The slow Ca2+-activated K+ current (sIAHP) was recorded in CA1 pyramidal neurons in hippocampal slices obtained from ovariectomized (OVX) or sham OVX (control) female rats. The sIAHP was significantly larger in cells from OVX rats than in cells from control rats. Superfusion with 5–100 nm 17β-estradiol (E2) caused a progressive decrease in the sIAHP in cells from OVX rats but not in cells from control rats. In slices from OVX rats injected with 10 μg of E2 24 and 48 hr before they were killed, superfusion with E2 did not modify the sIAHP. In neurons from OVX rats, but not in neurons from control rats, E2 significantly increased both the number of action potentials and the burst duration generated by depolarizing pulses. The inactive isomer 17α-estradiol had no effect. The impermeant protein conjugate E2—BSA was as effective as free E2 at decreasing the sIAHP. Ca2+ spikes were also depressed by E2 in neurons from OVX rats, but not in control rats. A decrease in the intracellular Ca2+ signal, correlating with the inhibition of the Ca2+ spike and sIAHP produced by E2, was observed only in neurons from OVX rats. Our results indicate that ovariectomy increases the sIAHP and depresses excitability, whereas bath application or priming with E2 decreases the sIAHP, thus promoting excitability. These effects of E2 on the sIAHP and excitability, which are stereospecific and presumably mediated by membrane-bound receptors, could contribute to the hormonal regulation of synaptic plasticity and epileptiform activity as well as to learning and cognitive abilities dependent on the function of hippocampal neural circuits.
- hyperpolarizing potassium current
- calcium-dependent potassium current
- calcium signal
- estrogen effects
Levels of circulating estrogen influence aspects of behavior that are not directly related to reproduction. Cognitive performance and learning ability in experimental animals (Fader et al., 1998; Daniel et al., 1999; Gibbs, 2000) and humans (Hampson, 1990b; Henderson et al., 1996; Asthana et al., 1999; Costa et al., 1999; Drake et al., 2000; Duka et al., 2000; Yaffe et al., 2000) vary concurrently with the reproductive cycle or with experimentally induced changes in estrogen levels. For example, administration of estrogen improves tests of spatial learning and navigation, tasks in which hippocampal neural circuits have been shown to be involved (Hampson, 1990a; Galea et al., 1995; Mead and Hampson, 1997). The precise mechanisms for these effects of estrogen are not fully understood. Reports from Moss' laboratory (Wong and Moss, 1991, 1992, 1994; Gu and Moss, 1998; Moss and Gu, 1999) suggest that the hormone regulates synaptic transmission. They described short-term and long-term effects of estradiol, all conducive to facilitated excitatory input, mediated at least in part by “fast” actions through putative membrane receptors. Moreover, estrogen can enhance long-term potentiation (LTP) (Córdoba Montoya and Carrer, 1997; Foy et al., 1999; Good et al., 1999), which could be a consequence of increased synaptic effectiveness resulting from stimulated dendritic spinogenesis, increased glutamate sensitivity, and/or a new balance of excitatory and inhibitory input (Rudick and Woolley, 2001) occurring in the hippocampus (for review, see McEwen et al., 2001).
In addition to improving synaptic transmission, estradiol could act to modulate the membrane mechanisms that control neuronal excitability and repetitive firing. In hippocampal pyramidal cells, action potentials are followed by a multicomponent afterhyperpolarization (AHP) comprising a fast AHP, a medium AHP, and a slow AHP (sAHP) (for review, see Storm, 1990; Sah and Davies, 2000). These AHPs act as a negative feedback regulating excitability and spike frequency adaptation and could be regulated by estrogen.
The following studies were designed to investigate whether estrogen could regulate the slow Ca2+-activated K+ current (sIAHP) that mediates the sAHP. We recorded the sIAHP of CA1 pyramidal neurons in hippocampal slices obtained from animals that had been ovariectomized (OVX) 4–5 weeks in advance and compared the responses with those of sham OVX rats. We show that neuronal excitability was markedly reduced, and that both the amplitude and area of the sIAHP were notably larger in OVX rats than in control rats. Furthermore, superfusion with estradiol significantly reduced the sIAHP and increased neuronal excitability in slices from ovariectomized animals. However, the sIAHP was unaffected by estradiol in animals previously exposed to estrogen (i.e., in both control and estradiol-primed ovariectomized rats). Furthermore, estradiol reduced the amplitude of depolarization-evoked Ca2+ spikes in slices from OVX rats. Finally, superfusion of estradiol in slices from ovariectomized rats but not from control rats reduced the depolarization-evoked intracellular Ca2+ elevation that correlated with the estradiol-mediated sIAHP reduction. We conclude that estrogen regulates the sIAHP and neuronal excitability, and that these effects are mediated by specific membrane receptors that control Ca2+ influx, thus modulating the intracellular Ca2+ signal.
Materials and Methods
OVX and sham OVX (control) female Wistar rats were used. Surgery was performed under cold anesthesia 2 d after birth (Yi and Barr, 1996). Pups were ovariectomized through a bilateral laparotomy; the skin was sutured and the incision was covered with the acrylic polymer Nobecutan (Inibsa Laboratorios, Barcelona, Spain) before they were returned to their mothers. In sham OVX animals, the ovaries were exposed and the skin was sutured. Animals were decapitated at 35–60 d of age, and brains were rapidly removed and submerged in ice-cold artificial CSF (ACSF). All experiments in this study conformed to international guidelines on the ethical use of animals, and every effort was made to minimize the suffering and number of animals used.
Transverse 300 μm slices of the hippocampus were cut with a vibratome (Pelco 101 Series 1000; Pelco, St. Louis, MO) and preincubated for 1 hr at room temperature in ACSF continuously bubbled with carbogen (95% O2, 5% CO2). The incubation ACSF contained (in mm): 124 NaCl, 2.6 KCl, 1.25 KH2PO4, 2 Mg2SO4, 26 NaHCO3, 2 CaCl2, and 10 glucose, pH 7.4. Slices were transferred to a 2 ml chamber fixed to an inverted microscope stage (Diaphot TMD; Nikon, Tokyo, Japan) and superfused continuously at 1 ml/min at room temperature (21–23°C) with ACSF containing 50 μm picrotoxin. Recordings in the whole-cell configuration of the “blind” patch-clamp technique used 4–7 MΩ pipettes filled with (in mm): 150 KMeSO4 (ICN Pharmaceuticals, Costa Mesa, CA), 10 HEPES, and 4 ATP-Na. Pipettes were connected to an Axoclamp-2A amplifier (Axon Instruments, Foster City, CA), and recordings were in the bridge-electrode or single-electrode current-clamp mode and single-electrode voltage-clamp mode. The series resistance was compensated to 60–80%, and capacitances were neutralized.
Data were low-pass filtered at 1 kHz (VBF4; Kemo, Beckenham, UK) and sampled at rates of >2 kHz with a 486 personal computer and a TL-1/DMA interface board (Axon Instruments). pClamp software (Axon Instruments) was used for experimental control, data acquisition, and analysis. Recording pipettes were positioned using a micromanipulator, and the CA1 soma layer was visualized directly. Pyramidal cells were recognized by their characteristic responses to depolarizing current pulses under current clamp (Borde et al., 1995, 2000). Only one neuron was studied per slice.
Experiments in both current-clamp or voltage-clamp modes were started subsequent to stabilization period of ∼5 min after access to the intracellular compartment. Two experimental protocols were used in the current-clamp mode. To evaluate passive membrane properties (Table 1; resting potential, membrane input resistance, and time constant), 10 successive 200 msec depolarizing pulses starting from 0.3 nA, and increasing in 0.08 nA steps, were delivered at rates of 1 sec -1 in current-clamp mode. Additionally, changes in cell excitability were evaluated in current-clamp mode by calculating the mean number of action potentials (APs) and the duration of bursts evoked by single 2 sec, 0.5 nA depolarizing current pulses delivered every 5–10 min while holding the membrane potential (Vm) at -60 mV (Table 1; action potential duration, adaptation, and burst duration).
To study the sIAHP, membrane currents were evoked by 200 msec depolarizing voltage commands, from a -60 or -50 mV holding potential (Vh) to +10 mV. To avoid possible interference between the responses, depolarizing pulses were delivered every 5–10 min. The sIAHP magnitude was quantified from the area under the current trace, measured 200 msec after the end of the pulse [when the amplitude of the medium IAHP (mIAHP) was negligible] (Martín et al., 2001). The mIAHP amplitude was measured ∼50 msec after the end of the pulse. To simultaneously monitor the access and membrane resistance and capacitance, the voltage-clamp protocol included a 70 msec, 5 mV hyperpolarizing pulse at the end of the recording 8 sec epoch (Fig. 1 A). Preliminary experiments showed that sIAHP was relatively small immediately after breaking the cell membrane but increased rapidly to stabilize in 10–15 min. For this reason, after establishing the whole-cell configuration, control recordings were obtained over ∼20 min. After obtaining control recordings (usually three to six), superfusion was started with ACSF containing 5, 10, or 100 nm 17β-estradiol (E2). Superfusion with E2 was maintained for 40 min and then replaced with normal ACSF. The E2 was dissolved in absolute ethanol and added to ACSF; final ethanol concentrations never exceeded 0.01%. E2 conjugated to bovine serum albumin [17β-estradiol-6-(O-carboximetil)oxima (E2–BSA)] was made up as a concentrated (100 μm) stock solution in ACSF, aliquoted, and frozen (-20°C) until use at a final concentration of 100 nm in ACSF. The concentration of BSA was matched in the control ACSF in experiments testing the effect of E2–BSA. Identical experimental protocols were used when studying the effect of 17α-estradiol.
Ca 2+ spikes were recorded in current-clamp mode in the presence of 0.5 or 1 μm tetrodotoxin (TTX; Tocris Cookson, Bristol, UK) and 5 mm tetraethylammonium (TEA). Ca 2+ spikes were generated by 0.5 sec, 0.3 nA depolarizing current pulses while holding the Vm at -60 mV.
Measurement of intracellular Ca2+ variations was performed on hippocampal slices obtained as described above. Pyramidal cells in the CA1 region were visualized under an BX50WI microscope (Olympus Optical, Tokyo, Japan) equipped with infrared and differential interference contrast imaging devices, and with a 40× water immersion objective. Patch pipettes were filled with the standard internal solution containing 10 μm Fluo-3 (Molecular Probes, Eugene, OR). Cells were illuminated with a xenon lamp at 490 nm using a monochromator Polychrome II (T.I.L.L. Photonics, Planegg, Germany). Fluorescence intensity was collected by a photomultiplier tube (model R928; Hamamatsu Photonic, Bridgewater, NJ) from a variable rectangular window (side: 25–50 μm) that included the apical dendrite and most of the neuronal soma. The fluorescence signal collected was integrated using the photometry system (T.I.L.L. Photonics) (Martín et al., 2001). Intracellular calcium variations were recorded under voltage-clamp conditions. After obtaining at least two control records, slices were superfused for 40 min with 100 nm E2 or 100 nm 17α-estradiol and records were obtained every 10 min. The voltage-clamp command was as described previously. Data are expressed as means ± SEM. Statistically significant differences were estimated with the Student's t test, unless indicated otherwise.
Drugs were obtained from Sigma (St. Louis, MO) unless specified otherwise.
No significant differences were found in resting Vm (input), membrane resistance, membrane capacitance, or duration of evoked APs in a representative sample of CA1 pyramidal neurons recorded from OVX (n = 41) and control (n = 28) rats (Table 1). Although all neurons tested from OVX or control rats showed marked spike frequency adaptation during the 2 sec depolarizing current pulses of 0.5 nA, the mean number of APs was smaller and the burst duration was briefer in OVX rats compared with control rats (Table 1; p < 0.05 and p < 0.01, respectively), indicating a decreased neuronal excitability in OVX rats (Fig. 1A).
Effects of E2 on the sIAHP
In neurons from control rats, the sIAHP peaked at 452 ± 35 msec (n = 28) after termination of the depolarizing pulse, whereas in neurons from OVX rats, the sIAHP peaked at 613 ± 77 msec (n = 41; p = 0.03). Both peak amplitude (OVX, 46 ± 5 pA; control, 24 ± 5 pA; p = 0.02) and area under the curve (OVX, 108 ± 20 nA × msec, n = 41; control, 36 ± 9 nA × msec, n = 28; p = 0.005) of the sIAHP were significantly larger in cells from OVX rats than in cells from control rats (Fig. 1B,C). The decay of the sIAHP could be fitted to a single exponential function, and no significant differences were observed in the time constant of the sIAHP decay measured in control versus OVX rats (2745 ± 271 and 3105 ± 279 msec, respectively).
Superfusion with 5–100 nm E2 caused a progressive decrease in the sIAHP in cells recorded in slices from OVX rats. This effect became significant after 20 min of superfusion with E2 (repeated-measures ANOVA; df = 2, 28; F = 7.177; p = 0.003) and stabilized thereafter up to the end of the superfusion with E2 (56 ± 7% of control values; n = 20) (Fig. 2). When superfusion with normal ACSF was resumed after E2, the sIAHP remained at depressed values. Indeed, in nine cells in which stable recordings could be obtained after 40 min of superfusion with normal ACSF, the sIAHP was 58 ± 11% of control values (data not shown).
In slices obtained from control rats, superfusion with 100 nm E2 for 40 min produced no significant decrease in the sIAHP (75 ± 15% from control values; n = 12) (Fig. 2). To determine whether priming with estrogen would change the response observed after superfusion with E2, OVX rats were injected with 10 μg of E2 24 and 48 hr before they were killed. In slices obtained from these rats, the mean sIAHP area was not statistically different from control rats (47 ± 10 nA × msec, n = 6; 36 ± 9 nA × msec, n = 28, respectively), and superfusion with E2 did not significantly modify the sIAHP (Fig. 2) (81 ± 21% from controls; n = 6). Therefore, E2 regulates the sIAHP of OVX rats without modifying the sIAHP in rats that have been exposed to estrogen previously (i.e., both control and E2-primed OVX rats).
Likewise, the mIAHP amplitude was also significantly higher in neurons from OVX rats (83 ± 12 pA; n = 41) than from control rats (39 ± 11 pA; n = 28; p = 0.01) (Figs. 1B,2A). Furthermore, E2 superfusion reduced the amplitude of the mIAHP in neurons from OVX rats (60 ± 7% from control values; n = 20; p = 0.001; sign test), but not in neurons from control rats (84 ± 12% from control values; n = 12), indicating similar effects of estrogen on both sIAHP and mIAHP (see Figs. 2A, 3, 6A,C). The present study focused on the sIAHP, and changes in the mIAHP were not considered further.
Effects of E2 on neuronal excitability
The sIAHP isaCa2+-dependent K+ current that plays a key role in the control of neuronal excitability (Storm, 1990; Borde et al., 1995; Sah and Davies, 2000). We therefore investigated the consequences of E2-mediated regulation of the sIAHP on the electrical excitability of CA1 pyramidal neurons.
During current-clamp recordings in neurons from OVX rats, superfusion with E2 increased both the number of APs (from 3.9 ± 0.4 to 5.7 ± 1.0; n = 20; p = 0.003; sign test) and the burst duration (from 148 ± 34 to 242 ± 60 msec; n = 20; p = 0.001; sign test) during depolarizing pulses presented at the same Vm (Fig. 3). In neurons from control rats, superfusion with E2 had no effect on the number of APs (6.2 ± 0.6 and 5.9 ± 0.6 before and after 40 min of E2 superfusion, respectively; n = 14) or burst duration (290 ± 58 msec in controls and 310 ± 75 msec in the presence of E2; n = 14). As described above, in control rats, the number of APs and the burst duration were larger than in OVX rats, as expected from the different amplitudes of the sIAHP.
No significant changes in the holding current or input resistance were found after 40 min of E2 perfusion in either control (108 ± 5 and 102 ± 2%, respectively, from control values; n = 12) or OVX rats (110 ± 5 and 98 ± 1%, respectively, from control values; n = 20). These results indicate that E2 can effectively regulate the excitability of neurons through the modulation of the sIAHP.
E2 acts via specific membrane receptors
To investigate whether the inhibitory effect of E2 on the sIAHP was mediated through a specific receptor, the effect of superfusion with the inactive isomer 17α-estradiol (100 nm) was tested. No significant change in the sIAHP was observed after 17α-estradiol treatment (n = 4) (Fig. 4A).
To determine whether penetration of E2 through the cell membrane was necessary for the steroid to be effective, we analyzed the actions of the membrane impermeant protein conjugate E2–BSA on the sIAHP in neurons from OVX rats. As shown in Figure 4B, the sIAHP area was decreased after superfusion with 100 nm E2–BSA (n = 5; ANOVA; df = 5, 16; F = 5.563; p = 0.005). Indeed, the sIAHP area was reduced 63 ± 11% (n = 5) from control values by 40 min of superfusion with E2–BSA, which was not significantly different from the reduction induced by E2 (Fig. 2) (56 ± 7%; n = 20), indicating that E2–BSA was as effective as free E2 at decreasing the sIAHP. These results indicate that the effect of E2 on the sIAHP is stereospecific and suggest that the receptor mediating this effect is located at the membrane of CA1 pyramidal neurons.
E2 reduces depolarization-evoked Ca2+ spikes
We also studied the possible cellular mechanisms involved in the depression of the sIAHP. Because the sIAHP is a Ca2+-dependent current, we investigated the possibility that the reduced sIAHP may be caused by E2 modulation of Ca2+ influx, which was evaluated by the amplitude and area of Ca2+ spikes. In current-clamp mode, after superfusing the slice with TTX and TEA, depolarizing current pulses evoked spikes of >100 msec duration and 80–100 mV amplitude (Fig. 5A). These spikes were stable for at least 60 min and were suppressed by perfusion with 100 μm Cd2+ (n = 2; data not shown). Perfusion with 100 nm E2 caused a significant decrease in amplitude and area (n = 5; ANOVA; df = 4, 16; F = 6.82; p = 0.002) of the Ca2+ spikes (Fig. 5A,B).
E2 reduces the depolarization-evoked intracellular Ca2+ signal
We also studied the possibility that reduction of Ca2+ influx may affect the intracellular Ca2+ signal paralleling the modifications of the sIAHP. As expected, membrane depolarization evoked a rapid increase in the Ca2+ signal that slowly decayed during the sIAHP deactivation (Martín et al., 2001). As described above, although the sIAHP in control rats was not significantly affected by superfusion with 100 nm E2, in OVX rats, the sIAHP was decreased after E2 treatment (Fig. 6A,C). A consistent decrease in the Ca2+ signal, which paralleled the inhibition of the sIAHP produced by E2, was observed in cells (n = 8) from OVX rats (Fig. 6D), whereas no significant decrease in the Ca2+ signal was observed in cells (n = 4) from control rats (Fig. 6B).
Statistical analysis (by ANOVA) indicated a significant treatment (control vs OVX) effect (df = 1, 10; F = 6.28; p = 0.03) and a significant interaction between time and treatment (df = 4, 40; F = 4.91; p = 0.003). Post hoc analysis indicated significant differences (control vs OVX; p < 0.001) starting at 20 min (Fig. 6E). These results imply that E2 inhibited the depolarization-evoked Ca2+ signal in OVX rats. Furthermore, the E2-induced inhibition of the Ca2+ signal and the sIAHP showed a strong linear relationship (r = 0.99) (Fig. 6F, filled symbols), suggesting that the E2-evoked inhibition of the Ca2+ signal was responsible for the modulation of the sIAHP by E2. Moreover, the mean effects of E2 on the Ca2+ signal and on the Ca2+ spike area (measured in current-clamp conditions in different cells) (Fig. 5) could also be accurately fitted to a linear regression (r = 0.97) (Fig. 6F, open symbols), suggesting that the E2-induced reduction of the depolarization-evoked Ca2+ spikes was the limiting factor for the E2-mediated inhibition of the intracellular Ca2+ variations.
Together, these results indicate that E2 regulates neuronal excitability by modulating the sIAHP via regulation of the influx of Ca2+ through voltage-gated Ca2+ channels.
The results described above indicate that manipulation of circulating E2 levels affects the sIAHP of CA1 pyramidal neurons. First, CA1 pyramidal neurons in OVX rats show a notably larger sIAHP than cells from normal rats. This difference is also reflected in the excitability of neurons, because a greater number of APs and longer bursts were evoked by membrane depolarization in neurons from control rats. Second, CA1 pyramidal neurons respond to E2 in quite a different manner when applied in control or OVX rats. Superfusion with E2 in neurons obtained from control rats had no effect on the sIAHP, whereas in OVX rats, E2 produced a significant and sustained reduction of the sIAHP, accompanied by increased excitability. These effects were stereospecific, because 17α-estradiol was ineffective.
Priming OVX rats with E2 before they were killed prevented the effect of superfused E2. The action of superfused E2 on the sIAHP appears to be mediated by membrane-bound receptors, because an impermeant conjugate of the steroid was as effective as the free form. The amplitude and area of Ca2+ spikes evoked under TTX were reduced by E2 superfusion in slices from OVX rats, suggesting a modulation of Ca2+ influx by E2. In addition, a reduction of the intracellular Ca2+ signal evoked by membrane depolarization correlated with the reduction of the sIAHP produced by estrogen superfusion, supporting the hypothesis that the main reason for the smaller sIAHP is a reduction of the influx of Ca2+, which leads to a reduced activation of Ca2+-dependent K+ channels.
Many studies have investigated the effects of gonadal steroids on hippocampal function. Previous reports focused on the transynaptic and receptor-mediated effects, showing that E2 facilitates excitatory responses of CA1 pyramids (Wong and Moss, 1992, 1994; Woolley et al., 1997), and that pyramidal neurons from adult OVX animals primed with E2 fire repetitively in response to stimulation (Wong and Moss, 1992), thus probably contributing to the increased bursting activity that characterizes cognitive-related hippocampal activity (Larson and Lynch, 1986; Huerta and Lisman, 1993).
The level of “spontaneous” neuronal activity and synaptic responses is considerably affected by the magnitude of the sIAHP, because by hyperpolarizing and shunting the membrane after a series of APs, the sIAHP greatly reduces excitability and synaptic efficacy (Borde et al., 1999). As this work was in preparation, Kumar and Foster (2002) reported that E2 reduced AHP in OVX rats; this result is hereby confirmed and directly explained by the decreased sIAHP observed in our experiments. The increase in the sIAHP found in OVX animals and its reduction by E2 can help explain the well known depressing effects of ovariectomy on many CNS functions and the compensating effects of E2 treatment. For example, the increase in the sIAHP may explain the difficulty in inducing LTP in OVX animals, because reduced excitability will make the needed coincidence of presynaptic and postsynaptic activity to meet the Hebbian rule less likely (Brown and Chattarji, 1995). Furthermore, treatment with estrogen has been shown to increase synaptic plasticity, facilitating the induction of and potentiating the LTP (Córdoba Montoya and Carrer, 1997; Foy et al., 1999). We found that superfusion with E2 reduced the sIAHP in cells from OVX rats, increasing excitability and decreasing spike frequency adaptation. The effects of ovariectomy and of E2 restitution on synaptic plasticity could be partially explained by estrogen modulation of the sIAHP.
The sIAHP is a K+ conductance activated by an increase in intracellular free Ca2+, mediated at least in part through L-type Ca2+ channels (Sah, 1996; Borde et al., 1999) and Ca2+-dependent Ca2+-release from intracellular stores (Tanabe et al., 1998; Borde et al., 1999). There are then several mechanisms that could be affected by the lack of estrogen to produce the observed increase in sIAHP in OVX rats and its reduction by superfused E2. The lack of estrogen could increase K+ conductances, and superfusion with E2 could decrease those conductances. As far as we know, the pharmacological and single-channel studies necessary to determine whether E2 can directly affect K+ channel characteristics in the hippocampus have not been conducted.
We show an E2-mediated modulation of the Ca2+ signal, and that this modulation correlates with the E2-mediated regulation of the Ca2+ spike and the sIAHP. The steroid could control Ca2+ permeability of the membrane and/or Ca2+ release from intracellular stores, because both sources contribute to activation of K+ channels (Tanabe et al., 1998; Borde et al., 1999; Shah and Haylett, 2000). Estrogen receptor-deficient mice showed increased expression of the cardiac L-type calcium channel (Johnson et al., 1997), and a similar augmented expression in CA1 pyramidal neurons of OVX rats could explain the observed increase in sIAHP. As a matter of fact, we obtained evidence that this may be the case, because superfusion of slices from OVX rats with E2 decreased Ca2+ influx. Although a direct regulation of the intracellular Ca2+-release mechanisms and sIAHP channels by E2 cannot be totally excluded, the strong correlation between the E2-induced changes in Ca2+ signal, the sIAHP, and the Ca2+ spike supports the possibility that the regulation of Ca2+ influx through voltage-gated Ca2+ channels by E2 is the limiting factor responsible for the observed effects. This interpretation is also supported by the similar modulation observed in the sIAHP and mIAHP. However, additional detailed analyses are required to identify the E2-sensitive mechanism responsible for the Ca2+ decrease, and whether the correlation between the modulation of the Ca2+ signal and sIAHP reflects a cause—effect link.
We have obtained evidence that the reduction of the sIAHP is mediated by E2 receptors of the “membrane” type, because an albumin-conjugated membrane-impermeable E2 construct had the same effect as permeable estrogen. Evidence has been obtained suggesting that these rapid effects are mediated by a membrane-bound estrogen receptor (Gu and Moss, 1998), possibly of the α-type (Razandi et al., 1999). The exact meaning of the “membrane effects” may have to be reappraised, considering recent evidence for a new pathway of receptor-mediated endocytosis of membrane-bound E2–BSA (Moats and Ramirez, 2000).
Numerous clinical (Newmark and Penry, 1980) and experimental (Woolley, 1999) studies have demonstrated that E2 can facilitate seizure activity. Apart from the demonstrated effects of E2 on synaptic function in CA1 neurons (Woolley and Schwartzkroin, 1998), because the synaptic regulation of the sIAHP in those cells has been proposed to be involved in epileptogenesis (Martín et al., 2001), the modulation of the sIAHP by E2 could contribute to the initiation and/or maintenance of seizures by predisposing hippocampal circuitry to epileptiform activity. Our results add the possibility of intrinsic cellular ionic mechanisms controlling neuronal excitability to help explain systemic changes known to take place in the CNS as a consequence of variations in the circulating levels of E2.
In conclusion, our results indicate that ovarian secretions regulate the sIAHP, ovariectomy increases the sIAHP, and bath application or priming with E2 decreases the sIAHP. E2 regulates the sIAHP by controlling Ca2+ influx, but the precise mechanism of this modulation remains to be studied. This regulation would be a contributing factor to the “excitatory” effects of estrogen and the regulation of synaptic plasticity. The physiological and behavioral consequences of this contribution should be of considerable interest, particularly because the hippocampus plays a key role in learning and memory related to the spatial abilities of adult organisms.
This work was supported by Dirección General de Investigación Cientifico y Técnica, Ministerio de Educación y Cultura (MEC) of Spain Grant PM98-0113 and Comunidad Autónoma de Madrid Grant 08.5/00361998 to W.B. and by grants from Agencia Córdoba Ciencia and Consejo Nacional de Investigaciones Científicas y Técnicas (H.F.C.). While on sabbatical leave, H.F.C. was supported by MEC of Spain.
Correspondence should be addressed to Washington Buño, Instituto Cajal, Avenida Doctor Arce 37, Madrid 28002, Spain. E-mail:.
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