p ~~AD______

EFFECT OF CHOLINERGIC PERTURBATIONS ONNEUROMOTOR-COGNITIVE PERFORMANCE

ANNUAL REPORT

N

E.H. ELLINWOOD, JR.A.M. NIKAIDO

S. GUNTAN D.G. HEATHERLY(NI K. NISHITA DT C

DTIC.! 1 ELECTE'SEPTEMBER 1, 1986 EECT

Supported by

U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMANDFort Detrick, Frederick, Maryland 21701-5012

Contract No. DAMD17-85-C-5207

Duke University Medical CenterDurham, North Carolina 27710

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The findings in this report are not to be construed as anofficial Department of the Army position unless so designated

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(if applicable)Duke University Medical CenteI

6c. ADDRESS (City, State, and ZIP Code) 7b. ADDRESS (City, State, and ZIP Code)

Durham, NC 27710

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63764A 63764D995 AB 19611. TITLE (Include Security Classification)(U) Effect of Cholinergic Perturbations on Neuromotor-Cognitive Performance

12. PERSONAL AUTHOR(S)

E.H. Ellinwood, Jr., A.M. Nikaido, S. Gunta, D.G. Heatherly, and K. Nishita13a. TYPE OF REPORT 13b. TIME COVERED 14. DATE OF REPORT (Year, Month, Day) 15. PAGE COUNT

AnnualI FROM 8/1/85 TO 7/31/86 1986 September 1 67

16. SUPPLEMENTARY NOTATION

17. COSATI CODES L SUBJECT TERMS '(C-ontiue -FIELD GROUP SUB-GROUP- Antimuscarinic pharmacokinetic-pharmacodynamic relationship

05 10 cognitive, neuromor, physiological measures, stress06 15 hormones . T If

19. ABSTRA r (Continue on reverse if necessary and identify by block-number)

,jtropine (d, 1-hyoscyamine), an antimuscarinic drug, is widely usedas an antidote for anticholinesterase poisoning, as pre-operativemedication and for the treatment of conditions, such as peptic ulcers and

Parkinsonism. A series of studies was designed to examine thepharmacokinetic-pharmacodynamic relationship of atropine for various

cognitive and neuromotor tasks and several physiological measures. In

the first study which has been completed, single doses of placebo and0.5, 1.0 and 2.0 mg of atropine were administered intramuscularly during

four test sessions to eight healthy young male volunteers accQrding to a

Latin square design balanced for order of dose administration.\ Thepsychomotor and cognitive tasks included standing steadiness, reactiontime, wheel tracking, visual tracking, divided attention and memory

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19. Abstract (continued)

tasks. Physiological processes were measured by assessing heart rate,blood pressure and body temperature. Plasma levels of stress hormones,ie. arginine vasopressin, adrenocorticotropic hormone, cortisol,catecholamines and growth hormone were also monitored. Thepharmacokinetics of atropine were best described by a simple one-compartment model with very rapid first order absorption. The meanelimination half-life, volume of distribution, and clearance we-e 2.04

(SD=0.23) hr, 132.85 (SD=18.92) L and 45.26 (SD=5.03) L/hr, respe.2tively.The atropine doses used in the present study significantly affected onlysitting heart rate and growth hormone levels. Preliminary resultsindicate a highly similar time course for the changes in plasma atropinelevels and plasma growth hormone values during the drug sessions. Theimplications of the findings from Experiment I were discussed andrecommendations for future experiments were presented.

Accession For

MTIC TAB

Unamounoed 0IFvUstiniOatlo-

Distribution/Availability Codes

Foreword

For the protection of human subjects the investigatorshave adhered to policies of applicable Federal Law 45CFR46.

m m I I iI

4

Table of Contents

Abstract . . . . . . . . . . . . . . . . . . . . . . . . 2Foreword . . . . . . . . . . . . . . . . . . . . . . . . 3

I. Introduction . . . . . . . . . . . . . . . . . . 5II. Experiment 1: Method . . . . . . . . . . . . . . 7III. Experiment 2: Method . . . . . . . . . . . . . . 13IV. Results . . . . . . . . . . . . . . . . . . .. . 13V. Discussion . . . . . . . . . . . . . . . . . . . . 38VI. Recommendations . . . . . . . . . . . . . . . . . 41

VII. Literature Cited . . . . . . . . . . . . . . . . . 43

Table 1. Testing Schedule for the Sensitivity Session . 9Table 2. Testing Schedule for the Drug Session . . . . 10Table 3. Average Pharmacokinetic Parameters for Atropine

Intramuscular Administration in Experiment 1 . 33Table 4. Atropine Plasma Levels for the Sensitivity

Sessions of Experiments 1 and 2 . . . . . . . 34Figure 2 . . . . . . . . . . . . . . . . . . . . . . . 15Figure 2 . .. a.. . ..... . . .ye . . . . . . 16Figure 3 . .. r.... . . . ... . ... ... . ... 17Figure t G . . . . . . . . .. . . . . . . . . . . . . . 18Figure 5 C . .a......... . . ... . . .s . . 19Figure 6 Asasfr.. onorn. omns..... 20Figure 7 . . . . . . . . . . . . . 21Figure 8 . . ... . . . . . . . . . . . . . . . . . . . 22Figure 9 . . . . . . . . . . . . . . . . . . . . . . . 23Figure 10 . . . . . . . . . . . . . . . . . . . . . . 24Figure 11 . . . . . . . . . . . . . . . . . . . . o.. . 26Figure 12 . . . . . . . . . . . . . . . . . . . . .. . 27Figure 13 . . . . . . .. . . . . . . . . . . . .. . . 28Figure 14 . . . . . . . . . . . . . . . . . . . . . . . 29Figure 15 . . . . . . . . . . . . . . . . o. . . . .. . 30Figure 16 . . . . . . . . . o. . . . . . . . . . . .. . 31Figure 17 . . . . . . . . . . . . . . . . . . . . . . . 35Figure 18 . . . . . . . . . . . . . . . . . . . . . . . 36Figure 19 . . . . . . . . . . . . . . . . . . . . . . .o 37Figure 20 . . . . . . . . . . . . . . . . .o . . . . 39APPENDIX A. Task Procedures and Analyses . .. . .. , 47APPENDIX B. Procedure for Plasma Collection of Atropine

and the Growth Hormones . . . o. . .. .. 62APPENDIX C. Radioimmunoassay of Atropine in Plsa .. 63APPENDIX D. Assays for Neuroendocrine hormones . . .. 65

5

I. Introduction

Atropine sulfate, a well-known anticholinergic agent, hasbeen used for many years in a broad range of clinicalapplications at moderate doses (1). At higher doses it is usedas an antidote for anticholinesterase organophosphorus poisons,including insecticides. Atropine has been distributed sincethe 1950's to military personnel as self-aid therapy when underchemical attack (2). In light of the highly stressful andcomplex nature of the tasks required of the modern armedforces, a detailed and comprehensive description of themagnitude and time-course of the effect of higher doses cfatropine on different cognitive and neuromotor capacities isimportant for a complete understanding of potential unwantedside effects. Moreover, Headley (2) lists a number ofnontherapeutic or even recreational situations in whichanticholinergic drugs may be misused by medically untrainedindividuals in the absence of organophosphorus nerve gas.

Previous studies have noted differential effects on theperipheral and central nervous systems for antimuscarinicdrugs. Peripheral effects include bradycardia and tachycardia,a reduction in systolic blood pressure, a decrease in theactivity of sweat glands and in saliva flow, an increase anddecrease in skin and oral temperature, pupil dilation andaccommodation paresis (2, 3, 4, 5, 6). While dose-dependenteffects have been reported for both peripheral and centralatropine effects (6), there have been frequent claims in theclinical literature that larger therapeutic doses, ranging from2 to 3 mg, produce significant peripheral anticholinergiceffects but rarely induce central nervous system (CNS) toxicity(7, 8, 9, 10). Many of the early studies, however, werecharacterized by low doses, small samples of young men,one-time testing and little statistical data analysis (2).

Recent studies have demonstrated that impairment of CNSfunctioning is detectable at much lower doses of atropine whenhighly sensitive measures of cognitive and psychomotorperformance are used (6, 11, 12). Seppala and Visakorpi (6)have conducted the most comprehensive comparison to date of theeffects of 0.85 and 1.7 mg of atropine on a large number ofautonomic and behavioral variables. The authors reported notonly dose-related effects but also differential impairment timecourses for the peripheral and central processes. Similarly,Shader and Greenblatt (10) noted a more prolonged duration ofeffect for cycloplegia and mydriasis than for sensoriumchanges. Other studies have demonstrated impairment onmathematical ability, simple reaction time, coordination,divided attention, flicker recognition, standing steadiness andthe use of common tools, especially at the higher doses (6, 13,14, 15).

6

The irfluence of anticheliner;ic Irugs or memory hasorcbably been more intensely examined than 2ny other conitiveor psychomotor process. Fairly coni3tent findings fromvarious studies indicate that the antichrlinergic drugs showmarked impairment in the acquisition or encoding of materialinto memory, less effct on memory retrieval, and, at moderatedoses, little effect on retrieval from memory of materiallearned prior to drug administration (11, 12, 16). Theseeffects are shared by other psychotropic drugs affecting memory(for example, diazepam) and thus may be non-specificallyrelated to the anticholinergic properties (11). There is alsoevidence that nonverbal visual memory is relatively spared (11,17) and that recent memory recall can be improved with cueing(16) which suggests that subjects can be trained to enhancetheir performance under the influence of anticholinergic drugs.

Although atropine has been used therapeutically for manyyears, there is relatively little information on therharmacokinetics of this drug and on the relationship betweenits pharmacokinetic and pharmacodynamic properties. A fewstudies have reported the excretion of 50% of a parenteral doseby the kidney after 4 hours (18) and complete excretion within24 hours (18, 19). Thus atropine has a rapid eliminationhalf-life of 2 to 4 hours (1, 20). Recently published dataalso indicate that atropine is extensively distributed andbound to tissue and that the distribution kinetics isdose-dependent (20, 21).

While a few studies have examined thepharmacokinetic-pharmacodynamic relationship for physiologicalvariables (20, 21), there has been no attempt to model thisimportant relationship for cognitive and psychomotorperformance. Adams et al. (21) found a high correlation (r =0.84) between heart rate and estimated tissue level ofatropine. Using an approach that fitted both pharmacokineticand pharmacodynamic data to an integrated kinetic-dynamicmodel, Hinderling et al. (20) determined that the atropineeffects on heart rate and saliva flow were related nonlinearlyto the drug concentration in the peripheral compartment.

The present study was an initial effort to describe thepharmacckinetic-oharmaccdyn.amic relaticnship of atrcoine forvaricus cognitive and neuromotor tasks. Specifically, theobjectives of the study included the determination of (1) thesensitivity of different performance tasks and physiologicalmeasures to cholinergic perturbation, (2) the time-course ofthe behavioral and physiological impairment, (3) theconcentration-response relationship across time for thedifferent task and physiological response measures, (4) thecomparative impairment on the cognitive, neuromotor andphysiological measures, and (5) individual variability inatropine responsivity. This report primarily presents theresults of an experiment which investigated the effects of 0.5,

7

1.0 and 2.0 mg doses of atropine on the cognitive, neuromotcrand physiological responses of 8 young, healthy men. We arecurrently completing a seccnd study which uses a similarexperimental protocol to administer 1.0, 2.0 and 4.0 mg of

atropine to another group cf 8 young males and have started tocollect data on a third sample of 8 young men using the threehigher atropine dosages.

II. Experiment 1: Method

Subjects

Eight normal male volunteers, who were between the agesof 21 and 29 years and within + 10% of ideal height-weight

ratios, participated in the study. Prior to starting theexperiment, the subjects were screened for any serious physicaland psychological problims and for any history of drug abusewith a physical and psychiatric evaluation, blood tests,

urinalysis, glaucoma tests and electrocardiogram. The bloodtests included chemistry (with hepatic) profile and completeblood count, and the urinalysis included a urine screen formarijuana. The Vocabulary, Block Design and Digit SymbolSubstitution subtests of the Wechsler Adult IntelligenceScale-Revised (22) and the Minnesota Multiphasic PersonalityInventory (MMPI) were also administered. Periodic urinescreens for marijuana were conducted prior to the scheduledtests session days. After completion of the study, thesubjects were given a post-study physical examination.Informed written consent was obtained from each subject afterthe purpose and procedure of the study as well as the potentialphysiological and psychological effects of atropine wereexplained.

None of the participants had any serious physical ormental disorder or were taking anticholinergic or CNS-activedrugs during their participation in the study. All of thesubjects reported that they were nonsmokers and did not drinkalcohol excessively. The mean scaled scores for theVocabulary, Block Design and Digit Symbol Substitution subtestswere 12.9 (± 1.6), 12.4 (± 1.7) and 11.7 (± 3.2), respectively.

~J~mnce Taks

The effects of atropine over time on fifteen cognitiveand neuromotor tasks were measured in the present study.Detailed descriptions of the procedures, response measurementsand data analysis programs for the tasks are presented inAppendix A. Different tasks were combined to create three testbatteries which required varying periods of time to complete.The ultra-short (briefest) battery consisted of just one task,continuous subcritical tracking (SCT). The medium batteryincluded four tests presented in the following order: standingsteadiness in the eyes open condition, digit symbol

substitution (DSS), SCT and abbreviated digit symbc! memorytasks. Tr the long battery, the sequence of the thirteen taskswas standing steadiness with visual feedback and in eyes openand eyes closed conditions, DSS, SCT, continuous perfcrmance,divided attention, digit symbol substitution with short terr-memory test, digit-keypad reaction time, sudden displacementeye saccade, central pendulum eye trackirg, lateral pendulumeye tracking and memory encoding. In addition to the threetest batteries, a concrete noun memory task was used to assessthe atropine effect on verbal memory processes.

Several physiological parameters were measuredperiodically during the sensitivity and test sessions. Heartrate, oral temperature and blood pressure were takenapproximately every 15 minutes (see Tables 1 and 2 for actusltimes of measurement). Heart rate was monitored continouslyusing a 7830A Hewlett Packard heart monitor equipped wit h abeat-to-beat audio output for easy identification of the onsetof bradycardia or tachycardia. The beats per minute (bpm)heart rate was determined by counting the auditory beats for aduration of 30 seconds and multiplying by 2. Heart rate wastaken while the subject was sitting and standing and documentedfor subsequent time course analysis. This method wascross-checked with the measurement of heart rate obtained bypalpitation to ensure proper equipment functioning. Inaddition, ... polygr:ph recordings were made periodicallyduring the test sessions and continously during any periods oftachycardla for detection and identification of arrhythmias.

Diastolic and systolic blood pressure were bothdetermined manually with a blood pressure cuff and stethoscope.Blood pressure was taken by all experimenters durinq practicesessions to ensure that interexperimenter variations were keptto a minimum. Temperatures, in degrees Fahrenheit, were takenorally using a digital converter and a test probe. The testprobes were calibrated with a glass Celsius laboratorythermometer throughout the range of body temperatures and werefound to vary within one degree Celsius. Room temperature wasalso measured and documented during the test session to ensurethat fluctuations in body temperature were not caused bychanrges in the temperature of the room atmosphere. Test roomtemperature was maintained within a range of 70 to 75 degreesFahrenheit.

fleuroge~orn-e Measures

In addition to the blood samples for the atropine assay,plasma samples were drawn during the sensitivity session forthe measurement of vasopresqi" (AVP), adrenor-;ticotropichormone (ACTH), cortisol and the catecholamines prior to and at40, 100, 160 and 400 min following drug administration. During

Table 1

Testing Schedule for the Sensitivity Session

Time (min) Procedurea, b

-90 BreakfastCatheter insertion, attachment of electrodes

-30 BP, HR, temperature, medium task batteryBlood sample

0 Atropine (0.25 mg) injectiun2 Medium task battery

20 BP, HR, temperature, medium task batterySBlood sample

Atropine (0.25 mg) injection42 BP, HR, temperature, medium task battery60 BP, HR, temperature, medium task battery80 BP, HR, temperature, medium task battery100 Blood sample

Atropine, (1.0 mg) injection102 BP, HR, temperature, medium task battery120 BP, HR, temperature, medium task battery140 BP, HR, temperature, medium task battery160 Blood sample

BP, HR, temperature, medium task battery180 BP, HR, temoerature, medium task battery200 BP, HR, temperature, medium task battery220 BP, HR, temperature223 Lunch280 BP, HR, temperature, medium task battery340 BP, HR, tcmperature, medium task battery400 Blood sample

BP, HR, temperature, medium task battery

a Physiological measures included recording of blood pressure

(BP), heart rate (HR) and oral temperture.

b The medium task battery is described in the text.

10

Table 2

Testing Schedule for the Drue Session

Time (min) Procedurea

-105 Breakfast, catheter insertion, attachment ofelectrodes

- 60 Long task battery, blood sample, BP, HRtemperature, Concrete Noun Learning Trial 1

0 Drug administration10 Blood sample, ultra-short battery15 Medium task battery20 Blood sample, BP, HR, temperature after sway task35 Medium task battery40 Blood sample, BP, HR, temperature after sway task55 Medium task battery60 Blood sample, BP, HR, temperature after sway task75 Medium task battery80 Blood sample, BP, HR, temperature after sway task95 Medium task battery100 Blood sample, BP, HR, temperature after sway task114 Concrete Noun Recall Trial 1115 Medium task battery120 Blood sample, BP, HR, temperature after sway task133 Concrete Noun Learning Trial 2135 Long task battery160 Blood sample, BP, HR, temperature165 Long task battery190 Blood sample, BP, HR, temperature195 POMS210 Medium task battery228 Concrete Noun Recall Trial 2, BP, HR, temperature230 Long task battery255 Blood sample, BP, HR, temperature260 Lunch290 BP, HR, temperature330 BP, HR, temperature, medium task battery350 Long task battery375 Blood sample, BP, HR, temperature380 Medium task battery400 Long task battery425 Blood sample, BP, HR, temperature430 Medium task battery447 BP, HR, temperature450 Concrete Noun Final Recall and Recognition Tests

a Physiological measures and long task battery are explained in

Table 1 and the text.

11

each test session AVP, ACTH, cortisol and catecholamine plasmasamples were collected or'or to and at 35 and 75 min after drug_njectior. Additional measurements of AVP and ACT! were takenat 135 min post drug administration. Assessment of the 5rcwth1hc.-mne levels were conducted at the same testiv time points

as for the determination of atropine drug concentration. Thepurpose for monitoring the potential changes in theseneuroendocrine variables was to explore the questicn of (1)whether atropine alters the plasma concentration of stresshormones and (2) whether the impairment effects of atropinenteracts with changes in the stress hormone levels. A

detailed description of the blood sample collecticn procedurefor atropine and the different hormones is contained inAppendix B.

Procedure

Initially subjects were trained on the behavicral tas:sjurina three or four 2-hour sessions until they shewed noevidence of substantial improvement in their oerformarcescores. After the training phase, a sensitivity session .asconducted to screen for individuals who might be extremelysensitive to the effects of atropine. Each subject thencompleted four drug sessions which were scheduled at 2-weekintervals.

Except for the differences meitioned below, the sameexperimental protocol was used for the sensitivity and testsessions. The subjects were instructed to sleep their normalnumber of hours the night before the test day and to consume noalcohol or drugs during the previous 24 hours. They reoortedto the laboratory at 07:30 and were given a light breakfast.An obturated intravenous teflon catheter for drawing bloodsamples was inserted in the forearm. Baseline measurements ofthe performance and physiological measures and predrug plasmasamples were then collected prior to the administration ofatropine.

Approximately 1 hr after breakfast, a single injection ofthe drug was administered. During the sensitivity session,additional injections were given at UQ min and 1 hr and LIO -inafter the init:al injection. The testing schedules for thesersitivity and drug sessions are descrited in Tables 1 and 2,respectively, which list the times at which blood samcles andassessments cf the performance anc physiological measures weretaken following drug administration. All subjects ate astandard lunch, consisting of a meat (usually turkey) sandwichand noncaffeinated soda.

Druz

Individual syringes, containing atropine sulfate orplacebo, were prepared by the Duke University Medical Center

12

Pharmacy for the sensitivity and tests sessions. Theconcentraticn of atroone was 1.0 mg/ml. For each sensitiv'tysession, three separate doses of atropine were injectedintramuscularly (I,M.) in the ventral aspect of the upperthigh. The dosing schedule was 0.25 mg at 0 min, follcwed ty0.25 mg at 40 min and 1.0 mg at 1 hr and 40 minutes after theinitial injection.

During each of the four drug sessions, a single dose ofplacebo or one of three doses of atropine, 0.5, 1.0 or 2.0 mw,was given to each subject according to a Latin square designbalance, for order of dose administration. Placebo consistedof 2 ml of bacteriostatic water. Similarly, the atropine doseswere diluted with sufficient quantities of bacteriostatic waterto produce a volume of 2 ml. Using a double-blind procedure,the drug was injected i.M. in the same location as for thesensitivity session.

Atropine assay. Our objective was to set up a sensitiveassay procedure for the detection of atropine in plasma thatwould allow us to delineate more definitively thepharmacokinetic parameters of atropine in humans. it tookabout 6 weeks to modify the radioimmunoassay (RIA) method ofthe Walter Reed Army Institute to a disequilibrium RIA methodin order to achieve greater sensitivity. The latter techniqueis described in greater detail in Appendix C.

Neuroinea ing horoe _". Different assay procedureswere used to determine the levels of AVP, ACTH, cortisol andthe cathecolamines. Each assay method is explained in AppendixD.

The plasma atropine concentration data for individualsubjects were analyzed using both compartmental andnon-compartmental methods. For the compartmental analysis,initial estimates of the parameters and compartment-!configuration were evaluated using ESTRIP (23). Parametervalues were further defined using SAS (2'4) Total Dlasmaclearance (CLp), maximum concentration (Cnax), time to reachconcentration maximum (tmax), volume of distributicn (V), meanabsorption time (MAT), absorption rate constant (ka),absorption half-life (tl/2,ka), elimination rate constant (K)and elimination half-life (tl/2,B or tl/2,K) were calculated inthe usual manner (22).

The units of measurement for the performance tasks areexplained in Appendix A. Due to the number of comparisons,Bonferroni t-tests were used to compare statistically theperformance of atropine vs. placebo conditions at each testing

13

time point. The t-tests were conducted to determine whetherany of the three atropine dosages significantly impaired thebehavioral or physiological scores in comparison to the placebccondition.

III. Experiment 2: Method

Subjects

Eight young males with characteristics similar to thoseof the subjects in Experiment 1 have been recruited for thesecond study. As in the Experiment 1, all the subjects weregiven various screening tests and written informed consent wasobtained. Eight subjects have completed the sensitivitysessions for Experiment 2. Three of the volunteers have alsodone all four of the drug sessions, while the remaining fivemen are currently at varying stages of finishing theirparticipation in the study. Finally, we have started torecruit and collect data on a third group of 8 young men whowill also be participating in Experiment 2. Thus, the totalsample size for the second experiment will be 16 subjects.

frocedure

Basically the performance tasks and physiologicalmeasures as well as the experimental protocol were identicalfor Experiments 1 and 2. The major change in the second studyinvolved the doses administered during the sensitivity and testsessions. For the sensitivity session of Experiment 2, theinitial dose was 0.5 mg at 0 min, followed by 1.0 mg at 40 minand 1.5 mg at 1 hr and 40 min after the first injection. Theatropine doses for the drug sessions were 1.0, 2.0 and U.0 nrg.In addition, a computerized method of recording heart rate wasintroduced in the second experiment (see Appendix A for adescription of the procedure). The only results from thesecond study that will be presented are the pharmacokineticanalyses of the atropine drug levels for the sensitivitysession since this is the only part of the experiment that hasbeen completed by the 8 subjects.

IV. Results

C ~ iv e Ta_.ks

In general, the dose response curves for performance onthe cogniti'e and neuromotor tasks showed little drug effect atthe doses adilinistered in the first study, that is, 0.5, 1.0and 2.0 mg. The results of the t-tests indicate that therewere no significant differences between the scores of the threeatropine doses and placebo for any of the behavioral tests. Inorder to provide a more meaningful perspective for theinterpretation of the trends noted, the atropine effects on the

14

SCT, DSS and sway with eyes open tasks for 2.0 mg were comparedto the effects of 1.0 and 2.0 rrg of alprazolam (Xanax), astandard anxiolytic drug commonly used with clinicalpopulations (Figures 1 to 3). The 1.0 mg dose of alorazolam isthe highest therapeutic dosage usually prescribed, and 2.0 mgis two times this hirhest recommended prescribed dose.Similarly, the usual clinical pre-anesthetic dose of atropireis 0.8 mg, and 2 mg would be approximately twice that dose. Adescription of the experimental procedure and results of thestudy that generated the alprazolam data have been presentedpreviously by Ellinwood et al. (26).

As clearly shown in Figures 1, 2 and 3, the magnitude ofthe effect of 2 mg of atropine on the SCT, body sway with eyesopen and DSS tasks, respectively, is much less than that foreither dose of alprazolam. Since the atropine and alprazolamstudies were accomplished with different subjects and thesample size of the atropine study was relatively small, directstatistical comparisons between the alprazolam 2nd atropineresults were not conducted for Experiment 1. Wihen we haveadded the data for the remaining 16 subjects tested at thehigher L.O mg dose to the present atropine subject cohort, wewill be able to conduct more systematic statistical analysesand comparisonS of the atropine effects with those of a varietyof other psychotropic drugs. This approach will provide a moreappropriate perspective for the evaluation of the magnitude ofthe atropine impairment.

Figures 4 through 9 illustrate the time course of theperformance of the 8 subjects in Experiment 1 on a variety ofcognitive and neuromotor tasks after I.M. injection of thethree atropine doses and placebo but without the comparisondrug, alprazolam. Similar impairment profiles over time wereobserved for the remaining tasks that are not shown. Althoughnone of the neuromotor or cognitive effects was significantlyaffected by the atropine treatment, there were indications ofdose-related trends in some cases. A currently unexplainedphenomenon is the occurrence of a second impairment peak at 210min post drug administration for the SCT task (Figure 4). Assoon as the data for the 1.0, 2.0, and 4.0 mg study (Experiment2) have been collected, the dose-response andconcentration-response relationships for the cognitive andneuromotor performance tasks will be re-analyzed and reported.In addition, the question of whether the late irnairment peakobserved for the 2.0 mg dose on the SCT task is related eitherto atropine pharmacokinetic or pharmacodynamic processes or tosome other random variable will be examined.

There was the expected dose-dependent increase in sittingheart rate for all the atropine doses, which lasted forapproximately 2 hr (Figure 10). There was very little change

15

200

160

ME 140-AN

Su 1208CRIT 100ICAL so

TR

CK 60-\

40-

I I

ii !

20 -

0 100 200 300 O0 Soo 600 00

KIN POST AOINISTRATION

Figure I. Performance on the SCT task for atropine (Experiment1) and alprazolam. The atropine dose was 2.0 mg (.) and thealprazolam doses were 1.0 mg (.) and 2.0 mg (J). The placebosessions for the atropine and alprazolam studies are indicated by.1 and Z, respectively. Performance is represented as unitchanges from the predrug score (delta score). The units ofmeasurement for the task are described in Appendix A. A smoothspline fitting procedure was used to generate the curves whichconnect sequential time points. Zero minutes represent thepredrug measurement of the task. (The alprazolam curves aretaken from Ellinwood et al. (25, 26)).

16

200

175

150

ISO

ME 125AN

Sw ooAY

EY 7SES

0P soEN

, ',, rF'0 25-X, , _ 7 '" . ,W '4

-. ".,, ".?"/ "

-25- .

-50i i . . . I " " i . . I " p -

a 60 120 10 240 300 360 120

MIN POST OMINISTRATION

Figure 2. Performance on the sway with eyes open task foratropine (Experiment 1) and alprazolam. The atropine dose was2.0 mg (A ) and the alprazolam dose was 1.0 mg (,). The placebosessions for the atropine and alprazolam studies are indicated by1 and .2, respectively. Performance is represented as deltascores. The delta score, units of measurement and curves aredescribed in Figure 1. (The alprazolam curve is taken fromEllinwood et al. (25)).

17

50-

40

35NEA

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0

G 25 :T

S 20-YM

L IL

P%0 1o - \ A

E , \,, 1'"R 4

-10

O, I

0 100 208 300 400 Soo 600 700

MIN POST AMINISTRRTION

Figure 3. Performance on the DSS task for atropine (Experimet1) and alprazolam. The atropine dose was 2.0 mg (') and thealprazolam doses were 1.0 mg (3) and 2.0 mg (r). The placebc

sessions for the atropine and alprazclar studies are indicated by1 and ., respectively. Performance is represented as deltascores. The delta score, units of measurement and curves are

explained in Figure 1. (The alprazolam curve is taken fromEllinwood et al. (25, 26)).

18

25 0 -

225

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II II 7.5 I /I\

AL "w 4 ,

A xC 2.5-S- ~ 'KN0 0.0

-25.S

0 60 120 * 180' 240 300 360 420

MIN POST ADMINISTRATION

Figure 4I. Performance on the SCT task after administration ofplacebo (pl) and 0.5 (JL), 1.0 Q!) and 2.0 (fl) mg of atropine forthe eight subjects in Experiment 1. Performance is representedas delta scores. The delta scores, units of measurement for the

task and the curves are the same as in Figure 1.

9 19

30-

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R MIA 1L • ' ; k " \

P

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Figure 5. Performance on the DSS task following I.M. injectionof placebo (.E) and 0.5 (L), 1 .0 (M) and 2.0 (B) mg of atropinefor the eight subjects in Experiment 1. Performance isrepresented as delta scores. The delta scores, units ofmeasurement for the task and the curves are described in "Figure

1.u-

20

12-

10-

MEA 8-N

D

ST bANCE

FR0N

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EN 2R

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0 so 100 ISO 200 250 300 350 400

MIN POST NPUNISThRTION

Figure 6. Performance on the sway with visual feedback task forplacebo (P-) and 0. 5 (L) , 1 .0 (M) and 2. 0 (B) mg of atrop ine f orthe eight subjects in Experiment 1 . Performance is representedas delta scores.An explanation of the delta scores, units ofmeasurement for the task and the curves are presented in Figure1.

2 1M M M E

30-

27-

2q

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0RY

0VERA 12-LL

0 9

ER

6-

-- t

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0 so 00 ISO 200 2S0 300 350 400

PIM POST APUNISTRATION

Figure 7. Performance on the recall test of the digit symbtolsubstitution with short term memory task during placebo (a) and0.5 (L~), 1 .0 (11) and 2.0 (11) mg atropine drug sessions for theeight subjects in Experiment 1. Performance is represented asdelta scores.The delta scores, units of measurement for the taskand the curves are described in Figure 1.

145

130-

MEA 115SN

T

KE 85.Y

0

REACT 55I

T 40-

E

10 - - - -

0 50 100 IS0 200 250 300 350 400

MIN POST ADMINISTRATION

Figure 8. Performance on the digit-keypad reaction time task forthe placebo (1!) and atropine 0.5 (L), 1.0 (M) and 2.0 (Ei) mg.treatment for the eight subjects in Experiment 1. Performance isrepresented as delta scores.The delta scores, units ofmeasurement for the task and the curves are explained in Figure

23

250

225

M 200E

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S 17SACCAo 150IS

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. .. . . . . . . . . ..I . . . . . I . . . . . I . . . . . . . . ..-. .

0 SO 100 ISO 200 250 300 350 00

MIN POST AMINISTRATION

Figure 9. Performance on the sudden displacement eye saccadetask after I.M. injection of placebo (F) and 0.5 (W), 1.0 (M) and2.0 (E) mg of atropine for the eight subjects in Experiment 1.Performance is represented as delta scores.A description of thedelta scores, units of measurement for the task and the curvesare presented in Figure 1.

24

30-

t

E I

. I I

E I

L

ft 0k ll

ft - ,, . '

-0 ,.

I '5'

-i " ' ,

- L

OUW PW5 \W0/T!W

Figure 10. Sitting heart rate measurements (beats per min)during the placebo (E) and 0.5 WL), 1.0 () and 2.0 () mgatropin~e test sessions for the eight subjects in Experiment 1.The delta scores and the curves are described in Figure 1. Theresponse measure is explained in the text.

25

in systolic or diastolic blood pressure, and oral temperaturedid not change significantly over time. Since there was nosignificant effect on the cognitive-neuromotor behaviors,sitting heart rate was selected as the pharmacodynamic variablewhich was used to explore the relationship between thepharmacokinetic and pharmacodynamic properties of atropine.This analysis is discussed further in a later section.Pharmacokinetic-pharmacodynamic modeling of the cognitive andneuromotor behaviors will be conducted as soon as thebehavioral and drug concentration data for Experiment 2 becomeavailable and are analyzed.

Neurcendocrine fhanges

Stress hormone levels were also measured pericdicallyfollowing the various atropine doses during Experiment 1.Plasma AVP, ACTE and cortisol show few, if any, systematicchanges across time (Figures 11 to 13). Plasma catecholamineassays have yet to be completed. On the other hand, based cnthe preliminary results of 4 subjects in Experiment 1, growthhormone appears to demonstrate a time course of responsivitythat is very similar to the onset and offset of the atropineplasma levels during the drug treatment sessions (Figure 14).The parallel ascending and descending trends of the changes inatropine drug concentration and growth hormone values acrosstime is further depicted as a hysteresis curve in Figure 15.

Growth hormone secretion is under the control ofhypothalamic influences, particularly somatostatin and growthhormone releasing hormone (GHRH). These hormones are underdirect neurotransmitter control, and different stimuli activategrowth hormone secretion by distinct central mechanisms. Forexample, stress, physical and psychological, increases growthhormone secretion. Anticholinergic drugs, such as atropine,produce an inhibitory effect on growth hormone secretion whenit is stimulated by stress (as in our experimental testingsituation) and sleep, but not when stimulted by hypcglycemia.The atropine-growth hormone relationship is under furtherstudy. We will be sampling more frequently for this and otherhormones and examining more closely in future experiments therelationship of stress hormones to performance and to theeffects of atropine on behavioral and physiclogical variables.

Lra.P In:e Fbrara_q Ir-t ic

Atropine plasma concentration values for Experiment 1have been determined for six subjects at the 1 mg dose and forfour subjects at the 2 mg dose. Mean drug concentration levelsare plotted as a function of time following I.M. administrationof 0.65 mg and 1.30 mg of atropine as base (equivalent to 1.0mg and 2.0 mg of atropine sulfate monohydrate) in Figure 16.Application of the F-test indicated that plasma atropineconcentration vs. time profiles for individual subjects were

26

30-

25-

LAS 20

P4A

S

P i5RESS

L IC0

VE

L.I

% 0 4 0 8 0 2 4 6

th sam meho as decrbe in0 Figur 1.0 16

27

20-

is-

p

AS

a 10-CTN

E

EL

0 20 40 60 so 100 120 140 160

MUN POST A"1ISTATXON

Figure 12. Plasma ACTH levels for the placebo QE) and 0.5 WL1 ,1.0 (h) and 2.0 (hl) mg doses of atropine during the test sessionsof the eight subjects in the first study prior to and at 35, 75and 135 min following drug injection. An explanation of thecurves Is presented in Figure 1.

28

40-

30-

PLASM

C V . . .o

T _+--- - -

S0L

LEvEL

10.

0 20 40 60 s0 100 120 140 160

IM POST MWNISTRRTI0N

Figure 13. Plasma cortisol levels for the placebo (f) and 0.5(L), 1.0 (M) and 2.0 (fl) mg doses of atropine prior to and at 35and 75 min after drug administration for the test sessions of theeight subjects in Experiment 1. The curves are described inFigure 1.

29

9-

ATR0P!N 7E

C0NCENTRA 5.TI

0 - E%N

A qN0

EF 3-FECTp 2-

ERCEN 4T

0 2 3 S

MMS POST AO LNISTATION

Figure 14. The time course of mean plasma atropine concentration(closed circle) and mean -effect percent (open square) followingI.M. injection of 2.0 mg of atropine sulfate for four subjects inExperiment 1. Mean effect percent (E%) represents the reductionof the plasma growth hormone levels over time. The times ofmeasurement shown are 0 hr (predrug) and 0.167, 0.333, 0.667,1.0, 2.0, 3.167, 4.25, and 6.25 hr after drug administration.Construction of the curves are explained in Figure 1.

30

90-

0.667 hr

80-

70. 1.0 hr

60- 0.333 hr

EF

E SOCT

p 2.0 hrER qOCENT

300.167 hr

3.167 hr21)

10- 4.25 hr

6.25 hr

0 hr

o q 5 6 7 8 9

MEAN ATROPINE PLASiA CONCENTRATION (NG/ML)

Figure 15. A clockwise hysteresis loop showing the relaticnshipbetween the mean plasma atropine concentration and effect percentfor the data shown in Figure 14. A smooth spline fittingprocedure was used to draw the curve and arrows indicate thedirection of the consecutive time points at which plasma samplesfor the atropine and growth hormone assays were drawn. Effectpercent is described in Figure 14.

31

9

MEAN

ATR

0p1 6NE

LASMA 2 2mg

T

0NCENTR I IgA

Io 2N

NG

ML

0 2 3 57

9OJRS POST WINISTRRTION

Figure 16. The time courses of the mean plasma atropineconcentration following I.M. injection of 1.0 (closed circle) and2.0 (open square) mg of atropine sulfate. The 1.0 and 2.0 mgcurves are based on the analyses of data for six and foursubjects, respectively, from Experiment 1. The spline fittedcurves are described in Figure 1.

32

best described by a cre-compartment model with very rapid firstorder absorption. Fased on the estimates calculated for theindividuals, preliminary average values for the kineticparameters are summarized in Table 3. The mean tl/2,k ofatropine in plasma was 2.04 hr. The mean observed Cmax andtmax values for the 1.0 mg and 2.0 mg dose levels were LI.91ng/ml at 0.333 hr and 8.67 ng/ml at 0.667 hr, respectively.The Cmax determined from the kinetic parameters in Table 3 forthe 1.0 mg and 2.0 mg doses were 5.29 and 8.41 ng/ml,respectively. The average value of the apparent volume ofdistribution (V/F) was 132.85 L. The mean value of themodel-estimated plasma clearance was 45.16/hr. The area underthe atropine plasma vs. time curves (AUC), as calculated by themodel independent method, increased linearly (17.58± 3.11ng hr/ml for the 1 mg dose and 30.96 ± 5.71 ng'hr/ml for the2.0 mg).

S ly~~ LgiQn w ith miili lD 4r g ;r _QrcTable 4 lists the mean plasma atropine concentrations for thesensitivity sessions of the eight subjects (ID = A to H) in thefirst study. Atropine was administered I.M. as follows: 0.25mg at 0.0 min (09:00), followed by 0.25 mg at 40 min (09:40)and 1.0 mg at 1 hr and 40 min (10:40) after the first dose. Inaddition, the mean drug levels for the sensitivity sessions ofthe eight subjects (ID = I to P) in the second study arepresented in the same table. The doses used were 0.5 mg at 0.0min (09:00), followed by 1.0 mg at 40 min (09:40) and 1.5 mg at1 hr and 40 min (10:40) after the initial injection. Theobserved mean atropine concentration values in Table 4 andsimulated curves based on the estimated pharmacokineticparameters from Table 3 are presented for the sensitivitysession data of Experiments 1 and 2 in Figures 17A and 17B,respectively.

As discussed earlier, consideration of thepharmacckinetic-pharmacodynamic relationship for atropine willbe restricted to the sitting heart rate measure in the firststudy, since this physiological variable was the orimary onethat showed any Zianificant impairment for the atrcoine dcsesused. Average change in heart rate and mean olasma atropineconcentration are plotted as a function of time for the 1.0 and2.0 mg doses in Figures 18A and 18P, respectively. Absoipticnrate constants based on heart rite data (tl/2,ka=2.16 hr- ) andplasma values (tl/2,ka=2.93 hr- ) were calculated independentlyfor the 2.0 mg dose and were almost identical (Figure 18B).

A comparison of the relationship between heart rate andatropine plasma concentration during the terminal phase of thecurve in Figure 18B is illustrated in Figure 19. Mean changesin heart rate for the final four testing time points wererepresented as a function of drug levels and the linear

13

Table 3

Average Pharmacokinetic Parametersa for Atropine

Following Intramuscular Administration in Experiment 1

Pharmacokinetic Parameters Meana ± SD

ka (hr-1) 2.93 ± 0.15tl/2,ka (hr) 0.238 + 0.03

K (hr- I 0.34 + 0.05t1/2,K (hr) 2.04 + 0.23

MRT (hr) 2.96 + 0.41MAT (hr) 0.38 ± 0.02tmax (1 mg dose) (hr) 0.333 ± 0.18tmax (2 mg dose) (hr) 0.667 + 0.22Cmax (1 mg dose) (ng/ml) 8.67 ± 1.32Crmax (2 mg dose) (ng!rl) 4.91 ± 0.76CLp/F (L/hr) 45.16 + 5.03V/F (L) 132.85 ± 18.92

a The Harmonic mean was calculated for t/2,ka and

ka = absorption rate constanttI/2,ka = absorption elimination half-life

K = elimination rate constantti/2,K = elimination half-life

MRT = Mean residence timeMAT = Mean absorption timeCmax maximum plasma concentrationtmax Time to reach CmaxCLp = Total plasma clearanceF = systemic availabilityV = Volume of distribution

34

Table 4

Atropine Plasma Levels for the Sensitivity

Sessions of Experiments 1 and 2

Plasma Atropine Concentration (ng/ml)

Time (h) / 'Plasma Sample Number

Subject -- - - - - - - - - - - - - - - - - - - - - - - - - - - -ID 0.0/01 0.667/02 1.667/03 2.667/04 7.0/05

YA01 0.0 1.38 2.32 6.61 1.49

fB01 0.0 1.62 2.57 7.58 1.92YCO1 0.0 1.02 2.01 5.24 0.95YD01 0.0 1.40 2.20 7.01 1.70YE01 0.0 2.01 3.12 8.34 2.45YF01 0.0 1.42 2.15 6.42 1.17YGO1 0.0 0.83 1.48 4.96 0.88YHO1 0.0 1.36 2.25 7.49 1.48

Mean 0.0 1.38 2.26 6.71 1.51SD 0.0 0.36 0.47 1.16 0.52

YI01 0.0 2.76 6.88 12.40 2.91YJ01 0.0 3.31 8.02 14.95 4.12YK01 0.0 2.70 6.56 12.88 2.68YL01 0.0 1.92 4.95 10.33 1.87YM01 0.0 2.05 5.83 11.86 2.42YN01 0.0 1.82 6.12 12.55 2.95YO01 0.0 4.55 8.84 13.90 4.42YPO1 0.0 3.05 7.86 10.86 1.97

Mean 0.0 2.77 6.88 12.47 2.92SD 0.0 0.90 1.29 1.51 0.93

35

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Figure 19. The best fitting linear regression model describingthe relationship between mean change in sitting heart rate andatropine plasma concentration for the final four testing timepoints of Figure 18B after I.M. injection of 2.0 mg. The unitsof measurement and delta scores are explained in Figure 1 and thetext. The equation for this line is shown on the plot. HR=meanheart rate expressed as bpm. A=mean atropine plasmaconcentration.

reressicn rocedure was used to deter ine tre best fitti",throuh these points. "he eouaticn of the least sauares

repression line is as fellows:

HP = -3.20 + 3.325 C

where HR is the average chanae in heart rate (bcm.) and C is theaverage plasma atropine concentration (ng/ml). Thecorrelation (r=0.992, p<0.01) between the heart rate and druglevel means suggests a very high deFree of correspondencetetween these two measures. Finally, Figures 20A and 20Bindicate a clockwise hysteresis relationship between rean heartrate change and average plasma atrocine concentration forsuccessive testing time intervals for 1.0 and 2.0 7q,respectively.

V. Discussion

The oharmacokinetic results of Experiment 1 indcicEte thatatropine is absorbed rapidly following I.M. administration withpeak drug concentrations appearing at 0.333 hr for the 1.0 mgdose and at 0.667 hr for the 2.0 mg dose. The discrepancy intmax may be caused by the binding of drug molecules to thetissue at the administration site for the higher dose;consequently, the 2.0 mg dose required a Ionger tire todiffuse. However, the tmax observed in the present study forboth doses was comparable to values found in the literature forsimilar dosages (27, 28). Moreover, the elimination half-lifeof 2.04 hr obtained in Experiment 1 corresponded to the valuesof 2 hr and 2.5 to 3 hr reported by others (29-31). Unliketmax, the terminal phase half-life was not dose-dependent.

The disposition kinetics of atropine appears to bedescribed best by a one-compartment model. The large volume ofdistribution (132.85 L) in Experiment 1 is consistent with theapplication of the one-ccmpartment model to estimate theatropine pharmacokinetic parameters. Some authors have evenreoorted a distribution half-life that is as ranid as 1 min(32) The I.M. clearance of atrcine was about L15.16 L/hr,which remained fairly constant for the 1.0 :r and 2.0 £ dcsen...ere .as rc nuaesticn of a ron-linear relaticnshir -etweenthe pharmacckinetics zf the 1.0 and 2.0 m doses of the firststudy.

"*Ione cf the dose levels in the first study inducedsignificant or substantial impairment on any of the cognitiveand neuromotor tasks. Results from previous studies obtainedsimilar results for comparable doses, particularly with complextasks (2, 5). Since there is evidence that the CNS effects ofatropine are dose-dependent (6), higher doses will probablyproduce significant impairment of cognitive-neuromotor

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7erformance. n crder to test thi _yothesi, we arecurrently conducting further studies in which we areadministering 14 m of atrccine tc young! heathy. male F.

Heart rate and growth hormone were the only variablesthat were affected substantially by the atropine doses used inExperiment 1. There was a very high correlation between clasraatropine levels and heart rate charge. The observed tmax fcrthis physiological variable corresponded to the tmax ofatropine Cmax (Figures 18A and 18B). Acccrding to previousstudies (33, 34), intermediate dose levels of atropine increaseheart rate, whereas low doses often transiently decrease theheart rate. Our results suggest that for 0.5, 1.0 and 2.0 rmof atropine, there is a direct relationship between increasesin both atropine level and heart rate. Tnterestingy,between

lU0 and 230 min after drue administration, the 0.5 -a doseprcduced a decrease in heart rate below the ccrresccndint levelof the heart rate for the clacebo condition. At LiC and VLOmin post drug both 0.5 and 1.0 mg of atropine induced lcwerheart rates than the placebo.

The clockwise hysteresis loop in Figure 20A and 20B forsitting heart rate and drug concentration is pathognomic ofacute tolerance at a moderate level. The possible mechanismsfor this phenomenon include changes in receptor sensitivity ornumber, the accumulation of an unknown metabolite (20) whichantagonizes the action of atropine at the receptor site, orreceptor kinetics and adaptation (26). Ellinwood et al. (26)reported a considerably greater degree of hysteresis for theeffect of diazecam on behavioral performance, sugg-sting thatthere would be a higher tendency for acute tolerance to develccwith cognitive-neuromotor impairment. An important cuestior,therefore, is whether a similar degree of acute tolerance willbe observed at higher doses for the atropine effect as fordiazepam. This question will be considered in the analysis ofthe performance data from Experiment 2, which will be using a amg dose (twice the highest dose in Experiment 1).

There was very little hysteresis in the relaticnshi:between grcwth hormone and atropine plasma levels, and the datasuggests a close correscondence in the chares cver -e .-values for these two variatles during the drur sessicr. Asdiscussed earlier, two comDeting nrocesses may be influencin;_the activation of the stress hormones. On the one hard,stressful situations, such as the test session, stimulate theproduction of these hormones. On the other hand,anticholinergic drugs, including atropine, have an inhibitoryeffect on the same hormones. A more complete understanding ofthe interaction between a stressful environment, the use ofantimuscarin c drugs and the activation or inhibition of thestress hormones may contribute si'nificantly to elucidating thenature of the effect of atropine on cognitive and neuromotorperformance, especially since the activation of different

41

hormones in respcnse to stress is an interral part cf the totalbehavioral response pattern being measured durin; the testsessicrs.

VI. Recommendations

The results of the present study indicate that 0.5, 1.0and 2.0 n'g of atropine did not significantly impair performanceon the cognitive and neuromotor tasks used in the presentstudy. The lack of effect may be due to a number of factors:1) there may be considerable interindividual variability inperformance; 2) the atropine doses used in Experiment 1 may belocated in the lower asymptotic portion of the dose-responsecurve; and 3) atropine inhibits activation of the stresshormones and may actually facilitate improvement in performanceat the lower doses. In order to address these issues we clarto conduct the following studies:

1. We are currently conducting a study which includes the useof a higher dose of atropine, that is, 1.0, 2.0 and 4.0 mg, to16 young healthy males. This second study serves two primarypurposes. First, the 4 mg dose will allow us to determine themagnitude of impairment induced by higher atropine doses. Thisis important since the doses of Experiment 1 may represent thelower asymptotic part of the dose-response curve, and the 4 mgdose will allow us to sample from the linear segment of thedose-response curve. Secondly, by increasing the number ofsubjects we will be able to determine the degreeofinterindividual variability in the effect of atropine oncognitive and neuromotor performance. If there is considerablevariance in sensitivity to the drug effect, then individualsshould be screened for unusual sensitivity to adverse sideeffects.

2. We plan in the next year to continue development ofadditional cognitive and neuromotor tasks for possibleinclusion in our drug task battery, as described in ouroriginal proposal. Thus far, we have completed development ofthe sudden disolacement eye saccade, continuous alternation eyesaccade and nystagmometry tasks and are currently workirg crthe hand tremor, hand raoid alternation, finger ranidalternation, pupil diareter, near point of vision, dichcticlistening and visual signal detection in a noisy backgroundtasks. Using the basic experimental protocol described forExperiments 1 and 2, we will also determine the sensitivity ofthe new tasks to the different doses of atropine. We areparticularly interested in tasks that may be sensitive to thelower doses. We will continue to compare the drug effects onthe behavioral tasks with those on the physiological measuresas well as examine the relationship of the pharmacokinetic andpharmacodynamic aspects of atropine for the new tasks.

22

. The imrlications cf the influence of atrovine on theactivaticn of the stress hormones will he more co~rr.etelyunderstocd when we have comcleted our investigation of theeffect cf the higher 4 mg atropine dose on these and otherhormones. Therefore, we will continue to measure and analyzethe relationship of the stress hormones, such as AVP, ACTH andgrowth hormones, to atropine induced impairment and/crimprovement in the studies that will be conducted within thenext year. The study of the influence of atrocire on thestress hormones will also provide the needed baseline data forthe assessment of the blocking action of atropine oncholinergic agonists when that phase of the project is incperation.

43

VII. Literature Cited

1. Weiner N (1985) Atropine, scopolar-ine and relatedantimuscarinic drugs. In: Gilman AG, Gilman LS, RallTW and Murad F (eds) Goodm aa glnla Thephabs 2I rpjolo .j kiht a, 7th ed.MacMillan, New York, pp 130-144.

2. Headley DB (1982) Effects of atropine sulfate andpralidoxine chloride on visual, physiological,performance, subjective, and cognitive variables inman: A review. MiLit Med 147:122-132.

3. Baker R, Adams A, Jampolsky A, Brown B,Haegerstrorr-Portnoy G, Jones R (1983) Effects ofatropine on visual performance. 111 * ! i8:530-53'

U. Hinderling PH, Gundert-Remy U, Schmidlin 0, Heinzel G(1985a) Integrated pharmacokinetics andpharmacodynamics of atropine in healthy humans II:Pharmacodynamics. I Pharm Sci 74:711-717.

5. Seppala T, Visakorpi R (1983a) Effect of atropine onshooting: A field trial. Mj1. Mjg 148:673-675.

6. Seppala T, Visakorpi R (1983b) Pschophysiologicalmeasurements after oral atropine in man. ActsPharmag91 Tpzicol 52:68-74.

7. Collumbine H, McKee WHE, Creasey N14 (1955) The effectsof atropine sulphate upon healthy male subjects. QuartI LLP Fx hyzi_ 40:309-319.

8. Innes IR, Nicherson M (1970) Drugs inhibiting theaction of acetycholine on structures innervated bypostganglionic parasympathetic nerves (Antimuscarinicor atropinic drugs). In: Goodman LS and Gilman A (eds)The pharmacological basis pf tt 1 th ed.MacMillan, New York, 524-548.

9. Longc VG (1966) Behavioral and electroencephalogranhlceffects of atropine and related comDounds. PhtgrMALQRea 18:105-116.

10. Shader RI, Greenblatt DJ (1972) Belladonna alkaloidsand synthetic anti-cholinergics: Uses and toxicity.In: Shader RI (ed) f-y~ihrQ gg~pL.atcns f Medicalr . Raven Press, New York, 103-147.

11. Ghoneim MM, Mewaldt SP (1975) Effects of diazepam andscopolamine on storage, retrieval and organizationprocesses in memory. !:yCgaMac ]a (Berlin)

44

44:257-262.

12. Petersen RC (1977) Scopolamine induced learninFfailures in man. Psvchp.;brzfa-Ql-E.Y (Berlin)52:282-289.

13. Holland P, Kemp KH, Wetherell (1978) Some effects of 2mg i.m. atropine and 5 mg i.m. diazepam, separately andcombined, on human performance. Fr jnzgz =Brit -. p5y _ Seog xy. 4th-6th January,1978.

14. Linnoila M (1973) Drug effects on psychomotor skillsrelated to driving: Interact-on of atropine,glycopyrrhonium and alcohol. mr- . Phacol6:107-112.

15. Moylan-Jcnes PJ (1969) The effect of a large dose ofatropine upon the performance of routine tasks. Fr JPharmago 37:301-395.

16. Caine ED, Weingartner H, Ludlow CL, Cudahy EA, Wehry S(1981) Qualitative analysis of scopolamine-inducedamnesia. Fsvchophgrmaco.o9- (Berlin) 74:74-80.

17. Adam N (1973) Effects of general anesthetics pn memoryfunctions in man. I JQ Psio 83:294-305.

18. Kalser SC, McLain PL (1970) Atropine metabolism in man.l.n fbirmacol Thor 11:214-227.

19. Gosselin RE, Gabourel JD, Willis JH (1960) The fate ofatropine in man. Clin Phrmgg_. :Ebr 1:598-603.

20. Adams RG, Verma P, Jackson AJ, Miller RL (1982) Plasmapharmacokinetics of intravenously administered atropinein normal human subjects. J fiin Phprma- 22:477-481.

21. WUechsler D (1981) Wechsler 1 lt intel1igences r1 = ? y i - a59P. The Psychological Corporation.Cleveland.

22. Brown RD, Manno JE (1978) STRP, a Basic computerprogram for obtaining initial polyexponential parameterestimates. f PharS 67: 1687-1692.

23. SAS UserIs Guide1 Statistics. 1982 (1982) SASInstitute, Cary, NC.

24. Ellinwood EH, Nikaido AM, Hatherly DG, Bjornsson, TD(1986) Benzodiazepine pharmacodynamics: Evidence forbiophase rate limiting mechanisms. fsyQb.QDharmacoloay(in press).

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25. Ellinwood EH, Jr., Heatherly DG, Nikaido AM, BjornssonTD, Kilts C (1985) Comparative Pharmacokinetics andpharmacodynamics of lorazepam, alprazolam and diazepam.PsQchopharmacoloav 86: 392-399.

26. Metcalfe RF (1981) A sensitive radioreceptor assay foratropine in plasma. Biiche P DBeh 30:209-212.

27. Berghem L, Bergman U, Schildt B, Sorb B (1980) Plasmaatropine concentrations determined by radioimmunoassayafter single dose intravenous and intramuscularadministration. Br I Anj.ash 52:597-601.

28. Gundert-Remy U, Schmidlin 0, Hinderling PH (1980)Pharmacokinetics and pharmacodynamics of atropine inhealthy humans. Naunvnbfrc_bhieebe A Phrmhako!311 Suppl., 75.

29. Virtanen R, Kanto J, Iisalo E (1980) Radioimmunoassayfor atropine and 1-hyoscyamine. ACta Pharmacol Toxicol47:208-212.

30. Hayden PW, Larson SM, Lakshminarayan S (1979) Atropineclearance from human plasma. L Nuel B.d 20:366-367.

31. Hinderling PH, Gundert-Remy U, Schmidlin 0 (1985)Integrated pharmacokinetics and pharmacodynamics ofatropine in healthy humans. I: Pharmacokinetics..harm 5sJ 74:703-710.

32. Morton HJ, Thomas EJ (1958) Effect of atropine on heartrate. Lancet 2:1313-1315.

33. Hayes WJ Jr, Copelan HW, Ketchum JS (1971) Effects oflarge intramuscular doses of atropine on cardiacrhythm. glln Pharmacl 1h_ 12:482-486.

34. Paivio A, Yuille JC, Madigan SA. (1968) Concreteness,imagery, and meaningfulness values for 925 nouns. IE p ch L(zhgj MonograL Suppl 76:1-25.

35. Ysewijn-Van Brussel KARN and DeLenheer AP (1985)Development and evaluation of a radioimmunoassay forArg 8-vasopressin, after extraction with Sep-Pak C18.CUin fhl= 31:861-863.

36. Robertson GL, Mahr EA, Athar S, Sinha T (1973)Development and clinical application of a new methodfor the radioimmunoassay of arginine vasopressin inhuman plasma. .L Clin Invest 52:2340-2352.

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37. Kilts CD, Gooch MD, KnoDes KD (1984) Ouantitation cfplasma catecholamines by on-line trace enrichment highperformance liquid chromatography with electrochemicaldetection. J Neurosci M 11:257-273.

38. Krishnan KRR, Carroll BJ, Manepalli AN, Ritchie JC,Pelton S, Nemeroff CB (1986) Dissociation of ACTH andcortisol secretion. J f-Ain EndQrfl g Ma d

39. Murphy BEP (1967) f .lin ndocInol n Metab27:973-990.

47

APPENDIX A

Performance Tasks and Analyses

In order to assess changes in performance atrelatively frequent intervals, especially during theperiod immediately after drug administration, twoshorter batteries consisting of selected performancetasks are used to estimate more precisely the rapidityof the onset of impairment. Thirteen tasks used toassess different types of cognitive-neuromotorimpairment are included in the long battery. The threetesting batteries are as follows: (1) an ultra-shortbattery consisting of only the subcritical trackingtask; (2) a medium battery, including standingsteadiness with eyes open, digit symbol substitution,subcritical tracking, and abbreviated digit symbolmemory tasks; and (3) a long battery consisting ofstanding steadiness with visual feedback, standingsteadiness with eyes open, and standing steadiness witheyes closed, digit symbol substitution, subcriticaltracking, continuous performance, divided attention,digit symbol substitution with short term memory,digit-keypad reaction time, sudden displacement eyesaccade, central pendulum eye tracking, and lateralpendulum eye tracking tasks.

During the first year of the present project majorchanges have been introduced in the procedure programsof the subcritical tracking, continuous performance anddivided attention tasks and in the analyses programs ofthe standing steadiness with eyes open and eyes closed,digit symbol substitution with short term memory,abbreviated digit symbol memory, subscritical tracking,continuous performance, divided attention, centralpendulum eye tracking, lateral pendulum eye trackingand concrete noun memory tasks. A new task, suddendisplacement eye saccade, was also added to the longtask battery. A computerized procedure for recordingheart rate has been created and is being used inExperiment 2.

STANDING STEADINESS TASK - EYES OPEN OR CLOSED

Aparatus

The sway table consists of a 45.5 x 45.5 x 1 cmsteel platform which is 1 cm above the floor. An 11 x18.5 x 0.5 cm board is mounted in the center to serveas as indexing mechanism for the placement of thesubject's feet. Four strain gauges are used totransduce side-to-side and fore-and-aft movement into avarying voltage. These quantities are digitized and

stored for subsequent analysis.

Instrutions

This test will measure your ability to stand steadywhile staring at a fixed point, and with your eyesclosed. When properly positioned on the sway table,with your hands at your sides, you will be instructedto look straight ahead and hold your position until youare told to relax. Following a 10 second rest period,you will be instructed to look straight ahead, closeyour eyes, and hold your position until you are told torelax. The object in both of these tasks is to standas steady as you can with as little side-to-side andfore-and-aft movement as possible.

Procedure

The subject is placed in a standing position on thetable, placing the long edges of the index board alongthe inside surface of the feet. Tape marks are used tojudge the position fore and aft. When properlypositioned, the subject is instructed to place hishands to his sides, look straight ahead with eyes open,and focus on a designated point until instructed torelax. Following a 10 second relaxation period, thesubject is instructed to place his hands at his sides,look straight ahead, close his eyes, and hold hisposition until instructed to relax. Thirty seconds ofdata are recorded for each task.

Anlyis

The standing steadiness data for both eyes open andeyes closed is analyzed using Fast Fourier transformsso that the spectral characteristics may ultimately beexamined. In a preliminary analysis it was determinedthat the low frequency bands (below 2.5 Hz) containedthe activity of greatest interest, and most statisticalanalyses are restricted to them. Fore-aft andport-starboard data are analyzed separately. A singleoverall measure of sway is fashioned by taking the rootof the sum of the low frequency power in bothdimensions.

STANDING STEADINESS TASK WITH VISUAL FEEDBACK

Apparatus

The sway table has been described in the previoussection, Standing Steadiness Task. The display screenis also used in this task to provide visual feedback to

49

the subject about his performance. The screen measures96 cm high and 127 cm wise. The sway table is alignedwith the right edge of the screen, and places thesubject 1.5 m from the center of the screen.

Instructions

This test will measure your ability to stand steadywith visual feedback of how well you are doing. Theprocedure is the same as the Standing Steadiness task,except the object is to keep the snake-like marker onthe screen as close to the center of the circle aspossible by making slight shifts in body weight.

Procedure

The subject positions himself on the sway table asin the Standing Steadiness task, except that he facesthe display screen instead of looking at a designatedpoint. The subject is instructed to hold as still aspossible for 5 sec, during which time the sway tableself-calibrates and a mean x-y position of thesubject's resting center is determined. Following thecalibration period a circle 56 cm in diameter is drawnin the center of the screen and a "+" is displayed tomark the resting center. As the subject sways on thetable his position is sampled at a rate of 60 times asecond. A dot is drawn on the screen at each sampling,indicating the subject's position relative to hiscalibrated center. A series of 30 dots, or the datacollected for 0.5 sec, is continually displayed. Thisproduces a "snake-like marker" that travels about thescreen showing the subject how well he is maintaining asteady position on the sway table. All measurementsare recorded as deviations from the subject's meancenter and are displayed on the screen as deviationsfrom the center of the circle. In this fashion thes,.reen acts as a mobile window that is centered overthe subject's calibrated resting center. The tasklasts for 60 sec and all data are recorded and stored.

Analysis

The measures of merit are: mean x (port-starboard),mean y (fore-aft), mean deviation from center andmaximum deviation from center.

DIGIT SYMBOL SUBSTITUTION TASK

This task involves the presentation on the displayscreen of a code consisting of a row of numbers from 1

50

to 9, and a row of symbols just below the row ofnumbers. The rows are positioned at 7 and 15 cm belcwthe top of the screen, respectively. Each number isassociated by its columnal relationship with aparticular symbol (a number positioned over a symbol).A symbol from the code is presented in the center ofthe display field, 50 cm from the top of the screen.The subject is seated approximately 110 cm from thescreen, and employs the response keypad to enter theappropriate digit associated with the symbol. Thekeypad is attached as an extension to the right armrestof the chair in which the subject is seated. Theconfiguration of the keypad digits, from left to rightare, beginning with the top row, 1, 2, 3; middle row,4, 5, 6; bottom row, 7, 8, 9.

l r-ctions

You will observe the sequence of numbers from 1 to9. Below each number is a symbol that corresponds withthe number. One at a time, a symbol will be presentedin the middle of the screen, and you are to enter thenumber associated with the symbol via the keypad, whichhas the same numbers, I to 9. Enter the number as fastas you can, as the speed, as well as the accu. acy ofyour response, will be recorded.

Procedure

The numbers of the code are digits 1 through 9.The symbols are the same as in the Wechsler AdultIntelligence Scale- Revised (WAIS). The symbols arealways presented in the same sequence on every test.The code, however, is changed in relationship to thedigit numbers. This change is made at the beginning ofeach test. In each test the subject is presented 108trials, and is required to enter the correspondingnumber associated with the symbol. The number enteredcauses the trial symbol to be erased and the numberdisplayed for 0.25 seconds just above the postion ofthe erased symbol. The duration of the task, althoughdependent on the subject's response rate, averages 2.0minutes. If the subject does not respond within 20sec, a warning message is displayed to the experimenterand the subject is verbally urged to continue.

The data are analyzed for the number of correctresponses, the average response time, the standarddeviation in the response times, and the "power" (the

51

quotient of the number correct by the mean responsetimtt. During the initial trials, this task involvesheavy reliance upon visual association while memory isimportant by its end. Consequently, the performancemeasures are computed separately for the first, second,third, and fourth quartiles, as well as overallmeasures at all time periods.

DIGIT SYMBOL SUBSTITUTION TASK WITH SHORT TERM MEMORY TEST

The apparatus used for this task is the same as

that used for the Digit Symbol Substitution task.

Instructions

This task is very similar to the Digit SymbolSubstitution task. However, the task is divided intofour equal parts, or quartiles. Each quartile consists

of 27 presentations, which you will respond to just asin the Digit Symbol Substitution task. After the 27presentations, the code table at the top of the screenwill disappear and each symbol will appear one at atime at the center of the screen. You are to respondby entering the appropriate number on the keypad thatcorrespot ds to the symbol presented. You will bescored on the number of correct associations you make.Although you will not be scored on your reaction timefor this part of the test, it is necessary that yourespond as soon as possible so the test will not bedelayed. Following the first quartile, the second,third and fourth quartiles will be presented in thesame manner, with memory tests given at the end of each

group of presentations.

fzrocedure

This task is very similar to the Digit Symbol

Substitution task, except that it is interrupted at theend of each quartile by a memory test. Follcwing 27presentations, the code table disappears. One at atime, the symbols are displayed in the middle of the

screen and the subject keys in the corresponding numberby memory. After the memory test, the code tablereappears. This sequence is repeated four times. Eachsymbol is presented three times per quartile, and onetime per memory test. The duration of the task dependson two factors; one, the amount of time it takes toperform the task with the code table present which,although dependent on the subject's response rate,averages about 2.0 minutes; and two, the amount of time

52

it takes to respond to the memory part of the taskwithout the code table present, which varies a greatdeal (up to several seconds for each of the ninesymbols presented). This task, like the Digit SymbolSubstitution task, offers the experimenter a warningmessage if the subject does not respond during a 20 secinterval following symbol presentation.

The data are analyzed for the number of correctresponses, the average response time, the standarddeviation in the response times, and the "power" (thequotient of the number correct by the mean responsetime). The performance measures are computedseparately for the first, second, third, and fourthquartiles, as well as overall measures at all timeperiods. This task involves many factors (i.e. motorcoordination, eye-hand coordination, scanning ability,qhort-term memory); thus, it will be necessary to do amore in depth analysis of this task after the initialanalysis. The in-depth analysis will compare: (1)Keyboard Reaction Task, (2) Digit Symbol Substitutionwithout memory task, (including i, explanation of thelearning curve), and (3) Digit Symtol Substitution withMemory task. By combining this data, it will bepossible to separate out the aforementioned variousfactors. It will then be possible to look at howdifferent pharmacologic agents affect the relativecontributions of memory (learning), simple keyboardreaction time, and residual psychomotor performanceeffect (e.g., eye scan time) on the Digit SymbolSubstitution Task.

ABBREVIATED DIGIT SYMBOL SUBSTITUTION TASK WITH MEMORY

Apparatuz

The apparatus for this task is the same as thatused for the Digit Symbol Substitution with M'emorytask.

This task is equivalent to the first quartile ofthe Digit Symbol Substitution with Memory task.

Procedure

This task is equivalent to the first quartile ofthe Digit Symbol Substitution with Memory task.

53

Ana! Y1 is

The data from both the Digit Zymbol Substitutionand the memory portions of the task are analyzed forthe number of correct responses, the average responsetime, the standard deviation in the response times, andthe power.

MEMORY ENCODING TASK

Two previously described tasks, Digit SymbolSubstitution with Memory and Abbreviated Digit SymbolSubstitution with Memory, are designed to assess thesubject's ability to recall from "short-term memcry'the nine WAIS symbols learned during the course of thetest. This memory encoding task is a test of theirability to recall those same symbols from "long-termmemory". The use of this test along with the DigitSymbol Substitution with Memory allows for comparativetesting of several components of memory and learningwhich we have found to be more sensitive to drugimpairment than other tests.

Apparat i

The apparatus for this task consists of a sheet of

paper containing a row of numbers 1-9, and a pencil.

InsLLQ .icns

Twenty minutes following the completion of theDigit Symbol Substitution with Memory task you will beasked to recall the Digit Symbol Substitution CodeTable from that test. We are interested in Long TermMemory under natural conditions, so do not practice orrehearse the material during the other tests. Do thebest you can without letting it interfere with yourperformance on your other tasks.

Twenty minutes after the completion of a D: :Symbol Substitution with Memory task the subject ispresented with a sheet of paper containing only a rowof numbers 1 to 9. The subject then writes under eachnumber the symbol which had been associated with it inthe recently-completed Digit Symbol Substitution withMemory task. Duri;.g the 20 min interval between theDigit Symbol Substitution with Memory task and thismemory encoding task the subject is engaged inadditional psychomotor testing which should prevent hispracticing the number-symbol relationships.

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The number of correct and incorrect responses isrecorded and stored for later analysis.

SUBCRITICAL TRACKING TASK

Apparatus

A 1 cm wide bar is displayed from top-to-bottom inthe center of the screen. A portion of the bar, 40 cmfrom the top of the screen and 24 cm in length, movesright or left across the full width of the screen. Thedistance from the screen to the top of the wheel is 57cm. The wheel, 40 cm in diameter, is connected to a 10turn pot 10 K ohm variable resistor.

Inst rutitn

The aim of this task is to maintain the moving barin the center of the screen, in line with the fixedbars running from the top and bottom of the screen,with the steering wheel which controls the position ofthe bar. If the bar moves to the right, for example,turn the whee. to the left or in the oppositedirection;if the bar moves to the left of the center,move the wheel to the right. As a hint to controllingthe bar, attempt to change the wheel in a smooth motionrather than in a fast jerky motion. You are scored onthis task by your ability to keep the tracking bar inthe center of the screen.

Procedure

During the first twenty seconds the level ofdifficulty (lambda) increases to a value of two. Dataare collected for 1 min during which the difficultylevel remains at lambda 2. During the next 20 sec thelevel of difficulty increases to lambda 3. Data areagain collected for 1 min during the period at whichthe difficulty remains at lambda 3. The level ofdifficulty is multiplied by the steering input(position of the pot) and added to the position of thebar, resulting in the bar being repositioned. Asimplified equation of this relationship is: Change inbar position = (bar position + pot position) x level ofdifficulty (lambda). A new bar position is calculatedfrom this equation 60 times a second. For example, ifthe bar moves to the right of the center line, and thepot is not turned in the opposite direction, then thebar will move further to the right. If the level ofdifficulty is increased, the bar will be displaced at a

55

higher rate. Data are recorded for three minutes.

A subject's performance on this task is analyzedboth in terms of his success in maintaining the barnear the center of the screen, and in terms of thecharacter of the steering inputs he makes. The squareroot of the mean squared error is quoted as a measureof tracking success both for easier and the moredifficult halves of the task. Steering wheel motion issubjected to Fourier transformations and the powerspectra condensed into bands one Hz wide. The lowerthree of these bands, which contain the bulk of theactivity are examined usually for drug dependentvariations both for lambda 2 and lambda 3 difficultylevels.

CONTINUOUS PERFORMANCE TASK

Numbers (1-9) 13.5 cm tall and 9.5 cm wide aredisplayed 42 cm below the top of the screen. Thesubject utilizes the response keypad described for theDigit Symbol Substitution task.

Instructions

Each number displayed will be replaced by anothernumber. You are to respond as fast as you can, as soonas you recognize whether the new number is different,in terms of being odd or even, from the prior number.That is, if the prior number was odd and the new numberis even then you would respond. If the new number isodd, then you would not respond even though it maydiffer in value from the prior number. To review theprocedure, you are to respond when either an odd numberfollows an even number or when an even number follcwsan odd number. You are not to respond when an evenfollows an even or an odd follows an odd.

Procedure

One hundred-eight randomly-selected digits (1 - 9)are individually displayed on the screen. Each of thefirst 54 digits is shown for a duration of 2.0 seconds,then followed immediately by the next digit. Each ofthe second 54 digits is displayed for a duration of1.33 seconds, then followed immediately by the nextdigit. The 1.33 sec trials follow the 2 sec trialswithout break or interruption. The duration of the

56

task is three minutes. The sequence of digits isselected from a random number table. In the sequenceof 108 numbers, there are 54 appropriate responses and54 inappropriate (false positive) responses. Thesubject may enter his response by pressing any one ofthe keys on the keypad. The subject's reaction time isrecorded, as well as the total number of appropriateand inappropriate responses.

Analysis

The number of inappropriate responses is subtractedfrom the number of appropriate responses beforerecording the "number correct." The mean responsetime, standard deviation in response times, and powerscore are computed using the correct responses.

DIVIDED ATTENTION TASK

This task is a combination of the SubcriticalTracking task and the Continuous Performance task. TheContinuous Performance portion of the task is theperipheral, secondary, or distractor task.Consequently, the duration of presentation of the 108digits remains constant; that is, the digits aredisplayed at a rate of one every 1.67 sec. Inaddition, the location of the odd-even digit display ismoved closer to the top of the screen so as not tointerfere with the tracking bar. The total duration ofthe task is 3 mins.

DIGIT-KEYPAD REACTION TIME TASK

Apparatu5

One hundred randomly-selected digits (1 - 9) aredisplayed individually in the center of the screen, 53cm from the top. The size of the digits are 6.5 cmtall by 4.5 cm wide. The subject uses the keypad(previously described in Digit Symbol Substitution) toenter the same number as apppears on the screen.

Instructions

This test is designed to measure simple reactiontime. A number will flash onto the screen and you areto respond as quickly as possible by pressing the samenumber on the keypad. You will be evaluated on bothaccuracy and speed.

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Procedure

One hundred randomly selected digits (1 - 9) arepresented individually to the subject. The subject isasked to enter into the keypad the same number asappears on the screen. This causes the trial number tobe erased and the subject's response to be displayedfor 0.25 see just above the location of the erasednumber. Immediately thereafter, another random numberis displayed. This task, like the Digit SymbolSubstitution task, offers the experimenter a warningmessage if the subject does not respond within a 15 secinterval following number presentation.

Analyss

The subject's responses are analyzed for the numbercorrect, average response time, standard deviation inresponse times and power.

CENTRAL PENDULUM TRACKING

Apparatus

A bar 15 cm long is displayed in the center of thescreen, 40 cm from the top of the screen. The subjectobserves the bar movement with his head rested againstthe chair's headrest, located 110 cm from the screen.EOG electrodes are placed as described for the SuddenDisplacement Saccade test. The electrodes areconnected to a Grass amplifier with filter settingsadjusted to: high frequency setting of 3 KHz, and lowfrequency of 0.1 Hz. These Grass amplifiers, locatedin the experimental chamber, are used to amplify thesignal to reduce artifact in transmission to thepolygraph outside the chamber.

Instructions

You are to rest your head against the headrest andfollow the moving bar with only your eyes.

f cedure

The bar moves in a 20 degree arc with a cycleperiod of two seconds (right-left-right). The top ofthe bar moves in the same plane as the bottom of thebar, rather than with respect to the imaginary apex ofthe pendulum. The task is 64 sec in duration.

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Analysis

We have implemented an analysis of smooth-pursuiteye movements based upon a statistical method publishedand evaluated by Lindsey, Holzman, Haberman and Yasillo(1978). This method generates a single figure of meritwhich reflects the quality of eye tracking movementsover any specified time interval. The figure of meritquoted is the natural logarithm of the signal to noiseratio, in (S/N), where the signal and noise powers areestimated over the time interval of interest.

The analysis proceeds in two steps, correspondingto the computation of signal and noise powers,respectively. First, the amount of signal present inthe EOG data is determined by doing a harmonicregression on a sinusoid of 0.375 Hz. This representsthe degree to which the eye movements correspond to theactual stimulus motion, and gives a measure of thesignal power S. Next this sinusoidal component issubtracted from the EOG data so that only noiseremains. A fast Fourier transform is then applied toreduce the effects of various artifacts. Finally, thenoise N is computed by summing the power in the rangefrom 0.7 to 8.0 Hz.

LATERAL PENDULUM TRACKING

Apparatu z

A 15 cm bar is displayed 40 cm from the top of thescreen at angle of 40 degrees from the center of thesubject's -sual field. The subject rests his headagainst the head rest, turns his head in the directionof a light source on the left edge of the screen, andfollows the bar movement with his eyes. EOG electroderecording is the same as those described in the SuddenDisplacement Saccade test Apparatus section.

Instructicns

With your head against the head rest, turn yourhead so that you are facing the red light on the leftedge off the screen. Moving your eyes only, you are tofollow the moving bar.

Procedure

The bar moves in a 20 degree arc with a cycleperiod of two seconds, as described in the precedingtask, except the center of the are is located 40.degrees from the center of the subject's visual field.The task duration is 64 seconds.

59

Data are analyzed using the procedure described inthe Pendulum Tracking section.

The apparatus utilized in this task is identical tothat in Pendulum Tracking, except for the bar positionwhich has been horizontally displaced.

CONCRETE NOUN MEMORY TASK

This task examines primarily long-term or secondaryverbal memory functioning. The learning and recalltrials will assess the effects of the drugs on bothstorage and retrieval.

On each test day the subjects will be shown twolists of concrete nouns, consisting of ten words each.The words were selected from a list of nouns publishedby Paivio, Yuille, and Madigan (34). All the words hadhigh ratings (above 6.0) on the attributes ofconcreteness, imagery, meaningfulness, andThorndike-Forge frequency.

Instructions

For Learning Trials

I am about to show you a list of words which Iwould like for you to remember. I will ask you torecall the words later. However, when I show you eachword, I am going to ask you either to name a categoryin which the noun belongs, or to tell me how manyletters are in the word. Do not say the word, butmerely answer my question. Later on, when you areasked to remember the words, you will not be askedabout the categories in which you placed them

For Recall Trial 1 (114 min after Learning Trial 1)

Tell me all the words that you can remember fromthe list that you learned before you got the drug thismorning.

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For Recall Trial 2 (95 min after Learning Trial 2)

Tell me all the words that you can remember fromthe list that you learned a short time ago, between thetesting batteries.

For Final Recall and Recognition Test (450 min afterLearning Trial 1)

Tell me all the words that you can remember fromboth of the word lists that you learned today. (Afterthe subject has listed all of the words that he canremember, the Recognition Test is done). I am going toshow you a set of words. Say "Yes" if you rememberbeing shown the word today, and "No" if you do not.

Erocedure

Immediately prior to drug administration in themorning, the subject is shown a series of concretenouns. The nouns are presented individually on indexcards. Upon presentation of each noun, the subject isasked either to categorize the noun, or to tell theexperimenter how many letters it contains. Theexperimenter records the subject's responses withoutgiving any feedback. After 114 min, the subject isasked to recall as many words as possible from thelist, without having to remember his earlier responses.

A second list is presented 19 min later, with thesame instructions. The subject is asked to recall thesecond list 95 min after its presentation.

At the end of the testing day, approximately sevenhours after the presentation of the first list ofwords, the subject is asked to recall as many words aspossible from the two lists. After that, a list offorty words is shown to the subject, one at a time.The subject is asked whether each word was shown to himat any time during that test session. The recognitionlist consisted of the 20 words that were presentedduring the test session and 20 words that were notpresented on that day.

Analysis

The data consist of the number of correct andincorrect responses as well as the number of wrongresponses from previously learned word lists for eachlearning plus recall and recall only trial. Overallmean number correct and standard deviation will becalculated for each test day.

61

HEART RATE MONITORING TEST

A new program has been incorporated into the testschedule after each battery in order to measure andrecord the changes in heart rate across the course ofthe day. The heart rate program was designed tominimize human error in measuring tachycardia, and tocreate a permanent data set which can be analyzedlater. The program accomplishes this by integratingthe Digital 11/23 computer with the Hewlett Packard7830A Heart Monitor. The monitor converts the ECGsignal into separate components such as the wave form,the audio output, and the heart rate bar graph. Thebar graph is the result of a beat to beatcardiotachometer in the heart monitor which measuresthe time interval between successive R-wave componentsin the ECG and then integrates the data into aprojected heart rate per minute.

The 11/23 computer converts these results from ananalog to a digital form, and samples at a rate of 100times a second for 60 seconds. The 600 data points arethen converted by a scaling factor to correspond todata determined by an independent measurement of theheart rate by pulse over a range of 0 to 150 beats perminute with a standard deviation of plus or minus 1beat per minute. The final form of the data is printedto the screen showing average heart rate over fivequartiles and a total average heart rate per minute.Finally, the permanent data sets of raw data with the600 data points, and the summary data with the 5quartiles and total average heart rate are stored inseparate files like the data from the battery tasks.

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APPENDIX B

Procedure for Plasma Collection for Atropine and the Growth Hormones

This outline describes the procedure for thecollection of blood samples for atropine and differenthormones. Blood samples are collected in two differentcontainers: (1) Green top tubes containing lithiumheparin anticoagulant for the atropine, growth hormone,cortisol and catecholamine samples, and (2) purple toptubes containing EDTA anticoagulant for the argininevasopressin (AVP) and adrenocorticotropic hormone(ACTH) samples. This procedure is necessary becauseheparin may destroy certain peptides such as AVP andACTH. In addition, the vacutainers are kept on iceprior to and after blood collection until the plasmasample is frozen. Since atropine is the compound withthe highest priority, the phlebotomist draws theatropine sample first (a safeguard in case the catheterbecomes clotted and further sampling at that time isprevented). Soon after the blood samples are collectedthey are centrifuged for 10 minutes at 4 degrees C and2500 rpm and the following procedures are followed forthe different types of samples.

AUL and ACMi: At least 1.0 ml of plasma is necessaryfor the assay of either AVP or ACTH. The plasma ispipetted and transferred to a polypropylene tubelabeled "AVP" or "ACTH,1" as appropriate. For eachassay, 0.2 ml (200 ul) of 1.0 N HCl per 1.0 ml ofplasma is added to the tube. Then the tubes arevortexed and then frozen rapidly on dry ice.

Atropine., Growth Hormone., £QEiU .a_ Catecholamines:A minimum of 1.0 ml of plasma is needed for each assayof atropine, growth hormone or catecholamines; 0.5 mlof plasma is needed for the cortisol assay. For theassay of atropine or growth hormone, 1.0 ml of plasmais placed into two separate polypropylene tubes whichare appropriately labeled and then frozen rapidly ondry ice. For the catecholamine assay 1.0 ml of plasmais transferred to a polypropylene tube labeled "CAT,"and 0.2 ml of a Glutathione solution is added. TheGultathione solution is prepared by adding 500 mg ofreduced glutathione to '0 ml of 200 nM HC1. The tubeis then vortexed and frozen rapidly on dry ice. Forthe cortisol assay, 0.5 ml of plasma is transferred toa polypropylene tube labeled "CORT." If osmolality isto be measured, the sample is refrigerated. Otherwise,it is frozen rapidly on dry ice.

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APPENDIX CRadioimmunoassay of Atropine in Plasma

A. Equipment:

The standards and samples are counted on aTRI-CARB 460 CD Liquid Scintillation System(Hewlett-Packard).

B. Reagents:

The atropine antibody dilution (1:1600) is preparedby adding 10 ul of antibody to 15.99 ml of 0.01 Mphosphate buffered saline (pH 7.5) in order to have50-70% binding capacity. Radioligand (3H-atropinesulfate, specific activity = 87 Ci/mol) is diluted inbuffered saline to make 4000 dpm counts per 20 ulsolution.

C. Standard Solutions:

A stock solution of atropine sulfate monohydrateis prepared with a concentration of 1.16 ug/ml(equivalent to 800 ug/ml atropine) in buffered saline.This solution is then further diluted with bufferedsaline in order to have atropine concentrations rangingfrom 62.5 pg to 4 ng in a 10 ul volume. Drug-freeplasma (0.99 ml) is added to each tube containing 10 ulof the atropine solutions to produce standard plasmasolutions ranging in concentration from 62.5 pg/ml to 8ng/ml.

D. Quality Control Solutions:

A quality control solution (500 ng/ml) is preparedby adding 6.25 ml stock solution (800 ng/ml) to 3.75 mlbuffered saline. This solution is further diluted withbuffered saline to prepare atropine concentrations of0.5 ng and 2.5 ng in a 10 ul volume. Drug-free plasma(0.99 ml) is added to each test tube containing 10 u!of the above solutions to five quality control plasmasolutions, 0.5 ng/ml, 2.5 ng/ml and 5 ng/ml,respectively.

E. Assay Procedure:

To a pyrex tube (12 X 75 mm) containing 50 ul ofplasma sample, quality control or standard, are added330 ul buffered saline and 100 ul atropine antibodysolution. The tubes are vortexed and then incubatedovernight at 4oC.. On the following day, a 20 ul volumeof radioligand is added to each tube, vortexed and thenincubated overnight at 4oC.

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The antibody-bound atropine is separated from freeatropine by adding 500 ul saturated ammonium sulfatesolution. The reaction tubes are vortexed, allowed toincubate 15 minutes at 4oC and a precipate is thenobtained by centrifugation at 3000 rpm for 20 minutes.The precipitate is washed once with 1 ml of a 50%

saturated ammonium sulfate solution, and then thecentrifugation is repeated immediately. Theprecipitate is dissolved in 1 ml distilled water andtransferred to a counting vial containing 8 ml ofAquasol. The test tubes are washed once with 2 ml ofAquasol, the washing being added to the same countingvial. The samples, quality control or standards, arecounted on the Hewlett Packard counter for 4 minutes.

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APPENDIX DAssays for Neuroendocrine Hormones

Radioimmunoassay for Vasopressin: Plasma vasopressinis assayed following the Sep-Pak extraction modifiedfrom Ysewijn and DeLeenheer (34) by theradioimmunoasaay technique developed by Robertson (35),antiserum supplied by Dr. Tj. B. Van WimersmaGreidanus, synthetic standards from Bachem ChemicalCo., and iodinated vasopressing prepared in thislaboratory from the same source. Vasopressin isiodinated by a modification of the Chloramine Ttechnique. Plasma samples are adjusted to pH 2.0 byaddition of iN HCl (0.2 ml/ml plasma), and adsorbedonto prewashed C-18 columns (Sep-Pak). Columns arewashed with 10 ml of 0.4% trifluoroacetic acid, and thevasopressin is eluted with 5 ml of a 40:60 mixture ofmethanol and 0.4% fluoroacetic acid. Samples are driedon a Savant evaporator, and reconstituted in assaybuffer (0.05 M phosphate buffer, pH 7.5 containing 0.01M EDTA, 100 KIU/ml aprotinin and 1% normal rabbitserum. Standards are extracted from vasopressin-freeplasma in an identical fashion.

Extracted samples are assayed by RIA at 4 degreesCelsius using delayed addition of trace to improvesensitivity and a second antibody precipitationtechnique. Samples are assayed in a final volume of0.4 ml containing 4,000 cpm 125 I-vasopressin, 0.1 mlof antiserum (final concentration 1:80,000) and 50 ulsample or standard. Thd standard curve is run from 0.1to 100 pg/tube. Samples are incubated with tracer for24 hrs and then for 24 hrs with antiserum before theaddition of second antibody. Goat antirabbit serum isadded, samples are centrifuged 24 hrs later, andpellets counted in a Packard gamma counter. The ED90is 0.15-0.5 ng/tube, and the ED50 ranges from 2-5pg/tube. The sensitivity of the assay is 0.5 pg/mlplasma.

Catecholamine Assay: Plasma levels of norepinephrine(NE) and epinephrine (E) for human studies will beconducted by high-pressure liquid chromatography (HPLC)with electrochemical detection (36). Catecholaminesfrom 1 ml of plasma are pH adjusted by addition of 1 mlTris buffer (1 M, pH 8.6), adsorbed onto alumina,washed with 0.01 M Tris, pH 8.6 and eluted with 200 ulof 0.2 N percholoric acid (PCA). The entire aliquot isinjected into the HPLC system, which utilizes a"trace-enrichment" method for sample concentration andpurification. Samples are first adsorbed onto a cationexchange column and washed with mobile phase of low

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ionic strength (0.02 M citrate, 0.02 M potassiumacetate, 0.5 mM EDTA, a final pH of 3.5). The samplesare separated by reverse-phase chromatography on a C18bonded 5 um microparticulate silica (Spherisorb CDS)with a mobile phase of higher ionic strength (0.02 Mcitrate, 0.2 M potassium acetate, 5 mM EDTA, 1.25 mMoctanesulfonate as ion pair reagent, pH 5.5).Catecholamines are detected with a TI-5A glassy carbonworking electrode maintained at +600 mV vs an Ag/AgCIreference electrode in conjunction with an LC-4Aamperometric controller (Bioanalytical System Inc.).An internal standard (3, 4 dihydroxybenzylamine isadded at the beginning of the procedure. Standardcurves are generated in catecholamine-free plasma.

ACTH iad Cortisol Assays: Plasma ACTH (37) andcortisol (38) will be assayed by RIA utilizing methodsdeveloped in the Psychiatry Department's ClinicalPsychobiology Laboratory by James Ritchie, Jr.