140 J
dopaminergic DAergic pathways: 1 Tuberoinfundibular libitum. Animal procedures were approved by the Florida
DAergic TIDA neurons arise from throughout the arcuate State University Animal Care and Use Committee. Rats
nucleus and terminate on the primary capillary loops of the were bilaterally ovariectomized OVX under Halothane
long portal vessels within the external zone of the median anesthesia and subsequently divided into three groups. Ten
eminence ME [22]. Long portal vessels transport DA days following OVX, rats in group one estrogen-treated
released from
TIDA neurons
to the
AL. 2
received an injection of estrogen 20 mg, i.p., Sigma, St Tuberohypophyseal DAergic THDA neuron cell bodies
Louis, MO at 10.00 h on day zero and corn oil at 12.50 h are located in the rostral portion of the arcuate nucleus and
on day one. Rats in group two received an injection of terminate in both the intermediate IL and neural NL
estrogen 20 mg, i.p., Sigma at 10.00 h on day zero and an lobes of the pituitary gland [6]. III Periventricular-hypo-
injection of progesterone 5 mg, i.p., Sigma at 12.50 h on physeal DAergic PHDA neurons originate in the periven-
day one. Rats in group three received an injection of corn tricular nucleus of the hypothalamus and terminate exclu-
oil 200 ml, i.p., vehicle at 10.00 h on day zero and 12.50 sively in the IL [23,24]. DA released from THDA and
h on day one. Five rats from each treatment group were PHDA neurons is transported to the AL through short
sacrificed by rapid decapitation every 2 h from 09.00 to portal vessels.
21.00 h on day one. Rats decapitated at 13.00 h on day 1 The increasing concentration of estradiol in serum on
had received corn oil or progesterone 10 min before the afternoon of diestrus-2 of the estrous cycle of the rat
decapitation. The ME, AL, IL, and NL from each animal stimulates the proestrous increase of PRL in serum [40].
were dissected and individually stored in cryogenic vials Immunoneutralization of endogenous estrogen during the
with 250 ml of homogenization buffer [0.2 N perchloric afternoon of diestrus-2 prevents the increase of PRL on the
acid, 26 mM EGTA, 700 pM dihydroxybenzylamine afternoon of proestrus [20,40]. In addition to stimulating
DHBA, internal standard] at 2408C until day of assay. the secretion of PRL [41], estrogen diminishes and or
Trunk blood was collected, allowed to clot, centrifuged, reverses the inhibitory response of lactotrophs to DA
and serum was removed and stored at 2408C until the [8,12,19,34], recruits cells within the pituitary gland to
concentration of PRL was measured by RIA. produce [31] and secrete [7,18,26] PRL and increases
transcription of the PRL gene [30–32,36,38,50–53]. Un- like estrogen, the role of progesterone in the regulation of
2.2. HPLC-EC the secretion of PRL is not as thoroughly defined. How-
ever, when administered in concert with estrogen, proges- ME, AL, IL, and NL samples were thawed, homogen-
terone is a potent stimulator of the secretion of PRL [10]. ized, and sonicated. Twenty microliters of homogenate
It has previously been shown that the activity of were removed for protein assay. The remaining sample
dopaminergic neurons terminating in the ME [42] and the was centrifuged 10 min 10003g. The supernatant was
concentration of DA in portal vessels is decreased [5] prior filtered through a 0.2 mm nylon microfiltration unit Os-
to the peak of PRL on the afternoon of proestrus. In monics, Livermore, CA, and then placed into autosampler
addition, we previously reported that the concentration of vials.
DA and DOPAC decreases in the AL and IL, but not the The concentration of DA and DOPAC in each sample
NL prior to increased secretion of PRL on the afternoon of was measured using high performance liquid chromatog-
proestrus [16]. Moreover, it has been shown that ovarian raphy coupled to electrochemical detection HPLC-EC, as
steroids regulate biosynthesis [2,3,28,49,55] and release previously described [15–17,39]. Twenty microliters of
[13,14] of DA from TIDA neurons. each sample was injected by an autosampler WISP 710
In this study we have described the effects of ovarian Autosampler, Waters Corp., Milford, MA. Mobile phase
steroids on the activity of all three populations of hypo- consisting of 75 mM sodium dihydrogen phosphate mono-
thalamic neuroendocrine dopaminergic neurons. Changes hydrate EM Science, Gibbstown, NJ, 1.7 mM 1-octane
in the concentrations of DA and DOPAC in the ME, AL, sulfonic acid Fisher Scientific, 100 ml l triethylamine
IL, and NL and the concentration of PRL in serum were Aldrich, Milwaukee, WI, 25 mM EDTA Fisher Sci-
monitored following administration of estrogen and es- entific, 6 acetonitrile EMScience, titrated to pH 3.0
trogen plus progesterone in ovariectomized rats. with phosphoric acid Fisher Scientific, was delivered by
a dual piston pump Kratos Analytical Instruments, Ram- sey, NJ at 600 ml min. Water was purified on a Milli-Q
2. Materials and methods system Millipore, Bedford, MA to 18 MV and polished
with a C Sep-Pak Millipore.
18
2.1. Animals and in vivo steroid treatments Catecholamines were separated on a reverse phase C
18
column MD-150, Dimensions 15033 mm, particle size 3 Female Sprague–Dawley rats 200–250 g; Charles
mm, ESA inc., Chelmsford, MA, oxidized on a con- River, NC were housed under 12 h of illumination
ditioning cell E: 1300 mV, ESA 5010 Conditioning Cell, initiated at 06.00 h with water and rat chow available ad
ESA inc. and then reduced on a dual channel analytical
J .E. DeMaria et al. Brain Research 879 2000 139 –147
141
cell E : 255 mV, E : 2185 mV, ESA 5011 High 3. Results
1 2
Sensitivity Analytical Cell, ESA inc.. The change in current on the second analytical electrode was measured by
3.1. Concentration of PRL in serum a coulometric detector ESA Coulochem II, ESA Inc. and
recorded using Baseline 810 software Waters Corp.. DA The concentration of PRL in serum is shown in Fig. 1.
and DOPAC were identified on the basis of their peak retention times RT511.0 and 6.5 min, respectively. The
amount of catecholamine in each sample was estimated by comparison to the area under each peak generated by
known amounts of each catecholamine. The amount of DHBA RT57.5 min recovered was compared to the
amount of DHBA added as internal standard and corrected for any loss of sample usually ,5. The sensitivity of
the assay was 30 pg of DA and 33 pg of DOPAC.
2.3. Protein assay The amount of protein in each sample was measured
using a modified form of the Pierce BCA Protein Assay Kit Pierce, Rockford, IL. Homogenate 10 ml from
sonicated tissue samples was aliquoted into 96-well plates Corning, Corning, NY in duplicate and 200 ml of BCA
solution was added to each well. The plate was incubated for 30 min at 608C, and the absorbance of each well was
measured at 562 nm by a plate spectrophotometer Molec- ular Devices, Palo Alto, CA. Unknowns were compared
against standards of bovine serum albumin.
2.4. Radioimmunoassay RIA The concentration of PRL in serum was determined by
RIA as previously described [21] with materials supplied by Dr Albert F. Parlow and the National Hormone and
Pituitary Program. Serum concentrations of PRL were expressed as ng ml in terms of the rat PRL RP-3 standard
in assays whose sensitivity averaged 1 ng ml. The inter- assay coefficient of variation was 10 and the intra-assay
coefficient of variation was 5 for both assays.
2.5. Data analysis Differences in the DOPAC DA ratio, the concentration
of DA in the AL, and the concentration of PRL in serum within individual treatment groups were compared statisti-
cally using a one-way ANOVA. Tukey’s honestly signifi- cant difference test was used as a post-hoc test for all data
sets. Values are considered significant at P,0.05.
Differences in the DOPAC DA ratio, the concentration of DA in the AL, and the concentration of PRL in serum
between treatment groups were compared statistically
Fig. 1. The concentration of PRL in the serum of OVX A, estrogen-
using a two-way ANOVA. Tukey’s honestly significant
B, and estrogen plus progesterone- C treated rats. Each point
difference test was used as a post-hoc test for all data sets.
represents the mean6S.E.M. n55. Points with dissimilar letters are
Values are considered significant at P,0.05.
significantly different. P,0.05.
142 J
The concentration of PRL in serum of OVX rats is low and unchanging for most of the day with the exception of a
25 increase from baseline P,0.05 occurring between 15.00 and 17.00 h Fig. 1A. Unlike the steady and
relatively unchanging levels of PRL in OVX rats, replace- ment of ovarian steroids significantly elevates baseline
secretion of PRL. The concentration of PRL in serum of estrogen-treated rats is significantly increased P,0.05 by
15.00 h, peaks by 17.00 h at a concentration three-fold greater than baseline, and returns to low levels by 21.00 h
Fig. 1B. The concentration of PRL in serum of estrogen plus progesterone-treated rats significantly increases by
19.00 h at a concentration five-fold greater than baseline, and diminishes to low levels thereafter Figs. 1C.
3.2. TIDA neuronal activity: DOPAC DA turnover in the ME
The turnover of DA in the ME of OVX A, estrogen- B and estrogen plus progesterone- C treated rats is
shown in Fig. 2 as the mean DOPAC DA ratio6S.E.M. In this and subsequent figures the serum PRL concentrations
shown in Fig. 1 are duplicated for comparison. DA turnover is significantly increased P,0.05 by 13.00 h in
OVX rats Fig. 2A, diminishes to low levels by 15.00 h, returns to baseline by 17.00 h and subsequently increases
by 21.00 h Fig. 2A. Injection of estrogen to OVX rats decreases the turnover of DA in the ME between 09.00 and
11.00 h P,0.05, delays the increase in DA turnover seen in OVX rats until 15.00 h concomitant with the initial
increase in the secretion of PRL, and prevents the late evening increase in turnover of DA observed in OVX rats
by 21.00 h Fig. 2B. The DOPAC DA ratio in estrogen plus progesterone-treated rats increases significantly P,
0.05 by 13.00 h similar to OVX rats, but unlike OVX rats is depressed through the late afternoon coincident with an
increase in serum PRL Fig. 2C.
3.3. PHDA neuronal activity: DOPAC DA turnover in the IL
The turnover of DA in the IL of OVX rats increases by 11.00 h P,0.05, returns to low levels by 17.00 h,
coincident with the increase of serum PRL, and again increases P,0.05 through 21.00 h Fig. 3A. In contrast
to the turnover of DA in the IL of OVX rats, the turnover of DA in the IL of estrogen-treated rats is unchanged
between 09.00 and 15.00 h Fig. 3B. The turnover of DA in the IL of estrogen-treated rats subsequently increases
P,0.05 by 17.00 h, coincident with the increase of serum PRL, and returns to low levels concomitant with the
Fig. 2. The concentration of PRL in serum and the DOPAC DA ratio in the median eminence of OVX A, estrogen- B, and estrogen plus
levels of PRL in serum Fig. 3B. This is nearly a mirror-
progesterone- C treated rats. Circles indicate mean concentration of
image of the pattern of PRL secretion in OVX rats.
PRL in serum6S.E.M. Significant differences in the concentration of
Estrogen plus progesterone treatment significantly in-
PRL are as indicated in Fig. 1. Squares are mean DOPAC DA
creases P,0.05 DA turnover by 13.00 h, which then
ratio6S.E.M. Points with different letters are significantly different P,
decreases thereafter through 17.00 h concomitant with the
0.05.
J .E. DeMaria et al. Brain Research 879 2000 139 –147
143
initiation of the increase of PRL in serum Fig. 3C. This pattern is similar in timing, but differs in magnitude from
that of OVX rats. However, unlike OVX rats, the turnover of DA in estrogen plus progesterone-treated rats remains
low and unchanging in the IL thereafter Fig. 3C.
3.4. THDA neuronal activity: DOPAC DA turnover in the NL
The turnover of DA in the NL of OVX rats increases P,0.05 twice; first by 13.00 h, prior to the increase of
PRL in serum, and the second by 19.00 h, during the waning phase of the peak of serum PRL Fig. 4A. Unlike
OVX rats, estrogen treatment causes a decrease P,0.05 in the activity of THDA neurons by 11.00 h. However,
similar to changes in OVX rats, the turnover of DA in the NL of estrogen plus progesterone-treated rats returns to
high levels by 13.00 h Fig. 4B. The turnover of DA in the NL of estrogen-treated rats decreases P,0.05 steadi-
ly thereafter to low levels at 21.00 h coincident with the increase of PRL in serum Fig. 4B. During the morning,
estrogen plus progesterone-treated rats present a DA turnover profile similar to that of the estrogen-treated rats
Figs. 4B, C. However, following injection of progester- one at 13.00 h, DA turnover is significantly P,0.05
depressed and remains low through 21.00 h Fig. 4C, prior to and coincident with the increase of PRL in serum.
3.5. Concentration of DA in the anterior lobe The concentration of DA in the AL of OVX rats
increases slightly from 09.00 to 15.00 h Fig. 5A. Coincident with the slight increase of PRL in serum at
17.00 h, the concentration of DA in the AL decreases to morning levels P,0.05 and remains low until it increases
significantly P,0.05 again at 21.00 h Fig. 5A. Al- though of greater magnitude in estrogen-treated rats, the
patterns of DA in the AL are similar between 09.00 h and 13.00 h in the OVX and estrogen-treated groups Fig. 5A,
B. In both groups, the concentration of DA in the AL significantly decreases P,0.05 coincident with the in-
crease in the concentration of PRL in serum at 15.00 h and remains low through 19.00 h Fig. 5B. Concomitant with
the return of PRL to baseline levels, the concentration of DA in the AL increases to levels similar to those of the
morning Fig. 5B. Similar to the concentration of DA in the AL of OVX rats, the concentration of DA in the AL of
estrogen plus progesterone-treated rats is unchanged through 17.00 h and then subsequently decreases through
21.00 h P,0.05. However, in estrogen plus progester-
Fig. 3. The concentration of PRL in serum and the DOPAC DA ratio in
one-treated rats, the rate of decrease is greater than that
the intermediate lobe of OVX A, estrogen- B, and estrogen plus
seen in the AL of OVX rats. The concentration of DA in
progesterone- C treated rats. Circles indicate mean concentration of
the AL of estrogen and progesterone-treated rats remains
PRL in serum6S.E.M. Significant differences in the concentration of
low through 21.00 h while the secretion of PRL increases
PRL are as indicated in Fig. 1. Squares are mean DOPAC DA
Fig. 5C; P,0.05, unlike the concentration of DA in the
ratio6S.E.M. Points with different letters are significantly different P, 0.05.
AL of estrogen-treated rats, which increased at 21.00 h.
144 J
4. Discussion
