Glutamate AMPA/Kainate Receptors, not GABAA

Receptors, Mediate Estradiol-Induced SexDifferences in the Hypothalamus

Brigitte J. Todd,1* Jaclyn M. Schwarz,1,2 Jessica A. Mong,2,3

Margaret M. McCarthy1,2,4

1 Department of Physiology, University of Maryland, Baltimore School of Medicine, Baltimore,Maryland 21201

2 Department of Program in Neuroscience, University of Maryland, Baltimore School of Medicine,Baltimore, Maryland 21201

3 Department of Pharmacology and Experimental Therapeutics, University of Maryland,Baltimore School of Medicine, Baltimore, Maryland 21201

4 Department of Psychiatry, University of Maryland, Baltimore School of Medicine, Baltimore,Maryland 21201

Received 24 March 2006; accepted 26 June 2006; accepted 28 July 2006

ABSTRACT: Sex differences in brain morphology

underlie physiological and behavioral differences between

males and females. During the critical perinatal period for

sexual differentiation in the rat, gonadal steroids act in a

regionally specific manner to alter neuronal morphology.

Using Golgi-Cox impregnation, we examined several pa-

rameters of neuronal morphology in postnatal day 2

(PN2) rats. We found that in the ventromedial nucleus of

the hypothalamus (VMN) and in areas just dorsal and just

lateral to the VMN that there was a sex difference in total

dendritic spine number (males greater) that was abolished

by treating female neonates with exogenous testosterone.

Dendritic branching was similarly sexually differentiated

and hormonally modulated in the VMN and dorsal to the

VMN. We then used spinophilin, a protein that positively

correlates with the amount of dendritic spines, to investi-

gate the mechanisms underlying these sex differences. Es-

tradiol, which mediates most aspects of masculinization

and is the aromatized product of testosterone, increased

spinophilin levels in female PN2 rats to that of males. Mus-

cimol, an agonist at GABAA receptors, did not affect spi-

nophilin protein levels in either male or female neonates.

Kainic acid, an agonist at glutamatergic AMPA/kainate

receptors, mimicked the effect of estradiol in females.

Antagonizing AMPA/kainate receptors with NBQX pre-

vented the estradiol-induced increase in spinophilin in fe-

males but did not affect spinophilin level in males.

' 2007Wiley Periodicals, Inc. Develop Neurobiol 67: 304–315, 2007

Keywords: sexual differentiation; estradiol; GABA;

glutamate; dendritic spine; spinophilin

INTRODUCTION

Male and female brains exhibit fundamental morpho-

logical differences, which are thought to underlie sex

differences in physiology and behavior. These dimor-

phisms may be at the cellular level, including differ-

ences in dendritic spine density (Matsumoto and

Arai, 1986; Munoz-Cueto et al., 1991; Mong et al.,

*Present address: Department of Neuroscience, Albert EinsteinCollege of Medicine, 1300 Morris Park Avenue, F113, Bronx, NewYork 10461.

Correspondence to: B.J. Todd (btodd@aecom.yu.edu).Contract grant sponsor: NIH; contract grant number: MH52716.

' 2007 Wiley Periodicals, Inc.Published online 12 January 2007 in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/dneu.20337

304

1999), neurite branching (Kawashima and Takagi,

1994), or astrocyte complexity (Mong et al., 1996;

Amateau and McCarthy, 2002b). Other dimorphisms

exist on a multicellular level. These include projec-

tions from one brain area to another, such as that

from the bed nucleus of the stria terminalis to the

anteroventral periventricular nucleus (Ibanez et al.,

2001) or in volume of nuclei, such as the sexually

dimorphic nucleus of the preoptic area (Gorski et al.,

1978) or the medial nucleus of the amygdala

(Mizukami et al., 1983; Cooke et al., 1999). To

explore the relationship between sex differences in

the morphometry of the brain and the control of sex-

specific physiology and behavior, it is essential to

understand the mechanisms by which sex differences

in morphometry are established.

The laboratory albino rat provides an excellent

model for mechanistic study of sex differences in the

brain. Adults exhibit highly differentiated sexual

behavior, which is determined by differential steroid

hormone exposure during a perinatal sensitive win-

dow. This sensitive period of brain organization in

the rat begins on embryonic day (ED) 18 and extends

to postnatal day (PN) 10. The sensitive period is

operationally defined by the onset of testicular andro-

gen synthesis in males at ED 18 and the loss of sensi-

tivity to exogenous androgens in females by around

PN10 (see for review (McCarthy and Albrecht, 1996;

Simerly, 2002; De Vries, 2004). In the brain, neuro-

nal aromatase converts testosterone to estradiol

(Naftolin et al., 1971; Ryan et al., 1972), and it is pri-

marily this steroid that induces many of the perma-

nent effects on the developing neuroarchitecture.

We have previously shown that testosterone and

its aromatized product, estradiol, exert region-specific

effects upon dendritic spine density and dendrite

branching in the developing hypothalamus and pre-

optic area, but effects are at times opposite in direc-

tion or selective to only a subregion of the diencepha-

lon (Mong et al., 1999; Amateau and McCarthy,

2004). Thus, even in such a small brain region as the

diencephalon, the effects of gonadal steroids in the

neonatal brain are tightly regulated and regionally

diverse.

Dendritic spines are a primary site of excitatory

synapses (Colonnier, 1968; Sorra and Harris, 2000)

and provide the technical advantage of being rela-

tively easy to count on dendrites subjected to Golgi/

Cox impregnation or visualized in some other man-

ner. This allows for determining spine density on a

length of dendrite, as well as the total number of

spines per dendrite. We have recently exploited an

additional easily quantified and reliable protein maker

of these spines, spinophilin, which is enriched in the

head and neck of the dendritic spine (Allen et al.,

1997; Feng et al., 2000). Use of Western immuno-

blotting reveals a sex difference in baseline levels of

spinophilin in the POA and mediobasal hypothalamus

of newborn rats, and that estradiol increases the level

of spinophilin in females to that of males in both

these regions (Amateau and McCarthy, 2002a; Todd

et al., 2005). The POA is a brain region centrally

involved in the expression of male sexual behavior

whereas the mediobasal hypothalamus, and in partic-

ular the ventromedial nucleus, is a critical region for

the expression of female sexual behavior (Meisel and

Sachs, 1994; McCarthy and Albrecht, 1996).

The mechanisms by which estradiol exerts its

effects on the developing brain remain incompletely

described. Two strong candidates are the amino acids

GABA and glutamate. Estrogen-receptive GABAer-

gic neurons are found in the hypothalamus (Flugge

et al., 1986) and male newborn rats have hypothala-

mic levels of mRNA for glutamic acid decarboxylase

(GAD; the rate limiting enzyme in GABA synthesis)

and GABA that are twice as high as females (Davis

et al., 19961999). Moreover, differentiation of sexual

behavior can be disrupted by decreasing neonatal

GAD with antisense oligodeoxynucleotides against

GAD mRNA (Davis et al., 2000) and GABA acting

via GABAA receptors mediates the effects of estra-

diol on astrocyte morphology in the developing arcu-

ate nucleus (Mong et al., 2002). In immature neurons,

GABA is an excitatory neurotransmitter (Obrietan

and van den Pol, 1995; Gao and van den Pol, 2001),

switching to inhibitory during the course of develop-

ment. Estradiol both delays this switch, extending the

duration of time during which GABA is excitatory, as

well as enhances the magnitude of each depolarizing

event (Perrot-Sinal et al., 2001), with consequences

for downstream signaling of the phosphorylated form

of cyclic AMP response element binding protein that

may provide a developmental divergence point in

sexual differentiation (Auger et al., 2001). Thus,

GABA would seem a likely mediator of estradiol-in-

duced changes in dendritic spine density in the devel-

oping hypothalamus. However, in the neighboring

preoptic area, glutamate, acting at least in part via the

AMPA/kainate receptor, mediates estrogen-induced

increases in dendritic spine density (Amateau and

McCarthy, 2002a), thus also making glutamate a

likely mediator of estradiol-induced dendritic spine

density changes in the mediobasal hypothalamus.

In a previous study from this group (Mong et al.,

1999), testosterone increased dendritic branching but

had no effect on spine density of VMN neurons.

Subsequent to that, we found that estradiol treatment

increased spinophilin protein in the mediobasal

Estrogen Increases Spinophilin in Hypothalamus 305

Developmental Neurobiology. DOI 10.1002/dneu

hypothalamus (Todd et al., 2005), suggesting spine

density and spinophilin levels are not always corre-

lated. This lack of correlation could be due to a

change in spine number but not spine density or that

the dissection used for the Western blot analysis

encompassed more than the VMN proper, to which

the Golgi analysis was restricted. Thus, to further

describe the morphology of dendrites in and around

the VMN, and their regulation by testosterone/

estradiol, we revisited the sections used in the previ-

ous study and subjected them to a more thorough

analysis by counting total number of dendritic spines

per neuron, as well as density, and measuring den-

drite length and frequency of dendritic branching. To

address whether differences between the two studies

could be due to the hypothalamic dissection for im-

munoblotting, we also sampled two regions outside

of the VMN region described in our previous paper.

We went on to use Western immunoblotting to inves-

tigate whether gonadal steroids were regulating den-

dritic spine number via the actions of excitatory

amino acids.

METHODS

Animals

Female Sprague-Dawley rats (220–250 g; Charles River

Laboratories, Wilmington, MA) were singly housed in our

animal facility and maintained under a reverse 12:12 light/

dark schedule (lights on at 2300) with food and water freely

available. They were mated and pregnancy was confirmed

by presence of sperm in a vaginal lavage. Animals were

allowed to deliver naturally. Cages were checked regularly

for the presence of pups and the morning of delivery desig-

nated postnatal day 0 (PN0). All procedures were approved

by the IACUC of the University of Maryland Baltimore.

In Vivo Manipulations

All drugs were obtained from Sigma (St. Louis, MO) unless

otherwise indicated. Pups received injections of one or

combinations of the following treatments, all administered

subcutaneously: (1) 0.1 cc sesame oil, (2) 100 �g testoster-

one proprionate in 0.1 cc sesame oil, (3) 100 �g estradiol

benzoate in 0.1 cc sesame oil, (4) 0.1 cc 0.9% saline, (5)

5 �g of the selective GABAA receptor agonist, muscimol in

0.1 cc saline, (6) 2.5 �g of the AMPA receptor agonist,

kainic acid in 0.05 cc saline, and (7) 1 �g of the AMPA re-

ceptor antagonist NBQX in 0.1 cc saline. Animals were

treated on PN0 and PN1 and euthanized on PN2, approxi-

mately 48 h after birth. We have previously shown that

changes in spinophilin protein consequent to neonatal hor-

mone manipulation are apparent by PN2 (Amateau and

McCarthy, 2002a). For any given experiment, pups from

at least four litters were used, and all treatment groups were

represented in every litter.

Golgi Staining

Neonatal males and females were treated on PN0 and PN1

with oil or testosterone (females only). After neonates were

overdosed with pentobarbital and transcardially perfused

with cold 0.9% saline, brains were removed and placed in

30 ml of Golgi-Cox solution (1:1 solution of 5% K2Cr2O7

and 5% HgCl2 added to 5% K2CrO4 in a 4:10 ratio). Forty-

eight hours later, the solution was renewed, and the brains

remained in impregnation solution for 20 days before being

placed in 30% sucrose solution for 3 days, cut on a vibro-

tome at 100 �m, and mounted on 2% gelatin-subbed glass

slides. Slices were counterstained with methylene blue to

distinguish anatomical landmarks. These sections had been

analyzed and results reported in a previous study (Mong

et al., 1999) but were more expansively analyzed here by a

new investigator (JMS) in consultation with the previous in-

vestigator (JAM).

Morphological Analysis

Golgi-Cox-impregnated neurons were analyzed as de-

scribed in a previous publication from our lab (Mong et al.,

1999). Slides were numerically coded, and the reader was

blind to the experimental group. Anatomically matched sec-

tions (two to three) were analyzed from each animal for all

groups. Five neurons were analyzed from each of three dif-

ferent regions; including the VMN, an area just lateral to

the VMN, and an area just dorsal to the VMN; all regions

which would be included in a freehand dissection of the

mediobasal hypothalamus (see Fig. 1). These regions would

not include any other nuclei of the hypothalamus, but would

contain projections to or from the VMN (Millhouse, 1973;

Nishizuka and Pfaff, 1989). Neurons that appeared well

impregnated were marked for analysis. A 20� Nikon objec-

tive was used for this selection procedure so that the ob-

server would not be biased by the appearance of the spines

on the dendrites. During Golgi-Cox impregnation, the axon

does not exhibit complete staining, and so any fully stained

process was considered to be a dendrite. Neurons deter-

mined not to be fully impregnated upon further inspection

were not analyzed and new neurons were selected for anal-

ysis. Criteria for neuron selection also included the ability

of the experimenter to distinguish an individual neuron.

Selected neurons were analyzed through the different focal

planes of the section with a 100� oil-immersion objective

using the Neurolucida system (MicroBrightField, Colches-

ter, VT). The following morphological features were meas-

ured: total number of primary dendrites per neuron, primary

dendrite length (in �m), dendritic spine density (1/�m),

total number of spine-like processes per dendrite, the num-

ber of primary dendrites that branch versus those that do

not branch, and the number of branches per primary den-

drite. Spines were defined as any protrusion less than 5 �min length and branches were defined as any protrusion

306 Todd et al.

Developmental Neurobiology. DOI 10.1002/dneu

greater than 5 �m in length. Analyses of spine-like pro-

cesses incorporated the complete dendrite from soma to dis-

tal end and included all processes extending from each neu-

ron examined. Each parameter was measured in five neu-

rons and a mean per animal used for statistical analysis.

Western Immunoblotting

Pups were euthanized on PN2 and the medial basal hypothal-

amus dissected, flash frozen in isopentane, and stored at

�808C before being homogenized in 400 �L of lysis buffer

[pH 7.5; 0.8% Tris-HCl, 0.9% NaCl, 1% Tergitol NP-40,

1 mM phenylmethylsulfonyl fluoride, and protease inhibitors

(1 �g/mL)], centrifuged for 30 min at 9000 rpm and the su-

pernatant collected. Protein levels in the supernatant were

measured by Bradford protein assay for standardization of

protein loading during Western blotting. Ten micrograms of

protein from each animal was electrophoresed in separate

lanes on an 8–16% Tris-glycine precast SDS-polyacrylamide

gel (Invitrogen, San Diego, CA) and transferred to a PVDF

membrane (Bio-Rad, Hercules, CA). Membranes were bloc-

ked in 5% nonfat milk powder (Bio-Rad) in TBS with 0.1%

Tween-20 (TTBS; Bio-Rad) for 1 h at room temperature and

then incubated with rabbit anti-rat spinophilin/neurabin II

polyclonal IgG (1:1000; Upstate Biotechnology) in TTBS

for 3 h. Following a 30 min incubation with goat anti-rabbit

HRP-conjugated IgG (New England Biolabs, Beverly, MA),

spinophilin bands were visualized with the Phototype chemi-

luminescence system (New England Biolabs). Blots were

exposed on Hyperfilm-ECL (Amersham, Arlington Heights,

IL). The spinophilin protein was detected as a band of rela-

tive molecular mass of 120 kDa, and the integrative grey-

scale pixel area density (iad) was captured with a CCD

camera and quantified using NIH Image software. Western

blots were compared both within and between gels. When

results were across several gels, bands were quantified as a

ratio of iad with the iad of control groups serving as the com-

parison. This standardization allowed for comparison of mul-

tiple groups across multiple films.

Figure 1 Regions of morphological analysis and representative examples of dendrites. Neurons

were analyzed in the VMN and surrounding regions (A). Top rectangle shows sampling region for

VMN-d, left hand square shows sampling region for VMN-l, and region within the darkly stained

nucleus shows sampling region for VMN. (B) Examples of dendritic spines (arrowheads). (C) An

example of the termination of a dendrite (arrowhead). Scale bars: (A) 200 �m; (B) and (C) 20 �m.

[Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Estrogen Increases Spinophilin in Hypothalamus 307

Developmental Neurobiology. DOI 10.1002/dneu

Statistics

All results except the presence of branching on the primary

dendrite were analyzed using either unpaired Student’s

t-test if two groups were being compared or one way

ANOVA followed by the Fisher PLSD post hoc test if more

than two groups were compared. The presence of branching

on the primary dendrites was analyzed using a �2 test. All

statistical tests used p < 0.05 as the criterion for signifi-

cance.

RESULTS

Dendrite Morphology

To accurately construct a picture of dendrite mor-

phology in the VMN and surrounding areas, we ana-

lyzed the following parameters: (1) number of dendri-

tic spines per primary dendrite, (2) length of primary

dendrites, (3) dendritic spine density, (4) number of

primary dendrites per neuron, (5) number of primary

dendrites that branch, and (6) number of branches per

primary dendrite.

Dendritic Spines. There was an overall effect of group

on dendritic spine number on primary dendrites in the

VMN (F(2,12) ¼ 6.8; p < 0.05), VMN-l (F(2,12) ¼ 6.74;

p < 0.05), and VMN-d (F(2,12) ¼ 8.59; p < 0.01). Post-

hoc testing revealed a sex difference in total number

of dendritic spines per neuron in each of these areas,

with males having up to twice the total number of

dendritic spines as females (see Fig. 2). Moreover,

when females received testosterone for the first two

days of life, their dendritic spine number per neuron

was indistinguishable from those of the males.

Dendritic spine density was not significantly different

between groups in any area (VMN: F(2,12) ¼ 1.39,

p ¼ 0.3; VMN-l: F(2,12) ¼ 1.28, p ¼ 0.314; VMN-d:

F(2,12) ¼ 3.16, p ¼ 0.08; Table 1).

Dendrite Length. All dendrites were measured on

each neuron selected. There were no significant differ-

ences in dendrite length between groups in any area

(VMN: F(2,12) ¼ 2.91; p ¼ 0.09; VMN-l: F(2,12) ¼ 0.73,

p ¼ 0.5; VMN-d: F(2,12) ¼ 3.48; p ¼ 0.09; Table 1), al-

though in every region there was a trend (p ¼ 0.09) for

the female group to have the shortest dendrite length,

and the female þ T to have the longest, with the male

group intermediate.

Dendrite Number. There was no significant differ-

ence in primary dendrite number between groups in

any area (VMN: F(2,12) ¼ 0.56, p ¼ 0.58; VMN-l

Figure 2 Dendritic spine number is sexually differenti-

ated in PN2 rats. Dendritic spines were counted along the

entire length of the dendrite. Data represent means 6 SEM.

In all three regions, male neurons had significantly more

spines than females. When females were treated with tes-

tosterone on PN0 and PN1, the spine number was indistin-

guishable from males. (*Significantly different from control

females, ANOVA; p < 0.05).

308 Todd et al.

Developmental Neurobiology. DOI 10.1002/dneu

F(2,12) ¼ 0.11, p ¼ 0.90; VMN-d: F(2,12) ¼ 1.57, p ¼0.25; Table 1).

Dendrite Branching. We calculated the percentage

of primary dendrites that branch in each area. In the

VMN (�2 ¼ 16.18, 2DF, p < 0.001) and VMN-l

(�2 ¼ 15.17, 2DF p < 0.001), there was a significant

difference between groups in the percent of primary

dendrites that branch (Table 1). We then determined

the number of branches per dendrite, excluding from

analysis those dendrites that had no branches (see

Fig. 3). In the VMN, there was an overall group effect

on mean number of branches per dendrite (F(2,12) ¼8.58; p < 0.01). Post-hoc analysis revealed that VMN

neurons of males and females treated with testoster-

one had significantly more branches than control

females (Fisher PLSD p < 0.05). There were no sig-

nificant sex or treatment differences in average num-

ber of branches per primary dendrite in the VMN-l or

VMN-d.

Spinophilin

GABAA Receptor Activation. Animals were treated

with 5 �g of muscimol s.c. for the first two days of

life. Activation of GABAA receptors by muscimol

increases phosphorylation of CREB in male neonates

(Auger et al., 2001). Phosphorylation of CREB is

necessary for establishment of excitatory spine syn-

apses in hippocampal cultures (Murphy and Segal,

1997). We used a smaller dose of muscimol than that

shown to increase CREB phosphorylation because

animals were treated over the course of two days and

as such could not tolerate the higher dose used in the

previous study (Auger et al., 2001). The current dose

was based on its ability to alter hippocampal develop-

ment (Nunez et al., 2003); however, there was no ef-

fect of treatment with the GABAA receptor agonist,

muscimol, on hypothalamic spinophilin level in males

or females [Fig. 4(A)].

AMPA/Kainate Glutamate Receptor Activation. We

have previously shown that glutamate in part medi-

ates the estradiol-induced increase in spinophilin in

the developing preoptic area (Amateau and McCar-

thy, 2002a). In the mediobasal hypothalamus, kainic

acid, an AMPA/kainate glutamate receptor agonist,

mimicked the effect of estradiol in that it increased

spinophilin in the female hypothalamus to that of

males and significantly higher than control females

[F(2,16) ¼ 5.54; p < 0.01; Fig. 4(B)]. Moreover, co-

administration of estradiol with NBQX, an AMPA/

kainate receptor antagonist, completely blocked the

effect of estradiol, resulting in hypothalamic spino-

philin levels indistinguishable from control females

[F(4,25) ¼ 28.4; p < 0.001; Fig. 4(C)]. Administration

of NBQX to neonatal male rats did not alter spinophi-

lin levels [Fig. 4(D)].

DISCUSSION

Differential exposure to gonadal hormones during a

critical period of perinatal development underlies the

Table 1 Morphological Parameters of Neurons in Regions of the Ventromedial Nucleus in PN2 Females,

Males, and Females Treated with Testosterone

Parameter Examined Female Male Female þ T �2 Value

VMN

Dendrite length (�m) 60.42 6 5.81 67.876 4.71 78.676 3.98

Spine density (1/�m) 0.15 6 0.01 0.176 0.01 0.146 0.009

No. primary dendrites 3.00 6 0.57 3.756 0.45 3.316 0.57

Primary dendrite branching (%) 37 68 61 16.18 (p < 0.001)

VMN-l

Dendrite length (�m) 59.5 6 5.07 67.86 4.71 69.16 6.29

Spine density (1/�m) 0.16 6 0.01 0.196 0.02 0.186 0.03

No. primary dendrites 3.20 6 0.33 3.086 0.17 3.066 0.06

Primary dendrite branching (%) 37 (63) 70 (73) 53 (45) 15.17 (p < 0.001)

VMN-d

Dendrite length (�m) 46.50 6 3.22 53.926 3.19 58.356 2.22

Spine density (1/�m) 0.15 6 0.009 0.206 0.15 0.196 0.019

No. primary dendrites 3.85 6 0.23 3.456 0.12 3.686 0.12

Primary dendrite branching (%) 30 (76) 47 (90) 40 (50) 4.66 (p ¼ 0.1)

Animals were injected with 0.1 cc sesame oil or testosterone (100 �g) on the day of birth and 24 h later. Data are presented as means 6SEM. There were no significant differences between sex or treatment in the parameters measured with the exception of the percentage of pri-

mary dendrite branching in the VMN and VMN-l.

Estrogen Increases Spinophilin in Hypothalamus 309

Developmental Neurobiology. DOI 10.1002/dneu

vast majority of sexual dimorphisms in the brain. In

the absence of testicularly-derived testosterone,

(which is both reduced to 5�-dihydrotestosterone andaromatized to estradiol) the brain develops a predom-

inantly female phenotype. Thus, male pups castrated

at birth grow up to exhibit female-typical lordosis

behavior when hormone-primed and placed with a

male rat. Moreover, the same rats have impaired

mounting, intromission, and ejaculation behavior

when paired with a sexually receptive female (Booth,

1977; Roffi et al., 1987). Complete restoration of

these male-typic sex behaviors requires activation of

androgen and estrogen receptors during the perinatal

critical period (Booth, 1977). Likewise, female rats

given testosterone at birth have impaired female sex

behavior in adulthood, but increased mounting and

intromission-like behaviors in the presence of activa-

tional testosterone.

The perinatal circulating hormonal milieu has per-

manent influences on the developing neuronal substrate,

which presumably mediates the observed changes in

adult behavior. Notable of these are the effects of estra-

diol, the aromatized product of testosterone, on dendri-

tic spine density. It is well established that estradiol

influences spine density in the adult, both in the

hippocampus (Woolley et al., 1990; Woolley and

McEwen, 1992) and in the ventromedial hypothalamus

(Frankfurt and McEwen, 1991; Segarra and McEwen,

1991; Calizo and Flanagan-Cato, 2000). Recently,

investigators have turned to measurements of spine

associated proteins, such as syntaxin, synaptophysin

(presynaptic), and spinophilin (postsynaptic). Changes

in levels of these proteins correlate well with changes

in dendritic spines as measured by more direct meth-

ods. Hence, estradiol increases syntaxin, synaptophy-

sin, and spinophilin in the CA1 region of the hippo-

campus in adult rats (Brake et al., 2001) and rhesus

monkeys (Choi et al., 2003). In the neonate, estradiol

increases spinophilin in the preoptic area and posi-

tively correlates with an increase in spines quanti-

fied by counting of Golgi/Cox impregnated dendrites

(Amateau and McCarthy, 2002a; Amateau and

McCarthy, 2004). Thus, we and others (Brake et al.,

2001; Alves et al., 2002; Choi et al., 2003; Hao

et al., 2003) have used spinophilin as an indirect

measure of dendritic spine density. Spinophilin reg-

ulates spine formation (Feng et al., 2000) and is con-

centrated in the head and neck of dendritic spines

(Allen et al., 1997) although light staining can be

found throughout the dendrite (see Amateau and

McCarthy, 2002a). Because the protein bundles

actin filaments (Satoh et al., 1998), it may play a key

role in reorganizing spines, and thus affect the loca-

tion and density of axospinous synapses.

Figure 3 Dendritic branching is sexually differentiated in

PN2 rats. Data represent means 6 SEM. In the VMN (3A),

males and testosterone-treated females had significantly

more dendritic branches than control females. In regions

just lateral to the VMN (VMN-I) or just dorsal to the VMN

(VMN-d) there were no significant differences between

groups. (*Significantly different from control females,

ANOVA; p < 0.05).

310 Todd et al.

Developmental Neurobiology. DOI 10.1002/dneu

Figure 4 Estradiol increases spinophilin in the neonatal hypothalamus via activation of AMPA/

kainate receptors. Neonatal rats were treated on PN0 and PN1 with one or a combination of drugs

or hormones. Each lane of the Western immunoblot represents tissue from one animal. Data in

graphs represent means 6 SEM. GABAA receptor activation with muscimol (5 �g) had no effect

on spinophilin in the hypothalamus of either males or females (A). Estradiol benzoate (EB; 100 �g)increased spinophilin in the hypothalamus of females, and this effect was mimicked by kainic acid

(2.5 �g; B). The effect of EB in females was blocked by NBQX, an AMPA/kainate receptor antag-

onist (C). NBQX alone had no effect in males (D). (* in B, significantly different from saline con-

trol; in C, significantly different from male oil and female EB, ANOVA; p < 0.05).

Estrogen Increases Spinophilin in Hypothalamus 311

Developmental Neurobiology. DOI 10.1002/dneu

The hypothalamus is a heterogeneous, sexually di-

morphic structure that can be subdivided into several

nuclei, many of which are responsible for regulation

of reproductive behavior in the adult. Estrogen recep-

tors are present in the hypothalamus throughout the

critical period of sexual differentiation (Vito and Fox,

1981) and estradiol, aromatized from testicular testos-

terone, is necessary for sexual differentiation of the

rat brain (McEwen et al., 1977). The ventrolateral

VMN is important for female sex behavior in adult-

hood and contains a high concentration of estrogen

receptors. However, it has previously been shown

that the effects of estrogen on the sexual differentia-

tion of the VMN are due to synapses on VMN neu-

rons from cell groups outside of the mediobasal hypo-

thalamus (Nishizuka and Pfaff, 1989). In this same

study, they have determined that *68% of the synap-

tic connections from VMN neurons project within the

VMN to both the ventrolateral VMN and dorsomedial

VMN. We might therefore expect the effects of estra-

diol on dendritic morphology to extend throughout

the entire VMN. Therefore, in this study, we sampled

from neurons throughout the entire VMN, including

both the dorsomedial portion (VMN-d) and the ven-

trolateral portion (VMN-l) as well as the ventrome-

dial (VMN). Previous work has shown that VMN

neuronal projections extend beyond the VMN to

areas both lateral and dorsal within the medial basal

hypothalamus (Millhouse, 1973). We chose these

regions for analysis to encompass regions of the

hypothalamus, which would receive direct input from

VMN neurons as well.

Consistent with our previous report (Mong et al.,

1999), there was no sex difference or hormonal mod-

ulation of spine density on VMN neuron dendrites.

However, males and females treated with testosterone

had a higher total number of spines than vehicle-

treated females. The greater dendritic branching in

males and females treated with testosterone compared

to control females was also consistent with the previ-

ous finding and may be the basis by which total den-

dritic spine number increases, without increasing den-

dritic spine density. In contrast to the increase in total

spine number seen in neonates, in adults testosterone

replacement in gonadectomized males decreases den-

dritic length and branching, and dendritic spine num-

bers are unchanged with a resultant increase in den-

dritic spine density (Danzer et al., 2001). In adult

females dendritic spine density is increased by estra-

diol or estradiol plus progesterone (Frankfurt et al.,

1990).

Having determined that the total number of den-

dritic spines in the mediobasal hypothalamus is

greater in males and increased by perinatal exposure

to testosterone, we sought to elucidate the down-

stream mechanism. Estradiol exerts the same effect as

testosterone (Todd et al., 2005), consistent with the Ar-

omatization Hypothesis of sexual differentiation. Es-

tradiol increases phosphorylation of CREB (Murphy

and Segal, 1996; Zhou et al., 1996) to increase dendri-

tic spine density on hippocampal neurons (Murphy

and Segal, 1997). The GABAA receptor agonist, mus-

cimol, also increases phosphorylation of CREB in sex-

ually dimorphic brain regions of the newborn male

rat pup, including the VMN (Auger et al., 2001), and

estradiol enhances both the magnitude and duration

of depolarizing GABAA receptor activation and the

consequent increase in intracellular calcium (Perrot-

Sinal et al., 2003). Surprisingly, despite this wealth of

converging evidence that GABA might mediate estra-

diol’s effects on dendritic spines, we found no effect

of muscimol treatment on spinophilin levels in either

male or female hypothalamus. This same dose has

been previously used by our group to induce excito-

toxic damage to the developing hippocampus (Nunez

et al., 2003), ameliorating against the possibility that

the dose or duration of exposure were insufficient to

activate GABAA receptors. Thus we are left with the

tentative conclusion, albeit based on negative results,

that GABA is not a mechanistic determinant of estra-

diol-induced increases in dendritic spine number in

the developing hypothalamus.

In a previous study, estradiol treatment increased

spinophilin in the neonatal preoptic area and activa-

tion of AMPA/kainate receptors was a major, but not

the only, component of this response (Amateau and

McCarthy, 2002a, 2004). In the current study, kainic

acid, which acts at both AMPA and kainate receptors,

increased spinophilin protein in the hypothalamus of

females and was as potent as estradiol. When the

AMPA receptor antagonist, NBQX, was coadminis-

tered with estradiol, spinophilin levels were indis-

tinguishable from controls, indicating that AMPA/

kainate receptor activation by glutamate is critical for

estradiol-induced increases in spinophilin (and pre-

sumably dendritic spine number). AMPA/kainate

receptors play a crucial role in dendritic spine forma-

tion. Overexpression of the GluR2 subunit of the

AMPA/kainate receptor increases dendritic spine size

and density in hippocampal neurons, and initiates

spine formation on GABAergic interneurons, which

do not normally have spines (Passafaro et al., 2003).

AMPA/kainate receptor activation is also necessary

for maintenance of dendritic spines in the hippocam-

pus (McKinney et al., 1999). The use of spinophilin

protein level as an endpoint in this study does not dis-

tinguish whether estradiol is initiating spine forma-

tion or promoting spine maintenance, but the obser-

312 Todd et al.

Developmental Neurobiology. DOI 10.1002/dneu

vation that NBQX had no effect on baseline hypo-

thalamic spinophilin level in females or males im-

plies that in this region, activation of AMPA/kainate

receptors is involved in the former phenomenon. Par-

ticularly in males, the fact that NBQX does not affect

spinophilin level suggests that spine formation was

initiated in response to the prenatal testosterone

surge, and as such remains unaffected by subsequent

blockade of AMPA/kainate receptors. This contrasts

with females treated with a combination of estradiol

and NBQX, where the AMPA/kainate receptor antag-

onist can act to inhibit formation of spines at the time

of estradiol exposure. An important next step in these

studies will be to manipulate glutamate receptor acti-

vation in the perinatal period, and investigate whether

we see similar effects on dendritic spine number and

branching in the VMN as we do with testosterone. At

the moment, studies concerning glutamate receptor

activation in the developing brain and its effects on

dendritic morphology are largely excitotoxic studies.

We report here a potential role for activation of the

AMPA/kainate receptor in normal hormone-induced

differentiation of the brain.

In summary, we have shown that estradiol plays a

critical role in increasing total number of dendritic

spines and a crucial dendritic protein, spinophilin, in

the developing hypothalamus. Estradiol’s actions are

mediated by activation of AMPA/kainate receptors.

This may be one mechanism by which differential hor-

mone exposure establishes sex differences in the

developing hypothalamus. What is left unanswered by

these studies is the mechanism by which gonadal ste-

roids regulate sex differences in dendrite length and

branching. Focal adhesion kinase (FAK) has been

implicated in neurite extension and axonal branching

of cultured neurons (Ren et al., 2004; Rico et al.,

2004). We have found that newborn female hypothala-

mus has higher levels of FAK protein than males

(Speert and McCarthy, unpublished observation), sug-

gesting a promising avenue for future mechanistic ex-

ploration of sex differences in morphometry.

We thank Danielle Rodman for conscientious technical

assistance.

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