5-HT2A Receptor Knockout Mice Show Sex-Dependent Differences following Acute Noribogaine Administration.

Noribogaine (noribo) is the primary metabolite from ibogaine, an atypical psychedelic alkaloid isolated from the root bark of the African shrub Tabernanthe iboga. The main objective of this study was to test the hypothesis that molecular, electrophysiological, and behavioral responses of noribo are mediated by the 5-HT2A receptor (5-HT2AR) in mice. In that regard, we used male and female, 5-HT2AR knockout (KO) and wild type (WT) mice injected with a single noribo dose (10 or 40 mg/kg; i.p.). After 30 min., locomotor activity was recorded followed by mRNA measurements by qPCR (immediate early genes; IEG, glutamate receptors, and 5-HT2AR levels) and electrophysiology recordings of layer V pyramidal neurons from the medial prefrontal cortex. Noribo 40 decreased locomotion in male, but not female WT. Sex and genotype differences were observed for IEG and glutamate receptor expression. Expression of 5-HT2AR mRNA increased in the mPFC of WT mice following Noribo 10 (males) or Noribo 40 (females). Patch-clamp recordings showed that Noribo 40 reduced the NMDA-mediated postsynaptic current density in mPFC pyramidal neurons only in male WT mice, but no effects were found for either KO males or females. Our results highlight that noribo produces sexually dimorphic effects while the genetic removal of 5HT2AR blunted noribo-mediated responses to NMDA synaptic transmission.

HDAC superfamily promoters acetylation is differentially regulated by modafinil and methamphetamine in the mouse medial prefrontal cortex.

Dysregulation of histone deacetylases (HDAC) has been proposed as a potential contributor to aberrant transcriptional profiles that can lead to changes in cognitive functions. It is known that METH negatively impacts the prefrontal cortex (PFC) leading to cognitive decline and addiction whereas modafinil enhances cognition and has a low abuse liability. We investigated if modafinil (90 mg/kg) and methamphetmine (METH) (1 mg/kg) may differentially influence the acetylation status of histones 3 and 4 (H3ac and H4ac) at proximal promoters of class I, II, III, and IV HDACs. We found that METH produced broader acetylation effects in comparison with modafinil in the medial PFC. For single dose, METH affected H4ac by increasing its acetylation at class I Hdac1 and class IIb Hdac10, decreasing it at class IIa Hdac4 and Hdac5. Modafinil increased H3ac and decreased H4ac of Hdac7. For mRNA, single-dose METH increased Hdac4 and modafinil increased Hdac7 expression. For repeated treatments (4 d after daily injections over 7 d), we found specific effects only for METH. We found that METH increased H4ac in class IIa Hdac4 and Hdac5 and decreased H3/H4ac at class I Hdac1, Hdac2, and Hdac8. At the mRNA level, repeated METH increased Hdac4 and decreased Hdac2. Class III and IV HDACs were only responsive to repeated treatments, where METH affected the H3/H4ac status of Sirt2, Sirt3, Sirt7, and Hdac11. Our results suggest that HDAC targets linked to the effects of modafinil and METH may be related to the cognitive-enhancing vs cognitive-impairing effects of these psychostimulants.

Novel fluorescent-based reporter cell line engineered for monitoring homologous recombination events.

Homologous recombination (HR) faithfully restores DNA double-strand breaks. Defects in this HR repair pathway are associated with cancer predisposition. In genetic engineering, HR has been used extensively to study gene function and it represents an ideal method of gene therapy for single gene disorders. Here, we present a novel assay to measure HR in living cells. The HR substrate consisted of a non-fluorescent 3' truncated form of the eGFP gene and was integrated into the AAVS1 locus, known as a safe harbor. The donor DNA template comprised a 5' truncated eGFP copy and was delivered via AAV particles. HR mediated repair restored full-length eGFP coding sequence, resulting in eGFP+ cells. The utility of our assay in quantifying HR events was validated by exploring the impact of the overexpression of HR promoters and the siRNA-mediated silencing of genes known to play a role in DNA repair on the frequency of HR. We conclude that this novel assay represents a useful tool to further investigate the mechanisms that control HR and test continually emerging tools for HR-mediated genome editing.

Acute Regulation of the Arousal-Enhancing Drugs Caffeine and Modafinil on Class IIa HDACs In Vivo and In Vitro: Focus on HDAC7.

Psychostimulant drugs, such as modafinil and caffeine, induce transcriptional alterations through the dysregulation of epigenetic mechanisms. We have previously demonstrated that acute modafinil administration is accompanied by multiple changes in the expression of histone deacetylases (HDACs) within the mouse medial prefrontal cortex (mPFC). Herein, we compared alterations in class IIa HDACs in the mouse mPFC and dorsal striatum (DS) after a single exposure to each psychostimulant. We treated male C57BL/6 mice with modafinil (90 mg/kg, i.p.), caffeine (10 mg/kg, i.p.), or vehicle and evaluated locomotor activity. Following, we examined hdac4, hdac5, and hdac7 mRNA expression using qRT-PCR and HDAC7, pHDAC7, and pHDACs4/5/7 using Western blot. Last, we explored generalized effects in N2a cell line using modafinil (100 µM and 1 mM) or caffeine (80 µM and 800 µM). Our results indicate that modafinil had greater effects on locomotor activity compared with caffeine. qRT-PCR experiments revealed that modafinil decreased hdac5 and hdac7 mRNA expression in the DS, while caffeine had no effects. In the mPFC, modafinil increased hdac7 mRNA expression, with no effects observed for caffeine. Western blot revealed that within the DS, modafinil induced increases in HDAC7, pHDAC7, and pHDACs4/5/7 protein expression, while, in the mPFC, caffeine induced decreases in HDAC7, pHDAC7, and pHDACs4/5/7 protein levels. In vitro studies revealed that modafinil increased hdac4, hdac5, and hdac7 mRNA levels in N2a, while caffeine only increased hdac5 at a higher dose. These findings support the notion that modafinil and caffeine exert distinct regulation of class IIa HDAC family members and that these transcriptional and translational consequences are region-specific.

The effects of single-dose injections of modafinil and methamphetamine on epigenetic and functional markers in the mouse medial prefrontal cortex: potential role of dopamine receptors.

METH use causes neuroadaptations that negatively impact the prefrontal cortex (PFC) leading to addiction and associated cognitive decline in animals and humans. In contrast, modafinil enhances cognition by increasing PFC function. Accumulated evidence indicates that psychostimulant drugs, including modafinil and METH, regulate gene expression via epigenetic modifications. In this study, we measured the effects of single-dose injections of modafinil and METH on the protein levels of acetylated histone H3 (H3ac) and H4ac, deacetylases HDAC1 and HDAC2, and of the NMDA subunit GluN1 in the medial PFC (mPFC) of mice euthanized 1 h after drug administration. To test if dopamine (DA) receptors (DRs) participate in the biochemical effects of the two drugs, we injected the D1Rs antagonist, SCH23390, or the D2Rs antagonist, raclopride, 30 min before administration of METH and modafinil. We evaluated each drug effect on glutamate synaptic transmission in D1R-expressing layer V pyramidal neurons. We also measured the enrichment of H3ac and H4ac at the promoters of several genes including DA, NE, orexin, histamine, and glutamate receptors, and their mRNA expression, since they are responsive to chronic modafinil and METH treatment. Acute modafinil and METH injections caused similar effects on total histone acetylation, increasing H3ac and decreasing H4ac, and they also increased HDAC1, HDAC2 and GluN1 protein levels in the mouse mPFC. In addition, the effects of the drugs were prevented by pre-treatment with D1Rs and D2Rs antagonists. Specifically, the changes in H4ac, HDAC2, and GluN1 were responsive to SCH23390, whereas those of H3ac and GluN1 were responsive to raclopride. Whole-cell patch clamp in transgenic BAC-Drd1a-tdTomato mice showed that METH, but not modafinil, induced paired-pulse facilitation of EPSCs, suggesting reduced presynaptic probability of glutamate release onto layer V pyramidal neurons. Analysis of histone 3/4 enrichment at specific promoters revealed: i) distinct effects of the drugs on histone 3 acetylation, with modafinil increasing H3ac at Drd1 and Adra1b promoters, but METH increasing H3ac at Adra1a; ii) distinct effects on histone 4 acetylation enrichment, with modafinil increasing H4ac at the Drd2 promoter and decreasing it at Hrh1, but METH increasing H4ac at Drd1; iii) comparable effects of both psychostimulants, increasing H3ac at Drd2, Hcrtr1, and Hrh1 promoters, decreasing H3ac at Hrh3, increasing H4ac at Hcrtr1, and decreasing H4ac at Hcrtr2, Hrh3, and Grin1 promoters. Interestingly, only METH altered mRNA levels of genes with altered histone acetylation status, inducing increased expression of Drd1a, Adra1a, Hcrtr1, and Hrh1, and decreasing Grin1. Our study suggests that although acute METH and modafinil can both increase DA neurotransmission in the mPFC, there are similar and contrasting epigenetic and transcriptional consequences that may account for their divergent clinical effects.

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

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.