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Endocrinology Vol. 140, No. 2 660-666
Copyright © 1999 by The Endocrine Society

ARTICLES

Rapid Action of 17ß-Estradiol on Kainate-Induced Currents in Hippocampal Neurons Lacking Intracellular Estrogen Receptors1

Qin Gu, Kenneth S. Korach and Robert L. Moss

Department of Physiology, University of Texas Southwestern Medical Center (Q.G., R.L.M.), Dallas, Texas 75235; Laboratories for Reproductive Biology, University of North Carolina (K.S.K.), Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: Dr. Robert L. Moss, Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9049. E-mail: rmoss{at}mednet.swmed.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
17ß-Estradiol can potentiate kainate-induced currents in isolated hippocampal CA1 neurons. The action of estrogen was rapid in onset, steroid and stereospecific, and reversible. The potentiation could be mimicked by 8-bromo-cAMP, an activator of protein kinase A. As the hippocampus expresses both isoforms of the intracellular estrogen receptor (ER{alpha} and ERß), the role of ERs in the rapid action of 17ß-estradiol remains elusive. Here we report that the rapid action of 17ß-estradiol is independent from the classical ER activation in the modulation of membrane excitability. Under whole cell voltage clamp recording configuration, 17ß-estradiol-induced potentiation was observed in both wild-type and the ER{alpha} gene knockout mice. The perfusion or incubation of ICI 182,780, which blocks both ER{alpha} and ERß, did not affect estrogen potentiation in either group. Further study showed that adenosine 3',5'-cyclic-monophosphothioate Rp-isomer, a specific inhibitor of protein kinase A, completely blocked the potentiation observed with the application of 17ß-estradiol in ER{alpha} gene knockout mice. Our results provide evidence that a distinct estrogen-binding site exists, which appears to be coupled to {alpha}-amino-3-hydroxyl-5-methyl-4-isoxazole proprionic acid/kainate receptors by a cAMP-dependent phosphorylation process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGEN has profound effects on the development and ongoing modulation of the nervous system. Most of the actions are via the genomic pathway, in which estrogen binds to intracellular estrogen receptors (ER) (1, 2, 3). The ER exists as two isoforms, ER{alpha} and ERß, that differ in the C-terminal ligand-binding domain and in the N-terminal trans-activation domain (1, 4, 5, 6). The interaction of the estrogen receptor complex with the hormone response elements in the genome enhances or suppresses transcription and protein synthesis. Studies have shown that the genomic effect of estrogen is of long latency and that it takes more than 30 min (usually hours to days) to occur. A novel pathway for the action of gonadal steroids on the central nervous system is indicated by studies demonstrating rapid (within 2–3 min) and reversible changes in membrane excitability after steroid application (7, 8). Although little is known about the molecular identity of the binding site of the rapid action, there is evidence that some rapid action may involve membrane or intracellular receptors that are coupled to ion channels and transmitter receptors by second messengers (9, 10, 11, 12). Some results also suggest synergistic or opposing interactions of rapid and genomic pathways in estrogen affecting membrane excitability (13, 14).

In the hippocampus, 17ß-estradiol has been shown to potentiate kainate-induced currents in dissociated CA1 neurons with a short latency (15). The action of estrogen is steroid and stereospecific (i.e. testosterone and 17{alpha}-estradiol are inactive) and reversible upon the removal of the steroid. The current-voltage plot and the dose-response curves of kainate-induced currents show that the application of 17ß-estradiol increases the conductance rather than affects the kinetics of the kainate-induced current. These data suggest that there is no direct allosteric interaction of estrogen with {alpha}-amino-3-hydroxyl-5-methyl-4-isoxazole proprionic acid (AMPA)/kainate receptors. As 8-bromo-cAMP, a membrane-permeable cAMP analog, can mimic the effect of 17ß-estradiol on the kainate-induced current, 17ß-estradiol may bind to distinct membrane or intracellular sites that are coupled to a cAMP-dependent phosphorylation process.

Previous studies have demonstrated that hippocampal neurons contain ERs. A comparative study of ER distribution indicated that messenger RNA (mRNA) for both forms of the ER ({alpha} and ß) was expressed in the hippocampus, where small numbers of cells have been shown to contain ER{alpha} mRNA, whereas a relatively large number of CA1 cells express ERß mRNA (16). An immocytochemical study has shown that there are ER-immunoreactive cells located in CA1 region and that there are no sex differences in either the number or the immunostaining intensity of ER-immunoreactive cells in the hippocampus (17). The presence of ERs in the hippocampus leaves open the possibility that the potentiation of kainate-induced currents by 17ß-estradiol involves ERs. The present study was designed to explore this possibility as well as to examine the possibility of interactions between rapid and genomic pathways in 17ß-estradiol potentiation. We examined the action of 17ß-estradiol on isolated CA1 neurons from mice in which ER{alpha} has been genetically knocked out (ERKO). The development of the ERKO mouse was accomplished by inserting a neomycin-encoding sequence into exon 2 of the mouse ER{alpha} gene. The neomycin insert inhibits proper transcription and translocation of the ER{alpha} gene by its premature stop codons and polyadenylation sequence and functionally inhibits expression (4). To investigate the involvement of ERß in the action of 17ß-estradiol on kainate-induced currents, ICI 182,780 (ICI) was employed in some experiments. ICI exerts its pure antagonism by blocking both ER{alpha} and ERß transcriptional activity (18).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Twenty female mice, 10 ERKO and 10 wild type (WT), were time bred at the NIEHS. The mice were 4 weeks old upon arrival and were housed in a temperature-controlled room on a 12-h light, 12-h dark cycle (lights off at 1300 h Central Standard Time). The animals were grouped (4–5/cage) and received ad libitum access to food and water. All animal experimentation was conducted in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals (NIH Publication 85–23, revised 1985).

Preparation of acutely dissociated neurons
CA1 hippocampal neurons were acutely dissociated using a modification of procedures reported by Kay and Wong (19). Mice were decapitated via a guillotine. Hippocampi were removed from the brain, quickly blocked, and placed in cold PIPES-saline solution: NaCl, 120 mM; KCl, 5.0 mM; CaCl2, 1.0 mM; MgCl2, 1.0 mM; D-glucose, 25 mM; and piperazine-N-N'-bis(2-ethane sulfonic acid) (PIPES), 20 mM; pH 7.4. The blocked tissue was cut on a Vibratome (Technical Products International, Inc., St. Louis, MO) into sections approximately 450 µm thick while being bathed in a 4 C oxygenated PIPES-saline solution. The slices were placed in a petri dish on a black surface, and punches were made in the CA1 area with a capillary tube. The punches were incubated at room temperature (20–22 C) in PIPES-saline solution with 1.5 mg/ml protease (Sigma Chemical Co., St. Louis, MO). The incubation medium was stirred slowly and smoothly with 95% O2-5% CO2 blown at its surface. After 30–45 min of enzymatic digestion, punches were rinsed three times with oxygenated PIPES-saline and triturated with a fire-polished Pasteur pipette for mechanical dissociation. The cell suspension was then plated into the central concave area of a slide containing the standard extracellular solution: NaCl, 140 mM; KCl, 3.0 mM; CaCl2, 2.0 mM; and HEPES, 10 mM; pH 7.3. All chemicals were obtained from Sigma Chemical Co.

Whole cell patch clamp recordings
Whole cell recordings were performed under the voltage clamp mode according to standard technique (20). Both conventional as well as perforated whole cell patch clamp recordings were employed in isolated CA1 hippocampal neurons. The dissociated neurons were approximately 30–40 µM in diameter and were visualized with a Nikon inverted, phase contrast microscope (Nikon, Melville, NY) equipped with Nomarski optics. The electrode resistance was typically 2–5 M{Omega} in bath solution. The standard internal solution for the recording electrode consisted of the following: CsCl, 140 mM; NaCl, 4.0 mM; EGTA, 10 mM; HEPES, 10 mM; and CaCl2, 1 mM. The internal solution was adjusted to pH 7.3 with CsOH. The holding potential was -60 mV. In studying the current-voltage relations, the holding potential was varied from -70 to +50 mV. Access resistance was compensated (80%) electronically and monitored periodically.

Puffer electrode and chemical application
A multiple barrel pipette with a total diameter of 10 µM was used to puff individual substances on the dendrite of the recorded dissociated CA1 hippocampal neuron. Ejection of each chemical could be made separately with a picosprizer unit (General Valve Corp., Fairfield, NJ). Kainate, 17ß-estradiol, 17ß-estradiol, and ICI 182,780 were assigned randomly to one of the barrels. Adenosine 3',5'-cyclic-monophosphothioate Rp-isomer (Rp-cAMPS), a specific inhibitor of protein kinase A (PKA), was obtained from L. C. Laboratories (Woburn, MA), and ICI 182,780 was obtained from Tocris (Baldwin, MO). Kainate currents were induced by pulses of ejection (20 msec; 0.1–1.0 psi) of kainate at the dendrite of the CA1 neuron. The application was repeated once every 30 s and commenced immediately after the patch was ruptured. The effects of the different drugs on the kainate-induced currents were tested by extracellularly perfusing the cell for 3 min.

Data analysis
Whole cell currents were recorded under voltage clamp configuration with an Adams/List EPC-9 amplifier (ALA, Great Neck, NY), sampled at 2 kHz, and filtered at 2.3 kHz. Data were digitized and stored on an Atari Mega 4 computer (Atari Corp., Sunnyvale, CA). Analysis of whole cell current records was performed with an Atari data analysis program. Peak currents were normalized as I/I0, where I represents the amplitude of kainate-induced currents at any testing time point, and I0 is the initial value at the beginning of the recording. The percent change in the amplitude of kainate-induced currents was determined according to the formula (Idrug/Io - 1)100%, where Idrug represents the peak amplitude of the kainate current in the presence of the test drug. Current-voltage data were obtained by subtraction of leak currents from currents recorded in the presence of agonists at each potential. All quantitative data are expressed as the mean ± SEM; n indicates the number of cells tested. Statistical analysis was performed using paired or unpaired Student’s t test. Results were considered significant only for P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The animals used in these studies were the offspring of heterozygous breeding pairs. Genotyping of the animals was performed using a modification of the method described in Lubahn et al. (21). A single reaction PCR analysis was employed using genomic DNA extracted from the tail biopsy samples. Primer pairs generating products of 239 and 555 bp for endogenous and disrupted ER genes were used. In ERKO mice, the expression of ER{alpha} gene is disrupted, whereas the ERß is apparently still present (18, 22). Both sexes of these animals from the ERKO group are infertile and demonstrate a variety of phenotypic changes (4, 5, 23).

The role of ER{alpha} in the potentiation of kainate-induced currents by 17ß-estradiol in ERKO and WT mice was examined under whole cell, voltage clamp recording configuration. Application of kainate (100 µM; 20 msec) elicited inward currents in dissociated hippocampal CA1 neurons from both WT and ERKO mice. The kainate-induced current showed similar characteristics in the time course and amplitude of the response in the WT group (306 ± 118 pA; n = 43) and the ERKO group (281 ± 107 pA; n = 58). 17ß-Estradiol application (50 nM; 3 min) increased the amplitude of kainate-induced currents in the WT mice (by 32.0 ± 4.0% in 6 of 18 neurons tested) and in ERKO mice (by 30.0 ± 7.2% in 8 of 22). The potentiation occurred within 3 min of application of 17ß-estradiol and was gradually reversed after removal of the chemical. This potentiation is similar to that previously observed in rats (15, 24). No significant differences were observed in the 17ß-estradiol potentiation of kainate-induced currents between the WT and ERKO mice (Fig. 1Go).



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  		Figure 1. Potentiation of kainate-induced currents by 17ß-estradiol on hippocampal CA1 neurons from WT and ERKO mice. Kainate was applied by pulses of pressure ejection (100 µM; 20 msec) onto dissociated CA1 neurons. The pulse was repeated every 30 sec. Kainate-induced currents were recorded under whole cell voltage clamp configuration. The holding potential was -60 mV. In the presence of 17ß-estradiol (50 nM; 3 min), the amplitude of kainate-induced currents was enhanced in a neuron from a WT mouse (•). The action of 17ß-estradiol was rapid in the onset and reversible upon removal of the steroid. A similar pattern of potentiation was observed during the application of 17ß-estradiol in the neuron from the ERKO mouse ({circ}). Actual current traces selected from the records at specific time points (arrows) are displayed in the upper portion of the graph.

 
Current-voltage (I-V) curves of kainate-induced currents from ERKO mice showed a similar reversal potential (~0 mV) and linearity in the absence and presence of 17ß-estradiol (n = 3). However, the slope of the I-V curve was significantly increased in the neurons exposed to 17ß-estradiol (P < 0.05; Fig. 2AGo). Similar findings were observed in hippocampal neurons from WT mice (Fig. 2BGo).



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  		Figure 2. I-V curves obtained in the absence ({circ}) and presence (•) of 17ß- estradiol (50 nM) in a CA1 hippocampal neuron. A, In the neuron from an ERKO mouse, I-V curves in the presence and absence of 17ß-estradiol are both linear, with nearly identical reversal potential. As determined from the slope of the current-voltage relation, the conductance of the membrane to kainate-induced currents increased in the presence of 17ß-estradiol. B, Similar I-V relationships were also observed in the neuron from a WT mouse. Individual kainate-induced currents used to construct the plots are shown to the right of the graphs. The current traces are obtained at different holding potentials from -70 mV to +50 mV (including 0 mV) in 20-mV steps.

 
The results suggest that potentiation of kainate-induced currents by 17ß-estradiol in hippocampal neurons is independent of ER{alpha}. To confirm and extend these findings, experiments were conducted by applying the compound ICI to the neurons from ERKO or WT mice.

As illustrated in Fig. 3Go, coapplication of ICI (100 nM; 5 min) with 17ß-estradiol did not abolish the potentiation of kainate- induced currents by 17ß-estradiol in the cells from the ERKO group. Similar potentiated amplitudes were observed in the absence (32 ± 8%) and presence (30 ± 5%) of ICI (n = 10). In the presence of ICI, 17ß-estradiol potentiation exhibited no significant difference between the ERKO and WT groups in terms of the short latency, amplitude, and reversibility. In another set of experiments, neurons from ERKO mice were preincubated with ICI (100 nM; ~30 min) before testing the effect of 17ß-estradiol. The results are summarized in Table 1Go. Kainate-induced currents displayed no obvious differences in amplitude between the preincubated and control groups. 17ß-Estradiol potentiated kainate-induced currents in the presence of the ICI compound in the same pattern as that observed in the control experiments. Collectively, these findings indicate that pharmacological blockage of ERß and ER{alpha} had no observable effect on the potentiation of kainate-induced currents by 17ß-estradiol.



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  		Figure 3. 17ß-Estradiol potentiation of kainate-induced currents in the presence of the compound ICI 182,780 in a hippocampal neuron from an ERKO mouse. Application of 17ß-estradiol (50 nM; 3 min) caused a reversible potentiation of kainate-induced currents. This potentiation was not abolished by coapplication of ICI 182,780 (100 nM), a potent antagonist of both ER{alpha} and ERß. Representative current traces from sample points (arrows) are shown and compared in the upper portion of the figure.

 

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  		Table 1. Effects of ICI 182,780 on kainate-induced currents and 17ß-estradiol potentiation in neurons from ERKO mice

 
The results from the above experiments suggested the existence of binding sites for 17ß-estradiol potentiation that are genetically or pharmacologically different from ER{alpha} or ERß. In rat hippocampal neurons, 17ß-estradiol potentiation can be mimicked by a cAMP analog and can be modulated by a phosphodiesterase inhibitor (isobutylmethylxanthine). By application of Rp-cAMPS (50 µM), a specific inhibitor of PKA, the potentiation of kainate-induced currents could be blocked (15). These previous findings strongly argue that these distinct binding sites were coupled to a cAMP-dependent phosphorylation process. This hypothesis was verified to some extent in the present experiment using ERKO mice. The administration of Rp-cAMPS (50 µM) completely blocked the potentiation observed with application of 17ß-estradiol (Fig. 4Go). Thus, the mechanism involved in 17ß-estradiol potentiation of kainate-induced currents is the same in ERKO mice.



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  		Figure 4. Effect of Rp-cAMPS on 17ß- estradiol potentiation of kainate-induced currents in a neuron dissociated from the ERKO mouse. In the neuron responsive to 17ß-estradiol (50 nM; 3 min), application of Rp-cAMPS (50 µM) gradually blocked the action of 17ß-estradiol on kainate-induced currents. Representative current traces selected at specific time points (arrows) are displayed at the top of the figure. The current traces in 1 and 4 are artificially separated to allow visualization of the identical current amplitudes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Intracellular estrogen receptors are members of a large superfamily that includes receptors for retinoids, T4, and vitamin D (25, 26). Although some rapid actions of estrogen on the membrane excitability cannot be explained by the genomic mechanism due to their short latency, intracellular ERs have been shown to be involved in certain nongenomic actions or in the interactions of genomic and nongenomic mechanisms. In hypothalamic opioidergic neurons, for example, estrogen acts through an intracellular receptor to uncouple {gamma}-aminobutyric acidB and µ-opioid receptors from a K+ channel. This results in more inhibition in the presynaptic cell and decreased excitability in the postsynaptic GnRH cell. Rather than through the genomic pathway, in this case estrogen acts rapidly via the intracellular ER to phosphorylate a component of the coupling of the transmitter receptor to the ion channel (27). On the other hand, an interaction between genomic and nongenomic mechanisms contributes to lactotroph excitation. In this case, estrogen decreases the efficacy of tonic inhibitory dopaminergic input to the cell by a rapid decoupling of the dopamine receptor from its channels and by a long term down-regulation of the number of dopamine receptors (13, 14). The expression of both isoforms of ER in hippocampal neurons raised the question as to whether intracellular ERs play a role in the rapid action, or whether the genomic mechanism has an impact on the nongenomic action of 17ß-estradiol.

One of the feasible approaches in testing these possibilities is to use the mutant mouse line without functional ER{alpha}. As the expression of the ER{alpha} gene has been suppressed, the pathways activated by the ER{alpha} are presumably blocked. In addition, the response of a organ to estrogen depends not only on its activation of the receptors, but also its effects during early development (28). The strength of using ERKO mice over other approaches is that the ERKO mice are deprived of estrogen’s action on brain both developmentally and in adulthood. Studies have shown that female ERKO mice have ovaries containing immature and atrophic follicles, immature reproductive tracts, and an inability to display sexual receptivity (22, 23). As elevated circulating levels of estradiol and androgen in plasma hormones have been found in the ERKO mice, the cell dissociation procedure used in the present study eliminated this condition.

In the present study, kainate-induced currents exhibited no observable differences in neurons from ERKO and WT mice. In the presence of 17ß-estradiol, the amplitude of kainate- induced currents in the ERKO group increased to about 30%, which is similar to that observed in the WT mice. Similarities were also found in terms of the short latency and reversibility of the potentiation and in the percentage of the cells responsive to 17ß-estradiol. It suggests that suppression of ER{alpha} expression does not alter the signaling pathway by which 17ß-estradiol enhances kainate-induced currents.

By employing ICI compounds in the genetically deficit ER{alpha} mouse line, ERß can be pharmacologically blocked. ICI 182,780 and ICI 164,384 belong to a series of 7{alpha}-alkylamide analogues of 17ß-estradiol. Studies have shown that ICI compounds are pure antiestrogens by directly binding to the estrogen receptor. The kinetic parameters of this interaction are similar to those for the binding of estradiol. The binding of estradiol results in a steroid-receptor complex that can be transformed to a form with increased affinity for DNA. However, the complex formed with the ICI compound suppresses the transformation process. Therefore, the application of ICI compounds should induce a complete blockage of the transcriptional effect of estrogen (29, 30). The specificity and potency of the ICI compounds have also been evaluated by several studies. ICI 182,780 and ICI 164,384 block both functional domains (AF-1 and AF-2) of the receptor {alpha} or ß. ICI 182,780 is more potent than ICI 164,384. When added at 10 nM and above, ICI 182,780 can lower estrogen-induced ERß activity even below its basal level (18). A recent study in our laboratory has shown that incubation of vomeronasal tissue with 17ß-estradiol for 15 min caused an increase in c-fos mRNA expression measured at 60 min. The effect of 17ß-estradiol was evident as low as 10 pM. Western blot analysis revealed the presence of ERs in the tissue. The response to 17ß-estradiol remained the same in vomeronasal tissue from ERKO mice. If the tissue was preincubated with ICI 182,780 for 15 min, however, administration of 17ß-estradiol induced no increase in c-fos mRNA expression (our unpublished observations). The data indicate that ERß or other ER variants play a role in the modulation of vomeronasal organ function. Based on the accumulated evidence, the concentration and duration of ICI 182,780 used in the present experiment were effective in blocking the actions of estrogen through ERß. Application of ICI in the present study, however, resulted in no observable differences in the potentiation of kainate- induced currents by 17ß-estradiol. This suggests that the binding site for estrogen potentiation of kainate-induced currents in hippocampal neurons is distinct from those for either of the two genomic ER isoforms.

The reversal potentials of the current-voltage curves suggest the involvement of nonspecific cationic channels. The application of 17ß-estradiol did not change the reversal potential or the linearity of the curves. The conductance of AMPA/kainate receptor channels, however, was significantly increased by 17ß-estradiol in both ERKO and WT groups. There was no statistically significant difference in the increase in conductance between the two groups. Wong and Moss have shown that 17ß-estradiol did not affect the parameters of the kainate receptor channels in excised patch from hippocampal neurons, suggesting that there was no direct allosteric interaction between estrogen with the AMPA/kainate receptors (31). This hypothesis was supported by the data from a kainate dose-response study performed under whole cell recording configuration. Here, 17ß-estradiol, without altering their kinetics, increased the amplitude of kainate-induced currents (15).

As the time course of 17ß-estradiol potentiation of kainate-induced currents in hippocampal neurons is too rapid to be explained by the classical genomic pathway for steroid action, a nongenomic binding site, especially a membrane binding site, might be responsible for this event. The fact that Rp-cAMPs blocks the effect of 17ß-estradiol implies possible involvement of PKA-dependent phosphorylation downstream in the signal processing.

To date, binding studies have not identified specific estrogen membrane receptors. However, a number of studies using immunocytochemical techniques have indicated the presence of estrogen-binding sites in the plasma membrane (32, 33, 34, 35). Watson’s laboratory has demonstrated a subpopulation of binding sites in the plasma membrane by using antibodies directed against a peptide representing the hinge region of intracellular ER. The membrane binding sites mediate a rapid release of PRL in GH3/B6 rat pituitary tumor cells. The confocal scanning laser microscopy of cells labeled live with the antipeptide antibody further supports a membrane localization of ER. The monoclonal antibodies H226 and H222 and the polyclonal antibody, ER21, immunohistochemically label membrane proteins in immunoselected GH3/B6 cells. Each of these antibodies recognizes a unique epitope on intracellular ER: NH2-terminal to the DNA-binding region, within the steroid-binding region, and NH2-terminal end, respectively. The results suggest that the membrane binding sites bear structural similarities to the intracellular ER. Coincubation of cells with anti-ER antibody and the fluorescent estrogen-BSA conjugate reveals that these labels colocalize on the cell surface. Although our present results also suggest distinct sites, especially membrane binding sites, for 17ß-estradiol action on hippocampal neurons from the ERKO mice, the site of action appears to be genetically and pharmacologically different from that of the classic intracellular ER. Further study is required to characterize the site to which 17ß-estradiol initially binds in modulation of membrane excitability.


   Acknowledgments
 
We are grateful to Carol Dudley for her valuable advice and assistance throughout the project. We also thank Cindy Patterson for her secretarial assistance.


   Footnotes
 
1 This work was supported by NIH Grant RO1-MH47418 awarded to R.L.M. Back

Received August 14, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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