Brain Choline Acetyltransferase Activity in Chronic, Human Users of Cocaine

Home , Choline, Cocaethylene

ORIGINAL RESEARCH ARTICLE Brain choline acetyltransferase activity in chronic, human users of cocaine, methamphetamine, and heroin SJ Kish1, KS Kalasinsky2, Y Furukawa1, M Guttman1, L Ang3,LLi4, V Adams5, G Reiber6, RA Anthony6, W Anderson7, J Smialek4 and L DiStefano1

1Human Neurochemical Pathology Laboratory, Centre for Addiction and Mental Health, Toronto, Canada; 2Division of Forensic Toxicology, Armed Forces Institute of Pathology, Washington, DC, USA; 3Department of Pathology (Neuropathology), Sunnybrook Hospital, Toronto, Canada; 4Department of Pathology, University of Maryland, Baltimore, MD; 5Ofﬁce of the Hillsborough County Medical Examiner, Tampa, FL; 6Northern California Forensic Pathology, Sacramento, CA; 7Ofﬁce of the Medical Examiner of District 9, Orlando, FL, USA

Cognitive impairment has been reported in some chronic users of psychostimulants, raising the possibility that long-term drug exposure might damage brain neuronal systems, including the cholinergic system, which are responsible for normal cognition. We measured the activity of choline acetyltransferase (ChAT), the marker enzyme for cholinergic neurones, in autopsied brain of chronic users of cocaine, methamphetamine, and, for comparison, heroin. As compared with the controls, mean ChAT levels were normal in all cortical and subcortical brain areas examined. However, the two of 12 methamphetamine users, who had the highest brain/blood drug levels at autopsy, had a severe (up to 94%) depletion of ChAT activity in cerebral cortex, striatum, and thalamus. Based on the subjects examined in the present study, our neurochemical data suggest that brain cholinergic neurone damage is unlikely to be a typical feature of chronic use of cocaine, methamphetamine, or heroin, but that exposure to very high doses of methamphetamine could impair, at least acutely, cognitive function requir- ing a normal nucleus basalis cholinergic neuronal system. Reduced brain ChAT might be explained in part by a hyperthermia-related mechanism as low ChAT levels have also been observed in brain of some patients with neuroleptic drug-associated hyperthermia. Studies of cognitive and brain cholinergic status in high dose users of MA are warranted. Keywords: methamphetamine; cocaine; heroin; acetylcholine; choline acetyltransferase; cognitive disorder; nucleus basalis; dopamine

Introduction mation appears to be available on the influence of long- term exposure of this drug on cognitive status. Cognitive impairment can be associated with long-term Much experimental animal and clinical data suggest use of the psychostimulant drug cocaine. In this regard, typically modest neuropsychological deficits on tests that brain cholinergic neurones originating in the sensitive to damage to executive system functioning, nucleus basalis brain area and terminating in the cer- verbal learning, and memory have been reported in ebral cortex subserve aspects of executive system func- 11–13 some chronic cocaine users.1–8 However, the structural tion, especially attentional processes, which have cause of the deficits (eg, drug toxicity to specific brain been reported to be affected in some psychostimulant 4,6,7 areas, global microvascular damage, etc) has not yet users. As a first step in determining whether long- been determined. Furthermore, in the absence of pro- term exposure to psychostimulants might damage the spective studies, it is not possible to determine nucleus basalis cholinergic system, and thereby con- whether such neuropsychological test deficits might tribute to such cognitive deficits, we measured the have preceded use of the drug.9 In the case of the psy- activity of choline acetyltransferase (ChAT), the chol- chostimulant methamphetamine, although acute high inergic marker synthetic enzyme, in autopsied cerebral dose exposure to this psychostimulant can produce cortex, as well as in other brain areas, of chronic impaired cognitive status in the human,10 no infor- human users of cocaine, amphetamine, and, for comparison, in brain of a group of users of heroin. We report that brain ChAT levels are normal in all three drug abuse groups with the exception of those users of Correspondence: SJ, Kish, PhD, Human Neurochemical Pathology methamphetamine who had been exposed to very high Laboratory, Centre for Addiction and Mental Health, 250 College doses of the drug. Street, Toronto, Ontario, Canada M5T 1R8. E-mail: kishsȰcs. clarke-inst.on.ca Received 8 June 1998; revised 17 July 1998; accepted 20 July 1998 Brain ChAT in human drug users SJ Kish et al 27 Patients and methods drug of abuse in blood and brain, suggesting that each subject had used the drug during the 72 h preceding Postmortem brain material from a total of 12 controls, death. Clinical information was obtained by the medi- 11 users of cocaine, 12 users of methamphetamine, and cal examiners using a questionnaire format and nine users of heroin was obtained from medical exam- through structured telephone interviews with the next iner offices in the US and Canada. As shown in Table 1, of kin. No formal neuropsychological testing had been the mean ages and postmortem time (interval between conducted on the drug users. death and freezing of the brain) between the control Clinical, toxicological, and pathological data, includ- and drug abuse groups did not differ significantly (one Ͼ ing suspected cause of death, have been previously way analysis of variance (ANOVA); P 0.05). At auto- published for the 11 cocaine users14 and 12 users of psy, one half-brain was fixed in formalin fixative methamphetamine.15 For the heroin users the sus- whereas the other half was immediately frozen at − ° pected causes of death were acute narcotic intoxication 80 C until biochemical analysis. Samples of cardiac for 11 of the 12 heroin users and atherosclerotic cardio- blood were obtained from all of the drug users and vascular disease with acute narcotic intoxication as a from the control subjects for drug screening. Scalp hair contributing factor in one subject. Brain neuropatho- samples for drug analyses could be obtained from nine logical analyses of the heroin users disclosed no sig- of the 12 control subjects, seven of the 11 cocaine nificant abnormalities with the exception of mild ven- users, seven of the 12 methamphetamine users, and tricular dilatation (n = 1) and mild diffuse gliosis in eight of the nine heroin users. Levels of drugs of abuse diencephalon and lower brain stem (n = 1). in blood and other bodily fluids were measured by the local medical examiner whereas drug analyses in brain and hair samples were conducted at the Armed Forces Brain dissection for neurochemical analysis Institute of Pathology (KK; Washington, DC, USA). Cerebral cortical subdivisions were excised using the Brodmann classification. Following dissection of the Control subjects cerebral cortical subdivisions, approximately 2.5 mm- Autopsied brain was obtained from 12 neurologically thick coronal sections of the brain were taken begin- normal subjects who died from a variety of causes ning with the anterior tip of the head of the caudate (gunshot wound to chest (2), chest trauma (2), cardio- nucleus to the tail of the caudate. Subcortical brain vascular disease (5), perforated vena cava (1), leukemia areas were dissected using the Atlas of Riley16 and as (1), and drowning (1)). All subjects tested negative for previously described.17,18 drugs of abuse in blood, autopsied brain, and, in the seven cases for which hair was available, sequential Neurochemical analyses hair samples. ChAT activity in brain homogenates was determined by minor modifications of the radioenzymatic pro- Drug users cedure of Fonnum and coworkers.19 Activities of cit- The subjects for the cocaine, methamphetamine, and rate synthase20 and glyceraldehyde-3-phosphate heroin groups were selected from a large group of dehydrogenase21 in brain homogenates were deter- potential cases who met the following criteria: (1) pres- mined by spectrophotometric procedures. ence of cocaine and/or metabolite benzoylecgonine, or methamphetamine, or heroin metabolites (6-acetylmorphine, morphine, or morphine glucuronide), on toxi- Measurement of drugs of abuse cology screens in blood or urine, autopsied brain (see Levels of cocaine and its metabolites benzoylecgonine, below), and, if available, scalp hair; (2) absence of other ecgonine methyl ester, norcocaine, and cocaethylene drugs of abuse in bodily fluids with the exception of were determined by a gas chromatography/mass spec- ethanol; (3) evidence from the case records of use of trometry (GC-MS) procedure as previously described.14 the primary drug for at least 1 year prior to death; and Concentrations of methamphetamine and ampheta- (4) absence of neurological illness or, at autopsy, brain mine were determined by GC-MS.15 For the measure- pathology unrelated to use of the drug. Most of the ment of heroin metabolites, a GC-MS analysis was per- potential subjects were rejected because of a known formed using a Varian 3400 gas chromatograph history of significant polydrug abuse or the presence of equipped with a Satum 3 ion-trap mass spectrometer other drugs of abuse in blood or brain at autopsy. Alco- as the detector. A 5% phenyl-methylpolysiloxane hol was known to have been used by eight of the 11 capillary column (Hewlett Packard, HP-5MS, 30 m × cocaine users and all 12 of the heroin users as evi- 0.25 mm, 0.25 ␮m film thickness) was used for the denced by review of the case records and by presence chromatographic separation with helium gas flow of in bodily fluids or brain of ethanol or cocaethylene (the 1 ml min−1 (200°C). Tandem MS was performed by res- transesterification product of cocaine and ethanol). onant excitation and monitoring of daughter ion spec- Ethanol was not detected in bodily fluids of any of the tra. Electron ionization was employed throughout and methamphetamine users. With the exception of one the following selected ions were monitored: m/z 324, cocaine user in which cocaine was detected in urine 340, 356 for 6-acetylmorphine derivative, m/z 287, 401, but not blood, all of the other users of cocaine, 414 for morphine derivative, and m/z 237, 346, 358 for methamphetamine, and heroin, tested positive for the d3-codeine derivative. Brain ChAT in human drug users SJ Kish et al 28 0.26 0.20 0.23 0.41 ± ± ± ± , cocaine 0.32 2.61 0.33 1.79 0.29 2.07 0.43 2.02 ± ± ± ± 1.7 2.66 3.1 1.92 2.1 2.13 3.6 1.72 ± ± ± ± 0.10 15.2 0.08 13.3 0.09 16.7 0.13 17.0 ± ± ± ± 0.08 1.28 0.11 1.12 0.08 1.36 0.07 1.13 ± ± ± ± 0.09 1.02 0.13 0.80 0.11 1.04 0.12 0.90 ± ± ± ± 0.10 1.41 0.10 1.20 0.10 1.54 0.09 1.25 ± ± ± ± 4.5 1.50 7.9 1.34 7.0 1.76 5.2 1.42 ± ± ± ± S.E. No statistically significant differences were observed between control subjects and the drug users (one ± 4.4 56.8 6.2 48.1 3.5 58.6 4.6 61.7 ± ± ± ± Caudate Putamen area 10 area 21 area 17 area 7b Nacs NPM NL 1.8 44.7 2.0 37.3 1.9 40.5 2.1 47.4 ± ± ± ± g protein) represent mean

␮ 0.05). Abbreviations: PM (postmortem time; interval between death and freezing of the brain); cerebral cortical areas 10 (frontal); 21 (temporal); 3.0 15.4 2.6 14.8 1.9 18.7 1.7 13.1 Ͼ ± ± ± ± P Subject characteristics and choline acetyltransferase (ChAT) activity in autopsied brain of control subjects and in users of methamphetamine (MA) way analysis of variance; 17 (occipital); 7b (parietal); Nacs (nucleus accumbens); NPM (medial pulvinar nucleus of the thalamus); NL (nucleus lateralis of the thalamus). Values (pmol per 10 min per (11) Heroin(9) 37.8 (12) MA(12) Cocaine 32.5 32.8 Control 32.9 and heroin Group(n) Age (yrs) PM (h) Brain region Table 1 Brain ChAT in human drug users SJ Kish et al 29 Results and heroin groups. For the methamphamine users statistically significant negative correlations (−0.65 to Table 1 shows the activities of ChAT in autopsied brain −0.81; P Ͻ 0.05) were observed between brain ChAT of the drug users. As compared with the controls, mean activity and methamphetamine plus amphetamine lev- ChAT activity was normal in all brain areas examined els in all of the brain areas examined with the excep- of the drug users (one way ANOVA; P Ͼ 0.05). Examin- tion of the frontal cortex (−0.13; P Ͼ 0.05). However, ation of the individual subject values (see Figure 1) examination of the individual subject values revealed revealed that enzyme activity in the drug users was that the significant negative relationship between within or close to the limits of the normal control range amount of methamphetamine exposure and ChAT with the exception of two methamphetamine users activity was primarily due to the two cases (cases a and (designated cases a and b (cases No. 5 and No. 1, b) who had the highest brain and blood drug levels and respectively, in Ref 15) who had markedly to severely very low ChAT activities (see Figure 2 for occipital reduced (up to 94% depletion) ChAT levels which cortex). After exclusion of these two cases from the cor- were below the lower limit of the range of the controls relational analysis a trend for a negative relationship in all areas examined with the exception of the frontal between total methamphetamine levels and ChAT cortex of case b. activity was observed in all brain areas examined (r = Spearman rank correlations were performed to deter- − 0.30 to −0.66), with the exception of the frontal cortex mine whether there might be a relationship between (r = 0.27), which was statistically significant for the amount of drug taken recently and enzyme activity. As caudate nucleus only (r =−0.66, P Ͻ 0.05). an index of recent drug use, brain levels of ‘total’ To determine whether the enzyme reduction in the cocaine (cocaine plus metabolites benzoylecgonine, two methamphetamine users with low brain ChAT ecognine methyl ester, norcocaine, cocaethylene), might be explained by a non-specific phenomenon, methamphetamine (methamphetamine plus activities of two additional enzymes which are stable amphetamine) and heroin metabolites (6-acetylmor- postmortem in human brain and unrelated to ChAT phine plus morphine plus morphine glucuronide) were and to each other, glyceraldehyde 3-phosphate calculated. For the cocaine and methamphetamine dehydrogenase and citrate synthase, were measured in users, drug levels were determined in all brain areas the occipital cortex, a region of brain in which ChAT with the exception of the nucleus accumbens, whereas activity was severely depleted in both cases (case a: in the heroin group, drug levels were determined only in the occipital cortex, the latter which provided values used for correlations for all brain areas in this group. Complete details of the brain regional distribution of the drugs of abuse will be reported elsewhere. No statistically significant correlations were observed between brain ChAT activities and drug levels in the cocaine

Figure 1 Normalized (expressed as % control mean) activities of choline acetyltransferase (ChAT) in autopsied brain of Figure 2 Relationship between brain levels of ‘total’ individual control subjects (solid circles) and in the two high methamphetamine (MA; methamphetamine plus dose methamphetamine users (cases a and b). Note that the amphetamine) vs activity of choline acetyltransferase (ChAT) brain ChAT activities of cases a and b are below the lower in autopsied occipital cortex (area 17) of the 12 methampheta- limit of the control range in most brain areas. Abbreviations: mine users. Note that cases a and b, who both had the highest CN (caudate nucleus); PUT (putamen); cerebral cortical areas brain methamphetamine levels, showed the lowest ChAT 10 (frontal): 21 (temporal); 17 (occipital); 7b (parietal); Nacs activities. The Spearman rank correlation was −0.70 (nucleus accumbens); NPM (medial pulvinar nucleus of the (P Ͻ 0.02) for all 12 cases and −0.48 (P Ͼ 0.05) with cases a thalamus); NL (nucleus lateralis of the thalamus). and b removed from the analysis. Brain ChAT in human drug users SJ Kish et al 30 −75%; case b: −90%). As shown in Table 2, activity of cerebral cortical (temporal/parietal) ChAT activities are both ‘control’ enzymes was within or close to the limits typically decreased by about 50–60%.20 of the control range with the exception of citrate syn- Although mean brain ChAT activity was normal in thase activity in case a, in which activity of the enzyme all three groups of drug users, two subjects, both was reduced by about 50% of the mean control value. methamphetamine users, had severely reduced ChAT levels in most of the brain areas examined (see cases a, b; Figure 1). The clinical and forensic characteristics Discussion of these two subjects are shown in Table 3. As these The major ﬁnding of our investigation is that brain individuals had received, at least recently, as inferred ChAT activity is generally normal in chronic users of from brain and blood methamphetamine concen- psychostimulant drugs and in users of heroin with the trations, the highest drug doses of all of the metham- exception of methamphetamine users who are exposed phetamine users examined (Figure 2), it seems reason- to very high doses of the drug. able to conclude that a brain ChAT depletion might A limitation of our study is the lack of detailed infor- be restricted to those methamphetamine users who are mation on the actual amount of drug taken on a daily exposed only to very high doses of the drug. Limited basis by each of the subjects. In this regard, although forensic information is available on the blood levels our forensic drug analysis of blood and brain provides of methamphetamine associated with fatalities due to a general assessment of the amount of drug taken methamphetamine intoxication, with the interpret- recently (ie, within approximately 72 h), and that of ation of the available data complicated by factors such sequential hair samples (for those cases in which hair as tolerance and postmortem diffusion of the drug. In was available) conﬁrms that the subjects must have our study, blood levels of the two subjects having very used the drug chronically, it was not possible to obtain low brain ChAT levels (7 mg L−1; 12.5 mg L−1) fall retrospective data detailing the precise amounts of within the wide range of those associated with drug taken over the last years before death. It is pos- methamphetamine-related fatalities (0.6–40.0 mg sible that our neurochemical data may have been con- L−1).22,23 founded somewhat by the use of other drugs of abuse In principle, as ChAT is the marker enzyme for chol- during the years before death. In this regard, although inergic neurones, low ChAT in cases a and b could be blood drug analysis revealed that none of the users of explained by an actual loss of cholinergic neurones methamphetamine had detectable levels of ethanol at resulting from chronic use of methamphetamine. How- autopsy, ethanol could be detected in all of the heroin ever, although no quantitative morphometric studies users and in most of the users of cocaine. were conducted, extensive qualitative microscopic To the extent that the subjects in our investigation neuropathological examination of the brain, including are representative of typical, chronic, drug users, our the nucleus basalis brain area of cases a and b dis- neurochemical data suggest that substantial damage to closed no evidence of cell loss or gliosis. In addition, the nucleus basalis cholinergic system, or to other our observation that, in these two subjects, the brain brain cholinergic neurones neurochemically assessed ChAT depletion was generally near-total and in our investigation (eg, striatal cholinergic regionally widespread makes the possibility of a near- interneurones) is not a typical feature of chronic use of total loss of brain cholinergic neurones unlikely. More psychostimulants or heroin in the human. This is in likely, the reduced brain enzyme levels in cases a and contrast to the marked degeneration of brain nucleus b is explained by the acute toxic effects of high dose basalis cholinergic neurones in the dementing illness methamphetamine on ChAT itself, an enzyme well of Alzheimer’s disease, in which, in our laboratory, recognized for its stability in postmortem human brain.24 This does not appear to be a direct inhibitory effect of residual brain methamphetamine on ChAT Table 2 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity determined in vitro, as concentrations of MA and citrate synthase activities in occipital cortex of control (0.4 mM) 20 times that of the brain methamphetamine subjects and in two high dose users of methamphetamine concentration resulting from the residual brain (cases a and b) methamphetamine of case b (the subject with the highest brain methamphetamine concentration) had no GAPDH activity Citrate synthase effect on ChAT activity in frontal cortical homogenates activity of control subjects (n = 3; data not shown). Although we could not obtain information on the clinical state ± ± Controls 1.72 0.06 196 7 of either subject immediately following the last taking (1.54–1.92) (149–228) of the drug, we speculate that both users probably Case a 1.61 109 developed methamphetamine-induced hyperthermia, Case b 1.51 226 as may occur following high doses of methamphetamine,10 and that the ChAT enzyme had been inacti- Control values represent mean ± S.E. of 7 (GAPDH; ␮mol vated, perhaps irreversibly, by the hyperthermia. This min−1 mg−1 protein) or 12 (citrate synthase; nmol min−1 mg−1 possibility is in fact supported by our previous obser- protein) controls with range of control values provided in par- vation of a regionally widespread brain ChAT entheses. depletion in several patients who died in severe hyper- Brain ChAT in human drug users SJ Kish et al 31 Table 3 Subject characteristics of two chronic methamphetamine (MA) users who had very low brain activities of choline acetyltransferase

Case number Case a Case b

Age (years) 20 34 Sex, race male, caucasian female, caucasian Postmortem time (h) 21 14 Estimated interval between last MA use and death (h) 1 16 Route of MA administration nasal oral Estimated amount/pattern of recent MA use unknown $10 per day/usually daily use (when available) taken in the morning Estimated duration of MA use (years) 1 10 Co-existing medical illnesses none known none; subject considered ‘quite healthy’ Social problems arrested for selling subject was unemployed illicit drugs Brain neuropathological status no abnormalities no abnormalities Blood MA level (mg L−1) 7.0 12.5 Brain (caudate nucleus) MA level (ng mg−1 tissue) 41.4 56.5 MA hair analysis hair not available 41 ng mg−1 MA; 37 ng mg−1 amphetamine Suspected cause of death MA intoxication MA intoxication

Cases a and b were originally described in Ref 15 as cases 5 and 1, respectively. thermia associated with neuroleptic drug use.25 In this Acknowledgements regard, our finding that the activities of the ‘control’ This study was supported by US NIH NIDA DA 07182 enzymes in cases a and b were normal to marginally to SK. reduced could also be explained by general hyperthermic damage, but with the ChAT enzyme being References much more susceptible to this damage. Our neurochemical data suggest that although a 1 Manschreck TC, Schneyer ML, Weisstein CC, Laughery J, Rosenthal J, Celada T et al. Freebase cocaine and memory. Comprehensive brain cholinergic deficit is unlikely to be a typical fea- Psychiatry 1990; 31: 369–375. ture of use of psychostimulants or of heroin, a brain 2 Ardila A, Rosselli M, Strumwasser S. Neuropsychological deficits ChAT depletion could occur in methamphetamine in chronic cocaine abusers. Int J Neurosci 1991; 57: 73–79. users who overdose on the drug. 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