Neuroscience & Medicine, 2013, 4, 134-139
http://dx.doi.org/10.4236/nm.2013.43021 Published Online September 2013 (http://www.scirp.org/journal/nm)
Unilateral Fimbria/Fornix Transection Prevents the
Synaptoplastic Effect of Dehydroepiandrosterone in the
Hippocampus of Female, but Not Male, Rats
Ari L. Mendell1, Neil J. MacLusky1*, Csaba Leranth2,3
1Department of Biomedical Sciences, Ontario Vete rinary Colleg e, University of Guelph, Guelph, Canada; 2Department of Obstetrics,
Gynecology & Reproductive Sciences, Yale University School of Medicine, New Haven, USA; 3 Department of Neurobiology, Yale
University School of Medicine, New Haven, USA.
Received May 29th, 2013; revised June 25th, 2013; accepted July 4th, 2013
Copyright © 2013 Ari L. Mendell et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Dehydroepiandrosterone (DHEA), the most abundant adrenal androgen in primates, is also synthesized from cholesterol
in the brain. Like testosterone, DHEA induces spine synapse formation in the hippocampus. In female rats, this re-
sponse is blocked by co-administration of an inhibitor of aromatase, the enzyme responsible for estrogen biosynthesis.
In males, by contrast, the hippocampal synaptic response to DHEA is unaffected by treatment with an aromatase in-
hibitor. We hypothesized that this sex difference might reflect differential dependence of the hippocampal responses on
subcortical afferents from the basal forebrain. To test this hypothesis, we examined the effects of unilateral fimbria/
fornix transection (FFX) on DHEA-induced synapse formation in the cornu ammonis 1 (CA1) hippocampal subfield of
gonadectomized female and male rats. In ovariectomized females, CA1 spine synapse density after DHEA treatment
was reduced by more than 60% ipsilateral to FFX. In males, however, unilateral FFX transection had no effect on spine
synapse density after DHEA treatment. These results suggest that sex differences in the dependence on local estrogen
biosynthesis of the CA1 synaptic resp onse to an drog en may at least in part be th e resu lt of sex differences in the relative
contributions of afferents to the hippocampus from the basal forebrain.
Keywords: Dehydroepiandrosterone; Subcortical Mediation; Synaptic Plasticity; Hippocampus; Sex Differences
1. Introduction
Gonadal hormones play an essential role in the mainte-
nance of spine synapses in the CA1 subfield of the hip-
pocampus. Both estrogens [1-4] and androgens [5] regu-
late hippocampal spine synapse density in female rats,
whereas only androgens regulate synapse density in
males [6]. The mechanisms by which gonadal hormones
exert their actions on synapses in the hippocampus are
incompletely characterized.
Dehydroepiandrosterone (DHEA) is the most abundant
circulating androgen produced by the adrenal glands in
primates, and there is a significant declin e in DHEA lev-
els with age [7,8]. A number of studies have indicated
that DHEA is also synthesized in the CNS [9-11] where
it may have direct effects on neuronal activity [12]. Al-
though it has relatively weak androgenic activity in non-
neural androgen target tissues, DHEA has been reported
to have positive effects on cognitive function [13], as
well as mood [14]. It has also been demonstrated that
DHEA has the ability to increase hippocampal spine
synapse density in gonadectomized female [15] and male
rats [16]. The mechanisms underlying these effects re-
main poorly understood .
In females, the synaptic effects of DHEA are blocked
by the aromatase inhibitor letrozole [15]. This suggests
that DHEA effects in the female hippocampus are similar
to those of testosterone, in which they are dependent on
conversion to estrogen [17]. In gonadectomized male rats,
however, letrozole has no effect on DHEA-induced in-
creases in hippocampal spine synapses [16], indicating
that there are sex differences in the mechanisms by
which these responses are produced.
Previous reports have shown that in females the effects
of estrogen [2,18] on the hippocampus are dependent on
*Corresponding a uthor.

Unilateral Fimbria/Fornix Transection Prevents the Synaptoplastic Effect of
Dehydroepiandroster o ne i n t he H ip p ocampus of Female, but Not Male, Rats 135
input from subcortical cholinergic neurons. Thus, tran-
section of the fimbria/fornix (FF) [2] or destruction of
cholinergic neurons in the medial septum and diagonal
band of Broca (MSDB) by intraventricular injection of
192 IgG-saporin [19] abolish the effects of estrogen on
CA1 spine synapse density. The MSDB region of the
brain contains high concentrations of aromatase [20]. We
hypothesized that the sex difference in the dependence
on aromatase activity of the hippocampal synaptic effects
of DHEA might at least in part be due to differences be-
tween males and females in the relative contributions
from subcortical input. If so, we might expect a profound
sex difference in the effects of eliminating this input on
the hippocampal synaptic response to DHEA. To test this
hypothesis, we examined the effect of unilateral fimbria/
fornix transection (FFX) on the effects of DHEA treat-
ment on hippocampal synaptic plasticity, in gonadecto-
mized male and female rats.
2. Materials and Methods
2.1. Animal Subjects
Adult male and female Sprague Dawley rats (60 - 70 days
old) were obtained from Charles River, Wilmington, MA,
USA. Animals were maintained in individual cages with
water and regular rat chow available ad libitu m, and kept
on a 12-hour light/dark cycle. All experiments included
as a part of this study conformed to relevant University
Animal Care and Use Committee guidelines and were in
compliance with both the National Institutes of Health
Guide for the Care and Use of Laboratory Animals and
the guidelines of the Canadian Council of Animal Care.
2.2. Surgical Procedures and Hormone
Treatment
Three male rats and three female rats were included in
this study. All rats were anesthetized for surgery using a
ketamine cocktail [3 ml/kg, i.m.; ketamine (25 mg/ml) +
xylazine (1.2 mg/ml) + acepromazine (0.03 mg/ml) in
saline (0.9%)].
The female rats were ovariectomized via small dorsal
flank incisions, and all males were orchidectomized. All
animals then underwent surgical unilateral transection of
the fimbria/fornix, as previously described [2,17]. Briefly,
the animals were fixed in a stereotaxic apparatus (David
Kopf Instruments, Tujunga, CA, USA), and, following
dorsal penetration and subsequent aspiration of the over-
lying cortical areas and corpus callosum, the right fim-
bria/fornix was completely transected under visual con-
trol. One week after surgery, all female and male rats
received DHEA treatment (Sigma Chemical Co. St.
Louis, MO), given in the form of two subcutaneous in-
jections (1 mg/day, in sesame oil) separated by 24 hours.
For this study, the intact side of the brain, contralateral
to fimbria/fornix transection, was used as the control. It
has previously been shown that DHEA administration
induces spine syn apses in both males and females to lev-
els indistinguishable from intact controls [15,16]. It has
also been shown that unilateral transection of the fimbria/
fornix has no significant effect on the synaptic response
to testosterone of the contralateral hippocampus [17].
Hence the response contralateral to FFX can be used as a
matched, individual control for the effects of the steroid,
in each animal. In the present study, the average spine
synapse density we observed for female rats contralateral
to FFX very closely matched previous observations of
ovariectomized females treated with DHEA [16] or tes-
tosterone [5], while the spine synapse density numbers
we observed ipsilateral to FFX were comparable, but
slightly lower than previously published density values
for ovariectomized females [2,5,16]. For the males, the
spine synapse density values on both sides of the hippo-
campus very closely matched previously published re-
ports of spine synapse density in either intact males, or
orchidectomized males following DHEA [16] or testos-
terone [6] treatment.
2.3. Tissue Processing
Two days following the second injection of DHEA, rats
were killed under deep ether anesthesia by transcardial
perfusion of heparinized saline, followed by a fixative
containing 4% paraformaldehyde and 0.1% glutaralde-
hyde in 0.1 M phosphate buffer (pH 7.35). The descend-
ing aorta was firmly clamped for the course of the perfu-
sion, in order to prevent access of the fixative to the
lower half of the body. Brains were removed and post-
fixed overnight in the same fixative used for perfusion,
without the glutaraldehyde. The hippocampi were dis-
sected out, and coronal vibratome sections (100 m)
were cut. The sections were then postfixed in 1% os-
mium tetroxide (30 min), dehydrated in ethanol (the 70%
ethanol contained 1% uranyl acetate) for 30 min, and flat
embedded in Araldite.
2.4. Synapse Counts
Spine synapse density was calculated using unbiased
stereological methods, as previously described [2,15,16].
Briefly, in order to assess any potential changes in the
volume of the tissue, a correction factor was first calcu-
lated, assuming that the treatment and surgical proce-
dures had no effect on the total number of pyramidal
cells in the hippocampus [16,21]. In all hippocampi, six
or seven disector pairs (pairs of adjacent 2 m toluidine
blue-stained semithin sections mounted on slides) were
analyzed using the technique of Braendgaard and Gun-
dersen [22]. The cell density value (D) for pyramidal

Unilateral Fimbria/Fornix Transection Prevents the Synaptoplastic Effect of
Dehydroepiandrosterone in t he H ipp oc ampus of Female, but Not Male, Rats
136
cells was calculated using the formula D = N/sT, where
N is the mean disector score across all sampling windows,
T is the thickness of the sections (2 m), and s is the unit
area of the window. Based on these values, a dimen-
sionless volume correction factor (kv) was introduced: kv
= D/D1, where D1 is the mean pyramidal cell density
across the groups of hippocampi.
Thereafter, pairs of consecutive serial ultrathin sec-
tions (reference and look-up) were cut from the Aral-
dite-embedded vibratome sections, from an area located
between the middle and upper third of the stratum radia-
tum of CA1 ( 300 - 50 0 m from the pyramidal cell layer;
for precise location, see [5]), and collected on Formvar-
coated single-slot grids. Digitized images were taken at a
magnification of ×9900 using a Tecnai 12 transmission
electron microscope furnished with an AMT Advantage
4.00 HR/HR-B CCD camera system, with the observer
blinded to the experimental treatment. At least five neu-
ropil fields were photographed on each electron micro-
scopic grid. With at least eight grids from each vibratome
section (five vibratome sections from each hippocampus
taken from different areas along the septo-temporal axis)
containing a minimum of two pairs of consecutive, serial
ultrathin sections, each animal provided a minimum of 5
× 8 × 5 = 200 or more neuropil fields. Identical regions
in reference and look-up sections were identified using
easily recognizable landmarks such as large myelinated
fibers, large dendrites or blood vessels with distinct ori-
entations, which were not changed significantly between
neighbouring sections. Areas occupied by potentially
interfering structures such as large dendrites, glial cells,
or capillaries were avoided, or subtracted from measured
areas using the NIH Scion Image software.
In order to obtain a comparable number of synaptic
numbers, unbiased for possible changes in synapse size,
the disector technique was used [23]. The digitized elec-
tron micrographs were printed using a laser printer, and a
reference grid superimposed onto the micrographs was
used to aid in calculation of spine synapse density. The
disector volume (volume of reference used for density
calculations) was the unit area of the reference grid mul-
tiplied by the distance between the upper faces of the
reference and look-up sections [22]. Section thickness
(average 0.075 m) was determined using the electron
scattering technique [24]. The calculated spine synapse
density values were then divided by the kv. This correc-
tion provided a normalized spine synapse density, ac-
counting for the density of pyramidal cells and for any
changes in hippocampal volume. Prior to data analysis,
the printed pictures were coded to ensure blind synapse
counting, and this code was not brok en until the analysis
was completed. Only those spine synapses that were
present in the reference section but not in the look-up
section, or vice versa, were counted [4].
2.5. Statistical Analysis
Spine synapse density counts were averaged for each
animal on each side of the hippocampus, and an overall
mean was then taken for each group. Values are pre-
sented as mean  S.E.M. Two-way ANOVA was used to
statistically analyze the data, followed by unequal vari-
ance, two-tailed t-tests for comparison of individual
group means. All statistical tests were performed using
Statview statistical software (SAS institute, Cary, NC,
USA), and statistical significance was set to p < 0.05
(two-tailed).
3. Results
The effect of DHEA on spine synapse density in the
stratum radiatum of CA1 following unilateral FFX in
females and males is demonstrated in Figure 1. Two-
Figure 1. Spine synapses in gonadectomized female and
male rats. Bar graphs show the number of spine synapses
(A) and the spine synapse density (B) in the CA1 stratum
radiatum of gonadectomized, unilaterally fimbria/fornix
transected (FFX), dehydroepiandrosterone (DHEA)-treated
female and male rats. DHEA treatment increased spine
synapses to normal and approximately equal levels in both
the contralateral and ipsilateral sides of the hippocampus to
FFX in male rats, and the contralateral side to FFX in the
females. The spine synapse density in the ipsilateral side to
FFX in females was not increased by DHEA tre atment, and
was significantly lower (~63.8%) compared to the contra-
lateral side [*represents P < 0.05. Bars represent mean 
S.E.M.].

Unilateral Fimbria/Fornix Transection Prevents the Synaptoplastic Effect of
Dehydroepiandroster o ne i n t he H ip p ocampus of Female, but Not Male, Rats 137
way ANOVA showed significant differences in synapse
density caused by FFX side (F = 35.863, p = 0.0003) and
sex (F = 22.261, p = 0.0015), as well as a significant in-
teraction effect of FFX side and sex (F = 30.426, p =
0.0006). In ovariectomized females, the number of CA1
spine synapses ipsilateral to FFX was significantly lower
(63.8%; t-test: p = 0.012; 1.577 × 109  0.043 × 109 syn-
apses) than the contralateral side (4.361 × 109  0.322 ×
109 synapses) following DHEA treatment. For orchidec-
tomized males receiving the same surgical procedure and
DHEA treatment, the numbers of spine synapses on both
sides of the hippocampus were indistinguishable, with no
significant difference observed (t-test: p = 0.771; 4.167 ×
109  0.319 × 109 on the contralateral side; 4.053 × 109 
0.164 × 109 on the ipsilateral side).
4. Discussion
These observations expand previous findings showing that
DHEA treatment increases hippocampal spine synapse
density, in both male [16] and female rats [15]. Here, we
demonstrate that the positive effect of DHEA on spine
synapses in hippocampal area CA1 in females is de-
pendent on afferents from subcortical structures via the
FF pathway. It is well established that estrogen increases
the density of hippocampal spine synapses in female rats
[1,3-5] as well as non-human primates [25,26]. In rats,
these responses are highly de- pendent on afferents from
estrogen-sensitive subcortical structures, including the
supramammillary area, MSDB and median raphe nuclei
[2,18]. Androgens also promote synaptogenic responses
in female rats, but the effects of both testosterone and
DHEA in females are almost completely blocked by pre-
treatment with an aromatase inhibitor [5,15]. Together
with the observations from the present study, these re-
sults suggest that the synaptic effect of DHEA in the fe-
male hippocampus may occur primarily through aroma-
tization of DHEA in the brain and effects of estrogen that
require intact subcortical afferents arriving in the hippo-
campus via the FF.
Previous work has demonstrated that the dose of
DHEA used here (1 mg/rat s.c., daily for 2 days) pro-
motes full recovery of spine synapses in the stratum ra-
diatum of CA1 of the hippocampus in orchidectomized
male rats, even though in males estradiol has no signifi-
cant effect on hippocampal spine synapse density [6,16].
The present data show that, in contrast to the situation in
females, the response to DHEA is unaffected by unilat-
eral transection of the fimbria-fornix, suggesting that the
mechanism by which DHEA produces its synaptic effects
in males not only does not involve intermediate estrogen
biosynthesis, it is also independent of subcortical media-
tion.
One hypothesis to explain these findings might be that,
in males, the actions of androgens on synapse density are
mediated locally, within the hippo campus itself, while in
females these responses involve a much larger contribu-
tion from estrogen-sensitive basal forebrain cholinergic
neurons. While such a model would be consistent with
the present data, other studies suggest that additional
factors are probably also involved. A large body of evi-
dence suggests that at least some of the effects of estra-
diol on hippocampal synaptic plasticity in females are
mediated via estrogen receptors located within the hip-
pocampus itself [27-29]. Previous work has demonstrated
that the synaptic effects of testosterone are partially
blocked by FFX in male rats [17]. Thus, at least for tes-
tosterone, some of the syn aptic effects in males appear to
involve sub-cortical afferent input. Testosterone is known
to regulate the basal forebrain cholinergic system: thus,
in orchidectomized males testosterone, but not estradiol,
increases choline acetyltransferase activity in the medial
preoptic area [30]. The absence of a response to estradiol
in males could potentially represent contributions from
both sexual differentiation of the responses to estradiol of
the basal forebrain cholinergic system and the lack of
local, rapid signaling effects of estrad iol in the male hip-
pocampus [27,28,31]. Conversely, in males testosterone
could modulate hippocampal synaptic plasticity via both
modulation of afferent cholinergic input [17], and effects
mediated via local membrane androgen receptors, which
are present throughout the hippocampus [32,33] and have
been localized to pre-terminal axons, axon terminals, and
dendritic spines in CA1 [33]. Such a mechanism would
be consistent with the observation that testosterone in-
duced increases in hippocampal spine synapse density
are partially blocked by FF transection [17].
Why then are the effects of DHEA in males unaffected
by FFX? There are two possible explanations. Since the
androgenic potency of DHEA is lower than that of tes-
tosterone, its effects on the basal forebrain cholinergic
system may be weaker than those of testosterone, which
could explain why the hippocampal synaptic response to
DHEA is unaffected by FFX. Alternatively, DHEA and
testosterone may exert their effects via different receptor
mechanisms. Several studies have suggested that DHEA
and its sulfate conjugate can act on the hippocampus via
receptors distinct from those mediating the actions of
testosterone and estradiol [34], including direct effects on
mGluR5 metabotropic glutamate receptors in the hippo-
campus [34]. If such mechanisms are primarily responsi-
ble for mediating the synaptic effects of DHEA in males,
this could explain why th e response to DHEA in males is
unaffected by FF transection. Further work on the rela-
tive effects of DHEA on different components of the ba-
sal forebrain-hippocampal circuitry will be required to test
this hypothesis.

Unilateral Fimbria/Fornix Transection Prevents the Synaptoplastic Effect of
Dehydroepiandrosterone in t he H ipp oc ampus of Female, but Not Male, Rats
138
5. Conclusion
This study demonstrates that the effects of DHEA on
spine synapses in the CA1 subfield of the hippocampus
may involve different mechanisms in female and male
rats. The effects of the weak androgen DHEA on CA1
synapse density in the male are unaffected by FF transec-
tion, whereas the effects of this steroid on the same re-
gion of the female are largely dependent on subcortical
mediation. These findings highlight sex differences in the
importance of afferent input from subcortical structures
in regulating hippocampal synaptic plasticity
6. Acknowledgements
This work was supported by Natural Sciences and Engi-
neering Research Council of Canada Discovery Grant
197293-2007 (NJM) and National Institutes of Health
Grant ES017013 (CL). ALM is a recipient of an Ontario
Graduate Studentship.
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