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Proc Natl Acad Sci U S A. 2001 February 13; 98(4): 2089–2094. Published online 2001 February 6. | PMCID: PMC29386 |
Copyright © 2001, The National Academy of Sciences Psychology Galantamine: Effect on nicotinic receptor binding,
acetylcholinesterase inhibition, and learning Diana S. Woodruff-Pak,*†‡ Richard W. Vogel, III,* and Gary L. Wenk§ *Research and Technology Development, Albert Einstein Healthcare Network, Philadelphia, PA 19141; †Departments of Psychology and Diagnostic Imaging, Temple University, Philadelphia, PA 19122; and §Arizona Research Laboratories, Division of Neural Systems, Memory, and Aging, University of Arizona, Tucson, AZ 85724 Received November 1, 2000; Accepted December 11, 2000. Classical eyeblink conditioning is a well-characterized model
paradigm that engages the septohippocampal cholinergic system. This
form of associative learning is impaired in normal aging and severely
disrupted in Alzheimer's disease (AD). Some nicotinic cholinergic
receptor subtypes are lost in AD, making the use of nicotinic
allosterically potentiating ligands a promising therapeutic strategy.
The allosterically potentiating ligand galantamine (Gal) modulates
nicotinic cholinergic receptors to increase acetylcholine release as
well as acting as an acetylcholinesterase (AChE) inhibitor. Gal was
tested in two preclinical experiments. In Experiment 1 with 16 young
and 16 older rabbits, Gal (3.0 mg/kg) was administered for 15 days
during conditioning, and the drug significantly improved learning,
reduced AChE levels, and increased nicotinic receptor binding. In
Experiment 2, 53 retired breeder rabbits were tested over a 15-wk
period in four conditions. Groups of rabbits received 0.0 (vehicle),
1.0, or 3.0 mg/kg Gal for the entire 15-wk period or 3.0 mg/kg Gal
for 15 days and vehicle for the remainder of the experiment. Fifteen
daily conditioning sessions and subsequent retention and relearning
assessments were spaced at 1-month intervals. The dose of 3.0 mg/kg
Gal ameliorated learning deficits significantly during acquisition and
retention in the group receiving 3.0 mg/kg Gal continuously.
Nicotinic receptor binding was significantly increased in rabbits
treated for 15 days with 3.0 mg/kg Gal, and all Gal-treated rabbits
had lower levels of brain AChE. The efficacy of Gal in a learning
paradigm severely impaired in AD is consistent with outcomes in
clinical studies. It has long been
established that acetylcholine neurotransmission plays a crucial role
in learning and memory, and more recently, the cholinergic system has
been the focus of treatment for memory impairment in Alzheimer's
disease (AD). The demonstrated role of acetylcholine in modulating the
rate of learning in eyeblink classical conditioning in rabbits ( 1)
makes this model system useful in preclinical investigations of
cognition-enhancing drugs ( 2). More is known about the neural
structures and systems that are involved in eyeblink classical
conditioning than about any other learning and memory task. Although
the neural circuitry essential for acquisition and retention of the
conditioned eyeblink response resides in the cerebellum ( 3), the
hippocampus is engaged during delay eyeblink classical conditioning
( 4). In the delay procedure, a neutral stimulus such as a tone
conditioned stimulus (CS) is presented half a second before the onset
of a corneal airpuff eyeblink-eliciting unconditioned stimulus (US).
The organism learns to blink to the tone CS before the onset of the
airpuff US, and the learned response is called the conditioned response
(CR). It is our working hypothesis that selective loss of hippocampal
pyramidal cells ( 5) and disruption of the septohippocampal cholinergic
system in AD ( 6) impairs acquisition of delay eyeblink classical
conditioning in AD beyond the impairment observed in normal aging. The
hypothesis was supported ( 7, 8) and independently replicated ( 9). Audioradiographic and histochemical studies of human brain tissue
collected postmortem ( 10– 13) and brain imaging studies in living AD
patients ( 14) demonstrated specific loss of nicotinic cholinergic
receptors and almost complete sparing of muscarinic cholinergic
receptors in AD. Identification of nicotinic cholinergic receptors as
the receptors impaired in AD led us to test a nicotinic cholinergic
antagonist and nicotinic agonists in the animal model of eyeblink
classical conditioning. By using a very low-dosage level of
mecamylamine in young rabbits so that nicotinic cholinergic receptors
would be selectively inhibited, we demonstrated a role for nicotinic
cholinergic receptors in eyeblink conditioning because the acquisition
of CRs was severely disrupted ( 15). A synthesized analog of the marine
natural product anabasine ( 16) called GTS-21
[3-(2,4-dimethoxybenzylidene)anabaseine] has been found to
preferentially interact with α7 neuronal nicotinic receptors. Several
doses of GTS-21 were administered to older rabbits, and this drug
enabled older animals to produce significantly more CRs than did
vehicle-treated older rabbits ( 17). Administration of nicotinic cholinergic agonists has promise in
the treatment of cognition impairment in AD, but there are also some
problems with this therapeutic strategy. It is difficult to establish
the appropriate dose of a nicotinic cholinergic agonist, as higher-dose
levels may cause desensitization rather than increased activation of
nicotinic receptors ( 18). Additional problems include drug transport to
the targeted nicotinic cholinergic receptors and the target selectivity
of the receptor subtype. An alternative approach to drug treatment in
AD is the application of allosteric modulators of nicotinic receptors
( 18, 19). Allosteric modulators are drugs that interact with the
receptor through binding sites that are distinct from those for
acetylcholine and nicotinic agonists and antagonists. Because these
modulators are not directly involved in the neurotransmission process
they affect, they typically do not induce compensatory processes that
the agonists and antagonists induce. Thus, problems such as receptor
desensitization and down-regulation of expression can be avoided with
allosteric modulators. AD has been associated with a deficit in nicotinic cholinergic
neurotransmission. A means to up-modulate or potentiate the channel
activity of nicotinic receptors in response to acetylcholine is to use
allosterically potentiating ligands (APLs). Representative nicotinic
APLs are the plant alkaloids physostigmine, galanthamine, and codeine
and the neurotransmitter serotonin ( 20). Structural properties of APLs
are different from the structural properties of inhibitors of the
enzyme acetylcholinesterase (AChE), the type of drugs currently
approved to treat cognition impairment in AD. Compared with
conventional AChE inhibitors, galantamine (Gal) produces relatively
less AChE inhibition. Codeine does not interact with AChE at all. In
the covalent AChE inhibitor, physostigmine, removal of the carbamate
function has no effect on potency as an APL, but this treatment reduces
significantly the potency of physostigmine's AChE inhibition ( 20). The
category of APLs has been limited to physostigmine, galanthamine,
codeine, and serotonin on the basis of functional properties tested
with nicotinic cholinergic agonists and antagonists ( 20). Functionally
unique features of APLs include the ability to induce single-channel
activity indistinguishable from single-channel activity induced by
acetylcholine. Having demonstrated that the nicotinic cholinergic drug GTS-21
ameliorated learning deficits in older rabbits, we wanted to determine
whether the dual action of an APL would have even greater efficacy in
the classical eyeblink-conditioning model paradigm. A nicotinic APL,
Gal, was tested at doses of 0.0, 1.0, 2.0, 3.0, and 4.0 mg/kg ( 21).
In 10 daily sessions, 40 older rabbits were tested in the 750-ms
delay-conditioning paradigm. A dose of 3 mg/kg Gal was extremely
effective in improving conditioning in older rabbits, enabling them to
achieve learning criterion rapidly and to produce a very high
percentage of CRs. Trials to learning criterion, a measure that is
larger when learning is poorer, revealed a classical U-shaped response
curve with doses of 1.0 and 2.0 mg/kg Gal producing nonsignificant
effects over vehicle-treated rabbits, a dose of 3.0 mg/kg Gal
reducing the number of trials to learning criterion to a mean
significantly lower than vehicle-treated rabbits, and 4.0 mg/kg Gal
producing a nonsignificant effect. Older rabbits treated with 3.0
mg/kg Gal achieved learning criterion 40% faster than older rabbits
tested with the optimal dose of GTS-21. Results with a dose of 3.0 mg/kg Gal were striking, but they were
observed in a relatively small sample ( 21). We undertook the present
experiments to explore further the effect of 3.0 mg/kg Gal on
learning. There were three major aims: ( i) to examine
behavioral and pharmacological effects of the 3.0-mg/kg dose of Gal
by testing the drug in young as well as older rabbits; ( ii)
to compare behavioral and pharmacological effects of Gal in larger
groups of older rabbits at a dose that affected eyeblink conditioning
in a 2-wk experiment (3.0 mg/kg) and a dose that was not different in
its behavioral effect from vehicle (1.0 mg/kg); and ( iii)
to compare behavioral and pharmacological effects of short-term (3 wk
of 5 daily injections per wk) versus longer-term (15 wk of 5 daily
injections per wk) administration of 3.0 mg/kg Gal. We examined the
effects of Gal in older rabbits over a time period (15 wk) that would
simulate a human clinical trial, testing rabbits at monthly intervals
for retention and relearning for 3 months after initial acquisition. Subjects. A total of 85 female specific pathogen free New Zealand White
rabbits completed Experiments 1 and 2. Sixteen were young (4–6
months), and 69 were retired breeder rabbits. Birth dates of the
rabbits were recorded by the breeder (Covance, Denver, PA). Older
rabbits ranged in age from 15 to 43 months, with a mean of 29.1 months
(SD = 5.7). Mean weight of the young rabbits was 2.7 kg (SD =
0.4), and the range was 2.0 to 3.7 kg. Mean weight of the older rabbits
was 4.4 kg (SD = 0.5), and the range was 3.2 to 5.7 kg. Rabbits
were individually housed in stainless steel cages in an American
Association for the Accreditation of Laboratory Animal Care-accredited
animal facility. They had 24-h access to rabbit chow and tap water and
a 12/12-h light/dark cycle. Apparatus and Behavioral Conditioning Procedure. At least 24 h after arrival at the animal facility, rabbits were
adapted twice in Plexiglas restrainers for 1 h in sessions
separated by 24 h. After the second adaptation session, rabbits
were given a local ophthalmic anesthetic (proparacaine hydrochloride)
in the left eye so that a 6–0 nylon suture loop could be placed in the
temporal margin of the nictitating membrane (NM). A patch of fur (≈3
cm2) was shaved on the back to expose the skin
for s.c. injections. The classical conditioning equipment attached to the rabbit's head
included elastic eyelid retractors and a platform holding a minitorque
potentiometer (San Diego Instruments prototype model, San Diego, CA)
for NM movement measurement that was secured under the animal's muzzle
and behind the ears. The potentiometer was attached by a lever and a
thread to the nylon suture loop in the NM. Analog output from the
potentiometer was digitized and read into an IBM-PC-compatible system
described by Chen and Steinmetz ( 22). This system also controlled the
timing and presentation of conditioning stimuli. For classical conditioning, the CS was an 850-ms, 85-dB, 1-kHz tone,
followed 750 ms after its onset by a 100-ms, 3-psi (1 psi = 6.89
kPa) corneal airpuff US. The CS and US coterminated. The intertrial
interval was random, ranging between 20 and 30 s at 1-s intervals.
One training session lasted about 45 min. Rabbits were tested in
separate conditioning chambers four at a time. Each training session was controlled by a program written in C++
language ( 22) and run on an IBM-PC-compatible 386 computer. Data were
collected about the position of the NM in 3-ms bins during the trials.
A CR was scored if the NM was pulled back a minimum of 0.5 mm in the
interval between 25 and 750 ms after CS onset. The dependent measure,
learning criterion, was scored as the number of training trials it took
the animal to produce eight CRs within nine consecutive trials. CR
amplitude was scored as the mean NM amplitude in the interval between
25 and 750 ms after CS onset. Response latency was the latency of a
response of 0.5 mm or greater in the CS or US period. Initially,
response latency is over 750 ms (after US onset). As learning occurs,
response latency shortens to less than 750 ms (becoming a CR). Data
were collected in RAM and saved to a hard drive, and individual data
summaries for each of the four rabbits run simultaneously were printed
at the end of each session. Rabbits in the explicitly unpaired condition were treated in a fashion
identical to rabbits tested in the paired condition with the exception
that the 850-ms, 85-dB, 1-kHz tone CS and 100-ms, 3-psi corneal
air-puff US were never paired. In the unpaired condition, rabbits
received a total of 90 stimuli, 45 tone CSs, and 45 corneal air-puff
USs. Each stimulus was presented at an intertrial interval that was
random and ranged between 20 and 30 s. Thus, the duration of the
session and the intertrial interval were identical in the paired and
unpaired sessions, and all rabbits were tested four at a time. Pharmacological Analyses. Plasma and brain AChE. From each rabbit's ear vein, 3–5 ml of blood was removed 15 min after
the 16th injection of drug or vehicle. Blood was processed in a Jouan
CT4.22 centrifuge (Cedex, France) for 60 min at 3,000 rpm and frozen at
−80°C. Young rabbits were killed after 15 days of conditioning and
drug treatment with an overdose (70–100 mg/kg) of pentobarbital
injected in the ear vein. Older drug-treated rabbits in Experiment 1
were killed 15 wk after training began, having received only 15 daily
injections of 3.0 mg/kg Gal during eyeblink conditioning. Older
rabbits in Experiment 2 received injections 5 days a wk for 15 wk of
0.0, 1.0, or 3.0 mg/kg Gal or 15 daily injections of 3.0 mg/kg Gal
and injections 5 days a wk for the remaining 12 wk of vehicle. After
sacrifice, animals were immediately decapitated. The brain of each
animal was removed rapidly and frozen at −80°C. Plasma and frozen
tissue were shipped on dry ice from Philadelphia to Tucson and
immediately stored at −70°C until assayed. Sections from
parietal/occipital cortex were prepared for neurochemical analysis of
AChE activity according to the colorimetric method of Ellman et
al. ( 23) by using a Beckman Coulter DU 640 spectrophotometer
equipped with a Peltier temperature controller. The incubation solution
contained the butyrylcholinesterase inhibitor tetraisopropyl
pyrophosphoramide (iso-OMPA) at a final concentration of 100 μM to
measure AChE activity specifically. Brain nicotine receptor-binding studies. Sections from sensorimotor cortex were homogenized and prepared for
analysis according to the method of Flores et al. ( 24).
Membrane suspensions were incubated (60 min at 4°C) with
[ 3H]epibatidine (0.1–10 nM) in a 50-mM
NaKHPO 4 (pH 7.4) buffer, in a final volume of 1
ml, with or without unlabeled nicotine (0.1 mM) to define specific
binding. [ 3H]Epibatidine was used to label the
number of α4β2 nicotinic acetylcholine receptors. Separation of
bound ligand from free was performed by filtering the samples through
Whatman GF/C filters that had been presoaked with 0.3%
polyethylenimine. The filters were washed three times with the buffer,
and the radioactivity trapped on the filters was counted in a
scintillation counter. All assays were performed in triplicate. The
Bmax and the
Kd were determined by saturation
experiments with six different concentrations of labeled ligand. Data
were analyzed by using the PHARM/PCS 4.2 program
(MCS, Philadelphia, PA). Kd and
Bmax values were determined after
transformation of the data to fit the Rosenthal equation. Drugs. Galantamine was purchased from Tocris Cookson (Ballwin, MO). The
vehicle solution was sterile saline. Drugs were freshly prepared in
solution each wk and injected s.c. a minimum of 15 min before
behavioral testing to ensure that peak blood levels of Gal were
attained during the testing session. Rabbits were tested between 15 and
30 min after injections, and behavioral testing was completed a maximum
of 1 h and 15 min after injection. Research Design. In Experiment 1, 3.0 mg/kg Gal or vehicle was injected s.c. daily for
15 days in young and older rabbits 15–30 min before sessions of
eyeblink classical conditioning to examine behavioral and
pharmacological effects. In Experiment 2, doses of 0.0, 1.0, and 3.0
mg/kg Gal were injected s.c. to older rabbits as indicated in Table
1. Equal volumes were injected into the
animals, including vehicle-treated animals, based on weight.
| Table 1Research design of Experiment 2: Acquisition 5 days/week
for 3 weeks; 3 monthly retests of retention and
relearning |
Experiment 1. Behavioral analyses. The dependent measure of trials to a learning criterion of eight CRs in
nine consecutive trials was evaluated in a 2 (drug dose) by 2 (age)
ANOVA. The effect of Gal was statistically significant,
F (1, 28) = 11.44, P < 0.002.
The effect of age was also statistically significant, F
(1, 28) = 8.45, P < 0.007. The Gal by
age-interaction effect approached statistical significance,
F (1, 28) = 3.21, P = 0.084
(Fig. A). Post hoc
analysis of the significant Gal effect indicated that a dose of 3.0
mg/kg Gal facilitated learning in young (P <
0.01) as well as older rabbits (P < 0.01). Post
hoc analysis of the significant age effect indicated that
vehicle-treated older rabbits took more trials to attain learning
criterion than vehicle-treated younger rabbits (P
< 0.05).
Separate 2 (drug dose) by 2 (age) by 15 (training sessions)
repeated-measures ANOVAs were carried out for three dependent measure
assessments of learning (percentage of CRs, CR amplitude, and response
latency). Gal had a significant effect on all three dependent measures:
F (1, 28) = 9.25, P < 0.005
for percentage of CRs (Fig. B); F (1,
28) = 5.26, P < 0.03 for CR amplitude; and
F (1, 28) = 12.70, P < 0.001
for response latency. The effect of age was significant for percentage
of CRs, F (1, 28) = 4.91, P <
0.04, but there was not a significant age effect for CR amplitude or
response latency. The drug dose by age-interaction effect did not reach
statistical significance with any dependent measure of learning. A 2
(drug dose) by 2 (age) ANOVA was carried out on an assessment of the
motor response, unconditioned response amplitude. The effect of age was
significant, F (1, 28) = 6.23,
P < 0.02, with older rabbits having greater
unconditioned response amplitude (mean of 7.4 and 7.3 mm for Gal and
vehicle, respectively) than young rabbits (mean of 6.2 and 6.2 mm for
Gal and vehicle, respectively) because of the larger NM of older
rabbits. No other effects were significant. Behavior and pharmacological relationships. Comparisons of plasma AChE, brain AChE, and nicotinic receptor
binding were carried out with one-way ANOVAs. There were statistically
significant group differences in plasma AChE, F (3,26)
= 11.40, P < 0.0001. Post hoc comparisons indicated
that the old Gal-treated rabbits had the lowest plasma AChE levels,
which were significantly lower than the levels in the young
vehicle-treated and young Gal-treated groups (P <
0.05). Young vehicle brains were not available for analyses, so
comparisons for brain AChE and nicotinic receptor binding were analyzed
for three groups. There was a significant difference in brain AChE
levels, F (2, 12) = 4.91, P < 0.03.
Young Gal-treated rabbits had the lowest brain AChE levels, whereas old
vehicle-treated rabbits had the highest levels. There was a significant
difference in brain nicotinic receptor binding
(Bmax values), F (2,
15) = 6.81, P < 0.01. Old rabbits treated with
Gal had the highest level of nicotinic receptor binding. Correlations between the behavioral measures of trials to learning
criterion and plasma AChE, brain AChE, and nicotinic receptor binding
were carried out. There was a statistically significant correlation
between brain AChE levels and trials to learning criterion,
r = 0.621, P = 0.007. Neither the
correlation between trials to learning criterion and plasma AChE nor
the correlations between trials to learning criterion and
Bmax or
Kd attained statistical significance. Experiment 2. Behavioral analyses. To compare the effects of various doses of Gal on the acquisition
of CRs, a one-way ANOVA using the dependent measure of trials to
learning criterion for the 4 treatment groups (0.0 Gal, 15 wk; 1.0 Gal,
15 wk; 3.0 Gal, 15 wk, 3.0 Gal, 3 wk) was carried out. There was a
significant difference among the groups, F (3, 49)
= 4.57, P = 0.007 (Fig.
A). Post hoc comparisons
using the Tukey honestly significant difference (HSD) test indicated
that rabbits in the 3.0 Gal, 15-wk and 3.0 Gal, 3-wk groups took
significantly fewer trials to attain learning criterion compared with
rabbits in the 1.0 Gal, 15-wk and 0.0 Gal, 15-wk groups. The difference
between 1.0 Gal and vehicle was not statistically significant.
Because the two groups of rabbits treated with 3.0 mg/kg Gal
during acquisition were similar in trials to learning criterion, the
groups were collapsed into one group of 24 rabbits for additional
analyses of behavioral acquisition data. Separate 3 (drug dose) by 15
(training sessions) repeated-measures ANOVAs were carried on for three
dependent-measure assessments of learning (percentage of CRs, CR
amplitude, and response latency) with a planned comparison between the
vehicle and 3.0 mg/kg groups. The effect of 3.0 mg/kg Gal was
significant for percentage of CRs and response latency,
F (1, 39) = 4.88, P = 0.033 and
F (1, 39) = 4.80, P = 0.034,
respectively (Fig. B). Percentage of CRs was
greater and response latency was shorter (more often preceding US
onset) for rabbits in the 3.0 mg/kg Gal group. As expected, all three
dependent measures changed significantly over training sessions,
indicating learning in all groups. The drug dose by training session
interaction effect was not significant for percentage of CRs and
response latency, but for CR amplitude the interaction effect
approached significance, F (14, 546) = 1.66,
P = 0.061. A one-way ANOVA used to compare drug
treatment on unconditioned response amplitude was not significant. To examine retention, percentage of CRs in the 20 CS-alone trials was
analyzed in a 4 (drug dose) by 3 (monthly retest) repeated-measures
ANOVA. The effect of drug dose was statistically significant,
F (3, 49) = 3.60, P = 0.020, as
was the drug dose by monthly retest interaction, F (6,
98) = 2.38, P = 0.035 (Table
2). Post hoc analysis of the significant
drug dose effect indicated that the group administered 3.0 mg/kg Gal
over the 15-wk period had significantly greater retention than did the
vehicle group in the 1-month retention session. As indicated in Table
2, the significant interaction resulted from better retention in the
second and third retention tests in several groups.
| Table 2Retention as measured by percentage of CRs to test trials
and relearning as measured by trials to learning criterion in three
1-month retests for the four drug treatment groups |
To examine relearning, a 4 (drug dose) by 3 (monthly retest)
repeated-measures ANOVA was carried out on the dependent measure of
trials to learning criterion. The monthly retest effect was
significant, F (2, 98) = 5.11,
P = 0.008. At each retest, reacquisition occurred
more rapidly than at the preceding retest (Table 2). The drug dose and
interaction effects did not attain statistical significance. Behavior and pharmacological relationships. Comparisons of plasma AChE, brain AChE, and nicotinic receptor binding
were carried out with one-way ANOVAs. There were statistically
significant group differences in plasma AChE, F (3, 48)
= 4.16, P = 0.011 (Fig.
). Post hoc comparisons using the Tukey
HSD test indicated that the 3.0 Gal, 3-wk group had significantly lower
plasma AChE levels than the vehicle-treated rabbits. Differences among
the three Gal-treated groups did not attain statistical significance in
the post hoc comparisons. The correlation between trials to learning
criterion and plasma AChE levels was low and did not approach
statistical significance, (r = −0.066,
P = 0.323).
Six brains from rabbits in the 3.0 Gal, 15-wk group and six brains from
vehicle-treated rabbits were analyzed for a different experiment, so
comparisons for brain AChE and nicotinic receptor binding had six
rabbits in the 3.0 mg/kg Gal group and 12 rabbits in the vehicle
group. A one-way ANOVA comparing brain AChE levels indicated a
significant difference in brain AChE levels, F (3, 38)
= 6.34, P = 0.001 (Fig. ). Post hoc comparisons using
the Tukey HSD test indicated that all three groups treated with Gal had
significantly lower brain AChE levels than vehicle-treated rabbits.
Differences among the three Gal-treated groups did not attain
statistical significance in the post hoc comparisons. The correlation
between trials to learning criterion and brain AChE levels approached
but did not attain statistical significance, r = 0.224,
P = 0.082. The saturation-binding experiments using
[3H]epibatidine to label the α4β2 nicotinic
acetylcholine receptors produced Bmax
and Kd values. A one-way ANOVA
comparing the brain nicotinic receptor-binding value,
Bmax, indicated a significant effect,
F (3, 38) = 6.95, P = 0.001. Post hoc
comparisons using the Tukey HSD test indicated that the 3.0 Gal, 3-wk
group differed significantly (P < 0.05) from the
values for the other three groups, 3.0 Gal, 15 wk, 1.0 Gal, 15 wk, 0.0
Gal, 15 wk. Fig. shows a Scatchard
analysis of these results comparing the 3.0 Gal, 3-wk and 0.0 Gal,
15-wk groups. The correlation between trials to learning criterion and
Bmax for the 24 rabbits in the 3.0
Gal, 3-wk and vehicle groups was −0.268 (P = 0.11),
and the correlation between trials to learning criterion and
Bmax for older rabbits in all four
groups was −0.099 (P = 0.271).
Galantamine at a dose of 3.0 mg/kg was effective in facilitating
learning. In Experiment 1, the drug improved learning significantly in
young as well as in older rabbits. Among the many cognition-enhancing
drugs we have tested in 4-month-old rabbits (BMY-21502, donepezil,
GTS-21, nefiracetam, nimodipine), Gal is the only drug that has
facilitated learning in young rabbits. Young animals acquire CRs at
close to ceiling levels (around 400 training trials), making it more
difficult to demonstrate a significant effect. In the present study,
the mean number of trials to criterion for young vehicle-treated
rabbits was 445 trials (SD = 130). The 3.0-mg/kg dose of Gal
enabled young rabbits to achieve learning criterion in a mean of 297
trials (SD = 166), and old rabbits treated with 3.0 mg/kg Gal
achieved criterion in 401 trials (SD = 192). The 3.0-mg/kg dose
of Gal caused older rabbits to learn at the same rate as young
vehicle-treated rabbits. In Experiment 2, 3.0 mg/kg Gal affected the rate of learning early in
the acquisition process. On average, old rabbits treated with 3.0
mg/kg Gal learned on training day 4 or 5; old rabbits treated with
1.0 mg/kg Gal learned on training day 6 or 7; and old rabbits treated
with vehicle learned on training day 9 or 10. Because all rabbits were
trained for 15 sessions, the groups were relatively equal at the end of
acquisition. The significant effect of the 3.0-mg/kg dose of Gal on acquisition
extended to retention in the case of the group continuously injected
with 3.0 mg/kg Gal. When they were tested for retention at 1-, 2-,
and 3-month intervals after acquisition, the continuously injected
group treated with 3.0 mg/kg Gal showed significantly greater
retention at the 1-month retest (52% CRs versus 17% CRs for
vehicle-treated rabbits). The significant retention effect did not
occur in the group treated with 3.0 mg/kg Gal only for the 15 days of
acquisition training. Indeed, the group treated continuously with 1.0
mg/kg Gal had a numerically higher retention score in the 1-month
retest than did the group treated with 3.0 mg/kg Gal for 15 days.
There was no significant drug dose effect on relearning. Associated with the facilitated learning in Experiments 1 and 2 were
statistically significant correlations between learning and brain (but
not plasma) AChE levels. Greater inhibition of brain AChE correlated
significantly with faster acquisition. It should be noted that this
correlation was between learning that was tested more than 12 wk before
the blood was sampled and the brain was removed. The drug that had been
administered to the animal was injected 15 min before blood was
sampled; the animal was killed, and the brain was removed. Thus, the
AChE levels probably reflect the characteristic response of the
individual animals, and this is why the correlation between brain AChE
level and learning is significant. Similarly, nicotinic receptor
binding was assessed in the brains removed after 15 wk of treatment,
whereas learning was affected by Gal dose in the first 3 wk of the
experiment. We assume that nicotinic receptor-binding increases
occurred during the first 3 wk of the experiment when learning was
improved, but we did not examine rabbit brains at that time. This
assumption is based on the reported findings that treatment with
nicotinic receptor agonists could increase cortical and hippocampal
nicotinic receptor number after only 10 days of treatment in rats ( 25)
and between 2 to 4 wk in mice ( 26). Furthermore, in the experiment with
rats, the elevation in nicotinic receptor number correlated with the
rate of acquisition in the Morris water-maze task ( 25). Plasma levels of AChE in Experiment 1 were lowest in older rabbits
treated with Gal, and these levels were significantly lower than plasma
AChE in both groups of young rabbits, suggesting an age effect. The
lower levels of AChE observed in Gal-treated old rabbits in the present
study might be due to the fact that aged animals are more vulnerable to
the effects of AChE inhibitors. In a recent study, the inhibitors
donepezil and tacrine produced greater decline in AChE activity in the
brains of aged rats, as compared with young rats, and this decline
might have been related to the higher concentrations of these drugs
achieved within the brain of older animals ( 27). In addition, the
degree of AChE inhibition achieved in the different aged animals might
also be related to the endogenous level of AChE enzyme. The
age-related decline in AChE activity varies across different brain
regions and rodent strains ( 28– 30), and specific molecular forms of
the enzyme may be more vulnerable than others ( 31). Using [ 3H]epibatidine to label the α4β2
nicotinic acetylcholine receptors produced
Bmax values indicating that nicotinic
binding was elevated significantly in older rabbits treated with 3.0
mg/kg Gal in both Experiments 1 and 2. The higher-dose Gal therapy
initially induced a significant up-regulation of nicotinic sites.
However, after long-term therapy for 15 wk, the response to Gal showed
that tolerance and up-regulation of nicotinic sites had attenuated by
the time of sacrifice. A similar tolerance to the effects of chronic
nicotinic agonist treatment was observed for locomotor depression in
mice after 7 wk of therapy ( 26). These findings are consistent with a report ( 32) that also demonstrated
a similar effect of chronic Gal therapy on nicotinic receptor density.
Although Barnes et al. ( 32) did not find a significant
improvement in spatial memory in their aged rats, they did find a
significant positive correlation between the durability of long-term
potentiation and the Bmax of nicotinic
receptors within the hippocampus that was induced by chronic Gal
therapy. Taken together with the results of the current study, these
data suggest that chronic Gal therapy can effectively and consistently
increase the density of nicotinic receptors in selected brain regions
that are involved in learning and memory. It is our conclusion that
this increase in nicotinic receptor number, and the resultant changes
in electrophysiological indicators of neural plasticity ( 32), may
underlie aspects of the cognitive benefits produced by long-term
therapy with Gal in humans with AD. We thank Michael Ewers, Tara Orlando, Michelle Pak, and Isagani
Santos for assistance with animal data collection. This research was
supported by grants from Janssen Pharmaceutica (to D.S.W.-P. and
G.L.W.) and by U.S. Public Health Service Contract Grant AG10546 (to
G.L.W.). | | APL | allosterically potentiating ligand | | | AChE | acetylcholinesterase | | | AD | Alzheimer's disease, CR, conditioned
response | | | CS | conditioned stimulus | | | Gal | galantamine | | | NM | nictitating
membrane | | | US | unconditioned stimulus |
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