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[Display omitted] Previous work from our laboratory showed that cotinine, a nicotine metabolite, reverses three nicotine-induced behavioral effects in freshwater planarians: motility decrease, seizure-like movements, and withdrawal-like behaviors. The present work explored whether cotinine, a nicotine metabolite, antagonized the nicotine-induced effects on planarian motility in a concentration-dependent manner. We found that nicotine decreased planarian motility at nicotine concentrations above 60 μM but increased planarian velocity at concentrations equal to or below 50 μM, in agreement with previous data. Cotinine did not affect planarian motility at a concentration range between 250 and 2750 μM. Furthermore, we found that cotinine alleviated the 100 μM nicotine-induced motility decrease in a concentration-dependent manner and reversed the low nicotine concentration motility increase, albeit in a concentration-independent manner. The apparent concentration-dependent alleviation of >60 μM nicotine-induced motility decrease by cotinine suggests an orthosteric relationship between nicotine and cotinine. On the other hand, the evident concentration-independent cotinine alleviation of the increase in motility induced by 50 μM nicotine suggests an allosteric relationship. Our data is consistent with the existing literature about the relationship between nicotine and cotinine in various models, reinforcing the case for the usefulness of the planarian model in pharmacological studies. [ABSTRACT FROM AUTHOR]

Planarian flatworms undergo continuous internal turnover, wherein old cells are replaced by the division progeny of adult pluripotent stem cells (neoblasts). How cell turnover is carried out at the organismal level remains an intriguing question in planarians and other systems. While previous studies have predominantly focused on neoblast proliferation, little is known about the processes that mediate cell loss during tissue homeostasis. Here, we use the planarian epidermis as a model to study the mechanisms of cell removal. We established a covalent dye-labeling assay and image analysis pipeline to quantify the cell turnover rate in the planarian epidermis. Our findings indicate that the ventral epidermis is highly dynamic and epidermal cells undergo internalization via basal extrusion, followed by a relocation toward the intestine and ultimately digestion by intestinal phagocytes. Overall, our study reveals a complex homeostatic process of cell clearance that may generally allow planarians to catabolize their own cells. [Display omitted] • Planarian epidermal turnover rates differ between dorsal and ventral • The ventral epidermis is a highly dynamic tissue with a half-life of 4.5 days • Epidermal cells are eliminated via basal extrusion, not outward shedding • Internalized epidermal cells are taken up and digested by gut phagocytes Although planarians are known to continuously renew all cell types, how they remove old cells is poorly understood. By developing imaging assays to quantify and observe cell turnover in the epidermis, the authors find that epidermal cells undergo basal extrusion and that the internalized cells are digested by intestinal phagocytes. [ABSTRACT FROM AUTHOR]

One approach to elucidating the principles of regeneration is to investigate mechanisms that regenerate a target organ. Planarian eyes are discrete, visible structures that are dispensable for viability, making them powerful for studying the logic of regeneration. Fate specification in eye regeneration occurs in stem cells (neoblasts), generating eye progenitors. Eye progenitor production is not responsive to the presence or absence of the eye, with regeneration explained by constant progenitor production in the appropriate positional environment. Eye progenitors display coarse spatial specification. A combination of eye-extrinsic cues and self-organization with differentiated eye cells dictate where migratory eye progenitors target. Finally, guidepost-like cells influence regenerating axons to facilitate the restoration of eye circuitry. These findings from the eye as a case study present a model that explains how regeneration can occur. [ABSTRACT FROM AUTHOR]

• Histamine functions as a punisher in vertebrate models. • We investigated if planarians would develop conditioned place aversion to histamine. • Planarians exposed to histamine in a dark environment spent more time in the light. • Histamine did not completely overcome innate preference for the dark. Although histamine functions as a punisher in vertebrate models, its potential aversive effects in invertebrates has gone largely unexamined. We investigated if planaria would develop conditioned place aversions to histamine. In the absence of any training, planaria readily move away from a light source. However, planaria will develop conditioned place preferences for lighted areas if those areas are paired with many of the same psychoactive drugs that produce conditioned place preferences in vertebrates. We confined planaria in the darkened side of a petri dish containing histamine, and they subsequently spent more time in the light than planaria confined initially to the darkened side of a petri dish containing fresh water only. This occurred whether we tested planaria individually or in groups. Pairing histamine with the light side of the petri dish had inconsistent effects, and histamine did not affect behavior under a motility assay. Although histamine altered the planaria behavior, it did not completely overcome the planaria's innate preference for darkened areas. Results add to a growing body of research showing continuity between planaria and vertebrates in the behavioral effects of psychoactive chemicals. [ABSTRACT FROM AUTHOR]

Photomotor response assay (PMR) is very useful in an ecotoxicological context because it allows evaluation of behavioral response to potential toxic compounds. However, a lack of procedure standardization makes results comparison difficult between labs and organisms. Here, we aimed to propose five different tools to standardize the PMR procedure so that it may be applied to all model species, regarding: (1) the minimum total sample size, (2) the acclimation period, (3) the number and duration of light and dark phases alternation, (4) the measured behavior, and (5) the statistical analysis. As an example of procedure application, we analyzed the effect of an exposure to the antibiotic gentamicin on the locomotion behavior during PMR in an invertebrate species: the asexual freshwater planaria Schmidtea mediterranea. We encourage future studies using PMR to follow these five tools to improve data analysis and results comparability. • PMR allows evaluation of behavioral response to potential toxic compounds. • Literature highlights the lack of procedure standardization between labs/organisms. • We proposed five tools to standardize PMR procedure. • Standardized PMR procedure was tested on gentamicin-exposed planaria S. mediterranea. • Standardized PMR procedure is suitable for ecotoxicological studies in invertebrates. [ABSTRACT FROM AUTHOR]

The objective of the present study was to develop a model of avoidance learning and its extinction in planarians (Schmidtea mediterranea). Based on previous experiments showing conditioned place preference, we developed a procedure to investigate conditioned place avoidance (CPA) using shock as an unconditioned stimulus (US) and an automated tracking system to record the animals' behaviour. In Experiment 1, we assessed the unconditioned properties of different shock intensities by measuring post shock activity. In two subsequent experiments we investigated CPA using different designs, surfaces as conditioned stimuli (CSs; rough and smooth), and different US intensities (5 V and 10 V). In general, we observed the successful development of CPA. However, CPA was stronger with higher shock intensities, and we found that, in our preparation, a rough surface is best at entering into an association with the shock than a smooth surface. Finally, we also observed extinction of CPA. The evidence of CPA and its extinction in flatworms validates the planaria as a pre-clinical model for the study of avoidance learning, a hallmark of anxiety disorders. • Planarians are sensitive to different shock intensities. • Planarians shocked in a distinctive context develop conditioned place avoidance. • Context non-reinforced exposure results in extinction of the avoidance response. • Planarians can be used as an invertebrate model to study avoidance behaviour. [ABSTRACT FROM AUTHOR]

Freshwater planarian are emerging as a valuable in vivo model for (eco) toxicological studies, but the lack of harmonization of procedures between laboratories remains a challenge. This study aimed to optimize, automate and select the best behavioural tests and analyse the potential of different planarian species as models for toxicological assessment. We implemented four tests: exploration, photomotor response, Tapping and Planarian Light Dark Test, on different planaria species using the DanioVision system. We conclude that the exploration assay performed in 24 well-plate at 10,000 lux is the one that is robust and reliable for toxicological studies with planaria. Dugesia japonica and Schmidtea mediterranea have proved to be sensitive models for toxicological screening of organophosphorus pesticides through behavioural analysis. Under necessary experimental conditions, the motility baseline in controls, for both species allowed the detection of behavioural changes, making both good models for behavioural testing in (eco) toxicological context. • Freshwater planarians are a promising in-vivo (eco)toxicological screening tool • Optimization of behavioural tests and selection of the best planarian species • Exploration test is robust and reliable for toxicological assessment of OPs on planaria • D.japonica and S.mediterranea are both good models for behavioural testing in toxicological context [ABSTRACT FROM AUTHOR]

Methylisothiazolinone (MIT) is a common biocide used in cosmetic and industrial settings. Studies have demonstrated that MIT is a human sensitizer, to the extent that in 2013 MIT was named allergen of the year. Recently, we showed that MIT exposure in Xenopus laevis (the African clawed frog) inhibits wound healing and tail regeneration. However, it is unknown whether MIT affects these processes in other animals. Here, we investigated the effects of MIT exposure in planaria—non-parasitic freshwater flatworms able to regenerate all tissues after injury. Using a common research strain of Dugesia japonica , we determined that intact planarians exposed to 15 μM MIT displayed both neuromuscular and epithelial-integrity defects. Furthermore, regenerating (head and tail) fragments exposed to 15 μM MIT failed to close wounds or had significantly delayed wound healing. Planarian wounds normally close within 1 h after injury. However, most MIT-exposed animals retained open wounds at 24 h and subsequently died, and those few animals that were able to undergo delayed wound healing without dying exhibited abnormal regeneration. For instance, head regeneration was severely delayed or inhibited, with anterior structures such as eyes failing to form in newly produced tissues. These data suggest that MIT directly affects both wound healing and regeneration in planarians. Next, we investigated the ability of thiol-containing antioxidants to rescue planarian wound closure during MIT exposure. The data reveal both n -acetyl cysteine and glutathione were each able to fully rescue MIT inhibition of wound healing. Lastly, we established MIT toxicity levels by determining the LC 50 of 5 different planarian species: D. japonica, Schmidtea mediterranea , Girardia tigrina, Girardia dorotocephala, and Phagocata gracilis . Our LC 50 data revealed that concentrations as low as 39 μM (4.5 ppm) are lethal to planarians, with concentrations of just 5 μM inhibiting wound healing, and suggest that phylogeny is predictive of species toxicity levels. Together these results indicate MIT may have broad wound healing effects on aquatic species in general and are not limited to X. laevis alone. Future studies should investigate the impact of MIT on wound healing in other organisms, including non-aquatic organisms and mammals. [ABSTRACT FROM AUTHOR]

The robust regenerative capacity of planarian flatworms depends on the orchestration of signaling events from early wounding responses through the stem cell enacted differentiative outcomes that restore appropriate tissue types. Acute signaling events in excitable cells play an important role in determining regenerative polarity, rationalized by the discovery that sub-epidermal muscle cells express critical patterning genes known to control regenerative outcomes. These data imply a dual conductive (neuromuscular signaling) and instructive (anterior-posterior patterning) role for Ca 2 + signaling in planarian regeneration. Here, to facilitate study of acute signaling events in the excitable cell niche, we provide a de novo transcriptome assembly from the planarian Dugesia japonica allowing characterization of the diverse ionotropic portfolio of this model organism. We demonstrate the utility of this resource by proceeding to characterize the individual role of each of the planarian voltage-operated Ca 2 + channels during regeneration, and demonstrate that knockdown of a specific voltage operated Ca 2 + channel ( Ca v 1B ) that impairs muscle function uniquely creates an environment permissive for anteriorization. Provision of the full transcriptomic dataset should facilitate further investigations of molecules within the planarian voltage-gated channel portfolio to explore the role of excitable cell physiology on regenerative outcomes. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. [ABSTRACT FROM AUTHOR]

Placenta specific protein 8 (Plac8) has been well studied in vertebrates, yet little is known in invertebrates. In this study, a novel Plac8 from the planarian Dugesia japonica was identified and its functions in immune responses and development were characterized. Our results show that Djplac8 was expressed in the pharynx, epidermis and intestine of intact adult planarian. The expression of Dj Plac8 increased significantly upon lipopolysaccharide (LPS) challenge, and inhibited the growth of the Gram-negative bacteria ( Escherichia coli and Pseudomonas aeruginosa ), suggesting the role of Plac8 in immune response. Spatial and temporal expression and distribution of DjPlac8 mRNA in regenerated planarians indicates that DjPlac8 was mainly expressed in the pharynx. In situ hybridization also revealed the elevated expression of the DjPlac8 gene in the embryonic pharynx, germ band and parenchyma cells, indicating an important role in embryonic development of D. japonica . When DjPlac8 was deactivated by RNA interference-mediated knockdown, the head of planarians underwent abnormal development. In summary, we identified Dj Plac8 as a novel multifunctional protein that plays essential roles in immune response and development of planarians. [ABSTRACT FROM AUTHOR]

Phospholipid scramblases (PLSCRs) are the conserved calcium-binding, type II transmembrane proteins synthesized in all eukaryotic organisms. In mammals, these proteins play essential roles in various physiological processes, especially in the immune responses. However, the existence of PLSCRs and their biological functions in planarian are still unknown at present. In this study, a new member of PLSCRs was identified in planarian Dugesia japonica ( D. japonica ), named DjPLSCR . The sequence analysis revealed that it contains an opening reading frame consisting of 726 bp encoding a putative protein of 241 amino acids with a predicted molecular mass of ~ 28.7 kDa and an isoelectric point of 6.21. Whole-mount in situ hybridization showed that mRNAs of DjPLSCR are predominantly expressed in adult and regenerative pharynx which is an important organ of immune system in planarians. Importantly, we found that the transcription level of DjPLSCR was significantly upregulated when planarians were stimulated with the pathogen-associated molecular patterns [polyinosinic-polycytidylic acid, lipopolysaccharide, peptidoglycan and β-glucan], suggesting that DjPLSCR is involved in the immune response upon pathogen invasion. Our findings provide the first experimental insights into the characteristics and potential functions of PLSCR in planarians. [ABSTRACT FROM AUTHOR]

Due to technical limitations, there have been minimal studies performed on thermal preferences and thermotactic behaviors of aquatic ectotherm species commonly used in ecotoxicity testing. In this work, we demonstrate an innovative, purpose-built and miniaturized electrothermal array for rapid thermal preference behavioral tests. We applied the novel platform to define thermal preferences in multiple invertebrate and vertebrate species. Specifically, Dugesia notogaea (freshwater planarians), Chironomus tepperi (nonbiting midge larvae), Ostracoda (seed shrimp), Artemia franciscana (brine shrimp), Daphnia carinata (water flea), Austrochiltonia subtenuis (freshwater amphipod), Physa acuta (freshwater snail), Potamopyrgus antipodarum (New Zealand mud snail) and larval stage of Danio rerio (zebrafish) were tested. The Australian freshwater water fleas, amphipods, snail Physa acuta as well as zebrafish exhibited the most consistent preference to cool zones and clear avoidance of zones >27 °C out of nine species tested. Our results indicate the larval stage of zebrafish as the most responsive species highly suitable for prospective development of multidimensional behavioral test batteries. We also showcase preliminary data that environmentally relevant concentrations of pharmaceutical pollutants such as non-steroidal anti-inflammatory drug (NSAID) ibuprofen (9800 ng/L) and insecticide imidacloprid (4600 ng/L) but not anti-depressant venlafaxine (2200 ng/L) and (iv) anticonvulsant medications gabapentin (400 ng/L) can perturb thermal preference behavior of larval zebrafish. Collectively our results demonstrate the utility of simple and inexpensive thermoelectric technology in rapid exploration of thermal preference in diverse species of aquatic animals. We postulate that more broadly such technologies can also have added value in ecotoxicity testing of emerging contaminants. [Display omitted] • Behavior-modifying pharmaceutical micropollutants are an increasing global risk. • Impact of pollutants on thermal preference behaviors is greatly understudied. • Miniaturized Peltier technology allows rapid analysis of thermotactic behaviors. • New technique reveled zebrafish as suitable for thermotactic biotests. • Ibuprofen and imidacloprid perturb zebrafish thermal preference behavior. [ABSTRACT FROM AUTHOR]

Nicotine is one of the most addictive drugs abused by humans. Our laboratory and others have demonstrated that nicotine decreases motility and induces seizure-like behavior in planarians (pSLM, which are vigorous writhing and bending of the body) in a concentration-dependent manner. Nicotine also induces withdrawal-like behaviors in these worms. Cotinine is the major nicotine metabolite in humans, although it is not the final product of nicotine metabolism. Cotinine is mostly inactive in vertebrate nervous systems and is currently being explored as a molecule which possess most of nicotine’s beneficial effects and few of its undesirable ones. It is not known whether cotinine is a product of nicotine metabolism in planarians. We found that cotinine by itself does not seem to elicit any behavioral effects in planarians up to a concentration of 1 mM. We also show that cotinine antagonizes the aforementioned nicotine-induced motility decrease and also decreases the expression of nicotine-induced pSLMs in a concentration-dependent manner. Also cotinine prevents the manifestation of some of the withdrawal-like behaviors induced by nicotine in our experimental organism. Thus, we obtained evidence supporting that cotinine antagonizes nicotine in this planarian species. Possible explanations include competitive binding of both compounds at overlapping binding sites, at different nicotinic receptor subtypes, or maybe allosteric interactions. [ABSTRACT FROM AUTHOR]

Summary A large population of proliferative stem cells (neoblasts) is required for physiological tissue homeostasis and post-injury regeneration in planarians. Recent studies indicate that survival of a few neoblasts after sublethal irradiation results in the clonal expansion of the surviving stem cells and the eventual restoration of tissue homeostasis and regenerative capacity. However, the precise mechanisms regulating the population dynamics of neoblasts remain largely unknown. Here, we uncovered a central role for epidermal growth factor (EGF) signaling during in vivo neoblast expansion mediated by Smed-egfr-3 ( egfr-3 ) and its putative ligand Smed-neuregulin-7 ( nrg-7 ). Furthermore, the EGF receptor-3 protein localizes asymmetrically on the cytoplasmic membrane of neoblasts, and the ratio of asymmetric to symmetric cell divisions decreases significantly in egfr-3 ( RNAi ) worms. Our results not only provide the first molecular evidence of asymmetric stem cell divisions in planarians, but also demonstrate that EGF signaling likely functions as an essential regulator of neoblast clonal expansion. [ABSTRACT FROM AUTHOR]

Summary Regeneration starts with injury. Yet how injuries affect gene expression in different cell types and how distinct injuries differ in gene expression remain unclear. We defined the transcriptomes of major cell types of planarians—flatworms that regenerate from nearly any injury—and identified 1,214 tissue-specific markers across 13 cell types. RNA sequencing on 619 single cells revealed that wound-induced genes were expressed either in nearly all cell types or specifically in one of three cell types (stem cells, muscle, or epidermis). Time course experiments following different injuries indicated that a generic wound response is activated with any injury regardless of the regenerative outcome. Only one gene, notum , was differentially expressed early between anterior- and posterior-facing wounds. Injury-specific transcriptional responses emerged 30 hr after injury, involving context-dependent patterning and stem-cell-specialization genes. The regenerative requirement of every injury is different; however, our work demonstrates that all injuries start with a common transcriptional response. [ABSTRACT FROM AUTHOR]

Camu-camu ( Myrciaria dubia (Kunth) Mc. Vaugh (Myrtaceae)) has shown potential for food and human health applications due to its rich bioactive functional properties linked to high antioxidant activity. The main objective of this study was to identify antioxidant-linked bioactive phenolic profiles in spray-dried and freeze dried camu-camu pulp and to evaluate potential functionality associated with anti-hyperglycemia and anti-hypertension properties using in vitro enzyme assay models. Further, antimicrobial properties and planarian-based cellular protection and regeneration were also investigated. Phenolic compounds, such as ellagitannins, ellagic acid, quercetin glycosides, syringic acid and myricetin were detected in camu-camu by LC–TOF-MS. Freeze dried camu-camu powder showed superior phenolic-linked antimicrobial properties and higher hyperglycemia relevant enzyme inhibitory activity compared to the spray dried samples. Besides that, freeze-dried powders and spray-dried (6% gum arabic at 120 °C) powders were more effective against prokaryotic Staphylococcus aureus and showed higher inhibition than ampicillin. Cellular regeneration of eukaryotic planarian model was stimulated with camu-camu powders when compared to control and showed potential of camu camu for redox-linked cellular protection and rejuvenation. Overall camu-camu rich in phenolic bioactive profiles showed superior antidiabetic and antimicrobial properties and has potential as part of dietary strategies in the management of early stages of type 2 diabetes and associated complications. [ABSTRACT FROM AUTHOR]

The SPSB family is comprised of four highly conserved proteins, each containing a C-terminal SOCS box motif and a central SPRY domain. Presently, Spsb genes have been found in mammals and in a few invertebrates, however, the specific functions of these genes are still unknown. In this study, we identified a Spsb gene from the planarian Dugesia japonica and termed it DjSpsb . The temporal and spatial expression patterns of DjSpsb were examined in both intact and regenerative animals, and expression levels were also quantified in response to various stressors. The results show that (1) DjSpsb is highly conserved in evolutionary history in metazoans and is at closer relationship to Spsb1 , Spsb2 and Spsb4 ; (2) DjSpsb mRNA is mainly expressed in the head and also throughout head regeneration processes, particularly, its expression up-regulated observably on day 5 after amputation; (3) DjSpsb is also expressed in the testes and yolk glands; (4) DjSpsb expression is induced by high temperature and ethanol but inhibited by high doses of ionic liquids. The date suggests that the DjSpsb gene might be active in central nervous system (CNS) formation and functional recovery during head regeneration, and it is also involved in the development of germ cells and stress responses in the planarians. [ABSTRACT FROM AUTHOR]

Advances in stem cell biology have led to the derivation of diverse cell types, yet challenges remain in creating complex tissues and functional organs. Unlike humans, some animals regenerate all missing tissues and organs successfully after dramatic injuries. Studies of organisms with exceptional regenerative capacity, like planarians, could complement in vitro studies and reveal mechanistic themes underlying regeneration on the scale of whole organs and tissues. In this review, we outline progress in understanding planarian organ regeneration, with focus on recent studies of the nervous, digestive, and excretory systems. We further examine molecular mechanisms underlying establishment of diverse cell fates from the planarian stem cell pool. Finally, we explore conceptual directions for future studies of organ regeneration in planarians. [ABSTRACT FROM AUTHOR]

The purpose of the present study was to determine the effects of exposure to cocaine and mephedrone on conditioned place preference (CPP) and locomotion in the flatworm planaria. Planaria were treated with either cocaine or mephedrone at 1 or 10 μM. Planaria were exposed to 15 min of drug in their non-preferred place (either a rough- or smooth-floored petri dish) on alternate days, and were exposed to normal water in their preferred place on the following day. There were 5 days of conditioning to drug. Planaria were then tested for CPP on day 2, 6 and 13 after withdrawal. We found that animals exhibited CPP to cocaine at both 1 and 10 μM, but not to mephedrone. When examining locomotor activity we found that neither cocaine nor mephedrone treatment showed any evidence of evoking increased motility or locomotor sensitisation. Hypo-motility was seen on the first day of conditioning at concentrations of 10 μM for both cocaine and mephedrone, but had disappeared by the last day of conditioning. Examining chronic withdrawal, only 10 μM mephedrone had a significant effect on motility, decreasing locomotion on day 2 of withdrawal. Taken together we have shown that cocaine evoked CPP in planaria. We have also shown withdrawal depressing effects of mephedrone on motility. [ABSTRACT FROM AUTHOR]

Using specific neurobehaviors as endpoints, previous studies suggested that planarian neurotransmission systems could be targets of Cd neurotoxicity. However, direct evidence for disturbed neurotransmission systems by Cd in treated planarians is still lacking. In planarians, dopamine (DA) and serotonin (5-HT) play critical roles in neuromuscular function, but little is known about their metabolic degradation. Therefore, in this study, we attempted to determine the appearances of DA, 5-HT, and their metabolic products in the freshwater planarian Dugesia japonica , characterize the activity of enzymes involved in their metabolism, and investigate the effects of Cd on planarian 5-HTergic and DAergic neurotransmission systems. Only DA, 5-HT, and 5-hydroxyindole-3-acetic acid (5-HIAA) were found in planarian tissues. Further enzymatic study revealed the activity of planarian monoamine oxidase (MAO) but not catechol-O-methyl transferase (COMT). These findings suggest that planarian MAO catalyzes the metabolism of 5-HT into 5-HIAA. However, DA metabolites from the MAO-involved metabolic pathway were not found, which might be due to a lack of COMT activity. Finally, in Cd-treated planarians, tissue levels of 5-HT and DA were decreased and MAO activity altered, suggesting that planarian neurotransmission systems are disturbed following Cd treatment. [ABSTRACT FROM AUTHOR]

Prenatal exposure to ethanol affects neurodevelopmental processes, leading to a variety of physical and cognitive impairments collectively termed Fetal Alcohol Spectrum Disorders (FASD). The molecular level ethanol-induced alterations that underlie FASD are poorly understood and are difficult to study in mammals. Ethanol exposure has been shown to affect regulation and differentiation of embryonic stem cells in vitro , suggesting that in vivo effects such as FASD could arise from similar alterations of stem cells. In this study, we hypothesize that ethanol exposure affects head regeneration and neuroregeneration in the Schmidtea mediterranea planarian. S. mediterranea freshwater flatworms have remarkable regenerative abilities arising from an abundant population of pluripotent adult somatic stem cells known as neoblasts. Here, we evaluated the mobility-normalized photophobic behavior of ethanol-exposed planaria as an indicator of cognitive function in intact and head-regenerating worms. Our studies show that exposure to 1% ethanol induces a delay in the reacquisition of behavior during head regeneration that cannot be attributed to the effect of ethanol on intact worms. This suggests that the S. mediterranea planarian could provide insight into conserved neurodevelopmental processes that are affected by ethanol and that lead to FASD in humans. [ABSTRACT FROM AUTHOR]

More than 90% of individuals who use cocaine also report concurrent ethanol use, but only a few studies, all conducted with vertebrates, have investigated pharmacodynamic interactions between ethanol and cocaine. Planaria, a type of flatworm often considered to have the simplest 'brain,' is an invertebrate species especially amenable to the quantification of drug-induced behavioral responses and identification of conserved responses. Here, we investigated stereotypical and environmental place conditioning (EPC) effects of ethanol administered alone and in combination with cocaine. Planarians displayed concentration-related increases in C-shaped movements following exposure to ethanol (0.01-1%) (maximal effect: 9.9±1.1 C-shapes/5 min at 0.5%) or cocaine (0.1-5 mM) (maximal effect: 42.8±4.1 C-shapes/5 min at 5 mM). For combined administration, cocaine (0.1-5 mM) was tested with submaximal ethanol concentrations (0.01, 0.1%); the observed effect for the combination was enhanced compared to its predicted effect, indicating synergism for the interaction. The synergy with ethanol was specific for cocaine, as related experiments revealed that combinations of ethanol and nicotine did not result in synergy. For EPC experiments, ethanol (0.0001-1%) concentration-dependently increased EPC, with significant environmental shifts detected at 0.01 and 1%. Cocaine (0.001-1 μM) produced an inverted U-shaped concentration-effect curve, with a significant environmental shift observed at 0.01 μM. For combined exposure, variable cocaine concentrations (0.001-1 μM) were administered with a statistically ineffective concentration of ethanol (0.0001%). For each concentration of cocaine, the environmental shift was enhanced by ethanol, with significance detected at 1 μM. Cocaethylene, a metabolite of cocaine and ethanol, also produced C-shapes and EPC. Lidocaine (0.001-10 μM), an anesthetic and analog of cocaine, did not produce EPC or C-shaped movements. Evidence from planarians that ethanol produces place-conditioning effects and motor dysfunction, and interacts synergistically with cocaine, suggests that aspects of ethanol neuropharmacology are conserved across species. [ABSTRACT FROM AUTHOR]

Here we review the examples of great longevity and potential immortality in the earliest animal types and contrast and compare these to humans and other higher animals. We start by discussing aging in single-celled organisms such as yeast and ciliates, and the idea of the immortal cell clone. Then we describe how these cell clones could become organized into colonies of different cell types that lead to multicellular animal life. We survey aging and longevity in all of the basal metazoan groups including ctenophores (comb jellies), sponges, placozoans, cnidarians (hydras, jellyfish, corals and sea anemones) and myxozoans. Then we move to the simplest bilaterian animals (with a head, three body cell layers, and bilateral symmetry), the two phyla of flatworms. A key determinant of longevity and immortality in most of these simple animals is the large numbers of pluripotent stem cells that underlie the remarkable abilities of these animals to regenerate and rejuvenate themselves. Finally, we discuss briefly the evolution of the higher bilaterians and how longevity was reduced and immortality lost due to attainment of greater body complexity and cell cycle strategies that protect these complex organisms from developing tumors. We also briefly consider how the evolution of multiple aging-related mechanisms/pathways hinders our ability to understand and modify the aging process in higher organisms. [ABSTRACT FROM AUTHOR]

Abstract: Recent data suggest that opioid receptors are involved in the development of nicotine physical dependence in mammals. Evidence in support of a similar involvement in an invertebrate (Planaria) is presented using the selective opioid receptor antagonist naloxone, and the more receptor subtype-selective antagonists CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) (μ, MOR), naltrindole (δ, DOR), and nor-BNI (norbinaltorphimine) (κ, KOR). Induction of physical dependence was achieved by 60-min pre-exposure of planarians to nicotine and was quantified by abstinence-induced withdrawal (reduction in spontaneous locomotor activity). Known MOR and DOR subtype-selective opioid receptor antagonists attenuated the withdrawal, as did the non-selective antagonist naloxone, but a KOR subtype-selective antagonist did not. An involvement of MOR and DOR, but not KOR, in the development of nicotine physical dependence or in abstinence-induced withdrawal was thus demonstrated in a sensitive and facile invertebrate model. [Copyright &y& Elsevier]

Planaria are known for their ability to completely regenerate upon fissioning or experimental amputation. Yet, metabolic costs of regeneration have not been directly measured in planaria. Our goal was to establish the relationships between oxygen consumption (V̇O2), regeneration, and reproductive mode for asexual and sexual strains of Schmidtea mediterranea. We hypothesized that V̇O 2 would vary by regeneration day for both sexual and asexual S. mediterranea , reflecting different costs of tissue reconstruction, but with an additional cost for regenerating sexual organs. Testes regeneration and body mass, as indicators of regeneration progress, and routine mass-specific V̇O 2 as a function of maturity, regeneration, and reproductive mode, were measured over a 22-day regeneration period. Testes growth was highest in sexually mature adults, ~1/2 that in 14-day post-amputation sexual adults, and not detectable in juveniles and hatchlings. Mass-specific routine V̇O 2 in sexuals was highest in mature controls at ~23 μl O 2 /g/h, but only half that in juveniles, hatchlings, and 14 day post-amputation adults. Both intact and 14-day post-amputation asexuals had a mass-specific routine V̇O 2 of ~10–12 μl O 2 /g/h. The sum of V̇O 2 of all amputated sections was ~100% higher than pre-amputation levels in the first 6 days of regeneration in asexuals, but not sexuals. There was no significant difference in V̇O 2 of head, middle, and tail sections during regeneration. Overall, the highest metabolic costs associated with regeneration occurred during the initial 1–6 days of regeneration in both strains, but regeneration costs for sexual structures were not reflected in major V̇O 2 differences between sexual and asexual strains. [Display omitted] • Little is known of metabolic costs of regeneration in sexual or asexual planaria. • Oxygen consumption of intact planaria and regenerating pieces was measured. • The highest metabolic costs appear in the first 6 days of regeneration. • Metabolic costs are higher in sexually mature planaria with testes. [ABSTRACT FROM AUTHOR]

Highlights: [•] Decapitated planarians lose most of their behavioral responses to cocaine exposure. [•] Decapitated planarians fully respond to nicotine exposure. [•] Regenerating planarians recover their cocaine sensitivity. [ABSTRACT FROM AUTHOR]

Abstract: Sucrose produces physical dependence and reinforcing effects in rats. We hypothesized that similar effects could be demonstrated in planarians, the earliest animal with a centralized nervous system. We used two assays, one that quantifies withdrawal responses during drug absence as a reduction in motility and another that quantifies reinforcing effects using a conditioned place preference (CPP) design. In withdrawal experiments, planarians exposed to sucrose (1%) for 60min and then tested in water for 5min displayed reduced motility compared to water controls. Acute or continuous sucrose (1%) exposure did not affect motility. CPP experiments used a biased design to capitalize upon planarians' natural preference for the dark (pretest, sucrose conditioning in the light, posttest). Planarians conditioned with sucrose (1%) displayed a greater preference shift than sucrose-naïve planarians. Glucose (0.1, 1%), but not the non-digestible disaccharide lactulose (0.1, 1%), also produced a greater preference shift than water-exposed planarians. Development of sucrose-induced CPP was inhibited when sucrose (1%) conditioning was conducted in combination with dopamine receptor antagonists SCH 23390 (1μM) or sulpiride (1μM). These results suggest that the rewarding and reinforcing effects of sugar are highly conserved across species and that planarians offer an invertebrate model to provide insight into the pharmacological effects of sucrose and related sweeteners. [Copyright &y& Elsevier]

Summary: Planarians grow and regenerate organs by coordinating proliferation and differentiation of pluripotent stem cells with remodeling of postmitotic tissues. Understanding how these processes are orchestrated requires characterizing cell-type-specific gene expression programs and their regulation during regeneration and homeostasis. To this end, we analyzed the expression profile of planarian intestinal phagocytes, cells responsible for digestion and nutrient storage/distribution. Utilizing RNA interference, we identified cytoskeletal regulators required for intestinal branching morphogenesis and a modulator of bioactive sphingolipid metabolism, ceramide synthase, required for the production of functional phagocytes. Additionally, we found that a gut-enriched homeobox transcription factor, nkx-2.2, is required for somatic stem cell proliferation, suggesting a niche-like role for phagocytes. Identification of evolutionarily conserved regulators of intestinal branching, differentiation, and stem cell dynamics demonstrates the utility of the planarian digestive system as a model for elucidating the mechanisms controlling postembryonic organogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Harmine is a β-carboline compound that targets glutamatergic, monoaminergic, and GABAergic pathways underlying drug addiction. We compared the efficacy of harmine against different psychoactive drugs using an invertebrate (planarian) assay designed to quantify ‘C-shape’ responses. Harmine itself (0.01–10μM) did not produce C-shapes. However, when applied over the same concentration range, harmine significantly inhibited C-shapes elicited by cocaine, with a concentration of 0.1μM producing almost 90% inhibition. Consistent with its putative actions, harmine produced a similar, though less efficacious, inhibition of C-shapes elicited by the substituted amphetamines methamphetamine and mephedrone (4-methylmethcathinone) but was much less effective against nicotine. When tested in the presence of the glutamate transporter inhibitor dihydrokainate (DHK) (0.1, 1μM), harmine (0.1μM) efficacy against cocaine-induced C-shapes was significantly reduced. Harmine also attenuated C-shapes elicited by N-methyl-d-aspartate (NMDA) and by glutamate itself. The present data suggest that harmine displays preferential efficacy against different addictive substances (cocaine>amphetamines>nicotine) and, at least for cocaine, is dependent on the glutamate system. [Copyright &y& Elsevier]

Abstract: The presence of adult pluripotent stem cells and the amazing regenerative capabilities make planarian flatworms an extraordinary experimental model to assess in vivo the effects of substances of both natural and synthetic origin on stem cell dynamics. This study focuses on the effects of chelidonine, an alkaloid obtained from Chelidonium majus. The expression levels of molecular markers specific for stem or differentiated cells were compared in chelidonine-treated and control planarians. The use of these markers demonstrates that chelidonine produces in vivo a significant anti-proliferative effect on planarian stem cells in a dose-dependent fashion. In response to chelidonine treatment mitotic abnormalities were also observed and the number of cells able to proceed to anaphase/telophase appeared significantly reduced with respect to the controls. Our results support the possibility that chelidonine acts on cell cycle progression by inhibition of tubulin polymerization. These studies provide a basis for preclinical evaluation in vivo of the effects of chelidonine on physiologically proliferating stem cells. [Copyright &y& Elsevier]

Abstract: The early embryo is often a two-dimensional surface. The fate map is the subdivision of this surface into regions which give rise to parts of the phenotype. It is shown for Drosophila that the fate map is generated by the spontaneous and sequential formation of Turing-Child (TC) eigenfunction patterns. These patterns are recorded by the maternal-effect genes. The addition of the nodal lines of the TC patterns yields the correct number, positions, sequences and symmetries of regional boundaries. A simplest nontrivial ‘homeotic transformation’ is suggested and explained. A single mutation converts a region in one end of the fate map to a mirror-symmetric image of a nonadjacent region in the other end of the fate map, and this is attributed to the geometry of the TC patterns. This geometry also determines the initial shape of the zygotic gene expression. The vision of William Bateson that biological form is shaped like Chladni''s patterns in acoustics and music is justified. A similar sequence of TC patterns occurs in the normal development of all organisms, and it is suggested that artificial intervention which completes the full sequence of TC patterns can be useful in the context of regenerative medicine and this is illustrated with the sea urchin. [Copyright &y& Elsevier]

Abstract: We recently reported that the natural cyclic lactone, parthenolide, and related analogs prevent the expression of behavioral effects induced by cocaine in planarians and that parthenolide''s γ-lactone ring is required for this effect. In the present work, we tested a series of alkyl γ-lactones with varying chain length (1–8 carbons) to determine their ability to antagonize the planarian motility decrease induced by 200μM cocaine. Alkyl lactones with up to a 4-carbon alkyl chain did not affect planarian motility or antagonized the cocaine-induced motility decrease; only the compound γ-nonalactone (a γ-lactone with a 5-carbon chain) was able to prevent the cocaine-induced behavioral patterns, while alkyl lactones with longer carbon chains failed to prevent the cocaine-induced effects. Thus, we conclude that the optimal structural features of this family of compounds to antagonize cocaine''s effect in this experimental system is a γ-lactone ring with at a 5-carbon long functional group. [Copyright &y& Elsevier]

Abstract: Background: Nicotine is one of the world''s most addictive substances and the primary reason that humans inhale tobacco smoke. The pharmacological effects of nicotine can be investigated in planarians, aquatic flatworms that possess an integrated neural network including cephalic ganglia that some consider the earliest ‘brain’ and spinal cord. Here, we tested the hypothesis that nicotine exposure elicits mammalian-like behaviors in planarians. Methods: Planarian motility and stereotypy (C-shape hyperkinesias) were quantified following acute nicotine exposure. During repeated nicotine exposure, we investigated the presence of withdrawal, tolerance, behavioral sensitization, and environmental place conditioning. Results: Acute nicotine exposure increased stereotypical activity and elicited biphasic effects on motility. A low concentration (0.01mM) increased motility whereas higher concentrations (0.3–10mM) elicited the opposite effect. Planarians exposed to nicotine (0.03mM) for 60min and then tested in water displayed reduced motility that was not observed during exposure to water, acute nicotine, or continuous nicotine. Nicotine-treated planarians withdrawn from the drug for 3 days before being challenged with nicotine displayed behavioral sensitization at low concentrations (0.1, 0.3mM) but tolerance at higher concentrations (1, 3mM). Planarians conditioned with nicotine in the ambient light (non-preferred environment) displayed a reduction in their natural preference for a dark environment. Conclusions: The present results suggest nicotine elicits mammalian-like effects in planarians, including decreased motility and increased stereotypy following acute administration and abstinence-induced withdrawal, behavioral sensitization, tolerance, and place conditioning during repeated exposure. [Copyright &y& Elsevier]

Abstract: The therapeutic effects elicited by photobiostimulation in the near infrared range may be associated with increased proliferation rate of particular cell-types. The present study utilized commercial light emitting diodes to investigate the effects of low-level near-infrared radiation on the proliferation rate of stem cells in amputated planarian. Whole and amputated animals were exposed to either ambient diurnal lighting, darkness, white light, red light, or near-infrared (880nm) light. Irradiation was consistent for the duration of the experiments and was provided using commercial 5mm light emitting diodes (∼1.0mW/m2 in power density and ∼0.01J/cm2 in radiant exposure). Compared to other groups amputated planarian exposed to near-infrared displayed increased mobility by the 3rd day of exposure (F (4,26) =4.31, p <0.04, η 2 =41%). Higher densities of stem cells were measured in these worms 84h post injury (F (4,72) =4.78, p <0.01, η 2 =21%). These findings suggest that non-coherent light sources with power-densities about 1000 times lower than contemporary low-power laser settings remain effective in generating photobiostimulation effects and warrants further investigation on stem-cell proliferation induced by near-infrared light emitting diodes. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Planaria present a unique model organism for studying primitive central nervous systems. The major mammalian excitatory neurotransmitters, glutamate and aspartate, have previously been measured in planaria via high pressure liquid chromatography (HPLC). A faster extraction and analysis procedure using capillary electrophoresis (CE) was developed which confirms the presence of these amino acids in single planaria homogenates. Method: Following homogenization and centrifugation of individual planaria in hydrochloric acid/acetonitrile, glutamate and aspartate were derivatized with naphthalene-2, 3-dicarboxaldehyde (NDA). The labeled amino acids were measured using capillary electrophoresis with laser-induced fluorescence (CE-LIF). Results: CE-LIF electropherograms were generated in less than 1min. The mean±S.D. amounts of glutamate and aspartate were 1200±500 and 1900±700pmol/mg-planarian (n =22), respectively. Spiked average recoveries of glutamate and aspartate were 96% and 91%, respectively. Discussion: The high-throughput method provides the ability to quantitate changes in excitatory neurotransmitters under developmental or stimulatory conditions. The capability to monitor multiple neurotransmitter levels offers the opportunity to correlate behavioral responses with biochemical changes in planaria. [ABSTRACT FROM AUTHOR]

Abstract: Antibiotics containing a β-lactam ring (e.g. ceftriaxone) display anti-glutamate effects that underlie their efficacy in animal models of central nervous system (CNS) diseases [Rothstein JD, Patel S, Regan MR, Haenggeli C, Huang YH, Bergles DE, Jin L, Dykes Hoberg M, Vidensky S, Chung DS, Toan SV, Bruijn LI, Su ZZ, Gupta P, Fisher PB (2005) Nature 433:73–77]. We hypothesized that the structurally related β-lactamase inhibitors (clavulanic acid, tazobactam)—which also contain a β-lactam ring—will mimic ceftriaxone efficacy in an invertebrate (planarian) assay designed to screen for anti-seizure activity [Rawls SM, Thomas T, Adeola M, Patil T, Raymondi N, Poles A, Loo M, Raffa RB (2009) Pharmacol Biochem Behav 93:363–367]. Glutamate or cocaine administration produced planarian seizure-like activity (pSLA). Glutamate- or cocaine-induced pSLA was inhibited by ceftriaxone, clavulanic acid, or tazobactam, but not by the non-β-lactam antibiotic vancomyocin. The present findings indicate β-lactamase inhibitors display efficacy, and mimic ceftriaxone activity, in an invertebrate anti-seizure screen. These results suggest β-lactamase inhibitors—particularly ones such as clavulanic acid that display enhanced brain penetrability, oral bioavailability, and negligible anti-bacterial activity—might offer an attractive alternative to direct antibiotic therapy for managing CNS diseases caused by increased glutamate transmission and provide a solution to the growing concern that ceftriaxone will be of only limited utility as a CNS-active therapeutic because of its intolerable side effects. [ABSTRACT FROM AUTHOR]

Abstract: Using an adaptation of published behavioral protocols, we determined that acute exposure to the cholinergic compounds nicotine and carbamylcholine decreased planarian motility in a concentration-dependent manner. A tobacco cembranoid (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R-cembranoid), also decreased planarian motility. Experiments in the presence of 1 µM 4R-cembranoid did increase the IC50 for nicotine- but not carbamylcholine-induced decrease in planarian motility. When planarians were exposed for 24 h to either nicotine or carbamylcholine at concentrations near their respective IC50 values and then transferred to plain media, nicotine-exposed, but not carbamylcholine- or cembranoid-exposed worms displayed withdrawal-like distress behaviors. In experiments where planarians were pre-exposed to 100 µM nicotine for 24 h in the presence of 1 µM 4R-cembranoid, the withdrawal-like effects were significantly reduced. These results indicate that the 4R-cembranoid might have valuable applications for tobacco abuse research. This experimental approach using planarians is useful for the initial screening of compounds relevant to drug abuse and dependence. [Copyright &y& Elsevier]

In the past decade, the planarian has become an increasingly tractable invertebrate model for the investigation of regeneration and stem cell biology. Application of a variety of techniques and development of genomic reagents in this system have enabled exploration of the molecular mechanisms by which pluripotent somatic stem cells called neoblasts replenish, repair, and regenerate planarian tissues and organs. Recent investigations have implicated evolutionarily conserved signaling pathways in the re-establishment of anterior–posterior (A–P), dorsal–ventral (D–V), and medial–lateral (M–L) polarity after injury. These studies have significantly advanced our understanding of early events during planarian regeneration and have raised new questions about the mechanisms of stem cell-based tissue repair and renewal. [Copyright &y& Elsevier]

Abstract: In order to simplify the design and implementation of a stereo vision system, catadioptric instruments have been used to capture stereo images with a single camera. These catadioptric stereo systems not only provide radiometric advantages over traditional two-camera stereo, but also reduce the complexity and cost of acquiring stereoscopic video. Although much research has been done on the design of the catadioptric stereo system, little attention has been paid to analyze the planar catadioptric stereo (PCS) system based on the epipolar geometry. In this paper, we investigated characteristics of a selected PCS system and proved that it can be approximated by affine epipolar geometry. This affine model reduces the number of parameters in the fundamental matrix from seven in the conventional stereo system to only four in the PCS system. Experimental results verify that estimation of the fundamental matrix for a PCS system can be more robust, precise, and much easier to implement with the affine model. Furthermore, rectification of the image pair based on the affine fundamental matrix can achieve better performance with much less geometric distortion. These significant advantages confirm the usefulness of an affine fundamental matrix model for the selected PCS system. [Copyright &y& Elsevier]

Complex anatomical form is regulated in part by endogenous physiological communication between cells; however, the dynamics by which gap junctional (GJ) states across tissues regulate morphology are still poorly understood. We employed a biophysical modeling approach combining different signaling molecules (morphogens) to qualitatively describe the anteroposterior and lateral morphology changes in model multicellular systems due to intercellular GJ blockade. The model is based on two assumptions for blocking-induced patterning: (i) the local concentrations of two small antagonistic morphogens diffusing through the GJs along the axial direction, together with that of an independent , uncoupled morphogen concentration along an orthogonal direction, constitute the instructive patterns that modulate the morphological outcomes, and (ii) the addition of an external agent partially blocks the intercellular GJs between neighboring cells and modifies thus the establishment of these patterns. As an illustrative example, we study how the different connectivity and morphogen patterns obtained in presence of a GJ blocker can give rise to novel head morphologies in regenerating planaria. We note that the ability of GJs to regulate the permeability of morphogens post-translationally suggests a mechanism by which different anatomies can be produced from the same genome without the modification of gene-regulatory networks. Conceptually, our model biosystem constitutes a reaction-diffusion information processing mechanism that allows reprogramming of biological morphologies through the external manipulation of the intercellular GJs and the resulting changes in instructive biochemical signals. • Intercellular gap junctions (GJs) regulate the permeability of signaling molecules. • We describe the effects of GJ blocking on instructive morphological patterns. • Our biosystem is a reaction-diffusion processor that allows reprogramming of morphologies. • We provide new biophysical insights that are testable in the lab, e.g. with planaria. [ABSTRACT FROM AUTHOR]

Abstract: Freshwater planarians are an emerging model in which to study regeneration at the molecular level. These animals can regenerate a complete central nervous system (CNS) in only a few days. In recent years, hundreds of genes expressed in the nervous system have been identified in two popular planarian species used by several laboratories: Dugesia japonica and Schmidtea mediterranea. Functional analyses of some of those neural genes have allowed the process of CNS regeneration to begin to be elucidated in those animals. However, additional work is required to characterize the different neuronal populations. Thus, the identification or generation of antibodies that act as markers for specific neuronal cell types would be extremely useful not only in obtaining a more detailed characterization of the planarian nervous system but also for the analysis of phenotypes obtained by RNA interference. Here, I have used five different antibodies to describe different neuronal populations in the freshwater planarian S. mediterranea. This study represents a first step in characterizing the organization of the nervous system of this species at the cellular level. [Copyright &y& Elsevier]

Planarian neoblasts are somatic stem cells that have the potential to be used in genotoxicity assays due to their proliferative nature, sensitivity to genotoxic agents, and experimental accessibility. Two freshwater planarian species, Girardia tigrina and Girardia schubarti, were used to develop a neoblast-based micronucleus (MN) assay to assess genotoxicity. Intact or regenerating planarians were exposed to γ-rays, methyl methanesulphonate (MMS), or cyclophosphamide (CP), and neoblast MN frequency was measured. Exposure to the clastogens had no detectable effect on the MN frequency of intact planarian neoblasts. However, for regenerating individuals, active neoblast proliferation was induced by decapitation, and γ-ray doses as low as 0.5Gy, and MMS and CP concentrations as low as 0.8μM and 100mM, respectively, induced a significant increase in MN frequency. Exposure to higher doses of γ-rays consistently resulted in detectable increases in MN frequency. For MMS and CP, concentrations of up to 1.6μM and 200mM, respectively, resulted in significant increases in MN frequency, but exposures to higher concentrations led to a decrease to non-significant levels, possibly due to cytotoxic effects of MMS and CP. After completion of regeneration, the MN frequencies returned to those of non-exposed controls, indicating that the neoblast MN assay for regenerating G. tigrina or G. schubarti reflects chromosomal damage caused by acute exposure to clastogenic agents. Upon standardization, this assay may represent an interesting alternative that allows damage caused to freshwater organisms by potentially genotoxic environmental pollutants to be monitored. [Copyright &y& Elsevier]

Abstract: A two-point set is a subset of the plane which meets every planar line in exactly two-points. We discuss the problem “What are the topological symmetries of a two-point set?”. Our main results assert the existence of two-point sets which are rigid and the existence of two-point sets which are invariant under the action of certain autohomeomorphism groups. We pay particular attention to the isometry group of a two-point set, and show that such groups consist only of rotations and that they may be chosen to be any subgroup of having size less than . We also construct a subgroup of having size that is contained in the isometry group of a two-point set. [Copyright &y& Elsevier]

Abstract: Planarians display a concentration-related reduction in locomotor activity when amphetamine, cocaine, cannabinoid, or benzodiazepine exposure is abruptly discontinued. In the present study, we tested the hypothesis that abrupt discontinuation of methamphetamine would also cause withdrawal-like behavior in planarians and that the withdrawal-like behavior would be prevented by nociceptin, which has been shown to modulate the effects of methamphetamine in mammals. We observed a concentration-related reduction of locomotor behavior when planarians exposed to methamphetamine (0.1–100μM) were tested in drug-free water. The withdrawal-like behavior was abolished when methamphetamine (10μM)-exposed planarians were placed into water containing nociceptin (10μM) or when planarians co-exposed to methamphetamine (10μM) and nociceptin (10μM) were placed into drug-free water. The effects of nociceptin were abolished in the presence of a nociceptin receptor antagonist, JTC-801 (1μM). Planarians did not display a change in locomotor behavior during exposure to nociceptin (10μM) or JTC-801 (1μM) by themselves. These results (1) reveal a functional interaction between nociceptin and methamphetamine in planarians and (2) provide evidence that nociceptin blocks methamphetamine-induced withdrawal-like behavior in planarians through a JTC-801-sensitive mechanism. [Copyright &y& Elsevier]

Abstract: Ceftriaxone (a β-lactam antibiotic) has recently been identified as having the rare ability to increase the expression and functional activity of the glutamate transporter subtype 1 (GLT-1) in rat spinal cord cultures. GLT-1 has been implicated in diverse neurological disorders and in opioid dependence and withdrawal. It has been speculated that it might also be involved in the physical dependence and withdrawal of other abused drugs, but demonstration of this property can be difficult in mammalian models. Here, we demonstrate for the first time using a planarian model that ceftriaxone attenuates both the development of physical dependence and abstinence-induced withdrawal from cocaine, amphetamine, methamphetamine, and a benzodiazepine (clorazepate) in a concentration-related manner. These results suggest that physical dependence and withdrawal from several drugs involve a common – β-lactam-sensitive – mechanism in planarians. If these findings can be shown to extend to mammals, β-lactam antibiotics might represent a novel pharmacotherapy or adjunct approach for treating drug abuse or serve as a template for drug discovery efforts aimed at treating drug abuse, recovery from drug abuse, or ameliorating the withdrawal from chronic use of therapeutic medications. [Copyright &y& Elsevier]

Abstract: Here we report the prevention and reversal of cocaine-induced behaviors in planarian worms by parthenolide and two related cyclic sesquiterpene lactones (SL), costunolide and santonin. Using established protocols, we studied two cocaine-induced behavioral effects in planaria; the induction of motility decrease and the induction of C-like hyperkinesia. Cocaine, parthenolide, costunolide, santonin, and a lactone-less cyclic sesquiterpene, β-eudesmol, decreased planarian motility in a concentration-dependent manner. Only cocaine induced C-like hyperkinesia. At concentrations that did not show any motility decrease, partenolide, costunolide and santonin, but not β-eudesmol, significantly reduced the cocaine-induced motility decrease and C-like hyperkinesia, in a concentration-dependent manner. Furthermore, partenolide, costunolide and santonin were able to rescue planaria from C-like hyperkinesia, after the worms were exposed to cocaine. Conversely, cocaine at a concentration that did not show any measurable effects (10 µM), was able to alleviate the SL-, but not the β-eudesmol-induced motility decrease. Liquid Chromatography/Mass Spectrometry experiments demonstrated that cocaine does not interact directly with any of the cyclic sesquiterpenoids, which suggests specific biochemical targets for these compounds in planarians. Our data suggests a common binding site for cocaine and the sesquiterpene lactones in planarians. [Copyright &y& Elsevier]

Abstract: Infrared spectra (4000–50cm−1) have been recorded for three isotopomers of aminodifluorophosphine, H2NPF2, H2 15NPF2 and D2NPF2, of the gases. The Raman spectra of all three molecules were recorded of the liquids. To support the vibrational assignment MP2(full) ab initio calculations with the 6-31G(d) basis set were carried out to predict the fundamental vibrational frequencies, infrared intensities, Raman activities, depolarization values and infrared band contours. Additional ab initio calculations employing a variety of basis sets with and without diffuse functions have been used to predict the conformational stability, structural parameters and centrifugal distortion constants. These predictions are compared to previously reported experimental values when available. The adjusted r 0 structural parameters have been obtained by systematically fitting the MP2(full)/6-311+G(d) predicted values with the previously reported rotational constants for five isotopomers obtained from the microwave study. The difference in the two NH distances is 0.003Å which is much smaller than the value of 0.021Å previously reported. The bonding around the nitrogen atom is effectively planar which is consistent with the previously reported structural information from the microwave study but differs from the reported slightly pyramidal bonding obtained in the electron diffraction investigation. The adjusted r 0 heavy atom distances and angles are: r(PF)=1.587(3); r(NP)=1.649(3)Å; ∠FPF=94.7(5); ∠NPF=100.6(5)°. Vibrational assignments are given for H2NPF2, H2 15NPF2 and D2NPF2 which are supported by ab initio MP2/6-31G(d) calculations. These results are compared to the corresponding quantities of some similar molecules. [Copyright &y& Elsevier]

Abstract: The ring puckering in the four-membered 3-chloro-1,3-thiaphosphetane 3-oxide, its 3-sulfide derivative and 1,3-dithietane 1,1-dioxide hetero rings were investigated by DFT and ab initio MP2 calculations using a 6-311+G∗∗ basis set. From the calculations at both levels of theory the asymmetric ring puckering potential energy curves in the two phosphetanes were consistent with an almost single minimum that corresponds to a pseudo-axial configuration (Ps in axial position) with a puckering angle of about 15–20°. In the case of the dithietane dioxide the symmetric ring puckering potential was consistent at the B3LYP level with a flat minimum that corresponds to a planar ring but at the MP2 level with a double minimum with a very low barrier of about 185cal/mol to ring planarity. The potential functions that describe the ring puckering in the three molecules were derived at the B3LYP/6-311+G∗∗ and the MP2/6-311+G∗∗ levels of calculations. The vibrational wavenumbers were calculated and the potential energy distributions PED among the symmetry coordinates of the normal modes were computed for the stable conformers of each of the molecules. Complete vibrational assignments were provided on the basis of the calculated PED values. The experimental infrared and Raman spectra of the two phosphetane molecules were simulated and compared to the calculated ones. [Copyright &y& Elsevier]

Copper is a common environmental contaminant, which is particularly toxic to living organisms when in high concentrations. To monitor environmental contamination by Cu2+ and other heavy metals, well characterized bioindicator organisms and standardized assays are needed. As a first step toward this end, we have analysed Cu2+ effects upon Girardia tigrina freshwater planarians, based on the assessment of mobility, regeneration performance, micronucleus (MN) frequency in regenerating animals, and reproductive performance. These four biomarkers provided complementary information on Cu2+ toxicity, teratogenicity, mutagenicity and chronic (>96h of exposure) effects, respectively. The LC50 was calculated for newborn, adult and regenerating planarians, and values of 12±0.02mgl−1, 42±0.08mgl−1, 48±0.13mgl−1, respectively, were obtained after 96h of exposure. Mobility, for intact adults, and time of regeneration and MN frequency, for regenerating animals, were significantly affected by Cu2+ concentrations as low as 0.10mgl−1. MN assay for regenerating G. tigrina neoblasts showed higher sensitivities than MN assays performed with other bioindicator freshwater organisms, such as moluscs or fish. Chronic exposure effects were clearly evidenced by assessment of reproductive performance, with significant reduction in fecundity and fertility rates upon exposure to Cu2+ concentrations as low as 0.05mgl−1. Therefore, G. tigrina can be regarded as a useful bioindicator species for the detection and evaluation of Cu2+ effects upon freshwater invertebrates, allowing insights on the effects of Cu2+ (and possibly other heavy metals) in a freshwater environment. [Copyright &y& Elsevier]