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.

Contribution of histone acetylation to the serotonin-mediated long-term synaptic plasticity in terrestrial snails.

Serotonin plays a decisive role in long-term synaptic plasticity and long-term memory in mollusks. Previously, we demonstrated that histone acetylation is a regulatory mechanism of long-term memory in terrestrial snail. At the behavioral level, many studies were done in Helix to elucidate the role of histone acetylation and serotonin. However, the impact of histone acetylation on long-term potentiation of synaptic efficiency in electrophysiological studies in Helix has been studied only in one paper. Here we investigated effects of serotonin, histone deacetylases inhibitors sodium butyrate and trichostatin A, and a serotonergic receptor inhibitor methiothepin on long-term potentiation of synaptic responses in vitro. We demonstrated that methiothepin drastically declined the EPSPs amplitudes when long-term potentiation was induced, while co-application either of histone deacetylase inhibitors sodium butyrate or trichostatin A with methiothepin prevented the weakening of potentiation. We showed that single serotonin application in combination with histone deacetylase blockade could mimic the effect of repeated serotonin applications and be enough for sustained long-lasting synaptic changes. The data obtained demonstrated that histone deacetylases blockade ameliorated deficits in synaptic plasticity induced by different paradigms (methiothepin treatment, the weak training protocol with single application of serotonin), suggesting that histone acetylation contributes to the serotonin-mediated synaptic plasticity.

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.

Histone deacetylase inhibitors rescue the impaired memory in terrestrial snails.

It is becoming increasingly clear that the long-term plasticity can be regulated via histone modifications. Many studies demonstrated the role of histone acetylation in acquisition, maintenance, and extinction of long-term memory. Nonetheless, the role of histone acetylation in memory reinstatement following its disruption by antimnemonic treatments was not studied in details. In terrestrial snails, we examined effects of the histone deacetylases inhibitors (HDACi) sodium butyrate (NaB) and trichostatin A (TSA) on reinstatement of the context fear memory impaired by antimnemonic agents such as protein synthesis blocker anisomycin (ANI) + reminding or a specific inhibitor of protein-kinase Mζ, zeta inhibitory peptide (ZIP). It was observed that both NaB and TSA applications restored the ANI-impaired context memory regardless of memory reactivation, while a combination of NaB or TSA plus memory reactivation (or additional training) was necessary for the effective reinstatement of the ZIP-impaired memory. Additionally, NaB injections significantly facilitated development of long-term memory in animals with weak memory, while no effect was observed in animals with strong memory. The data obtained confirmed the assumption that histone acetylation is a critical regulatory component of memory development and reinstatement.

Increase in serotonin precursor levels reinstates the context memory during reconsolidation.

In the present study, we tested possible ways of modification of the context long-term memory using the reconsolidation as a tool. Recently, using a depletion of the serotonin content, it was shown that the reinforcing neurotransmitter serotonin is necessary for successful repeated reconsolidation of context memory in terrestrial snails Helix lucorum (Balaban et al. in Sci Rep 6:36933, 2016), and in the present study, we investigated effects of serotonin increase in memory maintenance by injection of the serotonin precursor 5-hydroxytryptophan (5-HTP). We studied reinstatement of the context memory after its impairment during reconsolidation with a protein synthesis blocker anisomycin (ANI) or with a specific inhibitor of protein-kinase Mζ (ZIP). It was observed that applications of 5-HTP alone, known to increase the release of serotonin, or reactivation of memory alone did not restore the ZIP- or ANI-impaired context memory, while combination of the 5-HTP + reactivation of memory effectively reinstated the context memory. The data obtained confirmed the assumption that serotonin/reinforcing transmitter is a part of successful reconsolidation necessary for memory maintenance, demonstrated possible ways of long-term memory regulation during the reconsolidation process.

Adaptive Changes in the Vestibular System of Land Snail to a 30-Day Spaceflight and Readaptation on Return to Earth.

The vestibular system receives a permanent influence from gravity and reflexively controls equilibrium. If we assume gravity has remained constant during the species' evolution, will its sensory system adapt to abrupt loss of that force? We address this question in the land snail Helix lucorum exposed to 30 days of near weightlessness aboard the Bion-M1 satellite, and studied geotactic behavior of postflight snails, differential gene expressions in statocyst transcriptome, and electrophysiological responses of mechanoreceptors to applied tilts. Each approach revealed plastic changes in the snail's vestibular system assumed in response to spaceflight. Absence of light during the mission also affected statocyst physiology, as revealed by comparison to dark-conditioned control groups. Readaptation to normal tilt responses occurred at ~20 h following return to Earth. Despite the permanence of gravity, the snail responded in a compensatory manner to its loss and readapted once gravity was restored.

Cued memory reconsolidation in rats requires nitric oxide.

It is well-known that the reactivation of consolidated fear memory under boundary conditions of novelty and protein synthesis blockade results in an impairment of memory, suggesting that the reactivated memory is destabilized and requires synthesis of new proteins for reconsolidation. We tested the hypothesis of nitric oxide (NO) involvement in memory destabilization during the reconsolidation process in rats using memory reactivation under different conditions. We report that administration of NO-synthase selective blockers 3-Br-7-NI or ARL in the conditions of reactivation of memory under a protein synthesis blockade prevented destabilization of fear memory to the conditioned stimulus. Obtained results support the role of NO signaling pathway in the destabilization of existing fear memory triggered by reactivation, and demonstrate that the disruption of this pathway during memory reconsolidation may prevent changes in long-term memory.

Impairment of the serotonergic neurons underlying reinforcement elicits extinction of the repeatedly reactivated context memory.

We analyzed changes in the activity of individually identifiable neurons involved in the networks underlying feeding and withdrawal behaviors in snails before, during, and after aversive learning in vitro. Responses to food in the "reinforcing" serotonergic neurons involved in withdrawal changed significantly after training, implying that these serotonergic cells participate in the reactivation of memory and are involved in the reconsolidation process. In behavioral experiments it was shown that impairment of the functioning of the serotonergic system with the selective neurotoxin 5,7-DiHT did not change the memory, when tested once, but resulted in a complete extinction of the contextual memory after repeated reactivation of memory. Conversely, the cued memory to a specific type of food was significantly reduced but still present. Thus, we conclude that it is only for the context memory, that participation of the "reinforcing" serotonergic neurons in memory retrieval may be the gate condition for the choice between extinction/reconsolidation.

Homolog of protein kinase Mζ maintains context aversive memory and underlying long-term facilitation in terrestrial snail Helix.

It has been shown that a variety of long-term memories in different regions of the brain and in different species are quickly erased by local inhibition of protein kinase Mζ (PKMζ), a persistently active protein kinase. Using antibodies to mammalian PKMζ, we describe in the present study the localization of immunoreactive molecules in the nervous system of the terrestrial snail Helix lucorum. Presence of a homolog of PKMζ was confirmed with transcriptomics. We have demonstrated in behavioral experiments that contextual fear memory disappeared under a blockade of PKMζ with a selective peptide blocker of PKMζ zeta inhibitory peptide (ZIP), but not with scrambled ZIP. If ZIP was combined with a "reminder" (20 min in noxious context), no impairment of the long-term contextual memory was observed. In electrophysiological experiments we investigated whether PKMζ takes part in the maintenance of long-term facilitation (LTF) in the neural circuit mediating tentacle withdrawal. LTF of excitatory synaptic inputs to premotor interneurons was induced by high-frequency nerve stimulation combined with serotonin bath applications and lasted at least 4 h. We found that bath application of 2 × 10(-6) M ZIP at the 90th min after the tetanization reduced the EPSP amplitude to the non-tetanized EPSP values. Applications of the scrambled ZIP peptide at a similar time and concentration didn't affect the EPSP amplitudes. In order to test whether effects of ZIP are specific to the synapses, we performed experiments with LTF of somatic membrane responses to local glutamate applications. It was shown earlier that serotonin application in such an "artificial synapse" condition elicits LTF of responses to glutamate. It was found that ZIP had no effect on LTF in these conditions, which may be explained by the very low concentration of PKMζ molecules in somata of these identified neurons, as evidenced by immunochemistry. Obtained results suggest that the Helix homolog of PKMζ might be involved in post-induction maintenance of long-term changes in the nervous system of the terrestrial snail.

Nitric oxide is necessary for labilization of a consolidated context memory during reconsolidation in terrestrial snails.

Nitric oxide (NO) is known to be involved in associative memory formation. We investigated the influence of blocking NO function on the reconsolidation of context memory in terrestrial snails (Helix lucorum L.). After a 10 day session of electric shocks in one context only, context memory in snails was observed in test sessions as the significant difference of amplitudes of withdrawal responses to tactile stimuli in two different contexts. After a 1 day rest, a session of 'reminding' was performed, preceded by injection in different groups of the snails with either vehicle or combination of the protein synthesis blocker anisomycin (ANI) with one of the following drugs: the NO scavenger carboxy-PTIO, the NO-synthase inhibitors N-omega-nitro-L-arginin, nitroindazole and NG-nitro-L-arginine methyl ester hydrochloride, or the NO donor S-nitroso-N-acetyl-DL-penicillamine. Testing the context memory at different time intervals after the reminder under ANI injection showed that the context memory was impaired at 24 h and later, whereas the reminder under combined injection of ANI and each of the NO-synthase inhibitors used or the NO scavenger showed no impairment of long-term context memory. Injection of the NO donor S-nitroso-N-acetyl-DL-penicillamine with or without reminder had no effect on context memory. The results obtained demonstrated that NO is necessary for labilization of a consolidated context memory.