DNA Methylation Inhibition Reversibly Impairs the Long-Term Context Memory Maintenance in Helix.

This work aims to study the epigenetic mechanisms of regulating long-term context memory in the gastropod mollusk: Helix. We have shown that RG108, an inhibitor of DNA methyltransferase (DNMT), impaired long-term context memory in snails, and this impairment can be reversed within a limited time window: no more than 48 h. Research on the mechanisms through which the long-term context memory impaired by DNMT inhibition could be reinstated demonstrated that this effect depends on several biochemical mechanisms: nitric oxide synthesis, protein synthesis, and activity of the serotonergic system. Memory recovery did not occur if at least one of these mechanisms was impaired. The need for the joint synergic activity of several biochemical systems for a successful memory rescue confirms the assumption that the memory recovery process depends on the process of active reconsolidation, and is not simply a passive weakening of the effect of RG108 over time. Finally, we showed that the reactivation of the impaired memory by RG108, followed by administration of histone deacetylase inhibitor sodium butyrate, led to memory recovery only within a narrow time window: no more than 48 h after memory disruption.

Detailed Analysis of Dorsal-Ventral Gradients of Gene Expression in the Hippocampus of Adult Rats.

We performed RNA sequencing of the dorsal and ventral parts of the hippocampus and compared it with previously published data to determine the differences in the dorsoventral gradients of gene expression that may result from biological or technical variability. Our data suggest that the dorsal and ventral parts of the hippocampus differ in the expression of genes related to signaling pathways mediated by classical neurotransmitters (glutamate, GABA, monoamines, etc.) as well as peptide and Wnt ligands. These hippocampal parts also diverge in the expression of axon-guiding molecules (both receptors and ligands) and splice isoforms of genes associated with intercellular signaling and cell adhesion. Furthermore, analysis of differential expressions of genes specific for astrocytes, microglia, oligodendrocytes, and vascular cells suggests that non-neuronal cells may also differ in the characteristics between hippocampal parts. Analysis of expression of transposable elements showed that depletion of ribosomal RNA strongly increased the representation of transposable elements in the RNA libraries and helped to detect a weak predominance of expression of these elements in the ventral hippocampus. Our data revealed new molecular dimensions of functional differences between the dorsal and ventral hippocampus and points to possible cascades that may be involved in the longitudinal organization of the hippocampus.

Sodium butyrate as a selective cognitive enhancer for weak or impaired memory.

Several recent studies showed that memory can be modulated by manipulating chromatin modifications using histone deacetylase (HDAC) inhibitors during memory formation, consolidation, and reconsolidation. We used a context fear conditioning paradigm with minimal non-painful current as a reinforcement, what elicited alertness to the context and freezing during tests in rats. Such paradigm resulted in a relatively weak memory in significant part of the rats. Here, we demonstrate that intraperitoneal administration of the HDAC inhibitor sodium butyrate immediately following memory reactivation, produced memory enhancement in rats with weak memory, however, not in rats with strong memory. Additionally, we investigated the ability of the HDAC inhibitor sodium butyrate to restore the contextual memory impaired due to the blockade of protein synthesis during memory reactivation. The results obtained evidence that the HDAC inhibitor sodium butyrate reinstated the impaired contextual memory. This enhancement effect is consistent with other studies demonstrating a role for HDAC inhibitors in the facilitation of contextual fear.

Experiments with Snails Add to Our Knowledge about the Role of aPKC Subfamily Kinases in Learning.

Protein kinase Mζ is considered important for memory formation and maintenance in different species, including invertebrates. PKMζ participates in multiple molecular pathways in neurons, regulating translation initiation rate, AMPA receptors turnover, synaptic scaffolding assembly, and other processes. Here, for the first time, we established the sequence of mRNA encoding PKMζ homolog in land snail Helix lucorum. We annotated important features of this mRNA: domains, putative capping sites, translation starts, and splicing sites. We discovered that this mRNA has at least two isoforms, and one of them lacks sequence encoding C1 domain. C1 deletion may be unique for snail because it has not been previously found in other species. We performed behavioral experiments with snails, measured expression levels of identified isoforms, and confirmed that their expression correlates with one type of learning.

Responses of Withdrawal Interneurons to Serotonin Applications in Naïve and Learned Snails Are Different.

Long-term changes in membrane potential after associative training were described previously in identified premotor interneurons for withdrawal of the terrestrial snail Helix. Serotonin was shown to be a major transmitter involved in triggering the long-term changes in mollusks. In the present study we compared the changes in electrophysiological characteristics of identifiable premotor interneurons for withdrawal in response to bath applications of serotonin (5-HT) or serotonin precursor 5-hydroxytryptophan (5-HTP) in preparations from naïve, neurotoxin-injected or associatively trained snails. It was found that 5-HT or 5-HTP applications caused a significant decrease of membrane potential in premotor interneurons of naïve snails, associatively trained snails and snails with impaired serotonergic system by injection of a selective neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) 1 week before the experiments. Applications of 5-HT or 5-HTP did not cause significant changes in the action potential (AP) threshold potential of these neurons in naïve snails. Conversely, applications of 5-HT or 5-HTP to the premotor interneurons of previously trained or 5,7-DHT-injected snails caused a significant increase in the firing threshold potential in spite of a depolarizing shift of the resting membrane potential. Results demonstrate that responsiveness of premotor interneurons to extracellularly applied 5-HT or 5-HTP changes for days after the associative training or serotonin depletion. Similarity of the effects in trained and 5,7-DHT-injected animals may be due to massive release of serotonin elicited by 5,7-DHT injection. Our results suggest that serotonin release due to aversive conditionining or elicited by the neurotoxin administration triggers similar changes in resting membrane potential and AP threshold in response to bath applications of 5-HT or its precursor 5-HTP.

Modulation of defensive reflex conditioning in snails by serotonin.

Highlights Daily injection of serotonin before a training session accelerated defensive reflex conditioning in snails.Daily injection of 5-hydroxytryptophan before a training session in snails with a deficiency of serotonin induced by the "neurotoxic" analog of serotonin 5,7-dihydroxytryptamine, restored the ability of snails to learn.After injection of the "neurotoxic" analogs of serotonin 5,6- and 5,7-dihydroxytryptamine as well as serotonin, depolarization of the membrane and decrease of the threshold potential of premotor interneurons was observed. We studied the role of serotonin in the mechanisms of learning in terrestrial snails. To produce a serotonin deficit, the "neurotoxic" analogs of serotonin, 5,6- or 5,7-dihydroxytryptamine (5,6/5,7-DHT) were used. Injection of 5,6/5,7-DHT was found to disrupt defensive reflex conditioning. Within 2 weeks of neurotoxin application, the ability to learn had recovered. Daily injection of serotonin before a training session accelerated defensive reflex conditioning and daily injections of 5-HTP in snails with a deficiency of serotonin induced by 5,7-DHT restored the snail's ability to learn. We discovered that injections of the neurotoxins 5,6/5,7-DHT as well as serotonin, caused a decrease in the resting and threshold potentials of the premotor interneurons LPa3 and RPa3.