State Key Laboratory of Drug Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai, P.R. China
(J.W.Y., L.M.W., and X.C.T.); and
Kunming Institute of Zoology, Chinese
Academy of Sciences, Kunming, Yunnan, P.R. China (J.X.C.)
Our previous studies demonstrated that huperzine A, a reversible and
selective acetylcholinesterase inhibitor, exerts beneficial effects on
memory deficits in various rodent models of amnesia. To extend the
antiamnesic action of huperzine A to nonhuman primates, huperzine A was
evaluated for its ability to reverse the deficits in spatial memory
produced by scopolamine in young adult monkeys or those that are
naturally occurring in aged monkeys using a delayed-response task.
Scopolamine, a muscarinic receptor antagonist, dose dependently
impaired performance with the highest dose (0.03 mg/kg, i.m.) producing
a significant reduction in choice accuracy in young adult monkeys. The
delayed performance changed from an average of 26.8/30 trials correct
on saline control to an average of 20.2/30 trials correct after
scopolamine administration. Huperzine A (0.01-0.1 mg/kg, i.m.)
significantly reversed deficits induced by scopolamine in young adult
monkeys on a delayed-response task; performance after an optimal dose
(0.1 mg/kg) averaged 25.0/30 correct. In four aged monkeys, huperzine A
(0.001-0.01 mg/kg, i.m.) significantly increased choice accuracy from
20.5/30 on saline control to 25.2/30 at the optimal dose (0.001 mg/kg
for two monkeys and 0.01 mg/kg for the other two monkeys). The
beneficial effects of huperzine A on delayed-response performance were
long lasting; monkeys remained improved for about 24 h after a
single injection of huperzine A. This study extended the findings that huperzine A improves the mnemonic performance requiring working memory
in monkeys, and suggests that huperzine A may be a promising agent for
clinical therapy of cognitive impairments in patients with Alzheimer's disease.
 |
Introduction |
Alzheimer's
disease (AD) is a slowly progressive neuropsychiatric illness,
principally characterized by memory deficits. There is a substantial
body of experimental work in animals and humans suggesting that the
cholinergic mechanism plays an essential role in AD (Davies and
Maloney, 1976
; Bartus et al., 1982; Coyle et al., 1983).
Dysfunction in cholinergic mechanisms may contribute to age-related
memory impairments. The retrograde loss of the cholinergic system from
the basal forebrain is the most common and the most severe
neurochemical consequence of the disease (Susan, 1997). The
cholinergic neuron clusters of the basal forebrain innervate the
hippocampus and areas of association in the cortex involved in higher
processes such as long-term memory, working memory, and attention. In
these structures, the concentration of choline acetyltransferase (ChAT)
decreased, accompanied by the impaired ability of high-affinity choline
transport and synthesis of acetylcholine (ACh). The severity of memory
impairments seen in AD is consistent with dysfunction of the
cholinergic system (Coyle et al., 1983). Many attempts have been made
to correct the cholinergic deficiency at various levels of cholinergic
functioning to reduce, if not cure, some of the major cognitive
disturbances of AD patients. Some cholinomimetic agents have been shown
to improve age-related cognitive impairments. Among various
cholinomimetic drugs, the acetylcholinesterase (AChE) inhibitor as a
palliative agent in the treatment of AD has been the most promising so
far (Parnetti et al., 1997). Physostigmine and tacrine have shown some
clinical efficacy in AD patients (Mohs et al., 1985; Summers et al.,
1986). They are not, however, ideal drugs for clinical use due to the
short duration of action, the low bioavailability, and the frequent
side effects with physostigmine (Winblad et al., 1991) and
dose-dependent hepatotoxicity of tacrine (Watkins et al., 1994).
Thus the search for a new cholinesterase inhibitor (ChEI) with
properties that could overcome the limitations in the use of
physostigmine and tacrine is still ongoing (Giacobini, 1997).
In addition to the central cholinergic system, other transmitter
systems such as the monoaminergic system are thought to participate in
causing dementia in AD patients (Palmer and DeKosky, 1993). There is
evidence of interaction between cholinergic and monoaminergic systems
in the control of cognitive cortical function (Riekkinen et al., 1990).
The positive clinical effect of ChEIs such as tacrine has been related
to stimulation of both cholinergic and monoaminergic systems (Alhainen
et al., 1993). Therefore, the nootropic effects of ChEIs may involve
cholinergic mechanisms as well as monoaminergic mechanisms.
Huperzine A, a Lycopodium alkaloid isolated from the Chinese
herb Huperzia serrata (Thunb) Trev, is a reversible and
selective AChE inhibitor. The experiments showed that huperzine A can
produce a long-term inhibition of AChE activity in rat brains and a
sustained increase of ACh levels in the central nervous system (Tang et al., 1989). Compared with physostigmine, tacrine, and galanthamine, the
AChE inhibitory effect of huperzine A is more potent, its selectivity
for AChE other than butyrylcholinesterase is better, and its duration
of inhibition is longer; its bioavailability is higher but the side
effects are less (for review see Tang, 1996). It has been reported that
huperzine A can produce a dose-dependent increase of other transmitters
such as norepinephrine (NE) and dopamine (DA) in the rat cortex with
either systemic or local intracerebral administration (Zhu and
Giacobini, 1995). The previous studies in rodents showed that huperzine
A improves performance in a variety of paradigms including spatial
memory tasks (Tang, 1996) such as Y-maze (Tang et al., 1986; Lu et al.,
1988) and the radial-arm maze (Xiong and Tang, 1995; Cheng et al.,
1996). The duration of improving effects of huperzine A on learning and memory retention processes was longer than that of physostigmine or
tacrine (Tang et al., 1994).
Scopolamine, a muscarinic receptor antagonist, has been shown in
numerous studies to impair learning and memory under a variety of
testing conditions, not only in small animals (Spencer and Lal, 1973),
but also in monkeys (Ogura and Aigner, 1993; Rupniak et al., 1989) and
in human (Ghoneim and Mewaldt, 1977; Rusted and Warburton, 1988); some
of these impairments reflect neuropsychological similarities with the
demented states in patients with AD (Molchan et al., 1992). The aged
monkey is also a good candidate for studies of AD, because its
behavioral impairments are similar to those that are characteristic of
elderly human (Bartus, 1979). In particular, the similarities of
neurochemical changes in aged monkeys with those in humans indicate
that the aged monkeys may be a useful model for investigation of the
age-associated transmitter abnormalities which are similar to those
that occur in human (Wenk et al., 1989).
The aim of this study was to extend the findings as to whether
huperzine A can improve the memory impairments in aged monkeys with a
naturally occurring ACh decrease and in young monkeys with an
experimental disruption of cholinergic system using scopolamine. The
chemical structure of huperzine A is shown in Fig.
1.
| |
Materials and Methods |
Subjects.
The subjects in this study consisted of eight
rhesus monkeys (Macaca mulatta). Four young female
monkeys (three were approximately 6-7 years old; one was approximately
4 years old) were used to evaluate the effect of huperzine A on
scopolamine-induced memory impairments. The four aged monkeys (two
females and two males approximately 16-18 years old) were used to
study the effect of huperzine A on age-related memory deficits. Because
actual birth dates were unavailable, ages were estimated on the basis
of prior breeding and behavioral testing records, dental records, and
general appearance. Rhesus monkeys in captivity have been reported to live 20 to 25 years and longer. All young adult subjects were drug-naive, whereas all aged subjects had prior behavioral testing experience, but none had been involved in drug tests in the 1 year
preceding the present investigation. All animals were housed individually under standard laboratory conditions. Feeding occurred immediately after cognitive testing. Daily supplements of fruits and
vitamins were also given. Water was available ad libitum.
Delayed-Response Testing.
Monkeys were tested in the
Wisconsin General Testing Apparatus. The test tray contained a left and
a right food well spaced 15 cm apart. An opaque screen was lowered to
separate the monkey from the test tray. For testing sessions, the test
panel was attached to the home cage. While testing was in progress, the
light in panel was on so that the monkey could see clearly what
happened in the panel. Highly palatable food rewards (e.g., peanuts,
raisins, or sugar chips) were used during testing to minimize the need for dietary regulation. The monkeys were tested daily at the same time
of day in a quiet room by a trained observer. Using these conditions,
no problems with motivation were evident.
The monkeys had previously been trained on the two-well,
delayed-response task. During delayed response, the animal watched as
the experimenter baited one of two food wells. The food wells were then
covered with identical cardboard plaques, and an opaque screen was
lowered between the animal and the test tray for a specified delay. At
the end of this delay, the screen was raised and the animal was allowed
to choose. Reward was quasi-randomly distributed between the left and
right wells over the 30 trials that made up a daily test session.
During the initial training phase, delays were held constant during a
daily session and were gradually increased from 0 s according to a
step-wise procedure over the 1000 trials.
Following the 1000 trials, the monkeys were prepared for drug testing.
To observe the effects of drug on memory capacity, the animals were
trained on a variable delayed-response task in which five different
delay lengths were distributed over the 30 trials that made up the
daily test session. For four aged monkeys, delays were adjusted until
the animals exhibited stable baseline performance of approximately 67%
correct. For example, the range of delays for aged monkey no. 35 was 0, 6, 12, 18, and 24 s. All aged subjects performed perfectly at 0-s
delays and exhibited increasing difficulty with progressively longer
delays, a pattern consistent with memory impairment. In young adult
monkeys, delays were chosen to produce performance levels of about 90%
correct out of 30 trials. For example, the range of delays was 0 to
20 s for monkey no 19 and 0 to 8 s for monkey no. 36. The 0-s
delay consisted of lowering the screen and immediately raising it
again. Once performance was demonstrated to be stable at this baseline, drug treatment was initiated.
Drug Administration.
Huperzine A (provided by the Department
of Phytochemistry, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences) and scopolamine hydrobromide (Sigma Chemical Co.,
St. Louis, MO) were both dissolved in sterile 0.9% saline before
injection. Scopolamine or saline and huperzine A or saline were
injected i.m. 30 min and 20 min, respectively, before delayed-response
testing. The injection volume was kept constant at 0.1 ml/kg
irrespective of dose.
The doses of scopolamine were 0.01, 0.02, and 0.03 mg/kg; huperzine A
was coadministered with the highest dose of scopolamine (0.03 mg/kg) to
young adult monkeys. At this dose of scopolamine, all the young
subjects exhibited significant memory impairments so that there was
enough room to test the effects of huperzine A. The doses of huperzine
A were 0.001, 0.01, 0.1, and 0.2 mg/kg for young subjects and 0.0001, 0.001, 0.01, and 0.1 mg/kg for aged subjects. A wide range of doses was
selected to ensure the optimal dose within it for each monkey.
Drugs were administered no more than twice per week (Monday-Saturday),
and at least 3 days separated test sessions. Control injections of
saline alone were given the day before each drug testing to assure that
the performance was back to the baseline level. The experimenter
testing the monkeys was unaware of the drug treatment conditions.
Data Statistics.
Delayed-response performance on drug was
compared with matched placebo (saline) control performance for the same
week. Because the animals served as their own controls, statistical
analyses employed repeated measures designs: one-way analysis of
variance with repeated measures (1-ANOVA-R), and, if appropriate,
followed by post hoc tests. The level of significance was
P < .05.
| |
Results |
Effects of Scopolamine on Delayed-Response Performance in Young
Adult Monkeys.
Scopolamine at the doses of 0.01, 0.02, and 0.03 mg/kg produced a dose-related impairment in the performance of young
monkeys [1-ANOVA-R: F(3,9) = 6.66, P = .0116]
with the highest dose of scopolamine causing a significant disruption
of choice accuracy in all of the young animals [F(1,3) = 43.78, P = .0070] (Fig. 2).
After the 0.03-mg/kg dose, performance at all retention intervals was
impaired but the magnitude of this effect increased as the retention
interval lengthened (Fig. 3) [9.7 ± 4.7%, 10.3 ± 7.2%, 25.7 ± 8.7%, 30.5 ± 14%,
and 33.3 ± 2.5% decreases at A (0 sec), B, C, D, and E delays
respectively]. 1-ANOVA-R suggested no significant effect of
scopolamine at 0-s delays [F(1,3) = 4.42, P = .1263], but significant effect was found at the longest delays
[F(1,3) = 182.65, P = .0009]. At the highest dose
of scopolamine (0.03 mg/kg), some signs of the side effects of
cholinergic antagonism, such as a slower rate of chewing than usual and
pupillary dilation, were observed.
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Fig. 2.
Effects of scopolamine on delayed response task in
young adult monkeys (n = 4). Saline or scopolamine
administered i.m. 30 min before testing. Values represent mean ± S.E.M. number of trials correct out of a possible 30 trials.
*p < .05 versus saline control.
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Fig. 3.
Effects of scopolamine (0.03 mg/kg,  ) compared
with saline ( ) on delayed response performance for each of five
delay intervals (A, B, C, D, and E) used in each testing session in
young adult monkeys (n = 4). The A delay was always
0 s; the B, C, D, and E delays were incrementally increasing
delays (e.g., 5, 10, 15, and 20 s) individually selected for each
animal to produce an overall baseline performance of about 67% correct
(see Materials and Methods). Values represent mean ± S.E.M. number correct out of a possible six trials at each delay
interval.
|
|
Effects of Huperzine A on Scopolamine-Induced Deficit of Delayed
Performance in Young Adult Monkeys.
Huperzine A markedly improved
the delayed-response performance of scopolamine-treated monkeys (Fig.
4A) [1-ANOVA-R: F(4,12) = 14.3, P = .0002]. The dose-response curve was
bell-shaped with the maximum improvements at 0.1 mg/kg [15 ± 2.9% increase, 1-ANOVA-R: F(1,3) = 27.0, P = .0138, compared with scopolamine control]. Neither the lowest nor the
highest doses had effects [F(1,3) = 2.45, P = .23 for 0.001 mg/kg; F(1,3) = 2.46, P = .21 for 0.2 mg/kg] (e.g., young monkey no. 36, Fig. 4B). The beneficial effects of
huperzine A were most evident at the longest delays [27.1 ± 2.5% increases, F(1,3) = 10.50, P = .048].
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Fig. 4.
Effects of huperzine A on the deficit of
delayed-response performance induced by scopolamine in young adult
monkeys. Saline or huperzine A was administered i.m. 20 min before
testing. Scopolamine was administered 30 min before testing. A,
huperzine A produced a dose-related improvement in the delayed-response
performance of young monkeys (n = 4). Values
represent mean ± S.E.M. number of trials correct out of a
possible 30 trials. +p < .05 versus saline
control; *p < .05 versus scopolamine control. B,
effects of monkey no. 36. Values represent mean number of trials
correct out of a possible 30 trials.
|
|
Effects of Huperzine A on Delayed-Response Performance in Aged
Monkeys.
Administration of huperzine A to aged monkeys produced a
significant effect on delayed-response performance [1-ANOVA-R: F(4,12) = 11.26, P = .0005]. As can be seen in Fig.
5A, huperzine A produced a bell-shaped
dose-response curve similar to the one described above in
scopolamine-treated young monkeys. There were variances between the
performance of four aged subjects. Of these four doses (0.0001
0.1
mg/kg), the best dose was 0.001 mg/kg for two monkeys, and for the
remaining two animals the best dose was 0.01 mg/kg (e.g., monkey no.
34, Fig. 5B). The improvements following the best doses were most
apparent at two longer delays (Fig. 6)
[F(1,3) = 16.94, P = .026; F(1,3) = 12.63, P = .0380, respectively]. However, performance at
0-s delays did not change between monkeys on saline and on huperzine A
[F(1,3) = .36, P = .59]. These results are consistent with changes in cognitive performance rather than a nonspecific performance variable, which would be expected to disrupt performance after the 0-s delay control trials.
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Fig. 5.
Effects of huperzine A in aged monkeys. Saline or
huperzine A was administered i.m. 20 min before testing. A, huperzine A
produced a dose-related improvement in the delayed-response performance
of aged monkeys (n = 4). Values represent mean ± S.E.M. number of trials correct out of a possible 30 trials. B,
effects of huperzine A in monkey no. 32. Values represent mean number
of trials correct out of a possible 30 trials.
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Fig. 6.
Effects of huperzine A at the best doses compared
with saline control on delayed-response performance for each of the
five delayed intervals (A, B, C, D, and E) used in each testing session
in aged monkeys (n = 4). The number of trials
correct on saline was subtracted from the number of trials correct on
huperzine A; this difference score was then multiplied by 3.3% because
each trials constituted 3.3% of the total number of trials: [(number
correct huperzine A - number correct saline) × 3.3%]. Values in the
figure represent mean ± S.E.M. *p < .05 versus saline control.
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During the sessions conducted 24 h after huperzine A injection at
the dose of 0.01 mg/kg and 0.1 mg/kg, the improving performance remained evident (Fig. 7). Moreover,
these long-lasting beneficial effects were possibly dose dependent [24
h after 0.1 mg/kg, 10.8 ± 0.82% increases F(1,3) = 172.166, P = .0010; 24 h after 0.01 mg/kg, 5.5 ± 2.23% increases, F(1,3) = 12.24, P = .0395]. But performance had returned to baseline level by the sessions conducted 48 h after injection [F(1,3) = 1.47, P = 1.000;
F(1,3) = 3.000, P = .1817, respectively]. Huperzine A
was tolerated by all the monkeys even at the highest doses. No adverse
signs were observed.
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Fig. 7.
Effects of huperzine A [0.1-0.01 mg/kg] on
delayed-response tasks in aged monkeys (n = 4).
Performance was measured 20 min and 24 and 48 h after i.m.
injection of huperzine A, respectively. The number of trials correct on
saline was subtracted from the number of trials correct on huperzine A;
this difference score was then multiplied by 3.3% because each trials
constituted 3.3% of the total number of trials: [(number correct
huperzine A - number correct saline) × 3.3%]. Values in the figure
represent mean ± SEM. *p < .05 versus saline
control.
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| |
Discussion |
Scopolamine has been used as a pharmacological tool for
understanding pathological impairments such as AD, because it produces amnesiac effects similar to those identified in AD (Sakhakian et al.,
1987). In this study, scopolamine dose dependently impaired spatial working memory of young adult monkeys in delayed-response tasks, consistent with the previous reports using other paradigms of
delayed-response tasks in monkeys (Rupniak et al., 1989; Ogura and
Aigner, 1993), indicating that spatial working memory processes are
dependent upon the integrity of the brain cholinergic system. The
capacity to perform these tasks requires the bilateral integrity of the
dorsolateral prefrontal cortex (PFC) at both short and long delays and
the hippocampus mainly at long delays (>15 sec) (Goldman-Rakic, 1987),
which receive a massive projection of cholinergic axons originating in
basal forebrain. So reduced-choice accuracy caused by scopolamine is
due to disruption of the cholinergic system in PFC and the hippocampus
through blockade of muscarinic postsynaptic receptors in synaptic
clefts. The fact that there was a more significant decrease at longer
delays than at shorter delays, although choice accuracy at all
retention intervals decreased after scopolamine, showed that the major
effects appear to be directly on memory processes.
Huperzine A significantly improved the performance of
scopolamine-treated monkeys, producing a bell-shaped dose-response
curve, similar to previous findings in rodents (Xiong and Tang, 1995; Cheng et al., 1996). The bell-shaped dose-response curve is common with
most drugs that have been reported to exert cognitive enhancing actions; the precise mechanisms of this effect remain to be
established. Smaller doses of huperzine A stimulate cognitive function
through increasing ACh levels, whereas larger doses mask it via an
unknown mechanism. Huperzine A produced AChE inhibition in whole brain or brain regions in a dose-dependent manner following peripheral administration (Tang et al., 1994; Wang and Tang, 1998). Because huperzine A shows no significant affinity for muscarinic receptors (Tang et al., 1989), no evident pre- and postsynaptic effects (Lin et
al., 1997), and no effect on ChAT (Tang et al., 1994), its effects on
spatial memory in tasks are due primarily to the dose-dependent
increase in ACh resulting from direct AChE inhibition. The degree of
ACh elevation after huperzine A is selectively highest in frontal and
parietal cortex and there are smaller increases in other brain regions
(Tang et al., 1989). Considering that ACh is particularly low in the
cerebral cortex of AD patients (Bowen et al., 1983), this particularly
regional specificity may constitute a therapeutic advantage.
It has been observed that in aged monkeys the number of nicotinic and
muscarinic type-1 receptors decreased only slightly with aging, which
suggests that postsynaptic indicators of cholinergic function are only
mildly impaired with aging. However, the level of ChAT decreased
significantly with aging (Wenk et al., 1989). It is mainly because of
the loss of cholinergic neurons in forebrain basal nucleus, which
projected into the PFC and hippocampus. These age-related changes may
underlie a decline in cognitive abilities. Similar to the results found
in scopolamine-treated monkeys, the improving efficacy of huperzine A
in aged monkeys also exhibited a bell-shaped response. However, the
optimal dose is smaller than that for scopolamine-treated monkeys.
High-choice accuracy at 0-s delays (about 95%) and unaffected visual
discrimination (Bartus and Dean, 1979) shows that the deficits in aged
monkeys did not appear to be due to problems with perceptual sensory
processing but with memory. The increased ACh concentration induced by
huperzine A supplemented the impaired ability of ACh synthesis caused
by the decreased level of ChAT, so that the cholinergic transmission is
restored close to normal in a certain period. On the other hand,
biochemical, electrophysiological, and behavioral studies have
indicated an interaction between the cholinergic and noradrenergic systems, as well as the dopaminergic system (Decker and McGaugh, 1991),
which influences learning and memory (Kruglikov, 1982). After
administration of huperzine A, NE and DA levels were significantly increased over baseline for several hours, whereas ACh levels reach a
maximum. These increases in NE and DA levels may be related to the
increase of extracellular ACh levels through subcortical mechanism (Zhu
and Giacobini, 1995). Therefore, the effects of huperzine A on memory
may be associated with the increased levels of NE and DA, which
decreased significantly with aging in monkeys (Wenk et al., 1989).
In this study, the performance of all aged subjects after
administration of huperzine A was improved. But wide variations in the
most-effective dose of huperzine A in aged subjects were observed. This
finding is consistent with various levels of loss in the cholinergic
system in aged monkeys, suggesting that in clinical therapy the optimal
doses of huperzine A must be selected according to pathological
situation of the patients with AD. Their overall response as a group,
however, was less variable than that of physostigmine-treated aged
monkeys in earlier studies (Bartus, 1979), in which some subjects did
not benefit from physostigmine at all. The performance of all aged
subjects after administration of huperzine A was improved. In the
present study, a consistent finding was the long-lasting effect of
huperzine A on delayed-response tasks. The performance was
significantly different as compared with saline control even 24 h
after a single injection of huperzine A (0.1, 0.01 mg/kg) and
showed a possible dose-dependent manner. The facts that the terminal
half-life of huperzine A was 288 min in humans (Qia et al., 1995), and
that huperzine A could produce a long-term inhibition of AChE activity
in brain (Wang and Tang, 1998), suggested that the long-lasting effects
of huperzine A on cognitive function might simply result from its AChE inhibition.
Compared with rodents, rhesus monkeys may be more sensitive to
pharmacological manipulation of central cholinergic systems (Matsuoka
and Aigner, 1997). In addition, primates are able to perform many
complex behavioral tasks identical to those impaired in human amnesiac
states, including dementia (Freedman and Oscar-Berman, 1986; Sakhakian
et al., 1988), in which the delayed-response performance is commonly
used to test the mental status of nonhuman primates. Memory-impaired
humans show significant performance deficits when tested by this kind
of task (Rice, 1987), and the fact that these tasks are sensitive to
the impairments associated with human memory loss, supports the
validity of using nonhuman primates performing delayed-response tasks
as models for development of drugs designed to improve human memory.
Huperzine A could improve memory deficits either induced by scopolamine
in young adult monkeys or occurring naturally in aged monkeys. These
findings extend our previous studies in rodents, in which huperzine A
markedly reversed the memory impairments induced by central cholinergic
blockade with scopolamine treatment, lesions of the nucleus basalis
magnocellularis, or aging (Lu et al., 1988; Cheng et al., 1996; Xiong
et al., 1998). Taken together, these results can confirm that huperzine
A is a promising candidate for clinical evaluation as treatment for AD.
We acknowledge professor Da Yuan Zhu for preparation of
huperzine A and Hua Xian Zhang for technical assistance in testing the monkeys.
AD, Alzheimer's disease;
ChEI, cholinesterase
inhibitor;
ChAT, choline acetyltransferase;
NE, norepinephrine;
DA, dopamine;
PFC, prefrontal cortex;
1-ANOVA-R, one-way analysis of
variance with repeated measures.
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Abstract
Introduction
