Single and combined effects of cadmium, microplastics, and their mixture on whole-body serotonin and feeding behaviour following chronic exposure and subsequent recovery in the freshwater leech, Nephelopsis obscura.

Microplastics and metals are contaminants detected in many freshwater systems globally. Interactions of microplastics with other contaminants including cadmium poses potential threats to the health of aquatic organisms including Nephelopsis obscura, a predatory leech species that is widespread and serves important ecological and economic roles. The feeding biology of N. obscura has been well-described, including that serotonin regulates feeding behaviour. Further, exposure to cadmium has been found to cause decrease whole-body concentrations of serotonin. The influence that microplastic contamination and co-contamination of cadmium and microplastics has on N. obscura is unknown. The present study had three objectives: (1) to determine if water or sediment contaminated with cadmium, microplastics, or their mixture resulted in greater cadmium uptake by N. obscura, (2) to assess effects of chronic (21-day) exposure of N. obscura to waterborne cadmium, microplastics, and their mixture on bioaccumulation of cadmium, concentrations of serotonin, and feeding behaviour (latency to feeding, time spent feeding, and distance moved), and (3) to reassess the bioaccumulation of cadmium, concentrations of serotonin, and feeding behaviour following transfer to an uncontaminated environment for a one-week recovery period. This study revealed that access to and presence of sediment is protective against cadmium uptake and that cadmium is more readily accumulated from waterborne sources, even in environments where both sediment and surface water are contaminated. After 21-days of exposure to waterborne cadmium, microplastics, and their mixture, accumulation of cadmium, decreased concentrations of serotonin, and impaired feeding behaviours were greatest in leeches from the co-exposures compared to leeches from either single contaminant exposure group. Finally, after one week of depuration and recovery in freshwater following the 21-day exposures, concentrations of serotonin and feeding behaviour were restored in individuals from the microplastic exposure; however, cadmium-exposed individuals continued to show decreased concentrations of serotonin and behavioural deficits. The co-exposure of leeches to cadmium and microplastics resulted in additive effects to serotonin synthesis and feeding behaviour; however, this study demonstrated that leeches were able to recover from microplastic toxicity within a week whereas cadmium toxicity persisted.

Effect of Unloading Condition on the Healing Process and Effectiveness of Platelet Rich Plasma as a Countermeasure: Study on In Vivo and In Vitro Wound Healing Models.

Wound healing is a very complex process that allows organisms to survive injuries. It is strictly regulated by a number of biochemical and physical factors, mechanical forces included. Studying wound healing in space is interesting for two main reasons: (i) defining tools, procedures, and protocols to manage serious wounds and burns eventually occurring in future long-lasting space exploration missions, without the possibility of timely medical evacuation to Earth; (ii) understanding the role of gravity and mechanical factors in the healing process and scarring, thus contributing to unravelling the mechanisms underlying the switching between perfect regeneration and imperfect repair with scarring. In the study presented here, a new in vivo sutured wound healing model in the leech (Hirudo medicinalis) has been used to evaluate the effect of unloading conditions on the healing process and the effectiveness of platelet rich plasma (PRP) as a countermeasure. The results reveal that microgravity caused a healing delay and structural alterations in the repair tissue, which were prevented by PRP treatment. Moreover, investigating the effects of microgravity and PRP on an in vitro wound healing model, it was found that PRP is able to counteract the microgravity-induced impairment in fibroblast migration to the wound site. This could be one of the mechanisms underlying the effectiveness of PRP in preventing healing impairment in unloading conditions.

Two distinct electrophysiological mechanisms underlie extensive depolarization elicited by 2,4 diaminobutyric acid in leech Retzius neurons.

Recent studies suggest that 2,4-DABA, a neurotoxic excitatory amino acid present in virtually all environments, but predominantly in aquatic ecosystems may be a risk factor for development of neurodegenerative diseases in animals and humans. Despite its neurotoxicity and potential environmental importance, mechanisms underlying the excitatory and putative excitotoxic action of 2,4-DABA in neurons are still unexplored. We previously reported on extensive two-stage membrane depolarization and functional disturbances in leech Retzius neurons induced by 2,4-DABA. Current study presents the first detailed look into the electrophysiological processes leading to this depolarization. Intracellular recordings were performed on Retzius neurons of the leech Haemopis sanguisuga using glass microelectrodes and input membrane resistance (IMR) was measured by injecting hyperpolarizing current pulses through these electrodes. Results show that the excitatory effect 2,4-DABA elicits on neurons' membrane potential is dependent on sodium ions. Depolarizing effect of 5·10-3 mol/L 2,4-DABA in sodium-free solution was significantly diminished by 91% reducing it to 3.26 ±â€¯0.62 mV and its two-stage nature was abrogated. In addition to being sodium-dependent, the depolarization of membrane potential induced by this amino acid is coupled with an increase of membrane permeability, as 2,4-DABA decreases IMR by 8.27 ±â€¯1.47 MΩ (67.60%). Since present results highlight the role of sodium ions, we investigated the role of two putative sodium-dependent mechanisms in 2,4-DABA-induced excitatory effect - activation of ionotropic glutamate receptors and the electrogenic transporter for neutral amino acids. Excitatory effect of 5·10-3 mol/L 2,4-DABA was partially blocked by 10-5 mol/L 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) a non-NMDA receptor antagonist as the first stage of membrane depolarization was significantly reduced by 2.59 ±â€¯0.98 mV (40%), whilst second stage remained unaltered. Moreover, involvement of the sodium-dependent transport system for neutral amino acids was investigated by equimolar co-application of 5·10-3 mol/L 2,4-DABA and L-alanine, a competitive inhibitor of this transporter. Although L-alanine exhibited no effect on the first stage of membrane depolarization elicited by 2,4-DABA, it substantially reduced the second stage (the overall membrane depolarization) from 39.63 ±â€¯2.22 mV to 16.28 ±â€¯2.58 mV, by 58.92%. We therefore propose that the electrophysiological effect of 2,4-DABA on Retzius neurons is mediated by two distinct mechanisms, i.e. by activation of ionotropic glutamate receptor that initiates the first stage of membrane depolarization followed by the stimulation of an electrogenic sodium-dependent neutral amino acid transporter, leading to additional influx of positive charge into the cell and the second stage of depolarization.

[Effects of MT on growth and gonad development of natural overwintering Whitmania pigra].

The objectives of study were to explore the effects of exogenous methyltestosterone( MT) on the growth and gonadal development of overwintering Whitmania pigra. Before overwintering,0. 1,1. 0,10. 0,100. 0,150. 0 µg·L-1 of MT were added to the aquaculture water for 6 weeks. The changes of growth performance,gonad index,endogenous steroid hormones level and internal quality were measured after hibernate for 60 days. Then the tissue slice technique was used to observe the spermary( ovary) of Wh. pigra.The results showed that the body weight,survival rate and gonadal index increased first and then decreased with the increase of exogenous MT concentration; the male gland index was found the highest at the concentration of MT 10. 0 µg·L-1 and the female gland index was the highest at the concentration of MT 1. 0 µg·L-1. The survival rate of Wh. pigra peaked at the concentration of MT 10. 0 µg·L-1.The weight reaches a peak at a concentration of MT 100. 0 µg·L-1( P<0. 05). The number of primary spermatocytes in the testis was negatively correlated with the concentration of exogenous MT. The number of secondary spermatocytes and sperm cells increased first and then decreased. The concentration of secondary spermatocytes was the highest when the concentration of MT was 100. 0 µg·L-1.The number and volume of oocytes in the ovary and the yolk granules increased first and then decreased with the increase of exogenous MT concentration,and the highest was observed at the MT concentration of 100. 0 µg·L-1. The endogenous steroid hormone of Wh.pigra increased first and then decreased with the increase of exogenous MT concentration. The concentration of androgen and progesterone was the highest in MT 100. 0 µg·L-1 treatment( P<0. 05),and the concentration of estrogen was found the highest in MT 10. 0 µg·L-1 treatment( P<0. 05). After adding exogenous MT,Wh. pigra moisture content,acid-insoluble ash content,p H and anti-thrombin activity met the quality criterion of medicinal Wh. pigra in Chinese Pharmacopoeia( 2015 edition). In conclusion,the short-term addition of 1. 0-100. 0 µg·L-1 exogenous MT before hibernation can promote the growth,the development of sperm cells and the antithrombin activity of Wh. pigra.

Medical leech therapy in plastic reconstructive surgery.

The use of Hirudo medicinalis in clinical practice has increased in recent years. The primary indication in plastic surgery has traditionally been venous congestion. However, other reported clinical applications were in varicose veins, thrombophlebitis, and osteoarthritis. In this review, we summarize recent data elucidating the role that medicinal leeches play in the field of plastic surgery.

Hirudotherapy in veterinary medicine.

The saliva of medicinal leeches, e.g., Hirudo medicinalis and Hirudo verbana commonly used in hirudotherapy, contains more than 100 bioactive substances with various therapeutic effects, including anticoagulant, vasodilator, thrombolytic, anti-inflammatory and anaesthetic properties. Recently, leeches have been used very successfully in veterinary medicine to treat many diseases of animals, especially dogs, cats and horses. The most common indications for the use of leeches are hip and elbow dysplasia, acute and chronic arthritis, diseases associated with inflammation of tendons, ligaments, and fascia, diseases of the vertebrae and the treatment of scars. Leech therapy is a painless procedure which takes an average of 30 to 120 minutes, the time being dependent on the size of the animal. All leeches used in medical procedures should originate only from certified biofarms. The maintenance of sterile conditions for the culture, transport and storage of medical leeches is very important to protect animals from microbial infections. Hirudotherapy is successfully used in veterinary medicine, especially when traditional treatment is not effective, the effects of treatment are too slow, or after surgery, when the tissues may be threatened by venous congestion.

Medicinal leech therapy in pain syndromes: a narrative review.

Medicinal leech therapy is used in a variety of conditions; most of which have pain as a major symptom. Its mode of action relies on the injection of leech saliva into patients' tissues during the process of blood withdrawal. Leech saliva contains active ingredients with anti-inflammatory, thrombolytic, anti-coagulant and blood- and lymph-circulation enhancing properties. A specific analgesic substance within the leech saliva is yet to be identified. Pain relief from leech therapy is rapid, effective and long-lasting in many conditions. This review compiles studies and case reports that provide clinical evidence for leech therapy's analgesic effects.

Barbronia weberi (Clitellata, Hirudinida, Salifidae) has ovary cords of the Erpobdella type.

The organization of the ovaries in representative of the Salifidae (Hirudinida, Erpobdelliformes) was studied at the ultrastructural level for the first time. Like in other leeches, the ovaries of Barbronia weberi are composed of an outer envelope (i.e., an ovisac made up of two coelomic epithelia, muscle cells, and connective tissue) and several internal units, which are broadly similar to the ovary cords found in representatives of the Erpobdellidae. There are usually 6-8 ovary cords that are twisted or cambered with a narrow apical part and a broader, irregularly shaped distal end in each ovisac of B. weberi. Each ovary cord is built from somatic and germ-line cells and the latter tend to form multicellular cysts that are equipped with a central cytoplasmic core (cytophore). There are two morphologically different subpopulations of germ-line cells: oocytes and more numerous nurse cells. Growing oocytes form protuberances on the ovary cord surface and eventually detach from the cord and float freely in the ovisac lumen, whereas the other components of germ-line cysts (i.e., nurse cells and cytophore) degenerate. It should be pointed out that there is a prominent gradient of germ-cell development along the long axis of the cord. The somatic cells form the ovary cord envelope (the so-called spongiosa cells) and also penetrate the spaces between germ-line cells. Both kinds of the somatic cells, that is, those forming the cord envelope and the somatic cells that are associated with oocytes (follicular cells) have a well-developed system of intercellular channels. Additionally, one prominent somatic cell, the apical cell, was found at the apical tip of each ovary cord. Because the aforementioned features of ovary cords found in B. weberi are very similar (with a few minor exceptions) to the ovary cords that have been described in Erpobdella octoculata and E. johanssoni, we propose the term "ovary cords of the Erpobdella type" for them. Our results support a close phylogenetic relationship between Salifidae and Erpobdellidae.

Mechanisms contributing to the dopamine induction of crawl-like bursting in leech motoneurons.

Dopamine (DA) activates fictive crawling behavior in the medicinal leech. To identify the cellular mechanisms underlying this activation at the level of crawl-specific motoneuronal bursting, we targeted potential cAMP-dependent events that are often activated through DA(1)-like receptor signaling pathways. We found that isolated ganglia produced crawl-like motoneuron bursting after bath application of phosphodiesterase inhibitors (PDIs) that upregulated cAMP. This bursting persisted in salines in which calcium ions were replaced with equimolar cobalt or nickel, but was blocked by riluzole, an inhibitor of a persistent sodium current. PDI-induced bursting contained a number of patterned elements that were statistically similar to those observed during DA-induced fictive crawling, except that one motoneuron (CV) exhibited bursting during the contraction rather than the elongation phase of crawling. Although DA and the PDIs produced similar bursting profiles, intracellular recordings from motoneurons revealed differences in altered membrane properties. For example, DA lowered motoneuron excitability whereas the PDIs increased resting discharge rates. We suggest that PDIs (and DA) activate a sodium-influx-dependent timing mechanism capable of setting the crawl rhythm and that multiple DA receptor subtypes are involved in shaping and modulating the phase relationships and membrane properties of cell-specific members of the crawl network to generate crawling.

Marine leech Ozobranchus margoi parasitizing loggerhead turtle (Caretta caretta) in Rio Grande do Sul, Brazil / Sanguessugas Ozobranchus margoi parasitando uma tartaruga cabeçuda (Caretta caretta) no Rio Grande do Sul, Brasil

This paper reports the finding of several Ozobranchus margoi (Annelida: Hirudinea) parasitizing a loggerhead turtle (Caretta caretta) that was found in the municipality of Tavares, state of Rio Grande do Sul, southern Brazil. Since this parasite is considered to be a vector of chelonid herpesvirus 5 (ChHV-5), the leeches collected were tested for the presence of this virus. All the specimens were negative on PCR analysis. Although O. margoi is considered to be a common sea turtle parasite, this is the first official record describing collection of this parasite from a loggerhead turtle in southern Brazil, within the country's subtropical zone. This finding draws attention to the presence of this parasite and to the risk of leech-borne infectious diseases among turtles found along the coast of southern Brazil.

Este artigo relata a descoberta de vários exemplares de Ozobranchus margoi (Annelida Hirudínea) parasitando uma tartaruga cabeçuda (Caretta caretta) encontrada no município de Tavares, Rio Grande do Sul, sul do Brasil. Uma vez que esse parasito é considerado vetor do chelonid herpesvirus 5 (ChHV 5), as sanguessugas foram testadas para a presença deste vírus. Todas as amostras foram negativas pela análise de PCR. Embora o O. margoi seja considerado um parasito comum de tartarugas marinhas, este é o primeiro registro oficial que descreve a coleta deste parasita em uma tartaruga cabeçuda no sul do Brasil, dentro da zona subtropical do país. Este achado chama a atenção para a presença deste parasita e para o risco de sanguessugas transmitirem doenças infecciosas em tartarugas no litoral sul do Brasil.

Chromoblastomycosis after a leech bite complicated by myiasis: a case report.

BACKGROUND: Chromoblastomycosis is a chronic mycotic infection, most common in the tropics and subtropics, following traumatic fungal implantation. CASE PRESENTATION: A 72 year-old farmer was admitted to Luang Namtha Provincial Hospital, northern Laos, with a growth on the left lower leg which began 1 week after a forefoot leech bite 10 years previously. He presented with a cauliflower-like mass and plaque-like lesions on his lower leg/foot and cellulitis with a purulent tender swelling of his left heel. Twenty-two Chrysomya bezziana larvae were extracted from his heel. PCR of a biopsy of a left lower leg nodule demonstrated Fonsecaea pedrosoi, monophora, or F. nubica. He was successfully treated with long term terbinafin plus itraconazole pulse-therapy and local debridement. CONCLUSIONS: Chromoblastomycosis is reported for the first time from Laos. It carries the danger of bacterial and myiasis superinfection. Leech bites may facilitate infection.

Salivary transcriptome of the North American medicinal leech, Macrobdella decora.

A variety of bioactive proteins from medicinal leeches, like species of Hirudo , have been characterized and evaluated for their potential therapeutic biomedical properties. However, there has not previously been a comprehensive attempt to fully characterize the salivary transcriptome of a medicinal leech that would allow a clearer understanding of the suite of polypeptides employed by these sanguivorous annelids and provide insights regarding their evolutionary origins. An Expressed Sequence Tag (EST) library-based analysis of the salivary transcriptome of the North American medicinal leech, Macrobdella decora, reveals a complex cocktail of anticoagulants and other bioactive secreted proteins not previously known to exist in a single leech. Transcripts were identified that correspond to each of saratin, bdellin, destabilase, hirudin, decorsin, endoglucoronidase, antistatin, and eglin, as well as to other previously uncharacterized predicted serine protease inhibitors, lectoxin-like c-type lectins, ficolin, disintegrins and histidine-rich proteins. This work provides a lens into the richness of bioactive polypeptides that are associated with sanguivory. In the context of a well-characterized molecular phylogeny of leeches, the results allow for preliminary evaluation of the relative evolutionary origins and historical conservation of leech salivary components. The goal of identifying evolutionarily significant residues associated with biomedically significant phenomena implies continued insights from a broader sampling of blood-feeding leech salivary transcriptomes.

Properties of cannabinoid-dependent long-term depression in the leech.

Previously, a cannabinoid-dependent form of long-term depression (LTD) was discovered at the polysynaptic connection between the touch mechanosensory neuron and the S interneuron (Li and Burrell in J Comp Physiol A 195:831-841, 2009). In the present study, the physiological properties of this cannabinoid-dependent LTD were examined. Increases in intracellular calcium in the S interneuron are necessary for this form of LTD in this circuit. Calcium signals contributing to cannabinoid-dependent LTD are mediated by voltage-dependent calcium channel and release of calcium from intracellular stores. Inositol triphosphate receptors, but not ryanodine receptors, appear to mediate this store-released calcium signal. Cannabinoid-dependent LTD also requires activation of metabotropic serotonin receptors, possibly a serotonin type 2-like receptor. Finally, this form of LTD involves the stimulation of nitric oxide synthase and a decrease in cyclic adenosine monophosphate signaling, both of which appeared to be downstream of cannabinoid receptor activation. Based on these findings, the cellular signaling mechanisms of cannabinoid-dependent LTD in the leech are remarkably similar to vertebrate forms of cannabinoid-dependent synaptic plasticity.

Biodiversity and ecology of Hirudinea (Annelida) from the Natural Reserve of Isla Martín García, Río de la Plata, Argentina / A Biodiversidade e a Ecologia de Hirudinea (Annelida) da Reserva Natural da Ilha Martín García, na Argentina

The Island of Martin Garcia lies at the confluence of the Uruguay and Paraná Rivers (upper Río de la Plata). This island is an outcrop of the crystalline basement. Due to basalt exploitation the island exhibits several ponds covered by carpets of free-floating macrophytes. Seven major environmental variables were measured: water and air temperature, percentage of oxygen saturation, dissolved oxygen, electrical conductivity, total dissolved solids and pH. Eleven leech species were found, Helobdella striata, H. diploides, H. adiastola and H. hyalina were new records. UPGMA clustering of species based on their occurrence in different ecological conditions revealed three main species groups. Canonical Correspondence Analysis (CCA) explained 97.2 percent of the correlation between species and environmental variables. H. triserialis shows the widest range of tolerance, H. hyalina shows positive relationships to conductivity; H. diploides shows a high correlation with dissolved oxygen, H. striata, H. lineata, and S. similis are negatively correlated with water temperature, and H. simplex is positively correlated with pH. Relationships between the species richness (S) and the sampling sites were negatively correlated with water temperature and positively correlated with dissolved oxygen. Leech biodiversity from the water bodies of Martín García Island, shows a great diversity of species and a wide plasticity regarding the characteristics of the environmental factors considered.

A Ilha Martín García encontra-se na afluência dos Rios Uruguay e Paraná (no Rio de la Plata superior) e constitui um afloramento do maciço cristalino de Brasília. Por causa da exploração do basalto, formaram-se lacunas que apresentam tapetes de vegetação flutuantes e macrófitas enraizadas. Mediram-se sete variáveis físico-químicas da água: temperatura do ar e da água, porcentagem de saturação de oxigênio, oxigênio dissolvido, condutividade elétrica, sólidos totais dissolvidos e pH. Encontraram-se onze espécies de sanguessuga, como Helobdella striata, H. diploides, H. adiastola e H. hyalina que constituem novos registros para a ilha. A análise de agrupamento baseado na ocorrência das espécies em condições ecológicas diferentes revelou três grupos principais. A análise canônica da correspondência sugere que a distribuição das espécies relaciona-se às variáveis ambientais consideradas, 97,2 por cento da correlação entre as espécies e as variáveis se distribui no eixo dois do diagrama de ordenamento. As variáveis com maior flutuação foram a condutividade e o pH. H. triserialis foi a espécie mais tolerante às variáveis ambientais; H. hyalina se relacionou positivamente com a condutividade; H. diploides teve alta correlação com o oxigênio dissolvido; H. striata, H. lineata e Semiscolex similis relacionaram-se negativamente com a temperatura da água e H. simplex, positivamente com o pH. As relações entre a riqueza de espécies (S) e os lugares de amostras foram negativas com a temperatura da água e positivas com o oxigênio dissolvido. Os corpos da água da Ilha Martín García mostram uma grande biodiversidade de espécies de sanguessuga, assim como uma ampla ductilidade com relação aos fatores ambientais considerados.

Identification, isolation and expansion of myoendothelial cells involved in leech muscle regeneration.

Adult skeletal muscle in vertebrates contains myoendothelial cells that express both myogenic and endothelial markers, and which are able to differentiate into myogenic cells to contribute to muscle regeneration. In spite of intensive research efforts, numerous questions remain regarding the role of cytokine signalling on myoendothelial cell differentiation and muscle regeneration. Here we used Hirudo medicinalis (Annelid, leech) as an emerging new model to study myoendothelial cells and muscle regeneration. Although the leech has relative anatomical simplicity, it shows a striking similarity with vertebrate responses and is a reliable model for studying a variety of basic events, such as tissue repair. Double immunohistochemical analysis were used to characterize myoendothelial cells in leeches and, by injecting in vivo the matrigel biopolymer supplemented with the cytokine Vascular Endothelial Growth Factor (VEGF), we were able to isolate this specific cell population expressing myogenic and endothelial markers. We then evaluated the effect of VEGF on these cells in vitro. Our data indicate that, similar to that proposed for vertebrates, myoendothelial cells of the leech directly participate in myogenesis both in vivo and in vitro, and that VEGF secretion is involved in the recruitment and expansion of these muscle progenitor cells.

Cell dynamics during cocoon secretion in the aquatic leech, Theromyzon tessulatum (Annelida: Clitellata: Glossiphoniidae).

One distinguishing feature of clitellate annelids is the presence of specialized segments comprising the clitellum, whose primary function is to secrete a cocoon. Using histological analyses, we have documented cell types (I-V) and cellular processes associated with cocoon secretion in the aquatic leech, Theromyzon tessulatum. Our data indicate that the bulk of the cocoon's biomass arises from precursor cells of a single type that hypertrophy and proliferate approximately 1 week prior to egg laying, and then differentiate into either of two cell types (i.e., Type II or Type III) depending on their position within the clitellum. Type II cells are concentrated along the lateral edges and venter of the clitellum and secrete alcian blue-staining granules that form opercula (i.e., glue-like material that seals both cocoon ends), while Type III cells populate the dorsal midline and secrete azocarmine-staining granules that build the cocoon wall. Both cell types occupy spaces between deep muscle layers and extend long-neck tubules to the surface epithelium as they fill with granules a few days prior to egg laying. Other cell types appear to make minor contributions to the cocoon (e.g., Type I, Type IV) or have supporting or signaling roles (e.g., Type V). Our observations suggest that post-translational modification (i.e., glycosylation) of the same core protein(s) distinguishes the granules of Type II/III cells, and that the default state of the Type II/III precursor may be evolutionarily linked to secretory cells in basal polychaetes.

Forskolin induces NMDA receptor-dependent potentiation at a central synapse in the leech.

In vertebrate hippocampal neurons, application of forskolin (an adenylyl cyclase activator) and rolipram (a phosphodiesterase inhibitor) is an effective technique for inducing chemical long-term potentiation (cLTP) that is N-methyl-d-aspartate (NMDA) receptor (NMDAR)-dependent. However, it is not known whether forskolin induces a similar potentiation in invertebrate synapses. Therefore, we examined whether forskolin plus rolipram treatment could induce potentiation at a known glutamatergic synapse in the leech (Hirudo sp.), specifically between the pressure (P) mechanosensory and anterior pagoda (AP) neurons. Perfusion of isolated ganglia with forskolin (50 muM) in conjunction with rolipram (0.1 muM) in Mg(2+)-free saline significantly potentiated the P-to-AP excitatory postsynaptic potential. Application of 2-amino-5-phosphonovaleric acid (APV, 100 muM), a competitive NMDAR antagonist, blocked the potentiation, indicating P-to-AP potentiation is NMDAR-dependent. Potentiation was blocked by injection of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA, 1 mM) into the postsynaptic cell, but not by BAPTA injection into the presynaptic neuron, indicating a requirement for postsynaptic elevation of intracellular Ca(2+). Application of db-cAMP mimicked the potentiating effects of forskolin, and Rp-cAMP, an inhibitor of protein kinase A, blocked forskolin-induced potentiation. Potentiation was also blocked by autocamtide-2-related inhibitory peptide (AIP), indicating a requirement for activation of Ca(2+)/calmodulin-dependent kinase II (CaMKII). Finally, potentiation was blocked by botulinum toxin, suggesting that trafficking of glutamate receptors also plays a role in this form of synaptic plasticity. These experiments demonstrate that techniques used to induce cLTP in vertebrate synapses also induce NMDAR-dependent potentiation in the leech CNS and that many of the cellular processes that mediate LTP are conserved between vertebrate and invertebrate phyla.

Oogenesis in four species of Piscicola (Hirudinea, Rhynchobdellida).

Piscicola has a pair of elongated sac-shaped ovaries. Inside the ovaries are numerous small somatic cells and regularly spherical egg follicles. Each follicle is composed of three types of cells: many (average 30) germ cells (cystocytes) interconnected by intercellular bridges in clones (cysts), one intermediate cell, and three to five outer follicle cells (envelope cells). Each germ cell in a clone has one intercellular bridge connecting it to the central anucleate cytoplasmic mass, the cytophore. Each cluster of germ cells is completely embedded inside a single huge somatic follicle cell, the intermediate (interstitial) cell. The most spectacular feature of the intermediate cell is its development of a system of intracytoplasmic canals apparently formed of invaginations of its cell membrane. Initially the complex of germ cell cluster + intermediate cell is enclosed within an envelope composed of squamous cells. As oogenesis progresses the envelope cells gradually degenerate. All the germ cells that have terminated their mitotic divisions are of similar size and enter meiotic prophase, but one of the cystocytes promptly starts to grow faster and differentiates into the oocyte, whereas the remaining cystocytes withdraw from meiosis and become nurse cells (trophocytes). Numerous mitochondria, ER, and a vast amount of ribosomes are transferred from the trophocytes via the cytophore toward the oocyte. Eventually the oocyte ingests all the content of the cytophore, and the trophocytes degenerate. Little vitellogenesis takes place; the oocyte gathers nutrients in the form of small lipid droplets. At the end of oogenesis, an electron-dense fibrous vitelline envelope appears around the oocyte, among short microvilli. At the same time, electron-dense cortical granules occur in the oocyte cortical cytoplasm; at the end of oogenesis they are numerous, but after fertilization they disappear from the ooplasm. In the present article we point out many differences in the course of oogenesis in two related families of rhynchobdellids: piscicolids and glossiphoniids.

Serotonin delays habituation of leech swim response to touch.

Serotonin, acting through a cAMP-signaling pathway, delayed habituation to criterion of the leech's swim response to touch. This delay was reversed by crushing the connective between serotonin-exposed and serotonin-naive ganglia, and correlated with an increase in spontaneous impulse activity in this connective. We suggest that increased activity in intersegmental interneurons may play a role in maintaining swim responsiveness when concentrations of serotonin are elevated.

Reduced axon sprouting after treatment that diminishes microglia accumulation at lesions in the leech CNS.

The role of mammalian microglia in central nervous system (CNS) repair is controversial. Microglia accumulate at lesions where they act as immune cells and phagocytize debris, and they may secrete neurotrophins, but they also produce molecules that can be cytotoxic, like nitric oxide (NO). To determine the importance of microglial accumulation at lesions on growth of severed CNS axons in the leech (Hirudo medicinalis), in which axon and synapse regeneration are notably successful even when isolated in tissue culture medium, microglial migration to lesions was reduced. Pressure (P) sensory neurons were injected with biocytin to reveal the extent of their sprouting 24 hours after lesioning. To reduce microglia accumulation at lesions, cords were treated for 3.5 hours with 3 mM ATP or 2 mM N(omega)-nitro-L-arginine methyl ester (L-NAME) or 50 microM Reactive blue-2 (RB2) beginning 30 minutes before injury. Lesioned controls were either not treated with drug or treated 3 hours later with one of the drugs, after the migration and subsequent accumulation of most microglia had occurred, but before the onset of axon sprouting, for a total of seven separate conditions. There was a significant reduction in total sprout lengths compared with controls when microglial accumulation was reduced. The results suggest that microglial cells are necessary for the usual sprouting of injured axons.