Hypothalamic-Pituitary-Adrenocortical Axis

Responses to Physostigmine: Effects of Alzheimer' s

Disease and Gender
Elaine R. Peskind, Murray A. Raskind, Dane Wingerson, Marcella Pascualy,
Leon J. Thal, Dorcas J. Dobie, and Charles W. Wilkinson

We asked whether hypothalamic-pituitary-adrenocortical (HPA ) axis responses to a cholin-

ergic stimulus are blunted in patients with Alzheimer's disease (AD) of mild to moderate
severity. Such a finding would be consistent with a central cholinergic deficiency early in the
course of AD. To address this question, we measured the plasma adrenocorticotropic hormone
(ACTH), beta-endorphin-like immunoreactivity (f3E-LI), and cortisol responses to the cho-
linesterase inhibitor physostigmine in 10 healthy normal older subjects (age = 71 + 2 years)
and 11 outpatients with probable AD (age = 72 +- 2 years; Mini Mental State Exam score =
19 + 2). Cortisol concentrations were higher in AD subjects throughout the study, but AD and
normal older subjects had similar robust ACTH, f~E-LI, and cortisol responses to physostig-
mine. In all subjects combined, women had greater ACTH, f3E-LI, and cortisol responses to
physostigmine than did men. Plasma physostigmine concentrations did not differ between
groups. These results suggest that female gender enhances the magnitude of HPA axis
responses to cholinergic stimulation in older humans; however, the HPA axis response to
physostigmine does not appear to reflect central cholinergic deficiency in the early stages of
AD.

Key Words: Cortisol, corticotropin, beta-endorphin, physostigmine, Alzheimer's disease,

gender

BIOL PSYCHIATRY 1996;40:61-68

Introduction inhibitor physostigmine in male patients with advanced

Alzheimer's disease (AD) compared to normal older men
We previously reported a blunted beta-endorphin-like
(Raskind et al 1989). We interpreted this finding as
immunoreactivity ([3E-LI) response to the cholinesterase
decreased responsiveness in AD of the hypothalamic-
pituitary-adrenocortical (HPA) axis to central cholinergic
From the Geriatric Research, Education, and Clinical Center, and American Lake
stimulation and as a possible manifestation of the presyn-
Veterans Affairs Medical Centers, Seattle, WA, and Department of Psychiatry aptic cholinergic deficiency demonstrated in AD postmor-
and Behavioral Sciences (ERP, MAR, DW, MP, DJD, CWW), and San Diego
Veterans Affairs Medical Center and Department of Neurosciences (LJT),
tem brain tissue (Davies and Maloney 1976; Whitehouse
University of California San Diego, San Diego, California. et al 1982; Bird et al 1983).
Address reprint requests to Elaine R. Peskind, MD, GRECC (182B), Seattle VA
Medical Center, Seattle WA 98108.
This present study again tested the hypothesis that HPA
Received March 22, 1995; accepted June 14, 1995. axis responsiveness to physostigmine is blunted in AD, to

© 1996 Society of Biological Psychiatry 0006-3223/96/$15.00

SSDI 0006-3223(95)00318-B
62 BIOLPSYCHIATRY
1996;40:61 68
address methodologic and clinical issues raised by our
previous study. In that study we had used only [3E-LI, an
indirect measure of adrenocorticotropic hormone (ACTH)
release from the pituitary corticotroph, as the measure of
HPA axis responsiveness to physostigmine. Although
[3E-LI and ACTH are generally released in equimolar
quantities following central cholinergic stimulation (Risch
et al 1983), plasma [3E-LI measurements may not reflect
ACTH release in all circumstances (Kjaer et al 1992). In
the current study we measured plasma ACTH in addition
to plasma [3E-L1. We also assessed adrenocortical respon-
siveness by measuring plasma cortisol. The second issue
was that physostigmine concentrations were not measured
in our earlier study. Thus we could not exclude the
possibility that pharmacokinetic effects of AD on phy-
sostigmine disposition could have contributed to differen-
tial neuroendocrine responses between groups. Therefore,
in this study we measured plasma concentrations of
physostigmine at three time points following physostig-
mine infusion.
A third issue involved the clinical relevance of our
earlier study to the status of brain cholinergic activity in
subjects participating in recent large therapeutic outcome
trials of the cholinesterase inhibitor tacrine in AD (Davis
et al 1992; Farlow et al 1992; Knapp et al 1994). These
outcome trials were restricted to AD patients with mild to
moderate disease whose mean Mini Mental State Exam
(MMSE) (Folstein et al 1975) scores were 16, 18, and 19,
respectively. The AD subjects in our earlier study (Ras-
kind et al 1989) had much more advanced disease as
indicated by a mean MMSE score of 9. This mean MMSE
is below the lowest MMSE score allowed for inclusion in
any of the three tacrine clinical trials. It may be incorrect
to extrapolate evidence supporting impaired cholinergic
responsivity derived from our advanced AD patients to the
AD subjects with mild to moderate dementia for whom
cholinesterase inhibitor therapy has been approved and
recommended (Winker 1994). We therefore studied the
HPA axis responses to intravenous physostigmine in new
groups of normal older and AD subjects. We selected a
sample of AD patients who had dementia of mild to
moderate severity (mean MMSE score = 19) comparable
to those participating in recent therapeutic trials of tacrine
(Davis et al 1992; Farlow et al 1992; Knapp et al 1994).
Finally, women were included among both the AD and
normal older subjects in the current study. Previously, we
had studied only men. Inclusion of women allowed us to
gather preliminary data to address the possibility of a
gender effect on HPA axis responsiveness in later life.
Enhanced HPA axis responsiveness in older women has
received support from two recent studies (Greenspan et al
1993; Heuser et al 1994).
E.R. Peskind et al
Methods
Subjects
The study was approved by the Human Subjects Review
Committee of the University of Washington and informed
consent was obtained from all subjects. For the AD
subjects, informed consent was also obtained from the
legal guardian or next of kin. Participants included 10
healthy older subjects (71 _+ 2 years; five men, five
women) and 11 otherwise healthy older subjects with AD
(72 + 2 years; eight men, three women). AD subjects met
National Institute of Neurological and Communicative
Disorders and Stroke (NINCDS) criteria for probable AD
(McKhann et al 1984) and had a mean score (-+ standard
error) on the MMSE of 19 - 2. Normal older subjects
scored 29 or 30 on the MMSE and had no history or other
evidence of cognitive deterioration from their normal level
of function. All subjects were carefully screened by a
geriatric psychiatrist to exclude persons with depression,
dysthymia, or other current psychiatric disorders or symp-
toms. All subjects were in good general health as docu-
mented by medical history, physical examination, com-
plete blood count, and standard blood chemistries.
Specifically, there was no evidence of past or present renal
disease, hepatic disease, thyroid disease, organic heart
disease, or other diseases known to affect the HPA axis. In
addition, all subjects had no evidence or history of past or
present psychiatric or neurologic disorders (other than
AD), and all were nonsmokers free of chronic medications
for at least 2 months prior to study. All subjects were
normotensive (less than 135 mm Hg systolic and 90
mm Hg diastolic), and weighed within 125% of ideal
body weight (1983 tables, Metropolitan Life Insurance
Company).
Experimental Procedures
Studies were performed in a clinical research unit at the
Seattle Veterans Affairs Medical Center between 0800 and
1100 hours. Subjects fasted from midnight prior to study.
After the subject assumed a supine position, an intrave-
nous catheter was inserted into an antecubital vein of each
arm. Subjects maintained the supine position for the
duration of the study and the intravenous catheters were
kept patent with a slow (50 mL per hour) infusion of
normal saline. One catheter was used for the physostig-
mine infusion and the other for obtaining blood samples.
Thirty and thirty-five minutes after catheter insertion,
blood samples were obtained for hormone measurements.
The mean of these two measurements was considered the
baseline. Forty minutes after catheter insertion (time 0),
physostigmine (.0125 mg per kg) in 50 mL normal saline
was infused over 10 min. Blood samples were obtained at
HPA Axis Responses in AD
15, 30, 45, 60, 90, 105, and 120 rain after time 0 for
measurements of cortisol, ACTH, and 13E-LI. Additional
blood samples were obtained at 15, 45, and 105 min for
physostigmine measurements, which were obtained in all
but one normal older subject and two AD subjects. Blood
samples for cortisol, ACTH, and [3E-LI were collected in
chilled glass tubes containing ethylenediaminetetraacetic
acid (EDTA) and aprotinin, placed on ice immediately
after collection and cold centrifuged within 1 hour. Blood
samples for plasma physostigmine concentrations were
collected in polypropylene tubes containing Na 2 EDTA
and .05 mg neostigmine bromide, centrifuged immediately
after collection and frozen on dry ice. All plasma samples
were stored at - 8 0 ° C within 90 min of collection.
Physostigmine may induce nausea, which itself may
stimulate the HPA axis (Lewis et al 1984). Therefore,
nausea and related symptoms following physostigmine
were quantified at each blood sampling time point using a
four-point scale. On this scale, 0 indicated no nausea or
other gastrointestinal sensations, 1 indicated gastrointesti-
nal sensations but no nausea, 2 indicated nausea, and 3
indicated emesis.
Chemical Assays
Cortisol was measured by radioimmunoassay in unex-
tracted plasma as previously described (Raskind et al
1982). Samples were diluted with phosphate buffer and
heated for 20 rain at 80°C to denature binding globulins.
Cortisol antiserum was obtained from ICN Biomedicals
(Costa Mesa, CA). The detection limit for cortisol with a
100-~L sample is 10 pg/mL. Intra- and interassay coeffi-
cients of variation are 4.6% and 10.2%, respectively.
ACTH was measured by radioimmunoassay (RIA) as
previously described (Radant et al 1992). The assay used
IgG ACTH antiserum (IgG Corporation, Nashville, TN),
I-125-labeled ACTH (ICN Biomedicals, Costa Mesa, CA),
and human ACTH-1-39 standards (Peninsula Laborato-
ries, Belmont, CA). Stripped plasma was used as a diluent
for both standards and samples to avoid the nonspecific
binding effects often attributed to the use of ovine serum
albumin in ACTH assay buffers. By using the stripped
plasma to dilute standards and samples, parallel dilution
curves are obtained with unknown plasma samples. The
detection limit for ACTH with a 50-1xL sample is 4 pg/mL.
Intra- and interassay coefficients of variation are 6.3% and
11.5 %, respectively.
[3E-LI was measured by RIA as previously described
(Dorsa and Bottemiller 1983). Plasma samples (1 mL)
were acidified with HC1 and applied to octadecylsilyl
silica cartridges (Sep-Pak C18, Waters Associates, Mil-
ford, MA) that were preconditioned with 5 mL methanol
BIOLPSYCHIATRY 63
1996;40:61 68
and 10 mL water. Cartridges were washed with 10 mL 4%
acetic acid and extracts were eluted with 4 mL reagent
alcohol: 4% acetic acid (24:1). Extracts were dried under
vacuum (Speed-Vac Concentrator, Savant, Hicksville,
N J). Recovery of unlabeled human [3E using this method
is 80.2 + 3.2%. Beta-lipotropin ([3LPH) is retained during
this extraction procedure. Cross-reactivity of the antiserum
with porcine [3LPH is 60% on a molar basis. The sensi-
tivity of the assay is 3 pg/mL. The coefficient of variation
within assays is 6.4% and between assays is 10.4%.
For all neuroendocrine parameters, all samples from a
single subject were measured in the same assay. Approx-
imately equal numbers of subjects from the normal older
and AD groups were measured in any given assay.
Physostigmine concentrations were measured by high-
performance liquid chromatography (HPLC). Physostig-
mine was extracted from the 2-mL plasma samples and
standards by adding 100 IxL of 40 ng/mL N-methyl
physostigmine solution (internal standards), 1 mL o f . 1 M
NaHCO3-NaCO 3 pH 8.0 buffer, and 6 mL of methyl
t-butyl ether (MTBE, Fisher Scientific Inc.). The samples
were shaken for 15 rain and then centrifuged at 3000 × g
for 15 min. The top solvent layer was removed to a clean
tube to which was added 250 t~L of .0IN HC1. The MTBE
layer was evaporated and the remaining HC1 solution was
transferred to an injection vial. HPLC was performed on
all samples using a Waters Model 6000A pump with a
Bio-Rad AS-48 automatic sampler, Shimadzu RF-530
fluorescence spectrometer, Bio-Rad 3392A integrator, and
an Alltech Spherisorb 5IX silica column 250 mm × 4.6
mm. The mobile phase consisted of 65% acetonitrile, 25%
methanol, and 10% .1M NH4NO 3 buffer pH 8.0 (all
solvents from Fisher Scientific, Inc., Santa Clara, CA)
pumped at a flow rate of 1.4 mL/min. The injection
volume was 200 p~L and the physostigmine and N-methyl
physostigmine were detected using a fluorescence setting
of 254 nm for excitation and 360 nm for emission.
Quantitation of the physostigmine concentration was cal-
culated by the ratio of the height of the physostigmine
peak to the height of the N-methyl physostigmine peak
and compared to a standard curve of spiked plasma
containing 5, 2, 1, .5, .1, and .05 ng/mL physostigmine.
Quality control was provided by running a separate series
of standards at the beginning and end of the samples
consisting of 3, .75, a n d . 15 ng/mL physostigmine.
Statistical Analyses
Variables are expressed as mean -+ standard error of the
mean (SEM). The significance of differences between
groups for ACTH, [3E-LI, and cortisol responses to phy-
sostigmine was evaluated by analysis of variance
64 BIOL PSYCHIATRY E.R. Peskind et al
1996:40:61-68

( A N O V A ) with repeated measures. A differential response 300.

between groups was indicated by a significant group by
time interaction at p < .05 (two-tailed). Post h o c t tests 250.
were used to evaluate the significance of differences
between groups at specific time points if significant group 200-
by time interactions or main group effects were demon-
I-
strated. In addition, the neuroendocrine secretory re- o
<
150.
sponses to physostigmine were estimated by calculating <
the area under the curve (AUC) by the trapezoidal method II/J
i 100
<
(Dixon 1987) for the elevation of ACTH, [3E-LI, and ..I
¢1.
cortisol levels minus the baseline levels. AUCs were then 5O
compared between groups by t test for unpaired samples.
' i i t
Differences were considered significant when p < .05, 6 ' 3'o s'o do 1~'0
two-tailed.

Results 100.

ACTH, [3E-LI, and cortisol responses to physostigmine in

80.
normal older subjects and AD subjects are presented in
Figure 1. The neuroendocrine responses to physostigmine z
did not differ between these groups [group by time 60.
m
interactions for ACTH: F(7,133) = 1.68, p = ns; for 0
a
[3E-LI: F(7,133) = 1.47, p = ns; and for cortisol F(7,133) Z 40.
= .87, p = ns]; however, cortisol concentrations were
higher in A D than in normal older subjects [group effect, <
=Z 20-
F(1,19) = 4.41, p < .05]. There were no significant group
effects for A C T H or [3E-LI. Neuroendocrine responses as 5
L infusion
estimated by A U C did not differ between A D and normal ~) ' 3'0 ' 6'0 ' 9'0 1~0
older subjects.
Plasma physostigmine concentrations following infu-
sion did not differ between A D and normal older subjects
35-
at 15 min (2.76 ± .17 vs. 3.72 ± .80 ng/mL, t = 1.16,
p = ns), 45 rain (.98 ± .14 vs. 1.17 ± .25 ng/mL, t = .74, 30-
¢t
p = ns), or 105 rain (.47 ± .21 v s . . 2 9 - .08 ng/mL, t =
:=L 25-
.84, p = ns). There was an increase in nausea ratings v
,.I
following physostigmine [time effect, F(7,133) = 10.66, O
¢1 20.
p < .001], but nausea responses did not differ between
n-
groups [group by time interaction, F(7,133) = .38, p = O 15.
O
ns].
Because endocrine responses to physostigmine did not 10.
differ between normal older and A D subjects, these groups el 5,
were combined to evaluate the effects of gender on these
infusion
responses. Neuroendocrine responses estimated as A U C 0
b 3'0 6'0 ' 9'o ' 1~,0
are presented in Figure 2. A C T H A U C was higher in
MINUTES
women than in men (t = 3.01, p < .01), as were [3E-LI
AUC (t = 2 . 8 5 , p < .01) and cortisol A U C (t = 2.18, p < Figure 1. Mean (_+ SEM) plasma ACTH, beta-endorphin-like
.05). A N O V A also supported greater neuroendocrine re- immunoreactivity, and cortisol responses to a 10-min physostig-
sponses in older women than in older men [group × time mine infusion in 10 old normal (open circles) and 11 Alzheimer's
disease (AD) subjects (closed circles). AD greater than old, p <
interaction for ACTH: F(7,133) = 3.64, p < .01; for
.05, at individual time points following ANOVA demonstration
13E-LI: F(7,133) = 2.24, p < .05; for cortisol: F(7,133) = of significant group effect.
4.21, p < .001]. There were no significant differences in
physostigmine concentrations between older women and
older men at 15 min (3.66 ± .83 ng/mL vs. 2.92 ± .34
HPA Axis Responses in AD BIOLPSYCHIATRY 65
1996;40:61 - 68

Discussion
o The results of this study did not support our major
m hypothesis of blunted HPA axis responses to physostig-
mine in AD patients with mild to moderate disease. Both
2oooo AD subjects and healthy normal older controls had robust
--8-** and very similar ACTH, BE-LI and cortisol responses to
1oooo intravenous physostigmine, a drug that stimulates the HPA
axis at a brain level (Risch et al 1983; Hasey and Hanin
1990). Therefore, in these AD subjects in the earlier stages
of their disease, the HPA axis responses to physostigmine
do not appear to reflect the presynaptic cholinergic lesion
demonstrated in AD postmortem brain tissue (Davies
~ 10000 and Maloney 1976; Whitehouse et al 1982; Bird et al
1983).
0 These results in AD subjects with mild to moderate
o disease differed from the reduced BE-LI responses to
physostigmine we observed previously in AD patients
•-O-O*
with advanced disease (Raskind et al 1989). It is possible

[- O
O
that the presynaptic cholinergic deficiency in the currently
studied AD subjects with mild to moderate disease was not
severe enough to result in decreased HPA axis responsive-
o
M L ,
FEMALE ness to a cholinesterase inhibitor; however, studies in
neocortical tissue obtained at diagnostic brain biopsy from
m
AD patients with mild to moderate disease (DeKosky et al
1992; Francis et al 1985) indicate a substantial presynaptic
2OO0 cholinergic deficiency in AD patients with disease severity
w
similar to those who participated in the current study. It
E 1500 also is possible that the failure to demonstrate reduced
HPA axis responsivity in the current study represents
1000 8 successful compensation for the presynaptic cholinergic
<
O deficiency in the earlier stages of AD; however, compen-
satory upregulation of postsynaptic muscarinic or nicotinic
.o
cholinergic receptors has not been demonstrated in AD
(Flynn et al 1991; Weinberger et al 1991; Whitehouse et al
8 o 1986).
MALE FEMALE
Although the discrepant BE-LI findings between our
Figure 2. ACTH, beta-endorphin-like immunoreactivity, and earlier study (Raskind et al 1989) and the current study
cortisol responses [expressed as area under the curve (AUC)] to
10-min physostigmine infusion in male as compared to female may represent true differences of HPA axis responsiveness
subjects from combined older normal (open circles) and Alzhei- to cholinergic stimulation between AD subjects with
mer's disease (closed circles) subjects. *p < .05, **p < .01, advanced disease and AD subjects in the earlier stages of
female subjects greater than male subjects. AD, technical differences in the 13E RIA between studies
must also be considered. In our earlier study, a silicic acid
(powdered glass) extraction method was used to remove
[3LPH from plasma prior to assay. Thus, the RIA detected
ng/mL, t = .88, p = ns), 45 min (1.06 -+ .19 vs. 1.06 _+ only BE despite using a BE antibody with substantial
.18 ng/mL, t = .01, p = ns), or 105 min (.44 + .11 vs..32 cross-reactivity with [3LPH. In recent years, we have had
-+ .07 ng/mL, t = .6, p = ns). Nausea ratings did not differ difficulty obtaining silicic acid that provided acceptable
between groups [group × time interaction, F(7,133) = recovery; we therefore have changed to an extraction
.84, p = ns]. Three of the eight older women were taking procedure that is very reliable but does not separate BLPH
supplemental estrogen. Their neuroendocrine responses from BE. Therefore, the RIA in the current study measured
closely approximated those of the five older women not BE-LI composed of both BE and BLPH. Given these
taking estrogen. differences in the BE-LI RIA procedures between studies,
66 BIOLPSYCHIATRY
1996;40:6 l - 68
it still remains possible that the combined [3E and [3LPH
response to physostigmine demonstrated in the current
study does not differ between older normal and AD
subjects with mild to moderate disease, but that the
proportion of the combined "[3E-LI" comprised of [3E was
reduced in these AD subjects.
Complicating the rationale for using HPA axis respon-
siveness as a reflection of the cholinergic deficiency in AD
are our observations that human aging per se is associated
with enhanced responsiveness to physostigmine. For ex-
ample, even the blunted [3E-LI responses in advanced AD
subjects compared to normal older subjects (Raskind et al
1989) were larger than we have observed in two separate
groups of normal young subjects studied in our laboratory
(Raskind et al 1990; Peskind et al 1995). Thus, even
advanced AD subjects retain to some degree the aging-
associated enhancement of HPA axis responsiveness to
physostigmine.
Although the current study does not suggest altered
HPA axis responsiveness at or above the level of the
pituitary in AD patients with mild to moderate disease, the
higher cortisol concentrations despite equivalent ACTH
concentrations in AD compared to normal older subjects
are compatible with enhanced responsiveness of the HPA
axis at the level of the adrenal cortex. Similar increased
plasma and urinary cortisol concentrations in AD subjects
have been reported by others (Davis et al 1986; Masugi et
al 1989; Martignoni et al 1990; Maeda et al 1991; Rolandi
et al 1992). In two of these studies, elevated plasma
cortisol occurred concomitantly with normal plasma
ACTH (Masugi et al 1989) or normal plasma [3E (Rolandi
et al 1992). To our knowledge, a study in AD of adreno-
cortical sensitivity to exogenous ACTH has not been
performed. Increased cortisol concentrations but normal
ACTH concentrations also are consistent with decreased
sensitivity to cortisol feedback inhibition of the HPA axis
in AD at either a pituitary or suprapituitary level. De-
creased sensitivity to glucocorticoid feedback inhibition
by the synthetic glucocorticoid dexamethasone has been
demonstrated repeatedly in AD (Raskind et al 1982; Spar
References
Bird TD, Stranahan S, Sumi SM, Raskind MA (1983): Alzhei-
mer's disease: Choline acetyl transferase activity in brain
tissue from clinical and pathologic subgroups. Ann Neurol
14:284-793.
Bodnoff SR, Humphreys AG, Lehman JC, Diamond DM, Rose
GM, Meaney MJ (1995): Enduring effects of chronic corti-
costerone treatment in spatial learning, synaptic plasticity,
and hippocampal neuropathology in young and mid-aged rats.
J Neurosci 15:61-69.
E.R. Peskind et al
and Gerner 1982; Greenwald et al 1986; Gottfries et al
1994; Miller et al 1994). The elevated plasma cortisol in
AD also could represent decreased cortisol clearance in
AD. The effect of AD on cortisol clearance to our
knowledge has not been investigated.
The enhanced cortisol response to physostigmine in
older women is consistent with two recent studies of
pituitary and adrenocortical responses to corticotropin-
releasing hormone (CRH). Greenspan et al (1993) reported
markedly enhanced cortisol but not ACTH responses to
CRH in older women compared to older men. Heuser et al
(1994) similarly found higher cortisol responses and a
trend toward higher ACTH responses to CRH in older
women than in older men. Our observed effect of gender
on HPA axis responsiveness to physostigmine in later life
must be considered preliminary until larger numbers of
subjects are studied.
This study did not demonstrate a presynaptic cholin-
ergic deficiency in vivo in AD patients with mild to
moderate disease; however, the demonstration of in-
creased cortisol concentrations in AD and the apparent
increased HPA axis responsiveness in older women may
have implications for a number of disorders of later life. A
growing body of literature suggests that excess glucocor-
ticoids endanger hippocampal pyramidal neuronal survival
(Landfield et al 1981; Sapolsky et al 1985, 1990; Stein-
Behrens et al 1994; Bodnoff et al 1995) and may contrib-
ute to the pathophysiology of such important medical
disorders of the elderly as AD and osteoporosis (Resnick
and Greenspan 1989).
This investigation was supported by the Department of Veterans Affairs
and NIH Grants AG-8419 and AG-5136.
The authors wish to acknowledge Elizabeth Colasurdo and Richard
Vertz for their technical assistance, Sally Swedine for data management
and statistical analysis, and Susan Martin for manuscript preparation. The
authors also wish to thank Sandra Linauts and Lois Knaffier and the
laboratory staff of the Tacoma-Pierce County Blood Bank for providing
out-of-date blood for use in the ACTH assays.
Davies P, Maloney AJF (1976): Selective loss of central cholin-
ergic neurons in Alzheimer's disease. Lancet ii:1043.
Davis KL, Davis BM, Greenwald BS, Mohs RC, Mathe AA,
Johns CA, Horvath TB (1986): Cortisol and Alzheimer's
disease: I. Basal studies. Am J Psychiato' 143:300-305.
Davis KL, Thal LJ, Gamzu ER, Davis CS, Woolson RF, Gracon
SI, Drachman DA, Schneider LS, Whitehouse PJ, Hoover
TM, Morris JC, Kawas CH, Knopman DS, Earl NL, Kumar
V, Dooly RS (1992): A double-blind, placebo-controlled
HPA Axis Responses in AD
multicenter study of tacrine for Alzheimer's disease. N Engl
J Med 327:1253-1259.
DeKosky ST, Harbaugh RE, Schmitt FA, Bakay RAE, Chui HC,
Knopman DS, Reeder TM, Shetter AG, Senter HJ, Markes-
bery WR, and the Intraventricular Bethanecol Study Group
(1992): Cortical biopsy in Alzheimer's disease: Diagnostic
accuracy and neurochemical, neuropathological, and cogni-
tive correlations. Ann Neurol 32:625-632.
Dixon WJ (1987): BMDP Statistical Software Manual. Berkeley,
CA: University of California Press, p 52.
Dorsa DM, Bottemiller LA (1983): Vasopressin-like peptides in
the rat brain: Immunologic and chromatrographic behavior
and their response to water deprivation. Regul Pept 6:393-
403.
Farlow M, Gracon SI, Hershey LA, Lewis KW, Sadowsky CH,
Dolan-Ureno J (1992): A controlled trial of tacrine in Alzhei-
mer's disease. JAMA 268:2523-2529.
Flynn DD, Weinstein DA, Mash DC (1991): Loss of high affinity
agonist binding to muscarin receptor in Alzheimer's disease:
Implications for failure of cholinergic replacement therapies.
Ann Neurol 29:256-262.
Folstein MF, Folstein SE, McHugh PR (1975): Mini-mental
state: A practical method for grading the cognitive state of
patients for the clinician. J Psychiatr Res 12:189-198.
Francis PT, Palmer AM, Sims NR, Bowen DM, Davison AN,
Esiri MM, Neary P, Snowden JS, Wilcock GK (1985):
Neurochemical studies of early-onset Alzheimer's disease. N
Engl J Med 313:7-11.
Gottfries CG, Balldin J, Blennow K, BrSne G, Karlsson I,
Regland B, Wallin A (1994): Regulation of the hypothalamic-
pituitary-adrenal axis in dementia disorders. Ann NYAcad Sci
746:336 -344.
Greenspan SL, Rowe JW, Maitland LA, McAloon-Dyke M,
Elahi D (1993): The pituitary-adrenal glucocorticoid response
is altered by gender and disease. J Gerontol 48:M72-M77.
Greenwald BS, Mathe AA, Mohs RC (1986): Cortisol and
Alzheimer's disease: II. Dexamethasone suppression, demen-
tia severity, and affective symptoms. Am J Psychiatry, 143:
442-448.
Hasey G, Hanin I (1990): Plasma corticosterone is increased and
correlated with brain acetylcholine in physostigmine- but not
in neostigmine-treated rats. Psychoneuroendocrinology 15:
357-369.
Heuser IJ, Gotthardt U, Schweiger U, Schmider J, Lammers C-H,
Dettling M, Holsboer F (1994): Age-associated changes of
pituitary-adrenocortical hormone regulation in humans: Im-
portance of gender. Neurobiol Aging 15:227-231.
Kjaer A, Knigge U, Flemming WB, Warben J (1992): Histamine-
and stress-induced secretion of ACTH and [3-endorphin:
Involvement of corticotropin-releasing hormone and vaso-
pressin. Neuroendocrinology 56:419-428.
Knapp MJ, Knopman DS, Solomon PR, Pendlebury WW, Davis
CS, Gracon SD (1994): A 30 week randomized controlled
trial of high dose tacrine in patients with Alzheimer's disease.
JAMA 271:985-998.
Landfield P, Baskin R, Pitler T (1981): Brain aging correlates:
Retardation by hormonal-pharmacological treatments. Sci-
ence 214:581-584.
Lewis DA, Sherman BA, Kathol RG (1984): Analysis of the
BIOL PSYCHIATRY 67
1996:40:61 68
specificity of physostigmine stimulation of adrenocortico-
tropin in man. J Clin Endocrinol Metab 58:570-573.
Maeda K, Tanimoto K, Terada T, Shintani T, Kakigi TA (1991):
Elevated urinary free cortisol in patients with dementia.
Neurobiol Aging 12:161-163.
Martignoni E, Petraglia F, Costa A, Bono G, Genazzini AR,
Nappi G (1990): Dementia of the Alzheimer type and
hypothalamus-pituitary-adrenocortical axis: Changes in cere-
brospinal fluid corticotropin releasing factor and plasma
cortisol levels. Acta Neurol Scand 81:452-456.
Masugi T, Ogihara T, Sakaguchi K, Otsuka A, Tsuchiya Y,
Morimoto S, Kumahara Y, Saeki S, Nishide M (1989): High
plasma levels of cortisol in patients with senile dementia of
the Alzheimer's type. Meth Find Exp Clin Pharmacol 11:
707-710.
McKhann G, Drachman D, Folstein M, Katzman R, Price D,
Stadlan EM (1984): Clinical diagnosis of Alzheimer's dis-
ease. Neurology 34:939-944.
Miller AH, Sastry G, Speranza AJ Jr, Lawlor BA, Mohs RC,
Ryan TM, Gabriel SM, Serby M, Schmeidler J, Davis KL
(1994): Lack of association between cortisol hypersecretion
and nonsuppression on the DST in patients with Alzheimer's
disease. Am J Psychiatry 151:267-270.
Peskind ER, Raskind MA, Wingerson D, Pascualy M, Thai L J,
Dobie DJ, Veith RC, Dorsa DM, Murray S, Sikkema C, Galt
SA, Wilkinson CW (1995): Enhanced hypothalamic pituitary
adrenocortical axis responses to physostigmine in normal
aging. J Gerontol 50A:M114-M120,
Radant A, Peskind ER, Wilkinson CW, Veith RC, Dorsa DM,
Leake RD, Ervin MG, Raskind MA (1992): Neurohypophy-
seal and pituitary-adrenocortical responses to the alpha~
agonist methoxamine in man. Neuroendocrinology 55:361-
366.
Raskind MA, Peskind E, Rivard MF, Veith R, Barnes R (1982):
Dexamethasone suppression test and cortical circadian
rhythm in primary degenerative dementia. Am J Psychiatry
13:1468-1471.
Raskind MA, Peskind ER, Veith RC, Risse SC, Lampe TH,
Borson S, Gumbrecht G, Dorsa DM (1989): Neuroendocrine
responses to physostigmine in Alzheimer's disease. Arch Gen
Psychiatry 46:535-540.
Raskind MA, Peskind ER, Veith RC, Wilkinson CW, Federighi
D, Dorsa DM (1990): Differential effects of aging on neu-
roendocrine responses to physostigmine in normal men. J
Clin Endocrinol Metab 70:1420-1425.
Resnick NM, Greenspan SL (1989): "Senile" osteoporosis re-
considered. JAMA 261:1025-1029.
Risch SC, Kalin NH, Janowsky DS, Cohen RM, Pickar D,
Murphy DL (1983): Corelease of ACTH and [3-endorphin
immunoreactivity in human subjects in response to central
cholinergic stimulation. Science 222:77-79.
Rolandi E, Gandolfo C, Franceschini R, Cataldi A, Garibaldi A,
Barreca T (1992): Twenty-four-hour beta-endorphin secre-
tory pattern in Alzheimer's disease, Neuropsychobiology
25:188-192.
Sapolsky RM, Krey LC, McEwen B (1985): Prolonged glucocor-
ticoid exposure reduces hippocampal neuron number: Impli-
cations for aging. J Neurosci 5:1222-1227.
Sapolsky RM, Uno H, Rebert CS, Finch CE (1990): Hippocam-
68 BIOL PSYCHIATRY
1996;40:61-68
pal damage associated with prolonged glucocorticoid expo-
sure in primates. J Neurosci 10:2897-2902.
Spar JE, Gerner R (1982): Does the dexamethasone suppression
test distinguish dementia from depression? Am J Psychiatry
139:238-240.
Stein-Behrens B, Mattson MP, Chang I, Yeh M, Sapolsky R
(1994): Stress exacerbates neuron loss and cytoskeletal pa-
thology in the hippocampus. J Neurosci 14:5373-5380.
Weinberger DR, Gibson R, Coppola R, Jones DW, Molchan S,
Sunderland T, Berman KF, Reba RC (1991): The distribution
of cerebral muscarinic acetylcholine receptors in vivo in
patients with dementia. Arch Neurol 48:169-176.
E.R. Peskind et al
Whitehouse PJ, Price DL, Struble RG, Clark AW, Coyle JT,
DeLong MR (1982): Alzheimer's disease: Loss of neurons in
the basal forebrain. Science 215:1237-1239.
Whitehouse PJ, Martino AM, Antuono PG, Lowenstein
PR, Coyle JT, Price DL, Kellar KJ (1986): Nicotinic acetyl-
choline binding sites in Alzheimer's disease. Brain Res 371:
146-151.
Winker MA (1994): Tacrine for Alzheimer's disease: Which
patient, what dose? JAMA 271:1023-1024.