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.

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.

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.

A new cellular type in invertebrates: first evidence of telocytes in leech Hirudo medicinalis.

Telocytes, a peculiar cell type, were recently found in vertebrates. Hence this cell system has been reported as ubiquitous in the bodies of mammals and interpreted as an important player in innate immunity and tissue regeneration, it is reasonable to look for it also in invertebrates, that rely their integrity solely by innate immunity. Here we describe, at morphological and functional level, invertebrate telocytes from the body of leech Hirudo medicinalis (Annelida), suggesting how these cells, forming a resident stromal 3D network, can influence or participate in different events. These findings support the concepts that leech telocytes: i) are organized in a cellular dynamic and versatile 3D network likewise the vertebrate counterpart; ii) are an evolutionarily conserved immune-neuroendocrine system; iii) form an immuno-surveillance system of resident cells responding faster than migrating immunocytes recruited in stimulated area; iv) communicate with neighbouring cells directly and indirectly, via cell-cell contacts and soluble molecules secreted by multivesicular bodies; v) present within neo-vessels, share with immunocytes the mesodermal lineage; vi) are involved in regenerative processes. In conclusion, we propose that HmTCs, integrating so different functions, might explain the innate immune memory and can be associated with several aged related diseases.

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.

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.

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.

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.

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.

Acetyl-l-carnitine prevents serotonin-induced behavioural sensitization and dishabituation in Hirudo medicinalis.

Several studies suggest that acetyl-l-carnitine (ALC) might influence learning processes. Along this line of investigation, we have previously shown that ALC impaired sensitization and dishabituation induced by nociceptive stimulation of the dorsal skin of the leech Hirudo medicinalis, in the behavioural paradigm of the swim induction (SI). In previous works we showed that 5HT was involved in both sensitization and dishabituation of SI acting through the second messenger cAMP. In this work, we have reported that for given doses and temporal ranges ALC was able to block sensitization and to impair dishabituation mimicked by the injection of 5-HT or 8Br-cAMP, a membrane permeable analogue of cAMP. Our results show that a single treatment with 2mM ALC was the most effective concentration to block the onset of sensitization induced by 5-HT injection and its major effects occurred 11 days after ALC treatment. 2mM ALC also blocked sensitization induced by 8Br-cAMP injection, whereas, ALC did not completely abolish dishabituation induced by 5-HT or 8Br-cAMP injection at the tested concentrations and at every time point.

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.

Maturation of peptide-positive synaptic arbors in the medicinal leech requires rhythmic target activity.

The formation and refinement of synaptic connections are dependent on the activity that emerges from nascent synaptic connections. Such activity has the effect of regulating the production and release of specific neurotransmitters. To determine the role of activity in regulating the production of peptide-positive synapses, we used antibodies against Phe-Met-Arg-Phe-NH2 and acetylated α-tubulin as well as intracellular injections of Neurobiotin to examine varicosities belonging to heart excitor (HE) neurons on the heart tubes of medicinal leeches, Hirudo spp. We found that the production of peptide-positive varicosities increased considerably during the last week of embryogenesis, which coincided with the emergence of rhythmic activity of the heart tube. When we compromised central input to HE neurons with bicuculline or by surgical ablation of the central pattern generator during early embryogenesis, we found that activity in the heart tubes and its rhythmicity were greatly diminished. Furthermore, the activity of HE neurons had also lost its rhythmicity and appeared tonic, and production of peptide-positive varicosities was substantially reduced as well. Partial surgical ablations that preserved rhythmic activity in the heart tube while disrupting heart tube innervation by some HE neurons still resulted in peptide-positive varicosity production. Taken together, our results suggest that postsynaptic rhythmic activity of the heart tube is necessary and sufficient for the development and maturation of peptide-positive synapses.

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.

Differentially expressed genes in Hirudo medicinalis ganglia after acetyl-L-carnitine treatment.

Acetyl-L-carnitine (ALC) is a naturally occurring substance that, when administered at supra-physiological concentration, is neuroprotective. It is involved in membrane stabilization and in enhancement of mitochondrial functions. It is a molecule of considerable interest for its clinical application in various neural disorders, including Alzheimer's disease and painful neuropathies. ALC is known to improve the cognitive capability of aged animals chronically treated with the drug and, recently, it has been reported that it impairs forms of non-associative learning in the leech. In the present study the effects of ALC on gene expression have been analyzed in the leech Hirudo medicinalis. The suppression subtractive hybridisation methodology was used for the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts in the leech nervous system after ALC treatment. The method detects differentially but also little expressed transcripts of genes whose sequence or identity is still unknown. We report that a single administration of ALC is able to modulate positively the expression of genes coding for functions that reveal a lasting effect of ALC on the invertebrate, and confirm the neuroprotective and neuromodulative role of the substance. In addition an important finding is the modulation of genes of vegetal origin. This might be considered an instance of ectosymbiotic mutualism.

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.