Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611-3008
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ABSTRACT |
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Power, John M., M. Mathew Oh, and John F. Disterhoft. Metrifonate Decreases sIAHP in CA1 Pyramidal Neurons In Vitro. J. Neurophysiol. 85: 319-322, 2001. Metrifonate, a cholinesterase inhibitor, has been shown to enhance learning in aging rabbits and rats, and to alleviate the cognitive deficits observed in Alzheimer's disease patients. We have previously determined that bath application of metrifonate reduces the spike frequency adaptation and postburst afterhyperpolarization (AHP) in rabbit CA1 pyramidal neurons in vitro using sharp electrode current-clamp recording. The postburst AHP and accommodation observed in current clamp are the result of four slow outward potassium currents (sIAHP, IAHP, IM, and IC) and the hyperpolarization activated mixed cation current, Ih. We recorded from visually identified CA1 hippocampal pyramidal neurons in vitro using whole cell voltage-clamp technique to better isolate and characterize which component currents of the AHP are affected by metrifonate. We observed an age-related enhancement of the slow component of the AHP tail current (sIAHP), but not of the fast decaying component of the AHP tail current (IAHP, IM, and IC). Bath perfusion of metrifonate reduced sIAHP at concentrations that cause a reduction of the AHP and accommodation in current-clamp recordings, with no apparent reduction of IAHP, IM, and IC. The functional consequences of metrifonate administration are apparently mediated solely through modulation of the sIAHP.
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INTRODUCTION |
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There is extensive evidence that
acetylcholine (ACh) is an important neuromodulatory transmitter
affecting hippocampal-dependent learning and memory (Hagan and
Morris 1988; Kaneko and Thompson 1997
). In
addition, Alzheimer's disease (AD) and aging-related cognitive
impairments have been correlated with a loss of cholinergic function
(Disterhoft et al. 1999
; Kasa et al.
1997
; Muir 1997
). Consequently,
enhancement of ACh effectiveness with procholinergic drugs, especially
cholinesterase inhibitors (ChEI), has been a major thrust of AD
treatment. Metrifonate, which is transformed nonenzymatically to
0,0-dimethyl 2,2-dichlorovinyl phosphate producing long-lasting
inhibition of both acetylcholinesterase and butyrylcholinesterase (Nordgren et al. 1978
), has been shown to improve
cognitive performance in AD patients (Cummings et al.
1998
; Morris et al. 1998
; Pettigrew et
al. 1998
) and, in our hands, to enhance hippocampal-dependent learning in aging rabbits (Kronforst-Collins et al.
1997
).
ACh modulates conductances in hippocampal neurons that mediate spike
frequency adaptation (accommodation) and the postburst afterhyperpolarization (AHP) (Halliwell 1990;
Lancaster and Adams 1986
; Madison et al.
1987
). The cholinergic reduction of the AHP and accommodation
is of particular interest since the AHP and accommodation are reduced
in CA1 and CA3 hippocampal pyramidal neurons after hippocampally
dependent trace eyeblink conditioning (Moyer et al.
1996
, 2000
; Thompson et al.
1996b
). Accommodation and AHP are enhanced in CA1 neurons from
animals at ages that show learning deficits (Landfield
and Pitler 1984
; Moyer et al. 1992
,
2000
). Metrifonate reduces the AHP and accommodation in CA1 neurons in young and aging rabbits due to the muscarinic action of
increased ambient ACh levels (Oh et al. 1999
).
The postburst AHP (medium and slow AHP) and accommodation are regulated
by at least four classes of outward potassium currents (IC,
IM,
sIAHP, and
IAHP), in addition the AHP is
influenced by the mixed cation current,
Ih (Maccaferri et al.
1993; Stocker et al. 1999
; Storm
1990
). Of these currents,
IM and
sIAHP are reduced by ACh in CA1
hippocampal neurons with ACh being 10-fold more potent at reducing
sIAHP than
IM in young rats (Madison et al. 1987
). Recently we have shown an age-associated enhancement of the sIAHP and have hypothesized
that enhancement of sIAHP is integrally involved in age-related learning deficits (Power et al. 1999
). The following experiments were undertaken to
determine whether metrifonate reduces the postburst AHP and
accommodation by reducing the sIAHP.
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METHODS |
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Hippocampal slices (300 µm) were prepared from young (2-3 mo)
and aging (>31 mo) female New Zealand White rabbits using procedures identical to those previously published (Oh et al.
1999). Animal use procedures were approved by Northwestern
University's Animal Care and Use Committee according to the standards
of the US Department of Agriculture. Whole cell patch-clamp recordings
were made from the soma of visually identified CA1 pyramidal neurons
(Dodt and Zieglgansberger 1990
) perfused with 31°C
artificial cerebral spinal fluid (ACSF, composition, in mM: 124 NaCl, 3 KCl, 1.3 MgSO4, 1.24 NaH2PO4, 2.4 CaCl2, 26 NaHCO3, and 10 D-glucose). Patch electrodes (2-4 M
) were filled with
(in mM) 2 K-ATP, 10 HEPES, 140 KMeSO4, and 10 KCl; pH 7.25, 280 ± 10 mOsm. The use of
MeSO4
in place of Cl
reduces
the rundown of the slow AHP current over time (Zhang et al.
1994
).
Recordings were sampled at 5-10 kHz and filtered at 2 kHz using an
Axopatch 1C amplifier (Axon Instruments, Foster City, CA) and
controlled by custom software. Only neurons with an action potential
amplitude >90 mV, membrane resistance >60 M, and a stable series
resistance <0.15 M
were considered healthy and used in the data
set. The AHP was evoked by a 100-ms voltage step to 0 mV from a holding
potential of
55 mV. The
55-mV holding potential should eliminate
the influence of Ih, which activates at more hyperpolarized potentials. The medium
(IAHP,
IM, and
IC) and slow
(sIAHP) AHP components were separated
using a curve peeling technique (Lancaster and Adams
1986
; Womble and Moises 1993
) (Fig. 1). The
sIAHP amplitude was also determined
1 s after pulse offset, after medium AHP currents are no longer
active (Sah 1996
; Stocker et al. 1999
).
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Voltage-clamp measurements were taken immediately before and 20 min
after application of metrifonate (young 50 µM; aging 100 µM). These
concentrations of metrifonate were found to reduce the AHP and
accommodation in sharp electrode current-clamp recordings comparably
between young and aging neurons without inducing a bursting behavior
(Oh et al. 1999). Sodium and potassium channel blockers
were omitted to achieve the same cholinergic activity levels present in
our previous current-clamp study (Oh et al. 1999
). In
some neurons, current-clamp measures of the AHP were also taken. In
current clamp, the postburst AHP was evoked from a
68-mV holding
potential using a 100-ms depolarizing current step that elicited four
action potentials.
Age-related differences were analyzed with ANOVA. Drug-related effects were analyzed using paired t-tests. The correlation between current-clamp and voltage-clamp measures was tested using Fisher's r to z test.
Metrifonate was a gift from Bayer (West Haven, CT). KMeSO4 was purchased from ICN (Aurora, OH). All other drugs were purchased from Sigma (St. Louis, MO).
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RESULTS |
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Age effects
The AHP measures and passive membrane properties are given in
Table 1. The current-clamp AHP tended to
be enhanced in neurons from aging animals [F(18, 1) = 4.09, P = 0.0583] as had been previously observed
(Moyer et al. 1992; Oh et al. 1999
). In
voltage-clamp mode, a 55-mV, 100-ms voltage step from a holding
potential of
55 mV evoked a biphasic outward tail current in most
neurons that slowly decayed back to baseline (Fig. 1). This tail
current was separated into a fast
(Ifast) and a slow
(Islow) decaying component
representing the medium (IAHP,
IM, and
IC) and slow (sIAHP) AHP. The extrapolated
amplitude of Islow [F(18,
1) = 6.56, P = 0.0196] and the tail current
measured 1 s after pulse offset (1s_AHP) [F(20,
1) = 15.85, P = 0.0007] showed age related
enhancements. There was no age-associated change in
Ifast. The 1s_AHP current was
correlated with the current-clamp AHP area (r =
0.453, P = 0.0441) and duration (r = 0.527, P = 0.0155). Neurons from aging animals had a
more hyperpolarized resting potential (RMP) [F(19, 1) = 5.206, P = 0.0342] and a decreased membrane
resistance (Rm) [F(19,
1) = 5.40, P = 0.0313].
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Metrifonate effects
Bath application of metrifonate caused a reduction of the postburst AHP (Table 1). Metrifonate also reduced the sIAHP, measured as either Islow or 1 s after pulse offset, in neurons from both young and aging animals (Table 1, Fig. 2). Metrifonate did not produce a significant reduction in Ifast, nor did the effect of metrifonate on the fast component differ from that of saline controls.
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DISCUSSION |
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An age-associated enhancement of the slow
(sIAHP), but not the fast
(IAHP,
IC, and
IM), component of the AHP tail current was observed. Furthermore metrifonate decreased the slow tail component
(sIAHP) in CA1 neurons from both young
and aging animals at doses that reduce postburst AHP and accommodation
in sharp-electrode current-clamp recordings (Oh et al.
1999). We did not observe a metrifonate-induced reduction in
the fast component of the AHP tail current. ACh is more potent at
reducing sIAHP than
IM (Madison et al.
1987
). However, since we were unable to adequately peel the fast component of the AHP in every instance and the relative contribution of IM to the fast tail
component was not determined, it is unclear whether metrifonate reduced
IM as well as
sIAHP.
Metrifonate was shown to be less potent at reducing the postburst AHP
and accommodation of CA1 neurons of aging than young rabbits in our
previous current-clamp studies that evaluated dose-response relationships in the two age groups (Oh et al. 1999).
Metrifonate doses that produced a comparable reduction of the postburst
AHP in young and aging neurons (Oh et al. 1999
), caused
a similar reduction of the sIAHP. Thus
metrifonate appears to modulate neuronal excitability similarly in
neurons from young and aging subjects. It is likely that the
age-related potency differences are due to reduced levels of endogenous
ACh release observed in aging subjects in vivo (Vannucchi et al.
1997
).
In summary, metrifonate reduces the AHP, in particular, the
sIAHP. The AHP and accommodation are
reduced after hippocampally dependent trace eyeblink conditioning
(Moyer et al. 1996; Thompson et al.
1996b
). The sIAHP is enhanced
in CA1 neurons from aging rabbits (Power et al. 1999
), a
group that is learning-impaired (Thompson et al. 1996a
).
Metrifonate facilitates learning in aging rabbits
(Kronforst-Collins et al. 1997
). Similar results were obtained with the M1 muscarinic agonist CI-1017, and the L-type calcium
channel blocker, nimodipine. Both of these compounds reduce the AHP and
accommodation in vitro and facilitate acquisition of trace eyeblink
conditioning when administered to aging rabbits (Deyo et al.
1989
; Moyer et al. 1992
; Weiss et al.
2000
). As a whole, these data support our hypothesis that the
sIAHP is integrally involved in
age-related learning impairments and suggest that the functional
consequences of metrifonate administration are mediated through
modulation of the sIAHP. More
generally, they suggest that the ability of a drug to reduce the
sIAHP may be a good predictor of that
compound's ability to facilitate learning, especially in aging subjects.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health Grants AG-08796 and MH-11737 and by Bayer Inc.
Present address of J. M. Power: Division of Neuroscience, John Curtin School of Medical Research, Australian National University, ACT 2601, Australia.
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FOOTNOTES |
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Address for reprint requests: J. F. Disterhoft, Dept. of Cell and Molecular Biology, Northwestern University Medical School, Searle 4-427, 303 E. Chicago Ave., Chicago, IL 60611-3008 (E-mail: jdisterhoft{at}northwestern.edu).
Received 27 December 1999; accepted in final form 22 September 2000.
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REFERENCES |
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