Xavierd’AnglemontdeTassigny,1,2Ce ´lineCampagne,1,2Be ´ne ´dicteDehouck,1,2Danie `leLeroy,1,2GayR.Holstein,3
Jean-ClaudeBeauvillain,1,2Vale ´rieBue ´e-Scherrer,1,2andVincentPrevot1,2
SchoolofMedicine,InstitutdeMe ´decinePre ´dictiveetdeRechercheThe ´rapeutique,59046Lillecedex,France,and3DepartmentofNeurology,MountSinai
synthase (nNOS) and glutamate NMDA receptors via the scaffolding protein postsynaptic density-95 (PSD-95) in the hypothalamic
the physical association between nNOS and NMDA receptor NR2B subunit in the preoptic region of the hypothalamus. We found that
nNOS to PSD-95 and the magnitude of NO release in the preoptic region. Finally, temporary and local in vivo suppression of PSD-95
synthesis by using antisense oligodeoxynucleotides leads to inhibition of nNOS activity in the preoptic region and disrupted estrous
ling reproductive function). In conclusion, our findings identify a novel steroid-mediated molecular mechanism that enables the adult
Nitric oxide (NO) and its downstream signaling cascades are
critical to various cellular functions in the brain such as apopto-
sis, differentiation, development, synaptic plasticity, and neuro-
which travels readily across cellular membranes and enters pre-
synaptic sites, is capable of coordinating neuronal inputs in a
restricted brain volume delimited by its half-life and diffusion
constant (Prast and Philippu, 2001). Because NO cannot be
anisms that regulate its synthesis with respect to time and space
are crucial in determining its biological effects. Hence, precise
regulation of NO synthase (NOS) activity during fluctuating
as well as the avoidance of neuronal damage or death. Accord-
ingly, there are several endogenous mechanisms modulating
neuronal NOS (nNOS) (Boehning and Snyder, 2003), which
likely constitutes the predominant source of NO in neurons
(Bredt and Snyder, 1990) and concentrates inside postsynaptic
dendritic spines (Burette et al., 2002). One mechanism for the
regulation of nNOS activity may reside in the differential cou-
pling of this Ca2?-activated enzyme to glutamate NMDA recep-
tor channels by the scaffolding protein postsynaptic density-95
(PSD-95), which acts as an adaptor protein, thereby physically
and functionally coupling NMDA receptors to nNOS (Christo-
pherson et al., 1999; Sattler et al., 1999; Aarts et al., 2002).
Estrogens have profound effects on brain structure and phys-
been shown to play a key role in the cyclic increases in dendritic
spine density and synaptogenesis in the rat hippocampus
Emson (Medical Research Council, Laboratory for Molecular Research, Cambridge, UK) for his generous supply of
Correspondence should be addressed to Dr. Vincent Prevot, Inserm U837, Ba ˆtiment Biserte, Place de Verdun,
TheJournalofNeuroscience,June6,2007 • 27(23):6103–6114 • 6103
wise, estrogen treatment in gonadectomized female rats controls
dynamic changes in spine density in the hippocampus (Gould et
al., 1990) and hypothalamus (Calizo and Flanagan-Cato, 2000).
These findings raise the possibility that, in addition to neuronal
levels of estrogens that occur during the reproductive cycle may
result in ongoing, cyclic fluctuation in protein–protein interac-
mechanism for their control of NO production within the brain
(Weiner et al., 1994; Pu et al., 1996).
Using the preoptic region of the hypothalamus (central to
endogenous estrogen regulates the state of activation of nNOS
through changes in NMDA receptor/PSD-95/nNOS association
and whether cyclic variations in the estrogen levels of the region
assembly. In addition, we determined the magnitude of NO re-
lease in preoptic region explants obtained from adult female rats
els. Finally, we investigated whether local suppression of PSD-95
expression in the preoptic region in vivo impacts hypothalamic
function. Our results indicate that cyclic changes in female sex
hormones are indeed associated with marked variations in the
activity of a major brain neuronal signaling pathway and in a
model system that is critical to species survival.
Sprague Dawley female rats (Janvier, Saint-Berthevin, France) weighing
250–300 g were used for NO-production measurements, immunopre-
cipitation, Western blots from tissue explants, immunohistofluores-
room with controlled photoperiods (14 h of light and 10 h of darkness)
(2016; Harlan France, Gannat, France) (Odum et al., 2001). Vaginal
smears were examined daily, and only rats that exhibited at least two
consecutive 4 d estrous cycles were used for experiments. Diestrus I and
day of proestrus was characterized by the predominance of nucleated
by large numbers of clustered cornified squamous epithelial cells. For
primary cell culture from the hypothalamic preoptic region, an entire
litter of Sprague Dawley rats born the day of the culture setup [postnatal
day 0 (P0)] were used. All experiments were performed in accordance
with the European Communities Council Directive of November 24,
1986 (86/609/EEC), regarding mammalian research.
Amperometric measurements of NO release from preoptic
Female rats were decapitated at diestrus II 16 h (Di16h) or at proestrus
16 h (Pro16h); after rapid removal of the brain, the meninges and optic
chiasm were discarded, and the preoptic region was isolated under a
binocular magnifying glass with Wecker’s scissors (Moria, Antony,
external border of the medial preoptic area (MPO); dorsal, the internal
border of anterior commissures; the anteroposterior limits were ?0.95
to ?0.51 mm from bregma, according to the Swanson (1996) atlas. The
total dissection time was ?3 min from decapitation. After dissection,
explants were washed twice in modified Krebs’–Ringer’s bicarbonate/
glucose buffer, pH 7.4, in an atmosphere of 95% O2–5% CO2and im-
mersed in microfuge tubes containing 800 ?l of the same medium. Each
vot et al., 1999; Knauf et al., 2001). Calibration of the electrochemical
sensor was performed before each experiment according to the manu-
nor, S-nitroso-N-acetyl-D,L-penicillamine (Sigma, Saint-Quentin Falla-
vier, France). The concentration of NO gas in solution was measured in
real time, at a sampling rate of six per second (Prevot et al., 1999), using
a computer-interfaced DUO-18 (World Precision Instruments) for data
acquisition. The experimental values were transferred to SigmaPlot and
uation. Before NO-efflux measurements, a baseline was obtained by
in Krebs’–Ringer’s bicarbonate/glucose buffer at 35°C for 30 min in the
absence of tissue. The sensor used to monitor NO was then placed in an
nipulator (Carl Zeiss-Eppendorf, Hambourg, Germany). Preoptic re-
gion explants were then submitted to five 30 min incubation periods at
35°C, including a 1 h recovery period and a 90 min data acquisition
and replaced with 600 ?l of fresh prewarmed oxygenated Krebs’–Ring-
monitored by the amperometric probe, the NOS inhibitor N-acetyl-L-
A drop in NO flux reflects a shallower gradient, stemming from dimin-
ished production at the source.
Assay of NOS activity
NOS activity was determined by measuring the formation of nitrite pro-
duced in samples during a timed reaction using the BIOXYTECH nitric
oxide synthase assay kit (OXIS International, Portland, OR). Experi-
homogenates processed according to the manufacturer’s instructions.
Spectrophotometric quantitation of nitrite was performed at 540 nm
using Greiss reagents.
Primary neuron-containing cultures from the preoptic region
After decapitation and removal of the brain, the preoptic region was
gen, Carlsbad, CA). Each explant was cut into five to six smaller pieces
and incubated for 1 h at 37°C and a 5% CO2atmosphere in DMEM
containing papain (33 U/ml, 3126; Worthington-Cooper, Lakewood,
NJ), deoxyribonuclease I (DNase I; 125 U/ml, D4527; Sigma), and
L-cysteine (2.5 mM; Sigma) for papain activation. Papain-incubated tis-
sues were washed twice in DMEM with the trypsin inhibitor ovomucoid
(1.54 mg/ml, 109878; Roche Diagnostics, Somerville, NJ), DNase I (125
end the enzymatic reaction. The fragments were crushed through a 20
?m nylon mesh (Sefar America, Kansas City, MO), and the dissociated
neuronal-defined medium consisting of Neurobasal-A medium without
phenol red (12340-015; Invitrogen) with 2% (v/v) B-27 supplement
(17504-044; Invitrogen), 1% (v/v) Glutamax (35050-038; Invitrogen),
and 2% (v/v) antibiotics (penicillin/streptomycin, 10378-016; Invitro-
gen). Cells were counted with a hemacytometer (Thoma Cell, Marien-
field, Germany). For estradiol treatments, 2 ? 106cells were plated in
10-cm-diameter poly-L-lysine (molecular weight, ?300,000; P5899;
Sigma)-coated dishes (Falcon). For oligodeoxynucleotide (ODN) treat-
ments, 1 ? 106cells per well were seeded into poly-L-lysine-coated six-
well plates (Falcon). Primary cell cultures used for immunofluorescence
experiments were plated onto 12-mm-diameter poly-L-lysine-coated
coverslips in 24-well plates (Falcon) with a density of 7 ? 105cells per
well. Primary cultures were maintained in an incubator at 37°C and 5%
was changed after 2 d of culture and subsequently three times per week.
Antibodies used for coimmunoprecipitation and Western blot experiments.
ting and 1 ?g/750 ?l for immunoprecipitation), rabbit polyclonal anti-
endothelial NOS (eNOS) antibody (sc-654; 1:500 for immunoblotting
and 1 ?g/750 ?l for immunoprecipitation), and the goat polyclonal an-
6104 • J.Neurosci.,June6,2007 • 27(23):6103–6114 d’AnglemontdeTassignyetal.•EstrogenandnNOSCouplingtoNMDAReceptors
vitro, estrogen upregulates the synthesis of PSD-95 via the Akt/
the shape of the hippocampal dendritic spine (Li et al., 2004)
where PSD-95 is present in high amounts. Perhaps the nNOS
to dynamic events at the postsynaptic density. One plausible
2000) to PSD-95 through spine formation, which would require
remodeling of the actin cytoskeleton (Hering and Sheng, 2001).
din E2-mediated activation of glutamate AMPA-kainate recep-
tors (Amateau and McCarthy, 2002). These studies suggest that
such phenomenon could occur within the hypothalamus to
modulate the nNOS subcellular localization.
Until now, increased coupling of nNOS to NMDA receptors
by PSD-95 has been associated primarily with pathological con-
ditions, particularly those involving excitotoxicity (Sattler et al.,
PSD-95/NMDA receptor ternary complex assemblies vary dur-
ing fluctuating physiological conditions in discrete brain areas
critical to fundamental physiological processes. In addition, our
findings reveal a distinct role for estrogen in controlling nNOS/
PSD-95/NMDA receptor complex formation in the neuroendo-
crine brain and raise the intriguing possibility that such an effect
of estrogen on protein–protein interactions may be broadly ap-
plied in the CNS to control nNOS activity.
Aarts M, Liu Y, Liu L, Besshoh S, Arundine M, Gurd JW, Wang YT, Salter
MW,TymianskiM (2002) Treatmentofischemicbraindamagebyper-
turbing NMDA receptor- PSD-95 protein interactions. Science
AkamaKT,McEwenBS (2003) Estrogenstimulatespostsynapticdensity-95
rapid protein synthesis via the Akt/protein kinase B pathway. J Neurosci
(1997) Gapped BLAST and PSI-BLAST: a new generation of protein da-
tabase search programs. Nucleic Acids Res 25:3389–3402.
Amateau SK, McCarthy MM (2002) A novel mechanism of dendritic spine
plasticity involving estradiol induction of prostaglandin-E2. J Neurosci
Beauvillain JC, Tramu G (1980) Immunocytochemical demonstration of
LH-RH, somatostatin, and ACTH-like peptide in osmium-postfixed,
Bhat GK, Mahesh VB, Ping L, Chorich L, Wiedmeier VT, Brann DW (1998)
hormone secretion in the rat. Endocrinology 139:955–960.
Blaustein JD (1992) Cytoplasmic estrogen receptors in rat brain: immuno-
cytochemical evidence using three antibodies with distinct epitopes. En-
Blaustein JD, Lehman MN, Turcotte JC, Greene G (1992) Estrogen recep-
tors in dendrites and axon terminals in the guinea pig hypothalamus.
Boehning D, Snyder SH (2003) Novel neural modulators. Annu Rev Neu-
Bonavera JJ, Sahu A, Kalra PS, Kalra SP (1993) Evidence that nitric oxide
may mediate the ovarian steroid-induced luteinizing hormone surge: in-
volvement of excitatory amino acids. Endocrinology 133:2481–2487.
Bouret S, Croix D, Mariot M, Loyens A, Prevot V, Jegou S, Vaudry H, Beau-
villain JC, Mitchell V (2002a) Galanin modulates the activity of pro-
opiomelanocortin neurons in the isolated mediobasal hypothalamus of
the male rat. Neuroscience 112:475–485.
Bouret S, Prevot V, Takumi T, Beauvillain JC, Mitchell V (2002b) Regula-
tion by gonadal steroids of the mRNA encoding for a type I receptor for
TGF-beta in the female rat hypothalamus. Neuroendocrinology 76:1–7.
Brann DW, Mahesh VB (1991) Endogenous excitatory amino acid involve-
ment in the preovulatory and steroid-induced surge of gonadotropins in
the female rat. Endocrinology 128:1541–1547.
Bredt DS, Snyder SH (1990) Isolation of nitric oxide synthetase, a
calmodulin-requiring enzyme. Proc Natl Acad Sci USA 87:682–685.
Bredt DS, Glatt CE, Hwang PM, Fotuhi M, Dawson TM, Snyder SH (1991)
Nitric oxide synthase protein and mRNA are discretely localized in neu-
ronal populations of the mammalian CNS together with NADPH diaph-
orase. Neuron 7:615–624.
HuangF,XiaH,PetersMF,FroehnerSC,BredtDS (1996) Interactionof
nitric oxide synthase with the postsynaptic density protein PSD-95 and
alpha1-syntrophin mediated by PDZ domains. Cell 84:757–767.
BuretteA,ZabelU,WeinbergRJ,SchmidtHH,ValtschanoffJG (2002) Syn-
aptic localization of nitric oxide synthase and soluble guanylyl cyclase in
the hippocampus. J Neurosci 22:8961–8970.
Calizo LH, Flanagan-Cato LM (2000) Estrogen selectively regulates spine
rons. J Neurosci 20:1589–1596.
Chakraborty TR, Ng L, Gore AC (2003) Colocalization and hormone regu-
lation of estrogen receptor alpha and N-methyl-D-aspartate receptor in
the hypothalamus of female rats. Endocrinology 144:299–305.
Chen HJ, Rojas-Soto M, Oguni A, Kennedy MB (1998) A synaptic Ras-
GTPase activating protein (p135 SynGAP) inhibited by CaM kinase II.
ChristophersonKS,HillierBJ,LimWA,BredtDS (1999) PSD-95assembles
lent neuronal NO synthase PDZ domain. J Biol Chem 274:27467–27473.
Clancy B, Cauller LJ (1998) Reduction of background autofluorescence in
brain sections following immersion in sodium borohydride. J Neurosci
Czaja K, Ritter RC, Burns GA (2006) N-methyl-D-aspartate receptor sub-
unit phenotypes of vagal afferent neurons in nodose ganglia of the rat.
J Comp Neurol 496:877–885.
El-Husseini AE, Craven SE, Chetkovich DM, Firestein BL, Schnell E, Aoki C,
Bredt DS (2000) Dual palmitoylation of PSD-95 mediates its vesiculo-
tubular sorting, postsynaptic targeting, and ion channel clustering. J Cell
Eliasson MJ, Blackshaw S, Schell MJ, Snyder SH (1997) Neuronal nitric ox-
ide synthase alternatively spliced forms: prominent functional localiza-
tions in the brain. Proc Natl Acad Sci USA 94:3396–3401.
Garthwaite J, Charles SL, Chess-Williams R (1988) Endothelium-derived
relaxing factor release on activation of NMDA receptors suggests role as
intercellular messenger in the brain. Nature 336:385–388.
Gould E, Woolley CS, Frankfurt M, McEwen BS (1990) Gonadal steroids
regulate dendritic spine density in hippocampal pyramidal cells in adult-
hood. J Neurosci 10:1286–1291.
Gyurko R, Leupen S, Huang PL (2002) Deletion of exon 6 of the neuronal
The hDLG-associated protein DAP interacts with dynein light chain and
neuronal nitric oxide synthase. Genes Cells 5:905–911.
HerbisonAE (1998) Multimodalinfluenceofestrogenupongonadotropin-
releasing hormone neurons. Endocr Rev 19:302–330.
Herbison AE, Pape JR (2001) New evidence for estrogen receptors in
gonadotropin-releasing hormone neurons. Front Neuroendocrinol
Herbison AE, Simonian SX, Norris PJ, Emson PC (1996) Relationship of
ovariectomized and intact female rat. J Neuroendocrinol 8:73–82.
Hering H, Sheng M (2001) Dendritic spines: structure, dynamics and regu-
lation. Nat Rev Neurosci 2:880–888.
Huang YZ, Won S, Ali DW, Wang Q, Tanowitz M, Du QS, Pelkey KA, Yang
DJ, Xiong WC, Salter MW, Mei L (2000) Regulation of neuregulin sig-
naling by PSD-95 interacting with ErbB4 at CNS synapses. Neuron
(2006) Enhancement of nitric oxide production by association of nitric
oxide synthase with N-methyl-D-aspartate receptors via postsynaptic
d’AnglemontdeTassignyetal.•EstrogenandnNOSCouplingtoNMDAReceptorsJ.Neurosci.,June6,2007 • 27(23):6103–6114 • 6113
fluorescence imaging using nitric oxide sensitive dye. J Neurochem
Kalra P, McCann SM (1973) Involvement of catecholamines in feedback
mechanisms. Prog Brain Res 39:185–198.
Keilhoff G, Reiser M, Stanarius A, Aoki E, Wolf G (2000) Citrulline immu-
nohistochemistry for demonstration of NOS activity in vivo and in vitro.
Nitric Oxide 4:343–353.
Kim E, Niethammer M, Rothschild A, Jan YN, Sheng M (1995) Clustering
of Shaker-type K? channels by interaction with a family of membrane-
associated guanylate kinases. Nature 378:85–88.
Kim JH, Liao D, Lau LF, Huganir RL (1998) SynGAP: a synaptic RasGAP
that associates with the PSD-95/SAP90 protein family. Neuron
Knauf C, Prevot V, Stefano GB, Mortreux G, Beauvillain JC, Croix D (2001)
Evidence for a spontaneous nitric oxide release from the rat median em-
inence: influence on gonadotropin-releasing hormone release. Endocri-
KornauHC,SchenkerLT,KennedyMB,SeeburgPH (1995) Domaininter-
action between NMDA receptor subunits and the postsynaptic density
protein PSD-95. Science 269:1737–1740.
LemoineS,LeroyD,WarembourgM (2005) Progesteronereceptoranddo-
an immunohistochemical triple-label analysis. J Chem Neuroanat
Li C, Brake WG, Romeo RD, Dunlop JC, Gordon M, Buzescu R, Magarinos
AM,AllenPB,GreengardP,LuineV,McEwenBS (2004) Estrogenalters
noreactivity and spatial memory in female mice. Proc Natl Acad Sci USA
Maggi A, Ciana P, Belcredito S, Vegeto E (2004) Estrogens in the nervous
system: mechanisms and nonreproductive functions. Annu Rev Physiol
Mahesh VB, Brann DW (2005) Regulatory role of excitatory amino acids in
reproduction. Endocrine 28:271–280.
Martinelli GP, Friedrich Jr VL, Holstein GR (2002) L-citrulline immuno-
staining identifies nitric oxide production sites within neurons. Neuro-
McCann SM, Haens G, Mastronardi C, Walczewska A, Karanth S, Rettori V,
YuWH (2003) Theroleofnitricoxide(NO)incontrolofLHRHrelease
McEwen B, Akama K, Alves S, Brake WG, Bulloch K, Lee S, Li C, Yuen G,
Milner TA (2001) Tracking the estrogen receptor in neurons: implica-
tions for estrogen-induced synapse formation. Proc Natl Acad Sci USA
son M, He Y, Ramsay MF, Morris RG, Morrison JH, O’Dell TJ, Grant SG
(1998) Enhanced long-term potentiation and impaired learning in mice
with mutant postsynaptic density-95 protein. Nature 396:433–439.
Odum J, Tinwell H, Jones K, Van Miller JP, Joiner RL, Tobin G, Kawasaki H,
Deghenghi R, Ashby J (2001) Effect of rodent diets on the sexual devel-
opment of the rat. Toxicol Sci 61:115–127.
Ojeda SR, Terasawa E (2002) Neuroendocrine regulation of puberty (Pfaff
D, Arnold A, Etgen A, Fahrbach S, Moss R, Rubin R, eds), pp 589–659.
New York: Elsevier.
Okamura H, Yokosuka M, Hayashi S (1994) Estrogenic induction of
receptor immunoreactivity in the female rat. J Neuroendocrinol
Prast H, Philippu A (2001) Nitric oxide as modulator of neuronal function.
Prog Neurobiol 64:51–68.
Prevot V, Dutoit S, Croix D, Tramu G, Beauvillain JC (1998) Semi-
quantitative ultrastructural analysis of the localization and neuropeptide
content of gonadotropin releasing hormone nerve terminals in the me-
dian eminence throughout the estrous cycle of the rat. Neuroscience
Prevot V, Croix D, Rialas CM, Poulain P, Fricchione GL, Stefano GB, Beau-
villain JC (1999) Estradiol coupling to endothelial nitric oxide stimu-
via a membrane receptor. Endocrinology 140:652–659.
Pu S, Xu B, Kalra SP, Kalra PS (1996) Evidence that gonadal steroids mod-
ulate nitric oxide efflux in the medial preoptic area: effects of N-methyl-
D-aspartate and correlation with luteinizing hormone secretion. Endo-
Pu S, Kalra PS, Kalra SP (1998) Ovarian steroid-independent diurnal
rhythm in cyclic GMP/nitric oxide efflux in the medial preoptic area:
possible role in preovulatory and ovarian steroid-induced LH surge.
J Neuroendocrinol 10:617–625.
SatoS,BrahamCS,PutnamSK,HullEM (2005) Neuronalnitricoxidesyn-
thase and gonadal steroid interaction in the MPOA of male rats: co-
localization and testosterone-induced restoration of copulation and
nNOS-immunoreactivity. Brain Res 1043:205–213.
Sattler R, Xiong Z, Lu WY, Hafner M, MacDonald JF, Tymianski M (1999)
Specific coupling of NMDA receptor activation to nitric oxide neurotox-
icity by PSD-95 protein. Science 284:1845–1848.
Savchenko A, Barnes S, Kramer RH (1997) Cyclic-nucleotide-gated chan-
nels mediate synaptic feedback by nitric oxide. Nature 390:694–698.
Scannevin RH, Huganir RL (2000) Postsynaptic organization and regula-
tion of excitatory synapses. Nat Rev Neurosci 1:133–141.
Scordalakes EM, Shetty SJ, Rissman EF (2002) Roles of estrogen receptor
alpha and androgen receptor in the regulation of neuronal nitric oxide
synthase. J Comp Neurol 453:336–344.
Simerly RB (2005) Wired on hormones: endocrine regulation of hypotha-
lamic development. Curr Opin Neurobiol 15:81–85.
SmithMS,FreemanME,NeillJD (1975) Thecontrolofprogesteronesecre-
tion during the estrous cycle and early pseudopregnancy in the rat: pro-
lactin, gonadotropin and steroid levels associated with rescue of the cor-
pus luteum of pseudopregnancy. Endocrinology 96:219–226.
SwansonLW (1996) Structureoftheratbrain.Amsterdam:ElsevierScience
Urbanski HF, Ojeda SR (1990) A role for N-methyl-D-aspartate (NMDA)
receptors in the control of LH secretion and initiation of female puberty.
Valtschanoff JG, Weinberg RJ (2001) Laminar organization of the NMDA
receptor complex within the postsynaptic density. J Neurosci
Wang HG, Lu FM, Jin I, Udo H, Kandel ER, de Vente J, Walter U, Lohmann
SM,HawkinsRD,AntonovaI (2005) Presynapticandpostsynapticroles
of NO, cGK, and RhoA in long-lasting potentiation and aggregation of
synaptic proteins. Neuron 45:389–403.
Weiner CP, Lizasoain I, Baylis SA, Knowles RG, Charles IG, Moncada S
(1994) Induction of calcium-dependent nitric oxide synthases by sex
hormones. Proc Natl Acad Sci USA 91:5212–5216.
Wintermantel TM, Campbell RE, Porteous R, Bock D, Grone HJ, Todman
MG, Korach KS, Greiner E, Perez CA, Schutz G, Herbison AE (2006)
back to gonadotropin-releasing hormone neurons and fertility. Neuron
Woolley CS, McEwen BS (1992) Estradiol mediates fluctuation in hip-
Zar JH (1984) Biostatistical analysis. Englewood Cliffs, NJ: Prentice Hall.
6114 • J.Neurosci.,June6,2007 • 27(23):6103–6114 d’AnglemontdeTassignyetal.•EstrogenandnNOSCouplingtoNMDAReceptors