Probing Peripheral and Central Cholinergic System Responses

Probing peripheral and central cholinergic system responses

Claudio A. Naranjo, MD; Jeanne Fourie, MSc; Nathan Herrmann, MD; Krista L. LanctBt, PhD; Catherine Birt, MD; Kenneth K. Yau, BSc

Naranjo - Psychopharmacology Research Program, Sunnybrook and Women's College Health Sciences Centre, and Departments of Pharmacology, Psychiatry and Medicine, University of Toronto; Fourie, Yau- Psychopharmacology Research Program, Sunnybrook and Women's College Health Sciences Centre; Herrmann - Psychopharmacology Research Program and Department of Psychiatry, Sunnybrook and Women's College Health Sciences Centre, and Department of Psychiatry, University of Toronto; Lanctot - Department of Psychiatry, Sunnybrook and Women's College Health Sciences Centre, and Departments of Pharmacology and Psychiatry, University of Toronto; Birt - Department of Ophthalmology, Sunnybrook and Women's College Health Sciences Centre and University of Toronto, Toronto, Ont.

Objective: The pharmacological response to drugs that act on the cholinergic system of the iris has been used to predict deficits in central cholinergic functioning due to diseases such as Alzheimer's disease, yet correlations between central and peripheral responses have not been properly studied. This study assessed the effect of normal aging on (I) the tropicamide-induced increase in pupil diameter, and (2) the reversal of this effect with pilocarpine. Scopolamine was used as a positive control to detect age-dependent changes in central cholinergic functioning in the elderly. Design: Randomized double-blind controlled trial. Participants: Ten healthy elderly (mean age 70) and 9 young (mean age 33) volunteers. Interventions: Pupil diameter was monitored using a computerized infrared pupillometer over 4 hours. The study in- volved 4 sessions. In I session, tropicamide (20 pL, 0.01%) was administered to one eye and placebo to the other. In another session, tropicamide (20 p,L, 0.01 %) was administered to both eyes, followed 23 minutes later by the application of pilocarpine (20 p,L, 0.1 %) to one eye and placebo to the other. All eye drops were given in a randomized order. In 2 separate sessions, a single dose of scopolamine (0.5 mg, intravenously) or placebo was administered, and the effects on word recall were measured using the Buschke Selective Reminding Test over 2 hours. Outcome measures: Pupil size at time points after administration of tropicamide and pilocarpine; scopolamine-induced impairment in word recall. Results: There was no significant difference between elderly and young volunteers in pupillary response to tropicamide at any time point (p > 0.05). The elderly group had a significantly greater pilocarpine-induced net decrease in pupil size 85, 125, 165 and 215 minutes after administration, compared with the young group (p < 0.05). Compared with the young group, the elderly group had greater scopolamine-induced impairment in word recall 60, 90 and 120 minutes after administration (p < 0.05). Conclusion: There is an age-related pupil-

Correspondence to: Dr. Claudio A. Naranjo, University of Toronto, Psychopharmacology Research Program, Sunnybrook and Women's College Health Sciences Centre, Sunnybrook Campus, Room F3 27, 2075 Bayview Ave., Toronto ON M4N 3M5; fax 416 480-4708; [email protected] Medical subject headings: aged; cholinergic agonists; cholinergic antagonists; iris; miosis; muscarinic agonists; muscarinic antagonists; pilocarpine; scopolamine; tropicamide J Psychiatry Neurosci 2000;25(4):325-36. Submitted May I1, 1999 Revised Dec. 2, 1999 Accepted Dec. 13, 1999 © 2000 Canadian Medical Association

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lary response to pilocarpine that is not found with tropicamide. Thus, pilocarpine may be useful to assess variations in central cholinergic function in elderly patients.

Objectif: On a utilise la reaction pharmacologique a des medicaments qui agissent sur le systeme cholinergique de l'iris pour prevoir des deficits de la fonction cholinergique centrale causes par des maladies comme la maladie d'Alzheimer, mais on n'a pas etudie suffisamment les liens entre les reactions centrale et peripherique. Au cours de cette etude, on a evalue 1'effet du vieillissement normal sur (I) 1'elargissement du diametre de la pupille provoque par la tropicamide et (2) l'inversion de cet effet au moyen de la pilocarpine. On a utilise de la scopolamine comme temoin positif pour detecter les changements lies a l'ige de la fonction cholinergique centrale chez les personnes agees. Conception: Etude contr6lee randomisee a double insu. Participants: Dix personnes agees en bonne sante (age moyen de 70 ans) et neuf personnes plus jeunes (age moyen de 33 ans) qui se sont portees volontaires. Interventions: On a contr6le le diametre de la pupille au moyen d'un pupillometre a infrarouge informatis6 pendant quatre heures. L'etude a comporte quatre seances. Au cours d'une seance, on a applique de la tropicamide (20 pL, 0,01 %) dans un ceil et un placebo dans I'autre. Au cours d'une autre seance, on a applique de la tropicamide (20 pL, 0,01 %) dans les deux yeux et, 23 minutes plus tard, on a applique de la pilocarpine (20 pL, 0,1 %) dans un oeil et un placebo dans l'autre. Toutes les gouttes oculaires ont ete administrees au hasard. Au cours de deux seances distinctes, on a administre une seule dose de scopolamine (0,5 mg, par voie intraveineuse) ou un placebo et mesure les effets sur le rappel des mots au moyen du test de rappel selectif de Buschke pendant deux heures. Principales mesures de resultats: Diametre de la pupille a certains moments apres I'administration de tropicamide et de pilocarpine; deficit du rappel de mots provoque par la scopolamine. Resultats : On n'a pas constate de difference significative entre les volontaires ages et les jeunes quant a la reaction pupillaire a la tropicamide a n'importe quel moment (p > 0,05). Les sujets ages ont presente une diminution nette beaucoup plus importante du diametre de la pupille provoquee par la pilocarpine 85, 125, 165 et 215 minutes apres I'application, comparativement aux sujets jeunes (p < 0,05). Comparativement aux sujets jeunes, les sujets ages ont presente un deficit beaucoup plus important du rappel provoqu6 par la scopolamine 60, 90 et 120 minutes apres I'administration (p < 0,05). Conclusion: 11 y a un lien entre la reaction pupillaire a la pilocarpine liee a l'age qui n'existe pas dans le cas de la tropicamine. La pilocarpine peut ainsi etre utile pour evaluer des variations de la fonction cholinergique centrale chez les patients ages.

According to the cholinergic hypothesis of memory ergic antagonist.12 A classic study conducted in 1974 function, certain changes in memory are associated demonstrated that young normal subjects treated with with a decline in central cholinergic function.' The cen- scopolamine exhibited significant decreases in perfor- tral cholinergic system is known to play an important mance of certain cognitive tests.5 However, this role in learning, cognition and memory.2' This has been decreased level of cognition was not significantly dif- demonstrated in various studies involving both human ferent from elderly controls at baseline. It has also been and animal models, in which centrally acting choliner- shown that the cholinesterase inhibitor physostigmine gic agonists have been used to augment cognitive func- can block most of the cognitive deficits associated with tion and antagonists have been used to impair it.7 scopolamine treatment, whereas amphetamines and There have also been reports of decreased central other stimulants do not block these deficits.13 This result cholinergic function in several disease states character- suggests that the decline in cognitive function seen in ized by deficits in cognitive and intellectual function- aging, like the effects of scopolamine, may be caused by ing, such as Alzheimer's disease, Parkinson's disease, a decrease in central cholinergic system functioning. Down's syndrome and Korsakoff's syndrome.8 Currently, there are no pharmacologically based, Normal aging causes disruptions in the central noninvasive peripheral probes to monitor or predict cholinergic system. Choline uptake and acetylcholine central cholinergic function. Such peripherally acting synthesis and release have been found to be decreased probes would be advantageous because, unlike central- in the brains of aged rodents in comparison with young ly acting cholinergic probes, such as scopolamine, they animals.9 Studies have also shown that there are would be noninvasive and would not cause adverse changes in the binding of specific ligands for muscarinic central cholinergic effects. Recently, some evidence has receptor subtypes in elderly rats.","1 These changes have suggested that changes in the central cholinergic system been modelled with scopolamine, a muscarinic cholin- may be accompanied by changes in peripheral cholin-

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ergic function. For example, adults with Down's syn- 28 to rule out significant cognitive impairment.24 Sub- drome over the age of 30 have memory impairment, jects had good to excellent health, as assessed by the dementia and neuropathologic lesions (e.g., neurofibril- General Health Questionnaire,25 and underwent a gener- lary tangles) characteristic of central cholinergic al ocular examination, including slit-lamp biomicros- deficit."4 In these individuals, the cholinergic antagonist copy, by an ophthalmologist (C.B.). An electrocardio- atropine caused a significantly greater increase in heart gram was obtained from each elderly subject and was rate than in control subjects,15 while topically applied read by a cardiologist before the study. Exclusion crite- atropine and tropicamide (a muscarinic antagonist) eye ria were contraindications to any of the study drugs drops caused a significantly greater increase in pupil (tropicamide, pilocarpine or scopolamine), including a diameter."6"7 shallow anterior ocular chamber, history of intraocular Alzheimer's disease (AD) is another disorder in surgery, cloudy cornea, glaucoma, any sign of cataracts, which there is a widespread decrease in central cholin- or significant cardiac abnormality. Subjects continued to ergic function and a pattern of brain lesions similar to receive any concomitant medications during the study, that in Down's syndrome.'4 One group has observed and these medications were assessed by interviews with that patients with AD exhibit a significantly greater the subjects and their physicians when necessary. increase in pupil size in response to tropicamide eye Subjects abstained from alcohol, smoking and caffeine- drops than age-matched controls.'8 However, many containing food or beverages for at least 12 hours before groups have attempted and failed to replicate that and during test sessions, and were advised to get a good result.'9 A few groups have used a slightly different night's rest before each session. All subjects gave written approach, observing the pupillary response to pilo- informed consent and received compensation for their carpine, a muscarinic agonist.211 In contrast to the tropi- participation in the study. This study was approved by camide literature, all of the studies of pilocarpine indi- the Research Ethics Board of the Sunnybrook and cate that it may be sensitive to changes in central cho- Women's College Health Sciences Centre. linergic function, as patients with AD showed a hypersensitive response to pilocarpine in comparison with Study design age-matched controls. This study was performed to develop a peripheral For each of 4 study sessions, subjects arrived at the probe that would be able to detect age-related changes Psychopharmacology Research Laboratory at 10 a.m. in central cholinergic function. The objective of the The laboratory light conditions were standardized to an study was to assess the effect of normal aging on illumination of 600 lux. Sessions followed a balanced (1) tropicamide-induced increase in pupil diameter, and randomized double-blind study design and were and (2) the reversal of this effect with pilocarpine in separated by a minimum of 72 hours. During one ses- healthy elderly and young volunteers. We used scopo- sion, subjects were administered tropicamide (20 jL eye lamine as a positive control to detect age-dependent drop, 0.01%) to one eye and placebo (sodium chloride, changes in central cholinergic system function in the 20 ,uL eye drop, 0.9%) to the other eye, in a randomized elderly. order. Pupil diameter was measured twice at baseline and at 17 time points over a period of 240 minutes by a Methods computerized infrared pupillometer. In another session, tropicamide (20 ,L, 0.01%) was administered to Subjects both eyes, followed 25 minutes later by the application of pilocarpine (20 ,uL, 0.1%) to one eye and placebo to We recruited 10 elderly (older than 55 years) and 9 the other eye, also in a randomized order. Pupil diame- young (18 to 40 years) healthy volunteers through ter was measured at 13 time points over a 215-minute advertisements in local newspapers. Demographic period after pilocarpine administration. All eye drops information, including education, weight, ethnic back- were instilled using a precision micropipette into the ground and cognitive status, was collected before the lower conjunctival cul-de-sac with the subject supine. In study. Cognitive status was assessed with the Mini- addition, following administration of each set of eye Mental State Examination (MMSE)23 before the study, drops, the investigator pressed his or her fingers on the and the subjects' scores were required to be greater than subject's nasolacrimal duct for 1 minute.

...... Mmg ...... 4~~~~~~~~~~~~~~~~~~~~~~~~~~.~~~~~~~~~~~~~~K'9~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4...... ~ 4~~~~~~~~~ ~ ~ ~ ~~~~~~~~~~~...... In 2 separate test sessions, an indwelling catheter was The digit symbol substitution subtest (DSST) of the inserted into the cephalic vein of the less dominant fore- WAIS was also performed at each time point to assess arm. After baseline assessments, scopolamine (0.5 mg) psychomotor function.31 For the DSST, subjects were or placebo was administered intravenously via slow presented with a set of symbols and matching numbers injection over 2 minutes. Cognitive function was mea- and were asked to draw the correct symbol below each sured at baseline and at 30, 60, 90 and 120 minutes after number during a 90-second interval. drug administration. At these time points, pupil diame- Subject-rated sedation was assessed at each time point ter was also measured. using a 20-cm visual analogue scale that ranged from "not at all" at the left end to "extremely" at the right.32 Assessment of scopolamine effect Pupillometry Scopolamine-induced impairment of memory and leaming was assessed by a modified Buschke Selective A previously validated infrared pupillometer connect- Reminding Test, which has been used in previous stud- ed to a personal computer was used to measure pupil ies of scopolamine by other investigators.26 This test diameter.3 The apparatus consisted of 4 infrared diodes detects the cognitive impairment induced by scopo- that illuminated the eye, an infrared-sensitive video lamine in both young and elderly individuals.27-29 camera, and a small computer-controlled display televi- Buschke Selective Reminding Test. The test was per- sion for the eye. Each captured image was processed to formed at each of the 5 assessment time points and con- determine the edge of the pupil, and the maximal diam- sisted of a 12-word list in which the words have the eter value out of 40 computer-generated diameter mea- same frequency of use as they do in the English lan- surements was recorded. During pupillometry, the sub- guage and were categorically unrelated.26P Equivalent ject was seated in an armchair, with the chin placed on versions of the word lists were constructed and a dif- an adjustable chin-rest connected to the camera stand ferent set was used at each time point after scopolamine while the eyes were fixated on a target at a standardized or placebo administration. All 12 words were read to distance. the subject (1 word every 3 seconds), and the subject was asked to recall as many words a possible during a Data analysis 55-second timeframe. This was followed by a 30-second pause, after which the investigator read only the words Data were analyzed using the Statistical Analysis that were not recalled by the subject during the previ- System (SAS), version 6.11 (SAS Institute Inc., Cary, ous recall period. The subject was again asked to recall NC, 1996). The data on net increases in pupil size after as much of the 12-word list as possible. This procedure challenge with tropicamide and placebo were ex- was repeated 7 times (for a total of 8 recall attempts). pressed at each time point as [pupil diameter after However, if the subject recalled all 12 words in 2 suc- tropicamide minus the corresponding pupil diameter cessive trials, the test was discontinued and maximum after placebo] x 100/[pupil diameter after placebo at score (12) was assigned for each of the remaining recall that time point]. For the analysis of the pilocarpine data, trials. The total number of words recalled in the 8 trials the net decrease in pupil size was expressed as [pupil was used as a measure of memory/learning at each diameter after placebo minus the corresponding pupil time point. diameter after pilocarpine] x 100/[pupil diameter after Visuospatial praxis, psychomotor function, and subject- placebo at that time point]. The pupil diameter of the rated sedation. Visuospatial praxis was assessed with a treated eye was compared with the placebo-matched block-construction subtest of the Wechsler Adult pupil diameter at each given time point to account for Intelligence Scale (WAIS).31 The test was administered the potential contribution of tonic sympathetic activity at baseline and at 90 minutes after administration of to pupil diameter, which would affect both eyes equal- scopolamine or placebo. The blocks used in each test ly, over the course of the study session. The tropicamide were identical, with 2 red sides, 2 white sides and 2 and pilocarpine data were tested for significant differ- sides that are red and white. The subject was asked to ences by 2-way repeated measures analysis of variance make specific designs with the blocks during a specified (ANOVA), with 2-way interaction. The 2 variables in limited time interval. the ANOVA were age and time, where time was the

11ill"?;11"MI "111,11- %111.fflmm.g.,-%!. MR-1 lfiffi NO ;RP ...... -M ff RUN,, 0 -o sssffil4m .'.i§ RH SO IMEM MgHN4ffiN4MiM " IN M M..UmsP.M..Illm.1 Mr, fln: ffir ENR., WN 'M plfli;i SHE, M-09 MUMMMOl-, MM repeated-measures variable. The scopolamine data young subjects (Korean and Afghani descent). All sub- were analyzed using a 3-way repeated measures jects had good to excellent health as assessed by the ANOVA, with 3-way interaction. The variables in this General Health Questionnaire.-- Other characteristics ANOVA were age, treatment and time, with time as the are given in Table 1. None of the elderly subjects repeated-measures variable. When ANOVA was signif- screened were found to be ineligible for the study icant (p < 0.05), further multiple comparisons were per- because of ocular conditions. formed at each time point using Student's t-test with Bonferroni correction, which maintained the signifi- Tropicamide effect on pupil size cance level at p < 0.05. The time course of changes in pupil size after tropi- Results camide administration in the elderly and young subjects is shown in Fig. 1. The ANOVA found a significant Subjects time effect (F = 39, p < 0.05) on pupil size, but there was no significant effect of age, or age and time (F = 1.34, p Ten elderly (mean age 70, standard error of the mean > 0.05) on pupillary response to tropicamide. The mean [SEMI 2 years, range 59 to 79; years of education 14 SEM peak increase in pupil diameter was observed 50 min- 1; weight 75 SEM 5 kg) and 9 young (mean age 33 SEM utes after tropicamide administration and was not sig- 2, range 18 to 40; years of education 17 SEM 1; weight 73 nificantly different between the 2 age groups (p > 0.05). SEM 5 kg) healthy volunteers were recruited. There was no significant difference between elderly and young Pilocarpine effect on pupil size subjects in education and weight, or in cognitive status (elderly mean MMSE score 29 SEM 0.2; young 30 SEM The time course of the pilocarpine effect on pupil size, 0.3, p > 0.05). All subjects were of European descent following the tropicamide-induced increase in pupil except for 1 elderly subject (Caribbean descent) and 2 size, is presented in Fig. 2. The pilocarpine-induced net

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decrease in pupil size was faster and larger in the elder- subjects. Sex, iris colour and ethnic background did not ly group than in the young group (p = 0.003). The affect pilocarpine effect on pupil size (p > 0.05). ANOVA found a significant effect of time (F = 19.01, p < 0.05) as well as age and time (F = 2.51, p < 0.05) on pupil Scopolamine effect on cognitivefunction response to pilocarpine. The peak pilocarpine effect on pupil size was observed at the 85-minute time point. The The time course of scopolamine-induced impairment in elderly subjects had a significantly greater (p < 0.05) word recall for the Buschke Selective Reminding Test is mean net decrease in pupil size at the 85-,125-, 165- and shown in Fig. 3. The ANOVA found a significant effect 215-minute time points (34% SEM 2.1%, 30.6% SEM of time (F = 7.18, p < 0.05) as well as age, and time and

3.0%, 28.9% SEM 2.9%, 23.9% SEM 2.4%, respectively, treatment (F = 3.06, p < 0.05) on word recall after scopo- compared with placebo) compared with the young sub- lamine administration. The impairment in word recall jects (20.3% SEM 5.2%, 15.8% SEM 5.4%, 14.9% SEM was greater in the elderly subjects than in the young 3.0%, 8.8% SEM 4.9%, respectively, compared with subjects 60 (elderly 41.7% SEM 10.8% of baseline; young placebo). There was no significant difference in pupil 58.7% SEM 9.0%), 90 (elderly 43.1% SEM 12.5%; young size between the 2 groups at baseline (5.2 mm for the 60.6% SEM 7.8%), and 120 (elderly 47.1% SEM 12.7%; young group and 4.8 mm for the elderly group, p > 0.05). young 103.0% SEM 7.6%) minutes after scopolamine The mean absolute decrease in pupil size for the elderly administration (p < 0.05). There were no significant dif- group at the 85-, 125-, 165-, and 215-minute time points ferences in visuospatial praxis, DSST scores, subject- was 1.5, 1.3, 1.1 and 0.8 mm, respectively, compared rated sedation, and scopolamine effect on pupil size with 0.7,0.4,0.3 and 0.1 mm, respectively, for the young between the elderly and the young subjects (p > 0.05).

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Fig. 1: Time course of tropicamide effect on pupil diameter in elderly and young subjects (means and standard error bars).

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Sex and ethnic background did not significantly influ- pupil response to tropicamide in normal elderly and ence the scopolamine effects (p > 0.05). young subjects. The effect of a standard dose of tropicamide (20 gL, 0.01 %) on pupil diameter was measured Relation between peripheral using a computerized infrared pupillometer over a long and central cholinergic probes timeframe (240 minutes) in order to assess the tropicamide effect carefully. Only a few of the previous stud- The effect of scopolamine-induced impairment on word ies examining tropicamide have examined the time recall 90 minutes after administration for each individual course of the pupil-dilating effect beyond 60 minutes, as subject is illustrated in Fig. 4A. The effect of pilocarpine- the peak effect is reached 30 to 45 minutes after appli- induced net decrease in pupil diameter at the 125-minute cation of the drug. time point for each individual subject is presented in Fig. Our results indicate that tropicamide does not elicit 4B. All subjects demonstrated both concomitant memory possible functional changes in the cholinergic system in impairment due to scopolamine and decreased pupil the iris due to normal aging in 2 groups with known diameter due to pilocarpine administration, except sub- differences in central cholinergic function. This is in jects 1 and 11. However, there was no significant linear agreement with the other studies that did not detect dif- correlation between the 2 responses (r = 0.13, p = 0.60). ferences in central cholinergic function between patients with AD and normal age-matched controls.19 A Discussion previous study suggested that an increased pupil response to dilute tropicamide might be a diagnostic This study is the first to compare the time course of market for AD."8 That study found a mean 23% increase

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in pupil diameter from baseline in patients with AD number of studies that may have also detected cholin- compared with a 5% increase in healthy age-matched ergic differences between patients with AD and age- controls. However, studies attempting to replicate those matched controls, as those studies observed a signifi- results were unable to distinguish between the 2 cantly larger miotic response to pilocarpine in the groups, as the responses of both the control groups and patients with AD.2022 However, there were limitations patients with AD in later studies were similar to the in the methodology of these studies. These include a responses in patients with AD in the original study."9 As lack of placebo controls, a lack of a computerized pupil- a whole, these results indicate that it is still uncertain lometer, and the assessment of pupil diameter at a sin- whether pupillary response to tropicamide can distin- gle time point. This study improves on the previous guish between the different levels of central cholinergic studies by using a randomized placebo-controlled system activity in healthy elderly and young subjects, design and a computerized pupillometer to record and in healthy elderly subjects and patients with AD. more frequent measurements over a longer timeframe. Pilocarpine induced a net decrease in pupil diameter The tropicamide-pilocarpine challenge test was that was faster and greater in the elderly group than in designed to capitalize on the homeostatic cholinergic the young group. This result indicates that pilocarpine control of pupil size in such a way that changes in diam- may be a peripheral marker for the age-related decline eter would be maximized. It was expected that initial in central cholinergic function. This supports a limited dilation of the pupil with tropicamide would allow for

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Fig. 3: Time course of changes in word recall after scopolamine administration in elderly and young subjects, compared with placebo (means and standard error bars). *significant difference between the elderly and the young subjects.

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greater miotic changes due to administration of pilo- are not previously dilated with tropicamide. The maxi- carpine. While this study found that the magnitude of mum percentage decrease of pupil size from baseline the change was increased, the length of time required was mean 29.6% SEM 4.03% for pilocarpine challenge for contraction of the pupil was lengthened by the ini- alone (at 45 minutes after administration) compared tial dilation with tropicamide. Our group has also with a mean 29.7% SEM 3.06% for tropicamide-pilo- examined the possibility that pilocarpine administra- carpine challenge (at 85 minutes after administration). tion alone may be a peripheral marker for changes in When comparing individual responses, the net differ- central cholinergic function. To determine the useful- ence between the 2 procedures was mean 0.95% SEM ness of this procedure, we performed a challenge test 0.50%. A paired t-test showed that there was no signifi- with pilocarpine alone on 4 of the healthy elderly sub- cant difference in the pupil response to pilocarpine ver- jects and compared individual results from both meth- sus tropicamide followed by pilocarpine for elderly ods (data not published). The pilocarpine challenge test adults (p > 0.05). Furthermore, using only pilocarpine is employs the same techniques as as the tropica- advantageous because it requires much less time than mide-pilocarpine challenge test except that the pupils tropicamide and pilocarpine, and is a much simpler test

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Fig. 4: Individual changes in word recall after scopolamine administration (A) and pilocarpine effect on pupil diameter (B) in the elderly (I -10) and the young (I 1 -19) subjects. Subjects are arranged in decreasing order of impairment on word recall induced by scopolamine in A. The same arrangement of subjects is used in B.

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.- of central cholinergic function. This has led our group drugs. In light of this controversy, it is conceivable that to use pilocarpine alone in our current studies involving the use of another eye drop, with an established selec- elderly subjects and patients with AD. tivity, would result in a linear correlation between cen- As a whole, these results raise the question of why tral and peripheral probes. Second, the instrument used individuals with decreased central cholinergic function- to measure cognitive impairment, the Buschke Selective ing appear to show a hypersensitive response to pilo- Reminding Test, may not have been the ideal tool for carpine and not to tropicamide. Some possible explana- this purpose. The literature indicates variation in the tions include (1) differences in absorption due to differ- length of the word lists and the words themselves used ences in the molecular size and charge of the 2 drugs, (2) for different experimental designs. The word lists con- differences in mechanisms of action between agonists structed for this study may not have been sensitive to and antagonists, and (3) differences in receptor speci- the magnitude of central cognitive deficit. Furthermore, ficity of pilocarpine and tropicamide for various mus- the responses to the test may not be correlated to the carinic receptor subtypes and changes in various sub- peripheral probe, and another cognitive test may have type populations in aging. Alternative explanations been a more appropriate choice. A third possibility is include altered corneal permeability, dryness of eyes, simply that there is no relation between the central and tear production, blink rate, colour of the iris, and genet- peripheral cholinergic systems. However, in light of ic factors, all of which could affect the absorption of eye this study and others, it remains likely that a relation drops and thus the concentration of the 2 drugs at the between peripheral and central responses does exist, receptor level. but was not detected by the design of this study. For The elderly have been shown to be more sensitive to example, in the elderly group, 3 subjects had idiosyn- the cognitive effects of scopolamine than young indi- cratic responses: one had an early and accelerated viduals. It has been suggested that this may be due to response to pilocarpine; the second had an exaggerated central cholinergic system deficit associated with response to scopolamine (floor effect); and the third had aging.4 This study assessed the effects of scopolamine no response to scopolamine. Thus, the responses of 2 on memory in the elderly and the young groups and patients may be an artifact generated by using scopo- confirmed that the elderly group did indeed have an lamine as a model,,and the third is unexplained. When increased sensitivity to scopolamine, as demonstrated these subjects were removed from the analysis, we by a greater impairment in word recall. found a significant correlation between the responses (r No significant linear correlation was found between =0.66, p < 0.05). individual responses to scopolamine and the corre- Further evidence to support the link between central sponding decrease in pupil diameter in response to and peripheral cholinergic systems is the identification pilocarpine. There are several possible explanations as of AD pathology in the Edinger-Westphal nucleus (EW) to why we were unable to detect such a relation in this of patients with AD. The EW nucleus is known to have study. First, it may be explained by differences in recep- a role in controlling pupillary constriction and may tor pharmacology. The density and distribution of mus- mediate between the sympathetic and parasympathetic carinic receptor subtypes within different brain regions innervation of the iris musculature.35 The discovery of a are varied compared with each other and with the iris. peripheral marker for the central cholinergic system has In addition, changes in muscarinic receptor subtypes many possible implications in the area of cognitive peripherally in the iris may not mirror those occurring decline related to decreased central cholinergic func- centrally. There have not been any studies characteriz- tion. However, the potential for a pilocarpine eye drop ing changes in muscarinic receptors in the brain in test as a diagnostic tool for AD has yet to be deter- aging, although several such studies have been con- mined. We see our peripheral probe as an index of cen- ducted in patients with AD. As well, no published stud- tral cholinergic function and not as a potential diagnos- ies have examined the changes in muscarinic receptor tic tool for AD. One may argue that this distinction is subtypes in the iris in aging or AD. Similarly, scopo- merely one of semantics; however, the pathology of AD lamine and pilocarpine do not have the same mus- is not limited to cholinergic deficits, as evidence also carinic receptor subtype specificity. Interestingly, shows disruptions in neurochemical markers in the though, literature exists to support both as M2-selective noradrenergic, serotonergic, dopaminergic and hista- agents, although other studies list them as non-selective minergic systems.? Because of the heterogeneity of

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this disease, a probe of the central cholinergic system Kopell B. Physostigmine: improvement of long term memory may lack the specificity needed to be useful as a diag- process in normal humans. Science 1978;201:272-4. 8. Kopelman M, Corn T. Cholinergic "blockade" as a model for nostic test for AD. cholinergic depletion. Brain 1988;111:1079-110. We believe that this procedure could be better used to 9. Vannucchi M, Scali C, Kopf S, Pepeu G, Casamenti F. Selective monitor central cholinergic decline in the subset of AD muscarinic antagonists differentially affect in vivo acetylcholine release and memory performances of young and aged patients with severe cholinergic pathology. It may also rats. Neuroscience 1997;79:837-46. prove useful in predicting treatment response to cholin- 10. Araujo D, Lapchak P, Meaney M, Collier B, Quirion R. Effect of ergic therapies for AD. This includes treatments such as aging on nicotinic and muscarinic autoreceptor function in the the cholinesterase inhibitors, which increase the activity rat brain: relationship to presynaptic cholinergic markers and binding sites. J Neurosci 1990;10:3069-78. of the endogenous central cholinergic system.39 As well, 11. Aubert I, Rowe W, Meaney M, Gauthier S, Quirion R. it has been suggested that a minimal level of central Cholinergic markers in aged cognitively impaired Long-Evans cholinergic function must be present for effective treat- rats. Neurosci 1995;67:277-92. 12. Brown J, Taylor P, Hardman J, Limbird L, Molinoff P, Ruddon ment.1 Our probe may determine when there is enough R, Gilman A, editors. Goodman and Gilman's the pharmacological cholinergic damage that it is appropriate to discontinue basis of therapeutics. Toronto: Pergamon Press; 1998. p. 146-51. treatment. 13. Mewaldt S, Ghoneim M. The effects and interactions of scopo- From our results, we conclude lamine, physostigmine and methamphetamine on human that administration of memory. Pharmacol Biochem Behav 1979;10:205-10. dilute tropicamide eye drops is unable to detect poten- 14. Wisniewski K, Wisniewski H, Wen G. Occurrence of neu- tial age-related changes in the peripheral cholinergic ropathological changes and dementia of Alzheimer's disease in system of the iris. This is the first study to indicate that Down's syndrome. Ann Neurol 1985;17:278-82. 15. Harris W, Goodman R. Hyper-reactivity to atropine in Down's pilocarpine may detect changes in the peripheral syndrome. N Engl J Med 1968;270:407-10. cholinergic system due to normal aging. The result with 16. Sacks B, Smith S. People with Down's syndrome can be distin- pilocarpine may lead the way for the potential useful- guished on the basis of cholinergic dysfunction. J Neurol ness of this drug for tests in AD and other disorders of Neurosurg Psychiatry 1989;52:1294-5. 17. Berg J, Gillian-Brandon M, Kriman B. Atropine in mongolism. the central cholinergic system. Lancet 1959;2:441-2. 18. Scinto L, Daffner K, Dressler D, Ransil B, Rentz D, Weintraub Acknowledgements S, et al. A potential noninvasive neurobiological test for Alzheimer's disease. Science 1994;266:1051-4. 19. Kardon R. Drop the Alzheimer's drop test. Neurology 1998;50: Supported by a research grant from the Ontario Mental 588-91. Health Foundation (Grant #96-06). Jeanne Fourie was 20. Kaneyuki H, Mitsuno S, Nishida T, Yamada M. Enhanced supported by a University of miotic response to topical dilute pilocarpine in patients with Toronto Open Fellowship. Alzheimer's disease. Neurology 1998;50:802-4. Dr. Krista L. Lanctot was supported by the Alzheimer 21. Idiaquez J, Alvarez G, Villagra R, Martin R. Cholinergic super- Society of Canada Research Program. We are grateful to sensitivity of the iris in Alzheimer's disease. J Neurol Neurosurg Ms. Ruth Croxford for assistance in data analysis. Psychiatry 1994;57:1544-5. 22. Katz B, Rimmer S, Klauber M. Cholinergic supersensitivity of the iris sphincter in Alzheimer's disease. Ophthalmology 1988; References 96:134. 23. Folstein M, Folstein S, McHugh P. "Mini-mental state": A prac- 1. Drachman D, Reisberg B, editors. Alzheimer's disease: the stan- tical method for grading cognitive state of patients for the clin- dard reference. New York: The Free Press, 1983. p. 342-3. ician. J Psychiatry Res 1975;12:189-98. 2. Muir J, Everitt B, Robbins T. AMPA-induced excitotoxic lesions 24. Tombaugh T, McIntyre N. The Mini-Mental State Exam: a com- of the basal forebrain: a significant role for the cortical cholin- prehensive review. J Am Geriatr Soc 1992;40:922-35. ergic system in attentional function. J Neurosci 1994;14:2313-26. 25. Ware J, Sherbourne D. The MOS 36-item shortform health sur- 3. Dunnett S. The role and repair of forebrain cholinergic systems vey (SF-36): conceptual framework and item selection. Med in short-term memory. Studies using the delayed matching-to- Care 1992;30:473-83. position task in rats. Adv Neurol 1993;59:53-65. 26. Buschke H. Selective reminding for analysis of memory and 4. Squire L, Knowlton B, Musen G. The structure and organiza- learning. J Verb Learn Verb Behav 1973;12:543-50. tion of memory. Ann Rev Psychol 1993;44:453-95. 27. Sunderland T, Tariot P, Weingartner H, Murphy D, Newhouse 5. Drachman D, Leavitt J. Human memory and the cholinergic P, Mueller E. Pharmacological modeling of Alzheimer's dis- system. Arch Neurol 1974;30:113-21. ease. Prog Neuropsychopharmacol Biol Psychiatry 1986;10:599-610. 6. Bartus R. Evidence for a direct cholinergic involvement in the 28. Broks P, Preston G, Traub P, Poppleton P, Ward C, Stahl S. scopolamine-induced amnesia in monkeys: evidence of con- Modelling dementia: effects of scopolamine on memory and current administration of physostigmine and methylphenidate attention. Neuropsychologia 1988;26:685-700. with scopolamine. Pharmacol Biochem Behav 1978;9:833-6. 29. Molchan S, Martinez R, Hill J, Weingartner H, Thompson K, 7. Davis K, Mohs R, Tinklenberg J, Pfefferbaum A, Hollister L, Vitiello B. Increased cognitive sensitivity to scopolamine with age and a perspective on the scopolamine model. Brain Res Rev 35. Scinto LFM, Wu CK, Firla KM, Daffner KR, Saroff D, Geula C. 1992;17:215-26. Focal pathology in the Edinger-Westphal nucleus explains 30. Thorndike E, Lorge I. The teacher's word book of 30,000 words. pupillary hypersensitivity in Alzheimer's disease. Acta Neuro- New York: Columbia University Teacher's College Bureau of pathol 1999;97:557-64. Publication; 1944. 36. Herrmann N, Lanct6t KL. From transmitters to treatment: the 31. Wechsler D. A manual for the Wechsler adult intelligence scale. pharmacotherapy of behavioural disturbances in dementia. New York: Psychological Corporation; 1955. Can J Psychiatry 1997;42:51S-64S. 32. Sellers E, Naranjo C, Giles H, Frecker R, Beeching M. 37. Francis PT, Cross AJ, Bowen DM, Terry RD, Katsman R, Bick Intravenous diazepam and oral ethanol interaction. Clin KL, editors. Alzheimer's disease. New York: Raven Press, 1994. p. Pharmacol Ther 1980;28:638-45. 247-61. 33. Kaplan HL. Rapid computerized determination of pupil diam- 38. Reinikainen KJ, Soininen H, Riekkinen PJ. Neurotransmitter eter from imperfect video images. Problems of Drug Dependence, changes in Alzheimer's disease: implications to diagnostics 1994, Proceedings of the 56th Annual Scientific Meeting, The College and therapy. J Neurosci Res 1990;27:576-86. on Problems of Drug Dependence, Inc., Volume II. Rockville (MD): 39. Rogers S, Friedhoff L, the Donepezil Study Group. The efficacy National Institute on Drug Abuse; 1995. and safety of donepezil in patients with Alzheimer's disease: 34. Flicker C, Ferris F, Serby M. Hypersensitivity to scopolamine in results of a US multicentre, randomized, double-blind, place- the elderly. Psychopharmacology 1992;107:437-41. bo-controlled trial. Dementia 1996;7:293-303.

CANADIAN COLLEGE OF NEUROPSYCHOPHARMACOLOGY COLLEGE CANADIEN DE NEUROPSYCHOPHARMACOLOGIE Heinz Lehmann Award (Donated by Hoffmann-La Roche Limited) For Outstanding Contributions to Neuropsychopharmacology The Heinz Lehmann Award is designed to recognize outstanding contributions in the field of research in neuropsychopharmacology in Canada. The Award consists of $5000 and a suitably engraved plaque. The Heinz Lehmann Award shall be presented annually for work done primarily in Canada by Canadian scientists, unless there is, in the view of the Awards Committee, no qualified nominee. Eligibility shall include individuals from academic insti- tutions, foundations, governmental, industrial and research organizations, regardless of age or sex. The decision of the Awards Committee shall be based on originality and uniqueness of approach to clinical or laboratory research that had led to new, significant neuropsychopharmacological knowledge of concepts, or to the development of new therapeutic agents for the treatment of mental diseases. This should be evidenced by contributions based either on a specific piece of research or on a large body of neuropsychopharmacological research done over a period of years. The Awardee is expected to give a lecture based on his/her research at the Annual Meeting of the CCNP, and to contribute a manuscript, based on the lecture, to the CCNP official journal, the Journal of Psychiatry & Neuroscience, no later than six months after the lecture. Nomination for 2001 Heinz Lehmann Award The names of nominees should be received by Dr. Andrew Greenshaw by November 30th, 2000. Supporting documentation must be received by December 31st, 2000. For each award, this documentation shall consist of: 1. Six copies of a two-page summary prepared by the sponsor describing the nominee's work and its importance in further- ing the field of neuropsychopharmacology. 2. Six copies of manuscripts and reprints considered by the sponsor to be pertinent to the nomination and which highlight the nominee's work. 3. Six copies of the nominee's curriculum vitae and list of publications. 4. Six copies of a brief biographical sketch of the candidate prepared by the sponsor. Formal presentation of the Award will be made to the recipient during the Annual Meeting of the College. Note: The Heinz Lehmann Research Award may be given for basic research or clinical research in alternate years. The 2001 Award will be presented for basic research. Please send the name of the nominee and a short supporting letter to: Dr. Andrew J. Greenshaw President - CCNP University of Alberta, Department of Psychiatry lEl.01, 8440-112 St. Edmonton AB T6G 2B7 Deadline for receipt of initial nomination and short supporting letter is November 30, 2000.