Metrifonate Decreases sIAHP in CA1 Pyramidal Neurons In Vitro

John M. Power, M. Mathew Oh, and John F. Disterhoft

Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611-3008


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega ) 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 MOmega , and a stable series resistance <0.15 MOmega 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).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1. Age-associated changes in the afterhyperpolarization (AHP) tail current. A typical biphasic outward tail current (Itail) decaying back to baseline is shown in A. A single exponential was fitted to the late portion (400-3,000 ms) of the AHP and extrapolated to pulse offset (Islow). Islow was subtracted from the total tail, and a 2nd exponential curve was fitted to the residual current and Ifast (inset). Peeling was successful in 13 of 16 young and 4 of 5 aging neurons. An age-associated enhancement was observed in the slow component (B), but not the fast component (C).

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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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].


                              
View this table:
[in this window]
[in a new window]
 
Table 1. Biophysical properties of CA1 neurons from young and aging rabbits before and after bath application of metrifonate

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.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2. Bath application of metrifonate caused a reduction of the sIAHP. Representative tail currents are shown before and after the bath application of metrifonate. The inset bar graph shows the percent reduction (mean ± SE) of the sIAHP measured 1 s after pulse offset after metrifonate application in young (young 50 µM; n = 12) and aging neurons (aging 100 µM; n = 5). Also shown in the bar graph is the sIAHP reduction observed when normal saline (n = 4) was perfused over the same 20-min time period. The saline perfusate served as a control for normal rundown. The 30% reduction of the sIAHP in metrifonate is greater than the 4% reduction observed in saline controls [ANOVA: 1s_AHP F(15, 1) = 9.616, P = 0.0085; Islow F(12, 1) = 8.060, P = 0.0149].


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society