Same same, but different: exploring the enigmatic role of the pituitary adenylate cyclase-activating polypeptide (PACAP) in invertebrate physiology.

Evidence has been accumulating that elements of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) system are missing in non-chordate genomes, which is at odds with the partial sequence-, immunohistochemical-, and physiological data in the literature. Multilevel experiments were performed on the great pond snail (Lymnaea stagnalis) to explore the role of PACAP in invertebrates. Screening of neuronal transcriptome and genome data did not reveal homologs to the elements of vertebrate PACAP system. Despite this, immunohistochemical investigations with an anti-human PAC1 receptor antibody yielded a positive signal in the neuronal elements in the heart. Although Western blotting of proteins extracted from the nervous system found a relevant band for PACAP-38, immunoprecipitation and mass spectrometric analyses revealed no corresponding peptide fragments. Similarly to the effects reported in vertebrates, PACAP-38 significantly increased cAMP synthesis in the heart and had a positive ionotropic effect on heart preparations. Moreover, it significantly modulated the effects of serotonin and acetylcholine. Homologs to members of Cluster B receptors, which have shared common evolutionary origin with the vertebrate PACAP receptors, PTHRs, and GCGRs, were identified and shown not to be expressed in the heart, which does not support a potential role in the mediation of PACAP-induced effects. Our findings support the notion that the PACAP system emerged after the protostome-deuterostome divergence. Using antibodies against vertebrate proteins is again highlighted to have little/no value in invertebrate studies. The physiological effects of vertebrate PACAP peptides in protostomes, no matter how similar they are to those in vertebrates, should be considered non-specific.

Characterization of corazonin signaling in a molluscan model species, Lymnaea stagnalis.

In recent years, new concepts have emerged regarding the nomenclature, functions, and relationships of different peptide families of the gonadotropin-releasing hormone (GnRH) superfamily. One of the main driving forces for this originated from the emerging evidence that neuropeptides previously called molluscan GnRH are multifunctional and should be classified as corazonin (CRZ). However, research articles still appear that use incorrect nomenclature and attribute the same function to molluscan CRZs as vertebrate GnRHs. The aim of the present study was to further support the recent interpretation of the origin and function of the GnRH superfamily. Towards this goal, we report the characterization of CRZ signaling system in the molluscan model species, the great pond snail (Lymnaea stagnalis). We detected a CRZ-receptor-like sequence (Lym-CRZR) by homology-searching in the Lymnaea transcriptomes and the deduced amino acid sequence showed high sequence similarity to GnRH receptors and CRZ receptors. Molecular phylogenetic tree analysis demonstrated that Lym-CRZR is included in the cluster of molluscan CRZRs. Lym-CRZR transiently transfected into HEK293 cells was found to be localized at the plasma membrane, confirming that it functions as a membrane receptor, like other G protein-coupled receptors. The signaling assays revealed that the previously identified Lym-CRZ neuropeptide stimulated intracellular Ca2+ mobilization in a dose-dependent manner, but not cyclic AMP production, in HEK293 cells transfected with Lym-CRZR. Finally, we demonstrated a wide tissue distribution of Lym-CRZR. These results suggest that Lym-CRZ is a multifunctional peptide and provide further insights into the evolution of the GnRH neuropeptide superfamily. The present study also supports the notion that previously termed molluscan "GnRH" should be classified as "CRZ".

Issues, challenges, directives, and limitations concerning the improvement of environmental risk assessment of pharmaceutically active compounds.

Nowadays, when tons of different chemicals including pharmaceutically active compounds (PhACs) are known to be released into the environment, applying adequate risk assessment relating to the protection of the ecosystem is very important. A broad body of scientific papers expresses a growing demand for improvement of the method(s) for ecological/environmental risk assessment (ERA). Although certain issues about ERA often emerge in the community, most of them cannot be considered as real problems and its methodology was developed keeping several limitations in mind. Nevertheless, the current approaches can be improved in order to better serve the intended purposes. For example, there is a lack of an integrated, manageable ecotoxicological database. It is not uncommon for basic, but extremely important, influencing factors such as time of exposure, interactions between different compounds, and characteristics of different habitats to be ignored. Discussing under the basic regulatory framework used in the EU, this correspondence paper deals with these and other examples to present the current features of ERA, identify gaps in process and application, and propose possible improvements/directives.

Identification, presence, and possible multifunctional regulatory role of invertebrate gonadotropin-releasing hormone/corazonin molecule in the great pond snail (Lymnaea stagnalis).

In the last years, our interpretation of the origin and function of the gonadotropin-releasing hormone (GnRH) neuropeptide superfamily has changed substantially. A main driver for these conceptual changes came from increased investigations into functions and evolutionary lineage of previously identified molluscan GnRH molecules. Emerging evidence suggests not only reproductive, but also diverse biological effects of these molecules and proposes they should most likely be called corazonin (CRZ). Clearly, a more global understanding requires further exploration of species-specific functions and structure of invGnRH/CRZ peptides. Towards this goal, we have identified the full-length cDNA of invGnRH/CRZ peptide in an invertebrate model species, the great pond snail Lymnaea stagnalis, termed ly-GnRH/CRZ, and characterized the transcript and peptide distribution in the central nervous system (CNS) and peripheral organs. Our results are consistent with previous data that molluscan GnRHs are more related to CRZs and serve diverse functions. Hence, our findings support the notion that peptides originally termed molluscan GnRH are multifunctional modulators and that nomenclature change should be taken into consideration.

Investigation of estrogen activity in the raw and treated waters of riverbank infiltration using a yeast estrogen screen and chemical analysis.

Exposure to various endocrine disrupting chemicals (EDCs) can lead to adverse effects on reproductive physiology and behavior in both animals and humans. An adequate strategy for the prevention of environmental contamination and eliminating the effects of them must be established. Chemicals with estrogenic activity were selected, and the effectiveness of their removal during the purification processes in two drinking water treatment plants (DWTPs) using riverbank infiltrated water was determined. Thirty-five water samples in two sampling campaigns throughout different seasons were collected and screened with a yeast estrogen test; furthermore, bisphenol A (BPA), 17ß-estradiol (E2) and ethinyl-estradiol (EE2) content were measured using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Our results confirm that estrogenic compounds are present in sewage effluents and raw surface river water of DWTPs. Very low estrogen activity and pg/L concentrations of BPA and E2 were detected during drinking water processing and occasionally in drinking water. Based on this study, applied riverbank filtration and water treatment procedures do not seem to be suitable for the total removal of estrogenic chemicals. Local contamination could play an important role in increasing the BPA content of the drinking water at the consumer endpoint.

Complex molecular changes induced by chronic progestogens exposure in roach, Rutilus rutilus.

In our previous study, we measured 0.23-13.67ng/L progestogens (progesterone, drospirenone, levonorgestrel) in natural waters in the catchment area of the largest shallow lake of Central Europe, Lake Balaton. Progestogen contaminations act as potent steroids with mixed progestagenic, androgenic and mild estrogenic effects that is why our aim was to investigate the morphological and molecular effects of mixture of progesterone, drospirenone, and levonorgestrel in environmentally relevant (10ng/L) and higher (50 and 500ng/L) exposure concentrations in common roach, Rutilus rutilus. Steroids (e.g. progestogens) and the protein deglycase DJ-1 chaperon molecule aim the same target molecules in cells, therefore, we hypothesized that a relationship may exist between progestogens and DJ-1. Furthermore, our other aim was to follow the changes of signal molecules of different biological function due to progestogen treatment in serum and brain. Adult roaches were exposed to 10, 50 and 500ng/L of mixture of progestogen for 42 days and their somatic indices (brain-somatic, liver-somatic, gonadosomatic and kidney-somatic) were measured. Vitellogenin (VTG) expression (estrogen effect) or inhibition (androgen effect) in fish is a widely used biomarker so we measured its changes in liver by ELISA. To determine the quantity and to map the spatial distribution of DJ-1 chaperon protein the brain and liver tissues were analyzed by ELISA and immunohistochemistry. Furthermore, we also studied molecular alterations: a) in the serum by measuring cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride concentrations and b) in brain homogenate using a cell stress array kit (26 protein). The somatic index of liver and kidney significantly in all the treated groups, whereas the gonadosomatic index of 500ng/L treated group showed significant decrease compared to control animals. VTG level increased significantly in 500ng/L progestogen treated group. Since the concentration of DJ-1 significantly increased in brain and liver in all progestogen treatment groups, the DJ-1 protein could be able to a more sensitive marker than VTG. Serum LDL and cholesterol levels of exposed fish were significantly decreased. DJ-1 was mediated through the stimulation of the expression of LDL-receptor which facilitates reuptake subsequently. In summary, our observations unfolded new data about molecular alterations induced by the combined action of environmental progestogens. In addition, the DJ-1 chaperon protein as a possible biomarker helped to trace the abiotic chemical environmental contaminations, like progestogens in the freshwater ecosystems.

Exposure to anticoagulant rodenticides in steppe polecat (Mustela eversmanii) and European polecat (Mustela putorius) in central Europe.

Poisoning caused by coumarin-type anticoagulant rodenticides (ARs) stands as the predominant method for controlling rodents globally. ARs, through secondary poisoning, pose a significant threat to predators due to their lethal and sublethal effects. We examined the concentration of accumulated ARs in liver samples of mostly road-killed steppe polecats (Mustela eversmanii) and European polecats (M. putorius) collected throughout Hungary between 2005 and 2021. The steppe polecat samples were found mainly from Eastern Hungary, while European polecats from Western Hungary. We measured the concentration of six residues by HPLC-FLD. Our analysis revealed the presence of one first-generation and four second-generation ARs in 53% of the steppe polecat (36) and 39% of the European polecat (26) samples. In 17 samples we detected the presence of at least two AR compounds. Although we did not find significant variance in AR accumulation between the two species, steppe polecats displayed greater prevalence and maximum concentration of ARs, whereas European polecat samples exhibited a more diverse accumulation of these compounds. Brodifacoum and bromadiolone were the most prevalent ARs; the highest concentrations were 0.57 mg/kg and 0.33 mg/kg, respectively. The accumulation of ARs was positively correlated with human population density and negatively correlated with the extent of the more natural habitats in both species. To the best of our knowledge, this is the first study to demonstrate anticoagulant rodenticide exposure in steppe polecats globally, and for European polecats in Central European region. Although the extent of AR accumulation in European polecat in Hungary appears comparatively lower than in many other European countries, the issue of secondary poisoning remains a serious problem as these ARs intrude into food webs. Reduced and more prudent usage of pesticides would provide several benefits for wildlife, included humans. However, we advocate a prioritization of ecosystem services through the complete prohibition of the toxicants.

Impact of land use-induced soil heterogeneity on the adsorption of fluoroquinolone antibiotics, tested on organic matter pools.

The effects on the adsorption of fluoroquinolone antibiotics of long-term soil heterogeneity induced by land-use were investigated. Three different land use areas with their two organic matter (OM) pools were tested for the adsorption of three antibiotics widely detected in the environment (ciprofloxacin, norfloxacin, ofloxacin). The soils were separated into two size fractions, > 63 µm fraction and < 63 µm fractions for the fast and slow OM pools, respectively. Any effect of land use on adsorption was only observed in the slow pool in the increasing order: arable land, grassland, and forest. The composition of the soil organic matter (SOM) did influence adsorption in the slow pool, but not in the bulk soilsThis was, because: 1) the ratio of the slow pool was low, as in forest, 2) the ratio of the slow pool was high but its adsorption capacity was low due to its SOM composition, as in arable land and grassland. Soils containing a large slow SOM pool fraction with aliphatic dominance were found to be more likely to adsorb micropollutants. It is our contention that the release of contaminated water, sludge, manure or compost into the environment should only be undertaken after taking this into consideration.

Comparative local analysis of metabolites, lipids and proteins in intact fish tissues by LAESI mass spectrometry.

Direct mass spectrometric analysis of animal tissues is an emerging field enabled by recent developments in ambient ion sources. Label-free in situ analysis of metabolites, lipids, and peptides/proteins from intact tissues in whole fish specimens of different gender and age were performed by laser ablation electrospray ionization (LAESI) mass spectrometry (MS). Hypertrophied glandular tissue (gill gland) of adult male Aphyocharax anisitsi (bloodfin tetra) was compared with gill tissues in females of the same species. Comparison of a large number of sample-specific ions was aided by a multivariate statistical method based on orthogonal projections to latent structures discriminant analysis. More than 200 different ions were detected in the mass spectra corresponding to primary metabolites, hormones, lipids and peptides/proteins. The gill tissues of the sexually mature males exhibited multiply charged ions in the 6+ to 10+ charge states corresponding to a protein with a molecular weight of 11 380 Da. This protein was present only in the mature male gill glands but absent in the corresponding area of the female and immature male specimens. An additional nine proteins were detected by LAESI-MS in both the male and female gill tissues.

Copper-transporting ATPases throughout the animal evolution - From clinics to basal neuron-less animals.

Copper-transporting ATPases are a group of heavy metal-transporting proteins and which can be found in all living organisms. In animals, they are generally referred to as ATP7 proteins and are involved in many different physiological processes including the maintaining of copper homeostasis and the supply of copper to cuproenzymes. A single ATP7 gene is present in non-chordate animals while it is divided into ATP7A and ATP7B in chordates. In humans, dysfunction of ATP7 proteins can lead to severe genetic disorders, such as, Menkes disease and Wilson's disease, which are characterized by abnormal copper transport and accumulation, causing significant health complications. Therefore, there is a substantial amount of research on ATP7 genes and ATP7 proteins in humans and mice to understand pathophysiological conditions and find potential therapeutic interventions. Copper-transporting ATPases have also been investigated in some non-mammalian vertebrates, protostomes, single-cellular eukaryotes, prokaryotes, and archaea to gain useful evolutionary insights. However, ATP7 function in many animals has been somewhat neglected, particularly in non-bilaterians. Previous reviews on this topic only broadly summarized the available information on the function and evolution of ATP7 genes and ATP7 proteins and included only the classic vertebrate and invertebrate models. Given this, and the fact that a considerable amount of new information on this topic has been published in recent years, the present study was undertaken to provide an up-to-date, comprehensive summary of ATP7s/ATP7s and give new insights into their evolutionary relationships. Additionally, this work provides a framework for studying these genes and proteins in non-bilaterians. As early branching animals, they are important to understand the evolution of function of these proteins and their important role in copper homeostasis and neurotransmission.

A novel and unique refraction-based optical recording system for pharmacological investigations on isolated muscle preparations.

In the field of neuroscience and ecotoxicology, there is a great need for investigating the effect(s) of a variety of different chemicals (e.g., pharmacologically active compounds, pesticides, neurotransmitters, modulators) at different biological levels. Different contractile tissue preparations have provided excellent model systems for in vitro pharmacological experiments for a long time. However, such investigations usually apply mechanical force transducer-based approaches. Thus, a rapid, easy, cheap, digital, and reproducible in vitro pharmacological method based on an effective, 'non-invasive' (compared to the force-transducer approaches), refraction-based optical recording approach and isolated heart preparations was developed.•A versatile and unique refraction-based optical recording system with a Java application was developed.•The recording system was tested and validated on isolated heart preparations obtained from the widely used invertebrate model organism, the great pond snail (Lymnaea stagnalis).•The recording system illustrates the progression of technology from the mechanical force transducer system and can represent a suitable tool in ecotoxicology or neuroscience.

Presence, variation, and potential ecological impact of microplastics in the largest shallow lake of Central Europe.

The presence of microplastics (MPs) in the global ecosystem has generated a rapidly growing concern worldwide. Although their presence in the marine environment has been well-studied, much less data are available on their abundance in freshwaters. MPs alone and in combination with different chemicals has been shown to cause acute and chronic effects on algae and aquatic invertebrate and vertebrate species at different biological levels. However, the combined ecotoxicological effects of MPs with different chemicals on aquatic organisms are still understudied in many species and the reported data are often controversial. In the present study, we investigated, for the first time, the presence of MPs in Lake Balaton, which is the largest shallow lake of Central Europe and an important summer holiday destination. Moreover, we exposed neonates of the well-established ecotoxicological model organism Daphnia magna to different MPs (polystyrene [3 µm] or polyethylene [≤ 100 µm]) alone and in combination with three progestogen compounds (progesterone, drospirenone, levonorgestrel) at an environmentally relevant concentration (10 ng L-1) for 21 days. The presence of 7 polymer types of MPs in the size range of 50-100 µm was detected in Lake Balaton. Similarly to the global trends, polypropylene and polyethylene MPs were the most common types of polymer. The calculated polymer-independent average particle number was 5.5 particles m-3 (size range: 50 µm - 100 µm) which represents the values detected in other European lakes. Our ecotoxicological experiments confirmed that MPs and progestogens can affect D. magna at the behavioral (body size and reproduction) and biochemical (detoxification-related enzyme activity) levels. The joint effects were negligible. The presence of MPs may lead to reduced fitness in the aquatic biota in freshwaters such as Lake Balaton, however, the potential threat of MPs as vectors for progestogens may be limited.

Studies on a widely-recognized snail model species (Lymnaea stagnalis) provide further evidence that vertebrate steroids do not have a hormonal role in the reproduction of mollusks.

Experiments were carried out to determine whether, as with other mollusks that have been studied, the snail, Lymnaea stagnalis, can absorb, esterify and store vertebrate steroids that are present in the water. We also carried out experiments to determine whether neural tissues of the snail could be immunohistochemically stained with an antibody to human aromatase (a key enzyme that catalyzes the conversion of testosterone [T] to 17ß-estradiol [E2]); and, if so, to determine the significance of such staining. Previous studies on other mollusks have reported such staining and have proposed this as decisive evidence that mollusks have the same steroid synthesis pathway as vertebrates. We found that snails absorb, esterify and retain esterified T, E2, progesterone and ethinyl-estradiol (albeit with an absorption rate about four times slower, on a weight basis, than the mussel, Mytilus edulis). We also found that not only anti-human aromatase, but also anti-human nuclear progesterone receptor (nPR) and anti-human gonadotropin-releasing hormone antibodies immunohistochemically stained snail neural cells. However, further experiments, involving gel electrophoretic separation, followed by immunostaining, of proteins extracted from the neural tissue, found at least two positively-stained bands for each antibody, none of which had masses matching the human proteins to which the antibodies had been raised. The anti-aromatase antibody even stained the 140 kDA ladder protein used as a molecular weight marker on the gels. Mass spectrometric analysis of the bands did not find any peptide sequences that corresponded to the human proteins. Our findings confirm that the presence of vertebrate-like sex steroids in molluscan tissues is not necessarily evidence of endogenous origin. The results also show that immunohistochemical studies using antibodies against human proteins are grossly non-specific and likely to have little or no value in studying steroid synthesis or activity in mollusks. Our conclusions are consistent with the fact that genes for aromatase and nPR have not been found in the genome of the snail or of any other mollusk. Our overarching conclusion, from this and our previous studies, is that the endocrinology of mollusks is not the same as that of humans or any other vertebrates and that continuing to carry out physiological and ecotoxicological studies on mollusks on the basis of this false assumption, is an unconscionable waste of resources.

Chronic Effects of Carbamazepine, Progesterone and Their Mixtures at Environmentally Relevant Concentrations on Biochemical Markers of Zebrafish (Danio rerio).

The impact of pharmaceuticals on non-target organisms in the environment is of increasing concern and study. Pharmaceuticals and other pollutants are often present as mixtures in an environmental compartment. Studies on the toxicological implications of these drugs on fish, particularly as mixtures at environmentally relevant concentrations, are very limited. Thus, this study aimed to evaluate the chronic effects of the anticonvulsant drug carbamazepine (CBZ) and progesterone (P4) at environmentally relevant concentrations, individually and in binary mixtures, applying a suite of biomarkers at the molecular level in zebrafish (Danio rerio). The effects on biotransformation enzymes 7-ethoxyresorufin O-deethylase (EROD) and glutathione-S-transferase (GST), antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidases (GPxSe and GPxTOT), and glutathione reductase (GR), and markers of damage, such as DNA strand breaks (DNAsb), lactate dehydrogenase (LDH), lipid peroxidation (LPO), and vitellogenin-like proteins (VTG), were evaluated. Analyses of the biochemical markers indicated that a synergistic dose-ratio-dependent effect of CBZ and P4 in zebrafish occurs after chronic exposure regarding VTG, biotransformation enzymes (EROD, GST), and oxidative stress marker (DNAsb). The results suggest a synergistic effect regarding VTG, thus indicating a high risk to the reproductive success of fish if these pharmaceuticals co-occur.

Pharmaceuticals in water and sediment of small streams under the pressure of urbanization: Concentrations, interactions, and risks.

Small streams are crucial but vulnerable elements of ecological networks. To better understand the occurrence of pharmaceutically active compounds (PhACs) in streams, this study focused on the occurrence, distribution, and environmental risk of 111 PhACs and 7 trace elements based on a total of 141 water and sediment samples from small streams located in the urbanizing region of Budapest, Hungary. Eighty-one PhACs were detected in the aqueous phase, whereas sixty-two compounds were detected in the sediment. Carbamazepine (CBZ) was the most frequently identified PhAC in water, and was found in 91.5% of all samples. However, the highest concentrations were measured for lamotrigine (344.8 µg·L-1) and caffeine (221.4 µg·L-1). Lidocaine was the most frequently occurring PhAC in sediment (73.8%), but the maximum concentrations were detected for CBZ (395.9 ng·g-1) and tiapride (187.7 ng·g-1). In both water and sediment, more PhACs were found downstream of the wastewater treatment plants (WWTPs) than in the samples not affected by treated wastewater, even though no relationship was observed between the total amount of treated wastewater and the number of detected PhACs. The PhAC concentrations were also independent of the distance from the WWTP effluents. PhAC-polluted samples were detected upstream of the WWTPs, thereby suggesting the relevance of diffuse emissions in addition to WWTP outlets. The most frequently detected PhACs in the sediment were usually also present in the water samples collected at the same place and time. The varying concentrations of PhACs and the fluctuating water-sediment properties resulted in a lack of correlation between the general chemical properties and the concentrations of PhACs, which makes it difficult to predict PhAC contamination and risks in urbanized small streams. The environmental risk assessment indicated that diclofenac had the highest risk in the sampling area.

A homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide is both necessary and instructive for the rapid formation of associative memory in an invertebrate.

Similar to other invertebrate and vertebrate animals, cAMP-dependent signaling cascades are key components of long-term memory (LTM) formation in the snail Lymnaea stagnalis, an established experimental model for studying evolutionarily conserved molecular mechanisms of long-term associative memory. Although a great deal is already known about the signaling cascades activated by cAMP, the molecules involved in the learning-induced activation of adenylate cyclase (AC) in Lymnaea remained unknown. Using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy in combination with biochemical and immunohistochemical methods, recently we have obtained evidence for the existence of a Lymnaea homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) and for the AC-activating effect of PACAP in the Lymnaea nervous system. Here we first tested the hypothesis that PACAP plays an important role in the formation of robust LTM after single-trial classical food-reward conditioning. Application of the PACAP receptor antagonist PACAP6-38 around the time of single-trial training with amyl acetate and sucrose blocked associative LTM, suggesting that in this "strong" food-reward conditioning paradigm the activation of AC by PACAP was necessary for LTM to form. We found that in a "weak" multitrial food-reward conditioning paradigm, lip touch paired with sucrose, memory formation was also dependent on PACAP. Significantly, systemic application of PACAP at the beginning of multitrial tactile conditioning accelerated the formation of transcription-dependent memory. Our findings provide the first evidence to show that in the same nervous system PACAP is both necessary and instructive for fast and robust memory formation after reward classical conditioning.

Persistent sodium current is a nonsynaptic substrate for long-term associative memory.

Although synaptic plasticity is widely regarded as the primary mechanism of memory [1], forms of nonsynaptic plasticity, such as increased somal or dendritic excitability or membrane potential depolarization, also have been implicated in learning in both vertebrate and invertebrate experimental systems [2-7]. Compared to synaptic plasticity, however, there is much less information available on the mechanisms of specific types of nonsynaptic plasticity involved in well-defined examples of behavioral memory. Recently, we have shown that learning-induced somal depolarization of an identified modulatory cell type (the cerebral giant cells, CGCs) of the snail Lymnaea stagnalis encodes information that enables the expression of long-term associative memory [8]. The Lymnaea CGCs therefore provide a highly suitable experimental system for investigating the ionic mechanisms of nonsynaptic plasticity that can be linked to behavioral learning. Based on a combined behavioral, electrophysiological, immunohistochemical, and computer simulation approach, here we show that an increase of a persistent sodium current of this neuron underlies its delayed and persistent depolarization after behavioral single-trial classical conditioning. Our findings provide new insights into how learning-induced membrane level changes are translated into a form of long-lasting neuronal plasticity already known to contribute to maintained adaptive modifications at the network and behavioral level [8].

Progestogen-induced alterations and their ecological relevance in different embryonic and adult behaviours of an invertebrate model species, the great pond snail (Lymnaea stagnalis).

The presence of oral contraceptives (basically applying estrogens and/or progestogens) poses a challenge to animals living in aquatic ecosystems and reflects a rapidly growing concern worldwide. However, there is still a lack in knowledge about the behavioural effects induced by progestogens on the non-target species including molluscs. In the present study, environmental progestogen concentrations were summarised. Knowing this data, we exposed a well-established invertebrate model species, the great pond snail (Lymnaea stagnalis) to relevant equi-concentrations (1, 10, 100, and 500 ng L-1) of mixtures of four progestogens (progesterone, drospirenone, gestodene, levonorgestrel) for 21 days. Significant alterations were observed in the embryonic development time, heart rate, feeding, and gliding activities of the embryos as well as in the feeding and locomotion activity of the adult specimens. All of the mixtures accelerated the embryonic development time and the gliding activity. Furthermore, the 10, 100, and 500 ng L-1 mixtures increased the heart rate and feeding activity of the embryos. The 10, 100, and 500 ng L-1 mixtures affected the feeding activity as well as the 1, 10, and 100 ng L-1 mixtures influenced the locomotion of the adult specimens. The differences of these adult behaviours showed a biphasic response to the progestogen exposure; however, they changed approximately in the opposite way. In case of feeding activity, this dose-response phenomenon can be identified as a hormesis response. Based on the authors' best knowledge, this is the first study to investigate the non-reproductive effects of progestogens occurring also in the environment on molluscan species. Our findings contribute to the global understanding of the effects of human progestogens, as these potential disruptors can influence the behavioural activities of non-target aquatic species. Future research should aim to understand the potential mechanisms (e.g., receptors, signal pathways) of progestogens induced behavioural alterations.

Environmental risk assessment of pharmaceuticals at a seasonal holiday destination in the largest freshwater shallow lake in Central Europe.

The presence of pharmacologically active compounds (PhACs) in surface waters poses an environmental risk of chronic exposure to nontarget organisms, which is a well-established and serious concern worldwide. Our aim was to determine the temporal changes in ecological risk quotient (RQ) based on the concentrations of 42 PhACs from six sampling sites on seven sampling dates in the water of a freshwater lake in Central Europe preferentially visited by tourists. Our hypothesis was that the environmental risk increases during the summer holiday season due to the influence of tourists. Different experimental toxicological threshold concentrations and seasonal measured environmental concentrations of 16 PhACs were applied to ecological risk assessment. RQs of 4 dominant PhACs (diclofenac, estrone [E1], estradiol [E2], and caffeine) indicated high ecological risk (RQ > 1) for freshwater ecosystems. Additionally, our results confirmed the assumptions that the high tourist season had a significant impact on the calculated RQ; however, these results are mainly due to the concentration and temporal change of particular PhACs, including diclofenac (5.3-419.4 ng/L), E1 (0.1-5.5 ng/L), and E2 (0.1-19.6 ng/L). The seasonal dependent highest RQs changed as follows: 9.80 (June 2017; E2), 1.23 (August 2017; E1), 0.43 (November 2017; E1), 0.51 (April 2018; E1), 5.58 (June 2018, diclofenac), 39.50 (August 2018; diclofenac), and 30.60 (October 2018; diclofenac).

The Great Pond Snail (Lymnaea stagnalis) as a Model of Aging and Age-Related Memory Impairment: An Overview.

With the increase of life span, normal aging and age-related memory decline are affecting an increasing number of people; however, many aspects of these processes are still not fully understood. Although vertebrate models have provided considerable insights into the molecular and electrophysiological changes associated with brain aging, invertebrates, including the widely recognized molluscan model organism, the great pond snail (Lymnaea stagnalis), have proven to be extremely useful for studying mechanisms of aging at the level of identified individual neurons and well-defined circuits. Its numerically simpler nervous system, well-characterized life cycle, and relatively long life span make it an ideal organism to study age-related changes in the nervous system. Here, we provide an overview of age-related studies on L. stagnalis and showcase this species as a contemporary choice for modeling the molecular, cellular, circuit, and behavioral mechanisms of aging and age-related memory impairment.