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.

Expression of p53 target genes in the early phase of long-term potentiation in the rat hippocampal CA1 area.

Gene expression plays an important role in the mechanisms of long-term potentiation (LTP), which is a widely accepted experimental model of synaptic plasticity. We have studied the expression of at least 50 genes that are transcriptionally regulated by p53, as well as other genes that are related to p53-dependent processes, in the early phase of LTP. Within 30 min after Schaffer collaterals (SC) tetanization, increases in the mRNA and protein levels of Bax, which are upregulated by p53, and a decrease in the mRNA and protein levels of Bcl2, which are downregulated by p53, were observed. The inhibition of Mdm2 by nutlin-3 increased the basal p53 protein level and rescued its tetanization-induced depletion, which suggested the involvement of Mdm2 in the control over p53 during LTP. Furthermore, nutlin-3 caused an increase in the basal expression of Bax and a decrease in the basal expression of Bcl2, whereas tetanization-induced changes in their expression were occluded. These results support the hypothesis that p53 may be involved in transcriptional regulation during the early phase of LTP. We hope that the presented data may aid in the understanding of the contribution of p53 and related genes in the processes that are associated with synaptic plasticity.

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.

Regulation of S100B gene in rat hippocampal CA1 area during long term potentiation.

In the present study we investigated the regulation of S100B expression during tetanization-induced hippocampal long term potentiation, one of the best characterized forms of synaptic plasticity. Tetanization resulted in time-dependent change in S100B gene expression and protein content in hippocampal CA1 area. We analyzed the promoter region of the rat S100B gene and identified response elements for the tumor suppressor p53. ChIP assay revealed that p53 could bind to putative p53-binding sites of the S100B promoter. The time-dependent recruitment of p53 to its putative binding sites in the S100B gene promoter paralleled the time-course change of S100B mRNA and protein levels. Thus, these results strongly support the view that S100B gene may be a target of p53. Moreover, we demonstrated that the increase of S100B protein content was accompanied with the decrease of p53 protein content, and it seems that the decrease is regulated on post-translational level. Thus, our results may help to understand the physiological function of the p53-S100B-p53 loop in the process of synaptic plasticity.

A Comparison of the Dynamics of S100B, S100A1, and S100A6 mRNA Expression in Hippocampal CA1 Area of Rats during Long-Term Potentiation and after Low-Frequency Stimulation.

The interest in tissue- and cell-specific S100 proteins physiological roles in the brain remains high. However, necessary experimental data for the assessment of their dynamics in one of the most important brain activities, its plasticity, is not sufficient. We studied the expression of S100B, S100A1, and S100A6 mRNA in the subfield CA1 of rat hippocampal slices after tetanic and low-frequency stimulation by real-time PCR. Within 30 min after tetanization, a 2-4 fold increase of the S100B mRNA level was observed as compared to the control (intact slices) or to low-frequency stimulation. Subsequently, the S100B mRNA content gradually returned to baseline. The amount of S100A1 mRNA gradually increased during first hour and maintained at the achieved level in the course of second hour after tetanization. The level of S100A6 mRNA did not change following tetanization or low-frequency stimulation.