Egr2 induction in spiny projection neurons of the ventrolateral striatum contributes to cocaine place preference in mice
Psychostimulant addiction is characterized by life-long behavioral abnormalities , driven by circuit-specific modulation of gene expression ( Nestler , 2014; Nestler and Lüscher , 2019; Salery et al . , 2020; Steiner , 2016 ) . Induction of immediate-early gene ( IEG ) transcription in the nucleus accumbens ( NAc ) and dorsal striatum ( DS ) are hallmarks of psychostimulant exposure ( Berke et al . , 1998; Caprioli et al . , 2017; Chandra and Lobo , 2017; Gao et al . , 2017b; Gerfen , 2000; Gonzales et al . , 2020; Guez-Barber et al . , 2011; Hope et al . , 1994; Moratalla et al . , 1996; Mukherjee et al . , 2018; Nestler et al . , 1993; Nestler , 2001; Nestler and Aghajanian , 1997; Piechota et al . , 2010; Turm et al . , 2014 ) . As such , IEG induction has been utilized to support the identification of functional neuronal assemblies mediating the development of cocaine-elicited behaviors ( ‘cocaine ensembles’; Bobadilla et al . , 2020; Cruz et al . , 2013 ) . Within these striatal structures , the principal neuronal type is the spiny projection neuron ( SPN ) , which is comprised of two competing subtypes , defined by their differential expression of dopamine receptors . Expression of the D1R dopamine receptor is found on direct-pathway neurons , responsible for action selection by promoting behavioral responses , while D2R-expressing indirect pathway neurons are responsible for action selection through behavioral inhibition ( Kreitzer and Malenka , 2008; Lipton et al . , 2019 ) . In the striatum , the cellular composition of cocaine ensembles varies by domain: Fos-expressing cocaine ensembles in the NAc are enriched for D1R expression ( Koya et al . , 2009 ) , while in the DS , IEG expression and psychostimulant-responsive ensembles are spatially segregated to the medial striatum ( MS ) and ventrolateral striatum ( VLS ) , encompassing both D1R+ and D2R+ neurons in the MS , and enriched for D1R expression in the VLS ( Caprioli et al . , 2017; Cruz et al . , 2015; Gonzales et al . , 2020; Li et al . , 2015; Rubio et al . , 2015; Steiner and Gerfen , 1993 ) . The VLS subregion partially overlaps with a lateral striatum segment enriched for Gpr155 expression , defined in recent molecular striatal subdivisions ( Märtin et al . , 2019; Ortiz et al . , 2020 ) . Depending on the history of prior cocaine exposure , a unique pattern of IEG induction is observed across brain structures ( Mukherjee et al . , 2018 ) . This transcriptional code was characterized addressing a handful of transcripts within bulk tissue , warranting a comprehensive study of the induced gene expression programs across key structures of the reward circuitry . Here we comprehensively describe gene programs in progressive stages of cocaine experience across multiple brain structures , analyze the spatial and cell-type-specific patterns of IEG expression within prominently recruited brain regions , and functionally link induced gene expression to the development of cocaine preference . Taking an unbiased approach to the identification of the cellular and molecular modifications underlying the development of cocaine-elicited behaviors , we analyzed dynamics of cocaine-induced transcription across five structures of the reward circuitry . Of these , the most prominently induced gene programs were in the DS . Addressing the spatial segregation of these transcriptional programs within the DS ( studying 759 , 551 individual cells by multiplexed single-molecule fluorescence in-situ hybridization ) , we investigated the dynamics of cell-specific recruitment within the two striatal subdomains engaged by cocaine , the MS and VLS . While both D1R+ and D2R+ neurons in the MS were engaged transcriptionally throughout the development of cocaine sensitization , the recruitment of D1R+ neurons in the VLS fluctuated depending on the history of cocaine exposure . The IEG Egr2 , which we find to be the most robustly induced following cocaine experience , serves as a prominent marker for these VLS ensembles . We therefore addressed the function of VLS Egr2+ ensembles , as well the role of VLS expressed Egr2-transcriptional complexes , in the development of cocaine seeking . Our results identify the VLS as a hub of dynamic transcriptional recruitment by cocaine and define a role for Egr2-dependent transcriptional regulation in VLS D1R+ neurons in the development of cocaine seeking .
In order to characterize brain-wide gene expression programs corresponding to the development of psychostimulant sensitization , we exposed mice to cocaine ( 20 mg/kg , i . p . ) acutely , or repeatedly ( five daily exposures ) , as well as to a cocaine challenge ( acute exposure following 21 days of abstinence from repeated exposure to cocaine ) ( Figure 1A ) . We then profiled transcription ( applying 3′-RNA-seq ) within key brain structures of the reward circuitry ( limbic cortex = LCtx , nucleus accumbens = NAc , dorsal striatum = DS , amygdala = Amy , lateral hypothalamus = LH; see Figure 1—figure supplement 1 for the delineation of brain tissue dissected; Supplementary file 1 and Figure 1—figure supplement 2 for a description of the samples sequenced ) at 0 ( not exposed to cocaine on day of sample collection ) , 1 , 2 or 4 hr post-cocaine exposure ( Figure 1A ) . Mice exhibited increased locomotion upon acute exposure to cocaine , further increasing following repeated exposure and maintained after abstinence and challenge re-exposure , typical of locomotor sensitization to this intermediate cocaine dose ( Figure 1B , F8 , 312 = 178 . 9 , p<0 . 0001 , ANOVA ) . Experience impacts gene transcription at multiple timescales ( Clayton et al . , 2020; Mukherjee et al . , 2018; Nestler and Lüscher , 2019; Rittschof and Hughes , 2018; Sinha et al . , 2020; Yap and Greenberg , 2018 ) . Whereas the expression of inducible genes peak and decay on a time scale of minutes-to-hours following stimulation , baseline shifts in brain-wide gene expression programs are also observed following more prolonged periods ( days to weeks ) ( Clayton et al . , 2020 ) , presumably implementing , supporting , and maintaining the modified behavioral output ( Sinha et al . , 2020 ) . We initially focused on baseline shifts in gene expression , comparing naïve mice ( never exposed to cocaine ) to mice exposed repeatedly to cocaine , as well as to mice following 21 days of abstinence from repeated cocaine exposure ( Figure 1C; Figure 1—figure supplement 3A; refer to Supplementary file 2 for list of differentially expressed genes and normalized counts ) . Differentially expressed genes ( DEGs ) included both upregulated and downregulated genes across all brain regions analyzed , with prolonged abstinence driving the most extreme shifts in expression ( Figure 1—figure supplement 3B , C ) . While gene-expression shifts following repeated exposure to cocaine were prominent in the DS , abstinence-induced changes were more prominent in the NAc and LCtx ( Figure 1—figure supplement 3C ) . KEGG analysis demonstrated that DEGs were enriched for synaptic genes and disease pathways ( Figure 1—figure supplement 3D ) . To provide insight into the cellular mechanisms affected by repeated drug exposure and abstinence , we implemented Gene Ontology ( GO term ) enrichment analysis ( Figure 1C , Figure 1—figure supplement 4 , see Supplementary file 3 for definition of clusters and DEGs included within them ) . Gene clusters associated with synaptic plasticity , myelin , and proteostasis demonstrated shifts in expression across multiple brain structures , whereas a cluster of genes associated with structural plasticity appeared more specific to striatal structures ( DS and NAc ) . Noteworthy gene clusters that displayed modified expression were involved in cell–cell communication; glutamate-induced plasticity; synaptic vesicle formation , transport , and fusion; actin filament components; and projection morphogenesis . Notably , the expression of protein folding genes was coordinately upregulated across structures , while myelin components were coordinately downregulated ( Figure 1—figure supplement 4 ) . These results exemplify the dramatic shifts of transcription occurring in the brain in response to repeated cocaine exposure , potentially supporting maladaptive neuroplasticity driving drug addiction ( Bannon et al . , 2014; Lull et al . , 2008 ) . Inducible transcription supports the development of plastic changes following psychostimulant experience ( Alberini , 2009; Han et al . , 2019; McClung and Nestler , 2008; Nestler and Lüscher , 2019 ) . We therefore assessed the inducible transcription response at 1 , 2 , or 4 hr following acute , repeated , or challenge cocaine exposure , observing robust IEG induction across all brain structures studied ( Figure 1D ) . The largest number of induced genes , as well as the highest fold induction levels , were found in the DS ( Figure 1D , E; refer to Supplementary file 4 for the identities of genes induced in each structure and cocaine condition ) . To what extent do the transcription programs induced in the different structures share common attributes ? To query the overlap in the identity of genes induced and their temporal induction patterns following the different schedules of cocaine exposure , we graphed the induced genes , color coding them according to their time of peak induction ( 1 , 2 , or 4 hr following cocaine ) ( Figure 1F ) . Thus , for example , if a gene was commonly induced across structures with a peak at 1 hr across cocaine regimens , this would be evident as a contiguous vertical green line . This graph reveals aspects of the logic of these inducible transcription programs , whereby ( 1 ) genes induced following the different cocaine schedules largely maintain the same temporal structure , i . e . , if the peak induction of a given gene was observed at a defined time point in one program , its peak induction time was maintained across other programs; ( 2 ) following repeated cocaine exposure , we observe a substantial dampening of the transcriptional response in the DS , which recovers following cocaine challenge , recapitulating a significant proportion of the acute cocaine gene program; ( 3 ) all gene programs largely represent subcomponents of the program induced by acute cocaine in the DS . We further visualized the overlap in the identity of genes induced in the different structures using Venn diagrams ( Figure 1G ) , illustrating that the overlap stems principally from the immediate component of the transcriptional program ( peaking at 1 hr following cocaine ) , while transcripts induced at 2 or 4 hr following cocaine diverged between structures . Focusing on the most robust programs , induced in the DS , we found that gene clusters enriched at the 1 hr time points are related primarily to transcriptional regulation and synapse-to-nucleus signal transduction , while clusters related to modification of neural morphology and function were enriched at later time points ( Figure 1H; refer to Supplementary file 5 ) . Taken together , these results highlight robust transcriptional adaptations in the DS , positioning it as a major hub of cocaine-induced plasticity . Furthermore , our results illustrate the utilization of a conserved set of genes during the early wave of transcription following experience , followed by divergence of subsequent transcription , possibly to support region-specific mechanisms of plasticity ( Hrvatin et al . , 2018; Walker et al . , 2018 ) . Our observation of dynamic transcriptional responses to repeated cocaine exposure in the DS ( Figure 1 ) motivated us to address the cellular and spatial distribution of this transcriptional plasticity . Recently , using single-molecule fluorescence in-situ hybridization ( smFISH ) , we reported region-specific rules governing the recruitment of striatal assemblies following a single acute exposure to cocaine ( Gonzales et al . , 2020 ) . We now revisited this spatial analysis , applying smFISH to expand the investigation of the striatal distribution of the IEGs Arc , Egr2 , Fos , and Nr4a1 throughout the development of cocaine sensitization ( Figure 2; Figure 2—figure supplements 1 and 2; results from Gonzales et al . , 2020 serve as a reference for the effects of acute cocaine exposure ) . Addressing an overview of induced expression of these IEGs , we observed robust induction of Arc , Egr2 , Nr4a1 , and Fos following acute cocaine exposure , which was dampened following repeated exposure to cocaine and reinstated following a challenge dose of cocaine , in-line with the results described in Figure 1 ( Figure 2A–C ) . To visualize the subdomains defined by IEG expressing cells , we applied 2D kernel density estimation on striatal sections following repeated and challenge cocaine and compared resulting patterns to those previously described following acute cocaine exposure ( Gonzales et al . , 2020 ) . The prominent recruitment of IEG expression in the VLS observed following acute cocaine exposure was dampened drastically after repeated cocaine exposure , and re-emerged upon cocaine challenge . In contrast to the findings in the VLS , dampening of IEG induction in the MS , while evident , was more modest ( Figure 2D ) . These results are quantified in Figure 2E , F . In the VLS , the fraction of robustly expressing cells of Egr2 increased to 46 ± 10% after acute cocaine , decreased to 21 ± 4% following repeated cocaine , and subsequently increased to 40 ± 11% upon cocaine challenge . Similar dynamics were observed for Fos , where the fractions of suprathreshold cells were observed to be 37 ± 10% , 21 ± 4% , and 34 ± 7% following acute , repeated , and challenge cocaine , respectively . In contrast , in the MS , the fraction of cells expressing Egr2 and Fos increased to 42 ± 8% and 40 ± 7% after acute cocaine , modestly decreased to 33 ± 3% and 35 ± 3% after repeated cocaine , and regained elevated induction of 43 ± 4% and 40 ± 4% following challenge cocaine ( Figure 2E [mean ± SD]; Egr2 VLS F2 , 66 = 21 . 4 , p<0 . 0001; Fos VLS F2 , 30 = 4 . 9964 , p=0 . 01; Egr2 MS F2 , 66 = 6 . 4 , p=0 . 002; Fos MS F2 , 30 = 3 . 1 , p=0 . 06; ANOVA followed by Tukey’s test; for detailed statistics refer to Supplementary file 6 ) . With reference to expression levels , acute , repeated , and challenge cocaine-mediated puncta/cell expression in the VLS was observed to be 11 . 9 ± 3 . 8 , 3 . 9 ± 0 . 8 , 9 . 5 ± 3 . 4 for Egr2 and 6 . 8 ± 2 . 3 , 3 . 5 ± 0 . 5 , 5 . 9 ± 1 . 6 for Fos , respectively . Comparing these to the MS , the expression levels were observed to be 9 . 2 ± 2 . 2 , 6 . 5 ± 1 . 0 , and 9 . 5 ± 1 . 2 for Egr2 and 7 . 4 ± 1 . 5 , 6 ± 0 . 8 , and 7 . 5 ± 1 . 3 for Fos after acute , repeated , and challenge , respectively ( Figure 2F ) ( mean ± SD; Egr2 VLS F2 , 66 = 21 . 7 , p<0 . 0001; Fos VLS F2 , 30 = 4 . 9 , p=0 . 01; Egr2 MS F2 , 66 = 9 . 01 , p=0 . 0003; Fos MS F2 , 30 = 3 . 4 , p=0 . 04; ANOVA followed by Tukey’s test; for detailed statistics refer to Supplementary file 6 ) . A similar trend was evident for the expression of Arc and Nr4a1 in the VLS vs . the MS ( Figure 2—figure supplement 1A , B ) . Notably , the expression of different IEGs was highly correlated within individual cells , defining overlapping populations of neurons responsive to the cocaine experiences studied . Once recruited by cocaine , neurons committed to co-expression of multiple IEGs to virtually identical levels ( Figure 2—figure supplement 2; for detailed statistics , see Supplementary file 6 ) . These data demonstrate the coherent co-expression of multiple IEGs within striatal assemblies during the development of behavioral sensitization to cocaine , likely to support mechanisms of long-term plasticity within these ensembles . In sum , the history of cocaine experience is reflected in the differential transcriptional recruitment of striatal subdomains , dampening drastically in the VLS following repeated exposure . Striatal Drd1+-neurons are implicated in promoting actions , while Drd2+-neurons are implicated in the tempering and refinement of action selection ( Bariselli et al . , 2019 ) . Differential IEG induction in Drd1+ vs Drd2+ expressing SPN ensembles is expected to shed light on the relative contribution of plasticity within each cell type to the development of cocaine behaviors . We have previously reported that acute exposure to cocaine induces Egr2 expression in both Drd1+ and Drd2+ neurons in the MS , while more selectively inducing Egr2 expression in Drd1+-neurons in the VLS ( Gonzales et al . , 2020 ) . Extending this analysis to repeated and challenge cocaine exposures and with additional IEGs , we observed robust dampening of the induction of Egr2 and Fos in VLS Drd1+ neurons following repeated exposure to cocaine , while upon cocaine challenge , prominent induction was again evident , especially in Drd1 SPNs . ( Figure 3A–C , Figure 3—figure supplement 1 ) . In contrast , in the MS , subtle dampening was observed and Egr2 and Fos expression maintained consistent correlation to Drd1 and Drd2 expression throughout acute , repeated , and challenge cocaine exposures ( Figure 3C , Figure 3—figure supplement 1; for reference of Drd1 and Drd2 levels in MS and VLS , see Figure 3—figure supplement 2 , Supplementary file 6 for statistics ) . Thus , the observed attenuated transcriptional recruitment in the DS can be attributed to selective dampening of IEG induction , primarily within VLS Drd1+ neurons . This specialization in transcriptional plasticity likely underlies differential roles of the striatal subregions and cells within them in supporting behavioral modification induced by cocaine experience . The greater enrichment of Egr2 induction within VLS neurons suggests a causal role for this neuronal population in supporting cocaine conditioned behaviors . To address the role of VLS Egr2+ neurons in cocaine seeking , we bilaterally injected Cre-dependent inhibitory hM4Di DREADD ( VLS-Egr2hM4Di ) , targeting the VLS of Egr2-Cre knock-in mice . In these mice , an Egr2 allele is substituted for Cre ( Voiculescu et al . , 2000 ) , supporting the expression of Cre recombinase in neurons expressing Egr2 . DREADD hM4Di-mediated selective inhibition of the VLS Egr2-expressing neuronal ensembles was achieved by administration of clozapine-N-oxide ( CNO ) ( Atlan et al . , 2018; Terem et al . , 2020 ) . Control mice were either transduced with viruses expressing hM4Di , similar to the experimental group , and exposed to saline ( Figure 3E ) or transduced with viruses conditionally expressing mCherry and exposed to CNO ( Figure 3G , for expression domains , see Figure 3F , Figure 3—figure supplement 3A , B ) . In an initial experiment , we transduced two groups of mice with AAV-DIO-hM4Di to the VLS . Three weeks later , we ran a cocaine conditioned-place preference ( CPP ) experiment , in which mice were conditioned over three alternate days to a cocaine-associated context , while on the day following each conditioning session their side preference was tested ( conditioning – days 2 , 4 , 6; tests – days 1 , 3 , 5 , 7; Figure 3D , ‘Design 1’ ) . In control mice expressing hM4Di and exposed to saline prior to cocaine conditioning sessions , CPP developed following a single conditioning session and was reinforced following additional conditioning sessions ( Figure 3E ) . The experimental group , which was exposed to CNO ( 5 mg/kg; i . p . ) 30 min prior to cocaine conditioning , also displayed CPP following the initial exposure; however , in this group , the preference decayed with additional conditioning , such that following the third conditioning session , CPP in this group was significantly different from the control group ( preference score saline vs . CNO Test3: p<0 . 05 , t = 2 , df = 8 . 6; one-tailed t-test; Figure 3E ) . We interpret these results as suggesting that the first cocaine conditioning session induced expression of Cre within VLS Egr2+ neurons , supporting the accumulation of functional hM4Di within these neurons to a CNO-responsive complement by the third conditioning session , resulting in diminished conditioned-place preference . In a subsequent experiment , we implemented conditioning to the cocaine-associated context for three consecutive days prior to performing a preference test and exposed both experimental ( hM4Di-expressing ) and control ( mCherry-expressing ) groups to CNO ( 10 mg/kg; i . p . ) prior to cocaine conditioning session ( Figure 3D , ‘Design 2’ ) . We found that both groups demonstrated CPP ( Figure 3G; paired t-test on % time spent on drug paired side; pVLS-Egr2mCherry < 0 . 00001 , t = −11 . 362 , df = 6; pVLS-Egr2hM4Di < 0 . 003 , t = −4 . 1232 , df = 6 ) . However , mice in which VLS Egr2+ neurons were inhibited ( VLS-Egr2hM4Di ) displayed lower preference for the drug paired context ( preference score ) compared to control mice ( VLS-Egr2mCherry ) ( Figure 3G; p<0 . 05 , t = 1 . 95 , df = 9 . 4 , one-tailed t-test ) . Importantly , no differences in locomotion were observed between groups on conditioning or test days ( p=0 . 93 , F4 , 48 = 0 . 19 , ANOVA; Figure 3—figure supplement 3C ) . We therefore conclude that VLS Egr2+-expressing neurons contribute to the development of cocaine-seeking behavior , with no obvious impact on locomotor aspects of cocaine-driven behavior . Salient experiences in general , and specifically exposure to cocaine , are thought to modify future behavior through induced gene expression responses , leading to stable changes in cell and circuit function ( Nestler and Lüscher , 2019; Robison and Nestler , 2011 ) . We hypothesized that the induction of Egr2 by cocaine within VLS neurons may play a causal role in cocaine-induced modification of behavior . To assess a potential link between the expression of Egr2 and cellular plasticity responsible for such behavioral modification , we ran an additional CPP experiment , following bilateral viral transduction of the VLS neurons with AAV-eGFP ( VLSGFP ) , or a dominant-negative ( S382R , D383Y ) isoform of Egr2 ( VLSDNEgr2; Figure 3H , Figure 3—figure supplement 4A , B ) . The dominant-negative mutation disrupts the DNA-binding activity of Egr2 , while not interfering with the capacity of the protein to form heteromeric complexes with its natural binding partners , effectively inhibiting transcriptional activation of downstream genes regulated by Egr2 ( LeBlanc et al . , 2007; Nagarajan et al . , 2001 ) . Comparing the development of cocaine CPP , we found that both groups of mice developed CPP ( Figure 3I , paired t-test on % time spent on drug paired side; pVLS-GFP <0 . 001 , t = −4 . 9782 , df = 7; pVLS-DNEgr2 <0 . 05 , t = −2 . 2199 , df = 7 ) . However , VLSDNEgr2 developed lower CPP than VLSGFP mice ( Figure 3I , p<0 . 05 , t = 2 . 36 , df = 14 , unpaired t-test ) . No differences in locomotion were observed between the groups of mice ( p=0 . 7 , F4 , 56 = 0 . 54 , ANOVA; Figure 3—figure supplement 4C ) . These results assign a functional role to Egr2 induction , primarily within VLS Drd1+ neurons , in the development of conditioned-place preference to cocaine . To test the effect of disrupting Egr2 complexes may have on transcription , we analyzed the expression of Arc , Egr2 , and Nr4a1 in the VLS , MS , NAc , and LCtx . In the VLS , we observed the anticipated overexpression of Egr2 ( Figure 3—figure supplement 4D , p ( Egr2 ) <0 . 05 , t = −5 . 3616 , df = 2; two-tailed t-test , reflecting exogenous expression of the mutant gene ) , as well as blunted Arc and Nr4a1 expression ( Figure 3—figure supplement 4D , p ( Arc ) <0 . 01 , t = 6 , df = 2 . 8; p ( Nr4a1 ) <0 . 005 , t = 6 . 2 , df = 3 . 8; two-tailed t-test ) . We did not observe any clear differences in gene expression between groups within other structures , demonstrating the localized effect of our viral manipulation ( Figure 3—figure supplement 4E–G ) . These results demonstrate a role for cocaine-induced expression of Egr2 in the VLS in supporting the development of cocaine-seeking and suggest that inducible transcriptional complexes involving Egr2 are functional in facilitating drug-induced maladaptive plasticity .
Drugs of abuse such as cocaine are known to act on key brain circuits , modifying and biasing the future behavior of an individual toward increased drug seeking . In this study , we develop a comprehensive compendium of the transcriptional dynamics induced within key brain regions during the development of cocaine sensitization . We highlight the striatum as a major hub of plasticity , within which we identify differential transcriptional recruitment of neuronal ensembles by cocaine , dependent on striatal subdomain , identity of projection neurons and the history of cocaine exposure . Finally , we focus on a prominent cocaine-sensitive IEG , Egr2 , and show that Egr2-expressing SPNs in the VLS , and the expression of Egr2 within them , support drug-seeking behavior . Repeated exposure to cocaine , as well as abstinence , produces long-lasting functional changes in the reward circuit to drive the maladaptive modification of reinforced behavior ( Dong and Nestler , 2014; Everitt , 2014; Gremel and Lovinger , 2017; Hyman et al . , 2006; Kelley , 2004; Lüscher , 2016; Lüscher and Malenka , 2011; Nestler , 2013; Russo and Nestler , 2013; Salery et al . , 2020; Volkow and Morales , 2015; Wolf , 2016; Zahm et al . , 2010 ) . The imprinting of such potentially lifelong alterations in behavior driven by drug experience is supported by cocaine-induced modifications in gene expression ( McClung and Nestler , 2008; Nestler , 2002; Nestler and Lüscher , 2019; Salery et al . , 2020; Steiner , 2016; Steiner and Van Waes , 2013 ) . In this study , using an unbiased approach to screen gene expression , we resolved the transcriptional landscapes of distinct cocaine experiences across multiple reward-related brain circuits with broad temporal resolution . Our approach allowed us to describe transcripts modulated at updated baselines ( after a history of either repeated cocaine exposure or abstinence ) , as well as in the hours following exposure to distinct cocaine experiences . Baseline transcriptional changes in cortical and basal ganglia structures following defined cocaine schedules have been described previously in both rodents and humans ( Bannon et al . , 2005; Bannon et al . , 2014; Eipper-Mains et al . , 2013; Freeman et al . , 2010; Gao et al . , 2017a; Hurd and Herkenham , 1993; Lull et al . , 2008; Ribeiro et al . , 2017; Walker et al . , 2018 ) . Consistent with previous findings , we observed dynamic shifts in baseline gene expression in multiple categories potentially associated with neuronal plasticity ( synaptic genes; genes associated with projection morphogenesis , actin filament regulation; proteostasis and myelin ) . Interestingly , genes associated with neuronal morphology and synaptic function demonstrated unique patterns of shifts within different brain structures . For example , expression of genes such as Vamp , Pkrcg , Ncdn , Camk2b , Shank3 , and Syp were downregulated in the NAc following repeated cocaine exposure , while being upregulated in the DS . Such region-specific shifts in gene expression may support circuit-specific structural and functional modifications to cell assemblies ( Clayton et al . , 2020; Kyrke-Smith and Williams , 2018 ) . Myelin genes ( Plp1 , Mobp , Mbp , Mal , Pllp ) were downregulated across all structures studied ( LCtx , Amy , NAc , DS , LH ) , initially following repeated cocaine exposure , and further following abstinence , across all experimental mice . Conversely , genes associated with proteostasis ( e . g . , chaperones such as members of the CCT , Hsp40 , Hsp70 , and Hsp90 complexes ) were upregulated in concert across structures following cocaine abstinence . Notably , similar changes in myelin genes and genes associated with proteostasis have been described in both human and rodent studies ( Albertson et al . , 2004; Bannon et al . , 2014; García-Fuster et al . , 2012; Johnson et al . , 2012; Kovalevich et al . , 2012; Lull et al . , 2008; Narayana et al . , 2014 ) , but their functional implications remain unknown . Future investigation into the features of cocaine experience-related transcriptome is anticipated to provide targets for intervention , potentially supporting the reversal of brain function to a ‘cocaine-naive’ state . IEG expression is well accepted to be the substrate for long-term modulations supporting memory formation ( Alberini , 2009; Alberini and Kandel , 2015 ) . Although cocaine-induced IEG expression has been extensively characterized in rodents ( Berke et al . , 1998; Caster and Kuhn , 2009; Gao et al . , 2017a; Guez-Barber et al . , 2011; Moratalla et al . , 1996; Piechota et al . , 2010; Robison and Nestler , 2011; Savell et al . , 2020; Steiner , 2016; Valjent et al . , 2006; Zahm et al . , 2010 ) , these studies were mostly limited in the number of genes analyzed and restricted to isolated brain structures following specific drug regimens . Addressing the cocaine-induced transcriptome , we observed transcriptional recruitment of the LCtx , Amy , NAc , and DS , of which the DS was most prominent . Furthermore , the immediate-early transcriptional programs induced across other tissues largely consisted of subcomponents of DS programs . What does this imply ? We propose thathe overlapping fraction of induced genes is representative of a ‘core transcriptome’ that is consistently induced across many structures or cell types and only varies in the magnitude of their expression ( Hrvatin et al . , 2018; Savell et al . , 2020; Tyssowski et al . , 2018 ) . This core component predominantly corresponds to signaling molecules and transcriptional regulators ( the genes common across most programs are Arc , Arl4d , Btg2 , Ddit4 , Dusp1 , Egr2 , Egr4 , Fos , Fosb , Junb , Nr4a1 , Per1 , and Tiparp ) , likely responsible for transforming inducing signals into instructions for implementation of appropriate synaptic , cellular , and circuit-specific plasticity mechanisms by ‘effector’ genes . These downstream effector genes are induced in a secondary wave of transcription , corresponding to the significantly diversified gene response at 2–4 hr following cocaine ( Amit et al . , 2007; Clayton et al . , 2020; Gray and Spiegel , 2019; Hrvatin et al . , 2018; Mukherjee et al . , 2018; Tyssowski et al . , 2018; Yap and Greenberg , 2018 ) . Interestingly , a recent landmark study ( Savell et al . , 2020 ) utilized a multiplexed CRISPR strategy to drive co-expression of genes overlapping with many of the components of the putative ‘core transcriptome’ ( Btg2 , Egr2 , Egr4 , Fos , FosB , JunB , and Nr4a1 ) in the NAc and found that this manipulation increased SPN excitability and enhanced the development of cocaine sensitization . What might be the role of the transcriptional induction in the DS and its subsequent dampening ? It is becoming more broadly accepted that IEG induction serves to support long-term plasticity ( Chandra and Lobo , 2017; Clayton , 2000; Clayton et al . , 2020; Mukherjee et al . , 2018; Tyssowski and Gray , 2019 ) . The MS is defined as the ‘associative striatum’ and is associated with goal-directed behaviors , as well as defining the vigor of locomotor actions ( Balleine and O'Doherty , 2010; Balleine and Ostlund , 2007; Kravitz et al . , 2010; Lipton et al . , 2019; Nonomura et al . , 2018 ) . We propose that the cocaine-driven locomotor sensitization may be mediated by the balanced and largely maintained transcriptional induction within Drd1/Drd2 SPNs in the MS . The lateral ‘sensori-motor’ striatum is strongly associated with habit formation and compulsive drug seeking ( Lipton et al . , 2019; Yin et al . , 2004; Zapata et al . , 2010 ) . Moreover , the VLS receives selective sensorimotor afferents mapped to upper limb and orofacial cortical regions . Interestingly , behavioral stereotypies , primarily upper limb and orofacial , arise upon high-dose psychostimulant exposure ( Karler et al . , 1994; Murray et al . , 2015; Schlussman et al . , 2003 ) , and orofacial stereotypies have been induced following selective infusion of psychostimulants to the VLS ( Baker et al . , 1998; Delfs and Kelley , 1990; Rebec et al . , 1997; White et al . , 1998 ) . It is intriguing to consider the possibility that recruitment of plasticity mechanisms within VLS Drd1+ neurons supports the increased propensity to engage in orofacial stereotypies , while the subsequent dampening of cocaine-induced transcription within these neurons may indicate the ‘canalization’ of this limited action repertoire , at the expense of a broader behavioral repertoire . This topic will form the basis for future investigation . Infusion of psychostimulants into the VLS has also been shown to promote operant reinforcement and conditioned-place preference , implicating it in reward and reinforcement ( Baker et al . , 1998; Kelley and Delfs , 1991 ) . In order to query the role of the VLS IEG-expressing ensembles in the development of cocaine context association , we inhibited the activity of VLS Egr2+ neurons by conditional expression of hM4Di DREADDs , which curbed CPP . To directly investigate a role for VLS IEG induction on CPP behavior , we expressed a dominant-negative isoform of Egr2 ( in which the DNA-binding domain was inactivated ) in the VLS and observed a similar impact . Thus , to our knowledge , we provide the first functional implication of the VLS in cocaine seeking . Furthermore , we describe cellular dynamics of transcriptional recruitment of VLS IEG+ neurons ( primarily Drd1+ ) during the development of behavioral sensitization to cocaine . The development and execution of drug-seeking behavior is heavily context dependent ( Calipari et al . , 2016; Crombag et al . , 2002; Crombag et al . , 2008; Crombag and Shaham , 2002; Cruz et al . , 2014; Lee et al . , 2006; Rubio et al . , 2015 ) . Potentially , the dampening of sensorimotor VLS IEG induction following repeated cocaine could serve to ‘cement’ the initial context association , limiting behavioral flexibility and the capacity to revert context association , exacerbating the impact of contextual cues on drug seeking behavior ( Calipari et al . , 2016; Crombag and Shaham , 2002; Gipson et al . , 2013; Hyman , 2005; Phillips et al . , 2003; Shaham et al . , 2003; Volkow et al . , 2006 ) . Recently , we have shown that salient experiences are represented in the mouse brain by unique patterns of gene expression . Thus , the induction pattern of a handful of genes is sufficient to decode the recent experience of individual mice with almost absolute certainty . Of these , the IEG whose expression contributes most towards classification of the recent experience of individual mice is Egr2 ( Mukherjee et al . , 2018 ) . Egr2 is , furthermore , the gene most robustly induced by cocaine in the dorsal striatum ( Gonzales et al . , 2020; Mukherjee et al . , 2018; Supplementary file 4 ) and is a sensitive indicator of cocaine-engaged striatal cell assemblies ( Gonzales et al . , 2020 ) . In the current study , we initiated investigation into the role of Egr2 in promoting drug seeking . Previous studies have shown that Egr2 is crucial for normal hindbrain development , peripheral myelination , and humoral immune response and is implicated in diseases such as congenital hypomyelinating neuropathy , Charcot–Marie-Tooth disease , Dejerine–Sottas syndrome , as well as schizophrenia ( Boerkoel et al . , 2001; De and Turman , 2005; Li et al . , 2019; Morita et al . , 2016; Okamura et al . , 2015; Svaren and Meijer , 2008; Topilko et al . , 1994; Warner et al . , 1999; Warner et al . , 1998; Wilkinson , 1995; Yamada et al . , 2007 ) . In the central nervous system , Egr2 has been shown to be induced by seizure activity , kainic acid injection , LTP-inducing stimuli in hippocampal neurons , as well as following administration of several groups of drugs such as methamphetamine , cocaine , heroin , and alcohol ( Gao et al . , 2017a; Gass et al . , 1994; Imperio et al . , 2018; López-López et al . , 2017; Mataga et al . , 2001; Rakhade et al . , 2007; Saint-Preux et al . , 2013; Worley et al . , 1993 ) . However , the role Egr2 may play in encoding memory or drug-induced behavior remained unresolved . Our findings show that the activity of Egr2 is required for the full development of cocaine place preference , and highlight an additional member of the Egr family , alongside Egr1 and Egr3 , in drug-induced plasticity ( Bannon et al . , 2014; Chandra et al . , 2015; Moratalla et al . , 1992; Valjent et al . , 2006 ) . Egr2 has been implicated in the regulation of cell-specific gene expression in peripheral Schwann cells ( Jang et al . , 2006 ) and fibroblasts ( Fang et al . , 2011 ) , and disruptions to Egr2 DNA binding have been implicated in diseases of myelination and brain development . However , we are not aware of any study identifying the targets of Egr2 in the mature brain . We report downregulated expression of Nr4a1 and Arc following overexpression of dominant-negative Egr2 in the VLS . However , as we did not identify Egr2 binding sites within regulatory regions of Nr4a1 or Arc , we hypothesize that the impact of DN-Egr2 expression on Nr4a1 and Arc may be indirect , a point for future investigation . In conclusion , our study provides ( 1 ) a comprehensive description of brain-wide transcriptional dynamics , as well as spatial dynamics of SPN-specific IEG recruitment during the development of cocaine sensitization and ( 2 ) a demonstration of the role of VLS Egr2-expressing ensembles , as well as VLS expression of Egr2 , in the development of cocaine seeking . Future work will address the mechanisms supporting cell-type specificity of transcriptional induction , as well as the role of IEG-mediated plasticity mechanisms in VLS-dependent stereotypy and context association .
Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact , Ami Citri ( ami . citri@mail . huji . ac . il ) . This study did not generate new unique reagents .
Drug addiction develops due to brain-wide plasticity within neuronal ensembles , mediated by dynamic gene expression . Though the most common approach to identify such ensembles relies on immediate early gene expression , little is known of how the activity of these genes is linked to modified behavior observed following repeated drug exposure . To address this gap , we present a broad-to-specific approach , beginning with a comprehensive investigation of brain-wide cocaine-driven gene expression , through the description of dynamic spatial patterns of gene induction in subregions of the striatum , and finally address functionality of region-specific gene induction in the development of cocaine preference . Our findings reveal differential cell-type specific dynamic transcriptional recruitment patterns within two subdomains of the dorsal striatum following repeated cocaine exposure . Furthermore , we demonstrate that induction of the IEG Egr2 in the ventrolateral striatum , as well as the cells within which it is expressed , are required for the development of cocaine seeking .
The human brain is ever changing , constantly rewiring itself in response to new experiences , knowledge or information from the environment . Addictive drugs such as cocaine can hijack the genetic mechanisms responsible for this plasticity , creating dangerous , obsessive drug-seeking and consuming behaviors . Cocaine-induced plasticity is difficult to apprehend , however , as brain regions or even cell populations can react differently to the compound . For instance , sub-regions in the striatum – the brain area that responds to rewards and helps to plan movement – show distinct responses during progressive exposure to cocaine . And while researchers know that the drug immediately changes how neurons switch certain genes on and off , it is still unclear how these genetic modifications later affect behavior . Mukherjee , Gonzales et al . explored these questions at different scales , first focusing on how progressive cocaine exposure changed the way various gene programs were activated across the entire brain . This revealed that programs in the striatum were the most affected by the drug . Examining this region more closely showed that cocaine switches on genes in specific ‘spiny projection’ neuron populations , depending on where these cells are located and the drug history of the mouse . Finally , Mukherjee , Gonzales et al . used genetically modified mice to piece together cocaine exposure , genetic changes and modifications in behavior . These experiments revealed that the drive to seek cocaine depended on activation of the Egr2 gene in populations of spiny projection neurons in a specific sub-region of the striatum . The gene , which codes for a protein that regulates how genes are switched on and off , was itself strongly activated by cocaine intake . Cocaine addiction can have devastating consequences for individuals . Grasping how this drug alters the brain could pave the way for new treatments , while also providing information on the basic mechanisms underlying brain plasticity .