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Journal of Physiology - Paris 99 (2006) 180–192
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Pharmacological and molecular enhancement of learning in aging
and Alzheimer’s disease
John F. Disterhoft *, M. Matthew Oh
Department of Physiology and Institute for Neuroscience, Feinberg School of Medicine, Northwestern University, 303 E Chicago Avenue,
Chicago, IL 60611-3008, USA
Abstract
When animals learn hippocampus-dependent associative and spatial tasks such as trace eyeblink conditioning and the water maze,
CA1 hippocampal neurons become more excitable as a result of reductions in the post-burst, slow afterhyperpolarization. The calcium-activated potassium current that mediates this afterhyperpolarization is activated by the calcium influx that occurs when a series
of action potentials fire and serves as a modulator of neuronal firing frequency. As a result, spike frequency accommodation is also
reduced after learning. Neuronal calcium buffering processes change and/or voltage-dependent calcium currents increase during aging;
leading to enhancements in the slow afterhyperpolarization, increased spike frequency accommodation and age-associated impairments
in learning. We describe a series of studies done to characterize this learning-specific enhancement in intrinsic neuronal excitability and its
converse in aging brain. We have also combined behavioral pharmacology and biophysics in experiments demonstrating that compounds
that increase neuronal excitability in CA1 pyramidal neurons also enhance learning rate of hippocampus-dependent tasks, especially in
aging animals. The studies reviewed here include those using nimodipine, an L-type calcium current blocker that tends to cross the bloodbrain barrier; metrifonate, a cholinesterase inhibitor; CI1017, a muscarinic cholinergic agonist; and galantamine, a combined cholinesterase inhibitor and nicotinic agonist. Since aging is the chief risk factor for Alzheimer’s disease, a disease that targets the hippocampus
and associated brain regions and markedly impairs hippocampus-dependent learning, these compounds have potential use as treatments
for this disease. Galantamine has been approved by the USDA for this purpose. Finally, we have extended our studies to the TG2576
transgenic mouse model of Alzheimer’s disease (AD), that overproduces amyloid precursor protein (APP) and increases levels of toxic
b-amyloid in the brain. Not only do these mice show deficits in hippocampus-dependent learning as they age, but their hippocampal
neurons show a reduced capacity to increase their levels of intrinsic excitability with reductions in the slow afterhyperpolarization after
application of the muscarinic agonist carbachol. These TG2576 APP overproducing mice were crossed with BACE1 knockout mice, that
do not produce b-amyloid because cleavage of APP by the b-site APP cleaving enzyme 1 (BACE1) is a critical step in its formation. Not
only was hippocampus-dependent learning rescued in the bigenic TG2576-BACE1 mice, but the capacity of hippocampal neurons to
show normal enhancements of intrinsic excitability was restored. The series of studies reviewed here support our hypothesis that
enhancement in intrinsic excitability by reductions in calcium-activated potassium currents in hippocampal neurons is an important
cellular mechanism for hippocampus-dependent learning.
! 2005 Elsevier Ltd. All rights reserved.
Keywords: Afterhyperpolarization; BACE; Calcium channel; Cholinesterase inhibitor; Muscarinic; TG2576; Eyeblink conditioning
1. Introduction
One of the most troubling concomitants of aging for
many individuals is the impairment of learning and mem*
Corresponding author. Tel.: +1 312 503 7982; fax: +1 312 503 2090.
E-mail address: [email protected] (J.F. Disterhoft).
0928-4257/$ - see front matter ! 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jphysparis.2005.12.079
ory which often occurs even with ‘‘normal’’ aging. We have
been exploring the neuronal changes that occur during
aging and contribute to learning deficits. We have been
particularly interested in alterations of the slow outward
potassium currents, including calcium-mediated potassium
currents, as related to acquisition of hippocampus-dependent behavioral tasks and to age. The currents studied
J.F. Disterhoft, M.M. Oh / Journal of Physiology - Paris 99 (2006) 180–192
are the major components of the post-burst afterhyperpolarization and help determine the level of spike frequency
accommodation in hippocampal pyramidal neurons. Eyeblink conditioning and water maze place learning have
been combined with in vitro biophysical measures from
the hippocampal circuit of young and aging rabbits and
rats to define functional changes in hippocampal pyramidal
neurons in learning-intact as compared to learningimpaired animals. Although most of the work that we have
done has concentrated on normally aging animals, by
extension our research is relevant to understanding the
mechanisms of Alzheimer’s disease, a disease whose greatest risk factor is age and that has grown in importance as
the population is living longer on average. In an attempt
to contribute to the development of mechanism-based disease treatments, we have investigated a series of pharmacological manipulations that enhance learning in aging as
predicted by their effects on slow, calcium-activated potassium currents in hippocampal pyramidal neurons.
Two to four million people in the United States have
been estimated to suffer from Alzheimer’s disease in the late
1990s with this number predicted to climb steadily with
increasing life span (Brookmeyer et al., 1998; Hebert
et al., 2003). Although at present there is no cure for the
disease, there are five compounds that have been approved
by the US Food and Drug Administration for the treatment of the disease in the United States. Tacrine (Cognex"), donepezil (Aricept"), rivastigmine (Exelon") and
galantamine (Reminyl") are cholinesterase inhibitors that
prevent the breakdown of acetylcholine to its component
parts (acetylCoA and choline). Memantine (Namenda")
is an uncompetitive antagonist to the N-methyl-D-aspartate
(NMDA) receptor that is thought to help patients with
Alzheimer’s disease by limiting calcium entry into neurons
through the NMDA receptors. In this review, we will
discuss our experiences with four compounds (including
galantamine) that have been shown to ameliorate
age-related learning deficits.
We should first describe the behavioral tasks and the
in vitro biophysical measurement that we use in our work
and that form the basis for our behavioral pharmacological
studies. We have been particularly interested in behavioral
tasks that depend upon the hippocampus for their acquisition, given that such learning tasks are particularly likely to
be impaired during aging and especially Alzheimer’s disease. The hippocampus has been demonstrated to be critical for the ability of humans to form new declarative
memories that we commonly think of as cognitive or
conceptual learning in humans, in contrast to procedural,
sensorimotor learning. Therefore, we have concentrated
considerable effort on understanding the mechanisms associated with learning in hippocampal pyramidal neurons. In
experimental animals, many behavioral tasks that depend
on the hippocampus involve spatial or temporal learning.
One behavioral task that we have successfully used over
the years is trace eyeblink conditioning. In this task, the
subject is presented with a neutral, conditioning stimulus
181
Fig. 1. Percent of rats reaching a learning criterion of 70% late CRs at any
time during the trace 250 conditioning sessions. The percent of animals
who learned the trace eyeblink conditioning task decreased as a function
of age; the percent of animals classified as non-learners increased as a
function of age. The old group (27–29 months) demonstrates the age at
which learning deficits may be present in half of the population. Reprinted
from Knuttinen et al. (2001a), Copyright 2001, with permission from
Elsevier.
(usually a brief tone) that does not elicit an eyeblink
response prior to training. The conditioning stimulus is followed by a temporal gap (trace) before an unconditioned
stimulus (usually an airpuff to the cornea that always elicits
an eyeblink response) is presented. This Pavlovian task is a
very simple, but surprisingly, very difficult task for the
aging population to learn (Knuttinen et al., 2001a; Knuttinen et al., 2001b; Thompson et al., 1996a) (Fig. 1). Over
half of normal aging rabbits (Thompson et al., 1996a)
and rats (Knuttinen et al., 2001a) failed to acquire this
task; and those that did learn the task, did so at a slower
rate, as compared to the young animals (Thompson
et al., 1996a). Additionally, it has been repeatedly demonstrated that an intact hippocampus is a prerequisite for the
subjects to bridge the temporal gap (trace) between the
conditioning stimulus and the unconditioned stimulus
(McGlinchey-Berroth et al., 1997; Moyer et al., 1990;
Weiss et al., 1999).
A key in vitro biophysical measure that we examine
from hippocampal pyramidal neurons is the post-burst
afterhyperpolarization (AHP). The AHP is an intrinsic,
post-synaptic, membrane property of a neuron that serves
to limit the firing of action potentials during a long period
of membrane depolarization caused by direct current injection or by high frequency sustained excitation of the
neuron – a process known as spike-frequency adaptation
(accommodation). The slow, post-burst AHP is mediated
by a calcium-activated outward potassium current that is
mediated by the calcium influx associated with action
potentials (Hotson and Prince, 1980; Lancaster and
Adams, 1986; Landfield and Pitler, 1984; Power et al.,
2002; Schwartzkroin and Stafstrom, 1980; Storm, 1990).
The AHP and the currents underlying the AHP are greatly
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Fig. 2. The post-burst afterhyperpolarization (A) and the currents underlying it (B) are significantly enhanced in hippocampal pyramidal neurons from
aging animals as compared to that from young animals. Reprinted with permission from Power et al. (2002), Copyright 2002 by the Society for
Neuroscience.
enhanced in hippocampal pyramidal neurons from aging
animals as compared to those from the young (Kumar
and Foster, 2002; Landfield and Pitler, 1984; Moyer and
Disterhoft, 1994; Moyer et al., 1992; Power et al., 2002)
(Fig. 2). Thus, this enhanced AHP in neurons of aging subjects would make them less excitable and limit these neurons from firing at a high sustained rate.
We have examined the relationship between learned
behaviors and the AHP in a variety of contexts. We initially (Disterhoft et al., 1986) reported that the AHP was
reduced in CA1 hippocampal neurons from rabbits that
learned the delay eyeblink conditioning task as compared
to those from naı¨ve and pseudoconditioned rabbits
(Fig. 3). Pseudoconditioned rabbits received equal numbers
of the two stimuli as the trained rabbits, but the two stimuli
were presented in a random order and unpaired; i.e., the
CS and the US were never presented together in an associative fashion. We suggested that this reduction in the AHP
would increase neuronal excitability and should contribute
to the increased firing rate of single CA1 pyramidal neurons that had been observed in vivo during eyeblink conditioning (Berger et al., 1976; Berger and Thompson, 1977,
1978; McEchron and Disterhoft, 1997, 1999; Weiss et al.,
1996). This finding has led to numerous discoveries including that the alteration of the AHP is post-synaptic, and
that spike frequency accommodation is also reduced in
CA1 neurons after learning the delay eyeblink conditioning
task (Coulter et al., 1989). These findings have been
extended to trace eyeblink conditioning in rabbits (Fig. 4)
(Moyer et al., 1996; Thompson et al., 1996b) and rats
(Kuo et al., 2004; Oh et al., 1999a). Trace conditioning is
a version of the task that is hippocampus-dependent in
which a blank, trace period is inserted between the tone
CS and the air puff US. In addition, reductions in both
AHP and accommodation were observed in CA1 neurons
from aging animals that were trained and learned the trace
eyeblink conditioning task, such that the AHP and accommodation observed in these neurons were nearly identical
to that observed in CA1 neurons from young animals that
learned the task (Fig. 5) (Moyer et al., 2000). Aging animals that received more paired training trials but did not
learn, showed no reduction in the AHP or accommodation
as compared to naı¨ve animals. These findings support the
working hypotheses that the AHP and accommodation
of CA1 neurons from aging rabbits are potentially
‘‘plastic’’ and may be reduced; and that aging rabbits that
are trained but fail to acquire the task may have neurons
with too large an AHP to allow learning to occur and/or
reduced capacity for reducing the AHP. This hypothesis
is presumably relevant to explaining the mechanism for
age-associated learning deficits in other mammals, including humans.
2. Nimodipine, an L-type calcium channel antagonist
Fig. 3. AHP after one spike in a naı¨ve, pseudoconditioned, and
conditioned neuron. A 100-ms depolarizing current pulse sufficient to
elicit one action potential was injected into the cell. The AHP can be seen
by comparing the voltage response following the depolarizing pulse to the
baseline (indicated by the dashed line to facilitate comparison). The point
where the AHP was measured in these traces is indicated by the arrow.
Note that the AHP is considerably reduced in the conditioned as
compared to the naı¨ve and pseudoconditioned neuron. One trace is
illustrated for each neuron. Reprinted with permission from reference
Disterhoft et al. (1986).
Nimodipine is an L-type calcium channel antagonist
that readily crosses the blood brain barrier (van den Kerckhoff and Drewes, 1989). It has been demonstrated in
rabbits to enhance blood flow in the brain as a direct
consequence of vasodilation (Haws et al., 1983). Administration of nimodipine has been shown to be beneficial to
patients after an ischemic stroke (Gelmers, 1984); however,
this has been recently challenged (Horn et al., 2001). More
importantly, in double-blind clinical trials, nimodipine was
found to be beneficial in improving the cognitive deficits
observed in elderly patients with dementia (Ban et al.,
1990; Tollefson, 1990). Therefore, we investigated the
J.F. Disterhoft, M.M. Oh / Journal of Physiology - Paris 99 (2006) 180–192
183
Fig. 4. Acquisition of hippocampus-dependent trace eyeblink conditioning increased excitability of hippocampal CA1 pyramidal neurons. (A) Voltage
trace shows an overlay of recordings of the post-burst AHPs in CA1 neurons from a naive rabbit (Naive) and from trace-conditioned rabbits studied 24 h
after initial learning (Trace 24 h) or 24 h after receiving an additional training session given 14 d after initial learning (Retention). The resting membrane
potentials for these cells were approximately 66 mV, with action potentials truncated for visualization of the AHP. The AHP was measured for 5 s
beginning after a 100 ms depolarizing current injection (solid black line), with minimal current (!0.6 nA) required to reliably evoke a burst of four action
potentials. (B) Examples of typical accommodation responses in CA1 pyramidal cells from rabbits: 24 h after pseudoconditioning (Pseudo), 24 h after
acquisition of trace conditioning (Trace 24 h), and 24 h after receiving an additional training session 14 d after acquisition (Retention). Notice that
although the cell from the trace-conditioned rabbit fired more action potentials, accommodation was certainly not abolished (as evidenced by the increase
in interspike interval with time during the 800 ms depolarizing stimulus) but, rather, was significantly and transiently reduced after learning. Reprinted
with permission from reference Moyer et al. (1996), Copyright 1996 by the Society for Neuroscience.
potential benefits of calcium channel blockade, with nimodipine, in reversing the age-related learning deficit on the
trace eyeblink conditioning task; and also, investigated
whether the biophysical properties of hippocampal pyramidal neurons may have contributed to the amelioration of
the learning deficit.
Nimodipine reversed the age-related learning impairment of aged animals on the trace eyeblink conditioning
task. Treatment with nimodipine allowed the aged rabbits
to learn the task at a very similar rate as young rabbits
(Fig. 6) (Deyo et al., 1989; Straube et al., 1990). No significant impact was observed in young animals treated with
nimodipine. The behavioral rescue in aged animals may
in part be due to the enhanced neuronal activity of hippocampal pyramidal neurons in vivo, as administration of
nimodipine greatly enhanced the basal firing rate of CA1
pyramidal neurons (Thompson et al., 1990). Thus, we
examined the effects of nimodipine on the biophysical
properties of CA1 hippocampal pyramidal neurons
in vitro.
Nimodipine significantly enhanced the neuronal excitability of CA1 neurons in vitro by reducing the post-burst
AHP and the accommodation of these neurons. The normally enlarged post-burst AHP in CA1 neurons from aging
animals (Landfield and Pitler, 1984) was significantly
reduced by bath application of nimodipine (Fig. 7) (Moyer
et al., 1992). The concentration of nimodipine necessary to
significantly reduce the AHP and accommodation in neurons from aged animals was significantly less than that
needed to produce similar reductions in neurons from
young animals (Moyer et al., 1992). In addition, the
plateau phase of the calcium action potential (Moyer and
Disterhoft, 1994) and the currents underlying the AHP
(Power et al., 2002) in hippocampal CA1 neurons were also
significantly reduced by the bath application of nimodipine. More importantly, the concentration of nimodipine
that was bath applied to produce the significant enhancement of the CA1 neurons from aged animals in vitro was
similar to the dosage of nimodipine that was beneficial to
reverse the learning impairment in aged animals in vivo.
Therefore, the behavioral effects of nimodipine may be in
part be due to the enhanced activity of hippocampal
pyramidal neurons via reduced calcium entry through the
L-type calcium channel, which led to the significant
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Fig. 5. Acquisition of hippocampus dependent trace eyeblink conditioning increased excitability of aging rabbit hippocampal CA1 pyramidal
neurons. (A) Effects of trace conditioning on the size of the post-burst
AHP. (A1) Overlay of voltage recordings of the post-burst AHP in CA1
neurons from an aging naive rabbit (Naive), an aging rabbit that showed
<15% CRs after 15 sessions (Slow), and an aging trace-conditioned rabbit
(Trace). The resting membrane potentials of these cells were approximately 65 mV, with action potentials truncated for visualization of the
AHP. The AHP was measured for 5 s beginning after a 100 ms
depolarizing current injection (solid black line), with minimal current
(!0.6 nA) required to reliably evoke a burst of four action potentials. (A2)
Mean effects of trace eyeblink conditioning on post-burst AHP amplitude
in aging rabbit CA1 neurons. Notice that, after learning, the AHP was
significantly reduced compared with naive and slow-learning aging
controls. (B) Typical examples of accommodation responses in CA1
pyramidal cells from aging naive (Naive), aging slow-learning (Slow), and
aging trace-conditioned (Trace) rabbits. Although the cell from the traceconditioned rabbit fired more action potentials, accommodation was not
abolished, as evidenced by the increase in interspike interval with time
during the 800 ms depolarizing stimulus (solid black line), but rather was
significantly reduced after learning. The resting potentials of these cells
were approximately 67 mV. Reprinted with permission from reference
Moyer et al. (2000), Copyright 2000 by the Society for Neuroscience.
Fig. 6. Summary of the mean number of trials to reach the criterion of
eight CRs in any block of 10 trials in each of the trace-conditioned
treatment groups. Error bars indicate SEM (n = 6). Reprinted with
permission from reference Deyo et al. (1989), Copyright 1989 by the
American Association for the Advancement of Science.
Fig. 7. Nimodipine significantly enhanced neuronal excitability of CA1
neurons from aging rabbits by reducing the post-burst AHP (A) and spikefrequency accommodation (B). Reprinted with permission from reference
Moyer et al. (1992), Copyright 1992 by the American Physiological
Society.
reduction of the normally enlarged post-burst AHP of CA1
neurons in aged animals.
3. Metrifonate, a cholinesterase inhibitor
Metrifonate is an organophosphate compound that
undergoes nonenzymatic conversion to 0,0-dimethyl 2,2dichlorovinyl phosphate which produces the long lasting
inhibition of both acetylcholinesterase (AChE) and butyrylcholinesterase (Nordgren et al., 1978; Schmidt et al.,
1998). Treatments with metrifonate have reversed the
behavioral deficits observed in acquiring passive and active
avoidance, Morris water maze, and radial-arm maze tasks
by normal aging, medial-septum lesioned, or scopolaminetreated subjects (Dachir et al., 1997; Der Staay et al., 1996;
Itoh et al., 1997; Riekkinen et al., 1997; Riekkinen et al.,
1996). More importantly, in double-blind clinical trials,
the cognitive impairments observed in Alzheimer’s disease
patients were alleviated by metrifonate-treatment (Cummings et al., 1998; Morris et al., 1998; Pettigrew et al.,
1998). Thus, we were interested in observing metrifonate’s
effects on acquisition of trace eyeblink conditioning in
aging rabbits and on biophysical properties of CA1 hippocampal pyramidal neurons in vitro.
Chronic, oral treatment with metrifonate ameliorated
the learning deficit observed in aging rabbits (Fig. 8)
(Kronforst-Collins et al., 1997a,b). This amelioration was
dependent on AChE inhibition. However, the memory of
the task was not dependent on the AChE inhibition,
as the aging animals that learned the task still remembered the CS–US association even when the treatment with
metrifonate was stopped and the AChE activity returned to
J.F. Disterhoft, M.M. Oh / Journal of Physiology - Paris 99 (2006) 180–192
185
Fig. 8. Mean learning curves calculated for the three groups are expressed
as the percentage of conditioned responses (CRs) per training session
(M ± SEM). The 12- and 24-mg/kg groups demonstrated a significant
increase in percent conditioned responses over the course of training when
compared with the control group (p < 0.01). The 12- and 24-mg/kg groups
continued to demonstrate significantly increased levels of eyeblink
conditioning throughout retention testing after metrifonate treatment
was discontinued. There were no significant changes in performance of
any of the groups over the course of retention testing. The increased levels
of eyeblink conditioning during training and retention testing were not
due to generalized sensorimotor enhancement because there were no
significant differences between the mean CR amplitudes, CR peak
latencies, or unconditioned response amplitudes observed for the three
groups during training and retention testing. Reprinted with permission
from reference Kronforst-Collins et al. (1997b), Copyright 1997 by the
American Psychological Association.
Fig. 9. Bath application of metrifonate significantly decreased the
accommodation and the post-burst AHP in CA1 neurons from aging
subjects. An example of the effect of metrifonate on accommodation is
illustrated in (A)–(C) (same neuron). (A) depicts a typical response elicited
during the 800 ms depolarizing pulse obtained in baseline measures. (B)
illustrates a typical increase in number of action potentials elicited during
the accommodation pulse after the perfusate has been changed to 200 lM
metrifonate. (C) depicts a typical decrease in the number of action
potentials elicited after the perfusate has been changed to a combination
of 200 lM metrifonate and 1 lM atropine, indicating that the metrifonate
effect is muscarinic. An example of the AHP decrement observed after the
perfusate has been changed to a 100 lM metrifonate in CA1 neurons from
aging subjects is illustrated in (D) (scales for (B) and (C) are the same as
that of (A)). Reprinted with permission from reference Oh et al. (1999b),
Copyright 1999 by the Society for Neuroscience.
basal levels (Kronforst-Collins et al., 1997b). This demonstrated that modulation of cholinergic transmission was
essential for learning the task, but not for retrieval of the
learned association. Interestingly, the level of steady-state
AChE inhibition (40–60%) necessary for the amelioration
of the age-related learning deficit was achieved after
3 weeks of metrifonate treatment (Kronforst-Collins
et al., 1997a). This gradual buildup to the final, target
range of ChE inhibition was found to be beneficial (Cummings et al., 1998; Morris et al., 1998; Pettigrew et al.,
1998) and necessary for behavioral improvements in
humans (Becker et al., 1991).
Bath application of metrifonate dose-dependently
reduced the AHP and accommodation of CA1 hippocampal pyramidal neurons from both young and aging animals
(Oh et al., 1999b). These reductions of the AHP and
accommodation were effectively reversed by addition of a
muscarinic receptor antagonist, atropine, to the perfusate;
suggesting that the reductions of the AHP and accommodation involved modulation of muscarinic cholinergic
transmission (Fig. 9).
A key question that needed to be addressed was ‘could
the biophysical state of CA1 neurons be altered after
3 weeks of metrifonate treatment that may have led to
the behavioral amelioration of the learning deficit observed
in aging rabbits?’ Thus, we treated aging rabbits with
either metrifonate or saline for 3 weeks, after which the
Fig. 10. Chronic, oral treatment with metrifonate (12 mg/kg daily) in
aging subjects significantly reduced the accommodation in CA1 neurons.
A typical example of the differing response to an 800 ms depolarizing
current pulse used to obtain four action potentials in the first 100 ms
observed in CA1 neurons from chronically metrifonate- (top) or vehicletreated (bottom) subjects. Reprinted with permission from reference
Oh et al. (1999b), Copyright 1999 by the Society for Neuroscience.
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J.F. Disterhoft, M.M. Oh / Journal of Physiology - Paris 99 (2006) 180–192
biophysical properties of CA1 neurons from these animals
were compared. We found that CA1 hippocampal pyramidal neurons from aging rabbits chronically treated with
metrifonate had significantly reduced spike-frequency
accommodation as compared to that from vehicle-treated
rabbits (Fig. 10) (Oh et al., 1999b). Surprisingly, the
accommodation of CA1 neurons from chronically metrifonate-treated aging rabbits was similar to that observed in
neurons from naı¨ve, young rabbits. Thus, it appears that
3 weeks of metrifonate treatment produced a steady-state
inhibition of cholinesterase activity which, among many
things, altered the biophysical properties of CA1 pyramidal
neuronal of the aging subjects to a ‘young’ like state. This
change may have enabled these metrifonate treated aging
animals to learn the trace eyeblink conditioning task like
young animals.
4. CI-1017, a muscarinic receptor agonist
CI-1017, an oxime of 1-azabicyclo[2.2.1] heptan-3-one
with a 3-phenylpropargyl analogue substituent, is a muscarinic agonist designed to selectively activate the M1 receptor (Jaen et al., 1995; Tecle et al., 1998). The direct
stimulation of the muscarinic receptor may be beneficial,
because it does not depend on the presence of endogenous
acetylcholine in the brain for action like the cholinesterase
inhibitors. In addition, post-mortem examination of brains
from Alzheimer’s disease patients revealed that muscarinic
acetylcholine receptors appear to remain intact (Mash
et al., 1985; Pearce and Potter, 1991), although they may
not be all functional (Ferrari-DiLeo et al., 1995; Flynn
et al., 1995).
Animals given CI-1017 improved their performance on
the Morris water maze task (Schwarz et al., 1997; Symons
et al., 1988) and on a continuous performance task (Schwarz et al., 1997). In contrast, animals given the M1 antagonist pirenzepine were impaired on inhibitory avoidance
(Caulfield et al., 1993), water maze (Hagan et al., 1987;
Hunter and Roberts, 1988) and working memory (Ohno
et al., 1994) tasks, and representational memory (Messer
et al., 1987, 1990). Furthermore, a muscarinic receptor
antagonist, scopolamine, impaired acquisition of the trace
eyeblink conditioning task (Kaneko and Thompson,
1997). These data suggest that modulation of M1 receptors
impacts learning and memory. Thus, we were interested in
observing CI-1017’s effects on acquisition of trace eyeblink
conditioning in aging rabbits and on biophysical properties
of CA1 hippocampal pyramidal neurons in vitro.
CI-1017 ameliorated the learning deficit observed in
aged rabbits (Weiss et al., 2000). It significantly increased
the rate and amount of learning without any evidence of
pseudoconditioning (Fig. 11) (Weiss et al., 2000). Thus,
our data suggest that CI-1017 acts on associative sites to
increase the probability of evoking a CR, and not on
unconditioned reflex sites.
CI-1017 also enhanced the excitability of CA1 hippocampal pyramidal neurons from young and aging naive
Fig. 11. CI1017 significantly facilitated learning the trace eyeblink
conditioning task in aging rabbits. (A) A graph of the percent of trials
with CRs across 15 daily training sessions as a function of drug dose. (B)
The two higher doses (5 and 1 mg/ml) exhibited significantly more CRs
than the two lower doses (0 and 0.5 mg/ml). Data are means ± SE. Error
bars are omitted from (A) for clarity. Reprinted with permission from
reference Weiss et al. (2000), Copyright 2000 by the Society for
Neuroscience.
rabbits; via reductions of the AHP and accommodation
(Weiss et al., 2000). The AHP and accommodation reductions were reversed with addition of either atropine, a muscarinic receptor antagonist, or pirenzepine, a selective M1
muscarinic receptor antagonist, to the perfusate (Fig. 12).
These results suggest that M1 agonists ameliorate agerelated learning and memory impairments at least in part
by reducing the AHP and accommodation of hippocampal
pyramidal neurons, and that M1 agonists may be an effective therapeutic compound for reducing the cognitive deficits that accompany normal aging and/or Alzheimer’s
disease.
5. Galantamine, a cholinesterase inhibitor and an allosteric
potentiating ligand
Galantamine is a third generation cholinesterase inhibitor. Galantamine also potentiates the activity of nicotinic
acetylcholine receptors (nAChR) and, thus, is called an
allosteric potentiating ligand of nAChRs (Maelicke et al.,
2001). Galantamine treatment reversed learning impairments observed after various insults to the brain: nucleus
basalis magnocellularis lesion (Sweeney et al., 1988,
1990), ischaemia (Iliev et al., 2000), and ACh deficit due
to prolonged alcohol treatment (Iliev et al., 1999). Galantamine treatment also facilitated the acquisition of the delay
eyeblink conditioning task in aging rabbits (Woodruff-Pak
et al., 2001). More importantly, Alzheimer’s disease
patients treated with galantamine had their disease progression temporarily reversed and slowed as compared to
placebo treated patients (Raskind et al., 2000; Tariot
et al., 2000).
J.F. Disterhoft, M.M. Oh / Journal of Physiology - Paris 99 (2006) 180–192
187
Fig. 13. Aged galantamine-administered (Aged/Gal) rabbits performed
significantly more conditioned responses over the course of training
compared with aged saline control (Aged/Veh) rabbits. Aged/Gal and
young rabbits did not differ significantly on the final day of training. No
significant difference was observed between young galantamine-administered (Young/Gal) and young saline control (Young/Veh) rabbits.
Reprinted with permission from reference Weible et al. (2004), Copyright
2002 by Cold Spring Harbor Laboratory Press.
Fig. 12. Typical examples of the effects of CI-1017 on biophysical
properties from a single hippocampal CA1 pyramidal neuron from a
young naive rabbit. An 800 ms pulse was used to examine accommodation
after a burst of four action potentials. (A) Accommodation of the neuron
in aCSF. (B) Accommodation is reduced by the addition of CI-1017, i.e.,
the cell is more excitable. (C) The excitability change due to CI-1017 is
reversed by the addition of the muscarinic antagonist atropine. (D)
Examples of the post-burst AHP during control (aCSF), drug (CI-1017),
and reversal (CI-1017 plus atropine) conditions. Reprinted with permission from reference Weiss et al. (2000), Copyright 2000 by the Society for
Neuroscience.
We observed that galantamine ameliorated the agerelated learning impairment on the trace eyeblink conditioning task in rabbits (Fig. 13). Galantamine treated aged
rabbits met the learning criteria of 8 CRs in a 10 trial block
much quicker than the vehicle treated age-matched controls, requiring a similar number of trials as the young rabbits (Weible et al., 2004). Additionally, the properties and
timing of the eyeblink response resembled those of young
rabbits, which differed significantly as compared to agematched controls (Weible et al., 2004). These data suggest
that the learning deficits associated with decreased cholinergic transmission in the aging brain is offset by enhancing
nicotinic and muscarinic transmission with galantamine
treatment.
Preliminary findings from our laboratory demonstrate
that the post-burst AHP and accommodation of CA1 pyramidal neurons from young and aging rabbits are reduced
with bath application of galantamine (Oh et al., 2000).
Atropine, a muscarinic agonist, significantly reversed these
reductions; however, a nicotinic antagonist, a-bungarotoxin, had no effect. Additionally, galantamine significantly
enhanced the excitatory post-synaptic potentials (EPSP)
measured by Schaffer collateral stimulation. a-bungarotoxin blocked this EPSP enhancement, demonstrating that
a7 nAChRs are involved in this allosteric potentiation.
Several previous reports showed that nAChRs are involved
in enhancing neurotransmitter release throughout the brain
(Barazangi and Role, 2001; Dani, 2001; Lena et al., 1999;
Wonnacott, 1997); specifically, that a7 nAChRs are
involved in enhancing glutamate transmission in the hippocampus (Gray et al., 1996). Thus, the amelioration of learning deficit observed in aging subjects with galantamine
treatment (Weible et al., 2004; Woodruff-Pak et al., 2001)
may in part be due to the enhanced post-synaptic neuronal
excitability via muscarinic receptor activation, as well as,
the enhanced synaptic transmission via nicotinic receptor
activation.
6. Knocking out BACE1 improves learning in the Tg2576
Alzheimer’s mouse model
The b-amyloid (Ab) hypothesis of Alzheimer’s disease
has recently driven the search for the cure of the disease
(for review see Citron, 2004; Tanzi and Bertram, 2005). It
has been suggested that soluble, rather than insoluble, Ab
is the most important pathogenic factor in Alzheimer’s disease (Dodart et al., 2002; Selkoe, 2002; Walsh et al., 2002),
as behavioral deficits precede Ab plaque formation in mice
genetically engineered to overexpress the human form of
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J.F. Disterhoft, M.M. Oh / Journal of Physiology - Paris 99 (2006) 180–192
the disease (Dineley et al., 2002; Holcomb et al., 1999; Westerman et al., 2002). Within the past several years, the b-site
APP cleaving enzyme 1 (BACE1) (Ohno et al., 2004) has
become the primary therapeutic target candidate (Citron,
2004; Vassar et al., 1999). Thus, in collaboration with Robert Vassar and colleagues, we examined the potential rescue
of behavioral deficits observed in Tg2576 animals by eliminating the function of BACE1 by knocking it out in these
animals (Ohno et al., 2004).
Deletion of BACE1 prevented the behavioral deficit
observed in the Tg2576 animals (Ohno et al., 2004). The
bigenic animals with BACE1"/" and Tg2576 (BACE Æ
Tg2576) performed indistinguishably from their wild-type
littermates in the hippocampus-dependent social recognition and spontaneous alteration Y-maze tasks; whereas,
the Tg2576 animals were severely impaired (Fig. 14). The
behavioral rescue was corroborated with ELISA assays
that showed the levels of Ab were very similar between
the wild-type littermates and the BACE Æ Tg2576 animals.
Thus, we demonstrated for the first time that lowering
Ab levels by inhibiting BACE1 is beneficial for AD-associated memory impairments mediated through the hippocampus.
It has been suggested that Ab can inhibit cholinergic signal transduction independent of apparent neurotoxicity
(Auld et al., 1998; Huang et al., 2000; Kelly et al., 1996;
Zhong et al., 2003). Additionally, as reviewed above, the
cholinergic agonist CI1017 has been demonstrated to facilitate learning and enhance neuronal excitability, via reduction of the AHP. Thus, we examined the biophysical
properties of CA1 neurons from BACE Æ Tg2576 animals
to cholinergic stimulation by bath applying carbachol, a
cholinergic agonist. The capacity for post-synaptic plastic-
Fig. 14. BACE1 Null Mutation Rescues Memory Deficits in the Tg2576
Alzheimer’s Model. Social recognition memory assessed with a 3 h
intertrial delay (n = 10–20). The amount of investigation time during the
second exposure to the same juvenile mouse divided by that of the initial
investigation time · 100 (% investigation) was used as an index of social
recognition memory. Note that only the Tg2576+ group does not show a
reduction in spontaneous investigation to a familiar juvenile (approximately 100%) and thus is significantly impaired in this hippocampusdependent test. Each column represents the mean ± SEM. Significant
differences from wild-type group (**p < 0.01) and Tg2576+ group
(#p < 0.05), compared by ANOVA and post hoc Fisher’s PLSD test.
Reprinted from reference Ohno et al. (2004), Copyright 2004, with
permission from Elsevier.
Fig. 15. BACE1 Null Mutation Rescues Hippocampal Cholinergic
Dysfunction in the Tg2576 Alzheimer’s Model. (A) AHP in response to
a 100 ms depolarizing current injection sufficient to elicit a burst of 7
action potentials was recorded from hippocampal CA1 pyramidal cells.
Representative traces show the post-burst AHP before (control) and after
the application of 0.5 M carbachol (CCh). CCh at 0.5 M selectively
inhibits the slow component of AHP (sAHP) without affecting the peak
amplitude of AHP. Note that the effect of CCh on sAHP in Tg2576+
neurons is reduced as compared to hippocampal neurons from the other
three groups. (B) Summary bar graphs showing CCh-induced reduction in
sAHP measured by amplitudes at 1 s after pulse offset. The reduction of
Tg2576+ sAHP values following CCh application is less than that of wildtype and BACE1"/" Æ Tg2576+. Each column represents the mean ±
SEM of post-CCh sAHP expressed as % of control (pre-CCh) levels
(n = 5–10). Significant differences from wild-type group (*p < 0.05) and
Tg2576+ group (#p < 0.05), compared by ANOVA and post hoc Fisher’s
PLSD test. Reprinted from reference Ohno et al. (2004), Copyright 2004,
with permission from Elsevier.
J.F. Disterhoft, M.M. Oh / Journal of Physiology - Paris 99 (2006) 180–192
ity evidenced by reduction of the AHP in CA1 pyramidal
neurons was rescued in the BACE Æ Tg2576 animals. Carbachol significantly reduced the slow AHP in CA1 neurons
from BACE Æ Tg2576 and wild-type litter mate animals as
compared to those from Tg2576 animals (Fig. 15). Therefore, the behavioral rescue through BACE1 knockout in
Tg2576 animals may be due in part to the restored capacity
for post-synaptic neuronal excitability increases in hippocampal neurons via cholinergic modulation. We assume
that such a process is an important component of the cellular changes that occur during learning in wild type
animals.
7. Concluding statement
The results from our work led us to formulate this working hypothesis: the enhanced excitability of hippocampal
pyramidal neurons, via reductions in the slow AHP and
accommodation, are important cellular changes that
underlie hippocampus-dependent spatial and temporal
learning. In support of this hypothesis, we have demonstrated that a transient, but not permanent, reduction of
the AHP and accommodation is observed in hippocampal
pyramidal neurons of animals that learned a hippocampusdependent task (Kuo et al., 2004; Moyer et al., 1996, 2000;
Oh et al., 1999a, 2003; Thompson et al., 1996b). The postburst AHP in CA1 hippocampal pyramidal neurons from
aged animals is significantly enlarged as compared to that
in neurons from young animals (Kumar and Foster,
2002; Landfield and Pitler, 1984; Moyer et al., 1992,
2000; Oh et al., 1999b; Potier et al., 1992; Power et al.,
2002). Compounds that enhance excitability of CA1 neurons (by reducing the post-burst AHP and accommodation) ameliorate the learning impairment observed in
normal aging animals. It is very interesting to note that
chronic metrifonate treatment altered the biophysical properties of CA1 neurons from aging rabbits to that usually
observed in neurons from young, untreated animals (Oh
et al., 1999b). It is likely that alterations that we have
observed in CA1 hippocampal pyramidal neurons with
the cholinergic compounds are also occurring in other
pyramidal neurons throughout the neocortex. Furthermore, these compounds have been demonstrated to be beneficial in alleviating the cognitive deficits observed in
patients with Alzheimer’s disease.
The alteration of post-synaptic membrane properties
(more specifically, the relationship between AHP and
learning) is also being pursued in other laboratories.
Reduction of the AHP in piriform cortical neurons has
been demonstrated by Barkai and colleagues (Barkai and
Saar, 2001; Saar et al., 1998). Recently, Tombaugh et al.
(2005) elegantly demonstrated that there is a direct, inverse
relationship in aged rats between the size of the post-burst
AHP of CA1 neurons and performance on the water maze
task. They found that aged animals with pyramidal neuron
AHP amplitudes that are similar to that of neurons from
young animals were able to learn the location of the hidden
189
platform; whereas, animals with significantly large pyramidal neurons slow AHPs were unable to learn the task.
Thus, the post-burst AHP in hippocampal pyramidal neurons may be a cellular property that controls the capacity
to learn a hippocampus-dependent task.
In this abbreviated review, we discussed our experiences
with pharmacological compounds and a genetic manipulation that ameliorate the learning deficit observed in normal
aging and in a transgenic animal model of Alzheimer’s disease. A common denominator between normal aging and
the transgenic AD model is the altered biophysical properties of hippocampal pyramidal neurons of these animals as
compared to their respective counterparts (young and wildtype littermates). It remains to be seen if the rescue of
transgenic animals with BACE knockout prevents the
age-related enlargement of the post-burst AHP. The
post-burst AHP is an indicator of cellular excitability.
There are various neurotransmitter/neuromodulators that
have been shown to alter it; in addition to numerous biochemical pathways (Storm, 1990; Wu et al., 2002). Understanding how the AHP is modulated by these pathways will
help us come closer to understanding the cellular events
that take place during learning. This may, in turn,
help engineer future therapeutics to ameliorate the various learning deficits associated with normal aging and
dementia.
Acknowledgment
This work was supported by the National Institutes of
Health Grant R37 AG08796 and R01 MH047340.
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