Catecholamines in arthropods: A review

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C A T E C H O L A M I N E S IN A R T H R O P O D S :

A REVIEW

L.L. MURDOCK Fachbereich Biologic, Universit/it Konstanz, 775 Konstanz, Germany

(Received 7 Dec., I97O) ABSTRACT i. Knowledge about the physiological significance of catecholamines in arthropods is examined, with special emphasis on their roles as chemical ' messengers ' (i.e., as neurotransmitter substances, neurohormones, and hormones). 2. Dopamine and (to an extent not yet clear) noradrenaline are present in certain central and visceral nervous tissues in catecholamine-containing cells. Visceral neuromuscular systems and central nervous activity are responsive to applied catecholamines, and many drugs affecting catecholamine systems in vertebrates act in close parallel on insect systems. Though a messenger role for any arthropod catecholamine has not been rigorously proven, the weight of evidence for such a role is considerable. 3. Almost nothing is known about the metabolic pathways of ' messenger ' catecholamines in arthropods. There are indications that amine oxidase may not have a central Iole in catecholamine destruction. Metabolism of catecholamines in relation to cuticular synthesis, on the other hand, is much better understood. 4. It is pointed out that there must exist physiological/biochemical mechanisms to protect 'messenger' catecholamine systems from the many sympathomimetic amines produced in connexion with cuticular synthesis. 5. It is suggested that the enzyme systems synthesizing ' messenger ' catecholamines are not subject to variation in activity in response to ecdysone as are those synthesizing ' tanning ' catecholamines. 6. Evidence that adrenaline is present in arthropods is minimal, and is subject to serious doubt. ESPECIALLY in the past 5 years, evidence has been a c c u m u l a t i n g t h a t forcefully argues for a messenger role of c a t e c h o l a m i n e s in a r t h r o pods. Despite this, the i d e a t h a t catecholamines have neurohumoral, neurohormonal, or h o r m o n a l functions in these a n i m a l s is not yet w i d e l y recognized. T h e m a i n purposes o f this review a r e to e v a l u a t e the evidence, s u m m a r i z e existing knowledge, a n d to p o i n t o u t some of the gaps in o u r u n d e r s t a n d i n g o f c a t e c h o l a m i n e s as c h e m i c a l messengers in arthropods. I t has a l r e a d y been established t h a t a catecholamine, N-acetyldopamine (NADA), has a n i m p o r t a n t function in a r t h r o p o d s as a m a t e r i a l c o m p o n e n t in synthesis of the cuticle (for review see Sekeris a n d K a r l s o n , 1966 ) . I n view o f this, we c a n see t h a t a n y satisfactory evidence for a messenger function o f c a t e c h o l a m i n e s m u s t exclude the possibility t h a t the c a t e e h o l a m i n e is present to serve as c u t i c u l a r m a t e r i a l . Several e a r l y

studies d i r e c t e d towards i l l u m i n a t i n g messenger functions were done before it was k n o w n that a c a t e c h o l a m i n e is involved in c u t i c u l a r t a n n i n g ; the insight into a t a n n i n g role n o w p e r m i t s these studies to be r e - e v a l u a t e d . I f catecholamines p l a y b o t h t a n n i n g a n d messenger roles in a r t h r o p o d s , then we m u s t look for the physiological m e c h a n i s m s t h a t e n a b l e m e m b e r s of a class of c o m p o u n d s to p l a y s i m u l t a n e o u s l y two vital, different roles in the s a m e organism. Since we a r e c o n c e r n e d with the significance o f c a t e c h o l a m i n e s in a r t h r o p o d s , it m a y be instructive to m a k e a c o m p a r i s o n a n d ask ' W h a t is the significance o f c a t e c h o l a m i n e s in m a m m a l s ? ' The generally accepted answer has been s u m m e d u p b y G o r b m a n a n d Bern (I962) a n d T u r n e r (I966). D o p a m i n e is present in m a n y n e u r a l tissues. A l t h o u g h it is u n d o u b t e d l y i m p o r t a n t as a p r e c u r s o r for n o r a d r e n a l i n e (NA), it m a y have messenger functions on its own.

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regulated by the moulting hormone, ecdysone (Sekeris and Karlson, i962), which acts indirectly to increase the levels of tyrosinase (Karlson and Schweiger, i96i ) and DOPA decarboxylase activity (Karlson, Sekeris, and Sekeri, i962 ). Incorporation studies (Karlson, I96O ) and radiorespirometry (Murdock and others, I97O) showed that NADA biosynthesis accounts for the massive tyrosine utilization at the time of ecdysis. In insects so far studied, NADA biosynthesis is the dominant fate of tyrosine metabolism during the time that the new cuticle is being laid down. Its biosynthesis is suppressed at other times. A significant recent development is the finding that dopamine biosynthesis may occur in haemocytes (see Mills and Whitehead, i97o ) . CATECHOLAMINES AND CUTICULAR T A N N I N G It is too early to be sure that NADA is used Many years ago, Pryor (x94o) proposed for tanning of cuticle in all arthropod classes. that three main components participate in The gross structural similarity of cuticle cuticular tanning: (i) a tannable protein, throughout the phylum affords some ground (2) an oxidizing enzyme, and (3) an ortho- for suspecting that to be the case. The cuticle quinone precursor. Tanning occurs when of the spider Cupiennis salei (Ctenidae) is the enzyme oxidizes the precursor to an strikingly similar to that of insects (Birth, orthoquinone; the quinone, being highly i969). A circumstantial hint that NADA reactive, forms cross-links with cuticular pro- prevails in cuticle biosynthesis is the distein. The resulting matrix is light and strong covery that crustacean and insect moulting and contributes significantly to the mechan- hormones are, in some cases, identical (Horn, ical properties of the exoskeleton. Karlson Middleton, and Wunderlich, I966), being and Sekeris (I962a) proposed that JV-acetyl- polyhydroxylated steroids (for references see dopamine is the orthoquinone precursor. Adelung, I969). The only crustacean as yet Subsequent studies showed that jV-acetyl- studied, the crab Uca pugilator (Decapoda), dopamine is present in many species of can synthesize NADA from tyrosine (Suminsects including Calliphora erythrocephala (Dip- mers, i968 ). The decapod cuticle contains a tera) pupae (Karlson and Sekeris, I962b ) and pigmented layer (Lockwood, i968 ) which adults (Sekeris, 1964) ; Schistocerca gregaria appears to correspond to the tanned exo(Orthoptera) (Karlson and Herrlich, i965) ; cuticle of insects. The decapod cuticle is Tenebrio molitor (Coleoptera) and Drosophila similar in other respects, the principal differmelanogaster (Diptera) (Sekeris and Herrlich, ence being the large quantities of inorganic I966); eggs of Acheta domesticus (Orthoptera) ions. These ions may not serve as strengthen(Furneaux and McFarlane, i965a ) and eggs ing material because calcification does not of Teleogr21lus commodus (Orthoptcra), Gryl- harden or soften arthropod cuticles (Bailey, lodes sigillatus (Orthoptera), Gryllus bermudiensis i954). Probably they serve to increase the (Orthoptera), and Melanoplus bivittatus (Orth- specific gravity. Decapods are bottomoptera) (Furneaux and MacFarlane, I965b); dwellers and would need a specific gravity pupal Bombyx mori (Lepidoptera) (Tomino, greater than i in order to walk efficiently on I965) ; and in adults of Periplaneta americana the bottom. Recent measurements of the (Orthoptera) (Mills, Lake, Raymond, specific gravity of Astacus leptodactylus (Decaand Alworth, 1967; Murdock, Hopkins, poda) show a value of i. I I -~- O'O2 ( I O measureand Wirtz, I97O ). NADA biosynthesis is ments made in January) (Murdock, i97i ).

Noradrenaline is found in the CNS and is the neurotransmitter released from postganglionic sympathetic nerves; some of this substance reaches the circulation and participates in maintaining vasomotor tone. Thus, it acts both as a neurohumour and as a neurohormone. Adrenaline, on the other hand, acts mainly as a hormone. It is released into the circulation from the adrenal medulla. Once in the blood it exerts several effects, among them a pronounced hyperglycaemia resulting from elevated glycogenolysis in the liver and muscles, increased cardiac output, and inhibition of smooth muscle contractions. Adrenaline is often viewed as an emergency hormone that conditions the animal to respond effectively to certain acute stresses.

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SPECIFIC ANALYTICAL EVIDENCE

One of the main criteria for a functional role of a catecholamine, of course, is that the compound be present. Until a few years ago, the only evidence we had that catecholamines are present in arthropods came from analyses of questionable specificity. Beyond that it was not known that tanning-related catecholamines and tyramine are present in insects and that the levels of these cuticular precursors vary greatly in conjunction with the moulting cycle. So it is not particularly surprising that more than one ' adrenergic ' factor was turned up which was actually not a catecholamine at all, or which represented sympathomimetic factors synthesized for tanning. This early evidence for messenger functions of catecholamines in arthropods will be considered in some detail later, primarily in the hope that one or two of these ghostly 'factors ', which have been drifting around in the literature for m a n y years, will finally be exorcized. The first catecholamine analysis of insects was that of Wense (1938). He claimed to have isolated crystalline adrenaline from the mealworm, Tenebrio molitor. The apparent richness of that beetle as a source of adrenaline prompted attempts by Gregerman and Wald (i95~) to duplicate Wense's results. They failed. T h e y could not isolate adrenaline from Tenebrio, moreover, they could not even detect it by paper chromatography. Gregerman and Wald concluded that if adrenaline is present in the mealworm, it is there in minute amounts. Probably Wense isolated a mixture of catecholamines including NA, which could explain the sympathomimetic effects he observed (Ostlund, 1954). Considering the presence of tyramine, dopamine, and dV-acetyldopamine in Tenebrio in conjunction with cuticular tanning (Sekeris and Herrlich, 1966), Wense's results are readily understandable. Certainly the substance he isolated was not adrenaline. T h e most extensive study of arthropod catecholamines to date was made by C)stlund (1954). Using paper chromatography and pharmacological assay, he found dopamine, noradrenaline, and adrenaline in whole-body extracts of a Forficula species (Dermaptera),

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Vanessa urticae (Lepidoptera), Tenebrio molitor (Coleoptera), Musca domestica (Diptera), and Apis mellifica (Hymenoptera). The quantity of each catecholamine varied from species to species, and with the stage of development; on average, dopamine (about 8 lag. per g.) was present in io times the concentration of noradrenaline (about o.8 lag. per g.), which was present in concentrations at least a full order of magnitude higher than adrenaline (about o-o 4 ~tg. per g.). To learn about the gross distribution of the catecholamines, Ostlund sectioned Tenebrio larvae into anterior (head and thorax) and posterior (abdomen) portions, then extracted and analysed each separately. In the anterior region adrenaline was more concentrated than noradrenaline. In the posterior region the ratio was reversed. Dopamine was present in higher quantities (5-1o times in lag. per g.) in the posterior region. The meaning of these observations is not clear. Perhaps they reflect the association of the catecholamines with special tissues localized in the respective regions of the body. Further speculation seems unwarranted. Dresse, Jeuniaux, and Florkin (196o) applied the powerful fluorometric assay in a study of catecholamines in the silkworm, Bombyx mori (Lepidoptera), and found both adrenaline and noradrenaline. Consistent with Ostlund's (1954) findings in other species, the silkworm contained more noradrenaline than adrenaline. To learn if the titres of the two catecholamines changed during the course of a moult, three groups of immature Bombyx were analysed: larvae 284 ° hours after the last defaecation, prepupae, and pupae. Levels of adrenaline increased during the course of the moult and reached peak concentration in the pupae. Noradrenaline increased in the prepupae and fell sharply in the pupae. Since tanning of the pupal cuticle intervenes between the larval and pupal stages, it is probable that noradrenaline declined in concentration because it was incorporated during cuticular tanning. Since there was no drop in adrenaline during the transition (in fact there was a rise) it is unlikely that adrenaline is involved as a material component in tanning. This

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inference is reinforced when Ostlund's (i954) data on the honey bee arc more closely examined. During the transition of worker larvae to immature pupae the noradrenaline concentration fell from o.3o lag. per g. to o.045 lag. per g., a sixfold decrease. Adrenaline concentration, on the other hand, increased by a factor of 5 during the same period. A drop in NA was also seen with drones during the course of the moult, but in these the adrenaline levels also dropped. For an unexplained reason Ostlund did not report the amount of dopamine present in the immature pupae of either worker or drone bees although values were given for larvae, mature pupae, and adults. In all probability the dopamine was used in the formation of the pupal cuticle, so its concentration dropped below the limit of sensitivity of the analytical method. The recent discoveries on the role of N A D A in tanning of cuticle strongly suggest that biosynthesis of N A D A would have occurred. The parallel drop in NA suggests that at least part of its pool is used in cuticular synthesis, possibly via N-acetylnoradrenaline. The latter compound may occur in tanning of cockroach oothecae (Brunet, I965). Dresse and others (196o) tried to identify adrenaline and noradrenaline in silkworm nervous tissue but were frustrated by an unidentified substance in the extracts that interfered with the fluorimetric assay. The presence of the two catecholamines was, however, suggested by an indirect method. Larvae from which the brain and ventral ganglia had been removed contained only one-half to one-quarter as much adrenaline as larvae in which these tissues were left intact. This experiment is subject to the criticism that injuring an insect initiates oxidation of catechol derivatives as a part of the normal wound-healing process. Such oxidations could account for the observed drop in catecholamines in the operated animals. In any case, the possibility that at least part of the catecholamine pool is localized in the CNS was raised. The actual occurrence of adrenaline in arthropods will be discussed in a later section. In an investigation of the distribution of catecholamines in invertebrate phyla, yon

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Euler (1961) found that larvae of Pieris brassicae ~Lepidoptera) contained 0.33 lag. per g. noradrenaline. In 9 invertebrate species from the 7 phyla examined, this level of NA was by far the greatest. Levels of adrenaline in that insect were below the limits of detection. The crustacean, Carcinus maenas (Decapoda), contained no demonstrable adrenaline or noradrenaline. The first successful demonstration of a catecholamine in crustaceans and the first convincing evidence for any arthropod that nervous tissue is especially rich in a catecholamine was presented by Kerkut, Sedden, and Walker (I966). Using paper chromatography and spectrofluorometry, dopamine was found in the thoracic nerve mass of Carcinus maenas (Decapoda) in a concentration of 7"3 lag. per g. This observation led the authors to suggest a ' possible central transmitter role for dopamine in at least some of the crustacea '. NA and adrenaline were not found. That dopamine occurs in the CNS of this species was confirmed by Cottrell (1967) who reported a concentration of o. 5- i.o lag. per g. in the ventral ganglia. The presence of NA in Carcinus, and ofdopamine and NA in another crab, Hyas araneus, could not be established because of the presence of interfering material. Although only dopamine has so far been found in nervous tissue of crustacea, both NA and dopamine are present in the brain of the cockroach. Frontali and Haggendal (1969) found that Periplaneta americana brain tissue contained 2-5zko-6o lag. per g. dopamine and o.37zko.o4lag, per g. noradrenaline. No adrenaline was found. The relatively high values of dopamine suggest that it is not merely a precursor of NA but could also have a transmitter function. Noradrenaline has also been found in extracts of whole thoraces of the caddis fly, Anabolia nervosa (Bj6rklund, Falk, and Klemm, 197o ). Again the dopamine concentration (5" I and 2"4 lag. per g.) was higher than that of noradrenaline (o.75 and o-35 lag. per g.). No adrenaline was found. ENZYMES OF CATECHOLAMINEMETABOLISM I f catecholamines play messenger roles in arthropods, we expect to find the enzymes

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that catalyse their biosynthesis and destruction. Since the main site of release of the amines is probably nervous tissue, it is most likely that the biosynthetic enzymes are also localized in nerves. Most studies of catecholamine-metabolizing enzymes so far have used whole animals and have been made in conjunction with cuticular tanning. What these studies can tell us about the enzymes metabolizing messenger catecholamines is quite limited. A question that immediately occurs is, ' Are the enzymes that synthesize catecholamines for cuticular tanning identical with the enzymes that synthesize catecholamines for messenger functions ?' The first step in biosynthesis of catecholamines in mammals is the formation of dihydroxyphenylalanine (DOPA) from tyrosine, catalysed by tyrosine hydroxylase. The enzyme is specific for the L-form and appears to catalyse the rate-limiting step in catecholamine biosynthesis (Nagatsu, Levitt, and Udenfriend, I964). Many studies of tyrosine hydroxylating (monophenolase, cresolase) and oxidizing (diphenolase, catecholase) enzymes in insects have been made (Gilmour, i96x), but these have been largely concerned with cuticular tanning. It is sufficient for our purposes to note that insects and crustaceans can hydroxylate tyrosine and do so, especially at the time of the moult (see Sekeris and Karlson, i966 ). Whether there exists in arthropods a tyrosine hydroxylase-involved in the biosynthesis of DOPA to be used as a precursor for messenger catecholamines--as distinct from a tyrosinase which apparently produces D O P A as a precursor for NADA, remains to be learned. DOPA decarboxylase, the enzyme responsible for dopamine biosynthesis via the DOPA pathway, has been shown to be present in Calliphora larvae (Sekeris, I963) and Schistocerca (Karlson and Herrlich, I965). The enzyme is induced by ecdysone at the approach ofecdysis and is at maximal activity when the old cuticle is shed. Like the m a m malian decarboxylase, the insect enzyme requires pyridoxal phosphate. It differs in that it requires Fe 2+, and in substrate specificity. Colhoun (I963) found that many Periplaneta tissues--nervous system, corpora

Comp. gen. Pharmac.

cardiaca, flight and leg muscles--contain a decarboxylase. An enzyme with similar properties is present in houseflies (Colhoun, 1968 ). The housefly decarboxylase appears in the late egg stage and is present throughout the insect's further development. In the blowfly, however, the decarboxylase is lacking in the early third instar (Shaaya and Sekeris, 1965). Possibly the observed differences are the result of differing assay sensitivity. If catecholamines play messenger roles, decarboxylase activity would be expected in all stages (Colhoun, x968), although the total activity might be very small. Nevertheless, greatly increased decarboxylase activity could occur in conjunction with ecdysis. The decapod, Carcinus maenas, contains a decarboxylase acting on DOPA (Summers, I968 ). An amino-acid decarboxylase may function in regulation of the insect heart-beat by the corpus cardiacum hormone (Davey, I963). As with the tyrosine hydroxylating enzyme, it can only be said that arthropods so far examined have an enzyme that can catalyse biosynthesis of dopamine from DOPA. A study in progress shows that nervous tissue of Schistocerca gregaria, that have passed the imaginal ecdysis at least 3 weeks before the experiments, contains a decarboxylase acting on DOPA. Measurements were made with the radiochemical method as described by Tait (I97O) at 22 ° C., at the optimal pH, which is about 8.o. Brain (excluding optic lobes) decarboxylase activity was 2.64-o.16 (S.D.)pmoles per g. per hour while that of the ventral nerve-cord (excluding the suboesophageal gangli on) was o"76 ± o- I o ( S. D.) gmoles per g. per hour. It appears, then, that brain is a more concentrated source of the enzyme than ventral nerve cord. Clearly there is DOPA decarboxylase activity in animals far removed from the moult, a result in agreement with the observations of Colhoun (I968). It may perhaps be of interest to point out that choline acetylase in Schistocerca brain is approximately 2 orders of magnitude more active than DOPA decarboxylase. This should not be used as an argument against a role for DOPA decarboxylase, however, as the following consideration shows: a single

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Schistocerca adult brain weighs about 2. 5 mg. be ruled out that one or more of these It can then manufacture 6. 5 × lO -3 lamoles substances interfered with the fluoriof dopamine in an hour, or z.o lag. dop- metric assays in the earlier work, so that amine per hour. Assuming that the dop- another derivative was mistaken for noramine content of Schistocerca brain is the same adrenaline. T h e enzyme responsible for catalysing the as that of Periplaneta (2. 5 lag. per g.), a single biosynthesis of adrenaline from noradrenaline, Schistocerca brain would contain about 6.2 × lO -3 lag. dopamine. Assuming equal in vivo phenylethanolamine N-methyl transferase, and in vitro activity, this quantity of dopamine has never been demonstrated in arthropods. could be synthesized by the D O P A A role of catecholamines as messenger subdecarboxylase present in 22 seconds. In stances in an organism is usually associated other words, there is sufficient D O P A with an amine oxidase that destroys them or decarboxylase activity present to replenish their 0-methylated metabolites. This enzyme completely the dopamine store every 22 does not appear to destroy the catecholseconds. amines immediately after their release--as Particularly interesting is the recent dis- acetylcholinesterase destroys acetylcholine-covery by Whitehead (I969) that haemocytes but rather seems to act as a scavenger, of the Periplaneta m a y contain, instead of oxidizing catecholamines in cytoplasm (IverD O P A decarboxylase, tyrosine decarboxy- sen, I97o ) . This is suggested by two facts: lase. In this animal N A D A is the main pro- (I) amine oxidase is associated with mitoduct of tyrosine metabolism during hardening chondria which are intracellular, and (2) and darkening of the new cuticle (Murdock catecholamines are released into extraand others, i97o ) so there must also be a cellular spaces. Using a sensitive qualitative tyramine hydroxylase required for the syn- test Blaschko, Colhoun, and Frontali (I 96I) thesis of dopamine. It would appear that the detected amine oxidase in whole head, flight pathway to dopamine in insects m a y pass muscle, gastric caecae, midgut, and fat body through either tyramine or DOPA, or both, of Periplaneta americana. Especially high levels depending on the species. of the enzyme, comparable to those in the A substance with the chromatographic m a m m a l i a n liver, were found in Malpighian properties of noradrenaline is produced when tubules. The enzyme acted on [3-phenyldopamine is incubated with homogenates of ethylamine and dV-methyl-13-phenylethylTenebrio (Sekeris and Karlson, I966), which amine and so was potentially capable of suggests the presence ofdopamine-~-hydroxy- oxidizing catecholamines such as NA and lase in these insects. H a e m o l y m p h of freshly adrenaline. An expanded study confirmed moulted Periplaneta also contains a hydroxy- the earlier observations on Periplaneta but lase that converts tyramine to norsynephrine showed that another cockroach, Blaberus (Lake, Mills, and Brunet, I97O ). The further discoidalis, contained an enzyme oxidizing metabolism of norsynephrine has not been diamines but not monoamines. Five species clarified as yet, but one possibility is that it is of decapod crustacea were studied and no hydroxylated to noradrenaline. T h a t NA is monoamine oxidase could be discovered in one of its metabolites and that the NA so any of them (Boadle and Blaschko, I968). A produced is used during cuticle formation mitochondrial enzyme oxidizing tryptamine would be in full agreement with the observed was found in whole-body extracts of Tenebrio variations in NA during the course of a (Chaudhary, Srivastava, and Lemonde, i967). These observations suggest that some moult observed by Dresse and others (x96o) and Ostlund (1954), and with the high whole- arthropods can destroy aromatic monoamines body levels of NA in Pieris larvae reported by via oxidation and that others cannot. The von Euler (I96I). O n the other hand, it now absence of such an enzyme is, however, no appears that a considerable number of argument against a messenger role of catecholamine and tyramine derivatives m a y catecholamines because they can be removed be present in the whole insect, and it cannot from sites of action in other ways.

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In the light of the discovery that catechol O-methyl transferase ( C O M T ) is largely responsible for the destruction of m a m m a l i a n catecholamines (Axelrod, Enscoe, Serah, and Witkop, I958 ) and that this enzyme is competitively inhibited by catechol (Axelrod and Laroche, i959; Bacq, Gosselin, Dresse, and Renson, I959) it is intriguing to consider the effects of catechol on insects. Catechol increases the level of nervous activity in the cockroach nerve cord at I o - a M (Smyth, I959; Milburn and Roeder, I962 ). Injected into insects, it produces pronounced behavioural effects (Smyth, I959) to be discussed later. I f catecholamines play messenger roles in the insect CNS and if C O M T normally functions in their destruction, then exposure to catechol should slow or block the destruction of the catecholamines by inhibiting C O M T . T h e build-up ofsupranormal levels of the catecholamines would follow; the high level of catecholamines could then lead to abnormal patterns of neuronal firing, ultimately expressed as changes in behaviour. This hypothesis is necessarily speculative, because no studies of arthropod C O M T have been made. The effects of injected catechol m a y indicate that such a system is present, but it is perhaps just as likely that catechol has direct excitatory effects, acting as a sympathomimetic. MORPHOLOGICAL EVIDENCE

The development of the histochemical fluorescence technique by Falck (I962) and its application to arthropods during the last few years have provided trenchant evidence for a role of catecholamines in the nervous system of these animals. Not only does the method permit the discovery of catecholamine-containing cells, it also distinguishes between primary and secondary catecholamines. Ceils containing primary catecholamines are present in the three main parts of the cockroach brain (Frontali and Norberg, i966; Frontali, I968 ). Small, faintly fluorescent cells are seen near the periphery of the optic lobe of the protocerebrum. The aand [3-lobes of the corpora pedunculata are fluorescent and show transverse ' s t r i p e s ' of

Comp. gen. Pharmac.

more intense fluorescence. Other structures containing catecholamines are the intensely fluorescent central body, and part of the medulla which exhibits a semilunar zone of fluorescence. Several groups of two or more fluorescent cells are seen in the protocerebrum. Also, scattered through the protocerebrum are individual fluorescent fibres. It is noteworthy that the anterior pars intercerebralis, long known to be a site of elaboration of neurohormones, shows no fluorescence, so these neurosecretory cells contain no catecholamines. Large, brightly fluorescent cells are present in the deutocerebrum and its neuropile is crossed by a network of fluorescent fibres. The tritocerebrum contains a single pair of large catecholamine-containing cells; its neuropile also contains abundant fluorescent fibres. The suboesophageal ganglion contains a large pair of cells containing catecholamines, several small ones, and its neuropile is rich in fibres containing amines. Pretreatment of the cockroaches with a single dose of reserpine (5 ° gg. per g.) 2 or 4 days before removing the brain caused a depletion of the fluorescence in the neuropile but not in the cell-bodies. Repeated treatment with reserpine caused almost complete depletion of fluorescence both in the neuropile and the cell somata. T h a t D O P A m a y be the precursor of the contained catecholamines is suggested by the observation that injection of D O P A was followed by an increase in the intensity of fluorescence. I f a monoamine oxidase is responsible for the destruction of the catecholamines, then injection of an inhibitor of the enzyme should potentiate the fluorescence by slowing the destruction of the amines. Injection of nialamide alone was not fbllowed by an increase in fluorescence. Repeated treatment with nialamide and D O P A did result in a moderate increase. Frontali (I968) points out that the fluorescence of the ~t- and [Mobes of the corpora pedunculata must originate from fibres coming from outside the corpora pedunculata; in all probability from some of the scattered groups of cells located in other parts of the CNS. Since the central body and the ~-lobe of the corpora pedunculata receive rich innervation by catecholamine-

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T h a t the ventral ganglia of insects also containing neurons, and since these regions appear to be co-ordination centres of motor contain catecholamines was shown by Plotnipatterns, it appears that the amines ultimately vioka and Govrin (quoted in Klemm, 1968). serve as transmitters involved in motor co- Both individual fluorescence cells and fluorordination in the brain (Frontali and escing areas in the neuropile were observed in the metathoracic ganglion ofLocusta migraHaggendal, 1969). The distribution of catecholamine fluores- toria. Substantiation for the presence of cence in the adult caddis-fly brain is remark- catecholamines in insect ventral ganglia was ably similar to that in the brain of the provided by the observation of monoamine cockroach (Klemm, i968 ). Primary amine fluorescence in the three thoracic ganglia of fluorescence is seen in the optic lobe of the Anabolis nervosa (Bj6rklund and others, I97o ). protocerebrum, is especially intense in the Each ganglion contained two pairs of a- and [3-lobes of the corpus peduncu- brightly yellow-green fluorescent cell-bodies, latum and the central body, and is seen in as well as fluorescent fibres in the neuropile. the externa and interna medulla. Varico- Beyond that, fluorescent fibres were observed sities are also seen in the corpus ventrale and, to pass between ganglia. Dopamine was the unlike the cockroach, in the pars inter- principal catecholamine, with lesser amounts cerebralis. Green-fluorescing cells are present of noradrenaline. in the deutocerebrum and very sparsely in Catecholaminergic cells are not restricted the tritocerebrum. Catecholamine-contain- to the central nervous system. The ingluvial ing varicosities and 4 unipolar cells were seen ganglion of Schistocerca gregaria and Blaberus in the suboesophageal ganglion. No evidence cranifer shows intense green fluorescence after for amine-containing cells in sections of treatment with formaldehyde (Chanussot, peripheral nerves was found. K l e m m also Dando, Moulins, and Laverack, I969). The observed that catecholamines seem to be fluorescence is concentrated in the neuropile concentrated in primary association centres, and is that of a primary amine and not while the secondary association centres seem 5-HT. The authors point out that it is to be cholinergic. Cells containing primary perhaps significant that the foregut of the catecholamines are also present in the CNS two species examined is sensitive to catecholof the decapod, Astacus astacus (Elofsson, amines. Monoamines are present both in the Kauri, Nielsen, and Str6mberg, x966). neuropile and, as individually recognizable Catecholamine fibres were seen in the proto- ceils, in the periphery of the stomatogastric cerebrum but not in the deutocerebrum or ganglion of the lobster Homarus vulgaris (with a single exception) in the tritocerebrum. (Osborn and Dando, I97O ). Reserpine Such fibres were present in the eyestalk, the pretreatment effectively abolished the fluoresmedulla externa, the medulla interna, and the cence. The ganglion contains mostly secondmedulla internalis of the protocerebrum. In order motoneurons. Osborn and Dando general, fluorescent varicosities were found suggest that the amines could function as in areas presumed to be associative in func- transmitters between the motoneurons of the tion. Fluorescent cell-bodies and fibres were ganglion or as transmitters between the motoalso seen in the suboesophageal ganglion and neurons and the muscle cells of the foregut. in the first abdominal ganglion. As with the It was also pointed out that the location of cockroach, reserpine caused a depletion of the ganglion is appropriate for it to serve as a the catecholamines. Pretreatment with the site of release of neurohormones. monoamine oxidase inhibitor, nialimide, did Nerve-fibres containing catecholamines not cause a clear increase in fluorescence. also occur in the ' Grundplexus ' and ' EndT h e dominant catecholamine is thought to be plexus ' of the hindgut of Astacus (Elofsson dopamine, a view that is an agreement with and others, x968 ). Such fibres are relatively the finding of Kerkut and others (1966) of scarce but were seen among both circular dopamine in the CNS of another decapod, muscle and longitudinal muscle layers. They Carcinus maenas. are much more abundant in the outer part of

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the ' G r u n d p l e x u s ', more thick and less beaded than those seen in the ' Endplexus ', where they appear as varicosities. No sensory neurons were observed to show the green fluorescence, so the fibres containing the catecholamines must be either motor- or interneurons. Pretreatment with reserpine abolished the fluorescence. Microspectrofluorimetry indicated that NA is present in the cells but the presence of dopamine could not be ruled out. Groups of cells in the abdominal ganglia of Periplaneta americana appear to end in the median nerve neurohaemal organs (Smalley, 197o ) . When the animal is injected with tritiated dopamine, both the cell-bodies and the neurohaemal organs rapidly take up the label. Other cells in the ganglion do not do so, nor does the short nerve leaving the neurohaemal organ; therefore, the axons of the cells (three groups) in the preceding ganglion probably end in the neurohaemal organ. Uptake of catecholamines by catecholaminergic cells is a generally recognized characteristic of m a m m a l i a n systems. T h a t the ceils ending in the neurohaemal organs take up catecholamines m a y therefore indicate that the cells are, in fact, catecholaminergic, releasing a catecholamine into the blood as a neurohormone. The system described by Smalley m a y represent an example of a catecholamine playing a true role as a neurohormone in an arthropod. Evidence that catecholamines are normally produced and released by these cells is lacking. CATECHOLAMINES AND LIGHT PRODUCTION BY INSECTS

The exact mechanism by which luminescent insects control light production is still unknown. There is, however, little doubt that flashing in light-producing beetles is under nervous control (Wigglesworth, I965). The neurotransmitter substance released by nerves serving the light organs is probably a catecholamine. As was first observed by Kastle and McDermott (I9IO), adrenaline elicits a glow in isolated light organs of Photinus pyralis (Lampyridae). Later workers found that other sympathomimetic amines were also

Comp. gen. Pharmac.

effective. Picking up these leads, Smalley (I965) carried out a penetrating study using techniques of analytical pharmacology. When adrenaline, noradrenaline, or amphetamine is injected into adult male Photinus, glowing follows. Adrenaline or noradrenaline m a y be injected repeatedly into the same animal; each time there is enhanced glowing. This is not true for amphetamine; glowing occurs only after the first treatment. I f noradrenaline or adrenaline is injected into an amphetamine-treated animal, the animal glows. When the nerves serving the posterior lantern are severed and allowed time to degenerate, amphetamine triggers light production only in the anterior, undenervated light organ, but noradrenaline causes glowing in both lanterns. Smalley's interpretation is that amphetamine acts presynaptically to release the natural effector, while the externally applied catecholamine exerts its action directly on the post-synaptic cell. This conclusion is in good agreement with the known presynaptic effects of amphetamine in m a m mals, where it is thought to release noradrenaline and prevent its re-uptake (Burgen and Mitchell, 1968). Fireflies pretreated with reserpine exhibit diminished glowing upon injection with amphetamine. Since reserpine is known to deplete catecholamines in both vertebrates and arthropods (see above), this is exactly the result that would be expected if a catecholaminergic system is involved. Furthermore, the adrenergic blocking agents, yohimbine and dibenzyline, blocked or greatly increased the latency of the electrically induced slow flash, a result also consistent with a catecholamine-mediated system. Smalley (1965) showed that scintillations induced by the cholinesterase inhibitor, eserine, arise in the CNS; the drug had no effect on denervated organs. Similarly, other non-adrenergic drugs, 5-HT, atropine, eserine, y-aminobutyric acid (GABA), and DOPA, were ineffective on the isolated light organ of Photurus (Carlson, 1968a), supplying further reason to think of the organs as specifically catecholaminoceptive. Carlson essentially confirmed Smalley's observations with amphetamine, adrenaline, noradrenaline, and

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C A T E C H O L A M I N E S IN A R T H R O P O D S

reserpine. In addition, he found that dopamine and tyramine could also induce glowing, although much less effectively than adrenaline and noradrenaline. Monophenolic analogues ofcatecholamines are considerably more potent in inducing glowing of isolated larval light organs than are the catecholamines themselves (Carlson, I968b ). The most potent compound so far found, DE-synephrine, is 44 times more effective than DE-adrenaline and 66 times more effective than DE-noradrenaline. One monophenolic derivative, tyramine, acts postsynaptically on the light organ rather than by releasing amines from the presynaptic cells (Borowitz and Kennedy, 1968 ). These observations led Carlson (I969) to consider the possibility that the neurotransmitter substance is a monophenolic amine rather than a catecholamine. Other than the argument of potency, there is no support for this. Since m a n y synthetic drugs are far more potent than natural effectors [for example, methyldilvasene is io times more potent than acetylcholine on a m a m malian system (Burgen and Mitchell, 1968)] potency alone is a weak criterion. Carlson (I969) speculated: ' A monophenolic transmitter m a y be necessary so that it can be destroyed by a tyrosinase-type enzyme from the blood. ' This presupposes that the nature of the degrading enzyme somehow dictates the nature of the transmitter substance. The known actions of blood tyrosinase include hydroxylation, followed by oxidation to quinones. I f a blood tyrosinase destroyed a monophenolic amine by the pathways known at present, then it would first produce a catecholamine derivative--compounds that are known effectors on m a n y insect systems-and then quinones, which are themselves highly reactive and toxic. This seems most unlikely. Indeed, there is no evidence at all at present about how catecholamines naturally released in arthropods are destroyed. Carlson also speculated that the existence of a monophenolic transmitter m a y be related to the need to maintain a transparent cuticle. T h e assumption apparently is that if neurotransmission in the light organ were mediated by a catecholamine then the insect would be

263

unable to produce or maintain a transparent cuticle. The existence of enzymic and permeability barriers which can prevent the passage of a substance from one organ to another could easily solve this problem. Moreover, the quantities of catecholamines released by the nerves must be extremely small. It is difficult to imagine that this problem would arise. A counter-argument to the monohydroxyamine hypothesis is the repeated observation that certain insect nerves do indeed contain catecholamines. In view of these observations it seems more likely at present that the neuroeffector of the firefly lanterns is one of the common catecholamines. Application of the histochemical fluorescence technique to the nerves serving the lanterns m a y shed considerable light on this question. Strangely enough, the mechanism by which the neuro-effector triggers light production appears not to be via a classic permeability change/depolarization. Responses to catecholamines were not significantly different even if the larval light organs were bathed in various ionic media such as 0.32 M sucrose, o.I6 M choline chloride, or o. 16 M NaC1. Even exposure to high K ÷ did not cause glowing if the preparation had been pretreated with reserpine or amphetamine (Carlson, 1968a). The only effect of high K + was to release transmitter from the presynaptic cells. The effect of the presumed catecholamine transmitter may be to trigger glycogenolysis to provide energy to power the flash (Smalley, 1965). This is compatible with the known glycogenolytic effects of catecholamines in mammals; epinephrine does have glycogenolytic effects in insects (Bhakthan and Gilbert, 197i ). An alternative explanation was suggested by Carlson (I969) : the pyrophosphate produced via the catecholamine (or monohydroxy amine) acting on adenyl cyclase is the key for the flash. Pyrophosphate does appear to be an important intermediate in light production (see Gilmour, I964) , so pyrophosphate release could be the key control mechanism. Probably both glycogenolysis and pyrophosphate production are significant: glycogenolysis to supply substrate for A T P production

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(needed to maintain flashing) and pyrophosphate actually to trigger the flash. Whatever the actual mechanism, the system is an extremely interesting one and deserves more detailed study, especially on the biochemical level. EFFECTS ON NERVE TRANSMISSION AND SYNAPSES

Adrenaline and noradrenaline facilitate synaptic transmission in the desheathed sixth abdominal ganglion of P. americana (Twarog and Roeder, i957). At higher concentrations ( 1 o - S M ) , transmission becomes blocked. Ergotamine had no blocking action against presynaptic stimulation, however, which might be expected if catecholamine receptors were mediating synaptic transmission in the ganglion. Catecholamine sensitivity is undoubtedly present but the amounts required were very high (IO-4-IO -2 M). T h a t the sensory neurons synapsing in this ganglion are catecholaminergic thus seems doubtful. Since catecholamine-containing neurons do occur in the ventral nerve cord of insects it is not surprising that a CNS preparation responds to catecholamines with excitation. Dopamine at i o -4 M increased the level of activity in the ventral nerve cord of the cockroach (Milburn and Roeder, 1962 ). Besides dopamine, adrenaline, noradrenaline, epinine, and D O P A excite the ventral chain of P. americana and B. craniifer (Hodgson and Wright, 1963). These authors suggest ' t h a t epinephrine is probably the neurologically active substance among the group of related adrenergic compounds found in insects ', a conclusion based solely on the fact that adrenaline was the most potent of the compounds tested (see below). Gahery and Boistel (I965) found anteriorly propagated nerve impulses in the nerve cord of Periplaneta after bathing the desheathed last abdominal ganglion in 5 × I o-5 M dopamine. The amplitude and velocity of propagation of these potentials differ from potentials produced by stimulating the cereal nerves. The fibres carrying the dopamineinduced potentials do not synapse and appear to terminate in the mesothoracic ganglion. It would appear that these dopaminoceptive

Comp. gen. Pharmac.

fibres are interneurons. At the level tested dopamine had no effect on synaptic transmission in the terminal ganglion. It is interesting that a known arthropod interneuron is affected by catecholamines. The second-order, bipolar eccentric cell of the eye of Limulus is excited by adrenaline and noradrenaline to increase the number of spontaneous threshold potentials (Adolph, I966 ). In this preparation reserpine produced an initial depolarization possibly consistent with a release of catecholamines in the primary cell, then a weaker hyperpolarization. The discovery that dopamine is a very potent inhibitor of the crayfish stretch receptor (McGeer, McGeer, and McLennan, I961 ) seemed to raise the possibility that it is the transmitter of the I-neurons serving the organ. By contrast, however, Elliott and Florey (1956) reported that noradrenaline and adrenaline were completely inactive on stretch receptors of Cambarus virilus. A reinvestigation by McLennan and Hagen (I963) revealed a perplexing result: dopamine, although extremely potent in inhibiting the slowly adapting stretch receptors of the crayfishes Pacifastacus leniusculus and Procambarus clarkii, was quite impotent against Procambarus blandingi and Orconectespropinquus. The obvious lesson is that it is dangerous to generalize from the results of pharmacological experiments on a single species, even if the generalization is limited to the family of animals on which the experiments were made. Dopamine, which inhibits certain vertebrate brain neurons, reversibly blocks firing of spontaneously active neurons in the brain of the adult wood ant, Formica lugubris Zett (Steiner and Pieri, 1969). CATECHOLAMINES AND BEHAVIOUR

The concept that catecholamines in the arthropod brain serve as transmitters in brain co-ordination centres gains considerable support from studies of the effect of catecholamines on insect behaviour. Catechol, which presumably may act as an inhibitor of catecholamine O-methyl transferase in arthropods, or which may also mimic the common catecholamines by virtue of its O-dihydroxybenzene structure, has striking effects on

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insect behaviour. As noted by Smyth (I959) and Sittler (1962), moderate doses of catechol (about 40 pg. per animal) produce temporary convulsions when injected into Periplaneta americana. Smaller amounts (2o ~tg. per animal) cause the insect to assume positions resembling those occurring during ecdysis, and depress locomotor activity. Injections of as little as o.i ~g. of adrenaline (Barton-Browne, Dodson, Hodgson, and Kiraly, I96I) or 0. 3 ~g. per animal (Sittler, I962 ) produce inactivity or sluggishness in Periplaneta. Noradrenaline is less effective. These observations were confirmed and extended by Hodgson and Wright (I963). Injection of adrenaline, noradrenaline, and epinine but not catechol, DOPA, and several other catecholamines caused striking behavioural effects: antennal tremors, decreased activity, and altered coordination in Periplaneta americana and Blaberus craniifer. In females the active compounds produced abdominal contractions, wing fluttering, and oviposition behaviour. Dopamine produced increased activity without other behavioural effects. Hodgson and Wright (x963) noticed that newly moulted cockroaches were unresponsive to injected adrenaline. It now appears likely that this can be explained by uptake of the catecholamine into the cuticle along with the naturally produced tanning agents during the process of laying down the new cuticle. Noradrenaline injected into Drosophila melanogaster shortly after the imaginal ecdysis caused hyperactivity lasting about 2 hours, while dopamine-injected flies exhibited loss of coordination lasting about 3 hours (Jacobs, 197o). Reserpine and chlorpromazine, which interfere with normal eatecholamine and 5-HT storage and turnover, depress locomotor activity in the ant (Kostowski, Beck, and Mesaroy, 1965), producing tranquillizing effects seemingly parallel to those seen in mammals. Injection of adrenaline produced no apparent behavioural effects. Reserpine induced inactivity and unresponsiveness in Periplaneta (Frontali, x968 ). These observations clearly establish that injected catecholamines and associated drugs

265

elicit behavioural responses, and that this happens is entirely consistent with a presumed integrative transmitter function of the catecholamines in the CNS, derived from entirely different kinds of studies. CATECHOLAMINES AND METABOLISM

Noradrenaline and dopamine strongly inhibit CO 2 production from the carboxyl carbon of phenylalanine in Drosophila melanogaster at the time of the imaginal ecdysis (Jacobs, I97O ). This inhibition in all probability is the result of inhibiting the decarboxylase enzyme producing the amine precursor of the tanning agent. Sekeris (I 963) showed that another catecholamine, dVacetyldopamine, is a powerful inhibitor of D O P A decarboxylase from Calliphora. Shortly after the imaginal ecdysis the new cuticle is being tanned, so this effect of catecholamines may be rationalized as an effect on production of the tanning agent from phenylalanine. Jacobs made further observations of the effects of the two catecholamines on oxidative metabolism which may imply a more general metabolic effect. Dopamine significantly decreased CO 2 production from glucose but not from pyruvate in these same animals. Noradrenaline, on the other hand, depressed CO 2 production from pyruvate but increased it when glucose was the substrate. It seems fruitless at present to try to explain Jacobs's results as actions on particular enzymes in the glycolytic scheme and citricacid cycle. It is sufficient to note that NA and dopamine may affect these systems, and do so in different ways. The finding of Bhakthan and Gilbert ( 197 i) that adrenaline can stimulate glycogenolysis in cockroaches supplies another parallel between arthropod and vertebrate catecholaminergic systems. CATECHOLAMINE PHARMACOLOGY OF

A R T H R O P O D VISCERAL MUSCLE SYSTEMS

Catecholamines change the behaviour of many arthropod visceral muscle systems. This receptivity, of course, indicates that catecholamines might naturally function in controlling the activity of such systems, but it is by no means proof. The crayfish stretchreceptor organ, for example, responds to

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acetylcholine with excitation even though it receives no excitatory fibres (Florey, i967). Although the cell somata of certain gastropod central neurons are very sensitive to 5-HT, they are not known to form synapses (Gerschenfeld, i966 ). Chesher (197o) has shown that the ACh receptors in the m a m m a l ian urinary bladder that respond to exogenously applied ACh are different from those that respond to neurally released ACh. It appears that the former population of receptors plays a role in response to nerve-released ACh only during abnormal situations, such as when cholinesterase is inhibited. These observations reinforce the concept that receptivity does not necessarily mean a functional role for the receptors. A further difficulty with the two most popular arthropod visceral muscle preparations, heart and gut, is their complexity. Applied drugs m a y act on ganglion cells, at excitatory or inhibitory neuromuscular junctions, on sensory neurons or on neurosecretory cells, as well as have direct effects on muscle or neuronal excitability. Furthermore, there appear to be overlapping systems of control in these organs. T h a t arthropod heart and gut preparations respond to a spectrum of drugs is not, therefore, surprising. The cockroach heart, for example, is excited by cholinergic drugs, catecholamines and related drugs, by 5-hydroxytryptamine and derivatives, by a bewildering variety of peptides and various unknown small molecules in tissue extracts, even, it appears, by such a common molecule as uric acid (Fischer and Kapitza, I965). The catecholamine pharmacology of the insect heart has been reviewed in detail (Davey, i964; Jones, I964) and there is little to be gained now by a detailed reconsideration of these studies. It is sufficient to point out that catecholamines such as noradrenaline, adrenaline, and dopamine are generally found to be cardio-active, and that ergotamine, a blocking agent for catecholamines in vertebrate catecholaminergic systems, also blocks the action of adrenaline (Naidu, 1955) at least in the cockroach heart, a fact which points towards a specific adrenergic receptor. Compelling evidence for a role of a

Comp. gen. Pharmac.

catecholaminergic control system will come if the histochemical fluorescence technique for catecholamines should reveal the presence of catecholamine-containing neurons in the cardiac nerves. A considerable improvement in our understanding of the role of catecholamines will also come when the refined recording and denervation techniques of Miller and Metcalf (i968) are applied with a view to a role of catecholamines in control of the heart. These techniques have already made it clear that the sites of action of acetylcholine on the semi-isolated cockroach heart are the cardiac ganglion cells and not neuromuscular synapses. Dopamine also excites the cardiac ganglion cells (Miller, I968), but it apparently has not yet been tested on the denervated heart to determine if it acts at neuromuscular junctions. Adrenaline excites the heart of the spider Tegenaria atrica (Kadziela and Kokocifiski, 1966 ) and appears to act directly on the muscle of Limulus heart (Carlson, I9O5; quoted in Krijgsman and Krijgsman-Berger, i95I), unlike ACh which acts only on the ganglion cells. Similarly, adrenaline and noradrenaline usually excite the crustacean heart (Maynard, I96o ). At least in decap0d crustaceans ACh appears not to be the transmitter released by the ganglion cells which control heart frequency (Florey I963) , so it m a y be that some of the ganglion cells are adrenergic. Potent evidence regarding this possibility would be provided by a histochemical fluorescence study of crustacean or Limulus heart ganglia. It appears that receptivity of hearts to catecholamines is fairly general in the arthropods. Whether catecholamine messengers actually play a role in regulation of arthropod heart-beat remains to be shown. The reservations about the limitations of pharmacological results discussed above for the heart apply equally to such studies of gut preparations. Here again we find sensitivity to dopamine and noradrenaline in insect foreand hindgut (Freeman, I966 ) and in the crayfish hindgut (Florey, I96o; Murdock, 197x ). Catecholamines are generally excitatory. In these cases, however, there is trenchant corroborative evidence for a role of

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a catecholaminergic system. T h e ingluvial ganglion of Schistocerca or Blaberus, which innervates the insect foregut, does contain cells with the green fluorescence characteristic of a primary catecholamine (Chanussot and others, 1969). Such fluorescence is also seen in the stomatogastric ganglion of Homarus (Osborn and Dando, i97o ) and in the nerve plexus of the crayfish hindgut (Eloffson and others, I968 ). In all probability a catecholaminergic system is involved in control of arthropod alimentary tract peristalsis. CATECHOLAMINES AS ARTHROPOD CHROMATOPHOROTROPINS

Occasionally it has been proposed that catecholamines m a y act as chromatophorotropins in arthropods (e.g., Kopenec, i949). Arthropod chromatophores are not innervated, so the presumed catecholamines would be acting as neurohormones or hormones. Evidence for such a role is minimal and the observed effects of applied catecholamines probably stem from indirect actions, such as exciting the nervous system and triggering release of the true chromatophorotropin(s) which appear to be peptides (for review see Fingerman, I963). CATECHOLAMINES EFFECTORS

AS MALPIGHIAN

TUBULE

Orthopteroid Malpighian tubules appear not to be innervated and are excited to con-

tract at a more rapid rate by adrenaline (Cameron, i953; quoted in Davey, i964). I f this sensitivity can be substantiated, and if, in fact, the tubules are not innervated, then another interesting possibility of a role for catecholamines as a neurohormone in insects will be raised. To prove that this system functions it will only be necessary to prove that a catecholamine is released into the circulation at appropriate times in sufficient quantity to raise the rate of contraction. ADRENERGIC = FACTORS'

T h e fact that a tissue extract contains a factor that mimics the action of a catecholamine does not necessarily mean that the extract contains a catecholamine. Cameron

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(i953) extracted an adrenergic factor from Periplaneta corpora cardiaca and from whole Tenebrio molitor larvae that increased cockroach heart-beat rate and stimulated peristalsis of insect hindgut preparations. The active component (presumably only that from Tenebrio extracts) had the properties of an 0-diphenol but was distinct from adrenaline by paper chromatography. Cameron evidently assumed that the active factor from the corpora cardiaca was the same as that in Tenebrio extracts. This is not so. The corpora cardiaca activity must have been due to the presence of peptides active on visceral muscle systems. Other than the fact that they appear to be peptides, the nature of these hormones is not known; their existence is well established by the work of Davey (1962), Steele (i963) , and Natalizi and Frontali (I966) among others. Furthermore, Natalizi and Frontali (i966) showed that the cardioaccelerative activity of corpora cardiaca could be fully accounted for by the presence of the peptides. Moreover, the fluorescence microscopy technique gave no indication that catecholamine-containing cells are present in the corpus cardiacum. Similarly, the concept of active factors in extracts of whole Tenebrio stems from (i) the probable presence of catecholamines in the nervous systems; (2) the presence of the extracted cardioactive peptides from the corpora cardiaca; and (3) the presence of catecholamine made for tanning, such as dopamine, NADA, tyramine, and N-acetyltyramine (Ostlund, i954; Sekeris and Herrlich, I966 ). An adrenergic factor in cockroach corpora cardiaca was also reported by Barton-Browne and others (1961). Dual bioassay indicated activity intermediate between that of noradrenaline and adrenaline. Arguments against the presence of adrenaline in the corpora cardiaca will be discussed below. The possibility exists that bioactive peptides or other factors from insects can act on vertebrate pharmacological systems. An example is a powerful stimulant of m a m m a l i a n uterus extracted from insect gut (Barton-Browne, Hodgson, and Kiraly, 196 I) which has since been shown to be due to proteolytic activity in the extracts (Cobbin and Temple, 1969).

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DISCUSSION For lack of facts, our understanding of catecholamine systems in arthropods remains still somewhat out of focus. At this point it may be profitable to summarize and make a provisional general picture, to point out some of the major mysteries, and to examine the implications of having two catecholamine systems serving two different functional roles in the same animal simultaneously. Although relatively few chemical analyses of arthropod nervous tissue have been made, it is clear that significant quantities of dopamine occur in the CNS. Noradrenaline is also present, at least in the cockroach brain, but apparently in much lower concentration. A close inspection of the results of Ostlund (1954) and Dresse and others (i96o) suggests the existence of an additional pool of NA that waxes and wanes in conjunction with the moult. In other words, there may be a noradrenaline pool outside the nervous system which is involved in the process of sclerotization. This possibility is reinforced when Frontali and Haggendal's (i969) analyses of NA in Periplaneta brain are compared with von Euler's (I 96 I) of whole Pieris brassicae larvae. Values were : Periplaneta brain, o'37 p.g. per g. ; whole Pieris larvae, o.33 p.g. per g. Either Pieris larvae contain exceedingly high levels of NA in nervous tissue or there is another rich pool of noradrenaline, possibly representing a storage for later use in cuticular synthesis. The recent discovery of Lake and others (i97o) showing that a possible noradrenaline precursor, norsynephrine, can be synthesized in haemolymph from freshly moulted Periplaneta renders the idea even more likely. It must be emphasized, however, that the existence of a presumed noradrenaline pool outside the nervous system is predicated on the assumption that the analyses of Ostlund, Dresse and others, and von Euler truly reflect the NA content of the animals and not the content of some other amine such as norsynephrine or W-acetyldopamine. There is reasonable certainty that catecholamines such as dopamine, W-acetyldopamine, and probably others do, in fact, have a role in cuticular tanning. There may exist then, both

Comp. gen. Pharmac.

a ' messenger ' NA pool and a ' tanning ' NA pool in insects, just as there are ' messenger ' and ' tanning ' dopamine pools. Evidence for NA in other arthropods is practically nonexistent, but it seems reasonably safe to say it will be found when more sensitive and specific methods are applied. Catecholamines (principally dopamine) and, at least in some systems, noradrenaline are present in certain central and visceral neurons of arthropods. The central nervous system and various visceral muscle systems clearly respond to catecholamines. Drugs altering catecholamine systems in vertebrates appear to act in the same or similar ways in arthropods. There are indications that uptake of precursors and products occurs in arthropods as it does in vertebrates. In short, with the possible exception of the role of monoamine oxidase, arthropod catecholaminergic systems evidently closely parallel those of vertebrates. Although we cannot say that a transmitter role for a catecholamine has been rigorously proven, i.e., demonstration of release, identities of reversal potentials for nerve action and artificially applied catecholamines, etc., the evidence for a neurotransmitter role of catecholamines in arthropods is quite compelling. There are also some indications that catecholamines may function as neurohormones in arthropods. The uptake of dopamine by neurohaemal organs (Smalley, I97O ) and the optimal placement of the catecholamine - containing stomatogastric ganglion of the lobster (Osborn and Dando, 197 o) point towards a possible neurohormonal role. Routes of messenger catecholamines' synthesis in arthropods are unknown. Brain of Schistocerca contains an enzyme that decarboxylates D O P A (Murdock, this paper) which indicates that the route to dopamine may be similar to that in vertebrates. Frontali's (i 968) observation that injection of D O P A led to increases in primary catecholamine fluorescence in cockroach brain suggests that this precursor can be taken up and converted to dopamine, presumably directly by D O P A decarboxylase. The pathway to dot)amine in insect brain may be Tyr--,

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D O P A ~ d o p a m i n e as it is in vertebrate systems, but the evidence at present is only suggestive. Noradrenaline might be synthesized via norsynephrine or via dopamine. Norsynephrine is synthesized in haemolymph at the time of ecdysis in Periplaneta (Lake and others, I97O), but the wider significance of norsynephrine is not yet clear. Similarly, routes of messenger catecholamine degradation are unknown. It appears that the amine oxidase system m a y not be generally involved in this role because some arthropods seem to lack an enzyme that can degrade aromatic monoamines (Boadle and Blaschko, i968 ). Converging evidence for this comes from the histochemical fluorescence studies, which report either negligible or minimal increases in fluorescence intensity after treatment with monoamine oxidase inhibitors. There is as yet no solid evidence that the catechol O-methyl transferase system occurs in arthropods. Clearly the study of metabolic pathways of messenger catecholamines offers interesting possibilities because it will enable comparisons with the wellstudied vertebrates. For these studies it is absolutely essential that specific tissues from insects of known age be used. It is to be hoped that the days of grinding up insects whole for such studies are over. Besides their role as messenger substances, catecholamines are important as precursors for cuticular formation. Here our knowledge is almost restricted to insects. Such substances as DOPA, dopamine, tyramine, N-acetyltyramine, N-acetyldopamine, and norsynephrine and probably others are synthesized in the course of the complex events occurring during the laying down of the new cuticle after ecdysis. N-acetyldopamine seems to be the central catecholamine derivative involved and it appears that the pathway of NADA synthesis m a y vary from animal to animal. In some, e.g., the American cockroach, decarboxylation of tyrosine to tyramine is followed by hydroxylation to dopamine. In others, the main pathway is hydroxylation of tyrosine to DOPA, which is subsequently decarboxylated to dopamine. I t appears, in some cases, that both pathways m a y occur simultaneously in the same animal. A

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principal characteristic o f ' tanning ' catecholamines is that their synthesis is turned on and off in step with the moulting cycle. By contrast, it would seem unlikely that that messenger catecholamine pool should wax and wane in size, since this system must function more or less the same before, during, and after a moult. In the American cockroach, for example, extensive biosynthesis of NADA continues for at least 2 or 3 days after ecdysis. During this time the animals exhibit m a n y normal behavioural traits. The simultaneous occurrence of two functional catecholamine systems in the same animal raises an intriguing question: H o w are the arthropod's messenger catecholamine systems protected from the sympathomimetic amines produced during the period of cuticular tanning? I f the amines are synthesized in haemocytes (Whitehead, 1969; Mills and Whitehead, 197o), they may never be able to penetrate to potential catecholaminoceptive sites. Packaging, then, may actually represent a protective mechanism. Mills and Whitehead (197o) apparently think, however, that the newly synthesized dopamine m a y pass again into the haemolymph to be taken up by the epidermal cells, or, as an alternative possibility, that the haemocytes containing the tyrosine-metabolizing enzymes may burst as a result of osmotic tbrces generated by changes in permeability after the action of the diuretic hormone. Thus the enzymes enter the haemolymph and presumably catalyse the biosynthesis of dopamine there. In either of these cases dopamine would be produced in the haemolymph and a role for haemocytes in a protective mechanism has to be abandoned. Perhaps the passage of dopamine into the epidermal cells occurs in another way, such as the cells attaching to the epidermal layer basement m e m b r a n e and releasing it directly into the epidermal cells. T h a t the messenger catecholamine systems are functional and responsive even shortly after ecdysis is indicated by Jacobs's (197o) observation that injection of dopamine or noradrenaline into newly ecdysed Drosophila adults produces pronounced behavioural effects. This would indicate the need for some protective mechanism.

27o

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Comp. gen. Pharmac.

It would be valuable to know more about catecholamine derivatives that could have the responsivity of catecholaminoceptive been mistakenly identified as adrenaline. A systems both during and after completion fluorimetric study by von Euler (I 961) could of tanning, and to know more about not detect adrenaline in extracts of Pieris the pharmacological activity of NADA. brassicae larvae, and Gregerman and Wald Relationships between life-stage and cate- (1952) could not show it by paper chromatocholamine sensitivity of excitable systems graphy. No evidence exists for a methyl transappear not to have been considered. The ferase enzyme in arthropods that synthesizes enzymes synthesizing ' t a n n i n g ' catechol- adrenaline from noradrenaline. The histoamines are known to be ultimately under the chemical fluorescence studies of Frontali control of ecdysone (Karlson and Sekeris, (1968), K l e m m (1968), Elofl~on and others 1962b ). Probably the messenger catechol- (1966, 1968), and others unanimously report amine systems must go on with their functions failure to identify adrenaline-containing cells. during tanning as well as after it, so it would Frontali and Haggendal (1969) could find no seem that the synthesizing enzymes would adrenaline in their analyses of the cockroach not undergo the kind of variation in activity brain, nor could Bj6rklund and others (i 97 o) that the tanning enzymes do. These observa- in a study of Trichoptera. O f course it is tions do not necessarily mean that the impossible to prove that a compound is not messenger and tanning catecholamine systems present. Based on the above observations are completely independent. It m a y be that and arguments, it seems fair to say that there feedback from the sympathomimetic sub- is no convincing evidence that adrenaline stances produced for tanning releases or occurs in arthropods. modulates specific behaviour patterns associated with ecdysis via effects on the messenger ACKNOWLEDGEMENTS I am grateful to Professors T. L. Hopkins and systems. Studies of the interactions of these Ernst Florey for support and encouragement two systems will be most profitable. during various stages of this work. Is adrenaline synthesized in arthropods? The importance of the question warrants its being considered in some detail. Only two studies with modern methods have given REFERENCES positive indications. Ostlund (I954) com- ADELUNG, D. (I969), 'Die Ausschtittung und bined paper chromatography with dual Funktion yon H~iutungshormon w~ihrend eines Zwischenh~iutungsintervalls bei der Strandbioassay on fowl rectal caecum and cat's bloodkrabbe Carcinus maenas L.', Z. Naturf., 246, pressure. These procedures should have been 1447-I455. entirely satisfactory for assays of vertebrate ADOLVn, A. R. (1966), ' Excitation and inhibition tissues, for which they were developed. But of electrical activity in the Limulus eye by neurothey were used for extracts of whole insects pharmacological agents', in The Functional Organization of the Compound Eye (ed. BERNHARD, which, as we now know, contain a whole C. G.), pp. 465-48i. Oxford: Pergamon. gamut of sympathomimetics and possible AXELROD, J., ENSCOE, J. K., SERAn, S., and interfering substances, any one or combinaWITXOV, B. (I958), ' O-methylation, the princition of which could lead to mistaken identipal pathway for the metabolism of epinephrine and norepinephrine in the rat', Biochim. fication of adrenaline. Such compounds as Biophys. Acta, 27, 2 IO-°II. dopamine, N-acetyldopamine, tyramine, NJ., and LAROCHE, M. J. (z959), acetyltyramine, DOPA, norsynephrine as AXELROD, 'Inhibitors of O-methylation of epinephrine well as noradrenaline are present, and in and norepinephrine in vitro and in vivo ', Science, addition there are probably others yet N.Y., x3o, 800. unidentified. Such arguments apply also to BACQ, Z. M., GOSSELIN, L., DRESSE, A., and RENSON, J. (1959) , 'Inhibition of O-methylthe study of Dresse and others (196o) who transferse by catechol and sensitization to used the fluorimetric technique to assay epinephrine ', Science, N.Y., I3O, 453-454. whole-body extracts of silkworm larvae. BAILEY, S. W. (I954), 'Hardness of arthropod Silkworms are known to contain other mouthparts ', Nature, Lond., x73, 5o3.

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