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Dopamine and 5-hydroxytryptamine in the brain of the honeybee, apis mellifera: A review
0306-4492/88$3.00+ 0.00 0 1988Pergamon Press plc
Camp. fiiochem. Physioi. Vol. 9lC. No. I, pp. 133-137, 1988
Printed in Great Britain
DOPA~INE AND 5-HYDROXYTRYPTA~IN~ THE BRAIN OF THE HONEYBEE, APIS MELLJFERA: A REVIEW A. R. MERCER and D.
IN
FLANAGAN
Department of Zoology, University of Otago, Dunedin, New
Zealand
(Recehed 3 March 1988)
J. H. Welsh’s comparative studies drew attention to the extensive distribution of the catecholamines dopamine (DA) and noradrenaline (NA) and the indolalkylamine S-hydroxytryptamine (SHT, serotonin) in invertebrates (Welsh, 1968, 1972; Welsh and Moorhead, 1960). There is now convincing evidence that DA and 5-HT function as neurotransmitters, neuromodulators, and neurohormones in the invertebrate nervous system. For example, 5-HT plays an important role as a neuromodulator in the gill withdrawal reflex of the mollusc, Ap&.siu (Klein and Kandel, 1978); fluid secretion in the salivary gland of the fly, Cul~ip~oru, is regulated by 5-HT (Berridge, 1977; Berridge and Heslop, 1982), and DA appears to be a neurotransmitter in the salivary glands of many insect species (Evans, 1980; House and Ginsborg, 1982). Evidence for the roles of biogenic amines in the insect nervous system has been examined in a number of review articles, including those of Klemm (1976), Evans (1980), and Mercer (1987). Currently, more is known about the functions of DA and S-HT in the insect peripheral nervous system than the central nervous system, but with the development of highly sensitive assays for biogenic amines, the application of immunocytochemistry, and improvements in electrophysiologicai recording techniques, attention is now being focussed on central aminergic systems, and the role of biogenic amines in the insect brain. This paper reviews the distribution of DA and 5-HT in the brain of the honeybee, Apis mellifera, and examines evidence for the roles of biogenic amines in the brain of this insect.
ANATOMY OF THE BEE BRAIN
A brief outline of the anatomy of the bee brain is presented here for orientation purposes. A comprehensive description of the comparative neuroanatomy of the insect brain is provided by Mobbs (1985). The insect brain can be subdivided into 3 major regions; the protocerebrum, or midbrain, flanked by the optic lobes, the deutocerebrum which gives rise to the roots of the antenna1 nerves, and the tritocerebrum, a small region of neuropil that connects the
brain with the stomatogastric nervous system. The tritocerebrum has received little attention to date and will not be considered further in this discussion. The protocerebrum is dominated by the distinctive and highly structured mushroom bodies or corpora pedunculata. In the honeybee, each mushroom body consists of 2 cup-shaped calyces connected via a stalk (pedunculus) to 2 lobes, the alpha-lobe which projects anteriorly, and the beta-lobe which projects ventrally in the brain neuropil (Fig. la, b). Between the 2 mushroom bodies is a central complex consisting of the central body (Fig. la, b), 2 ventral noduli posterior to the central body, and the protocerebral bridge which lies in the posterior protocerebral neuropil. Three distinct ganglia, the lamina, lobula, and medulla make up the optic lobes which lie on either side of the protocerebrum (Fig. la). The primary olfactory centres of the brain, the antenna1 lobes (Fig. la), are situated in the deutocerebrum, which lies beneath the ventral margin of the protocerebral neuropil.
QUANTITATIVE DISTRIBUTION OF DA AND S-HT
The primary catecholamine, DA, was identified in whole adult honeybees by t)stlund in 1954 using chromatography and bioassay techniques. Using a fluorescence based assay, Welsh and Moorhead (1960) identified 5-HT in the head of the honeybee. From these early studies, however, it was not possible to determine whether or not DA and 5-HT were localised in the nervous system of the honeybee. High performance liquid chromatography (HPLC) using el~trochemical detection methods has been used more recently to measure DA, NA, and 5-HT in nervous tissues of the honeybee. Approximately 25 pmol DA, 1 pmol NA, and 21 pmol 5-HT were detected in the brain minus optic lobes (“cerebral ganglion”) of the bee (Mercer et al., 1983). In the optic lobe, approximately 8 pmol DA, 0.5 pmol NA, and IO pmol .5-HT were found. Most of the DA was located in the retina of the optic lobes, only very low levels were found in the lamina and lobula, and none was detected in the neuropil of the medulla. NA could not be detected in the antenna1 lobes of the bee brain, but approximately 1 pmol DA and 3 pmol 5-HT were found in this region of the brain. The levels of 133
134
Fig. I (a) Computer generated view of major structures in the brain of the honeybee reconstructed f rom The IOpn I sections. (Bar = 250 III”). (b) Frontal silver-stained section through the median protocerebrum. upper and lower parts of the central body can be identified, as can the [I-lobes and calyces of the with mush1 -oom bodies. (Bar = 100 /cm). (c) Section of the brain at a similar depth to that m (b) treated > Bright bands of fluorescence can be observed in both the g)Yox! flit acid for fluorescence histochcmistry. stalks of the mushrc ,om upper and lower regions of the central body, and in the P-lobes and peduncular bodie! 3. (Bar = 100pm). Abbreviations: x. alpha lobe of mushroom body: a.l., antenna1 lobe; p. 8-i lobe ofmu shroom body: c.b., central body of the central complex: c.b.(l), lower part of the central body: c.b .(u). upper part of the central body: I. lobula: la. lamina; l.c., lateral calyx of the mushroom body; mc.. met han calyx of the mushroom body: me. medulla: p. peduncular stalk of the mushroom body.
DA and 5HT in honeybee brain biogenic amines in the central nervous system (CNS) of the bee are very similar to those reported for the CNS of the cockroach and the locust (Evans, 1978; Dymond and Evans, 1979; Sloley and Owen, 1982).
DISTRIBUTION OF MONOAMINERGIC
135
5-HT-containing neurones that project into the central body and mushroom body neuropils of the bee brain also remains unclear. It is hoped that some of the aminergic neurones identified in the brain of the honeybee will prove to be accessible to detailed experimental analysis. The “deutocerebral giant” (see above) offers one exciting possibility.
NEURONES
An important prerequisite for determining the functions of DA and 5-HT in the insect brain is the identification of cells containing these amines. Catecholamines and indolalkylamines were first localised in the brain of the honeybee using the Falck-Hillarp fluorescence histochemistry technique (Elofsson and Klemm, 1972; Klemm, 1974). More recently, fluorescence histochemistry with glyoxylic acid has been used in conjunction with detailed neuroanatomical studies to investigate the distribution of fluorogenic amines in the bee brain (Mercer et al., 1983). After treatment with glyoxylic acid, fluorescent fibres can be seen throughout the protocerebrum of the brain, concentrated in the mushroom body and central body neuropils (Fig. Ic). Weak, rapidly fading fluorescence, characteristic of 5-HT, is found in the optic lobes, but long-lasting green fluorescence, typical of catecholamines, is not apparent in this region of the brain. Fibres in the antenna1 lobes which fluoresce after treatment with glyoxylic acid are restricted to the periphery of the “olfactory glomeruli”, where sensory neurones converge on local interneurones and on extrinsic fibres, including second order olfactory interneurones that project to the calyces of the mushroom bodies. Amine-containing cell bodies are located beneath the calyces of the mushroom bodies, in the lateral protocerebrum, between the protocerebrum and deutocerebrum, and in the posterior medial protocerebral rind. Identifying cell bodies of amine containing neurones and their projections through the brain neuropil has proved difficult using fluorescence histochemistry. To some degree this problem has been overcome by the application of immunocytochemical techniques. Fibres in the antenna1 lobes of the bee brain that stain with antibody raised to 5-HT, for example, have been found to be derived from a single interneurone that connects the antenna1 and dorsal lobes of the deutocerebrum with the suboesophageal ganglion and descends into the ventral nerve cord (Rehder et al., 1987). Rehder and coworkers refer to this neurone as the “deutocerebral giant”. Some 20-30 5-HT-immunoreactive cell bodies have been identified at the anterior ventral margin of the lobula and medulla in the optic lobes of the honeybee (Schurmann and Klemm, 1984). These cells project to the medulla and lamina of the optic lobes. 5-HT-immunoreactive neurones in the lamina show a striking overlap with fibres that stain with antibody raised to gamma-aminobutyric acid (GABA) (Bicker et al., 1987). Both the 5-HT- and the GABAimmunoreactive fibres derive from a set of at least 5 widefield tangential cells that invade the lamina at its posterior margin. 5-HT-immunoreactive fibres in the lobula of the honeybee originate in the lateral protocerebrum of the brain (Schiinnann and Klemm, 1984). The cell bodies of these neurones have not yet been identified. The location of the cell bodies of
ANALYSIS OF AMINE FUNCTION
Attempts to elucidate the physiological functions of biogenic amines within neuropils of the insect brain are hampered currently by insufficient knowledge of brain function in general in insects. The highly structured central body and mushroom body neuropils in the protocerebrum of the brain, for example, have been the subject of numerous studies over the years (see Mobbs, 1985) but the functions of these higher brain centres remain obscure. There is some evidence to suggest that DA and 5-HT modulate activity within the insect brain, perhaps controlling information being passed through conventional synapses, or regulating responses of target cells to classical transmitters in the brain. The number of monoamine-containing cell bodies identified in the brain of the honeybee is small in relation to the total number of brain neurones (8.5 x 105) estimated by Witthdft (1967). Many cells that contain DA or 5-HT in the insect brain appear to be widefield neurones that invade several regions of the brain neuropil (Klemm and Sundler, 1983; Schiirmann and Klemm, 1984) suggesting that DA and 5-HT may be involved in the simultaneous control of activity within widely separated areas of the brain neuropil. The distribution of monoamine-containing neurones in highly structured neuropils, such as the mushroom bodies, suggests that monoamines may control the activity of whole assemblies of neurones within the insect brain (see Evans, 1980). A modulatory role for DA is indicated also by the finding that DA injected into the mushroom bodies has long-term effects on the size of potentials, evoked by stimulation of the antennae with air or scent. recorded in the alpha-lobe of the mushroom bodies of the bee brain (Mercer and Erber, 1983). Unfortunately, little is known as yet about the receptors that mediate the effects of biogenie amines in the insect brain. Both DA and 5-HT have been implicated in the regulation of locomotor activity in insects (see Evans, 1980; Mercer, 1987). Muszynska-Pytel and Cymborowski (1978a) suggest, for example, that release of 5-HT at night from the central body neuropil of the cricket reduces activity of inhibitory cells in the pars intercerebralis of the cricket brain, thus increasing overall locomotor activity in this insect during the night phase. 5-HT levels in the brain of the cricket, however, show no direct correlation with circadian rhythms of locomotor activity in this insect (Muszynska-Pytel and Cymborowski, 1978b). In the brain of the cockroach, cyclic changes in DA content have been detected, and high levels of this amine correlated with maximum locomotor activity in the cockroach (Pree and Rutschke, 1983). The possible involvement of DA or 5-HT in the control of locomotor activity has not yet been examined in the honeybee. However,
A. R. MERCERand D.
136
diurnal changes in the activity of visual interneurones have been observed in the bee (Kaiser and SteinerKaiser, 1983). In Limbs, and in the gastropod mollusc Apl~xiu, 5-HT influences circadian changes in the sensitivity of the eye (Barlow et nl., 1977a. b: Eskin and Maresh, 1982; Eskin et al., 1982). It would be interesting to determine whether or not 5-HT identified in the optic lobes of the honeybee is involved in diurnal regulation of neuronal activity in this animal. The primary sensory centres of the insect brain are better understood than the higher brain centres of the protocerebrum. The distribution of catecholamineand indolalkylaminc-containing neurones in the optic lobes and antenna1 lobes of the honeybee suggest that DA and 5-HT control information within sensory processing channels of the bee brain (Mercer, 1987). Application of DA to the antenna] lobes of the honeybee reduces the size of potentials, evoked by stimulation of the antennae with puffs of air or scent, recorded in the alpha lobe of the mushroom bodies of the bee (Mercer et al., unpublished observation). DA applied to the antenna1 lobes also reduces the percentage of bees that respond to a conditioned olfactory stimulus paired to a food reward in a single conditioned olfactory stimulus paired to a food reward in a single conditioning trial (Macmillan and Mercer, 1987). Interpretation of these findings awaits further investigation of the effects of amines on output of information from the antenna1 lobes of the bee brain. and characterization of receptors that mediate the actions of biogenic amines in the brain of the honeybee. The honeybee provides an excellent model for neuroethological research. The field of honeybee neurobiology and behaviour has grown rapidly over recent years (see Menzel and Mercer, 1987). Developments in immunohistochemistry, and improvements in cell recording techniques are providing the tools for detailed analysis of the neurophysiology and neurochemistry of the bee brain. Application of these tools in studies closely coordinated with detailed neuroanatomical. biochemical, and pharmacological investigations of the bee brain will provide the key to eiucidating the roles played by biogenic amines in the insect brain. AcknnM/rdKemmrs-We would like to thank Dr Y. Usson for the use of his computer programme for 3-D reconstruction, and Mr D. Sanderson for assistance with the photography.
REFERENCES
Berridge M. J. (1977) Cyclic AMP, calcium and fluid secretion. In Transport of Ions and Water in Animals
(Edited by Gupta B. L., Moreton R. B.. Oschman J. L. and Wall 8. J.), pp‘ 225238. Academic Press, London, Berridge M. J. and Heslop J. P. (1982) Receptor mechanisms mediating the actions of 5-hydroxytryptamine. In Neuropharmacoiogy of Insects, pp. 260-270. Ciba Foundation Symposium 88. Pitman, London, Bicker G., Schafer S. and Rehder V. (1987) Chemical neuroanatomy of the honeybee brain. In Neurobiology and Behatior in Honeybees (Edited by Menzel R. and Mercer A. R.). Springer. Berlin.
FLANAGAN
Dymond G. R. and Evans P. D. (1979) Biogenic ammes in the nervous system of the cockroach, Periplunera americana: association of octopamine with mushroom bodies and dorsal unpaired median (DUM) neurones. Insecr B&hem.
9, 535.-545.
Elofsson R. and Klemm N. (1972) Monoamine-containing neurones in the optic ganglia of crustaceans and insect; Z. Zeliforsch. mikrosk. Anar. 133. 4755499. Evans P.-D. (1978) Octopamine distribution in the insect nervous system. J. Neurorhem. 30, 1009-1013. Evans P. D. (1980) Biogenic amines in the insect nervous system. Adr. Insect Physiol. 15, 317-473. House C. R. and Ginsborg B. L. (1982) Properties of dopamine receptors at a neuroglandular synapse. In Neurophurmacolog! ofInsecfs, pp. 32 -43. Ciba Foundation Symposium 88. Pitman. London. Klein K. and Kandel E. R. (1978) Presynaptic modulation of voltage-dependent Ca+ current: mechanism for behavioural sensitization in ApIwiu cui~fitrnica. Proc. nom. Acad. Sci. L’S;4 75, 351223516. Klemm N. (1974) Vergieichend-histochemische Untersuchungen iiber die Verteilung monoamine-haltiger Strukturen im Oberschlundganglion von Angehorigen verschiedener InsektenOrdnungen. Entomol. Germ. 1. 2 I -49. Klemm N. (1976) Histochemistry of putative transmitter substances in the insect brain. Prog. ~~eurobio/. 7.999169. Klemm N. and Sundler F. (1983) Organisation of catecholamine and serotonin-immunor~active neurons m the corpora pedunculata of the desert locust, Schistocerca gregaria Forsk. Neurosri. Lelf. 36, 13-17. Macmillan C. S. and Mercer A. R. (1987) An investigation of the role of dopamine in the antenna1 lobes of the honeybee. Apis meI(ifera. .I. camp. Physiol. A 160, 359-366. Menzel R. and Mercer Behavior of Honeybees.
A. R. (1987) ~etlrobioIog~ and Springer, Berlin. Mercer A. R. (1987) Biogenic amines in the insect brain. In Arthropod
Brain:
Its
Eroiution,
Structure
(Edited by Gupta B. L.), pp. 399414. Wiley York. Mercer A. R. and Erber J. (1983) The effects evoked potentials recorded in the mushroom bee brain. J. camp. Phyfiol. 151, 469-476. Mercer A. R.. Mobbs P. G.. Davenport A. P. D. (I 983) Biogenic amines in the brain of Apis mellifera.
Mobbs
Cell
and Functions
& Sons, New of amines on bodies of the P. and Evans the honeybee.
Tiss. Res. 234. 655677.
P. G. (1985)
Brain structure. In Comprehensiw Insect Physiology, Biochemtr/r>, and Pharmacology. Vol 5. Ntrrous System. Sirucrure and Motor Funcrkn (Edited by
Kerkut G. A. and Gilbert L. 1.). pp. 299 370. Pergamon Press, Oxford. Muszynska-Pytel M. and Cymborowski B. (1978a) The role of serotonin in regulation of the circadian rhythms of locomotor activity-in the cricket (A&eta domesricus L.). 11. Distribution of serotonin and variations in different brain structures. Comp. Biochem. Ph,~siol. 59C, 17 20. ~us~ynska-Pytel M. and C~borowski B. (1978b) The role of serotonin in regulation of the circadian rhythms of locomotor activity in the cricket (Achefa domesticus L.). I. Circadian variations in serotonin concentration in the brain and hemolymph. Comp. Biochem. Ph?jsiol. 59C, 13-15.
ostlund E. (1954) The distribution of catecholamines in lower animals and their effect on the heart. Acia ph_v.rio/. wand. 31, Suppl. I12 Pree J. and Rutschke E. (1983) Zur circadian Rhythmik des Dopamingehaltes in Gehirn von Periplanera americana L. Zool. Jh. Physial. 87, 455460. Rehder V., Bicker G. and Hammer M. (1987) Serotoninimmunoreactive neurons in the antenna1 lobes and suboesophageal ganglion of the honeybee. Cell Tiss. Res. 247, 59-66.
DA and 5HT in honeybee Schiirmann F. W. and Klemm N. (1984) Serotoninimmunoreactive neurons in the brain of the honeybee. J. cotnp. Neural. 225, 570-580. Sioley B. D. and Owen M. D. (I 982) The effects of reserpine on amine concentrations in the nervous system of the cockroach (Periplaneta americana). Insect Bioehem. 12, 469-476. Welsh J. H. (1968) Distribution of serotonin in the nervous system of various animal species. A&. Pharmac. 6A, 171-188.
brain
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Welsh J. H. (1972) Catecholamines in the Invertebrates. In Handbook of Experimental Pharmacology (Edited by Blaschko H. and Muscholl F.), Vol. 33, pp. 79109. Springer, Berlin. Welsh J. H. and Moorhead M. (1960) The quantitative distribution of 5-hydroxytryptamine in the invertebrates, especially in their nervous systems. J. Neurochem. 6, 146-169. Witthoft W. (1967) Absolute Anzahl und Verteilung der Zellen im Hirn der Honigbiene. Z. morphol. 61, 16CL184.
Camp. fiiochem. Physioi. Vol. 9lC. No. I, pp. 133-137, 1988
Printed in Great Britain
DOPA~INE AND 5-HYDROXYTRYPTA~IN~ THE BRAIN OF THE HONEYBEE, APIS MELLJFERA: A REVIEW A. R. MERCER and D.
IN
FLANAGAN
Department of Zoology, University of Otago, Dunedin, New
Zealand
(Recehed 3 March 1988)
J. H. Welsh’s comparative studies drew attention to the extensive distribution of the catecholamines dopamine (DA) and noradrenaline (NA) and the indolalkylamine S-hydroxytryptamine (SHT, serotonin) in invertebrates (Welsh, 1968, 1972; Welsh and Moorhead, 1960). There is now convincing evidence that DA and 5-HT function as neurotransmitters, neuromodulators, and neurohormones in the invertebrate nervous system. For example, 5-HT plays an important role as a neuromodulator in the gill withdrawal reflex of the mollusc, Ap&.siu (Klein and Kandel, 1978); fluid secretion in the salivary gland of the fly, Cul~ip~oru, is regulated by 5-HT (Berridge, 1977; Berridge and Heslop, 1982), and DA appears to be a neurotransmitter in the salivary glands of many insect species (Evans, 1980; House and Ginsborg, 1982). Evidence for the roles of biogenic amines in the insect nervous system has been examined in a number of review articles, including those of Klemm (1976), Evans (1980), and Mercer (1987). Currently, more is known about the functions of DA and S-HT in the insect peripheral nervous system than the central nervous system, but with the development of highly sensitive assays for biogenic amines, the application of immunocytochemistry, and improvements in electrophysiologicai recording techniques, attention is now being focussed on central aminergic systems, and the role of biogenic amines in the insect brain. This paper reviews the distribution of DA and 5-HT in the brain of the honeybee, Apis mellifera, and examines evidence for the roles of biogenic amines in the brain of this insect.
ANATOMY OF THE BEE BRAIN
A brief outline of the anatomy of the bee brain is presented here for orientation purposes. A comprehensive description of the comparative neuroanatomy of the insect brain is provided by Mobbs (1985). The insect brain can be subdivided into 3 major regions; the protocerebrum, or midbrain, flanked by the optic lobes, the deutocerebrum which gives rise to the roots of the antenna1 nerves, and the tritocerebrum, a small region of neuropil that connects the
brain with the stomatogastric nervous system. The tritocerebrum has received little attention to date and will not be considered further in this discussion. The protocerebrum is dominated by the distinctive and highly structured mushroom bodies or corpora pedunculata. In the honeybee, each mushroom body consists of 2 cup-shaped calyces connected via a stalk (pedunculus) to 2 lobes, the alpha-lobe which projects anteriorly, and the beta-lobe which projects ventrally in the brain neuropil (Fig. la, b). Between the 2 mushroom bodies is a central complex consisting of the central body (Fig. la, b), 2 ventral noduli posterior to the central body, and the protocerebral bridge which lies in the posterior protocerebral neuropil. Three distinct ganglia, the lamina, lobula, and medulla make up the optic lobes which lie on either side of the protocerebrum (Fig. la). The primary olfactory centres of the brain, the antenna1 lobes (Fig. la), are situated in the deutocerebrum, which lies beneath the ventral margin of the protocerebral neuropil.
QUANTITATIVE DISTRIBUTION OF DA AND S-HT
The primary catecholamine, DA, was identified in whole adult honeybees by t)stlund in 1954 using chromatography and bioassay techniques. Using a fluorescence based assay, Welsh and Moorhead (1960) identified 5-HT in the head of the honeybee. From these early studies, however, it was not possible to determine whether or not DA and 5-HT were localised in the nervous system of the honeybee. High performance liquid chromatography (HPLC) using el~trochemical detection methods has been used more recently to measure DA, NA, and 5-HT in nervous tissues of the honeybee. Approximately 25 pmol DA, 1 pmol NA, and 21 pmol 5-HT were detected in the brain minus optic lobes (“cerebral ganglion”) of the bee (Mercer et al., 1983). In the optic lobe, approximately 8 pmol DA, 0.5 pmol NA, and IO pmol .5-HT were found. Most of the DA was located in the retina of the optic lobes, only very low levels were found in the lamina and lobula, and none was detected in the neuropil of the medulla. NA could not be detected in the antenna1 lobes of the bee brain, but approximately 1 pmol DA and 3 pmol 5-HT were found in this region of the brain. The levels of 133
134
Fig. I (a) Computer generated view of major structures in the brain of the honeybee reconstructed f rom The IOpn I sections. (Bar = 250 III”). (b) Frontal silver-stained section through the median protocerebrum. upper and lower parts of the central body can be identified, as can the [I-lobes and calyces of the with mush1 -oom bodies. (Bar = 100 /cm). (c) Section of the brain at a similar depth to that m (b) treated > Bright bands of fluorescence can be observed in both the g)Yox! flit acid for fluorescence histochcmistry. stalks of the mushrc ,om upper and lower regions of the central body, and in the P-lobes and peduncular bodie! 3. (Bar = 100pm). Abbreviations: x. alpha lobe of mushroom body: a.l., antenna1 lobe; p. 8-i lobe ofmu shroom body: c.b., central body of the central complex: c.b.(l), lower part of the central body: c.b .(u). upper part of the central body: I. lobula: la. lamina; l.c., lateral calyx of the mushroom body; mc.. met han calyx of the mushroom body: me. medulla: p. peduncular stalk of the mushroom body.
DA and 5HT in honeybee brain biogenic amines in the central nervous system (CNS) of the bee are very similar to those reported for the CNS of the cockroach and the locust (Evans, 1978; Dymond and Evans, 1979; Sloley and Owen, 1982).
DISTRIBUTION OF MONOAMINERGIC
135
5-HT-containing neurones that project into the central body and mushroom body neuropils of the bee brain also remains unclear. It is hoped that some of the aminergic neurones identified in the brain of the honeybee will prove to be accessible to detailed experimental analysis. The “deutocerebral giant” (see above) offers one exciting possibility.
NEURONES
An important prerequisite for determining the functions of DA and 5-HT in the insect brain is the identification of cells containing these amines. Catecholamines and indolalkylamines were first localised in the brain of the honeybee using the Falck-Hillarp fluorescence histochemistry technique (Elofsson and Klemm, 1972; Klemm, 1974). More recently, fluorescence histochemistry with glyoxylic acid has been used in conjunction with detailed neuroanatomical studies to investigate the distribution of fluorogenic amines in the bee brain (Mercer et al., 1983). After treatment with glyoxylic acid, fluorescent fibres can be seen throughout the protocerebrum of the brain, concentrated in the mushroom body and central body neuropils (Fig. Ic). Weak, rapidly fading fluorescence, characteristic of 5-HT, is found in the optic lobes, but long-lasting green fluorescence, typical of catecholamines, is not apparent in this region of the brain. Fibres in the antenna1 lobes which fluoresce after treatment with glyoxylic acid are restricted to the periphery of the “olfactory glomeruli”, where sensory neurones converge on local interneurones and on extrinsic fibres, including second order olfactory interneurones that project to the calyces of the mushroom bodies. Amine-containing cell bodies are located beneath the calyces of the mushroom bodies, in the lateral protocerebrum, between the protocerebrum and deutocerebrum, and in the posterior medial protocerebral rind. Identifying cell bodies of amine containing neurones and their projections through the brain neuropil has proved difficult using fluorescence histochemistry. To some degree this problem has been overcome by the application of immunocytochemical techniques. Fibres in the antenna1 lobes of the bee brain that stain with antibody raised to 5-HT, for example, have been found to be derived from a single interneurone that connects the antenna1 and dorsal lobes of the deutocerebrum with the suboesophageal ganglion and descends into the ventral nerve cord (Rehder et al., 1987). Rehder and coworkers refer to this neurone as the “deutocerebral giant”. Some 20-30 5-HT-immunoreactive cell bodies have been identified at the anterior ventral margin of the lobula and medulla in the optic lobes of the honeybee (Schurmann and Klemm, 1984). These cells project to the medulla and lamina of the optic lobes. 5-HT-immunoreactive neurones in the lamina show a striking overlap with fibres that stain with antibody raised to gamma-aminobutyric acid (GABA) (Bicker et al., 1987). Both the 5-HT- and the GABAimmunoreactive fibres derive from a set of at least 5 widefield tangential cells that invade the lamina at its posterior margin. 5-HT-immunoreactive fibres in the lobula of the honeybee originate in the lateral protocerebrum of the brain (Schiinnann and Klemm, 1984). The cell bodies of these neurones have not yet been identified. The location of the cell bodies of
ANALYSIS OF AMINE FUNCTION
Attempts to elucidate the physiological functions of biogenic amines within neuropils of the insect brain are hampered currently by insufficient knowledge of brain function in general in insects. The highly structured central body and mushroom body neuropils in the protocerebrum of the brain, for example, have been the subject of numerous studies over the years (see Mobbs, 1985) but the functions of these higher brain centres remain obscure. There is some evidence to suggest that DA and 5-HT modulate activity within the insect brain, perhaps controlling information being passed through conventional synapses, or regulating responses of target cells to classical transmitters in the brain. The number of monoamine-containing cell bodies identified in the brain of the honeybee is small in relation to the total number of brain neurones (8.5 x 105) estimated by Witthdft (1967). Many cells that contain DA or 5-HT in the insect brain appear to be widefield neurones that invade several regions of the brain neuropil (Klemm and Sundler, 1983; Schiirmann and Klemm, 1984) suggesting that DA and 5-HT may be involved in the simultaneous control of activity within widely separated areas of the brain neuropil. The distribution of monoamine-containing neurones in highly structured neuropils, such as the mushroom bodies, suggests that monoamines may control the activity of whole assemblies of neurones within the insect brain (see Evans, 1980). A modulatory role for DA is indicated also by the finding that DA injected into the mushroom bodies has long-term effects on the size of potentials, evoked by stimulation of the antennae with air or scent. recorded in the alpha-lobe of the mushroom bodies of the bee brain (Mercer and Erber, 1983). Unfortunately, little is known as yet about the receptors that mediate the effects of biogenie amines in the insect brain. Both DA and 5-HT have been implicated in the regulation of locomotor activity in insects (see Evans, 1980; Mercer, 1987). Muszynska-Pytel and Cymborowski (1978a) suggest, for example, that release of 5-HT at night from the central body neuropil of the cricket reduces activity of inhibitory cells in the pars intercerebralis of the cricket brain, thus increasing overall locomotor activity in this insect during the night phase. 5-HT levels in the brain of the cricket, however, show no direct correlation with circadian rhythms of locomotor activity in this insect (Muszynska-Pytel and Cymborowski, 1978b). In the brain of the cockroach, cyclic changes in DA content have been detected, and high levels of this amine correlated with maximum locomotor activity in the cockroach (Pree and Rutschke, 1983). The possible involvement of DA or 5-HT in the control of locomotor activity has not yet been examined in the honeybee. However,
A. R. MERCERand D.
136
diurnal changes in the activity of visual interneurones have been observed in the bee (Kaiser and SteinerKaiser, 1983). In Limbs, and in the gastropod mollusc Apl~xiu, 5-HT influences circadian changes in the sensitivity of the eye (Barlow et nl., 1977a. b: Eskin and Maresh, 1982; Eskin et al., 1982). It would be interesting to determine whether or not 5-HT identified in the optic lobes of the honeybee is involved in diurnal regulation of neuronal activity in this animal. The primary sensory centres of the insect brain are better understood than the higher brain centres of the protocerebrum. The distribution of catecholamineand indolalkylaminc-containing neurones in the optic lobes and antenna1 lobes of the honeybee suggest that DA and 5-HT control information within sensory processing channels of the bee brain (Mercer, 1987). Application of DA to the antenna] lobes of the honeybee reduces the size of potentials, evoked by stimulation of the antennae with puffs of air or scent, recorded in the alpha lobe of the mushroom bodies of the bee (Mercer et al., unpublished observation). DA applied to the antenna1 lobes also reduces the percentage of bees that respond to a conditioned olfactory stimulus paired to a food reward in a single conditioned olfactory stimulus paired to a food reward in a single conditioning trial (Macmillan and Mercer, 1987). Interpretation of these findings awaits further investigation of the effects of amines on output of information from the antenna1 lobes of the bee brain. and characterization of receptors that mediate the actions of biogenic amines in the brain of the honeybee. The honeybee provides an excellent model for neuroethological research. The field of honeybee neurobiology and behaviour has grown rapidly over recent years (see Menzel and Mercer, 1987). Developments in immunohistochemistry, and improvements in cell recording techniques are providing the tools for detailed analysis of the neurophysiology and neurochemistry of the bee brain. Application of these tools in studies closely coordinated with detailed neuroanatomical. biochemical, and pharmacological investigations of the bee brain will provide the key to eiucidating the roles played by biogenic amines in the insect brain. AcknnM/rdKemmrs-We would like to thank Dr Y. Usson for the use of his computer programme for 3-D reconstruction, and Mr D. Sanderson for assistance with the photography.
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