Effects of calcium-activated potassium channel modulators on afterhyperpolarizing potentials in identified motor and mechanosensory neurons of the medicinal leech.

Calcium-activated potassium (KCa) channels contribute to multiple neuronal properties including spike frequency and afterhyperpolarizing potentials (AHPs). KCa channels are classified as KCa1.1, KCa2, or KCa3.1 based on single-channel conductance and pharmacology. Ca2+-dependent AHPs in vertebrates are categorized as fast, medium, or slow. Fast and medium AHPs are generated by KCa1.1 and KCa2 channels, respectively. The KCa subtype responsible for slow AHPs is unclear. Prolonged, Ca2+-dependent AHPs have been described in several leech neurons. Unfortunately, apamin and other KCa blockers often prove ineffective in the leech. An alternative approach is to utilize KCa modulators, which alter channel sensitivity to Ca2+. Vertebrate KCa2 channels are targeted selectively by the positive modulator CyPPA and the negative modulator NS8593. Here we show that AHPs in identified motor and mechanosensory leech neurons are enhanced by CyPPA and suppressed by NS8593. Our results indicate that KCa2 channels underlie prolonged AHPs in these neurons and suggest that KCa2 modulators may serve as effective tools to explore the role of KCa channels in leech physiology.

The stomatogastric nervous system of the medicinal leech: its anatomy, physiology and associated aminergic neurons.

Blood feeding is an essential and signature activity of the medicinal leech species Hirudo verbana. Despite keen interest in understanding the neuronal substrates of this behavior, a major component of the nervous system associated with feeding has remained overlooked. In this study, for the first time, we report on the presence and characteristics of five stomatogastric ganglia (STGs) comprising the visceral stomatogastric nervous system (STN) of the leech. Although a brief report was published by Ruth Hanke in 1948 indicating that a ring of three ganglia (not five) was associated with the cephalic ganglia, this information was never integrated into subsequent neurobiological studies of feeding. Here, the anatomical features of the STGs are described, as are the morphological and electrophysiological characteristics of neurons originating in them. We also determined that two of the five STGs (STG-1 and STG-3) each contained two relatively large (ca. 40 µm diameter) serotonergic neurons. The STN was also enriched with dopaminergic and serotonergic arborizations; however, no intrinsic dopaminergic somata were observed. The trajectory of the serotonergic large lateral (LL) neuron, a command-like cell for feeding, was documented to project directly to the STN and not to the jaw and pharyngeal musculature as previously reported, thus reopening the important question of how the LL cell activates and coordinates biting activity with pharyngeal swallowing. Additional studies revealed that the LL cell is excited by blood serum applied to the lip and is strongly inhibited by dopamine. These findings provide a new foundation for understanding the regulation and modulation of neural networks involved in feeding.

Responses to mechanically and visually cued water waves in the nervous system of the medicinal leech.

Sensitivity to water waves is a key modality by which aquatic predators can detect and localize their prey. For one such predator - the medicinal leech, Hirudo verbana - behavioral responses to visual and mechanical cues from water waves are well documented. Here, we quantitatively characterized the response patterns of a multisensory interneuron, the S cell, to mechanically and visually cued water waves. As a function of frequency, the response profile of the S cell replicated key features of the behavioral prey localization profile in both visual and mechanical modalities. In terms of overall firing rate, the S cell response was not direction selective, and although the direction of spike propagation within the S cell system did follow the direction of wave propagation under certain circumstances, it is unlikely that downstream neuronal targets can use this information. Accordingly, we propose a role for the S cell in the detection of waves but not in the localization of their source. We demonstrated that neither the head brain nor the tail brain are required for the S cell to respond to visually cued water waves.

Medicinal Leech CNS as a Model for Exosome Studies in the Crosstalk between Microglia and Neurons.

In healthy or pathological brains, the neuroinflammatory state is supported by a strong communication involving microglia and neurons. Recent studies indicate that extracellular vesicles (EVs), including exosomes and microvesicles, play a key role in the physiological interactions between cells allowing central nervous system (CNS) development and/or integrity. The present report used medicinal leech CNS to investigate microglia/neuron crosstalk from ex vivo approaches as well as primary cultures. The results demonstrated a large production of exosomes from microglia. Their incubation to primary neuronal cultures showed a strong interaction with neurites. In addition, neurite outgrowth assays demonstrated microglia exosomes to exhibit significant neurotrophic activities using at least a Transforming Growth Factor beta (TGF-ß) family member, called nGDF (nervous Growth/Differentiation Factor). Of interest, the results also showed an EV-mediated dialog between leech microglia and rat cells highlighting this communication to be more a matter of molecules than of species. Taken together, the present report brings a new insight into the microglia/neuron crosstalk in CNS and would help deciphering the molecular evolution of such a cell communication in brain.

Human recombinant RNASET2-induced inflammatory response and connective tissue remodeling in the medicinal leech.

In recent years, several studies have demonstrated that the RNASET2 gene is involved in the control of tumorigenicity in ovarian cancer cells. Furthermore, a role in establishing a functional cross-talk between cancer cells and the surrounding tumor microenvironment has been unveiled for this gene, based on its ability to act as an inducer of the innate immune response. Although several studies have reported on the molecular features of RNASET2, the details on the mechanisms by which this evolutionarily conserved ribonuclease regulates the immune system are still poorly defined. In the effort to clarify this aspect, we report here the effect of recombinant human RNASET2 injection and its role in regulating the innate immune response after bacterial challenge in an invertebrate model, the medicinal leech. We found that recombinant RNASET2 injection induces fibroplasias, connective tissue remodeling and the recruitment of numerous infiltrating cells expressing the specific macrophage markers CD68 and HmAIF1. The RNASET2-mediated chemotactic activity for macrophages has been further confirmed by using a consolidated experimental approach based on injection of the Matrigel biomatrice (MG) supplemented with recombinant RNASET2 in the leech body wall. One week after injection, a large number of CD68+ and HmAIF-1+ macrophages massively infiltrated MG sponges. Finally, in leeches challenged with lipopolysaccharides (LPS) or with the environmental bacteria pathogen Micrococcus nishinomiyaensis, numerous macrophages migrating to the site of inoculation expressed high levels of endogenous RNASET2. Taken together, these results suggest that RNASET2 is likely involved in the initial phase of the inflammatory response in leeches.

9-Phenanthrol modulates postinhibitory rebound and afterhyperpolarizing potentials in an excitatory motor neuron of the medicinal leech.

Postinhibitory rebound (PIR) responses in leech dorsal excitatory motor neurons (cell DE-3) are eliminated by Ca2+ channel blockers but also exhibit a strong dependence on extracellular Na+. These features could be explained by a voltage-gated Ca2+ current acting in concert with a Ca2+-activated nonspecific current (ICAN). In vertebrates, ICAN is associated with TRPM4 channels which are blocked selectively by 9-phenanthrol. Here, we show that 9-phenanthrol selectively inhibits a late phase of PIR and simultaneously enhances afterhyperpolarizing potentials (AHPs). Bath application of NNC 55-0396 or Cd2+ combined with ion substitution experiments indicate that a low-voltage-activated Ca2+ current plays a key role in generating PIR and that Ca2+ influx through low- or high-voltage-activated Ca2+ channels can trigger AHPs via activation of a Ca2+-dependent K+ current. We also demonstrate modulation of rebound responses by other ICAN blockers such as gadolinium and flufenamic acid, as well as the calmodulin antagonist W-7. We discuss how these results provide additional insights into the specific types of ionic currents underlying rebound responses of motor neuron DE-3 in the medicinal leech.

Locomotor and energetic consequences of behavioral thermoregulation in the sanguivorous leech Hirudo verbana.

Medicinal leeches (Hirudo verbana) thermoregulate with respect to their sanguivorous feeding behavior. Immediate postprandial preferences are for warmer than their initial acclimation temperature (Ta, 21°C, Petersen et al. 2011), while unfed leeches have a lower preferred temperature (Tpref, 12.5°C). This may reduce energy expenditure and defer starvation if feeding opportunities are limited. Energetic benefits may have an associated cost if low temperatures reduce mobility and the ability to locate further hosts. These costs could be limited if mobility is unimpaired at low temperatures, or if acclimation can restore locomotor performance to the levels at Ta. The transition from Ta to the unfed Tpref significantly reduced speed and propulsive cycle frequency during swimming, and extension and retraction rates during crawling. Aerobic metabolic rate was also reduced from 0.20±0.03Wkg-1 at Ta to 0.10±0.03Wkg-1 at Tpref. The Q10 values of 1.7-2.9 for energetic and swimming parameters indicate a substantial temperature effect, although part of the decline in swimming performance can be attributed to temperature-related changes in water viscosity. 6 weeks at Ta resulted in no detectable acclimation in locomotor performance or aerobic metabolism. The energetic savings associated with a lower Tpref in unfed leeches effectively doubled the estimated time until depletion of energy reserves. Given that some mobility is still retained at Tpref, and that acclimation is in itself costly, the energetic benefits of selecting cooler temperatures between feedings may outweigh the costs associated with reduced locomotor performance.

Role of the tissue free amino acids in adaptation of medicinal leeches Hirudo medicinalis L., 1758 to extreme climatic conditions.

The first comparison of the spectra of free amino acids in tissues of the medicinal leeches H. medicinalis from different climatic and geographical Eurasian areas has been performed. Adaptation of H. medicinalis to extreme climatic conditions occurs via intensification of the amino acid metabolism resulting from a significant increase in the content of essential amino acids. Accumulation of arginine, histidine, and lysine (3.6-, 3.9-, and 2.0-fold increases, respectively) has proved to play a special protective role in adaptation of H. medicinalis to the low positive temperatures.

Physiological, pharmacological and behavioral evidence for a TRPA1 channel that can elicit defensive responses in the medicinal leech.

Transient receptor potential ankyrin subtype 1 (TRPA1) channels are chemosensitive to compounds such as allyl isothiocyanate (AITC, the active component of mustard oil) and other reactive electrophiles and may also be thermodetectors in many animal phyla. In this study, we provide the first pharmacological evidence of a putative TRPA1-like channel in the medicinal leech. The leech's polymodal nociceptive neuron was activated by both peripheral and central application of the TRPA1 agonist AITC in a concentration-dependent manner. Responses to AITC were inhibited by the selective TRPA1 antagonist HC030031, but also by the TRPV1 antagonist SB366791. Other TRPA1 activators - N-methylmaleimide (NMM) and cinnamaldehyde (CIN) - also activated this nociceptive neuron, although HC030031 only inhibited the effects of NMM. The polymodal nociceptive neurons responded to moderately cold thermal stimuli (<17°C) and these responses were blocked by HC030031. AITC sensitivity was also found in the pressure-sensitive sensory neurons and was blocked by HC030031, but not by SB366791. AITC elicited a nocifensive withdrawal of the posterior sucker in a concentration-dependent manner that could be attenuated with HC030031. Peripheral application of AITC in vivo also produced swimming-like behavior that was attenuated by HC030031. These results suggest the presence of a TRPA1-like channel in the medicinal leech nervous system that responds to cold temperatures and may interact with the leech TRPV-like channel.

Detection and selective avoidance of near ultraviolet radiation by an aquatic annelid: the medicinal leech.

Medicinal leeches are aquatic predators that inhabit surface waters during daylight and also leave the water where they might be exposed to less screened light. Whereas the leech visual system has been shown to respond to visible light, leeches in the genus Hirudo do not appear to be as negatively phototactic as one might expect in order to avoid potential ultraviolet radiation (UVR)-induced damage. I used high intensity light emitting diodes to test the hypothesis that leeches could detect and specifically avoid near UVR (395-405 nm). Groups of unfed juvenile leeches exhibited a robust negative phototaxis to UVR, but had no behavioral response to blue or red and only a slight negative phototaxis to green and white light. Individual leeches also exhibited a vigorous negative phototaxis to UVR; responding in 100% of trials compared with modest negative responses to visible light (responding in ~8% of the trials). The responses in fed and unfed leeches were comparable for UVR stimuli. The responses depended upon the stimulus site: leeches shortened away from UV light to the head, and extended away from UV light to the tail. Electrophysiological nerve recordings showed that the cephalic eyes responded vigorously to UVR. Additionally, individual leech photoreceptors also showed strong responses to UVR, and a higher-order neuron associated with shortening and rapid behavioral responses, the S-cell, was activated by UVR, on both the head and tail. These results demonstrate that the leech can detect UVR and is able to discriminate behaviorally between UVR and visible light.

Physiological and behavioral evidence of a capsaicin-sensitive TRPV-like channel in the medicinal leech.

Transient receptor potential vanilloid (TRPV) channels are found throughout the animal kingdom, where they play an important role in sensory transduction. In this study, we combined physiological studies with in vivo behavioral experiments to examine the presence of a putative TRPV-like receptor in the medicinal leech, building upon earlier studies in this lophotrochozoan invertebrate. The leech polymodal nociceptive neuron was activated by both peripheral and central application of the TRPV1-activator capsaicin in a concentration-dependent manner, with 100 µmol l(-1) being the lowest effective concentration. Responses to capsaicin were inhibited by the selective TRPV1 antagonist SB366791. The polymodal nociceptive neuron also responded to noxious thermal stimuli (>40°C), and this response was also blocked by SB366791. Capsaicin sensitivity was selective to the polymodal nociceptor with no direct response being elicited in the mechanical nociceptive neuron or in the non-nociceptive touch- or pressure-sensitive neurons. Capsaicin also elicited nocifensive behavioral responses (withdrawals and locomotion) in a concentration-dependent manner, and these behavioral responses were significantly attenuated with SB366791. These results suggest the presence of a capsaicin-sensitive TRPV-like channel in the medicinal leech central nervous system and are relevant to the evolution of nociceptive signaling.

Involvement of nitric oxide through endocannabinoids release in microglia activation during the course of CNS regeneration in the medicinal leech.

The medicinal leech is notable for its capacity to regenerate its central nervous system (CNS) following mechanical trauma. Using an electrochemical nitric oxide (NO)-selective electrode to measure NO levels, we found that the time course of NO release in the injured leech CNS is partially under the control of endocannabinoids, namely, N-arachidonyl ethanolamide (AEA) and 2-arachidonyl glycerol (2-AG). Relative quantification of these endocannabinoids was performed by stable isotope dilution (2AGd8 and AAEd8) coupled to mass spectrometry in course of regeneration process or adenosine triphosphate (ATP) treatment. Data show that 2-AG levels rose to a maximum about 30 min after injury or ATP treatment, and returned to baseline levels 4 h after injury. In same conditions, AEA levels also rapidly (within 5 min) dropped after injury or ATP treatment to the nerve cord, but did not fully return to baseline levels within 4 h of injury. In correlation with these data, chemoattraction activities of endocannabinoids on isolated leech microglial cells have been shown in vitro and in vivo reflecting that control over NO production is accompanied by the controlled chemoattraction of microglia directed from the periphery to the lesion site for neuronal repair purposes. Taken together, our results show that in the leech, after injury concurrent with ATP production, purinergic receptor activation, NO production, microglia recruitment, and accumulation to lesion site, a fine imbalance occurs in the endocannabinoid system. These events can bring explanations about the ability of the leech CNS to regenerate after a trauma and the key role of endocannabinoids in this phenomenon.

The leech nervous system: a valuable model to study the microglia involvement in regenerative processes.

Microglia are intrinsic components of the central nervous system (CNS). During pathologies in mammals, inflammatory processes implicate the resident microglia and the infiltration of blood cells including macrophages. Functions of microglia appear to be complex as they exhibit both neuroprotective and neurotoxic effects during neuropathological conditions in vivo and in vitro. The medicinal leech Hirudo medicinalis is a well-known model in neurobiology due to its ability to naturally repair its CNS following injury. Considering the low infiltration of blood cells in this process, the leech CNS is studied to specify the activation mechanisms of only resident microglial cells. The microglia recruitment is known to be essential for the usual sprouting of injured axons and does not require any other glial cells. The present review will describe the questions which are addressed to understand the nerve repair. They will discuss the implication of leech factors in the microglial accumulation, the identification of nerve cells producing these molecules, and the study of different microglial subsets. Those questions aim to better understand the mechanisms of microglial cell recruitment and their crosstalk with damaged neurons. The study of this dialog is necessary to elucidate the balance of the inflammation leading to the leech CNS repair.

Specialized brain regions and sensory inputs that control locomotion in leeches.

Locomotor systems are often controlled by specialized cephalic neurons and undergo modulation by sensory inputs. In many species, dedicated brain regions initiate and maintain behavior and set the duration and frequency of the locomotor episode. In the leech, removing the entire head brain enhances swimming, but the individual roles of its components, the supra- and subesophageal ganglia, in the control of locomotion are unknown. Here we describe the influence of these two structures and that of the tail brain on rhythmic swimming in isolated nerve cord preparations and in nearly intact leeches suspended in an aqueous, "swim-enhancing" environment. We found that, in isolated preparations, swim episode duration and swim burst frequency are greatly increased when the supraesophageal ganglion is removed, but the subesophageal ganglion is intact. The prolonged swim durations observed with the anterior-most ganglion removed were abolished by removal of the tail ganglion. Experiments on the nearly intact leeches show that, in these preparations, the subesophageal ganglion acts to decrease cycle period but, unexpectedly, also decreases swim duration. These results suggest that the supraesophageal ganglion is the primary structure that constrains leech swimming; however, the control of swim duration in the leech is complex, especially in the intact animal.

Centrally patterned rhythmic activity integrated by a peripheral circuit linking multiple oscillators.

The central pattern generator for heartbeat in the medicinal leech, Hirudo generates rhythmic activity conveyed by heart excitor motor neurons in segments 3-18 to coordinate the bilateral tubular hearts and side vessels. We focus on behavior and the influence of previously un-described peripheral nerve circuitry. Extracellular recordings from the valve junction (VJ) where afferent vessels join the heart tube were combined with optical recording of contractions. Action potential bursts at VJs occurred in advance of heart tube and afferent vessel contractions. Transections of nerves were performed to reduce the output of the central pattern generator reaching the heart tube. Muscle contractions persisted but with a less regular rhythm despite normal central pattern generator rhythmicity. With no connections between the central pattern generator and heart tube, a much slower rhythm became manifest. Heart excitor neuron recordings showed that peripheral activity might contribute to the disruption of centrally entrained contractions. In the model presented, peripheral activity would normally modify the activity actually reaching the muscle. We also propose that the fundamental efferent unit is not a single heart excitor neuron, but rather is a functionally defined unit of about three adjacent motor neurons and the peripheral assembly of coupled peripheral oscillators.

The Hirudo medicinalis species complex.

Recently, Hildebrandt and Lemke (Naturwissenschaften 98:995-1008, 2011) argued that the taxonomic status of the three European medicinal leeches, Hirudo medicinalis Linnaeus 1758, Hirudo verbana Carena 1820, and Hirudo orientalis Utevsky and Trontelj (Parasitol Res 98:61-66, 2005) is "questionable" since "all three species interbreed in the laboratory". This statement is in conflict with data published by Elliott and Kutschera (Freshwater Reviews 4:21-41, 2011), indicating that these leeches, which are reciprocally copulating hermaphrodites, represent reproductively isolated biospecies. Here, I summarize evidence indicating that these three European taxa, plus the North African "dragon leech" (Hirudo troctina Johnson 1816), must be interpreted as a complex of closely related species, and that the economically most important taxon H. verbana is polymorphic.

Feeding-mediated distention inhibits swimming in the medicinal leech.

An animal's response to a stimulus depends on many factors such as age, hormonal state, experience, and its behavioral state. For example, an animal may suppress a behavior that is inappropriate or incompatible with its current state. In this study, we show that, as a medicinal leech feeds, the distention that it incurs inhibits its expression of swimming. Distention slows the swimming pattern and decreases the number of swim cycles elicited by a test electrical stimulation; large distentions inhibit swimming altogether. We have previously shown that the ingestive phase of feeding inhibits behaviors by presynaptic inhibition of mechanosensory neurons. Distention has its effects downstream (e.g., gating and central pattern generating interneurons) from these sensory neurons and thus represents a novel mechanism for choosing between conflicting behaviors during feeding. Because removing the leech's gut surgically did not eliminate the effects of body distention, we conclude that the receptors mediating the distention-induced suppression of swimming are likely to be located in the animal's body wall. Together with previous findings, these new data show that leeches rely on two different decision-making networks to ensure that a biologically important behavior is not disrupted by other behaviors.

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.

Small bite, large impact-saliva and salivary molecules in the medicinal leech, Hirudo medicinalis.

Blood-sucking leeches have been used for medical purposes in humans for hundreds of years. Accordingly, one of the most prominent species has been named Hirudo medicinalis by Carl Linne in 1758. Feeding on vertebrate blood poses some serious problems to blood-sucking ectoparasites, as they have to penetrate the body surface of the host and to suppress the normal reactions of the host to such injuries (swelling, pain, inflammation) to remain undetected during the feeding period. Furthermore, the parasites have to take measures to inhibit the normal reactions in host tissues to blood vessel damage, namely hemostasis and blood coagulation (platelet aggregation and activation, activation of thrombin and formation of fibrin clots). During evolution, leeches have acquired the ability to control these processes in their hosts by transferring various bioactive substances to the host. These substances are supposedly produced in unicellular salivary gland cells and injected into the wound at the feeding site through tiny salivary ductule openings in the jaws that the leech uses to slice open the host body surface and to cut blood vessels in the depth of the wound. This review summarizes current knowledge about the salivary gland cells and the biological effects of individual saliva components as well as hints to the potential usefulness of some of these compounds for medical purposes.