Monoamine-containing neurons in the optic ganglia of ... - Springer Link

Z. Zellforsch. 133, 475~499 (1972) © by Springer-Verlag 1972

Monoamine-Containing Neurons in the Optic Ganglia of Crustaceans and Insects* Rolf Elofsson Zoological Institute, Lund, Sweden Nikolai Klemm Institute ffir Angcwandte Zoologic, Wfirzburg, Deutschland Received May 18, 1972

Summary. With the fluorescence method of Falck and Hillarp, the presence and localization of monoaminergic neurons in the optic ganglia of several crustaceans and insects have been investigated. It was found that in both classes the monoaminergic terminals, when present, appeared (especially in the medullae externa and interna of the crustaceans and the medulla of the insects) in strata specific for each species. So far, the only monoamine (visualized by this technique) present in the crustacean optic ganglia is dopamine, whereas in the Insecta, the catecholamines dopamine and noradrenaline, and the indolamine, 5-hydroxytryptamine, are found in the optic lobe. But in the Insecta, different species show different content of these amines. Key words: Optic ganglia - - Monoamines - - Crustacea - - Insecta. Introduction The presence of m o n o a m i n e r g i c neurons in c r u s t a c e a n s a n d insects has been visualized b y t h e fluorescence m e t h o d of F a l e k a n d t I i l l a r p ( F a l c k a n d Owman, 1965; B j 6 r k l u n d , F a l e k a n d Owman, 1972) a n d r e p o r t e d in several r e c e n t investig a t i o n s (Elofsson, K a u r i , Nielsen, a n d S t r S m b e r g , 1966, 1968 ; Osborne a n d D a n d o , 1970 ; Cooke, Berlind, a n d Goldstone, 1970 ; Cooke a n d Goldstone, 1970; Goldstone a n d Cooke, 1971 ; F r o n t a l i a n d Norberg, 1966 ; P l o t n i k o v a , 1967 ; F r o n t a l i , 1968 ; K l e m m , 1968a, b, 1971a, b, in press; B j 6 r k l u n d , F a l e k , a n d K l e m m , 1970; Ceeh a n d K n o z , 1970; K l e m m a n d B j 6 r k l u n d , 1971). I n a few of these (Elofsson et al., 1966; K l e m m , 1968b, 1971a) a t t e n t i o n was d r a w n to t h e p e c u l i a r p a t t e r n disp l a y e d b y t h e m o n o a m i n e r g i e neurons in t h e optic ganglia. The f u n c t i o n a l implications for t h e visual process suggested b y t h e findings u r g e d us to gain a m o r e d e t a i l e d k n o w l e d g e of t h e localization of t h e m o n o a m i n e r g i c s t r u c t u r e s in t h e optic ganglia of m o r e r e p r e s e n t a t i v e s of t h e two classes. * This work was supported by grants 2760-3 and 2760-4 from the Swedish Natural Science Research Council (R.E.), by a fellowship from Deutsche Forschungsgemeinschaft, and a grant from the Swedish Medical Research Council B72-14X-712-D7B (N.K.). We are very grateful to the director of the Department of Histology, Faculty of Medicine, Lund, Professor Bengt Falck, who put all his facilities and knowledge at our disposal.

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1~. Elofsson and N. Klemm: Material and Methods

The following species were investigated: Crustacea

Insecta

Artemia salina L. (Anostraca)

Lepisma saccharina L. (Apterygota: Thysanura)

Neomysis integer Leach (Mysidacea)

Aeschna viridis Eversm. (Odonata: Anisoptera)

Leander ]abricii Rathke (Decapoda: Macrura)

Schistocerca gregaria Forsk&l (Orthoptera: Aerididae)

Spirontoearis lilljeborgii Danielssen (Decapoda: Macrura)

Aeheta domesticus L. (Orthoptera: Gryllidae)

Pandalus borealis KrSyer (Decapoda: Macrura)

Limnephilidae (Trichoptera)

P. montagui Leach (Decapoda: Macrura)

Spodoptera littoralis Bsd. (Lepidoptera: Noctuidae)

Crangon crangon Fabr. (Decapoda: Macrura) Callilghora vomitoria L. (Diptera: Muscidae) C. allmani Kinahan (Decapoda: Macrura)

Apis melli/ica L. (Itymenoptera)

Astacus astacus L. (Decapoda: Macrura) Pagurus bernhardus L. (Decapoda: Anomura)

Portunus depurator L. (Decapoda: Brachyura) Several of the species were reared for us or brought to us from other places by colleagues (Drs. B. Schricker, Zool. Inst. Freie Univ. Berlin, G. Geisthard and Dipl. Biol. R. Sartorius, Schwabenheim B.R.D., Drs. 1). Meurling and S.-O. Nielsen, Zool. Inst., Lund, Mr. R. Odselius and U. Norling, Zool. Inst., Lund, Mr. A. Lundquist, Zoofysiol. Inst., Lund, Dep. of Molecular Cytogenetics, Lund). We are grateful to these colleagues who enabled us to extend our investigations to encompass so many species.

The histological method used is the fluorescence microscopy method specific for monoamines developed by Falck and Hillarp (Falck and Owman, 1965; Bj6rklund, Falck and Owman, 1972). Small modifications were introduced (Klemm, 1968b). The paraffin preferred is the one reported by Bj6rklund, Falck and Klemm (1970). To obtain good results in the different species, paraformaldehyde of varying humidity was used (e.g. 30% for Lepisma and 90% for Spodoptera). Also, with the best results, marine animals show a diffusion of the monoamines, which is not the case with most land-living or freshwater animals. The terms green and yellow fluorescence, which are used throughout this investigation, denote the fluorescence that by the established criteria could be shown to be caused by the presence of certain hiogenic monoamines in the neurons. (Falck and Owman, 1965; Corrodi and Jonsson, 1967). In some instances, a characterization of the monoamines was undertaken with the aid of microspeetrofluorimetry. The excitation and emission curves of the fluorophores were measured with a modified Leitz microspectrofluorimeter and the differentiation between the fluorophores of the primary catecholamines dopamine and noradrenaline was performed according to Bj6rklund, Ehinger and Falck (1968) and revised by Bj6rklund et al. (1970). To get a higher fluorescence yield of the weakly fluorescent indole-compound, acidification with hydrochloric acid was used (BjSrklund and Stenevi, 1970).

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in the Optic Ganglia of Crustaceans and Insects

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The brain of Schistocerca was also incubated with the monoamine oxidase inhibitor, nialamid (Niamid R, Pfizer) in a saline solution according to Mordue and Goldsworthy (1969) (0.1 mg/ml). With this treatment, the fluorescence of 5-hydroxytryptamine neurons could be markedly improved (Klemm and Axelsson MS). After treatment according to the Falck-Hillarp method including paraformaldehyde, ordinary histological methods were tried on the same sections, i.a. silver impregnation, secretory stains and general hematoxyline stains. The aim was to correlate the monoaminergic structures with the stratification obtained by ordinary histological technique. However, little correlation was achieved.

Observations General Remarks. I t could be noted here as a general observation, pertaining both to crustaceans and insects, t h a t the number of monoaminergic layers described below could in some instances be more because the optimum conditions are difficult to attain with the technique. I n order to ensure a uniform treatment in the following descriptions, it is, first, implicit t h a t the eye-stalk or eyes are sectioned horizontally; second, a common terminology for spatial indications is used. Distal means against the ommatidia; proximal, against the brain. Crustacean eye-stalks are thought of as carried in the long axis of the animal, which is why medial and lateral are natural usage. The sessile eyes of the Insecta are mostly thought of as being oriented perpendicular to the long axis of the insect; thus medial and lateral, in this case, are synonymous with anterior and posterior, respectively. Crustacea. There are four ganglia in the malacostraean Crustaeea generally situated in the eye-stalk. These are the lamina ganglionaris receiving the axons from the retinula cells, the medulla externa further proximal, followed by the medulla interna, and finally medulla termina]is. The two latter are shown to be detached parts of the brain (Elofsson, 1969; Elofsson and Dahl, 1970). Nonmalacostraean crustaceans have only three ganglia; they lack the equivalent of medulla interna. Monoaminergie neurons have been found in all these ganglia. The medulla terminalis has no specific pattern discernible in the distribution of the monoaminergic terminals. I t much resembles the protocerebral distribution, although with some distinct areas (Elofsson et al., 1966). The medulla externa and interna, on the other hand, have a distinct and conspicuous stratification of the monoaminergie terminals, which are specially dealt with in this investigation. The lamina ganglionaris only rarely contains monoaminergie fibres. Monoaminergie perikarya are found in the cell body layers belonging to each ganglion. They are of globuli and of ordinary ganglion cell type. Only the former type is found connected with the medullae externae and internae. The excitation and emission spectra of the green monoamine compound present were taken in the case of Astacus astacus and were typical for a cateeholamine fluorophore (Exmax 410 :Emmax 475 nm) (Caspersson, tIillarp, and I~itzgn, 1966). Microspectrofluorimetric differentiation of the primary eateeholamine eompund showed, after treatment with hydrochloric acid, a shift of the excitation max. from 410 to 370-380 nm which was not changed after prolonged exposure to gaseous hydrochloric acid. These reactions point towards a dopamine-indueed fluorophore (BjSrkinnd et al., 1968). No other species were examined mierospectrofluorimetrieally. All species described below show the same green fluorescence in the monoaminergie neurons.

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Fig. 1. Artemia salina. Frontal section through the eye-stalk. Ommatidia (ore). Arrow points at fluorescent fibres in the medulla. Scale 50 Fig. 2. Artemia salina. Section close to that of Fig. 1 showing at arrow a varicose fibre running between the medulla and the medulla terminalis. Scale 50 Fig. 3. Neomysis integer. Horizontal section through the eyestalk. Ommatidia (ore), Lamina ganglionaris (/), Medulla cxterna (wee), Medulla interna (mi). Arrows point at the fluorescent layers. Only the distal is seen clearly in the medulla interna. Microphotograph through the courtesy of Mr. R. Aramant. Scale 50~

Artemia salina. This species is t h e o n l y r e p r e s e n t a t i v e of t h e n o n - m a l a e o s t r a c a n crustaceans i n v e s t i g a t e d . These h a v e no m e d u l l a i n t e r n a a n d t h e m e d u l l a t e r m i nalis is a d j a c e n t to t h e brain. Monoaminergic neurons are f o u n d in t h e m e d u l l a e x t e r n a , a l t h o u g h t h e y are few a n d give a w e a k green fluorescence, which is dispersed in t h e whole m e d u l l a (Fig. 1). There are t h u s no d i s t i n c t layers to be found. Monoaminergic p e r i k a r y ~ of globuli t y p e are p r e s e n t in t h e celLlayer covering t h e m e d u l l a m e d i a l l y . Varicose fibres are f o u n d in t h e t r a c t r u n n i n g to

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the medulla terminalis (Fig. 2). The brain shows a rather strong green fluorescence in distinct areas indicating the presence of monoaminergic neurons distributed approximately as in Astacu8 (Elofsson et al., 1966).

Neomysis integer. (Fig. 27A). Through the courtesy of Mr. Robert Aramant, we are allowed to quote from his unpublished observations the situation in this species. The medulla externa has three layers: one thin in the distal, and one in the proximal margin; there is also one thick, strongly fluorescent layer situated slightly proximal of the distal layer (Fig. 3). The medulla interna has one thick, strongly fluorescent layer just proximal of the distal margin, and three thinner layers lining up behind. They all occupy only the distal part of the whole medulla. Leander ]abrieii (Fig. 27B). The medulla externa has four fluorescent layers. The distal is broad and reaches from the distal surface quite a distance into the neuropile. The strongest fluorescence comes from the proximal part of it. There is a faint suggestion t h a t it could be subdivided into two or three layers, in which case, one or two thinner layers are situated distal to the strongly fluorescent proximal part. Further proximally in the medulla, there is one layer in the middle of it, whereafter one thin follows, and finally one in the proximal margin (Fig. 4). The medulla interna has six layers in a regular pattern. I n the distal margin, there is a moderately broad layer, followed by a thin layer, and further proximally, a layer of the same thickness as the first. This pattern is repeated once more proceeding proximally in the medulla so t h a t the last thick layer is situated in the proximal margin (Fig. 5).

Spirontoearis lilljeborgii (Fig. 27C). The medulla externa has only one single catecholamine-containing layer situated a little distal of the middle. The medulla interna contains three layers. There is one in the distal margin and one in the proximal margin. Both are fairly thick. I n addition, there is a faint, thin layer distal of the middle of the medulla (Fig. 6). Pandalus borealis (Fig. 27D). The medulla externa has two fluorescent layers. The distal shows rather strong fluorescence and is situated a distance from the distal margin. The proximal is less strong and runs in the proximal margin. There is, in some instances, a suggestion of a weakly fluorescent band in between the two clearly seen layers (Fig. 7). The medulla interna has five equally strong and thick layers spaced at regular intervals, the fhost being situated in the distal margin and the fifth in the proximal margin (Fig. 8). Pandalus montagui (Fig. 27E). The medulla externa has four layers of catecholamine-containing terminals in this species, which is closely related to the previous. The distal layer, situated behind the distal surface, displays a clear fluorescence, whereas the others are more diffuse. The proximal layer runs in the proximal margin, and the closest distal layer is rather thin. The two middle layers could correspond to the weakly fluorescent band sometimes found in P. borealis, but the material was too scanty to allow a more definite statement. The medulla interna shows the same pattern as t h a t of Pandalus borealis. Once a weak fluorescence in the lateral margin of the lamina ganglionaris was seen. I t appeared as two layers.

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Figs. 4---6

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Crangon crangon (Fig. 27F). The medulla externa has two relatively thick layers of fluorescent fibres situated in its distal half. The distal of them runs somewhat behind the distal margin and the proximal approximately in the middle of the medulla (Fig. 9). The medulla interna has three layers: one in the distal surface and one in the proximal margin. The third layer is situated somewhat behind the distal layer. Crangon allmani (Fig. 27 G). This species also has few fluorescent layers in the medullae. The medulla externa has two thick layers: one in the distal and one in the proximal margin. The medulla interna has one single layer in the distal margin (Fig. 10). Astacus astacus (Fig. 27tt). This species has three distinct layers of fluorescent fibres parallel with the distal surface of the medulla externa (not two as previously stated, Elofsson et al., 1966). The distal layer is situated some distance from the front margin. I t is broad and shows a strong fluorescence. The two proximal layers are thinner and their content of varicose fibres is evident. The anterior of these two lies almost in the middle of the ganglion and is thicker than the posterior, which is close to the proximal surface (Fig. 13). The medulla interna is less clearly organized in layers with regard to the monoaminergic fibres. There are two thick, strongly fluorescent layers: one close to the distal surface and one approximately in the middle of the medulla. Between them, there is one broad, diffuse, and weakly fluorescent layer. The proximal part of the medulla interna is occupied by probably two thick and diffuse layers of fluorescent fibres (Fig. 14). On rare occasions, we have seen green fluorescent terminals in the lamina ganglionaris. These have been concentrated in the lateral margin and conformed with the cartridge arrangement of this optic neuropile (Fig. 12). Microspectrofluorimetric measurements were made on the distal layer of the medulla externa. They all showed the presence of dopamine. Pagurus bernhardus (Fig. 27I). The medulla externa has one strongly fluorescent layer situated just distal of the middle of the medulla (Fig. 11). The medulla interna has five or six layers. The material has not allowed a more definite statement. They are approximately of the same thickness and regularly spaced. The distal layer is situated close to the distal margin. The lamina ganglionaris has two layers of fluorescent fibres. Their varicose appearance is fairly distinct in this case. Portunus depurator (Fig. 27 J). The medulla externa has one layer in the distal surface, one in the middle of the medulla, and one in its proximal surface. The medulla interna has five equally thick and spaced layers of catecholaminecontaining terminals.

Fig. 4. Leander ]abrieeii. Horizontal section through part of the eye-stalk. Abbreviations as in Fig. 3. Arrows point at the fluorescent layers. Scale 50 Fig. 5. Leander/abrieii. Section as Fig. 4 but showing in addition the medulla interna. Arrows point at the six fluorescent layers. Scale 100 Fig. 6. Spirontocar,is lill]eborgii. Horizontal section showing the medulla externa and medulla interna. Arrows point at fluorescent layers. Scale 100

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Fig. 7. Pandalus borealis. Horizontal section through the medulla externa. Arrows indicate the fluorescent layers. Scale 100


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Fig. 9. Crangon crangon. Horizontal section through the eye-stalk. Abbreviations as Fig. 3 with the medulla terminalis added (mr). The layers in the medulla externa, the only ones clearly visible in this section. Scale 100 tz Fig. 10. Crangon allmanni. Horizontal section through the eye-stalk. Abbreviations as in Fig. 3. Fluorescent layers in the medullae externa and interna. Scale 100 ~z Fig. 11. Pagurus bernhardus. Horizontal section through the eye-stalk showing the medullae externa and interna. Arrow points a t the single fluorescent layer in the medulla externa. The n u m b e r of layers in the medulla interna is not entirely clear. Scale 100 Fig. 8. Pandalus borealis. Horizontal section through the medulla interna. Arrows indicate the five fluorescent layers. Scale 100 ~z 33 Z. Zellforsch., Bd. 133

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Figs. 12--14

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The lamina ganglionaris contains probably two layers, although they are difficult to define. A cartridge arrangement within the layers conforming with the neurommatidial cartridges can be discerned. Insecta. Insects have three optic ganglia: the lamina, the medulla, and the lobula. The terminology adopted here is that of Bullock and Horridge (1965). I t is recognized t h a t the different parts of the lobula complex in different orders are not homologized. For the purpose of this investigation, we use terms used by others or simple spatial indications. All these optic ganglia contain monoaminergic neurons, although to a varying extent and of different kinds in different species. The lamina, and especially the medulla, show a patterned appearance of the monoaminergie neurons similar to that in the Crustaeea. The lobula only exceptionally reveals a pattern. Monoaminergie perikarya of ordinary ganglion cell type exist belonging to all ganglia, whereas those of globuli-type arc preferably found connected with the lamina and medulla. The characterization of the monoamines present has been made in this investigation in the case of Schistocerca gregaria and previously for Anabolia nervosa (Klemm and Bj6rklund, 1971). I t was found t h a t the green fluorescent monoamines were noradrenaline and dopamine. In addition, some of the neurons of the optic ganglia in some insects contain 5-hydroxytryptamine. I t should be remarked here that, in the case of insects, the presence of a tracheal system in the optic ganglia interferes with the interpretation of the monoaminergie struetures. With the method used, the tracheae display a green to yellow fluorescence (the colour varies in different insect species) which could easily be mistaken for monoaminergie neurons. A careful examination with regard both to structure and to characterization of the monoamines must be undertaken to avoid mistakes (Fig. 26). Lepisma saccharina. This single representative of the apterygote insects has monoaminergie neurons in the optic ganglia. They are restricted to the medulla. Here appear one broad layer in the proximal part and one or two thin layers distally. I t is a green fluorescence which also applies to the perikarya that are found. These are situated ventrally on the lateral side of the medulla (Fig. 16). The fluorescence further proximal cannot be related with certainty to a lobula, because this is not clearly defined in this species. Aeschna viridis (Fig. 28A). The lamina is occupied by green fluorescent monoaminergie fibres. These appear especially in the larvae in a regular neurommatidial pattern (Fig. 15A). I t should be noted that, in larval Aesehna, the portion of the lamina called "embryonic a r e a " by Zawarzin (1914) does not show any fluorescence (Fig. 15). These larvae are also the only ones t h a t show green fluorescence

Fig. 12. Astacus astacus. Horizontal section through part of the lamina ganglionaris showing

the fluorescent fibres sometimes seen (arrow). Scale 50 ~z Fig. 13. Astacus astacus. Horizontal section through the medulla externa showing the fluorescent layers (arrows). Scale 100 ~z Fig. 14. Astacus astacus. Horizontal section through the medulla interna showing the fluorescent layers (arrows). Scale 100 33*

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f

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tf

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in the outer chiasma (Fig. 15A). I t has not been positively observed in adult animals. The medulla has at least four green fluorescent layers. The two thin proximal layers show very distinct fluorescent fibres. One is situated in the proximal margin and one close to the middle of the medulla. From these two layers in the ventral part of the medulla, a joined bundle of fibres continues to the ventro-eandal part of the brain (Fig. 15B). Further distally, there are two broad layers situated very close to each other, both approximately in the middle of the medulla. Their fluorescence is less distinct than the previous two. The distal one of these two layers contains fine yellow fluorescent fibres interwoven between the green ones. There is one more layer in the distal half of the medulla with yellow fluorescent fibres (Fig. 15). Proximally on the ventral side of the medulla, there is a group of green and yellow fluorescent perikarya. All the four parts of the lobula (Zawarzin, 1914) show green fluorescence. Evenly distributed yellow fluorescent fibres have also been observed. I n the large p a r t of the lobula, the green fluorescence appears as three proximo-distM thin layers situated dorsally. Ventrally, these layers disappear and unite to a broad tract, which in turn unites with the tract from the medulla, and runs to the brain (Fig. 15 C). Single green fluorescent perikarya are situated on the proximo-ventrM side of the lobula. Schistocerca gregaria (Fig. 28B). The optic ganglia of this species contain all three monoamines encountered in insects. The proximal surface of the lamina is occupied by a layer of monoaminergic fibres emitting a green fluorescence. They are found to contain the primary cateeholamine, dopamine. I n addition, the lamina contMns a yellow, quickly vanishing fluorescence which is referred to terminals possessing an indolamine (Fig. 18). These are arranged in cartridges, as are the neurommatidia. Monoaminergic perikarya associated with the lamina are found laterally and medially. The former belong to the group to be mentioned in connexion with the medulla and the latter are a few, at least four, large nerve cell bodies. All emit a green fluorescence. The monoaminergic fibres of the medulla appear in five distinct layers. There is one thick layer well behind the distal surface, followed b y a thinner, strongly fluorescent layer approximately in the middle of the medulla. The proximal portion of the medulla contains three thin equally spaced layers. These are connected with one another b y sparse, proximo-distal fibres. All five layers display a green fluorescence, and the one in the strongly fluorescent second layer has been microspectrofluorimetrically characterized as a noradrenaline-eompound. I n addition, the two distal layers contain fibres with an indolamine which appears yellow in this method (Fig. 19). There is quite a number of monoaminergic

Fig. 15. Aeschna viridis, last instar larva. Horizontal section through the optic lobe. Lamina (la), embryonic portion of the lamina (e la), medulla (~n), lobula (lo), first chiasma (ch). Large arrows point at green fluorescent layers and small arrows at yellow layers in the medulla. Inset A shows the cartridge arrangement of the fluorescent fibres of the lamina. Inset B shows the tract to the two proximal layers in the medulla (arrow). Inset C shows the tract to the lobula (arrow). Scale 100 ~z

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Fig. 16. Lepisma saccharina. Frontal section through the optic lobe. Abbreviations as Fig. f5 a n d green fluorescent perikaryon (io). Large arrows point at the fluorescent layers in the medulla. Scale 50 ix Fig. 17. Schistocerca gregaria. Section through the medulla (preparation treated with nialamid). Arrow points a t the yellow fluorescent tract. The t r a c t abuts a green fluorescent portion of the medulla. Scale 100 ix Fig. 18. Schistocerca gregaria. Horizontal section through the optic lobe. Abbreviations as Fig. 15 a n d 16. Large arrows point at the green fluorescent layers in the lamina and the medulla (the two distal layers) a n d small arrow at the yellow fluorescent fibres in the lamina. The perikarya belong to the lateral group on the distal side of the medulla. Scale 100 fz


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perikarya associated with the medulla. On the lateral and medial margins of the m e d u l l a - - o n the distal side--there are cell aggregations containing m a n y perikarya which emit green fluorescence (Fig. 18). The lateral of these cell groups also contain perikarya belonging to the lamina, as mentioned above. The cell bodies in the medial group are less spaciously arranged than in the lateral. Ventrally, along the distal rim of the medulla, the mentioned aggregations of perikarya are connected with one another. This cell bridge contains a number of green fluorescent perikarya of globulitype. Further proximally, on the lateral and medial sides of the medulla, there are also monoaminergie perikarya. Laterally, there is one group with large green fluorescent perikarya. Medially, there are a few large, green ones. Dorsal of the latter, there is one group with at least 22 large yellow fluorescent perikarya. The latter have been analysed mierospeetrofluorimetrieally and contain 5-hydroxytryptamine. Most of their axons run to the medulla, but a few go to the lobula complex. The lobula complex eontMns mainly indolamine-eontaining fibres. Centrally, in the dorsM p a r t of the lobula, the indolaminergie fibres are arranged in three thick, approximately proximo-distM layers. This layering disappears as it proceeds ventrally. Finally, in the ventral part of the lobula, there are only green fibres coming from the lobula and continuing as a tract to the brain. There is no difference in the pattern of monoaminergie neurons of the optic ganglia between adult and larval Schistocerca. The latter were investigated from the first and second larval instar. I n the experiments with niMamid, there were no essential changes in the morphological pattern from that described above. However, the enhanced fluorescence allowed some observations on the eonnexions between perikarya and terminals which, with routine preparations, are invisible. Thus it was found t h a t monoaminergie axons from the distal group of perikarya situated on the medial side of the medulla in fact run to the medulla. Further, the 5-hydroxytryptamine containing perikarya on the lateral surface of the medulla give rise to two tracts running on the proximal side of the medulla and continuing along the medial and lateral sides to enter the two distal layers found to contain yellow fluorescent fibres. The latter tracts also contain green fibres at the height of the short sides of the medulla (Fig. 17, 19). Acheta domesticus (Fig. 28 C). Weakly yellow fluorescent fibres were sometimes found in the lamina in the distal margin indicating the presence of an indolamine. The medulla has at least six layers of green fluorescent terminals. One layer is situated in the distal margin. The next, proceeding proximally, is moderately broad and weakly fluorescent. The following layer is broad and strongly fluorescent and is situated approximately in the middle of the medulla. I t is irregular in outline and could be composed of at least three interconnected layers. The three following layers, of which the last is situated in the proximal margin, are moderately thick. The three proximal and tile three distal layers have constantly been found to diverge medially, and all layers are connected b y proximo-distal fibres (Fig. 20). There has also been found a non-fluorescent small portion of the medulla dorsally (Fig. 21). So far, green perikarya of globuli type belonging to the medulla have been found ventrally and medially on the distal margin of the medulla. There are two groups of yellow fluorescent perikarya: one situated dorso-medial of the

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Fig. 19


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Fig. 20. Acheta domesticus. Horizontal section through the optic lobe. Abbreviations as in Fig. 15. Large arrows point at the green fluorescent layers in the medulla. The second and third layers almost coalesce in this section. Scale 50 Fig. 21. Acheta domesticus. Sagittal section through the optic lobe showing the non-fluorescent portion of the medulla (arrow). Abbreviations as in Fig. 15. Scale 100 Fig. 22. Limnephilidae. Horizontal section through the optic lobe. Abbreviations as in Fig. 15. Large arrows point at the three layers in the medulla. Note the fluorescence in the lobula. Scale 50

Fig. 19. Schistocerca gregaria. Horizontal section through the optic lobe. Abbreviations as in Fig. 15. Large arrows point at the green fluorescent layers and small arrows at the yellow ones. Only one layer is seen in the lobula, tr green portion of the tract shown in Fig. 17. Green fluorescent perikarya belong to the medulla. Scale 50

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Fig. 23. Spodoptera littorali¢. Horizontal section through the medulla and the lobula. Large arrows point at the two green layers of the medulla. Scale 50 Fig. 24. Spodoptera Iittoralis. Section through the lamina. Small arrow points at the strongly yellow fluorescent proximal layer. Scale 50 Fig. 25. Calliphora vomitoria. Frontal section through the medulla and lobula. Large arrows point at the green fluorescent layers of the medulla. Scale 100 Fig. 26. Caltiphora vomitoria. Horizontal section through the optic lobe. Mote tile fluorescence in both parts of the ]obula. The tracheal system is conspicuous in this section (end of large T: s). Scale 100 tz


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medulla, continuing towards the lobula. The processes can be followed to all three optic ganglia. Despite this, no yellow fluorescence has been discovered in the medulla which must be due to the masking effect of the green fluorescence. The other group of yellow fluorescent neurons is situated ventrally of the medulla and shows much weaker fluorescence. The lobula contains diffusely distributed yellow fibres, but these can only be seen under favourable conditions. The proximal part of the lobula contains green fluorescent fibres which continue as a tract into the brain. Of the perikarya surrounding the lobula, there are large green ones both distally and proximally, and also on the dorsal side of it. There is also a dorsal group of yellow fluorescent perikarya which could belong to the dorso-medial group of the medulla. Limnephilidae (Fig. 28 D). We had at our disposal a trichopteran species of the family Limnephflidae (Integripalpia) which had not been closely examied. Usually, fluorescence is lacking in the lamina. However, in very few eases, there appeared a weak yellow-white fluorescence that indicated the possible presence of neurons containing an indolamine, which resembles the condition in the lamina of the Lepidoptera. The medulla has three green fluorescent layers: One situated in the distal margin. The next occupies almost half the medulla; it has a more distinctly fluorescent band in the middle of the layer (at the same time, situated in the middle of the medulla). Further proximally, there is one broad layer (Fig. 22). This often appears as two lines in the proximal margin with a typical varicose appearance. As in the Lepidoptera, the fluorescent layers occupy the whole medulla medially, whereas they disappear ventrally in the lateral half. The lobula has green monoaminergic fibres marginally. The monoaminergic pattern in the optic ganglia resembles those found earlier in other representatives of the Trichoptera (Molannct angustata Curt., Limnephilus politus Mc Lach., Anabolia nervosa Curt., Klemm, 1968b). Moreover, the distinctly fluorescent layer in the middle of the medulla of Anabolia nervosct was found to contain noradrenaline and dopamine (Klemm and Bj6rklund, 1971). Spodoptera littoralis (Fig. 28 E). This lepidopteran has monoaminergic neurons in all three optic ganglia. The lamina has a yellow-white fluorescence which indicates the presence of an indolamine in the neurons. There is a thin, strongly fluorescent layer in the proximal margin. The rest of the lamina in front of this is occupied by a thick layer with diffusely distributed fibres (Fig. 24). The medulla has probably two broad, green fluorescent layers which might be separated by a non-fluorescent part in the middle of the medulla. The distal margin of the proximal layer shows a stronger fluorescence. Interrupted streaks of fluorescence in the proximal margin could indicate a third thin layer (Fig. 23). There is a group of green fluorescent perikarya of globuli-type dorsally between the medulla and the lobula. The lobula has its medial half filled with green fluorescent varicose fibres. They are also found along the margins of the lobula plate. Calliphora vomitora (Fig. 28F). The lamina has no monoaminergie neurons. The medulla has three layers displaying green fluorescence. There is one strongly fluorescent layer in the middle of the medulla and two weakly fluorescent distally (Fig. 25).

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f~. Elofsson and N. Klemm: Monoamines in the Optic Ganglia

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Fig. 28 A--F. Schematic drawings of the optic ganglia of A, Aeschna viridis; B, Schistoeerea gregaria; C, Aeheta domestieus; D, Limnephilid; E, Spodoptera littoralis; F, Calliphora vomitoria. The optic ganglia are from the left; lamina, medulla and lobula. Green fluorescent layers arc indicated by coarse dots and yellow ones by fine dots. Large green fluorescent perikarya are represented by large filled circles and small (globulitype) by small filled circles. Yellow perikarya are shown as open circles. The proximo-ventral perikarya in Aesehna (A) are displaced medially to be seen

The lobula a n d lobula plate have green fluorescent fibres centrally. There is also a slight yellow fluorescence i n the lobula proper (Fig. 26). A p i s melli/ica. Several specimens of this species were investigated, b u t so far, no m o n o a m i n e r g i c n e u r o n s whatsoever were f o u n d i n the optic ganglia.

Fig. 27A--J. Schematic drawings of the optic ganglia of A, Neomy8is integer; B, Leander

/abricii; C, Spirontocaris lill]eborgii; D, Pandalus borealis; E, P. montagui; F, Crangon crangon; G, C. allmani; H, Astaous asta~us; I, Pagurus bernhardus; J, Portunus depurator. The optic ganglia are from the left; lamina ganglionaris, medulla externa, medulla interna and medulla terminalis. Green fluorescent layers are indicated by cross-hatching

496


Discussion The results concerning both crustaceans and insects can be concluded thus: 1. Monoaminergic neurons exist in the visual pathway, 2. these neurons appear very often in strata in the optic ganglia and form a pattern characteristic of each species. At present, it is difficult to formulate more conclusions of general application; this applies not only to conclusions concerning crustaceans and insects, but also to conclusions within each class. There is a difference between crustaceans and insects : catecholamine (of which dopamine has been demonstrated) is probably the only monoamine present in the crustaceans, whereas the insects have both the primary catecholamines, dopamine and noradrenaline, and the indolamine, 5-hydroxytryptamine. Concerning the insects, all these amines are not always present simultaneously in all species. The conspicuous patterned appearance of the monoamines invite a search for taxonomic and phylogenetic relations. The closely related species Crangon crangon and Crangon allmanni of the Crustacea show different patterns indicating t h a t the monoaminergic neurons of the optic ganglia could be differently arranged even within small taxonomic units. On the other hand, different trichopterid species of the Insecta are similar in this respect; also the orders Trichoptera and Lcpidoptera show far-reaching similarities. These variations call for a cautious attitude to phylogenetic speculations based on the pattern of these neurons. The variation in the pattern of monoaminergic neurons displayed in the present material is shown schematically in Figs. 27 and 28. The pattern of monoaminergic neurons could also have a simple correlation with the mode of life of the species. Supposing an improved visual function with m a n y layers of monoaminergic neurons, shallow water animals t h a t rely on visual stimuli, as in the case of crustaceans or similar eye-depending insects, should have m a n y of them. Other combinations are, of course, possible. But no such correlation between milieu or biology, when known, and content of monoaminergic neurons in the optic ganglia has been found in the present material. I t suffices to mention that the insects Apis and Aeschna are dependent on their eyes and have extremely different content of monoamines. Lack of easily resolved connexions, at the present state of knowledge, between the content and pattern of monoaminergic neurons in the optic ganglia and externally observable facts suggest a function of these neurons that has to be sought for in the physiological processes underlying vision. A comparison between the optic ganglia of one eye provides few criteria. In the Crustaeea, the number of layers with monoaminergic neurons usually increases in the ganglia proceeding proximally in the eyestalk from the lamina ganglionaris to the medulla interna. In the Insecta, a similar comparison does not make sense in the same way, as in most cases only the medulla shows the layers more clearly. I t is naturally desirable to identify the monoaminergic neurons in the optic ganglia. There are several good investigations on the neuronal topography and the characterization of neurons of the optic ganglia arrived at with the aid of metal impregnations, especially regarding the insects (Cajal and Sanchez, 1915, 1921 ; Zawarzin, 1914, 1924; Pflugfelder, 1937; Strausfcld and Blest, 1970; Strausfeld, 1970). The terminology and views adopted here are those of Strausfeld, who also


497

reviews the older investigations. I t is not intended, nor is it possible, to make a direct comparison between the neuronal studies and the monoaminergie distriburich. By way of an approach between the results of the two techniques, however, some hint at further research could emerge. Judging from the position of the monoaminergic perikarya, the amines could be contained, where they occur, in tangential and amacrine cells; two of the three overriding classes of neurons present in the optic ganglia. Provided, of course, t h a t monoaminergic neurons are at all visible with metal impregnation techniques. The branching of all neurons occur in distinct strata, which are seen in non-selective silver impregnations and are shown to be eight in the medulla of the Lepidoptera and Diptera. The layered appearance of the monoaminergic neurons suggests the possibility of attributing these layers to those obtained with silver techniques. I t must be remarked that the highly variable pattern shown by the monoaminergie neurons within different orders and even species calls for studies of one and the same species with all techniques, as no general criteria can be disclosed at present. Thus a two-step approach to reveal the identity of the monoaminergie neurons would involve, first, a comparison between monoaminergic distribution and medullary strata; and second, a comparison between medullary strata and neuronal topography. The situation in the Crustaeea is perhaps somewhat simpler concerning the monoaminergie neurons, but suffers the drawback of few penetrating neuronal studies. I t is impossible in this connexion to ignore the interesting findings of monoaminergic (adrenergie) neurons in the vertebrate retina. Naturally, this is mentioned as a morphological and perhaps a functional analogy without any further implications. Recent investigations with the fluorescence microscopical method of Falck and Hillarp have revealed the presence of up to three layers of monoaminergie terminals in the inner plexiform layer (Hgggendal and Malmfors, 1965; Ehinger, 1966; Ehinger and Falek, 1969) of several vertebrate classes (fishes, amphibians, reptiles, birds, and mammals). Most of these neurons are dopaminergie. I t should be noted t h a t the number of layers varies in different species (e.g. in the monkeys). However, the position of the layers within the inner plexiform layer seems to be relatively constant, and the variation appears as a disappearance of one or two of the three possible sites. This is in contrast to crustaceans and insects, where also the position within the optic ganglia varies. The function of the vertebrate adrenergic neurons in the retina remains unsolved, and the situation regarding the crustaceans and the insects is equally unclear. Until more morphological and experimental data are available, speculations about the function are unwarranted.

References Bj6rklund, 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., Falck, B., Klemm, N. : Microspectrofluorimetrie and chemical investigation of catecholamine-containing structures in the thoracic ganglia of Trichopter~. J. Insect. Physiol. 16, 1147-1154 (1970). Bj6rklund, A., Falck, B., Owman, Ch.: Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterization of biogenic amines. In: ~ethods of investigative and diagnostive endocrinology (ed. I. Kopin). Amsterdam: North Holland Publ. Co. 1972.

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BjSrklund, A., Stenevi, U. : Acid catalysis of the formaldehyde condensation reaction for sensitive histochemical demonstration of tryptamines and 3-methoxylatcd phenylethylamines. 1. Model experiments. J. ttistochem. Cytochem. 18, 794-802 (1970). Bullock, T. H., Horridge, G. A. : Structure and function in the nervous system of invertebrates II. San Francisco-London: W. H. Freeman & Co. 1965. Cajal, S. 1~., S£nchez, D. : Contribuci6n al conocimiento de los centros nerviosos de los insectos. Trab. Lab. Invest. biol. Univ. Madr. 13, 1-164 (1915). Cajal, S.R., S£nchez, D.: Sobre la structura de los centros opticos de los insectos. Rec. Chilena Hist. Nat. 25, 1-18 (1921). Caspersson, R., Hillarp, N.-A., Ritz6n, M. : Fluorescence microspectrophotometry of cellular catecholamines and 5-hydroxytryptamine. Exp. Cell Res. 42, 415-428 (1966). Cech, S., Knoz, J. : ~onoamine-containing structures in the nerve cord of some representatives of Diptera. Experientia (Basel) 26, 1125-1126 (1970). Cooke, I.M., Berlind, A., Goldstone, M. W.: Histochemical localization of monoamines in axons and terminals in a neurohemal organ: tests for interaction with peptide neurosecretion. J. gen. Physiol. 55, 141 (1970). Cooke, I.M., Goldstone, M . W . : Fluorescence localization of monoamines in crab neurosecretory structures. J. exp. Biol. 53, 651-668 (1970). Corrodi, H., Jonsson, G.: The formaldehyde fluorescence method for the histochemical demonstration of biogenic monoamines. J. Histochem. Cytochem. 15, 65-78 (1967). Ehinger, B. : Adrenergic retinal neurons. Z. Zellforsch. 71, 146-152 (1966). Ehinger, B., Falck, B. : Adrenergie retinal neurons of some new world monkeys. Z. Zellforsch. 100, 364-375 (1969). Elofsson, R.: The development of the compound eyes of Penaeus duorarum (Crustacea: Decapoda) with remarks on the nervous system. Z. Zellforsch. 97, 323-350 (1969). Elofsson, 1~., Dahl, E. : The optic neuropiles and chiasmata of Crustacea. Z. Zellforsch. 107, 343~60 (1970). Elofsson, R., Kauri, T., Nielsen, S.-O., StrSmberg, J.-O.: Localization of monoaminergic neurons in the central nervous system of Astacus astacus (Crustacea). Z. Zellforsch. 74, 464-473 (1966). Elofsson, R., Kauri, T., Nielsen, S.-O., Str6mberg, J.-O.: Catecholamine-containing nerve fibres in the hindgut of the crayfish Aastacus astacus L. (Crustacea: Decapoda). Experientia (Basel) 24, 1159-1160 (1968). Falck, B., Owman, Ch. : A detailed methodological description of the fluorescence method for the cellular demonstration of biogenic monoamines. Acta univ. Lund Sect. I I No 7, 1-23 (1965). Frontali, N. : Histochemieal localization of catecholamines in the brain of normal and drugtreated cockroaches. J. Insect. Physiol. 14, 881-886 (1968). Frontali, N., Norberg, K.-A.: Catecholamine containing neurones in the cockroach brain. Acta physiol, scand. 66, 243-244 (1966). Goldstone, M. W., Cooke, I. M. : Histochemical localization of monoamines in the crab central nervous system. Z. Zellforsch. 116, 7-19 (1971). Hi~ggendal, J., Malmfors, T. : Identification and cellular localization of the catecholamines in the retina and the chorioid of the rabbit. Acta physiol, scand. 64, 58-66 (1965). Klemm, N. : Monoaminerge Zellelemente im stomatogastrischen Nervensystem der Trichopteren (Insecta). Z. Naturforsch. 23, 1279-1280 (19683). Klemm, N. : l~onoaminhaltige Strukturen im Zentralnervensystem der Trichoptera (Insecta). Tell I. Z. Zellforsch. 92, 487-502 (1968b). Klemm, N. : Monoaminhaltige Strukturen im Zentralnervensystem der Trichoptera (Insecta). T e i l l I . Z. Zellforsch. 117, 537-558 (19713). Klemm, N. : 1VIonoaminhaltige Zellelemente im stomatogastrischen Nervensystcm und in den corpora cardiaca yon Schistocerca gregaria Forsk. (Insecta, Orthoptera). Z. Natufforsch. 26, 1085-1086 (1971b). Klemm, N. : Monoamine-containing nervous fibres in fore gut and salivary gland of the desert locust, Schistoeerca gregaria Forsk. (Orthoptera, Acrididae) Comp. Biochem. Physiol. in press.


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Klemm, N., Axelsson, S. : Determination of dopaminc, noradrenaline and 5-hydroxytryptamine in the brain of the desert locust, Schistocerca gregaria Forsk. (Insecta, Orthoptera). MS. Klemm, N., Bj6rklund, A.: Identification of dopamine and noradrenaline in nervous structures of the insect brain. Brain l~es. 26, 459464 (1971). Mordue, W., Goldsworthy, G. J. : The physiological effects of corpus cardiacum extracts in locusts. Gen. comp. Endocr. 12, 360-369 (1969). Osborne, N. N., Dando, M. R.: Monoamines in the stomatogastric ganglion of the lobster, Homarus vulgaris. Comp. Biochem. Physiol. 82, 327-331 (1970). Pflugfelder, 0. : Vergleichend-anatomische, experimentclle und embryologische Untersuchungen fiber das Nervensystem und die Sinnesorgane der Rhyncoten. Zoologica (Stu$tg.) 84, 1-102 (1937). Plotnikova, S. I. : The structure of the sympathetic nervous system of insects. In: Symposium on neurobiology of Invertebrates 1967, p. 59-68. Budapest: Publ. House Hungarian Acad. Sci. 1967. Strausfeld, N. J. : Golgi studies on insects II. The optic lobes of Diptera. Phil. Trans. B 258, 135-223 (1970). Strausfeld, N. J., Blest, A. D. : Golgi studies on insects I. The optic lobes of Lepidoptera. Phil. Trans. B 258, 81-134 (1970). Zawarzin, A . A . : Histologische Studien fiber Insekten IV. Die optischen Ganglien der Aeschnalarven. Z. wiss. Zool. 108, 175-257 (1914). Zawarzin, A. A. : Einige Bemerkungen fiber den Bau der optischen Zentren. Anat. Anz. 59, 551-559 (1924). Dr. Nikolai Klemm Institut ffir Angewandte Zoologie D-8700 Wiirzburg Bundesrepublik Deutschland

34 Z. Zellforsch., Bd. 133

Dr. Rolf Elofsson Zoological Institute S-22362 Lund Sweden