Materials and methods atlas of the rat brain [15]: nucleus tractus solitarius NTS,
106 M
significant increases in parasympathetic tone and barorefl- daily subcutaneous injections of either estrogen 17b-
ex sensitivity were observed following bilateral injection estradiol-3-sulfate, water soluble form; 5 mg kg; Sigma
of estrogen into the nucleus tractus solitarius and nucleus Chemical, St. Louis, MO, USA; n555 or physiological
ambiguus. Conversely, sympathetic tone was significantly saline 0.9; n553 over an additional 7 days. This
decreased following injection of estrogen into the nucleus estrogen replacement regimen produces sustained serum
tractus solitarius and rostral ventrolateral medulla [20]. In estrogen levels similar to that in an intact rat during
general, the apparent role of estrogen in cardiovascular proestrous 40–50 pg ml [19].
nuclei is to produce a shift in sympatho-vagal balance On the day of experimentation, animals were anaesthet-
toward the parasympathetic limb resulting in an enhance- ized with sodium thiobutabarbital Inactin; RBI, Natick,
ment of baroreflex function which has been shown to MA, USA; 100 mg kg; ip and instrumented to record
provide antifibrillatory protection on the heart [5]. blood pressure, heart rate and efferent vagal and renal
Investigation into the role of estrogen in cardiovascular nerve activities as described previously [17–19]. Blood
nuclei of female rats has been limited. In estrogen-replaced pressure and heart rate data were displayed and analysed
ovariectomized female rats, the cardiovascular responses to using
POLYVIEW PRO
32 data acquisition software Grass; glutamate injection in the bed nucleus of the stria ter-
Warwick, RI, USA. The multi-unit nerve activity was minalis were significantly enhanced when preceded by
amplified by a Grass model P55 preamplifier Grass with injection of estrogen [6]. Similar injections of estrogen in
a 100-Hz to 3-kHz bandpass and 60-Hz notch filter and non-estrogen replaced ovariectomized female rats had no
then displayed using the
POLYVIEW PRO
32 data acquisition effect on the glutamate-evoked responses [6] suggesting
system Grass. A sampling rate of 2000 s was used to that the effects of estrogen within the central nervous
record the raw nerve signal. Non-biological noise was system may in fact be dependent on levels of circulating
obtained by recording from the nerve after death and was estrogen in the peripheral vasculature. The present in-
subtracted from the nerve signal recorded during the vestigation will attempt to expand our understanding of the
experiment. A venous catheter PE-10 was inserted into role of estrogen within the central nervous system as well
the right femoral vein to permit administration of drugs. as the contribution of peripheral estrogen levels to the
Respiration was facilitated via the insertion of an endotra- neuromodulatory function of estrogen. Specifically, the
cheal tube and ventilation with room air on a Harvard effects of estrogen injection into several central autonomic
rodent ventilator 65 strokes per min; 2.5-ml tidal volume. nuclei on autonomic tone and baroreflex function will be
examined in estrogen-replaced and saline-replaced ovariec- 2.2. Central injections
tomized female rats. Changes in autonomic tone will be assessed by monitoring efferent vagal and renal nerve
Animals n593 were placed in a David Kopf Tujunga, activities. Baroreflex function will be assessed using
CA, USA stereotaxic frame and small burr holes were intravenous administration of a single dose of phenylep-
drilled bilaterally through the skull to permit stereotaxic hrine hydrochloride. Finally, co-injection of a potent and
insertion of a 30-gauge stainless steel, 1-ml Hamilton selective estrogen receptor antagonist, ICI 182,780 [10,25],
micro-syringe. A bilateral injection of either estrogen with estrogen into brain nuclei will be used to verify the
17b-estradiol-3-sulphate; Sigma-Aldrich; St. Louis, MO, specificity and receptor-mediated action of estrogen on
USA; 0.5 mM; 100 nl per side or a combination of autonomic tone and baroreflex function.
estrogen 0.5 mM and ICI 182,780 Tocris; Bristol, UK; 1 pM; 100 nl per side was made into each of the following
nuclei according to coordinates obtained from a stereotaxic
2. Materials and methods atlas of the rat brain [15]: nucleus tractus solitarius NTS,
nucleus ambiguus Amb, rostral ventrolateral medulla All experiments were carried out in accordance with the
RVLM, parabrachial nucleus PBN, central nucleus of guidelines of the Canadian Council on Animal Care and
the amygdala CNA and insular cortex IC. Control were approved by the University of Prince Edward Island
injections of physiological saline 0.9; 100 nl per side Animal Care Committee.
and ICI 182,780 were made into each nucleus prior to injection of estrogen and ICI 182,780 estrogen respective-
2.1. General surgical procedures ly.
Experiments were performed on a total of 108 female 2.3. Intrathecal injections
Sprague–Dawley rats Charles Rivers; Montreal, PQ weighing 250–275 g. All rats were anaesthetized with
A separate group of animals n515 was instrumented sodium pentobarbitol somnitol; 17 mg kg and valium
for the intrathecal administration of either estrogen 0.5 0.4 mg kg; ip and a bilateral ovariectomy was performed
mM; 1 ml or a combination of ICI 182,780 1 pM; 1 ml by ligation and dissection of the ovaries. A recovery period
and estrogen 0.5 mM by the insertion of a catheter PE of 1 week was permitted prior to the commencement of
10 through a small puncture in the atlanto-occipital
M .C. Saleh et al. Brain Research 879 2000 105 –114
107
membrane. The catheter was advanced caudally through Changes in nerve activity and phenylephrine-induced
the subarachnoid space placing the tip of the catheter cardiovascular responses were analysed by two-way
immediately rostral to the lumbar enlargement T To ANOVA for repeated measures followed by a Student–
12 .
inject, the catheter was slowly withdrawn over a distance Newman–Keul’s post hoc analysis Sigma Stat. In all
of 5 cm while making 200-nl injections at 1-cm intervals cases, differences were considered significant if P0.05.
between the lumbar enlargement T and the base of the
12
cervical spinal cord T Control injections of saline 2.6. Histology
1 .
0.9; 1 ml preceded intrathecal estrogen injection. Control injections of saline ICI 182,780 0.9; 1 pM; 1
At the end of each experiment animals were perfused ml preceded intrathecal injection of estrogen ICI 182,780.
transcardially with 0.9 saline followed by 10 formalin. The brains were removed and stored in 10 formalin until
2.4. Baroreflex testing and autonomic tone the location of micro-syringe tracks could be verified
measurements histologically in thionin-stained coronal sections 6100
mm. For verification of intrathecal catheter placement, 1 In all animals, the baroreflex was evoked by the
ml of blue ink was injected using the protocol described intravenous administration of phenylephrine hydrochloride
above and the presence of ink in the region of the base of PE; 0.1 mg kg 5 min prior to, and 5, 30, 60, 90 and 120
the cervical spinal cord T to the rostral tip of the lumbar
1
min following the central injection of estrogen and 5 min enlargement T was confirmed.
12
prior to, and 5, 30, and 60 min following the central injection of estrogen ICI 182, 780. As well, the baroreflex
was tested 5 min prior to and 5 and 30 min following