Amanda L. Brown

Down-regulated striatal gene expression for synaptic plasticity-associated proteins in addiction and relapse vulnerable animals

Reducing the likelihood of relapse represents one of the greatest obstacles in the successful treatment of cocaine addiction. Dysregulation of the synaptic plasticity processes long-term potentiation (LTP) and long-term depression (LTD) is thought to be associated with protracted relapse risk. To improve our understanding of the molecular mechanisms contributing to relapse vulnerability we trained rats (n=52) to self-administer cocaine and phenotyped animals as relapse-vulnerable or relapse-resilient using procedures adapted from Deroche-Gamonet et al. (Science 2004, 305, 1014-1017). Gene expression analysis, targeted at synaptic plasticity-related genes, revealed significant transcript down-regulation in the ventral and dorsal striatum of relapse-vulnerable animals compared to relapse-resilient controls. This included reduced expression of genes encoding proteins implicated in the dendritic translation of synaptic plasticity-related transcripts, the dynamic regulation and trafficking of ionotropic glutamate receptors important for LTP and LTD, along with neuronal surface receptors that initiate downstream signalling pathways associated with synaptic plasticity. Together, our data are consistent with recent reports of an inability to evoke LTD in the striatum of addiction-vulnerable rats. To our knowledge, this is the first study to demonstrate down-regulated synaptic plasticity-associated gene expression not only in the ventral striatum, where the majority of addiction-related synaptic plasticity studies have been conducted, but also in the dorsal striatum of animals categorized as relapse-vulnerable. As these neural correlates were elucidated using an approach incorporating individual behavioural differences, they potentially provide more relevant insight into addiction and assist the development of novel pharmacotherapies to treat relapse.

Region-specific changes in mitochondrial D-loop in aged rat CNS.

Impaired mitochondrial oxidative phosphorylation (OXPHOS) is considered a cause of aging. A reduction in mitochondrial DNA (mtDNA) replication and/or transcription may contribute to this OXPHOS diminution. Impairments in the displacement (D) loop, or non-coding, region of the mitochondrial genome, or accumulation of mtDNA mutations, may affect mtDNA replication and transcription. We determined the effects of age on the D-loop and on mtDNA deletion mutations in the spinal cord, medulla, midbrain, cerebellum, striatum, and cerebral cortex of Fischer 344 rats. D-loop, 7S DNA levels were reduced by 3-fold in striatum, 2.5-fold in cortex, and 2-fold in the spinal cord of older animals. We did not detect a population of mtDNA affected by the most prevalent known (ND4-containing) deletions, indicating they do not comprise a significant portion of total mtDNA. However, we detected an age-related and region-specific increase in the common deletion, which comprised 0.0003-0.0007% of total mtDNA. Mitochondrial genome copy number varied between regions, in addition to an overall 18% decrease with age across the whole brain. These results suggest the age-related decline in OXPHOS may be related to a reduction in D-loop function.

Improved RNA preservation for immunolabeling and laser microdissection

Microdissection techniques have the potential to allow for transcriptome analyses in specific populations of cells that are isolated from heterogeneous tissues such as the nervous system and certain cancers. Problematically, RNA is not stable under the labeling conditions usually needed to identify the cells of interest for microdissection. We have developed an immunolabeling method that utilizes a high salt buffer to stabilize RNA during prolonged antibody incubations. We first assessed RNA integrity by three methods and found that tissue incubated in high salt buffer for at least 20 h yielded RNA of similar quality to that for RNA extracted from fresh-frozen tissue, which is considered highest quality. Notably, the integrity was superior to that for RNA extracted from tissue processed using rapid immunolabeling procedures (5 min total duration). We next established that high salt buffer was compatible with immunolabeling, as demonstrated by immunofluorescent detection of dopamine neurons in the brain. Finally, we laser microdissected dopamine neurons that were immunolabeled using high salt buffer and demonstrated that RNA integrity was preserved. Our described method yields high quality RNA from immunolabeled microdissected cells, an essential requirement for meaningful genomics investigations of normal and pathological cells isolated from complex tissues.

Distinct miRNA expression in dorsal striatal subregions is associated with risk for addiction in rats.

Recently, we published data using an animal model that allowed us to characterize animals into two groups, addiction vulnerable and addiction resilient, where we identified that addiction/relapse vulnerability was associated with deficits in synaptic plasticity-associated gene expression in the dorsal striatum (DS). Notable was the strong reduction in expression for activity-regulated cytoskeleton-associated protein (Arc) considered a master regulator of synaptic plasticity. In the present study, we confirmed that Arc messenger RNA was significantly decreased in the DS, but importantly, we identified that this reduction was restricted to the dorsomedial (DMS) and not dorsolateral striatum (DLS). There is recent evidence of microRNA (miRNA)-associated posttranscriptional suppression of Arc and animal models of addiction have identified a key role for miRNA in the regulation of addiction-relevant genes. In further support of this link, we identified several differentially expressed miRNA with the potential to influence addiction-relevant plasticity genes, including Arc. A key study recently reported that miR-212 expression is protective against compulsive cocaine-seeking. Supporting this hypothesis, we found that miR-212 expression was significantly reduced in the DMS but not DLS of addiction-vulnerable animals. Together, our data provide strong evidence that miRNA promote ongoing plasticity deficits in the DS of addiction-vulnerable animals.

Development of a brief tool for monitoring aberrant behaviours among patients receiving long-term opioid therapy: The Opioid-Related Behaviours In Treatment (ORBIT) scale

BACKGROUND Early identification of problems is essential in minimising the unintended consequences of opioid therapy. This study aimed to develop a brief scale that identifies and quantifies recent aberrant behaviour among diverse patient populations receiving long-term opioid treatment. METHOD 40 scale items were generated via literature review and expert panel (N=19) and tested in surveys of: (i) N=41 key experts, and (ii) N=426 patients prescribed opioids >3 months (222 pain patients and 204 opioid substitution therapy (OST) patients). We employed item and scale psychometrics (exploratory factor analyses, confirmatory factor analyses and item-response theory statistics) to refine items to a brief scale. RESULTS Following removal of problematic items (poor retest-reliability or wording, semantic redundancy, differential item functioning, collinearity or rarity) iterative factor analytic procedures identified a 10-item unifactorial scale with good model fit in the total sample (N=426; CFI=0.981, TLI=0.975, RMSEA=0.057), and among pain (CFI=0.969, TLI=0.960, RMSEA=0.062) and OST subgroups (CFI=0.989, TFI=0.986, RMSEA=0.051). The 10 items provided good discrimination between groups, demonstrated acceptable test-retest reliability (ICC 0.80, 95% CI 0.60-0.89; Cronbachs alpha=0.89), were moderately correlated with related constructs, including opioid dependence (SDS), depression and stress (DASS subscales) and Social Relationships and Environment domains of the WHO-QoL, and had strong face validity among advising clinicians. CONCLUSIONS The Opioid-Related Behaviours In Treatment (ORBIT) scale is brief, reliable and validated for use in diverse patient groups receiving opioids. The ORBIT has potential applications as a checklist to prompt clinical discussions and as a tool to quantify aberrant behaviour and assess change over time.

Purity and enrichment of laser-microdissected midbrain dopamine neurons

The ability to microdissect individual cells from the nervous system has enormous potential, as it can allow for the study of gene expression in phenotypically identified cells. However, if the resultant gene expression profiles are to be accurately ascribed, it is necessary to determine the extent of contamination by nontarget cells in the microdissected sample. Here, we show that midbrain dopamine neurons can be laser-microdissected to a high degree of enrichment and purity. The average enrichment for tyrosine hydroxylase (TH) gene expression in the microdissected sample relative to midbrain sections was approximately 200-fold. For the dopamine transporter (DAT) and the vesicular monoamine transporter type 2 (Vmat2), average enrichments were approximately 100- and 60-fold, respectively. Glutamic acid decarboxylase (Gad65) expression, a marker for GABAergic neurons, was several hundredfold lower than dopamine neuron-specific genes. Glial cell and glutamatergic neuron gene expression were not detected in microdissected samples. Additionally, SN and VTA dopamine neurons had significantly different expression levels of dopamine neuron-specific genes, which likely reflects functional differences between the two cell groups. This study demonstrates that it is possible to laser-microdissect dopamine neurons to a high degree of cell purity. Therefore gene expression profiles can be precisely attributed to the targeted microdissected cells.

The prevalence of QT prolongation in a population of patients with substance use disorders

INTRODUCTION AND AIMS Drug induced QT prolongation occurs in patients with substance use disorders from prescription medications that prolong the QT, such as methadone. Knowing the prevalence of QT prolongation in this population is important for prescribers. This study aimed to investigate the prevalence of QT prolongation in patients with current substance use disorders. DESIGN AND METHODS We undertook a retrospective review of electrocardiograms (ECG) from patients with substance use disorders from an urban general hospital with a large drug and alcohol service and toxicology unit. ECGs were taken from patients seen by the alcohol and drug unit over three years. The QT interval was measured manually on each ECG and defined as abnormal if above the line on the QT nomogram. The QT was also heart rate corrected using Fridericias formula (QTcF) to investigate associated factors. RESULTS Nine of 446 (2.0%; 95% confidence interval 1.0-3.9%) patients had an ECG with a prolonged QT interval. Three were prescribed methadone for opiate dependence (80, 90 and 125 mg daily), one also with hypokalemia; one prescribed escitalopram with hypokalaemia/hypomagnesaemia; three more with hypokalaemia alone. Only two patients had a prolonged QT with no identifiable cause. There was no association between QTcF and sex (P = 0.34), but there was a statistically significant association with age (Pearson R = 0.19, 95% confidence interval 0.10-0.28, P < 0.0001). DISCUSSION AND CONCLUSIONS QT prolongation is rare in patients with substance use disorders and is most likely similar to the general population once cases related to methadone use and electrolyte abnormalities are excluded. [Scott AJ, Dunlop AJ, Brown A, Craig S. The prevalence of QT prolongation in a population of patients with substance use disorders. Drug Alcohol Rev 2017;36:239-244].

Temporally specific miRNA expression patterns in the dorsal and ventral striatum of addiction-prone rats

MicroRNAs (miRNAs) within the ventral and dorsal striatum have been shown to regulate addiction‐relevant behaviours. However, it is unclear how cocaine experience alone can alter the expression of addiction‐relevant miRNAs within striatal subregions. Further, it is not known whether differential expression of miRNAs in the striatum contributes to individual differences in addiction vulnerability. We first examined the effect of cocaine self‐administration on the expression of miR‐101b, miR‐137, miR‐212 and miR‐132 in nucleus accumbens core and nucleus accumbens shell (NAcSh), as well as dorsomedial striatum and dorsolateral striatum (DLS). We then examined the expression of these same miRNAs in striatal subregions of animals identified as being ‘addiction‐prone’, either immediately following self‐administration training or following extinction and relapse testing. Cocaine self‐administration was associated with changes in miRNA expression in a regionally discrete manner within the striatum, with the most marked changes occurring in the nucleus accumbens core. When we examined the miRNA profile of addiction‐prone rats following self‐administration, we observed increased levels of miR‐212 in the dorsomedial striatum. After extinction and relapse testing, addiction‐prone rats showed significant increases in the expression of miR‐101b, miR‐137, miR‐212 and miR‐132 in NAcSh, and miR‐137 in the DLS. This study identifies temporally specific changes in miRNA expression consistent with the engagement of distinct striatal subregions across the course of the addiction cycle. Increased dysregulation of miRNA expression in NAcSh and DLS at late stages of the addiction cycle may underlie habitual drug seeking, and may therefore aid in the identification of targets designed to treat addiction.

Effectiveness and cost-effectiveness of unsupervised buprenorphine-naloxone for the treatment of heroin dependence in a randomized waitlist controlled trial

BACKGROUND Access to opioid agonist treatment can be associated with extensive waiting periods with significant health and financial burdens. This study aimed to determine whether patients with heroin dependence dispensed buprenorphine-naloxone weekly have greater reductions in heroin use and related adverse health effects 12-weeks after commencing treatment, compared to waitlist controls and to examine the cost-effectiveness of this strategy. METHODS An open-label waitlist RCT was conducted in an opioid treatment clinic in Newcastle, Australia. Fifty patients with DSM-IV-TR heroin dependence (and no other substance dependence) were recruited. The intervention group (n=25) received take-home self-administered sublingual buprenorphine-naloxone weekly (mean dose, 22.7±5.7mg) and weekly clinical review. Waitlist controls (n=25) received no clinical intervention. The primary outcome was heroin use (self-report, urine toxicology verified) at weeks four, eight and 12. The primary cost-effectiveness outcome was incremental cost per additional heroin-free-day. RESULTS Outcome data were available for 80% of all randomized participants. Across the 12-weeks, treatment group heroin use was on average 19.02days less/month (95% CI -22.98, -15.06, p<0.0001). A total 12-week reduction in adjusted costs including crime of

QT interval prolongation in opioid agonist treatment: analysis of continuous 12-lead electrocardiogram recordings.

A5,722 (95% CI 3299, 8154) in favor of treatment was observed. Excluding crime, incremental cost per heroin-free-day gained from treatment was