The Expression of miRNAs Involved in Long-Term Memory Formation in the CNS of the Mollusk Helix lucorum.

Mollusks are unique animals with a relatively simple central nervous system (CNS) containing giant neurons with identified functions. With such simple CNS, mollusks yet display sufficiently complex behavior, thus ideal for various studies of behavioral processes, including long-term memory (LTM) formation. For our research, we use the formation of the fear avoidance reflex in the terrestrial mollusk Helix lucorum as a learning model. We have shown previously that LTM formation in Helix requires epigenetic modifications of histones leading to both activation and inactivation of the specific genes. It is known that microRNAs (miRNAs) negatively regulate the expression of genes; however, the role of miRNAs in behavioral regulation has been poorly investigated. Currently, there is no miRNAs sequencing data being published on Helix lucorum, which makes it impossible to investigate the role of miRNAs in the memory formation of this mollusk. In this study, we have performed sequencing and comparative bioinformatics analysis of the miRNAs from the CNS of Helix lucorum. We have identified 95 different microRNAs, including microRNAs belonging to the MIR-9, MIR-10, MIR-22, MIR-124, MIR-137, and MIR-153 families, known to be involved in various CNS processes of vertebrates and other species, particularly, in the fear behavior and LTM. We have shown that in the CNS of Helix lucorum MIR-10 family (26 miRNAs) is the most representative one, including Hlu-Mir-10-S5-5p and Hlu-Mir-10-S9-5p as top hits. Moreover, we have shown the involvement of the MIR-10 family in LTM formation in Helix. The expression of 17 representatives of MIR-10 differentially changes during different periods of LTM consolidation in the CNS of Helix. In addition, using comparative analysis of microRNA expression upon learning in normal snails and snails with deficient learning abilities with dysfunction of the serotonergic system, we identified a number of microRNAs from several families, including MIR-10, which expression changes only in normal animals. The obtained data can be used for further fundamental and applied behavioral research.

Stress early in life leads to cognitive impairments, reduced numbers of CA3 neurons and altered maternal behavior in adult female mice.

The hippocampus is a crucial part of the limbic system involved both in cognitive processing and in the regulation of responses to stress. Adverse experiences early in life can disrupt hippocampal development and lead to impairment of the hypothalamic-pituitary-adrenal axis response to subsequent stressors. In our study, two types of early-life stress were used: prolonged separation of pups from their mothers (for 3 hours/day, maternal separation, MS) and brief separation (for 15 minutes/day, handling, HD). In the first part of our study, we found that adult female mice (F0) who had experienced MS showed reduced locomotor activity and impairment of long-term spatial and recognition memory. Analysis of various hippocampal regions showed that MS reduced the number of mature neurons in CA3 of females, which is perhaps a crucial hippocampal region for learning and memory; however, neurogenesis remained unchanged. In the second part, we measured maternal care in female mice with a history of early-life stress (F0) as well as the behavior of their adult offspring (F1). Our results indicated that MS reduced the level of maternal care in adult females (F0) toward their own progeny and caused sex-specific changes in the social behavior of adult offspring (F1). In contrast to MS, HD had no influence on female behavior or hippocampal plasticity. Overall, our results suggest that prolonged MS early in life affects the adult behavior of F0 female mice and hippocampal neuronal plasticity, whereas the mothers' previous experience has effects on the behavior of their F1 offspring through disturbances of mother-infant interactions.

Failure of long-term memory formation in juvenile snails is determined by acetylation status of histone H3 and can be improved by NaB treatment.

BACKGROUND: Animals' capacities for different forms of learning do not mature simultaneously during ontogenesis but the molecular mechanisms behind the delayed development of specific types of memory are not fully understood. Mollusks are considered to be among the best models to study memory formation at the molecular level. Chromatin remodeling in developmental processes, as well as in long-term memory formation, was recently shown to play a major role. Histone acetylation is a key process in the chromatin remodeling and is regulated through the signaling cascades, for example MAPK/ERK. Previously, we found that MAPK/ERK is a key pathway in the formation of the food aversion reflex in Helix. Pretreatment with upstream ERK kinase inhibitor PD98059 prevented food avoidance learning in adult Helix. In contrast to adult snails, juveniles possess immature plasticity mechanisms of the avoidance reflex until the age of 2-3 months while the MAPK/ERK cascade is not activated after aversive learning. In the present study, we focused on the potential MAPK/ERK target--histone H3. METHODOLOGY/PRINCIPAL FINDINGS: Here we found that a significant increase in histone H3 acetylation occurs in adult animals after learning, whereas no corresponding increase was observed in juveniles. The acetylation of histone H3 is regulated by ERK kinase, since the upstream ERK kinase inhibitor PD98059 prevented the increase of histone H3 acetylation upon learning. We found that the injection of histone deacetylase inhibitor sodium butyrate (NaB) prior to training led to induction in histone H3 acetylation and significantly ameliorated long-term memory formation in juvenile snails. CONCLUSIONS/SIGNIFICANCE: Thus, MAPK/ERK-dependent histone H3 acetylation plays an essential role in the formation of food aversion in Helix. Dysfunction of the MAPK/ERK dependent histone H3 acetylation might determine the deficiency of avoidance behavior and long-term plasticity in juvenile animals. Stimulation of histone H3 acetylation in juvenile animals by NaB promoted avoidance plasticity.

Histone H3 Acetylation is Asymmetrically Induced Upon Learning in Identified Neurons of the Food Aversion Network in the Mollusk Helix Lucorum.

Regulation of gene expression is an essential step during long-term memory formation. Recently, the involvement of DNA-binding transcription factors and chromatin remodeling in synaptic plasticity have been intensively studied. The process of learning was shown to be associated with chromatin remodeling through histone modifications such as acetylation and phosphorylation. We have previously shown that the MAPK/ERK (mitogen-activated protein kinase/extracellular signal-regulated kinase) regulatory cascade plays a key role in the food aversion conditioning in the mollusk Helix. Specifically, command neurons of withdrawal behavior exhibit a learning-dependent asymmetry (left-right) in MAPK/ERK activation. Here, we expanded our molecular studies by focusing on a potential MAPK/ERK target - histone H3. We studied whether there is a learning-induced MAPK/ERK-dependent acetylation of histone H3 in command neurons RPa(2/3) and LPa(2/3) of the right and left parietal ganglia and whether it is asymmetrical. We found a significant learning-dependent increase in histone H3 acetylation in RPa(2/3) neurons but not in LPa(2/3) neurons. Such an increase in right command neurons depended on MAPK/ERK activation and correlated with a lateralized avoidance movement to the right visible 48 h after training. The molecular changes found in a selective set of neurons could thus represent a lateralized memory process, which may lead to consistent turning in one direction when avoiding a food that has been paired with an aversive stimulus.

Learning-induced lateralized activation of the MAPK/ERK cascade in identified neurons of the food-aversion network in the mollusk Helix lucorum.

The MAPK/ERK pathway plays an important role in the regulation of gene expression during memory formation both in vertebrates and invertebrates. In the mollusk Helix lucorum, serotonin induces activation of MAPK/ERK in the central nervous system (CNS) upon food aversion learning. Such learning depends on a neuronal network in which specialized neurons play distinct roles so that they may exhibit different activation levels of the MAPK/ERK pathway. Here we performed a comparative analysis of MAPK/ERK activation in single neurons of the food-aversion network, focusing both on command neurons, which mediate withdrawal behavior and process information pertaining to the unconditioned stimulus, and on neurons of the procerebrum, the mollusk's olfactory center, which process information from the conditioned stimulus. By means of Western blots designed to detect micro amounts of proteins, we determined MAPK/ERK activation in these neurons and found that after food aversion learning phospho-ERK levels increased significantly in RPa(2/3) command neurons of the right parietal ganglia and in the procerebrum. Such an increase was prevented by injection of PD98095, an inhibitor of the ERK upstream kinase (MEK-1). In contrast, no activation of MAPK/ERK was detected in similar conditions in the corresponding neurons of the left parietal ganglia LPa(2/3). This asymmetry was verified after serotonin application to the CNS in order to mimic learning. Our results thus show that learning involves synchronous and asymmetric serotonin-dependent MAPK/ERK activation. Such an asymmetry may reflect lateralization of memory processes in the mollusk brain.

Expression of MAP/ERK kinase cascade corresponds to the ability to develop food aversion in terrestrial snail at different stages of ontogenesis.

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) cascade plays an important role in gene expression regulation during memory formation in both vertebrates and invertebrates. MAPK/ERK regulates gene expression through phosphorylation of transcription factors binding to the regulatory elements SRE and CRE of target genes. Previously we reported that juvenile snails Helix lucorum differ from adult animals in a spectrum of transcription factors binding to DNA regulatory elements SRE and AP-1. In this study we analyzed the expression and activation of MAPK/ERK in CNS of H. lucorum during formation of the conditioned avoidance reflex at different stages of postnatal ontogenesis. Under conditions of learning, juvenile snails (aged 2-3 months) possessing immature mechanisms of avoidance reflex plasticity showed dramatically low level of phosphorylation and, correspondingly, low activation of MAPK/ERK in comparison to adult animals. Beside this, the MAPK/ERK cascade was not activated after 10 and 60 min after learning in juvenile snails in contrast to adults, while basal expression level of this kinase was similar in juveniles and adults. Low activation of MAPK/ERK cascade can cause a deficiency in phosphorylation of downstream transcription factors binding to SRE and thereby influence the expression of early response genes (particularly, of the family AP-1) and late response genes necessary for cellular and synaptic plasticity. These observations suggest that the MAPK/ERK regulatory cascade plays an essential role in the formation of conditioned avoidance reflexes in Helix. Low activation of this cascade might be one of the reasons for deficiency of long-term memory formation during avoidance learning in juvenile animals.