Central Anticholinergic Hypersensitivity in Aging P.G. Ray, PhD; K.J. Meador, MD; D.W. Loring, PhD; E.W. Zamrini, MD; X.-H. Yang, MD; J.J. Buccafusco, PhD

Although neurochemical reductions in cholinergic systems have been found to occur during aging, such changes d o not necessarily translate to functional deficits. The cognitive deficits of normal aging have been attributed in part to hypocholinergic function, but anticholinergic hypersensitivity in the elderly has not been systematically documented. To test the cholinergic hypothesis of aging, we investigated the effects of scopolamine on memory and attention in healthy young and elderly subjects. Treatments included intramuscular glycopyrrolate (0.0044 mg/kg) and scopolamine (0.002, 0.004, and 0.007 mgkg) in a randomized double-blind design. The test battery included the Selective Reminding Task (SRT), Digit Span, Paired Associates Learning (PAL), Symbol Digit Modalities Test (SDMT), and the Continuous Performance Task. Elderly controls were more impaired at lower scopolamine doses than were the young on SRT, PAL, and SDMT. These results demonstrate anticholinergic hypersensitivity and are consistent with decremental changes in cholinergic status during normal aging. (J Gerintr Psychintry Neiirol 1992;5:72-77).

T

he cognitive changes of aging are hypothesized to result in part from disruptions in the cholinergic neurotransmitter system.' When administered to young, healthy subjects, the anticholinergic drug scopolamine produces a pattern of memory* and cognitive deficits' that parallels those seen in normal aging, although some differences exi ~ t .Conflicting ~,~ results have been obtained regarding neurochemical changes in the cholinergic system of the aging but reductions in choline acetyltransferase activity, muscarinic receptor number, and choliner ic nerve terminal markers have been reported?-'' However, those neurochemical alterations do not necessarily translate directly to functional deficits. Despite the cholinergic hypothesis of aging, anticholinergic hypersensitivity has not been systematically investigated in the elderly. In

Received Jan 26, 1991. Received revised April 19, 1991. Accepted for publication July 23, 1991. From the Department of Neurology (Drs Ray, Meador, Loring, and Zamrini), and the Department of Pharmacology and Toxicology (Drs Yang and Buccafusco), Medical College of Georgia, Augusta, GA. Presented in part at the annual meeting of the American Academy of h'eurology, Miami, FL, April 1990. Address correspondence to Dr P.G. Ray, Department of Neurology, Medical College of Georgia, Augusta, GA 30912.

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the present study, we examined the effects of three dosages of scopolamine on tests of memory, attention, and other cognitive functions in healthy young and elderly subjects. Dose-response relationships were used to examine the relative sensitivity of each group to cholinergic blockade.

Methods

Subjects Eleven young (28 to 47 years old) and eight healthy elderly subjects (55 to 67 years old) participated as paid volunteers in the study. Five female and six male subjects with an average of 17 years of education made up the young group, and seven women and one man having an average of 14 years of education were included in the elderly group. A comprehensive history and a physical and neurologic examination with emphasis on mental status were obtained from all subjects prior to admission into the study. Volunteers having major medical or psychiatric illnesses were excluded. All subjects were free of centrally active medication. After an explanation of the procedures and relevance of the study, informed consent was obtained from all subjects.

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Anticholinergic Hypersensitivity

Procedures Neuropsychological tests were administered in four posttreatment testing periods. Drug treatments included intramuscular scopolamine (0.002, 0.004, and 0.007mgkg) and intramuscular glycopyrrolate (0.0044mgkg), a peripherally active anticholinergic drug which crosses the blood-brain barrier poorly. Posttreatment testing was performed 90 minutes after each injection during morning sessions separated by at least 72 hours. A randomized, double-blind procedure was used. Following the 30-minute test period, approximately 5 mL of blood was obtained by venipuncture for determination of the level of anticholinergic activity. The samples were placed on ice and then centrifuged at 3800 rpm for 10 minutes. Serum was drawn off and frozen until the assay was performed. Neuropsychological Tests A description of the tests follows in the order in which each was administered. The Selective Reminding Test (SRT) is a verbal learning test.” A list of 12 words was read to the subject, who was asked to recall the list. Those words not recalled were then reread to the subject and he or she was asked to recall the entire list. Six trials were obtained using this procedure. The consistent long-term retrieval score (CLTR) reflects the subject’s ability to recall items consistently across trials without prompting (maximum score, 72). The total number of words recalled across trials was assessed (maximum score, 72). A delayed recall test was administered 30 minutes after completion of the six trials (maximum score, 12). Different word lists were presented during each session. Forward and reverse digit spans were obtained as measures of attention and immediate memory processes (maximum score, 14). Paired Associates Learning (PAL)I6 is a lower level verbal memory test than the SRT. It involves the presentation and recall of 12 pairs of associated words, of which the first member of each pair is a category (eg, color-purple). After the entire list of word pairs was read, the subject was prompted with the first word of each pair, and asked to recall the associated word. The list was presented u p to five times as needed for correct recall of all word pairs on two consecutive trials. If the subject successfully completed the task prior to five trials, credit was given for the remaining trials. Scores indicate the total number of correct responses in five trials (maximum score, 60). A different word list was used in each session. The Symbol Digit Modalities Test (SDMT) in-

volves the written substitution of numbers for meaningless geometric designs as indicated by a key.17 Parallel forms of the test were constructed rearranging the association of symbols and numbers from the original format. Scores reflect the number of correct substitutions in 90 seconds. The Continuous Performance Test (CPT)’* was included as a measure of sustained visual attention. During this test, letters of the alphabet were randomly generated on a computer screen, and the subject was instructed to respond following each presentation of the letter X by depressing the space bar (CPT-X). In a more difficult variation of this task, the subject responded only to an X preceded by an A (CPT-AX). Scores reflect the percentage of correct responses in each 5-minute session. Determination of Anticholinergic Activify in Serum Scopolamine-like material in serum was determined according to the method of Dennison et al.I9 Whole brain from adult, male Wistar rats was homogenized in 50 mmol/L tris(hydroxymethy1)aminomethaneHCl, pH 7.4. A synaptosomal fraction was isolated by centrifugation and the pellet resuspended in buffer. The binding of [3H]-methylscopolamine was measured at room temperature using a standard filtration assay. Suspended membranes (100 pg protein) were incubated with 0.32 nmoVL [3H]-methylscopolamine in buffer, and nonspecific binding was determined in the presence of 10 pmol/L scopolamine. A standard curve was obtained by employing increasing concentrations of cold scopolamine (0.01 to 1 pmol/L). Incubation time was 60 minutes. Scopolamine-like material in serum samples (0.1 mL), performed in duplicate, was determined from the standard curve. Statistical Analyses Test scores were subjected to independent repeatedmeasures ANOVAs contrasting young and elderly controls across treatment conditions. Paired comparisons were made using Student’s f-test when there were significant interaction effects. Levels of scopolamine-like material in serum were compared using Student’s t-tes t.

Results Subjective Effects Drowsiness, mydriasis, and a dry mouth were the most common side effects experienced after scopol-

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amine administration and were present in all subjects, with an intensity that varied with dosage. Pupillary diameter increased approximately 0.5 mm across doses, while subjective descriptions of drowsiness and light-headedness on standing intensified with increasing dose but appeared parallel across the groups. All side effects frequently diminished within 2 to 3 hours after the session ended. However, the drowsiness, light-headedness, and mouth/ throat dryness persisted for several hours following high-dose scopolamine. In addition, one of the elderly experienced a period of mild, short-lived confusion following the high-dose test session. During the day .after the highest scopolamine dose, several subjects from each group expressed that "extra concentration" was needed to recall their testing performance or the routine activities of the following hours. Scopolamine Assay Scopolamine-like material was detected in serum samples from all subjects. There was no significant difference in the level of anticholinergic activity detected at each dose in the two groups (Table 1). Neuropsychological Tests Test scores are shown in Table 2. There was not a significant difference between baseline scores and those obtained following glycopyrrolate. ANOVAs were performed on all tests in the battery, using results from the randomized glycopyrrolate and scopolamine treatment sessions. When a significant group x treatment interaction term appeared, post hoc t-tests were applied comparing the glycopyrrolate and scopolamine conditions to reveal the dose at which impairment of performance occurred. After receiving scopolamine, significantly greater impairment of the elderly subjects as compared to the young was seen in the following tasks:

CLRT Score of the SeZectiue Reiiiirrdiiig Task.-The group x treatment interaction was significant (F[3,51] = 2.91; P < .04), and t-tests indicated that the elderly were significantly impaired, compared to their score following glycopyrrolate, at 0.004 mgkg (t = 3.405; df = 14; P < .005) and 0.007 mgkg scopolamine (t = 5.121; df = 14; P < .0005). The young were impaired at 0.007 mgkg only (t = 2.735; df = 20; P < .01). The elderly performed more poorly than the young at baseline (t = 1.926; df = 15; P < .05).

Total Recall of tlie Selective Reniirrdiiig Tmk.-The significant interaction term was (F[3,51] = 3.37; P < .03). Elderly scores declined significantly at 0.004 mgkg (t = 4.834; df = 14; P < .OOOS) and 0.007 mgkg (t = 2.521; df = 14; P < .025), while performance by the young was disrupted only at 0.007 mgkg (t = 3.121; df = 20; P < .005). Performance of the two groups was different at baseline (t = 2.289; df = 15; P < .025), with the elderly having lower scores. Delnyed Recall of the Selective Reiiriiidirig Task.-Delayed recall was statistically more impaired in the elderly. The interaction was significant (F[3,48] = 3.3; P < .03), and the elderly were impaired at 0.004 mg/kg (t = 3.563; df = 14; P < .005) and 0.007 mgkg (t = 4.902; df = 14; P < .OOOS). Paired Associates Lerrriiirig.-The interaction term was significant (F[3,51] = 3.61; P < .02), and impairment of performance in the elderly group occurred at the highest scopolamine dose (t = 1.868; df = 14; P < .05). Syiiibol Digit Modnlities Test.-The interaction term was (F[3,51] = 2.80; P < .05). The elderly were impaired at 0.007 mgkg (t = 2.358; df = 14; P < .025).

TABLE 1 Levels of Scopolamine-like Material in Serum Following Intramuscular Injections of Scopolamine and Glycopyrrolate Scopolamine-like Material, nrnoVL Elderly Young Scopolamine, m g k g 0.002 1.08 (0.21)+ 1.11(0.09) 0.00.1 1.27 (0.23) 1.40 (0.14) 0.007 1.31 (0.30) 1.36 (0.11) Glycopyrrolate, mgkg 0.0014 1.24 (0.19) 1.35 (0.14) *Mean (SD).

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Anticholinergic Hypersensitivity TABLE 2

Test Results for Scopolamine Treatment of Healthy Elderly (n = 8) and Healthy Young Control Subjects (n = 11) Test Baseline Glyco Scop Low Scop Medium Scop High SRT-CLTR* Elderly

Young SRT-Delayed recall+ Elderly Young SRT-Total recall' Elderly Young PAL+

35 (10)t 47 (13)

39 (12) 46 (11)

9 (2) 10 (2)

10 (1) 9 (2)

49 (6) 58 (Sj

52 (7) 57 (9j

34 (15) 45 (16)

31 (10) 47 (7) 50 (12) 57 (3)

Elderly

Young

SDMT+ Elderly

32 (9) 53 (10)

Young CPT-x Elderly Young CPT-AX Elderly Young Foreivard digit span

84 (18) 94 (8) 78 (24) 97 (4)

Elderly

Young

Backward

digit span Elderly

Young

7 (2)

7 (2)

7 (2)

7 (2)

6 (1)

8 (2) 9 (3) 9 (2) 9 (3) 9 (3) 'Tests reflecting anticholinergic hypersensitivity in the elderly group. tMean (SD). Glyco = glycopyrrolate; Scop = scopolamine; SRT '= Selective Reminding Test, CLTR = consistent long-term retrieval; PAL = Paired Associates Learning; SDMT = Symbol Digit Modalities Test; CPT = Continuous Performance Test.

At baseline, the elderly performed significantly less well than did the young (t = 4.082; df = 15; P < .0005). Performance of the elderly was not different from that of the young on some tests in the battery. These tests were:

Digit Synii.-Performance of the elderly and young was not significantly impaired by scopolamine. There were significant group effects for both the foreward (F[1,17] = 4.88; P < .04) and backward tests (F[1,17] = 5.88; P < .03). Perfortimiice Tnsk-Xs and AXs.-Treatment effects were significant for CPT-X (F[3,48] = 9.34; P < .0001), with both the young (t = 2.279; df = 20; P < ,025) and the elderly (t = 2.264; df = 12; Confiiiiroirs

P < .025) impaired at the highest dose. The interac-

tion term was significant for CPT-AX (F[3,48] = 2.96; P < .04), and t-tests revealed that the young were significantly impaired at 0.007 mg/kg (f = 1.974; df = 20; P < .05) compared to their scores after glycopyrrolate. A closer examination of the scores revealed that the young performed without error (10010) after receiving glycopyrrolate, and the group means of the young were significantly higher than those of the elderly after each treatment. Discussion The results of this study demonstrate that healthy elderly subjects are significantly more impaired by the anticholinergic drug scopolamine on tests of cognitive function than are young adults. Baseline

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scores revealed that performances by the two groups were significantly different on selected tests in the battery. However, be tween-group comparisons of scores at each dose indicated that these differences became even more marked after the subjects received scopolamine. Performance on the SRT and PAL indicated significantly greater effects of scopolamine on memory processes in the elderly as compared to the younger subjects. In fact, the strongest impairment induced by anticholinergic blockade in the elderly involved the learning and recall 0-f new words in the SRT. Decrements in elderly scores were apparent at the midrange and high doses, while PAL scores declined significantly only after the highdose test session. This finding was to be expected, based on the differences in cognitive demand for the two tasks. PAL is less demanding than SRT, since associated word pairs are formed during rehearsal and cues are given to prompt recall. Memory tasks that involve the storage and retrieval of information beyond the limits of immediate memory have previously been shown to be particular1 sensitive to disruption by anticholinergic drugs.'. Performance of the SDMT was also impaired in the elderly subjects after high-dose scopolamine, but no effect was seen in the young. The SDMT is a graphomotor coding task indicative of the efficiency of different cerebral mechanisms in both hemis p h e r e ~SDMT . ~ ~ is used as a measure for screening cerebral dysfunction of varying etiologies and is sensitive to the effects of centrally active drugs.22 Digit span is useful as a crude measure of attention (ie, immediate memory).= In agreement with previous reports, digit s an was not significantly impaired by scopolamine!20#21 The CPT requires sustained vigilance and is also associated with attentional mechanisms.'8 Performance of CPT-X was similarly affected in both the young and elderly subjects, with scores declining significantly after the highest dose. Although the elderly performed more poorly than did the young on CPT-AX after all treatments, their scores did not decline significantly across doses as did those of the young group. If, in fact, hypersensitivity to anticholinergic blockade is manifest during normal aging, why were the "attentional" tasks (ie, digit span and CPT) affected in essentially the same manner in both groups, while "memory" tasks were more strongly impaired in the elderly? Caine et alz4 found that a simple auditory vigilance task was not significantly impaired by scopolamine. The lack of cognitive demand coupled with a low event rate were considered to contribute to the insensitivity of the task to

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Or2'

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the central effects of s c o p ~ l a m i n e . ~The ~ , ~CPT-X ~ used in the present study had a minimal cognitive component, but a high event rate, while the CPT-AX was somewhat more cognitively demanding since it required "immediate" memory for order of presentation of the stimuli. Nevertheless, CPT proved to be fairly insensitive to anticholinergic blockade except in the high-dose situations. The use of a vigilance task of longer duration and/or requiring greater cognitive activation may prove useful in resolving whether the elderly are more sensitive to anticholinergic blockade for attentional tasks. Interpretations of the central actions of scopolamine have included both a direct impairment of memory processes and disruption of attentional mechanisms. Early studies suggested an interference during memory consolidawhile others havk forwarded the view that scopolamine's central actions disrupt the ability to control or maintain attention, and thereby modulate the encoding pro~ess.~'-~' Future research strategies should be designed to more closely assess the action of scopolamine on these aspects of information processing in young and elderly populations. The results of this study show increased sensitivity to scopolamine in the healthy elderly as compared to younger subjects. Although central cholinergic neurochemical markers are reduced in normal other neurochemical abnormalities have been reported in the aging brain.32-36 Thus, while it is unlikely that the cognitive deficits seen in normal aging are entirely due to changes in cholinergic neurotransmission, the results of cholinergic manipulations in the present study indicate decremental changes in the functional status of the central cholinergic system in the elderly. The results cannot be explained by age-related differences in absorption or metabolism, since serum levels of anticholinergic activity were the same in both groups at 2 hours postinjection. These findings have important implications for the use of medications affecting the cholinergic system in the elderly population. The selectivity of the present results to the cholinergic neurotransmitter system must be assessed in future studies using other centrally active drugs in similar populations. This study has demonstrated, however, hypersensitivity to the central effects of cholinergic blockade in the healthy elderly. Acknowledgments The investigators were supported by NIA/NIH grant KO8 AGO314 and by the Medical College of Georgia Research Institute. The authors wish to thank 0. Abney for technical assistance.

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Central anticholinergic hypersensitivity in aging.

Although neurochemical reductions in cholinergic systems have been found to occur during aging, such changes do not necessarily translate to functiona...
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