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Department of Neurobiology and Physiology and Northwestern University Institute for Neuroscience, Northwestern University, Evanston, Illinois 60208
Address all correspondence and requests for reprints to: Catherine S. Woolley, Department of Neurobiology and Physiology, Northwestern University, 2205 Tech Drive, Evanston, Illinois 60208. E-mail: cwoolley{at}northwestern.edu.
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Abstract |
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Introduction |
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-aminobutyric acid (GABA)ergic inhibitory neurotransmission (9). The distribution of estrogen receptors (ERs) in the hippocampus in vivo has been mapped and is consistent with the possibility of a direct effect of estrogen on GABAergic interneurons because a subset of these cells express a nuclear ER (10).
In previous studies, we took initial steps toward understanding the mechanism of estrogen action in the hippocampus by monitoring the time course of pyramidal cell activation and inhibition after estrogen treatment. We used immunohistochemistry for the immediate early gene, c-Fos, to assess the activation of pyramidal cells at various time points after estrogen treatment (11), and we used immunohistochemistry for 65-kDa isoform of glutamic acid decarboxylase (GAD65, the rate-limiting enzyme GABA synthesis) and whole-cell voltage-clamp recording GABAergic inhibitory postsynaptic currents to assess the inhibition of CA1 pyramidal cells after estrogen (8). These analyses demonstrated that a single treatment with estrogen induces phasic activation of hippocampal CA1 pyramidal cells, as evidenced by two sequential waves of c-Fos expression: one at 2 h, and another at 24 h (shown schematically in Fig. 1
). The second wave of pyramidal cell activation, at 24 h, coincides with transient suppression of GABAergic inhibition, as reflected by a reduction of GABAergic postsynaptic currents in pyramidal cells and suppression of GAD65 immunoreactivity in the dendritic layers of CA1 (Fig. 1).
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To gain further insight into the mechanism of estrogen activation and disinhibition of CA1 pyramidal cells, in the current study, we used c-Fos and GAD65 immunohistochemistry to determine whether the selective ER modulators (SERMs), tamoxifen (T) and CI628, can inhibit the first or second wave of pyramidal cell activation by estrogen, as well as the suppression of GAD65 immunoreactivity that parallels the second wave of activation. These SERMs were chosen because each has been shown to inhibit estrogen induction of dendritic spines in CA1; T inhibits spine induction in vitro (17), and CI628 inhibits spine induction in vivo (18). In a separate set of electrophysiological experiments, we used whole-cell voltage-clamp recording to gain further insight into the mechanisms of estrogen action that are sensitive to modulation by SERMs. Though T and CI628 generally are thought to act as antagonists of classical ERs, we tested whether T (or its potent metabolite, 4OH-T) or CI628 also can inhibit rapid estrogen-induced potentiation of excitatory postsynaptic currents (EPSCs) in CA1 pyramidal cells.
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Materials and Methods |
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Immunohistochemistry for c-Fos and GAD65
Rats were deeply anesthetized with Nembutal (80 mg/kg, ip) and perfused using 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). After perfusion, brains were removed, blocked, and postfixed overnight in the same solution at 4 C. The next day, brains were rinsed with 0.1 M PB and cyroprotected in 30% sucrose in PB and, the following day, were coronally sectioned (50 µm) through the hippocampus on a freezing microtome. Sections from each brain were stained immunohistochemically for c-Fos (polyclonal to human c-Fos, Oncogene Research Products, Cambridge, MA) or GAD65 (monoclonal to rat GAD, 65-kDa isoform; Chemicon, Temecula, CA) using the avidin-biotin-peroxidase method and visualized using 3,3' diaminobenzidine (for c-Fos labeling) or the peroxidase substrate kit (Vector SG, Vector Laboratories, Inc., Burlingame, CA, for GAD65 labeling) as described below.
Immediately after sectioning, tissue sections were rinsed in PB and treated with sodium borohydride to remove residual aldehydes, followed by H2O2 to inhibit endogenous peroxidase activity. After rinsing in 0.1 M Tris buffer (pH 7.4), sections were incubated in 0.5 M Tris-buffered saline containing 5% normal serum, 3% BSA, and 0.3% dimethylsulfoxide (DMSO). Sections were then incubated in primary antisera or antibody (1:5000 for c-Fos or 2 µg/ml for GAD65) solution containing 1% normal serum, 3% BSA, and 0.3% DMSO for 42 h at 4 C. Some sections from each brain were incubated without the primary, to assess nonspecific secondary antibody staining. After primary incubation, the sections were washed with 0.1 M Tris-buffered saline and incubated in biotinylated secondary antibody (antirabbit for c-Fos or antimouse for GAD65) solution containing 1% normal serum, 2% BSA, and 0.3% DMSO for 3 h, rinsed, and then incubated in Avidin-Biotin HRP Complex (Vector Laboratories, Inc. Elite Kit) for 3 h. For c-Fos staining, sections were rinsed and preincubated in Tris buffer (pH 7.6) containing 0.025% diaminobenzidine with nickel sulfate, for 20 min, and then 0.01% H2O2 was added, and the sections were incubated for an additional 20 min. Tissue for GAD65 labeling was incubated in Vector SG for 20 min. After these reactions, sections were rinsed, mounted onto subbed slides, dehydrated in graded ethanols, cleared in xylene, and coverslipped under Permount for later analysis.
Quantification of c-Fos-labeled nuclei and GAD65-labeled cell bodies
Unbiased estimates of the density of c-Fos-labeled nuclei in the pyramidal cell layer and GAD65-labeled cell bodies in str. radiatum and oriens of CA1 were obtained using the optical disector principle and random systematic sampling (19). For both left and right sides of 8 sections for each brain, 10 sectors for cell counting (150 x 100 µm = 15,000 µm2 in the pyramidal cell layer for c-Fos; 246 x 184 µm = 45,000 µm2 in the str. radiatum and oriens for GAD65) were chosen randomly. The starting point for counting was set at 5 µm below the surface and stepped down 5 times, at 2 µm per step, to a total depth of 10 µm. Labeled nuclei or cell bodies that were sharply in focus and inside the counting frame or that intersected the upper horizontal and right vertical were counted at each step using a x100, oil-immersion lens on an Olympus BX60 microscope (Olympus Optical, Tokyo, Japan) with a Dage DC330 camera (Dage MTI, Inc., Michigan City, IN) and Image-Pro Plus software (Media-Cybernetics, Silver Spring, MD). Mean densities of labeled nuclei or cell bodies in each layer of CA1 were calculated for each animal, and the data were analyzed statistically using ANOVA followed by Tukey post hoc comparisons.
Slice preparation and maintenance for electrophysiological recording
Rats used for electrophysiological recording were deeply anesthetized with Nembutal (80 mg/kg, ip) and transcardially perfused with ice-cold oxygenated artificial cerebrospinal fluid (ACSF) containing, in mM: 125 NaCl, 25 NaHCO3, 25 dextrose, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, and 2 CaCl2 (pH 7.5). The brain was removed, and 300-µm transverse dorsal hippocampal slices were cut into an ice-cold bath of oxygenated ACSF using an oscillating tissue slicer. Slices then were transferred to a holding chamber, where they remained submerged in oxygenated ACSF at 35 C for 30 min and then remained at room temperature until used for recording.
Whole-cell voltage-clamp recordings
Slices were transferred to a recording chamber mounted on a Zeiss Axioskop (Carl Zeiss, Oberkochen, Germany), where they were submerged in oxygenated ACSF at 35 C. Neurons were visualized with infrared differential interference video microscopy. Somatic whole-cell voltage-clamp recordings were obtained from CA1 pyramidal cells using patch electrodes with an open tip resistance of 3–5 M
. Pipette solution contained, in mM: 115 K-gluconate, 20 KCl, 10 Na2-creatinine phosphate, 10 HEPES, 2 EGTA, 2 MgATP, and 0.3 NaGTP (pH 7.3). A stimulating electrode (glass electrode with chlorided silver wire) was placed in the str. radiatum, approximately 150–250 µm from the recorded cell, halfway between the str. lacunosum and pyramidal cell layer. Synaptically evoked EPSCs were recorded at a holding potential of -70 mV in normal ACSF, ACSF containing 100 pM E, 10 nM-1 mM of each SERM, or 100 pM E plus 10 nM-1 mM SERM. Data were collected with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA), acquired using Data Pro software and analyzed using Igor Pro software (WaveMetrics, Inc., Lake Oswego, OR). Data were analyzed statistically using a Student’s t test.
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Results |
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; Ref. 11). Our approach in the current study was to use the SERMs, T and CI628, to test whether ER antagonists can block the first and/or second wave of pyramidal cell activation. To validate this approach, two control experiments were necessary. First, because other studies have shown that c-Fos can be induced more rapidly than 2 h (20, 21), we needed to establish whether 2 h is an appropriate time point at which to measure the initial induction of c-Fos by estrogen. Thus, to confirm that c-Fos is not significantly increased earlier than 2 h, we counted c-Fos immunolabeled nuclei in the dorsal CA1 pyramidal cell layer in OVX animals before any treatment, and at 1 h after oil or estrogen. This analysis showed that, although there is a trend toward increased c-Fos expression at 1 h after estrogen, the difference is not statistically significant (#Fos+ nuclei/1.5 x 103 µm, mean ± SEM: before treatment, 2.79 ± 0.67; 1 h O, 3.22 ± 0.71; 1 h E, 3.97 ± 0.74; 0.1>P > 0.05). Second, because SERMs, such as T, can act as ER agonists in some systems, it also was necessary to confirm that SERM treatment alone does not induce c-Fos in CA1 pyramidal cells. To accomplish this, we performed an additional control experiment in which we counted c-Fos immunolabeled nuclei in OVX rats 2 h after treatment with oil, estrogen, or T alone. We found that T had no effect on c-Fos expression (#Fos+ nuclei/1.5 x 103 µm, mean ± SEM: oil, 3.65 ± 0.9; estrogen, 6.86 ± 1.1; T alone, 3.51 ± 0.8), thereby confirming that T does not act as an ER agonist in our system.
Having validated our approach with the control experiments above, we then tested whether pretreatment with either of the SERMs, T or CI628, was capable of blocking the increase in c-Fos at 2 h after estrogen. We counted c-Fos immunolabeled nuclei in the dorsal CA1 pyramidal cell layer in animals treated only with oil or estrogen, and in oil- or estrogen-treated animals that were pretreated with vehicle or with T (Fig. 2) or CI628. Consistent with the results of Rudick and Woolley (11), estrogen increased c-Fos expression at 2 h, both in animals with no pretreatment (P < 0.01; not shown) and in vehicle-pretreated controls (P < 0.01; Fig. 3A). There was no difference in c-Fos expression between controls that received no pretreatment and those pretreated with vehicle (P > 0.1), confirming that the preinjection itself had no effect on c-Fos induction. Additionally, c-Fos also was increased by estrogen in animals that had been pretreated with either T (P < 0.05; Fig. 3A) or CI628 (P < 0.05, Table 1). Thus, neither SERM blocked the initial estrogen-induced increase in c-Fos expression at 2 h.
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; P > 0.1). Additionally, neither T (P > 0.1; Fig. 3B) nor CI628 (P > 0.1; Table 1) had any effect on c-Fos expression in the ventral hippocampus. Thus, despite the fact that many more CA1 pyramidal cells in the ventral than dorsal hippocampus express a nuclear ER (10, 13), these cells do not respond to estrogen with induction of c-Fos, as CA1 pyramidal cells in the dorsal hippocampus do.
Pyramidal cell activation by estrogen at 24 h
Next, we tested whether T or CI628 pretreatment blocks the second wave of c-Fos expression that occurs at 24 h after estrogen treatment. As in our analysis with 2-h treatment, we counted c-Fos immunolabeled nuclei in the dorsal (Fig. 4) and ventral CA1 pyramidal cell layer in animals treated only with oil or estrogen, and in oil- or estrogen-treated animals that were pretreated with vehicle or with T or CI628. Again consistent with the results of Rudick and Woolley (11), estrogen increased c-Fos expression at 24 h in the dorsal CA1 pyramidal cell layer (Fig. 5A; P < 0.01). No differences in c-Fos expression were observed between controls that received no pretreatment vs. vehicle pretreatment (P > 0.1; not shown). Additionally, and unlike the 2-h time point, estrogen also increased c-Fos expression in the ventral CA1 pyramidal cell layer (Fig. 5B; P < 0.01). In further contrast to the 2-h time point, pretreatment with either T or CI628 did block estrogen-induced c-Fos at 24 h in both dorsal and ventral CA1. The density of c-Fos-labeled nuclei in estrogen-treated animals that were pretreated with T was not different from that in oil-treated controls (P > 0.1) and was significantly lower than in animals treated with estrogen alone (P < 0.01; Fig. 5, A and B). Results with CI628 pretreatment were nearly identical to results with T (Table 1).
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; Ref. 8). To investigate whether T or CI628 block estrogen-induced disinhibition at 24 h, in addition to their blockade of pyramidal cell activation, we quantified GAD65 labeling in the dorsal and ventral CA1 str. radiatum and oriens in alternate sections from the same animals used for c-Fos labeling. Consistent with the results of Rudick and Woolley (8), estrogen decreased GAD65 labeling at 24 h, both in animals with no pretreatment (P < 0.05; not shown) and in vehicle-pretreated controls (Fig. 5, C–F; P < 0.05). As with c-Fos labeling, no differences in GAD65 staining were observed between controls that received no pretreatment vs. vehicle pretreatment (P > 0.1; not shown). Additionally, pretreatment with either T or CI628 blocked the estrogen-induced decrease in GAD65 staining. The density of GAD65-labeled cells in estrogen-treated animals that were pretreated with T was not different from that in oil-treated controls (Fig. 5, C–F; P > 0.10) and greater than in animals treated only with estrogen (Fig. 5, C–F; P < 0.05). Results with CI628 were nearly identical (Table 1). The parallel timing of increased c-Fos expression, indicating pyramidal cell activation, and decreased GAD65 staining (which correlates with a suppression of inhibition), suggests that the estrogen-induced activation of pyramidal cells at 24 h may be attributable, in part, to disinhibition of these cells. In support of this suggestion, a within-animal analysis of c-Fos and GAD65 labeling showed that these measures are significantly inversely correlated (r = -0.63; P < 0.05); the greater the decrease in GAD65 labeling in an animal, the greater that animal’s increase in c-Fos expression.
To more closely evaluate the timing of estrogen action to activate/disinhibit pyramidal cells at the 24-h time point, we performed an additional experiment in which animals first were treated with estrogen and then with vehicle or T 4 h after estrogen. We quantified both c-Fos-labeled nuclei in the CA1 pyramidal cell layer and GAD65-labeled cell bodies in the str. radiatum and oriens, 24 h after estrogen treatment. Interestingly, in contrast with the results with T pretreatment, T given 4 h after estrogen failed to block either the 24-h increase in c-Fos or decrease in GAD65 staining. c-Fos was increased (Fig. 6, A and B; P < 0.01) and GAD65 was decreased (Fig. 6, C–F; P < 0.05) both by estrogen alone and by estrogen followed by T. These data indicate that estrogen acts via a SERM-sensitive mechanism sometime within the first 4 h of treatment, to produce disinhibition and activation of CA1 pyramidal cells 24 h later.
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We used whole-cell voltage-clamp recording of CA1 pyramidal cell EPSCs to determine whether estrogen’s SERM-sensitive effects on hippocampal pyramidal cells might include EPSC potentiation. We found that 100 pM estrogen rapidly potentiated EPSC amplitude in 34 of 55 (62%) CA1 pyramidal cells recorded. In recorded cells that were estrogen-responsive, EPSC amplitude was increased within 2–3 min by an average of 24% (Fig. 7B; P < 0.05). Estrogen-induced potentiation of CA1 pyramidal cell EPSCs was similar in cells from gonadally intact or estrogen-primed animals (P > 0.10); however, as reported by others (23), estrogen priming increased the proportion of cells that showed potentiation (from 33–73%). Both the proportion of pyramidal cells that were responsive to estrogen and the magnitude of estrogen-induced potentiation were consistent with previous reports of rapid potentiation of synaptic responses by estrogen (14, 15, 23).
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; P > 0.1). CI628 also was capable of inhibiting estrogen potentiation of CA1 pyramidal cell EPSCs, but much higher concentrations of CI628 than 4OH-T were required to see complete blockade of the effect. At 10 µM, CI628 was not effective (5 estrogen-responsive cells tested), but 100 µM (5 estrogen-responsive cells tested) CI628 did block estrogen potentiation of the EPSC (not shown). Because estrogen’s ability to potentiate CA1 pyramidal cell EPSCs is limited to the first 20 min (approximately) after break-in to whole-cell recording mode (14), we confirmed, in each experiment, that estrogen potentiation of EPSCs was still detectable after washout of 4OH-T (Fig. 7D; P < 0.05) or CI628. These data demonstrate that rapid potentiation of EPSCs must be considered as a SERM-sensitive mechanism of estrogen action on CA1 pyramidal cells. |
Discussion |
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Although SERMs, such as T and CI628, generally are thought to act by blocking ER-regulated gene expression (i.e. by blocking nuclear ERs that act through a classical mechanism), in a separate set of in vitro electrophysiological experiments, we found that the active metabolite of T, 4OH-T, or CI628 also can block the rapid effect of estrogen to potentiate fast glutamatergic synaptic transmission, which does not involve changes in gene expression. Thus, rapid EPSC potentiation must be considered among the cellular mechanisms of estrogen action that are sensitive to modulation by SERMs.
As discussed in more detail below, our results reveal three novel insights into estrogen activation of CA1 pyramidal cells. First, we have dissociated the first and second waves of pyramidal cell activation mechanistically based on their differential sensitivity to SERMs and on the lack of a 2-h increase in ventral CA1. Second, our data suggest at least two mechanisms by which estrogen could produce the second wave of pyramidal cell activation at 24 h: an effect on locally projecting GABAergic interneurons that express classical ERs to consequently activate CA1 pyramidal cells through disinhibition, and/or a direct effect on CA1 pyramidal cells through nonclassical rapid potentiation of glutamatergic synaptic transmission. Third, neither of these mechanisms seems to be responsible for the first wave of pyramidal cell activation at 2 h.
Relationship between first and second phases of pyramidal cell activation
Our findings answer several questions about the relationship between the first and second waves of pyramidal cell activation by estrogen. For example, previously it was not known whether estrogen induces two waves of pyramidal cell activation sequentially in the same or different cells; and similarly, it was not known whether the first wave of activation might be a necessary precursor for the second. The data reported here dissociate the first and second waves of pyramidal cell activation by estrogen in three ways. First, our results argue against the possibility that the 2-h increase in c-Fos leads to the 24-h increase, because the 24-h increase occurs in the ventral hippocampus despite the lack of a 2-h increase in the same region. Second, given that the first wave of activation apparently is not required for the second wave to occur in the ventral hippocampus, it is unlikely that c-Fos is induced twice in the same cells, even in the dorsal CA1. Third, because T pretreatment blocked the increase in c-Fos at 24 h but not at 2 h, it is also clear that the second wave of activation is not the inevitable consequence of the first. Thus, estrogen must have some effect(s), in addition to activating pyramidal cells at 2 h, that is required for the second wave of activation to occur.
Estrogen activation of pyramidal cells at 24 h
T and CI628 each blocked c-Fos induction 24 h after estrogen, which suggests that classical ERs might be involved in the second wave of CA1 pyramidal cell activation. However, it is highly unlikely that classical ERs expressed in the pyramidal cells themselves produce this activation. The pattern of nuclear ER expression (i.e. ER that could act through a classical mechanism) is very different in the dorsal vs. ventral hippocampus. Very few, if any, dorsal CA1 pyramidal cells express a nuclear ER, whereas nuclear ER is expressed in a high proportion of ventral CA1 pyramidal cells (10, 13). Therefore, given that delayed estrogen-induced c-Fos and the SERM sensitivity of delayed c-Fos induction is very similar in dorsal vs. ventral CA1 pyramidal cells, a classical ER-mediated mechanism that affects both dorsal and ventral pyramidal cells similarly is likely to involve some other cell type.
GABAergic inhibitory interneurons are a likely target for estrogen action through a classical mechanism in the hippocampus. Nuclear ERs are expressed in at least a subset of GABAergic inhibitory interneurons in the hippocampus and, in contrast with ER expression in pyramidal cells, the pattern of ER expression in GABA neurons is very similar in the dorsal vs. ventral CA1 (known for ER
; Ref. 10). Thus, it is possible that estrogen acts through a classical mechanism directly on GABAergic neurons in CA1 to increase indirectly the activity of CA1 pyramidal cells through disinhibition, which then can be detected by induction of c-Fos. The observation that estrogen suppresses GAD65 immunoreactivity in GABAergic neurons in both the dorsal and ventral CA1, at the same time as activating CA1 pyramidal cells dorsally and ventrally, is consistent with this suggestion. Further support for a mechanistic link between increased c-Fos in CA1 pyramidal cells and decreased GAD65 in GABAergic neurons in CA1 comes from the observation that the degree of c-Fos induction and GAD65 suppression were inversely correlated in our within-animal analysis.
Though the SERM sensitivity of c-Fos induction at 24 h supports the suggestion that delayed activation occurs through a classical ER-mediated mechanism, it is also possible that a SERM-sensitive rapid estrogen effect is involved in delayed pyramidal cell activation by estrogen. Our electrophysiological analysis revealed that the T metabolite, 4OH-T, or CI628 can block at least one nonclassical rapid estrogen effect, potentiation of pyramidal cell EPSCs, and T also can modulate other nonclassical effects as well (e.g. Ref. 22). Thus, it is possible that the T sensitivity of c-Fos induction at 24 h is attributable, at least in part, to the dependence of the 24-h increase on a SERM-sensitive rapid estrogen effect, such as EPSC potentiation. Notably, classical and rapid mechanisms of estrogen action are not mutually exclusive; recent studies have suggested that some actions of classical ERs can be potentiated by preceding rapid effects of estrogen at the cell membrane (22, 24).
Estrogen activation of pyramidal cells at 2 h
Our data also lend insight into the involvement of nuclear ERs and/or rapid estrogen effects in the initial wave of pyramidal cell activation at 2 h. For example, we show that classically acting (i.e. nuclear) ERs are not involved in 2-h activation of pyramidal cells. First, T and CI628, which should block ER-regulated gene expression, do not block estrogen induction of c-Fos at 2 h. Second, estrogen fails to increase c-Fos at 2 h in the ventral hippocampus, where many pyramidal cells express a nuclear ER, but estrogen does increase c-Fos in CA1 pyramidal cells of the dorsal hippocampus, very few of which express a nuclear ER, either ER (10) or ERß (25).
These results suggest that a rapid estrogen effect, as opposed to a classical ER-mediated mechanism, is involved in the first wave of pyramidal cell activation by estrogen. We tested one likely candidate for a rapid estrogen effect in CA1 pyramidal cells, rapid EPSC potentiation, and ruled it out as a mechanism that could be responsible for estrogen induction of c-Fos at 2 h. Both 4OH-T and CI628 were capable of blocking rapid EPSC potentiation by estrogen in electrophysiological experiments, but T and CI628 each failed to block the 2-h increase in c-Fos in vivo. Estrogen has numerous other rapid effects that are reported to be insensitive to T, including modulation of neuronal calcium channels (26), increasing intracellular calcium (27) or cAMP (28, 29) levels, and activation of MAPK (30). Activation of MAPK, in particular, is a likely candidate for estrogen activation of CA1 pyramidal cells, because estrogen has been shown to induce c-Fos through activation of MAPK (30) and estrogen can activate MAPK in CA1 pyramidal cells of adult female rats (31).
Extrahippocampal afferents
Also important to note when considering possible mechanisms of estrogen’s effects on the hippocampus (at 2 or 24 h) is that such effects may involve direct action on estrogen-sensitive extrahippocampal afferents rather than, or in addition to, effects on neurons in the hippocampus itself. For example, estrogen could regulate hippocampal activity through direct effects on basal forebrain cholinergic neurons or serotonergic neurons in the raphe nuclei, both of which contain neurons that express nuclear ERs. Suppression of GAD65 at 24 h may not require action outside the hippocampus, because a similar effect of estrogen is observed in dissociated cultures of hippocampal neurons (9), which lack such afferents. However, other effects of estrogen on excitatory synaptic function in CA1, such as the estrogen-induced increase in NMDA receptor binding (32) or the increase in dendritic spine density (4) that occur 72 h after estrogen treatment, have been shown to be modulated by extrahippocampal afferents.
Future studies will be required to understand how classical and rapid effects of estrogen in hippocampal neurons and/or afferents to the hippocampus cooperate to activate hippocampal pyramidal cells and how this activation is related to estrogen’s subsequent effects on the structure and function of hippocampal synaptic connections.
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Footnotes |
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Abbreviations: ACSF, Artificial cerebrospinal fluid; DMSO, dimethylsulfoxide; E, 17ß-estradiol benzoate; EPSC, excitatory postsynaptic currents; ER, estrogen receptor; GABA, -aminobutyric acid; GAD, glutamic acid decarboxylase; NMDA, N-methyl D-aspartate; O, oil alone; OVX, ovariectomized; SERM, selective ER modulator; T, tamoxifen.
Received June 4, 2002.
Accepted for publication September 6, 2002.
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and GAD colocalization in the hippocampus of the adult female rat. J Comp Neurol 440:144–155[CrossRef][Medline]
and ERß exhibit unique pharmacologic properties when coupled to activation of the mitogen-activated protein kinase pathway. Endocrinology 142:2336–2342This article has been cited by other articles:
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C. N. Rudick, R. B. Gibbs, and C. S. Woolley A Role for the Basal Forebrain Cholinergic System in Estrogen-Induced Disinhibition of Hippocampal Pyramidal Cells J. Neurosci., June 1, 2003; 23(11): 4479 - 4490. [Abstract] [Full Text] [PDF]
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