Addictive substances mediate positive and negative states promoting persistent drug use. However, substrates for aversive effects of drugs remain elusive. We found that, in mouse lateral habenula (LHb) neurons targeting the rostromedial tegmental nucleus, cocaine enhanced glutamatergic transmission, reduced K+ currents and increased excitability. GluA1 trafficking in LHb was instrumental for these cocaine-evoked modifications and drug-driven aversive behaviors. Altogether, our results suggest that long-lasting adaptations in LHb shape negative symptoms after drug taking.

Genes are generally expressed from their two alleles, except in some particular cases such as random inactivation of one of the two X chromosomes in female mammals or imprinted genes which are expressed only from the maternal or the paternal allele. A lesser-known phenomenon is random monoallelic expression (RME) of autosomal genes, where genes can be stably expressed in a monoallelic manner, from either one of the parental alleles. Studies on autosomal RME face several challenges. First, RME that is based on epigenetic mechanisms has to be distinguished from biased expression of one allele caused by a DNA sequence polymorphism in a regulatory element. Second, RME should not be confused with transient monoallelic expression often observed in single cell analyses, and that often corresponds to dynamic bursting of expression. Thanks to analyses on clonal cell populations, the existence of RME in cultured cells is now well established. Future studies of RME in vivo will have to overcome tissue heterogeneity and certain technical limitations. Here, we discuss current knowledge on autosomal RME, as well as possible mechanisms controlling these expression patterns and potential implications for development and disease, drawing parallels with what is known for X-chromosome inactivation, a paradigm of random monoallelic expression.

Drugs of abuse, such as cocaine, induce changes in gene expression and epigenetic marks including alterations in histone posttranslational modifications in striatal neurons. These changes are thought to participate in physiological memory mechanisms and to be critical for long-term behavioral alterations. However, the striatum is composed of multiple cell types, including two distinct populations of medium-sized spiny neurons, and little is known concerning the cell-type specificity of epigenetic modifications. To address this question we used bacterial artificial chromosome transgenic mice, which express EGFP fused to the N-terminus of the large subunit ribosomal protein L10a driven by the D1 or D2 dopamine receptor (D1R, D2R) promoter, respectively. Fluorescence in nucleoli was used to sort nuclei from D1R-or D2R-expressing neurons and to quantify by flow cytometry the cocaine-induced changes in histone acetylation and methylation specifically in these two types of nuclei. The two populations of medium-sized spiny neurons displayed different patterns of histone modifications 15 min or 24 h after a single injection of cocaine or 24 h after seven daily injections. In particular, acetylation of histone 3 on Lys 14 and of histone 4 on Lys 5 and 12, and methylation of histone 3 on Lys 9 exhibited distinct and persistent changes in the two cell types. Our data provide insights into the differential epigenetic responses to cocaine in D1R-and D2R-positive neurons and their potential regulation, which may participate in the persistent effects of cocaine in these neurons. The method described should have general utility for studying nuclear modifications in different types of neuronal or nonneuronal cell types.addiction | psychostimulant drugs | chromatin | nucleus | fluorescence-activated cell sorting I n both humans and rodents, repeated self-administration of addictive substances can lead to a state of addiction characterized by a loss of control over drug use, with compulsive selfadministration despite its severe negative consequences (1). The addictive state is persistent, with drug craving and relapse occurring even after decades of abstinence. All addictive drugs share the ability to increase extracellular dopamine (DA) levels in the ventral striatum (2) and are thought to exert their longlasting effects by "hijacking" the normal reward systems, including the mesolimbic-striatal DA pathway and the nucleus accumbens (NAc) (3-5).A single exposure to psychostimulants triggers long-lasting alterations in behavior and synaptic physiology (6), revealing their powerful control of neuronal plasticity. Repeated exposures induce further changes that contribute to the occurrence of addiction (7-10). Growing evidence suggests that long-lasting changes in the properties of striatal neurons involve epigenetic processes (11). In particular, histone posttranslational modifications (PTMs), including phosphorylation, acetylation, and methylation play a role in promoting transcriptional alterations following cocaine administration ...

Environmental enrichment has multiple effects on behaviour, including modification of responses to psychostimulant drugs mediated by striatal neurons. However, the underlying molecular and cellular mechanisms are not known. Here we show that DARPP-32, a hub signalling protein in striatal neurons, interacts with adducins, which are cytoskeletal proteins that cap actin filaments' fast-growing ends and regulate synaptic stability. DARPP-32 binds to adducin MARCKS domain and this interaction is modulated by DARPP-32 Ser97 phosphorylation. Phospho-Thr75-DARPP-32 facilitates β-adducin Ser713 phosphorylation through inhibition of a cAMP-dependent protein kinase/phosphatase-2A cascade. Caffeine or 24-h exposure to a novel enriched environment increases adducin phosphorylation in WT, but not T75A mutant mice. This cascade is implicated in the effects of brief exposure to novel enriched environment on dendritic spines in nucleus accumbens and cocaine locomotor response. Our results suggest a molecular pathway by which environmental changes may rapidly alter responsiveness of striatal neurons involved in the reward system.

Long-lasting brain alterations that underlie learning and memory are triggered by synaptic activity. How activity can exert long-lasting effects on neurons is a major question in neuroscience. Signalling pathways from cytoplasm to nucleus and the resulting changes in transcription and epigenetic modifications are particularly relevant in this context. However, a major difficulty in their study comes from the cellular heterogeneity of brain tissue. A promising approach is to directly purify identified nuclei. Using mouse striatum we have developed a rapid and efficient method for isolating cell type-specific nuclei from fixed adult brain (fluorescence-activated sorting of fixed nuclei; FAST-FIN). Animals are quickly perfused with a formaldehyde fixative that stops enzymatic reactions and maintains the tissue in the state it was at the time of death, including nuclear localisation of soluble proteins such as GFP and differences in nuclear size between cell types. Tissue is subsequently dissociated with a Dounce homogeniser and nuclei prepared by centrifugation in an iodixanol density gradient. The purified fixed nuclei can then be immunostained with specific antibodies and analysed or sorted by flow cytometry. Simple criteria allow distinction of neurons and non-neuronal cells. Immunolabelling and transgenic mice that express fluorescent proteins can be used to identify specific cell populations, and the nuclei from these populations can be efficiently isolated, even rare cell types such as parvalbumin-expressing interneurons. FAST-FIN allows the preservation and study of dynamic and labile post-translational protein modifications. It should be applicable to other tissues and species, and allow study of DNA and its modifications.

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Most autosomal genes are thought to be expressed from both alleles, with some notable exceptions, including imprinted genes and genes showing random monoallelic expression (RME). The extent and nature of RME has been the subject of debate. Here we investigate the expression of several candidate RME genes in F1 hybrid mouse cells before and after differentiation, to define how they become persistently, monoallelically expressed. Clonal monoallelic expression is not present in embryonic stem cells, but we observe high frequencies of monoallelism in neuronal progenitor cells by assessing expression status in more than 200 clones. We uncover unforeseen modes of allelic expression that appear to be gene-specific and epigenetically regulated. This non-canonical allelic regulation has important implications for development and disease, including autosomal dominant disorders and opens up therapeutic perspectives.

Locomotor sensitization (LS) is an early behavioral adaptation to addictive drugs, driven by the increase of dopamine in the Nucleus Accumbens (NAc). However, the effect on accumbal population activity remains elusive. Here we used single cell calcium imaging in mice to record the activity of dopamine-1-receptor (D1R) and dopamine-2-receptor (D2R) expressing spiny projection neurons (SPNs) during cocaine LS. Acute exposure to cocaine elevated D1R SPN activity and reduced D2R SPN activity, albeit with high variability between neurons. During LS, the number of D1R and D2R neurons responding in opposite directions increased. Moreover, preventing LS by inhibition of the ERK signaling pathway decreased the number of cocaine responsive D1R SPNs, but had little effect on D2R SPNs. These results indicate that accumbal population dichotomy is dynamic and contains a subgroup of D1R SPNs that eventually drives LS. Insights into the drug-related activity dynamics provides a foundation for understanding the circuit-level addiction pathogenesis.