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The Journal of Neuroscience, June 1, 2003, 23(11):4479-4490
Previous Article | Next Article
A Role for the Basal Forebrain Cholinergic System in Estrogen-Induced Disinhibition of Hippocampal Pyramidal Cells
Charles N. Rudick,1 Robert B. Gibbs,2 andCatherine S. Woolley1 1 Department of Neurobiology and Physiology and Northwestern University Institute for Neuroscience, Northwestern University, Evanston, Illinois 60208, and 2 Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Abstract
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Abstract
Introduction
Estrogen transiently disinhibits hippocampal CA1 pyramidal cells in adult female rats and prolongs the decay time of IPSCs in these cells. Estrogen-induced changes in synaptic inhibition are likely to be causally related to subsequent enhancements in excitatory synaptic function in CA1 pyramidal cells. Currently, it is unknown how or on what cells estrogen acts to regulate synaptic inhibition in the hippocampus. We used whole-cell voltage-clamp recording of synaptically evoked IPSCs, spontaneous IPSCs, and miniature IPSCs in CA1 pyramidal cells to evaluate estrogen-induced changes in synaptic inhibition in ovariectomized rats that either were pretreated with the estrogen receptor (ER) antagonist tamoxifen or in which basal forebrain cholinergic neurons were eliminated by previous infusion of 192IgG-saporin toxin into the medial septum. We found that estrogen-induced disinhibition and prolongation of IPSCs are entirely dependent on a tamoxifen-sensitive ER. Estrogen-induced disinhibition is partially dependent on basal forebrain cholinergic neurons, but the prolongation of IPSCs is not at all dependent on these cells. Paired-pulse experiments and recordings of action potential-related spontaneous IPSCs suggest that estrogen-induced disinhibition is associated with a decrease in probability of release at GABAergic synapses, which decreases the amplitude of IPSCs produced by inhibitory neuron action potentials. Our findings lend novel insights into estrogen regulation of inhibitory synapses in the hippocampus and point to estrogen action on basal forebrain cholinergic neurons as critically involved in mediating the effects of estrogen in the hippocampus.
Key words: GABA; IPSCs; tamoxifen; ChAT; estradiol; 192IgG-saporin; CA1
Introduction
Estrogen regulates both inhibitory and excitatory synaptic input to CA1 pyramidal cells in the hippocampus of adult female rats. With a relatively short latency of 24 hr, estrogen decreases the amplitude of synaptically evoked GABAA receptor-mediated IPSCs and decreases the frequency, but not amplitude, of miniature IPSCs (mIPSCs) in these cells (Rudick and Woolley, 2001). The decrease in inhibitory synaptic function induced by estrogen is transient in that by 72 hr, inhibition is restored to control levels. In parallel with the restoration of inhibition, excitatory input to CA1 pyramidal cells is enhanced: dendritic spine and synapse density are increased (Woolley and McEwen, 1992
Estrogen also transiently alters the kinetics of CA1 pyramidal cell IPSCs at the same time that it decreases IPSC amplitude.
Twenty-four hours after estrogen, synaptically evoked IPSCs show a prolonged decay time, which is paralleled by the appearance of a subpopulation of mIPSCs with prolonged decay. By 72 hr, evoked IPSC decay has returned to control levels, and the subpopulation of prolonged mIPSCs has disappeared.
Currently, it is unknown how or where estrogen acts to regulate synaptic inhibition in the hippocampus. Estrogen can act through classical nuclear receptors, estrogen receptor (ER)-
and ER-
, and through "rapid effects" that may involve membrane or cytoplasmic receptors (Moss and Gu, 1999
37% of cholinergic neurons in the medial septum bind estrogen and express ER-
To begin to elucidate the mechanisms by which estrogen regulates synaptic inhibition in the hippocampus, we tested whether the ER antagonist tamoxifen can block the effects of estrogen on CA1 pyramidal cell IPSCs and the ability of estrogen to regulate IPSCs in animals in which basal forebrain cholinergic neurons were eliminated by infusion of 192IgG-saporin (Sap) toxin into the medial septum. Our findings demonstrate that estrogen regulation of hippocampal synaptic inhibition depends completely on tamoxifen-sensitive ERs and partly on basal forebrain cholinergic neurons. We propose models for estrogen action in the basal forebrain to disinhibit hippocampal pyramidal cells.
Materials and Methods
Animal treatments. Animal procedures were approved by the Northwestern University Animal Care and Use Committee. Adult female Sprague Dawley rats (180–220 gm) were housed on a 12 hr light/dark cycle with ad libitum access to food and water. All rats were bilaterally ovariectomized under ketamine (85 mg/kg)/xylazine (13 mg/kg) anesthesia using aseptic procedures. On the third day after surgery, rats were injected subcutaneously with either 10 µgof17In additional experiments, 192IgG-Sap (Chemicon International, Temecula, CA) was infused directly into the medial septum to selectively destroy basal forebrain cholinergic neurons before ovariectomy. 192IgG-Sap is a selective immunotoxin consisting of the ribosome-inactivating toxin saporin coupled to an antibody against the p75NTR. Infusing small quantities of Sap directly into the medial septum selectively destroys cholinergic projection neurons, with no apparent affect on GABAergic projections (Book et al., 1994
Preparation and maintenance of hippocampal slices. On each recording day, three or four animals each from different treatment groups within a particular type of experiment (e.g., tamoxifen pretreatment or Sap lesion) were coded, and the code was not broken until all data analysis for those animals was complete. Rats were anesthetized with Nembutal (80 mg/kg) and transcardially perfused with ice-cold oxygenated (95% O2/5% CO2) artificial CSF (ACSF) containing (in mM): 125 NaCl, 25 NaHCO3, 25 dextrose, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, pH 7.5. The brain was quickly removed and cooled in ice-cold oxygenated ACSF. For experiments with Sap- or Sal-infused animals, the rostral portion of each brain containing the medial septum was saved and frozen for later choline acetyltransferase (ChAT) immunohistochemistry. Using a Vibroslicer (Electron Microscopy Sciences, Fort Washington, PA), 300-µm-thick transverse hippocampal slices were cut into a bath of ice-cold oxygenated ACSF. Also, for Sap- or Sal-infused animals, slices interleaved with those used for electrophysiology were frozen for later analysis of ChAT activity. Slices for electrophysiology were transferred to a holding chamber where they remained submerged in oxygenated ACSF at 35°C for 30 min and then at room temperature (
Electrophysiological recording. Slices were transferred to a recording chamber mounted on a Zeiss Axioskop (Oberkochen, Germany) where they were submerged in oxygenated ACSF maintained at 35 ± 1°C. Neurons in the slice were visualized using infrared differential interference videomicroscopy (Hamamatsu, Hamamatsu City, Japan). Somatic whole-cell current- or voltage-clamp recordings were obtained from CA1 pyramidal neurons using patch electrodes made from thick-walled borosilicate glass (Garner Glass, Claremont, CA) pulled on a P-97 micropipette puller (Sutter Instrument Co., Novato, CA) with an open tip resistance of 3–5 M
in ACSF. Series resistance (average 13 ± 4.7 M
Synaptically evoked action potentials were recorded in current clamp using a pipette solution containing (in mM): 115 K-gluconate, 20 KCl, 10 Na2-creatinine phosphate, 20 HEPES, 2 EGTA, 2 MgATP, NaGTP, pH 7.3. Before recording, a cut in the slice was made between the CA1 and CA3 regions. A bipolar stimulating electrode was placed in the CA1 stratum radiatum
Synaptically evoked IPSCs, spontaneous IPSCs, and mIPSCs were recorded in voltage clamp using a pipette solution containing (in mM): 140 CsCl, 2 MgCl2, 10 HEPES, 2 EGTA, 2 MgATP, 0.3 NaGTP, 10 Na2-creatinine phosphate, 0.1% biocytin, pH 7.2–7.3. The bath contained 5 mM kynurenic acid to block glutamate receptor-mediated synaptic transmission. To record synaptically evoked IPSCs, a stimulating electrode (patch pipette with chlorided silver wire) was placed in the CA1 pyramidal cell layer 50–100 µm away from the recording electrode. Stimulus–response curves were generated at a holding potential of -70 mV by varying stimulus intensity from the minimum current necessary to evoke a postsynaptic response to the current that produced a maximal response; stimuli were delivered at 0.1 Hz. Synaptically evoked IPSCs were blocked by the GABAA receptor antagonist bicuculline (10 µM). The stimulus–response protocol was repeated at least three times per cell, and the average of peak synaptically evoked IPSC amplitude at each stimulus intensity was calculated for each cell. Synaptically evoked IPSC rise and decay times were obtained from maximal currents, and data were averaged per cell. Paired-pulse depression of synaptically evoked IPSCs was measured using a stimulus intensity that evoked a maximal current. Pairs of IPSCs were evoked with interstimulus intervals from 10 to 300 msec delivered with 20 sec between stimulus pairs. The paired-pulse protocol was repeated at least three times per cell, and the ratio of IPSC amplitude evoked by the second pulse versus the first pulse (P2/P1) was calculated for each cell at each interstimulus interval. Stimulus–response curves were analyzed statistically with repeated-measures ANOVA. Synaptically evoked IPSC rise time and time to 50% decay were analyzed statistically with ANOVA followed by Turkey post hoc comparisons. Paired-pulse depression was analyzed statistically with repeated-measures ANOVA (for experiments with multiple interstimulus intervals) or ANOVA followed by Tukey post hoc comparisons (for experiments with a single interstimulus interval).
Spontaneous IPSCs were recorded from CA1 pyramidal cells without synaptic stimulation, and data were obtained from the first 500 spontaneous events per cell. Subsequently, 5 µM TTX was added to the bath to reveal mIPSCs from the same cell. Miniature IPSCs were recognized as events at least 2 SD above the amplitude of noise, as determined by recording in kynurenic acid and bicuculline. Data were collected from the first 500 mIPSCs per cell. The frequency, mean amplitude, and rise and decay time of TTX-sensitive (i.e., action potential-related) spontaneous IPSCs and mIPSCs were analyzed statistically using ANOVA followed by Tukey post hoc comparisons. Amplitude and decay time histograms for action potential-related spontaneous IPSCs and mIPSCs were analyzed statistically using the Kolmogorov–Smirnov test.
In some experiments, exogenous GABA was applied directly to the soma of a recorded cell using a PV830 pneumatic picopump (World Precision Instruments, Sarasota, FL). GABA application was repeated at least 10 times with a 1 min interval between applications, and measurements of peak amplitude and decay time were averaged for each cell. The amplitude and time to 50% decay of GABA-evoked currents were analyzed statistically using Student's t test.
Immunohistochemistry for ChAT. ChAT immunohistochemistry was performed for each Sap- or Sal-infused animal to confirm cholinergic lesion in the Sap-infused animals. All tissue was coded, and the code was not broken until analysis of both immunohistochemical and enzyme activity assays for ChAT was complete. The rostral portion of each brain containing the medial septal area was fixed by immersion in 4% paraformaldehyde in 50 mM PBS for several hours and then in 4% paraformaldehyde in 15% sucrose at 4°C overnight. Brain tissue was coronally sectioned (40 µm) through the medial septal area using a freezing microtome. Sections were processed for immunohistochemical detection of ChAT as described previously (Gibbs, 1997
Briefly, sections were incubated in the primary antiserum (1:3500; Chemicon International) for 72 hr at 4°C. After primary incubation, sections were rinsed thoroughly and incubated in biotinylated secondary antibody (1:250, anti-goat IgG; Vector Laboratories, Burlingame, CA). The sections were rinsed and incubated in avidin–biotin HRP complex (1:500; Vector Elite Kit) for 1 hr. Next, the tissue was incubated for 10 min in Tris acetate solution containing 3–3'-diaminobenzidine (0.5 mg/ml), H2O2 (0.01%), and NiCl2 (0.032%). After the reaction, sections were rinsed, mounted onto subbed slides, dehydrated in graded ethanols, cleared in xylene, and coverslipped under DePex.
ChAT assay. Frozen, coded, hippocampal slices were thawed and dissociated by sonication in a medium containing EDTA (10 mM) and Triton X-100 (0.5%) and diluted to a concentration of 10 mg tissue per milliliter. An aliquot of each sample was used for the determination of total protein (Bradford, 1976
Briefly, three 5 µl aliquots of each sample were incubated for 30 min at 37°C in a medium containing [3H]acetyl-CoA (3.6 Ci/mmol, 30,000–40,000 dpm per tube, final concentration 0.25 mM acetyl CoA; Sigma, St. Louis, MO), choline chloride (10 mM), physostigmine sulfate (0.2 mM), NaCl (300 mM), sodium phosphate buffer, pH 7.4 (50 mM), and EDTA (10 mM). The reaction was terminated with 4 ml of sodium phosphate buffer (10 mM, pH 7.2) followed by the addition of 1.6 ml of acetonitrile containing 5 mg/ml tetraphenylboron. The amount of [3H]acetylcholine produced was determined by adding 8 ml of EconoFluor scintillation mixture (Packard Instruments, Meriden, CT) and counting total counts per minute (cpm) in the organic phase using an LKB beta counter. Background was determined using identical tubes to which no sample was added. For each sample, the three reaction tubes containing sample were averaged, and the difference between total cpm and background cpm was used to estimate the total amount of acetylcholine produced per sample. ChAT activity was then calculated for each sample as picomoles of acetylcholine produced per hour per microgram of protein. All samples were assayed at the same time using the same stock of [3H]acetyl-CoA, and the data were analyzed statistically using ANOVA followed by Tukey post hoc comparisons.
Results
Estrogen-induced disinhibition
Previously, we found that 24 hr of estrogen treatment significantly decreases the amplitude and prolongs the decay time of synaptically evoked IPSCs in CA1 pyramidal cells. Recordings of the mixed current (EPSC + IPSC) evoked by stimulation in the stratum radiatum suggested that these changes are associated with reduced disynaptic inhibition of CA1 pyramidal cells (Rudick and Woolley, 2001
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Figure 1. Estrogen treatment decreases the threshold for action potential firing in CA1 pyramidal cells. A, Plot of the minimum stimulus necessary to evoke an action potential in cells from oil-treated (O, open circles; n = 11 cells from 6 animals) and estrogen-treated (E, closed circles; n = 12 cells from 6 animals) groups. Action potential threshold was significantly lower for the cells in the estrogen-treated group (p < 0.01). B, Representative EPSPs and action potential from a cell in the O-treated group; stimuli for the traces shown were 40, 50, 60, and 70 µA (this cell first fired at 69 µA). C, Representative EPSPs and action potential from a cell in the E-treated group; stimuli for the traces shown were 40, 50, and 60 µA (this cell first fired at 51 µA).
Effects of tamoxifen on estrogen-induced changes in synaptic inhibition
We used the selective estrogen receptor modulator tamoxifen to investigate whether the effects of estrogen on synaptic inhibition of CA1 pyramidal cells depend on an ER. In these experiments, we replicated both the estrogen-induced decrease in IPSC amplitude and prolongation of IPSC decay and found that each is completely blocked by pretreatment with tamoxifen. Tamoxifen is generally an ER antagonist in brain, but also can act as an ER agonist in some systems. To validate use of tamoxifen as an ER antagonist, we confirmed in a separate study that tamoxifen does not act as an ER agonist in CA1 (Rudick and Woolley, 2003
Whole-cell voltage-clamp recordings were made using a CsCl-based internal solution and at a holding potential of -70 mV so that the GABAA receptor-mediated IPSC was an inward current. In the first series of experiments, synaptically evoked IPSCs recorded in cells from oil-treated or estrogen-treated animals that were pretreated either with vehicle (Fig. 2A,B, VO and VE) or with tamoxifen [TO or TE (Fig. 2C)] were used to generate stimulus–response curves (Fig. 2D). Twenty-four hours of estrogen treatment had two principal effects on evoked IPSCs: decreased amplitude and increased decay time. Peak amplitude of evoked IPSCs was 30% lower in VE than VO cells (p < 0.05). Pretreatment with tamoxifen had no effect on evoked IPSC amplitude in oil-treated controls: stimulus–response curves were virtually identical for VO and TO cells (p > 0.1). However, tamoxifen pretreatment did block the estrogen-induced decrease in IPSC amplitude. Stimulus–response curves for VE cells were significantly different from those for VO, TO, or TE cells (p < 0.05), which were not different from each other (p > 0.1).
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Figure 2. Tamoxifen blocks the estrogen-induced decrease in the amplitude of synaptically evoked IPSCs. All recordings were made with a CsCl-based internal solution at a holding potential of -70 mV so that GABAA-mediated IPSCs are inward currents. A, Representative individual traces of IPSCs evoked in a VO cell using 30, 50, 100, and 250 µA stimulating current. B, Representative individual traces of IPSCs evoked in a VE cell using the same stimulus intensities as in A. C, Representative individual traces of IPSCs evoked in a TE cell using the same stimulus intensities as in A. Evoked currents are blocked by bicuculline. D, Averaged stimulus–response curves for VO (open circles; n = 12 cells from 6 animals), VE (black circles; n = 12 cells from 6 animals), TO (light gray circles; n = 11 cells from 6 animals), and TE (dark gray circles; n = 13 cells from 6 animals) groups. *Peak IPSC amplitude is significantly reduced in VE cells compared with TE, TO, and VO cells (p < 0.05).
Estrogen also prolonged the decay time of evoked IPSCs. IPSC decay in CA1 pyramidal cells is best described by two time constants,
decay fast and
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Table 1. Kinetic parameters of synaptically evoked IPSCs
Previously, we demonstrated that the decrease in evoked IPSC amplitude was associated with a decrease in the frequency, but not amplitude, of TTX-insensitive mIPSCs recorded from the same cells (Rudick and Woolley, 2001
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Figure 3. Tamoxifen blocks the estrogen-induced decrease in the frequency of TTX-insensitive mIPSCs. All recordings were made with a CsCl-based internal solution at a holding potential of -70 mV so that GABAA-mediated mIPSCs are inward currents. A–C, Representative traces from four different VO cells (A), VE cells (B), and TE cells (C) showing mIPSCs. D, Bar graph of mean ± SEM frequency of mIPSCs in VO (open bars), VE (black bars), TO (light gray bars), and TE (dark gray bars) groups. Data were obtained from the first 500 mIPSCs from each of the same cells as in Figure 2 D. **Significant difference from TO and VO (p < 0.01) and from TE (p < 0.05).
Analysis of histograms for mIPSC amplitude and decay time from cells in each treatment group provided additional information about estrogen-induced changes in GABAergic synapses and confirmed complete blockade of the effects of estrogen by tamoxifen. Miniature IPSC amplitude histograms (Fig. 4A–C) show that although estrogen does not significantly alter the mean amplitude of mIPSCs, it does produce a statistically significant skewing toward larger amplitude mIPSCs (Fig. 4D) (p < 0.01). This shift toward larger currents is clear evidence that the estrogen-induced decrease in evoked IPSC amplitude is not caused by a decrease in the strength of individual GABAergic synapses, because this would produce a shift in the direction of smaller mIPSCs. Additionally, comparison of mIPSC decay time histograms (Fig. 4E–H) revealed a subpopulation of mIPSCs with prolonged decay time in VE cells that parallels the longer decay of evoked IPSCs in the same animals; mIPSCs with prolonged decay also are evident in the VE traces shown in Figure 3B. Although group data are shown in Figure 4, it is important to note that mIPSC decay time histograms were clearly bimodal in 28 of the 32 individual cells from estrogen-treated animals in this study. Pretreatment with tamoxifen did not affect the distributions of mIPSC amplitudes or decay times in control animals but completely blocked effects of estrogen on both measures. Miniature IPSC amplitude and decay time histograms for VE cells were significantly different from those for VO, TO, or TE cells (p < 0.01), which were not different from each other (p > 0.1). Together, these data demonstrate that estrogen acts through a tamoxifen-sensitive ER to regulate both disinhibition and prolongation of IPSCs in CA1 pyramidal cells.
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Figure 4. Tamoxifen blocks estrogen-induced changes in mIPSC amplitude and decay time distributions. A–C, mIPSC amplitude histograms for the VO group (A), VE group (B), and TE group(C).D, Cumulative histogram of mIPSC amplitudes for VO (opencircles), VE (blackcircles), TO (light gray circles), and TE (dark gray circles). Mean mIPSC amplitude is not significantly affected by estrogen, but note the shift toward larger amplitude mIPSCs in the VE group (p < 0.01) that is absent in the TE group. E–G, mIPSC decay time histograms for the VO group (E), VE group (F), and TE group (G). H, Cumulative histogram of mIPSC decay times for VO (open circles), VE (black circles), TO (light gray circles), and TE (dark gray circles). Mean mIPSC decay time is not significantly affected by estrogen, but note the subpopulation of longer decay time mIPSCs in the VE group (p < 0.01) that is absent in the TE group. Data were obtained from the same 500 mIPSCs per cell as in Figure 3D.
Paired-pulse depression and GABA-evoked currents
The decreases in evoked IPSC amplitude and mIPSC frequency in cells from estrogen-treated animals suggest that estrogen may decrease probability of release at GABAergic synapses. To investigate this possibility, we measured paired-pulse depression of evoked IPSCs (Fig. 5A), which is an indicator of probability of release. We found that paired-pulse depression was significantly reduced in cells from estrogen-versus oil-treated animals (p < 0.05); the difference was greatest with interstimulus intervals of ≤100 msec (Fig. 5B). For example, with a 50 msec interstimulus interval, the P2/P1 ratio in controls cells was 0.67 ± 0.05, indicating 33% depression of the second response, whereas P2/P1 was 0.95 ± 0.02 in cells from estrogen-treated animals, indicating only 5% depression. These data suggest that the decrease in evoked IPSC amplitude and mIPSC frequency induced by estrogen is caused, at least in part, by a functional change in GABAergic synapses that decreases the probability of GABA release. However, other factors such as receptor desensitization also may influence measures of paired-pulse depression.
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Figure 5. Estrogen decreases paired-pulse depression of evoked IPSCs in CA1 pyramidal cells. Recordings were made with a CsCl-based internal solution at a holding potential of -70 mV so that GABAA-mediated spontaneous IPSCs are inward currents. A, B, Representative traces of pairs of IPSCs from an O-treated cell (A) and an E-treated cell (B) evoked with a 50 msec interstimulus interval. C, Plot of P2/P1 ratios in the O-treated group (open circles; n = 11 cells from 6 animals) compared with the E-treated group (black circles; n = 13 cells from 6 animals). Note that P2/P1 is closer to 1.0 in cells from the E-treated group, indicating less paired-pulse depression (p < 0.05).
To evaluate the effect of estrogen treatment on postsynaptic and extrasynaptic GABA receptors, we measured the current evoked by application of GABA to the soma using a picopump (e.g., 1 sec application of 500 µM GABA). Experiments with exogenous GABA application revealed no differences in the amplitude or kinetics of GABA-evoked currents in cells from oil- (n = 8) and estrogen-treated (n = 9) animals (p > 0.1 on all measures; data not shown). These data indicate that estrogen does not produce a global (i.e., postsynaptic and extrasynaptic) increase in the decay time of GABA-evoked currents, but rather increases decay time of currents selectively at a subset of GABAergic synapses. This observation is consistent with the mIPSC decay time histograms, which show that only a subset of mIPSCs is prolonged in cells from estrogen-treated animals. However, because synaptic receptors may be only a fraction of those activated by exogenous GABA application, it is not possible to conclude from this negative result whether the effect of estrogen on IPSC decay time is presynaptic or postsynaptic.
Effects of cholinergic lesions on estrogen-induced changes in synaptic inhibition
Our results with tamoxifen indicated than an ER is involved in mediating the effects of estrogen on synaptic inhibition of CA1 pyramidal cells. As mentioned previously, nuclear ER-Several lines of evidence point to basal forebrain cholinergic neurons as likely candidates for mediating the effects of estrogen in the hippocampus. First,
To explore the possibility that basal forebrain cholinergic neurons are involved in estrogen-induced changes in synaptic inhibition of hippocampal neurons, we tested the ability of estrogen to disinhibit CA1 pyramidal cells in animals in which basal forebrain cholinergic neurons had been eliminated by previous infusion of 192IgG-Sap into the medial septum. For each animal in this study, we confirmed the effectiveness of Sap infusion on the basis of ChAT immunoreactivity in the basal forebrain and ChAT activity in hippocampal slices interleaved with those used for electrophysiology. These analyses showed that intraseptal infusions of Sap resulted in a substantial loss of ChAT-positive cells in the medial septum and diagonal band of Broca (Fig. 6A–C) and significantly decreased ChAT activity in the hippocampus by 85% (p < 0.01) (Fig. 6D).
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Figure 6. Infusion of 192IgG-saporin into the medial septum substantially reduces the number of cholinergic neurons in the basal forebrain and decreases ChAT activity in the hippocampus. A–C, Photomicrographs of ChAT-immunolabeled cells in Sal- and Sap-infused animals. Low magnification view of ChAT-labeled cells in a Sal-E control (A) and a Sap-E (B) animal. Note the significant reduction in ChAT labeling in the medial septum (MS) and vertical diagonal band (VDB) in Sap-E compared with Sal-E. C, High magnification view of ChAT-labeled cells in a Sal-E control. Scale bars: (in A) A, B, 400 µm; C, 50 µm. D, Bar graph of mean ± SEM. ChAT activity in hippocampal slices interleaved with those used for electrophysiology demonstrating effective reduction of cholinergic activity: Sal-O (open bars; n = 12 slices from 6 animals), Sal-E (black bars; n = 12 slices from 6 animals), Sap-O (light gray bars; n = 9 slices from 6 animals), and Sap-E (dark gray bars; n = 8 slices from 6 animals) groups. **Significant difference from Sal-O and Sal-E (p < 0.01).
Electrophysiological analysis demonstrated that the ability of estrogen to disinhibit CA1 pyramidal cells was significantly attenuated in Sap-lesioned animals, but it was not completely blocked. As before, we evaluated evoked IPSCs and mIPSCs. Removal of basal forebrain cholinergic neurons by itself had no effect on measures of synaptic inhibition in CA1; data from Sap-O and Sal-O animals were indistinguishable (p > 0.1 on all measures). As expected from previous experiments, estrogen decreased the peak amplitude of synaptically evoked IPSCs in Sal-infused animals by 35% (Fig. 7A)(p < 0.05). In Sap-lesioned animals, however, the estrogen-induced decrease in evoked IPSC amplitude was significantly attenuated in that peak IPSC amplitude was reduced by only 13% and was significantly different from all other groups (p < 0.05) (Fig. 7A). The effect of estrogen on paired-pulse depression of IPSCs also was reduced by Sap lesion (Fig. 7B). In Sal-infused controls, the P2/P1 ratio with a 50 msec interstimulus interval was 0.63 ± 0.05 Sal-O cells and 0.93 ± 0.06 Sal-E cells (p < 0.01), whereas in Sap-lesioned animals, estrogen did not significantly alter the P2/P1 ratio (0.64 ± 0.04 in Sap-O vs 0.72 ± 0.06 in Sap-E; p = 0.054). Similar to evoked IPSC amplitude, the frequency of mIPSCs in Sap-E animals was intermediate between Sal-E animals and Sal-O and Sap-O animals (p < 0.05; Sap-E vs all other groups) (Fig. 7C). Additionally, the skewing of mIPSC amplitudes toward larger currents was intermediate in Sap-E animals (data not shown). With the exception of paired-pulse experiments in which estrogen did not have a statistically significant effect in Sap-lesioned animals, data from the Sap-E cells were significantly different from cells in all other groups (p < 0.05). These results demonstrate that the basal forebrain cholinergic system plays a critical role in estrogen-induced disinhibition of hippocampal CA1 pyramidal cells. The observation that Sap lesion produced only a partial blockade of the effects of estrogen indicates that in addition to basal forebrain cholinergic neurons, other cells also are likely to be involved in estrogen-induced disinhibition in the hippocampus.
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Figure 7. 192IgG-saporin lesion significantly attenuates estrogen-induced disinhibition but does not completely block it. A, Averaged stimulus–response curves for synaptically evoked IPSCs in Sal-O (open circles; n = 12 cells from 6 animals), Sal-E (black circles; n = 12 cells from 6 animals), Sap-O (light gray circles; n = 9 cells from 6 animals), and Sap-E (dark gray circles; n = 8 cells from 6 animals) groups. B, Bar graph of mean ± SEM. P2/P1 ratio for evoked IPSCs in Sal-O, Sal-E, Sap-O, and Sap-E groups (50 msec interstimulus interval). Note that P2/P1 in the Sal-E group is closer to 1.0 than in other groups, indicating less paired-pulse depression in Sal-E. C, Bar graph of mean ± SEM frequency of mIPSCs for Sal-O, Sal-E, Sap-O, and Sap-E groups. *Significant difference from Sal-O, Sap-O, and Sap-E (p < 0.05); **significant difference from Sal-O and Sal-O (p < 0.01); significant difference from Sal-O, Sap-O, and Sal-E (p < 0.05; trend from Sap-O in B, p = 0.054).
Although we expected that cholinergic tone in an acute hippocampal slice, i.e., denervated from subcortical inputs, would be minimal, we performed additional experiments to confirm that the effects of estrogen on synaptic inhibition are not caused by an enhancement of presynaptic cholinergic function in the slice. We recorded paired-pulse depression and mIPSC frequency in cells from nonlesioned oil- (n = 6) and estrogen-treated (n = 4) animals before and after bath application of the nonselective muscarinic receptor antagonist atropine (1–5 µM) and found that neither measure was affected by atropine (p > 0.1; data not shown). These data indicate that estrogen-induced disinhibition in CA1 is not caused by a difference in cholinergic activity in the slice 24 hr after estrogen treatment.
Interestingly, in contrast to the effect of Sap lesion on estrogen-induced disinhibition, the ability of estrogen to prolong IPSC decay times was unaffected by elimination of basal forebrain cholinergic neurons. Synaptically evoked IPSCs were similarly prolonged in Sal-E and Sap-E cells (Fig. 8A, Table 2), and the subpopulation of prolonged mIPSCs was similarly present in Sal-E and Sap-E cells (Fig. 8B). Thus, analysis of Sap-lesioned animals demonstrated that the effects of estrogen on IPSC amplitudes versus decay times are separable, indicating that different mechanisms mediate these two effects of estrogen on synaptic inhibition of hippocampal CA1 pyramidal cells.
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Figure 8. 192IgG-saporin lesion has no effect on estrogen-induced prolongation of IPSC decay times. A, Bar graph of mean ± SEM decay times of synaptically evoked IPSCs in Sal-O (open bars), Sal-E (black bars), Sap-O (light gray bars), and Sap-E (dark gray bars) groups. B, Cumulative histogram of mIPSC decay time distributions for Sal-O (open circles), Sal-E (black circles), Sap-O (light gray circles), and Sap-E (dark gray circles). Note the leftward shift in mIPSC decay time in both the Sal-E and Sap-E groups (p < 0.01) that is not observed in the Sal-O and Sap-O groups. Data are from the same cells as in Figure 6. **Significant difference from oil-treated groups (p < 0.01).
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Table 2. Kinetic parameters of synaptically evoked IPSCs
Action potential-related spontaneous IPSCs
Each CA1 pyramidal cell receives inhibitory synaptic input from multiple GABAergic neurons, and each GABAergic neuron forms multiple inhibitory synapses with each pyramidal cell that it contacts (Buhl et al., 1994
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Figure 9. Estrogen decreases the amplitude, but not frequency, of action potential-related spontaneous IPSCs, and this decrease is blocked by 192IgG-saporin lesion. Recordings were made with a CsCl-based internal solution at a holding potential of -70 mV so that GABAA-mediated spontaneous IPSCs are inward currents. A, B, Representative traces of spontaneous IPSC events (mIPSCs and action potential-related IPSCs) from a Sal-O cell (A) and a Sal-E cell (B). Insets, Amplitude histograms for all spontaneous IPSC events in each group; note two peaks, corresponding to mIPSCs and action potential-related IPSCs. C, Bar graph of the mean ± SEM amplitude of action potential-related (i.e., TTX-sensitive) spontaneous IPSCs in Sal-O, Sal-E, Sap-O, and Sap-E groups. Note the estrogen-induced decrease in action potential-related spontaneous IPSC amplitude in Sal animals (p < 0.05) but not Sap animals (p = 0.09; Sap-E vs Sap-O). D, Bar graph of the mean ± SEM frequency of action potential-related spontaneous IPSCs in Sal-O, Sal-E, Sap-O, and Sap-E groups. Neither estrogen nor 192IgG-saporin lesion affects the frequency of these events. Data were collected from 326 ± 75 action potential-related spontaneous IPSCs in the same cells as in Figure 6. *Significant difference from Sal-O, Sap-O (p < 0.05).
Analysis of spontaneous IPSCs indicated that estrogen does not alter the number of inhibitory neurons in functional synaptic contact with CA1 pyramidal cells but that the current produced by each inhibitory neuron action potential, on average, is reduced. Similar to the effect of estrogen on evoked IPSC amplitude, estrogen treatment significantly decreased the mean amplitude of spontaneous IPSCs by 30% in cells from Sal-infused animals (Fig. 9C)(p < 0.05). As was the case with other measures of inhibition, Sap-lesion itself had no effect on the amplitude of spontaneous IPSCs in oil-treated controls (p > 0.10). Additionally, estrogen did not significantly alter the amplitude of spontaneous IPSCs in Sap-lesioned animals (p = 0.09 vs Sap-E vs Sap-O), although mean amplitude was slightly lower (by 9%) (Fig. 9C). Importantly, in contrast to the effect of estrogen on the amplitude of spontaneous IPSCs, estrogen had no effect on spontaneous IPSC frequency (Fig. 9D)(p > 0.10), nor was spontaneous IPSC frequency affected by the Sap lesion itself (Fig. 9D). Together, our spontaneous IPSC amplitude and frequency data are consistent with an estrogen-induced decrease in probability of GABA release that is distributed across multiple presynaptic neurons in contact with an individual CA1 pyramidal cell. Our results do not support the suggestion that estrogen substantially decreases probability of release at synapses from a subset of pre-synaptic inhibitory neurons, because this would be expected to decrease the frequency of spontaneous IPSCs. Interestingly, the lack of an effect of Sap lesion itself on spontaneous IPSC amplitude or frequency indicates that altering cholinergic tone in the hippocampus in vivo does not have a lasting effect on tonic inhibition of pyramidal cells, i.e., that is evident in a hippocampal slice.
Analysis of spontaneous IPSC decay times indicated that estrogen-induced prolongation of IPSCs also appears to be an effect that is distributed across presynaptic GABA neurons and not an effect on synapses formed by a subset of GABA neurons. One possibility suggested by analysis of mIPSC decay time histograms is that fast versus slow decay time mIPSCs might arise from distinct subsets of presynaptic inhibitory neurons. If this were the case, one would expect decay time histograms for action potential-related spontaneous IPSCs to be bimodal, similar to those for mIPSCs. Alternatively, if individual inhibitory neurons form a mixture of synapses with fast versus slow decay times, this would result in a unimodal decay time histogram for spontaneous IPSCs. Our findings point to the second possibility. First, consistent with the prolonged decay time of evoked IPSCs, mean decay time of spontaneous IPSCs was significantly greater in cells from Sal-infused, estrogen-treated animals compared with cells from oil-treated controls (p < 0.05) (Fig. 10A). Second, in contrast to the bimodal decay time histograms for mIPSCs from estrogen-treated animals, decay time histograms for spontaneous IPSCs in cells from estrogen-treated animals were unimodal in all cells recorded, but the distribution was broader and shifted toward prolonged decay times (Fig. 10C) compared with controls (Fig. 10B). These data suggest that individual inhibitory neurons form a mixture of synapses with fast versus slow decay times, which in concert produce a relatively prolonged IPSC in estrogen-treated animals. Also consistent with data from evoked IPSCs, Sap lesion had no effect on spontaneous IPSC decay time or the estrogen-induced increase in spontaneous IPSC decay time (Fig. 10A).
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Figure 10. Estrogen increases the decay time of action potential-related spontaneous IPSCs, but this increase is unaffected by 192IgG-saporin lesion. A, Bar graph of mean ± SEM decay time of action potential-related spontaneous IPSCs in Sal-O, Sal-E, Sap-O, and Sap-E groups. B, C, Decay time histograms for action potential-related spontaneous IPSCs in the Sal-O group (B) and Sal-E group (C). Note that mean decay time of action potential-related spontaneous IPSCs is significantly increased by estrogen and that the distribution of action potential-related spontaneous IPSC decay times is unimodal but broader and shifted toward prolonged currents in the Sal-E-treated cells (p < 0.05). *Significant difference from the O-treated groups (p < 0.05). Data are from the same action potential-related spontaneous IPSCs as in Figure 9, C and D.
Discussion
Our results provide five novel insights into the mechanism by which estrogen regulates synaptic inhibition of hippocampal CA1 pyramidal cells. (1) The effects of estrogen on pyramidal cell IPSCs result in disinhibition of these cells; (2) estrogen regulation of synaptic inhibition, both disinhibition and prolongation of IPSCs, is entirely dependent on a tamoxifen-sensitive ER; (3) estrogen-induced disinhibition is partially dependent on basal forebrain cholinergic neurons, whereas the prolongation of IPSCs is not at all dependent on these cells; (4) estrogen-induced disinhibition appears to involve a decrease in the probability of transmitter release at GABAergic synapses; and (5) estrogen-induced disinhibition is associated with a decrease in the synaptic current produced by action potentials in individual inhibitory neurons rather than a decrease in the number of inhibitory neurons in functional contact with each CA1 pyramidal cell. Because estrogen-induced changes in synaptic inhibition precede and are likely to be causally related to enhancement of excitatory synaptic structure and function in the hippocampus, these findings contribute to understanding the chain of events by which estrogen influences hippocampal circuitry.Estrogen-induced disinhibition
CA1 pyramidal cells are innervated by multiple GABAergic neurons, and each presynaptic GABA neuron forms multiple inhibitory synapses with individual postsynaptic pyramidal cells (Buhl et al., 1994ER requirement for estrogen-induced disinhibition
We found that estrogen-induced disinhibition of CA1 pyramidal cells is blocked completely by pretreatment with tamoxifen, indicating estrogen action through an ER. Estrogen could regulate synaptic inhibition of CA1 pyramidal cells by acting directly on ER-expressing cells within the hippocampus and/or by acting on ER-expressing cells in brain regions that project to the hippocampus, such as the medial septum in the basal forebrain.We used 192IgG-saporin infusion into the medial septum to test the possibility that basal forebrain cholinergic neurons are critically involved in estrogen-induced disinhibition of hippocampal CA1 pyramidal cells. As described earlier, the suggestion that estrogen acts directly within basal forebrain nuclei such as the medial septum to regulate changes in hippocampal function is supported by the observations that cholinergic neurons in the medial septum express ER-
The distribution of ER-expressing cells within the hippocampus suggests that estrogen also could act directly on a subset of hippocampal GABA neurons to regulate inhibition of CA1 pyramidal cells. Although pyramidal cells in CA1 do not express classical ERs, nuclear ER-
Models of estrogen-induced disinhibition involving the basal forebrain
The hippocampus receives both GABAergic and cholinergic input from basal forebrain regions such as the medial septum (Paxinos, 1995A second possibility is that estrogen acts directly on cholinergic septohippocampal projection neurons to increase acetylcholine release (Gibbs et al., 1997
Several alternatives to the models discussed above also exist. For example, although our data indicate that basal forebrain cholinergic neurons are involved in estrogen-induced disinhibition, they do not distinguish between a direct effect of estrogen on these cells and the possibility that basal forebrain cholinergic neurons play a permissive role in estrogen-induced disinhibition. Some noncholinergic neurons in the medial septum also express ER-
Estrogen action on IPSC decay time
We found that tamoxifen completely blocked the effect of estrogen to prolong IPSC decay time, indicating action through an ER, whereas elimination of basal forebrain cholinergic neurons had no effect on estrogen-induced prolongation of IPSCs. Estrogen does not appear to alter GABA receptor kinetics globally, as evidenced by no effect on GABA-evoked currents. Recordings of both GABA-evoked currents and mIPSCs suggest that estrogen acts to prolong currents specifically at a subset of GABAergic synapses.The prolongation of IPSC decay time could occur through postsynaptic changes in the subunit composition of GABAA receptors and/or changes in GABA transporter function, which could affect GABA uptake from the synaptic cleft. Precedents exist for each possibility. For example, regulation of the
Footnotes
Received Dec. 26, 2002; revised Mar. 13, 2003; accepted Mar. 17, 2003.This work was supported by National Institute of Neurological Disorders and Stroke Grant NS37324 (C.S.W.) and National Science Foundation Grant IBN9905676 (R.B.G.).
Correspondence should be addressed to Catherine S. Woolley, Department of Neurobiology and Physiology, Northwestern University, 2205 Tech Drive, Evanston IL 60208. E-mail: cwoolley{at}northwestern.edu.
Copyright © 2003 Society for Neuroscience 0270-6474/03/234479-12$15.00/0
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