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Low dopamine D2 receptor expression drives gene networks related to GABA, cAMP, growth and neuroinflammation in striatal indirect pathway neurons

Open AccessPublished:September 08, 2022DOI:https://doi.org/10.1016/j.bpsgos.2022.08.010

      Abstract

      Background

      A salient effect of addictive drugs is to hijack the dopamine reward system, an evolutionarily conserved driver of goal-directed behavior and learning. Reduced dopamine type-II receptor (D2R) availability in the striatum is an important pathophysiological mechanism for addiction that is both consequential and causal for other molecular, cellular, and neuronal network differences etiologic for this disorder. Here, we sought to identify gene expression changes attributable to innate low expression of the Drd2 gene in the striatum and specific to striatal indirect medium spiny neurons (iMSNs).

      Methods

      Cre-conditional, Translating Ribosome Affinity Purification (TRAP) was used to purify and analyze the translatome (ribosome-bound mRNA) of iMSNs from mice with low/heterozygous or wild-type Drd2 expression in iMSNs. Complementary electrophysiological recordings and gene expression analysis of postmortem brain tissue from human cocaine users were performed.

      Results

      Innate low expression of Drd2 in iMSNs led to differential expression of genes involved in GABA and cAMP signaling, neural growth, lipid metabolism, neural excitability, and inflammation. Creb1 was identified as a likely upstream regulator, among others. In human brain, expression of FXYD2, a modulatory subunit of the Na/K pump, was negatively correlated with DRD2 mRNA expression. In iMSN-TRAP-Drd2HET mice, increased Cartpt and reduced S100a10 (p11) expression recapitulated previous observations in cocaine paradigms. Electrophysiology experiments supported a higher GABA tone in iMSN-Drd2HET mice.

      Conclusion

      This study provides strong molecular evidence that in addiction inhibition by the indirect pathway is constitutively enhanced through neural growth and increased GABA signaling.

      INTRODUCTION

      Addictive drugs, particularly stimulants, are associated with increased dopamine release in the striatum (
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      The heritability of SUDs ranges from 0.39 for hallucinogens to 0.72 for cocaine (
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      ). Genome wide association studies have implicated the dopamine receptor gene DRD2 in alcohol and tobacco consumption (
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      ) and other psychiatric disorders (

      23andMe Research Team, Major Depressive Disorder Working Group of the Psychiatric Genomics Consortium, Howard DM, Adams MJ, Clarke T-K, Hafferty JD, et al. (2019): Genome-wide meta-analysis of depression identifies 102 independent variants and highlights the importance of the prefrontal brain regions. Nat Neurosci 22: 343–352.

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      GWAS Catalog (n.d.): DRD2 GWAS Catalog. Retrieved from https://www.ebi.ac.uk/gwas/genes/DRD2

      ), although not driven by a frequently studied polymorphism (DRD2/ANKK1 rs1800497) (
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      ).
      D2Rs are mostly expressed on indirect pathway medium spiny neurons (iMSNs), that mediate behavioral inhibition (“no go” signal). Conversely, dopamine D1 receptors (D1Rs) are mostly expressed on direct pathway MSNs (dMSNs) that mediate reward and the initiation of behavior (“go signal”) (
      • Kravitz A.V.
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      ). In the context of addiction, sensitization and conditioned place preference to psychostimulants are generally elicited by dMSNs and inhibited by iMSNs (
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      ). Unbalanced striatal direct and indirect pathway signaling are hypothesized to be key to the pathophysiology of SUD and other neuropsychiatric disorders such as schizophrenia and obsessive-compulsive disorder, and neurological conditions such as Parkinson's and Huntington's disease. Therapies for several of these conditions target dopamine neurotransmission and alter signaling balance, partly restoring function but at times leading to troubling side effects (
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      ). For example, Parkinson’s disease is treated with levodopa, a dopamine precursor, while dopamine D2R blockade is often used to treat both schizophrenia and Huntington’s disease. Despite its centrality to SUDs, there is currently no effective treatments that target dopamine signaling.
      We previously reported that iMSNs partially or fully deficient for Drd2 exhibit decreased dMSN excitability due to increased GABAergic collateral inhibition from iMSNs (
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      ). This could contribute to addiction by increasing input needed for the striatum to select and maintain behavior, biasing the brain to pursue high-reward drug-associated cues and ignore lower intensity natural rewards.
      In this study we investigate gene expression changes in iMSNs that may explain the enhanced influence of the indirect pathway due to reduced Drd2 expression. For this, we use a genetically modified mouse with innate low expression of Drd2 selectively in iMSNs and analyze its ribosome-bound transcriptome (translatome) with normal (two functional alleles, iMSN-TRAP-Drd2WT) or heterozygous/low (one functional allele, iMSN-TRAP-Drd2HET) expression of Drd2. iMSNs account for roughly ∼12% of all cells and ∼47% of all neurons in the striatum, where the glia/neuron ratio is ∼4:1 (
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      ). Further, iMSNs and dMSNs share most of their transcriptional signatures (
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      ). Therefore, to reliably detect and quantify medium and low abundance transcripts specific to iMSNs we used Translating Ribosome Affinity Purification (TRAP) (
      • Heiman M.
      • Kulicke R.
      • Fenster R.J.
      • Greengard P.
      • Heintz N.
      Cell type–specific mRNA purification by translating ribosome affinity purification (TRAP).
      ), followed by a low-input RNA sequencing strategy and analysis.

      MATERIALS AND METHODS

      Animals

      Experimental protocols were approved by the NIAAA Animal Care and Use Committee. Animals were group-housed and kept in a standard light-dark cycle (6:00 ON – 18:00 OFF). Mice of both sexes were used and counterbalanced in all experiments. For RNAseq and qPCR validation experiments (Ntotal=48 mice, age 2-5 months), Adora2a-Cre+/- Drd2loxP/wt (iMSN-Drd2HET) mice were crossed with Cre-conditional TRAP+/+ (JAX Stock #022367) to obtain Adora2a-Cre+/- TRAP+/- Drd2loxP/wt and Drd2wt/wt (iMSN-TRAP-Drd2HET and iMSN-TRAP-Drd2WT mice respectively). Adora2a-Cre (
      • Gerfen C.R.
      • Paletzki R.
      • Heintz N.
      GENSAT BAC Cre-Recombinase Driver Lines to Study the Functional Organization of Cerebral Cortical and Basal Ganglia Circuits.
      ) (GENSAT, 036158-UCD) was used to target both the Drd2 deletion (
      • Bello E.P.
      • Mateo Y.
      • Gelman D.M.
      • Noaín D.
      • Shin J.H.
      • Low M.J.
      • et al.
      Cocaine supersensitivity and enhanced motivation for reward in mice lacking dopamine D2 autoreceptors.
      ) (Drd2loxP, JAX020631) and the TRAP system specifically to iMSNs. The Drd2 gene was knocked out by a floxed deletion of exon 2, which contains the translation start codon. For electrophysiology experiments (N=13 mice, age 4-7 months), both the Adora2a-Cre control and iMSN-Drd2HET mice were crossed with Drd1a-tdTomato (JAX Stock # 016204).

      Translating Ribosome Affinity Purification (TRAP)

      TRAP isolation of ribosome-bound mRNA from iMSNs in mouse was performed as described (
      • Heiman M.
      • Kulicke R.
      • Fenster R.J.
      • Greengard P.
      • Heintz N.
      Cell type–specific mRNA purification by translating ribosome affinity purification (TRAP).
      ) with the following differences: Matrix per whole mouse striatum: 150μl Streptavidin MyOne T1 Dynabeads (Invitrogen, #65601) and 60μl Biotinylated protein L recombinant purified (Thermo Scientific, #29997). Briefly, whole mouse striata were collected (∼40 mg/sample), snap frozen into 1.5ml tubes and stored at -80°C; homogenization was performed with 500μl of tissue-lysis buffer, Fisherbrand RNase-Free Disposable Pellet Pestles individually wrapped (Fisher Scientific, #12-141-364) and pre-chilled and a Pellet Pestle Cordless Motor (Kimble, #749540-0000). TRAP isolation was performed in 3 batches of 8 samples each (N=24 samples; 1 sample = 1 mouse) for RNAseq, and 2 batches of 12 samples each for independent qPCR validations (N = 24 samples). One sample from the RNAseq and two samples from the qPCR were excluded due to low quality. “Total” (ribosome-bound) RNA was purified from positive TRAP fractions (∼50-200 ng totRNA/sample) using PicoPure RNA Isolation Kit (Thermo Fisher Scientific, #KIT0204), always including on-column DNase I treatment to remove genomic DNA contamination. The isolated iMSN mRNA from iMSN-TRAP-Drd2HET and iMSN-TRAP-Drd2WT mice is termed here iMSND2HET and iMSND2WT respectively.

      Sequencing

      The mRNA from 50-100 ng totRNA TRAP positive fractions was amplified and converted into double stranded cDNA using Ovation RNA-seq System V2 (NuGEN, #7102-32). Quantity and quality were assessed by Qubit (dsDNA HS Assay Kit, ThermoFisher Scientific, #Q32854) and Bioanalyzer (High Sensitivity DNA Kit, Agilent Technologies, #5067-4626) respectively. ∼200 ng double stranded cDNA was sheared to ∼200 bp fragments using Covaris microTUBEs (#520045) and sonicator (Covaris S2). Sequencing library and reagents were from IonTorrent by ThermoFisher Scientific unless otherwise specified. Ion Plus Fragment Library Kit (#4471252) and Ion Xpress Barcode Adapters 1-16 Kit (#4471250) were used to construct sequencing libraries. Quantity and quality of the sequencing libraries were assessed by Qubit, Bioanalyzer and Ion Library TaqMan Quantitation Kit (#4468802). Ion P1 Hi-Q sequencing 200 Kit (#A26433) and Ion P1 Hi-Q Template OT2 Kit (#A26434) were used to template sequencing beads, and Ion P1 Chip Kit v3 (#A26771) for sequencing in an Ion Torrent Proton sequencer.

      Differentially Expressed Gene (DEG) analysis

      An average of 27 M reads (± 8 SD) were obtained per sample. Reads were mapped to mouse reference genome (mm10) and gene expression was modeled by Generalized Linear Model (GLM) using CLC Genomics Workbench (QIAGEN Bioinformatics, version 10) in default settings. Protein coding genes (21950 genes) were filtered by expression of RPKM ≥ 2, resulting in 8,332 genes considered expressed. A batch effect driven by the TRAP purification was corrected using ComBat (
      • Johnson W.E.
      • Li C.
      • Rabinovic A.
      Adjusting batch effects in microarray expression data using empirical Bayes methods.
      ) (Sup Fig 1) from Bioconductor. EdgeR (
      • Robinson M.D.
      • McCarthy D.J.
      • Smyth G.K.
      edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.
      ) was used for differential gene expression (DEG) analysis. The expression of functional Drd2 mRNA in iMSND2HET (Figure 1D) was calculated by exon2/exon3-6 ratio, exons 3-6 being non-deleted in this construct and expressed normally. Nominal p-values (i.e., unadjusted/uncorrected for multiple comparison) are denominated “p”, and adjusted p-values “padj”. False Discovery Rate (FDR = padj) was used for DEG analysis.
      Figure thumbnail gr1
      Figure 1Enrichment of iMSN translatome signal in Drd2-low expression mice. A) Experimental design. Whole striatum was microdissected from mice with WT or HET expression of Drd2 and transgenic for a GFP-ribosomal subunit fusion protein conditionally expressed in Drd2 neurons (iMSNs) for cell-specific mRNA purification. Conditional expression of the TRAP system and the floxed Drd2 allele were driven by Adora2a’s promoter. Isolation of actively translating mRNA (translatome) from iMSNs was achieved by pull down of GFP-ribosomes-associated mRNA, followed by low input pre-amplification and sequencing. Differential gene expression from RNAseq samples was validated by qPCR (Sup ) on an independent set of mice and samples. B) Quantitative enrichment analysis of iMSN signal compared to total striatum by RNAseq shows a strong enrichment in iMSN translatome signal. C) Drd2 exon signal coverage and enrichment in iMSN samples vs total striatum, visualization on Integrative Genomics Viewer (IGV, (
      • Robinson J.T.
      • Thorvaldsdóttir H.
      • Winckler W.
      • Guttman M.
      • Lander E.S.
      • Getz G.
      • Mesirov J.P.
      Integrative genomics viewer.
      )). For comparison purposes, plots were set at the same scale. D) Expression level of the full-length, functional Drd2 mRNA calculated by mRNA exon coverage signal from the RNAseq data.

      Gene ontology enrichment analysis

      The DEG analysis of the 8,332 expressed genes was further analyzed with QIAGEN Ingenuity Pathway Analysis (IPA) (
      • Krämer A.
      • Green J.
      • Pollard J.
      • Tugendreich S.
      Causal analysis approaches in Ingenuity Pathway Analysis.
      ). Given the lack of nonsense mediated decay for the knocked-out allele of Drd2 mRNA (exon 2 deletion), its expression value was divided in half before import to account for expression of the functional isoform. An IPA core analysis (based on expression and fold change) was performed using default settings except for: Reference Set = User Dataset (8,332 genes), and Set Cutoff = 0.05 p-value (unadjusted or nominal). Benjamini-Hochberg (B-H = padj) was used for enrichment analysis.

      Data sharing

      Data deposited at NCBI: BioProject PRJNA865732 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA865732).
      qPCR
      Quantification by qPCR of Drd2 mRNA expression (Figure 1D) was calculated using Ct values of exon 2-3/exon 3-4 ratio and normalized to WT Drd2 expression.
      An independent set of 24 iMSN-TRAP-Drd2WT and -HET mice was used for qPCR validation (Sup Fig 2) of RNAseq gene expression results. qPCR samples were processed similarly to RNAseq samples until the cDNA/dsDNA was obtained. Then, 10ng of double stranded cDNA per well were used in Custom TaqMan Array Fast 96-well Plates (Thermo Fisher Scientific #4413261), multiplexing with GAPDH-VIC and the gene target in -FAM. TaqMan Fast Universal PCR Master Mix (#4352042) and the QuantStudio 7 Flex Real-Time PCR System were used to run the fast 96-well plates. Two samples were excluded due to low quality. ComBat correction was used to address the qPCR TRAP-batch effect using ΔCt values.
      Figure thumbnail gr2
      Figure 2Differential gene expression in iMSND2HET. A) RNAseq bioinformatic analysis overview. B) Volcano Plot showing effect size (expression fold change) between iMSND2HET and iMSND2WT against statistical significance (red: genes with padj ≤ 0.1; yellow: p < 0.05) of all expressed genes in iMSND2HET (RPKM ≥ 2, ∼8.3k genes). C) MA Plot showing effect size against mean level of expression (counts per million, CPM) in WTs.
      Gene expression from postmortem brain tissue from subjects with cocaine abuse history
      The gene expression levels for DRD2 and FXYD2 from an independent transcriptomics study (yet to be published) on postmortem human brain from the anterior caudate and nucleus accumbens (NAc) from subjects with a severe cocaine abuse history (N = 25) and age-matched unaffected controls (N = 25) (Figure 5C) were obtained from the University of Miami Brain Endowment Bank. Details on samples and psychiatric pathology of these individuals have been reported elsewhere (
      • Vaillancourt K.
      • Yang J.
      • Chen G.G.
      • Yerko V.
      • Théroux J.-F.
      • Aouabed Z.
      • et al.
      Cocaine-related DNA methylation in caudate neurons alters 3D chromatin structure of the IRXA gene cluster.
      ). Briefly, neuropathological specimens were obtained during routine autopsy. Total RNA was extracted from ∼100 mg frozen caudate or ventral striatum, including on-column DNase I treatment. ∼500 ng totRNA/sample were sequenced at the Broad Institute (Cambridge, MA) using Illumina TruSeq library construction including poly-A selection, run as 76 bp paired-end to a depth of ∼50 million reads and aligned against Ensemble transcript reference.
      Figure thumbnail gr5
      Figure 5FXYD2, a potentially inhibitory subunit of the Na/K Pump, in the context of addiction in mouse and human. A) Fxyd2 mRNA expression fold change in iMSND2HET relative to their WT counterparts, in both RNAseq and qPCR independent samples (p=5x106 and p<1x106 respectively). B) and C) Correlated expression of FXYD2 and DRD2 mRNA in mouse and human striatum. B) Mouse Fxyd2 and Drd2 mRNA expression correlation from qPCR on TRAP purified iMSND2HET and iMSND2WT. C) Human FXYD2 and DRD2 mRNA expression correlation from whole tissue RNAseq (FPKM) from postmortem caudate of individuals with severe cocaine abuse history (n=25, red dots) and controls (n=25, black dots). Pearson correlation values r and p are plotted. D) Fxyd2 expression in mouse striatum and cortex by RNAscope suggests colocalization with Drd1 (dMSNs) and Adora2a (iMSNs) expressing striatal neurons, as well as unlabeled cells. CTX = cortex; Str = striatum. E) Cell type expression of Fxyd2 in mouse striatum from published single cell transcriptome data (
      • Gokce O.
      • Stanley G.M.
      • Treutlein B.
      • Neff N.F.
      • Camp J.G.
      • Malenka R.C.
      • et al.
      Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq.
      ) shows iMSN, dMSN and interneuron expression. Values correspond to the mean expression.

      RNAscope

      Sagittal brain slices (∼12μm thick) of WT mouse were used to detect Drd1, Adora2a and Fxyd2 gene expression in striatum. RNAscope Multiplex Fluorescent Reagent V2 (ACD #323100) was used with dyes and dilutions as follows: 1:1500 Drd1(high expression gene)-Opal520, 1:1500 Adora2a(high expression gene)-Opal570 and 1:750 Fxyd2(low expression gene)-Opal690 (Akoya Bioscience #FP1487001KT, FP1488001KT and FP1497001KT respectively), following HybEZ II Hybridization System (#321721) protocols.

      Electrophysiology

      iMSN-Drd2HET mice or its control (Adora2a-Cre) crossed with Drd1a-tdTomato mice (N = 13) were subjected to five consecutive days of cocaine (15 mg/kg, intraperitoneal injection) or saline treatment. Recordings were performed 2-5 days after the last injection. Whole-cell voltage clamp recordings were measured from D1-MSNs, tdTomato positive neurons (N = 35, mean of 9 cells / 3 mice) in the NAc core. Sagittal brain slices (240 μm) were prepared in ice-cold cutting solution, and transferred to warm (31-33 °C) artificial cerebrospinal fluid bubbled with carbogen for 30 min. tdTomato+ dMSNs were held at -55 mV using glass electrodes (2.5–3.5 MΩ) filled with internal saline (pH ∼ 7.2, ∼300 mOsm). Data were acquired using Multiclamp 700B (Molecular Devices), filtered at 1 kHz, and digitized at 5 kHz. All data were analyzed using pClamp (Clampfit, v. 10.3). For solutions see key resources table.

      RESULTS

      Drd2-low iMSN translatome from a mouse model of addiction

      iMSN-specific ribosome-bound mRNA (translatome) from a genetically diminished Drd2 expression mouse model was isolated by TRAP, amplified and sequenced (Figure 1A). Cre expression was driven by an Adora2a promoter in animals with one (HET) or no (WT) floxed alleles in exon 2 (containing the start codon) of the Drd2 gene. The amplified and sequenced TRAP positive fractions showed strong enrichment for iMSN specific transcripts (Penk 7.8-fold, Drd2 6.8-fold, Adora2a 2.6-fold) and reduced levels of non-iMSN transcripts as compared to total striatum (Figure 1B-C, Sup Fig 3). Expression of functional Drd2 mRNA in iMSND2HET was approximately 60% of iMSND2WT (Figure 1D), whilst expression of Drd2 exons other than exon 2 was unaltered, indicating lack of nonsense mediated decay of Drd2 mRNA transcribed from this construct.
      Figure thumbnail gr3
      Figure 3Creb1 and others were predicted upstream regulators of the observed expression profile in iMSND2HET. A) Upstream regulators predicted to have driven the expression changes observed in overlapping and non-overlapping subsets of genes (see also Sup Table 3) from the extended DEG list in iMSND2HET. B) Gene network showing Creb1 and forskolin (exogenous drug) targets within our dataset.

      Differentially expressed genes (DEGs) in iMSND2HET

      A total of 43 protein coding genes were differentially expressed in iMSND2HET compared to iMSND2WT (padj ≤ 0.1, p = 5x10-4), 19 being upregulated and 24 downregulated (Figure 2A-C and Table 1). To assess reproducibility, we used qPCR on TRAP-purified iMSN fractions from 24 iMSN-TRAP-Drd2HET and WT mice (independent from the 24 mice used for RNAseq). The 38 genes assessed for differential expression by both RNAseq and qPCR on independent samples were strongly correlated in both statistical significance (r = 0.55, p = 2.7x10-4) and fold-change values (r = 0.85, p = 10-12) (Sup Fig 2), supporting the reliability of the RNAseq measurements.
      Table 1Differentially Expressed Genes (DEGs) in iMSND2HET. List of 20 genes up (top) and down (bottom) regulated in iMSND2HET (padj ≤ 0.1) with relevant function notes. FC = fold change; padj = p-value adjusted for multiple testing, FDR = False Rate Discovery.
      Gene symbolFCpadj (FDR)NotesReferences
      Fxyd22.204.E-13modulatory γsubunit of the Na/K pump; associated with schizophrenia and nicotine addiction(
      • Hu Y.
      • Fang Z.
      • Yang Y.
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      • Cheng F.
      • Wang J.
      Analyzing the genes related to nicotine addiction or schizophrenia via a pathway and network based approach.
      )
      Pde1c1.492.E-06phosphodiesterase that degrades cAMP/cGMP; intronic variant associated with smoking phenotypes by GWAS(
      • Hancock D.B.
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      Human Genetics of Addiction: New Insights and Future Directions.
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      • Uhl G.R.
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      Molecular genetics of nicotine dependence and abstinence: whole genome association using 520,000 SNPs.
      )
      Igfbp61.673.E-04growth factor that activates the MAPK signaling pathway; role in neurogenesis in the hippocampus(
      • Zhang J.
      • Moats-Staats B.M.
      • Ye P.
      • D’Ercole A.J.
      Expression of insulin-like growth factor system genes during the early postnatal neurogenesis in the mouse hippocampus.
      )
      Spon11.500.001secreted extracellular protein; role in neurite growth and axon guidance; genetic variant on its locus associated with cognitive decline by GWAS(
      • Feinstein Y.
      • Klar A.
      The neuronal class 2 TSR proteins F-spondin and Mindin: a small family with divergent biological activities.
      ,
      • Sherva R.
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      • et al.
      Genome‐wide association study of the rate of cognitive decline in Alzheimer’s disease.
      )
      Nrn1l2.120.002extracellular protein; enhances neurite growth and neuronal survival
      Gad11.230.010key enzyme of GABA synthesis
      Lypd11.340.010(aka Lynx2), a modulator of nicotinic acetylcholine receptors; KO mouse model displays increased anxiety-related behavior(
      • Anderson K.R.
      • Hoffman K.M.
      • Miwa J.M.
      Modulation of cholinergic activity through lynx prototoxins: Implications for cognition and anxiety regulation.
      ,
      • Tekinay A.B.
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      A role for LYNX2 in anxiety-related behavior.
      )
      Crem1.400.020cAMP-responsive element modulator; multiple alternative-splice isoforms acting as activators and repressors of transcription; paralog to Creb1; increased expression reduced impulsive action in an attention-deficit/hyperactivity disorder (ADHD) animal model(
      • Beauvais G.
      • Jayanthi S.
      • McCoy M.T.
      • Ladenheim B.
      • Cadet J.L.
      Differential effects of methamphetamine and SCH23390 on the expression of members of IEG families of transcription factors in the rat striatum.
      ,
      • Miller M.L.
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      • et al.
      IMAGEN Consortium
      Ventral striatal regulation of CREM mediates impulsive action and drug addiction vulnerability.
      )
      Nptx21.460.039neuronal pentraxin protein involved in synapse formation, specifically AMPA receptor clustering(
      • Chapman G.
      • Shanmugalingam U.
      • Smith P.D.
      The Role of Neuronal Pentraxin 2 (NP2) in Regulating Glutamatergic Signaling and Neuropathology.
      )
      Cartpt2.190.067CART (cocaine- and amphetamine-regulated transcript protein), which modulates cocaine and dopamine effects in the striatum(
      • Kuhar M.J.
      CART Peptides and Drugs of Abuse: A Review of Recent Progress.
      ,

      Jaworski JN, Jones DC (2006): The role of CART in the reward/reinforcing properties of psychostimulants. Peptides 27: 1993–2004.

      )
      Stc1-1.902.E-11glycoprotein involved in calcium/phosphate homeostasis
      Gpx6-2.112.E-07glutathione peroxidase; neuroprotective against oxidative and other forms of stress; in a Huntington Disease mouse model, Gpx6 upregulation in striatum improved behavioral and molecular phenotypes(
      • Aoyama K.
      Glutathione in the Brain.
      ,
      • Shema R.
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      Synthetic lethal screening in the mammalian central nervous system identifies Gpx6 as a modulator of Huntington’s disease.
      )
      Fam163b-1.623.E-06membrane protein of unknown function; implicated in tobacco use disorder and stress(
      • Quach B.C.
      • Bray M.J.
      • Gaddis N.C.
      • Liu M.
      • Palviainen T.
      • Minica C.C.
      • et al.
      Expanding the genetic architecture of nicotine dependence and its shared genetics with multiple traits.
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      • et al.
      A gene expression atlas for different kinds of stress in the mouse brain.
      )
      Cnih3-1.626.E-06cornichon family AMPA receptor auxiliary protein
      Gstm6-1.869.E-06glutathione transferase; neuroprotective against oxidative and other forms of stress(
      • Aoyama K.
      Glutathione in the Brain.
      )
      Slc24a3-1.597.E-05plasma membrane potassium-dependent sodium-calcium exchanger, a druggable target for treatment; intronic variant associated with externalizing behavior by GWAS(
      • Karlsson Linnér R.
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      • et al.
      Multivariate analysis of 1.5 million people identifies genetic associations with traits related to self-regulation and addiction.
      ,
      • Uhl G.R.
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      • et al.
      Molecular Genetics of Addiction and Related Heritable Phenotypes.
      )
      Id4-1.367.E-05transcription factor inhibitor; required for the correct timing of neural differentiation(
      • Bedford L.
      • Walker R.
      • Kondo T.
      • van Crüchten I.
      • King E.R.
      • Sablitzky F.
      Id4 is required for the correct timing of neural differentiation.
      )
      Cdh13-1.383.E-04(aka T-cadherin), a GPI-anchored cell adhesion molecule; inhibits neural growth and signals through Erk pathway; impacts GABAergic function; accumulative evidence of association with SUDs from GWAS(
      • Uhl G.R.
      • Drgon T.
      • Johnson C.
      • Li C.-Y.
      • Contoreggi C.
      • Hess J.
      • et al.
      Molecular Genetics of Addiction and Related Heritable Phenotypes.
      ,
      • Mossink B.
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      • Linda K.
      • Vitale M.R.
      • Zöller J.E.M.
      • et al.
      Cadherin-13 is a critical regulator of GABAergic modulation in human stem-cell-derived neuronal networks.
      ,

      Rivero O, Selten MM, Sich S, Popp S, Bacmeister L, Amendola E, et al. (2015): Cadherin-13, a risk gene for ADHD and comorbid disorders, impacts GABAergic function in hippocampus and cognition. Transl Psychiatry 5: e655–e655.

      ,
      • Takeuchi T.
      • Misaki A.
      • Liang S.-B.
      • Tachibana A.
      • Hayashi N.
      • Sonobe H.
      • Ohtsuki Y.
      Expression of T-Cadherin (CDH13, H-Cadherin) in Human Brain and Its Characteristics as a Negative Growth Regulator of Epidermal Growth Factor in Neuroblastoma Cells.
      )
      S100a10-1.680.036calcium-binding membrane protein; involved in neurotransmitter transport; associated with depression and cocaine reward(
      • Arango-Lievano M.
      )
      Mapk3-1.200.071kinase in the Mapk/Erk pathway involved in growth (
      • Dobbs L.K.
      • Kaplan A.R.
      • Lemos J.C.
      • Matsui A.
      • Rubinstein M.
      • Alvarez V.A.
      Dopamine Regulation of Lateral Inhibition between Striatal Neurons Gates the Stimulant Actions of Cocaine.
      )
      (
      • Chapman G.
      • Shanmugalingam U.
      • Smith P.D.
      The Role of Neuronal Pentraxin 2 (NP2) in Regulating Glutamatergic Signaling and Neuropathology.
      )
      Among the DEGs (padj ≤ 0.1), several have known pathway membership and/or functions, including cAMP signaling (Pde1c, Crem, Mapk3, Drd2), calcium signaling (Stc1, Slc24a3, Kcnip3), cellular growth (Id4, Cdh13, Igfbp6, Spon1, Nrn1l, Osbpl3, Crem, Ppm1l, Fgf11), inflammation (Stc1, Cdh13, Tac1, Scg2, S100a10, Hrh3) and behavior (Tac1, Crem, Sst, Hrh3, Mapk3, Adora2a, Mptx2). See Table 1 for reported functions of 20 DEGs, Sup Fig 3 for striatal cell-type expression, and Sup Table 1 for a complete gene list and values. Gad1, which encodes for a key GABA synthesis enzyme, was upregulated (FC = 1.23, padj = 0.01 and p = 2x10-5), and Gad2 also but at trend level (FC = 1.18, padj = 0.12 and p = 7x10-4). And Cartpt (FC = 2.19, padj = 0.07 and p = 3x10-4), associated with consumption of psychostimulants, was also up in iMSND2HET.

      Gene Ontology (GO) analysis

      To identify complex biological functions impacted by innate, low Drd2 expression in iMSNs we extended the list of genes of focus to include all those with suggestive or potential association (p ≤ 0.05, n = 474 genes). Of these 474 potentially differentially expressed genes, 214 were up-regulated and 260 were down-regulated (Figure 2) and will be referred to as the “extended DEG list”.
      To identify molecules that could explain gene expression changes observed in iMSND2HET we performed upstream regulator analysis in IPA. Known Creb1 targets were strongly enriched (padj = 4x10-10, 52 out of 474 genes, i.e., 11%) and Creb1 function was strongly predicted to be increased (z-score = 2.6) (Figure 3 and Sup Table 3). Creb1 is a transcription factor well-recognized to be activated by cAMP/PKA signaling, facilitate synaptic plasticity and play important roles in learning, long-term memory formation, and addiction (
      • Nestler E.J.
      Cellular basis of memory for addiction.
      ,
      • McPherson C.
      • Lawrence A.
      The Nuclear Transcription Factor CREB: Involvement in Addiction, Deletion Models and Looking Forward.
      ,
      • Carlezonjr W.
      • Duman R.
      • Nestler E.
      The many faces of CREB.
      ). Other upstream regulators also predicted to be responsible for subsets of the observed differential expression pattern included beta-estradiol responsive to hormones, IFNG, IL2 and IL1B cytokines, Ca++ and SNCA.
      While the statistical significance for enrichment of canonical pathways did not survive correction for multiple testing (Sup Table 4), “Glutamate dependent acid resistance” (conversion of glutamate to GABA by Gad enzymes, p = 0.003) and “cAMP-mediated signaling” (p = 0.03) remain likely candidates to be affected in this low Drd2 model. See Sup Fig 4 for iMSND2HET DEGs in these signaling pathways.
      Figure thumbnail gr4
      Figure 4Disease and function enrichment analysis in iMSND2HET. A) Top disease and function annotations significantly enriched in the extended DEG list in iMSND2HET. B) Gene network for “Behavior” related functions: “Learning”, “Cognition” and “Long-term memory”. C) Gene network for “Lipid Metabolism” related functions: “Release of fatty acid”, “Release of lipid”, “Release of eicosanoid”, “Concentration of GABA”, “Release of GABA” and “Concentration of lipid”. Functions in blue or orange indicate a predicted reduction or increase of function respectively. For a complete list of genes, annotations and values see Sup Table 5.
      Further gene ontology analysis of the extended DEG list revealed strong enrichment in genes with diverse functions, including: “Release of fatty acid and lipid” and “Inflammation of the nervous system” (Figure 4, Sup Fig 5 and Sup Table 5). Dysregulated lipid metabolism in iMSNs has been associated with reward-related psychopathologies (
      • Ducrocq F.
      • Walle R.
      • Contini A.
      • Oummadi A.
      • Caraballo B.
      • van der Veldt S.
      • et al.
      Causal Link between n-3 Polyunsaturated Fatty Acid Deficiency and Motivation Deficits.
      ). Enriched “Disease and Function” terms with strong predicted activation (z-score > 2) include “Epilepsy or neurodevelopmental disorder”, “Hypothermia” and “Secretion of catecholamine”, while functions with strong predicted inhibition (z-score < -2) include “Excitation of neurons”, “Formation of neointima” and “Immune-mediated inflammatory disease”.
      The gene network of Drd2 in iMSND2HET is enriched in cell-to-cell communication molecules
      To identify genes whose expression co-varied with Drd2 expression we performed an independent weighted gene co-expression network analysis (WGCNA). In line with the IPA analysis, and further highlighting the impact of Drd2 expression on iMSN’s functional output, the module containing Drd2 showed strong enrichment in genes involved in cell-to-cell communication, including ion transport, synaptic signaling and assembly, axon guidance, and matching molecular functions and cellular localizations (Sup Fig 6 and Sup Methods).
      Figure thumbnail gr6
      Figure 6Functional impairment of D2R mediates electrophysiologic response in striatum of mice with low D2R expression, and with evidence for enhanced GABA tone. Left, Recordings from MSNs in control Adora2a-Cre mice show changes in holding current in response to application of the D2-like agonist quinpirole 1 μM (green trace). This response is severely impaired in mice with low D2R expression (iMSN-Drd2HET) which show no changes in holding current after quinpirole. Note that there are changes in baseline holding current in mice with low Drd2, which are suggestive of enhanced GABAergic tone in iMSN-Drd2HET mice. Right, Cocaine pretreatment (5 days, 15 mg/kg) enhanced the average holding current in control mice from -38 to -52 pA but not in mice with low D2R expression, which show persistently smaller holding currents than control mice, consistent with increased GABAergic tone in mice with low D2R.
      Fxyd2, a potentially inhibitory subunit of the Na/K pump is upregulated in iMSND2HET
      Fxyd2 was one of the genes with strongest differential expression (both in magnitude and statistical significance, Figure 2B) in the iMSND2HET RNAseq and independent qPCR samples (Figure 5A). Fxyd2 encodes the modulatory γ-subunit of the ATP-dependent Na/K pump that is essential for membrane potential and therefore neural excitability. Fxyd2 is a phosphorylation target of PKA (
      • Cortes V.F.
      • Veiga-Lopes F.E.
      • Barrabin H.
      • Alves-Ferreira M.
      • Fontes C.F.L.
      The γ subunit of Na+, K+-ATPase: Role on ATPase activity and regulatory phosphorylation by PKA.
      ) and a member of the cAMP pathway (KEGG). In the brain, Fxyd2 function is poorly understood but it modulated neuropathic pain through inhibition of the Na/K pump in nociceptive neurons (
      • Ventéo S.
      • Laffray S.
      • Wetzel C.
      • Rivat C.
      • Scamps F.
      • Méchaly I.
      • et al.
      Fxyd2 regulates Aδ- and C-fiber mechanosensitivity and is required for the maintenance of neuropathic pain.
      ,
      • Wang F.
      • Cai B.
      • Li K.-C.
      • Hu X.-Y.
      • Lu Y.-J.
      • Wang Q.
      • et al.
      FXYD2, a γ subunit of Na+,K+-ATPase, maintains persistent mechanical allodynia induced by inflammation.
      ).
      We found that FXYD2 and DRD2 mRNA expression were inversely correlated in both mouse purified iMSNs (Figure 5B) and human postmortem caudate (whole tissue RNAseq) of individuals with severe cocaine abuse history and controls (Figure 5C). This correlation was validated by independent cohorts from public repositories (Sup Fig 7A). FXYD2 expression was not significantly different between cocaine and control groups (p = 0.11, Sup Fig 7B), however FXYD2 expression in both mouse iMSNs (Figure 2C) and human is relatively low, and several samples had non-detectable levels (Sup Fig 7B and Figure 5C, note axis scales for DRD2 vs FXYD2 FPKM). RNAscope staining (Figure 5D) together with publicly available single-cell RNAseq data from mouse striatum (
      • Gokce O.
      • Stanley G.M.
      • Treutlein B.
      • Neff N.F.
      • Camp J.G.
      • Malenka R.C.
      • et al.
      Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq.
      ) (Figure 5E) suggest Fxyd2 expression in striatal iMSNs, dMSNs and to a lesser extent in interneurons, with little or no expression in glial populations.
      Finally, no association approaching genome wide significance was observed (Sup Fig 7C) between FXYD2 genetic variations and lifetime overall alcohol use score in the NIAAA Clinical Center patient cohort (1181 cases and 546 controls). In publicly available knowledgebases such as Open Targets Genetics and GWAS Catalog, non-coding genetic variants near FXYD2 were associated with human putamen volume (
      • Luo X.
      • Mao Q.
      • Shi J.
      • Wang X.
      • Li C.-S.R.
      Putamen gray matter volumes in neuropsychiatric and neurodegenerative disorders.
      ,
      • Satizabal C.L.
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      • et al.
      Genetic architecture of subcortical brain structures in 38,851 individuals.
      ,
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      Understanding the genetic determinants of the brain with MOSTest.
      ).

      Heightened striatal GABA tone and impaired cocaine response in Drd2-low mice

      dMSNs receive inhibitory GABA synapses from iMSN axon collaterals, which are suppressed by activation of D2Rs by the agonist quinpirole (
      • Dobbs L.K.
      • Kaplan A.R.
      • Lemos J.C.
      • Matsui A.
      • Rubinstein M.
      • Alvarez V.A.
      Dopamine Regulation of Lateral Inhibition between Striatal Neurons Gates the Stimulant Actions of Cocaine.
      ,
      • Burke D.A.
      • Alvarez V.A.
      Serotonin receptors contribute to dopamine depression of lateral inhibition in the nucleus accumbens.
      ). Thus, electrophysiological recording of dMSNs provides a useful readout of GABA and D2R signaling in the striatum. We performed whole-cell voltage-clamp recordings from tdTomato+ dMSNs from the NAc core in sagittal brain slices of iMSN-Drd2HET and control mice. The D2R-like agonist, quinpirole (Figure 6, green trace) decreased the holding current in WT dMSNs (compared to baseline, gray trace) but exerted no effect in dMSNs from iMSN-Drd2HET mice. This loss of quinpirole-mediated response (in both saline and cocaine-treated animals) provides further evidence of the functional impact of D2R reduction (
      • Dobbs L.K.
      • Kaplan A.R.
      • Bock R.
      • Phamluong K.
      • Shin J.H.
      • Bocarsly M.E.
      • et al.
      D1 receptor hypersensitivity in mice with low striatal D2 receptors facilitates select cocaine behaviors.
      ). Further, the baseline average holding current of dMSNs in iMSN-Drd2HET was smaller than in controls (-24/-18 pA vs -38/-52 pA in saline and cocaine-treated groups respectively), indicating more hyperpolarized dMSNs in iMSN-Drd2HET mice compared to WT, consistent with a heightened inhibitory GABA tone.
      dMSNs in WT animals treated with repeated cocaine (15 mg/kg/day, 5 days) showed persistent depolarization at baseline compared to saline-treated controls (-52 pA vs -38 pA). More depolarized dMSN membrane potential after repeated cocaine suggests reduced GABA inhibition. This cocaine-induced depolarization was not seen in dMSNs of iMSN-Drd2HET mice. This suggests that D2Rs are required for mediating the effect of repeated cocaine in dMSNs, likely via suppression of striatal GABA release (
      • Dobbs L.K.
      • Kaplan A.R.
      • Lemos J.C.
      • Matsui A.
      • Rubinstein M.
      • Alvarez V.A.
      Dopamine Regulation of Lateral Inhibition between Striatal Neurons Gates the Stimulant Actions of Cocaine.
      ).

      DISCUSSION

      Reduced D2R availability in the striatum is both a consequence of SUD and an etiologic contributor. Innate low D2R expression in young/adult mice appears to simultaneously stimulate the “no go” pathway and inhibit the direct “go” pathway (
      • Dobbs L.K.
      • Lemos J.C.
      • Alvarez V.A.
      Restructuring of basal ganglia circuitry and associated behaviors triggered by low striatal D2 receptor expression: implications for substance use disorders: Restructuring of basal ganglia circuitry and associated behaviors.
      ). Here, we sought to better understand the long-term consequences of innately reduced Drd2 expression by analyzing the purified translatome (ribosome-bound transcriptome) of iMSNs from mice with wild type or heterozygous expression of Drd2 (selectively in iMSNs).
      Consistent with our previous findings of increased striatal GABA signaling in mice with D2R deficiency (
      • Dobbs L.K.
      • Kaplan A.R.
      • Lemos J.C.
      • Matsui A.
      • Rubinstein M.
      • Alvarez V.A.
      Dopamine Regulation of Lateral Inhibition between Striatal Neurons Gates the Stimulant Actions of Cocaine.
      ,
      • Lemos J.C.
      • Friend D.M.
      • Kaplan A.R.
      • Shin J.H.
      • Rubinstein M.
      • Kravitz A.V.
      • Alvarez V.A.
      Enhanced GABA Transmission Drives Bradykinesia Following Loss of Dopamine D2 Receptor Signaling.
      ), we found Gad1 and Gad2, key genes encoding GABA-synthesis enzymes, upregulated, and a predicted inhibition in “Stimulation of neurons” function (z-score = -2) by enrichment analysis in iMSND2HET. Electrophysiological recordings showed that dMSNs were more inhibited at baseline in iMSN-Drd2HET mice compared to WT controls.
      Possibly related to the increased GABA tone, Fxyd2, a potentially inhibitory subunit of the Na/K pump, was robustly upregulated in iMSND2HET. Its modulatory role is cell-type and environment dependent (
      • Mayan H.
      • Farfel Z.
      • Karlish S.J.D.
      Renal Mg handling, FXYD2 and the central role of the Na,K-ATPase.
      ), but was shown to be inhibitory in nociceptive neurons (
      • Ventéo S.
      • Laffray S.
      • Wetzel C.
      • Rivat C.
      • Scamps F.
      • Méchaly I.
      • et al.
      Fxyd2 regulates Aδ- and C-fiber mechanosensitivity and is required for the maintenance of neuropathic pain.
      ,
      • Wang F.
      • Cai B.
      • Li K.-C.
      • Hu X.-Y.
      • Lu Y.-J.
      • Wang Q.
      • et al.
      FXYD2, a γ subunit of Na+,K+-ATPase, maintains persistent mechanical allodynia induced by inflammation.
      ) where loss of Fxyd2 resulted in neural hyperpolarization (
      • Wang F.
      • Cai B.
      • Li K.-C.
      • Hu X.-Y.
      • Lu Y.-J.
      • Wang Q.
      • et al.
      FXYD2, a γ subunit of Na+,K+-ATPase, maintains persistent mechanical allodynia induced by inflammation.
      ). If Fxyd2 inhibits the Na/K pump also in iMSNs, its upregulation could result in increased iMSN excitability. This could be an adaptive response by iMSNs to counteract the heightened GABA tone.
      An evolutionarily conserved negative correlation between FXYD2 and DRD2 mRNA expression was observed in both our mouse model and human postmortem caudate with and without severe cocaine use history. This correlation could be explained by a genetic variant at the DRD2 locus affecting FXYD2 expression (i.e., expression quantitative trait locus, eQTL), which we could not test due to the limited detectability of FXYD2 in brain.
      iMSND2HET displayed differential expression of genes associated with cAMP signaling and cellular growth. Mapk3 (Erk), modulates processes that overlap with those of cAMP-dependent Pka. We have previously observed a shift from Pka towards the Erk/Mapk signaling pathway in dMSNs from iMSN-Drd2KO mice (
      • Dobbs L.K.
      • Kaplan A.R.
      • Bock R.
      • Phamluong K.
      • Shin J.H.
      • Bocarsly M.E.
      • et al.
      D1 receptor hypersensitivity in mice with low striatal D2 receptors facilitates select cocaine behaviors.
      ). Here, iMSND2HET showed downregulation of Mapk3 (Erk), suggesting an opposing shift in iMSNs.
      Creb1, a transcription factor well-known for its role in growth, learning and synaptic reinforcement, was predicted to be an upstream regulator (padj = 4x10-10) with increased activity (z-score = 2.6). There was no differential expression of the Creb1 gene at the time of sample collection, suggesting it exerted its effect at an earlier timepoint. Studies across development are needed to test the hypothesis of differential neural growth of iMSN over dMSNs in iMSN-Drd2HET mice.
      Several genes were associated with inflammation, “Inflammation of the nervous system” was among the most significantly enriched functions, and “Immune-mediated inflammatory disease” was predicted to be inhibited (Fig 4 and Sup Table 5). There is a growing body of evidence for a role of neuroinflammation (not accompanied by infiltration of peripheral immune cells) in several psychiatric disorders, including SUDs (
      • Dunn G.A.
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      • Kohno M.
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      • Moretti M.
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      • Visonà S.D.
      Drug Abuse-Related Neuroinflammation in Human Postmortem Brains: An Immunohistochemical Approach.
      ). Transcription profiling of rhesus macaques following long-term (∼100 days) cocaine self-administration revealed upregulation of neuroinflammation-related genes in the NAc but not in the ventral tegmental area (
      • Vallender E.J.
      • Goswami D.B.
      • Shinday N.M.
      • Westmoreland S.V.
      • Yao W.-D.
      • Rowlett J.K.
      Transcriptomic profiling of the ventral tegmental area and nucleus accumbens in rhesus macaques following long-term cocaine self-administration.
      ), showing differential neuroinflammation response to drugs of abuse across brain regions. Anti-inflammatory strategies to treat SUDs show promising results including improvements in behavioral and cognitive outcomes (
      • Liśkiewicz A.
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      ,
      • Namba M.D.
      • Leyrer-Jackson J.M.
      • Nagy E.K.
      • Olive M.F.
      • Neisewander J.L.
      Neuroimmune Mechanisms as Novel Treatment Targets for Substance Use Disorders and Associated Comorbidities.
      ,
      • Lwin T.
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      • Viwatpinyo K.
      • Chancharoen P.
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      Melatonin ameliorates methamphetamine-induced cognitive impairments by inhibiting neuroinflammation via suppression of the TLR4/MyD88/NFκB signaling pathway in the mouse hippocampus.
      ) but with negative as well as positive outcomes in early clinical trials (
      • Schwandt M.L.
      • Diazgranados N.
      • Umhau J.C.
      • Kwako L.E.
      • George D.T.
      • Heilig M.
      PPARγ activation by pioglitazone does not suppress cravings for alcohol, and is associated with a risk of myopathy in treatment seeking alcohol dependent patients: a randomized controlled proof of principle study.
      ).
      iMSND2HET also displayed upregulated Cartpt and downregulated S100a10, reproducing gene expression changes observed in mouse studies in cocaine paradigms. In the striatum, cocaine and amphetamine upregulate Cartpt mRNA expression and CART peptides, and co-administration of CART peptide and cocaine into the NAc reduced cocaine-induced locomotor activation (
      • Kuhar M.J.
      CART Peptides and Drugs of Abuse: A Review of Recent Progress.
      ,
      • Jaworski J.N.
      • Vicentic A.
      • Hunter R.G.
      • Kimmel H.L.
      • Kuhar M.J.
      CART peptides are modulators of mesolimbic dopamine and psychostimulants.
      ,

      Philpot K, Smith Y (2006): CART peptide and the mesolimbic dopamine system. Peptides 27: 1987–1992.

      ), suggesting a compensatory role for Cartpt. Conversely, mice with reduced S100a10 (p11), a small calcium-binding protein involved in neurotransmitter transport, have an enhanced cocaine conditioned place preference, while p11 overexpression in the NAc reduced it (
      • Arango-Lievano M.
      ). Mice with low p11 also exhibited depression-like behavior, and its restoration in the NAc recovered the phenotype (
      • Alexander B.
      • Warner-Schmidt J.
      • Eriksson T.M.
      • Tamminga C.
      • Arango-Lievano M.
      • Ghose S.
      • et al.
      Reversal of Depressed Behaviors in Mice by p11 Gene Therapy in the Nucleus Accumbens.
      ).
      Recent studies show gene expression biases of Drd2 expressing neurons across regions of the striatum (
      • Puighermanal E.
      • Castell L.
      • Esteve-Codina A.
      • Melser S.
      • Kaganovsky K.
      • Zussy C.
      • et al.
      Functional and molecular heterogeneity of D2R neurons along dorsal ventral axis in the striatum.
      ) and as compared to Drd1 neurons (
      • Montalban E.
      • Giralt A.
      • Taing L.
      • Schut E.H.S.
      • Supiot L.F.
      • Castell L.
      • et al.
      Translational profiling of mouse dopaminoceptive neurons reveals region-specific gene expression, exon usage, and striatal prostaglandin E2 modulatory effects.
      ). Among our DEGs Gstm6, Fam163b, Etl4, Spoon1 (
      • Puighermanal E.
      • Castell L.
      • Esteve-Codina A.
      • Melser S.
      • Kaganovsky K.
      • Zussy C.
      • et al.
      Functional and molecular heterogeneity of D2R neurons along dorsal ventral axis in the striatum.
      ) and S100a10 (
      • Puighermanal E.
      • Castell L.
      • Esteve-Codina A.
      • Melser S.
      • Kaganovsky K.
      • Zussy C.
      • et al.
      Functional and molecular heterogeneity of D2R neurons along dorsal ventral axis in the striatum.
      ,
      • Montalban E.
      • Giralt A.
      • Taing L.
      • Schut E.H.S.
      • Supiot L.F.
      • Castell L.
      • et al.
      Translational profiling of mouse dopaminoceptive neurons reveals region-specific gene expression, exon usage, and striatal prostaglandin E2 modulatory effects.
      ) were preferentially expressed in Drd2 neurons from dorsal striatum, while Lypd1, a modulator of nicotinic acetylcholine receptors, showed increased expression in NAc (
      • Puighermanal E.
      • Castell L.
      • Esteve-Codina A.
      • Melser S.
      • Kaganovsky K.
      • Zussy C.
      • et al.
      Functional and molecular heterogeneity of D2R neurons along dorsal ventral axis in the striatum.
      ,
      • Montalban E.
      • Giralt A.
      • Taing L.
      • Schut E.H.S.
      • Supiot L.F.
      • Castell L.
      • et al.
      Translational profiling of mouse dopaminoceptive neurons reveals region-specific gene expression, exon usage, and striatal prostaglandin E2 modulatory effects.
      ) and in D1s (
      • Montalban E.
      • Giralt A.
      • Taing L.
      • Schut E.H.S.
      • Supiot L.F.
      • Castell L.
      • et al.
      Translational profiling of mouse dopaminoceptive neurons reveals region-specific gene expression, exon usage, and striatal prostaglandin E2 modulatory effects.
      ). Further studies are needed to translate these observations into function.
      An intrinsic challenge of this study was the observed and expected small magnitude changes between groups, as they only partially differ in the expression of a modulatory signaling receptor (Drd2). Gene expression changes translate into function whether they are big or small in magnitude, but small magnitude differences increase the number of samples needed to statistically resolve them. Regarding technical strengths, low-input sequencing of TRAP positive fractions allowed us to study the iMSNs (∼12% of cells in the striatum) translatome, and reliably detect transcripts with medium and low levels of expression that would have been lost via whole-tissue RNAseq. This was critical to our research, particularly given the functionally opposing roles of dMSNs and iMSNs, while also sharing a highly overlapping molecular profile.
      In summary, we identified numerous differentially expressed genes in iMSNs driven by low D2R expression, modeling an observed trait of addiction in humans. We provide molecular evidence for enhanced GABA transmission. We identified enrichment in lipid metabolism, growth-related genes, cell-to-cell communication and synaptic components, that may reflect neural growth and/or increased maintenance given previous active growth. Striatal development in iMSND2HET, and by extension, in people with genetically encoded lower levels of D2R expression, may feature an increased number of inhibitory iMSN-dMSN axon collaterals and an increased number of GABAergic pre-synaptic vesicles and release. Thus, our results further support an addiction model in which low D2R expression drives changes in the striatal micro-circuitry, and help explain its contribution to an enhanced indirect pathway and SUD related behaviors.

      ACKNOWLEDGMENTS

      This research was supported by the Intramural Research Program of the National Institute of Alcohol Abuse and Alcoholism ( NIAAA ), National Institutes of Health ( NIH ), Projects ZIA-AA000301 for DG and ZIA-AA000421 for VAA, and the DA033684grant/ NIH /R01-DA033684-06" title="http://grantome.com/grant/ NIH /R01-DA033684-06"> R01 grant from NIDA , NIH “Epigenetic Marks of Cocaine Addiction” for DCM. We are also grateful to Kornel Schuebel for scientific assistance during this research, and to Christopher A. Harris for the support, scientific assistance, and comments on the manuscript.

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