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The Control of Salivary Glands in the Stick Insect, Carausius morosus.
Spilios Asimakopoulos
A thesis submitted in conformity with the requirements for the degree of Masters of Science
Graduate Department of the Department of Zoology University of Toronto
O Copyright by Spilios Asimakopoulos (1998)
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ABSTRACT The control of salivary glands in the stick insect, Cmausius morosus
Spilios Asimakopoulos Masters of Science, 1998
Department of Zoology, University of Toronto
Tmmunohistochemistry in Camsius morosus reveals that the salivary glands are
innervated by two paired neurons, the SNl and SN2, located in the suboesophageal ganglion.
The SNl stain for tyrosine hydroxylase-like immunoreactivity, while the SN2 stain for
serotonin-like immunoreactivity. Dopamine and serotonin cause a dose-dependent increase in
cyclic AMP levels in the salivary glands, indicating cyclic AMP may play a role as a second
messenger. A partial pharmacological profile ofthe receptor shows that the dopamine receptors
are similar to vertebrate Dl-like receptors, while the serotonin receptors are similar to vertebrate
5HT2 or 5HT, receptors. Electrical stimulation of the salivary nerve also elevates cyclic AMP
levels in the salivary glands, an elevation that can be partially inhibited by 0. lmM SCH-23390
and cyp ro hep t adine.
Immunohistochemistry also reveals that the salivary glands of C m s i u s receive
GABA-like immunoreactive projections fiom the salivary nerve. The GABA-like
immunoreactive axon branches over the acini, where many varicosities are seen along the length
of the axon. GABA potentiates dopamine-induced increases in cyclic AMP content of the
salivary glands, although it has no intrinsic activity. The potentiation is completely blocked
when the salivary glands are preincubated in 2x104M 2-hydroxysactofen, a potent GAB&
channel blocker. It would appear that GABA may sewe as a neuromodulator in the salivary
glands of Carausius.
Acknowledgments
Ian Orchard, for giving me the opportunity to discover the pleasure and frustration that
comes with research, for being supportive through the whole process and for being
understanding when I wasn't sure what I wanted to do in the &hue. THANK YOU.
Megumi, Rod and V~cki, for making my stay in the lab a . enjoyable experience, for
their help and support with my research and for tolerating my bad habits! Thanks.
Angela Lange and Les Buck, who were always forthcoming with helpll advice.
Declan, for his help when I was starting out.
To the Tobe lab and its adopted members, thanks for making the experience fun and
very interesting!
To my friends, who never quite understood what I was doing, but thought it was cool
any ways. Thanks.
To my family for all their support, and a special thank you to my mother for being very
helpful and understanding when it wasn't always easy to be.
Organization of the Thesis
Chapter II was published inBiogenic Amines (Asimakopoulos S. and Orchard I. (1998).
Biogenic Amines 14. 143- 1 62) and was jointly authored with Dr. Ian Orchard. Chapter III is
being prepared for publication. All the experiments were performed by myself. Dr. Ian
Orchard, as my supervisor, rendered invaluable aid in the form of discussions and suggestions,
editorial comments on manuscripts and financial support for all research projects.
I. GENERAL INTRODUCTION
Salivary Glands, General
Salivary glands, also known as labial glands, are found in most insects, although they
appear to be absent from many species of Coieoptera (Srivastava. 1959).The primary role of
salivary glands is to aid in the digestion of food, both internally and externally. Salivary glands
produce saliva, which is composed of water, ions and proteins, the proteins being mainly
digestive enzymes such as amylases and invertases (House and Ginsborg, 1985). The saliva
also serves a lubricating function for the mouthparts involved in feeding. Salivary glands
perform other functions in some insect; they produce vasodilators and anticoagulants in th5
mosquitoe, (Gardiner, 1972) and produce silk in larval Iepidoptera (Kafatos, 1968).
Salivary glands are classified as tubular or acinar based on their structure (House and
Ginsborg, 1985). Examples of tubular glands are found in the blowfly, whereas examples of
acinar glands are found in the cockroach, locust and stick insect. Tubular salivary glands are
formed of simple tubules that have epithelial layers that are one cell thick (House and Ginsborg,
1985). Different functions are performed in distinct areas of the tubules. The tubules open to
a salivary duct which extends to the preorai cavity where saliva is secreted. Acinar salivary
glands have groups of cells that form acini, which secrete salivary products into a network of
ducts. The ducts coalesce to form a main salivary duct- which opens into the preoral cavity.
There are distinct cell types within the acini which perform different functions, such as fluid
production and enzyme production (Kendal, 1969; Whitehead, 197 1; Bland and House, 197 1;
Just and Waltz, 1996).
Salivary glands are controlled neurohormonally andlor by direct innervation. Many
insects receive innervation from the suboesophageal ganglion via the salivary nerve (Baptist,
1941; Whitehead, 1971; Altman and Ken, 1979). Some insects, such as the cockroach, receive
innervation from the stomatogastric nervous system (Davis, 1985). In addition, insects may also
receive innervation from the median-transverse nervous system, as found in the locust (Myers
and Evans, 1985; Fuse et al., 1996). Only a few insect salivary gland systems have been
characterized in any detail, and the following is a detailed description of each of these systems.
Blowfly Salivary Glands
The blowfly, Calliphora erythrocephala, has tubular salivary glands, which have been
studied in depth. The glands are comprised of two tubes that extend from the abdomen into
the thorax (House and Ginsborg, 1985). Abdominal sections of the tube secrete water, ions a ~ d
protein; thoracic sections of the tube secrete only water and ions; a small "clear region" appears
to reabsorb ions, while the duct cells probably do not alter the content of the saliva (House and
Ginsborg, 1985). The glands are not directly innervated, but they appear to be neurohormonally
controlled. Serotonin appears to be the primary neurohorrnone involved in the control of
blowfly salivary glands. Serotonin causes a dose-dependent increase in the rate of salivary
secretion (Berridge and PateI, 1968), activates potassium and chloride currents in the salivary
glands (Bemidge et al., 1975), and also elevates cyclic AMP levels(Hes1op and Benidge, 1980).
Fain and Benidge (1979) showed that serotonin activates the phospholipase C second
messanger pathway in the blowfly salivary glands. This evidence indicates that serotonin is
likely a primary control compound in the blowfly salivary glands.
FMRFamide related peptides (FalWs) may also play a role in the control of blowfly
salivary glands. Three FaRPs isolated from the thoracic ganglia of the blowfly (Culliphora
vornitoria), have been shown to induce secretion from the salivary glands (Duve et ul., 1992).
Cockroach Salivary Glands
The cockroach has acinar salivary glands composed of different cell types. Peripheral
cells mainly secrete water and ions, whereas central cells secrete mainly enzymes (lust and
Waltz, 1994). The duct cells in cockroach appear to be able to alter the composition of saliva
as it passes along the duct (House and Ginsborg, 1985). The cockroach also has a salivary
reservoir, that stores saliva prior to being released, which has some small groups of muscle
fibers associated with it (Sutherland and ChiIlseyzn, f 968).
The salivary glands of the cockroach appear to receive dual innervation, from both the
suboesophageal ganglion, and the stomatogastric nerve (Willey, 196 1). Two neurons, salivary
neuron one (SNI) and salivary neuron two (SN2), whose cell bodies lie within the
suboesophageal ganglion, project their axons to the salivary glands through the salivary nerve
(Klernm, 1972). SNl appears to contain the neurotransmitter doparnine (Gifford et al., 199 1 ;
Elia et al., 1994), while it is still unknown what neurotransmitter is contained in SN2. It does
not appear to be dopamine or serotonin. The salivary nerve also contains several smaller axons
(Whitehead, 1971), which appear to contain serotonin (Davis, 1985). These smaller axons,
which comprise the sattelite nervous system (SNS) project along the nerve close to its surface,
and have many varicosities associated with them (Davis, 1985). Whitehead (l971), using
rnethylene blue staining, showed that the salivary nerve gives off branches to anterior regions
of the salivary duct. Interestingly, the ducts do not appear to receive projections from tyrosine
hydroxylase-like immunoreactive axons (Elia et ul.., 1994). Tyrosine hydroxylase is the rate
limiting enzyme in the formation of catecholamines. Since adrenaline and noradrenaline are
either not present, or present in very small quantities in insects (Evans, 1980), tyrosine
hydroxylase-like staining is indicative of the presence of dopamine. It is not clear, therefore,
what neuroactive chemicals may be associated with the projections to the duct.
Electrical stimulation of the salivary nerve has physiological effects on the salivary
glands. Stimulation hyperpolarizes the membrane potential of salivary gland cells (House,
1973), and stimulates fluid secretion (Smith and House, 1977).
Branches from the stomatogastric nerve project to the salivary glands, where they result
in smaller axons that have many varicosities dong their length m t e h e a d , 197 1 ; Davis, 1985).
These axons stain positively for serotonin-like irnmunoreactivity (Davis, 1985). The salivary
reservoirs also receive innervation from both SN1 @lia et al., 1994), and from the
stomatogastric nerve (Davis, 1985). FMRFamide-Like irnrnunoreactivity has been described in
the salivary glands and reservoir in the cockroach, Diploptera punctata (Fuse et al., 1998).
Doparnine produces many physiolo~cd effects in the salivary glands of the cockroach.
Dopamine has been shown to stimulate fluid secretion (Bowser-Riley and House, 1976),
increase CAMP levels in the salivary glands (Grewe and Kebabian, 1982), and hyperpolarize
the membrane potential of salivary gland cells in Nauphoeta cinerea (Ginsborg et aL, 1974;
Blackman et al., 1979 ). It has aIso been shown to stimdate fluid secretion in Periplaneta
americana (Just and Waltz, 1996). Serotonin has been shown to stimulate fluid secretion in
Periplaneta (Just and Waltz, 1996) and Nauphoeta (Bowser-Riley and House, 1976), and
hyperpolarize the membrane potential of salivary gland cells in Nauphoeta (House, 1973).
Dopamine is a more potent secretogogue than serotonin in both Periplaneta and Nauphoeta.
In Nauphoeta, the threshold for secretion with serotonin is 1000 times greater than that of
dopamine, and adrenaline and noradrenaline are also more potent secretagogues than serotonin,
although there is no evidence of adrenergic innervation (Bowser-Riley and House, 1976).
The hyperpolarization response to salivary nerve stimulation and dopamine application
in Narcphoeta has been shown to be reversible at the expected equilibrium potential of
potassium (Ginsborg et al., 1974; Blackrnan et at., 1979). Thus, the hyperpolarization response
is likely due to an increased potassium conductance. The increased potassium conductance
appears to be calcium dependent (Ginsborg et al., 1980ab), but does not rely on extracellular
calcium (Ginsborg et al., 1980a). Thus, the calcium is likely released from an intracellular store
as is found associated with the endoplasmic reticulum.
Serotonin and doparnine appear to play different roles in the stimulation of fluid
secretion in the cockroach. Serotonin appears to stimulate the production of the protein
component of saliva in the central cells, whiIe dopamine stimulates the production of the fluid
component of the saliva from peripheral cells in Periplaneta ( Just and Waltz, 1996). This may
explain the different potencies of doparnine and serotonin in causing fluid secretion from the
salivary glands.
A pharmacological profile of the dopamine receptors in the salivary glands has been
established (Evans and Green, 1990a ; Evans and Green, 1990b). Vertebrate antagonists and
agonists were used to inhibit and mimic the hyperpolarization response and the increased
adenylate cyclase activity response. The receptors are pharmacologically similar to vertebrate
Dl-like receptors, which are also positively linked to adenylate cyclase. The pharmacological
evidence suggests that both the hyperpolarization response and the increase in adenylate cyclase
activity are mediated by the same receptor (Evans and Green, 1990b).
Locust Salivary Glands
The salivary gland systems of two species of locust, Schistocerca gregaria and Locusta
migratoria have been studied. The locust is similar to the cockroach in having acinar salivary
glands (Kendall, 1969). The salivary glands are composed of several cell types. The zymogenic
(central) and parietal (peripheral) cells appear to be responsible for the production of saliva
(Kendall, 1969). Salivary secretions pass through the microvilli of the zymogenic cells into the
lumen of the salivary ducts (Kendall, 1969). The salivary glands receive innervation from the
suboesophageal ganglion (Altman and Kien, 1979), and from the transverse nerves of the
prothoracic and mesothoracic ganglia (Myers and Evans, 1985; Baines and Tyrer, 1989; Fuse
et al., 1996).
The SNI and SN2 in the suboesophageal ganglion of the locust project to the salivary
glands through nerve 7b (salivary nerve) (Altman and Kien, 1979). Glyoxylic acid treatment,
radioenzymatic assays and high-performance liquid chromatography (HPLC) analysis have
shown that the SNI contains dopamine, while the SN2 contains serotonin (Gifford et al., 1991).
Imrnunohistochemistry has also shown that the SNL contains tyrosine hydroxylase-like
irnrnunoreactivity which is indicative of the presence of doparnine (Orchard et nL, 1992; Ali et
al., 1993). Imrnunohistochemistry has also confirmed the presence of serotonin in the SN2 of
the locust (Ali et al., 1993). The salivary ducts do not appear to receive tyrosine hydroxylase-
like or serotonin-like immunoreactive projections (Ali et aL, 1993). There is also
irnrnunohistochemical evidence that GABA may be colocalized within the SN2 of Locusta
(Watkins and Burrows, 1989), although its function is unknown.
Stimulation of the salivary nerve increases fluid secretion from the salivary glands of
Schistocerca (Baines and Tyrer, 1989). It has also been shown that stimulation of the salivw
nerve in Locurta leads to increased cyclic AMP levels in the salivary glands (Ali et a[., 1993).
Dopamine and serotonin can produce a variety of physiological responses in the salivary glands
of the locust. Dopamine and serotonin have been shown to cause fluid secretion from the
salivary glands of Schistocerca, with serotonin being the more potent secretagogue (Baines and
Tyrer, 1989). Doparnine and serotonin increase cyclic AMP levels in the salivary glands of
Locusta in a dose-dependent manner (Ali et al, .1993). Dopamine and serotonin have similar
dose-response curves for cyclic AMP elevation, although dopmine has a lower threshold (Ali
et al., 1993). Vertebrate agonists and antagonists were used to partially characterize the
doparnine and serotonin receptors involved in the cyclic AMP response. The dopamine
receptors are pharmacologicaily similar to vertebrate D, receptors, and the serotonin receptors
are similar to vertebrate 5HT2 receptors (Ali and Orchard, 1994).
The salivary glands also receive innervation from the transverse nerves of the prothoracic
and mesothoracic ganglia (Myers and Evans, 1985; Baines et aL, 1989; Fuse et al., 1996).
These transverse nerves stained positively for anti-FMRFarnide antisera (Myers and Evans,
1985; Fuse et al., 1996) suggesting that there may be FaRP innervation of the salivary glands.
HPLC analysis shows that FaRPs are present in the salivary glands of Locusta and Schistocercn
(Fuse et aL, 1996; Baines et al., 1989). AFRFamide and GQERNFLRFamide were partially
characterized in the salivary glands of Locusta, but did not affect cyclic AMP or cyclic GMP
levels in the salivary glands (Fuse e? al., 1996). Stimulation of the prothoracic posterior
transverse nerve has been shown to increase fluid secretion in Schistocerca (Eiaines and Tyrer,
1989), however the increase in fluid secretion was dependent upon an intact salivary nerve.
When the salivary nerve was cut, the volume and duration of fluid secretion was greatly
diminished during prothoracic transverse nerve stimulation (Baines and Tyrer, 1989). It has
also been shown that certain FaRPs can enhance fluid secretion in the presence of serotonin or
with salivary nerve stimulation. It appears that FMRFamide related peptides may play a
significant modulatory role in the control of salivation in the locust.
An axon from an octopaminergic dorsal unpaired median neuron (DUMlb) in the
metathoracic ganglion, projects to the salivary glands in locusts (Braunig et aL, 1994).
However, octopamine does not affect salivary fluid secretion or cyclic AMP levels in the
salivary glands of the locust (Baines and Tyrer, 1989; Ali et al., 1993). The role, if any, of
octopamine in locust salivary glands remains unknown.
Stick Insect Salivary Glands
The stick insect, Carausius morosus has acinar salivary glands in common with the
cockroach and locust. The salivary glands receive axonal projections from the SNl and SN2
in the suboesophageal ganglion (Ali and Orchard, 1996). Irnrnunohistochemistry indicates that
the SNl likely contains dopamine, while the SN2 likely contains serotonin. The stick insect
provides a good preparation to study the control of salivary glands in insects. The glands are
large, easily dissected, and are easily penetrated by rnicroelectrodes. These factors aid in the
study of physiological properties, especially electrophysiological effects caused by
neurotransmitters.
This study will look at the physiological effects of neurotransmitters on the salivary
glands of Carausius rnorosus, and look at the pharmacological properties of the receptors
mediating these effects. The focus will be on dopamine and serotonin, which are known to be
involved in salivary gland control in other insects. The study will also examine GABAergic
innervation of the salivary glands, and the possible physiological effects of GABA. The
salivary glands of Carausius rnorosus provide a good model system to study the control of
salivary glands in insects.
REFERENCES
Ali D.W. and Orchard I. (1994). Characterization of dopamine and serotonin receptors on the salivary glands of the locust, Locusta migratoria. Biogenic Arnines 10. 195-2 12.
Ali D.W. and Orchard I. (1996). Immunohistocheinical localization of tyrosine hydroxylase in the ventral nerve cord of the stick insect, Carausius rnorosus, including neurons innervating the salivary glands. Cell Tissue Res. 285.453-462.
Ali D.W., Orchard I. and Lange A.B. (1993). The aminergic control of locust (Locusta migratoria) salivary glands: Evidence for dopaminergic and serotonergic innervation. J. Insect Physiol. 39. 623-632.
Altman I.S. and Kien J. (1979). Suboesophageal neurons involved in head movements and feeding in locusts. Proc. R. Soc. Lond. B 205.209-227.
Baines R.A. and Tyrer N.M. (1989). The innervation of locust salivary glands. II. Physiology of excitation and modulation. J. Comp. Physiol. A 165.4074 13.
Baines R.A., Tyrer N.M. and Mason J.C. (1989). The innervation of locust salivary glands. I. Innervation and analysis of transmitters. J. Comp. Physiol. A 165. 395405.
Baptist B.A. (1941). The morphology and physiology of the salivary glands of Herniptera- Heteroptera. Q. J. Microsci. Sci. 83, 91-139.
Berridge M.J. and Pate1 N.G. (1968). Insect salivary glands stimulation of fluid secretion by 5-hydroxytyramine and adenosine-3,5-monophosphate. Science 162, 462-463.
Berridge M. J., Lindley B .D . and Prince W.T. (1975). Membrane permeability changes during stimulation of isolated salivary glands of Calliphora by 5 - h y d r o x ~ p tarnine. J. Physiol. h d . 244, 549-567.
Blackman J.G., Ginsborg B.L. and House C.R. (1979). On the effect of ionophoretically applied dopamine on the salivary gland cells of Nauphoera cinerea. J. Physiol. Lond. 287, 67-80.
Bowser-Riley F. and House C.R. (1976). The actions of some putative neurotransmitters on the cockroach salivary gland. J. exp. B i d 64. 665-676.
Braunig P., Stevenson P.A. and Evans P.D. (1994). A locust octopamine-irnmunoreactive dorsal unpaired median neurone forming tenninal networks on sympathetic nerves. J. exp. Bid. 192, 225-238.
Davis N.T. (1985). Serotonin-immunoreactive visceral nerves and neurohemal system in the cockroach Periplaneta arnen'cana (L.). Cell Tissue Res. 240, 593-600.
Duve H., Johnson A.H., Sewell J.C., Scott A.G., Orcard L, Rehfeld J.F. and Thorpe A. (1992). Isolation, stucture, and activity of Phe-Met-Arg-Phe-NHz neuropeptides (designated calliFMRFamides) from the blowfly Calliphora vornitoria. Proc. Natl. Acad. Sci. USA 89,2326-2330.
Elia A.J., Ali D.W. and Orchard I (1994). Irnmunochemical staining of tyrosine hydroxylase(TI3)-like material in the salivary glands and ventral nerve cord of the cockroach, Periplaneta americana (L.). J. Insect Physiul. 40. 67 1-68 3.
Evans P.D. (1980). Biogenic amines in the insect nervous system. Adv. Insect Physiol. 15. 3 17-473.
Evans A.M. and Green K.L. (1990a). The action of dopamine receptor antagonists on the secretory response of the cockroach salivary gland in vitro. Comp. Biochern. Physiol. 97C. 283-286.
Evans A.M. and Green K.L. (1990b). Characterization of the doparnine receptor mediating the hyperpolarization of cockroach salivary gland acinar cells in vitro. Br. J. Phamacol. 101. 103-108.
Fain J.N. and Berridge M.J. (1 979). Relationship between hormonal activation of phosphatidylinositol hydrolysis, fluid secretion and calcium flux in the blowfly salivary gland. Biochem. J. 178,4558.
Fust M., Ali D.W. and Orchard I. (1996). The distribution and partial characterization of FMRFamide-related peptides in the salivary glands of the locust, Locusta migratoria. Cell Tissue Res. 284,425-433.
Fuse M., Bendena W.G., Donly C., Tobe S.S. and Orchard I. (1998). In situ hybridization analysis of Leucornyosuppressin mRNA expression in the cockroach, Diploptera punctata. J . Cornp. Neurol. 395, 328-34 1.
Gardiner M.S. (1 972). The biology of invertebrates. New York: McGraw-Hill.
Gifford A.N., Nicholson R.A. and Pitman R.M. (1991). The dopamine and S- hydroxytlyptamine content of locust and cockroach salivary neurones. exp. Biol. 161. 405-4 14,
Ginsborg B.L., House C.R. and Silinsky E.M. (1974). Conductance changes associated with the secretory potential in the cockroach salivary gland. J. Physiol. Lond. 236,723-73 1.
Ginsborg B.L., House C.R. and Mitchell M.R. (1980a). On the role of calcium in the electrical responses of cockroach salivary gland cells to dopamine. J. Physiol. Lond. 303,325- 335.
Ginsborg B.L., House C.R. and Mitchell M.R. (1980b). A calcium readmission response recorded from Nauphoeta salivary gland acinar cells. J. Physiol. Lond. 304,437-447.
Grewe C.W. and Kebabian J.W. (1982). Dopamine stimulates production of cyclic AMP by the salivary gland of the cockroach, Nauphoeta cinerea. Cell. Mol. Neurobiol. 2,65-69.
Heslop J.P. and Berridge M.J. (1980). Changes in cyclic AMP and cyclic GMF concentrations during the actions of 5-hydroxytryptamine on an insect salivary gland. Biochem. J. 192, 247-255.
House C.R. ( 1973). An electrophysiological study of neuroglandular transmission in the isolated salivary glands of the cockroach. J. Exp. Biol. 58.29-43.
House C.R. and Ginsborg B.L. (1985). Salivary Gland. In: Comprehensive Insect Physiology, Biochemistry Ntd Phamacology, G.A. Kerkut and L.I. Gilbert (Eds). Pergarnon Press, 195-224.
Just F. and Waltz B. (1 994). Salivary glands of the cockroach, Periplunetu americana: new data from light and electron microscopy. J. Morphol. 220, 35-46.
Just F. and Waltz B. (1996). The effects of serotonin and dopamine on salivary gland secretion by isolated cockroach salivary glands. L exp. Biol. 199,4074 13.
Kafatos F.C. (1968). The labial gland: a salt-secreting organ of saturnid moths. J. exp. Biol. 48, 435-453.
Kendall M.D. (1969). The fine structure of the salivary glands of the desert locust Schistocerca gregaria Fors3 . Z. Zellforsch. 98,3 99-420.
Klemm N. (1972). Monoamine-containing nervous fibres in foregut and salivary gland of the desert locust, Schistocerca gregaria Forskril (Orthoptera, Acrididae) . Comp. Biochem. Physiol. 43A, 207-2 1 1.
Myers C.M. and Evans P.D. (1985). The distribution of bovine pancreatic polypeptide/ FMRFamide-like immunoreactivity in the ventral nervous system of the locust. J. Comp. Neurol. 234, 1-16.
Orchard I., Lange A.B. and Brown B .B. (1992). Tyrosine hydroxylase-like immunoreactivity in the ventral nerve cord of the locust (Locusta migratoria), including neurones innervating the salivary glands. J. Imect Physiol. 38, 19-27.
Smith R.K. and House C.R. (1977). Ion and water transport by isolated cockroach salivary glands. Experientia 33, 1182-1 183.
Srivistava U.S. (1959). The maxillary glands of some Coleoptera. Proc. R. ent. Soc. Lond. 34, 57-62.
Sutherland D.J. and Chillseyzn J.M. (1968). Function and operation of the cockroach salivary reservoir. J. Insect Physiol. 14,2 1-3 1.
Watkins B.L. & Burrows M. (1989). GABA-like immunoreactivity in the suboesophageal ganglion of the locust Schistocerca gregaria. Cell Tissue Res. 258. 53-63.
Whitehead A.T. (197 1). The innervation of the salivary gland in the american cockroach: light and electron microscope observations. J. Morph. 135.483-506.
Willey R.B. (1961). The morphology of the stomadeal nervous system in Peripluneta americana (L) and other Blattaria. J. Morph. 108, 219-26 1.
II. The aminergic control of salivary glands in the stick insect, Carausius morosus.
ABSTRACT
Immunohistochernistry in Carausius morosus reveals that the salivary glands are
innervated by two paired neurons, the SN1 and SN2, located in the suboesophageal ganglion.
The SNI stain for tyrosine hydroxylase-like imrnunoreactivity, while the SN2 stain for
serotonin-like immunoreactivity. Tyrosine hydroxylase is the rate limiting enzyme in the
formation of catecholamines, and irnmunoreactivity to it is indicative of dopamine presence in
insects (Evans 1980, and Ali and Orchard 1996). Both neurons project axons through the
salivary nerve, and branch over the acini. Immunohistochernistry and biocytin filling shows that
the salivary ducts are targets for branches from the salivary nerve. Doparnine and serotonin
cause a dose-dependent increase in cyclic AMP levels in the salivary glands, indicating cyclic
AMP may play a role as a second messenger. Increases in cyclic AMP induced by dopamine
and serotonin, can be inhibited by vertebrate dopaminergic and serotonergic receptor
antagonists respectively. The rank order of potency of dopaminergic antagonists (based on IC,,
values) of SCH-23390 > flupenthixol > chlorpromazine > butaclamol, suggests the presence of
receptors similar to vertebrate D,-like receptors. The rank order of potency of serotonergic
receptor antagonists of spiperone > ketanserin > rnianserin > cyproheptadine, suggests the
presence of receptors similar to vertebrate 5HT2 or 5HT, receptors. Electrical stimulation of
the salivary nerve also elevates cyclic AMP levels in the salivary glands, an elevation that can
be partially inhibited by 0. IrnM SCH-23390 and cyproheptadine.
INTRODUCTXON
Dopamine and serotonin are biogenic arnines that are found in a variety of vertebrates
and invertebrates, and can serve roles as neurotransmitters, neurohormones and
neuromodulators. These biogenic arnines have been shown to play an important role in the
control of salivary glands in a number of insects (Berridge, 1970; House, 1973; Baines and
Tyrer, 1989; Ali et al., 1993). Insect salivary glands are useful preparations for the study of
aminergic control mechanisms. Dopamine stimulates fluid secretion in the cockroach (Bowser-
Riley and House, 1976), and locust (Baines and Tyrer, 1989). Dopamine increases cyclic AMP
(CAMP) levels in salivary glands of cockroach (Grewe and Kebabian, 1982) and locust (Ali et
al., 1993), and is capable of hyperpolarizing resting membrane potential in salivary gland cells
of cockroach (House, 1973). Serotonin has also been shown to stimulate fluid secretion in the
blowfly (Berridge, 1970) and in the locust (Baines and Tyrer, 1989). Serotonin also increases
CAMP in the salivary glands of locust (Ali et al., 1993), and hyperpolarizes the resting
membrane potential of cockroach salivary gland cells (House, 1973).
In the locust (Locusfa migratoria) the salivary glands are innervated by nerve 7b
(salivary nerve) from the suboesophageal ganglion (Klemm, 1972). Cobalt backfilling of the
nerve has shown that it contains two axons (Altman and Kien, 1979) arising from two neurons
within the suboesophageal ganglion, which have been named salivary neurons 1 and 2 (SNl
and SN2) respectively. Imrnunohistochemistry has shown that SNI likely uses dopamine as a
neurotransmitter, while SN2 uses serotonin (Ali et al., 1993). The salivary gland cells have also
been suggested to use CAMP as a second messenger in response to released dopamine and
serotonin from the SN1 and SN2 (Ali et al., 1993). Studies of cockroach also indicate that SN1
contains dopamine (Gifford et al., 199 1; Elia et al., 1994), although the neurotransmitter used
by SN2 remains uncertain. Initial studies in the stick insect, Carausius morosus, have shown
that the salivary glands resemble those in the locust (Mi and Orchard, 1996). Carausius
salivary glands are innervated by SNl and SN2 neurons that appear to contain the same
neurotransmitters as Locusta.
Doparnine and serotonin receptors in the salivary glands of locust and cockroach have
been pharmacologically characterized with the use of vertebrate receptor antagonists. Vertebrate
dopamine receptors are characterized as being DL-like or D,-like based on pharmacological
properties, these types are further divided into subtypes based on molecular structure (Gingrich
and Caron, 1993). There are seven types of serotonin receptors classified as 5HTL through
5HT,, based on structural and pharmacological properties (Bard et al., 1993; Plassat et al.,
1993; Shen et al., 1993). The dopamine and serotonin receptors in locusts are similar to
vertebrate Dl-like and 5HT2 receptors, and appear to be coupled to adenylate cyclase (Ali and
Orchard, 1994). Receptors mediating the secretory and hyperpolarization response in the
cockroach are also similar to D, and 5HT2 receptors of vertebrates (Evans and Green, 1990a;
Evans and Green 1990b)
The aim of this present study is to examine the innervation of the salivary glands of
Carausius, and to characterize the mode of action of dopamine and serotonin in the glands.
Irnrnunohistochernistry of the salivary glands verified previously described innervation by SN 1
and SN2, and was used to study projection to the salivary ducts. The effect of dopamine and
serotonin on CAMP leveIs in the salivary glands was determined, and a pharmacologicd study
of dopamine and serotonin receptors was conducted using vertebrate receptor antagonists and
agonists. The salivary nerve was stimulated and recorded from in order to confirm the presence
of two axons, and to examine the effects of stimulation on CAMP levels in the salivary glands.
I I M A ~ ~ AND METHODS
Animals
Adult female Carausius morosus were taken from a parthenogenic colony maintained
at 22°C under a 12 hour light: 12 hour dark regime. Insects were fed oak and Ficus benjamina
leaves.
Tmrnunohistochernistry
Immunohistochernistry was performed on isolated suboesophageal ganglia and saiivary
glands which were dissected under physiological saline (15 rnM NaCI; 18 rnM KCI; 50 mM
MgCl,; 7.5 m CaCI,; 184mM Glucose; Tris-HC1 2 m, pH 6.6), and fixed in 2%
paraformaldehyde in Millonig's buffer for lh. The tissues were washed several times in
phosphate-buffered saline (PBS; lOmM phosphate buffer, pH 7.2 containing 0.9% NaCI) for
4-6h, then incubated in 4% Triton X-100 and 10% normal goat serum in PBS for 1 h at room
temperature. Preparations were then processed for either tyrosine hydroxylase-like or serotonin-
like irnrnunoreactivity. For tyrosine hydroxylase-like imrnunoreactivity, tissues were incubated
for 48 h at 8°C with mouse monoclonal antibody generated against tyrosine hydroxylase
(Incstar Corp., Stillwater, MN, U.S.A.) diluted 1500 in phosphate-buffered saline containing
0.4% Triton X-100 and 2% normal goat serum. The tissues were washed for 6-8h in PBS, then
incubated for 24 h at 8°C in a 1 :200 dilution of fluorescein isothiocyanate wC)-labelled goat
anti-mouse immunoglobulin G (Jackson Immunoresearch Labs, West Grove, PA, U.S.A.) in
PBS containing 10% normal goat serum. Tissues were then washed in PBS for 18 h, mounted
and viewed in 5% n-propyl gallate in glycerol, pH 7.3.
Serotonin-like immonoreactivity was examined using similar methods. The primary
antiserum was a 1: 1000 dilution of a rabbit anti-serotonin antiserum (Incstar Corp.). The
secondary antibody was a sheep anti-rabbit immunoglobulin G labelled with Cy3 (Sigma
ChemicaI Co., St. Louis, MO, USA).
Mounted preparations were viewed through a Nikon OPTIPHOT-2 microscope with a
Bio-Rad (Richmond, CA ,USA) View Scan DVC-250 confocal imaging system.
Biocytin Filling
Salivary glands, including ducts and nerves, were dissected from adult insects and
placed in a pool of saline. The proximal portion of the salivary nerve was removed from the
surface of the salivary duct, while the remainder of the nerve was still attached to the duct. The
cut end of the salivary nerve was draped over a mineral oil well and placed in a pool of distilled
water for 2 min. The distilled water was replaced with 15% biotin in physiological saline, and
the preparation was covered and incubated for 48 hours. The tissues were washed several times
with PBS, then fixed in 2% paraformaldahyde in Millonig's buffer for 1 h. The tissues were
then washed several times in PBS for 2 h, then incubated in 4% Triton X-100 in PBS for 1 h.
The tissues were washed several times with PBS, then incubated in a 1 :200 dilution of strep-
avidin labelled with Cy3 (BIOICAN, Mississauga, ON, Canada) in PBS with 10% normal goat
serum. The preparation was washed several times in PBS for 6 h, then mounted in 5% n-propyl
gallate in glyceroI.
Cyclic AMP measurements
Salivary glands were dissected under physiological saline and assayed for the effect of
various agents on CAMP levels. Individual pairs of glands provided 8 tissue samples, which
were incubated at room temperature in physiological saline containing 0SmM 3-isobutyl- 1-
methylxanthine (IBMX), along with various concentrations of dopamine, serotonin or receptor
agonists for 10 rnin. When investigating the effects of various receptor antagonists, the tissues
were incubated in the antagonist and IBMX for Smin prior to the addition of dopamine or
serotonin. At the end of the incubation period, the reaction was stopped by the addition of
5 0 0 ~ 1 boiling 0.05M sodium acetate buffer, (pH 6.2) followed by 5min boiling. The samples
were stored at -20°C until further use. The tissues were sonicated, then centrifuged at 8800g
for 10min. The supernatant was removed for cyclic AMP measurement, whereas the pellet was
dissolved in 5 0 ~ 1 1.0 N sodium hydroxide for protein determination. Cyclic AMP levels were
measured by radioimrnunoassay (see Lange and Orchard, 1986) using a commercially available
kit (Mandel Scientific Co., Guelph, ON, Canada). The pratein content of the salivary glands
was measured using the Bio-Rad (Bio-Rad, Richmond, CA, USA) protein assay using gamma
globulin as standard.
Neurophysialogy
Salivary glands and duct, with the salivary nerve still attached were placed in a 4 0 0 ~ 1
pool of physiological saline containing 0SmM IBMX. The cut end of the salivary nerve was
drawn into a suction electrode and stimulated, while a recording suction electrode was used to
record from the salivary nerve within the salivary glands. The nerve was stimulated at 4Hz for
Smin with 0.5 msec square pulses. Control experiments used pulses of subthreshold voltage
in the other salivary gland of the same insect. To test the effects of receptor antagonists, the
salivary glands were preincubated in IBMX and the antagonist for 5min prior to stimulation.
At the completion of the experiment the saline pool and tissue were added to 6 0 0 ~ 1 of boiling
0.05M sodium acetate buffer, followed by 5 rnin of boiling. CyclicAMP and protein
measurements were carried out as described above.
Chemicals
The following drugs were obtained from Research Biochemical Inc. (Natick, MA,
USA): (*)SKF-82958, (*)SKF-383 93, (+)-SCH-23 3 90, (*)-butaclamol, flupenthixol,
spiperone, cyproheptadine, mianserin, methysergide, ketanserio. Dopamine, serotonin and
IBMX were obtained fiom Sigma Chemical Co. (St. Louis, MO, USA).
RESULTS
Immunohistochemisiry rmd biocytin filling
Tyrosine hydroxylase-like immunoreactivity, which is indicative of the presence of
dopamine in insects (Evans, 1980; Ali and Orchard 1996), is shown in Fig. 1. Fig. la shows
staining in the suboesophageal ganghon of C. morosus, which reveals a number of cell bodies.
Located anteriorly and medially are the large cell bodies of the SNI neurons that innervate the
salivary glands. They are the only positively-stained neurons with peripherally projecting axons
found within the suboesophageal ganglion. The axons exit contralaterally through the salivary
nerve, which is attached to the salivary duct by connective tissue. The axon branches to
coincide with the branching of the salivary duct (Fig. Ib). The axon continues to branch with
the duct within the salivary glands, and projects to each acinus where nerve terminals are seen
innervating the cells of the salivary gland (Fig. 1 c). Fig 1 d is a high magrufication image of the
salivary duct, showing that the duct itself is covered with nerve terminals positive for tyrosine
hydroxylase-like staining. Fig. 2a shows sero tonin-like imrnunoreacfivty in the sub oesophageal
ganglion. Many cell bodies are present, including the paired SN2 cell bodies located posteriorly
and centrally in the ganglion. The SN2 axon can be seen leaving the ganglion through the
salivary nerve in the anterior region of the suboesophageal ganglion. Fig. 2b is a higher
magdication image of the SN2 cell body showing the path of the axon as it
Figure 1
Tyrosine hydroxylase-like immunoreactive staining of suboesophageal ganglion and salivary
glands in Carausius. (a) Suboesophageal ganglion showing SNl cell body (thick arrow), and
axon (thin arrow) projecting contralaterally, and leaving the ganglion through the salivary nerve.
Bar: 50pm. (b) Axon of SN1 branching (thin arrow) as it projects along the salivary duct (thick
arrow). Bar: 50prn. (c) Acini of the salivary glands showing branching of the SNl axon
(arrow) within the glands. Bar: 50pm. (d) Surface of salivary ducts showing tyrosine
hydroxylase-like irnrnunoreactivity (arrow). Bar: 25pm.
Figure 2
Serotonin-like imrnunoreactive staining of suboesophageal ganglion and salivary glands in
Carausius. (a) Suboesophageal ganglion showing SN2 cell body (black arrow), and axon
leaving the ganglion through the salivary nerve (white arrow). Bar: 50pm. (b) High
magnification image showing cell body of SNI (thick arrow), and the path of it's axon (thin
arrows). Bar: 50pm. (c) Branching of the SN2 axon (arrow) towards the accini of the salivary
glands. Bar: 50pm. (d) Surface of salivary duct showing serotonin-like immunoreactive
terminals (arrow). Bar: 25pm.
Figure 3
Tyrosine hydroxylase-like irnrnunoreactive staining, and biocytin backfilling showing branching
of salivary nerve to innervate the salivary duct. (a) Tyrosine hydroxylase-like staining showing
a branch (long arrow) from the SNl axon projecting to the salivary duct and further branching
to innervate it. The nerve tenninais of one of the branches can be seen (short arrow). Bar:
25,um. (b) Biocytin forwardfill of the salivary nerve showing innervation of the salivary duct
(short arrow). The salivary nerve is the bright and out of focus object (long arrow). Bar: 25pm.
projects anteriorly towards the salivary nerve. The staining pattern on the duct and in the
sdivary glands is very similar to the tyrosine hydroxylase-like staining. Fig 2c shows the axons
branching to innervate the acini in the salivary glands. The salivary duct also has nerve
terminals with serotonin-like staining (Fig. 2d). To positively identify the origin of the nerve
terminals on the sdivary duct, confocal microscopy and biocytin filling techniques were used.
Using high magnification on a confocal microscope, some branches from the SN1 and SN2
axons could be seen projecting onto the salivary duct, although the branches were often difficult
to observe. Fig 3a shows one such branch of the SN1 axon with tyrosine hydroxylase-like
staining. Confirmation that the axons in the salivary nerve branch onto the salivary duct was
obtained by biocytin filling. Fig 3b shows the results of biocytin filIing of the salivary nerve
towards the glands, with the ducts intact, where filled nerve terminals are visible on the surface
of the duct.
Cyclic AMP Content
Both dopamine and serotonin increased CAMP levels in the salivary glands of Carausius
in the presence of KJ3MX. Fig 4 is a dose-response curve for the increase of CAMP in the
salivary glands when dopamine was applied. The increase in CAMP was dose-dependent, and
followed a sigmoidal relationship when CAMP was plotted against the log of dopamine
concentration. The threshold for CAMP elevation occurred between 0. lpM and OSpM, while
maximal elevation occurred at 50pM. The half maximal response occured at approximately
at 10 p M dopamine. Serotonin also caused a dose-dependent increase in CAMP in the salivary
glands (Fig 5). The relationship was sigmoidal when CAMP was plotted against the log of
serotonin concentration. The threshold for CAMP elevation was about 0.05pM, while maximal
Figure 4
CyclicAMP contentimg protein of salivary glands of adult Carausius foIIowing incubation for
different concentrations of doparnine and 0.5rnM IBMX. The IBMX control value is 62.5 & 5.2,
and aI1 value are means A S .E. (n>8).
1 o - ~ I 0-8 I o - ~ I 0-6 I o - ~ I 04
Dopamine Concentration (M) log scale
Figure 5
CyclicAMP content/mg protein of salivary glands of adult Carausius folowing incubation for
different concentrations of serotonin and 0.5mM IBMX. The IBMX control value is 56.3 m 3.2,
and a11 values are means A S.E. (n>8).
I o - ~ I o4 I o - ~ I o-= I o - ~
Serotonin Concentration (M) log scale
Table I
Action of doparninergic antagonists on dopamine-induced accumulation of CAMP in salivary
glands. Changes from basal levels are expressed as a percent. Drugs tested at lpM in the
absence (change from basal level) or presence of 1pM dopamine, with 0.5pM IBMX included
in all incubations. IC,, represents the concentration of antagonist needed to inhibit the effects
of lpM doparnine by 50%. (n>5)
Percent effect on basal percent inhibition of 1% Drug levels of CAMP response to lo4 M doparnine (M)
SCH-23390 -26.5%
Flupenthixol - 18.7%
Chiorpromazine -46.5%
ButacIamol - 26.5%
Tabie II
Action of dopaminergic agonists on CAMP content of salivary glands. CyclicAMP values are
expressed as mean f S.E. (n=8). Drugs tested at 1pM in the presence of OSrnM IBMX.
CAMP (pmol/mg protein)
percent response relative to ~ o - ~ M doparnine
IBMX control
Dopamine ( 1 O"M)
SKF-82958
SKF-38393
+ significantly different from control values at P~0.05 (student's t-test)
Table III
Action of serotonergic antagonists on serotonin-induced accumulation of CAMP in salivary
glands. Changes from basal levels are expressed as a percent (n=5). Drugs tested at 1pM in
the absence (change from basal level) or presence of 0.5pM serotonin, with 0.5pM IBMX
included in dl incubations. IC, represents the concentration of antagonist needed to inhibit the
effects of lpM serotonin by 50%.
Percent effect on basal percent inhibition of IC 50 Drug levels of CAMP response to 5x 10“ serotonin (M)
-- - --- - -
Spiperone -30.2% 63.5% 6 . 5 ~ loA7
Ketansarin -38.5% 62.5% 6 . 8 ~ lo-'
Mianserin -42.0% 56.9% 8. lxlo-'
Cyproheptadine - 28.7% 7.3% 2 . 8 ~ lod
elevation occurred at 10 pM. Fifty percent of maximal elevation occurred at approximately at
0.3pM serotonin.
Characterization of Dopamine and Serotonin Receptors
The effects of certain vertebrate doparninergic antagonists on the increase of CAMP in salivary
glands due to doparnine is shown in Table I. SCH-23390, flupenthixol, and butaclamol are D ,- like receptor antagonists, while chlorpromazine is a non-specific dopaminergic antagonist, and
they all inhibited the dopamine-induced increase of CAMP in the salivary glands. SCH-23390,
the most potent antagonist, completely inhibited the increase in CAMP when tested at a
concentration of lpM. It also had the lowest IC, value, at 30nM. Flupenthixol,
chlorpromazine, and butaclamol had higher IC,, values of 38nM, 50nM, and 720nM
respectively. These drugs also decreased the basal level of CAMP in the salivary glands. The
rank order of potency of these drugs was SCH-23390, > flupenthixol > chlorpromazine >
butaclamol. The effects of two Dl-like dopaminergic agonists were tested on the salivary
glands (Table Q. SKF-82958, at a concentration of lpM, induced a statistically significant
elevation of CAMP in the salivary glands, which was 19.5% of the elevation induced by
10,uMdoparnine. SKF-38393 did not yield a significant change from control values of CAMP.
Table III shows the effects of certain SHT, antagonists on the serotonin-induced
elevation of CAMP in the salivary glands. Spiperone, the most potent antagonist (IC,,=
0.65,uM), reduced the CAMP elevation induced by 0.5pM serotonin, by 63.5%, at a
concentration of 1pM. Ketansarin (IC,p 0.68pM) and rnianserin (IC,= 0.8 lpM) also inhibited
the serotonin-induced elevation of CAMP by 62.5% and 56.9% respectively, when tested at
1 Cyproheptadine was less potent, having an IC,, v,due of 2.8pM. The rank order of
Figure 6
Typical extracellular recording from the salivary nerve (7b) during stimulation of the same
nerve. Initial downward deflection is caused by the stimulus artifact (sa). Two action potentials
(ap) are recorded when the salivary nerve is stimulated supramaximally.
Figure 7
Increase in CAMP contenvmg protein of sdivary glands of adult Carausius folowing
stimulation of the salivary nerve. The nerve was stimulated at 4 Hz, and in a separate
experiment the salivary glands were preincubated for 5min in 0. lrnM SCH-23390 and
cyproheptadine. IBMX A refers to the paired control of the stimulation experiment. IBMX B
refers to the paired control of the stimulation with antagonists experiment. All values are means
+ S.E. (n>8).
potency of the drugs was spiperone > ketansarin > miansarin > cyproheptadine. All the
antagonists were able to lower basal levels of CAMP in the salivary glands.
Stimulation of the Salivary Nerve
Extracellular electrodes were used to stimulate the salivary nerve close to its exit fiom the
suboesophageal ganglion, while extracellular recording electrodes were used to record fiom the
salivary nerve in the region of the salivary glands. Fig 6 is a typical recording fiom the salivary
nerve, showing the presence of two distinct action potentials. One action potential had a
consistently shorter latency ~ o r n the stimulus artifact, and a larger amplitude.
The salivary nerve was stimulated at a f?equency of 4H2, and the effect on CAMP
content in the salivary glands was determined. Fig 7 shows that stimulation of the nerve
resulted in a 9 1% increase in CAMP content over IBMX control values. When the salivary
glands were preincubated in 0. lmM SCH-23390 and cyproheptadine and stimulated at 4H2, the
increase in CAMP content was only 36% over control values.
DISCUSSION
Immunohistochemistry is a powefil technique in the identification of neurons and the
neurotransmitters, neuromodulators or neurohormones these neurons may utilize. Antibodies
may not only be directed towards possible neuroactive substances, but also towards enzymes
in the pathway of their formation. Tyrosine hydroxylase is the first and rate-limiting enzyme
in the formation of catecholamines. Since it has been shown that insects produce very Little
adrenaline or noradrenaline (Evans, 198O), positive tyrosine hydroxylase-like staining is likely
indicative of the presence of dopamine.
Immunohistochemistry shows that the innervation of the salivary glands in Carausius
is very similar to that in Locusta rnigratoria (Mi et al., 1993). In both insects, tyrosine
hydroxylase-like immunoreactivity is found in the SNl cell bodies, and serotonin-like
immunoreactivity is found in the SN2 cell bodies. These cell bodies are located in the
suboesophageal ganglia. The SNI axons project through the contralateral salivary nerves, and
the SN2 axons project through the ipsilateral salivary nerves. These axons branch repeatedly
and project over the acini of the salivary glands. The SN1 in Periplaneta americana dso show
positive tyrosine hydroxylase-like staining (Elia et d., 1994), but in contrast, the SN2 do not
appear to contain serotonin or tyrosine hydroxylase (Gifford et al, 1991; Elia et al., 1994).
Furthermore, the salivary nerves in Periplaneta contain several smaller axons in addition to the
SNI and SN2 axons (Whitehead, 1971). Electrical stimuiation of the salivary nerve in
Carausius induced only two distinct sizes of action potentials confirming the presence of only
two axons, as indicated by cobalt backfilling of the salivary nerve (Ali & Orchard, 1996). One
action potential also consistently had a smaller latency, and a larger amplitude than the other
action potential, illustrating one axon to be significantly larger in diameter than the other one.
In the locust Schistocerca gregaria, the axons of SNl and SN2 have significantly different
diameters, and the SNI has a larger amplitude action potential when recorded extracellularly
(Baines et d., 1989).
The salivary ducts of Carausius possess terminals which are tyrosine hydroxylase-like
and serotonin-like imrnunoreactive. In contrast, the salivary ducts of Locusra do nc t appear to
receive projections from the dopaminergic or serotonergic axons (Ali et al., 1993). In some
instances, the axons originating from the salivary nerve of Curausius were visible
irnrnunohistochemically, but more often, biocytin forwardfilling of the salivary nerve into the
salivary gland was necessary to determine the origin of the terminals. The projections to the
ducts were visible when the salivary nerve was filled. Therefore the terminals on the salivary
ducts appears to originate from branches of the salivary nerve, and may mean broader functions
for catecholamines in some insect salivary glands.
Doparnine and serotonin each cause a dose-dependent increase in CAMP content in the
salivary glands of Caratrsius in the presence of IBMX. This indicates that CAMP may play a
role as a second messenger in the salivary glands in response to the release of doparnine and
serotonin from SNl and SN2. Dopamine and serotonin were found to have a similar effect on
the salivary glands of Locusta rnigratoria (Ali et al., 1993). Dopamine and serotonin also
increase secretion in the salivary glands of the locust, Schistocerca gregaria (Baines and Tyrer,
1989). Doparnine increases CAMP content in the salivary glands of the cockroach, Narcphoeta
cinerea (Grewe and Kebabian, 1982), and also stimulates fluid secretion (Evans and Green,
1990), an effect mimicked by exogenous CAMP (Gray et aI., 1984). Serotonin elevates CAMP
and also stimulates fluid secreation in the salivary glands of the blowfly Calliphora
erythrocephala (Berridge, 1970). Cyclic AMP therefore appears to play an important role in
the signal transduction mechanism for control of salivation in insects.
A partial characterization of dopamine and serotonin receptors was performed on the
salivary glands of Carausius morosus. The current data support the idea that the dopamine
receptors on the salivary glands are pharmacologically similar to vertebrate DL-like receptors.
Vertebrate Dl-like receptors are positively linked to adenylate cyclase leading to the production
of CAMP (Gingrich and Caron, 1993). Receptor antagonists specific for vertebrate Dl-like
receptors inhibited the elevation of CAMP caused by bathing in dopamine. This lends support
to the idea that dopamine is being released from SNL, binds to Dl-like receptors on the salivary
gland cells, and elevates CAMP within them.
The rank order of potency of doparnine receptor antagonists in Carausius morosus is
SCH-23390 > flupenthixol > chlorpromazine > butaclamol. This is similar to the rank order
of potency in locust salivary glands, namely SCH-23390 > butaclamol> flupenthixol, (Ali and
Orchard, 1994). Chlorpromazine, which is a non-specific doparnine antagonist, was ineffective
in Locusta (Ali and Orchard, I994), but was an effective antagonist in Carausius salivary
glands. The other antagonists are all specific Dl-Like receptor antagonists, and were all effective
inhibitors of doparnine-induced elevation of CAMP in Carausius. Cockroach salivary glands
appear to have receptors similar to D, (now refered to as D,-like receptors) vertebrate receptors
mediating the secretory response as well (Evans and Green, 1990a). The rank order of potency
of the antagonists for the secretory response are chlorpromazine > SCH-23390 > haloperidol
>> metocloprarnide. It is believed that the same D,-type @,-like) receptor is involved in the
hyperpolarization response in cockroach salivary glands (Evans and Green, 1990b), even though
it appears CAMP is not involved as a second messenger (Gray et al., 1984).
Interestingly, both the doparninergic and the serotonergic antagonists were able to
decrease basal levels of CAMP. This may indicate that there is a basal Ievel of dopamine and
serotonin release onto the salivary glands. A similar situation has been described for
octopaminergic neurons innervating the lateral oviducts of Locusta, where arninergic
antagonists lower basal levels of CAMP (Orchard and Lange, 1986).
Dopamine agonists were not very effective at elevating CAMP in the salivary glands of
Carausius morosus. The selective Dl-like receptor agonist SKF-82958 did elevate CAMP
content in the salivary glands significantly, but the increase was only 19.5% of that induced by
10pM doparnine. Pharmacological profile studies of receptors in other insect preparations have
also revealed the ineffectiveness of vertebrate agonists (AIi and Orchard, 1994; Baines and
Downer, 1991). The ineffectiveness of the agonist may not only be due to the agonist binding
site being different from the natural amine, but also to divergence in receptors from the
vertebrate counterpart.
The pharmacological profile of serotonin receptors in Carausius suggests the presence
of 5m2-type or 5HT, receptors. Recent research has expanded the number of serotonin
subtypes that have been characterized. The antagonists used in this study are 5HT2-type
receptor antagonists, however they also bind to SHT, receptors. 5HT7 receptors are positively
linked to adenylate cyclase in vertebrates (Bard et al., 1993; Plassat et al., 1993; Shen et al..
19931, whereas SHT,-type receptors are known to stimulate the phospholipase C second
messenger pathway (Humphrey et al., 1993). From research in mammalian systems it has been
shown that rnethysergide,spiperone, mianserin, cyproheptadine and ketanserin show affinity for
the 5HT, receptor, and some have also been shown to stimulate adenylate cyclase activity (Bard
et al., 1993; PIassat et al., 1993; Shen et al., 1993). 5HT, and 5HT, receptors are also known
to be positively linked to adenylate cyclase, however they show much lower affinity for 5HT,-
type antagonists (Hoyer et a[., 1993). Thus, the serotonin receptor is pharmacologically similar
to 5HT2-type and 5HT, recep tors, but is linked to the same second messenger system that 5HT,.
It is possible that the receptor in the salivary glands may be similar to vertebrate 5HT2-type
receptors, yet be linked to adenylate cyclase. Additional pharmacological data is needed to be
better able to characterize the serotonin receptor in the salivary glands of Carausius. The
pharmacological data supports the idea that serotonin is released from SN2, binds to receptors
on the salivary gland cells, and elevates CAMP in the cells. There are many examples of
serotonin receptors being classified as 5HT2 receptors in insects, even though they are
positively Linked to adenylate cyclase. (Berridge and Heslop, 198 1 ; Barret and Orchard, 1990;
Baines and Downer, 1991; Ali and Orchard, 1994). It may be possible that these receptors may
also be similar to vertebrate 5HT7 receptors. The response to serotonin in the salivary glands
of Locusta can be blocked by a number of serotonin antagonists which have the following order
of potency; spiperone > cyproheptadine > mianserin > rnethysergide > ketanserin (Ali and
Orchard, 1994). The order of potency found in Carausius salivary glands is, spiperone >
ketanserin > rnianserin > cyproheptadine, and supports the presence of SHT,-type or 5HT,
receptors in the salivary glands.
Stimulation of the salivary nerve leads to an increase of CAMP content in the salivary
glands of Carausius. This increase in CAMP can be partially inhibited by preincubating the
glands in 0. lrnM SCH-23390 and cyproheptadine, indicating that doparnine and serotonin are
probably being released from the SNI and SN2. It is interesting that the elevation in CAMP
could not be completely inhibited. A similar result was obtained when nerve 7b in Locusta was
stimdated and CAMP measured (Mi and Orchard, 1994). Considering that the receptor
antagonists were capable of Iowering basal levels of CAMP, and relatively high concentrations
were used, it is unlikely that the residual elevation is due to insufficient blocking by the
antagonists. However, it may be possible that cyproheptadine was not able to completely block
serotonin receptors, even when the concentration used was 0. I rnM. It is also possible that the
residual elevation in CAMP is due to another substance that is released when the salivary nerve
is stimulated. For example, there is immunohistochernical evidence that GABA may be
colocalized in the SN2 of the locust Schistocerca gregaria (Watkins and Burrows, 1989).
Carausius salivary glands provide a good model system for understanding the
arninergic control of salivary glands in insects. Future studies will provide a better
understanding of the control of salivary glands, and of the events involved in secretion.
REFERENCES
Ali D.W. and Orchard I. (1994). Characterization of dopamine and serotonin receptors on the salivary glands of the locust, Locusta migratoria. Biogenic Arnines 10. 1 95-2 1 2.
AIi D.W. and Orchard I. (1996). Immunohistochemical localization of tyrosine hydroxylase in the ventraI nerve cord of the stick insect, Carausius morosus, including neurons innervating the salivary glands. Cell Tissue Res. 285.453-462.
Ali D.W., Orchard I. and Lange A.B. (1993). The aminergic control of locust (Locusta migratoria) salivary glands: Evidence for dopaminergic and serotonergic innervation. J. Insect Physiol. 39.623-632.
Altman J.S. and Kien J. (1979). Suboesophageal neurons involved in head movements and feeding in locusts. Proc. R. Soc. Lond. B 205.209-227.
Baines R.A. and Tyrer N.M. (1989). The innervation of locust salivary glands. II. Physiology of excitation and modulation. J. Comp. Physiol. A 165.407-4 1 3.
Baines R.A. and Downer R.G.H. (1 99 1). Pharmacological characterization of a 5- hydroxytryptamine-sensitive recep todadenylate cyclase complex in the mandibular closer muscles of the cricket, Gryllus domestics. Arch. Insect Biochem. Physiol. 16. 153-163.
Baines R.A., Tyrer N.M. and Mason J.C. (1 989). The innervation of locust salivary glands. I. Innervation and analysis of transmitters. J. Comp. Physiol. A 165. 395-405.
Bard J.A., Zgombick I., Adharn N., Vaysse P., Branchek T.A. and Weinshank R.L. (1993). Cloning a novel human receptor @FIT,) positively liked to adenylate cyclase. J. Biol. Chem. 268.23422-23426.
Barrett M. and Orchard I. (1990). Serotonin-induced elevation of CAMP levels in the epidermis of the blood-sucking bug, Rhodnius prolixus. J. Insect Physiol. 36.625-633.
Berridge M.J. (1970). The role of 5-hydroxytxyptamine and cyclic AMP in the control off fluid secretion by isolated salivary glands. J. exp. B i d . 53. 171-1 86.
Bemdge M.J. and Heslop J.P. (1981). Separate 5-hydroxytqptamine receptors on the salivary gland of the blowfly are linked to the generation of either cyclic adenosine 3 ' ,5 ' -monophosphate or calcium signals. Br. J. P h a m c . 73, 729-73 8.
Bowser-Riley F. and House C.R. (1976). The actions of some putative neurotransmitters on the cockroach salivary gland. J. exp. Bid . 64. 665-676.
Elia A.J., Ali D.W. and Orchard I (1994). IrnmunochemicaI staining of tyrosine hydroxylase(TH)-like material in the salivary glands and ventral nerve cord of the cockroach, Periplaneta arnericana (L.). J. Insect Physiol. 40.67 1-683.
Evans P.D . (1980). Biogenic amines in the insect nervous system. Adv. Insect Physiol. 15. 3 17-473.
Evans A.M. and Green K.L. (1990a). The action of dopamine receptor antagonists on the secretory response of the cockroach salivary gland in vitro. Comp. Biochem. Physiol. 97C. 283-286.
Evans A.M. and Green K.L. ( l99Ob). Characterization of the doparnine receptor mediating the hyperpolarization of cockroach salivary gland acinar cells in vitro. Br. J. Phamacol. 101. 103-108.
Gifford A.N., Nicholson R.A. and Pitman R.M. (1991). The dopamine and 5- hydroxytryptamine content of locust and cockroach salivary neurones. J. exp. Biol. 161. 405-4 14.
Gingrich J.A. and Caron M.G. (1993). Recent advances in the molecular biology of doparnine receptors. Annu. Rev. Neurosci. 16. 299-32 1.
Gray D.C., Ginsborg B.L. and House C.R. (1984). Cyclic AMP as a possible mediator of doparnine stimulation of cockroach gland cells. Quart. J. Exp. Physiol. 69. 17 I - 186.
Grewe C.W. and Kebabian J.W. (1 982). Doparnine stimulates production of cyclic AMP by the salivary gland of the cockroach, Nauphoeta cinerea. Cell. Mol. Neurobiol. 2. 65-69.
House C.R. (1973). An electrophysiological study of neuroglandular transmission in the isolated salivary glands of the cockroach. J. Exp. Biol. 58. 29-43
Hoyer et al. (1993). International union of pharmacology classification of receptors for 5- hydroxytxyptamine (serotonin). Phamacol. Rev. 46. 157.
Klemm N. (1972). Monoaminecontaining nervous fibres in foregut and salivary gland of the desert locust, Schistocerca gregaria Forskrl (Orthoptera, Acrididae) . Comp. Biochem. Physiol. 43A. 207-2 1 1.
Lange A.B. and Orchard I. (1986). Identified octopaminergic neurons modulate contractions of locust visceral muscle via adenosine 3', 5'-monophosphate (cyclic AMP). Brain Res. 363. 340-349.
Orchard I. and Lange A.B. (1986). Pharmacological profile of octoparnine receptors on the lateral oviducts of the locust, Locusta rnigratoria. J. Insect Physiol. 32. 741-745.
Plassat J.L., Amlaiky N., Hen R. (1993). Molecular cloning of a marnalian serotonin receptors that activates adenylate cyclase. Mol. Pharmacul. 44.229-236.
Shen Y., Monsma F.J., Metcalf M.A., Jose P.A., Hamblin W. and Sibley D.R. (1993). Molecular cloning and expression of a 5-hydroxytryptamine, serotonin receptor subtype. J. Biol. Chern. 268. 18200-18204.
Watkins B.L. & Burrows M. (1989). GABA-like imrnunoreactivity in the suboesophageal ganglion of the locust Schistocerca gregaria. Cell Tissue Res. 258. 53-63.
Whitehead A.T. (1971). The innervation of the salivary gland in the arnerican cockroach: light and electron microscope observations. J. Morph. 135.483-506.
ID. A possible role for GABA in the salivary glands of the stick insect,
Carausius rnorosus.
ABSTRACT
Immunohistochemistry reveals that the salivary glands of Carausius morosus receive
GAB A-like irnmunoreactive projections from the salivary nerve. The GAB A-like
irnrnunoreactive axon branches over the acini, where many varicosities are seen along the length
of the axon. h one preparation a second process was seen in the salivary nerve that appeared
to possess neurohaemal-like terminals. GABA potentiates dopamine-induced increases in
cycIic AMP content of the salivary glands, although it has no intrinsic activity. The threshold
for potentiation occurs at about 10-'M GABA. The potentiation is completly blocked when the
salivary glands are preincubated in 2x lo4M Zhydroxysaclofen, a potent GABA, channel
blocker. It would appear that GABA may serve as a neuromodulator in the salivary glands of
Carausius.
INTRODUCTION
Insect salivary glands have served a useful role for the study of arninergic control
mechanisms (for review see House and Ginsborg, 1985). Doparnine and serotonin have been
shown to play an important role in the control of salivary glands in a number of insects
(Berridge, 1970; House, 1973; Baines and Tyrer, 1989; Ali et al., 1993). Dopamine stimulates
fluid secretion in the cockroach (Sowser-Riley and House, 1976), and the locust (Baines and
Tyrer, 1989). Dopamine also increases cyclic AMP levels in the salivary glands of the
cockroach (Grewe and Kebabian, 1982) and the locust (Ali et al., 1993), and is capable of
hyperpolarizing the membrane potential of acinar cells of the salivary glands in the cockroach
(House, 1973). Serotonin has also been shown to stimulate fluid secretion in the blowfly
(Berridge, 1970) and in the locust (Baines and Tyrer, 1989). Serotonin also increases cyclic
AMP levels in the salivary glands of the locust(A1i et aL, 19931, and hyperpolarizes the
membrane potential of acinar cells in the salivary glands of the cockroach (House, 1973).
In the stick insect, Carausius morosus, the salivary glands receive axonal projections
from salivary neuron one (SNI) and salivary neuron two (SN2) (Ali and Orchard, 1996;
Asimakopoulos and Orchard, 1998). Immunohistochernistry has shown that SNl is tyrosine
hydroxylase-like immunoreactive, while SN2 is serotonin-like immunoreactive (Ali and
Orchard, 1996; Asimakopoulos and Orchard, 1998). Tyrosine hydroxylase is the rate limiting
enzyme in the formation of catecholamines. Since adrenaline and noradrenaline are absent or
present only in very low levels in insects (Evans, 1980), tyrosine hydroxylase-like
immunoreactivity is indicative of the presence of dopamine in insects. The cell bodies of SN1
and SN2 are located in the suboesophageal ganglion, and send axonal projections to the salivary
glands through the salivary nerve (Ali and Orchard, 1996; Asimakopoulos and Orchard, 1998).
Dopamine and serotonin increase cyclic AMP levels in the salivary glands of Carausius
in a dose-dependent manner (Asimakopoulos and Orchard, 1998). Electrical stimulation of the
salivary nerve also increases cyclic AMP levels in the salivary glands; this increase can be
blocked by dopamine and serotonin receptor antagonists (Asimakopoulos and Orchard, 1998).
A partial pharmacological characterization shows that the dopamine receptors are similar to
vertebrate D,-like receptors, while the serotonin receptors are similar to vertebrate SKI',-type
and 5HT, receptors (Asimakopoulos and Orchard, 1998).
There is immunohistochemical evidence that y -amino butyric acid (GAB A) is present
in the SN2 of the locust (Watkins and Burrows, 1989). However, since this discovery there has
been no progress in determining GABAYs role in the control of insect salivary glands. GABA
has, however, been shown to potentiate dopamine-induced fluid secretion in the ixodid tick
(Lindsay and Kaufman, 1985). In this study, we use imrnunohistochemistry to determine if
GABA is present in the salivary gland system of Carausius. We also examine for any effect
of GABA on the cyclic AMP content in the salivary glands of Carausius.
MATERIALS AND METHODS
Animals
Adult female Carausius rnorosus were taken from a parthenogenic colony maintained
at 22°C under a 12 hour light: 12 hour dark regime. Insect were fed Romaine lettuce.
Imnzunohistochemistry
The suboesophageal ganglia and salivary glands were fixed using Boer's GPA fixative
(Boer et al., 1979). The fixative was injected into the abdomen and head of the insect with a
hypodermic needle. The insect was opened with a mid-dorsal incision, and further fixed for
three hours at 4°C. The preparation was then washed several times with phosphate buffered
saline (PBS; lOmM phosphate buffer, ph 7.2 containing 0.9% NaCl), and the suboesophageal
ganglion and salivary glands were dissected and washed in PBS for two hours at room
temperature. The tissues were then incubated in 4% Triton X-100 and 10% normal goat serum
in PBS for 1 hour at room temperature. Tissues were then incubated for 48h at 8°C with a
guinea pig monoclonal antibody generated against GABA (Incstar Corp., Stillwater, MN,
U.S.A.) diluted 1: 1000 in PBS containing 0.4% Triton X-100 and 2% normal goat serum. The
tissues were washed in PBS, then treated for 30 minutes with 0.45M sodium borohydride in
SST (0.1M Tris HcI/).3M NaCl), ph 7.4, containing 0.5% Triton X- 100, to remove background
fluorescence. The tissues were washed in PBS for two hours at room temperature, then
incubated for 18 hours at 8°C in a 1:200 dilution of biotin labeled sheep anti immunoglobulin
G antiserum, in PBS with 10% normal goat serum. The tissues were then washed in PBS for
5 hours at room temperature, then incubated for 18 hours at 8 "C in a 1 :200 dilution strep-avidin
labeled with Cy-3, in PBS with 10% normal goat serum. Tissues were then washed in PBS for
18 hours, mounted and viewed in 5% n-propyl gallate in glycerol, ph 7.3.
Mounted preparations were viewed through a Nikon OPTIPHOT-2 microscope with a
B io-Rad (Richmond, CAPS A) View Scan DVC-250 confocal imaging system.
Cyclic AMP Measurements
Salivary glands were dissected under physiological saline (1 5 rnM NaCl; 18 rnM KCI;
50 mM MgCI,; 7.5 m CaCl,; L84mM Glucose; Tris-HCI 2 m, pH 6.6) and assayed for the effect
of various agents on cyclic AMP levels. Individual pairs of glands provided 8 tissue samples,
which were incubated at room temperature in physiological saline containing 0.5rnM 3-isobutyl-
1-methylxanthine (IEiMX), along with various concentrations of dopamine, serotonin and 2-
hydroxysaclofen for 10 min. When investigating the effects of 2-hydroxysaclofen, the tissues
were incubated in the antagonist and IBMX for Smin prior to the addition of dopamine or
serotonin. At the end of the incubation period, the reaction was stopped by addition of 500p1
boiling 0.05M sodium acetate buffer, (ph 6.2) foIlowed by 5 rnin boiling. The samples were
stored at -20°C until further use. The tissues were sonicated, then centrifuged at 8800g for
IOmin. The supernatant was removed for cyclic AMP measurement, whereas the peIlet was
dissolved in 5 0 ~ 1 1.0 N sodium hydroxide for protein determination. Cyclic AMP levels were
measured by radioirnrnunoassay (see Lange and Orchard,' 1986) using a cornrnercially available
kit (Mandel Scientific Co., Guelph, ON, Canada). The protein content of the salivary glands
was measured using the Bio-Rad protein assay using gamma globulin as standard.
RESULTS
hrnunohistochernistry
The immunohistochernical protocol used gave strong staining in nerves associated with
the salivary glands, however cell bodies in the suboesophageal ganglion and other sections of
the ventral nerve cord did not stain. The lack of central staining may be explained by GABA
being produced in axonal endings near the site of its release. It is also possible that there were
penetration limitations of the fixative and antibodies due to the whole mount preparation used.
GABA-like irnmunoreactivity in the salivary glands of Carausius is shown in Figure 1.
Figure la shows a GABA-like irnmunoreactive axon in the salivary nerve which runs along the
salivary duct. The salivary ducts themselves do not appear to receive projections from GABA-
like immunoreactive axons. The GABA-like immunoreactive axon follows the salivary duct
Figure 1
GABA-Iike imrnunoreactive staining of salivary gIands in Carausius. (a) Axon within the
salivary nerve (arrow), which runs adjacent to the salivary duct. (b) Axon branching (arrow)
in the vicinity of the salivary glands. (c) Large varicosities (arrows) at the endings of the axon
branches on the acini. (d) Varicosities (arrows) within the salivary nerve.
to the salivary glands where it branches and sends projections to individual acini (Figure 1 b).
The axons continue to branch within the acini, and end in many varicosities (Figure lc). An
interesting feature was seen in only one preparation. A very small process was seen in the
salivary nerve beside the main axon. This process had many large varicosities along its length
(Figure Id).
Cyclic AMP Content
Since it had been shown previously that cyclic AMP likely serves as a second messenger
for doparnine and serotonin in the salivary glands of Cnrausius (Asimakopoulos and Orchard,
1998), we looked for possible effects of GABA on the cyclic AMP content of the salivary
glands. GABA did not increase cyclic AMP content in the salivary glands when tested at lo4
M (Figure 2). However, when L O ~ M GABA is applied with 5x104M dopamine, there was a
significant increase in cyclic AMP content in the salivary glands in comparison to 5 x 1 0 " ~
dopamine alone. GABA increased the cyclic AMP content by 74% over the doparnine control
value. When the salivary glands were incubated with 104M GABA and 10-'M serotonin, there
was no significant change in the cyclic AMP content over the serotonin control value.
Figure 3 is a dose-response curve for the increase in cyclic AMP content in the salivary
glands when GABA is applied in the presence of 10"M dopamine. Although there is a slight
increase in cyclic AMP content when ~ O - ~ M to 10"M GABA was applied, this was not a
statistically significant increase (t-test, p=0.05). The threshold for cyclic AMP elevation was
about 10-'M. The cyclic AMP increase was statistically significant over doparnine control
values when 10"M and 1 0 4 ~ of GABA was applied to the salivary glands (t-test, p= 0.05).
For GABA to have an effect on cyclic AMP levels in the presence of doparnine, it would
be anticipated that its effects may be elicited by a GABA, receptor, which is known to be
Figure 2
Effect of GABA on the cyclic AMP content of salivary glands in Carausius. All chemicals
tested in the presence of OSmM IBMX, and all values are mean & S .E. ( ~ 4 ) . DA=dopamine,
5HT= serotonia.
lBMX 1 O ~ M 5x1 0% 5x1 O - ~ M 1 O - ~ M 5HT 1 O - ~ M 5HT control GABA DA DA +I O ~ M
+1 O+M GABA
GABA
' significantly different from each other (student's t-test)
Figure 3
Cyclic AMP contenumg protein of salivary glands of adult Carazcsius following incubation in
different concentrations of GABA, 1pM dopamine and 0.5rnM IBMX. The IBMX control
value is 42.1 t 2.4, and the dopamine control vaIue is 186.7 & 13.6. All values are mean &
S.E.(n26).
Figure 4
Effects of 2-hydroxysaclofen on GABA potentiation of dopamine induced cyclic AMP elevation
in the salivary glands of Carausius. All chemicals tested in the presence of 0.5m.M IBMX, and
all values are mean + S.E.M. (n=5). DA = dopamine, HS = 2-hydroxysaclofen,
G = GABA.
l BMX control
* significantly different from 10; M dopamine (stuteent's t-test) * significantly differentfrom 10 M dopamine + 10 M GABA (student's t-test) " significantly different from IBMX control (student's t-test)
coupled to G proteins. For this reason we tested the effects of a vertebrate GAB& receptor
blocker on the GABA-evoked potentiation of dopamine-induced cyclic AMP elevation in the
salivary glands. Figure 4 shows that when the salivary glands are incubated in 1 04M GABA
and lodM dopamine, there is a significant (t-test, p= 0.05) 55% increase in cyclic AMP levels
in comparison to 1 04M dopamine alone. This increase in cyclic AMP is completely blocked
when the salivary glands are preincubated for 5 min in 2 x 1 0 ~ ~ 2-hydoxysaclofen, which is a
potent vertebrate GABA, receptor antagonist (blocker did not effect dopamine stimulated cyclic
AMP increase). When the glands were preincubated in O.2mM 2-hydroxysaclofen incubated
only in IBMX, there is a significant 63% decrease from IBMX control values of cyclic AMP.
DISCUSSION
The salivary glands of Carausizis receive GABA-like immunoreactive axonal
projections &om the salivary nerve. It has been previously shown that the salivary nerve
contains only two axons, originating from the SNl and SN2 (Ali and Orchard, 1996;
Asimakopoulos and Orchard, 1998). This suggests that GABA is colocalized with dopamine
or serotonin in one of these neurons. The GABA-like irnmunoreactivity in Cmausius is similar
to both the tyrosine hydroqlase-like staining and serotonin-like staining. Since the
immunohistochemical technique used did not stain cell bodies in the ventral nerve chord, it is
not possible to determine which neuron contains GABA. Double labeling for GABA with
either tyrosine hydroqlase or serotonin would have allowed us to determine which neuron
contains GABA However, the fixation process used for GABA is very different fiom those
used to fix tyrosine hydroxylase or serotonin, which made attempts to double label the
GABAergic axon unsuccessll. In the locust, Schistocerca gregmia, it has been shown that
GABA is colocalized with serotonin in the SN2 (Watkins and Burrows, 1989). Future studies
using an antibody against glutamic acid decarboxylase may allow us to stain the cell body of
the GABAergic neuron.
Interestingly, the salivary ducts of Carausius do not appear to receive GABA-like
innervation. It has been shown that the ducts receive tyrosine hydroxylase-like and serotonin-
like projections from the SN 1 and SN2 (Asirnakopoulos and Orchard, 1998). Therefore, there
may be differences in the neurotransmitters released in different termind regions of the same
neuron. Since the salivary ducts and acinar cells likely have different functions, it is reasonable
to expect them to be under different neuron4 control. Further study is needed in order to obtain
a better understanding of the function and control of the salivary ducts.
An interesting feature in one of the preparations was staining of an additional
GABAergic process within the salivary nerve. The process was very fine, but it had large
varicosities along its length which may be indicative of release sites. It is likely that the fine
axon is a branch of either the SN1 or SN2, since it has been shown that the salivary nerve
contains only two axons (Ali and Orchard, 1994). Neurohaemal sites have been characterized
previously in insect saiivary gland preparations. There are several small serotonergic processes
that are neurohaemal in appearance within the salivary nerve of both the cockroach, Periplaneta
americana (Davis, 1985), and the locust, Locusta migratoria (Braunig, 1987). There are also
FMRFarnide related peptide-like immunoreactive neurohaemal sites on the surface of the
salivary nerve in Loc~uta (Fuse et al., 1996). The role of such neurohemal sites in the control
of salivary glands has not been elucidated, although activity in the satellite nervous system of
Lmusta has been correlated to feeding (Schactner and Braunig, 1995).
GABA usually serves as an inhibitory neurotransmitter in the brain of vertebrates and
the central nervous system of insects (Harrow et al., 199 1). GABA has also been shown to have
an inhibitory role in salivary gland secretion in the rat (Shida et aL, 1995). However, GABA
may play an excitatory role in the control of salivary glands of Carausius. GABA done, does
not affect the cyclic AMP content of Carausius salivary glands, but in the presence of
dopamine, GABA does result in an increase in cyclic AMP levels in comparison to doparnine
alone. A similar synergistic effect was not observed with serotonin. The threshold of
potentiation occurred at about 10"M GABA. A similar effect has been seen in the salivary
glands of the tick, Amblyomma hebraeurn, where GABA (at concentrations as low as 1U6M)
increases salivary gland secretion in the presence of doparnine, but has minimal intrinsic activity
(Lindsay and Kaufman, 1985). Though salivary secretion has not been studied in Carausius,
cyclic AMP may act as a second messenger in the secretion process (Asimakopoulos and
Orchard, 1998). Doparnine has been shown to increase cyclic AMP levels in the salivary glands
of the tick (Sauer et al., 1979), and cyclic AMP stimulates fluid secretion from the salivary
glands (Needham and Sauer, 1975; Needharn and Sauer, 1979).
The potentiation effect may be explained by GABA modulating the dopamine receptor
allosterically. GABA may bind to a site on or near the dopamine receptor, leading to an
increased level of cyclic AMP production. This would explain why GABA has no intrinsic
effects on cyclic AMP values.
The tick salivary gland system has control characteristics that are different than those
of Carausius. Spiperone and other butyrophenones show a similar potentiation effect as GABA
in the salivary glands of the tick, but have no intrinsic activity (Wong and Kaufman, 198 1).
Spiperone and other butyrphenones are known to be potent antagonist of D, receptors in the
mammalian central nervous system and peripheral systems (Gingrich and Caron, 1993).
Spiperone has been shown to inhibit serotonin-induced elevation of cyclic AMP levels in the
salivary glands of Carausius, however its effects on dopamine receptors have not been tested
(Asimakopoulos and Orchard, 1998). In Locusta salivary glands, which are under similar
aminergic control to Carausius salivary glands, spiperone has been shown to have no effect on
dopamine-induced elevation of cyclic AMP (Ali and Orchard, 1994). It is postulated that
spiperone and GABA may act through the same receptor site in the tick, since their potentiation
effects can be blocked by the GABA antagonists picrotoxin and bicuculline (Lindsay and
Kaufman, 1985). In Carausius, the potentiation of GABA was inhibited when the glands were
preincubated in 0.2mM 2-hydroxysaclofen, which is a potent GABA, vertebrate receptor
antagonist. Baclofen, an agonist of vertebrate GABA, receptors, did not potentiate doparnine
induced fluid secretion in the tick (Lindsay and Kaufman, 1985). Thus, there appear to be
differences in the GABA receptors that mediate the potentiation response in Carausius and the
tick. The GABA receptors in the tick appear to be pharmacoIogically similar to vertebrate
GABA, receptors, while the GABA receptors in Carausius may be similar to vertebrate
GABA, receptors. Further pharmacological data is needed in order to better characterize the
GABA receptor in the salivary glands of Carausius.
Interestingly, 2-hydroxysaclofen was able to decrease basal levels of CAMP. This may
indicate that there is a basal level of both GABA and doparnine release onto the salivary glands
in vitro. Dopaminergic and serotonergic antagonists also lower basal levels of cyclic AMP in
Carausius (Asimakopoulos and Orchard, 1998). A similar situation has also been described for
octoparninergic neurons innervating the lateral oviducts of Locusta, where aminergic
antagonists lower basal levels of CAMP (Orchard and Lange, 1986).
In conclusion, we have shown that the salivary glands of Carausius may receive
GABAergic innervation, and GABA appears to potentiate dopamine-induced cyclic AMP
increases in the salivary glands. Further work is needed to determine which neuron that is
responsible for the GAB Aergic projections to the salivary glands, and to better understand the
physiological roles for these projections
REFERENCES
Ali D.W. and Orchard I. (1996). Immunohistochernical localization of tyrosine hydroxylase in the ventral nerve cord of the stick insect, Carausius morosus, including neurons innervating the salivary glands. Cell Tissue Res. 285.453-462.
Ali D.W., Orchard I. and Lange A.B. (1993). The arninergic control of locust (Locusta rnigratoria) salivary glands: Evidence for dopaminergic and serotonergic innervation. J. Insect Physiol. 39.623-632.
Asimakopoulos S. and Orchard I. (1998). The aminergic control of salivary glands in the stick insect, Carausius morosus. Biogenic Arnines 14. 143- 162.
Baines R.A. and Tyrer N.M. (1989). The innervation of locust salivary glands. II. Physiology of excitation and modulation. J. Cornp. Physiol. A 165.407-413.
Boer H.H., Schot L.P.C., Roubos E.W., ter Maat A., Ladder J.C. and Reichelt R. (1979). ACTH-like immunoreactivity in two electrotonically coupled giant neurons in the pond snail Lymnaea stagnalis. Cell Tissue Res. 202. 23 1-240.
Bowser-Riley F. and House C.R. (1976). The actions of some putative neurotransmitters on the cockroach salivary gland. J. exp. Biol. 64. 665-676.
Davis N.T. (1985). Serotonin-immunoreactive visceral nerves and neurohemal system in the cockroach Periplaneta americana (L.). Cell Tissue Res. 240, 593-600.
Evans P.D. (1980). Biogenic amines in the insect nervous system. Adv. Insect Physiol. 15.
Fuse M., Ali D.W. and Orchard I. (1996). The distribution and partial characterization of FMRFarnide-related peptides in the salivary glands of the locust, Locusta rnigratoria. Cell Tissue Res. 284,425-43 3.
Grewe C.W. and Kebabian J.W. (1982). Doparnine stimulates production of cyclic AMP by the salivary gland of the cockroach, Nauphoeta cinema. Cell. Mol. Neurobiol. 2,6569-
Harrow LD., Gration K.A.F. and Evans N.A. (1991). Neurobiology of arthropod parasites. Parasitology 102. S59-S69.
House C.R. (1973). An electrophysiological study of neuroglandular transmission in the isolated salivary glands of the cockroach. J. f ip . Biol. 58.29-43.
House C.R. and Ginsborg B.L. (1985). Salivary Gland. In: Comprehensive Insect Physiology, Biochemistry and Pharmacology, G.A. Kerkut and L.I. Gilbert (Eds). Pergamon Press, 195-224.
Lindsay P.J. and Kauiman W.R. (1985). Potentiation of salivary fluid secretion in ixodid ticks: a new receptor system for y-arninobutyric acid. Can. J. Physiol. Pharmacol. 64. 1 1 19- 1126.
Needharn G.R. and Sauer J.R. (1975). Control of fluid secretion by isolated salivary glands of the lone star tick. J. Insect. Physiol. 21. 1893-1898.
Needharn G.R. and Sauer J.R. (1 979). Involvement of calcium and cyclic AMP in controlling ixodid tick salivary fluid secretion. J. Parasitol. 65.53 1-542.
Orchard I. and Lange A.B. (1986). Pharmacological profile of octopamine receptors on the lateral oviducts of the locust, Locusta migrutoria. J. Insect Physiol. 32. 741-745.
Sauer J.R., McSwain J.L., Bowman A.S. and Essenberg R.C. (1995). Tick salivary gland physiology. Annu. Rev. Entomol. 40. 245-267.
Schacter J. and Braunig P. (1995). Activity pattern of suboesophageal ganglion cells innervating the salivary glands of the locust Locusta rnigratoria. J. Comp. Physiol. 176. 491-501.
Shida T., Kondo E., Ueda Y., Takai N., Yoshida Y., Araki T., Kiyarna H. and Tohyama M. (1995). Role of amino acids in salivation and the localization of their receptors in the rat salivary gland. Mol. Brain. Res. 33. 26 1-268.
Watkins B.L. & Burrows M. (1989). GABA-like immunoreactivity in the suboesophageal ganglion of the locust Schistocerca gregaria. Cell Tissue Res. 258. 53-63.
Wong D.L.P. and Kaufman W.R. (198 1). Potentiation by spiperone and other botyrophenones of fluid secretion by isolated salivary glands of ixodid ticks. Eur. J. Pharmacol. 73. 163-173.
IV. GENERAL DISCUSSION
The insect salivary gland preparation has been well studied over the past thirty years.
It has served as a preparation for the study of the control of a secretory organ, and for the
elucidation of the secretion process itself (for review see House and Ginsborg, 1985). The god
of this project was to obtain a better understanding of the control of an insect salivary gland, and
to study amine-amine interactions, as well as interactions between arnines and other neuroactive
substances. Carausius salivary giands provide a good model system to study these interactions.
In this section I will summarize what has been learned using the salivary glands of Carausius,
and how it relates to our present understanding of the topic.
h the fmt chapter of this thesis, I have confirmed that the salivary glands of Carausius
receive projections from the SN1 and SN2 via the salivary nerve. The cell bodies of the SN1
and SN2 are located in the suboesophageal ganglion, and the neurons stain positively for
tyrosine hydroxylase and serotonin respectively. Tyrosine hydroxylase is the rate limiting
enzyme in the formation of catecholamines in vertebrates, and tyrosine hydroxylase-like
imrnunoreactivity is indicative for the presence of doparnine in insects (Ali and Orchard, 1996).
Dopamine and serotonin increase cyclic AMP levels in the salivary glands of Carausius in a
dose-dependent manner. The elevation of cyclic AMP is mediated through distinct receptors.
The dopamine receptors are pharmacologically similar to vertebrate D,-like receptors, and the
serotonin receptors are similar to both vertebrate 5HT, and 5HT, receptors. Stimulation of the
salivary nerve also increases cyclic AMP content in the salivary nerves, an effect which can be
partially blocked by doparnine and serotonin receptor antagonists. Dopamine and serotonin
also hyperpolarize the membrane potential of acinar cells in the salivary glands (unpublished
results).
In the second chapter of this thesis I have shown that the salivary glands also receive
GABA-like projections from the salivary nerve. Since the salivary nerve of Carausius is known
to contain only the axons of the SNZ and SN2 (AIi and Orchard, 1996), GABA must be
colocalized in one of these neurons. GABA potentiates dopamine-induced increases in cyclic
AMP content in the salivary glands, but has no intrinsic activity. This potentiation can be
blocked by 2-hydroxysaclofen, a potent GABA, receptor antagonist.
The salivary gland system of Caraurius shows many similarities to other salivary gland
systems that have been studied. The salivary glands of the locust and cockroach receive
innervation from the SNl and SN2 (Altman and Kien, 1979; Klemm, 1972), as do many other
insects that have been studied. Doparnine increases cyclic AMP levels in the salivary glands
of the locust and cockroach (Ali et al., 1993; Grewe and Kebabian, 1982)- while serotonin also
increases cyclic AMF levels in the locust (Ali et al., 1993). Dopamine and serotonin
hyperpolarize the membrane potential of acinar cells in the locust and cockroach
(Asimakopoulos and Orchard, unpublished observations; House, 1973). Furthermore, the
pharmacological characteristics of the dopamine and serotonin receptors mediating
physiological responses in the locust and cockroach are similar to receptors found in Caruusius
(Ali and Orchard, 1994; Evans and Green, 1990a; Evans and Green 1990b).
There does appear to be a general theme in the control of salivary glands of many of the
insects studied. Dopamine and serotonin are usually involved, although they may arise from
different neuronal sources. Cyclic AMP appears to act as a second messenger in some of the
systems studied, although other second messenger pathways such as the phospholipase C
pathway may be involved (Fain and Berridge, 1979). However, there appear to be variations of
this general theme. Interestingly, the salivary ducts of Carausius appear to receive innervation
from both SNl and SN2, whiIe the ducts of Locusta do receive innervation (Whitehead, 197 I),
but this innervation was not tyrosine hydoxylase-like or serotonin-like positive. Some
GABAergic projections have been observed on the salivary ducts of the locust (unpublished
observations), while the ducts of Carausius do not appear to receive GABAergic projections.
These variations in innervation patterns between the two species may represent differing
physiological roles of the ducts. Much more study is needed to understand the role of the
salivary ducts, and how they are controlled.
One question of interest has been why are the salivary glands innervated by two neurons
that use two different neurotransmitters, but appear to serve the same role in the control of
salivary glands. One possibility is that the two neurons are active at different times, and this
has been shown to be the case in the locust (Schactner and Braunig, 1995). Furthermore it has
been shown that stimuIation of the submaxiIlary nerve in locust increases activity of the SNZ
but not the SN2 @.W. Ali, Ph.D. Thesis). Another possibilty involves the discovery of GABA
colocalized in the SN2 of locust (Watkins and Burrows, 1989) and one of the salivary neurons
of Carausius. Since the discovery of GABA in the SN2 of locust, there has been no elucidation
of the role that GABA may serve in this system. In the tick, it has been shown that GABA
potentiates dopamine-induced fluid secretion (Lindsay and Kaufman, 1985). In the present
study, we have shown that GABA potentiates doparnine-induced increases in cyclic AMP
content in the salivary glands. Cyclic AMP is believed to be a second messenger in the control
of salivation in Carausius (Asimakopoulos and Orchard, 1998). GABA may have a
neuromodulatory role in the control of salivary glands in Carausius. If GABA is colocalized
in the SN2 of Carausius as it is in the locust, then activity in the SN2 would influence
physiological effects caused by activity in the SNI. The neuromodualtion by GABA could
provide a finer and more complicated control of the salivary glands in different feeding
circumstances. GABA's neuromodulatory role also reveals that there are differences in how
dopamine and serotonin act in controlling the salivary glands of Carausius.
It is interesting that there are major differences in the actions of GABA between the tick
and Carausius, even though GABA appears to play a similar role in both. In the tick, the
potentiation is blocked by GABA, receptor anatagonists, while GABA, receptor agonist had
no effect (Lindsay and Kaufman, 1985). In Carausius the potentiation is blocked by 2-
hydroxysaclofen, a potent GABA, receptor antagonist. Thus, GABA may be working through
two different receptor types in these species, although a more complete pharmacological study
of the GABA receptor in Carausius is needed. This study has merely provided an introduction
to GABA's possible role in insect salivary glands, and provides some groundwork for future
study.
REFERENCES
Ali D.W. and Orchard 1. (1994). Characterization of dopamine and serotonin receptors on the salivary glands of the locust, Locusta rnigratoria. Biogenic Amines 10. 195-2 12.
Ali D.W. and Orchard I. (1996). Irnrnunohistochemical localization of tyrosine hydroxylase in the ventral nerve cord of the stick insect, Carausius morosus, including neurons innervating the salivary glands. Cell Tissue Res. 285453462.
Ali D.W., Orchard I. and Lange A.B. (1993). The aminergic control of locust (Locusta rnigratoria) salivary glands: Evidence for dopaminergic and serotonergic innervation. J. Insect Physiol. 39. 623-632.
Altman J.S. and Kien J. (1979). Suboesophageal neurons involved in head movements and feeding in locusts. Proc. R. Soc. Lond. B 205.209-227.
Asimakopoulos S. and Orchard I. (1998). The aminergic control of salivary glands in the stick insect, Carausius morosus. Biogenic Arnines 14. 143-162.
Evans A.M. and Green K.L. (l99Oa). The action of dopamine receptor antagonists on the secretory response of the cockroach salivary gland in vim. Comp. Biochem. Physiol. 97C. 283-286.
Evans A.M. and Green K.L. (1990b). Characterization of the doparnine receptor mediating the hyperpolarization of cockroach salivary gland acinar cells in vitro. Br. J. Pharmacol. 181- 103-108.
Fain J.N. and Berridge M.J. (1979). Relationship between hormonal activation of phosphatidylinositol hydrolysis, fluid secretion and calcium flux in the blowfly salivary gland. Biochem. J. 178,45-58.
Grewe C.W. and Kebabian J.W. (1982). Doparnine stimulates production of cyclic AMP by the salivary gland of the cockroach, Nauphoeta cinerea. Cell. Mol. Neurobiol. 2,65-69.
House C.R. (1973). An electrophysiological study of neuroglandular transmission in the isolated salivary glands of the cockroach. J. Exp. Biol. 58. 29-43.
House C.R. and Ginsborg B .L. (1 985). Salivary Gland. In: Comprehensive lmecr Physiology, Biochemistry and Phamacology, G.A. Kerkut and L.I. Gilbert (Eds). Pergamon Press, 195-224.
Klemm N. (1972). Monoamine-containing nervous fibres in foregut and salivary gland of the desert locust, Schistocerca gregaria Forskril (Orthoptera, Acrididae) . Comp. Biochem. Physiol. 43A, 207-21 1 .
Lindsay P.J. and Kaufman W.R. (1985). Potentiation of salivary fluid secretion in ixodid ticks: a new receptor system for y-aminobutyric acid. Can. J. Physiol. Phamacol. 64. 1 1 19- 1126.
Schactner J. and Braunig P. (1 995). Activity pattern of suboesophageal ganglion cells innervating the salivary glands of the locust Locusta migratoria. J. Comp. Physiol. 176. 491-501.
Watkins B.L. & Burrows M. (1989). GAE3A-like immunoreactivity in the suboesophageal ganglion of the locust Schistocerca gregaria. Cell Tissue Rex 258.53-63.
Whitehead A.T. (1971). The innervation of the salivary gland in the arnerican cockroach: light and electron microscope observations. J. Morph. 135.483-506.
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