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Psychiatry and Neurobehavioral Sciences, University of Virginia, Charlottesville, VAMedical Scientist Training Program, University of Virginia, Charlottesville, VA
Exercise has shown promise as a treatment for cocaine use disorder; however, the mechanism underlying its efficacy has remained elusive.
METHODS
We used a rat model of relapse (cue-induced reinstatement) and exercise (wheel running, 2-hr/day) coupled with RNA-sequencing to establish transcriptional profiles associated with the protective effects of exercise (during early, days 1-7, or throughout, days 1-14, withdrawal) versus non-effective exercise (during late withdrawal, days 7-14) against cocaine-seeking and sedentary conditions.
RESULTS
As expected, cue-induced cocaine-seeking was highest in the sedentary and late-withdrawal exercise groups; both groups also showed upregulation of a Grin1-associated transcript and enrichment of Drd1-Nmdar1 complex and glutamate receptor complex terms. Surprisingly, these glutamate markers were also enriched in the early- and throughout-withdrawal exercise groups despite lower levels of cocaine-seeking. However, a closer examination of the Grin1-associated transcript revealed a robust loss of transcripts spanning exons 9 and 10 in the sedentary condition relative to saline controls that was normalized by exercise during early- and throughout-withdrawal, but not late-withdrawal exercise, indicating these exercise conditions may normalize RNA mis-splicing induced by cocaine-seeking. Our findings also revealed novel mechanisms by which exercise initiated during early withdrawal may modulate glutamatergic signaling in dmPFC (e.g., via transcripts associated with non-NMDA glutamate receptors or those impacting signaling downstream of NMDA receptors), along with mechanisms outside of glutamatergic signaling such as circadian rhythm regulation and neuronal survival.
CONCLUSIONS
These findings provide a rich resource for future studies aimed at manipulating these molecular networks in order to better understand how exercise decreases cocaine-seeking.
More than 5.1 million individuals in the United States over the age of 12 reported using cocaine in 2020 and 1.3 million met the criteria for cocaine use disorder (CUD; 1). As the supply of cocaine continues to grow, the number of users and individuals with CUD is projected to increase (
United States Department of State (2017): International Narcotics Control Strategy Report, Drug and Chemical Control. Bureau for International Narcotics and Law Enforcement Affairs.
). Exercise has shown promise as a non-pharmacological intervention for substance use disorders in human and animal studies. In humans, clinical trials of exercise have revealed beneficial health and anti-craving effects (
Hallgren M, Vancampfort D, Hoang MT, Andersson V, Ekblom Ö, Andreasson S, et al. (2021): Effects of acute exercise on craving, mood and anxiety in non-treatment seeking adults with alcohol use disorder: An exploratory study. Drug Alcohol Depend 1;220:108506.
), with recent findings showing that drug-craving is immediately and persistently attenuated following an acute bout of aerobic exercise in individuals undergoing in-patient treatment for polysubstance use disorder (
). Similarly, animal studies show that exercise, such as voluntary running in a wheel, markedly decreases drug use and drug-seeking, and when available during withdrawal following drug self-administration, prevents the incubation of drug-craving, a phenomenon that occurs in both humans and animals and is believed to reflect an enhanced vulnerability to relapse (
Exercise during abstinence normalizes ultrastructural synaptic plasticity associated with nicotine-seeking following extended access self-administration.
Chronic wheel running-induced reduction of extinction and reinstatement of methamphetamine seeking in methamphetamine dependent rats is associated with reduced number of periaqueductal gray dopamine neurons.
Despite these encouraging findings, the efficacy of exercise in humans has been variable, and while lack of power and compliance issues undoubtedly contribute to the variability (
). For example, we showed that exercise (wheel running) during early (days 1-7) or throughout withdrawal (days 1-14) prevents the development of high levels of cocaine-seeking and persistently protects against cocaine-seeking as assessed on withdrawal day 15. In contrast, exercise during late withdrawal (days 8-14) was not effective at reducing cocaine-seeking responding on withdrawal day 15, suggesting that the timing of exercise during withdrawal is more important than amount or recency of exercise (19, exercise 2h/day; 21, exercise 1-6h/day; 22, exercise 1-24h/day; see 44,45 for reviews). It also suggests that effects in humans could be prolonged by initiating exercise during early withdrawal rather than after detoxification as it typically occurs. A better understanding of the molecular mechanisms that underlie these differential effects of exercise would also help guide the development of exercise in humans with CUD since such information will help define the exercise conditions necessary for achieving an anti-relapse effect.
), we started to isolate the molecular changes associated with effective versus non-effective exercise conditions focusing on neuroadaptations in the dorsomedial prefrontal cortex (dmPFC), which is known to be critically involved in cocaine-seeking and its incubation over withdrawal (
Prelimbic cortex is a common brain area activated during cue‐induced reinstatement of cocaine and heroin seeking in a polydrug self‐administration rat model.
). We focused on markers known to be regulated by exercise and/or drug-seeking including genes involved in brain-derived neurotrophic factor (BDNF; Bdnf exons I, IV, and IX and its receptor, Ntrk2), glutamate (AMPA type subunit 1, 2, and 3; Gria1, 2, and 3; NMDA type subunit 1; Grin1; mGlu1, 2, and 5; Grm1, 2, and 5), and dopamine signaling (D1 and D2 receptors; Drd1 and Drd2). mGlu1 and mGlu5 showed the most promise as a potential mechanism underlying the protective effect of exercise against cocaine-seeking given that dmPFC gene expression levels (Grin1 and Grm5) were positively associated with cocaine-seeking responses (or responding during extinction/reinstatement testing); site-specific activation of mGlu5 receptors during early withdrawal also mimicked the efficacy of exercise. However, blockade of these receptors during early withdrawal did not block the efficacy of exercise indicating that while dmPFC mGlu5 contribute to the incubation of cocaine-seeking, they are not necessary for the efficacy of exercise to reduce cocaine-seeking (
). Thus, the mechanism for the efficacy of exercise in reducing cocaine-seeking remains elusive.
The purpose of the present study was to use RNA-sequencing (RNA-seq) as an unbiased and hypothesis generating approach to explore transcriptional changes in the dmPFC associated with cocaine-seeking and the protective effects of exercise against cocaine-seeking. Although several studies have characterized transcriptional changes associated with cocaine self-administration and/or cocaine-seeking, the vast majority of these studies have used short-access conditions (1-2hr/day access to the drug), which are best for studying initial vulnerability to drug use (e.g., 86-90, but see 91). Notably, to our knowledge, RNA-seq has not been used to assess changes that occur in response to cue-induced cocaine-seeking following withdrawal from extended-access drug self-administration (i.e., ≥ 6-hr/day access to the drug), which is the gold-standard for inducing addiction-like features in animals, including enhanced drug-seeking/vulnerability to relapse (
). We focused on changes in transcripts rather than genes given mounting evidence indicating that RNA mis-splicing contributes to numerous human diseases (e.g., Parkinson’s, dilated cardiomyopathy;53), with preliminary support for a role in substance use disorder (e.g., alcohol use disorder,54). Based on our previous findings in male rats showing that cocaine-seeking is attenuated by exercise during early (days 1-7) or throughout withdrawal (days 1-14), but not by exercise during late withdrawal (days 8-14; 18-19), we predicted that the protective effects of exercise against cocaine-seeking would be revealed by isolating transcriptional changes induced by early and throughout exercise versus late and sedentary conditions (relative to saline).
METHODS AND MATERIALS
Subjects
Adult male Sprague-Dawley rats (Charles River; N=21) weighing approximately 380 g at the start of the study were used as subjects. These animals were representative subsets of the sedentary (5 of 8), early (4 of 14), throughout (4 of 8), late (4 of 10), and saline (5 of 10) groups we used in our previous study on the impact of early- versus late-withdrawal initiated exercise on cocaine-seeking and associated gene expression changes (as assessed using qPCR;19). All procedures were approved by the University of Virginia Animal Care and Use Committee and were conducted within guidelines set by the NIH.
Behavioral Procedure
The procedures used for housing, lever-pretraining, and surgical implantation of jugular catheters, cocaine self-administration during training and the extended-access period, exercise during withdrawal, and cue-induced cocaine-seeking testing are thoroughly described in our previous study (19; also see Figure 1). Briefly, rats were trained to self-administer cocaine (1.5 mg/kg/infusion) under a fixed-ratio 1 schedule with a maximum of 20 infusions available/day and, once acquired (20 infusions, 2 consecutive days), were given extended (24-h/day) access to the drug using a discrete trial procedure (2 trials/hr, 1.5 mg/kg/infusion) for 10 days. Two additional fixed-ratio 1 sessions were run to confirm patency before withdrawal. Then, rats were housed in polycarbonate cages without (sedentary; n=5) or with 2-hr/day access to a running wheel during early (days 1-7; n=4), late (days 8-14; n=4), or throughout (days 1-14; n=4) withdrawal. Saline controls underwent the procedures as those described for the sedentary cocaine group (n=4) except that they previous self-administered saline instead of cocaine. Rats were returned to their operant chambers on day 14 of withdrawal and then underwent relapse testing on withdrawal day15 using a within-session extinction/cue-induced reinstatement procedure (i.e., a minimum of six, 1-hr extinction sessions wherein responding was without consequence followed by a 1-hr reinstatement session where each response was reinforced with cocaine-associated cues which included a stimulus light and the sound of the pump).
Figure 1Summary of experimental events. Male rats were trained to self-administer cocaine. Following acquisition of cocaine self-administration (2 consecutive sessions wherein all 20 infusions available were obtained), rats were given 24-hr/day, extended-access to cocaine (1.5 mg/kg/infusion) under a discrete trail procedure for 10 sessions. Following the last cocaine self-administration session, rats were housed without (sedentary, n=5) or with access to wheel running (2- h/day) during early (days 1–7; early, n=4), late (days 8–14; late, n=4), or throughout (days 1–14; thru, n=4) a 14-day withdrawal period. Additional rats were given access to saline and housed without access to a wheel during “withdrawal” (saline, n=4). On day 15 of withdrawal, rats underwent extinction/reinstatement testing. On the morning following this 1-day test session, tissue was collected from the dorsomedial prefrontal cortex (dmPFC) and RNA-sequencing was performed. This graphical illustrations was made using BioRender (https://biorender.com/).
The morning following the test session, anesthetized rats were euthanized by rapid decapitation, and the dmPFC was dissected from 2-mm-thick coronal brain slices based on Paxinos and Watson (2007) coordinates (Bregma 3.2 mm). Brain tissue was rapidly frozen and stored at −80 °C until further processing. To generate RNA-seq libraries, total RNA was isolated from each sample using RNeasy®Lipid Tissue Mini Kit (Qiagen) and 5 μg of total RNA was processed for ribosomal RNA removal using the Illumina Ribo-Zero KIT. RNA-seq libraries were prepared using the Illumina ScriptSeq™ v2 RNA-seq Library Preparation Kit and purified using AMPure XP beads. The library quality and quantity was assessed by a 2100 Bioanalyzer prior to sequencing by the UVA Core facility. Datasets were assessed for quality and consistency using FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). Raw reads were then aligned to the rat rn6 genome using HiSAT2 specifying --rna-strandness FR (
). SAMtools was used to convert the resulting SAM files to BAM format, and transcripts were assembled and quantified with StringTie using the ENSEMBL Rattus_norvegicus_Rnor_6.0.85 annotations (
). The resulting count tables were then used for identification of differentially expressed transcripts. DESeq2 (v1.30.1) was used to normalize the raw counts and to perform differential expression analysis (
). The RNA-seq analysis was performed using the tidyverse (v1.3.1) and dplyr (v1.0.8) software packages and the term plots were made using ggplot2 (v3.3.3). The network maps were made using Cytoscape (v3.8.2). Volcano plots were created using the EnhancedVolcano (v1.8.0) R package (
Blighe, K., Rana, S., & Lewis, M. (2019): EnhancedVolcano: Publication-ready volcano plots with enhanced colouring and labeling. R package version 1.2.0.
To visualize transcript changes relative to saline control animals, the merged RNA-seq data sets were loaded on the Integrative Genomics Viewer (IGV) using the rat rn6 genome (
). Differential normalized read counts for each condition were generated by subtracting the merged saline reads. IGV coverage tracks were visualized from bigwig files of paired reads, which joins the two mates of the paired-end reads, and therefore span the entire range between the two mates. Exon numbering was generated by IGV using the NCBI RefSeq rat rn6 transcripts.
Data analysis
We first confirmed that the effects of exercise in these subsets of rats were similar to those observed previously in the larger sample of sedentary, early, throughout, and late groups (
). Specifically, univariate ANOVA was used to examine group differences in cocaine-seeking during extinction (total responses) and repeated measures ANOVA was used to examine group differences in cocaine-seeking during reinstatement relative to responses during the last extinction session. Repeated measures ANOVA was also used to verify that cocaine intake was similar between each of the cocaine groups during the extended-access phase and that levels of running were similar between each of the exercise groups during the seven-day exercise period (first seven days for throughout exercise). Post-hoc comparisons were Tukey-corrected and based on either one-tailed (for predicted differences; e.g., higher cocaine-seeking in sedentary versus early and throughout exercise) or two-tailed (non-predicted differences) distributions. The association between the efficacy of exercise to reduce cocaine-seeking responses and transcriptional changes (normalized counts) were examined using Benjamini-Hochberg corrected Pearson correlations.
RESULTS
Behavioral Results
Extended-Access Cocaine Self-Administration and Exercise
), results from these subsets of rats showed high levels of cocaine self-administration during the 10-day extended-access period with similar levels and patterns of intake between the groups (Figure 2A-B; group and group by session, P’s>0.05). Intake was highest in each of the groups during initial versus later sessions (session, F9,117=3.22, P<0.01; session 1 versus 10, P<0.05). Overall levels and patterns of running during withdrawal were also similar between the three exercise groups (Figure 2C; group and group by session, P’s>0.05). Levels of running progressively increased in each of the groups over the seven exercise sessions (session, F6,54=5.18, P<0.001; session 1 versus 7, P<0.01). Thus, prior to extinction/reinstatement testing, levels and patterns of cocaine intake and exercise were similar between the groups.
Figure 2Timing of exercise during withdrawal on cue-induced cocaine-seeking responding. A) Mean (±SEM) number of infusions for each of the 10 extended-access sessions, B) cocaine intake averaged across the extended-access period (mg/kg), C) distance run for the first 7 exercise sessions (km), and D) total number of responses made on the lever formerly associated with cocaine during extinction, and E) during the last extinction session versus the reinstatement session for male rats in the sedentary (n=5), early (n=4), thru (n=4), late (n=4), or exercise conditions. +: significantly different from session 10 (B), session 7 (C), and the last extinction session (E). *: significantly decreased versus sedentary and late. ˆ: significantly increased versus sedentary. Thru: Throughout.
), cocaine-seeking (or responding during extinction/reinstatement testing) in these subsets of rats was affected by the timing of exercise during withdrawal. For extinction, responses were significantly lower in early and throughout (Figure 2D; group, F3,17=20.30, P<0.001) as compared to both sedentary and late (P’s<0.01) and significantly higher in late as compared to sedentary (P<0.01). Similarly, reinstatement of cocaine-seeking in response to cocaine-associated cues was markedly attenuated in early and throughout versus sedentary and late (Figure 2E; group by session, F3,13=10.43, P<0.001). Specifically, while no group differences were observed during the last extinction session (P>0.05), a significant group effect was observed within the reinstatement session (F3,13=11.27, P<0.001, P<0.001), and post-hoc comparisons revealed significantly lower responses in early and throughout as compared to sedentary and late (P’s<0.05). Further analysis within each group revealed significantly higher responses during the reinstatement session versus the last extinction session for sedentary and late (P<0.05), but not for early and throughout (P>0.05). Thus, as with the larger dataset (
), results from these subsets of rats show that exercise initiated during early withdrawal, but not late withdrawal, reduces cocaine-seeking responding.
Molecular Results
Transcriptional Changes Associated with Cue-Induced Cocaine-Seeking
The analysis of transcriptional changes in the dmPFC of sedentary versus saline controls revealed 29 upregulated and 37 downregulated transcripts based on Padj=0.1 and Log2Fold change >1.3 (Figure 3A). Consistent with our previous findings (
Towers EB, Kilgore M, Bakhti-Suroosh A, Pidaparthi L, Abel J, Lynch WJ. Sex Differences in the Neuroadaptations Underlying Associated with Cocaine-Craving: A Focus on the Dorsomedial Prefrontal Cortex. Frontiers in Behavioral Neuroscience. Accepted
), one of the upregulated transcripts was Grin1 (ENSRNOT00000044246), which encodes the NR1 subunit of the NMDA receptor. Analysis of the upregulated transcript list using gene ontology (GO) and the comprehensive resource of mammalian protein complexes (CORUM, which was manually created using reliable experimental evidence and utilizes the specific genes that contribute to the formation of specific quaternary protein structures,64) revealed an enrichment of terms related to glutamate receptors, including glutamate receptor complex and Drd1-Nmdar1 complex, and macromolecule localization and organic substrate transport (Figure 3B). Interestingly, the downregulated transcript list revealed terms linked to the regulation of neurotransmission and the presynaptic active zone cytoplasmic component. Thus, cue-induced cocaine-seeking appears to be associated with genes related to increased glutamate/NMDA receptor signaling and decreased regulation of presynaptic neurotransmission.
Figure 3Cue-induced cocaine-seeking associated transcriptional changes in the dmPFC. A) Volcano plot depicting the differentially expressed transcripts in the dmPFC of male rats in the sedentary group (n=5) relative to the saline group (n=5), with statistically significant differences depicted in red (Padj<.1, log2fc<1.3). B) Bar plots depicting the enriched GO and CORUM terms using the differentially up- and downregulated transcripts in the cocaine versus saline comparison. Bars with the same shade of red represent terms gathered from the same database. Adjusted p-values (Padj) were calculated using DESeq2. Log2fc: Log2(Fold Change). GO: Gene ontology. CORUM: comprehensive resource of mammalian protein complexes.
Transcriptional Changes Associated with the Protective Effects of Exercise Against Cocaine-Seeking
A similar strategy was used to investigate transcriptional changes associated with cue-induced cocaine-seeking (versus saline) in the dmPFC of rats that had previously exercised during early, throughout, or late withdrawal (Figure 4A). Surprisingly, despite the similarity between early and throughout exercise with regard to cocaine-seeking, only 5 upregulated and 8 downregulated gene-associated transcripts overlapped between the groups, and of these, only 2 upregulated transcripts and 6 downregulated transcripts were specific to early and throughout (Figure 4B; Table 1). Notably, the Grin1 transcript was one of three overlapping upregulated transcripts common to each of the exercise groups. We also investigated transcripts that were similarly upregulated or downregulated in early and throughout, but not late, relative to sedentary. This analysis produced similar results as those relative to saline with only 5 upregulated and 4 downregulated gene-related transcripts unique to early and throughout exercise (Table 2).
Figure 4Transcriptional changes associated with the protective effects of exercise against cocaine-seeking. A) Volcano plots depicting the differentially expressed transcripts in the dmPFC of male rats in the early (n=4), thru (n=4), and late (n=4) exercise groups each relative to saline group (n=5), with statistically significant transcripts depicted in blue, purple, and pink for the early, thru, and late comparisons, respectively (Padj<.1, log2fc<1.3). Grin1 is labeled in all three volcano plots. B) Venn diagrams showing the overlap of the significantly up- and down-regulated transcripts in the early, thru, and late groups each compared to saline. The uniquely overlapping transcripts between early and thru exercise are shown. C) Bar plot depicting the enriched GO, WP, and CORUM terms using the significantly up- and down-regulated transcripts in the early exercise versus saline comparison. Bars with the same shade of blue represent terms gathered from the same database. D) Bar plot depicting the enriched GO, TF, and CORUM terms using the significantly up- and down-regulated transcripts in the thru exercise versus saline comparison. Bars with the same shade of purple represent terms gathered from the same database. E) Bar plot depicting the enriched GO and CORUM terms using the significantly upregulated transcripts in the thru exercise vs saline comparison. Bars with the same shade of pink represent terms gathered from the same database. No terms were enriched using the significantly downregulated transcripts. F) Saline-subtracted, normalized RNA-seq paired-read density for Grin1 in the sedentary, early, thru, and late exercise conditions were mapped to chromosome 3 of the rat rn6 genome using the Integrated Genome Viewer. Differential transcripts above saline (green) or below saline (red) are indicated relative to known RefSeq exon transcripts (blue). Padj values were calculated with DESeq2 using the Wald test for significance after fitting to a negative binomial linear model and performing the Benjamini-Hochberg procedure to limit false discoveries. Thru: Throughout. Padj: P-adjusted. Log2fc: Log2(Fold Change). GO: gene ontology. WP: WikiPathways. CORUM: comprehensive resource of mammalian protein complexes. TF: transcription factor.
Table 1Gene-associated transcripts that were similarly upregulated or downregulated in the early and throughout, but not late, exercise groups relative to saline controls.
Table 2Gene-associated transcripts that were similarly upregulated or downregulated in the early and throughout, but not late, exercise groups relative to sedentary controls.
To further assess similarities and differences in transcriptional profiles between the sedentary and exercise groups, we performed a GO, KEGG, CORUM, transcription factor database (TRANSFAC), and WikiPathways (WP) analysis on the differentially up- and down-regulated gene-associated transcripts in each exercise group relative to saline (Figure 4C-E). To our surprise, this analysis revealed that there were two shared terms common for each of the exercise groups, glutamate receptor complex and Drd1-Nmdar1 complex. It is notable that these two terms were also found in the sedentary versus saline comparison (see Figure 4C), suggesting that the efficacy of exercise is not mediated via normalization of glutamate/NMDA receptor levels in the dmPFC (but see IGV analysis below). Another notable finding from this analysis is that throughout exercise was uniquely associated with an upregulation of transcripts related to the transcription factor AP-2 (Figure 4D), which plays an important role in synaptic vesicle endocytosis and NMDA-stimulated AMPA receptor endocytosis (
). Finally, given the relative lack of overlapping transcripts and enrichment terms between the early and throughout exercise groups, it is likely that different mechanisms underlie the protective effects of exercise during early versus throughout withdrawal.
Given our previous work and work from others indicating a role for Grin1/NR1 in cocaine-seeking and the efficacy of exercise to reduce cocaine-seeking (
Towers EB, Kilgore M, Bakhti-Suroosh A, Pidaparthi L, Abel J, Lynch WJ. Sex Differences in the Neuroadaptations Underlying Associated with Cocaine-Craving: A Focus on the Dorsomedial Prefrontal Cortex. Frontiers in Behavioral Neuroscience. Accepted
), Grin1 transcription was visualized using IGV. This analysis revealed that the normalized mapped reads densities for the sedentary and exercise conditions varied significantly by exon (Figure 4F). For example, there was a robust loss of transcripts spanning exons 9 and 10 in the sedentary group relative to saline that was either countered or normalized with early and throughout, but not late exercise. Given that these effects mirror effects on cocaine-seeking, it is possible that early and throughout exercise may normalize the RNA mis-splicing induced by cocaine-seeking. Additionally, while throughout exercise normalized expression in these exon regions, early exercise led to an elevation of transcripts from these exon regions, indicating that, as suggested above, the mechanisms by which early and through withdrawal exercise exert their efficacy are different.
Transcriptional Changes Associated with the Protective Effects of Exercise Throughout Withdrawal Against Cocaine-Seeking
Given the relative lack of overlapping transcriptional profiles between the early and throughout exercise groups, we focused on characterizing the transcriptional profile associated with the protective effects of exercise throughout withdrawal cocaine-seeking (relative to sedentary). We also compared effects to those observed in late (relative to sedentary) as a negative control for throughout exercise since the last exercise session was the day before the cue-induced cocaine-seeking test for both groups, yet only throughout exercise decreased cocaine-seeking. This analysis revealed 28 upregulated gene-associated transcripts unique to throughout exercise (Figure 5A; Table 3) and further analysis of this transcript list using GO, KEGG, and CORUM revealed enrichment of terms related to neuron projection, cell cortex, growth hormone synthesis, secretion, and action, circadian entrainment, GnRH, cortisol, renin, and aldosterone secretion, and neurotransmitter synapses, including serotonergic, dopaminergic, cholinergic, and glutamatergic synapses (Figure 5B). We also confirmed that transcriptional changes for four of the gene-associated transcripts unique to throughout exercise (Adcy10, Septin6, Plcb2, and Ank2) corresponded to the efficacy of exercise to reduce cocaine-seeking such that higher expression levels (normalized counts) were predictive of significantly lower drug-seeking (Figure 5C; P’s<0.05). It is notable that three of these transcripts are involved in neuron projection (Ank2, Septin6, and Adcy10).
Figure 5Transcriptional upregulation associated with the protective effects of exercise throughout withdrawal against cocaine-seeking. A) Venn diagram depicting the shared significantly upregulated transcripts between the thru (n=4) versus sedentary (n=5) and late (n=4) versus sedentary (n=5) comparisons (Padj<.1, log2fc<1.3). B) Network map depicting the enriched GO, KEGG, and CORUM terms using the 28 transcripts uniquely upregulated transcripts in the thru vs sedentary comparison. Terms are in red ovals and genes are in blue circles. Lines connect the genes that contributed to each of the enriched terms. Red stars denote transcripts that had significant behavioral correlations. C) The normalized counts of Adcy10, Sept6, Plcb2, and Ank2 were negatively associated with cocaine-seeking responding as indicated by a significant correlation (*). Thru: Throughout. Padj: P-adjusted. Log2fc: Log2(Fold Change). GO: gene ontology. CORUM: comprehensive resource of mammalian protein complexes. *P<.05
The same analysis revealed 37 downregulated gene-associated transcripts unique to throughout exercise (Figure 6A; Table 4) as well as an enrichment of terms related to cytoplasm, the Trpc1-Stim1-Orai1 complex, and several transcription factors, including ZF5, E2F, and PLAG1 (Figure 6B). Interestingly, while E2F has been implicated in neuronal migration, it also has been shown to be a positive regulator of neuronal apoptosis (
). The normalized transcript counts of several of the transcripts unique to throughout exercise also corresponded to the efficacy of exercise. Specifically, reinstatement responses were positively associated with normalized transcript counts for Exoc3, Ggnbp2, Syt3, Trmt10a, Ldb1, Pfkb3, RGD1564855, and Nfxl1 (P’s<0.05; Figure 6C). These transcripts all intersected with at least one of the aforementioned enrichment terms. Thus, throughout exercise may serve to reduce cocaine-seeking by promoting neuronal synapses and hormone secretion and preventing neuronal apoptosis.
Figure 6Transcriptional downregulation associated with the protective effects of exercise throughout withdrawal against cocaine-seeking. A) Venn diagram depicting the shared significantly downregulated transcripts between the throughout (thru; n=4) versus sedentary (n=5) and late (n=4) versus sedentary (n=5) comparisons (Padj<.1, log2fc<1.3). B) Network map depicting the enriched GO, TF, and CORUM terms using the 37 transcripts uniquely upregulated transcripts in the thru vs cocaine sedentary comparison. Terms are in red ovals and genes are in green circles. Lines connect the genes that contributed to each of the enriched terms. Red stars denote genes that had significant behavioral correlations. C) The normalized counts of Ggnbp2, Syt3, Exoc3, Pfkb3, Tm10a, Ldb1, RGC156485, and Nfxl1 were negatively associated with cocaine-seeking responding as indicated by a significant correlation (*). Thru: Throughout. Padj: P-adjusted. Log2fc: Log2(Fold Change). GO: gene ontology. TF: transcription factor. CORUM: comprehensive resource of mammalian protein complexes. *P<.05
The present study used RNA-seq to identify the transcriptional changes in the dmPFC associated with cue-induced cocaine-seeking and its modulation by exercise. As expected (
), the sedentary group demonstrated high levels of cocaine-seeking responding which may be associated with increased glutamate signaling as indicated by the upregulation of a Grin1-associated transcript and an enrichment of Drd1-Nmdar1 complex and glutamate receptor complex terms. To our surprise, each of the exercise groups also showed an upregulation of the same Grin1-associated transcript and an enrichment of the same glutamate receptor-related terms (i.e., Drd1-Nmdar1 complex and glutamate receptor complex). While on the surface these findings suggest that the protective effects of exercise against cocaine-seeking are not mediated through normalization of glutamatergic signaling, particularly via gene-associated Grin1 transcripts, a closer examination revealed a robust loss of transcripts spanning exons 9 and 10 in sedentary controls (relative to saline) that was normalized with the early and throughout, but not late exercise, suggesting that exercise initiated during early withdrawal normalizes cocaine cue-induced RNA mis-splicing of Grin1. It is also notable that, despite similar protection against cocaine-seeking by early and throughout exercise, there was minimal overlap between these groups in their transcriptional profiles. Each of these findings are discussed further below.
As expected, cue-induced cocaine-seeking was high following extended-access cocaine self-administration and protracted withdrawal and appears to be associated with increased glutamate signaling in the dmPFC considering our findings showing upregulation of the Grin-1 associated transcript and an enrichment of terms related to Drd1-Nmdar1 complex and glutamate receptor complex. These findings are consistent with findings in both humans and animals showing a strong association between drug-craving/seeking and dmPFC hyperactivity (
Prelimbic cortex is a common brain area activated during cue‐induced reinstatement of cocaine and heroin seeking in a polydrug self‐administration rat model.
Towers EB, Kilgore M, Bakhti-Suroosh A, Pidaparthi L, Abel J, Lynch WJ. Sex Differences in the Neuroadaptations Underlying Associated with Cocaine-Craving: A Focus on the Dorsomedial Prefrontal Cortex. Frontiers in Behavioral Neuroscience. Accepted
) and others (e.g.,70-73), supporting the involvement of NMDA receptors in particular. Thus, targeting overactive glutamatergic signaling in the dmPFC, particularly via NMDA receptors, could serve as a potential intervention for drug-craving. Drug side effects would need to be considered when selecting an NMDA receptor antagonist for treatment, but it may be possible given that high doses of methadone, a full μ-opioid agonist that also acts as an NMDA receptor antagonist, are commonly prescribed for the use in humans. High doses of methadone of have also been shown to significantly reduce cocaine use in methadone maintenance treatment patients (
Surprisingly, a Grin1-associated transcript was upregulated in all the exercise groups along with the sedentary controls. While it is possible that this change was induced by extended-access cocaine self-administration independent of cue-induced cocaine-seeking (
Towers EB, Kilgore M, Bakhti-Suroosh A, Pidaparthi L, Abel J, Lynch WJ. Sex Differences in the Neuroadaptations Underlying Associated with Cocaine-Craving: A Focus on the Dorsomedial Prefrontal Cortex. Frontiers in Behavioral Neuroscience. Accepted
Expression of glutamatergic genes in healthy humans across 16 brain regions; altered expression in the hippocampus after chronic exposure to alcohol or cocaine.
), a more likely possibility is that exercise that effectively reduces cocaine-seeking/craving does so by normalizing signaling downstream of NR1/NMDA receptor signaling, or as suggested by the IGV analysis, by normalizing mis-splicing at Grin1. For example, our findings showing the loss of transcripts spanning exons 9 and 10 in the sedentary condition and normalization by early and throughout, but not late exercise is potentially significant considering that these exon regions are largely extracellular and include the first 5 amino acids of the ligated ion channel L-glutamate and glycine binding site. Therefore, splicing out these exons (and loss of the first 5 amino acids) could change the fidelity of NMDA receptor signaling (
). This highlights an area for future research that may provide insight on the mechanisms underlying CUD, since these splicing effects are often overlooked in RNA-seq studies and possibly missed by qPCR depending on the primers selected. It is also notable that the rigor of these findings is demonstrated by the fact that our RNA-seq results with Grin1 in the sedentary condition is consistent with previous gene expression findings from our group (
Towers EB, Kilgore M, Bakhti-Suroosh A, Pidaparthi L, Abel J, Lynch WJ. Sex Differences in the Neuroadaptations Underlying Associated with Cocaine-Craving: A Focus on the Dorsomedial Prefrontal Cortex. Frontiers in Behavioral Neuroscience. Accepted
Also, to our surprise, despite similar effects with regard to cocaine-seeking, there were few transcriptomic similarities between the early and throughout exercise conditions. Of the 146 transcripts that were differentially expressed in the early and throughout exercise groups (relative to saline), only 8 were shared between the two conditions. The functional classification analysis of the differentially expressed transcripts also revealed only 2 overlapping enrichment terms between the early and throughout exercise groups (i.e. Drd1-Nmdar1 complex and glutamate receptor complex), and both terms overlapped with the sedentary and late exercise groups. Together, these findings suggest that the mechanisms underlying the protective effects of exercise against cocaine-seeking are different for exercise during early versus throughout withdrawal; however, recency of exercise and methodological limitations, such as the homogeneous analyses of mRNA transcripts from a heterogeneous brain region with different cell- and projection-specific functions, may also have contributed to the differences observed. Thus, future research investigating the molecular changes induced by exercise during early, late, and throughout withdrawal (prior to reinstatement testing) or using techniques, such as single-cell RNA-seq, are necessary to examine these possibilities.
Our findings also reveal novel potential mechanisms by which exercise mediates its protective effects against cocaine-seeking. Specifically, exercise initiated during early withdrawal was associated with the downregulation of transcripts associated with presynaptic active zone cytoplasmic component and regulation of neurotransmitter release. Additionally, the comparison that isolated transcriptional changes specific to exercise throughout withdrawal (relative to sedentary controls) revealed an upregulation of terms associated with neurotransmitter synapses, including glutamatergic synapses. One of the transcript intersections for the glutamatergic synapse enrichment term was Plcb2, which is a phosphodiesterase that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to the second messenger inositol 1,4,5-triphosphate (IP3). Interestingly, IP3 is downstream of mGlu5 (
), which we showed previously to be necessary for the incubation of cocaine-seeking, but not for the efficacy to exercise to decrease cocaine-seeking (
). Thus, one possibility that we plan to address in future studies is that exercise exerts its efficacy downstream of mGlu5 via interactions with IP3.
The efficacy of exercise to reduce cocaine-seeking was also associated with terms associated with circadian rhythms and neuronal survival. There is a large literature showing that circadian rhythms play a critical role in substance use disorder, including drug-seeking/craving (
). Specifically, findings in rats show that circadian genes mutations enhance ethanol consumption, and that melatonin treatment reduces motivation for cocaine and drug-seeking (
). One interesting transcript in this regard is Adcy10, which contributed to the enrichment of the circadian entrainment term, was upregulated by throughout exercise (relative to sedentary controls), and was positively correlated with behavior. The downregulation of E2F-related transcripts, which contributed to the neuronal survival term, is also of interest considering that it was also correlated with the behavior; it has also been shown to prevent morphine and heroin induce apoptosis in cerebellar neurons (
Liu LW, Lu J, Wang XH, Fu SK, Li Q, Lin FQ (2013): Neuronal apoptosis in morphine addiction and its molecular mechanism. Int J Clin Exp Pathol. 2013;6(7):540.
). Our RNA-seq findings thus shed light on the potential pivotal role that circadian entrainment and neuronal survival may play in preventing cocaine-seeking responding, but, as with the glutamatergic findings, we will need to provide functional confirmation in our future studies.
In conclusion, these findings reveal possible glutamatergic mechanisms as well as other novel candidate mechanisms that may underlie cocaine-seeking and the efficacy exercise to reduce cocaine-seeking. We plan to use these findings to guide our future studies, which will be focused on determining the functional consequences of these molecular changes. Future studies also are needed to address mechanistic differences between early versus throughout withdrawal exercise since their protective effects against cocaine-seeking are likely mediated via different mechanisms. Finally, these findings suggest RNA mis-splicing as a potential mechanism underlying cocaine-seeking and highlight a need for further research on its role in CUD.
Substance Abuse and Mental Health Services Administration (2021): Detailed Tables: Results from the 2020 National Survey on Drug Use. Retrieved from https://www.samhsa.gov/data/report/2020-nsduh-detailed-tables
Role of Src Family Kinases in BDNF-mediated suppression of cocaine-seeking and prevention of cocaine-induced ERK, GluN2A, and GluN2B dephosphorylation in the prelimbic cortex.
Extended daily access to cocaine results in distinct alterations in Homer 1b/c and NMDA receptor subunit expression within the medial prefrontal cortex.
Environmental enrichment during forced abstinence from cocaine self-administration opposes gene network expression changes associated with the incubation effect.
Duttke SH, Montilla-Perez P, Chang MW, Li H, Chen H, Carrette LL, de Guglielmo G, George O, Palmer AA, Benner C, Telese F (2022). Glucocorticoid Receptor-Regulated Enhancers Play a Central Role in the Gene Regulatory Networks Underlying Drug Addiction. Genetic Analysis of Animal Models to Understand the Genomic Architecture of Substance Use Disorders.
This work was supported by R01 grants from NIDA (DA039093 and DA024716, WJL), funds from the Brain Institute at the University of Virginia (Lynch), and a Pharmacological Sciences Training Grant (5T32GM007055-47), an MSTP Training Grant (T32 GM007267), and a Wagner Fellowship from the University of Virginia (Towers).
Substance Abuse and Mental Health Services Administration (2021): Detailed Tables: Results from the 2020 National Survey on Drug Use. Retrieved from https://www.samhsa.gov/data/report/2020-nsduh-detailed-tables
United States Department of State (2017): International Narcotics Control Strategy Report, Drug and Chemical Control. Bureau for International Narcotics and Law Enforcement Affairs.
Hallgren M, Vancampfort D, Hoang MT, Andersson V, Ekblom Ö, Andreasson S, et al. (2021): Effects of acute exercise on craving, mood and anxiety in non-treatment seeking adults with alcohol use disorder: An exploratory study. Drug Alcohol Depend 1;220:108506.
Exercise during abstinence normalizes ultrastructural synaptic plasticity associated with nicotine-seeking following extended access self-administration.
Chronic wheel running-induced reduction of extinction and reinstatement of methamphetamine seeking in methamphetamine dependent rats is associated with reduced number of periaqueductal gray dopamine neurons.
Prelimbic cortex is a common brain area activated during cue‐induced reinstatement of cocaine and heroin seeking in a polydrug self‐administration rat model.
Blighe, K., Rana, S., & Lewis, M. (2019): EnhancedVolcano: Publication-ready volcano plots with enhanced colouring and labeling. R package version 1.2.0.
Towers EB, Kilgore M, Bakhti-Suroosh A, Pidaparthi L, Abel J, Lynch WJ. Sex Differences in the Neuroadaptations Underlying Associated with Cocaine-Craving: A Focus on the Dorsomedial Prefrontal Cortex. Frontiers in Behavioral Neuroscience. Accepted
Role of Src Family Kinases in BDNF-mediated suppression of cocaine-seeking and prevention of cocaine-induced ERK, GluN2A, and GluN2B dephosphorylation in the prelimbic cortex.
Extended daily access to cocaine results in distinct alterations in Homer 1b/c and NMDA receptor subunit expression within the medial prefrontal cortex.
Expression of glutamatergic genes in healthy humans across 16 brain regions; altered expression in the hippocampus after chronic exposure to alcohol or cocaine.
Liu LW, Lu J, Wang XH, Fu SK, Li Q, Lin FQ (2013): Neuronal apoptosis in morphine addiction and its molecular mechanism. Int J Clin Exp Pathol. 2013;6(7):540.
Environmental enrichment during forced abstinence from cocaine self-administration opposes gene network expression changes associated with the incubation effect.
Duttke SH, Montilla-Perez P, Chang MW, Li H, Chen H, Carrette LL, de Guglielmo G, George O, Palmer AA, Benner C, Telese F (2022). Glucocorticoid Receptor-Regulated Enhancers Play a Central Role in the Gene Regulatory Networks Underlying Drug Addiction. Genetic Analysis of Animal Models to Understand the Genomic Architecture of Substance Use Disorders.
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