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.

A circuit mechanism linking past and future learning through shifts in perception.

Long-term memory formation is energetically costly. Neural mechanisms that guide an animal to identify fruitful associations therefore have important survival benefits. Here, we elucidate a circuit mechanism in Lymnaea, which enables past memory to shape new memory formation through changes in perception. Specifically, strong classical conditioning drives a positive shift in perception that facilitates the robust learning of a subsequent and otherwise ineffective weak association. Circuit dissection approaches reveal the neural control network responsible, characterized by a mutual inhibition motif. This both sets perceptual state and acts as the master controller for gating new learning. Pharmacological circuit manipulation in vivo fully substitutes for strong paradigm learning, shifting the network into a more receptive state to enable subsequent weak paradigm learning. Thus, perceptual change provides a conduit to link past and future memory storage. We propose that this mechanism alerts animals to learning-rich periods, lowering the threshold for new memory acquisition.

The role of non-coding RNAs in the formation of long-term associative memory after single-trial learning in Lymnaea.

Investigations of the molecular mechanisms of long-term associative memory have revealed key roles for a number of highly evolutionarily conserved molecular pathways in a variety of different vertebrate and invertebrate model systems. One such system is the pond snail Lymnaea stagnalis, in which, like in other systems, the transcription factors CREB1 and CREB2 and the enzyme NOS play essential roles in the consolidation of long-term associative memory. More recently, epigenetic control mechanisms, such as DNA methylation, histone modifications, and control of gene expression by non-coding RNAs also have been found to play important roles in all model systems. In this minireview, we will focus on how, in Lymnaea, even a single episode of associative learning can activate CREB and NO dependent cascades due to the training-induced up- or downregulation of the expression levels of recently identified short and long non-coding RNAs.

Molecular and functional characterization of an evolutionarily conserved CREB-binding protein in the Lymnaea CNS.

In eukaryotes, CREB-binding protein (CBP), a coactivator of CREB, functions both as a platform for recruiting other components of the transcriptional machinery and as a histone acetyltransferase (HAT) that alters chromatin structure. We previously showed that the transcriptional activity of cAMP-responsive element binding protein (CREB) plays a crucial role in neuronal plasticity in the pond snail Lymnaea stagnalis. However, there is no information on the molecular structure and HAT activity of CBP in the Lymnaea central nervous system (CNS), hindering an investigation of its postulated role in long-term memory (LTM). Here, we characterize the Lymnaea CBP (LymCBP) gene and identify a conserved domain of LymCBP as a functional HAT. Like CBPs of other species, LymCBP possesses functional domains, such as the KIX domain, which is essential for interaction with CREB and was shown to regulate LTM. In-situ hybridization showed that the staining patterns of LymCBP mRNA in CNS are very similar to those of Lymnaea CREB1. A particularly strong LymCBP mRNA signal was observed in the cerebral giant cell (CGC), an identified extrinsic modulatory interneuron of the feeding circuit, the key to both appetitive and aversive LTM for taste. Biochemical experiments using the recombinant protein of the LymCBP HAT domain showed that its enzymatic activity was blocked by classical HAT inhibitors. Preincubation of the CNS with such inhibitors blocked cAMP-induced synaptic facilitation between the CGC and an identified follower motoneuron of the feeding system. Taken together, our findings suggest a role for the HAT activity of LymCBP in synaptic plasticity in the feeding circuitry.

A combined bioinformatics and LC-MS-based approach for the development and benchmarking of a comprehensive database of Lymnaea CNS proteins.

Applications of key technologies in biomedical research, such as qRT-PCR or LC-MS-based proteomics, are generating large biological (-omics) datasets which are useful for the identification and quantification of biomarkers in any research area of interest. Genome, transcriptome and proteome databases are already available for a number of model organisms including vertebrates and invertebrates. However, there is insufficient information available for protein sequences of certain invertebrates, such as the great pond snail Lymnaea stagnalis, a model organism that has been used highly successfully in elucidating evolutionarily conserved mechanisms of memory function and dysfunction. Here, we used a bioinformatics approach to designing and benchmarking a comprehensive central nervous system (CNS) proteomics database (LymCNS-PDB) for the identification of proteins from the CNS of Lymnaea by LC-MS-based proteomics. LymCNS-PDB was created by using the Trinity TransDecoder bioinformatics tool to translate amino acid sequences from mRNA transcript assemblies obtained from a published Lymnaea transcriptomics database. The blast-style MMSeq2 software was used to match all translated sequences to UniProtKB sequences for molluscan proteins, including those from Lymnaea and other molluscs. LymCNS-PDB contains 9628 identified matched proteins that were benchmarked by performing LC-MS-based proteomics analysis with proteins isolated from the Lymnaea CNS. MS/MS analysis using the LymCNS-PDB database led to the identification of 3810 proteins. Only 982 proteins were identified by using a non-specific molluscan database. LymCNS-PDB provides a valuable tool that will enable us to perform quantitative proteomics analysis of protein interactomes involved in several CNS functions in Lymnaea, including learning and memory and age-related memory decline.

Time dependent differential regulation of a novel long non-coding natural antisense RNA during long-term memory formation.

Long natural antisense transcripts (NATs) have been demonstrated in significant numbers in a variety of eukaryotic organisms. They are particularly prevalent in the nervous system suggesting their importance in neural functions. However, the precise physiological roles of the overwhelming majority of long NATs remain unclear. Here we report on the characterization of a novel molluscan nitric oxide synthase (NOS)-related long non-coding NAT (Lym-NOS1AS). This NAT is spliced and polyadenylated and is transcribed from the non-template strand of the Lym-NOS1 gene. We demonstrate that the Lym-NOS1AS is co-expressed with the sense Lym-NOS1 mRNA in a key neuron of memory network. Also, we report that the Lym-NOS1AS is temporally and spatially regulated by one-trial conditioning leading to long term memory (LTM) formation. Specifically, in the cerebral, but not in the buccal ganglia, the temporal pattern of changes in Lym-NOS1AS expression after training correlates with the alteration of memory lapse and non-lapse periods. Our data suggest that the Lym-NOS1AS plays a role in the consolidation of nitric oxide-dependent LTM.

Interneuronal mechanisms for learning-induced switch in a sensory response that anticipates changes in behavioral outcomes.

Sensory cues in the natural environment predict reward or punishment, important for survival. For example, the ability to detect attractive tastes indicating palatable food is essential for foraging while the recognition of inedible substrates prevents harm. While some of these sensory responses are innate, they can undergo fundamental changes due to prior experience associated with the stimulus. However, the mechanisms underlying such behavioral switching of an innate sensory response at the neuron and network levels require further investigation. We used the model learning system of Lymnaea stagnalis1-3 to address the question of how an anticipated aversive outcome reverses the behavioral response to a previously effective feeding stimulus, sucrose. Key to the switching mechanism is an extrinsic inhibitory interneuron of the feeding network, PlB (pleural buccal4,5), which is inhibited by sucrose to allow a feeding response. After multi-trial aversive associative conditioning, pairing sucrose with strong tactile stimuli to the head, PlB's firing rate increases in response to sucrose application to the lips and the feeding response is suppressed; this learned response is reversed by the photoinactivation of a single PlB. A learning-induced persistent change in the cellular properties of PlB that results in an increase rather than a decrease in its firing rate in response to sucrose provides a neurophysiological mechanism for this behavioral switch. A key interneuron, PeD12 (Pedal-Dorsal 12), of the defensive withdrawal network5,6 does not mediate the conditioned suppression of feeding, but its facilitated output contributes to the sensitization of the withdrawal response.

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.

Proactive and retroactive interference with associative memory consolidation in the snail Lymnaea is time and circuit dependent.

Interference-based forgetting occurs when new information acquired either before or after a learning event attenuates memory expression (proactive and retroactive interference, respectively). Multiple learning events often occur in rapid succession, leading to competition between consolidating memories. However, it is unknown what factors determine which memory is remembered or forgotten. Here, we challenge the snail, Lymnaea, to acquire two consecutive similar or different memories and identify learning-induced changes in neurons of its well-characterized motor circuits. We show that when new learning takes place during a stable period of the original memory, proactive interference only occurs if the two consolidating memories engage the same circuit mechanisms. If different circuits are used, both memories survive. However, any new learning during a labile period of consolidation promotes retroactive interference and the acquisition of the new memory. Therefore, the effect of interference depends both on the timing of new learning and the underlying neuronal mechanisms.

Subcellular Peptide Localization in Single Identified Neurons by Capillary Microsampling Mass Spectrometry.

Single cell mass spectrometry (MS) is uniquely positioned for the sequencing and identification of peptides in rare cells. Small peptides can take on different roles in subcellular compartments. Whereas some peptides serve as neurotransmitters in the cytoplasm, they can also function as transcription factors in the nucleus. Thus, there is a need to analyze the subcellular peptide compositions in identified single cells. Here, we apply capillary microsampling MS with ion mobility separation for the sequencing of peptides in single neurons of the mollusk Lymnaea stagnalis, and the analysis of peptide distributions between the cytoplasm and nucleus of identified single neurons that are known to express cardioactive Phe-Met-Arg-Phe amide-like (FMRFamide-like) neuropeptides. Nuclei and cytoplasm of Type 1 and Type 2 F group (Fgp) neurons were analyzed for neuropeptides cleaved from the protein precursors encoded by alternative splicing products of the FMRFamide gene. Relative abundances of nine neuropeptides were determined in the cytoplasm. The nuclei contained six of these peptides at different abundances. Enabled by its relative enrichment in Fgp neurons, a new 28-residue neuropeptide was sequenced by tandem MS.

A CREB2-targeting microRNA is required for long-term memory after single-trial learning.

Although single-trial induced long-term memories (LTM) have been of major interest in neuroscience, how LTM can form after a single episode of learning remains largely unknown. We hypothesized that the removal of molecular inhibitory constraints by microRNAs (miRNAs) plays an important role in this process. To test this hypothesis, first we constructed small non-coding RNA (sncRNA) cDNA libraries from the CNS of Lymnaea stagnalis subjected to a single conditioning trial. Then, by next generation sequencing of these libraries, we identified a specific pool of miRNAs regulated by training. Of these miRNAs, we focussed on Lym-miR-137 whose seed region shows perfect complementarity to a target sequence in the 3' UTR of the mRNA for CREB2, a well-known memory repressor. We found that Lym-miR-137 was transiently up-regulated 1 h after single-trial conditioning, preceding a down-regulation of Lym-CREB2 mRNA. Furthermore, we discovered that Lym-miR-137 is co-expressed with Lym-CREB2 mRNA in an identified neuron with an established role in LTM. Finally, using an in vivo loss-of-function approach we demonstrated that Lym-miR-137 is required for single-trial induced LTM.

Interneuronal mechanism for Tinbergen's hierarchical model of behavioral choice.

Recent studies of behavioral choice support the notion that the decision to carry out one behavior rather than another depends on the reconfiguration of shared interneuronal networks [1]. We investigated another decision-making strategy, derived from the classical ethological literature [2, 3], which proposes that behavioral choice depends on competition between autonomous networks. According to this model, behavioral choice depends on inhibitory interactions between incompatible hierarchically organized behaviors. We provide evidence for this by investigating the interneuronal mechanisms mediating behavioral choice between two autonomous circuits that underlie whole-body withdrawal [4, 5] and feeding [6] in the pond snail Lymnaea. Whole-body withdrawal is a defensive reflex that is initiated by tactile contact with predators. As predicted by the hierarchical model, tactile stimuli that evoke whole-body withdrawal responses also inhibit ongoing feeding in the presence of feeding stimuli. By recording neurons from the feeding and withdrawal networks, we found no direct synaptic connections between the interneuronal and motoneuronal elements that generate the two behaviors. Instead, we discovered that behavioral choice depends on the interaction between two unique types of interneurons with asymmetrical synaptic connectivity that allows withdrawal to override feeding. One type of interneuron, the Pleuro-Buccal (PlB), is an extrinsic modulatory neuron of the feeding network that completely inhibits feeding when excited by touch-induced monosynaptic input from the second type of interneuron, Pedal-Dorsal12 (PeD12). PeD12 plays a critical role in behavioral choice by providing a synaptic pathway joining the two behavioral networks that underlies the competitive dominance of whole-body withdrawal over feeding.

Reversal of age-related learning deficiency by the vertebrate PACAP and IGF-1 in a novel invertebrate model of aging: the pond snail (Lymnaea stagnalis).

With the increase of life span, nonpathological age-related memory decline is affecting an increasing number of people. However, there is evidence that age-associated memory impairment only suspends, rather than irreversibly extinguishes, the intrinsic capacity of the aging nervous system for plasticity (1). Here, using a molluscan model system, we show that the age-related decline in memory performance can be reversed by administration of the pituitary adenylate cyclase activating polypeptide (PACAP). Our earlier findings showed that a homolog of the vertebrate PACAP38 and its receptors exist in the pond snail (Lymnaea stagnalis) brain (2), and it is both necessary and instructive for memory formation after reward conditioning in young animals (3). Here we show that exogenous PACAP38 boosts memory formation in aged Lymnaea, where endogenous PACAP38 levels are low in the brain. Treatment with insulin-like growth factor-1, which in vertebrates was shown to transactivate PACAP type I (PAC1) receptors (4) also boosts memory formation in aged pond snails. Due to the evolutionarily conserved nature of these polypeptides and their established role in memory and synaptic plasticity, there is a very high probability that they could also act as "memory rejuvenating" agents in humans.

Susceptibility of memory consolidation during lapses in recall.

Memories that can be recalled several hours after learning may paradoxically become inaccessible for brief periods after their formation. This raises major questions about the function of these early memory lapses in the structure of memory consolidation. These questions are difficult to investigate because of the lack of information on the precise timing of lapses. However, the use of a single-trial conditioning paradigm in Lymnaea solves this problem. Here we use electrophysiological and behavioural experiments to reveal lapses in memory recall at 30 min and 2 h post conditioning. We show that only during these lapses is consolidation of long-term memory susceptible to interruption by external disturbance. These shared time points of memory lapse and susceptibility correspond to transitions between different phases of memory that have different molecular requirements. We propose that during periods of molecular transition memory recall is weakened, allowing novel sensory cues to block the consolidation of long-term memory.

Axonal trafficking of an antisense RNA transcribed from a pseudogene is regulated by classical conditioning.

Natural antisense transcripts (NATs) are endogenous RNA molecules that are complementary to known RNA transcripts. The functional significance of NATs is poorly understood, but their prevalence in the CNS suggests a role in brain function. Here we investigated a long NAT (antiNOS-2 RNA) associated with the regulation of nitric oxide (NO) production in the CNS of Lymnaea, an established model for molecular analysis of learning and memory. We show the antiNOS-2 RNA is axonally trafficked and demonstrate that this is regulated by classical conditioning. Critically, a single conditioning trial changes the amount of antiNOS-2 RNA transported along the axon. This occurs within the critical time window when neurotransmitter NO is required for memory formation. Our data suggest a role for the antiNOS-2 RNA in establishing memories through the regulation of NO signaling at the synapse.

Single electrode dynamic clamp with StdpC.

Dynamic clamp is a powerful approach for electrophysiological investigations allowing researchers to introduce artificial electrical components into target neurons to simulate ionic conductances, chemical or electrotonic inputs or connections to other cells. Due to the rapidly changing and potentially large current injections during dynamic clamp, problematic voltage artifacts appear on the electrode used to inject dynamic clamp currents into a target neuron. Dynamic clamp experiments, therefore, typically use two separate electrodes in the same cell, one for recording membrane potential and one for injecting currents. The requirement for two independent electrodes has been a limiting factor for the use of dynamic clamp in applications where dual recordings of this kind are difficult or impossible to achieve. The recent development of an active electrode compensation (AEC) method has overcome some of these prior limitations, permitting artifact-free dynamic clamp experimentation with a single electrode. Here we describe an AEC method for the free dynamic clamp software StdpC. The AEC component of StdpC is the first such system implemented for the use of non-expert users and comes with a set of semi-automated configuration and calibration procedures that facilitate its use. We briefly introduce the AEC method and its implementation in StdpC and then validate it with an electronic model cell and in two different biological preparations.

Dynamic clamp with StdpC software.

Dynamic clamp is a powerful method that allows the introduction of artificial electrical components into target cells to simulate ionic conductances and synaptic inputs. This method is based on a fast cycle of measuring the membrane potential of a cell, calculating the current of a desired simulated component using an appropriate model and injecting this current into the cell. Here we present a dynamic clamp protocol using free, fully integrated, open-source software (StdpC, for spike timing-dependent plasticity clamp). Use of this protocol does not require specialist hardware, costly commercial software, experience in real-time operating systems or a strong programming background. The software enables the configuration and operation of a wide range of complex and fully automated dynamic clamp experiments through an intuitive and powerful interface with a minimal initial lead time of a few hours. After initial configuration, experimental results can be generated within minutes of establishing cell recording.

Different circuit and monoamine mechanisms consolidate long-term memory in aversive and reward classical conditioning.

There has been considerable recent interest in comparing the circuit and monoamine-based mechanisms of aversive and reward-associative conditioning in a number of vertebrate and invertebrate model systems. The mollusc Lymnaea stagnalis provides a unique opportunity to explore changes in the neural and chemical pathways underlying these two different types of conditioning as its feeding circuitry has been thoroughly characterised. Animals can learn after a single trial to associate the same CS (amyl acetate) either with a punishment (quinine) or reward (sucrose), showing either a reduced or an elevated feeding response, respectively, to the CS. We previously showed that reward conditioning strengthened the direct excitatory pathway from the lips to the feeding central pattern generator in the buccal ganglia through the activation of feeding interneurons in the cerebral ganglia. Now we demonstrate that aversive conditioning enhances the strength of a different inhibitory pathway that suppresses feeding but has no effect on the excitatory pathway. Here we show that consolidation of long-term memory (LTM) in reward conditioning depends on dopamine but not octopamine. In contrast, aversive LTM depends on octopamine but not dopamine. Octopamine is the invertebrate equivalent of noradrenalin, so these results on the monoamine dependence of reward and aversive conditioning in Lymnaea resemble, at the transmitter receptor level, those in mammals but are the opposite of those in another invertebrate group, the insects.

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.