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I have examined the effects of serotonin on the generation of the motor program for swimming by the medicinal leech, Hirudo medicinalis, using both intact animals and isolated nerve cords. In normal saline, isolated nerve cords rarely produced episodes of the motor program for swimming except in response to electrical stimulation of swim-initiating interneurons or of segmental nerves; saline containing micromolar concentrations of serotonin caused isolated nerve cords to produce episodes of the swim motor program in the absence of electrical stimulation. Stimulation of serotonin-containing neurons also caused isolated nerve cords to produce the swim motor program but only if the volume of saline around the ganglia containing the stimulated neurons was small (less than 50 ~1); under such conditions, serotonin was detected in the saline bathing the stimulated neurons. Serotonin also was found in blood from the sinus that contains the nerve cord of the leech. The concentrations of serotonin were correlated with the behavior of the animals whose blood was sampled; animals with the highest levels swam significantly more than did animals with low or intermediate levels; animals with the lowest levels of serotonin in their blood swam very little and were generally inactive. The concentrations of serotonin found in leech blood (10 to 100 nM) were similar to the lowest concentrations of serotonin that elicited swimming from isolated nerve cords. Experimentally elevating the concentration of serotonin in leech blood caused increased swimming activity. These observations suggest that serotonin is a modulator of neuronal control of swimming by the medicinal leech. Biogenic amines influence the control of motor systems in many organisms. Motor areas of vertebrate brains, such as the basal ganglia, receive rich aminergic innervation, degeneration of which results in such motor abnormalities as parkinsonism (Hornykiewicz, 1966, 1973). In spinal cords, aminergic pathways may activate pattern generators for locomotion (Grillner, 1975). Invertebrate nervous systems, in which amines also activate motor programs (Granzow and Kater, 1977; Anderson and Barker, 1977; Livingstone et al., 1980), are useful for analyzing the cellular basis of aminergic actions. For example, studies of identified neural pathways in Aplysia have allowed detailed analyses of the mechanisms by which serotonergic neurons cause long term changes in synaptic transmission (Klein and Kandel, 1978; Kupfermann, 1979). ’ This work was supported by National Institutes of Health Grant NS14410 and National Science Foundation Grant BNS75-23567 to Dr. William B. Kristan, Jr. and by National Institutes of Health Postdoctoral Fellowship NS 06182-01 to A. L. W. I thank Dr. Kristan for his support, advice, and enthusiasm; Philip Lloyd for advice on the bioassay for serotonin; and Janis Weeks for her helpful criticism of this manuscript. This paper presents a study of the effects of serotonin on the neuronal generation of the motor program for swimming by the leech.” The nervous system of the leech is useful for studying the effects of serotonin on a central pattern generator for several reasons: the motor program for swimming can be produced by isolated chains of ganglia (Kristan and Calabrese, 1976), a number of the motor and interneurons that participate in the control of swimming have been identified (Stent et al., 1978; Weeks, 1980), interneurons that elicit swimming in response to sensory stimuli have been identified (Weeks and Kristan, 1978; Weeks, 1980, 1981), and each segmental ganglion contains a small number (9 in ganglia 1 to 3, 7 in ganglia 4 to 21) of identifiable serotonin-containing neurons (Lent et al., 1979; Marsden and Kerkut, 1969; Rude, 1969). Although intact leeches often initiate swims in the apparent absence of external stimuli, isolated nerve cords ’ Present address and that to which all reprint requests should be ,’ Terms such as “swim” and “swimming” will be used as shorthand sent: Department of Neurobiology, Harvard Medical School, 25 Shatfor longer phrases such as “motor program for swimming.” SII will be tuck Street, Boston, MA 02115. used as an abbreviation for swim-initiating interneuron. The <Journal of Neuroscience Effects of Serotonin on Leech Swimming 937 rarely swim except in response to stimulation of whole supernatants were brought to neutral pH with KHCOX nerves or of swim-initiating interneurons. The results immediately before loading them on small (0.4 X 3 cm) presented in this paper demonstrate that: ( 2) isolated columns of Sephadex G-10 (Pharmacia) that had been nerve cords do swim in the absence of external stimulaactivated by 0.5 M formic acid for 30 min. The columns tion if serotonin is applied to them (first shown by Weeks, had been washed with distilled water until the pH of the 1980) or if, under certain conditions, identified serotonereffluent was 7.0 before loading the samples. After sample gic neurons are stimulated; (2) stimulation of serotonerapplication, the columns were washed with 6 ml of disgic neurons causes release of serotonin into the fluid tilled water to remove acetylcholine, catecholamines, surrounding the ganglia containing the stimulated neuindoleamines, and peptides that might interfere with the rons; (3) serotonin is found in the blood that bathes the bioassay for serotonin. Serotonin then was eluted by a 3nerve cord (the nerve cord of a leech lies inside a blood ml wash of 0.5 M formic acid. The formic acid was sinus); and (4) the levels of serotonin in the blood of evaporated in the dark over NaOH pellets and CaS04 leeches are related to the tendency of the leeches to swim (Drierite) and the serotonin then was dissolved in 20 ~1 in the absence of external stimuli. Some of these results of snail physiological saline (Kerkut and Meech, 1966). have been published in an abstract (Willard, 1980). Recovery of known amounts of serotonin was routinely Materials and Methods 90 to 95%. Serotonin was measured by a bioassay (Lloyd, 1978; P. Electrophysiology. The procedures of Kristan and CalE. Lloyd, personal communication) that is based on the abrese (1976) were used for the maintenance of the positive inotropic effect of serotonin on snail hearts. leeches (Hirudo medicinalis) and for the electrophysioMature (3to B-gm) snails (Helix aspersa) were kept logical experiments on isolated nerve cords. Nerve cords active for at least 6 days by feeding them lettuce and were removed from animals and pinned in a dish of cucumber. Their hearts were removed and the ventricle physiological saline (Nicholls and Baylor, 1968; as modiwas cannulated to allow constant perfusion with snail fied by Weeks, 1981) kept at 16°C by a cooling system. saline. A tension transducer monitored the strength of Individual neurons were viewed with dark-field illumiindividual ventricular contractions. After 30 to 60 min, nation and were impaled with glass microelectrodes (40 the strength of individual contractions was constant but to 60 megohms, filled with 4 M potassium acetate) for could be increased transiently by pulsed additions of intracellular recording and stimulation. Glass-tipped sucserotonin to the perfusate. Calibration curves were obtion electrodes were placed on segmental nerves for extained by injecting known amounts of serotonin into the traceliular stimulation and recording. Semi-intact prepperfusate before and after injecting aliquots (10 ~1) of arations were made as described by Kristan et al. (1974). test solutions containing serotonin that had been parThe system of Ort et al. (1974) was used for numbering tially purified from leech blood. Two criteria were used the ganglia and for the nomenclature of the nerves and to determine whether a particular heart would be used neurons. to measure the amounts of serotonin recovered from Serotonin was applied to whole nerve cords by replacsamples of leech blood: ( 1) only hearts whose contractile ing the physiological saline with saline to which serotonin strength increased at least 5% to injections of 0.1 pmol of had been added immediately before use. To apply seroserotonin were used; (2) only hearts producing linear tonin to single ganglia or to pairs of ganglia within a increases in contractile strength to injections of saline nerve cord, small dams of Vaseline/mineral oil (95/5%, containing between 0.1 and 0.8 pmol of serotonin were w/w) were constructed so that the saline bathing the used. Solutions of the serotonin from leech blood often desired ganglion or ganglia could be changed indepenneeded to be diluted to lower the concentration to within dently of the saline bathing the rest of the nerve cord the linear range of the hearts. At least three and usually (see the schematic drawing in Fig. 3). To determine the five aliquots of each sample were tested; standard deviaminimum number of connected ganglia that would swim tions for these replicate aliquots were less than 10% of in the absence of electrical stimuli, ganglia were cut away the means. The concentration of serotonin in leech blood from whole cords until the remaining connected ganglia was calculated by dividing the total amount of serotonin no longer swam. recovered from the blood sample by the volume of the Serotonin assays. Leeches were stretched rapidly, vensample. tral side up, and pinned through their suckers. Their Experiments on intact animals. The behavioral prefbody walls were slit just anterior to the rear suckers to erences of individual leeches that had been kept in sepexpose the ventral sinus in which the nerve cord lies. arate 500-ml jars were observed for 10 to 60 min each Samples of blood (10 to 50 ~1) were collected with a day after placing them in a 5-gallon aquarium. In each Hamilton microsyringe and were diluted immediately aquarium, there were six test tubes and two jar lids on with a lo-fold excess of 0.1 M HCl to stabilize the serothe bottom and one jar lid at the air-water interface; this tonin. T

Effects of serotonin on the generation of the motor program for swimming by the medicinal leech