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. 2018 Mar 2:82:1-11.
doi: 10.1016/j.pnpbp.2017.12.009. Epub 2017 Dec 13.

Repeated methamphetamine and modafinil induce differential cognitive effects and specific histone acetylation and DNA methylation profiles in the mouse medial prefrontal cortex

Betina González  1 , Subramaniam Jayanthi  2 , Natalia Gomez  1 , Oscar V Torres  3 , Máximo H Sosa  1 , Alejandra Bernardi  1 , Francisco J Urbano  4 , Edgar García-Rill  5 , Jean-Lud Cadet  2 , Verónica Bisagno  6
Affiliations

Repeated methamphetamine and modafinil induce differential cognitive effects and specific histone acetylation and DNA methylation profiles in the mouse medial prefrontal cortex

Betina González et al. Prog Neuropsychopharmacol Biol Psychiatry. .

Abstract

Methamphetamine (METH) and modafinil are psychostimulants with different long-term cognitive profiles: METH is addictive and leads to cognitive decline, whereas modafinil has little abuse liability and is a cognitive enhancer. Increasing evidence implicates epigenetic mechanisms of gene regulation behind the lasting changes that drugs of abuse and other psychotropic compounds induce in the brain, like the control of gene expression by histones 3 and 4 tails acetylation (H3ac and H4ac) and DNA cytosine methylation (5-mC). Mice were treated with a seven-day repeated METH, modafinil or vehicle protocol and evaluated in the novel object recognition (NOR) test or sacrificed 4days after last injection for molecular assays. We evaluated total H3ac, H4ac and 5-mC levels in the medial prefrontal cortex (mPFC), H3ac and H4ac promotor enrichment (ChIP) and mRNA expression (RT-PCR) of neurotransmitter systems involved in arousal, wakefulness and cognitive control, like dopaminergic (Drd1 and Drd2), α-adrenergic (Adra1a and Adra1b), orexinergic (Hcrtr1 and Hcrtr2), histaminergic (Hrh1 and Hrh3) and glutamatergic (AMPA Gria1 and NMDA Grin1) receptors. Repeated METH and modafinil treatment elicited different cognitive outcomes in the NOR test, where modafinil-treated mice performed as controls and METH-treated mice showed impaired recognition memory. METH-treated mice also showed i) decreased levels of total H3ac and H4ac, and increased levels of 5-mC, ii) decreased H3ac enrichment at promoters of Drd2, Hcrtr1/2, Hrh1 and Grin1, and increased H4ac enrichment at Drd1, Hrh1 and Grin1, iii) increased mRNA of Drd1a, Grin1 and Gria1. Modafinil-treated mice shared none of these effects and showed increased H3ac enrichment and mRNA expression at Adra1b. Modafinil and METH showed similar effects linked to decreased H3ac in Hrh3, increased H4ac in Hcrtr1, and decreased mRNA expression of Hcrtr2. The specific METH-induced epigenetic and transcriptional changes described here may be related to the long-term cognitive decline effects of the drug and its detrimental effects on mPFC function. The lack of similar epigenetic effects of chronic modafinil administration supports this notion.

Keywords: Cognition; DNA methylation; Histone acetylation; Methamphetamine; Modafinil; Prefrontal cortex.

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Figures

Figure 1:
Figure 1:. Schematic representation of repeated drug treatments, behavioral analysisand tissue sampling.
A) Locomotor analysis performed before tissue sampling for all molecular assays. This administration protocol was performed for tissue sampling of different animals groups tested for i) western blot, ii) ChIP-PCR/5-mC determination and iii) RT-PCR experiments. PND: postnatal day. B) Novel Object Recognition (NOR) task. Drug- and vehicle-treated mice were habituated to the open field arena 5 min a day for 3 consecutive days. On day 4 of wash-out, mice performed a training session in which they were allowed to freely explore two equal objects for 10 min, and 24 hrs later (day 5) performed a 5-min retention session, where one of the familiar objects was replaced by a novel object.
Figure 2:
Figure 2:. Effect of repeated modafinil (MOD) and methamphetamine (METH) treatment on behavioral sensitization.
A) Locomotor activity evaluated as distance traveled for 30 min after drug injection on Day 1 and Day 7 of treatment. Two-way ANOVA with repeated measures-Bonferroni (N=24–28). *** p<0.001 vs Vehicle, $$$ p<0.001 vs the corresponding group on Day 1. B) Baseline locomotion and time spent in center of the locomotor arena for the habituation sessions (5 min, prior to drug injections) at day 1 and day 7 of treatment. * p<0.05 vs Vehicle.
Figure 3:
Figure 3:. Novel object recognition (NOR) task after repeated treatment with modafinil (MOD) and methamphetamine (METH).
A) Training and retention sessions: ANOVA-Bonferroni (N=7–8), * Different from Vehicle, # different from MOD, p<0.05. B) Total exploration time of both objects. C) Locomotion. D) Representative captions from Ethovision files showing the mice nose path on the retention session. + indicates objects position.
Figure 4:
Figure 4:. Effect of modafinil (MOD) and methamphetamine (METH) repeated treatment on total acetylated histone 3 and 4 and 5-methylcytocine (5-mC) in the mPFC.
A) Total levels of H3ac and H4ac after 7-day treatment (washout day 4). ANOVA-Bonferroni (N=6–8). B) Global 5-mC levels in genomic DNA were measured using a commercially available ELISA kit. ANOVA-Bonferroni (N=7). * different from Vehicle p<0.05, ** different from Vehicle p<0.01, # different from MOD p<0.05, ## different from MOD p<0.01.
Figure 5:
Figure 5:. Effect of repeated modafinil (MOD) and methamphetamine (METH) treatment on the enrichment of acetylated histones 3 (A) and 4 (B) at specific promoters.
Genes evaluated: dopamine receptors Drd1 and Drd2, alpha-adrenergic α(1)AR subunits Adra1a and Adra1b, orexin receptors Hcrtr1 and Hcrtr2, histamine receptors Hrh1 and Hrh3, and glutamate receptor AMPA subunit Gria1 and NMDA subunit Grin1. (N=8–10). * Different from Vehicle p<0.05 or ** p<0.01, # different from MOD p<0.05 or ## p<0.01.
Figure 6:
Figure 6:. Effect of repeated modafinil (MOD) and methamphetamine (METH) treatment on mRNA expression by RT-PCR. (N=5–6).
* Different from Vehicle p<0.05, # different from MOD p<0.05.
Figure 7:
Figure 7:. Modafinil (MOD) and methamphetamine (METH) shared and differential histone 3 and 4 acetylation and gene expression profiles in the mPFC.
Results summary showing in blue: MOD specific effects; in red: METH specific effects; in green: MOD and METH shared effects. Upward arrow indicates increase, downward arrow indicates decrease and dash indicates no change compared to vehicle-treated controls.
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