Molecular Psychiatry (1999) 4, 26–32 1999 Stockton Press All rights reserved 1359–4184/99 $12.00 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 com- pared 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 com- parison, 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-acetylmor- phine, 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