Differential effects of serotonin depletion on sensitization and dishabituation in the leech,Hirudo medicinalis

The goal of these experiments was to test the hypothesis that serotonin (5-HT) is involved in facilitation of the shortening reflex in the leech Hirudo medicinalis. For this reason, we have used the toxin 5-hydroxytryptamine (5,7-DHT) to deplete serotonin from the nervous systems of intact leeches and have assessed the effect on early facilitation, dishabituation, and sensitization of the touch-elicited shortening reflex using behavioral procedures previously developed in our lab (Boulis and Sahley, 1988). We find that 5,7-DHT lesions completely attenuate early facilitation and sensitization but only reduce dishabituation of the touch-elicited shortening reflex. Histological analyses of the ganglia from these leeches using glyoxilic acid staining procedures revealed an absence of staining in the Retzius cell of experimental leeches. All other serotonin-containing neurons showed glyoxilic acid staining comparable to that observed in the control leeches.     

Temporal correlation between neuronal tail ganglion activity and locomotion in the leech,Hirudo medicinalis

Intracellular and extracellular recordings were performed in the posterior ventral nerve cord of restrained crawling preparations of the medicinal leech,Hirudo medicinalis. Short-latency neuronal activities in the tail ganglion nerves correlated with different phases of crawling behavior. Eight neurons with characteristic activation patterns during crawling were identified morphologically and physiologically in the tail ganglia of 23 preparations. The axons of four of these neurons projected through posterior tail brain nerves; four ascending interneurons had projections in the connectives or in Faivre's nerve. These interneurons are suitable candidates for carrying information between the front end and the tail end of the animal to coordinate the behavioral components during a crawling step.     

Small cardioactive peptide-like immunoreactivity and its colocalization with FMRFamide-like immunoreactivity in the central nervous system of the leech Hirudo medicinalis

Summary The distributions of small cardioactive peptide (SCP)- and FMRFamide-like immunoreactivities in the central nervous system of the medicinal leech Hirudo medicinalis were studied. A subset of neurons in the segmental ganglia and brains was immunoreactive to an antibody directed against SCP_B. Immunoreactive cell bodies were regionally distributed throughout the nerve cord, and occurred both as bilaterally paired and unpaired neurons. The majority of the unpaired cells displayed a tendency to alternate from side to side in adjacent ganglia. A small number of neurons were immunoreactive only in a minority of nerve cords investigated. Intracellular injections of Lucifer yellow dye and subsequent processing for immunocytochemistry revealed SCP-like immunoreactivity in heart modulatory neurons but not in heart motor neurons. FMRFamide-like immunoreactivity was also detected in cell bodies throughout the central nervous system. A subset of neurons contained both SCP- and FMRFamide-like immunoreactivities; others stained for only one or the other antigen. These data suggest that an antigen distinct from FMRFamide is responsible for at least part of the SCP-like immunoreactivity. This antigen likely bears some homology to the carboxyl terminal of SCP_A and SCP_B.     

Mechanisms of postinhibitory rebound and its modulation by serotonin in excitatory swim motor neurons of the medicinal leech

Postinhibitory rebound (PIR) is defined as membrane depolarization occurring at the offset of a hyperpolarizing stimulus and is one of several intrinsic properties that may promote rhythmic electrical activity. PIR can be produced by several mechanisms including hyperpolarization-activated cation current (I_h) or deinactivation of depolarization-activated inward currents. Excitatory swim motor neurons in the leech exhibit PIR in response to injected current pulses or inhibitory synaptic input. Serotonin, a potent modulator of leech swimming behavior, increases the peak amplitude of PIR and decreases its duration, effects consistent with supporting rhythmic activity. In this study, we performed current clamp experiments on dorsal excitatory cell 3 (DE-3) and ventral excitatory cell 4 (VE-4). We found a significant difference in the shape of PIR responses expressed by these two cell types in normal saline, with DE-3 exhibiting a larger prolonged component. Exposing motor neurons to serotonin eliminated this difference. Cs⁺ had no effect on PIR, suggesting that I_h plays no role. PIR was suppressed completely when low Na⁺ solution was combined with Ca²⁺ -channel blockers. Our data support the hypothesis that PIR in swim motor neurons is produced by a combination of low-threshold Na⁺ and Ca²⁺ currents that begin to activate near –60 mV.     

Anatomical analysis of contacts between identified neurons that control heartbeat in the leech Hirudo medicinalis

Summary The rhythmic constriction of the heart tubes in the leech Hirudo medicinalis is controlled by an identified set of motor neurons (HE cells) and interneurons (HN cells) (reviewed by Calabrese and Peterson 1983). Electrophysiological recordings have indicated particular synaptic relationships among HE and HN cells. In the present study, the synaptic framework mediating the interactions among HE cells and HN cells was examined anatomically. Using light and electron microscopy of physiologically identified, HRP-injected cells, we have examined the zones of interaction and types of contacts between specific cells. HE cells, which have very fine, threadlike processes, interact with their contralateral homologues throughout most of the middle third of the ganglionic neuropil. When HE-cell neuntes come together, the apposed plasma membranes are rigidly parallel, separated by an intercellular gap of 6 nm, for up to 6 m. These specializations must form the structural basis for the strong electrical coupling observed (Peterson 1983) between HE-cell pairs. HE cells also emit from the main neurite a series of extremely fine processes that extend dorsally. These appear in the light microscope to contact processes of the ipsilateral HN cell of the same ganglion, and are also in a position to make contact with the axons of more anterior HN cells. The intraganglionic processes of HN cells, which are studded with large varicosities, ramify in part of the region of neuropil occupied by HE-cell processes, as well as more posteriorly. Contacts between HE and HN cells, which are known to be mostly inhibitory synaptic contacts, are seen in the electron microscope to be formed between medium-diameter HN processes, which are filled with clear round synaptic vesicles, and multiple fine tendrils of the HE cell that surround the HN process. Certain HN cells form reciprocal inhibitory synapses with their contralateral homologues. These contacts occur near the midline, sometimes in the major mass of neuropil and sometimes embedded in the extracellular material that ensheathes the neuropil. The contacts are between medium-and small-diameter profiles that are both filled with synaptic vesicles. Our findings indicate that various classes of physiological interactions among HE and HN cells are mediated by anatomically distinct types of contacts and, at least in some cases, are segregated from each other on the neuritic trees of the cells.     

Celebrity with a neglected taxonomy: molecular systematics of the medicinal leech (genus Hirudo)

The medicinal leech is the most famous representative of the Hirudinea. It is one of few invertebrates widely used in medicine and as a scientific model object. It has recently been given considerable conservation effort. Despite all attention there is confusion regarding the taxonomic status of different morphological forms, with many different species described in the past, but only two generally accepted at present. The results of the phylogenetic analysis of a nuclear (ITS2+5.8S rRNA) and two mitochondrial gene sequences (12S rRNA, COI) suggest that the genus Hirudo is monophyletic. It consists, apart form the type Hirudo medicinalis and the East Asian Hirudo nipponia, of three other, neglected species. All of them have already been described either as species or morphological variety, and can readily be identified by their coloration pattern. The type species is in weakly supported sister relation with Hirudo sp. n. (described as variety orientalis) from Transcaucasia and Iran. Sister to them stands Hirudo verbana from southeastern Europe and Turkey, which is nowadays predominantly bred in leech farms and used as 'medicinal leech.' The North African Hirudo troctina is the sister taxon to this group of Western Eurasian species, whereas the basal split is between H. nipponia and the Western Palaearctic clade.     

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     

Anaerobic metabolism in the leech (Hirudo medicinalis L.): direct and indirect calorimetry during severe hypoxia

Anaerobic metabolism in the limnic annelid Hirudo medicinalis L. was investigated by direct and indirect calorimetry. During long-term severe hypoxia, the rate of heat dissipation was reduced up to 13% of the aerobic rate. At the same time, the rate of ATP turnover was reduced to about 30% of the aerobic rate, indicating that metabolic depression is an important mechanism to ensure survival of the leech during environmental anaerobiosis. Heat dissipation during hypoxia was monitored under two experimental conditions, favouring either concomitant hypocapnia (continuous N₂ bubbling) or hypercapnia (self-induced hypoxia). The reduction in heat dissipation during hypocapnic hypoxia was less pronounced than during hypercapnic hypoxia, indicating that the different experimental conditions may influence anaerobic metabolism and the extent of metabolic depression. Biochemical analysis of known anaerobic substrates and endproducts provided the basis for indirect calorimetry during self-induced hypoxia. From changes in metabolites, the expected heat dissipation was calculated for initial (0–8 h) and long-term severe hypoxia (8–72 h). During the initial period, the calculated heat dissipation fully accounted for direct calorimetric determination. During long-term hypoxia, only 71% of the measured heat production could be explained from biochemical analysis of metabolites. Therefore, an additional unknown endproduct cannot be excluded, especially when anaerobic ammonia production and analysis of the carbohydrate balance are considered.Abbreviations APW artificial pond water - HPLC high-performance liquid chromatography - fw fresh weight - HP heat production - HD heat dissipation - MR metabolic rate     

Effects of transition metal ions on spontaneous electrical activity and chemical synaptic transmission of neurons in the medicinal leech

Leech neurons exposed to salines containing inorganic Ca²⁺-channel blockers generate rhythmic bursts of impulses. According to an earlier model, these blockers unmask persistent Na⁺ currents that generate plateau-like depolarizations, each triggering a burst of impulses. The resulting increase in intracellular Na⁺ activates an outward Na⁺/K⁺ pump current that contributes to burst termination. We tested this model by examining systematically the effects of six transition metal ions (Co²⁺, Ni²⁺, Mn²⁺, Cd²⁺, La³⁺, and Zn²⁺) on the electrical activity of neurons in isolated leech ganglia. Each ion induced bursting activity, but the amplitude, form, and persistence of bursting differed with the ion used and its concentration relative to Ca²⁺. All ions tested suppressed chemical synaptic transmission between identified motor neurons, consistent with block of voltage-dependent Ca²⁺ currents in these cells. In addition, a strong correlation between suppression of synaptic transmission and burst amplitudes was obtained. Finally, burst duration was increased and the rate of repolarization decreased in reduced K⁺ saline, as expected for pump-dependent repolarization. These results provide further support for the hypothesis that a novel form of oscillatory electrical activity driven by persistent Na⁺ currents and the Na⁺/K⁺ pump occurs in leech ganglia exposed to Ca²⁺-channel blockers. Accepted: 15 May 1997     

Molecular characterization and embryonic expression of innexins in the leech Hirudo medicinalis

Gap junctions are direct intercellular channels that permit the passage of ions and small signaling molecules. The temporal and spatial regulation of gap junctional communication is, thus, one mechanism by which cell interactions, and hence cell properties and cell fate, may be regulated during development. The nervous system of the leech, Hirudo medicinalis, is a particularly advantageous system in which to study developmental mechanisms involving gap junctions because interactions between identified cells may be studied in vivo in both the embryo and the adult. As in most invertebrates, gap junctions in the leech are composed of innexin proteins, which are distantly related to the vertebrate pannexins and are encoded by a multi-gene family. We have cloned ten novel leech innexins and describe the expression of these, plus two other previously reported members of this gene family, in the leech embryo between embryonic days 6 and 12, a period during which the main features of the central nervous system are established. Four innexins are expressed in neurons and two in glia, while several innexins are expressed in the excretory, circulatory, and reproductive organs. Of particular interest is Hm-inx6, whose expression appears to be restricted to the characterized S cell and two other neurons putatively identified as presynaptic to this cell. Two other innexins also show highly restricted expressions in neurons and may be developmentally regulated. Electronic Supplementary Material Supplementary material is available for this article at .     

Protease inhibitors in the alimentary tract of the medicinal leech Hirudo medicinalis: In vivo and in vitro studies

Summary In individual leeches the flux of labeled serum through the digestive tract was monitored to measure the rate of digestion. A mean value of 10 mg of the original serum (or 2–3 mg of the contents of the foregut) per individual per day was found, which was constant during 10 weeks. On average the serum remained in the intestinum for 20 days. Occurrence and concentrations of eglin and bdellin, specific proteinase inhibitors of Hirudo, were analyzed after various periods following the ingestion of a meal. In the foregut they were present immediately after feeding. Their quantities increased several-fold within a few weeks. In the intestinum the tests for these inhibitors were always negative. The inhibition of the proteolytic activity of intestinum preparations by eglin, bdellin, and foregut extract was tested in vitro. Using azo-albumin as (an unspecific) substrate, inhibition by eglin was maximally 25% and by bdellin 60%. When the quantitative relations presumably representing in vivo conditions were applied, only a slight inhibition of proteolysis occurred. A hypothetical role of the inhibitors in the preservation of the blood stored in the foregut is discussed.Abbreviations BIGGANA N-benzoyl-isoleucyl-glutaryl-glycyl-arginyl-4-nitroanilide - SAPPNA N-succinyl-alanyl-alanyl-prolyl-phenylalanyl-4-nitroanilide - TPCK tosyl-phenylalanyl-chloromethyl-detone - TRIS tris-(hydroxymethyl)aminomethane     

Distribution and status of the medicinal leech (Hirudo medicinalis L.) in Turkey

A survey of all the major potential habitats in western Turkey showed that medicinal leeches, Hirudo medicinalis L., are widely distributed over the country and are not rare. They occur in practically all suitable habitats and the only region where they were found to be absent is that of the large river deltas in the south of the country (Çukurova deltas, Göksu delta). There may be zoogeographic reasons for this (Taurus mountains barrier). The application of a semi-quantitative survey method using collecting efficiency (number of leeches collected per hour by a single person) allowed a rapid assessment to be made of its status in a large number of wetlands. Leech density varied considerably from wetland to wetland, and the results enabled a ranking of the Turkish wetlands to be made according to their importance for medicinal leeches. Taking both the leech density and the size of leech habitats into account, the largest populations were identified on the Black Sea coast (Kizilirmak delta, Yeilirmak delta and Karagöl Marshes near Sinop) and in inner and south-west Anatolia (Eber Gölü, Karamik and Sultan Marshes). Commercial exploitation for the pharmaceutical industry and for other purposes takes place at only a few places and does not appear to affect the population seriously. However, many populations are threatened by the draining of their habitats.     

Stimulus-Specific Hierarchy of Enhancer Elements Within the Rat Prodynorphin Promoter

Abstract: Induction of the prodynorphin gene occurs in a tissue-specific manner following different physiological stimuli. Using electrophoretic mobility shift assays, we studied the relative activity of the five major regulatory sites in the rat prodynorphin promoter. Prodynorphin cyclic AMP-responsive element 2 (DynCRE2), DynCRE3, and the noncanonical prodynorphin AP-1 (ncDynAP-1) regulatory sites control, in a coordinated manner, prodynorphin induction in the spinal cord after noxious stimulation, whereas prodynorphin up-regulation in supraoptic neurons is regulated predominantly by the ncDynAP-1. Conversely, prodynorphin transactivation in the ovaries upon gonadotrophin stimulation is controlled by DynCRE1 and DynCRE3. Our results support the idea that stimulus-specific changes in nuclear proteins establish a functional hierarchy among regulatory sites in the prodynorphin promoter and provide further insight in the molecular mechanisms that govern prodynorphin gene regulation.     

Cell proliferation in a peripheral target is required for the induction of central neurogenesis in the leech

Several days after the completion of the early phase of cell proliferation that generates most of the leech central nervous system, the pair of “sex ganglia” in the two reproductive segments of the midbody undergo a second period of neurogenesis that gives rise to several hundred peripherally induced central (PIC) neurons. This proliferative phase, which begins on embryonic day 17 (E17), is induced by the interaction of a few specific neurons in the sex ganglia with a peripheral target, the male genitalia, during a critical period that extends from E13 to E16. The central nervous system (CNS) determines the critical period, since the male genitalia have the capacity to induce PIC neurons beginning on E10 and continuing throughout embryogenesis. Here we first show, by injecting hydroxyurea into staged embryos to ablate dividing cells, that PIC neuron precursors begin to divide at a low rate before E17, during the critical period. Then, through a series of homochronic and heterochronic male organ transplantations combined with hydroxyurea treatment of hosts and/or donors, we show that cell proliferation is required in the target itself for it to be competent to induce PIC neurons. These observations demonstrate that a nerve connection can couple cell proliferation in a peripheral target to cell proliferation in the CNS, providing a novel means for size adjustment of a central neuronal population relative to a peripheral target. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 295–303, 1998     

Molecular cloning and sequencing of the Na+/K+-ATPase α-subunit of the medical leech Hirudo medicinalis (Annelida) – implications for modelling protostomian evolution

Within the past few years, the phylogenetic tree is discussed controversially regarding the position of the different bilaterian groups. There are two varying views of evolution: the classical one based on morphological structures where the annelids and arthropods are combined in the group of Articulata, and a new hypothesis based on molecular data sets, which divides the protostomian groups in Ecdysozoa (e.g. arthropods, and nematodes) and Lophotrochozoa (e.g. annelids, molluscs, and platyhelminthes). The Na⁺/K⁺-ATPase is a highly conserved protein and fulfils a very important role in physiology and maintaining the homeostasis of cells and can be found in almost all eukaryotic animals. Due to its similar molecular structure throughout the animal kingdom the Na⁺/K⁺-ATPase is an excellent marker for phylogenetic studies. Here we report the molecular cloning, sequencing and phylogenetic analysis of Na⁺/K⁺-ATPase complementary deoxyribonucleic acid (cDNA) of the medical leech Hirudo medicinalis . The cloned cDNA codes for a polypeptide of 1022 amino acids and possesses a predicted molecular mass of 113.33 kDa. Phylogenetic analysis of the complete Na⁺/K⁺-ATPase α -subunit of H. medicinalis and sequence data from other Na⁺/K⁺-ATPases supports the previously developed 'Ecdysozoa concept' with high posterior probabilities. A common clade comprising annelids and platyhelminthes can be defined, whereas nematodes are in a basal position at the arthropod stem line.     

Analysis of the Proenkephalin Second Messenger-Inducible Enhancer in Rat Striatal Cultures

Abstract: We have previously shown that in cell extracts from rat striatum, cyclic AMP response element (CRE) binding protein (CREB), rather than AP-1 proteins, preferentially interacts with the CRE-2 element of the proenkephalin second messenger-inducible enhancer, even under conditions in which AP-1 proteins are highly induced. Here we use primary striatal cultures to permit a more detailed analysis of CRE-2 function and protein binding in relevant neural cell types. By transfection we find that in primary striatal cultures, as in transformed cell lines, the CRE-1 and CRE-2 elements are required for significant induction by cyclic AMP. We report that cyclic AMP induction of the proenkephalin gene in striatal cultures is protein synthesis independent, excluding a role for newly synthesized proteins like c-Fos. We also show that cyclic AMP induces CREB phosphorylation and that phosphorylated CREB interacts strongly with CRE-2 and weakly with CRE-1. The predominant protein bound to CRE-1 is not CREB, however, and remains to be identified. Despite some prior predictions, we do not find a role for c-Fos in cyclic AMP regulation of proenkephalin gene expression in neurons.