Neuropharmacology 38 (1999) 65–71
Chronic treatment with oestradiol does not alter in vitro LTP in
subfield CA1 of the female rat hippocampus
D.J. Barraclough 1, C.D. Ingram, M.W. Brown *
Department of Anatomy, Uni6ersity of Bristol, School of Medical Sciences Bristol BS8 1TD, UK
Accepted 8 September 1998
Abstract
Population excitatory post-synaptic potentials (pEPSPs) were recorded in vitro from subfield CA1 of the hippocampus of female
rats which had been ovariectomized and treated for 14 days with either oil or 17b-oestradiol (10 mg:day). The currents applied
to the Schaffer collateral-commissural input necessary to induce threshold, maximum and 50% maximum pEPSP responses did
not differ between groups. Application of trains of pulses (0.1–1 s; 100 Hz) evoked post-tetanic and long-term (\60 min)
potentiation of pEPSP responses, the magnitude of which was related to stimulus duration in both groups. However, the degree
of potentiation induced by near-threshold (0.1, 0.15 and 0.2 s) and saturating (1 s) stimuli did not differ between groups. Thus,
despite reports that oestradiol can modulate synaptic spine density and glutamatergic and GABAergic components of the inputs
to CA1, these data suggest that chronic oestradiol treatment has no effect on either the excitability or induction of LTP in the
Schaffer collateral-commissural-CA1 pathway. © 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Long-term potentiation; Hippocampal formation; Steroid; Oestradiol; Hormone replacement therapy
1. Introduction
There is increasing evidence to suggest that memory
function is linked to the female reproductive state. For
example, women suffer from a novel form of amnesia
during pregnancy (Condon, 1987; Brindle et al., 1991;
Condon et al., 1991; Sharp et al., 1993), while oestrogen
hormone replacement therapy (HRT) given after the
menopause or bilateral ovariectomy, is associated with
improved memory (e.g. Kampen and Sherwin, 1994).
The causes of these variations are unknown, although,
at least in part, the effects may be due to altered
function of the hippocampus. The hippocampus is
thought to be important for some aspects of memory
(Squire, 1992) and has been found to contain messenger
RNA for steroid hormone receptors, including oestro-
gen (Pelletier et al., 1988; Simerly et al., 1990; Tohgi et
al., 1995). Changes in the levels of gonadal steroids
associated with different reproductive states may alter
the function of the hippocampus as these hormones can
freely cross the blood–brain barrier and, accordingly,
reproductive status may influence memory function by
this means. Indeed, gonadal steroids such as oestradiol
can affect many aspects of neural function (McEwen et
al., 1979), in particular, the structure and function of
the hippocampus (Gould et al., 1991). Thus, after bilat-
eral ovariectomy, chronic treatment with oestradiol re-
sults in an increase in the density of both dendritic
spines and synapses in stratum radiatum of subfield
CA1 of the hippocampus (Gould et al., 1990; McEwen
and Woolley, 1994; Woolley and McEwen, 1994; Wool-
ley et al., 1996). Oestradiol treatment also increases
dendritic spine density on hippocampal cells grown in
culture (Murphy and Segal, 1996; Murphy et al., 1998).
Furthermore, systemic oestradiol treatment increases
the density of NMDA receptors (Weiland, 1992a), NM-
DAR1 immunofluorescence (Gazzaley et al., 1996), and
post-synaptic sensitivity to NMDA receptor-mediated
synaptic transmission (Woolley et al., 1997) in the same
region. Recent work has indicated that while a-type
oestrogen receptors appear to be located primarily in
* Corresponding author. Tel.: 44-117-9287408; fax. 44-117-
9291687; e-mail: m.w.brown@bristol.ac.uk.
1 Current address: Department of Neurobiology and Anatomy,
University of Rochester Medical Center, 601 Elmwood Avenue, Box
603, Rochester, NY14642, USA. Tel.: 1-716-2753627; fax: 1-
716-4428766; e-mail: dominic@cvs.rochester.edu
0028-3908:99:$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S00 2 8 -3908 (98 )00157 -9

D.J. Barraclough et al. : Neuropharmacology 38 (1999) 65–7166
scattered interneurones throughout the hippocampus
(Weiland et al., 1997), b-type oestrogen receptors and
their associated mRNA are more widespread and are
located in both interneurones and pyramidal cells in
subfields CA1 and CA2 (Li et al., 1997; Shughrue et al.,
1997).
The effect of oestradiol treatment on synaptic density
and on NMDA-mediated transmission suggests that
such treatment should influence NMDA-dependent
long-term potentiation (LTP) as found in hippocampal
subfield CA1. LTP is currently the foremost model of
vertebrate synaptic plasticity and the mechanism in-
volved may provide a physiological substrate of learn-
ing and memory (Bliss and Collingridge, 1993).
Oestradiol treatment has recently been reported to facil-
itate LTP in vivo (Cordoba-Montoya and Carrer, 1997)
and LTP has been reported to vary across the oestrous
cycle (Warren et al., 1995), but there have been no
reports of the chronic effects of oestradiol treatment on
LTP studied in vitro. Accordingly, this study set out to
explore how long-term oestradiol treatment might influ-
ence LTP in the Schaffer collateral:commissural-CA1
pathway. The effects of oestradiol were sought as the
strength of high frequency stimulation (length of stimu-
lus train) was varied from below threshold for the
induction of LTP to a level sufficient to produce maxi-
mal LTP.
2. Methods
Experiments were performed on 27 virgin adult fe-
male Wistar rats (12–17-weeks-old) maintained in a
light (14 h light; 10 h dark, lights on at 05:00 h) and
temperature (19–23°C) regulated environment, with
free access to food and water. Bilateral ovariectomy
was performed on virgin animals under gaseous anaes-
thesia; 1.5% Halothane in O2 (0.5 l:min):N2O (1 l:min).
The animals were then assigned pseudo-randomly to
receive oestradiol replacement or control treatment. At
17:00–18:00 h on the day of surgery and at the same
time on the following 13 days, rats in the oestradiol-
treated group received a single s.c. injection of 10 mg
17-b-oestradiol-3-benzoate (Sigma) in 200 ml olive oil
into the scruff of the neck. Animals in the control,
vehicle-treated group, received equivalent injections of
oil. The experimenter was unaware of the status of each
animal during the steroid treatment and subsequent
recordings. The code was only broken for the final
statistical analysis of results. At the time of bilateral
ovariectomy, the treatment groups did not differ signifi-
cantly in mean age or body weight.
Between 11:00 and 14:15 h on the day after the last
injection, the animals were anaesthetised with
halothane and decapitated by someone other than the
experimenter (so that the treatment-induced weight dif-
ference was not revealed to the experimenter). The
experimenter then prepared transverse hippocampal
slices (400 mm) from the mid-septotemporal region of
the hippocampus, using a multibladed tissue chopper.
The slices were maintained at 3090.5°C in an interface
recording chamber (Haas et al., 1979) under a moist
atmosphere of 95% O2 and 5% CO2. The slices were
perfused at 0.5–1 ml:min with artificial cerebrospinal
fluid of composition (in mM): NaCl 124, KCl 3,
NaHCO3 26, NaH2PO4 1.25, MgSO4 1, D-glucose 10,
CaCl2 2. The slices were allowed a minimum of 1 h to
equilibrate after entering the interface chamber before
any attempt was made to detect evoked potentials.
Each animal contributed only a single set of recordings
from one slice.
Fibres of the Schaffer collateral:commissural path-
way were stimulated by brief electrical pulses (0.1 ms)
applied via a bipolar twisted wire stimulating electrode
(55 mm diameter, insulated nichrome wire) located in
the stratum radiatum near the junction of subfields
CA1 and CA3. Evoked population excitatory post-
synaptic potentials (pEPSPs) were monitored using a
lagged glass microelectrode (1.8–3.2 MV) filled with
aqueous NaCl (1 M, containing 1% pontamine blue).
This recording electrode was placed in the stratum
radiatum of subfield CA1 and moved so as to optimise
the response to low frequency (0.033 Hz) stimulation. If
it was not possible to obtain a pEPSP with maximum
amplitude of at least 1 mV, the slice was rejected and
another was tested.
The evoked potential between the microelectrode and
an indifferent silver electrode was conventionally am-
plified (system frequency response: 90.5 dB from 1–
3600 Hz), monitored, and analogue-to-digitally
converted (1401plus, CED, Cambridge, UK) at a sam-
pling rate of 12.5 kHz, and stored on a computer
(Viglen 486PC, running CED Spike2). The slice was
stimulated at 0.033 Hz for a minimum of 30 min at an
intensity necessary to produce pEPSP responses with
amplitudes 50% that of the maximum pEPSP response
(50% max. pEPSP intensity). After this stabilisation
period, the minimum current required to generate the
maximum amplitude pEPSP was again determined as
were the currents necessary to produce threshold re-
sponses and 50% max. pEPSP responses. The slice was
then stimulated at 0.033 Hz at 50% max. pEPSP inten-
sity until the amplitude of the responses remained
within 10% of the mean amplitude for a 35 min period.
Next the slice was stimulated with the first of four high
frequency trains (100 Hz for 0.1, 0.15, 0.20 and 1 s,
respectively) at 50% max. pEPSP intensity. After each
high frequency train, the low frequency stimulation
(0.033 Hz) was resumed and responses were monitored
for 75 min. The next high frequency train was then
applied. Low frequency stimulation (0.033 Hz) was
applied between each train and for 70 min after the