precise control of gene expression is essential for proper neuronal function and the integrity of the central nervous system . although several concerted mechanisms work together to control gene transcription [ 2 , 3 ] , dna methylation has drawn special interest as a cellular mechanism that is capable of adapting gene expression to environmental conditions . several studies have already established the importance of dna methylation both during development   and in adult animals , with a particularly emphasis on its involvement in learning processes and long - term potentiation ( ltp ) [ 6 , 7 ] . however , little is known regarding the mechanisms that regulate dna methylation and demethylation . this is particularly important in the adult nervous system , where the regulation of transcription can be quite dynamic and require rigorous temporal control [ 8 , 9 ] . in mammalian genomes , including that of humans , the addition of a methyl group occurs exclusively at a position 5 of the cytosine , located immediately before a guanosine ( cpg ) . an interesting fact is that only neurons , virtually absent in other cell types , exhibit multiple cph methylation sites , where h corresponds to another nucleotides , in a different context to the classical cpg dinucleotide . although the vast majority of cpgs in the mammalian genome are normally methylated and part of condensed chromatin , the regulation of gene expression through methylation / demethylation actively occurs at particular genomic regions that are enriched in sparsely methylated cpgs motifs that are known as cpgs islands . the process of dna methylation occurs through an enzymatic reaction that is catalyzed by the superfamily of dna methyltransferases ( dnmts ) . these enzymes transfer a methyl group from s - adenosylmethionine ( sam ) [ 14 , 15 ] to a cytosine , resulting in the formation of 5-methylcytosine ( 5mc ) . dnmt-3a and 3b catalyze de novo methylation , while dnmt1 is responsible for the maintenance of previously methylated sites in the adult brain . interestingly , dnmt1 is highly expressed in postmitotic neurons , suggesting an alternative role for dnmt1 . on the other hand , a recent study showed that azanucleosides inhibitors ( 5aza ) could induce dna damage , thus recruiting repair machinery and dnmt1 to double - strand cleavage sites , which could explain why these inhibitors can demethylate even in the absence of cell division . in contrast to dna methylation , the mechanism underlying demethylation involves the dna - repair system protein gadd45 and a family of proteins that includes oxygenase tetl , which oxidize 5mc to 5-hydroxymethyl , 5-formyl , or 5-carboxyl cytosine [ 8 , 20 ] . one of the main effectors of dna methylation - dependent gene regulation is methyl - cpg - binding protein 2 ( mecp2 ) , a transcriptional factor that reads the methylation of several genes and controls their expression by recruiting corepressors to their promotor region . the mecp2 gene is strongly expressed in the brain , and mutations in mecp2 have been associated with delayed neuronal maturation and neuropsychiatric disorders , including rett syndrome . in turn , mecp2 is dynamically regulated by neuronal activity mainly via the differential phosphorylation of key residues that modulate its affinity to its partners , which affects downstream gene expression and cellular responses to environmental variation [ 3 , 24 , 25 ] . few studies have explored the involvement of dna methylation and mecp2 modifications during the different temporal stages of processes that involve active gene regulation , such as synaptic plasticity . here , we approach this question by studying changes in the methylation of the reelin ( rln ) gene . this gene encodes an extracellular matrix protein that contacts postsynaptic dendritic spines via the very low - density protein receptor ( vldlr ) and the apolipoprotein e receptor 2 ( apoer2 ) . in the adult brain , rln is secreted by gabaergic interneurons and is critical for synaptic plasticity and memory formation [ 27 , 28 ] . several reports have suggested that the rln gene may be acutely regulated by dna methylation [ 29 , 30 ] and changes in the binding of mecp2 to the rln promoter . in acute hippocampal slices obtained from rats , the inhibition of dna methylation using azanucleosides inhibitors affected both the induction and the expression of schaffer collateral - ca1 pyramidal cell ltp that was induced using high frequency stimulation . we investigated the time window during which ltp is sensitive to azanucleosides inhibitors and the correlated dynamic changes in mecp2 phosphorylation and the methylation state of rln . male sprague dawley rats ( 21 days old ) were obtained from the animal facility of the university of valparaso . they were housed under standard conditions at a constant temperature and with a 12-hour light / dark cycle with food and water provided ad libitum . all experiments were performed in accordance with the guidelines of the bioethics committee of the university of valparaso for animal research for the treatment and care of animals . hippocampal slices ( 400  m ) were cut from rat brain tissues in ice - cold dissection buffer ( in mm : 212.7 sucrose , 2.6 kcl , 1.23 nah2po4 , 26 nahco3 , 10 dextrose , 3 mgcl2 , and 1 cacl2 , bubbled with a mixture of 5% co2 and 95% o2 ) . slices were incubated for 1  h at room temperature in artificial cerebrospinal fluid ( acsf , in mm : 124 nacl , 5 kcl , 1.25 nah2po4 , 26 nahco3 , 10 dextrose , 1.5 mgcl2 , and 2.5 cacl2 , continuously equilibrated with 5% co2 and 95% o2 ) . synaptic responses were evoked by stimulating the schaffer collaterals using concentric bipolar stimulating electrodes ( 0.2  ms ) , and field excitatory postsynaptic potentials ( fepsps ) were recorded using extracellular electrodes that were filled with acsf and placed in the ca1 stratum radiatum [ 32 , 33 ] . control responses were recorded using half - maximum stimulation intensity at a frequency of 0.033  hz . the stimulation consisted of four theta epochs that were delivered every 10  s. each epoch consisted of 10 trains of four pulses at 100  hz that were generated at a frequency of 0.5  hz [ 32 , 33 ] . when testing the effect of a pharmacological agent , recordings were made using slices from the same animal in two independent submersion - recording chambers ( 32  0.5c ) , one of which was superfused with vehicle - containing acst , while the other was superfused with drug - containing acsf . hippocampal slices were stabilized in oxygenated acsf ( 32c ) for 1  h and then incubated for 60  min with vehicle ( 0.001% dmso ) , actinomycin - d ( 25  m ) , to block transcription and vehicle ( 0.001% ch3cooh ) or 5-aza-2-deoxycytidine ( 5aza , 30  m ) to inhibit dna methylation . genomic dna was isolated from hippocampal ca1 microdissected tissues using a wizard genomic dna purification kit ( promega , madison , wi ) according to the manufacturer 's instructions . the dna was processed for bisulfite modifications , which indicates the conversion of nonmethylated cytosine into uracil while 5-methylcytosine remains unmodified . the bisulfite reaction was performed according to published protocols , which were modified to use small quantities of dna . briefly , dna in te buffer was denatured by adding naoh ( 3  m ) and then incubating the solution for 30  min at 42c . subsequently , sodium bisulfite ( 3.9  m , ph 5 ) , hydroquinone ( 10  mm ) , and nanopure h2o were added , and the solution was incubated at 55c for 16  h. the resulting modified dna was purified ( wizard clean - up de promega kit ) and then eluted using nuclease - free water . the modified and purified dna was used as a template for methylation - specific pcr ( msp ) targeting the intergenic region of the rln gene . -tubulin iv was used for normalization ( intergenic rln region primers : forward , 5-ggtgttaaatttttgtagtattggggac-3 , and reverse , 5-tccttaaaataatcc aacaacacgc-3. -tubulin iv primers : forward , 5-ggagagtaatatgaatga tttggtg-3 , and reverse , 5-catctccaactttccctaacctacttaa-3 ) . each reaction was amplified using the following program : one cycle at 95c for 3 minutes for initial denaturation ; 40 cycles consistent at 95c for 15 seconds for denaturation , 58.9c for 1 minute for annealing , and 72c for 30 seconds for extension . after completing 40 cycles , the amplified products were analyzed using electrophoresis on a 2% agarose gel that was stained with gelstar ( cambrex bio science rockland , inc . ) and then visualized under uv light . naive and tetanized hippocampal rat hippocampal slices were placed in 4% pfa/4% sucrose for 30 minutes and then placed in 30% sucrose . the slices were washed 3 times with pbs , embedded in medium for frozen tissue specimens ( oct ) and later sectioned at 30  m using a cryostat at 20c . free - floating sections were bathed in permeabilization / blocking buffer ( 0.7% triton x-100 ( pbs - tx ) , 0.1% sodium borohydride , and 10% goat serum ) overnight at 4c . the sections were later incubated with primary rabbit polyclonal antibodies against mecp2 that had been phosphorylated at ser-80 or at ser-421 ( dilution 1  :  200 , ecm biosciences ) or with a mouse monoclonal anti--tubulin iii antibody ( 1  :  500 , millipore ) overnight at 4c in 0.7% pbs - tx and 10% goat serum . after the sections were exposed to the primary antibodies , the sections were washed and incubated for two hours with donkey - anti - rabbit alexa fluor 546 , donkey - alexa fluor 488 anti - rabbit ( 1  :  200 ) , or donkey alexa fluor 488 anti - mouse ( 1  :  500 ) antibodies , depending on the primary antibody that was used . nuclei were stained using hoechst 33342 according to the manufacturer 's instructions ( molecular probes ) . images were obtained using a confocal microscope ( nikon eclipse c180i ) with 3 laser excitation lines and the following respective emission filters : 408  nm ( 450/35 ) , 488  nm ( 515/30 ) , and 543  nm ( 605/75 ) . fluorescence intensity was measured using the nis - elements software viewer 4.0 and the ez - c1 3.90 free viewer . the one - sample mann - whitney test was used to assess changes in the methylation state and expression of rln in the hippocampus and student 's t - test for analysis of mecp2 phosphorylation . a previous study showed that preexposing slices to dnmts capturers / inhibitors ( e.g. , 5aza and zebularine ) for 20 minutes before the induction of ltp resulted in an immediate and significant reduction in both the induction and the expression of l - ltp , suggesting that dnmts play an important role in both dna methylation and synaptic plasticity . to more specifically test the effect of blocking / capturing dnmt during ltp , we incubated slices with 5aza ( 30  m ) twenty minutes after tbs to avoid disrupting ltp induction . interestingly , exposing tetanized slices to 5aza resulted in significantly less ltp than those observed in the slices treated with vehicle and near baseline values at 1  h after drug application ( figure 1(b ) , white circles ) without a significant influence on basal synaptic transmission in the absence of tbs ( figure 1(c ) ) . given that l - ltp involves the activity - dependent regulation of gene expression [ 3638 ] , we studied the effects of dna methylation during the period when l - ltp is sensitive to inhibitors of transcription . in agreement with previous studies [ 38 , 39 ] , we found that blocking gene transcription using actinomycin - d ( 25  m ) impaired l - ltp without attenuating e - ltp and that synaptic transmission returned to baseline values at 2 - 3  h after tetanization ( figure 2(a ) ) . in contrast , in the vehicle - treated slices , ltp was maintained for three hours . surprisingly , superfusing slices with 5aza two hours after tbs had no effect on ltp ( figure 2(b ) ) , suggesting that l - ltp is modulated by dna methylation only during its early phases . the activation of gene transcription is associated with the loss of dna methylation at regulatory sequences [ 4 , 40 , 41 ] . we therefore expected that , within the two hours during which dmnt inhibition / capturing was able to block ltp , changes in dna methylation influenced gene transcription . to test this hypothesis this area of the gene is required for its neuronal activity - dependent transcription ( figure 3(a ) ) . in replicating cells , 5aza forms an irreversible complex dna - dnmt , which captures dnmt in the genome , which in turn inhibits dna methylation . 5aza is one of the azanucleosides inhibitors with the highest potency and effectiveness , used in clinical trials approved by the fda for the treatment of myelodysplastic syndrome [ 42 , 43 ] . the low effectiveness of sam competitive inhibitor compared with azanucleoside inhibitors was our reason for choosing 5aza to be used in our study , which despite the potential cytotoxic effects causes dna demethylation in neurons through a mechanism that is not yet fully established . to determine how reliable our detection was in the msp analysis , we first characterized changes in the methylation status of the rln gene in adult hippocampus slices that were treated with 5aza . microdissected ca1 tissues showed a robust decrease in methylated dna in response to inhibition with dnmt ( figures 3(b ) and 3(c ) ) . we also tested the specificity of our procedure by sequencing the amplified pcr product ( upper sequence ) and comparing it to the ncbi database rln gene ( lower sequence ) . this gave us an identity of 86% ( figure 3(d ) ) , a value that can be explained by the bisulfite modifications of unmethylated cytosines ( figure 3(d ) , black arrowheads ) . this activity was prevented at methylated cytosines in specific cpgs ( figure 3(d ) , black boxes ) . we next investigated whether rln undergoes acute changes in its methylation status in response to tbs - induced hippocampal ltp . a significantly lower amount of methylated dna was observed in microdissected ca1 tissues obtained from slices in which a robust ltp was induced ( > 2  h , figure 4(a ) ) than in naive slices ( figures 4(b ) and 4(d ) , naive = 1.0  0.046 ; l - ltp = 0.698  0.048 ; n = 5 animals , p < 0.05 , mann - whitney test ) . in combination with the decrease in the dna methylation of the rln gene following tbs - induced ltp , there were also more rln mrna transcripts in the treated slices than in the naive slices ( data not shown ) . these results demonstrate that the expression of l - ltp involves changes in the methylation of the rln gene and a correlated increase in its transcription . as expected , blocking / capturing dmnt using 5aza twenty minutes after ltp induction resulted in the methylation of the analyzed gene being significantly reduced to a level that was lower than was observed in the tetanized slices ( figures 4(c ) and 4(e ) ; veh = 1.0  0.081 ; l - ltp/5aza = 0.3945  0.0279 , n = 3 animals ; p < 0.05 ; mann - whitney test ) . to understand how neuronal activity can influence the transcription level of genes involved in ltp expression through dna methylation , we studied the levels at which mecp2 was phosphorylated at its serine 80 ( mecp2-s80 ) and serine 421 ( mecp2-s421 ) residues , both of which are known to be controlled by neuronal activity and to regulate its binding to methylated and unmethylated regions in the genome . slices were exposed to different experimental conditions and then tested with antibodies that specifically recognize the phosphorylated residues at mecp2-s80 or mecp2-s421 . immunoreactivity for mecp2-s80 was weaker in tetanized slices , which showed stable l - ltp that lasted over two hours , than in naive slices ( figures 5(a ) and 5(b ) ) , while mecp2-s421 reactivity was stronger ( figures 6(a ) and 6(b ) ) . colocalization with hoechst nuclear stain showed that antibody reactivity was limited to the nuclear region ( figures 5(a ) , 5(c ) , 6(a ) , and 6(c ) ) . incubating slices from different experimental groups with high concentrations of the immunogenic peptide to which the appropriate antibody was raised resulted in a strong decrease in fluorescence to barely detectable levels ( figures 5(c ) and 6(c ) ) . these results demonstrate the specificity of the detection method . finally , to show that the fluorescent nuclear profiles of the mecp2-s80 and mecp2-s421 antibodies were not due to an unspecific somatic signal , we compared the profiles to patterns that were observed when we used an antibody raised against -tubulin iii . these data showed that the mecp2 protein was in all cases confined to the nucleus ( figure 7(a ) ) . because 5aza is able to modify the expression of mecp2   and because dna methylation is a phenomenon that is closely associated with the ability of mecp2 to recognize these changes , we next assessed whether the presence of 5aza alters ltp- induced phosphorylation patterns . we found that , at two hours after the l - ltp induction protocol was applied , the level of immunoreactivity for s80 was lower in the tetanized slices incubated in the presence of 5aza ( figure 7(b ) ) , while the level of immunoreactivity for s421 was higher ( figure 7(c ) ) than in the vehicle - exposed slices that were not treated with 5aza . in this study , we shed light on the dynamic process through which gene expression is controlled by dna methylation during synaptic plasticity . the early phases of ltp ( e - ltp ) do not require gene transcription . however , previous studies have shown that inhibiting / capturing dnmt prior to the induction of ltp has a robust effect on e - ltp . consistent with these findings , our results show that exposure to 5aza twenty minutes after tetanization ( to avoid interfering with the induction and early phases of ltp ) resulted in significantly less ltp ( figure 1(b ) ) . recent studies have shown that dynamic methylation / demethylation cycles are involved in the transcriptional regulation of the trefoil factor 1 gene by o - estrogens in mcf-7 human cells [ 46 , 47 ] . these data indicate that cyclical changes in the dna methylation status of a gene can be a critical component of the complex machinery that controls its transcription and could be an active participant as a mechanism for activity - induced plasticity . our data demonstrate that there is a critical time window during which dna methylation processes can affect ltp maintenance . this window is temporally correlated with the time during which gene transcription is required for the late phase of ltp . these studies suggest that electrical stimulation protocols that induce plasticity activate a complex mechanism that regulates dna methylation , which can be disturbed only during the early stages of the process , before transcriptional dependency . we have shown using hippocampal slices that the methylation of the rln gene decreases in response to tetanic stimulation , an effect that persists for at least two hours after the induction of ltp . consistent with our results , pharmacological ltp induced with phorbol ester resulted in rapid demethylation of the rln promoter . newer genome - wide methods for analyzing dna methylation status have revealed that neuronal activity induces rapid and active dna modifications in brain genes that are associated with synaptic plasticity in vivo . interestingly , slices superfused with 5aza twenty minutes after the induction of ltp exhibited even less methylation than the slices incubated in the absence of an inhibitor of dnmts , suggesting that there is a synergistic effect between inhibitors of dnmt and tetanic stimulation . consistent with our data , inhibiting / capturing dnmt1 blocked hippocampus - dependent memory formation in a contextual fear - conditioning paradigm . moreover , one hour after training , animals showed significantly less rln gene methylation than the controls and returned to baseline within 24  h of training . altogether , these data suggest that the aberrant dna methylation of critical genes may explain why ltp is lost in tissues incubated with 5aza . moreover , these data highlight the highly sensitive nature of dna methylation processes during the early stages of ltp maintenance . in fact , evidence indicates that perturbing epigenetic regulatory mechanisms can have devastating effects on neuronal functions . compelling evidences have shown that dna methylation in neurons appears to be governed by different rules than other cell types , as has been suggested by other authors . important evidence supporting an unusual methylation mechanism in neurons is that brain exhibits high levels of 5-hydroxymethylation , a modification that leads to dna demethylation in the absence of cell division . we found that , two hours after the application of tetanic stimulation , the s421 residue of mecp2 was phosphorylated , while the s80 residue was less phosphorylated compared to a resting condition . furthermore , 5aza does not affect phosphorylation patterns in tetanized slices , suggesting that the mechanisms affected by the inhibitor are not related to changes in its phosphorylation . different forms of synaptic plasticity might be explained , at least in part , as interplay between calcium - dependent phosphorylation and dephosphorylation events [ 52 , 53 ] . since the first report that showed that neuronal depolarization resulted in the calcium - dependent phosphorylation of mecp2 and its subsequent release from regulatory regions of genes such as bdnf , remarkable progress has been made in exploring the roles of posttranslational modifications of mecp2 , some of which activate or inhibit transcription . in particular , we studied the phosphorylation of s421 , which is selectively expressed in neuronal tissues   and is modified by calcium influx and the subsequent activation of calcium / calmodulin - dependent protein kinase iv [ 44 , 53 ] . consistent with our data , a recent study showed that a hippocampal - dependent behavioral task increased the phosphorylation of s421 . although it was thought that the phosphorylation of s421 was related only to its selective detachment to dna , more detailed genomic distribution analyses of phospho - s421 have revealed that , under both resting and stimulated conditions , mecp2 is not released from the target sequences in the dna . therefore , the additional phosphorylation events that have been described for mecp2 must necessarily also involve the regulation of dna binding because neural activity modifies other residues on mecp2 . because it has been shown that the dephosphorylation of s80 does not necessarily coincide with the phosphorylation of s421 or vice versa , we studied the effect of the s80 residue , which is the most constitutively phosphorylated residue in resting neurons and is dephosphorylated by neuronal activity [ 44 , 53 ] . in contrast to s421 , we found that tetanic stimulation also activates unidentified calcium - dependent phosphatases that dephosphorylate the s80 residue and that this is a critical event during synaptic plasticity . functionally , the phosphorylation of s80 does not affect the overall subcellular localization of mecp2 , but it has a strong impact on the affinity of this protein for dna [ 3 , 53 ] . finally , the data presented in this work raise a number of new questions that must be addressed in the future , and although the mechanisms by which the azanucleosides inhibit dna methylation are not fully understood , its use in the future will continue [ 7 , 35 , 54 ] providing valuable information about dna methylation in synaptic plasticity , learning , and memory .
although the importance of dna methylation - dependent gene expression to neuronal plasticity is well established , the dynamics of methylation and demethylation during the induction and expression of synaptic plasticity have not been explored . here , we combined electrophysiological , pharmacological , molecular , and immunohistochemical approaches to examine the contribution of dna methylation and the phosphorylation of methyl - cpg - binding protein 2 ( mecp2 ) to synaptic plasticity . 
 we found that , at twenty minutes after theta burst stimulation ( tbs ) , the dna methylation inhibitor 5-aza-2-deoxycytidine ( 5aza ) impaired hippocampal long - term potentiation ( ltp ) . 
 surprisingly , after two hours of tbs , when ltp had become a transcription - dependent process , 5aza treatment had no effect . by comparing these results to those in naive slices , we found that , at two hours after tbs , an intergenic region of the rln gene was hypomethylated and that the phosphorylation of residue s80 of mecp2 was decreased , while the phosphorylation of residue s421 was increased . as expected , 5aza affected only the methylation of the rln gene and exerted no effect on mecp2 phosphorylation patterns . in summary 
 , our data suggest that tetanic stimulation induces critical changes in synaptic plasticity that affects both dna methylation and the phosphorylation of mecp2 . 
 these data also suggest that early alterations in dna methylation are sufficient to impair the full expression of ltp .