INTRODUCTION

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An influential hypothesis of brain aging states that age-related changes in neuronal function, viability, and ultimately cognition are attributable to the gradual dysregulation of Ca²⁺ homeostasis (Disterhoft et al. 1994; Foster and Norris 1997; Khachaturian 1994; Landfield 1994; Thibault et al. 1998; Verkhratsky and Toescu 1998). Altered Ca²⁺ regulation is thought to be due, at least in part, to increased Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channels (VDCCs). L-channels have been implicated in the increased afterhyperpolarization (AHP), altered synaptic plasticity, and shift in protein kinase and phosphatase activity during brain aging (Disterhoft et al. 1996; Foster 1999; Foster et al. 2001; Landfield and Pitler 1984; Norris et al. 1998a,b; Shankar et al. 1998; Thibault and Landfield 1996). Indeed, several of these processes appear to be linked since L-channel antagonists block induction of long-term depression (LTD) and reduce the AHP by limiting Ca²⁺ influx. In turn, the reduced AHP facilitates the induction of long-term potentiation (LTP) in aged animals (Norris et al. 1998a). The results of these studies suggest that treatments directed at Ca²⁺ regulation or Ca²⁺-dependent processes may prove beneficial in preventing cognitive decline during aging (Foster 1999).

Clinical data indicate that estrogen replacement therapy in women can delay cognitive decline associated with brain aging (Birge et al. 2001; Hogervorst et al. 2000; Sherwin 1999). The mechanism for gonadal steroid modulation of brain function is unclear and may involve a number of different genomic and nongenomic mechanisms. Recent evidence suggests that estradiol can alter Ca²⁺-dependent processes through nongenomic mechanisms. For example, estradiol rapidly modifies synaptic strength and Ca²⁺-dependent synaptic plasticity of the hippocampus, blocking LTD and facilitating the induction of LTP (Cordoba Montoya and Carrer 1997; Foy et al. 1999; Good et al. 1999; Sharrow et al. 2002; Vouimba et al. 2000; Warren et al. 1995). The influence of estradiol on hippocampal synaptic function is rapid and can be observed in the presence of estrogen receptor antagonists and in estrogen receptor knockout animals, indicating a nongenomic mechanism (Fugger et al. 2001).

Thus the effects of estradiol on Ca²⁺-dependent processes such as synaptic plasticity are opposite those observed during aging and similar to those observed following L-channel blockade (Foster 1999; Sharrow et al. 2002). The results suggest that estradiol may influence hippocampal synaptic plasticity through altered Ca²⁺ regulation including a reduction in the AHP. The current study examined the effects of 17-estradiol benzoate (EB) on the AHP recorded from hippocampal CA1 neurons in aged and young ovariectomized rats. Furthermore, to delineate the possible role of L-type Ca²⁺ channels in mediation of EB effects, experiments were also performed in the presence of the L-channel antagonist, nifedipine.

	METHODS

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Subjects

Procedures involving animal subjects have been reviewed and approved by University of Kentucky Institutional Animal Care and Use Committee. Female Fischer 344 rats of two ages, young (5-8 mo) and aged (22-24 mo), were group housed (2 per cage), maintained on a 12:12 h light:dark schedule, and provided ad libitum access to water and a casein-based rat chow (Cincinnati Lab Supply, Cincinnati, OH), which is low in phytoestrogens.

Surgery

Female rats were ovariectomized under ketamine:xylazine anesthesia (50 mg:5 mg/kg body weight). Rats were handled for 5 min a day for 1 wk prior to surgery. Bilateral incisions were made to expose the ovaries and ovaries were cleared from the fat tissue and dissected out. On days 3 and 5 post surgery vaginal lavage was performed to insure that the estrous cycle had stopped (Sharrow et al. 2002).

Hippocampal slice preparation

Rats were overdosed with CO₂ (8-28 days after ovariectomy) and hippocampi were dissected out. Hippocampal slices (~400 µm) were cut parallel to the alvear fibers using a Vibratome (Technical Products International Inc., St. Louis, MO). Slices were then transferred to a standard interface-recording chamber that was continuously perfused (1 ml/min) with oxygenated artificial cerebrospinal fluid (ACSF) containing the following (in mM): 124 NaCl, 2 KCl, 1.25 KH₂PO₄, 2 MgSO₄, 2 CaCl₂, 26 NaHCO₃, and 10 glucose. Slices were maintained at 30-32°C and humidified air (95% O₂-5% CO₂) was blown over the slices.

Intracellular recording

Microelectrodes were pulled from thin-wall 1.0-mm microfiber-filled borosilicate capillaries using a Flaming/Brown horizontal micropipette puller (Sutter Instruments, San Rafael, CA). The resistance of microelectrodes when filled with 3 M potassium acetate ranged from 50 to 100 M. Microelectrodes were visually positioned in the CA1 pyramidal cell layer using a dissecting microscope (SZH10, Optical Elements Corp., Washington, DC). The signals were amplified using an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA), and recordings were sampled at 1 kHz and stored on computer disk for off-line analysis (Data Wave Technologies, Longmont, CO).

Only neurons with resting membrane potential (RMP) less than 60 mV, an input resistance >20 M, and an action potential amplitude rising 70 mV above the point of spike initiation were included in the analysis. Cells' RMP was maintained between 63 and 75 mV with current injection. Voltage deflections resulting from hyperpolarizing current pulses (100 ms, 0.2 nA) were used to determine input resistance. Depolarizing current pulses (100 ms, 0.1-1.2 nA) were delivered every 20 s through the intracellular electrode to elicit a sodium spike bursts of two to four action potentials. The AHP was measured as the difference between the membrane potential during the 100-ms period immediately before the onset of the depolarizing current and the membrane potential 500 ms after the offset of the depolarizing current.

In some cases, nifedipine was dissolved in a small amount of DMSO and added to regular ACSF to obtain a desired concentration of nifedipine (10 µM) and a level of DMSO of no more than 0.005%. Application of DMSO alone had no effect on the AHP (data not shown). Nifedipine was applied for 30 min before commencement of recording. EB was initially dissolved in a small amount of ethanol and diluted by ACSF to a final ethanol concentration of 0.001% and EB of 100 pM. Slices were either perfused with 100 pM EB, or EB was rapidly applied in the vicinity of the cell through the broken tip of a glass pipette. The tip was placed within 1 mm of the recording pipette, and the drug was observed to "weep" across the slice (Blalock et al. 1999). Because of the light sensitivity of EB and nifedipine, all experiments were conducted in a darkened room.

For each cell, the average of 5 to 10 AHPs was calculated at the three different spike levels (2, 3, and 4 spikes). For analysis of drug or hormone effects, all AHPs were normalized relative to the mean for the control condition within each spike level. A one-way analysis of variance (ANOVA), repeated across the three spike levels, was used to determine age or treatment effects. Post-hoc analyses were conducted using Scheffe's F tests, with significance set at P < 0.05. Where stated, n represents the number of cells used in each set of experiments.

	RESULTS

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A total of 50 recorded cells were acceptable according to our criteria. In several cases, the same cell was recorded under different conditions, such as in the presence and absence of EB. No significant differences in the intrinsic membrane properties (input resistance, resting membrane potential, and spike amplitude) were observed between the two age groups or due to the various treatment conditions (Table 1). Previous research has demonstrated that there is an increase in the AHP amplitude with advanced age (Disterhoft et al. 1993; Landfield and Pitler 1984; Pitler and Landfield 1990) and this was confirmed in the current study. Figure 1A shows an example of the AHP elicited by depolarizing current pulses to evoke four action potentials from CA1 pyramidal cells of young and aged ovariectomized female rats. A repeated measure ANOVA on the AHP amplitude indicated a significant increase in AHP amplitude with increasing number of action potentials [F(2,26) = 22.96, P < 0.0001] and a significant age-related increase in the AHP [F(1,26) = 18.37, P < 0.001]. Scheffe post-hoc comparisons indicated that the AHP was significantly increased in aged rats for each spike level (Fig. 1B).

View larger version (14K): [in this window] [in a new window]   Fig. 1. The afterhyperpolarization (AHP) amplitude is increased in aged ovariectomized female rats. A: representative intracellular voltage records from CA1 pyramidal cells in aged and young rats are shown after a train of 4 action potentials elicited by a 100-ms pulse of depolarizing current. Note that in this and subsequent figures, action potentials are truncated or removed to better show the AHPs. B: mean AHP amplitude (mV) recorded in neurons of aged (filled bars, n = 7) and young (open bars, n = 8) rats for the three spike levels, from 2 to 4 spikes. Pound signs indicate a significant increase (P < 0.05) in AHP amplitude in aged rats compared with young rats. C: percentage change in the AHP relative to controls following application of 17-estradiol (EB; 100 pM). EB reduced the AHP amplitude in aged ovariectomized female rats (filled bars, n = 5). In general, application of EB reduced the AHP in young ovariectomized female rats (open bars, n = 6). However, the EB-mediated decrease in the AHP was highly variable and did not reach significance. Asterisk indicates a significant (P < 0.05) decrease in AHP amplitude relative to the mean for the control condition within each spike level. In this and subsequent figures, error bars represent ±SE.

For slices from aged rats, direct application of EB resulted in a decrease in the AHP that could be observed within 5-10 min of EB application (Fig. 2). An ANOVA indicated that the decrease was significant [F(1,20) = 5.01, P < 0.05] (Fig. 1C). Post-hoc comparisons indicated that EB reduced the AHP at the two- and three-spike level and the reduction in the AHP approached significance (P = 0.09) for the four-spike level. In contrast, while application of EB tended to reduce the AHP in young rats, the reduction was highly variable and failed to reach significance (Fig. 1C). Finally, while EB washout was not systematically investigated, our initial observations (n = 3) indicate that reversal required 30 min.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Illustration of the rapid reduction in the AHP amplitude following EB application. The data points represent the means for 6 cells from aged animals. For each cell, depolarization was set to elicit 4 spikes and the amplitude of the AHP collected during the 5 min prior to EB application was used as a baseline for calculating the percentage change over time. In general cells exhibited a reduction in the AHP within the first 5-10 min following EB application (arrow).

To determine whether the differential ability of EB to reduce the AHP might be related to differences in Ca²⁺ regulation and the AHP amplitude, the external Ca²⁺ concentration was raised to 4 mM for slices from young animals. Under these conditions, an increase in the AHP was observed compared with slices from young rats bathed in the 2 mM Ca²⁺ medium, and an ANOVA indicated a significant [F(1,34) = 17.76, P < 0.001] effect of Ca²⁺ level on the AHP (Fig. 3A). Scheffe post-hoc comparisons revealed that the AHP increase was significant at each spike level. In young rats, under condition of elevated Ca²⁺, application of EB was associated with a significant [F(1,30) = 7.69, P < 0.05] reduction in the AHP amplitude, which could be observed for each spike level (Fig. 3B). Finally, application of the ethanol vehicle alone did not mimic the effect of EB on the AHP (94% of baseline, n = 3, data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 3. EB reduces the AHP in young ovariectomized female rats under conditions of elevated Ca2+. A: elevation of Ca2+ concentration in the recording medium from 2 mM (open bars, n = 8) to 4 mM (filled bars, n = 11) is associated with a marked increase in the AHP amplitude. B: under conditions of elevated Ca2+, EB (100 pM) reduced the AHP measured as the percentage change relative to control (n = 6). C: examples of AHPs, elicited by 4 spikes in elevated Ca2+ conditions alone (Control), and following application of EB. Pound signs indicate a significant difference (P < 0.05) in AHP amplitude for slices recorded in 4 mM Ca2+ relative to slices recorded in 2 mM Ca2+. Asterisks indicate a significant decrease (P < 0.05) in AHP amplitude relative to the control condition.

The age-related increase in the AHP is thought to involve increased Ca²⁺ influx through L-channels (Disterhoft et al. 1996; Thibault and Landfield 1996). In an attempt to determine the role of L-type Ca²⁺ channels in mediating the EB-mediated AHP reduction, the ability of L-channel blockade to occlude EB effects was examined. The AHP was recorded following addition of the L-type Ca²⁺ channel antagonist nifedipine (10 µM) to the bath (n = 10). Subsequently, EB (100 pM, n = 6) was applied through a weeper pipette. Figure 4 illustrates the percentage decrease in the AHP for nifedipine alone and for application of EB in the presence of nifedipine. An ANOVA indicated a significant interaction [F(4,40) = 3.89, P < 0.01] between the three spike levels and the three treatment conditions, control, nifedipine, and nifedipine + EB. Post-hoc tests indicated that, for each spike level, the AHP was significantly reduced for nifedipine and nifedipine + EB compared with controls. While the response to nifedipine + EB was generally reduced relative to nifedipine alone, this difference did not reach significance (P = 0.09).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Role of L-channels in the EB-mediated decrease in the AHP amplitude. A: nifedipine (10 µM) application decreased the AHP for aged rats (filled bars, n = 10). In the presence of nifedipine, the AHP amplitude exhibited a nonsignificant tendency toward a further decrease following application of EB (open bars, n = 6). B: illustration of treatment effects on the AHP amplitude elicited by 4 spikes in the same cell from an aged animal. The largest AHP was observed under control conditions (Control). Adding EB (100 pM) to the bath reduced the AHP (EB) and occluded the ability of L-channel blockade (Nifedipine + EB) to further decrease the AHP. Asterisks indicate a significant (P < 0.05) decrease in the AHP relative to the ACSF control condition.

In a second series of studies, the AHP was elicited by four spikes in cells of aged animals during perfusion with control ACSF recording media; the recording media was then switched to ACSF containing EB (100 pM), followed by switching to recording media containing EB (100 pM) + nifedipine (10 µM). The AHP responses elicited by four spikes were recorded for 15 min after each switching of the recording media, until the response stabilized. Only cells that exhibited four spikes across all conditions were included in the analysis (n = 5). Addition of EB to the bath reduced the AHP to 66 ± 6% of control. Subsequently switching to EB + nifedipine resulted in an AHP that was 62 ± 10% of the control level. A repeated measures ANOVA indicates an effect of treatment [F(2,8) = 10.38, P < 0.01] and post-hoc comparisons indicated that the AHP was reduced under both treatment conditions. However, no difference was observed between EB and the addition of nifedipine in the presence of EB, indicating that EB occluded any further reduction due to addition of the L-channel antagonist.

	DISCUSSION

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The main finding of the present study is that EB can decrease the AHP in CA1 pyramidal neurons. The effect of EB was particularly robust in aged animals under control conditions of 2 mM Ca²⁺ recording media. The age difference was due, at least in part, to the larger AHP observed in aged animals, since an EB-mediated reduction in the AHP could be observed in young animals under conditions that enhance the AHP amplitude. The AHP is a Ca²⁺-dependent process and previous studies demonstrate that Ca²⁺ action potentials, macroscopic Ca²⁺ currents, and the Ca²⁺-dependent AHP are consistently increased and prolonged in hippocampal CA1 pyramidal neurons in aged male rats (Kerr et al. 1989; Landfield and Pitler 1984; Pitler and Landfield 1990) and aged female rabbits (Disterhoft et al. 1993; Moyer et al. 1992; Moyer and Disterhoft 1994). The current study confirms an age-related increase in the AHP and extends the results to include female rats.

Changes in the AHP and subsequent cell excitability following EB treatment have been reported for various excitable cells and in other brain regions (Inoue et al. 1999; Israel and Poulain 2000; Schiess et al. 1988). One possible mechanism for EB effects on the Ca²⁺-dependent AHP is through inhibition of L-type Ca²⁺ channels (Jiang et al. 1992; Kurata et al. 2001; Mermelstein et al. 1996; Sheldon and Argentieri 1995; Zhang et al. 1994). The finding that EB could occlude the reduction in the AHP due to subsequent L-channel blockade is consistent with the idea that EB inhibits L-type Ca²⁺ channels. Several studies have provided evidence for the idea that rapid EB effects in the hippocampus mediated through a membrane-associated estrogen receptor, which regulates protein kinase/phosphatase signaling pathways. For example, a recent study in hippocampal cell cultures suggests that EB can reduce Ca²⁺ influx by modulating the dihydropyridine site and verapamil-binding site of Ca²⁺ channels (Kurata et al. 2001), which in turn could influence the activity of Ca²⁺-dependent enzymes. Indeed, the activity of the Ca²⁺-dependent phosphatase, calcineurin, is reduced following treatment with EB (Sharrow et al. 2002) or L-channel blockade (Foster et al. 2001). Alternatively, other studies suggest that EB acts on G proteins within the membrane to influence the balance of kinase/phosphatase activity (Gu and Moss 1996), and this altered activity could reduce L-channel function through a shift in L-channel phosphorylation state (Norris et al. 2002).

The age-related increase in the AHP is thought to involve an increase in the number of L-type Ca²⁺ channels (Thibault and Landfield 1996), which might explain the age-related differences in the EB-mediated reduction in the AHP. In contrast, the fact that EB tended to further reduce the AHP during L-channel blockade suggests that other mechanisms may also be involved. These other mechanisms could include direct actions on K⁺ channels (Valverde et al. 1999) or indirect effects due to activity of second messenger systems (Kelly et al. 1999). However, previous work indicates that EB injections do not alter K⁺ currents in CA1 pyramidal cells (Joels and Karst 1995). Alternatively, EB may have prevented Ca²⁺ influx from other VDCCs (Zhang et al. 1994) or influenced release of Ca²⁺ from intracellular stores (Beyer and Raab 1998), which would normally contribute to the level of intracellular Ca²⁺ and the AHP amplitude (Jacobs and Meyer 1997; Sandler and Barbara 1999).

Regardless, the ability of EB to rapidly reduce the AHP has important implications for understanding EB effects on hippocampal functions. The amplitude of the AHP can modify cell excitability and the threshold for induction of Ca²⁺-dependent synaptic plasticity (Foster 1999). In aged animals a decrease in the AHP due to blockade of K⁺ channels or L-channels is associated with facilitation of LTP induction and L-channel antagonism also impairs induction of LTD (Norris et al. 1998a). A similar shift in synaptic plasticity can be observed immediately following EB application (Foy et al. 1999; Sharrow et al. 2002), suggesting that EB rapidly shifts synaptic plasticity threshold through similar mechanisms.

In contrast to rapid, short-term effects, CA1 pyramidal cells also exhibit prolonged changes in excitability in response to the history of hormone exposure. In young animals, sexually dimorphic responsiveness of hippocampal field potentials to EB application depends on the history of circulating gonadal steroids (Foy et al. 1984). Furthermore, EB responsiveness is a function of the presence/absence of the -estrogen receptor, indicating that gonadal steroids may be acting through genomic mechanisms to influence excitability (Fugger et al. 2001). In the current study the AHP amplitude for aged ovariectomized females was similar to that recorded under similar conditions (i.e., 2 mM Ca²⁺ and Mg²⁺) in aged male rats (Landfield and Pitler 1984). Nonetheless, the AHP amplitude in young ovariectomized females was reduced compared with those reported in young males. The smaller AHP may relate to the tendency for a larger RMP in the present study. In addition to the influence of extracellular Ca²⁺, the AHP amplitude is a function of the RMP, with smaller responses observed for RMPs nearer to the K⁺ reversal potential (Potier et al. 1992). In addition, it is possible that AHP amplitude exhibits sexually dimorphic differences due to the transcription of Ca²⁺ regulatory genes according to the history of circulating gonadal steroids. For example, EB treatment is associated with a shift in the expression of Ca²⁺ regulatory genes including VDCCs and N-methyl-D-aspartate receptors (Adams et al. 2001; Johnson et al. 1997), which could result in a rearrangement of Ca²⁺ homeostasis.

In conclusion, our results indicate that EB can rapidly reduce the AHP of ovariectomized rats. The effect of EB on the AHP may have important implications for understanding hormone effects on physiological processes (e.g., synaptic plasticity, cell excitability, and Ca²⁺ homeostasis) and changes in cognition during aging or across the estrus cycle that are thought to depend on these physiological processes.