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Neuronal Control of Mucus Secretion by Leeches: Toward a General Theory for Serotonin CHARLES M. LENT Department of Cellular and Comparative Biology, State University of New York, Stony Brook, New York 11790 SYNOPSIS. The large Rctzius cells are serotonin-containing neurons whose impulse activity controls the secretion of mucus from the skin of leeches. Serotonin elicits the secretion of mucus without any apparent synaptic transfers in either the central or peripheral nervous systems. Such a secretogogue function may be more general as serotonin controls the secretion of mucus from the gastrointestinal tract of mammals and from the ciliated gills of bivalved molluscs. Furthermore, the qualitative and quantitative distribution of serotonin in molluscs, annelids, arthropods, and vertebrates corresponds approximately with mucosecretory structures. Serotonin appears also to control other secretory functions in some of these animals. It is proposed therefore that serotonin might often function in controlling secretion. INTRODUCTION Each of the 21 segmental ganglia comprising the ventral nerve cord in leeches contains approximately 175 bilaterally symmetrical pairs of neurons. The "kolossal" cells of Retzius (1891) are the largest pair in each of these neural assemblages (Fig. I). These large neurons can be identified not only in ganglia of various species of leeches (Lent, 1973a), but also in their "brains" which consist of fused segmental ganglia (Wilson and Lent, 1973). An elegant utilization of autoradiography, chromatography, and microspectrofluorometry (Rude et al., 1969) demonstrated conclusively that Retzius cells contain serotonin (5-hydroxytryptamine, 5-HT) in high concentrations. In addition, these neurons contain up to 70 times more aromatic acid clecarboxylase, the enzyme catalyzing the synthesis of 5-HT from 5-HTP (5-hydroxytryptophan), than other large neurons in the leech ganglion (Coggeshall et al., 1972). Thus, the Retzius cell might be a model serotonergic neuron. And given the nature of a symposium, I shall treat it as such a neuron and proceed I thank Gunther S. Stem for helpful discussions and critical ideas for the research reported herein. I am also grateful for the technical assistance of Ellis Story, Ginna Davidson, Sharon Swift, and Trey Parrish. Supported in part by PHS Grant CM 17866 (to G.S.S.) and in part by N'SK Grant GB39614. 931 FIG. 1. The large paired Retzius cells in a segmental ganglion of the leech. (From Retzius, 1891.) from its function in the Jeech to suggest that serotonin might function similarly in other animals. THE FUNCTION OF RETZIUS CELLS IN LEECHES As their large size (50 to 80 ^m) renders them experimentally accessible, Retzius cells have been subjected to histochemical 932 CHARLES M. LENT (Ehinger et al., 1968), electron-microscopic (Gray and Guillery, 1963), neuropharmacological (Kerkut and Walker, 1967), microchemical (Rude et al., 1969), and electrophysiological studies (Hagiwara and Morita, 1962). Despite these studies however, the functional role of these large neurons has remained unknown until recently (Lent, 19736). It had been postulated on the bases of the studies cited above that Retzius cells function as inhibitory motor neurons since (i) their function must be intra- rather than inter-segmental as their processes do not enter the longitudinal connectives; (ii) their axonal branches project to the body wall via all the major ipsilateral ganglionic roots; (iii) action potentials travel centrifugally along these axons; and (iv) the perfusion of leech muscle fibers with 5-HT reduces the amplitude of their excitatory junction potentials. I have executed a variety of experiments designed to reveal effects of Retzius cell impulse activity on the musculature of the leech as a test of the validity of this suggestion (Lent, 19736). The results of these experiments were uniformly negative. For example, simultaneous intracellular recordings from Retzius cells and individual muscle fibers failed to show any correlation between neuronal action potentials and either inhibitory or excitatory postjunctional potentials in the fibers of longitudinal, circular, oblique, or dorso- ventral muscles. Moreover, bursts of impulses induced in the Retzius cells by current injection caused neither visible relaxations nor contractions. When the longitudinal or circular muscles were made to contract by stimulating the longitudinal nerve connective, alterations of the impulse activity of the Retzius cells did not affect the rate of tension increase, the maintenance of tonus, or the rate of relexation of either muscle layer. In view of the intrinsic likelihood of Retzius cells having a peripheral function, and the failure to demonstrate any effects on the musculature, I examined the possibility that Retzius cells might govern the secretion of mucus from the large glands which lie between the skin and muscle layers (Lent, 19736). The 30- to 80-ju.m glands are either tubular or globose (Fig. 2) and many axonal processes terminate near them. Leeches secrete large amounts of mucus as do many aquatic animals and, although a precise role for this slime has not been formulated, it is thought to function in sucker adhesion and skin cleansing. Two preliminary experiments demonstrated that the secretion of mucus from the skin of the leech is under neural control. Firstly, one or two segments of body wall in an otherwise intact leech were severed from the ventral nerve cord by cutting ganglionic roots. Visual inspection indicated that the Dermis" Globose Glands- Circular Muscle Tubular Glands Longitudinal Muscle Dorso-ventral Muscle Botryoidal Tissue FIG. 2. A cross section of leech body wall illus- trating the position of the mucous glands. (After Bhatia, 1941.) LEECHES, SEROTONIN, AND SECRETION Body wall Dorsal mid line Innervated segment Mucus field 933 Ganglion L Microelectrode Retzius cells Suction electrode Connective FIG. 3. Diagrammatic representation of the preparation and experimental apparatus. Retzius cell impulse activity is generated by passing current through the microelectrode and the efferent impulses are monitored by the suction electrode. The ganglionic and body wall compartments are separated by a petroleum jelly dam. The vertical calibration bar represents 25 mV for the intracellular trace A and 100 ^V for the extracellular trace B. The time calibration bar is 1 sec. During stimulation, 8 to 9 annuli secrete mucus and therefore the effector field of the Retzius cell overlaps adjacent segments (each with y annuli) about 2 in each direction. (After Lent, 19736.) denervated, and only the denervated, segmental skin remained free of slime. Secondly, the longitudinal connective in another group of leeches was cut near its midpoint and either the anterior or posterior cut end was stimulated by means of a suction electrode. The stimulated half animals—whether anterior or posterior—invariably secreted more mucus than the unstimulated control halves. The results presented below show that the neurons which control the secretion of mucus are the Retzius cells. Quantitative experiments on the relation of mucus secretion to Retzius cell activity were conducted on a preparation from the medicinal leech, Himdo medicinalis. This preparation consists of a single ganglion connected unilaterally by its roots to a corresponding half body wall extending slightly over three segments (15 annuli) in length (Fig. 3). The ganglion, pinned to a layer of transparent Sylgard resin (Dow Chemical) and viewed with transmitted light, is separated from the body wall by a dam of petroleum jelly, in order that the ganglion and wall can be bathed in salines of different composition. The body wall is usually bathed in leech physiological saline (Nicholls and Baylor, 1968), whereas the ganglion is usually bathed in saline whose Mg-+ concentration is raised to 20-mM. Thus, while the high Mg-+ in the ganglion compartment abolishes chemical synaptic transmission in the central nervous system of this preparation (Stuart, 1970)—presumably by inhibiting the release of neurotransmitters from axon terminals—the saline in the body wall compartment permits release of transmitters in the periphery. This procedure has the advantage that, being deprived of synaptic inputs, the Retzius cells lose most of their spontaneous impulse activity until depolarized experimentally. Furthermore, any increase in the rate of mucus secretion can be attributed to a direct effector action of Retzius cells on the periphery rather than any synaptic activation of other central neurons. Since leeches often secrete copious amounts of mucus during their dissection, the preparation is removed from the animal in a high Mg2+ saline. This dissection procedure reduces neural activity which in turn restricts somewhat the loss of glandular stores of mucus prior to an experiment. The Retzius cell innervating the body wall is impaled with a micropipette con- 934 CHARLES M. LENT taining 4-M potassium acetate. Depolarizing current pulses are passed into the cell by means of a balanced bridge circuit which allows the same electrode simultaneously to detect the impulse activity, which in turn is monitored on an oscilloscope, recorded on a penwriter, and summed automatically on an electronic counter. The efferent impulse activity from the ganglion was also monitored by means of a suction electrode attached to the dorsal branch of the posterior root, usually at the cut end of the contralateral root, but occasionally en passant on the intact ipsilateral root. Since the pair of Retzius cells is so strongly coupled by an electrotonic junction that their impulses are nearly synchronous (Hagiwara and Morita, 1962), either side provides a good measure of the impulses traveling from the central cell body to the innervated half body wall. The secreted mucus is assayed by taking advantage of its adsorption of carmine red. For this purpose, the skin is inundated with an excess of carmine suspended in saline at an approximate concentration of 75 mg/ ml. After 5 min, the unadsorbed carmine is removed by gentle washing with saline. The mucus together with its adsorbed carmine is then collected with a suction pipette, transferred into a test tube, and sedimented into a pellet in a clinical centrifuge. After removing the supernatant fluid, the pellet is resuspended in a fixed volume of diluted sulfuric acid which dissolves the carmine. The concentration of carmine is assayed colorimetrically by measuring intensity of red color in solution and this is taken to be proportional to the amount of mucus. As the rate of mucus secretion by different preparations is variable (about twofold), results are reported as percentages of the maximum amount of mucus secreted by a particular preparation. The Retzius cell was depolarized by current pulses sufficient to produce different numbers of impulses during successive 15min periods. The rate of mucus secretion is roughly proportional to the number of impulses (Fig. 4) rising at least eightfold from the control period (when the 500 impulses were spontaneous) to the period of maxi- ~ '00 - £60 r 0 2 4 6 3 Retzius cell impulses (10 per 15 min) FIG. 4. Percent mucus secreted as a function of Retzius cell impulses during 15-min stimulation periods. The depolarizing pulses were delivered at 0.1 Hz and each lasted 4.5 sec. The current injected determined the total impulse number. (After Lent, 197S/>.) mum stimulus depolarizations (when 6000 impulses were generated). The maximum amount of mucus secreted by this preparation corresponded to a weight of about 10 mg. In this experiment, the number of impulses was increased in successive stimulation periods in order to insure that any exhaustion of mucus reserves during the experiment would result in an underestimate of the dependence of the rate of mucus secretion on impulse activity. As it takes a few minutes for the accelerated rate of mucus secretion to return to its low spontaneous level, successive stimulation periods were separated by intervals of at least 10 min. In other experiments, the number of impulses was decreased in successive stimulation periods. Since here, too, the rate of mucus secretion corresponded to the total impulse number, one must conclude that these results (Fig. 4) cannot be attributed to any cumulative effect of successive collections of mucus in the assay procedure. Thus, these results demonstrate that the impulse activity of Retzius cells controls the rate of mucus secretion by the dermal glands of leeches. No increases in the rates of mucus secretion with impulse activity were observed when the body wall was bathed in a high Mg2+ saline, which suggests that the Retzius cell exerts its effect by the release of 5-HT from its axon terminals in the body wall. To test this possibility, I examined the LEECHES, SEROTONIN, AND SECRETION 935 these large neurons. Nor do the data allow any decision as to whether the control of mucus secretion is mediated by a synaptic or indirect route such as neurosecretion or diffusion through tissue spaces. An indirect route appears more likely as very few 5-HTcontaining axons terminate directly upon the mucous glands (Yaksta-Sauerland and Coggeshall, 1973). O " 10~ 5 10" 4 10"3 10" 2 5-Hydroxytryptamine (M) FIG. 5. Per-cent mucus secreted by deganglionated body wall of leech as a function of 5-HT concentration. The data are from a single experiment on 5 half sections of body wall each 1.5 segments long and bathed in 20 mM Mg2+. (After Lent. 19736.) A GENERAL FUNCTION FOR SEROTONIN? The literature on 5-HT (Garattini and Shore, 1968; Page, 1968; Barchas and Usdin, 1973) contains several lines of evidence which suggest that the mediation of mucus secretion by serotonin may not be restricted to leeches, but may be more widespread. The parallel to the neuron-serotonin-secretion process _n leeches is especially evident in preparations from two diverse groups of animals: the gastro-intestinal tract of mammals and the ciliated gills of bivalve molluscs. effect that the direct application of 5-HT onto the body wall has on mucus secretion. Preliminary experiments showed that a drop of concentrated 5-HT applied to the skin results in the immediate formation of a glob of mucus in the treated area. For a more quantitative study of this effect, equalsized deganglionated sections of body wall (lateral halves, three segments long) were The mammalian gastro-intestinal tract each bathed in high Mg2+ saline to which different amounts of 5-HT had been added. The abundant serotonin of the gastroAfter 45 min, the mucus was collected and intestinal (G-I) tract is localized within its 3 assayed. Exposure of the body wall to 10~ enterochromaffin cells at concentrations up M and 10~2 M 5-HT increased the rate of to 10 jw,g/g—nearly 100 times more concenmucus secretion more than threefold over trated than in neural tissue (Feldberg and the control with no added 5-HT, while in- Toh, 1953; Pletscher et al., 1955). The G-I termediate concentration produced sub- mucous membranes both synthesize and maximal effects (Fig. 5). release this serotonin (Blaschko, 1958). The The finding that the direct application of addition of serotonin to the stomach of 5-HT to the body wall causes mucus secre- either dogs (Smith, 1958; Menguy, 1967) or tion offers further support for the inference guinea pigs (Wilson, 1958) stimulates the that the Retzius cell controls the secretory secretion of copious mucus. Intravenous adprocess without synaptic transfers within ministration of serotonin to dogs stimulates the central nervous system, because the only the secretion of so large a volume of G-I axons in the ganglionic roots containing mucus (up to a 700% increase) as to induce this neurochemical are those of the Retzius vomiting and defecation (White and Magee, cells (Ehinger et al., 1968; Marsden and 1958). Furthermore, the serotonergic secreKerkut, 1969). Since the applied 5-HT tion of G-I mucus is probably under neural mediates the secretion of mucus in the pres- control, as stimulation of the vagus nerve ence of Mg2+, the 5-HT from the Retzius results in the release of 5-HT from the cells most likely activates the mucous glands stomach (Bulbring and Gershon, 1968) and directly without any synaptic transfers gastric mucus secretion follows hypothalathrough a peripheral nerve plexus. These mic stimulation (Leonard et al., 1967). data do not demonstrate that the control of Serotonin has often been implicated in mucus secretion is the only effector role for increasing G-I tract motility; however, in 936 CHARLES M. LENT the experiments reported above, either atropine or hexamethonium blocked the motility induced by the 5-HT without altering its secretory effects. This implies, of course, that the serotonin is activating either other neurons or different receptor sites which in turn control the gut musculature. Thus, the mammalian gut is rich in serotonin which induces its epithelium to secrete mucus. Here too then, as in the body wall of the leech, serotonin appears to mediate mucus secretion. Additional inferential support for serotonin subserving the secretion of a protective coat of mucus is provided by the experimental application of reserpine to the gastric mucosa of rats (Rasanen, 1968). Reserpine is effective both in depleting gastric enterochromaffin cells of their serotonin and in producing ulcer-like lesions in the treated areas. These studies suggest that G-I serotonin directly stimulates secretion of a lubricating mucus and indirectly stimulates the movements of the bowels; therefore, the answer to the query of Collier (1958), "Is HT nature's remedy for constipation?", is very likely—yes! The ciliary cpithclia of molluscs Bivalved molluscs trap their food in a sheel of mucus which is continually secreted, moved across the surface of large, paired gills by ciliary activity, and then ingested together with entrapped food particles. The frequency of the ciliary metachronal waves on each of these gills is controlled by the impulse activity of the branchial nerve, which when cut reduces ciliary activity (Aeillo, 1960) and when stimulated increases the ciliary activity (Aeillo, 1970) of nearly all species examined. Serotonin is suggested to be the transmitter responsible for this cilio-excitation, not only because it can be measured in the gills (1.5 /xg/g in Mytilus), but also because the addition of serotonin to these gills increases the frequency of their ciliary movements two to three times (Gosselin et al., 1962). Furthermore, the gills of these bivalves enzymatically decarboxylate 5-HTP to 5-HT, which in turn stimulates cilio-excitation, from which it follows that cilio-excitation by 5-HTP has a slower onset and a longer duration than excitation by serotonin. Treatment of the gills with BOL (bromolysergic acid diethylamide, an inhibitor believed to block serotonin receptor sites) interferes with cilio-excitation produced either by neural stimulation or by addition of 5-HT (Aeillo, 1970)—implying that both these types of excitation possess a common receptor. The central ganglia of bivalves contain higher concentrations of 5-HT (up to 60 jttg/g) than neural tissues of the many other species examined (Welsh and Moorhead, 1960). The innervation of the bivalve gill has been investigated by fluorescence histochemistry—a technique which utilizes a reaction between the serotonin in freeze-dried tissues and formaldehyde producing a fluorophore which emits a characteristic yellow color unlike the greener colors from other monoamines (Falck et al., 1962). Pharmacological agents can either enhance or deplete particular monoamines, and these techniques yield evidence suggesting that the yellow fluorescent axons in the bivalve branchial nerve contain serotonin, enter the gill filaments, and innervate the ciliated epithelial cells (Paparo, 1972). The evidence presented above strongly suggests that the neuronal release of serotonin excites the gill cilia of bivalves. Remembering that a major function of these motile organelles is the propulsion of a mucous sheet, I have conducted some qualitative experiments in order to ascertain whether neuronally-released serotonin also controls the secretion of mucus from the gills of bivalved molluscs. The neural control of secretion was investigated by transection and stimulation of the branchial nerve in the ribbed mussel, Modiolus dcmissus. The distal end of the cut nerve was stimulated through a suction electrode and the amount of mucus secreted was visually compared (by the application of powdered carmine) to that of the control gills on the unstimulated half of the mussel. In all cases, branchial nerve stimulation produced obvious increases in the rates of mucus secretion (and ciliary activity) by the gills of this LEECHES, SEROTONIN, AND SECRETION bivalved mussel. Experiments measuring the effects of 5-HT on mucus secretion were conducted by exposing isolated pairs of gills from Modiolus and Mytilus edulis to 5-HT (10~3 M) in artificial sea water (Instant Ocean, Eastlake, Ohio). The gills of both species secreted more mucus than control gills without added 5-HT. Therefore, the serotonin in the neural tissues of bivalves stimulates not only cilio-excitation, but also the secretion of the large volumes of mucus for which they are so well known. Such a concomitant function for serotonin presumably has the advantage to the mollusc of maintaining a uniformly thick layer of mucus which is propelled across the gill surfaces at various velocities dictated by neural activity. PHYLOGENETIC DISTRIBUTION OF SEROTONIN The experiments cited above demonstrate that neuronally released serotonin mediates the secretion of mucus by preparations from three phylogenetically distant species: leech skin, mammalian gastric mucosa, and clam gill. Such observations lead to the inference that serotonin may often function as a mediator of secretion. If this suggestion is valid, then the qualitative and quantitative phylogenetic distribution patterns of serotonin ought to correspond with muco-secretory structures. Indeed, it does, as the quantities of serotonin within the neural tissues of the major taxa correlate approximately with their relative mucussecreting abilities. Thus, molluscs and annelids—animals whose outer surfaces are composed of active mucous epithelia—have the highest amounts of serotonin within their nervous systems, while the arthropods with their cuticular exoskeletons have the lowest amounts. The vertebrates, which are intermediate in their mucus-secreting abilities, have intermediate amounts of 5-HT in their nervous systems (Welsh, 1968). Further monoamine oxidase, the enzyme which inactivates 5-HT can be detected in annelids, molluscs, echinoderms, and lampreys—all known slime producers (Blaschko, 1958). A detailed examination 937 of the available data on molluscs, annelids, arthropods, and vertebrates follows. Mollusca The concentration of serotonin within the nervous systems of the three major classes nf molluscs correlates approximately with their relative mucus-secreting ability. Thus, bivalves with 10 to 60 jxg 5-HT/g of nerve tissue produce more mucus than cephalopods (1 to 4 jxg/g) and the gastropods are intermediate between them with 2 to 10 ng/g (Welsh and Moorhead, 1960). Fluorescence histochemistry indicates that in bivalves both gills (see above) and some kidney cells contain serotonin (Sweeney, 1968). The escargot, Helix, has a pair of large cerebral serotonergic neurons (Bardessono et al., 1972) whose axons terminate in the body wall near the lip (Penreath and Cottrell, 1972)—a structure known to secrete much of the mucus lubricating the foot (Bullough, 1958). Large amounts of serotonin are found also in the muricid gastropod hypobranchial glands—organs which produce large amounts of a shelldissolving secretion when these marine snails "drill" their bivalve prey (Carriker, 1961; Welsh, 1970). Very high amounts of serotonin are associated also with the salivary glands of Octopus; however, it cannot now be decided whether this serotonin stimulates release of a secretion used to drill through the shells of molluscan prey (Arnold and Arnold, 1969; Wodinsky, 1969) or whether the serotonin is used as a toxin (a role which is further discussed below). Thus, in molluscs, the amounts and sites of serotonin seemingly correspond with mucus or with secretory structures. Annelida All three classes of annelids have similar quantities of serotonin within their neural tissues (3 to 10 /*g/g). The central neurosomata contain serotonin, fluoresce a brilliant yellow-gold, and send their axons peripherally in polychaetes (Clark, 1966), earthworms (Rude, 1966), and leeches (Ehinger et al., 1968; Marsden and Kerkut, 938 CHARLES M. LENT be stimulated also by 5-HT at 10~7 M (House, 1973) even though its actual transmitter appears to be dopamine (House et al., 1973). Many crustaceans are bioluminescent, and in the euphausiid Meganyctiphanes serotonin is able to both directly stimulate and increase the sensitivity of their light organs (photophores) to other forms of stimulation (Kay, 1965). Many photophores are glandular structures only slightly modified from mucous glands and other crustaceans emit their extra-corporeal bioluminescent secretions cojointly with mucus from unmodified glands—apparently lending cohesiveness to the luminescent chemicals (Nicol, 1962). The venom glands of many arthropods— scorpions, spiders, wasps, and hornets, but not bees—contrast with most of their tissues by containing massive amounts of 5-HT (up to 19 mg/g dry weight; Welsh, 1970). A role for serotonin in venoms is widespread, however, and it can be found highly conArthropoda centrated in the salivary glands of Octopus, Arthropods usually have less than 0.2 jxg the labial poisons of the Gila monster, and of serotonin per gram of neural tissue—a the poisonous skin secretions of the toad fact which correlates with the low secretory Bnfo (Garattini and Valzelli, 1965). Many activity of their hypodermis. Thus, the of these venoms produce intense pain as crayfish Astacus has but a single pair of does serotonin, and the venom of the Gila yellow fluorescent neurons (Osborne and monster produces symptoms similar to the (Elofsson et al., 1966). However, the lobster effects of injected sertonin, vomiting, salivastomatogastric ganglion contains several tion, and defecation (Minton and Minton, yellow fluorescent axons (Osborne and 1969); however, no inference can be drawn Dando, 1970) whose axons presumably in- regarding the function of serotonin in toxnervate the muco-secretory intestine. The ins. At the very least there is some assoneurosecretory pericardial organs (Cooke, ciation of the serotonin in arthopods with 1964) of decapod crustaceans contain 100 to secretory activity. 200 times more serotonin than their ordiVertebrata nary neural ganglia (Welsh, 1970). Serotonin can be detected within the High concentrations of serotonin are brain of blowflies and their salivary glands are sensitive to it—more so in fact than any present within these vertebrate structures: related chemical species tested by Berridge G-I tract, pulmonary epithelium, pancreas, (1972). These glands respond to 5-HT in thyroid, pineal gland, spleen, mast cells, concentrations as low as 10~10 M with in- and platelets. Furthermore, chromaffin cells creases in their rates of fluid secretion of can be identified by histochemistry in the 20 to 40 times (Berridge and Patel, 1968). biliary tract, pancreatic duct, prostate This serotonergic salivation is mediated by gland, and vaginal epithelium of mammals a Ca2+-dependent, cyclic AMP mechanism (Hakanson, 1970). Many of these structures (Berridge and Prince, 1972). The secretion are secretory. of saliva by the glands of the cockroach can A secretagogue function for serotonin in 1969). These yellow axons terminate most often in the body wall, and in the polychaete Glycera large yellow terminals can be seen within the skin (Manaranche and L'Hermite, 1973). A yellow fluorescence is often noted also within the tissues of the nephridia, pharynx, and intestinal tract. Thus, the amounts and distribution of serotonin within the bodies of annelid worms correlate somewhat with mucosecretory structures, and further, for each class there are experimental findings which support the general hypothesis. The data reported above on leeches form the nucleus for this hypothesis. In polychaetes, Lawry (1967) demonstrated that stimulation of the parapodial nerves in Harmathoe causes the discharge of its mucous glands. Additionally, the topical application of drops of concentrated 5-HT to the deganglionated skin of earthworms elicits a localized secretion of mucus (Lent, unpublished). 939 LEECHES, SEROTONIN, AND SECRETION the G-I tract was detailed above, and there are data suggesting a similar function for the serotonin within the pulmonary tree. Foreign particles are removed from the respiratory epithelium on a sheet of mucus propelled by ciliary activity. The epithelial mucous glands are under neural control (Schlesinger, 1973) and ciliary activity of isolated rabbit iiddiea is modestly accelerated by 10~9 M 5-HT. Even lower concentrations of 5-HT are cilio-excitatory if the trachea is pretreated with reserpine (Tsuchiya and Kensler, 1959). The fine structural organization of pulmonary epithelium in rabbit occasioned the proposal that neuro-epithelial bodies (serotonincontaining cells) might modulate mucus secretion (Layweryns et al., 1973). Thus, this muco-ciliary epithelium in mammals may be excited by serotonin as is the mucociliary epithelium of the molluscan gill (see above). Serotonin is implicated in a variety of other secretory functions in mammals including the following: 1) the initiation of milk secretion from the mammary glands of virgin rats (Meites et al., 1959). 2) an increase in the amylase and protein secreted with saliva from rabbit parotid glands (Kojima et al., 1973). 3) stimulation of excess salivation by goats (Andersson et al., 1966). 4) being essential in the secretion of insulin from the pancreatic islet cells (Lernmark, 1971). 5) secretion of both ACTH (Verdesca et al., 1961) and a substance which reduces tetrazolium blue (Rosenkranz and LaFerte, 1960) from adrenal glands, organs where the secretagogue function of serotonin is Ca2+~ dependent (Douglas, 1965). CONCLUSION Many data are consonant with the proposal that serotonin often functions by controlling secretion. This thesis in no way argues against other serotonergic functions, such as relaxing the "catch" of molluscan tonic muscles (Twarog, 1967) or as a neuro- ACh E3=5-HT ED'SECRETION PERIPHERY B FIG. 6. Anatomical relationships between serotonergic cells and mucous glands. A, The unicellular mucous glands in the brain of Nepthys. (After Clark, 1!)56.) B, A central serotonergic neuron controlling peripheral mucous glands. C, Extrinsic serotonergic control of mucous glands. The peripheral seiotoneigic cells are controlled by central neurons. transmitter within the central nervous system (e.g., Greschenfeld, 1971). The neuronal control of mucus secretion has been a common theme throughout this paper; however, there are diverse morphological relationships of the serotonergic cell and mucous gland to the central nervous system and the periphery. In some animals, such as the polychaete worm, Nepthys, cell bodies of unicellular mucous glands reside within the brain where they are presumably excited and their secretions are then carried outside the body by long tubular lumina (Fig. 6/4). The mucous glands are situated in the periphery in mollusca and most annelids. Serotonergic neurons within the central nervous system control these glands through peripheral axons (Fig. 6B). The leech Retzius cell is such a neuron and it is excited by cholinergic neurons (Kerkut and Walker, 1967). In the G-I tract and pulmonary epithelium of mammals, the serotonergic cells are themselves in the periphery and closely associated with the mucous glands (Fig. 6C). The activity of other central neurons (which may be cholinergic, e.g., the Vagus nerve and gastric secretion, 940 CHARLES M. see above) modulate the activity of the serotonergic cells, indirectly controlling the secretion of mucus. This thesis for serotonin functioning often as a secretagogue should provide an impetus for definitive research designed to answer questions such as these: 1) By what mechanisms does serotonin control mucous glands? 2) Do these mechanisms depend upon Ca2+ and cyclic AMP? 3) What biophysical mechanisms underlie the secretory process? This thesis should be easily testable. If it is not found coherent, we can discard or modify it. However, if it is even moderately accurate, we can begin formulating answers to Irwing Page's (1968) question on serotonin: "What does it do?" REFERENCES Aiello, E. 1960. Factors affecting ciliary activity in the gill of the mussel, Mytilus edulis. Physiol. Zool. 33:120-135. Aiello, E. 1970. Nervous and chemical stimulation of gill cilia in bivalve molluscs. Physiol. Zool. 43-60-70. Andresson, B., M. Jobin, and K. Olsson. 1966. Serotonin and temperature control. Acta. Physiol. Scand. 67:50-56. Arnold, J. M., and K. O. Arnold. 1969. Some aspects of hole boring predation by Octopus vulgaris. Amer. Zool. 9:991-996. Barchas, J., and E. Usdin (Ed.). 1973. Serotonin and behavior. Academic Press, New York. Bardessono, R., E. Giacobini, and M. Stepita-Klauco. 1972. Neuronal localization of monoamines in the cerebral ganglia of the snail Helix pomatia. Brain Res. 47:427-437. Berridge, M. J. 1972. The mode of action of 5-hydroxytryptamine. J. Exp. Biol. 56:311-321. Berridge, M. J., and N. G. Patel. 1968. Insect salivary glands: stimulation of fluid secretion by 5-hydroxytryptamine and adenosine-3',5'-monophosphate. Science 162:462-463. Berridge, M. J., and \V. T. Prince. 1972. The role of cyclic AMP and calcium in hormone action. Insect Physiol. 9:1-49. Bhatia, M. L. 1941. Hirudinaria (the Indian cattle leech). Ind. Zool. Mem. 3. Lucknow. Blaschko, H. 1958. Biological inactivation of 5-hydroxytryptamine, p. 50-57. In G. P. Lewis [ed.], 5-Hydroxytryptamine. Pergamon Press, New York. Biilbring, E., and M. D. Gershon. 1968. Serotonin participation in the vagal inhibitory pathway to the stomach. Advan. Pharmacol. 6A:323-333. Bullough, W. S. 1958. Practical invertebrate LENT anatomy. 2nd. ed. St. Martin's Press, New York. Carriker, M. R. 1961. Comparative functional morphology of boring mechanisms in gastropods. Amer. Zool. 1:236-266. Clark, M. E. 1966. Histochemical localization of monoamines in the nervous system of the polychaete Nepthys. Proc. Roy. Soc. London B 165: 308-325. Clark, R. B. 1956. On the origin of neurosecretory cells. Ann. Sci. Natur. Zool. 118:199-207. Coggeshall, R. E., S. A. Dewhurst, D. Weinreich, and R. E. McCaman. 1972. Aromatic acid decarboxylase and choline acetylase activities in a single identified 5-HT containing cell of the leech. J. Neurobiol. 3:259-265. Coggeshall, R. E., and D. W. Fawcett. 1964. The fine structure of the central nervous system of the leech, Hitudo medicinalis. J. Neurophysiol. 27:229-289. Collier, H. O. J. 1958. The occurrence of 5-hydroxytryptamine in nature, p. 5-19. In G. P. Lewis [ed.], 5-Hydroxytryptamine. Pergamon Press, New York. Cooke, I. M. 1964. Electrical activity and release of neurosecretory material in crab pericardial organs. Comp. Biochem. Physiol. 13:353-366. Douglas, W. W. 1965. Calcium-dependent links in stimulus-secretion coupling in the adrenal medulla and neurohypophysis, p. 267-289. In U. S. von Euler, S. Rosell, and B. Uvnas [ed.], Mechanisms of release of biogenic amines. Pergamon Press, New York. Ehinger, B., B. Falck, and H. E. Myhrberg. 1968. Biogenic amines in Hirudo medicinalis. Histochemie 15:140-149. Elofsson, R., T. Kauri, S. -O. Nielsen, and J. -O. Stromberg. 1966. Localization of monoaminergic neurones in the central nervous system of Astacus astacus L'inne (Crustacea). Z. Zellforsch. Mikroskop. Anat. 74:464-473. Falck, B., N. -A. Hillarp, G. Thieine, and A. Torp. 1962. Fluorescence of catecholamines and related compounds condensed with formaldehyde. Histochem. Cytochem. 10:348-354. Feldberg, W., and C. C. Toh. 1953. Distribution of 5-Hydroxytryptamine (serotonin, enteramine) in the wall of the digestive tract. J. Physiol. (London) 119:352-362. Garattini, S., and P. A. Shore [ed.]. 1968. Biological role of indolealkylamine derivatives. Advances in pharmacology. Vols. 6A and 6B. Academic Press, New York. Garattini, S., and L. Valzelli. 1965. Serotonin. Elsevier, New York. Gerschenfeld, H. M. 1971. Serotonin: two different inhibitory actions on snail neurons. Science 171: 1252-1254. Gosselin, R. E., and K. E. Moore, and A. S. Milton. 1962. Physiological control of molluscan gill cilia by 5-hydroxytryptamine. J. Gen. Physiol. 46:277296. Gray, E. G., and R. W. Guillery. 1963. An electron microscopical study of the ventral nerve cord of LEECHES, SEROTONIN, AND SECRETION the leech. Z. Zellforsch. Mikroskop. Anat. 60: 826-849. Hagiwara, S., and H. Morita. 1962. Electrotonic transmission between two nerve cells in leech ganglion. J. Neurophysiol. 16:740-756. Hakanson, R. 1970. New aspects of the formation and function of histamine, 5-hydroxytryptamine, and dopamine in gastric mucosa. Acta. Physiol. Scand. Suppl. 340:1-134. House, C. R. 1973. An eletrophysiological study of neuroglandular transmission in isolated salivary glands of the cockroach. J. Exp. Biol. 58:29-43. House, C. R., and B. L. Ginsborg, and E. M. Silinsky. 1973. Dopamine receptors in cockroach salivary gland cells. Nature New Biol. 245:63. Kay, R. H. 1965. Light-stimulated and lightinhibited bioluminescence of the euphausiid Meganyctiphanes norvegica (G. O. Sars). Proc. Roy. Soc. London B 162-365. Kerkut, G. A., and R. J. Walker. 1967. The action of acetylcholine, dopamine, and 5-hydroxytryptamine on the spontaneous activity of the cells of Retzius of the leech, Hirudo medicinalis. Brit. J. Pharmacol. Chemother. 30:644-654. . Kojima, S., M. Ikeda, and A. Tsujimoto. 1973. Effect of serotonin, glucagon, and other hormones on amylase secretion from rabbit parotid glands. Jap. J. Pharmacol. 23:588-591. Lauweryns, J. M., H. Cokelaere, and P. Theunynck. 1973. Serotonin producing neuroepithelial bodies in rabbit respiratory mucosa. Science 180:410-413. Lawry, J. V. 1967. Structure and function of the parapodial cirri of the polynoid polychaete, Harmuthue. '/,. Zellforsch Mikroskop. Anat. 82: 345-361. Lent, C. M. 1973a. Retzius cells from segmental ganglia of four species of leeches: comparative neuronal geometry. Comp. Biochem. Physiol. 44A: 35-40. Lent, C. M. 1973fc. Retzius cells: neuroeffectors controlling mucus release by the leech. Science 179:693-696. Leonard, A. S., R. B. Gilsdorf, J. M. Pearl, E. T. Peter, and W. P. Ritchie. 1967. Hypothalamic influence on gastric blood flow, cell counts, acid, and mucus secretion—factors in ulcer provocation, p. 149-165. In T. K. Shnitka, J. A. L. Gilbert, and R. C. Harrison [ed.], Gastric secretion, mechanisms and control, Pergamon Press, New York. Lernmark, A. 1971. The significance of 5-hydroxytryptamine for insulin secretion in the mouse. Horm. Metab. Res. 3:305-309. Manaranche, R., and P. L'Hermite. 1973. Etude des amines biogenes de Glycera convoluta K. (annelide polychete). Z. Zellforsch Mikroskop. Anat. 137:21-36. Marsden, C. A., and G. A. Kerkut. 1969. Fluorescence microscopy of the 5-HT and catecholamine containing cells in the central nervous system of the leech Hirudo medicinalis. Comp. Biochem. Physiol. 31:851-862. Meites, J., C. S. Nicoll, and P. K. Talwalker. 1959. Effects of reserpine and serotonin on milk secre- 941 tion and mammary gland growth in the rat. Proc. Soc. Exp. Biol. Med. 101:563-565. Menguy, R. 1967. Regulation of gastric mucus secretion, p. 177-185. In T. K. Shnitka, J. A. L. Gilbert, and R. C. Harrison [ed.]. Gastric secretion, mechanisms and control. Pergamon Press, New York. Minton, S. A., and M. R. Minton. 1969. Venomous reptiles. Scribner's. New York. Nicholls, J. G., and D. A. Baylor, 1968. Specific modalities and receptive fields of sensory neurons in CXS of the leech. J. Neurophysiol. 16:740756. Nicol, J. A. C. 1962. Animal luminescence. Advan. Comp. Physiol. Biochem. XX:217-273. Osborne, N. N., and M. R. Dando. 1970. Monoamines in the stomatogastric ganglion of the lobster Homarus vulgaris. Comp. Biochem. Physiol. 32:327-331. Page, I. 1968. Serotonin. Year Book Medical, Chicago, Illinois. Paparo, A. 1972. Innervation of the lateral cilia in the mussel. Mytilus edulis. L. Biol. Bull. 143:592604. Penreath, V. \\\, and G. A. Cottrell. 1972. Selective uptake of 5-hydroxytryptamine by axonal processes in Helix pomatia. Nature New Biol. 239:213214. Pletscher, A., P. A. Shore, and B. B. Brodie. 1955. Serotonin release as a possible mechanism of reserpine action. Science 122:374. Rasanen, T. 1968. Protection of the gastric mucosa against lesions caused by reserpine through degranulation of mucosal mast cells, p. 248-249. In L. S. Semb and J. Myren [ed.], The physiology of gastric secretion. Williams and Wilkins, Baltimore, Maryland. Retzius, G. 1891. Zur Kenntnis des centralen Nervensystems der Wiirmer. Das Nervensystem der Annulaten. Biol. Untersuch. N. F. 2:1-28. Rosenkrantz, H., and R. O. Laferte. 1960. Further observations on the relationship between serotonin and the adrenal. Endocrinology 66:832-841. Rude, S. 1966. Monoamine-containing neurons in the nerve cord of Lumbricus terrestris. J. Comp. Neurol. 128:397-412. Rude, S., R. E. Coggeshall, and L. S. VanOrden. 1969. Chemical and ultrastructural identification of 5-hydroxytryptamine in an identified neuron. J. Cell. Biol. 41:832-854. Schleslinger, R. B. 1973. Mucociliary interaction in the tracheobronchial tree and environmental pollution. Bioscience 23:567-573. Smith, A. N. 1958. The effect of 5-hydroxytryptamine on acid gastric secretion, p. 183-190. In G. P. Lewis [ed.], 5-Hydroxytryptamine. Pergamon Press, New York. Stuart, A. E. 1970. Physiological and morphological properties of motor neurones in the central nervous system of the leech. J. Physiol. (London) 208:627-646. Sweeney, D. C. 1968. The anatomical distribution of monoamines in a freshwater bivalve mollusc, 942 CHARLES M. Sphaerium sulcalum (L.). Comp. Biochem. Physiol. 25:601-613. Tsuchiya, M., and C. J. Kensler. 1959. The effects of autonomic drugs and 2-amino-2-methyl propanol, a choline antagonist, on ciliary movement. Fed. Proc. 18:453. Twarog, B. M. 1967. The regulation of catch in molluscan muscle. J. Gen. Physiol. 50:157-169. Verdesca, A. S., C. D. Westermann, R. S. Crampton, W. C. Black, R. I. Xedeljkovic, and J. G. Hilson. 1961. Direct adrenocoitical stimulatory effect of serotonin. Amcr. J. Physiol. 201:1065-1067. Welsh, J. H. 1968. Distribution of serotonin in the nervous system of various animal species. Advan. Pharmacol. 6A:171-188. Welsh, J. H. 1970. Phylogenetic aspects of the distribution of biogenic amines, p. 75-94. In J. J. Blum [ed], Biogenic amines as physiological regulators. Prentice-Hall, Englewood Cliffs, New Jersey. LENT Welsh, J. H., and M. Moorhead. 1960. The quantitative distribution of 5-hydioxytryptamine in the invertebrates, especially in their nervous systems. J. Neurochem. 6:146-169. White, T. T., and D. 1". Magee. 1958. The influence of serotonin on gastric mucin production. Gastroenterology 35:289-291. Wilson, A. H., and C. M. Lent. 1973. Electrophysiology and anatomy of the laige paired neurons in the subesophageal ganglion of the leech. Comp. Biochem. Physiol. 46A:301-309. Wilson, C. W. M. 1958. Discussion, p. 191-193. In G. P. Lewis [ed.], 5-hydroxytryptaminc. Pergamon Pi ess, New York. Wodinsky, J. 1969. Penetration of the shell and feeding on gastropods by Octopus. Amer. Zool. 9:997-1010. Yaksta-Sauerland, B. A., and R. E. Coggeshall. 1973. Neuromuscular junctions in the leech. J. Comp. Neurol. 151:85-100.