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The Journal of Neuroscience, November 1, 1996, 16(21):6830 – 6838 Differential Regulation of NMDAR1 mRNA and Protein by Estradiol
in the Rat Hippocampus
Adam H. Gazzaley,1 Nancy G. Weiland,3 Bruce S. McEwen,3 and John H. Morrison1,2
1Laboratories for Neurobiology of Aging, F ishberg Research Center for Neurobiology, New York, New York 10029-6574,2Department of Geriatrics and Adult Development, The Mount Sinai School of Medicine, New York, New York 10029, and3Laboratory of Neuroendocrinology, Rockefeller University, New York, New York 10021 Estradiol treatment increases the number of NMDA receptor sity levels in comparison with nonsteroid treated ovariecto- binding sites, and changes evoked synaptic currents in a man- mized rats within the somata and dendrites of CA1 pyramidal ner consistent with a steroid-induced functional enhancement cells and, to a lesser extent, within the granule cell somata of of NMDA receptors in rat hippocampus. In this study, we the dentate gyrus. In contrast, such alterations in immunofluo- investigate the cellular mechanisms of estradiol-induced NMDA rescence intensity occur without concomitant changes in receptor regulation at the protein and mRNA levels in ovariec- mRNA hybridization levels. Thus, these data suggest that es- tomized rats treated with ovarian steroids using immunocyto- chemical and in situ hybridization techniques. Confocal laser transcriptional regulation of the NMDAR1 subunit protein. The scanning microscopy was used to quantify alterations in immu- increase in immunofluorescence intensity may reflect an in- nofluorescence intensity levels of NMDAR1 subunit proteins crease in the concentration of the subunit protein, which could within neuronal somata and dendrites of discrete hippocampal account for estrogen-induced changes in pharmacological and fields, whereas in parallel, in situ hybridization was used to physiological properties of the NMDA receptor.
examine NMDAR1 mRNA levels in corresponding hippocampal Key words: excitatory amino acid receptors; NMDAR1; im- regions. The data indicate that estradiol treatment in ovariec- munocytochemistry; in situ hybridization; estrogen; CA1; con- tomized rats significantly increases immunofluorescence inten- focal microscopy Ovarian steroids affect brain regions and behaviors that are not Sato et al., 1989) and memory and learning (Morris et al., 1986; directly associated with reproductive functions (McEwen et al., Bliss and Collingridge, 1993).
1995). However, the mechanisms by which these effects are pro- Few studies have directly addressed the mechanisms underlying duced have not yet been determined. NMDA receptors estrogen regulation of NMDARs. Autoradiographic analysis has (NMDARs), a subtype of ionotropic glutamate receptors revealed that NMDAR agonist binding sites are increased in the (Moriyoshi et al., 1991), are implicated as mediators of effects of dendritic layer of CA1 in response to estradiol treatment in estradiol on morphological plasticity and related physiological and ovariectomized rats (Weiland, 1992). However, because an alter- cognitive processes in the brain. For example, in the CA1 field of ation in receptor stoichiometry or regulation by modulatory li- rat hippocampus, estradiol treatment after ovariectomy increases gands can alter ligand binding, these results do not determine dendritic spine density (Gould et al., 1990) and synapses (Woolley whether steroidal treatment induced an increase in NMDA re- and McEwen, 1992) on pyramidal cells via a mechanism depen- ceptor subunit protein levels in these neurons. Additionally, no dent on NMDAR activation (Woolley and McEwen, 1994). Ad- studies have investigated the effect of ovarian hormone treatment ditionally, intracellular recordings reveal that estradiol treatment on NMDAR subunit mRNA levels in hippocampal neurons.
increases the duration of EPSPs in a subpopulation of CA1 Therefore, the focus of this study is to elucidate the cellular neurons, an alteration suggestive of an increased NMDAR con- mechanisms by which hippocampal NMDARs are regulated by tribution (Wong and Moss, 1992). Less direct evidence of ovarian steroids. Through the use of quantitative confocal laser NMDAR involvement in estrogen-induced neural modifications scanning microscopy (CLSM), we have recently demonstrated includes estrogen's role in facilitating seizure induction (Terasawa that the immunocytochemically localized cytoplasmic pool of the and Timiras, 1968; Backstrom, 1976; Buterbaugh and Hudson, NMDAR subunit 1 (NMDAR1), an obligatory subunit of the 1991) and memory and learning enhancement (Philips and Sher- NMDAR complex (Monyer et al., 1992; Nakanishi, 1992), is win, 1992a,b; Luine, 1994; Singh et al., 1994) in both humans and modifiable in hippocampal neurons during aging (Gazzaley et al., experimental animals. This connection is based on the NMDAR's 1996a) and in response to deafferentation (Gazzaley et al., 1996b).
important role in seizure-triggering mechanisms (Gilbert, 1988; These data suggest that alterations in cytoplasmic receptor pro-tein levels is a component of the neuronal response strategy to Received June 17, 1996; revised July 25, 1996; accepted Aug. 16, 1996.
various conditions. To investigate the role of estradiol and pro- This work was supported by the Charles A. Dana Foundation and National gesterone in regulating NMDARs at the protein level, we per- Institutes of Health Grants AG-06647 (J.H.M.), NS-30105 (N.G.W.), and NS-07080(B.S.M.). We thank Dr. George Huntley for helpful comments on this manuscript.
formed a CLSM evaluation of NMDAR1 immunofluorescence Correspondence should be addressed to Dr. John H. Morrison, Fishberg Research intensity in the somata and dendrites of the dentate gyrus and Center for Neurobiology, The Mount Sinai School of Medicine, P.O. Box 1065, OneGustave L. Levy Place, New York, NY 10029-6574.
CA1 and CA3 hippocampal fields of ovariectomized rats and Copyright q 1996 Society for Neuroscience 0270-6474/96/166830-09$05.00/0 ovariectomized rats treated with estradiol and estradiol plus pro- Gazzaley et al. • Estrogen Regulation of NMDAR1 J. Neurosci., November 1, 1996, 16(21):6830 – 6838 6831
gesterone. A CLSM analysis was selected for this study, because it associated protein 2 (MAP2) (Huber and Matus, 1984) at a concentration is performed on structurally intact tissue and permitted the eval- of 4.6 and 2.5 mg/ml, respectively, in PBS for 48 hr. Sections were thenwashed three times in PBS, transferred to biotinylated anti-mouse IgG uation of discrete cell groups and intracellular compartments, (Vector Laboratories, Burlingame, CA) for 2 hr, washed again in PBS, which would not have been possible by biochemical methods such and transferred to FITC-conjugated avidin (Vector Laboratories) for 1 as a Western blot analysis of homogenized tissue extracts. Addi- hr. Sections were then mounted and coverslipped with Vectashield (Vec- tionally, to determine whether changes observed at the protein tor Laboratories) to reduce fluorescence quenching.
level reflect changes at the level of gene transcription, we exam- CLSM and quantitative immunocytochemical evaluation. Quantitative CLSM analysis was performed on three sections from each rat brain, for ined NMDAR1 mRNA levels by quantitative in situ hybridization each of the two antibodies, with a Zeiss LSM 410 inverted confocal analysis in the same hippocampal regions.
microscope (Thornwood, NY). The investigator was blinded throughoutthe evaluation as to which sections were from which experimental group.
MATERIALS AND METHODS
The quantitative analysis performed in this study was adapted from aprevious study that quantified relative differences in immunofluorescence Animal and tissue processing. Forty-three young adult Sprague Dawley intensity levels (Gazzaley et al., 1996b). The confocal parameters were rats (Charles River, Wilmington, MA), weighing ;250 gm, were main- established at the beginning of the study and remained constant through- tained in a temperature- and light-controlled environment with a light (14 out. An Argon/Krypton laser was used to excite FITC at 488 nm. A 90% hr)/dark (10 hr) cycle (lights on at 0500 hr). Animals were treated in neutral density filter was used to attenuate the light, and a confocal accordance with the principles and procedures of the National Institutes aperture pinhole setting of 17 was set digitally. The image was visualized of Health Guide for the Care and Use of Laboratory Animals, and all with a Zeiss Plan-Neofluar 633/1.25 NA oil immersion objective. For surgeries were performed under Metofane anesthesia. Sixteen rats were each antibody, a contrast/brightness setting was selected that yielded a used in the immunocytochemical analysis. All 16 rats were ovariecto- high-resolution image for both bright and dim sections without exceeding mized for 1 week, at which time 10 rats received SILASTIC capsules a maximal pixel intensity of 255. For the NMDAR1 analysis, a separate containing 180 mg of 17-b estradiol/sesame oil, and the remaining 6 were contrast/brightness setting was established for somata and dendrites sham operated. Two days later at 1000 hr, five of the estradiol-treated because of a considerable intensity difference between them that pre- animals were injected subcutaneously with progesterone (1 mg in 0.3 ml vented the use of a single setting. All of the settings were kept constant oil/rat), and all remaining animals were injected with oil. This procedure throughout the analysis to yield unbiased measurements for each set of resulted in the following groups of ovariectomized rats: six sham plus oil (OVX), five estradiol plus oil (OVX1E), and five estradiol plus proges- For the study of somatic immunofluorescence intensity, six fields were terone (OVX1E1P). Five hours after progesterone or oil injection (1500 randomly selected on each section within a centrally located region of the hr), the animals were deeply anesthetized with Metophane and transcar- suprapyramidal blade of the dentate gyrus granule cell layer and the dially perfused with cold 1% paraformaldehyde in 0.1 M PBS followed by middle portion of the CA1 and CA3 pyramidal cell layer. The dendritic cold 4% paraformaldehyde in PBS. The brains were removed, post-fixed immunofluorescence intensity study consisted of six randomly selected in 4% paraformaldehyde, and sectioned at 40 mm on a vibratome (OTS dendritic fields, on each section, from the middle molecular layer of the 3000, Electron Microscopy Sciences, Fort Washington, PA). The sections dentate gyrus and the stratum radiatum of CA1 adjacent to the somatic were stored in PBS with 0.1% sodium azide at 48C.
regions analyzed in the somatic immunofluorescence analysis. All den- Twenty-seven rats were used for the in situ hybridization study. All rats dritic fields scanned were the same size (3832 mm2) and all were selected were ovariectomized for 1 week and then adrenalectomized. Eighteen at a distance of ;70 mm from the principal cell layers. Each field was rats were then treated with SILASTIC capsules containing 180 mg of 17-b scanned only once, to reduce fluorescence quenching, and at the same estradiol/sesame oil, whereas the other nine were sham operated. Two predetermined z axis distance from the surface of the section. Scanning days later at 1000 hr, the animals were either injected subcutaneously was performed with a two line average for a total scan time of 4.52 sec and with progesterone (1 mg. in 0.3 ml oil/rat) or oil, resulting in the following an electronic zoom factor of 3.28, which increased the resolution to groups of ovariectomized rats: nine sham plus oil (OVX), nine estradiol 0.0081 mm/pixel. Each digitized image consisted of a 512 3 512 3 eight plus oil (OVX1E), and nine estradiol plus progesterone (OVX1E1P).
bit pixel array, in which every pixel was assigned a gray level intensity The animals were killed by decapitation 5 hr after progesterone or oil value ranging from 0 to 255. An image-analysis program (Zeiss) was used injection (1500 hr), and the brains were removed, frozen on dry ice, and to determine the average pixel intensity of each field. To remove the stored at 2708C.
negative contribution of unlabeled portions of the field to the average In situ hybridization. In situ hybridization was performed using previ- field intensity (i.e., nuclei and unlabeled regions between soma and ously published sequences of oligonucleotides (Oligos Etc., Wilsonville, dendrites), a photometric offset was used to establish a pixel intensity OR) complementary to rat cDNA encoding subunit residues between threshold below which a pixel would have no contribution to the average putative transmembrane domains I and II, encoding amino acids 566 – pixel intensity of the field. The threshold was set by viewing the image at 580, and recognizing all published splice variants (Monyer et al., 1992). A a display magnification of 23 and manually increasing the thresholding search of the GenBank database indicated that there is no significant value until a blue display, designating the thresholded area, completely homology among any of these sequences and known mammalian gene occupied the unstained nuclei and abutted somatic and dendritic profiles sequences. Oligonucleotides were 39 end-labeled with terminal trans- (Fig. 1). Thus, the average pixel intensity of the portion of the field above ferase (Boehringer Mannheim, Indianapolis, IN) using a 2:1 molar ratio threshold represents the immunofluorescence intensity within either the of [a-35S] dATP:cDNA (1200 –1400 Ci/mmol; New England Nuclear, dendritic segments or the somata of the principal cell layers.
Boston, MA). Unincorporated nucleotides were removed using NucTrap Data analysis. For the CLSM immunocytochemical analysis, an inten- push columns (Stratagene Cloning Systems, La Jolla, CA). Hybridization sity value was computed for each animal in each of the three regions by was performed as described previously, (Orchinik et al., 1994). Briefly, determining the mean of the 24 individual field values (6 field values sections were fixed in 4% paraformaldehyde, acetylated, hybridized with obtained from each of three sections). For both the immunocytochemical saturating concentrations of labeled oligonucleotides overnight at 428C, and in situ hybridization analyses, a mean value for each group was washed to a final stringency of 0.13 SSC (13 5 0.15 M NaCl and 0.015 obtained from the individual values determined for each animal, in CA1, M sodium citrate) at 558C for 1 hr, dehydrated, and exposed to Hyperfilm- CA3, and the dentate gyrus. Percent difference was determined by com- bmax (Amersham, Arlington Heights, IL) for 24 hr.
paring the steroid-treated ovariectomized rats with the nontreated ovari- Quantitative in situ hybridization analysis. The films were analyzed by ectomized rats [i.e., ((OVX1E) 2 (OVX))/(OVX) or ((OVX1E1P) 2 measuring the optical density of specific regions of the hippocampus (OVX))/(OVX)]. All the data were analyzed with a one-way ANOVA, at using an automated paint function that covered the region of interest a significance level of ,0.05, and a Sheffe's post hoc test.
(Imaging Research, St. Catherines, Ontario, Canada). Bilateral measure-ments were taken from four sections per animal, nine animals per group in the principal cell layer of CA1, CA3, and the suprapyramidal layer ofthe dentate gyrus. Background was measured from the corpus callosum NMDAR1: somatic immunofluorescence intensity
and subtracted from the total optical density.
NMDAR1 immunolabeling was evaluated in three groups of rats: Immunocytochemistry. Three nonadjacent sections from the rostral hippocampus of each rat were incubated with monoclonal antibodies to ovariectomized rats (OVX) and ovariectomized rats that had both the NMDAR1 subunit (54.1) (Siegel et al., 1994) and microtubule- been treated with either estradiol (OVX1E) or estradiol plus 6832 J. Neurosci., November 1, 1996, 16(21):6830 – 6838
Gazzaley et al. • Estrogen Regulation of NMDAR1 significantly lower magnitude than that recorded in the CA1 field,as determined by a statistical comparison of the CA1/DG ratioamong the three groups, which revealed a significant increase inthe ratio in the steroid-treatment groups as compared with theOVX group. As in the CA1 subfield, there was no significantdifference between the OVX1E and the OVX1E1P groups.
Quantitative analysis of the somata of the CA3 pyramidal cells revealed no difference in immunofluorescence intensity levelsamong the three groups (Figs. 2g– i, 4a).
NMDAR1: dendritic immunofluorescence intensity
To investigate the cytoplasmic pool of the NMDAR1 subunitprotein within the dendrites of the two regions where a somaticintensity increase was observed, we analyzed fields of dendriticsegments in the CA1 stratum radiatum and the dentate gyrusmolecular layer. Quantitative analysis revealed a statistically sig-nificant increase in intensity within the dendritic segments of theCA1 field when comparing both the OVX1E and theOVX1E1P groups with the OVX group (35 and 32%, respec-tively) (Figs. 3a–c, 4b). There was no significant intensity differ-ence between the two steroid-treated groups in the CA1 den-drites. Analysis of the dentate gyrus dendritic fields revealed nosignificant difference among the three groups, but there was atrend toward an increase in both steroid-treated groups compared CLSM images of NMDAR1 immunolabeled CA1 pyramidal with the OVX group (Fig. 4b).
cell somata (A, B) and dendrites (B, C) before (A, C) and after (B, D)intensity thresholding, as indicated by the blue color overlay. After the MAP2: somatic immunofluorescence intensity
thresholding procedure, all unlabeled portions of the field (in blue) have To access whether the NMDAR1 intensity change may have been no contribution to the average intensity of the field.
the result of a more general increase in protein production, anidentical quantitative analysis was performed after immunocyto- progesterone (OVX1E1P). This steroid treatment paradigm has chemical staining with a monoclonal antibody to MAP2. MAP2 been used previously by Weiland and Orchinik (1995) and results was selected because it is localized specifically within the soma in serum estrogen levels of ;20 pg/ml and progesterone levels of and dendrites of all principal cells in the hippocampus, and there 60 ng/ml, which mimic preovulatory hormone levels. Nonovariec- are no reports of estradiol-induced changes in MAP2 cytoplasmic tomized rats were not used in this study, because estrogen levels concentration. Quantitative and qualitative analysis revealed no fluctuate throughout the estrous cycle. In animals from all three significant differences in immunofluorescence intensity or distri- groups, NMDAR1 immunofluorescence was present within the bution among the three groups in the somata of any hippocampal cell bodies and dendrites of the hippocampal principal neurons, a field analyzed (Figs. 5a– i, 6).
general pattern consistent with earlier descriptions of NMDAR1immunolabeling in the male rat hippocampus (Petralia et al., NMDAR1 mRNA labeling
1994). Confocal images of pyramidal cells within the CA1 and To determine whether a change in immunofluorescence intensity CA3 subfields and granule cells of the dentate gyrus revealed a levels of the NMDAR1 protein corresponded to an alteration in patchy intracellular distribution of immunofluorescence through- gene transcription, in situ hybridization analysis of NMDAR1 out the somatic and dendritic cytoplasm, whereas nuclei con- mRNA levels was performed. Hybridization signal was localized tained no labeling (see Figs. 2, 3). There were no observable within the somata of the dentate gyrus granule cells and the qualitative differences in overall distribution and intracellular pyramidal cells of the CA fields (Fig. 7), and no overt differences pattern of immunolabeling between OVX rats and steroid-treated were observed in the overall distribution of hippocampal mRNA labeling in comparison with that described previously in the rat Immunofluorescence intensity levels of the cytoplasmic pool brain (Moriyoshi et al., 1991). Densitometric analysis of the films of receptors within the somata of the CA1 and CA3 pyramidal determined that there was no significant difference in silver grain cell layers and the dentate gyrus granule cell layer were ob- intensity in the principal cell layers of CA1, CA3, and the dentate tained by CLSM quantitative analysis. All statistical compari- gyrus across the three groups (Fig. 8).
sons were made among the three groups, within a given hip-pocampal subfield. In the CA1 subfield of either the OVX1E or OVX1E1P group, quantitative data revealed a significant In the present study, quantitative confocal microscopic evaluation intensity increase within the pyramidal cell somata of 52.2% in of NMDAR1 immunofluorescence intensity revealed that both comparison with the OVX group (Fig. 2a–c; see also Fig. 4a).
estradiol and estradiol plus progesterone treatment in ovariecto- No difference in intensity levels was evident between the two mized rats induced a significant intensity increase within the somata and dendrites of CA1 pyramidal cells in comparison with In the dentate gyrus, a smaller but statistically significant inten- nonsteroid treated ovariectomized rats. A smaller, although sta- sity increase was observed within the somata of the granule cells tistically significant, increase was also observed within the somata in OVX1E (31.3%) and OVX1E1P (33.5%) rats relative to the of the dentate gyrus granule cells of steroid-treated animals in OVX rats (Figs. 2d–f; Fig. 4a). This intensity increase was of a comparison with nontreated ovariectomized animals but not Gazzaley et al. • Estrogen Regulation of NMDAR1 J. Neurosci., November 1, 1996, 16(21):6830 – 6838 6833
Examples of CLSM images of NMDAR1 immunolabeled somata in CA1 (A–C), the dentate gyrus (D–F ), and CA3 (G–I ) of OVX (A, D, G), OVX1E (B, E, H ), and OVX1E1P (C, F, I ) rats. Note the presence of punctate staining within the cytoplasm surrounding the unlabelednuclei (see Discussion). When comparing the CA1 fields, an increase in the somatic intensity of staining is evident in the OVX1E and OVX1E1Prats (B, C) as compared with the OVX rats (A). This increase in the steroid-treated ovariectomized rats is also apparent in the dentate gyrus (E,F compared with D), although to a lesser extent. There is no obvious difference in intensity levels among the three groups in the CA3 field (G–I ).
DG, Dentate gyrus; OVX, ovariectomized rats; OVX1E, estradiol-treated ovariectomized rats; OVX1E1P, estradiol plus progesterone-treatedovariectomized rats. Scale bars, 10 mm.
within their dendrites. Because there was a trend toward an Interpretation of NMDAR1 immunofluorescence
intensity increase in the granule cell dendrites, the absence of a significant change may have been the result of an inability to The use of confocal microscopy yields high-resolution, cross- detect small intensity increases because of a degree of variability sectional images of neurons, which when coupled with gray-level inherent in this technique. Additionally, there were no detectable intensity quantification and a photometic offset, enabled us to differences in intensity levels within CA3 pyramidal cell somata of obtain intensity measurements within major cellular compart- animals from all three groups. In situ hybridization analysis re- ments. Several factors contribute to the validity of the immuno- vealed no accompanying detectable alterations in NMDAR1 fluorescence intensity results as representing significant alter- mRNA levels in any hippocampal subfield in steroid-treated rats ations between nontreated ovariectomized rats and steroid- as compared with OVX rats.
treated ovariectomized rats. First, methodological variability was 6834 J. Neurosci., November 1, 1996, 16(21):6830 – 6838
Gazzaley et al. • Estrogen Regulation of NMDAR1 Bar graphs depicting NMDAR1 immunofluorescence intensity measurements in the somata (A) and dendrites (B) of the CA1, DG, andCA3 fields of the hippocampus. For somatic intensity measurements (A),there is a significant increase in both CA1 and the dentate gyrus whencomparing OVX1E and OVX1E1P rats with OVX rats. Based on theresults of the somatic intensity measurements (A), the CA1 and the DGdendritic fields were quantified (B). In B, note that there are significantincreases only in the dendritic intensity measurements of steroid-treatedrats as compared with the OVX rats in the CA1 subfield, although thereis a trend toward increase in the dentate gyrus. Values represent mean 6SEM for six OVX rats and five OVX1E and OVX1E1P rats (24measurements per rat). *p , 0.05, **p , 0.0001 compared with OVXgroup; ANOVA and Sheffe's test.
Examples of CLSM images of NMDAR1 immunolabeled den- our results. Similar to our qualitative CLSM observations, elec- drites in the CA1 subfield of an OVX rat ( A), an OVX1E rat (B), and an tron microscopic descriptions of rat hippocampal neurons re- OVX1E1P rat (C ). Note the presence of punctate staining within thecytoplasm of the dendritic segments (see Discussion) and the increased vealed a patchy distribution of NMDAR1 immunocytochemical intensity of staining within the CA1 dendrites of the steroid-treated deposition throughout the somatodendritic cytoplasm (Petralia et ovariectomized rats (B, C ) rats compared with the nonsteroid-treated al., 1994). In the somata and dendrites, these patches were asso- ovariectomized rats (A). Scale bar, 10 mm.
ciated with bundles of microtubules and the surface of mitochon-dria as well as with rough endoplasmic reticulum, golgi apparatus, reduced by rigidly controlling tissue processing, immunofluores- and the nuclear envelope in somata (Petralia et al., 1994). The cent staining, confocal parameters, and analysis design. As a association of concentrations of NMDAR1 subunits with these result, variability was minimized and intensity changes that were subcellular structures suggests that the cytoplasmic patches rep- both consistent and of a substantial magnitude yielded statistically resent the synthesis, processing, and transport pools of the pro- significant results. Additionally, the positive findings were region- tein. Previous CLSM investigations have demonstrated that im- ally specific and occurred with greatest magnitude in CA1, the munoreactive intensity can reflect protein concentration (Good et hippocampal field, in which estrogen-induced morphological al., 1992; Dodge et al., 1993), suggesting that our data may also changes have been observed (Gould et al., 1990; Woolley et al., represent alterations in protein concentration within these cyto- 1990; Woolley and McEwen, 1992, 1994). Our results also corre- plasmic pools. Given both an increase in NMDA agonist binding late with an increased number of NMDAR agonist binding sites sites at the membrane (Weiland, 1992) and an increase in synaptic (Weiland, 1992) and a suggested functional increase in NMDAR- activity consistent with an enhancement of the NMDAR (Wong mediated synaptic activity (Wong and Moss, 1992) in the CA1 and Moss, 1992), alterations in the cytoplasmic pool of receptors neurons after estradiol treatment. Lastly, no intensity differences appear to be reflected at the synaptic level.
were observed in any hippocampal field after an identical analysiswith a monoclonal antibody to MAP2, arguing against a more Mechanism of estrogen regulation of the NMDAR
general increase in protein production.
Consideration of both the immunofluorescence intensity and in Immunoelectron microscopic descriptions of the ultrastructural situ hybridization data together suggests that alterations in distribution of the NMDAR1 subunit aids in the interpretation of Gazzaley et al. • Estrogen Regulation of NMDAR1 J. Neurosci., November 1, 1996, 16(21):6830 – 6838 6835
Examples of CLSM images of MAP2-immunolabeled somata in CA1 ( A–C ), the dentate gyrus (D–F ), and CA3 (G–I ) of OVX rats (A, D, G), OVX1E rats (B, E, H ), and OVX1E1P rats (C, F, I ). Note that there are no obvious differences in intensity levels when comparing the differentexperimental groups within any hippocampal subfield. Scale bars, 10 mm.
transcriptional regulation of the NMDAR1 protein. Reasonable were chronically treated with an NMDAR antagonist (Follesa and possibilities of post-transcriptional regulation include an increase Ticku, 1996) and the hippocampus of rats treated chronically with in rate of protein translation and/or post-translational modifica- ethanol (Trevisan et al., 1994; Follesa and Ticku, 1995) exhibited tions such as an alteration in the rate of protein degradation.
increased levels of NMDAR1 protein with no detectable change Given the association of NMDAR1 protein with microtubules in in NMDAR1 mRNA levels.
the dendrites, intensity increases in the CA1 dendrites may be the The mechanism of regulation suggested by these results is result of increased dendritic transport. It is possible that the in situ somewhat different from the classical cellular mechanism of ste- hybridization technique is not sensitive enough to detect subtle roid hormone regulation of gene transcription. This is indicated changes in mRNA levels; however, post-transcriptional control of by previous observations that suggest that estrogen regulation of NMDAR1 protein expression has been demonstrated previously NMDARs may be mediated by trans-synaptic interactions (Wei- in PC12 cells, in which NMDAR1 mRNA is transcribed but not land, 1992; Woolley and McEwen, 1993; Woolley and McEwen, translated (Sucher et al., 1993). Additionally, two examples of 1994). Indeed, putative intracellular estrogen receptors identified post-transcriptional regulation of the NMDAR1 subunit have by autoradiography (Loy et al., 1988) and immunocytochemistry been recently documented. Both cultured cortical neurons that (Don Carlos et al., 1991; Weiland et al., 1996) are never found in 6836 J. Neurosci., November 1, 1996, 16(21):6830 – 6838
Gazzaley et al. • Estrogen Regulation of NMDAR1 Bar graphs depicting MAP2 immunofluorescence intensity Bar graphs depicting the quantification of NMDAR1 mRNA measurements in the somata of the CA1, dentate gyrus, and CA3 fields of labeling in the CA1, dentate gyrus, and CA3 subfields of the hippocampus.
the hippocampus. Quantitative analysis revealed no statistically significant There were no statistically significant differences in optical density mea- differences among the three groups when comparing within a hippocampal surements in any of the subfields among the three groups. Values repre- field. Values represent mean 6 SEM for six OVX rats, five OVX1E rats, sent mean 6 SEM for nine OVX rats, nine OVX1E, and nine and OVX1E1P rats (24 measurements per rat).
OVX1E1P rats.
protein regulation remains to be elucidated. The subcellular dis-tribution of the NMDAR1 subunit has been demonstrated to becontrolled by specific amino acid sequences that are locatedwithin a C-terminal exon that is subject to alternative splicing(Ehlers et al., 1995). The antibody used in this study detects allNMDAR1 splice variants. Future studies using splice variant-specific antibodies are necessary to determine whether estrogen-induced regulation is NMDAR1 splice variant-specific. Addition-ally, a possible stoichiometric change in the NMDAR complex inCA1 is suggested by disparate results obtained using differentagonist versus antagonist NMDAR binding ligands (Weiland,1992). A stoichiometric change may also account for our deter-mination of a subtle intensity increase in the dentate gyrus,whereas autoradiographic analysis revealed no change in NMDAagonist binding and a sight decrease in NMDA noncompetitiveantagonist binding in the dentate gyrus (Weiland, 1992). TheNMDAR is a heteromeric complex of several subunits, most likelyconsisting of an NMDAR1 subunit and one or more of fourdifferent NMDAR2 subunits that affect function and ligand bind-ing characteristics (Monyer et al., 1992; Hollmann and Heine- Photomicrographs of film autoradiograms show NMDAR1 mann, 1994). Differential regulation of NMDAR subunits will be mRNA hybridization in the hippocampus of an OVX rat (A), an OVX1E investigated after the development of antibodies specific for rat (B), and an OVX1E1P rat (C). Note that there is no overt difference NMDAR2 subunits.
in hybridization distribution or intensity in the hippocampal subfieldsamong rats from different treatment groups.
Estradiol has an important role in cognitive function in exper- CA pyramidal neurons but rather are found in interneurons in the imental animals (Luine, 1994; Singh et al., 1994) and in main- CA1, subiculum, and dentate gyrus subfields. Interestingly, very taining certain memory functions in surgically postmenopausal few such estrogen-sensitive interneurons are found in the CA3 women given estradiol replacement therapy, which are com- region (Loy et al., 1988), in which we could find no estrogen effect promised in women not given estradiol (Philips and Sherwin, on NMDAR1 immunofluorescence intensity in the present study.
1992a). Additionally, in some women, specific memory func- An estrogen-mediated increase in the expression of NMDARs tions were found to co-vary with sex steroid plasma concentra- and an increase in the density of excitatory spine synapses on CA1 tions across the menstrual cycle (Philips and Sherwin, 1992b).
pyramidal neurons that occur in an NMDA-dependent manner Considering the well-established role of the hippocampus (Woolley and McEwen, 1994) thus may be reflections of the same (Zola et al., 1986; Alvarez et al., 1995) and NMDARs (Morris underlying mechanism. This mechanism may involve a trans- et al., 1982; Bliss and Collingridge, 1993) in learning and synaptic control of the excitability of pyramidal neurons via estro- memory formation, estrogen may affect memory by maintaining gen receptors in inhibitory interneurons, or it may reflect actions dendritic spines (Gould et al., 1990), excitatory synapses of estrogen more directly on the excitability of pyramidal neurons (Woolley and McEwen, 1992), and NMDARs in specific hip- themselves by an as-yet undefined membrane mechanism. In pocampal neuronal populations via mechanisms suggested in either case, estrogen-induced changes in neuronal excitability may this study. We have previously characterized an intradendritic increase functional demands on the pyramidal neurons, leading to alteration in NMDAR1 immunofluorescence intensity within increased post-transcriptional expression of NMDAR1.
the dentate gyrus of aged female monkeys that revealed a A more detailed characterization of estrogen-induced NMDAR decrease in intensity within dendritic segments of the outer Gazzaley et al. • Estrogen Regulation of NMDAR1 J. Neurosci., November 1, 1996, 16(21):6830 – 6838 6837
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