Egr2 induction in spiny projection neurons of the ventrolateral striatum contributes to cocaine place preference in mice

  1. Diptendu Mukherjee
  2. Ben Jerry Gonzales
  3. Reut Ashwal-Fluss
  4. Hagit Turm
  5. Maya Groysman
  6. Ami Citri  Is a corresponding author
  1. The Edmond and Lily Safra Center for Brain Sciences, Israel
  2. Institute of Life Sciences, The Hebrew University of Jerusalem, Israel
  3. Program in Child and Brain Development, Canadian Institute for Advanced Research, MaRS Centre, Canada
6 figures, 1 table and 8 additional files

Figures

Figure 1 with 4 supplements
Transcriptional profiling resolves the dynamics of cocaine-induced gene expression within major nodes of the reward circuitry.

(A) Scheme describing the cocaine sensitization paradigm and time points (0, 1, 2, 4 hr) at which samples were obtained for analysis of gene expression following acute (0 = cocaine naïve); repeated …

Figure 1—figure supplement 1
Boundaries of dissected brain structures.

Illustration of the brain tissue collected for RNA extraction and sequencing analysis. Red shaded boxes represent the area dissected from 400-micron coronal sections at specific rostral (+) or …

Figure 1—figure supplement 2
Quality control analysis of RNA-seq experiments.

(A) Histograms of mean usable read depth of 3′-RNA-seq libraries. n = 194 libraries. (B) Principal component analysis (PCA) demonstrates clustering of RNA-seq libraries according to brain region, …

Figure 1—figure supplement 3
Repeated cocaine administration and abstinence induce baseline shifts in gene transcription.

(A) Gene expression profiles of repeated cocaine treated and abstinent mice within different brain regions. Heatmaps reflect log2 (fold change) of genes demonstrating differential expression between …

Figure 1—figure supplement 4
Repeated cocaine exposure and abstinence alters the expression of gene clusters related to neuroplasticity.

Heatmaps depict the expression levels of genes which are modulated between naïve mice and mice following repeated cocaine or abstinence in different brain regions clustered according to GO terms …

Figure 2 with 2 supplements
Dynamic IEG induction in subregions of the striatum accompany the development of cocaine sensitization.

(A) Scheme of a coronal section of the dorsal striatum (DS) (+0.52 ± 0.1 mm from Bregma) corresponding to the region assayed by multicolor smFISH for cocaine-induced IEG expression. (B) …

Figure 2—figure supplement 1
Cocaine dynamically modulates cellular IEG expression in the VLS and MS.

(A) Dot plot of the fraction of cells positive for expression of Arc and Nr4a1 in the VLS and MS (threshold: Arc = 11, Nr4a1 = 12 puncta/cell) following acute, repeated, and challenge cocaine …

Figure 2—figure supplement 2
Coherence of cocaine-induced IEG expression.

(A) Arc, Egr2, and Nr4a1 are coherently induced in cells of the VLS and MS. Coherence refers to the correlated co-expression of multiple IEGs within individual cells. Each dot depicts the expression …

Figure 3 with 4 supplements
Induction of Egr2 in VLS neurons contributes to the acquisition of cocaine reward.

(A) Representative 40× images showing Egr2 expression within Drd1+ and Drd2+ SPNs in the VLS (left) and MS (right) following acute, repeated, and challenge cocaine exposures compared to controls. (B,…

Figure 3—figure supplement 1
Induced IEGs are correlated to Drd1 expression in the VLS and to both Drd1 and Drd2 expressions in the MS.

(A) Egr2 induction in the MS does not exhibit prominent cell-type bias and is modestly affected by the history of cocaine experience. Scatter plots show cellular Egr2 expression with Drd1 or Drd2

Figure 3—figure supplement 2
Drd1 and Drd2 receptor expression in the VLS and MS.

(A) Dot plots depicting the fraction of Drd1 vs Drd2 expressing cells in the VLS and MS in control (gray) and cocaine (red) conditions following distinct cocaine experiences. n = 6 sections from …

Figure 3—figure supplement 3
DREADD inhibition of VLS Egr2+ cells does not affect locomotion.

(A, B) Verification of AAV transduction in the VLS of transgenic Egr2-CRE mice tested in experiment depicted in Figure 3F-G. Example of AAV-DIO-hM4Di-mcherry infections in the VLS (A) and summary of …

Figure 3—figure supplement 4
Disruption of Egr2 function in the VLS does not affect locomotor behavior or IEG expression outside of VLS.

(A, B) Verification of AAV-GFP and AAV-DNEgr2 infection in the VLS. Example of AAV-DNEgr2 infection in the VLS (A). (B) Summary of infection from individual mice of VLSDNEgr2 and VLSGFP groups …

Author response image 1
Efficacy and efficiency of transduction of hM4Di infection of CLEgr2+ neurons.

(A) Representative traces of whole-cell current clamp recordings from CLEgr2+ neurons expressing the hM4Di DREADD, in the presence or absence of CNO (clozapine-N-oxide; 1 μM). 50-250 pA current …

Author response image 2
hM4Di-CNO inhibits cocaine-induced Fos induction in Egr2+ claustral neurons.

The claustrum of Egr2-CRE mice was transduced with AAV viruses conditionally expressing hM4Di. 3 weeks later, following habituation to ip saline injections, mice were injected (i.p. 10mg/kg) with …

Author response image 3
hM4Di-CNO inhibits cocaine-induced Fos induction in VLS Drd1+-neurons.

The VLS of Drd1+-CRE mice was transduced with AAV viruses conditionally expressing hM4Di (red). Following habituation to ip saline injections, mice were injected with CNO (i.p. 10 mg/kg), and 30 …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)Wild-type C57BL/6OLAHSD miceThe Harlan LaboratoryNA
Strain, strain background (Mus musculus)Egr2-Cre knock in miceThe Jackson LaboratoryCat# 025744
RRID: IMSR_JAX:025744
Recombinant DNA reagentAAV2-hSyn-DIO-hM4d(Gi)- mCherryAddgeneCat# 44362-AAV2
RRID: Addgene_44362
1.15 dilution
Recombinant DNA reagentAAV2-hSyn-DIO-mCherryUNC vector core facilityN/A1.15 dilution
Recombinant DNA reagentAAVdj-CMV-eGFPELSC vector core facilityN/A1.15 dilution
Recombinant DNA reagentAAVdj-CAG-DNEgr2-IRES-GFPELSC vector core facilityN/A1.15 dilution
Recombinant DNA reagentPlasmid with dominant negative mutant Egr2 (S382R,D383Y)Jeffrey Milbrant, Washington UniversityN/A
Chemical compound, drugClozapine-N-oxide (CNO)Sigma–AldrichCat # C0832-5MG
Chemical compound, drugCocaineHadassah Hospital PharmacyN/A
Commercial assay, kitFluorescent Multiplex
Reagent Kit
Advanced Cell Diagnostics RNAscopeCat # 320850
Commercial assay, kitNEBNext Ultra II Non-Directional RNA Second-Strand Synthesis ModuleNew England BiolabsCat # E6111L
Commercial assay, kitKAPA Hifi Hotstart ReadyMixRocheCat # KK-KK2601-2 07958927001
Commercial assay, kitMinElute Gel Extraction KitQiagenCat # 28604
Commercial assay, kitNEBNext Library Quant Kit for IlluminaNew England BiolabsCat # E7630L
Commercial assay, kitHigh-sensitivity DNA kitAgilent TechnologiesCat # 5067–4626
Commercial assay, kitNextSeq 500 High Output V2 kitsIlluminaCat # FC-404–2005
Commercial assay, kitSMARTScribe Reverse TranscriptaseTakaraCat # 639536
Sequence-based reagent (smFISH)Probe-Mm-Drd1a-C2Advanced Cell Diagnostics RNAscopeCat # 406491-C2
Sequence-based reagent (smFISH)Probe-Mm-Drd1a-C3Advanced Cell Diagnostics RNAscopeCat # 406491-C3
Sequence-based reagent (smFISH)Probe-Mm-Drd2-C2Advanced Cell Diagnostics RNAscopeCat # 406501-C2
Sequence-based reagent (smFISH)Probe-Mm-Egr2Advanced Cell Diagnostics RNAscopeCat # 407871
Sequence-based reagent (smFISH)Probe-Mm-Fos-C3Advanced Cell Diagnostics RNAscopeCat # 316921-C3
Sequence-based reagent (smFISH)Probe-Mm-Arc-C3Advanced Cell Diagnostics RNAscopeCat # 316911-C3
Sequence-based reagent (smFISH)Probe-Mm-Nr4a1-C2Advanced Cell Diagnostics RNAscopeCat # 423341-C2
Sequence-based reagent
(RNA-seq)
Primers for first-strand synthesisThis paperN/ACGATTGAGGCCGGTAATACGACTCACTATAGGGGCGACGTGTGCTCTTCCGATCTNNNNNNNNNNNNNNNTTTTTTTTTTTTTTTTTTTTN
Sequence-based reagent
(RNA-seq)
Forward primer with P5-Read1 sequenceThis paperNAAATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTG
Sequence-based reagent
(RNA-seq)
Reverse primer with P7-Read2 sequenceThis paperNACAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
Software, algorithmRR studiohttps://rstudio.com/products/rstudio/
Software, algorithmImageJNational Institutes of Healthhttps://imagej.nih.gov/ij/
RRID:SCR_003070
Software, algorithmCellProfilerBroad Institutehttps://cellprofiler.org/
RRID:SCR_007358
Software, algorithmPrism7GraphPadhttps://www.graphpad.com
RRID:SCR_002798
Software, algorithmEthovision XTNoldushttps://www.noldus.com/ethovision-xt RRID:SCR_000441
Software, algorithmPhotoshop and IllustratorAdobehttps://www.adobe.com/in/creativecloud/catalog/desktop.html?promoid=PTYTQ77P&mv=other
Other0.9% NaclCat # 3642828
OtherIsofluranePiramal Critical CareCat # AWN34014604
OtherMicrotome (7000 smz2)Camden Instrumentshttps://www.emsdiasum.com/microscopy/products/equipment/vibrating_microtome.aspx
OtherStereoscopeOlympusCat # N1197800
OtherTissueLyser LTQiagenCat # 69980
OtherSuperfrost Plus slidesThermoFisher ScientificCat # J1800AMNZ
OtherHermes high-definition cell-imaging systemWiscanhttps://idea-bio.com/products/wiscan-hermes/
OtherSomnoSuite Low-Flow Anesthesia SystemKent Scientific
Corporation
https://www.kentscientific.com/products/somnosuite/
OtherFine drill burrRWD Life ScienceCat # 78001
OtherMicrosyringe (33G)HamiltonCat # 65460–05
Other3M Vetbond tissue Adhesive3M (Ebay)Cat # 8017242664
OtherIsofluranePiramal Critical CareCat # AWN34014604
OtherTri-ReagentSigma–AldrichCat # T9424
OtherOCT embedding mediumScigen Scientific GardenaCat # 23-730-625
OtherACD RNAscope fresh frozen tissue pretreatmentAdvanced Cell Diagnostics RNAscopeCat # 320513
OtherDAPISigma–AldrichCat # 10236276001
OtherLab Vision PermaFluor Aqueous Mounting MediumThermoFisher ScientificCat # TA-030-FM
OtherdNTPsNew England BiolabsCat # N0447s
OtherMnCl2Sigma–AldrichCat # 244589–10G
OtherSPRI magnetic beadsBeckman CoulterCat # A63881
Other1 M Tris–HCI, pH 8.0ThermoFisher ScientificCat # 15568025
OtherSDS Solution (10%)Biological IndustriesCat # 01-890-1B

Additional files

Supplementary file 1

Distribution of sequencing libraries analyzed in this study.

https://cdn.elifesciences.org/articles/65228/elife-65228-supp1-v2.xlsx
Supplementary file 2

Normalized reads of baseline shifted genes (tabs corresponding to individual structures).

https://cdn.elifesciences.org/articles/65228/elife-65228-supp2-v2.xlsx
Supplementary file 3

Clusters of Gene Ontology (GO) analysis and corresponding genes.

https://cdn.elifesciences.org/articles/65228/elife-65228-supp3-v2.xlsx
Supplementary file 4

Normalized reads of induced genes (tabs correspond to structures).

https://cdn.elifesciences.org/articles/65228/elife-65228-supp4-v2.xlsx
Supplementary file 5

Gene Ontology annotation of cocaine-induced genes in the dorsal striatum.

https://cdn.elifesciences.org/articles/65228/elife-65228-supp5-v2.xlsx
Supplementary file 6

Statistical analysis.

https://cdn.elifesciences.org/articles/65228/elife-65228-supp6-v2.xlsx
Supplementary file 7

Distribution of cell numbers among replicates in smFISH analysis.

https://cdn.elifesciences.org/articles/65228/elife-65228-supp7-v2.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/65228/elife-65228-transrepform-v2.docx

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