Benzoylecgonine

De-identified postmortem human brain specimens were collected during the routine autopsy process as described in detail previously (Albertson et al, 2004 (link); Albertson et al, 2006 ; Bannon and Whitty, 1997 (link); Johnson et al, 2011 (link), Johnson et al, 2012 (link); Zhou et al, 2014 (link)). In brief, the cause and manner of death were determined by forensic pathologists following medico-legal investigations evaluating the circumstances of death including medical records, police reports, autopsy results, and toxicological data (Karch, 2002 ). Subject inclusion in the cocaine cohort (n=10) was based on determination of cocaine abuse as the cause of death, a documented history of drug abuse, and a positive toxicology for cocaine and/or the cocaine metabolite benzoylecgonine but negative for other drugs of abuse or CNS medications at time of death. No significant correlation was observed between levels of cocaine or its metabolites and the abundance of individual transcripts studied (not shown). Control subjects (n=10) died as a result of cardiovascular disease or gunshot wound, had no documented history of drug abuse, and tested negative for cocaine and other drugs of abuse (with the exception of a single subject with a subintoxicating level (0.06 g/dl) of ethanol). Samples were not screened for the presence of nicotine or metabolites. Exclusion criteria for the study included a known history of neurological or psychiatric illness, death by suicide, estimated postmortem interval exceeding 20 h, evidence of neuropathology (eg, encephalitis or stroke), or chronic illness (eg, cirrhosis, cancer, HIV, or prolonged hospitalization). To reduce the variance between groups unrelated to drug abuse, each cocaine abuse subject was matched to a control subject based on race, gender, and age before inclusion in the study (most study subjects were African American and all were males). The final groups (Table 1) did not differ with regard to any of these parameters or with regard to well-established measures of sample quality and perimortem agonal state (ie, brain pH and RNA integrity number (RIN); Schroeder et al, 2006 (link); Stan et al, 2006 (link)).
The methodologies used for the analyses of gene expression have also been described previously (Johnson et al, 2011 (link); Johnson et al, 2012 (link)). In brief, intact brains were sectioned transversely at the level of the posterior diencephalon and mid-pons to obtain a tissue block encompassing the entire human midbrain (corresponding approximately to plates 51–56 of DeArmond et al, 1989 ). Fresh-frozen blocks were subsequently cryostat-sectioned to a thickness of 250 μm and slide-mounted. Guided by the presence of neuromelanin, DA cell-enriched regions of the substantia nigra (including the dorsal and ventral tiers, pars lateralis) and ventral tegmental area (including the interfascicular, central linear, paranigral, and parabrachial nuclei), identified as previously described (Bannon and Whitty, 1997 (link); Whitty et al, 1997 (link)), were finely dissected and pooled into a single sample from each subject. RNA was isolated, quantified, and assessed for integrity. A pilot microarray experiment comparing this dissection method with samples obtained using a more standard block dissection of ventral midbrain found an enrichment of DA cell-specific transcripts (eg, TH: 2.7-fold) but not of transcripts indicative of glutamate neurons (VGLUT2: 0.2-fold), GABA neurons (VGAT: 1.2-fold), neurons overall (NSE: 0.7-fold), astroglia (GFAP: 1.2-fold), oligodendrocytes (MBP: 1.0-fold), or microglia (CD68: 1.0-fold).
Microarray procedures were performed at the Keck microarray facility (Yale Center for Genome Analysis) as previously described (Johnson et al, 2011 (link), 2012 (link)). HT-12 BeadChips (Illumina, San Diego, CA) were hybridized with cRNAs generated from each specimen, scanned on an Illumina Iscan, and loaded into Illumina BeadStudio to evaluate spiked-in controls before quantile normalization of the data. Raw and normalized data have been deposited in the NCBI-GEO repository (GSE54839). Normalized microarray data were imported into MultiExperiment Viewer (MeV) (http://www.tm4.org/mev/). Hierarchical clustering of the expression profiles of sample replicates intentionally processed at different times ruled out microarray chip and batch effects (Supplementary Figure 1). Subsequent hierarchical clustering of all subject samples (20 samples × technical triplicates=60 total profiles) revealed only three potential outliers (one triplicate each from three cases); after their removal, the remaining replicates (17 triplicates and 3 duplicates) clustered precisely according to the sample of origin (Pearson r=0.99, two-tailed P<0.0001) and were thus averaged to obtain a single expression profile for each subject. A total of 16,301 of the 48,761 probes represented on the microarray were detected (P⩽0.05) in the majority of subjects analyzed (⩾11/20). A subset of differentially expressed genes identified by microarray was subsequently validated by quantitative real-time polymerase chain reaction PCR (qPCR) as previously described (Johnson et al, 2011 (link), 2012 (link))(primer sequences listed in Supplementary Table 1).