Identification of dopamine and noradrenaline in nervous structures of the insect brain

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taentification of dopamine and noradrenaline in nervous structures of the insect brain I n the m a m m a l i a n n e r v o u s system, the biogenic m o n o a m i n e s d o p a m i n e , n o r a d r e n a l i n e , a n d 5 - h y d r o x y t r y p t a m i n e occur i n t r a n e u r o n a l l y . These substances are f o u n d in specific n e u r o n a l systems a n d are a c c u m u l a t e d in the p e r i k a r y a and especially in the a x o n a l terminals, as d e m o n s t r a t e d with the fluorescence m i c r o s c o p i c technique o f F a l c k a n d H i l l a r p (see refs. 13 a n d 21), a n d there is g o o d evidence t h a t at least the catecholamines function as n e u r o t r a n s m i t t e r s in vertebrates (for discussion, see refs. 12 a n d 20). T h e catecholamines a n d 5 - h y d r o x y t r y p t a m i n e also occur in i n v e r t e b r a t e n e r v o u s systems o f widely v a r y i n g degrees o f d e v e l o p m e n t 1°,11,25,26,3° (for f u r t h e r references, see ref. 7) where they are m a i n l y localized in a x o n terminals a n d cell bodies o f n e u r o n s s,14,eT. T h a t these m o n o a m i n e s thus m a y act as transmitters also in the n e r v o u s systems in m a n y p h y l a o f invertebrates is s u p p o r t e d by experimerttal evidence (see review by Tauc29). A m o n g the invertebrates, the nervous system o f insects is characterized by a c o n c e n t r a t i o n o f the nervous elements into well-defined structures, i.e. ganglia, thus representing a high s t r u c t u r a l d e v e l o p m e n t . C a t e c h o l a m i n e s have been d e m o n s t r a t e d in m a n y a d u l t insects: b o t h d o p a m i n e a n d n o r a d r e n a l i n e have been isolated f r o m whole animals 26 (Apis mellifica L.), f r o m t h o r a x 4 (Trichoptera) a n d f r o m ganglion G1Tr

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Fig. 1. Schematic representation of catecholamine-containing structures in the cerebral ganglion of Trichoptera. The structures are drawn into one frontal plane. Stippled areas indicate where catecholamine-containing varicose fibres are found. The densely stippled areas have a dense supply of catecholamine fibres, whereas the widely stippled areas have only a sparse one. The catecholaminecontaining cell bodies are indicated as filled circles, a = a-lobe; fl = fl-lobe; C.c. = corpus centrale; C.v. = corpus ventrale; D = deutocerebrum; La = lamina; Lo lobula; M = medulla (m = catecholamine-containing middle line); N = nodulus; P -= pedunculus; S.c. = stratus caudalis; T.v. = tractus ventralis. GI-GIV denote catecholamine-containing cell groups. Data compiled from Klemm 24.

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Fig. 2. A, Excitation (left) a n d e m i s s i o n (solid curve right) characteristic of the d o p a m i n e fluorophore, recorded f r o m the fl-lobe. , After f o r m a l d e h y d e t r e a t m e n t only; .... After s u b s e q u e n t exposure to HC1 for a b o u t 10 rain. - - - -, After an additional exposure to HC1 for 18 r a i n . B, Excitation (left) a n d e m i s s i o n (solid curve right) characteristic for the n o r a d r e n a l i n e fluorophore, recorded f r o m the stratus caudalis. - - , After f o r m a l d e h y d e t r e a t m e n t only; . . . . , After a s u b s e q u e n t exposure to HCI for 27 rain. All spectra are corrected i n s t r u m e n t a l values a n d expressed as relative q u a n t a versus wavelength. Brain Research, 26 (1971) 459-464

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Fig. 3. Sagittal section of the cerebral ganglion. Dopamine-containing varicose fibres in a longitudinal arrangement throughout the a-lobe (a). Catecholamine fluorescence is also seen in the neuropile (n). a autoftuorescent pigment granules in the ganglion cell layer; c -- cuticle; f = fat body; nl = neurilemma. Fig. 4. Horizontal section of the cerebral ganglion showing dopamine fluorescence in the neuropile of the central body (C.c.), and dopamine and noradrenaline fluorescence in the fibres of the stratus caudalis (S.c.). For further abbreviations, see Fig. 3. cerebrale and ganglion suboesophageale 16 (Periplaneta americana). Furthermore, with the histochemical fluorescence technique, catecholamines have been found to be localized in different nervous structures within the central nervous system 4,15,17,23,27. Since thus both dopamine and noradrenaline have been isolated from insect tissues, it was thought of interest to investigate whether both these catecholamines occur intraneuronally, and whether they occur in separate neuronal structures. In a previous study 4, using microspectrofluorometric analysis, we found that dopamine was localized in perikarya and terminals in the thoracic ganglia of the Caddis fly (Trichoptera). In the present study, the microspectrofluorometric analysis - - in combination with chemical determinations - - was also carried out on the more complex structural arrangement found in the ganglion cerebrale of this insect. Adult male and female Trichoptera (Anabolia nervosa Curt.) were used. For the histochemistry, the heads were cut off, quenched in liquid propane-propylene cooled by liquid nitrogen, freeze-dried, treated with formaldehyde gas and processed according to the Falck-Hillarp method la. The sections were analysed in the fluorescence microscope and in a modified Leitz fluorescence microspectrograph a, as previously described 4. Specimens not treated with formaldehyde were used as controls for the specificity of the fluorescence studied. For biochemicalfluorometric analysis ofmonoamines, the heads of about 330 animals were pooled in 3 samples: two samples of about 110 heads each were used for determination of dopamine, noradrenaline, and adrenaline according to Bertler et al. 2 and Hfiggendal ag, and about 110 heads were used for the determination of 5-hydroxytryptamine according to Bertler 1.

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In the biochemical determinations, 3.52 and 3.26 /zg of dopamine/g of wet tissue and 0.32 and 0.29 #g of noradrenaline/g were found in the extracts of whole heads, but no adrenaline or 5-hydroxytryptamine could be detected. In the fluorescence microscope, specific fluorescence with the spectral characteristics of the catecholamine fluorophores was observed in cell bodies and in areas of nerve terminals in various regions of the cerebral ganglion. These catecholamine structures have been described in detail elsewhere22, 24 and are shown diagrammatically in Fig. 1. The fluorescent structures of high fluorescence intensity were analysed microspectrofluorometrically for dopamine and noradrenaline. Spectral characteristics of the dopamine fluorophore (Fig. 2A) were recorded from cell bodies situated in Group II (GII in Fig. 1), from terminals located in the a- and {/-lobes (a and fl, Fig. 3), in both parts of the central body (C.c., Fig. 4), in the stratus caudalis (S.c., Fig. 4; see ref. 24), and in the middle layer of the optical medulla (m). The noradrenaline type of spectrum (Fig. 2B) was recorded from fluorescent terminals in the stratus caudalis and in the middle layer of the optical medulla. Although dopamine was the predominating neuronal catecholamine in Trichoptera, noradrenaline also occurred intraneuronally. The noradrenaline terminals were intermingled with dopamine terminals, and no isolated noradrenaline structures could be found. Although the microspectrofluorometric recordings demonstrated a specific localization of noradrenaline and dopamine to different terminals within the stratus caudalis and the optical medulla, light microscopy does not allow any conclusion concerning the arrangement of the noradrenaline and the dopamine terminals within these structures. It was not possible to relate the catecholamine-containing perikarya with the catecholamine terminal systems, since no direct continuity was observed between the perikarya and the terminals in the brain. Also, the localization of the cell bodies of the noradrenaline neurons is not known since only dopaminecontaining perikarya were identified in the microspectrograph. It should be pointed out, however, that many perikarya in the brain had such a low fluorescence intensity that they could not be analysed in the microspectrograph. Sekeris and Karlsson 2s have suggested that the high concentration of dopamine found in whole larvae of Tenebrio molitor 25 is due to the formation of dopamine in the cuticle during the sclerotization process. However, this study has demonstrated that dopamine has a wide intraneuronal distribution in the brain and that it occurs to a greater extent than noradrenaline. It is also notable that we found a roughly similar ratio between the concentrations of dopamine and noradrenaline in the whole head of Trichoptera as Frontali and Hfiggenda116 found in the brain of the cockroach. Thus it seems reasonable that the major portion of the dopamine in the head of the adult is localized in neurones. No 5-hydroxytryptamine was detected either chemically in the head or histochemically in the brain of Trichoptera. However, it is possible that 5-hydroxytryptamine occurs in the nervous system of many insects 6,9,1°,1s,~°, and perikarya containing a specific yellowish fluorescence have been observed in the stomatogastric nervous system of insectsS, 22. Thus these structures could store 5-hydroxytryptamine, although no reliable evidence of this has yet been obtained. Brain Research, 26 (1971) 459-464

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Conclusions. By means o f chemical a n d histochemical analytical techniques, b o t h d o p a m i n e a n d n o r a d r e n a l i n e have been f o u n d in the b r a i n o f Trichoptera, having a n int r a n e u r o n a l localization. Both catecholamines are stored in nerve terminals, a n d the occurrence of these two types o f t e r m i n a l in various structures o f the b r a i n has been studied. Thus these two putative t r a n s m i t t e r substances have an i n t r a n e u r o n a l storage in the highly developed a n d complex insect n e r v o u s system similar to that f o u n d in m a n y species of other phyla. This work was s u p p o r t e d by grants f r o m the Swedish Medical Research C o u n c i l (No. B70-14X-56-06 a n d B70-14X-712-05). Institute of Anatomy and Histology, Department of Histology, University of Lurid, Lurid (Sweden)

NIKOLAI KLEMM ANDERS BJORKLUND

1 BERTLER,ilk., Effect of reserpine on the storage of catecholamines in brain and other tissues, Acta physiol, scand., 51 (1961) 75-83. 2 BERTEER,ilk., CARLSSON,A., ROSENGREN,E., AND WALDECK,B., A method for the fluorimetric determination of adrenaline, noradrenaline and dopamine in tissues, Kungl. Fysiogr. Siillsk. Lund FOrh., 28 (1958) 121-123. 3 BJ/3RKLUND, A., EHINGER, B., AND FAECK, B., A method for differentiating dopamine from noradrenaline in tissue sections by microspectrofluorimetry, J. Histochem. Cytochem., 16 (1968) 262-270. 4 BJORKEUND,A., FAECK,B., ANDKEEMM,N., Microspectrofluorimetric and chemical investigation of catecholamine-containingstructures in the thoracic ganglia of Trichoptera (Insecta), J. Insect Physiol., 16 (1970) 1147-1154. 5 CffANUSSOT,M. B., DANDO,J., MOUEINS,M., ET LAVERACK,M. S., Mise en dvidence d'une amine biog6ne dans le syst~me nerveux stomatogastrique des Insectes: I~tude histoch6mique et ultrastructurale, C. R. Acad. Sci. (Paris), 268 (1969) 2101-2104. 6 COLHOUN,E. H., The physiological significance of acetylcholine in insects and observations upon other pharmacologically active substances, Advanc. Insect Physiol., 1 (1963) 1-47. 7 COTTRELL,G. A., AND LAVERACK,M. S., Invertebrate pharmacology, Ann. Rev. Pharmaeol., 8 (1968) 273-299. 8 DANE, E., FALCK, B., VON MECKLENBURG,C., MYHRBERG,H., AND ROSENGREN,E., Neuronal localization of dopamine and 5-hydroxytryptamine in some mollusca, Z. Zellforsch., 71 (1966) 489-498. 9 DAVEY,K. G., The control of visceral muscles in insects, Advanc. Insect Physiol., 2 (1964) 219-245. 10 ERSPAMER,V., Occurrence of indolealkylaminesin nature. In Handbuch der eaperimentellen Pharmakologie, Springer, Berlin, 1966, p. 132. 11 EUEER,U. S. VON, Occurrence of catecholamines in acrania and invertebrates, Nature (Lond.), 190 (1961) 170-171. 12 EULER,U. S. VON,Adrenergic neuroeffector transmission. In G. H. BOURNE(Ed.), The Structure and Function of Nervous Tissue, VoL 2, Academic Press, New York, 1969, p. 724. 13 FAECK,B., AND OWMAN,CH., A detailed methodological description of the fluorescence method for the cellular demonstration of biogenic monoamines, Acta Univ. Lund, II, 7 (1965) 1-23. 14 FALCK,B., AND OWMAN,CH., Histochemistry of monoaminergic mechanisms in peripheral neurons. In U. S. YON EUEER, S. ROSELEAND B. UVN.~S(Eds.), Mechanisms of Release of Biogenic Amines, Pergamon Press, Oxford, 1966, pp. 59-72. 15 FRONTAEI,N., Histochemical localization of catecholamines in the brain of normal and drugtreated cockroach, J. Insect PhysioL, 14 (1968) 881-886. 16 FRONTALI,N., AND HAGGENDAE,J., Noradrenaline and dopamine content in the brain of the cockroach Periplaneta americana, Brain Research, 14 (1969) 540-542. 17 FRONTALI,N., AND NORBERG,K.-A., Catecholamine containing neurons in the cockroach brain, Acta physiol, scand., 66 (1966) 243-244. Brain Research, 26 (1971) 459-464

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18 GERSCH, M., FISCHER,F., UNGER, H., UND KABITZA,W., Vorkommen von Serotonin im Nerveia~ system von Periplaneta americana L. (insecta), Z. Naturforsch., 16 (1961) 351-352. 19 H-AGGENDAL,J., An improved method for the fluorometric determination of small amounts of adrenaline and noradrenaline in plasma and tissues, Acta physiol, scand., 59 (2963) 242-254. 20 HEBB, C., CNS at the cellular level: Identity of transmitter agents, Ann. Rev. Physiol., 32 (1970) 165-192. 21 HILLARP, N. ,~., FUXE, K., AND DAHLSTR()M,A., Central monoamine neurons. In U. S. yon EULER, S. ROSELL AND B. UVN.~S (Eds.), Mechanisms of Release of Biogenic Amines, Pergamon Press, Oxford, 1966, pp. 31-57. 22 KLEMM, N., Monoaminerge Zellelemente im stomatogastrischen Nervensystem der Trichoptera (Insecta), Z. Naturforsch., 23b 0968) 1279-1280. 23 KLEMM, N., Monoaminhaltige Strukturen im Zentralnervensystem der Trichopteren (Insecta). Teil I, Z. Zellforsch., 92 (1968) 487-502. 24 KLEMM, N., Monoaminhaltige Strukturen im Zentralnervensystem der Trichopteren (Insecta). Tell II, to be published 1971. 25 (~STLUND,E., Adrenalin, noradrenaline and hydroxytryptamine in extracts from insects, Nature (Lond.), 172 (1953) 1042-1043. 26 [~STLUND,E., The distribution of catecholamines in lower animals and their effect on the heart, Actaphysiol. stand., 31, Suppl. 112 (1954) 1-67. 27 PLOTNIKOVA,S. N., AND GOVYRIN, W. A., Distribution of catecholamine-containing nerve elements in some representatives of Protostomia and Coelenterata, Arch. anat. gistol, embrioL, 50 (1966) 79-87. (In Russian.) 28 SEKERIS, C. E., AND KARLSSON, P. In G. H. ACHESON (Ed.), Second Symposium on Cateeholamines, Pharmacol. Rev., 18 (1966) 89-95. 29 TAUC, L., Transmission in invertebrate and vertebrate ganglia, Physiol. Rev., 47 (1967) 521-593. 30 WELSH, J. H., AND MOORHEAD,M., The quantitative distribution of 5-hydroxytryptamine in the invertebrates especially in their nervous system, J. Neurochem., 6 (1960) 146-169. (Accepted December 8th, 1970)

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