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

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 as a learning model.

Sirt1 Regulates p53 Stability and Expression of Its Target S100B during Long-Term Potentiation in Rat Hippocampus.

Induction of long-term potentiation in rat hippocampus was followed by short-term activation of transcription factor p53 and its subsequent degradation. We studied the effects of EX-527 (inhibitor of deacetylase Sirt1, a negative regulator of p53) and pifi thrin-β (inhibitor of p53-dependent transcription) on the levels of p53 protein and mRNA of its target gene S100B during long-term potentiation. Pifi thrin-β limited the increase in S100B mRNA content after tetanization, which confi rmed signifi cant contribution of p53 in the regulation of S100B during long-term potentiation.

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.

[Regulation of S100B expression during long term potentiation].

In this study, contributions of intracellular regulatory cascades in the induction of S100B expression in rat hippocampal CA1 area during long term posttetanic potentiation (LTP) were estimated. The activation of transcription factor p53 (positive regulator of S100B transcription) by nutlin-3 increased the basal content of S100B mRNA up to 151% of the control level, which was significantly lower than its content in tetanized slices (280%). Therefore, p53 seems to be not unique transcription factor upregulating S100B expression during LTP.

Mdm2-dependent regulation of p53 expression during long-term potentiation.

Induction of long-term posttetanic potentiation in rat hippocampal CA1 filed was followed by a decrease in the content of transcription factor p53 against the background of unchanged level of p53 mRNA, which implies activation of negative regulators of p53. Mdm2 is an important regulator of p53. We studied the effects of Mdm2 inhibitor nutlin-3 on p53 expression during generation of long-term posttetanic potentiation.

Expression of Bcl2 family genes in the early phase of long-term potentiation.

Expression of Bcl2 family genes was studied during the early phase of long-term potentiation in the CA1 field of rat hippocampal slices. The level of Bax mRNA and protein increased, while the content of Bcl2 mRNA and protein decreased 30 min after tetanization of the Schaffer collaterals. Our results suggest that proteins of the Bcl2 family play a role in the mechanisms of synaptic plasticity.

Induction of S100B gene expression in long-term potentiation in the hippocampal CA1 field depends on activity of NMDA receptors.

The effects of NMDA receptor blocker MK-801 on the increase in S100B protein mRNA content induced by long-term posttetanic potentiation in the hippocampal sections were studied. The level of S100B mRNA after 30-min tetanization in the presence of 10 μM MK-801 constituted 132% of the basal level, which was significantly (226%) lower than the control level. Hence, gene expression, induced by long-term posttetanic potentiation, in the glial cells (similarly as in the neurons) depended significantly on NMDA receptors.

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.

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.

Time course of expression of "early" genes during long-term posttetanic potentiation in rat hippocampal CA1 field.

The expression of egr-1, junB, c-jun, and c-fos genes in rat hippocampal CA1 field was studied by the real-time PCR 30, 60, and 120 min after induction of long-term posttetanic potentiation. The content of egr-1, junB, and c-jun mRNA gradually increased and doubled 120 min after tetanization. The increase in c-jun mRNA level lagged behind the increment of egr-1 and junB.

[Neuronal plasticity and gene expression].

Neuronal plasticity--a fundamental feature of brain--provides adequate interactions with dynamic environment. One of the most deeply investigated forms of the neuronal plasticity is a long-term potentiation (LTP)--a phenomenon underlying learning and memory. Signal paths activated during LTP converge into the nuclear of the neuron, giving rise to launch of the molecular-genetic programs, which mediate structural and functional remodeling of synapses.

Expression of S100B and S100A6 genes during long-term posttetanic potentiation in the hippocampus.

The expression of S100B and S100A6 mRNA in CA1 region of rat hippocampal sections was studied after tetanizing stimulation. The level of S100B expression increased 2-4-fold in comparison with the control after 30 min and gradually returned to the basal level 120 min after tetanization. The level of S100A6 mRNA was very low and did not change after tetanization.

Epileptiform activity in the hippocampus of mice with different predisposition to pinch catalepsy.

Experiments on hippocampal slices of CBA mice showed that the probability of spontaneous epileptiform discharges recorded in the field CA1 pyramidal layer is higher in animals predisposed to catalepsy compared to mice with low predisposition to catalepsy. Presumably, some factors determining predisposition to catalepsy modulate synchronization of neuronal activity in the hippocampus; this suggests using hippocampal slices as a model for studies of neurophysiological mechanisms of hereditary predisposition to catalepsy.

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.

Comparative analysis of the activation of MAP/ERK kinases in the CNS of animals with different learning abilities.

Western blot analysis was used to study the activation of MAP/ERK protein kinases responsible for controlling gene expression via phosphorylation of transcription factors CREB and ELK-1 in native common snails and animals with impaired abilities to form long-term types of conditioned aversive reflexes. Different periods of the formation of this reflex were found to be characterized by different levels of activation of MAP-ERK kinases. The extents of activation of MAP-ERK kinase cascade were different in ganglia (parietal-visceral, cerebral, and pedal) with different roles in the formation of this reflex.

[Comparative analysis of MAP/ERK-kinase activation in the CNS of animals with different capability for learning].

With the Western-blot analysis, we studied the activation of MAP/ERK protein kinases (regulating genes expression by the phosphorylation of transcription factors (TF) CREB and ELK-1) in native Helix pomatia and in animals with disturbed abilities to form long-term conditioned avoidance reflexes. Different stages of this reflex formation were shown to be characterized by different levels of MAP/ERK kinases activation, the latter being different in CNS's regions (visceral complex of ganglia, cerebral and pedal ganglia) playing different roles in the reflex formation. The dynamics of activation was wave-shaped with peaks at 10 min and 4 h.

Formation of AP-1 transcription factors during learning in Helix.

Gel shift assays were used to study the formation of transcription factors of the AP-1 family in the CNS of Helix during the acquisition of a conditioned defensive reflex based on food aversion. Increases in the DNA-binding activity of AP-1 factors were seen 1-3 h after training. Modeling of "learning" in an in situ system (incubation of the CNS in the presence of serotonin and the Ca2+ ionophore A23187 or the protein kinase C activator phorbol ester (TPA)) also increased the DNA-binding activity of this transcription complex.

[Formation of transcription factors AP-1 during learning in Helix].

An increase in the DNA-binding activity for the AP-1-family factors was shown to occur after food aversion conditioning. Learning experiments in vitro enhanced the DNA-binding activity. Effects of simultaneous action of serotonine and calcium on activation of transcriptional AP-1 factors was suppressed by inhibitor of calcium/calmodulin-dependent protein-kinases KN62 as well as by inhibitor of MAP-kinases PD98059.

[The activation of the transcription factors of the CRE and AP-1 families correlates with the development of plasticity in Helix].

By means of gel retardation assay the DNA-binding activity of transcription factors of CRE and AP-1 families was determined in adult and juvenile (incapable for conditioning) Helix snails. The presence of DNA-binding factors of the CRE and AP-1 families was shown in adult snails. The active factors only of the CRE family were present in juveniles.

Role of G-proteins and second messenger systems in the plasticity of a defensive reflex in the common snail.

Enzymatic methods were used to demonstrate an increase in the activity of G-proteins and protein kinase A in the brain of the common snail at early stages of learning. There were no differences in the activity of G-proteins in the brain between young (unable to learn) and adult snails. Snail brain protein kinase C activity was unchanged compared to controls 20-40 minutes after the end of the training procedure.

Conditioned defensive reflex in the edible snail (molecular-genetic aspects).

The expression of the early genes c-fos and c-jun were studied by blot hybridization in the central nervous system of the edible snail at the consolidation stage of a conditioned defensive reflex, with the aim of investigating genomic activity in neurons during learning. The c-fos gene was shown to be present in the Helix central nervous system, and its expression was shown to increase significantly during learning. Superinduction of the c-fos gene was observed in the presence of cycloheximide, a protein synthesis inhibitor.

[The role of G-proteins and second messenger systems in the plasticity of the avoidance reflex in the snail].

An increase in the activity of G-proteins and protein kinase A was shown in the brain of Helix at the early stages of learning. The protein kinase C activity did not differ from the baseline in 20-40 min after training. There was no difference between the brain G-protein activities in the young (unable to learn) and adult snails.

[The conditioned defensive reflex in the snail (the molecular genetic aspects)].

A considerable increase in the RNA amount in the course of learning was revealed in the Heix mollusk. The same chemicals were found to induce expression of the c-fos both in higher vertebrated and in the snail thus suggesting similar mechanisms of the regulation. The c-jun in the Helix CNS seems to take no part in the learning process.

[The neurochemical correlates of plasticity].

Water-soluble proteins in the CNS of Helix pomatia were studied by the method of disk electrophoresis in polyacrylamide gel. The protein Rf 0.58 proved to be absent in young snails, while in adults its amount varied from 3% (naive snails) to 6-10% (trained animals) of the proteins separated by the method used.