Epigenetic regulation of gene expression occurs in a cell type-specific manner. Current cell-type specific neuroepigenetic studies rely on cell sorting methods that can alter cell phenotype and introduce potential confounds. Here we demonstrate and validate a Nuclear Tagging and Translating Ribosome Affinity Purification (NuTRAP) approach for temporally controlled labeling and isolation of ribosomes and nuclei, and thus RNA and DNA, from specific central nervous system cell types. Analysis of gene expression and DNA modifications in astrocytes or microglia from the same animal demonstrates differential usage of DNA methylation and hydroxymethylation in CpG and non-CpG contexts that corresponds to cell type-specific gene expression. Application of this approach in LPS treated mice uncovers microglia-specific transcriptome and epigenome changes in inflammatory pathways that cannot be detected with tissue-level analysis. The NuTRAP model and the validation approaches presented can be applied to any brain cell type for which a cell type-specific cre is available.

23Epigenetic regulation of gene expression occurs in a cell type-specific manner. Current cell-type 24 specific neuroepigenetic studies rely on cell sorting methods that can alter cell phenotype and 25 introduce potential confounds. Here we demonstrate and validate a Nuclear Tagging and 26Translating Ribosome Affinity Purification (NuTRAP) approach for temporally controlled labeling 27 and isolation of ribosomes and nuclei, and thus RNA and DNA, from specific CNS cell types. 28Paired analysis of the transcriptome and DNA modifications in astrocytes and microglia 29 demonstrates differential usage of DNA methylation and hydroxymethylation in CG and non-CG 30 contexts that corresponds to cell type-specific gene expression. Application of this approach in 31 LPS treated mice uncovers microglia-specific transcriptome and epigenome changes in 32 inflammatory pathways that cannot be detected with tissue-level analysis. The NuTRAP model 33 and the validation approaches presented can be applied to any CNS cell type for which a cell 34 type-specific cre is available. 36Significant advances are being made in understanding the epigenome and its relationship with 37 gene expression in the brain 1-3 . However, the lack of approaches for paired analysis of DNA and 38RNA profiles at the cell type-specific level within the same animal is a significant limitation for the 39 field, given that epigenetic processes differ across CNS cell types at the level of chromatin 40 organization and DNA modifications 1,4 . Obtaining enriched cell populations by flow sorting 41 requires cell surface markers but these markers can change with experimental conditions and cell 42 sorting causes molecular, morphological, and functional changes, such as cell activation, that 43 could confound studies 3,5,6 . Single cell approaches 7 may overcome some of the challenges of 44 cell sorting but the scale of such studies, partial genomic coverage, restriction to only certain types 45 of endpoints, and continued potential for brain dissociation artifacts are limitations. 46This has led to development of transgenic labeling approaches to isolate RNA or DNA from 47 specific cell types. Ribosome labeling and RNA isolation methods, such as Translating Ribosome 48Affinity Purification (TRAP 8 ), and ribosome tagging (RiboTag 9 ), are gaining acceptance across 49 neuroscience studies examining the transcriptome. Similar approaches have been developed to 50 transgenically tag and allow isolation of nuclei and thus DNA (Isolation of Nuclei TAgged in 51 Specific Cell Types, INTACT) 10 . However, using separate transgenic mouse strains for DNA and 52RNA endpoints is a complicated and resource intensive approach. 53Here we describe an approach where Nuclear Tagging and Translating Ribosome Affinity 54Purification (NuTRAP) 11 is combined with well-established cell-specific inducible cre-recombinase 55 expressing systems 12,13 to perform paired transcriptomic and epigenomic analyses of specific 56 CNS cell types in a temporally controllable manner from a single mouse. ...

Dopamine neurons in the substantia nigra (SN) and ventral tegmental area (VTA) play a central role in the reinforcing properties of abused drugs including methamphetamine and cocaine. Chronic effects of psychostimulants in the SN/VTA also involve non-dopaminergic transmitters, including glutamate and the stress-related peptide corticotropin-releasing factor (CRF). In the SN/VTA, astrocytes express a variety of membrane-bound neurotransmitter receptors and transporters that influence neurotransmission. CRF receptor type 2 (CRF2) activity in the VTA is important for stressinduced relapse and drug-seeking behaviour, but the localization of its effects is incompletely understood. Here, we first identified CRF2 transcript in astrocytes of the SN/VTA using RNA-Seq in Aldh1l1;NuTRAP mice and confirmed it using in situ hybridization (RNAscope) in wild-type mice. We then used immunofluorescence to quantify the astrocytic marker protein S100β, glial-specific glutamate/aspartate transporter GLAST, and CRF2 in the SN/VTA following 12 days of treatment (i.p.) with methamphetamine (3 mg/kg), cocaine (10 mg/kg), or saline. We observed a significant decrease in GLAST immunofluorescence in brains of psychostimulant treated mice compared with saline controls. In addition, we observed increased labelling of CRF2 in drug treated groups, a decrease in the number of S100β positive cells, and an increase of co-staining of CRF2 with both S100β and tyrosine hydroxylase (dopamine neurons). Our results suggest a significant interaction between CRF2, GLAST, and astrocytes in the midbrain that emerges with repeated exposure to psychostimulants. These findings provide rationale for future investigation of astrocyte-based strategies for altering cellular and circuit function in response to stress and drug exposure.

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World-wide, nearly 40% of the adult population is classified as clinically obese. Central inflammation is highly correlated with obesity and increases morbidity and deterioration of health. The hypothalamus is a brain region that governs many facets of energy homeostasis, and melanocortins in the hypothalamus both decrease feeding and increase metabolism via melanocortin-4 receptors (MC4Rs). Although MC4Rs are present on neurons and astrocytes (aMC4R) previous work has focused almost exclusively on the neuronal population with the contribution of aMC4R on these processes largely unknown. Our objective was to determine the effects of hypothalamic aMC4R deletion on central and peripheral inflammation, as well as feeding and body weight homeostasis. Adult MC4R fl/fl mice were microinjected with an astrocyte-specific promoter driving Cre-expression (AAV-GFAP-GFP-Cre) or AAV-control (AAV-GFAP-GFP; n=4-7/group/sex) to produce a hypothalamic knock-down of aMC4R (KD). Body weight and composition were monitored throughout the study, and indirect calorimetry was conducted at 1 and 4 weeks after AAV injection. Acquisition of operant self-administration of palatable food was also examined. Mice were euthanized 7-8 weeks post AAV injection and brain and tissue samples were collected. We observed a significant increase in body weight, feeding, and energy balance in the KD group compared to control group. Inflammation was significantly increased centrally in KD mice within the hypothalamus, but not peripherally within serum. Additionally, aMC4R KD mice trended towards an increased reward learning for palatable food. This is the first demonstration that hypothalamic aMC4R, independent of neuronal MC4R, is important in modulating inflammation as well as contributing to energy balance. These results provide an integral understanding of the aMC4R system that will provide the foundation for future studies investigating the role of aMC4R in various disease states.