Serotonin modulates muscle function in the medicinal leech Hirudo verbana

The body wall muscles of sanguivorous leeches power mechanically diverse behaviours: suction feeding, crawling and swimming. These require longitudinal muscle to exert force over an extremely large length range, from 145 to 46 per cent of the mean segmental swimming length. Previous data, however, suggest that leech body wall muscle has limited capacity for force production when elongated. Serotonin (5-HT) alters the passive properties of the body wall and stimulates feeding. We hypothesized that 5-HT may also have a role in allowing force production in elongated muscle by changing the shape of the length-tension relationship (LTR). LTRs were measured from longitudinal muscle strips in vitro in physiological saline with and without the presence of 10 μM 5-HT. The LTR was much broader than previously measured for leech muscle. Rather than shifting the LTR, 5-HT reduced passive muscle tonus and increased active stress at all lengths. In addition to modulating leech behaviour and passive mechanical properties, 5-HT probably enhances muscle force and work production during locomotion and feeding.     

Chemical and thermal stimuli have short-lived effects on the retzius cell in the medicinal leech

During the appetitive phase of feeding, hungry leeches detect a prey by the integration of signals perceived by different sensory systems. Earlier reports suggested that chemical or thermal sensory stimulation of the lip was associated with increased afferent activity in cephalic nerves connecting the lip to the central nervous system. These authors further suggested that this activity was relayed to Retzius cells in segmental ganglia, which then released serotonin to initiate and control all aspects of feeding behavior. In this study, we show that chemosensory or thermal activation of the lip lasting for at least 5 min produces a distinct signal in the cephalic nerves consisting of action potentials of low amplitude. These small amplitude signals are clearly distinguishable from the large action potentials evoked by mechanosensory stimuli applied to the same area of the lip. Both types of sensory stimuli also evoke an increase in the firing frequency of the Retzius cells in segmental ganglia. However, the response recorded in the nerves and the Retzius cells during a maintained stimulus is not constant but decreases with an exponential time course. These results agree with our earlier observations on a semi-intact feeding preparation in which we showed that the firing frequency of the Retzius cell decreased as soon as the leech began to ingest its meal. Therefore, our data provide further evidence suggesting that it is unlikely that heat or chemical cues maintain the Retzius cell in an active state throughout the consummatory phase of feeding.     

Neuronal control of swimming in the medicinal leech

Summary The cell bodies and function of twelve neurons whose impulse pattern is clearly related to that of the swimming rhythm were identified in the segmental ganglion of the leech. These include excitatory and inhibitory motor neurons of the dorsal and ventral longitudinal muscles and the excitatory flattener motor neuron of the dorsoventral muscles. During swimming the membrane potential of these cells oscillates between a depolarized and a hyperpolarized phase. The activity of this ensemble of cells is sufficient to account for the contractile rhythm of the swimming animal. The following connections were found between these motor neurons. Electrotonic junctions link: (1) bilaterally homologous cells; (2) excitors of the dorsal longitudinal muscles; (3) excitors of the ventral longitudinal muscles; (4) inhibitors of both dorsal and ventral longitudinal muscles. The dorsal inhibitors project via an inhibitory pathway to the dorsal excitors, and the ventral inhibitor projects via an inhibitory pathway to the ventral excitors. The membrane potential oscillation of the excitors is at least partly attributable to the phasic inhibitory synaptic input which they receive from the inhibitors. The excitatory shortener motor neuron of the entire longitudinal musculature is maintained in an inactive state during swimming. This control is achieved by rectifying electrotonic junctions linking this neuron to the dorsal and ventral excitors. These junctions allow passage of only depolarizing current from the shortener to the dorsal and ventral excitors and of only hyperpolarizing current in the reverse direction. Furthermore, both dorsal and ventral inhibitors project via inhibitory pathways to the shortener neuron.We are greatly indebted to Ann Stuart for advice and help in this study, and for communicating to us some unpublished findings. We thank Elizabeth Mullenbach for excellent technical assistance.This research was supported by grant GB 31933 X from the National Science Foundation, and by Public Health Service Research grant GM 17866 and Training Grant GM 01389 from the Institute for General Medical Sciences.     

Neuronal control of heartbeat in the medicinal leech

Summary The leech heartbeat consists of a constriction-dilation rhythm of two lateral heart tubes extending over the length of the body. The beats of the segmental sections of these two tubes are coordinated in such a manner that the heart tube of one body side produces a frontward peristaltic wave while the heart tube on the other body side produces nearly concerted constrictions. This rhythm is metastable, in that left and right heart tubes alternate between peristaltic and concerted constriction modes, with a given mode lasting for tens or hundreds of beat cycles.The constriction-dilation cycles of the segmental heart tube sections are controlled by a set of rhythmically active motor neurons, the heart excitors, or HE cells. A bilateral pair of HE cells is located in all but the two frontmost and the two rearmost segmental ganglia of the ventral nerve cord. Each HE cell innervates via excitatory synapses the circular muscle fibers in the wall of the ipsilateral heart tube section. The activity cycle of the HE cells consists of an active phase, during which they are depolarized and produce a burst of impulses, and an inactive phase during which they are repolarized by a burst of inhibitory synaptic potentials. The intersegmentally coordinated activity cycles of the HE cell set are maintained in an isolated ventral nerve cord. Hence the generation of the heart excitor rhythm does not require sensory feedback.We are indepted to Amy Kelly and King-Wai Yau for advice on the use of the intracellular staining technique and to John Kretz for calling to our attention the existence of an afferent impulse burst rhythm emanating from denervated heart tube preparations. We thank Georgia Harper for excellent technical assistance. This research was supported by Grant GB 31933X from the National Science Foundation and NIH research grants GM17866 and Training Grant GM 01389 from the Institute of General Medical Sciences.     

Modulation of swimming behavior in the medicinal leech

The effects of serotonin on the electrical properties of swim-gating neurons (cell 204) were examined in leech (Hirudo medicinalis) nerve cords. Exposure to serotonin decreased the threshold current required to elicit swim episodes by prolonged depolarization of an individual cell 204 in isolated nerve cords. This effect was correlated with a more rapid depolarization and an increased impulse frequency of cell 204 in the first second of stimulation. In normal leech saline, brief depolarizing current pulses (1 s) injected into cell 204 failed to elicit swim episodes. Following exposure to serotonin, however, identical pulses consistently evoked swim episodes. Thus, serotonin appears to transform cell 204 from a gating to a trigger cell.Serotonin had little effect on the steady-state currentvoltage relation of cell 204. However, serotonin altered the membrane potential trajectories in response to injected current pulses and increased the amplitude of rebound responses occurring at the offset of current pulses. These changes suggest that serotonin modulates one or more voltage dependent conductances in cell 204, resulting in a more rapid depolarization and greater firing rate in response to injected currents. Thus, modulation of intrinsic ionic conductances in cell 204 may account in part for the increased probability of swimming behavior induced by serotonin in intact leeches.Abbreviations AHP afterhyperpolarizing potential - DCC discontinuous current clamp - DP dorsal posterior nerve - G2 segmental ganglion 2 - PIR postinhibitory rebound - RMP resting membrane potential     

Modulation of swimming behavior in the medicinal leech

Expression of swimming in the medicinal leech (Hirudo medicinalis) is modulated by serotonin, a naturally occurring neurohormone. Exogenous application of serotonin engenders spontaneous swimming activity in nerve-cord preparations. We examined whether this activity is due to enhanced participation of swim motor neurons (MNs) in generating the swimming rhythm. We found that depolarizing current injections into MNs during fictive swimming are more effective in shifting cycle phase in nerve cords following serotonin exposure. In such preparations, the dynamics of membrane potential excursions following current injection into neuronal somata are substantially altered. We observed: 1) a delayed outward rectification (relaxation) during depolarizing current injection, most marked in inhibitory MNs; and 2) in excitor MNs, an enhancement of postinhibitory rebound (PIR) and afterhyperpolarizing potentials (AHPs) following hyperpolarizing and depolarizing current pulses, respectively. In contrast, we found little alteration in MN properties in leech nerve cords depleted of amines. We propose that enhanced expression of swimming activity in leeches exposed to elevated serotonin is due, partly, to enhancement of relaxation, PIR and AHP in MNs. We believe that as a consequence of alterations in cellular properties and synaptic interactions (subsequent paper) by serotonin, MNs are reconfigured to more effectively participate in generating and expressing the leech swimming rhythm.Abbreviations AHP Afterhyperpolarizing potential - DCC Discontinuous current clamp - DE Dorsal excitor motor neuron - DI Dorsal inhibitor motor neuron - IPSP Inhibitory postsynaptic potential - MN Motor neuron - PIR Postinhibitory rebound - VE Ventral excitor motor neuron - VI Ventral inhibitor motor neuron     

Ultrastructure of the water-movement-sensitive sensilla in the medicinal leech

Behavioral and physiological experiments have shown that medicinal leeches are able to detect low amplitude surface waves, and further, that the transduction of this stimulus modality occurs primarily, if not exclusively, at the annular sensilla (Young, Dedwylder, and Friesen, 1981; Friesen, 1981). Here we examine the morphology of these specialized sensory structures using light, scanning electron, and transmission electron microscopes. We found that three types of ciliated sensory cells occur at the sensilla: (1) a uniciliate cell, with an axial cilium that projects at least 12 μm beyond the cuticle; (2) a multiciliate cell with from two to four grouped cilia that extend 1–3 μm beyond the cuticle; and (3) a second multiciliate cell, whose cilia project parallel to the body surface but remain within the cuticle. The cilia of all three cell types arise from the cuplike depressions which form the apices of slender, elongated cells (approximately 2 μm diameter × 50 μm length). A complexly interconnected ring of microvilli surrounds the cilium of the uniciliate cells. The morphology of the uniciliate cells closely resembles the structure of vibration-sensitive sensory neurons found in other species. We propose, based on previous results and our new findings, that the uniciliate receptor cells are the sensillar movement receptors which mediate leech sensitivity to water movements.     

The effects of feeding regime on the growth and reproduction of the medicinal leech Hirudo medicinalis

1. The feeding frequency, the size of meals, the number of meals required to attain reproductive maturity and the number of meals taken between iteroparous reproductive bouts were determined in the laboratory under optimal conditions for the medicinal leech Hirudo medicinalis fed exclusively on mammalian (bovine) blood. In addition the number of bouts of reproduction and the numbers of cocoons and hatchlings per cocoon produced were determined.
2. The average time for H. medicinalis to reach reproductive maturity at 20°C was 289 days, at an average wet biomass of 8143 mg with two–nine separate bouts of cocoon production. The number of meals to first reproduction was 8.9 (mean meal size of 3066.7 mg), with a significant correlation between total mass of blood ingested and the numbers of reproductive bouts and number of cocoons produced. Mean lifetime cocoon production per individual was 12.43, with 3.9 hatchlings per cocoon.
3. The significant positive relationships between ingestion, fecundity and developmental rate observed support the hypothesis that declining abundances of field populations of H. medicinalis are the result of lower available energy for growth, reflecting leeches now feeding predominantly on amphibian blood of lower energetic value than mammalian blood.     

The effects of feeding regime on the growth and reproduction of the medicinal leech Hirudo medicinalis

1. The feeding frequency, the size of meals, the number of meals required to attain reproductive maturity and the number of meals taken between iteroparous reproductive bouts were determined in the laboratory under optimal conditions for the medicinal leech Hirudo medicinalis fed exclusively on mammalian (bovine) blood. In addition the number of bouts of reproduction and the numbers of cocoons and hatchlings per cocoon produced were determined.
2. The average time for H. medicinalis to reach reproductive maturity at 20°C was 289 days, at an average wet biomass of 8143 mg with two–nine separate bouts of cocoon production. The number of meals to first reproduction was 8.9 (mean meal size of 3066.7 mg), with a significant correlation between total mass of blood ingested and the numbers of reproductive bouts and number of cocoons produced. Mean lifetime cocoon production per individual was 12.43, with 3.9 hatchlings per cocoon.
3. The significant positive relationships between ingestion, fecundity and developmental rate observed support the hypothesis that declining abundances of field populations of H. medicinalis are the result of lower available energy for growth, reflecting leeches now feeding predominantly on amphibian blood of lower energetic value than mammalian blood.     

Modulation of swimming activity in the medicinal leech by serotonin and octopamine

1. The monoamines serotonin (5-HT) and octopamine (OA) enhance the expression of swimming activity in the medicinal leech (Willard, 1981; Belanger and Orchard, 1988). We explored further the effects of these monoamines and related agents on swimming activity observed in isolated leech nerve cords. 2. We confirmed that swimming activity is induced reversibly following exposure of the nerve cord to 5-HT (50 microM); the half-maximal rate of swimming activity develops in about 15 min. Swimming activity returns to control levels about 30 min after drug washout. 3. Swim-induction by 5-HT is blocked by the presence of 10 microM cyproheptadine (a 5-HT antagonist). 4. Although apparently less effective than 5-HT, OA application to nerve cords also induced swimming activity. 5. Depletion of endogenous amines from nerve cords by acute exposure to reserpine (10-150 microM) blocked stimulus-evoked swimming activity within 4 hr. 6. Subsequent application of 5-HT (50 microM) or OA (100 microM) reinstated stimulus-evoked swimming and induced repeated episodes of non-triggered swimming activity. 7. Application of cAMP and cAMP analogs, as well as phosphodiesterase inhibitors (theophylline and IBMX), mimicked the effects of the monoamines, suggesting that 5-HT and OA may activate swimming activity by increasing neuronal cAMP. 8. We obtained episodes of swim-like activity from individual, isolated ganglia exposed to 5-HT or OA. Such episodes were usually brief, with variable cycle period. 9. We conclude that individual nerve cord ganglia contain the complete neuronal circuitry required to generate the rudiments of swimming activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of RFamide neuropeptides in the medicinal leech.

Using a four-step reverse phase HPLC separation and RIA, five RFamide peptides were purified from CNS extracts of the leech Hirudo medicinalis. YMRFamide, FMRFamide, YLRFamide, FLRFamide, and GGKYMRFamide were identified by a combination of antiserum specificity in RIA, Edman degradation, and mass spectrometry. At least three of these five endogenous peptides can modulate neuromuscular interactions in the leech (38). FMRFamide-like immunoreactivity was selectively released from neural processes on isolated heart tubes in the presence of calcium and depolarizing levels of potassium.     

The segmentation of the gnathobdellid leeches with special reference to the Indian leech Hirudinaria and medicinal leech Hirudo

Large number of annuli in Hirudinea are not true segments, and in the absence of spacious bodycavity and septa in adult no decision was taken regarding limit of a somite, until Gratiolet 1862 recognised a segment by colour marking, repetition of nephridial openings, and especially by the presence of segmental receptors, distinguishing first annulus of a segment. Whitman 1884 gave precision to these determinations and analyzed morphology of leeches to logical completeness. He recognised that though Hirudinaria and Hirudo have 102 body annuli and posterior sucker, true segments are only 26 plus 7. Castle ('00) and Moore ('00) proposed a new scheme of segmentation, with segmental receptor bearing annulus, as central annulus of a complete somite, with nerve ganglion, like that of other annelids, in center of a segment. They orientated everything roundabout the ganglion without noticing distorted fate of organ system. In this paper both the views are compared. Morphological and embryological studies reveal that the annulus bearing the segmental receptors in uniformly first annulus of all segments, including incomplete segments at the two extremities, with nerve ganglion in first annulus of the segment. Clitellum occupies three natural segments, IX, X, XI; crop caeca, nephridia, testis sacs, haemocoelomic channels and “rhomboidal figures” formed by ventrolaterals, all make a complete unit, well integrated in such segment. Conclusive evidence comes from the presence of septa at the level of each nerve ganglion in embryos of Hirudinaria. These observations corroborate Gratiolet and Whitman's view.     

Chemical and thermal stimuli have short‐lived effects on the Retzius cell in the medicinal leech

During the appetitive phase of feeding, hungry leeches detect a prey by the integration of signals perceived by different sensory systems. Earlier reports suggested that chemical or thermal sensory stimulation of the lip was associated with increased afferent activity in cephalic nerves connecting the lip to the central nervous system. These authors further suggested that this activity was relayed to Retzius cells in segmental ganglia, which then released serotonin to initiate and control all aspects of feeding behavior. In this study, we show that chemosensory or thermal activation of the lip lasting for at least 5 min produces a distinct signal in the cephalic nerves consisting of action potentials of low amplitude. These small amplitude signals are clearly distinguishable from the large action potentials evoked by mechanosensory stimuli applied to the same area of the lip. Both types of sensory stimuli also evoke an increase in the firing frequency of the Retzius cells in segmental ganglia. However, the response recorded in the nerves and the Retzius cells during a maintained stimulus is not constant but decreases with an exponential time course. These results agree with our earlier observations on a semi‐intact feeding preparation in which we showed that the firing frequency of the Retzius cell decreased as soon as the leech began to ingest its meal. Therefore, our data provide further evidence suggesting that it is unlikely that heat or chemical cues maintain the Retzius cell in an active state throughout the consummatory phase of feeding. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 304–311, 2000     

Quantitative effects of a neurotoxin upon serotonin levels within tissue compartments of the medicinal leech

The serotonin (5-hydroxytryptamine, 5-HT) content of tissue compartments in the medicinal leech, Hirudo medicinalis, was measured by means of high-pressure liquid chromatography coupled with electrochemical detection (HPLC-EC). Each segmental ganglion contains 21.3 +/- 2.9 (9) pmol 5-HT [X +/- SEM (N)]. The pharynx contains 7.1 +/- 1.1 (9) pmol 5-HT/mg wet weight; the salivary glands 3.2 +/- 0.9 (10), ventral body wall 2.0 +/- 0.2 (11), and vasofibrous tissue 1.2 +/- 0.2 (11). The blood of hungry leeches contains 8.7 +/- 1.9 (7) nM 5-HT while that of well-fed leeches is 2.2 +/- 0.4 (6) nM. Leeches were injected with the cytotoxic analog of serotonin, 5,7-dihydroxytryptamine (5,7-DHT) producing selective lesions of the peripherally projecting serotonin-containing neurons, and which in turn abolished their feeding behavior. The serotonin content of the pharynx and ganglia of these toxin-treated leeches were lowered significantly. The serotonin levels within the body wall and salivary glands were not altered significantly by the toxin treatment, but the levels within the vasofibrous tissue and blood were elevated substantially.     

The role of glutamate in swim initiation in the medicinal leech

Antagonists were used to investigate the role of the excitatory amino acid,l-glutamate, in the swim motor program ofHirudo medicinalis. In previous experiments, focal application ofl-glutamate or its non-NMDA agonists onto either the segmental swim-gating interneuron (cell 204) or the serotonergic Retzius cell resulted in prolonged excitation of the two cells and often in fictive swimming. Since brief stimulation of the subesophageal trigger interneuron (cell Tr1) evoked a similar response, we investigated the role of glutamate at these synapses. Kynurenic acid and two non-NMDA antagonists, 6,7-dinitroquinoxaline-2,3-dione (DNQX) and Joro spider toxin, effectively suppressed (1) the sustained activation of cell 204 and the Retzius cell following cell Tr1 stimulation and (2) the monosynaptic connection from cell Tr1 to cell 204 and the Retzius cell, but did not block spontaneous or DP nerve-activated swimming. Other glutamate blockers, including -d-glutamylaminomethyl sulfonic acid,l(+)-2-amino-3-phosphonoproprionic acid and 2-amino-5-phosphonopentanoic acid, were ineffective. DNQX also blocked both indirect excitation of cell 204 and direct depolarization of cell Tr1 in response to mechanosensory P cell stimulation. Our findings show the involvement of non-NMDA receptors in activating the swim motor program at two levels: (1) P cell input to cell Tr1 and (2) cell Tr1 input to cell 204, and reveal an essential role for glutamate in swim initiation via the cell Tr1 pathway.     

Persistent inward currents in cultured Retzius cells of the medicinal leech

Current-clamp studies of cultured leech Retzius cells revealed inward rectification in the form of slow voltage sags in response to membrane hyperpolarization. Sag responses were eliminated in Na⁺-free saline and blocked by Cs⁺, but not Ba²⁺. Voltage clamp experiments revealed a Cs⁺-sensitive inward current activated by hyperpolarization negative to −70 mV. Cs⁺ decreased the frequency of spontaneous impulses in Retzius cells of intact ganglia. Plateau potentials were evoked in Retzius cells following block of Ca²⁺ influx with Ni²⁺ and suppression of K⁺ currents with internal tetraethylammonium. Plateau potentials continued to be expressed with Li⁺ as the charge carrier, but were eliminated when Na⁺ was replaced with N-methyl-d-glucamine. A persistent Na⁺ current with similar pharmacology that activated positive to −40 mV and reached its peak amplitude near −5 mV was identified in voltage-clamp experiments. Inactivation of the persistent Na⁺ current was slow and incomplete. The current was revealed by slow voltage ramps and persisted for the duration of 5-s voltage steps. Persistent Na⁺ current may underlie Na⁺-dependent bursting recorded in neurons of intact ganglia exposed to Ca²⁺-channel blockers. Accepted: 22 September 1998     

Postactivation inhibition of spontaneously active neurosecretory neurons in the medicinal leech

Spontaneously active neurosecretory neurons in vertebrate and invertebrate nervous systems share similarities in firing frequencies, spike shapes, inhibition by the transmitters they themselves release and postactivation inhibition, an intensity-dependent period of suppressed spontaneous generation of action potentials following phases of high-frequency activity. High-frequency activation of spontaneously active serotonin-containing Retzius cells in isolated ganglia of the leech Hirudo medicinalis induced prolonged membrane hyperpolarisations causing periods of postactivation inhibition of up to 33 s. The duration of the inhibitory periods was directly related to both the number and rate of spikes during activation and was inversely proportional to a cell’s spontaneous firing frequency. The periods of postactivation inhibition remained unaffected by both serotonin depletion through repeated injections of 5,7-dihydroxytryptamine and suppressing the afterhyperpolarisation following each action potential with tetraethylammonium (TEA), iberiotoxin or charybdotoxin, suggesting that neither autoinhibition by synaptic release of serotonin nor calcium-activated potassium channels contribute to the underlying mechanism. In contrast, the postactivation inhibitory period was significantly affected both by differential electrical stimulation of the same Retzius cells via microelectrodes filled with molar concentrations of either Na⁺-acetate or K⁺-acetate, and by partial inhibition of Na⁺/K⁺-ATPase with ouabain. Thus, postactivation inhibition in Retzius cells results from prolonged hyperpolarising activity of Na⁺/K⁺-ATPase stimulated by the accumulation of cytosolic Na⁺ during phases of high-frequency spike activity.     

Predicting efficiency of selection on reproductive traits in the medicinal leech Hirudo medicinalis L

A set of selection measures for increasing reproduction efficiency in Hirudo medicinalis has been developed. The optimal values of reproductive traits corresponding to the highest progeny number were determined and recommended. The probability of correlated selection response in traits "number of threads in a cocoon" and "weight of threads" was estimated. Based on earlier results on phenotypic variation and heritability of reproductive traits in medicinal leech, efficiency of different selection modes was predicted.