Estradiol Increases Spine Density and NMDA-Dependent Ca2+ Transients in Spines of CA1 Pyramidal Neurons From Hippocampal Slices

Lucas D. Pozzo-Miller,1 Takafumi Inoue,2 and Diane Dieuliis Murphy1

 1Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892; and  2Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Tokyo 108, Japan


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Pozzo-Miller, Lucas D., Takafumi Inoue, and Diane Dieuliis Murphy. Estradiol increases spine density and NMDA-dependent Ca2+ transients in spines of CA1 pyramidal neurons from hippocampal slices. To investigate the physiological consequences of the increase in spine density induced by estradiol in pyramidal neurons of the hippocampus, we performed simultaneous whole cell recordings and Ca2+ imaging in CA1 neuron spines and dendrites in hippocampal slices. Four- to eight-days in vitro slice cultures were exposed to 17beta -estradiol (EST) for an additional 4- to 8-day period, and spine density was assessed by confocal microscopy of DiI-labeled CA1 pyramidal neurons. Spine density was doubled in both apical and basal dendrites of the CA1 region in EST-treated slices; consistently, a reduction in cell input resistance was observed in EST-treated CA1 neurons. Double immunofluorescence staining of presynaptic (synaptophysin) and postsynaptic (alpha -subunit of CaMKII) proteins showed an increase in synaptic density after EST treatment. The slopes of the input/output curves of both alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) postsynaptic currents were steeper in EST-treated CA1 neurons, consistent with the observed increase in synapse density. To characterize NMDA-dependent synaptic currents and dendritic Ca2+ transients during Schaffer collaterals stimulation, neurons were maintained at +40 mV in the presence of nimodipine, picrotoxin, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). No differences in resting spine or dendritic Ca2+ levels were observed between control and EST-treated CA1 neurons. Intracellular Ca2+ transients during afferent stimulation exhibited a faster slope and reached higher levels in spines than in adjacent dendrites. Peak Ca2+ levels were larger in both spines and dendrites of EST-treated CA1 neurons. Ca2+ gradients between spine heads and dendrites during afferent stimulation were also larger in EST-treated neurons. Both spine and dendritic Ca2+ transients during afferent stimulation were reversibly blocked by D,L-2-amino-5-phosphonovaleric acid (D,L-APV). The increase in spine density and the enhanced NMDA-dependent Ca2+ signals in spines and dendrites induced by EST may underlie a threshold reduction for induction of NMDA-dependent synaptic plasticity in the hippocampus.


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The function of dendritic spines has been studied using various structural, biophysical, and computational approaches (Shepherd 1996). Dendritic spines may act as independent compartments with a diffusional barrier that allows for biochemical gradients to be generated between spines and dendrites (Koch and Zador 1993; Svoboda et al. 1996; Wickens 1988). In this model, voltage-gated ionic influx and/or signaling molecules generated by synaptic activation of membrane receptors coupled to second-messenger cascades would be restricted to activated spines and therefore used for integrative computation of synaptic inputs (Llinás 1995). Synaptically mediated, compartmentalized intracellular Ca2+ transients in CA1 hippocampal dendritic spines (Alford et al. 1993; Müller and Connor 1991; Petrozzino et al. 1995; Yuste and Denk 1995) have been postulated to participate in the induction of N-methyl-D-aspartate (NMDA)-dependent long-term synaptic modifications, such as long-term potentiation (LTP) (Bliss and Collingridge 1993).

Dendritic spine density in hippocampal CA1 pyramidal neurons increases during the estrous cycle coincident with the estrogen peak (Woolley et al. 1990), and 17beta -estradiol (EST) augments hippocampal spine density in vivo (Woolley and McEwen 1994) and in vitro (Murphy and Segal 1996). Here, we show that CA1 pyramidal neurons from postnatal hippocampal slices retain the ability to increase their dendritic spine density in response to estradiol treatment in vitro, as observed in embryonic hippocampal neurons in dissociated cultures (Murphy and Segal 1996). Because LTP is enhanced as estradiol levels are higher during the estrous cycle (Warren et al. 1995), and EST facilitates the induction of LTP in ovariectomized (OVX) awake rats (Córdoba Montoya and Carrer 1997), increases in dendritic spine density may underlie enhanced hippocampal synaptic plasticity. To investigate the physiological consequences of the EST-induced increase in spine density, we performed simultaneous whole cell recordings and digital imaging of NMDA-dependent intracellular Ca2+ transients in spines and dendrites of CA1 pyramidal neurons in hippocampal slices during high-frequency Schaffer collaterals stimulation.


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Slice cultures

Hippocampal slices (500 µm thick) from postnatal day 7 rats were prepared with a custom-designed wire slicer (Katz 1987) and maintained in vitro in a 36°C, 5% CO2 humidified incubator, as previously described (Petrozzino et al. 1995; Pozzo-Miller et al. 1993, 1995, 1996). All tissue culture reagents were obtained from GIBCO BRL (Gaithersburg, MD). Four- to eight-days in vitro (div) organotypic slice cultures were exposed to EST (0.2 µg/ml; Sigma, St. Louis, MO) for an additional 4- to 8-day period. Control and EST-containing media were replaced every 48 h.

Dendritic spine labeling, confocal imaging, and quantification

Eight- to 16-div control and EST-treated slices were fixed overnight in 4% paraformaldehyde in PBS. After rinsing in PBS, small droplets (<10 µm) of a saturated solution of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI; Molecular Probes, Eugene, OR) in fish oil were microinjected with a patch pipette into the CA1 region using a picospritzer (PLI-100; Medical Systems, Greenvale, NY). After 12-24 h at 4°C in the dark, z-sections from labeled dendritic segments were collected in a laser-scanning confocal microscope (Zeiss, Thornwood, NY; ×100, 1.4 NA oil immersion objective). Dendritic projections shorter than 1-3 µm were quantified off-line as dendritic spines (Murphy and Segal 1996). Due to the high density of labeled dendrites, the single sections from the z-section projections were used to count the number of spines for measured lengths of dendrite (10-µm segments), ensuring that each spine was counted only once by following its course in the z-section reconstructions. Spine counts were performed in a total of 164 dendritic segments (72 apical and 92 basal) from control slices, and in 112 segments from EST-treated slices (98 apical dendrites and 14 basal dendrites).

Double immunofluorescent staining and confocal imaging

Eight to 16-div control and EST-treated slices were fixed overnight in 4% paraformaldehyde in PBS. After incubation in blocking and permeabilization buffer containing 10% serum in PBS with 0.1% saponin, slices were exposed to primary antibodies against synaptophysin (Zymed, South San Francisco, CA) and the alpha -subunit of Ca2+/calmodulin-dependent protein kinase 2 (CamKII, Boeringer-Mannheim, Indianapolis, IN) for 1 h at room temperature. After incubation in FITC- and rhodamine-conjugated secondary antibodies for 1 h at room temperature, slices were finally mounted and sealed using Vectashield (Vector Laboratories, Burlingame, CA). Imaging was performed in a confocal laser scanning microscope (Zeiss, ×100, NA 1.4 oil immersion objective).

Simultaneous whole cell recording and Ca2+ imaging

Eight- to 16-div control and EST-treated slice cultures were continuously perfused (2 ml/min) in an immersion-type slice chamber with artificial cerebrospinal fluid (ACSF) at room temperature containing (in mM) 124 NaCl, 2 KCl, 1.3 MgSO4, 1.24 KH2PO4, 17.6 NaHCO3, 2.5 CaCl2, and 10 D-glucose; 310-320 mOsm; equilibrated with 95% O2-5% CO2. Nimodipine (20 µM in 0.05% DMSO; RBI, Natick, MA) and picrotoxin (50 µM; Sigma) were routinely included in the ACSF, while D,L-2-amino-5-phosphonovaleric acid (D,L-APV; 100 µM; Sigma) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 µM in 0.1% DMSO; RBI) were added when indicated.

CA1 pyramidal neurons in the ~200-µm-thick slice cultures were visualized by differential interference contrast (DIC) optics with infrared illumination (centered at 770 nm, 40 nm bandwidth; Omega Optical, Brattleboro, VT) in a fixed-stage upright microscope (Zeiss Axioskop FS) using a water-immersion ×63 objective (Zeiss Achroplan, 0.9 NA), a charge-coupled device (CCD) camera (C2400; Hamamatsu, Hamamatsu City, Japan), and video monitor (Dodt and Zieglgänsberger 1990). Somatic whole cell recordings were performed as described (Petrozzino et al. 1995; Pozzo-Miller et al. 1993, 1995, 1996) using unpolished patch pipettes pulled from thin-wall borosilicate glass capillary (WPI, Sarasota, FL), and containing (in mM) 120 Cs+-gluconate, 10 NaHEPES, 17.5 CsCl, 10 NaCl, 2 Mg-ATP, 0.2 Na-GTP, 5 QX-314, 0.5 hexapotassium salt of the Ca2+-sensitive dye bis-fura-2 (Kd = 525 nM; Molecular Probes); 280-290 mOsm; pH 7.2 (final resistance 4-8 MOmega ). Neurons were voltage clamped in the continuous, single-electrode mode of an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA). Excitatory postsynaptic currents (EPSCs) were elicited with a bipolar stainless steel electrode (WPI), or a patch pipette filled with ACSF, positioned over the Schaffer collaterals in CA1 stratum radiatum, 100-200 µm away from the dye-filled neuron. Square current pulses of 100 µs were delivered by a computer-triggered stimulus isolation/constant-current unit (Isolator 11; Axon Instruments). High-frequency afferent stimulation consisted of 1-s trains at 50 Hz. Voltage (Vh) and current (I) signals were digitized at 10 kHz (ITC-16; Instrutech, Great Neck, NY) for display and analysis.

The fluorescent Ca2+-sensitive dye bis-fura-2 was excited with 357- and 380-nm monochromatic light (12 nm bandwidth) using a galvanometric scanner-mounted grating (<1.5 ms wavelength change; Polychrome I; TILL Photonics, Planegg, Germany), under computer control (PCI-1200; National Instruments, Austin, TX). Fluorescent dye emission (dichroic beamsplitter mirror >= 420 nm, emission longpass filter >= 450 nm) was detected with a 12-bit, liquid-cooled, CCD camera (PXL-37; Photometrics, Tucson, AZ) operating at -25°C in frame-transfer mode (12-28 ms frame read-out interval for 100 × 200 pixel subarrays with 3 × 1 pixel binning). Single frames obtained at the calibrated isosbestic point (357 nm; Ca2+-insensitive wavelength) were acquired before and after a fast sequence of 10-20 frames at 380 nm excitation (Ca2+-sensitive wavelength) to generate ratio images. Synchronized afferent stimulation (1 s at 50 Hz) was delivered after 2 prestimulus frames of the fast sequence at 380 excitation, and continued thereafter. Regions of interests (ROIs; <4 × 6 pixels) were defined within spine heads and adjacent dendrites. Only primary or secondary dendrites, 50-100 µm from the soma, were selected to ensure that recordings were performed from spines and adjacent dendritic segments that reached steady-state dye concentration and appropriate signal-to-noise ratio in reasonably short times (20-30 min) after whole cell break-in. This also allowed better voltage clamp of synaptic currents originated from electrotonically proximal regions. ROIs over the slice but outside the dye-filled neuron were used for background estimation. Free cytosolic Ca2+ concentration was calculated from background-subtracted average fluorescence intensities within ROIs by the isosbestic ratio method (Grynkiewicz et al. 1985; Neher and Augustine 1992). Synchronized electrical and optical recording, afferent stimulus trigger, and control of the cooled CCD camera trigger and the galvanometric monochromator was performed using a single Power Macintosh computer (9500/120 MHz, Apple Computer, Cupertino, CA) running custom-developed software.

All data are expressed as means ± SE. Statistical differences were assessed by the Student's t-test. P < 0.05 was considered as significant.


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CA1 pyramidal neurons from postnatal hippocampal slices maintained in organotypic culture develop dendritic spines resembling those observed in vivo (Fig. 1, A and B). In DiI-labeled CA1 neurons from 8- to 16-div control slices (Fig. 1A), spine density was 11 ± 0.5 (SE) spines per 10 µm of dendritic length (n = 72 apical and 92 basal dendrites from 3 slices). In slices exposed for 4-8 days to 0.2 µg/ml EST (starting at 4-8 div; Fig. 1B), spine density was significantly larger, reaching 21 ± 0.4 spines per 10 µm of dendritic length (n = 98 apical and 14 basal dendrites from 5 slices; t-test, P < 0.0001; Fig. 2A). Because the same effect of EST on spine density was observed in apical and basal dendritic segments, all data were pooled for statistical analysis.



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Fig. 1. Estradiol increases dendritic spine and synaptic density in CA1 neurons in hippocampal slice cultures. A: reconstructed projections from z-sections (0.5 µm) collected in a laser-scanning confocal microscope from 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI)-labeled dendrites in a control slice. Arrowheads indicate examples of dendritic spines (length 1-3 µm) used for quantification. B: DiI-labeled dendrites in a 17beta -estradiol (EST)-treated slice. Scale bar for A and B, 5µm. C: confocal image of the CA1 region of a control slice immunolabeled with an antibody against synaptophysin. Note the localization of synaptophysin-positive profiles in the apical dendritic region of CA1 (sr, stratum radiatum). D: confocal image of the CA1 region of a EST-treated slice immunolabeled with an antibody against synaptophysin. Note the increased immunoreactivity compared with the control slice (C). E: confocal image of the CA1 region of a control slice double-labeled with synaptophysin (green) and the alpha -subunit of CaMKII (red). Yellow represents overlapping of immunopositive profiles for both pre- and postsynaptic markers. F: confocal image of the CA1 region of a EST-treated slice double-labeled with synaptophysin (green) and the alpha -subunit of CaMKII (red). Yellow represents overlapping of immunopositive profiles for both pre- and postsynaptic markers. Note that the ratio of pre-to postsynaptic immunostaining was not different after EST treatment. Scale bar for C-F, 10 µm. sp, stratum pyramidale; sr, stratum radiatum.



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Fig. 2. Estradiol increases spine density and peak N-methyl-D-aspartate (NMDA)-dependent Ca2+ transients in spines and dendrites of CA1 neurons of hippocampal slice cultures. A: estradiol increases dendritic spine density in CA1 neurons. Dendritic spine density was calculated from z-sections collected in a laser-scanning confocal microscope from DiI-labeled dendrites. Numbers in parentheses indicate the number of slices. Asterisk indicates significantly larger than controls (P < 0.05; Student's t-test). B: representative alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and NMDA-mediated postsynaptic currents in control and EST-treated slice cultures. AMPA-mediated excitatory postsynaptic currents (EPSCs) were evoked at resting potentials (-80 mV) in the presence of picrotoxin (50 µM). NMDA-dependent PSCs were evoked at depolarized potentials (+40 mV) after addition of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 µM). The L-type Ca2+ channel blocker nimodipine (20 µM) was also included in the bath to reduce voltage-dependent Ca2+ influx evoked by the step depolarization to +40 mV. C: estradiol enhances maximum NMDA-mediated spine and dendritic Ca2+ transients elicited during high-frequency Schaffer collateral stimulation. NMDA-mediated Ca2+ transients were evoked under voltage clamp at depolarized potentials (+40 mV) in the presence of CNQX (20 µM), picrotoxin (50 µM), and nimodipine (20 µM), and recorded with patch pipettes containing Cs+-based intracellular solution, QX-314 (5 mM), and bis-fura-2 (0.5 mM). Peak Ca2+ values were measured within spine heads and adjacent dendrites [<4 × 6 pixels regions of interests (ROIs)] at 150-200 ms from stimulus onset (12- to 28-ms frame interval). Numbers in parentheses indicate the number of spines and dendrites. Asterisks indicate significantly larger than controls (P < 0.05; Student's t-test).

Consistent with the increased spine density, and therefore membrane surface area, cell input resistance (Rn) was lower in EST-treated CA1 neurons. The contribution of spontaneous synaptic activity to Rn was eliminated by addition of picrotoxin, CNQX, and D,L-APV to block GABAA, alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and NMDA receptors, respectively. GABAB receptor activity was blocked by intracellular application of QX-314 and Cs+. Under voltage clamp at -80 mV in the whole cell configuration with patch pipettes containing Cs+ and QX-314 to block K+ and Na+ channels, respectively, control CA1 pyramidal neurons had input resistances of 368 ± 45 MOmega (n = 9 cells), whereas EST-treated neurons had input resistances of 230 ± 15 MOmega (n = 13 cells; t-test, P = 0.0035).

Do newly formed spines induced by EST in the postnatal hippocampal slices establish synapses with functional presynaptic partners? The density of presynaptic terminals assayed by immunostaining of synaptophysin was indeed higher in EST-treated slices (Fig. 1, C and D). Furthermore, synapse density was also increased by EST, as evidenced by double immunofluorecent staining of presynaptic (synaptophysin) and postsynaptic (alpha -subunit of CaMKII) proteins (Fig. 1, E and F). The ratio of pre- to postsynaptic immunostaining (yellow represents the overlapping staining in Fig. 1, E and F) was not different after EST treatment, consistent with the hypothesis that new spines have presynaptic partners. As suggested by these results, and other structural studies (Murphy and Segal 1996; Woolley and McEwen 1992), newly formed dendritic spines induced by estradiol may represent functional synaptic contacts. To test this hypothesis, input/output (I/O) curves were constructed for the AMPA and NMDA components of the excitatory postsynaptic currents (EPSC) (Hestrin et al. 1990). Figure 2B shows AMPA-mediated EPSCs evoked at resting potentials (-80 mV) in the presence of picrotoxin (50 µM), and NMDA-dependent EPSCs evoked at depolarized potentials (+40 mV) after addition of CNQX (20 µM); the L-type Ca2+ channel blocker nimodipine (20 µM) was also included in the bath to reduce voltage-dependent Ca2+ influx evoked by the step depolarization to +40 mV. The I/O curves of the AMPA EPSCs had a tendency to exhibit steeper slopes in EST-treated slices than in control slices (2.27 n = 7 vs. 0.68 n = 3). Similarly, the I/O curve of the NMDA EPSCs exhibited a trend of steeper slopes in EST-treated slices (2.67 n = 3 cells vs. 1.57 n = 4). Taken together, these structural and physiological observations are consistent with an increase of active synapses on spines containing functional AMPA and NMDA receptors.

To investigate the effect of increased spine density on NMDA-dependent spine and dendritic Ca2+ transients during synaptic activity, simultaneous voltage clamp and Ca2+ imaging was performed in CA1 neurons using the Ca2+ indicator bis-fura-2. Only clearly resolvable, laterally projecting spines in primary or secondary dendrites, 50-100 µm from the soma, were included in the following analysis. To minimize voltage-gated Ca2+ influx into spines and dendrites during high-frequency stimulation of Schaffer collaterals, CA1 neurons were dialyzed with Cs+ and QX-314 and then steadily depolarized (soma voltage +40 mV) in the presence of 20 µM nimodipine. At these potentials, EPSCs are outward flowing, and negative voltage changes (toward the NMDA receptor reversal potential at 0 mV) would occur at poorly clamped synaptic sites in spines and dendrites during high-frequency afferent stimulation. Holding the cells at depolarized potentials would reduce the likelihood of activation of voltage-gated Ca2+ channels and, together with the blockade of AMPA (20 µM CNQX) and GABAA (50 µM picrotoxin) receptors, allows for isolation of NMDA-dependent Ca2+ transients (Malinow et al. 1994; Perkel et al. 1993; Pozzo-Miller et al. 1996). After an initial widespread elevation when Vh was stepped to +40 mV, Ca2+ concentration returned to levels that, although higher than those at resting potential, were within the range allowing transients to be measured without saturation of bis-fura-2. No differences in resting Ca2+ levels of CA1 spines were observed between control (282 ± 13 nM, n = 16, 7 cells) and EST-treated neurons (296 ± 25 nM, n = 19, 8 cells; t-test, P = 0.6543). Similar results were obtained for CA1 dendrites (284 ± 11, n = 22, 7 control cells vs. 275 ± 12 nM, n = 27, 8 EST cells; t-test, P = 0.595).

Under the above conditions, Schaffer collateral stimulation (1 s, 50 Hz) evoked outward EPSCs of similar amplitude (1.7 ± 0.5 nA control cells vs. 1.3 ± 0.2 nA EST cells; t-test, P = 0.4929). The associated spine and dendritic Ca2+ transients were detected within 20-25 ms from the stimulus onset and exhibited a faster slope in spine heads than in adjacent dendritic segments (arrowheads in Fig. 3). Ca2+ transients reached maximum levels at 100-200 ms from the stimulus onset in both compartments (Fig. 3). In control CA1 cells (Fig. 3A), maximum Ca2+ elevations at 100-200 ms from the stimulus onset were larger in spine heads, reaching 933 ± 72 nM in spines and 585 ± 51 nM in the adjacent dendrites (n = 16 spine/dendrite pairs from 7 cells; t-test, P = 0.0004). Similar Ca2+ spine/dendrite gradients were observed in EST-treated CA1 cells (Fig. 3B), with peak Ca2+ elevations reaching 1,436 ± 147 nM in spines and 972 ± 109 nM in the adjacent dendrites (n = 19 spine/dendrite pairs from 8 cells; t-test, P = 0.0156). Synaptic Ca2+ transients observed under these conditions were reversibly blocked by 100 µM D,L-APV, further confirming that they were mediated by activation of synaptic NMDA receptors (Figs. 3 and 4). D,L-APV also reversibly reduced the faster component of the EPSCs, leaving a postsynaptic current previously characterized as a nonselective cationic conductance mediated by G protein-coupled metabotropic glutamate receptors (Pozzo-Miller et al. 1995).



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Fig. 3. NMDA-dependent Ca2+ transients in spines and dendrites of CA1 pyramidal neurons in slice cultures during high-frequency stimulation of Schaffer collaterals. A: control CA1 pyramidal neuron. B: estradiol-treated CA1 pyramidal neuron. Left panels: fluorescence images at 357-nm excitation. Middle (prestimulus), right (150 ms from stimulus onset), and far right [150 ms from stimulus onset in D,L-2-amino-5-phosphonovaleric acid (D,L-APV)] panels are pseudocolor Ca2+ maps calculated from pixel-by-pixel ratio of background-subtracted fluorescence images at 357- and 380-nm excitation. Intracellular Ca2+ concentration traces were calculated from the dotted ROIs shown in the 357-nm fluorescence images after background subtraction from a ROI over the slice but outside the dye-filled cell (not shown). White arrowheads indicate the 2 spines from which Ca2+ concentration traces are shown. Membrane voltage and current traces (at 10 kHz) were acquired synchronously with Ca2+ concentration traces (at 12- to 28-ms frame interval) and are shown with the same time base. Black arrowheads show the onset of Schaffer collateral stimulation (1 s at 50 Hz). ACSF contained CNQX (20 µM), picrotoxin (50 µM), and nimodipine (20 µM). Patch pipettes contained Cs+-based intracellular solution, QX-314 (5 mM), and bis-fura-2 (0.5 mM).



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Fig. 4. Ca2+ transients in CA1 spines and dendrites during Schaffer collaterals stimulation are sensitive to D,L-APV. A: control slices. B: estradiol-treated slices. Recording conditions as in Fig. 3. Numbers in parentheses indicate the number of spines and dendrites. Asterisks indicate significantly larger than responses in CNQX, picrotoxin, and nimodipine (P < 0.05; Student's t-test).

Maximum Ca2+ elevations in dendrites during afferent stimulation were larger in EST-treated CA1 cells (554 ± 39 nM, n = 22 dendrites from 7 control cells vs. 893 ± 87 nM, n = 27 dendrites from 8 EST cells; t-test, P = 0.0019; Fig. 2C). This observation is consistent with the prediction that a larger number of spines expressing functional Ca2+-permeable NMDA receptors permit larger Ca2+ influx into dendritic compartments. Surprisingly, peak Ca2+ elevations within spine heads were also larger in EST-treated CA1 cells (933 ± 72 nM, n = 16 spines from 7 control cells vs. 1,436 ± 147 nM, n = 19 spines from 8 EST cells; t-test, P = 0.0066; Fig. 2C). Due to this significant difference in peak spine Ca2+ elevations, spine/dendrite Ca2+ gradients had a tendency to be larger in EST-treated CA1 neurons (349 ± 40 nM, n = 16 spine/dendrite pairs from 7 control cells vs. 487 ± 83 nM, n = 19 spine/dendrite pairs from 8 EST cells; t-test, P = 0.1664).


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The current report presents direct structural and physiological evidence that newly formed dendritic spines of CA1 pyramidal neurons induced by estradiol in postnatal hippocampal slices represent functional postsynaptic compartments expressing AMPA and NMDA receptors. In addition to an increase in dendritic spine and synapse density, the sensitivity to AMPA and NMDA receptor-mediated synaptic input, and the NMDA-dependent Ca2+ transients in spines and dendrites are enhanced in EST-treated CA1 pyramidal neurons. Our results correlate well with recent observations that LTP is enhanced during proestrous, when EST levels are highest (Warren et al. 1995), and that EST treatment facilitates the induction of LTP in awake OVX rats (Córdoba Montoya and Carrer 1997). These observations strongly suggest that increases in dendritic spine density, together with enhanced NMDA-dependent spine and dendritic Ca2+ signaling, may underlie augmented hippocampal synaptic plasticity. Because the duration of the in vitro EST treatment in the present report exceeds the EST peak during the rodent estrous cycle by 3-7 days, our observations most likely reflect organizational rather than activational EST effects during the adult estrous cycle and, as such, may be relevant to synaptic development and/or plasticity.

Compartmentalized NMDA-mediated Ca2+ transients in CA1 spines have been reported in earlier current-clamp experiments (Müller and Connor 1991; Petrozzino et al. 1995). Since then, several experimental approaches have been used to minimize voltage-dependent Ca2+ influx and isolate dendritic Ca2+ signals mediated by influx through NMDA receptor channels during synaptic stimulation (Alford et al. 1993; Malinow et al. 1994; Perkel et al. 1993; Pozzo-Miller et al. 1996). In the present configuration the somatic membrane was steadily depolarized to +40 mV under whole cell voltage clamp. If membrane voltage control were to fail during afferent stimulation in the presence of nimodipine, CNQX, and picrotoxin, the membrane potential would hyperpolarize toward 0 mV, the reversal potential of the remaining glutamatergic currents. This hyperpolarization would reduce the likelihood of activation of voltage-activated Ca2+ channels. In addition, the dihydropyridine Ca2+ channel antagonist nimodipine was included in the ACSF cocktail to reduce the steady-state Ca2+ influx through noninactivating Ca2+ channels, thus maintaining the background Ca2+ levels low. Furthermore, Ca2+ imaging experiments were always performed on primary dendrites, 50-100 µm from the cell body, ensuring adequate voltage clamp of synaptic currents originated from electrotonically proximal regions. Ca2+ transients still occur in spines and adjacent dendrites under these stringent conditions and, together with the associated EPSCs, were reversibly inhibited by D,L-APV, confirming that they are mediated by the synaptic activation of NMDA receptors. These observations have been recently confirmed in layer V cortical pyramidal neurons using local photolysis of caged glutamate to mimic presynaptic release and allowing complete pharmacological blockade of voltage-dependent Ca2+ channels (Schiller et al. 1998).

The potential limitations of our experimental approach (unknown voltage clamp of the spine membrane potential; altered activity of the Na+/Ca2+ exchanger and prolonged Ca2+ influx through NMDA receptors with altered kinetics at depolarized potentials; contribution of Ca2+ release from intracellular stores) do not affect the primary conclusion of our imaging experiments: synaptic activation under conditions of minimal voltage-gated Ca2+ influx and maximal NMDA receptor activation is enhanced in CA1 pyramidal neuron spines and dendrites from EST-treated slices. This result is consistent with the observation of increased spine and synapse density and I/O slope of AMPA and NMDA currents. Consistently, increased NMDA binding sites (Weiland 1992), and protein levels of NMDAR1 (Gazzaley et al. 1996), associated with increased spine/synapse density and sensitivity of CA1 cells to NMDA-dependent synaptic input (Woolley et al. 1997) have been reported in the hippocampus of EST-treated OVX animals. However, potential effects of EST on voltage-gated Ca2+ channel activity cannot be excluded. The possibility of differences in Ca2+ buffering and/or sequestration could reflect another potential effect of estradiol as a growth factor, namely the modulation of dendritic organelles functioning as intracellular Ca2+ stores and/or sinks, such as the smooth endoplasmic reticulum (Pozzo-Miller et al. 1997).

The larger Ca2+ transients in dendritic shafts mediated by synaptic activation of NMDA receptors in EST-treated slices observed in our study are likely due to enhanced NMDA-mediated Ca2+ influx into dendritic compartments from a higher density of spines expressing functional Ca2+-permeable NMDA receptors. The higher Ca2+ levels reached within spines in EST-treated slices could represent attenuated diffusion from spine heads into dendritic shafts due to the higher Ca2+ concentration of the latter compartment brought up from a higher density of spines with Ca2+-permeable NMDA receptors. Alternatively, estradiol-induced spines may express a higher density of NMDA receptors in their postsynaptic densities, or these receptors may have larger Ca2+ permeability or single-channel properties (but see Wong and Moss 1994). Either enhanced Ca2+ release from intracellular stores (Pozzo-Miller et al. 1996), or impaired Ca2+ sequestration and/or buffering, may also contribute to enhanced spine Ca2+ elevations in EST-treated slices.

An increase in plasma membrane surface area by the addition of new dendritic spines would induce a decrease in cell input resistance (Rn). Woolley et al. (1997) reported that there were no significant differences in Rn between CA1 cells from OVX and EST-treated OVX rats, although a negative correlation between spine density and Rn was statistically significant. Since in that study (Woolley et al. 1997) the spine density increased by only 22-28% in EST-treated OVX rats, we conceive that the much larger increases in spine density observed in the EST-treated slice cultures (almost 100%) could account for the statistically significant differences in Rn in our study. The contribution of increased dendritic length to reduced Rn was not ruled out in our study, although this parameter was not affected by EST in OVX rats (Woolley and McEwen 1994). In addition, the measurements of Rn were performed under conditions in which background synaptic activity and its potential contribution to Rn were completely eliminated. CA1 neurons were voltage clamped at -80 mV in the presence of nimodipine, picrotoxin, CNQX, and D,L-APV to block high-voltage activated L-type Ca2+ channels, GABAA, AMPA, and NMDA-type glutamate receptors, respectively. Furthermore, patch pipettes were filled with Cs+-based intracellular solution containing QX-314 to block K+ and Na+ channels, as well as GABAB receptor activity. The significant decrease in Rn is therefore consistent with the observed increase in membrane surface area provided by the addition of new dendritic spines in CA1 neurons from EST-treated slices.

We have also provided evidence of higher density of synaptic contacts by double immunofluorescent staining of synaptophysin and alpha -CaMKII in hippocampal slice cultures after EST treatment, reminiscent of similar effects in primary cultures of embryonic hippocampal neurons, estimated by immunofluorescence staining of synaptophysin (Murphy and Segal 1996) and synaptotagmin (Murphy, unpublished observations). An increase of presynaptic release by EST could also account for the observed enhancement of I/O curves and Ca2+ signaling and is consistent with the lower threshold for LTP induction in EST-OVX and proestrous animals.

Estradiol has been implicated in cognitive function in both experimental animal models (Luine 1994) and postmenopausal women (Sherwin 1996). Because NMDA receptors and the hippocamal formation have been strongly linked to LTP as a cellular model of learning and memory (Bliss and Collingridge 1993), estradiol may exert its effects in cognitive functions via the induction of new dendritic spines with enhanced NMDA-mediated Ca2+ signaling mechanisms.


    ACKNOWLEDGMENTS

We acknowledge Drs. Thomas S. Reese and S. Brian Andrews for support and comments on the manuscript, the Light Imaging Facility of the National Institute of Neurological Disorders and Stroke, National Institutes of Health, for use of the confocal laser scanning microscope, and David Ide (National Institute of Mental Health, NINDS Research Services Branch) for building the brain slicer.

This work was supported by the NIH Intramural Research Program.

Present address of D. D. Murphy: Dept. of Pathology and Laboratory Medicine, University of Pennsylvania, 3rd Floor Maloney, 3600 Spruce St., Philadelphia, PA 19104.


    FOOTNOTES

Present address and address for reprint requests: L. D. Pozzo-Miller, Dept. of Neurobiology, University of Alabama at Birmingham, CIRC 429, 1719 Sixth Ave. South, Birmingham, AL 35294.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4 June 1998; accepted in final form November 1998.


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ABSTRACT
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