maximum at 375 nm with a smaller peak or shoulder at 330 nm and sometimes a shoulder in the spectrum at 410 nm; emission maximum at 475 nm). The emission maximum coincides with that of the catecholamines and DOPA (475 nm). The excitation maximum (375 nm) directly after formaldehyde treatment, however, differs from that of the catecholamines and DOPA (410nm), but is similar to the excitation maximum displayed by these catechol derivatives at acid pH. The spectral characteristics of the fluorophore in the sensory cells might therefore theoretically be explained by an acid pH in the cells. This seems improbable, however, and it is suggested that the phenomenon is due to the presence of unknown catechol derivatives. Analyses of the pH-dependent spectral changes indicate that the presumed catechol derivative in Tealiafelina is fl-hydroxylated, whereas that in Artemia salina is not. Key words: Catecholamines - Sensory neurons - Coelenterates - Crustaceans - Microspectrofluorometry.
Introduction
Sensory neurons assumed to contain catecholamines have been reported in sea anemones, planarians, nematodes, various annelids, molluscs, and one crustacean (Dahl et al., 1963; Clark, 1966; Rude, 1966; Plotnikova and Govyrin, 1967; Send offprint requests to: Doc. Dr. Rolf Elofsson, Zoological Institute, Helgonaviigen 3, S-22362 Lund,
Sweden
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Myhrberg, 1967, 1971; Plotnikova and Kuzmina, 1968; Kuzmina, 1968; Sweeney, 1968; Sulston et al., 1975; Lure, 1975; Aramant and Elofsson, 1976). 5-HTcontaining sensory cells have so far been reported only in planarians (Welsh and Williams, 1970). Only in one case has a catecholamine in sensory cells been described in close detail (Ehinger and Myhrberg, 1971). Spectral analyses have indicated that the sensory cells of earthworm (Lumbricus terrestris) contain dopamine. In the course of a microspectrofluorometric survey of invertebrate sensory cells presumed to be catecholaminergic some diverging results were found. Neurons in the tentacles of sea anemones (Coelenterata) and in the cavity receptor of the crustacean Artemia salina displayed spectral characteristics of formaldehydeinduced fluorescence that deviated from those of dopamine, noradrenaline, adrenaline, or dopa.
Material and Methods Artemia salina L. (Anostraca) was reared to the adult stage from commercially available eggs in artificial sea water (Dohse Aquaristik) on Artemia-futter (Dohse Aquaristik). The sea anemones Metridium senile and Tealia felina were obtained from fiords outside Bergen through the courtesy of Dr. Tor Samuelsson, Bergen Sea Aquarium, Norway and from Helsing6r Marine Biological Station, Denmark, thanks to Dr. Lars Hagerman. In depletion experiments Artemia was allowed to swim in artificial sea water containing reserpine (Serpasil | CIBA, 0.125 mg/ml) for 24 to 48 h. Some animals were transferred to normal sea water and kept alive for up to 9 days. Fluorescence Microscopy. Tentacles of the sea anemones and whole Artemia were frozen, freeze-dried, and treated with formaldehyde gas in accordance with the specific method for cellular demonstration of biogenic monoamines described by Falck and Hillarp (Falck, 1962; Bj6rklund et al., 1972) using minor modifications of the method as described by Klemm (1968), Bj6rklund et al. (1970) and Elofsson and Klemm (1972). The best results were obtained with paraformaldehyde stored at a relative humidity of 50 per cent. The microscopical work was preformed with a Leitz fluorescence microscope equipped as described by Bj6rklund et al. (1972 b). Electron Microscopy. Heads of Artemia were fixed in a glutaraldehyde/paraformaldehyde mixture (Karnovsky, 1965) in cacodylate buffer for 4 h. Postfixation was performed in 2 ~o osmium tetroxyde in the same buffer for 1 he Pieces of coelenterate tentacles were fixed in 2.5 ~ glutaraldehyde in phosphate buffer for 1 h and postfixed in ~ ~ osmium tetroxide for ~ h or in 2 ~ potassium permanganate in veronal/acetate buffer for I h. Fixation was followed by dehydration in an alcohol series and embedding in Vestopal W. Ultrathin sections were examined on formvar-coated grids in a Philips EM 300 and a Zeiss EM 10 electron microscope. Microspectrofluoromeo3;. Excitation and emission spectra of the fluorophores were recorded in a modified Leitz microspectrofluorometer (Bj6rklund et al., 1972 b). For further characterization of the fluorophores, the paraffin sections ofA rtemia salina were then exposed to HCI vapor (10-30 min) and/or to NH 3 vapor (10-30 min) and the deparaffinized sections of Tealiafelina exposed to HCI vapor (30 s 1 rain) and/or to NH 3 vapor (10 20 s) at room temperature. The excitation and emission spectra of the tissue fluorophores were compared with spectra obtained simultaneously from histochemical protein models containing dopamine treated with formaldehyde vapor as above. These models were prepared by dissolving dopamine to a concentration of I mg/ml in a 2 ~ buffer solution (pH 7.0) of human serum albumin (AB Kabi, Stockholm, Sweden). The solution was sprayed in droplet form onto cover glasses, air-dried, and then formaldehyde-treated. All spectra were corrected according to the previously described procedures (Bj6rklund et al., 1968). and are expressed as relative quanta versus wave length.
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Results
Artemia salina Morphology of the Cavity ReceptorOrgan. The terms denominating the organ have changed over the years along with the interpretation of its function. Names such as frontal organ and X-organ are among the older terms. The recent term, cavity receptor organ, is based on the fact that the organ was found on morphological grounds to be a receptor of presumed chemosensory function (Elofsson and Lake, 1971). The organ is a paired structure arising medially from the dorsal margin of the brain (Fig. 1). It runs on both sides of the nauplius eye anteriorly to the cuticle. The cavity receptor organ consists of bipolar neurons along the whole of its course (Fig. 2). The nuclei are situated at all levels. Distally, each dendrite carries two unbranched cilia which are housed in a small cavity beneath the cuticle. This cavity is a space resulting from the detachment of the epidermal cell from the cuticle. The distal portion of the dendrite and part of the cilia thus pierce the epidermal cells and also a large accompanying cell adjacent to the epidermis. Proximally the organ sends its axons into a part of the brain called the medulla terminalis, a region which in crustaceans either forms part of the brain, as in this case, or is situated within the eyestalks. Microscopy and Depletion Experiments. The fluorescence histochemical method of Falck and Hillarp demonstrated intensely green-fluorescent bipolar neurons in the
L Fig. 1. Schematic drawing of the head ofArtemia salina. The brain (br), compound eye (ce), nauplius eye (he), and cavity receptor organ (cro) are hatched. The stumps of the first antenna (a) are indicated. The rectangular box marks the portion enlarged in Figure 2 Fig. 2. Schematic drawing of the cavity receptor organ (ero). The nauplius eye (ne) and nuclei of the cells of the cavity receptor organ are hatched. Cuticle (cu) and epidermis (ep) enclose the cavity (car). The cilia of the sensory cells pierce the accompanying cell (ace) and the epidermis. The organ enters the brain (br)
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Fig. 3 A and B. Micrographs showingthe green-fluorescentA sensorycells in the basal portion of the cavity receptor organ of Artemia salina (arrow) (Scale 50 gm) and B sensory cells (fine arrow) and basiepithelial nerve net (thick arrow) in Tealiafelina (Scale 50 lam)
cavity receptor organ (Aramant and Elofsson, 1976) (Fig. 3 a). The neurons were found ultrastructurally to contain dense core vesicles (about 65 nm in diameter, Fig. 4 a)(Elofsson and Lake, 1971) which were tentatively ascribed to the presence of a catecholamine (Aramant and Elofsson, 1976). In animals exposed to reserpine for 24 h and then processed for fluorescence microscopy, the bipolar neurons displayed no fluorescence. The animals remained healthy in reserpinized sea water for a longer period, but 24 h proved sufficient to cause disappearance of the fluorogenic substances. In animals exposed to reserpine for 24h and then transferred to normal sea water for up to nine days no reappearance of the fluorescence took place. In contrast, reserpinized animals examined by electron microscopy after 24 h did not differ from normal animals with regard to the number or appearance of the dense core vesicles. Microspectrofluorometry. The fluorophore in the green-fluorescent cells in the cavity receptor organ showed an excitation maximum at 375 nm with a small
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Fig. 4 A-C. Electron micrographs showing the dense core vesicles in A a dendrite o f a sensory cell in the cavity receptor organ of A rtemia salina (Scale 1 ~tm), B a cell in the epidermis of Metridium senile (Scale 1 ~tm), and C an axon within the basiepithelial nerve net of Metridium senile (Scale 0.5 lam)
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Fig. 5. Excitation (left) and emission (right) spectra recorded in a sensory cell in the cavity receptor organ of Artemia salina: directly after formaldehyde treatment; .......... after subsequent exposure of the paraffin sections to HCI vapor for 10 min; . . . . . . after exposure of the paraffin sections, previously acidified as above, to N H 3 vapor for 10 min. For comparison, the excitation and emission spectra of dopamine enclosed in histochemical protein droplets and treated with formaldehyde alone are shown
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secondary peak or shoulder at 330nm, and often a shoulder in the excitation spectrum at 410 nm. The emission maximum was at 475 nm (Fig. 5). After exposure of the paraffin sections to HC1 vapor for 10-30min (Bj6rklund et al., 1968, 1972 a) similar excitation peaks at 330 and 375nm, respectively, were recorded, the shoulder at 410nm having disappeared. After treatment with N H 3 vapor (1030 min) the excitation peak moved to 415 nm, sometimes with a small secondary peak at about 335 nm. Neither acidification nor alkalinization of the sections caused any change in the emission spectra. Green-fluorescent cells in the brain, belonging to cell groups 1 and 5 (Aramant and Elofsson, 1976), and the dopaminecontaining droplet models showed similar excitation and emission spectra (excitation maximum at 410nm and emission maximum at 470nm) after the standard formaldehyde treatment (Fig. 5).
Sea Anemones Microscopy. Intensely green-fluorescent bipolar cells with axons extending into the basiepithelial nerve net were disclosed by the Falck-Hillarp histochemical method in the sea anemones Metridium senile and Tealinafelina (Fig. 3 b), confirming the findings of Dahl et al. (1963). Ultrastructurally the present investigation has shown dense core vesicles in epidermal cells (Fig. 4 b) and in the basiepithelial nerve net (Fig. 4 c). Recent electron microscopical investigations of the coelenterate nervous system and sensory cells (Westfall, 1971 ; Peteya, 1975) have established the presence o f dense core vesicles in sensory cells and in the basiepithelial nerve net at polarized synapses of neuromuscular and neuronematocyte junctions. According to Peteya (1975) the size of the vesicles is 60-90nm, which accords with our findings in the
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nerve net. Westfall (1971) gives the size 120-200nm for the dense core vesicles, which is the size range for the cell body vesicles in the present investigation. On the accepted grounds that dense core vesicles represent the storage site of catecholamine transmitter substances (H6kfelt, 1968), it has been suggested that the sensory cells and terminals of the coelenterates containing these vesicles contained an adrenergic transmitter substance. In the case o f the sensory cells Peteya (1975) suggested that these were the fluorescent bipolar cells found by Dahl et al. (1963). Because of the complicated spatial relationships within the epidermis including the basiepithelial nerve net, even modern techniques have failed to demonstrate any functional contacts between sensory cells and presumed target cells. The theory of a sensory motor connection proposed by Dahl et al. (1963) is still tenable with regard to the new ultrastructural findings, although direct evidence is lacking. Another type of sensory motor contact was found by Westfall (1973 a, b), who demonstrated in Hydra littoralis a remarkable omnipotent neuron which she called sensory motor - interneuron and which establishes synapses on epithelio-muscular and other cells. The role of the neuron is believed to be chemosensory owing to its basal position in the epidermis. The fairly complicated behavioral repertoire of coelenterates and their acknowledged responsiveness to many kinds of external stimuli (Lentz, 1968) suggest that several diversified sensory pathways exist, and both alternatives, single or combined, are thus feasible.
Microspectrofluorometry. The spectral characteristics of the fluorescence in the sensory cells were evaluated in Tealiafelina, and were shown to be similar to those in the cavity receptor organ of Artemia salina directly after formaldehyde treatment (excitation maximum at 375 nm with a smaller peak or shoulder at 330 nm and sometimes a shoulder in the spectrum at 410 nm; emission maximum at 475 rim) (Fig. 6). As with Artemia salina, alkalinization of the Tealiafelina specimens induced an excitation maximum change to 410 nm. In contrast, after acidification of the
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tissue sections from Teal&felina there was a relative rise of the peak at 330 nm not seen with the sections from Artemia salina. In some cases this peak was even higher than that at 375 nm. This spectral changes is characteristic for /%hydroxylated catecholamines (see Discussion).
Discussion
Microspectrofluorometric analysis is today an indispensable tool for the identification and characterization of intracellular substances demonstrated by fluorescence histochemical techniques (see Bj6rklund et al., 1975, and others). Although it must be accompanied by identification of the compound in question by biochemical analytical techniques, spectral analysis provides a unique means for the detection of substances in individual cells. In the present investigation, microspectrofluorometric analysis was done to characterize the formaldehyde-induced fluorescence in some invertebrate receptor neurons. When considering the identity of the fluorogenic compound, the following should be borne in mind. Firstly, only compounds fulfilling certain molecular requirements become fluorescent in the histochemical reaction. Secondly, the fluorophores formed display characteristic and reproducible excitation and emission spectra. This means that the very fact that fluorophores have been formed allows certain conclusions about the molecular structure of the intracellular compound. Furthermore, for identification, direct comparisons can be made between the spectral characteristics of the tissue fluorophores and those of fluorophores formed from known compounds in histochemical protein models. The sensory cells in the invertebrates investigated became strongly fluorescent after formaldehyde treatment. With the knowledge of the molecular requirements for fluorophore formation in mind, this means that the fluorogenic compound or compounds must be a 3-hydroxylated, primary or secondary phenylethylamineor a related amino acid (Fig. 7), or a primary or secondary indolylethylamineor a related amino acid (Corrodi and Jonsson, 1967; Bj6rklund et al., 1975). The sensory cells in Artemia salina and Tealiafelina showed similar excitation and emission spectra directly after formaldehyde treatment (Figs. 5, 6) (excitation maximum at 375 nm, often with a small secondary peak or shoulder at 330 nm and sometimes a shoulder at 410 nm; emission maximum at 475 nm). The formaldehyde-inducedfluorophores from biogenic monoamines and related compounds have been divided into different groups on the basis of their emission peak maxima (Bj6rklund et al., 1971). The emission maximum of the fluorescence in the sensory cells (475 nm) places the fluorophore in the group comprising the catecholamines, DOPA and cysteinyldopa in which the emission maximum lies between 475 and 490 nm. However, directly after formaldehyde treatment, the excitation peak maximum of the cellular fluorescence (375 nm) is clearly different from the excitation maxima of the above compounds (410-420 nm). One characteristic feature of the catecholamine fluorophores is that they exhibit a pH-dependent tautomerism (Fig. 8) between a non-quinoidal (at acid pH) and quinoidal form (at neutral pH) (Corrodi and Hillarp, 1964; Jonsson, 1966; Bj6rklund et al., 1968, 1972 a). This is reflected in characteristic changes, notably of
New Catecholaminesin Invertebrate SensoryNeurons
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Fig. 7. Labeling of positions in phenylethylaminederivatives Fig. 8. The pH-dependenttautomerism exhibited by 6-hydroxylateddihydroisoquinolinefluorophores formedfrom 3-hydroxylatedphenylethylaminesand related aminoacids after formaldehydetreatment. The dopamine fluorophore shown in the figure is in its non-quinoidatform (/) at acid pH, and in its quinoidal form (I/) at neutral pH their excitation spectra (main excitation peak at 370 nm at acid pH, and at 410 nm at neutral and alkaline pH). This pH-dependent tautomerism, which is fully reversible, is dependent on the presence of the 3-hydroxyl group on the catecholamine molecule, and only catecholamines and other phenylethylamines with this substituent have been shown to exhibit such spectral changes upon variations in pH (Jonsson, 1966). The fluorescence in the invertebrate sensory cells also showed a pH-dependent shift which could be similar to a tautomeric shift indicative of 3-hydroxylated phenylethylamines. Thus, after exposure to HC1 vapor the excitation peak maximum was at 370-375 nm with a secondary peak or shoulder at 330nm. Alkalinization by exposure to N H 3 vapor involved a change of the excitation maximum to 410-415 nm with a small peak at 330nm. When comparing the spectral characteristics of the fluorescence in the sensory cells with those of the catecholamine fluorophores, it is noteworthy that after acidification and alkalinization of the specimens the excitation and emission spectra show close correspondence. On the other hand, directly after formaldehyde treatment the excitation spectra differ greatly, the cell fluorescence excitation spectrum being consistent with a non-quinoidal form o f fluorophore. However, it should be noted that some of the excitation spectra recorded directly after formaldehyde treatment also show a small peak or shoulder at 410 nm, indicating the presence also of a portion of the quinoidal form of fluorophore. Thus, both the fluorescence yield and the spectral characteristics of the compound in the sensory cells after formaldehyde treatment are consistent with a 3hydroxylated phenylethylamine derivative. Comparison with all 3-hydroxylated phenylethylamine derivatives and related amino acids tested in histochemical models indicates that the excitation and emission spectra o f the cellular fluorescence tally only with those of the fluorophores of catecholamines and D O P A (see, for example, Bj6rklund and Falck, 1973). It should be noted that cysteinyldopa has a higher excitation maximum (420 rim) than the fluorescence in the sensory cells after alkalinization (Agrup et al., 1977). If the cellular fluorescence is indeed due to the presence of a catechol derivative, how is the predominance of the non-quinoidal form of fluorophore directly after
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formaldehyde treatment to be explained? One explanation would be that the p H in the sensory ceils is below 7, and therefore most of the fluorophores formed in the histochemical reaction have been directly converted to their non-quinoidal, acid form. The intracellular fluorescence could then be due to the presence ofdopamine, noradrenaline, adrenaline, or the amino acid DOPA. The exact pH at which the different catecholamine fluorophores shift from the quinoidal to the non-quinoidal form is not known. Jonsson (1966) has shown that for the dopamine fluorophore, the quinoidal form predominates in the pH-range 6-10. The possibility that the spectral phenomena could be explained by a pH lower than 6 in the sensory cells cannot be denied, but it does not seem very likely. Another explanation would be that the sensory cells contain a previously unknown catechol derivative which, in contrast to the well-known catecholamines and DOPA, remains in its non-quinoidal form directly after formaldehyde treatment, i.e. near neutral pH. The existence of a new catechol derivative is supported by results from paper chromatographic analysis of extracts of tentacular tissue of Tealiafelina and Metridium senile (unpublished results). These analyses have proved the presence of D O P A and cysteinyldopa. In addition, however, three different spots, fluorescing upon formaldehyde treatment and showing a positive catechol reaction with K 3FeCN6 , were found on the chromatograms. One of these spots might contain the unknown intracellular catechol derivative. Carlyle (1969) reported the presence of D OPA, dopamine and noradrenaline in whole animals ofActinia equina, and Lenicque et al. (1977) found DOPA, dopamine and 5-HT in the oral zone of Melridium senile. In this laboratory the sensitive gas chromatographic-mass spectrometric technique has been applied on tentacular tissue of Metridium senile and Tealia felina (unpublished observations). The presence of D O P A was confirmed but no dopamine or noradrenaline was found; nor was 5-HT detected in fluorometric analyses. No significant change in the excitation spectrum of the cellular fluorescence in Artemia saIina was recorded after prolonged HC1 treatment. On the other hand, after HC1 treatment of the Tealiafelina specimens an increase of peak at 330 nm was recorded, and in some cases this peak was the predominating one. This type of spectral change resulting from HC1 exposure is characteristic of the fluorophores of fl-hydroxylated catecholamines, e.g. noradrenaline, and is due to a conversion of the dihydroisoquinoline fluorophores to fully aromatic isoquinolines (Corrodi and Jonsson, 1965; Bj6rklund et al., 1968, 1972a). The spectra after HC1 treatment thus seem to indicate that the presumed catechol derivative in the sensory cells of Tealia felina has a fl-hydroxyl group, whereas that in Artemia salina has no such substituent.
Acknowledgements. The work was supported by the followinggrants: the Swedish Medical Research Council (04)(-712 and 04X-56), the Swedish Natural ScienceResearch Council (2760-009), the U.S. Public Health Service(NS 06701-11), and the Magnus Bergvall Foundation. References
Agrup, G., Bj6rklund, A., Falck, B., Jacobsson, S., Lindvall, O., Rorsman, H., Rosengren, E.: Fluorescence histochemical demonstration of Dopa thioethers by condensation with gaseous formaldehyde. Histochemistry(in press) (1977)
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Aramant, R., Elofsson, R.: Distribution ofmonoaminergic neurons in the central nervous system of nonmalacostracan crustaceans. Cell Tiss. Res. 166, 1-24 (1976) Bjfrklund, A., Ehinger, B., Falck, B.: A method for differentiating dopamine from noradrenaline in tissue sections by microspectrofluorometry. J. Histochem. Cytochem. 16, 263-270 (1968) Bj6rklund, A., Ehinger, B., Falck, B. : Analysis of fluorescence excitation peak ratios for the cellular identification of noradrenaline, dopamine or their mixtures. J. Histochem. Cytochem. 20, 56-64 (1972a) Bj6rklund, A., Falck, B.: Cytofluorometry of biogenic monoamines in the Falck-Hillarp method. Structural identification by spectral analysis. In: Fluorescences techniques in cell biology (A.A. Thaer and M. Semetz, eds.). Berlin-Heidelberg-New York: Springer 1973 Bj6rklund, A., Falck, B., Klemm, N.: Microspectrofluorometric and chemical investigation of catecholamine-containing structures in the thoracic ganglia of Trichoptera. J. Insect Physiol. 16, 1147-1154 (1970) Bj6rklund, A., Falck, B., Lindvall, O.: Microspectrofluorometric analysis of cellular monoamines.after formaldehyde or glyoxylic acid condensation. In: Methods in brain research (P.B. Bradley, ed.). London: John Wiley and Sons 1975 Bj6rklund, A., Falck, B., Owman, Ch.: Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterization of biogenic amines. In: Methods of investigative and diagnostic endocrinology (S.A. Berson, ed.), Vol. 1, The thyroid and biogenic amines (J.E. Rall, I.J. Kopin, eds.), pp. 318-368. Amsterdam: North-Holland Publ. Co. 1972 Bj6rklund, A., Falck, B., Stenevi, U.: Microspectrofluorometric characterization of monoamines in the central nervous system: evidence for a new neuronal monoamine-like compound. Progr. Brain Res. 34, 63-73 (1971) Carlyle, R.F.: The occurrence of catecholamines in the sea anemone Actinia equina. Brit. J. Pharmac. Chemother. 36, 182 P (1969) Clark, M.E.: Histochemical localization of monoamines in the nervous system of the polychaete Nephthys. Proc. roy. Soc. B 165, 308-325 (1966) Corrodi, H., Hillarp, N.-A.: Fluoreszenzmethoden zur histochemischen Sichtbarmachung yon Monoaminen. 2. Identifizierung des fluoreszierenden Produktes aus Dopamin und Formaldehyd. Heir. chim. Acta 47, 911-918 (1964) Corrodi, H., Jonsson, G.: Fluorescence method for the histochemical demonstration of monoamines. 4. Histochemical differentiation between dopamine and noradrenaline in models. J. Histochem. Cytochem. 13, 484-487 (1965) Corrodi, H., Jonsson, G.: The formaldehyde fluorescence method for the histochemical demonstration of biogenic amines. A review on the methodology. J. Histochem. Cytochem. 15, 65-78 (1967) Dahl, E., Falck, B., von Mecklenburg, C., Myhrberg, H.: An adrenerglc nervous system in sea anemones. Quart. J. micr. Sci. 104, 531-534 (1963) Ehinger, B., Myhrberg, H.E.: Neuronal localization of dopamine, noradrenaline, and 5-hydroxytryptamine in the central and peripheral nervous system ofLumbricus terrestris (L.). Histochemie 28, 265275 (1971) Elofsson, R., Klemm, N.: Monoamine containing neurons in the optic ganglia of crustaceans and insects. Z. Zellforsch. 133, 475 499 (1972) Elofsson, R., Lake, P.S.: On the cavity receptor organ (X-organ or organ of Bellonci) of Artemia salina (Crustacea, Anostraca). Z. Zellforsch. 121,319-326 (1971) Falck, B.: Observations on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta physiol, scand. 56, Suppl. 197, 1-25 (1962) H6kfelt, T.: In vitro studies on central and peripheral monoamine neurons at the ultrastructural level. Z. Zellforsch. 91, 1-74 (1968) Jonsson, G.: Fluorescence studies on some 6,7-substituted 3,4-dihydroisoquinolines formed from 3hydroxytyramine (dopamine) and formaldehyde. Acta chem. scan& 20, 2755-2762 (1966) Karnovsky, M.J.: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137A (1965) Klemm, N.: Monoaminhaltige Strukturen im Zentralnervensystem der Trichoptera (Insecta). Teil I. Z. Zellforsch. 92, 487-502 (1968) Kuzmina, L.V.: Distribution of biogenic monoamines in the nervous system of the body segment of the leech Hirudo medicinalis (in Russian) (E.M. Kreps, ed.), Physiology and Biochemistry of invertebrates. J. Evol. Biochem. Fisiol., Suppl. (1968)
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Lenique, P.M., Toneby, M.I., Doumenc, D.: Demonstration of biogenic amines and localization of monoamine oxidases in the sea anemone Metridium senile (Linn6). Comp. Biochem. Physiol. 56 C, 31-34 (1977) Lentz, T.L.: Primitive nervous systems. New Haven Connecticut: Yale Univ. Press 1968 Lure, B.L.: Monoamine-containing neurons of the planarian Polyeelis nigra (Turb.). Moscow Univ. Biol. Sci. Bull. 30, 69-76 (1975) Myhrberg, H.: Monoaminergic mechanisms in the nervous system of Lumbricus terrestris (L.). Z. Zellforsch. 81, 311-343 (1967) M yhrberg, H.: Ultrastructural localization of monoamines in the epidermis ofLumbrifus terrestris (L.). (L.). Z. Zellforsch. 11"/, 139-154 (1971) Peteya, DJ.: The ciliary-cone sensory cell of anemones and cerianthids. Tiss & Cell 7, 243-252 (1975) Plotnikova, S.I., Govyrin, V.A.: Distribution of catecholamine-containing nerve elements in some representatives of Protostomia and Coelenterata (in Russian). Arch. anat. gistol, embriol. 50, 79-87 (1967) Plotnikova, S.I., Kuzmina, L.V.: Distribution of catecholamine-containing nervous elements in planaria Dendrocoelum lacteum (Turbellaria) (in Russian). (E.M. Kreps, ed.), Physiology and biochemistry of invertebrate. J. Evol. Biochim. Fisiol., Suppl. (1968) Rude, S.: Monoamine-containing neurons in the nerve cord and body wall of Lumbricus terrestris. J. comp. Neurol. 128, 397-412 (1966) Sulston, J., Dew, M., Brenner, S.: Dopaminergic neurons in the nematode Caenorhabditis elegans. J. comp. Neurol. 163, 215 226 (1975) Sweeney, D.C.: The anatomical distribution of monoamines in a freshwater bivalve mollusc, Sphaerium sulcatum (L.). Comp. Biochem. Physiol. 25, 601-613 (1968) Welsh, J.H., Williams, J.D.: Monoamine-containing neurons in Planaria. J. comp. Neurol. 138, 103-116 (1970) Westfall, J.A.: Ultrastructural evidence for neuromuscular systems in coelenterates. Amer. Zool. 13, 237246 (1973 a) Westfall, J.A.: Ultrastructural evidence for a granule-containing sensory-motor-interneuron in Hydra littoralis. J. Ultrastruct. Res. 42, 268-282 (1973 b) Wesffall, J.A., Yamataka, S., Enos, P.D.: Ultrastructnral evidence of polarized synapses in the nerve net of Hydra. J. Cell Biol. 51, 318 323 (1971)
Accepted May 22, 1977
Cell and Tissue Research 9 by Springer-Verlag 1977
Evidence for New Catecholamines or Related Amino Acids in Some Invertebrate Sensory Neurons R. Elofsson, B. Falck, O. Lindvall, and H. Myhrberg Department of Zoology and Department of Histology, University of Lund, Sweden
Summary. In certain sensory neurons of many different invertebrate species, including the sea anemones. Metridium senile and Tealiafelina and the crustacean Artemia salina, fluorophores are formed during the course of the fluorescent histochemical technique of Falck-Hillarp. The presumed catecholamine nature of the neuronal fluorogenic compound was investigated by microspectrofluorometry, and the spectral characteristics of the fluorescence in the taxonomically different species was found to be very similar (excitation maximum at 375 nm with a smaller peak or shoulder at 330 nm and sometimes a shoulder in the spectrum at 410 nm; emission maximum at 475 nm). The emission maximum coincides with that of the catecholamines and DOPA (475 nm). The excitation maximum (375 nm) directly after formaldehyde treatment, however, differs from that of the catecholamines and DOPA (410nm), but is similar to the excitation maximum displayed by these catechol derivatives at acid pH. The spectral characteristics of the fluorophore in the sensory cells might therefore theoretically be explained by an acid pH in the cells. This seems improbable, however, and it is suggested that the phenomenon is due to the presence of unknown catechol derivatives. Analyses of the pH-dependent spectral changes indicate that the presumed catechol derivative in Tealiafelina is fl-hydroxylated, whereas that in Artemia salina is not. Key words: Catecholamines - Sensory neurons - Coelenterates - Crustaceans - Microspectrofluorometry.
Introduction
Sensory neurons assumed to contain catecholamines have been reported in sea anemones, planarians, nematodes, various annelids, molluscs, and one crustacean (Dahl et al., 1963; Clark, 1966; Rude, 1966; Plotnikova and Govyrin, 1967; Send offprint requests to: Doc. Dr. Rolf Elofsson, Zoological Institute, Helgonaviigen 3, S-22362 Lund,
Sweden
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Myhrberg, 1967, 1971; Plotnikova and Kuzmina, 1968; Kuzmina, 1968; Sweeney, 1968; Sulston et al., 1975; Lure, 1975; Aramant and Elofsson, 1976). 5-HTcontaining sensory cells have so far been reported only in planarians (Welsh and Williams, 1970). Only in one case has a catecholamine in sensory cells been described in close detail (Ehinger and Myhrberg, 1971). Spectral analyses have indicated that the sensory cells of earthworm (Lumbricus terrestris) contain dopamine. In the course of a microspectrofluorometric survey of invertebrate sensory cells presumed to be catecholaminergic some diverging results were found. Neurons in the tentacles of sea anemones (Coelenterata) and in the cavity receptor of the crustacean Artemia salina displayed spectral characteristics of formaldehydeinduced fluorescence that deviated from those of dopamine, noradrenaline, adrenaline, or dopa.
Material and Methods Artemia salina L. (Anostraca) was reared to the adult stage from commercially available eggs in artificial sea water (Dohse Aquaristik) on Artemia-futter (Dohse Aquaristik). The sea anemones Metridium senile and Tealia felina were obtained from fiords outside Bergen through the courtesy of Dr. Tor Samuelsson, Bergen Sea Aquarium, Norway and from Helsing6r Marine Biological Station, Denmark, thanks to Dr. Lars Hagerman. In depletion experiments Artemia was allowed to swim in artificial sea water containing reserpine (Serpasil | CIBA, 0.125 mg/ml) for 24 to 48 h. Some animals were transferred to normal sea water and kept alive for up to 9 days. Fluorescence Microscopy. Tentacles of the sea anemones and whole Artemia were frozen, freeze-dried, and treated with formaldehyde gas in accordance with the specific method for cellular demonstration of biogenic monoamines described by Falck and Hillarp (Falck, 1962; Bj6rklund et al., 1972) using minor modifications of the method as described by Klemm (1968), Bj6rklund et al. (1970) and Elofsson and Klemm (1972). The best results were obtained with paraformaldehyde stored at a relative humidity of 50 per cent. The microscopical work was preformed with a Leitz fluorescence microscope equipped as described by Bj6rklund et al. (1972 b). Electron Microscopy. Heads of Artemia were fixed in a glutaraldehyde/paraformaldehyde mixture (Karnovsky, 1965) in cacodylate buffer for 4 h. Postfixation was performed in 2 ~o osmium tetroxyde in the same buffer for 1 he Pieces of coelenterate tentacles were fixed in 2.5 ~ glutaraldehyde in phosphate buffer for 1 h and postfixed in ~ ~ osmium tetroxide for ~ h or in 2 ~ potassium permanganate in veronal/acetate buffer for I h. Fixation was followed by dehydration in an alcohol series and embedding in Vestopal W. Ultrathin sections were examined on formvar-coated grids in a Philips EM 300 and a Zeiss EM 10 electron microscope. Microspectrofluoromeo3;. Excitation and emission spectra of the fluorophores were recorded in a modified Leitz microspectrofluorometer (Bj6rklund et al., 1972 b). For further characterization of the fluorophores, the paraffin sections ofA rtemia salina were then exposed to HCI vapor (10-30 min) and/or to NH 3 vapor (10-30 min) and the deparaffinized sections of Tealiafelina exposed to HCI vapor (30 s 1 rain) and/or to NH 3 vapor (10 20 s) at room temperature. The excitation and emission spectra of the tissue fluorophores were compared with spectra obtained simultaneously from histochemical protein models containing dopamine treated with formaldehyde vapor as above. These models were prepared by dissolving dopamine to a concentration of I mg/ml in a 2 ~ buffer solution (pH 7.0) of human serum albumin (AB Kabi, Stockholm, Sweden). The solution was sprayed in droplet form onto cover glasses, air-dried, and then formaldehyde-treated. All spectra were corrected according to the previously described procedures (Bj6rklund et al., 1968). and are expressed as relative quanta versus wave length.
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Results
Artemia salina Morphology of the Cavity ReceptorOrgan. The terms denominating the organ have changed over the years along with the interpretation of its function. Names such as frontal organ and X-organ are among the older terms. The recent term, cavity receptor organ, is based on the fact that the organ was found on morphological grounds to be a receptor of presumed chemosensory function (Elofsson and Lake, 1971). The organ is a paired structure arising medially from the dorsal margin of the brain (Fig. 1). It runs on both sides of the nauplius eye anteriorly to the cuticle. The cavity receptor organ consists of bipolar neurons along the whole of its course (Fig. 2). The nuclei are situated at all levels. Distally, each dendrite carries two unbranched cilia which are housed in a small cavity beneath the cuticle. This cavity is a space resulting from the detachment of the epidermal cell from the cuticle. The distal portion of the dendrite and part of the cilia thus pierce the epidermal cells and also a large accompanying cell adjacent to the epidermis. Proximally the organ sends its axons into a part of the brain called the medulla terminalis, a region which in crustaceans either forms part of the brain, as in this case, or is situated within the eyestalks. Microscopy and Depletion Experiments. The fluorescence histochemical method of Falck and Hillarp demonstrated intensely green-fluorescent bipolar neurons in the
L Fig. 1. Schematic drawing of the head ofArtemia salina. The brain (br), compound eye (ce), nauplius eye (he), and cavity receptor organ (cro) are hatched. The stumps of the first antenna (a) are indicated. The rectangular box marks the portion enlarged in Figure 2 Fig. 2. Schematic drawing of the cavity receptor organ (ero). The nauplius eye (ne) and nuclei of the cells of the cavity receptor organ are hatched. Cuticle (cu) and epidermis (ep) enclose the cavity (car). The cilia of the sensory cells pierce the accompanying cell (ace) and the epidermis. The organ enters the brain (br)
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Fig. 3 A and B. Micrographs showingthe green-fluorescentA sensorycells in the basal portion of the cavity receptor organ of Artemia salina (arrow) (Scale 50 gm) and B sensory cells (fine arrow) and basiepithelial nerve net (thick arrow) in Tealiafelina (Scale 50 lam)
cavity receptor organ (Aramant and Elofsson, 1976) (Fig. 3 a). The neurons were found ultrastructurally to contain dense core vesicles (about 65 nm in diameter, Fig. 4 a)(Elofsson and Lake, 1971) which were tentatively ascribed to the presence of a catecholamine (Aramant and Elofsson, 1976). In animals exposed to reserpine for 24 h and then processed for fluorescence microscopy, the bipolar neurons displayed no fluorescence. The animals remained healthy in reserpinized sea water for a longer period, but 24 h proved sufficient to cause disappearance of the fluorogenic substances. In animals exposed to reserpine for 24h and then transferred to normal sea water for up to nine days no reappearance of the fluorescence took place. In contrast, reserpinized animals examined by electron microscopy after 24 h did not differ from normal animals with regard to the number or appearance of the dense core vesicles. Microspectrofluorometry. The fluorophore in the green-fluorescent cells in the cavity receptor organ showed an excitation maximum at 375 nm with a small
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Fig. 4 A-C. Electron micrographs showing the dense core vesicles in A a dendrite o f a sensory cell in the cavity receptor organ of A rtemia salina (Scale 1 ~tm), B a cell in the epidermis of Metridium senile (Scale 1 ~tm), and C an axon within the basiepithelial nerve net of Metridium senile (Scale 0.5 lam)
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secondary peak or shoulder at 330nm, and often a shoulder in the excitation spectrum at 410 nm. The emission maximum was at 475 nm (Fig. 5). After exposure of the paraffin sections to HC1 vapor for 10-30min (Bj6rklund et al., 1968, 1972 a) similar excitation peaks at 330 and 375nm, respectively, were recorded, the shoulder at 410nm having disappeared. After treatment with N H 3 vapor (1030 min) the excitation peak moved to 415 nm, sometimes with a small secondary peak at about 335 nm. Neither acidification nor alkalinization of the sections caused any change in the emission spectra. Green-fluorescent cells in the brain, belonging to cell groups 1 and 5 (Aramant and Elofsson, 1976), and the dopaminecontaining droplet models showed similar excitation and emission spectra (excitation maximum at 410nm and emission maximum at 470nm) after the standard formaldehyde treatment (Fig. 5).
Sea Anemones Microscopy. Intensely green-fluorescent bipolar cells with axons extending into the basiepithelial nerve net were disclosed by the Falck-Hillarp histochemical method in the sea anemones Metridium senile and Tealinafelina (Fig. 3 b), confirming the findings of Dahl et al. (1963). Ultrastructurally the present investigation has shown dense core vesicles in epidermal cells (Fig. 4 b) and in the basiepithelial nerve net (Fig. 4 c). Recent electron microscopical investigations of the coelenterate nervous system and sensory cells (Westfall, 1971 ; Peteya, 1975) have established the presence o f dense core vesicles in sensory cells and in the basiepithelial nerve net at polarized synapses of neuromuscular and neuronematocyte junctions. According to Peteya (1975) the size of the vesicles is 60-90nm, which accords with our findings in the
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nerve net. Westfall (1971) gives the size 120-200nm for the dense core vesicles, which is the size range for the cell body vesicles in the present investigation. On the accepted grounds that dense core vesicles represent the storage site of catecholamine transmitter substances (H6kfelt, 1968), it has been suggested that the sensory cells and terminals of the coelenterates containing these vesicles contained an adrenergic transmitter substance. In the case o f the sensory cells Peteya (1975) suggested that these were the fluorescent bipolar cells found by Dahl et al. (1963). Because of the complicated spatial relationships within the epidermis including the basiepithelial nerve net, even modern techniques have failed to demonstrate any functional contacts between sensory cells and presumed target cells. The theory of a sensory motor connection proposed by Dahl et al. (1963) is still tenable with regard to the new ultrastructural findings, although direct evidence is lacking. Another type of sensory motor contact was found by Westfall (1973 a, b), who demonstrated in Hydra littoralis a remarkable omnipotent neuron which she called sensory motor - interneuron and which establishes synapses on epithelio-muscular and other cells. The role of the neuron is believed to be chemosensory owing to its basal position in the epidermis. The fairly complicated behavioral repertoire of coelenterates and their acknowledged responsiveness to many kinds of external stimuli (Lentz, 1968) suggest that several diversified sensory pathways exist, and both alternatives, single or combined, are thus feasible.
Microspectrofluorometry. The spectral characteristics of the fluorescence in the sensory cells were evaluated in Tealiafelina, and were shown to be similar to those in the cavity receptor organ of Artemia salina directly after formaldehyde treatment (excitation maximum at 375 nm with a smaller peak or shoulder at 330 nm and sometimes a shoulder in the spectrum at 410 nm; emission maximum at 475 rim) (Fig. 6). As with Artemia salina, alkalinization of the Tealiafelina specimens induced an excitation maximum change to 410 nm. In contrast, after acidification of the
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tissue sections from Teal&felina there was a relative rise of the peak at 330 nm not seen with the sections from Artemia salina. In some cases this peak was even higher than that at 375 nm. This spectral changes is characteristic for /%hydroxylated catecholamines (see Discussion).
Discussion
Microspectrofluorometric analysis is today an indispensable tool for the identification and characterization of intracellular substances demonstrated by fluorescence histochemical techniques (see Bj6rklund et al., 1975, and others). Although it must be accompanied by identification of the compound in question by biochemical analytical techniques, spectral analysis provides a unique means for the detection of substances in individual cells. In the present investigation, microspectrofluorometric analysis was done to characterize the formaldehyde-induced fluorescence in some invertebrate receptor neurons. When considering the identity of the fluorogenic compound, the following should be borne in mind. Firstly, only compounds fulfilling certain molecular requirements become fluorescent in the histochemical reaction. Secondly, the fluorophores formed display characteristic and reproducible excitation and emission spectra. This means that the very fact that fluorophores have been formed allows certain conclusions about the molecular structure of the intracellular compound. Furthermore, for identification, direct comparisons can be made between the spectral characteristics of the tissue fluorophores and those of fluorophores formed from known compounds in histochemical protein models. The sensory cells in the invertebrates investigated became strongly fluorescent after formaldehyde treatment. With the knowledge of the molecular requirements for fluorophore formation in mind, this means that the fluorogenic compound or compounds must be a 3-hydroxylated, primary or secondary phenylethylamineor a related amino acid (Fig. 7), or a primary or secondary indolylethylamineor a related amino acid (Corrodi and Jonsson, 1967; Bj6rklund et al., 1975). The sensory cells in Artemia salina and Tealiafelina showed similar excitation and emission spectra directly after formaldehyde treatment (Figs. 5, 6) (excitation maximum at 375 nm, often with a small secondary peak or shoulder at 330 nm and sometimes a shoulder at 410 nm; emission maximum at 475 nm). The formaldehyde-inducedfluorophores from biogenic monoamines and related compounds have been divided into different groups on the basis of their emission peak maxima (Bj6rklund et al., 1971). The emission maximum of the fluorescence in the sensory cells (475 nm) places the fluorophore in the group comprising the catecholamines, DOPA and cysteinyldopa in which the emission maximum lies between 475 and 490 nm. However, directly after formaldehyde treatment, the excitation peak maximum of the cellular fluorescence (375 nm) is clearly different from the excitation maxima of the above compounds (410-420 nm). One characteristic feature of the catecholamine fluorophores is that they exhibit a pH-dependent tautomerism (Fig. 8) between a non-quinoidal (at acid pH) and quinoidal form (at neutral pH) (Corrodi and Hillarp, 1964; Jonsson, 1966; Bj6rklund et al., 1968, 1972 a). This is reflected in characteristic changes, notably of
New Catecholaminesin Invertebrate SensoryNeurons
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Fig. 7. Labeling of positions in phenylethylaminederivatives Fig. 8. The pH-dependenttautomerism exhibited by 6-hydroxylateddihydroisoquinolinefluorophores formedfrom 3-hydroxylatedphenylethylaminesand related aminoacids after formaldehydetreatment. The dopamine fluorophore shown in the figure is in its non-quinoidatform (/) at acid pH, and in its quinoidal form (I/) at neutral pH their excitation spectra (main excitation peak at 370 nm at acid pH, and at 410 nm at neutral and alkaline pH). This pH-dependent tautomerism, which is fully reversible, is dependent on the presence of the 3-hydroxyl group on the catecholamine molecule, and only catecholamines and other phenylethylamines with this substituent have been shown to exhibit such spectral changes upon variations in pH (Jonsson, 1966). The fluorescence in the invertebrate sensory cells also showed a pH-dependent shift which could be similar to a tautomeric shift indicative of 3-hydroxylated phenylethylamines. Thus, after exposure to HC1 vapor the excitation peak maximum was at 370-375 nm with a secondary peak or shoulder at 330nm. Alkalinization by exposure to N H 3 vapor involved a change of the excitation maximum to 410-415 nm with a small peak at 330nm. When comparing the spectral characteristics of the fluorescence in the sensory cells with those of the catecholamine fluorophores, it is noteworthy that after acidification and alkalinization of the specimens the excitation and emission spectra show close correspondence. On the other hand, directly after formaldehyde treatment the excitation spectra differ greatly, the cell fluorescence excitation spectrum being consistent with a non-quinoidal form o f fluorophore. However, it should be noted that some of the excitation spectra recorded directly after formaldehyde treatment also show a small peak or shoulder at 410 nm, indicating the presence also of a portion of the quinoidal form of fluorophore. Thus, both the fluorescence yield and the spectral characteristics of the compound in the sensory cells after formaldehyde treatment are consistent with a 3hydroxylated phenylethylamine derivative. Comparison with all 3-hydroxylated phenylethylamine derivatives and related amino acids tested in histochemical models indicates that the excitation and emission spectra o f the cellular fluorescence tally only with those of the fluorophores of catecholamines and D O P A (see, for example, Bj6rklund and Falck, 1973). It should be noted that cysteinyldopa has a higher excitation maximum (420 rim) than the fluorescence in the sensory cells after alkalinization (Agrup et al., 1977). If the cellular fluorescence is indeed due to the presence of a catechol derivative, how is the predominance of the non-quinoidal form of fluorophore directly after
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formaldehyde treatment to be explained? One explanation would be that the p H in the sensory ceils is below 7, and therefore most of the fluorophores formed in the histochemical reaction have been directly converted to their non-quinoidal, acid form. The intracellular fluorescence could then be due to the presence ofdopamine, noradrenaline, adrenaline, or the amino acid DOPA. The exact pH at which the different catecholamine fluorophores shift from the quinoidal to the non-quinoidal form is not known. Jonsson (1966) has shown that for the dopamine fluorophore, the quinoidal form predominates in the pH-range 6-10. The possibility that the spectral phenomena could be explained by a pH lower than 6 in the sensory cells cannot be denied, but it does not seem very likely. Another explanation would be that the sensory cells contain a previously unknown catechol derivative which, in contrast to the well-known catecholamines and DOPA, remains in its non-quinoidal form directly after formaldehyde treatment, i.e. near neutral pH. The existence of a new catechol derivative is supported by results from paper chromatographic analysis of extracts of tentacular tissue of Tealiafelina and Metridium senile (unpublished results). These analyses have proved the presence of D O P A and cysteinyldopa. In addition, however, three different spots, fluorescing upon formaldehyde treatment and showing a positive catechol reaction with K 3FeCN6 , were found on the chromatograms. One of these spots might contain the unknown intracellular catechol derivative. Carlyle (1969) reported the presence of D OPA, dopamine and noradrenaline in whole animals ofActinia equina, and Lenicque et al. (1977) found DOPA, dopamine and 5-HT in the oral zone of Melridium senile. In this laboratory the sensitive gas chromatographic-mass spectrometric technique has been applied on tentacular tissue of Metridium senile and Tealia felina (unpublished observations). The presence of D O P A was confirmed but no dopamine or noradrenaline was found; nor was 5-HT detected in fluorometric analyses. No significant change in the excitation spectrum of the cellular fluorescence in Artemia saIina was recorded after prolonged HC1 treatment. On the other hand, after HC1 treatment of the Tealiafelina specimens an increase of peak at 330 nm was recorded, and in some cases this peak was the predominating one. This type of spectral change resulting from HC1 exposure is characteristic of the fluorophores of fl-hydroxylated catecholamines, e.g. noradrenaline, and is due to a conversion of the dihydroisoquinoline fluorophores to fully aromatic isoquinolines (Corrodi and Jonsson, 1965; Bj6rklund et al., 1968, 1972a). The spectra after HC1 treatment thus seem to indicate that the presumed catechol derivative in the sensory cells of Tealia felina has a fl-hydroxyl group, whereas that in Artemia salina has no such substituent.
Acknowledgements. The work was supported by the followinggrants: the Swedish Medical Research Council (04)(-712 and 04X-56), the Swedish Natural ScienceResearch Council (2760-009), the U.S. Public Health Service(NS 06701-11), and the Magnus Bergvall Foundation. References
Agrup, G., Bj6rklund, A., Falck, B., Jacobsson, S., Lindvall, O., Rorsman, H., Rosengren, E.: Fluorescence histochemical demonstration of Dopa thioethers by condensation with gaseous formaldehyde. Histochemistry(in press) (1977)
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Aramant, R., Elofsson, R.: Distribution ofmonoaminergic neurons in the central nervous system of nonmalacostracan crustaceans. Cell Tiss. Res. 166, 1-24 (1976) Bjfrklund, A., Ehinger, B., Falck, B.: A method for differentiating dopamine from noradrenaline in tissue sections by microspectrofluorometry. J. Histochem. Cytochem. 16, 263-270 (1968) Bj6rklund, A., Ehinger, B., Falck, B. : Analysis of fluorescence excitation peak ratios for the cellular identification of noradrenaline, dopamine or their mixtures. J. Histochem. Cytochem. 20, 56-64 (1972a) Bj6rklund, A., Falck, B.: Cytofluorometry of biogenic monoamines in the Falck-Hillarp method. Structural identification by spectral analysis. In: Fluorescences techniques in cell biology (A.A. Thaer and M. Semetz, eds.). Berlin-Heidelberg-New York: Springer 1973 Bj6rklund, A., Falck, B., Klemm, N.: Microspectrofluorometric and chemical investigation of catecholamine-containing structures in the thoracic ganglia of Trichoptera. J. Insect Physiol. 16, 1147-1154 (1970) Bj6rklund, A., Falck, B., Lindvall, O.: Microspectrofluorometric analysis of cellular monoamines.after formaldehyde or glyoxylic acid condensation. In: Methods in brain research (P.B. Bradley, ed.). London: John Wiley and Sons 1975 Bj6rklund, A., Falck, B., Owman, Ch.: Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterization of biogenic amines. In: Methods of investigative and diagnostic endocrinology (S.A. Berson, ed.), Vol. 1, The thyroid and biogenic amines (J.E. Rall, I.J. Kopin, eds.), pp. 318-368. Amsterdam: North-Holland Publ. Co. 1972 Bj6rklund, A., Falck, B., Stenevi, U.: Microspectrofluorometric characterization of monoamines in the central nervous system: evidence for a new neuronal monoamine-like compound. Progr. Brain Res. 34, 63-73 (1971) Carlyle, R.F.: The occurrence of catecholamines in the sea anemone Actinia equina. Brit. J. Pharmac. Chemother. 36, 182 P (1969) Clark, M.E.: Histochemical localization of monoamines in the nervous system of the polychaete Nephthys. Proc. roy. Soc. B 165, 308-325 (1966) Corrodi, H., Hillarp, N.-A.: Fluoreszenzmethoden zur histochemischen Sichtbarmachung yon Monoaminen. 2. Identifizierung des fluoreszierenden Produktes aus Dopamin und Formaldehyd. Heir. chim. Acta 47, 911-918 (1964) Corrodi, H., Jonsson, G.: Fluorescence method for the histochemical demonstration of monoamines. 4. Histochemical differentiation between dopamine and noradrenaline in models. J. Histochem. Cytochem. 13, 484-487 (1965) Corrodi, H., Jonsson, G.: The formaldehyde fluorescence method for the histochemical demonstration of biogenic amines. A review on the methodology. J. Histochem. Cytochem. 15, 65-78 (1967) Dahl, E., Falck, B., von Mecklenburg, C., Myhrberg, H.: An adrenerglc nervous system in sea anemones. Quart. J. micr. Sci. 104, 531-534 (1963) Ehinger, B., Myhrberg, H.E.: Neuronal localization of dopamine, noradrenaline, and 5-hydroxytryptamine in the central and peripheral nervous system ofLumbricus terrestris (L.). Histochemie 28, 265275 (1971) Elofsson, R., Klemm, N.: Monoamine containing neurons in the optic ganglia of crustaceans and insects. Z. Zellforsch. 133, 475 499 (1972) Elofsson, R., Lake, P.S.: On the cavity receptor organ (X-organ or organ of Bellonci) of Artemia salina (Crustacea, Anostraca). Z. Zellforsch. 121,319-326 (1971) Falck, B.: Observations on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta physiol, scand. 56, Suppl. 197, 1-25 (1962) H6kfelt, T.: In vitro studies on central and peripheral monoamine neurons at the ultrastructural level. Z. Zellforsch. 91, 1-74 (1968) Jonsson, G.: Fluorescence studies on some 6,7-substituted 3,4-dihydroisoquinolines formed from 3hydroxytyramine (dopamine) and formaldehyde. Acta chem. scan& 20, 2755-2762 (1966) Karnovsky, M.J.: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137A (1965) Klemm, N.: Monoaminhaltige Strukturen im Zentralnervensystem der Trichoptera (Insecta). Teil I. Z. Zellforsch. 92, 487-502 (1968) Kuzmina, L.V.: Distribution of biogenic monoamines in the nervous system of the body segment of the leech Hirudo medicinalis (in Russian) (E.M. Kreps, ed.), Physiology and Biochemistry of invertebrates. J. Evol. Biochem. Fisiol., Suppl. (1968)
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Lenique, P.M., Toneby, M.I., Doumenc, D.: Demonstration of biogenic amines and localization of monoamine oxidases in the sea anemone Metridium senile (Linn6). Comp. Biochem. Physiol. 56 C, 31-34 (1977) Lentz, T.L.: Primitive nervous systems. New Haven Connecticut: Yale Univ. Press 1968 Lure, B.L.: Monoamine-containing neurons of the planarian Polyeelis nigra (Turb.). Moscow Univ. Biol. Sci. Bull. 30, 69-76 (1975) Myhrberg, H.: Monoaminergic mechanisms in the nervous system of Lumbricus terrestris (L.). Z. Zellforsch. 81, 311-343 (1967) M yhrberg, H.: Ultrastructural localization of monoamines in the epidermis ofLumbrifus terrestris (L.). (L.). Z. Zellforsch. 11"/, 139-154 (1971) Peteya, DJ.: The ciliary-cone sensory cell of anemones and cerianthids. Tiss & Cell 7, 243-252 (1975) Plotnikova, S.I., Govyrin, V.A.: Distribution of catecholamine-containing nerve elements in some representatives of Protostomia and Coelenterata (in Russian). Arch. anat. gistol, embriol. 50, 79-87 (1967) Plotnikova, S.I., Kuzmina, L.V.: Distribution of catecholamine-containing nervous elements in planaria Dendrocoelum lacteum (Turbellaria) (in Russian). (E.M. Kreps, ed.), Physiology and biochemistry of invertebrate. J. Evol. Biochim. Fisiol., Suppl. (1968) Rude, S.: Monoamine-containing neurons in the nerve cord and body wall of Lumbricus terrestris. J. comp. Neurol. 128, 397-412 (1966) Sulston, J., Dew, M., Brenner, S.: Dopaminergic neurons in the nematode Caenorhabditis elegans. J. comp. Neurol. 163, 215 226 (1975) Sweeney, D.C.: The anatomical distribution of monoamines in a freshwater bivalve mollusc, Sphaerium sulcatum (L.). Comp. Biochem. Physiol. 25, 601-613 (1968) Welsh, J.H., Williams, J.D.: Monoamine-containing neurons in Planaria. J. comp. Neurol. 138, 103-116 (1970) Westfall, J.A.: Ultrastructural evidence for neuromuscular systems in coelenterates. Amer. Zool. 13, 237246 (1973 a) Westfall, J.A.: Ultrastructural evidence for a granule-containing sensory-motor-interneuron in Hydra littoralis. J. Ultrastruct. Res. 42, 268-282 (1973 b) Wesffall, J.A., Yamataka, S., Enos, P.D.: Ultrastructnral evidence of polarized synapses in the nerve net of Hydra. J. Cell Biol. 51, 318 323 (1971)
Accepted May 22, 1977
