Axonal projections of the Retzius cells to the dorsal segmental root in the leech Hirudo medicinalis

The presence of RC's axon running in the dorsal root of the leech Hirudo medicinalis was studied by means of electrophysiological and neuroanatomical techniques. Electrical stimulation of the dorsal root gave invariably the antidromic invasion of the Retzius cell. This effect disappears after crushing the dorsal root between the stimulating electrode and the ganglion, and hence is not due to stimulus spread to the nearby posterior root. Anatomical evidence for an axon collateral running in the dorsal root was provided by catecholamine histofluorescence of the axonal branches of the RCs and by Lucifer Yellow intracellular injection into the RCs.     

The role of the sexual organs in the generation of postembryonic neurons in the leech Hirudo medicinalis

In adult Hirudo medicinalis, the segmental ganglia (SG) of the fifth and sixth body segments contain a few hundred more cells than the other segmental ganglia of the body (Macagno, 1980). These ganglia innervate the sex organs and are known as the sex SG. As shown here, these cells are stained by neuron-specific antibodies. Cell counts at several developmental stages reveal that the extra cells are added exclusively to the sex SG, and that this addition occurs gradually and almost entirely postembryonically (embryogenesis ends at 30 days of development). Hence, we refer to them as PE cells. Until 16 days of development, deleting the male genitalia or disconnecting them from the sex ganglia results in a complete absence of the PE cells. From 16 days onward, the generation of the PE cells is independent of the male genitalia. No PE cells appear in non-sex SG that innervate male genitalia transplanted to ectopic locations at 10 days or later, indicating that as early as about 10 days of development sex SG are different from other SG in their ability to acquire PE cells. These data suggest that the PE cells are generated within the sex SG, in a process that is triggered by an interaction with the male genitalia and mediated through the innervation of the organs by these ganglia.     

Seasonal variation of serotonin content and nonassociative learning of swim induction in the leech Hirudo medicinalis

Summary It is possible to obtain habituation of swim induction by stimulating the leech with repetitive light electrical trains. After obtaining this simple form of nonassociative learning, it is also possible to potentiate its response by a series of nociceptive skin brushings (dishabituation). Serotonin applied to the animal is the only neurotransmitter found to mimick dishabituation. We have observed that in the period April–June most animals did not exhibit potentiation of the swimming response after nociceptive stimulation while injection of serotonin mimicked dishabituation as in the animals treated in the period October–March. We have seen correlation between the changes in nonassociative learning and the seasonal variation of serotonin levels in segmental ganglia. This finding strengthens the hypothesis of serotonin as the neurotransmitter mediating dishabituation in swim induction of the leech.Abbreviations AHP afterhyperpolarization - HPLC high pressure liquid chromatography     

Synaptic effects elicited in the Retzius cells of the leech Hirudo medicinalis by stimulation of the segmental roots

Electrical stimulation of the segmental roots of each ganglion of Hirudo medicinalis, elicits in both Retzius' cells inhibitory and excitatory effects. The IPSP and EPSP are chemical in nature, being dependent on the membrane potential, and suppressed by high Mg++. Selective inactivation of one RC shows that the responses of the contralateral RC are not due to electrotonic coupling between the two cells, but to synaptic actions impinging upon the membrane of both RCs. The two synaptic potentials appear to be mediated by two set of fibres with a different threshold to electrical stimulation. Their actions on the RCs appear to be polysynaptic on the basis of central latency. Simultaneous stimulation of two roots shows evidence for occlusion for IPSP and summation for EPSP, confirming the polysynaptic nature of the effects. The possible functional significance of the inhibitory and excitatory pathways, is discussed.     

Apparent intracellular Mg2+ buffering in neurons of the leech Hirudo medicinalis

The apparent intracellular Mg2+ buffering, or muffling (sum of processes that damp changes in the free intracellular Mg2+ concentration, [Mg2+](i), e.g., buffering, extrusion, and sequestration), was investigated in Retzius neurons of the leech Hirudo medicinalis by iontophoretic injection of H+, OH-, or Mg2+. Simultaneously, changes in intracellular pH and the intracellular Mg2+, Na+, or K+ concentration were recorded with triple-barreled ion-selective microelectrodes. Cell volume changes were monitored measuring the tetramethylammonium (TMA) concentration in TMA-loaded neurons. Control measurements were carried out in electrolyte droplets (diameter 100-200 microm) placed on a silver wire under paraffin oil. Droplets with or without ATP, the presumed major intracellular Mg2+ buffer, were used to quantify the pH dependence of Mg2+ buffering and to determine the transport index of Mg2+ during iontophoretic injection. The observed pH dependence of [Mg2+](i) corresponded to what would be expected from Mg2+ buffering through ATP. The quantity of Mg2+ muffling, however, was considerably larger than what would be expected if ATP were the sole Mg2+ buffer. From the decrease in Mg2+ muffling in the nominal absence of extracellular Na+ it was estimated that almost 50% of the ATP-independent muffling is due to the action of Na+/Mg2+ antiport.     

Recovery after anaerobic metabolism in the leech (Hirudo medicinalis L.)

Medicinal leeches (Hirudo medicinalis L.) responded to self-induced hypoxia (72 h) with typical anaerobic metabolism characterized by a decrease in adenylate energy charge, utilization of the substrates glycogen and malate, and accumulation of the main anaerobic endproducts succinate and propionate. Propionate was also excreted into the medium. Ammonia excretion was suppressed. Aerobic recovery resulted in a profound O₂ debt. Resynthesis of ATP was completed within 30 min. Disposal of succinate and restoring of malate required 2–3 h, and clearance of propionate and recharging of glycogen 6–12 h. Ammonia excretion did not exceed normoxic rates and excretion of propionate during recovery accounted for only 10% of total propionate accumulated during hypoxia. It is postulated that the clearance of succinate and propionate involves oxidation but also resynthesis of malate and glycogen. During hypoxia and recovery blood osmolality remained constant. The Na⁺ and Cl^- ion concentrations in blood, the decrease of which was nearly equimolar during hypoxia, were re-established following different time-courses. Na⁺ concentration returned to normoxic levels after 2–3 h. The delayed increase in Cl^- concentration, however, correlating with 6–12 h necessary to clear blood propionate, is interpretated as an anion regulating effect.Abbreviations AEC adenylate energy charge; fw, fresh weight - HPLC high-performance liquid chromatography - SCCA shortchain carboxylic acids     

Effects of egg removal on maternal aggression, biogenic amines, and stress indicators in ovigerous lobsters (Homarus americanus)

In many disparate taxa, including crayfish, a freshwater decapod crustacean, the presence of one's offspring has been shown to be an important variable in the level of maternal aggression. Ovigerous American lobsters (Homarus americanus) show a territorial advantage against nonmaternal females, even though no posthatch care is provided. The eggs are attached to the pleopods (swimmerets) throughout embryogenesis. We evaluated the effect of stripping ovigerous H. americanus females of their eggs on maternal territoriality. In one treatment group, maternal females were stripped of all eggs. Twelve days later, including being individually isolated for an additional 48 h in a test tank, each resident was serially intruded upon by 4 non-maternal conspecific females. An identically treated control group of ovigerous female residents was left intact prior to the serial intrusions. Various biochemical parameters of the stripped and unstripped animals were measured before and after the experimental treatment (or control). The behavioral modulators measured were the biogenic amines, octopamine and serotonin, and the stress indicators were a heat-shock protein (HSP70), methyl farnesoate, and crustacean hyperglycemic hormone. The intact ovigerous residents showed a significant territorial advantage over the intruders, whereas the contest outcomes of stripped residents were reduced to chance. The presence of eggs in maternal H. americanus is therefore necessary for the maintenance of the shelter-related territorial advantage. However, no significant differences in any of the biochemical parameters were observed between or within treatment conditions. It appears that these amines are not prominently involved in the mechanism of maternal aggression, and that these changes in territorial defense are not simply due to changes in general stress as a result of stripping the females of their eggs.     

Effects of egg removal on maternal aggression,biogenic amines,and stress indicators in ovigerous lobsters (Homarus americanus)

In many disparate taxa, including crayfish, a freshwater decapod crustacean, the presence of one’s offspring has been shown to be an important variable in the level of maternal aggression. Ovigerous American lobsters (Homarus americanus) show a territorial advantage against nonmaternal females, even though no posthatch care is provided. The eggs are attached to the pleopods (swimmerets) throughout embryogenesis. We evaluated the effect of stripping ovigerous H. americanus females of their eggs on maternal territoriality. In one treatment group, maternal females were stripped of all eggs. Twelve days later, including being individually isolated for an additional 48?h in a test tank, each resident was serially intruded upon by 4 non-maternal conspecific females. An identically treated control group of ovigerous female residents was left intact prior to the serial intrusions. Various biochemical parameters of the stripped and unstripped animals were measured before and after the experimental treatment (or control). The behavioral modulators measured were the biogenic amines, octopamine and serotonin, and the stress indicators were a heat-shock protein (HSP70), methyl farnesoate, and crustacean hyperglycemic hormone. The intact ovigerous residents showed a significant territorial advantage over the intruders, whereas the contest outcomes of stripped residents were reduced to chance. The presence of eggs in maternal H. americanus is therefore necessary for the maintenance of the shelter-related territorial advantage. However, no significant differences in any of the biochemical parameters were observed between or within treatment conditions. It appears that these amines are not prominently involved in the mechanism of maternal aggression, and that these changes in territorial defense are not simply due to changes in general stress as a result of stripping the females of their eggs.     

Phenology of the medicinal leech, Hirudo medicinalis L., in north-western Turkey

The seasonal occurrence of medicinal leeches was studied at two lakes in the Black Sea coastland of north-western Turkey. The number of leeches attracted by a collector per hour was taken as an indicator for population density. Leeches were most abundant in May/June, and were almost absent during the cold season (November – March). Leeches with a weight of less than 1 g occurred principally between July and October with a peak in July. It is thought that these individuals represent newly hatched leeches. Although both study areas are close to each other, significant differences were found in respect to mean leech size and the seasonal frequency of leeches (both total number and certain age classes). It is suggested that leech biology is greatly dependent on factors such as microclimate and the availability of hosts, and no general conclusions on phenology can be drawn. There is therefore no scientific basis for restricting commercial leech collecting to certain months of the year.     

Osmotic and ionic regulation during hypoxia in the medicinal leech, Hirudo medicinalis L.

The concentrations of inorganic and organic ions and osmolality in the blood of the medicinal leech, Hirudo medicinalis, were determined during normoxia and hypercapnic and hypocapnic hypoxia. In normoxic animals, the blood sodium concentration was 124.5 +/- 4.2 mmol/l and the total cation concentration was 132.2 +/- 4.3 mEq/l (mean +/- S.D.). Major anionic compounds were chloride (40.8 +/- 1.6 mmol/l), bicarbonate (8.4 +/- 1.3 mmol/l), and organic anions (42.5 +/- 2.3 mEq/l). Among the latter, malate accounts for 30.4 +/- 2.2 mEq/l. The nature of the remaining anion fraction, which balances cation and anion concentrations in leech blood, remains unknown. Within 96 h of hypercapnic hypoxia, the amount of organic osmolytes in leech tissue increased from the control level of 56.6 +/- 9.1 to 158.3 +/- 19.5 mumol/g dry weight. An even higher amount of organic acids was accumulated within 96 h of hypocapnic hypoxia (218.0 +/- 53.7 mumol/g dry weight). A possible reason for this is that lactate, which is a major end-product of hypocapnic hypoxia, cannot be excreted to the external medium as easily as propionate. The accumulation of blood organic acids generating osmotic stress in the animals was compensated by an equimolar decrease in sodium and chloride ion concentrations. In hypercapnic animals these changes resulted in a constant osmotic concentration of the blood (200 mosmol/kg H2O) during the experimental period. Between 24 and 96 h of hypocapnic hypoxia, however, the increase in the osmotic gradient between animal and medium was correlated with further net water uptake and the obvious deterioration of the volume- and ion-regulatory mechanisms in these animals.     

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.     

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.     

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.     

Cell-and tissue-specific expression of putative protein kinase mRNAs in the embryonic leech,Hirudo medicinalis

Protein kinases play important roles in various cellular interactions underlying metazoan development. To complement existing analyses of protein kinase function in the development of members of the three phyla, Chordata, Arthropoda, and Nematoda, we have begun to examine the cell-and tissue-specific localization of protein kinases in another metazoan phylum, the Annelida. For this purpose, we used the polymerase chain reaction to amplify putative protein kinase catalytic domain cDNAs from the medicinal leech, Hirudo medicinalis. This strategy allowed us to identify 11 cytoplasmic and receptor tyrosine kinase catalytic domains, and 2 cytoplasmic serine/threonine kinase catalytic domains. Using these cDNAs as probes for nonradioactive whole-mount in situ hybridization, we examined the embryonic expression pattern of each of the corresponding putative kinase mRNAs. As has been found in other species, most of the Hirudo protein kinase mRNAs were expressed in a highly specific manner in certain embryonic cells and tissues. We found both neuron-and glia-specific kinases within the nervous system, as well as kinases expressed in non-nervous tissues, such as the haemocoelomic, muscular, and excretory systems. These kinase cDNAs encode proteins likely to be critical for proper development, and can be used as cell-and tissue-specific histological probes for the analysis of Hirudo embryogenesis.     

5-Hydroxytryptamine-like immunoreactivity in the peripheral and central nervous systems of the leech Hirudo medicinalis

Summary Chains of segmental ganglia and various peripheral tissues from the leech (Hirudo medicinalis) were screened as whole-mount preparations for the presence of 5-hydroxytryptamine-like immunoreactivity. The gut was richly supplied with immunoreactive nerve fibres. Plexus of fibres, numerous of which were varicose, were found in the crop, with many immunopositive nerve cell bodies in the posterior region and a few in the anterior region. The intestine contained a few longitudinally oriented nerve fibres, while the rectum contained a dense network of non-varicose and varicose fibres. Fine immunopositive fibres were associated with the lateral blood vessel and reproductive organs. Many immunopositive nerve fibres ran in each of the paired connectives linking the segmental ganglia, and two fine varicose fibres were seen in Faivre's nerve. At least two immunopositive processes left each lateral segmental nerve and branched repeatedly, with many varicosities on the distal branches. The dorso-ventral and longitudinal body wall muscles both contained immunoreactive fibres, the plexus being more dense in the former muscle. The possible roles of the immunoreactive nerve fibres demonstrated in the various tissues of the leech have been discussed in relation to the known peripheral effects of serotoninergic neurone stimulation in the leech and to the actions of exogenously applied 5-hydroxytryptamine in these and other invertebrate tissues.     

Effect of salt and volume loading on the circulation in the leech,Hirudo medicinalis L.

Summary The effects of increased fluid volume in the closed vascular system on circulation were studied in the leech (Hirudo medicinalis) by intravascular pressure recordings and blood flow measurements.Significant increases in blood volume were achieved by crop loading with hyposmotic (72 mOsmol·kg^–1 H₂O) or hyperosmotic (300 mOsmol·kg^–1 H₂O) salt solutions or by infusion of isosmotic saline (200 mOsmol·kg^–1) into the vascular system.During the high-pressure (HIP) phase, which maintains the rear-to-front circulation, systolic blood pressure in the heart was not affected. An increase in systolic pressure in the heart was observed during the low-pressure (LOP) phase, which supplies the segmental circulation. Heart rate was not changed by crop loading with hyposmotic saline or by vascular infusion. Heart rate decreased after crop loading with hyperosmotic saline. Blood flow rate in the dorsal vessel was increased by crop loading with hyposmotic saline, but not after crop loading with hyperosmotic saline. In all cases the diameter of the dorsal vessel was not affected. A possible mechanism controlling blood pressure and blood flow in the vascular system is discussed.Abbreviations HIP-phase high-pressure phase - LOP-phase low-pressure phase - CNS central nervous system     

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 .