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.</p

Research data for 'A circuit mechanism linking past and future learning through shifts in perception'

Data for paper published in Science Advances (24 Mar 2023, Vol 9, Issue 12) A spreadsheet separated into tabs that contain all datasets for each related figure and supplementary figure. The datasets include behavioural measures of learning and the perception of training, and measures of neuronal activity based on electrophysiological recordings.    The tabs with datasets they contain are as follows:   TAB 1 relating to: Fig. 1. Previous strong learning enhances subsequent weak learning. Also Fig. S1, Fig. S2 and Fig. S3.  TAB 2 relating to: Fig. 2. Previous learning alters the perception of future training by shifting the network state. Also Fig. S4, Fig. S5, Fig. S6, Fig. S7 and Fig. S8.  TAB 3 relating to: Fig. 3. Learning-induced shift recorded in command-like interneurons controlling antagonistic behaviours.  TAB 4 relating to: Fig. 4. Characterization of perceptual control circuit that mediates competitive interactions between ingestion and egestion. Also Fig. S9.  TAB 5 relating to: Fig. 5. Pharmacological manipulation of perceptual control circuit substitutes for strong training and facilitates new memory acquisition in vivo. Also Fig. S10.  TAB 6 relating to: Fig. 6. Memory-linked shifts in perception generalize to an alternative training paradigm.  Abstract Long-term memory formation is energetically costly. Neural mechanisms that guide an animal to identify fruitful associations therefore have significant 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 which 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. </p

Research data for paper 'Time dependent differential regulation of a novel long non-coding natural antisense RNA during long-term memory formation'.

Behavioural data used in paper published in Scientific ReportsDataset for Fig. 4a: The results of the behavioural test of long-term memory (LTM) formation at 24 h after single-trial food reward classical conditioning. The data shown are difference scores calculated by subtracting the number of spontaneous feeding rasps counted in a two-minute period before the application of the conditioned stimulus from those counted in the two-minute period after the conditioned stimulus was applied.AbstractLong 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 spatially and temporally 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 well with the alternation 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.</p

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.</p

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 (MALDI-TOF) mass spectroscopy in combination with biochemical and immunohistochemical methods, recently we have obtained evidence for the existence of a Lymnaea homologue 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 multi-trial 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 multi-trial 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

Data from the behavioural experiments described in the paper: A CREB2-targeting microRNA is required for long-term memory after single-trial learning.

<b>Data for paper appearing in Scientific Reports. </b><div><br></div><div>Excel workbooks with unconditioned and conditioned feeding response data obtained in behavioural pharmacological experiments exploring the role of microRNAs in general and miR-137 in particular in long-term memory after single-trial classical conditioning.<br><br><b>Abstract from research paper</b><br>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 <i>Lymnaea stagnalis</i> 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 <i>Lym-CREB2</i> mRNA. Furthermore, we discovered that Lym-miR-137 is co-expressed with <i>Lym-CREB2</i> 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.</div

Dynamic control of a central pattern generator circuit: a computational model of the snail feeding network

Central pattern generators (CPGs) are networks underlying rhythmic motor behaviours and they are dynamically regulated by neuronal elements that are extrinsic or intrinsic to the rhythmogenic circuit. In the feeding system of the pond snail, Lymnaea stagnalis, the extrinsic slow oscillator (SO) interneuron controls the frequency of the feeding rhythm and the N3t (tonic) has a dual role; it is an intrinsic CPG interneuron, but it also suppresses CPG activity in the absence of food, acting as a decision-making element in the feeding circuit. The firing patterns of the SO and N3t neurons and their synaptic connections with the rest of the CPG are known, but how these regulate network function is not well understood. This was investigated by building a computer model of the feeding network based on a minimum number of cells (N1M, N2v and N3t) required to generate the three-phase motor rhythm together with the SO that was used to activate the system. The intrinsic properties of individual neurons were represented using two-compartment models containing currents of the Hodgkin–Huxley type. Manipulations of neuronal activity in the N3t and SO neurons in the model produced similar quantitative effects to food and electrical stimulation in the biological network indicating that the model is a useful tool for studying the dynamic properties of the feeding circuit. The model also predicted novel effects of electrical stimulation of two CPG interneurons (N1M and N2v). When tested experimentally, similar effects were found in the biological system providing further validation of our model

Research data for paper 'Proactive and retroactive interference with associative memory consolidation in the snail Lymnaea is time and circuit dependent'

Behavioural and electrophysiological data used in the figures for the paper by Crossley et al. published in Communications Biology June 2019 Abstract for paper 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.  </p

Research data for paper ‘Interneuronal mechanisms for learning-induced switch in a sensory response that anticipates changes in behavioural outcomes’.

Behavioural and electrophysiological data used in the figures for paper published in Current Biology 10 February 2021.Datasets:Fig. 1. Behavioural data obtained by testing animals before and after aversive classical conditioning.Fig. 2D. Data obtained by electrophysiologically testing the sucrose response of the interneurone PlB in semi-intact preparations.Fig. 2E. Data obtained by measuring the baseline membrane potential and firing frequency of the interneurone PlB in semi-intact preparations.Fig. 3D. Data obtained by electrophysiologically testing the sucrose response of the feeding motoneuron B3 during the application of sucrose, before and after the photoinactivation of the interneurone PlB.Fig. 4C. Data obtained by electrophysiologically testing the response of the interneurone PlB to the electrical stimulation of the interneurone PeD12.Fig. S1. 1. Behavioural data obtained by testing animals before and after control protocols for aversive classical conditioning.AbstractSensory 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 whilst the recognition of inedible substrates prevents harm. Whilst 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 behavioural switching of an innate sensory response at the neuron and network levels require further investigation. We used the model learning system of Lymnaea stagnalis [1-3] to address the question of how an anticipated aversive outcome reverses the behavioural response to a previously effective feeding stimulus, sucrose. Key to the switching mechanism is an extrinsic inhibitory interneuron of the feeding network, PlB (pleural buccal [4, 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 behavioural switch. A key interneuron, PeD12 (Pedal-Dorsal 12), of the defensive withdrawal network [5, 6] does not mediate the conditioned suppression of feeding but its facilitated output contributes to the sensitization of the withdrawal response. </div

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.</p