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Official websites use. Share sensitive information only on official, secure websites. Conceptualization, J. To identify addiction genes, we evaluate intravenous self-administration of cocaine or saline in 84 inbred and recombinant inbred mouse strains over 10 days. We integrate the behavior data with brain RNA-seq data from 41 strains. The self-administration of cocaine and that of saline are genetically distinct. We maximize power to map loci for cocaine intake by using a linear mixed model to account for this longitudinal phenotype while correcting for population structure. A total of 15 unique significant loci are identified in the genome-wide association study. A transcriptome-wide association study highlights the Trpv2 ion channel as a key locus for cocaine self-administration as well as identifying 17 additional genes, including Arhgef26, Slc18b1 , and Slco5a1. We find numerous instances where alternate splice site selection or RNA editing altered transcript abundance. Our work emphasizes the importance of Trpv2 , an ionotropic cannabinoid receptor, for the response to cocaine. Khan et al. By combining genetic mapping data with RNA sequencing data from the brain of 41 strains, increased expression of the ionotropic cannabinoid receptor Trpv2 is linked to decreased cocaine use. Cocaine use disorders are a significant health burden. In the United States, 2 million people use cocaine once a month or more, and greater than , individuals are dependent on the drug. Cocaine acts by blocking the reuptake transporters for dopamine, serotonin, and norepinephrine in presynaptic nerve terminals, thus increasing the concentrations of these neurotransmitters in the synaptic cleft. The rewarding effects of cocaine are largely mediated by increased dopaminergic neurotransmission in the limbic system, in particular the nucleus accumbens NAc and prefrontal cortex. Addiction to cocaine is a complex trait, with multiple environmental and genetic factors. There is evidence of overlap between the genetic risk factors for cocaine use and other addictive drugs, in particular cannabis. Properly powered genome-wide association studies GWASs of cocaine addiction in humans await ascertainment of adequate population sizes, likely tens to hundreds of thousands of individuals. Hurdles in obtaining sufficient numbers include difficulties in recruiting cocaine-dependent individuals, gene-environment interactions, and population and phenotypic heterogeneity. One genome-wide significant variant for cocaine use disorder has been identified in the FAM53B gene. Genetic studies in mice can provide useful insights into cocaine addiction, since control of environment and behavioral endpoints is easily obtained. One investigation evaluated cocaine self-administration in 39 strains of recombinant inbred BXD mice. Further evidence for a genetic basis of cocaine use was the observation that an acute dose of the drug caused significant differences in locomotor activity across 45 inbred mouse strains. Because the HMDP is genetically stable, it is possible to layer multiple phenotypes on the panel, providing ever more powerful insights. Genetic mapping revealed significant loci for cocaine IVSA on chromosomes 3 and 14 and another suggestive locus on chromosome 3. However, the power of the study was not completely realized since the genome scans used five sequential sets of binned 2-day intervals, rather than exploiting the full longitudinal nature of the datasets. In this study, we used a linear mixed model to analyze the same dataset and identify loci that affect longitudinal cocaine IVSA while correcting for population structure. We further extended the analyses of RNA-seq data by mapping cis and trans QTLs affecting transcript, spliceform, and editing abundance. Splicing and editing events that influenced transcript abundance were identified. We then used transcriptome-wide association studies TWASs to combine the results of the behavioral GWASs with the RNA-seq data, providing confirmatory support for genes underlying cocaine use while also suggesting additional genes. A total of four behavioral endpoints were evaluated: number of infusions, active lever presses, percent active lever presses, and inactive lever presses Table S1. Normalized data were used for all analyses. To evaluate the influences on behavior independent of genetic background, we used a linear mixed model implemented in lme4 with fixed effects of testing chamber, active lever left vs. The significant effect of testing chamber may reflect the fact that chambers were assigned non-randomly, to minimize the consequences of having multiple mice from the same strain in the same chamber. There was significantly higher self-administration of both cocaine left vs. Conversely, inactive lever presses were significantly higher when the active lever was on the right cocaine,leftvs. Even though significant, the relatively modest effect size of lever placement was similar for all covariates and reflected the large sample size. Males had significantly higher measures for all cocaine endpoints males vs. In contrast, sex had no significant effect on saline endpoints. Mice working for cocaine compared to saline showed significantly higher infusions, active lever presses, and percent active lever presses active lever presses, cocaine vs. In contrast, mice showed significantly higher inactive lever presses for saline than cocaine cocaine vs. Infusions and active lever presses for either cocaine or saline showed no significant effect of experimental day. A number of analyses suggested distinct genetics for cocaine and saline taking. We found significant correlations between infusates for behavioral measures averaged by strain cocaine vs. Each row and column represents a different day of testing. Gray key indicates saline or cocaine. Blue key indicates behavioral endpoint. B Broad sense heritability H 2 for cocaine and saline infusions over 10 days. F GWAS clustering. Dendrogram shows unsupervised clustering of columns. Rows represent SNPs maintained in genome order; chromosomes are indicated. Over the day period, all measures of self-administration showed significant H 2 , both for saline e. H 2 and h 2 were roughly consistent with estimates from human populations. The decrease in h 2 for cocaine inactive lever presses occurred simultaneously with a switch to the active lever Figure S1B , suggesting reduced additive variance as the cause of the decrease. To further explore the genetic basis for cocaine and saline IVSA, we performed a separate GWAS for each of the 10 days using the four behavioral endpoints. Further, there was a significantly higher correlation of GWAS results within infusate cocaine vs. Together, these observations indicate different genetic factors for cocaine and saline IVSA. To improve statistical power, we evaluated the longitudinal behavioral phenotypes using a linear mixed model implemented in GMMAT software. The model further incorporated fixed and random slopes of testing day as a continuous variable plus fixed covariate effects of age, sex, active lever position left or right , testing chamber, and cohort. Genetic relatedness was corrected via a random intercept derived from an SNP-based kinship matrix. A total of 17 significant loci were identified, of which 15 were unique Figure 2 , Table 1. D Inactive presses. Rik, AG21Rik. See also Figure S3. In the absence of other supporting evidence, candidate genes were nominated based on a combination of proximity and biological function. Negative distance, gene centromeric to SNP; positive, telomeric. We nominated plausible candidate genes for cocaine self-administration based on proximity to the behavioral loci and known roles in addiction, dopamine neurotransmission, or the brain. As expected, loci showed allelic differences in behavioral endpoints over the time course of the study Figure S3A. Quantile-quantile plots for longitudinal cocaine IVSA showed deviations from normality Figure S1E , reflecting longer linkage disequilibrium blocks in the HMDP compared to human, as well as the longitudinal nature of the phenotypes. Samples showed strong separation due to region, some separation due to batch, but little or no separation based on sex or infusate. This error rate is comparable to, or better than, other genome-scale studies. Changes in transcript and isoform abundance may be caused directly by the infusate or indirectly influenced by genetic background. Our study is nearly balanced with respect to infusate and mouse strain, so population structure is unlikely to be an appreciable confound. Nevertheless, to ensure that we identified expression changes independent of genetic background, we used a linear mixed model implemented in lme4qtl to correct for population structure via a kinship matrix. We included only one interaction term, which evaluated sex-dependent effects of cocaine on gene expression. Additional interactions could exhaust the power of the model and lead to unacceptable false positive and false negative rates. Per2 is a core circadian rhythm gene known to be regulated by cocaine that, in turn, alters the effects of cocaine on circadian phase shifts. Metabolic process was prominent, including nitrogen compound and organic substance metabolic process. A Normalized transcript abundance. Sa, saline; Co, cocaine. M, male; F, female. C Splicing. D Sashimi plot of cocaine-regulated splicing of Luc7l , exon 2. E Alternate splicing of Rap1gap , exon 6, affects transcript abundance. F RNA editing. See also Figures S3 , S4 , and S5. Bc1 is a non-coding gene whose RNA is transported to dendrites to regulate translation. To identify factors that regulate splicing or RNA editing, we again used the linear mixed model implemented in lme4qtl. A total of 31 exons showed significant differential splicing as a result of infusate cocaine vs. Both editing sites are in intronic Alu elements. The distance between the cis eQTLs and their corresponding genes was 0. This cutoff is consistent with previous studies using the HMDP. A Cis red and trans blue eQTLs. Red arrow, location of Runx2. Red horizontal line, cis eQTL significance threshold. Peak marker rs for both QTLs. Blue and red horizontal lines, respective significance thresholds. Individual samples are shown. H Allele effect of rs on Lsm6 expression. See also Figures S5 and S6. We identified hotspots for transcript abundance, in which a locus regulates many genes. We sought candidate genes for hotspots by looking for co-aligned cis eQTLs. Spliceforms regulated by genetic variants can affect transcript abundance as a result of changes in mRNA stability. RNA editing results in sequence changes that can affect transcript stability and abundance as well as coding sequence. This prediction was correct. Cis eQTLs were used to narrow down the candidate genes for the longitudinal behavioral loci. A total of The two most significant loci for cocaine infusions mapped to chromosome 11 at 60,, bp and 62,, bp Figures 2A , 5 , and S3A. The effect sizes of the two loci on infusions 0. A LocusZoom plot for cocaine infusions, showing loci harboring Drg2 and Trpv2. R 2 values convey linkage disequilibrium. Allele effect for saline not significant using longitudinal model. Saline allele effect is not significant. The TWAS approach nominates a gene for a trait if the gene possesses a cis eQTL and also shows significant correlation of its expression with the trait. Because TWAS employs genes rather than markers, there is decreased multiple hypothesis correction and thus increased statistical power. Linkage disequilibrium map is shown underneath. See also Figure S7 and Table S2. In some cases, TWAS suggested different genes than those nominated on the basis of proximity and biological plausibility Table 1. However, G3bp1 was , bp from the nearest behavioral QTL. In contrast, the nominated candidate gene for this QTL, Hint1 , was 7, bp from the locus. Although Hint1 had no significant cis eQTLs, we gave this gene precedence because of its proximity to the behavioral QTL and its known role in addiction. A similar situation exists for Pdyn and Cpxm1 , and Drg2 and Mief2. Drg2 and Trpv2 show significant linkage disequilibrium, but the linkage is less than perfect, leaving room for discordant TWAS results. Further, environment may affect Drg2 expression differently than Trpv2 , weakening any correlation of Drg2 expression levels with cocaine self-administration. Alternatively, Drg2 may exert its effects through amino acid variations rather than expression, although no such variants are currently known among the 37 sequenced inbred mouse strains. This software evaluates the posterior probability that the same SNP is causal for both GWAS and expression QTLs, while accounting for the uncertainty introduced by linkage disequilibrium. GWASs using individual days identified no significant loci, while GWASs using binned 2-day intervals identified two significant loci and one suggestive locus for infusions. Both QTLs are within credible linkage disequilibrium distances of loci identified using the longitudinal model. The suggestive 2-day QTL on chromosome 3 is 1,, bp centromeric to a longitudinal QTL for inactive lever presses, which has Spry1 as the candidate gene Table 1. The 2-day QTL on chromosome 14 is 8, bp telomeric to a longitudinal QTL for inactive lever presses, with Rnf17 as the candidate gene. Moreover, the performance of FOCUS is preserved in proxy tissues whose expression is correlated with the causative tissue. Cannabidiol shows promise as a treatment for cocaine use disorder, consistent with our observation that higher expression of Trpv2 is associated with lower cocaine IVSA. Consistent with a potential role in cocaine addiction, Drg2 knockout mice show decreased dopamine release in the striatum. Another locus for inactive lever presses was close to Pdyn. Candidate genes lacking additional supporting evidence in the current study are speculative. For example, the peak SNP for the locus on chromosome 12 regulating the number of active presses rs is actually located in the middle of the immunoglobulin heavy chain complex, near variable region gene 3—48 Ighv1— The nominated candidate gene, Vipr2 , is located 1,, bp telomeric to the SNP, and another gene, Ptprn2 , is 1,, bp telomeric. Although both genes are biologically plausible and within the credible distance for linkage disequilibrium, their candidacy should be treated with caution. Slc18b1 is a serotonin transporter, consistent with the role of cocaine in inhibiting the reuptake of this neurotransmitter. For example, Rnf17, Pdyn , and Hnrnpab , candidate genes for inactive lever presses Figure 6 and Table 1 , showed genetic interactions. Our study lacks experimental confirmation of the candidate genes. However, at least eight of the candidate genes have been evaluated in knockout mice for traits other than addiction Vipr2, Pdyn, Asic2, Hint1, Drg2, Spry1, Rnf17, Trpv2. The single-day GWAS and heritability data suggest that saline longitudinal loci will show illuminating differences with cocaine. However, a fuller picture awaits the completion of saline longitudinal GWASs, which are ongoing. Recently, a large number of new BXD RI strains have been constructed and genotyped, bringing the total to strains. Dose-dependent gene regulation by cocaine or saline could be evaluated in the lme4qtl linear mixed model using self-administration measures as continuous rather than categorical variables. Analysis of various developmental time points in other brain regions, such as amygdala or ventral tegmental area, may be more relevant. In contrast, reference trait analysis employs both environmental and genetic variation to relate transcript levels to phenotypes and can outperform TWASs. Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Desmond J. Smith DSmith mednet. This paper analyzes existing, publicly available data. The accession numbers for the datasets are listed in the key resources table. DOIs are listed in the key resources table. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. A total of 32 inbred and 52 recombinant inbred strains were evaluated. Target numbers were 3 males and 3 females for each strain for each of the two infusates. The actual number per strain was 5. The age of the mice was Mouse experiments were approved by the Binghamton University Institutional Animal Care and Use Committee and conformed to all relevant regulatory standards. Mice were subjected to either cocaine or saline IVSA over 10 consecutive daily sessions. Animals were confronted with two levers in the testing chambers, one of which gave an infusion of cocaine or saline active lever , the other of which did not inactive lever. A time-out period of 20 s occurred after an infusion, during which active lever presses were recorded but no infusion was given. Testing continued until 65 infusions were administered or 2 h elapsed, whichever came first. The amount of free base cocaine administered per infusion was 0. Consequently, the behavioral endpoints represent normalized cocaine doses. The concentration of sterile saline was 0. The infusion volume was 0. Four endpoints were analyzed; number of infusions, number of active lever presses, percentage of active lever presses and number of inactive lever presses. The first three endpoints evaluate the propensity of the mice for cocaine self-administration. Percent active lever presses control for locomotor activity by normalizing active lever presses to total lever presses. In contrast, inactive lever presses may measure either the aversive properties of the infusate or locomotor activity, whether intrinsic or modified by infusate. NAc core and shell and mFC were harvested from all mice 24 h after their final test session. For the remaining strains, we pooled samples of the same sex, yielding a total of 2 samples male or female for each infusate and brain region. Samples were pooled to save library construction costs, while preserving information on sex, infusate and brain region. The total number of RNA-seq samples was Sequencing used 75 bp paired-ends for cocaine with Reads were mapped as described 25 , 51 to mouse genome sequence build GRCm The effects of the covariates on the normalized behavioral phenotypes were evaluated using a linear mixed model implemented in lme4 with day of assay, sex, chamber number, active lever and age as fixed effects. Day of assay and age were treated as continuous variables. The model assessed population structure using strain as a random effect. Broad sense heritability H 2 was calculated using the same model as the behavioral covariates, but with day of assay omitted to evaluate H 2 on individual days. Additive heritability h 2 was calculated using the heritability package. Single nucleotide polymorphism SNP genotypes were obtained from the mouse diversity array. The model employed fixed and random effects of testing day as a continuous variable and also corrected for population structure using random effect of genotype via a kinship matrix. However, we were unable to find available software that would accomplish this goal while incorporating other necessary model features. Significance testing used the Wald test, because of its increased power compared to the score test. Conditional quantile normalization was used to normalize the data. Cis eQTLs were defined as residing within 2 Mb of the regulated gene. All editing sites were A to I. To identify expression changes independent of genetic background, we used a linear mixed model implemented in lme4qtl. For splicing and editing, transcript expression level was added as an additional fixed effect. Random effects used a kinship matrix to account for population structure and correct for regulatory effects due solely to genetic background. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Cell Rep. Published in final edited form as: Cell Rep. Find articles by Arshad H Khan. Find articles by Jared R Bagley. Find articles by Nathan LaPierre. Find articles by Carlos Gonzalez-Figueroa. Find articles by Tadeo C Spencer. Find articles by Mudra Choudhury. Find articles by Xinshu Xiao. Find articles by Eleazar Eskin. Find articles by James D Jentsch. Find articles by Desmond J Smith. Issue date Aug PMC Copyright notice. The publisher's version of this article is available at Cell Rep. Open in a new tab. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel. Rik f. Deposited data. Rau et al. Lee et al. Mouse genome sequence build GRCm Howe et al. Picardi et al. Bagley et al. Transcriptome, Gencode M Frankish et al. The Jackson Laboratory. Software and algorithms. Hormozdiari et al. Lippert et al. Mancuso et al. Gusev et al. Chen et al. Kruijer and White, Kim et al. Anders et al. Bates et al. Ziyatdinov et al. Pruim et al. R Core Team Dobin and Gingeras,

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How can I buy cocaine online in Koh Phi Phi

Official websites use. Share sensitive information only on official, secure websites. Astrocytes have become established as an important regulator of neuronal activity in the brain. Accumulating literature demonstrates that cocaine self-administration in rodent models induces structural changes within astrocytes that may influence their interaction with the surrounding neurons. Here, we provide evidence that cocaine impacts astrocytes at the functional level and alters neuronal sensitivity to astrocyte-derived glutamate. This is accompanied by increased prevalence of slow inward currents, a physiological marker of neuronal activation by astrocytic glutamate, in a subset of medium spiny neurons. Within, but not outside, of this subset, we observe an increase in duration and frequency of NMDA receptor-mediated synaptic events. Additionally, we find that the link between synaptic NMDA receptor plasticity and neuron sensitivity to astrocytic glutamate is maintained independent of drug exposure, and is observed in both cocaine and saline control animals. Therefore, our data indicate that cocaine self-administration promotes astrocyte-specific functional changes that can be linked to increased glutamate-mediated coupling with principal neurons in the nucleus accumbens. Such coupling may be spatially restricted as it does not result in a broad impact on network structure of local neuronal circuits. Astrocytes play diverse roles in regulation of synaptic transmission and have been proposed to coordinate synaptic plasticity and organization of neuronal microcircuits 1 , 2. The mechanisms underlying astrocytic release of neuromodulators, gliotransmission, remain subjects of active debate 8 — However, considering the lack of evidence for specialized gliotransmitter release sites and the fact that only the finest of astrocytic processes penetrate into the synaptic cleft, the bulk of gliotransmitter exocytosis is thought to occur outside the synapse 15 , Accordingly, neuronal response to gliotransmitters has been linked to the pool of extrasynaptic receptors. The electrophysiological hallmark of the NMDAR-mediated response to astrocytic glutamate are slow inward currents SICs , that are prominently distinguished from synaptic NMDAR-mediated events by their large amplitudes and slow kinetics 17 , 20 , SICs arise spontaneously in subpopulations of cells, but can also be elicited or inhibited by pharmacological, optical, and mechanical stimulation of astrocytes 18 , 19 , 22 — NMDA-mediated SICs may therefore serve as a useful proxy of neuronal sensitivity to glutamate released from the surrounding astrocytes. Indeed, a number of studies have highlighted the possibility that SICs may be involved in synchronization of local neuronal microcircuits in the hippocampus, the somatosensory cortex and the NAc 17 — 19 , 27 — Multiple behavioral outputs have been reported to result from astrocyte-selective manipulations. These include locomotor effects, sleep regulation, affective processing, memory encoding, and food intake Mounting evidence indicates that substance use triggers changes within astrocytes that impact drug-evoked behavior. For example, chemogenetic activation of astrocytes has been shown to attenuate motivation for ethanol, an effect that may involve signaling through astrocytic gap-junctions Astrocyte-released adenosine has been found to depress excitatory synaptic transmission and regulate effects of amphetamine 32 and ethanol while reduced association of astrocyte processes with synapses may play a role in heroin seeking Mice with astrocytes lacking putative gliotransmitter release machinery displayed attenuated cocaine-induced reinstatement of conditioned place preference and cue-induced reinstatement of cocaine self-administration in a transgenic mouse model Despite evidence that astrocyte-specific manipulations may influence cocaine-seeking, it is not known how cocaine experience affects baseline levels of astrocyte activity and functional interactions with neuronal targets. The goal of this study was to evaluate whether a history of cocaine self-administration impacts measures of astrocyte signaling that may be linked to synaptic plasticity of medium spiny neurons MSNs in the NAc shell. Male Sprague-Dawley rats Rattus norvegicus weighing — g were obtained from Taconic Laboratories. Rats were individually housed with food and water available ad libitum in their home cages. All experimental procedures were performed during the light cycle. The experimental protocols were consistent with the guidelines issued by the U. Under isoflurane anesthesia 1. The catheter was routed subcutaneously to a mesh platform placed between the shoulder blades. Catheters were flushed daily with 0. The catheters were sealed with plastic obturators when not in use. Following catheter implantation, the rats were placed in a stereotaxic apparatus under isoflurane anesthesia. SV40, Addgene or astrocytes pZac2. At 14—20 days after virus microinjections, the rats began cocaine self-administration training. The rats were placed in operant chambers Med Associates and allowed to lever-press for intravenous cocaine infusions 0. Briefly, each cocaine infusion was followed by a s timeout period during which responses had no scheduled consequences. The rats were initially trained using a fixed ratio 1 FR1 schedule of reinforcement. When stable responding was achieved under the FR1 schedule, they were switched to an FR5 schedule. Rats self-administered cocaine for 14 days and were paired with yoked saline controls. The yoked animals received an infusion of saline every time their pair received cocaine. There were no consequences to lever pressing by the yoked saline animals. Twenty-four hours after the last behavioral session, the rats were deeply anesthetized with isoflurane and decapitated. DNQX did not have a significant effect on any of the measured SIC parameters and these recordings were pooled with DNQX-free recordings for analysis unless otherwise indicated in the text. Brain slices were viewed under an upright microscope Olympus BX51WI with infrared differential interference contrast optics and a 40x water-immersion objective. The intracellular solution contained the following in mM : potassium gluconate, 2 MgCl 2 , 2. Delayed rectifier currents were measured at current peak. The amplitude of spontaneous excitatory postsynaptic currents sEPSCs was computed from an average of 50— current traces. Mean sEPSC frequencies were analyzed from 10—20 s long trace segments. Videos were analyzed offline with ImageJ and Matlab Mathworks. Individual cells were manually isolated as regions of interests ROIs. Amplitude, half-width, and frequency of calcium transients were analyzed with custom-written Matlab scripts. An undirected adjacency matrix was then constructed for each imaged field containing values of phi coefficients between ROI pairs. Adjacency matrices were used to calculate eight measures of network structure for each imaged field ROIs are nodes in this analysis. Communities: the number of non-overlapping groups maximally connected to each other, but minimally connected to other groups. Transitivity: the probability that, if one node is connected to two others, those two nodes are also connected to each other. Assortativity: coefficient that measures the extent to which nodes of similar strength connect to each other. Density: the ratio of existing connections relative to all possible connections in a network; 6. Modularity: the tendency of a network to be segregable into communities with dense intracommunity connections and sparse intercommunity connections; 7. Characteristic path length: the average shortest path between any two nodes in a network. Global efficiency: the inverse of the characteristic path length. More details on calculation, interpretation, and applicability of these and other measures to brain networks can be found in several excellent reviews 48 , All analyses were done in Excel or GraphPad Prism 8. The color scale is in arbitrary units A. Arrows indicate time points t1—t3 represented by image frames to the left. We evaluated eight measures of network organization with graph theory tools previously used for analysis of neuronal networks. Cocaine self-administration at 24 hours of withdrawal significantly decreased modularity and increased efficiency of astrocyte networks within slices as compared to the saline-yoke population Fig. None of these significant differences were preserved at 14 days of withdrawal from cocaine self-administration Fig. Instead, 14 days of withdrawal was associated with significant decreases in network transitivity and assortativity compared to networks from the respective saline-yoke population, producing disassortative networks Fig. Further implications of these findings are presented in the Discussion. A A table of network measures for astrocytes in the NAc shell of cocaine and yoked saline rats 24 hours after the last operant session. B Graphical representations of astrocytic networks in a single slice from a yoked saline or a cocaine-experienced rat at 24 hours of withdrawal. Density is the ratio of existing connections to all possible connections. Color represents connection strength phi-coefficient. Two communities are shown in the saline condition and three communities in the cocaine condition dashed squares. Intra-community connections are inside the dashed squares and all colored slots outside of dashed squares indicate inter-community connections. C A table of network measures for astrocyte in the NAc shell of cocaine and yoked saline rats 14 days after the last operant session. D Graphical representations of astrocytic networks in a single slice from a yoked saline or a cocaine experienced rat at 14 days of withdrawal. Buffering of extracellular potassium is one of the main functions attributed to astrocytes throughout the brain. Astrocytic potassium channels are known to be involved in regulation of astrocytic calcium signaling 51 , astrocytic resting membrane potential 52 , and astrocytic glutamate release In whole-cell patch-clamp recordings, we observed weak rectification of K ir amplitude in both saline and cocaine conditions at 24 hours of withdrawal from cocaine. There was no main effect of cocaine exposure across the entire range of evoked K ir current amplitudes Fig. Similarly, we observed no main effect of cocaine exposure on the amplitude of outward delayed rectifier K dr currents Fig. Together, these results show that neither the current rectification attributed to the astrocytic voltage-gated K ir and K dr currents nor the passive indicators of astrocyte membrane excitability are affected by cocaine self-administration. A Representative traces and B current-voltage relationship of currents attributed to K ir channels in NAc shell astrocytes from saline control and cocaine-experienced rats. C Representative traces and D current-voltage relationship of currents attributed to K dr channels in NAc shell astrocytes from saline control and cocaine-experienced rats. We next examined whether a combination of changes in some functional signatures of astrocyte activity i. Increased prevalence of SICs could be driven by increased number of astrocytic glutamate release events which may be reflected as an increase in SIC frequency. However, the mean frequency of SICs was not affected by cocaine self-administration saline: 0. Increased concentration of glutamate per release event may also increase probability of detecting SICs especially in the case of small-amplitude events that are not likely to saturate the pool of available NMDA receptors. If this were the case, an increase in SIC amplitude should also be expected. However, the mean amplitude of SICs was not significantly altered by cocaine experience saline: These results indicate that short-access cocaine self-administration increases functional coupling between neurons and astrocytes in the NAc shell such that greater number of neurons within the local circuit are responsive to astrocyte-released glutamate. Greater astrocyte-neuron functional coupling is not due to cocaine-induced increase in amount of astrocyte-released glutamate or increase in frequency of astrocytic glutamate release events. C-D SIC frequency and amplitude are not affected by cocaine self-administration. Circles indicate individual MSNs. Black horizontal bars indicate distribution means and standard errors, respectively. Since astrocytic glutamate release underlying the SICs has been proposed to co-ordinate neuronal microcircuit organization in a number of brain regions 17 — 19 , 27 , we followed up with exploration of network structure using graph theory methods as we had done for astrocytic networks. Despite the intriguing relationships that we report for astrocytic networks, none of the eight measures of neuronal network interactions that we examined showed significant differences between saline and cocaine conditions Table 1. Measures of network structure calculated for MSNs in the NAc shell of rats trained to self-administer cocaine and their saline-yoke counterparts. Our data indicate that cocaine self-administration is linked to a number of novel functional adaptations at NAc shell astrocytes. Second, we identify cocaine-induced plasticity of astrocytic networks supporting the hypothesis of structured communication between astrocytes. Finally, we report that increased glutamate-mediated neuroglial coupling at 24 hours of withdrawal from cocaine is not associated with altered structure of local MSN circuits in the NAc shell. Extensive literature supports the argument that history of drug exposure is a critical determinant of the molecular and behavioral adaptations to cocaine 62 — In our experiments, we controlled for the environmental stimuli lights, syringe pump sounds, etc. This design did not allow for isolation of operant training effects since the saline animals were not given the opportunity to control action-outcome contingencies. As the NAc shell is part of a network that processes not only reward signals, but salient environmental cues, environmental changes irrespective of cocaine experience could conceivably impact cellular activity. Here we examined functional networks, that is, networks whose connections are determined on the basis of correlated activity of their constituent cells. This would be predicted to decrease the fraction of cells with correlated activity, leading to reduced network density. However, we found that network density is increased after cocaine at 24 hours of withdrawal, but is similar between cocaine and saline groups at 14 days of withdrawal. These observations suggest that functional network alterations induced by cocaine may not be solely attributable to changes in activity of single cells. We propose that cocaine-induced increase in connection density may explain the finding of reduced network modularity at 24 hours of withdrawal. Modularity measures the tendency of a network to be segregable into communities of highly connected nodes that have minimal inter-community connections. Greater connection density can contribute to the breakdown of modularity if new connections are between communities or randomly distributed between and within communities cf. This finding indicates that cocaine self-administration generates less structured more random astrocyte functional networks. Increased network density may also contribute to decreased path length, the number of connections that lie between any two cells in a network which, in turn, would increase network efficiency calculated as the inverse of the characteristic path length. Such coordinated activity would be expected to increase connection density and decrease network modularity as observed in our analyses, since higher correlations among all cells in the network would increase the number of functional connections and make segregation of the network into discrete modules less likely. We found no significant differences in network density, modularity, and efficiency between saline and cocaine groups after 14 days of withdrawal. This finding, again, suggests that cessation of daily exposure to the operant chamber and non-contingent saline infusions may alter astrocyte responses independent of cocaine. Significant differences in NAc astrocyte network assortativity and transitivity between the saline and cocaine groups were observed at 14 days of withdrawal. Networks comprised of nodes that are preferentially connected to other nodes with similar numbers of connections are referred to as assortative and have a positive assortativity value. Conversely, networks comprised of nodes that are preferentially connected to nodes with different numbers of connections either less or more are described as disassortative. Cocaine self-administration followed by withdrawal, thus, produced strongly disassortative astrocyte networks. Lower transitivity values can be related to this, as disassortative networks are likely to have reduced number of three-node interconnected loops, the basis of transitivity measurements. In neuronal networks, disassortativity has been proposed to improve network stability 71 which may indicate reduced sensitivity to weak stimuli Interestingly, disassortative neural networks have also been linked to increased synchronization of activity Network level relationships have not been previously explored among astrocytes in situ and it is too early to say whether changes that we observed here warrant similar conclusions with regard to stimulus sensitivity, synchronizability, etc. It is nevertheless notable that the absence of changes in network density at 14 days of withdrawal suggests that astrocyte network plasticity after cocaine may not be explainable solely by introduction or deletion of random connections. Network changes do not require structural remodeling 72 , however, evidence in support of structural plasticity for both neurons and astrocytes after cocaine exposure is extensive and the combined impact of structural and functional connectivity changes on astrocyte network activity is of great interest. Increased SIC prevalence could be attributed to increased probability of gliotransmitter release. However, available evidence indicates that extrasynaptic glutamate is decreased in the NAc following chronic cocaine 37 , While glutamate release events associated with SICs may not substantially contribute to ambient extracellular glutamate levels, our finding that the frequency of SICs was not altered by cocaine-experience suggests that the probability of astrocytic glutamate release also remained unchanged. SICs are mediated by NMDA receptors and those receptors located at extrasynaptic sites are ideally situated to detect glia-released glutamate 20 , We and others have previously reported that cocaine self-administration increased contribution of extrasynaptic NMDA receptors in the NAc shell 46 , 75 which is supported by cocaine-induced upregulation of non-canonical NMDA receptors often found at extrasynaptic sites 76 — Upregulation of GluN2B protein following cocaine self-administration has been previously reported although sensitivity of NMDAR-mediated currents to GluN2B antagonist, Ro 25—, remained equivalent between control and cocaine-experienced animals at short withdrawal time points 46 , 69 , 78 , indicating that increased GluN2B expression may not result in functional changes at the synapse. Irrespective of subunit composition, the presence of NMDA receptors at peri- and extra-synaptic sites may be sufficient to prolong decay time of synaptic currents as a result of synaptic glutamate spillover Altogether, our data indicate that cocaine-induced redistribution of synaptic NMDARs to extrasynaptic sites facilitates both detection of SICs and slower decay kinetics of synaptic currents in SIC-positive cells. A range of factors including cocaine dose, total cocaine intake, duration of withdrawal from cocaine and other behavioral parameters could impact neuroglial coupling. We have not evaluated synaptic NMDA receptors signaling or SICs at long withdrawal intervals, but a number of recent studies indicate that cocaine-induced increase in extrasynaptic NMDA signaling may be a transient phenomenon. For example, initial up-regulation of extrasynaptic NMDA receptors was found to dissipate by one month of withdrawal from cocaine 75 and 14 days of extinction training was not associated with differences in SIC frequency in the NAc Relative abundance of extrasynaptic NMDA receptors may also be synergistic with the effects of dendritic sprouting reported after cocaine since dendritic coverage has been shown to influence duration of synaptic transients 81 , Finally, the association between cocaine self-administration training and SIC prevalence may be amplified by changes in glutamate homeostasis. Both the cocaine-induced decrease in glial glutamate extrusion by system -xc exchangers 83 and the increase in glutamate transporter-mediated reuptake 46 may be expected to accumulate glutamate inside astrocytes and promote neuronal SICs with possible behavioral effects 64 , Cocaine exposure has been linked to activation of neuronal subpopulations in the NAc, identified via various characteristics 84 , Here, we present a novel observation that plasticity of synaptic NMDA receptor currents is associated with the presence of SICs, independent of cocaine experience. Neuroglial coupling may thus serve as a marker of synapses that display NMDA receptor plasticity. It remains unknown, however, whether changes in SIC-mediated neuroglial coupling are a precursor to or a consequence of NMDA receptor synaptic plasticity. This may have relevance to cocaine-linked behaviors, as astrocytic control over the relevant neurons may be implemented via precisely targeted, rather than non-discriminate global, interactions. The authors have no competing financial interests to disclose in relation to this work. The data that support the findings of this study are available from the corresponding author upon reasonable request. As a library, NLM provides access to scientific literature. Addict Biol. Published in final edited form as: Addict Biol. Lexington, KY. Find articles by A Neugornet. Find articles by R Neogi. Find articles by Mengfan Xia. Find articles by PI Ortinski. Issue date Nov. PMC Copyright notice. The publisher's version of this article is available at Addict Biol. Open in a new tab. Network measure Saline Cocaine Clustering global 0. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. 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